Variable Expression of Myoglobin Among the Hemoglobinless Antarctic Icefishes (Channichthyidae͞oxygen Transport͞phylogenetics)

Proc. Natl. Acad. Sci. USA Vol. 94, pp. 3420–3424, April 1997 Physiology

Variable expression of myoglobin among the hemoglobinless Antarctic icefishes (Channichthyidae͞oxygen transport͞phylogenetics)

BRUCE D. SIDELL*†‡,MICHAEL E. VAYDA*†,DEENA J. SMALL†,THOMAS J. MOYLAN*, RICHARD L. LONDRAVILLE*§, MENG-LAN YUAN†¶,KENNETH J. RODNICK*ʈ,ZOE A. EPPLEY*, AND LORI COSTELLO†

*School of Marine Sciences and Department of Zoology, †Department of Biochemistry, Microbiology and Molecular Biology, University of Maine, Orono, ME 04469

Communicated by George N. Somero, Stanford University, Pacific Grove, CA, January 13, 1997 (received for review October 24, 1996)

ABSTRACT The important intracellular oxygen-binding Icefishes exhibit several unique physiological features. Car- protein, myoglobin (Mb), is thought to be absent from oxi- diovascular adaptations to compensate for lack of hemoglobin dative muscle tissues of the family of hemoglobinless Antarctic in the circulation include lower blood viscosity, increased heart icefishes, Channichthyidae. Within this family of fishes, which size, greater cardiac output, and increased blood volume is endemic to the Southern Ocean surrounding Antarctica, compared with their red-blooded notothenioid relatives (2, there exist 15 known species and 11 genera. To date, we have 14–16). Combined with the high aqueous solubility of oxygen examined eight species of icefish (representing seven genera) at severely cold body temperature, these cardiovascular fea- using immunoblot analyses. Results indicate that Mb is tures are considered necessary to ensure that tissues obtain present in heart ventricles from five of these species of icefish. adequate amounts of oxygen carried in physical solution by the Mb is absent from heart auricle and oxidative skeletal muscle plasma. Although enhancing circulatory delivery of oxygen, of all species. We have identified a 0.9-kb mRNA in Mb- these adaptations do not assist intracellular movement of expressing species that hybridizes with a Mb cDNA probe oxygen within tissues. Heart ventricle (7, 14) and aerobic from the closely related red-blooded Antarctic nototheniid skeletal muscle (17) of icefishes contain among the highest fish, Notothenia coriiceps. In confirmation that the 0.9-kb mitochondrial densities (Ͼ40% of cell volume) of any verte- mRNA encodes Mb, we report the full-length Mb cDNA brate tissues. Consistent with this robust mitochondrial pop- sequence of the ocellated icefish, Chionodraco rastrospinosus. ulation, energy metabolism of the icefishes is highly and Of the eight icefish species examined, three lack Mb polypep- obligately aerobic (2), exacerbating the challenges to both tide in heart ventricle, although one of these expresses the Mb circulatory and intracellular delivery of oxygen. mRNA. All species of icefish retain the Mb gene in their Pale coloration of icefish tissues has led most researchers to genomic DNA. Based on phylogeny of the icefishes, loss of Mb assume that Mb is not present in icefish oxidative muscles expression has occurred independently at least three times (3–7). However, we collected three channichthyid species off and by at least two distinct molecular mechanisms during the Antarctic Peninsula, Chionodraco rastrospinosus, speciation of the family. Pseudochaenichthys georgianus, and Chaenodraco wilsoni, that displayed distinctly rose-colored hearts. By contrast, hearts of Icefishes (family Channichthyidae) of the Southern Ocean two other icefish species common to Peninsular waters, surrounding Antarctica are unique among vertebrate animals; Chaenocephalus aceratus and Champsocephalus gunnari, were all 15 species lack hemoglobin (1, 2) and, despite their highly pale yellow. Absorption spectrum of a clarified supernatant aerobic mode of metabolism, are believed also to lack the (40,000 ϫ g) from P. georgianus ventricle exhibited maxima at intracellular respiratory pigment, myoglobin (Mb) (3–7). Mb 530 and 580 nm, characteristic of oxymyoglobin. Although not normally is present in high concentration in aerobic muscle conclusive, these observations prompted us to ascertain tissues of vertebrate animals, where it functions both as an whether Mb is expressed in aerobic muscle tissues of icefishes. intracellular oxygen reservoir and to facilitate the transcellular diffusion of oxygen (8). MATERIALS AND METHODS The Perciform suborder Notothenioidei (which includes the Animals and Tissues. Live specimens of icefishes C. acera- icefishes) arose and evolved in coastal Antarctic waters during tus, C. rastrospinosus, C. gunnari, P. georgianus, C. wilsoni, and the last 25 million years (9, 10). Antarctica became isolated at red-blooded notothenioids Notothenia coriiceps and Gobiono- that time upon the opening of the Drake Passage and estab- tothen gibberifrons were collected by Otter Trawl off the south lishment of circumpolar currents that led to the rapid cooling shores of Low and Brabant Islands during the austral autumn of the Southern Ocean. At present, the ocean surrounding in 1991, 1993, and 1995. Pagetopsis macropterus was captured Antarctica is uniquely cold and thermally stable; water tem- off Brabant Island in March 1995. Cryodraco antarcticus tissues peratures around the Antarctic Peninsula fluctuate only be- were the kind gift of A. L. DeVries (University of Illinois) (the tween ϩ0.3 and Ϫ1.87ЊC annually (11, 12). Divergence of mitochondrial DNA sequences suggests that radiation of no- Abbreviations: Mb, myoglobin; PVDF, polyvinylidene difluoride. tothenioid families began 7 to 15 million years ago, but that Data deposition: The sequences reported in this paper have been speciation of channichthyid icefish began approximately 1 deposited in the GenBank database [accession no. U68350 (Notothenia coriiceps partial Mb cDNA sequence) and accession no. U70871 million years ago (13). (Chionodraco rastrospinosus full-length Mb cDNA sequence)]. ‡To whom reprint requests should be addressed. § The publication costs of this article were defrayed in part by page charge Present address: Department of Biology, University of Akron, Akron, payment. This article must therefore be hereby marked ‘‘advertisement’’ in OH 44325. ¶ accordance with 18 U.S.C. §1734 solely to indicate this fact. Present address: Department of Neurology, Harvard Medical School and Division of Neuroscience, The Children’s Hospital, Boston, MA Copyright ᭧ 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA 02115-5373. 0027-8424͞97͞943420-5$2.00͞0 ʈPresent address: Department of Biological Sciences, Idaho State PNAS is available online at http:͞͞www.pnas.org. University, Pocatello, ID 83209.

3420 Downloaded by guest on September 26, 2021 Physiology: Sidell et al. Proc. Natl. Acad. Sci. USA 94 (1997) 3421

specimen was captured from McMurdo Sound). Chionodraco hamatus tissues, from animals captured at Terra Nova Bay, were the kind gift of G. di Prisco and R. Acierno (Italian National Antarctic Program). Heart ventricle, auricle, pectoral adductor profundus, testes, and spleen tissues were dissected immediately upon sacrifice of the animals. Samples were frozen in liquid nitrogen, transported to the United States on dry ice, and stored at Ϫ70ЊC until isolation of protein and nucleic acids. Protein Extraction and Immunoblot Analysis. Soluble polypeptides were liberated from heart ventricles by homog- enization in 20 mM Hepes (pH 7.8) at 4ЊC and centrifuged at 10,000 ϫ g for 10 min. Protein in the supernatant was determined by BCA assay (Pierce). Polypeptides were dena- tured by boiling in the presence of 1% SDS͞1 mM 2-mercap- toethanol, separated by electrophoresis through 17% Tricine– SDS͞PAGE gels, and either electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Micron Sepa- rations, Westboro, MA) or stained using Coomassie brilliant blue R-250. For slot blots, supernatants were diluted in 1ϫ phosphate-buffered saline (PBS) (pH 7.4) and vacuum blotted onto PVDF membrane. Electroblot and slot-blot membranes were blocked by incubation overnight in 5% nonfat milk, washed in 1ϫ PBS, and incubated with a 1:500 dilution of mouse anti-human Mb monoclonal antibody (Sigma). This antibody displayed strong cross-reactivity with Mb isolated from red-blooded notothenioid species known to express the pigment. Bound antibody was detected by a rabbit anti-mouse IgG secondary antibody (1:1000 dilution) conjugated to alkaline phosphatase (Bio-Rad), and visualized by subsequent incubation in stabilized Western blue substrate (Promega). RNA Gel Blot Hybridization and PCR Amplification. Total RNA was isolated from finely ground, frozen heart ventricles by acid guanidinium–thiocyanate–phenol chloroform extraction (18). RNA concentrations were determined in triplicate by spectrophotometric analyses. Equal amounts (5 ␮g) of total RNA were resolved by electrophoresis through 1% agarose͞ formaldehyde gels (19) and blotted to GeneScreen Plus nylon membranes (DuPont). Blots were probed with a 329-bp segment of N. coriiceps Mb cDNA corresponding to codons 5–114 that was 32P-labeled by the random primer method (Boehr- inger Mannheim). Blots received two successive 30-min washes in 0.1ϫ SSC͞0.1% SDS at 63ЊC. The N. coriiceps partial Mb cDNA was obtained by reverse transcriptase-PCR (20); first- strand synthesis was primed by oligo(dT)–NotI primer adapter (Pharmacia), and amplification employed degenerate oligonucleotides based on conserved segments of tuna and carp Mb polypeptide sequences (Swiss-Prot accession nos. PO2205 and PO2204, respectively). Sequencing confirmed that the ampli- fied sequence encoded Mb (21). The distal two-thirds of the C. rastrospinosus Mb sequence was obtained by reverse transcriptase-PCR using the oligo(dT)–NotI primer adapter and non- degenerate primers based upon the N. coriiceps Mb sequence. The 5Ј proximal segment was obtained by linker-mediated FIG. 1. Presence of Mb polypeptide in Antarctic icefishes. (a) Soluble PCR (22) using the 5Ј rapid amplification of cDNA ends polypeptides were liberated from heart ventricles and resolved by SDS͞ system (CLONTECH) and a Mb-specific antisense oligonu- PAGE. Lanes: 1, protein molecular weight standards (Bio-Rad); 2, ␮ cleotide primer. Mb-specific oligonucleotides were used to human heart Mb standard (1 g; Biodesign International, Kennebunk- port, ME); 3, C. aceratus (35 ␮g total protein); 4, C. rastrospinosus (35 ␮g prime dideoxy cycle-sequencing reactions (Perkin–Elmer), total protein). (b) Duplicate gel to a above was electroblotted to PVDF which were resolved with an Applied Biosystems 373A auto- membrane for immunoblot analysis. The PVDF membrane was incubated mated DNA sequencer. with mouse anti-human Mb monoclonal antibody (Sigma). Bound anti- Southern Blot Hybridization. Genomic DNA was isolated body was detected by rabbit anti-mouse IgG antibody covalently linked to from testes (or spleen of P. macropterus) by proteinase K alkaline phosphatase (Bio-Rad) and subsequent incubation with stabi- digestion and phenol chloroform extraction as described by lized Western blue substrate (Promega). Only the molecular weight range Sambrook et al. (19). Ten micrograms of each genomic DNA containing Mb is shown. Lane assignments are described as in a above. sample was digested with 2 units of HindIII overnight. Re- (c) Slot immunoblot of soluble polypeptides from heart ventricles of eight striction fragments were resolved by electrophoresis through channichthyid icefishes (1A to 2C) and one red-blooded Antarctic nototheniid fish (2D). Equal amounts (5 ␮g) of total protein were loaded 0.7% agarose gels cast in 25 mM Tris-borate (pH 8.3) con- in each slot and incubated with antibody as described in b above. 1A, C. taining 1 mM EDTA, and transferred to Hybond-C nylon rastrospinosus; 1B, P. macropterus; 1C, P. georgianus; 1D, C. aceratus; 1E, membranes (Amersham) by capillary blot. Blots were hybrid- C. hamatus; 2A, C. gunnari; 2B, C. wilsoni; 2C, C. antarcticus; 2D, N. ized with the 32P-labeled, 329-bp N. coriiceps cDNA PCR coriiceps. Downloaded by guest on September 26, 2021 3422 Physiology: Sidell et al. Proc. Natl. Acad. Sci. USA 94 (1997)

FIG. 2. Interspecific pattern of Mb mRNA expression in Antarctic icefishes. (a) Northern blot analysis performed on total RNA extracted from heart ventricle. Mb mRNA (0.9 kb) was detected by hybridization with random-primed (Boehringer Mannheim) 32P-labeled N. coriiceps Mb cDNA probe. Lanes: 1, C. rastrospinosus;2,C. gunnari;3,P. georgianus;4,C. aceratus;5,C. antarcticus;6,P. macropterus;7,C. wilsoni;8,N. coriiceps. Analysis of C. hamatus and G. gibberifrons total RNA also indicated the presence of Mb mRNA in heart ventricle, but not in oxidative skeletal muscle tissues (data not shown). Relative abundance of Mb mRNAs depicted in the figure is representative of results obtained from several individuals of each species. (b) Equal loading of gels indicated by ethidium bromide staining of the 28S and 18S ribosomal RNA in total RNA samples used in a above.

product. Blots were washed twice in 0.2ϫ SSPE at 62ЊC and Mb cDNA (Fig. 3) is comprised of a short, 57 nucleotide 5Ј exposed to Kodak X͞AR5 film for 7–10 days with a tungsten untranslated leader, a 441 nucleotide coding sequence, a 78 intensifying screen. nucleotide 3Ј untranslated region proximal to the poly(A) tail, and includes a segment with 98% identity to the 329-bp N. RESULTS coriiceps Mb PCR product. The predicted coding sequence Immunoblot analyses (Fig. 1) show that Mb is present in the contains 147 amino acids; this is consistent with the mature 146 heart ventricles of at least five icefish species: C. rastrospinosus, residue Mb polypeptides of other teleosts from which the P. georgianus, C. wilsoni, C. antarcticus, and C. hamatus. Both N-terminal methionine is removed. The sequence flanking the polyclonal (not shown) and monoclonal antibodies (Fig. 1), initiator methionine is consistent with the Kozak consensus which recognize Mb in a broad variety of vertebrates, identi- sequence Ϫ3AynATGGϩ4 (26). When the complete (100%) fied a polypeptide of the expected size in these five icefish deduced Mb polypeptide from C. rastrospinosus is compared species. Teleost Mb is comprised of 146 amino acids (23), with with that of tuna (Thunnus albacares) Mb, 78% of the residues an apparent molecular size of approximately 16 kDa. The more are identical and an additional 9% are similar. Key structural slowly migrating human Mb with a molecular size of 17.8 kDa elements, such as his60 and his88, which mediate heme binding, contains 153 amino acids and is typical of mammalian Mbs (24, are conserved. This high degree of homology leaves no doubt 25). Heart ventricles of the notothenioid species contained a that some icefish species express Mb in cardiac ventricle. 0.9-kb mRNA (Fig. 2), which hybridized under high stringency Genomic DNA of all channichthyid species tested to date to a Mb cDNA probe isolated from the red-blooded Antarctic contain restriction fragments that hybridize to nototheniid Mb nototheniid N. coriiceps. Three icefish species, C. aceratus, C. cDNA (Fig. 4). Red-blooded nototheniid species N. coriiceps gunnari, and P. macropterus, lacked the immunoreactive and G. gibberifrons exhibited two or three Mb-specific restric- polypeptide (Fig. 1). C. aceratus and P. macropterus lacked tion fragments indicating that Mb is a single-copy or low-copy Mb-hybridizing mRNA (Fig. 2). However, C. gunnari individ- gene. Both C. aceratus and P. macropterus contained Mb- uals reproducibly exhibited low steady-state quantities of the specific fragments, although Mb mRNA was not detected in 0.9-kb Mb-hybridizing mRNA (Fig. 2) despite consistent ab- the heart ventricle of either species. Mb-hybridizing bands of sence of the Mb polypeptide in protein extracts from the same similar size were apparent in genomic DNA of the Mb- tissue samples. Neither immunoblot nor RNA gel blot hybrid- expressing channichthyid species C. rastrospinosus and C. ization detected Mb in the heart auricle or highly aerobic gunnari. These results indicate that loss of Mb expression in C. skeletal muscle (pectoral adductor profundus) of the icefishes aceratus and P. macropterus did not result from large-scale or red-blooded Antarctic nototheniids tested (data not deletion of the gene; deletion of the ␤-globin gene is the shown). This highly tissue-specific expression of Mb, confined apparent mechanism by which hemoglobin expression was lost exclusively to the heart ventricle, is unique among vertebrates in C. aceratus (27). and is a possible synaptomorphic character among notothenioid fishes. DISCUSSION Verification that the 0.9-kb mRNA encodes Mb was ob- Our data provide conclusive evidence that Mb is present and tained by sequencing C. rastrospinosus cDNA products pro- expressed in several species of Antarctic icefish that lack duced by reverse transcriptase-PCR and ligation-mediated hemoglobin. The expression of Mb is extremely tissue-specific PCR. The composite full-length sequence of C. rastrospinosus in both the icefishes and their red-blooded nototheniid rela- Downloaded by guest on September 26, 2021 Physiology: Sidell et al. Proc. Natl. Acad. Sci. USA 94 (1997) 3423

FIG. 3. Composite sequence of C. rastrospinosus Mb cDNA and deduced Mb primary structure.

tives. Mb expression is confined to the heart ventricle and Ability to express Mb in the heart ventricle appears to have absent from the primary aerobic skeletal muscle used for been lost by three independent mutational events during the labriform locomotion, the pectoral adductor profundus. Abil- evolution of icefishes. Comparison of Mb expression with a ity to express Mb in skeletal musculature was apparently lost morphologically based phylogenetic tree for the channichthy- early in the notothenioid lineage and prior to radiation of the ids (9, 28) indicates that three icefish species that lack Mb channichthyid icefishes. expression in the heart ventricle, C. aceratus, C. gunnari, and P. macropterus, occupy distinct clades; two of these clades contain relatives that express Mb (Table 1). Our recent phylogenetic analysis of mitochondrial DNA control region sequences also unambiguously supports the occurence of three independent losses of Mb expression (I. Kornfield, Y. K. Tam, M.E.V., and B.D.S., unpublished data). Furthermore, the observation that C. gunnari contains Mb mRNA, whereas the other two species do not, suggests at least two distinct molecular mechanisms for the loss of Mb: failure to synthesize the Mb polypeptide in C. gunnari, and failure to transcribe the Mb gene or process Mb transcripts in C. aceratus and P. macropterus. All three species contain remnants of the Mb gene in the genome, in distinct contrast to the apparent mechanism for the loss of hemoglobin expression within the family (27). These results suggest that loss of Mb expression is both a recent and recurrent event in the evolutionary history of icefishes. FIG. 4. Southern blot hybridization of notothenioid genomic DNA At present, it is unclear whether Mb plays a role in facilitated with 32P-labeled N. coriiceps Mb cDNA. Genomic DNA isolated from N. coriiceps (lane 1), G. gibberifrons (lane 2), C. rastrospinosus (lane 3), oxygen transport or storage at cold body temperatures. Main- C. gunnari (lane 4), P. macropterus (lane 5), and C. aceratus (lane 6) tenance of Mb expression in several icefish species could be were digested with HindIII, resolved by electrophoresis though a 0.7% either a vestigial characteristic or could indicate selective agarose gel, and hybridized with the random-primed Mb cDNA probe. retention of the hemoprotein for aiding intracellular oxygen Mobility of molecular size markers is shown at right. transport in the hearts of these species. Oxygen dissociation Downloaded by guest on September 26, 2021 3424 Physiology: Sidell et al. Proc. Natl. Acad. Sci. USA 94 (1997)

Table 1. Expression of Mb polypeptide and mRNA in aerobic muscles with respect to phylogenetic relationships among channichthyid species Total no. of Species Genus species examined Polypeptide RNA Champsocephalus 2 gunnari Ϫϩ

Pseudochaenichthyes 1 georgianus ϩϩ Neopagetopsis 1 Pagetopsis 2 macropterus ϪϪ

Dacodraco 1

Channichthys 1

Cryodraco 1 antarcticus ϩϩ Chionobathyscus 1 Chaenocephalus 1 aceratus ϪϪ

Chionodraco 3 rastrospinosus ϩϩ hamatus ϩϩ Chaenodraco 1 wilsoni ϩϩ All species contain discrete, single-copy genomic DNA fragments that hybridize to N. coriiceps Mb cDNA.

rates of mammalian and tuna Mbs are dramatically lengthened 10. Eastman, J. T. (1991) Am. Zool. 31, 93–109. at cold temperature, suggesting that a similar respiratory 11. DeWitt, H. H. (1971) Antarct. Map Folio Ser. 15, 1–10. pigment would not function at physiological temperatures of 12. Littlepage, J. L. (1965) in Antarctic Research Series Vol. 5: Biology icefishes (29–31). However, sequence differences observed of the Antarctic Seas II, ed. Llano, G. A. (Am. Geogr. Soc., New between the notothenioid and tuna Mbs may confer improved York), pp. 1–10. function at cold temperatures. If the latter possibility proves 13. Bargelloni, L., Ritchie, P. A., Patarnello, T., Battaglia, B., Lam- correct, it raises fascinating questions about how similar de- bert, D. M. & Meyer, A. (1994) Mol. Biol. Evol. 11, 854–863. mands for storage and diffusion of oxygen are met in the heart 14. Johnston, I. A., Fitch, N., Zummo, G., Wood, R. E., Harrison, P. & Tota, B. (1983) Comp. Biochem. Physiol. A 76, 475–480. ventricle of species without Mb and in the oxidative skeletal 15. Fitch, N. A., Johnston, I. A. & Wood, R. E. (1984) Respir. Physiol. muscles of all Antarctic notothenioid species examined. No 57, 201–211. obvious differences have been observed among channichthyid 16. Hemmingsen, E. A., Douglas, E. L., Johansen, K. & Millard, species in either mitochondrial densities or in intracellular R. W. (1972) Comp. Biochem. Physiol. A 43, 1045–1051. neutral lipid content, a factor that may play a role in aiding 17. Londraville, R. L. & Sidell, B. D. (1990) Respir. Physiol. 81, intracellular oxygen movement (16). Definitive resolution of 291–302. the importance of Mb in these unusual animals may be aided 18. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, 156– by physiological studies of heart performance and determina- 159. tion of the kinetics of oxygen binding and dissociation for the 19. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular hemoproteins from Antarctic icefishes. Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Plain- view, NY). We gratefully acknowledge the generous contribution of samples by 20. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S., Higucci, R., Dr. A. L. DeVries (University of Illinois) and Drs. G. di Prisco and R. Horn, R., Mullis, K. B. & Elrich, H. A. (1988) Science 239, Acierno (Italian National Antarctic Program). This study would not 487–491. have been possible without the excellent support provided by station 21. Vayda, M. E., Small, D. J. & Sidell, B. D. (1997) in VI SCAR personnel at the U.S. Antarctic Program’s Palmer Station, Antarctica, Antarctic Communities, eds. Battaglia, B., Valencia, J. & Walton, and the masters and crew of M͞V Polar Duke. The work was supported by U.S. National Science Foundation (NSF) Grants OPP 92-20775 and D. W. H. (Cambridge Univ. Press, Cambridge, U.K.), in press. 94-21657 to B.D.S. The University of Maine automated DNA se- 22. Trout, A. B. (1994) in PCR Technology: Current Innovations, eds. quencing facility was initiated by support through NSF EPSCoR Grant Griffin, H. G. & Griffin, A. M. (CRC, Boca Raton, FL), pp. EHR91-08766 and Maine Science and Technology Foundation Grant 141–146. 07G-GTSS930323. 23. Watts, D. A., Rice, R. H. & Brown, W. D. (1980) J. Biol. Chem. 255, 10916–10924. 1. Ruud, J. T. (1954) Nature (London) 173, 848–850. 24. Akaboshi, E. (1985) Gene 33, 241–249. 2. Hemmingsen, E. A. (1991) in Biology of Antarctic Fish, eds. 25. Blanchetot, A., Price, M. & Jeffreys, A. J. (1986) Eur. J. Biochem. diPrisco, G., Maresca, B. & Tota, B. (Springer, New York), pp. 159, 469–474. 191–203. 26. Kozak, M. (1986) Proc. Natl. Acad. Sci. USA 83, 2850–2853. 3. Hamoir, G. (1988) Comp. Biochem. Physiol. B 90, 557–559. 27. Cocca, E., Ratnayake-Lecamwasam, M., Parker, S. K., Ca- 4. Feller, G. & Gerday, C. (1987) Polar Biol. 7, 225–229. 5. Hamoir, G. & Gerardin-Otthiers, N. (1980) Comp. Biochem mardella, L., Ciaramella, M., di Prisco, G. & Detrich, H. W. Physiol. B 65, 199–206. (1995) Proc. Natl. Acad. Sci. USA 92, 1817–1821. 6. Walesby, N. J., Nicol, C. J. M. & Johnston, I. A. (1982) Br. 28. Iwami, T. (1985) Mem. Nat. Inst. Polar Res., Ser E 36, 1–69. Antarct. Surv. Bull. 51, 201–214. 29. Sato, F., Shiro, Y., Sakaguchi, Y., Izukda, T. & Hayshi, H. (1990) 7. Johnston, I. A. & Harrison, P. (1987) Fish Physiol. Biochem. 3, J. Biol. Chem. 265, 18823–18828. 1–6. 30. Dowd, M. K., Murali, R. & Seagrave, R. C. (1991) Biophys. J. 60, 8. Wittenberg, J. B. (1970) Physiol. Rev. 50, 559–636. 160–171. 9. Eastman, J. T. (1993) Antarctic Fish Biology: Evolution in a 31. Stevens, E. D. & Carey, F. G. (1981) Am. J. Physiol. 240, R151– Unique Environment (Academic, San Diego). R155. Downloaded by guest on September 26, 2021