Comparative and Physiology, Part A 147 (2007) 799–807 www.elsevier.com/locate/cbpa

Review Biochemical adaptations of notothenioid : Comparisons between cold temperate South American and New Zealand and Antarctic species☆ ⁎ Zulema L. Coppes Petricorena a, , George N. Somero b

a Faculty of Chemistry — Montevideo, Uruguay b Hopkins Marine Station, Department of Biological Sciences, Stanford University, Pacific Grove, CA 93950-3094, USA Received 17 June 2006; received in revised form 17 September 2006; accepted 29 September 2006 Available online 5 December 2006

Abstract

Fishes of the perciform suborder afford an excellent opportunity for studying the evolution and functional importance of diverse types of biochemical adaptation to temperature. Antarctic notothenioids have evolved numerous biochemical adaptations to stably cold waters, including antifreeze glycoproteins, which inhibit growth of ice crystals, and enzymatic proteins with cold-adapted specific activities (kcat values) and substrate binding abilities (Km values), which support metabolism at low temperatures. Antarctic notothenioids also exhibit the loss of certain biochemical traits that are ubiquitous in other fishes, including the heat-shock response (HSR) and, in members of the family , hemoglobins and myoglobins. Tolerance of warm temperatures is also truncated in stenothermal Antarctic notothenioids. In contrast to Antarctic notothenioids, notothenioid species found in South American and New Zealand waters have biochemistries more reflective of cold-temperate environments. Some of the contemporary non-Antarctic notothenioids likely derive from ancestral species that evolved in the Antarctic and later “escaped” to lower latitude waters when the Antarctic Polar Front temporarily shifted northward during the late Miocene. Studies of cold-temperate notothenioids may enable the timing of critical events in the evolution of Antarctic notothenioids to be determined, notably the chronology of acquisition and amplification of antifreeze glycoprotein genes and the loss of the HSR. Genomic studies may reveal how the gene regulatory networks involved in acclimation to temperature differ between stenotherms like the Antarctic notothenioids and more eurythermal species like cold-temperate notothenioids. Comparative studies of Antarctic and cold-temperate notothenioids thus have high promise for revealing the mechanisms by which temperature-adaptive biochemical traits are acquired – or through which traits that cease to be of advantage under conditions of stable, near-freezing temperatures are lost – during evolution. © 2006 Elsevier Inc. All rights reserved.

Keywords: ; Antifreeze glycoproteins; Heat-shock response; Notothenioid; Temperature adaptation

Contents

1. Geological and oceanographic drivers of evolution in notothenioid fishes ...... 800 2. Characteristics of the Antarctic fauna: the suborder Notothenioidei ...... 800 3. Non-Antarctic notothenioids ...... 801 4. Antifreeze glycoproteins ...... 801 5. Gene loss in stably cold waters: the heat-shock response ...... 802

☆ This paper is part of the 3rd special issue of CBP dedicated to The Face of Latin American Comparative Biochemistry and Physiology organized by Marcelo Hermes-Lima (Brazil) and co-edited by Carlos Navas (Brazil), Rene Beleboni (Brazil), Rodrigo Stabeli (Brazil), Tania Zenteno-Savín (Mexico) and the editors of CBP. This issue is dedicated to the memory of two exceptional men, Peter L. Lutz, one of the pioneers of comparative and integrative physiology, and Cicero Lima, journalist, science lover and Hermes-Lima's dad. ⁎ Corresponding author. E-mail address: [email protected] (Z.L. Coppes Petricorena).

1095-6433/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2006.09.028 800 Z.L. Coppes Petricorena, G.N. Somero / Comparative Biochemistry and Physiology, Part A 147 (2007) 799–807

6. Temperature adaptation of enzymatic proteins...... 803 7. Structural adaptations of muscle fibres: relationship between diameter and number ...... 804 8. Genetics of notothenioids: what has been lost during evolution in stably cold waters? ...... 805 Acknowledgements ...... 805 References ...... 805

1. Geological and oceanographic drivers of evolution in The stably cold and oxygen-rich waters found southward of notothenioid fishes the APF would be expected to serve as important effectors of evolution in the Antarctic marine biota. One would anticipate Fishes of the perciform suborder Notothenioidei afford an that during the approximately 40 million years of existence of excellent study system for examining how large-scale geologi- the ACC and APF, adaptations to temperature would have led to cal and oceanographic processes serve as drivers of evolution to extensive differentiation of organisms endemic to waters to the the physical environment. The formation of the Southern north or south of the APF. Studies of the major group of Ocean, which surrounds Antarctica and includes the great , members of the perciform suborder Notothe- embayments of the Weddell and Ross Seas, was marked by the nioidei, and their cold-temperate relatives in and creation of a large mass of water – the planet's fourth largest New Zealand, show this to be the case. The biochemical ocean – that is uniquely cold and thermally stable. The Southern differences between polar and cold-temperate notothenioids Ocean is covered by sea ice during the winter, from the reflect the gain of important adaptive traits in both groups and Antarctic coastline northward to approximately 60°S (Gordon, the loss of traits no longer needed for life in stably cold waters in 1988, 1999, 2003). The is much younger than Antarctic species. This short review discusses these key other oceans because of its origins as a result of plate tectonic differences and suggests new lines of studies, many of which activities over the past approximately 40–60 million years. Two are based on the new genomic technologies now becoming key events in the formation of the Southern Ocean were the available for fishes, that may contribute importantly to our opening of the Drake Passage between South America and the understanding of molecular evolution in protein-coding and Antarctic continent, which recent analyses suggest took place gene regulatory components of the genome. approximately 41 million years ago, and the formation of the Tasmanian Gateway, which is now thought to have occurred a 2. Characteristics of the Antarctic fish fauna: the suborder few millions years after the opening of the Drake Passage Notothenioidei (Scher and Martin, 2006). The separation of these southern landmasses permitted formation of the Antarctic Circumpolar Beginning in the early Miocene (25–22 million years ago), Current (ACC), the oceanographic feature of the Southern the Antarctic shelf was subject to a series of tectonic and Ocean that plays a pivotal role in establishing the thermal oceanographic events that undoubtedly altered faunal composi- conditions that have driven evolution of the Antarctic biota tion. Antarctica gradually became isolated and colder and (Eastman, 1993). The ACC is the ocean's largest current. It is expansion of the ice sheet led to destruction and disturbance of 21,000 km in length and transports 130 million cubic meters of inshore habitat by ice, as a consequence of repeated groundings water per second — 100 times the flow of all the world's rivers of parts of the ice sheet as far as the shelf break (Clarke and (Gordon, 1999). The Antarctic Polar Front (APF), the northern Johnston, 1996; Anderson, 1999). Loss of habitat and changes border of the ACC between 50°S and 60°S, prevents mixing of in the trophic structure of the ecosystem probably led to the the waters of the Southern Ocean with those of the Indian, local extinction of many of the components of the fish Pacific and Atlantic oceans. The APF thus acts as a cold “wall” fauna. Thus, the diversity of the fauna was reduced and, as that inhibits mixing of the fauna of the cold temperate ocean to Antarctica became increasingly isolated, new niches became the north with the cold-adapted fauna of the Southern Ocean. available to groups that were diversifying in situ (notothe- However, this “wall” may not be impenetrable at all depths, for nioids) or immigrating into (liparids and zoarcids) this recent studies suggest that “leakage” of invertebrates may occur developing cold-water ecosystem (Eastman, 2005). Little is in deep water (Clarke et al., 2005). known, however, about when the fauna became modern in Sea temperatures of the Southern Ocean have been well taxonomic composition. below 5 °C for 10 to 14 MY and they presently approach −2°C The first Antarctic notothenioids to be reported in the at the more southerly boundaries of the shelf (Littlepage, 1965). literature were collected near Kerguelen Island during the Annual variation in temperature of McMurdo Sound waters expedition of the Erebus and Terror under command of Sir (78°S) is between −1.9 °C and −0.5 °C (Hunt et al., 2003). In James Clark Ross (1839–1843). Prior to these collections, it is more northerly waters of the Antarctic Peninsula, summer doubtful that many believed that fishes could live in such a temperature reach +1.5 °C and winter temperatures are near harsh environment. In fact, the fish fauna of the Southern Ocean −1.8 °C (Dewitt, 1970). As the water column of the ACC/APF is limited in both species and higher taxonomic diversity and is very well mixed, temperature varies little with depth and contains only 313 species distributed among 50 families (Gon waters are close to complete oxygen saturation. and Heemstra, 1990; Eastman, 2000). Thus, although the Z.L. Coppes Petricorena, G.N. Somero / Comparative Biochemistry and Physiology, Part A 147 (2007) 799–807 801

Southern Ocean represents approximately 10% of the world Pseudaphritidae and Eleginopidae, with 12 species are non- ocean, it contains only 1.3% of the world's fish fauna (Eastman Antarctic. Among the other five notothenioid families, and McCune, 2000). Benthic fishes are the major component of , Harpagiferidae, , - the fauna with 213 species; higher taxonomic diversity of nidae and Channichthyidae, 15 species occur along the cool- benthic fishes is confined to 18 families (Eastman, 2000). Two temperate southern coast of South America and New Zealand perciform groups, the suborder Notothenioidei (notothenioids) (Eastman and Eakin, 2000). These cold-temperate species and the Zoarcidae (eelpouts), and the scorpaeniform family encounter water temperatures of approximately 5 °C–15 °C Liparidae () are the most speciose taxa, accounting (Johnston et al., 1998; Fields and Somero, 1998). for 87.4% of the species (Eastman, 2000). The fish fauna as a The evolutionary histories of the non-Antarctic notothe- whole is highly endemic; 88% endemism is found for species nioids are not fully established, but there is compelling confined to water south of the APF. If only the notothenioids are evidence, much of it of biochemical and molecular nature, considered, endemism rises to 97%, a very high percentage for a that supports an Antarctic ancestry for some of these species. marine group, because only 10% specific endemism is enough When the APF advanced ∼300 km to the north during the late for recognition of provinces (Eastman and McCune, 2000). Miocene (6.5–5.0 million years ago), cold water reached as far The suborder Notothenioidei dominates the Antarctic fish north as New Zealand (Kennett, 1982). Some notothenioids fauna. Notothenioids account for approximately 46% of the migrated along with the shifting APF and established total fish species known to occur south of the APF. More themselves in New Zealand and cold-temperate South Amer- significantly, they comprise as much as 90% of the biomass of ican waters. Other notothenioids, notably representatives of the fish captures around the continent (Ekau, 1990; Eastman, 2005). basal lineages like the Family Bovichtidae, presumably The suborder Notothenioidei comprises eight families and 122 diverged and became established in the brackish coastal water species. Five families and 96 species are Antarctic whereas of the southern continental blocks before the isolation of three families and 26 species are non-Antarctic (Eastman, Antarctica (Eastman and McCune, 2000). The lack of genes for 2005). There is no fossil record of these fishes, and the origin of antifreeze glycoproteins in members of the Bovichtidae, this group is ambiguous. According to Sidell (2000) all that can Pseudaphritidae and Eleginopidae, supports this scenario for be said with certainty is that, sometime between the Mid- the basal notothenioid lineages (Cheng and Detrich, in press). Tertiary and the present, there was a massive crash of species The presence or absence of genes encoding glycoprotein diversity that left a sluggish, demersal stock of ancestral antifreezes thus provides a window – albeit one that is cloudy notothenioids to colonize the vast Southern Ocean, which at that at times (see below) – into the evolutionary histories of time was not as cold as at present. The initial diversification of Antarctic and non-Antarctic notothenioids. some of the basal families took place during fragmentation of Gondwana 60–40 Mya when cooling of circumpolar waters 4. Antifreeze glycoproteins began (Eastman, 1993). Since the Mid-Miocene, geographical isolation and a chronically cold environment have resulted in We begin our comparison of Antarctic and non-Antarctic extreme stenothermality of extant species (Somero and DeVries, notothenioids with what is certainly the most striking difference 1967; Cheng and Detrich, in press; Podrabsky and Somero, between teleost fish that can and cannot survive in the presence 2006). The collapse in species diversity may be due in part to of ice — the occurrence in polar species of “antifreeze” decreases in water temperature; however, the slow geologic glycoproteins or proteins that inhibit the growth of ice crystals pace of ocean cooling has led Eastman and Clarke (1998) to (Cheng and DeVries, 1991; DeVries and Cheng, 2005; Cheng conclude that the loss of shallow water habitats that accom- and Detrich, in press). Based on solute concentrations in blood panied Antarctica's glaciation may have been of equal, if not and cells, notothenioids living in most regions of the Southern greater importance in causing the extinction. Whatever the Ocean spend their entire lives at body temperatures below the causes of the initial extinctions, subsequent radiations of the fish predicted colligative freezing point of their body fluids (Cheng fauna definitely are based on adaptations to the low tempera- and Detrich, in press). What keeps these fish in “liquid-state” is tures currently found in the Southern Ocean, as discussed in the a battery of antifreeze glycoproteins (AFGPs) that reduce the following sections of this review. freezing point – the temperature of ice crystal growth – to well below ambient sea water temperatures. Recent studies by Cheng 3. Non-Antarctic notothenioids and colleagues (2006) have toppled the prevailing paradigm concerning where AFGPs are produced. They showed that To appreciate the unique biochemical features of Antarctic hepatic synthesis, the initially proposed site of AFGP produc- notothenioids, it is important to contrast our knowledge of these tion, does not, in fact, occur; rather the production of these highly cold-adapted stenotherms with the information available critical molecules is restricted to pancreatic tissue and the about their cold-temperate relatives from South American and anterior portion of the stomach. From these sites of synthesis, New Zealand waters. Although most notothenioids are endemic AFGPs enter the entire gut cavity where the presence of ice to the Southern Ocean, a number of species are endemic to ingested during feeding creates an acute danger of lethal ice temperate areas north of the Antarctic Polar Front such as in formation (Cheng et al., 2006). How antifreezes move from the southern , Tasmania, New Zealand, and southern gut into the general circulation remains unknown and represents South America. Three small basal families, Bovichtidae, a critical question for study. Might there be a set of adaptations 802 Z.L. Coppes Petricorena, G.N. Somero / Comparative Biochemistry and Physiology, Part A 147 (2007) 799–807 for AFGP transport that are unique to the notothenioids that D. mawsoni lacks AFGPs (Cheng and Detrich, in press). It is have evolved freezing resistance? highly unlikely that D. eleginoides diverged before the The occurrence of AFGPs in non-Antarctic notothenioids evolutionary acquisition of the afgp gene, so the most from South American and New Zealand waters has been parsimonious explanation for the apparent absence of these examined in several studies (Cheng et al., 2003; for review, see genes in this cold-temperate nototheniid entails loss or severe Cheng and Detrich, in press). In two endemic New Zealand mutation of this gene following entry into cold-temperate notothenioids, angustata and Notothenia micro- waters. Study of the mechanisms of loss of afgp genes in the lepidota, two or three genes encoding AFGPs are present, cold-temperate congener of is clearly warranted. respectively, and low amounts of AFGPs can be detected in Puzzles also remain about the occurrence of afgp genes in blood (Cheng et al., 2003). This discovery indicates that these the , a large genus with 12 species, all of two cold-temperate New Zealand notothenioids are derived which are found in South American waters except for one, P. from an Antarctic ancestor, because there is strong evidence that guntheri, which occurs at the northern tip of the Antarctic the gene encoding AFGP arose only once and at a time that Peninsula. All of these species appear to lack afgp genes preceded the radiation of the notothenioid families with (Cheng and Detrich, in press). As in the case of D. eleginoides, Antarctic representatives (Eastman, 1993; Cheng, 1996; the absence of afgp genes in this genus is unlikely to reflect the Cheng et al., 2003). Thus, the most parsimonious explanation radiation of this group before the acquisition of the original afgp for the presence of AFGP-containing notothenioids in a cold- gene. Rather, loss of AFGP-encoding genes appears to be the temperate New Zealand habitat is that the species (or their likely mechanism, although this conjecture will require more ancestor(s)) were “escapees” from Antarctic waters that analysis using genomic technologies. Perhaps remnants of afgp followed the northern-moving APF to lower latitudes when genes will be found in the AFGP-lacking species and a deeper this type of oceanographic shift occurred, most recently during understanding of gene loss during adaptation to non-freezing the late Miocene (Eastman and McCune, 2000). temperatures by AFGP-containing species will be obtained. Two further differences between Antarctic and cold- Such discoveries would be an excellent complement to the temperate AFGP-containing notothenioids are the copy num- elegant work that has shown us how the AFGP-encoding genes bers of afgp genes and the numbers of AFGPs encoded by the initially arose (Chen et al., 1997; Cheng and Detrich, in press). polyprotein-encoding afgp genes. This gene family is thought to have arisen from a trypsinogen-like serine protease gene no 5. Gene loss in stably cold waters: the heat-shock response more than 14 million years ago, a date that is consistent with the freezing of the coastal Southern Ocean (10–15 million years Whereas Antarctic notothenioids are extraordinarily well- ago; Kennett, 1977) and the diversification of the 5 Antarctic adapted for life at near-freezing temperatures, they fare poorly notothenioid families 7–15 million years ago (Bargelloni et al., when confronted with elevated temperatures. Upper incipient 1994; Chen et al., 1997). The size of the AFGP gene family has lethal temperatures for several notothenioids from McMurdo increased in Antarctic notothenioids and the number of AFGP Sound acclimated to −1.9 °C were near 5–6 °C, marking these molecules encoded in a given antifreeze gene likewise has fish as extreme stenotherms (Somero and DeVries, 1967). increased in Antarctic species (Cheng et al., 2003). Both Nonetheless, a recent study showed that some capacity for evolutionary trends reflect the severity of the freezing threat that induced thermal tolerance is present in certain notothenioid Antarctic notothenioids face. The small number of afgp genes species (Podrabsky and Somero, 2006). Thus, for the in the New Zealand notothenioids and the small number of nototheniids bernacchii and Trematomus pennellii, AFGPs encoded by each of these genes suggest that these survival times at 14 °C increased from approximately 20 min in species reached New Zealand waters before the production of −1.9 °C laboratory-acclimated specimens to 60 and 140 min, AFGPs had reached the levels found in current Antarctic respectively, in specimens acclimated for 6–8 weeks to 4 °C. notothenioids. If this hypothesis is true, then the dating of the These results indicate that, although Antarctic notothenioids are evolution of the expansion of the afgp gene family in terms of among the most stenothermal species of known, they gene copy number and protein-encoding sites per gene can be do have some mechanisms for increasing their resistance to better understood. acute heat stress. The amino acid sequences of the AFGPs of Antarctic and Unlike all other fishes so examined, however, these New Zealand notothenioids also exhibit interesting differences. mechanisms fail to include the once-thought-to-be “ubiquitous” Thus, 6 of the 10 AFGPs found in N. angustata and 4 of the 11 heat-shock response (HSR) (Hofmann et al., 2000). Cells found in N. microlepidota contain amino acid substitutions that typically respond to heat stress with the synthesis of a group of are predicted to lead to a loss of antifreeze function. It remains highly conserved proteins termed heat shock proteins (Hsps) unclear as to why the New Zealand notothenioids continue to belonging to several size classes (Lindsquist and Craig, 1988). express afgp genes and why expression increases in the cold. Hsps, as members of a broad family of molecular chaperones, These, too, are questions for future study. function to minimize protein denaturation and aggregation It is interesting that the Patagonian tooth fish Dissostichus during heat stress, assist in renaturation of proteins that are eleginoides (Nototheniidae), which occurs from a latitude of partially denatured, and, in some cases, play a role in proteolytic 40°S off the coasts of South America to 60°S in the Antarctic degradation of irreversibly damaged proteins (Hochachka and and overlaps in distribution with its strictly Antarctic congener Somero, 2002). Using 35S-labeled methionine and cysteine Z.L. Coppes Petricorena, G.N. Somero / Comparative Biochemistry and Physiology, Part A 147 (2007) 799–807 803 and SDS-PAGE separation to detect the presence of different candidate for an evolutionary lesion. However, Buckley and size classes of newly synthesized proteins in T. bernacchii colleagues (2004) showed that HSF1 is present in cells of T. subjected to different levels of heat stress, Hofmann et al. bernacchii, so loss of this protein has not occurred. However, (2000) showed that no size class of Hsp exhibited increased unlike what has been observed in other species, elevated synthesis. Prior to this investigation, only a single study temperature had no effect on the ability of HSF1 to bind to the suggested that the HSR may not be ubiquitous: a study of gene regulatory region, the heat shock element, which congeners of Hydra showed that a species (H. oligactis) found modulates expression of Hsp-encoding genes. Further study in cool, thermally stable environments produced mRNA for will be needed to elucidate the precise alterations in the different Hsp70, but this message was unstable and did not get translated components of the HSR that lead to constitutive expression of into protein (Bosch et al., 1988; Gellner et al., 1992). In Hsps yet prevent heat-induced activation of the hsp genes. Antarctic notothenioids, in contrast, neither mRNA for Hsp70 In addition to differing in gene-regulatory characteristics, the nor Hsp70 protein increases in abundance following heat stress thermal optima of heat-shock proteins themselves differ (Hofmann et al., 2000; Place and Hofmann, 2004; Place et al., between Antarctic and cold-temperate nototheniids. Place and 2004). The lack of an HSR in Antarctic notothenioids belies the Hofmann (2005) studied a constitutively expressed heat-shock fact that constitutive expression of heat-inducible genes such as protein, Hsc70 (a “cognate” of Hsp70), purified from N. that encoding Hsp70 occurs in these fish (Place et al., 2004). angustata, T. bernacchii, and P. borchgrevinki. They measured Thus, unlike other fish, production of mRNA and protein abilities of Hsc70 to prevent thermal aggregation of lactate appears decoupled from thermal stress — or at least stress from dehydrogenase (LDH) and to refold chemically denatured LDH elevated temperatures. Place and colleagues propose that the over the temperature range of −2 °C to 45 °C. Hsc70 orthologs constitutive expression of Hsp-encoding genes in Antarctic of all three species were capable of refolding chemically notothenioids may, in fact, reflect thermal stress to protein denatured LDH in vitro over this temperature range. However, folding that arises from constraints of low temperatures on this the Hsc70 of the cold-temperate fish N. angustata out- process. Thus, if the kinetics of protein folding are slowed at performed the Hsc70s of the two Antarctic species at subzero temperatures, the dangers from aggregation of partially temperatures of 20 °C and higher; conversely, the Hsc70s of folded nascent polypeptides could be substantial. If this is the the Antarctic species out-performed the cold-temperate ortholog case, then high concentrations of molecular chaperones like at −2 °C. The Hsc70 orthologs of Antarctic and cold-temperate Hsp70 would be adaptive because cold-induced aggregation notothenioids could thus be an excellent study system for would be reduced. There are examples of cold-induced discerning how the temperature sensitivities of molecular induction of Hsps, so threats to protein folding from low chaperones evolve. extremes of temperature potentially exist (see Place et al., 2004, for review). Comparative study of the temperature-dependence 6. Temperature adaptation of enzymatic proteins of folding of nascent polypeptide chains is clearly warranted to determine whether reduced rates of protein maturation in The discovery that orthologous Hsc70s from Antarctic and extreme cold establish a need for enhanced levels of molecular cold-temperate notothenioids differ in thermal optima comple- chaperones. ments the findings of studies of temperature adaptation of The absence of an HSR in Antarctic notothenioids contrasts enzymatic proteins. Enzyme function is highly sensitive to with the responses of New Zealand notothenioids to heat stress temperature change, largely because of the balance that must be (Place et al., 2004; Hofmann et al., 2005). Two New Zealand maintained between flexibility and stability in discrete, endemics, variegatus and N. angustata, exhibited relatively mobile regions of the protein that are involved in abilities to up-regulate expression of Hsp70-encoding genes in catalytically important conformational changes (Fields and response to heat stress of 16 °C and 18.8 °C, temperatures near Somero, 1998; Hochachka and Somero, 2002; Fields and the upper end of their environmental temperature range. The Houseman, 2004). Molecular flexibility is needed to maintain occurrence of the HSR in N. angustata, a species that probably an appropriate catalytic rate, but stability is required to ensure is derived from an ancestor that “escaped” from Antarctic correct active site geometry for substrate recognition. waters, helps to put a date on the timing of the loss of the HSR in Fields and Somero (1998) compared the catalytic rate Antarctic species. Thus, if the northward movement of the APF constants (kcat) and Michaelis–Menten constants (Km,an occurred during the late Miocene, 6.5–5 million years ago, well index of substrate binding ability) in lactate dehydrogenase-A after the origin of the AFGP-encoding genes some 14 million orthologs (LDH-As) of Antarctic and South American notothe- years ago, the loss of the HSR would be a relatively recent event nioids. Each biogeographic group showed distinct differences. in the evolutionary histories of Antarctic notothenioids. In the Rates of LDH-A activity, as measured by kcat values, were on case of B. variegatus, the occurrence of the HSR is consistent average higher for the Antarctic species. Binding (Km of the PYR with the hypothesis that the Bovichtidae is a basal group that has substrate pyruvate (Km )) was strongly conserved at normal evolved under temperate conditions. body temperatures due to an intrinsically stronger binding PYR The mechanisms responsible for the lack of inducibility of (lower Km at a common temperature of measurement) for the Hsps in Antarctic notothenioids remain to be discovered. The LDH-A orthologs of the South American notothenioids. PYR transcriptional factor that is critical for expression of Hsp- Conservation of Km in LDH-A orthologs of differently encoding genes, heat-shock factor 1 (HSF1), is a logical thermally adapted vertebrates has previously been documented 804 Z.L. Coppes Petricorena, G.N. Somero / Comparative Biochemistry and Physiology, Part A 147 (2007) 799–807 for fishes (Yancey and Somero, 1978; Graves and Somero, The structure–function analyses that could be performed 1982; Coppes and Somero, 1988, 1990; Holland et al., 1997; through comparative studies of enzymes in cold-temperate and Johns and Somero, 2004) and other taxa (Yancey and Somero, Antarctic notothenioids offer exciting promise for furthering 1978). our understanding of how enzymes function and evolve. In Sequencing studies of the twelve notothenioid LDH-A particular, the discovery that notothenioids from Antarctic and orthologs revealed a number of insights into the evolution of South American habitats may attain adaptation through this enzyme in the notothenioid lineages. First, only a single modifying different sites in the amino acid sequence is helping notothenioid species, the South American Eleginops maclovi- to reveal the set of “evolutionary options” proteins possess for nus, had Histidine-75 in its sequence. This amino acid residue is adaptive modification of kinetic parameters. present in all non-notothenioid teleost LDH-As sequenced to date. The deduced amino acid sequence of the LDH-A of E. 7. Structural adaptations of muscle fibres: relationship maclovinus has eleven differences from the notothenioid between diameter and number consensus sequence, whereas other species' LDH-As differed by only one to four changes. The large divergence of the E. Skeletal muscle fibres are differentiated multicellular maclovinus sequence from other notothenioid sequences is structures specialized for contraction. The functional proper- consistent with the recent placement of this species into a new ties of a muscle will be strongly influenced by fibre diameter family (Eleginopidae) (see Fields and Somero, 1998). and fibre number. The maximum diameter of each muscle A second finding of this comparison helps to link amino acid fibre is related to ultimate body size and is probably limited substitutions to changes in enzyme function. The amino acid by diffusional constraints that stem from metabolic demand sequences of the active site regions of all of the orthologs are and temperature (Archer and Johnston, 1991). Fibre number identical, so the temperature-adaptive differences in kinetic may increase during post-embryonic stages through activation PYR properties (kcat and Km ) must arise from substitutions in of myogenic precursor cells, which proliferate before leaving regions of the enzyme that do not directly interact with substrate the cell cycle and fuse to form new myotubes (Johnston, or cofactor. The majority of changes in sequence occur in 2001). regions of the enzyme that influence the conformational Antarctic notothenioids have unusually large diameter fibres, flexibility of the parts of the enzyme that change shape during which can reach 100 μm in slow muscle and 600 μm in fast binding events. The cold-adapted LDH-As of Antarctic muscle (Battram and Johnston, 1991). Slow muscle fibres have notothenioids appear to have higher flexibility in these regions, relatively high densities of mitochondria (O'Brien et al., 2003), which allows lower energy (enthalpy) barriers to conforma- reaching 50% of fibre volume in some icefishes (Johnston, PYR tional changes and, hence, higher kcat and Km values. A 1989). These mitochondria are found in the central zone of even number of substitutions are capable of adapting kinetic the largest diameter slow fibres (Archer and Johnston, 1991). properties, such that within a biogeographic group, similar This localization is consistent with maintenance of adequate kinetic properties are found in orthologs with different primary oxygenation at the low body temperature of this species structures. For example, although the LDH-A orthologs of two (Egginton et al., 2002). Fast muscle fibres with diameters of of the South American species, Patagonotothen tessellata and 400 μm have also been reported in sub-Antarctic notothenioids PYR magellanica, have the same Km values, from the Beagle Channel, although a relatively restricted size they share none of the differences from the consensus sequence. range of fish was studied (Fernandez et al., 2000). A third finding, which reflects results seen in other studies of Johnston et al. (2003) studied the number of muscle fibres enzymatic adaptation to temperature (e.g., Holland et al., 1997; and fibre diameters in 16 species of notothenioids from three Johns and Somero, 2004), is that only a single amino acid geographical regions, Tierra del Fuego, South Georgia, and substitution may be sufficient to adaptively alter kinetic Antarctica, including representatives with benthic and secon- properties of enzymes. Fields and Houseman (2004) showed darily pelagic life styles. They estimated ancestral values for that altering one residue (number 233) from a glutamate to a body size and fibre number to explore how fibre number and methionine was sufficient to change an Antarctic LDH-A to a size changed during the evolutionary radiation of this group. variant with kinetic properties identical to those of an LDH-A They showed that the radiation has been associated with a from a warm-temperate fish. progressive loss in body size specific-maximum number of fast Lastly, the kcat values for the South American species offer fibres in the myotomal muscle of the more derived species. An insights into the evolutionary histories of these species. important consequence of the reduction in fibre number in the Although on average the kcat values for these species were more derived lineages of notothenioid fish is an increase in their lower than those for the Antarctic notothenioids, the kcat of the maximum diameter, which can reach 600 μm in some species, LDH-A of P. tessellata fell into the range of values for the depending on their final body size. Antarctic species. This may reflect the retention of a cold- The unusual evolutionary patterns in muscle fibre number adapted kcat in this species, which is derived from an ancestral and size in notothenioids can be appreciated by comparisons species that “escaped” from cold Antarctic waters and gave rise with other fish. Atlantic salmon with a standard length of 50– to the 13 extant Patagonotothen species. In contrast, the low 70 cm have 550,000 to 1,200,000 fast muscle fibres per trunk kcat of E. maclovinus may reflect the absence of cold adaptation cross section (Johnston et al., 2000), with a fibre diameter of in this species' evolutionary history. approximately 220 μm. In contrast, the sub-Antarctic species Z.L. Coppes Petricorena, G.N. Somero / Comparative Biochemistry and Physiology, Part A 147 (2007) 799–807 805

Chaenocephalus aceratus with a body length of 85 cm, has only anatomical, physiological and behavioral capacities of Ant- 12,700 fast muscle fibres per trunk cross section, and a fibre arctic notothenioids. diameter of 600 μm. The Patagonian notothenioid Eleginops Although at present we can provide, at best, only preliminary maclovinus, which reaches similar size to the C. aceratus,has answers to any of these questions about genetic losses during 164,000 fibres with a fibre diameter of approximately 490 μm evolution in stably cold waters, the exploitation of genomic (Johnston et al., 2003). Thus, although data are limited, technologies in the study of notothenioids may soon allow us to comparisons of fibre diameters in notothenioids with those of examine in fine detail the “genetic tool kits” of Antarctic and temperate and tropical fishes suggest that the large maximum non-Antarctic notothenioids (Peck et al., 2005). Sequencing and diameter of muscle fibres in notothenioid fishes, especially the annotating of Antarctic notothenioid genomes is under way, and more derived Antarctic lineages, is exceptional. we may soon know what protein-encoding genes and regulatory The factors that have selected for small numbers of large loci have been lost (or rendered dysfunctional) during evolution sized muscle fibres in derived Antarctic notothenioids, notably in the Southern Ocean. Complementary comparative studies of the Nototheniidae and Channichthyidae, are not fully under- the genomes of cold-temperate notothenioids could reveal stood, but may relate to the energy costs of life at near-freezing whether “escapees” from Antarctica migrated to temperate temperatures, especially the costs of producing large amounts of waters before some (or all) of these genetic lesions occurred. AFGPs (Johnston et al., 2003). The greater fibre number in Better defining the acclimatory capacities of Antarctic and cold- muscle of two South American notothenioids lacking anti- temperate notothenioids will allow firmer predictions to be freezes, and gobio, may made about the potential effects of climate change on this reflect the absence of an evolutionary period in cold Antarctic suborder of fish. waters by these fish. Analyses of gene expression using complementary DNA According to Egginton et al. (2002) the rate of oxygen (cDNA) microarrays (“gene chips”) offer another promising delivery to aerobic muscle fibres is a function of the fibre experimental strategy for elucidating the genetic capabilities of diameter together with factors that affect diffusion rate: notothenioids fishes. Studies of eurythermal fishes have temperature, distribution of mitochondria and lipid droplets revealed that hundreds of genes may shift expression during within the fibre as well as the overall metabolic demand thermal acclimation (Gracey et al., 2004; Podrabsky and (O'Brien et al., 2003). Adequate oxygen delivery to large Somero, 2004; Buckley et al., 2006). Microarray analysis of diameter muscle fibres is probably only possible because of the temperature-induced changes in gene expression of Antarctic very low metabolic demand in polar fishes at low temperature notothenioids is underway and is beginning to show similarities (Clarke and Johnston, 1996; Steffensen, 2002). Modeling and differences between these extreme stenotherms and the studies by Egginton et al. (2002) indicate that a low fibre more eurythermal fishes recently examined with this technology number and high maximum fibre diameter do not limit adequate (Buckley and Somero, in preparation). Absence of induction of oxygen flux at low body temperatures in notothenioids. Hsp-encoding genes is confirmed, but other stress-related genes Further comparisons of muscle fibre size and number in show qualitatively similar expression patterns to those seen in cold-temperate notothenioids, including New Zealand species temperate species. Thus, not all of the acclimatory abilities of that arose from ancestors that “escaped” from Antarctica could Antarctic fishes have been lost during millions of years of further clarify the roles that adaptation to cold has played in the evolution at cold, stable temperatures. The finding that evolution of skeletal muscle in notothenioids. notothenioids have the capacity to increase their resistance to acute heat shock suggests that some temperature-dependent 8. Genetics of notothenioids: what has been lost during gene regulatory abilities related to stress tolerance remain in the evolution in stably cold waters? “tool kits” of Antarctic notothenioids, even though the HSR has been lost (Podrabsky and Somero, 2006). How the genetic The discovery that Antarctic notothenioids have lost genetic repertoires of Antarctic notothenioids have changed and how information that likely is essential for life in warmer waters they differ from those of cold-temperate notothenioids repre- raises a number of questions about the evolutionary histories sents an exciting frontier in the study of this fascinating of notothenioids and the future prospects of these species in a suborder of fishes. warming world. What other types of genetic information have been lost, in addition to genes encoding hemoglobin and Acknowledgements myoglobin (for recent reviews of this topic, see Cheng and Detrich, in press; Sidell and O'Brien, 2006) and components We thank Dr. Bradley Buckley for his critical reading of this of the heat-shock response (Hofmann et al., 2000; Place et al., manuscript. 2004; Place and Hofmann, 2005)? Does loss of genetic information preclude acclimation to elevated temperatures References such as those predicted as a result of climate change? Have cold-temperate notothenioids retained this critical genetic Anderson, J.B., 1999. Antarctic Marine Geology. Cambridge University Press, Cambridge. information? These questions about genetic lesions comple- Archer, S.D., Johnston, I.A., 1991. Density of cristae and distribution of ment those asked by Montgomery and Clements (2000) in mitochondria in the slow muscles of Antarctic fish. Physiol. 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