Marine Genomics 2 (2009) 1118

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Marine Genomics
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m a r g e n

Review

HSP70 heat shock proteins and environmental stress in Antarctic marine organisms: A mini-review
Melody S. Clark , Lloyd S. Peck
Biological Sciences Division, British Antarctic Survey, Natural Environment Research Council, High Cross, Madingley Road, Cambridge, CB3 0ET, UK

a r t i c l e

i n f o

a b s t r a c t
The ability to understand and predict the effects of environmental stress on biodiversity is becoming increasingly important in our changing environment. Antarctic marine species are some of the most stenothermal on the planet and many inhabit the waters off the Antarctic Peninsula which is one of the areas where there is rapid regional climate change. Therefore these animals are highly vulnerable to changing environmental temperatures and clearly we need to understand the complexities of their response, not just at the individual species level, but also the implications for the ecosystem as a whole. Heat shock proteins have a long history of use in studies of organism stress responses and have frequently been proposed as potential universal molecular biomarkers, especially for non-model species. In this mini-review, the heat shock response and heat shock proteins (specically the HSP70 family) are examined in Antarctic marine species alongside their physiological capabilities and limits to answer a series of questions: do these animals have a heat shock response which includes the expression of HSP70 genes? What is the relationship between their heat shock response and physiological capabilities? Can HSP70 genes be used as molecular biomarkers for these species? Crown Copyright 2009 Published by Elsevier B.V. All rights reserved.

Article history: Received 2 October 2008 Received in revised form 3 February 2009 Accepted 2 March 2009 Keywords: Climate change Adaptation Temperature tolerance Temperature limit Stenotherm Extinction

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . 2. The HSP70 family of heat shock proteins . . . . . . 3. Antarctic sh and HSP70 genes . . . . . . . . . . . 4. Other Antarctic marine species and HSP70 genes. . . 5. The heat shock response and environmental challenge 6. Plasticity of response and trade-offs . . . . . . . . . 7. Conclusions . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 12 12 13 13 15 16 16

1. Introduction The earliest biologists, from Plato onwards recognised variation in nature as important. Lamarck (1809) suggested change in species was driven towards more complex states, but that there was also some adaptive effect of the environment. Darwin (1859) whilst fascinated with these initial theories used meticulous scientic observation to produce the theory of evolution and transform our thinking in this area. This is now the cornerstone of understanding how organisms

Corresponding author. E-mail address: mscl@bas.ac.uk (M.S. Clark).

respond to change with time. A multitude of scientists across the globe currently investigate how species respond to environmental change, and one of the main thrusts of this work is the response to stress, which can be addressed from a wide range of different standpoints from the ecological, with an interest in what sets colonisation and range boundaries (reviewed in Somero, 2002; Hofmann, 2005) through to the more applied elds of aquaculture and sh health (c.f. Varsamos et al., 2006). The publication of the latest IPCC report (IPCC, 2007) has lead to an increasing need to understand stress responses in order to predict the impact of climate change on our planet and the inherent biodiversity, which shapes our world. Hence, there is an added impetus to investigate species response to environmental challenge and how this may affect

1874-7787/$ see front matter. Crown Copyright 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.margen.2009.03.003

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ecosystem functioning. In this review, the terms environmental stress or stress will often be used and in this context are dened as an environmental factor causing a change in a biological system, which is potentially injurious (Hoffmann and Parsons, 1997) and which has some tness consequences (Bijlsma and Loeschcke, 1997) i.e. ultimately affects survival, but potentially also has other consequences such as longevity and reproductive capacity. Understanding such responses is particularly crucial for species, such as marine organisms, whose body temperature directly reects that of the environment (ectotherms). Cold-blooded Antarctic marine species face a range of unique environmental challenges of extreme low temperature and stability combined with the most intense seasonality in the world's oceans (Clarke, 1988; Peck et al., 2006a) that have resulted in the evolution of a suite of specic adaptations. The best known of these is antifreeze production in sh (DeVries and Wohlshlag 1969; Nicodemus et al., 2006). Others include slow rates of growth (Arntz et al., 1994; Peck et al., 1997; Peck, 2002), extended long periods needed to process meals (Clarke and Prothero-Thomas 1997; Peck, 1998), very long development periods (Bosch et al., 1988; Peck et al., 2006b), slow rates of activity (Peck et al., 2004) and large size (Chapelle and Peck 1999). These adaptations place Antarctic marine species at the extreme end of adaptations seen in marine species, and confer specic problems for them in the face of change. To provide an environmental context: globally, oceanic sea surface temperatures are predicted to rise on average by 2 C over the next 100 years (Murphy and Mitchell, 1995; IPCC 2007) but regional differences are already apparent, i.e. the Polar Regions (specically the Arctic and Antarctic Peninsula) where warming is happening more rapidly than most other places on the planet. Average Arctic air surface temperatures are increasing at approximately twice the global average (IPCC 2007). Regional climate change along the Antarctic Peninsula has been rapid with air temperature rises of up to 3 C and elevation in the surface layers of the Bellingshausen Sea of 1 C in 50 years (Meredith and King, 2005). Therefore the highest priority areas to study organism responses to environmental change must include those where change is happening at the fastest rate. Whilst marine animals from both Poles survive temperatures down to 1.9 C, there are important differences. Antarctic species have been effectively geographically isolated in a constantly cold environment for around 2522 Ma BP and contain a high percentage of endemic species (reviewed in Clarke and Johnston, 1996). In contrast the Arctic marine benthic fauna comprises a relatively young assemblage characterised by species from either the Pacic or Atlantic with notably few endemics (Dunton, 1992). Hence by comparing species, which inhabit similarly cold waters, specic regional evolution can be dissected from cold adaptation and temperature adaptability (c.f. Verde et al., 2007). Indeed, Antarctic species are more stenothermal than the Arctic biota, such that many suffer large-scale failure of essential biological functions with temperature rises of just 23 C (Peck et al., 2004; Peck, 2005), and the most temperature tolerant species appear only able to survive physiologically extended periods (months) at temperature around 6 C (Peck et al., 2008), but generally this gure is much lower (Peck et al., 2009b). This restricted thermal envelope, particularly when combined with the absence of the confounding effects of pollution (which affects most of the rest of the planet) means that Antarctic species provide the most efcient models for studying responses to warming seawater temperatures. Research into the response to environmental change can be carried out at different experimental levels from ecological observation (Walther et al., 2002; Aronson et al., 2007), behavioural, physiological (Peck et al., 2004) and cellular (molecular) (Clark et al., 2004; Peck et al., 2005). Whilst all contribute to understanding ecosystem function, the ability to predict the vulnerability of a species to stress is best achieved at the molecular level, as sub-lethal effects across a range of functions can be quantied. Hence there is now interest in developing molecular

biomarkers for environmental stress, with research to date, concentrated on the heat shock response and heat shock proteins/genes (HSPs), in particular the HSP70 family of heat shock proteins (Clark et al., in press). This is because in the almost a complete absence of sequence data for environmentally meaningful species or species key for ecosystem function, the use of highly conserved genes facilitates the development of a universal biomarker system. HSP70 genes are strongly conserved between species and are therefore relatively easy to clone using degenerate PCR techniques. Hence they have been proposed as strong candidates for such types of biomarkers (c.f. Tomanek and Sanford, 2003; Hamer et al., 2004). This mini-review will concentrate on research examining the contribution of the molecular HSP70 heat shock response in Antarctic marine species compared to their ability to adapt to changing environmental conditions. 2. The HSP70 family of heat shock proteins An organism's response to stress can take many forms (behavioural, biochemical etc.), most of which are species-specic. However, so far the only known universal response is the production of stress proteins (Gross, 2004). Again, these differ between species, but the most commonly studied are a family of proteins, commonly known as heat shock proteins (HSPs). Numerous families of heat shock proteins have been identied, the naming of which is related to their molecular weight in kiloDaltons. The most studied of these family members are the 70 kDa heat shock proteins, which comprise two main forms: Constitutive: present all the time (heat shock cognates e.g. HSC70) Inducible: expressed in response to external stimuli (heat shock proteins e.g. HSP70). Both types have been well studied and their roles as molecular chaperones documented. They are present in the normal cell state to aid in the folding of native polypeptides and their translocation to different cellular compartments (Feder and Hofmann, 1999; Hartl and HartlMeyer 2002). But, more importantly, as regards their role in the stress response, they assist mis-folded proteins to attain or regain their native states and also target degraded proteins and regulate their removal from the cell, thus preventing the formation of cytotoxic aggregates (Parsell and Lindquist, 1993; Hartl, 1996; Fink 1999). The classical activation of the inducible HSP70 genes is in response to elevated environmental temperatures (often called the heat shock response (HSR)) and has been described in most organisms examined to date. HSP production is, however, seen in response to a wide range of environmental insults including dehydration in plants (Schramm et al., 2008) and insects (Hayward et al., 2004), osmotic stress in crustaceans (Chang, 2004) and sh (Todgham et al., 2005) and heavy metal pollutants in oysters (Corporeau and Auffret, 2003) and amphipod crustaceans (Schill et al., 2003). The HSP70 response is tightly controlled by heat shock transcription factors (HSF) (reviewed in Morimoto, 1998), but HSP70s are also up-regulated in response to other external signals (Feder and Hofmann, 1999). In the environment, these proteins are generally induced in response to an external temperature rise of 510 C greater than the optimal growth temperature of an organism (Lindquist, 1986). So the interest in the heat shock response and Antarctic marine species was fuelled by the fact that the Southern Ocean has a very stable and narrow temperature range, where sudden temperature rises, even over a period of decades, of +5 C have not occurred for millions of years (Maresca et al., 1988). So if the threshold temperatures for HSP production are correlated with the temperature the species live at, are Antarctic animals a special case? 3. Antarctic sh and HSP70 genes The molecular characterisation of heat shock proteins started 20 years ago, with the identication of HSP70 genes in three

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Antarctic sh via hybridization of Southern and Northern blots using a heterologous Drosophila probe (Maresca et al., 1988). This eld of investigation remained dormant for ten years, before conrmation of the mRNA results (Carrat et al., 1998), but really took off in 2000 spear-headed by the work of Hofmann and co-workers. Improvements in molecular techniques provided more sophisticated tools for the analysis of gene expression and the early results provided tantalising evidence that maybe all was not quite what it seemed with the heat shock response in Antarctic sh. Hofmann et al. (2000) conclusively proved that the classical heat shock response was absent in Trematomus bernacchii. Further investigations on a range of Antarctic sh species showed that it was not just a lack of response to heat, but that the inducible form of HSP70 was unaffected by cadmium (a toxicant which causes protein denaturation and creates reactive oxygen species) and MG132 (an inhibitor of proteasome activity which results in the accretion of misfolded or damaged proteins) also failed to induce expression of this gene (Hofmann et al., 2000; Buckley et al., 2004). Therefore it was hypothesised that there was an irregularity in the HSP gene expression pathway, rather than a loss of temperature sensitivity per se. The inducer of HSP70, HSF1 was shown to have retained DNA binding activity with heat shock element (HSE) binding competency in T. bernacchii hepatocyte cultures and hence the logical conclusion was that a mutation had occurred in the promoter region of the HSP70 gene which prevented HSF1 binding and inducing transcription of this gene (Buckley et al., 2004). From studies on a series of New Zealand notothenioid congeners and a distantly related Antarctic sh species (Lycodichthys dearborni: zoarcid), this mutation was suggested to have occurred between 512 Mya during the period of non-bovichtid notothenioid radiation (Carpenter and Hofmann, 2000; Place et al., 2004; Hofmann et al., 2005). So the majority of Antarctic sh cannot up-regulate HSP70 in response to external stresses. This is further complicated by the fact that four related Antarctic species, T. bernacchii, Pagothenia borchgrevinki (Nototheniidae) Harpagifer antarcticus (Harpagiferidae) and L. dearborni (Zoarcoidei) permanently express the inducible form of HSP70 (Place et al., 2004; Place & Hofmann 2005a; Clark et al., 2008a), presumably in response to the problems of protein folding at low temperatures (Privalov, 1990). Antarctic marine species, in general show elevated levels of protein damage (Place et al., 2004; Todgham et al., 2007), with concommitant low levels of overall protein retention efciency (Fraser et al., 2007); clear indications of problems in protein homeostasis. Given these data, the obvious question is whether the permanent expression of HSP70 and lack of a heat shock response in Antarctic notothenioids is family-specic, and/or a consequence of adaptation to highly stable, cold Antarctic seawater temperatures and could therefore be a general phenomenon extending to other non-piscine Antarctic marine organisms. 4. Other Antarctic marine species and HSP70 genes At a similar time to the Antarctic sh work, LaTerza et al. (2001) reported another Antarctic marine species, this time a ciliate (Euplotes focardii) with a barely detectable inducible HSP70 response. This added weight to the argument that the lack of the classical heat shock response might be an adaptation to the cold stable Antarctic marine environment. Further investigations identied a different situation to the sh. In-depth sequence analysis of the 5 region of the E. focardii HSP70 gene revealed the presence of heat shock promoter elements (HSEs) and also StREs (Stress response elements), both of which confer the ability (in theory) to respond to stress. These data also run contrary to the earlier explanations of loss of HSP response in Antarctic sh. Furthermore a series of chemical agents (tributyltin, sodium arsenite and hydrogen peroxide) induced HSP70 gene expression in this organism Hence, there was clearly some mutation

in the capacity of HSF trans-acting factors to bind and activate the HSE and hence HSP70 expression (LaTerza et al., 2004, 2007). No additional species were investigated until 2007, when Park et al. (2007) reported the full length sequence of an inducible form of HSP70 in the clam (Laternula elliptica) and up-regulation of this gene at 10 C in digestive gland and gill tissue (3.6 fold and 4.6 fold induction respectively). A more comprehensive analysis was conducted on this species by Clark et al. (2008b), examining 3 HSP70 family members in 5 tissues over a range of temperatures (415 C). L. elliptica in fact possesses a duplication of the inducible HSP70 gene (HSP70A and HSP70B), both forms of which vary in their tissue expression and levels of induction (maximally 12.5 and 86 respectively). Such variation between isoform expression patterns and independent regulation is exactly what would be expected in line with the retention and evolution of duplicate genes via sub-functionalisation (Force et al., 1999). Interestingly, in agreement with the sh work, permanent expression of HSP70A and HSP70B plus GRP78 (78 kDa glucose regulated protein, an HSP70 family member) was detected. Similarly to Park et al. (2007), the induction temperature for these genes was between 810 C, far higher than that experienced by any species in Antarctic waters. This work on a bivalve mollusc was paralleled by work on the limpet (Nacella concinna), in which 4 members of the HSP70 family were investigated (HSP70A, HSP70B, GRP78 and HSC70). Again there was permanent expression of all of these genes (particularly HSC70) in control animals, but with a much clearer induction temperature of 15 C (Clark et al., 2008b). These abnormally high induction temperatures compared to experienced natural environmental temperatures were presumed to be the relic of a temperate ancestor, with no discernable adaptation of the threshold to the colder Antarctic waters, possibly because the stable temperatures did not necessitate selection pressure for change. However, the situation is more complex, as will become apparent below (c.f. Fig. 1). So, maybe the sh and the ciliate were extraordinary examples of the heat shock response. However, additional work by Clark et al. (2008c) identied two more Antarctic species, a sea star (Odontaster validus) and a gammarid (Paraceradocus gibber) which did not possess a discernable heat shock response, in spite of identifying several HSP70 gene family members in each species. Also these species showed no signicant permanent expression of the HSP70 family gene members which were surveyed (Fig. 1). GRP78 was not isolated in P. gibber and HSC70 was not cloned from O. validus, although it is more difcult to identify specic genes from multigene families using small gene fragments as opposed to full length sequences (c.f. Leignel et al., 2007). Previous work showed that it is not always the same HSP70 family member that is predominantly expressed constitutively, being HSC70 in N. concinna and GRP78 in L. elliptica (Clark et al., 2008a). So these genes need to be comprehensively identied in all species to prove whether constitutive expression of HSPs is a general requirement for survival at low temperatures. This statement has the caveat that standard experimental protocols identify all family members, including potentially novel subgroups (Leignel et al., 2007). Although the number of examples is limited, the distribution across taxa is vast: from a microbial eukaryote (E. focardii) through to several lower vertebrates (sh), there is no simple rule for possession and expression or loss of the classical heat shock response in Antarctic marine organisms. The question that then arises is, given the stenothermal nature of the Antarctic marine life, are those species that have a heat shock response (albeit seemingly activated at relatively high temperatures) better able to adapt to warmer seawater temperatures than those that do not? To date, such data do not exist in Arctic species and so the other question of general adaptation to extreme cold and lack of a classical heat shock response cannot be answered. 5. The heat shock response and environmental challenge Given the scale of the thermal challenges needed to induce the HSP70 heat shock response in the species already described (Table 1),

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Fig. 1. A: Antarctic Peninsula marine species tested for the heat shock response at both + 10 C and + 15 C. Red arrows below the species indicate presence of an inducible HSP70 heat shock response, whilst grey arrows indicate that no production of inducible HSP70s was recorded. B: Different stress experiments carried out on the limpet N. concinna. Red arrows above the animal indicate that these stresses elicited an HSP70 response, whilst grey arrows indicate that no HSP70 response was produced. C: Different stress experiments carried out on the clam Laternula elliptica signicance of the colour of the arrows above the animal, as for Fig. 1B.

there is clearly a question of environmental relevance. The experiments largely consisted of very short, acute thermal challenges e.g. 10 C for 2 h, with no period of gradually warming to the target temperature. Only T. bernacchii was surveyed after a period of acclimation (Carpenter and Hofmann, 2000; Hofmann et al., 2000), but this was still only for a period of 3 weeks. Antarctic brittle stars, Ophionotus victoriae, when held at +2 C are not capable of acclimation, whilst 100% survive 3 weeks, there are 96% losses within 68 days (Peck et al., 2009a). There was N 50% mortality of the T. bernacchii in the Carpenter and Hofmann (2000) study, and so the sh were not physiologically acclimated to 4 C, but were resisting the stress prior to death. Some acclimation work has been carried out on the species described in this review and in most cases the upper limit for acclimation is around 34 C. Interestingly, however, it is the sea star (O. validus) that appears the most robust. This species survives long-term (several months) and still feeds at 6 C. It is also capable of

maintaining activity up to 9 C in shorter-term trials (Peck et al., 2008). Several sh species have been shown to acclimate to temperatures up to 34 C (Lowe and Davison, 2005; Jin and DeVries, 2006; Gonzalez-Cabrera et al., 1995; Seebacher et al., 2005; Podrabsky and Somero, 2006; Robinson and Davison, 2008) with the most conclusive evidence presented for Pagothenia borchgrevinki which was sucessfully acclimated for 6 months (Robinson and Davison, 2008). Of the marine invertebrates studied in excess of O. victoriae, that is incapable of acclimating to 2 C, both the bivalve mollusc (Laternula elliptica) and the amphipod Paraceradocus gibber cannot acclimate to 3 C (S. Morley pers. comm.; M. Clark and L. Peck pers obs). The brachiopod Liothyrella uva can acclimate to 3 C but not 4 C (Peck 1989). The bivalve Yoldia eightsi, the limpet Nacella concinna and the sea urchin Sterechinus neumayeri have similar capacities and the amphipod Cheirimedon femoratus can acclimate to 4 C (Peck et al., 2009b). Overall very few Antarctic marine species appear able to

M.S. Clark, L.S. Peck / Marine Genomics 2 (2009) 1118 Table 1 Species surveyed for induction of the classical heat shock response and their physiological heat limits. Species Trematomus bernacchii Upper lethal temp (ULT) 6 C Acclimation temp (AT) N 4 C Inducible HSPs No Short-term heat shock assay conditions (HSP) (a) 10 C: 2 h (b) 4 C: 1, 2, 4, 8 & 12 h (c) 4 C: 21 days (d) 5 C: 22 days References

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Pagothenia borchgrevinki

6 C

4 C

No

4 C: 1, 2, 4, 8 & 12 h

Lycodichthys dearborni Harpagifer antarcticus Euplotes focardii Laternula elliptica

Not known Not known Not known 9 C

Not known 3 C Not known 2 C

Yes No No Yes

4 C: 1, 2, 4, 8 & 12 h 10 & 15 C: 2 h 6 C: 2, 4, 8, 12, 24 & 48 h 4, 8, 12, 15, 18, 20 & 25 C: 30 min or 4 h (a) 10 C: 6, 12, 24 & 48 h (b) 4, 6, 8, 10 &15 C: 2 h

Nacella concinna

9.5 C

4 C

Yes

10, 15, 20 C: 2 h

Odontaster validus

9 C

6 C

No

10 & 15 C: 2 h; 2 C & 6 C: 2, 6, 12, 48 & 168 h

Paraceradocus gibber

Not known

b 3 C

No

10 & 15 C: 2 h

ULT: Somero and DeVries, 1967 AT: Carpenter and Hofmann, 2000 HSPa: Hofmann et al., 2000 HSPb: Place and Hofmann, 2005a HSPc: Carpenter and Hofmann, 2000 HSPd: Hofmann et al., 2000 ULT: Somero and DeVries, 1967 AT: Robinson and Davison, 2008 HSP: Place and Hofmann, 2005a HSP: Place and Hofmann, 2005a AT: Clark (unpub) HSP: Clark et al., 2008a HSP: LaTerza et al., 2001 ULT: Peck, 2002 AT: S. Morley, pers comm HSPa: Park et al., 2007 HSPb: Clark et al., 2008b ULT: Peck, 1989 AT: Peck et al., 2009b HSP: Clark et al., 2008b ULT: Peck et al., 2008 AT: Peck et al., 2009a HSP: Clark et al., 2008c HSP: Clark et al., 2008c

Upper lethal temperature limits quoted are those where temperatures were raised 12 C every 57 days. Upper limits vary with rate of warming (Peck et al., 2009b).

acclimate and perform normal biological functions over periods of months at temperatures above 4 C, and many have limits signicantly below this, but once again the situation is complex. Such short-term experimental data does not negate the utility of the laboratory experiments in the heat shock investigations. Without them, it would not have been possible to clone, identify and develop assays for the HSP70 heat shock response in a series of non-model species. Whilst lack of sequence data hinders research into most animals, there is also the inherent problem of identifying a response above the background noise of individual variation in wild, noninbred species (c.f. Oleksiak et al., 2002; Clark et al., 2008c). Therefore the assay has to be robust and trends in data are more important than actual numerical increases in gene expression, as the dramatic response at 15 C for N. concinna is likely to be the exception rather than the rule. The fact that HSP70 genes have been duplicated and manitained in N. concinna and maintained in some other species (L. elliptica, P. gibber and O. validus) (Clark et al., 2008b,c) implies, according to the theory of sub-functionalisation (Force et al., 1999), that the genes are still present due to advantageous selective pressures. Indeed, both the two isoforms of HSP70 in L. elliptica and N. concinna show differential expression patterns in response to heat induction and tissue-specic expression in the case of L. elliptica (Clark et al., 2008b). Hence, why were these genes maintained, if seemingly unresponsive to thermal challenge? The answers may lie in responses to multiple stressors, potentially even damage from early ice nucleation in cells, but research into these factors is currently lacking. This is where the environmental sampling and more complex laboratory experiments can quantify responses to change. N. concinna is a common inter-tidal species and can be found anywhere from midtide level (Walker 1972) to depths greater than 110 m depth (Powell 1951, 1973). The initial experiments were carried out on sub-tidal animals (810 m depth) that normally experience a yearly water temperature range of between 1.86 C and +1 C. To compare this population and assay the effect of more complex stresses. Clark et al. (2008d) collected inter-tidal N. concinna over the period of several tidal cycles at different seasonal timepoints. In all three samplings, the inducible forms of HSP70 were permanently expressed at higher levels than the sub-tidal animals and HSP70A showed a signicant

induction in response to emersion (Clark et al., 2008d). The average foot temperature of these animals was only 3.3 C, far below the induction temperature of 15 C in the previous experiments (Clark et al., 2008d). Additional experiments indicate a small effect due to desiccation, which is exacerbated with heat, but none due to cold shock or immersion for several hours in fresh-water run-off (Clark and Peck, unpub) (Fig. 1). Longer term acclimation experiments at 2 C (1 month), which is close to, but under the upper temperature limit for 1 month acclimation for N. concinna without mortality (which survives 1 month at 3 C with no mortality, but has 50% mortality after 3 months (S. Morley, pers. comm), show elevated levels of all four HSP70 gene family members (HSP70A, HSP70B, GRP78 and HSC70) over this extended time period. Hence the heat shock response in chronic stress has different thresholds to acute experiments. Further work on the clam (L. elliptica) indicates that this species also induces HSP70 genes in response to stresses other than heat (Philipp and Clark, unpub) (Fig. 1). So there are more complex triggers of these genes than is obvious from the initial laboratory experiments. It is clear that these results in two species could be mirrored in the other species which to date have no proven classical heat shock response, but may still use HSP70 genes as part of their natural cellular stress response mechanism. 6. Plasticity of response and trade-offs What these data clearly demonstrate is the plastic nature of the induction and expression of the HSP70 response. This is not a new phenomenon, having previously been shown in species from lower latitudes via studies on, for example, thermal history, seasonality, inter-tidal vertical zonation and biogeography (Buckley et al., 2001; Tomanek, 2002; Osovitz and Hofmann, 2005). All these studies are based on environmental sampling, i.e. describing what was happening at a particular time point in wild sampled populations, given the prevailing habitat conditions. The objective of developing biomarkers for environmental stress is more complicated than just identifying a few genes. This is a new area of biological prediction; seawater temperatures are rising at a rate faster than in any period over the past million years or on record over the last Pleistocene glacial cycle (Zachos et al., 2001). So prediction of

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effects is intermediate between ecological observation of species boundaries (adaptation over ecological timescales) and relatively short-term experimental manipulation. The stress response is complex and there are clearly trade-offs between the requirement to combat the environmental insult and maintain normal homeostasis. With regard to HSPs, there is a demonstrable energetic cost associated with the production of heat shock proteins, overproduction may be cytotoxic and the system is proposed to have evolved under strong trade-offs (reviewed in Srensen and Loeschcke, 2007). Several of the Antarctic species surveyed here, already permanently express the inducible form of HSP70 and therefore further continued up-regulation of HSPs in response to environmental stressors may not be without signicant deleterious effects on other cellular processes. There have been some studies examining the thermal activity of HSC70 orthologues from temperate and Antarctic species and there has been at least a partial adaptation to the permanently cold conditions with higher expression levels of constitutive elements than expected (Place and Hofmann, 2005b,c), and this may be akin to the universal production of antifreeze in sh as a required adaptation for survival in Antarctic marine conditions. The pressing question arises as to how effective these adaptations are in the face of change? And whether such adaptations have occurred in other heat shock family members? 7. Conclusions To date, the classical heat shock response has been identied in all species studied with the exception of the Antarctic examples quoted here and the hydra (Hydra oligactis) (Bosch et al., 1988). HSP70 heat shock proteins are documented as being one of the major groups of proteins involved in stress responses (Gross, 2004) and therefore their induction and utility in Antarctic marine organisms is of great interest, given climate change predictions; predicted seawater temperatures along the Peninsula put Antarctic stenotherms at considerable future risk of seasonal exposure to ambient water temperatures that exceed those known to result in the loss of critical biological functions (Peck et al., 2004). Whilst the options are limited for these animals (Peck, 2005; Barnes and Peck, 2008), they provide ideal models with which to study the complexities of the temperature-related stress response. The thermal sensitivity of these species combined with a comprehensive understanding of their physiology and thermal limits and an absence of confounding anthropogenic inuences in their environment promote them for just such a purpose and the development of biomarkers for stress in temperate species living in a more complex environment. Laboratory work enables assays to be developed and the effects of different stresses to be examined individually, which then provides added value when examined in an environmental context, where multiple stressors are the rule rather than the exception. Clearly the body of work reviewed here indicate that HSP70 genes are not ideal candidates for stress response biomarkers, a situation analogous to that identied in sh species (Iwama et al., 2004), but the work does provide tantalising clues as to the complexity of the response with different stressors and over differing timescales. Hence more genes need to be identied and surveyed for their expression related to the stress response. The pluripotent nature of chaperone function has been known for some time (c.f. Morimoto, 1998; Gross, 2004). However, this understanding is improving with new studies on cell network interactions. There is increasing data on the promiscuity of chaperone interactions, with the proposal that they are important inter-modular elements of cellular networks, acting as multifunctional hubs (Korcsmros et al., 2007). Hence they aid in assembling the modular structure of the cell through low afnity temporary interactions (Csermely, 2004). As such, even subtle environmental perturbations are proposed as being able to profoundly affect not only chaperone production, but also the functions of the whole cell network (Korcsmros et al., 2007). So even though the Antarctic HSP70 genes are not necessarily potential

biomarkers, they are clearly pivotal in the stress response and more work is needed to examine the complex interactions of these proteins in marine ectotherms and indeed combine the very specic HSP70 work with that studying other types of heat shock protein (c.f. small heat shock proteins (Nakamoto and Vigh, 2007). This is where the increasing use of technologies such as EST libraries, Q-PCR, microarrays and 454 sequencing will become increasingly important. They can provide new resources to access these previously unknown genomes (c.f. Vera et al., 2008) on a massive scale. This will progress the current work from one or two candidate genes to potentially thousands. Work is urgently required on the transcription proling of such organisms under normal and stressful conditions using thousands of genes over a range of species as this will enable the comprehensive dissection of common gene pathways involved in the stress response. Additionally studying why some taxa are more resistant than others and whether some trophic guilds are more sensitive than others or more active species more temperature tolerant will enable us to produce assessments of organism stress in an ecosystem context. But, as always the genomics tools must be used alongside robust physiological and ecological studies to provide environmentally meaningful data, which in the case of Antarctic species means that the data can be extrapolated to the global context. References
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