Doris Abele
email: doris.abele@awi.de

Thomas Brey

Eva Philipp

Bivalve models of aging and the determination of molluscan lifespans

Keywords: Bivalve, longevity, aging, stress tolerance, heterozygosity, Antarctic, genes, metabolic rate depression

Bivalves are newly discovered models of natural aging. This invertebrate group includes species with the longest metazoan lifespan approaching 400 years, as well as species of swimming and sessile lifestyles that live just for 1 year. Bivalves from natural populations can be aged by shell growth bands formed at regular intervals of time. This enables the study of abiotic and biotic environment factors (temperature, salinity, predator and physical disturbance) on senescence and fitness in natural populations, and distinguishes the impact of extrinsic effectors from intrinsic (genetic) determinantes of animal aging. Extreme longevity of some bivalve models may help to analyze general metabolic strategies thought to be life prolonging, like the transient depression of metabolism, which forms part of natural behaviour in these species. Thus, seasonal food shortage experienced by benthic filter feeding bivalves in polar and temperate seas may mimic caloric restriction in vertebrates. Incidence of malignant neoplasms in bivalves needs to be investigated, to determine the implication of late acting mutations for bivalve longevity. Finally, bivalves are applicable models for testing the implication of heterozygosity of multiple genes for physiological tolerance, adaptability (heterozygote superiority), and life expectancy.

Bivalves: a diverse set of models for ageing studies and environmental recorders

Commonly used animal models for cellular and molecular mechanisms underlying the process of aging, such as Drosophila melanogaster, Caenorhabditis elegans or small rodents, are bred and reared under controlled laboratory conditions. Most of these aging models are short lived (days in C. elegans, weeks in D. melanogaster, <5 y in rodents) and, hence, may not show all the age-dependent changes of long lived species [START_REF] Reznik | New model systems for studying the evolutionary biology of aging: crustacea[END_REF][START_REF] Kirkwood | Evolution of ageing[END_REF]. Especially, short lived models are not representative of the negligible aging type [START_REF] Finch | Longevity, Senescence, and the Genome[END_REF][START_REF] Terzibasi | The short-lived Nothobranchius furzeri as a new model system for aging studies[END_REF]). Further, aging under controlled laboratory conditions may differ significantly from aging in the wild, and the applicability of the results to natural animal populations is often questionable. Good reasons to establish animal models that can be sampled from a range of environmental settings and scenarios, but at the same time provide information on their individual life history, including their chronological age [START_REF] Austad | The uses of intraspecific variation in aging research[END_REF].

Such new models are found among bivalve molluscs. Worldwide, approximately 20,000 species [START_REF] Pearse | Living invertebrates[END_REF]) of marine bivalves exist so that this class offers a rich diversity of lifestyles, adaptations to specific environmental conditions and corresponding strategies of ageing, albeit based on the same filter feeder blueprint in the majority of species. Some bivalve species, like the long lived ocean quahog, Arctica islandica or the blue mussel Mytilus edulis exhibit broad geographical distribution, with habitats spanning large latitudinal ranges and covering different climate zones. Other species like the hydrothermal vent mussel Bathymodiolus azoricus are restricted to a rather narrow environmental window and have specified their physiology for adaptation to one ecological setting. Mainly filter and deposit feeders, bivalves are of central importance for carbon flux in many ecosystems.

Often they have key species function with respect to habitat structuring and energy flow, such as the burrowing mud clams Laternula elliptica in the Antarctic, Hiatella arctica in the Arctic region, or bank forming species (mussels, oysters) in general.

Within the Bivalvia, we find species firmly attached to the substratum (e.g. Ostreidae, oysters, and Mytilidae, mussels), others that crawl with the help of their muscular foot (most species), and even facultative swimmers (e.g. Pectinidae, scallops). Infaunal species burrow in soft (e.g. Tellinidae, clams) and hard substrates (e.g. Teredinidae, shipworms) and can live at the sediment-water interface (e.g. Unioinidae, freshwater mussels), or burrow down to 100cm of sediment depth (e.g. Myidae, soft shell clams, or Hiatellidae, geoducks). Epifaunal species are encountered on all kinds of surfaces, from rocks to such exotic places as the spines of sea urchins (e.g. Lissarca notorcadensis on Antarctic cidaroids, [START_REF] Brey | Growth, reproduction and production of Lissarca notorcadensis (Bivalvia: Philobryidae) in the Weddell Sea, Antarctica[END_REF]. Bivalves inhabit a pressure range from 1 atm in the littoral to about 1000 atm in the deep sea; a temperature range from -1.8°C in polar waters (Antarctic clams) to >30°C in tropical seas and around deep sea hot vents (e.g. Vesicomyidae), and a salinity range from fresh water (e.g. Unionidae) to hypersaline waters (e.g. the cockle Fragum erratum at ±60 ‰, [START_REF] Morton | The biology and functional morphology of Fragum erugatum (Bivalvia: Cardiidae) from Shark Bay, Western Australia: the significance of its relationship with entrained zooxanthellae[END_REF].

Adult bivalve size ranges across several orders of magnitude, from micro-bivalves of a few mm and mg in size such as the Neoleptonidae to the giant clam Tridacna gigas with a length of 120 cm and weighing 220 kg. Bivalve maximum lifespans (MLSPs) range from 1y in small warm water clams [START_REF] Powell | Are molluscan maximum life spans determined by longterm cycles in benthic communities?[END_REF] to ± 400y in the ocean quahog Arctica islandica [START_REF] Schöne | Climate records from a bivalve Methuselah (Arctica oslandica, Mollusca; Iceland)[END_REF], the longest lived invertebrate known. Thus, bivalves offer a diversity of ageing models, from which we can select those that best suit a particular question or hypothesis.

The distinct advantage of bivalves compared to the classical aging-models, however, is their carbonate shell. The shell can serve the determination of individual age and, at the same time, it can archive changes of environmental conditions throughout the animal's lifetime.

Both the instantaneous shell growth rate and the composition of newly forming shell material are subject to extrinsic change, meaning they can be modified by environmental factors.

Shell growth rate is largely governed by temperature and food availability [START_REF] Richardson | Molluscs as archives of environmental change[END_REF]). Hence, bivalves, coming from systems with a distinct seasonality with regard to either one or both parameters, exhibit seasonal shell growth patterns that produce an annual growth banding, similar to trees. Once the annual shell band patterns are calibrated for a population, animals can be taken from the environment and individually aged. This enables the study of the impact of environmental drivers on the ecology and physiology of bivalves over time in natural populations from different climatic or ecological settings. Moreover, lunar, daily, or even tidal cycles are distinguishable in some fast growing species, at least a posteriori (e.g. [START_REF] Chauvaud | Shell of the Great Scallop Pecten maximus as a high-frequency archive of paleoenvironmental changes[END_REF][START_REF] Kanazawa | Environmental and physiological controls on shell microgrowth pattern of Ruditapes philippinarum (Bivalvia: Veneridae) from Japan[END_REF]. Further, archived banding patterns allow reconstruction of lifetime growth history, including long-term trends, such as climate driven decadal patterns (e.g. [START_REF] Schöne | Climate records from a bivalve Methuselah (Arctica oslandica, Mollusca; Iceland)[END_REF], as well as unique physical disturbance events caused by storms, predator attack, extreme temperatures or even incidents of low oxygen concentrations [START_REF] Witbaard | Long-term trends on the effects of southern North Sea beam trawl fishery on the bivalve mollusc Arctica islandica L. (Mollusca, Bivalvia)[END_REF][START_REF] Sejr | Growth and production of Hiatella arctica (Bivalvia) in a high-Arctic fjord (Young Sound, Northeast Greenland)[END_REF].

Thus, bivalve models of aging not only reveal their chronological age at capture, but also record environmental parameters important for the reconstruction of animal and population life history. This opens the field for a new ecophysiological approach to study principles of aging in marine and freshwater systems, and will perhaps enable a clearer distinction of the effects of intrinsic (genetically fixed) and extrinsic (environmental) factors on organismal life expectancy and fitness.

ROS formation and antioxidant strategies in bivalves

Reactive oxygen species (ROS) released by electron transport systems of the mitochondria and the endoplasmatic reticulum are well established to be important drivers of aging in senescent, post-mitotic animal cells and tissues [START_REF] Harman | Free Radical Theory of aging: effect of free radical inhibitors on the mortality rate of male LAF1 mice[END_REF][START_REF] Barja | Free radicals and aging[END_REF]). If not controlled on low levels by antioxidant systems (enzymes and low molecular antioxidants), ROS cause damage to cellular lipids and proteins, as well as to mitochondrial and nuclear DNA, resulting in accelerated senescence.

Bivalves are excellent models showing how different lifestyles modulate the balance between ROS production and antioxidant defence involved in the determination of the maximum lifespan (Fig 1): Pectinids have a high scope for activity, adaptive for burst swimming. To reduce oxidative stress during frequent exhaustive swimming, they need many energetically well coupled mitochondria with low oxygen radical output per mitochondrion. Indeed, pectinids have the lowest rates of mitochondrial in-vitro ROS production measured so far in any molluscan species [START_REF] Abele | Marine invertebrate mitochondria and oxidative stress[END_REF]). It appears that control of ROS formation on low levels is extremely important in these actively swimming animals, respiring fully oxygenated seawater. Mobile pectinids are still relatively short lived compared to sessile molluscs, indicating that even these low ROS amounts per mitochondrion produce enough wear and tear in active swimmers. We found a significant decrease in mitochondrial number with age in queen scallops, which could relate to the fact that damaged mitochondria are removed from their tissues, in this contrasting the mud clams like Mya arenaria, where respiratory efficiency in mitochondrial isolates declined dramatically with animal age [START_REF] Philipp | Physiological ageing in a polar and a temperate swimming scallop[END_REF] and see Fig. 2). Short lifetime of scallops may also reduce the need for life-protecting antioxidant activity, and scallops range rather low in antioxidants compared to other molluscs (black dots in Fig. 3). The opposite is found in the ocean quahog, Arctica islandica, the longest-lived of sessile mud clams. The animals exhibit low aerobic scope and moderate ROS production rates per mitochondrion, but have the double to 10-fold catalase activity (depending on tissue) than other bivalves. MLSP is, however, 20-30 times longer in A. islandica than other mud clams, indicating moderate ROS formation and higher levels of antioxidants may be only a part of the long life program of the ocean quahog. Based on the current data, a sessile benthic lifestyle (Fig. 1, left) would involve moderate to high ROS formation rates, seasonal adjustment of the proton leak (= non-phosphorylating proton flux through inner mitochondrial membrane) with higher leak for mild uncoupling in summer in order to prevent thermally induced ROS formation and oxidative damage. Extremely long MLSPs as in the ocean quahog seem to further require high antioxidant potential.

The ocean quahog and the pearl clam: models for extreme lifespan and negligible aging

Life expectancy of more than 100 years are known for only a dozen out of the total 1.5 millions of animal species on earth [START_REF] Ziuganov | Life Span Variation of the Freshwater Pearl Shell: A Model Species for Testing Longevity Mechanisms in Animals[END_REF]. Among endotherms, only humans and presumably some whales reach such venerable ages (see [START_REF] Finch | History and prospects:symposium on organisms with slow aging[END_REF].

Astonishingly enough, the aquatic ectotherms hold all age records [START_REF] Mangel | Environment and longevity: The demography of the growth rate[END_REF], even if we exclude colonial organisms with asexual reproduction from our considerations, such as the polyp Hydra which seemingly does not age at all [START_REF] Martinez | Mortality patterns suggest lack of senescence in Hydra[END_REF]. Although an attractive hypothesis [START_REF] Cailliet | Age determination and validation studies of marine fishes: do deep-dwellers live longer?[END_REF]), extreme longevity is neither an exclusive, nor a characteristic trait of deep sea fauna and the longest life expectancies are found among shallow water species [START_REF] Mangel | Environment and longevity: The demography of the growth rate[END_REF]. Two bivalves are the actual record holders: the ocean quahog Arctica islandica with a reported maximum lifespan (MLSP) of 374y around Iceland [START_REF] Schöne | Climate records from a bivalve Methuselah (Arctica oslandica, Mollusca; Iceland)[END_REF]) and the freshwater pearl shell clam Margaritifera margaritifera with an MLSP of 190 y in the Arctic [START_REF] Ziuganov | Life Span Variation of the Freshwater Pearl Shell: A Model Species for Testing Longevity Mechanisms in Animals[END_REF]. Other long lived shallow water species like the hard clam Eurhomalea exalbida from the South Patagonian shelf [START_REF] Lomovasky | Individual age and connective tissue lipofuscin in the hard clam Eurhomalea exalbida[END_REF], the Pacific mussel Crenomytilus grayanus [START_REF] Zolotarev | The lifespan of bivalves from the sea of Japan and the sea of Okhotsk[END_REF], and the fossil Cucullea raea from Eocene Antarctic [START_REF] Buick | 100 years in the dark: Extreme longevity of Eocene bivalves in Antarctica[END_REF] have centenarian lifespans, and indicate that cold water temperatures and presumably food limitations during winter at high latitudes may be favourable for longevity [START_REF] Buick | 100 years in the dark: Extreme longevity of Eocene bivalves in Antarctica[END_REF]. Extremely long-lived bivalves share common and characteristic life history features. The first is an extremely slow and seemingly indeterminate growth [START_REF] Finch | History and prospects:symposium on organisms with slow aging[END_REF] in Arctic and Subarctic climates, compared to sympatric species from warmer habitats [START_REF] Ziuganov | Life Span Variation of the Freshwater Pearl Shell: A Model Species for Testing Longevity Mechanisms in Animals[END_REF]. A second is the late onset of reproduction which then continues into old age without a post-reproductive phase [START_REF] Thorarinsdottir | Size and age at sexual maturity and sex ratio in ocean quahog, Arctica islandica (Linnaeus, 1767), off northwest Iceland[END_REF][START_REF] Mangel | Environment and longevity: The demography of the growth rate[END_REF][START_REF] Ebert | Longevity and lack of senescence in the red sea urchin Strongylocentrotus franciscanus[END_REF].

Icelandic A. islandica can clearly be regarded as a prototype example for imperceptible/ negligible senescence. The animals mature between 10 and 32y of age, and mature specimens maintain constant levels of several antioxidant factors, including the enzymes SOD and catalase, as well as the glutathione concentration and redox state in gill and mantle tissues over at least 150y. Higher antioxidant activities and glutathione (GSH) levels are seen during an initial phase of active growth in young animals, whereupon values decline rapidly until the age of maturation and then remain stable [START_REF] Abele | Imperceptible senescence -Ageing in the ocean quahog Arctica islandica[END_REF]. Post maturation antioxidant defence levels are up to 10-fold above those found in other bivalve species with shorter life. As a consequence, oxidative damage accumulation is extremely low with protein carbonyl accumulation amounting to only 50% of the lowest values measured in shorter lived bivalves [START_REF] Strahl | Physiological aging in the Icelandic population of the ocean quahog Arctica islandica[END_REF]. A recent comparison of fluorescent age pigment (lipofuscin) in mantle tissue of A. islandica populations from different geographic regions, differing distinctly with respect to longevity, showed that individuals of the extremely long lived Icelandic population, indeed, have the lowest lipofuscin accumulation rates (unpublished data of L. Basova). Thus, aside from its well developed antioxidant defence system, low natural levels of ROS formation and presumably a high capacity of cellular damage repair and autodigestion of severely damaged macromolecules might be important life prolonging factors in the ocean quahog. However, autophagy and apoptosis in these animals and their role in bivalve aging still wait to be addressed.

Arctica islandica displays a characteristic behaviour in spontaneously burrowing into the sediment, which is not regulated by external triggers and therefore occurs at irregular and individually determined intervals. The normal position of A. islandica is just below the sediment surface, so that the very short siphons reach into the water column. Occasionally, the animals burrow a couple of cm deeper into the sediment and, by closing their siphons and shell valves, expose their tissues to hypoxic and even completely anoxic conditions for 1 to 5 days [START_REF] Taylor | Burrowing behaviour and anaerobiosis in the bivalve Arctica islandica (L.)[END_REF] and our own observations). During these periods, the animals enter a metabolically depressed state ("metabolic rate depression", MRD) down to 10% of their normoxic metabolic activity (Taylor 1976: heart beat;Oeschger and Storey 1993: down regulation of glycolytic enzyme activity) which allows them to survive on anaerobic energy production alone. Similarly, Ziuganov and colleagues reported pearl shell clams to be capable of "altering metabolic rate independently of temperature" and thereby "enter and exit diapause" [START_REF] Ziuganov | Life Span Variation of the Freshwater Pearl Shell: A Model Species for Testing Longevity Mechanisms in Animals[END_REF][START_REF] Ziuganov | Arctic and southern freshwater pearl mussel Margaritifera margaritifera with long and short life span as a model system for testing longevity[END_REF]). This energy saving behaviour could theoretically slow senescence in several ways in the bivalves. Firstly, it involves a slow down of metabolic ROS production in the depressed state, which, in oxyconforming species not prone to oxidative stress during hypoxia-reoxygenation cycles, could be beneficial [START_REF] Abele | Marine invertebrate mitochondria and oxidative stress[END_REF]). On the contrary, frequent hypoxia-reoxygenation cycles in ectotherms may trigger antioxidant protection (Hermes-Lima and Zenteno-Savin 2002). Another important mechanism is the induction of a "famine-like" state [START_REF] Ziuganov | Life Span Variation of the Freshwater Pearl Shell: A Model Species for Testing Longevity Mechanisms in Animals[END_REF] during shell closure, bound to involve up-regulation of autophagic processes of cellular selfdigestion. So far, molluscan autophagic genes are unspecified, and the function of pro-and antiautophagic factors, like glucagon and insulin still wait to be investigated in these animals.

Metabolic rate depression (MRD) in bivalves resembles the state of caloric restriction (CR), recognized as life prolonging intervention in several metazoans. It is important to note that both, depression of metabolic rate in hypoxia tolerant animals as much as CR, which after an initial transition phase is completely aerobic [START_REF] Lin | Calorie restriction extends yeast life span by lowering the level of NADH[END_REF], could result in prolongation of life expectancy. [START_REF] Buick | 100 years in the dark: Extreme longevity of Eocene bivalves in Antarctica[END_REF] proposed that one of the processes conferring an extension of lifespan in bivalves from high latitudes could be the seasonal limitations of light and food availability. Periodic times of low food intake might induce autophagic activity and possibly apoptosis in these animals, clean their cellular biochemical background from oxidized intermediates, and eliminate damaged cells and mitochondria.

Regional and seasonal cold climates obviously favour frequent metabolic rate depression in bivalves like Margaritifera margaritifera [START_REF] Ziuganov | Arctic and southern freshwater pearl mussel Margaritifera margaritifera with long and short life span as a model system for testing longevity[END_REF]), Laternula elliptica [START_REF] Morley | Hypoxia tolerance associated with activity reduction is a key adaptation for Laternula elliptica seasonal energetics[END_REF] or Arctica islandica [START_REF] Taylor | Burrowing behaviour and anaerobiosis in the bivalve Arctica islandica (L.)[END_REF]. Slower growth, increased stress hardiness and shell repair in the Arctic and subarctic compared to warm adapted populations in Spain (M. margaritifera) or in the North Sea (A. islandica) support this hypothesis. Specifically, Ziuganov and colleagues (2000) found shell repair to function 30-times faster in Arctic pearl shells than in specimens collected in Catalonian rivers. Survival of specimens with severe injury of the anterior adductor muscle was 4-times better in the Arctic than in Spain. Thus, temporary depression of metabolic rate, self-induced or through an environmental signal, may shift resources from growth to damage repair and cellular renewal in long-lived Arctic specimens, allowing for a marked extension of lifespan in the cold.

Surf clams and bay scallops: models for short lifespan and semelparous reproduction

Whereas the determination of long lifespans is always biased by the impracticality to sample the oldest of all specimens in a population, short bivalve lifespans under 2y are relatively easily determined by mark-and-recapture field studies. Another possibility is to detect onset of senescent mass mortality as cohorts of known age approach death [START_REF] Bricelj | Resource allocation and population genetics of the bay scallop, Argopecten irradians irradians: effect of age and alloenzyme heterozygosity on reproductive output[END_REF]. As in longer lived bivalves, lifespan in short lived species can differ between geographical and climatic zones [START_REF] Bricelj | Comparative physiology of young and old cohorts of bay scallop Argopecten irradians irradians (Lamarck): mortality, growth, and oxygen consumption[END_REF][START_REF] Cardoso | Population dynamics and secondary production of the wedge clam Donax hanleyanus (Bivalvia: Donacidae) on a high-energy, subtropical beach of Brazil[END_REF]. [START_REF] Powell | Are molluscan maximum life spans determined by longterm cycles in benthic communities?[END_REF] listed MLSPs of 53 bivalves and 48 gastropod snails. Their compilation indicates two families with conspicuously short life expectancy: the scallops, Pectinidae, particularly species within the genus Argopecten and surf or wedge clams, Donacidae. Donacidae colonize tropical and warm temperate sandy beaches, deserted by most macrofauna, where they then achieve very high densities (12.000 ind/m2 Wilson 1999). They are characterized by high growth rates, high production to biomass ratios (P/B: 6-7:1, see [START_REF] Wilson | Population dynamics and energy budget for a population of Donax variabilis (Say) on an exposed South Carolina beach[END_REF]), short lifespans of 1-2 y (Cardoso and Veloso 2003) and semelparous reproduction. Moreover, surf clams are characterized by a "supreme ability as rapid burrowers" [START_REF] Wilson | Population dynamics and energy budget for a population of Donax variabilis (Say) on an exposed South Carolina beach[END_REF], which perform seasonal as well as daily migrations up and down the beach with the tides. Donax species from tropical beaches are shorter lived and display higher spontaneous mortality rates under experimental conditions than temperate Donax species [START_REF] Cardoso | Population dynamics and secondary production of the wedge clam Donax hanleyanus (Bivalvia: Donacidae) on a high-energy, subtropical beach of Brazil[END_REF].

Published data on Donax physiology and biochemistry are limited. [START_REF] Wilson | Population dynamics and energy budget for a population of Donax variabilis (Say) on an exposed South Carolina beach[END_REF] measured temperature dependent respiration (10 -45°C at 5°C intervals) in Donax variabilis from South Carolina. His data document very efficient seasonal temperature compensation between 10 and 20°C in winter and suggest that the animals maintain high year round metabolic rates. A compilation of aquatic invertebrate respiration by T. Brey (unpubl. data) shows that Donacidae and Pectinidae (see next paragraph) have significantly higher metabolic activity than long lived Arcticidae (A. islandica). High standard metabolic rates correspond to high scopes for growth and activity in short lived clams, and altogether this indicates relatively higher mitochondrial density in Donax cells. However, neither mitochondrial densities nor mitochondrial respiration or marker enzyme activities have so far been measured. Antioxidant enzyme activities reported for Mediterranean Donax trunculus mid gut gland amounted to 2-12 U SOD mg -1 protein and between 0.05 and 0.15 U catalase mg -1 protein [START_REF] Angel | Catalase activity in macro-and microorganisms as an indicator of biotic stress in coastal waters of the eastern Mediterranean Sea[END_REF]. Whereas the SOD activities are in the same range as in other bivalves (Abele and Puntarulo 2004: Tab. 2;[START_REF] Philipp | Physiological ageing in a polar and a temperate swimming scallop[END_REF]), catalase activity is extremely low in Donax trunculus digestive gland. Low ROS detoxification and whole year high metabolic rates speak for low investments into cellular protection mechanisms and, according to [START_REF] Williams | Pleitropy, natural selection, and the evolution of senescence[END_REF], are typical for semelparous animals, which invest into reproduction rather then into prolonged somatic fitness. This means that after spawning, animals survive for limited periods of days and weeks with high probability for predation.

Both burrowing infauna bivalves, Donax and Arctica, belong to the same ecotype and elegantly document adjustment of physiological and biochemical antioxidant parameters to different life history and reproductive strategies (see also chapter 2, Fig. 1

of this review).

A second group of short lived, semelparous bivalves, scallops of the Argopecten family, have lifespans of less than 2 years, and only few specimens survive for a second annual breeding cycle [START_REF] Williams | Growth of the saucer scallop, Amusium japonicum balloti Habe, in central eastern Queensland[END_REF][START_REF] Bricelj | Comparative physiology of young and old cohorts of bay scallop Argopecten irradians irradians (Lamarck): mortality, growth, and oxygen consumption[END_REF]. Scallops belong to a different ecotype, namely that of motile swimming bivalves and populations are often under strong pressure from predation and commercial exploitation. In populations of the bay scallop Argopecten irradians only few specimens survive through the first winter [START_REF] Bricelj | Comparative physiology of young and old cohorts of bay scallop Argopecten irradians irradians (Lamarck): mortality, growth, and oxygen consumption[END_REF][START_REF] Bricelj | Resource allocation and population genetics of the bay scallop, Argopecten irradians irradians: effect of age and alloenzyme heterozygosity on reproductive output[END_REF]. Bricelj and coworkers (1987) investigated traits of senescence in caged A. irradians at Long Island by comparing young and old cohorts before and following spawning in late spring and summer. Mass occurrence of natural mortality in caged specimens shortly after spawning was not a consequence of reproductive efforts, and animals which survived through the winter continued somatic growth, although at lower rates and at significantly diminished oxygen uptake (~ 30% less MO 2 ) during the second year.

None of these senescent animals survived longer than 23 months and instead succumbed to a second year mass mortality event, concurrent with mass mortality in the younger cohort.

Decreased metabolic rates in the second year may reflect reduced mitochondrial densities in the cells of aged animals. This was recently shown to happen in queen scallops, Aequipecten opercularis, from the Irish Sea, with an MLSP of 8y. Between 2 ± 0.5y and 4 ± 0.5y of age, the investigated A. opercularis lost 30 % of their muscle fibre mitochondria and also a consistent 30% of citrate synthase activity per unit wet weight. This loss of mitochondria in A. opercularis corresponded to the age dependent decline of ATP concentration and whole adenylate pool in this longer lived, iteroparous scallop (Philipp et al. 2008). Interestingly the respiratory capacity of mitochondria isolated from young and older queen scallops revealed only minor loss of function with size and age, as compared to longer lived mud clams [START_REF] Philipp | Physiological ageing in a polar and a temperate swimming scallop[END_REF][START_REF] Philipp | Size and age dependent changes in adductor muscle swimmimng physiology in the scallop Aequipecten opercularis[END_REF]. We termed this phenomenon the "superman effect" and proposed that it might be their strategy to maintain the remaining mitochondria in good shape, than to retain high numbers of poorly functioning mitochondria per cell in the swimming muscle over their entire life.

Similar patterns of physiological aging in short lived A. irradians and 3-times longer lived queen scallops raise doubts about the trueness of semelparity in short lived bay scallops. As pointed out by [START_REF] Estabrooks | The possible role of telomers in the short life span of the bay scallop, Argopecten irradians irradians (Lamarck1819)[END_REF], there seems to be no selective advantage, justifying such early death in bay scallops, as, in contrast to the Pacific salmon, young scallops do not benefit from enrichment of nutrients and improved feeding conditions from dying older cohorts. To the contrary, Estabrooks, conducting investigations of telomere frequency in short lived bay scallops (A. irradians, MLSP 2y) and longer lived ostion scallops (A.

purpuratus, MLSP 6-8y) reached the conclusion that bay scallops represent a case of "interrupted iteroparity", caused by an evolutionary loss of genomic material resulting in fewer telomeres perhaps related to loss of key chromosomal material in this species over evolutionary time scales.

Age effects on metabolic and antioxidant capacities in bivalves from different climate regions

Several studies of moderately long-lived bivalves (up to 20 years) document a decline of growth rates as well as respiratory and filtration capacities with age, albeit independent of changes of body mass (see data compilation in Table 1). [START_REF] Sukhotin | Influence of age and size on pumping activity and stress resistance in the marine bivalve Mytilus edulis[END_REF] and [START_REF] Sukhotin | Age-dependence of metabolism in mussels Mytilus edulis (L.) from the White Sea[END_REF] analyzed the blue mussel, Mytilus edulis, from temperate, sub-arctic and fully Arctic habitats. They documented decreasing respiration and filtration rates in a subarctic White Sea population above 6 years of animal age, indicating that blue mussels either lose or reduce respiratory capacities and functional activity over lifetime. The decline of specific respiration (µmol O 2 g -1 tissue wet weight h -1 ) was even more pronounced when data were normalized to a standard size in all age groups. This makes sense, because growth rates in mussel settlements are highly variable, and each age cluster encompasses a wide range of animal sizes. However, it also enables size independent evaluation of age effects on respiration and other parameters in the M. edulis model and excludes the possibility that any age dependent change is merely an allometric effect, where larger animals have lower specific parameter activity. [START_REF] Sukhotin | Age-dependence of metabolism in mussels Mytilus edulis (L.) from the White Sea[END_REF] and [START_REF] Sukhotin | Growth metabolism and lipid peroxidation in Mytilus edulis L.: Age and size effects[END_REF] analyzed citrate synthase activity in gills and whole body tissue of sub-arctic M. edulis from the White Sea and found no clear indication for an age dependent change of the mitochondrial marker enzyme in this species. However, direct measurement of respiratory capacity in mitochondrial isolates of mud clams and a scallop (Fig. 3, Tab. 1) document loss of respiratory efficiency and of mitochondrial energetic coupling (RCR: respiratory control rate which indicates the intensity of mitochondrial energetic coupling) over animal lifetime (Philipp et al. 2005b;[START_REF] Philipp | Physiological ageing in a polar and a temperate swimming scallop[END_REF]. The intensity of the decrease of mitochondrial respiration and phosphorylation differed significantly between the species and was most pronounced in the North Sea soft shell clam Mya arenaria over an age range from 2 to 10y.

Comparison of temperate and Antarctic mud clam mitochondria revealed more rapid "quality loss" in mitochondria of the North Sea clam: RCR and mitochondrial membrane potential declined 6-fold more rapidly, whereas proton leak increased 5-times faster with age than in L. elliptica from the Antarctic. The percent rate of state 4 (uncoupled respiration without ADP) and state 3 (coupled respiration in the presence of ADP) H 2 O 2 formation by the isolated mitochondria increased only in the temperate mud clam, whereas it remained low and constant over 25 years of lifespan in the Antarctic species (Philipp et al. 2005b). Thus, the superior longevity in the polar clam, which otherwise resembles the North Sea clam in lifestyle (sedentary benthic infauna), feeding mode (particle filtration), and shell size (max ±100mm), seems to be based on its potential to control the intensity of mitochondrial ROS formation, presumably through a higher proton leak which exerts a mild uncoupling effect on the mitochondrial membranes [START_REF] Brand | Uncoupling to survive? The role of mitochondrial inefficiency in ageing[END_REF].

Another important antioxidant protection regarding the mitochondrial ROS formation is the radical scavenging glutathione system (Table 1). An age dependent diminishment of parameters of the glutathione system was recorded in temperate Mytilus edulis from the Plymouth area [START_REF] Canesi | Age-related differences in glutathione metabolism in mussel tissues (Mytilus edulis L.)[END_REF]: performed measurements in digestive gland and gills of total glutathione, glutathione reductase, glutathione transferase and -glu-cys synthetase activity). In a similar approach, our team recorded reduction of the glutathione tissue concentration over lifetime in temperate and polar scallops [START_REF] Philipp | Physiological ageing in a polar and a temperate swimming scallop[END_REF] in mantle tissue) and in the Antarctic mud clam Laternula elliptica (Philipp et al. 2005a in mantle tissue). The GSSG:GSH ratio, however, decreased only in scallops, the temperate Aequipecten opercularis and the polar Adamussium colbecki over age, whereas it remained constant in L. elliptica. Scallops maintained generally less oxidized GSSG:GSH ratios compared to the mud clams, and it may be adaptive for these active species to preserve high GSH levels for quenching ROS during burst swimming. [START_REF] Philipp | Size and age dependent changes in adductor muscle swimmimng physiology in the scallop Aequipecten opercularis[END_REF] showed that during exercise [GSH] and pH rapidly decline in the adductor muscle of queen scallops, A. opercularis, which markedly affects the cellular redox ratio. Further, the decrease in tissue GSSG:GSH ratios over lifetime in scallops suggests that the age dependent net loss of glutathione was not due to progressive oxidation, but rather to an effective removal of oxidized GSSG from the tissues. A high GSSG:GSH ratio is problematic as it can impact on signal transduction, DNA and RNA synthesis, protein synthesis, enzyme activation, and even regulation of the cell cycle.

The temperate soft shell clam Mya arenaria was the only bivalve in which we found GSSG to accumulate with age, the concentration doubling in mantle tissue within 6 years of lifetime from 30 to over 80 nmol GSSG g -1 wet weight. Within the same period, the overall glutathione concentration (GSH + 2GSSG) increased from 200 to 400 nmol g -1 wet weight in M. arenaria mantle tissue. Together with the decrease in mitochondrial respiration and coupling, increased conversion of oxygen to ROS and accumulation of GSSG with age, increases in the overall glutathione concentration in mantle tissue suggest that prooxidant processes intensify, and higher antioxidant protection is needed in aging soft shell clams.

Both burrowing mud clams, Mya arenaria from the North Sea and Arctica islandica from the Iceland population had very low glutathione levels around 300 nmol g -1 wet weight in mantle tissue. In A. islandica the concentration remained constant between 36-196 years of age, following maturation of the animals [START_REF] Abele | Imperceptible senescence -Ageing in the ocean quahog Arctica islandica[END_REF]). Thus, these burrowing mud clams preserve glutathione at constant (or even increasing) levels over lifetime, as opposed to epibenthic scallops or blue mussels (Table 1), in which glutathione levels decrease over lifetime in mantle and adductor muscle.

Another interesting question regarding polar and temperate ectotherm longevity is whether or not changes involved in physiological cold adaptation may directly impinge on the process of ageing. According to [START_REF] Porter | Allometry of mitochondrial proton leak: influence of membrane surface area and fatty acid composition[END_REF], proton leak increases as a function of inner mitochondrial membrane surface area (cristae density) and can be modulated by the degree of unsaturated fatty acids in the membrane. Higher numbers of mitochondria and higher cristae densities are found in cold adapted ectotherms, helping them to overcome diffusion problems and compensate for the decline in metabolism at cold temperatures. A comparison of the ratio of cytochrome oxidase/citrate synthase activity (COX/CS) in temperate and polar mud clams indeed suggests that polar species have higher mitochondrial cristae densities (Philipp et al. 2005b). Thus, on the one hand, higher proton leak which, theoretically, mitigates ROS formation and slows aging, may indeed be a consequence of cold adaptation in polar and especially Antarctic ectotherms with low aerobic scopes. This brings up the question whether mild uncoupling of the mitochondria and high glutathione levels in polar compared to temperate mud clams are primarily a consequence of physiological cold adaptation. Alternatively L. elliptica may have evolved higher proton permeability of the inner membrane and store more glutathione in order to prolong lifespan as a feature supporting sustainable "population management" in the cold. In other words, do polar clams need longer life time to insure survival of the stock, or does a different lipid composition render polar clam membranes leaky as a "by-effect of cold adaptation"? The long lifespan may be adaptive for survival of ectotherms in polar habitats, where temperature slows not only adult growth and development, but moreover protracts molluscan gametogenesis and embryonic development, and possibly delays hatching of mature trochophora larvae from the eggs [START_REF] Peck | Very slow development in two Antarctic bivalve mollsucs, the infaunal clam Laternula elliptica and the scallop Adamussium colbecki[END_REF]).

Genes and lifespan in bivalves

Within the metazoan kingdom, extreme differences of species longevity, from a few days to a hundreds of years reflect the genetic basis of MLSP biodiversity. However, if life expectancy is partly in our genes, this raises the question of which genetic traits exactly support longevity, and whether or not the same genes are essentially involved in all metazoans (see also [START_REF] Austad | An experimental paradigm for the study of slow aging organisms[END_REF]? Furthermore, what renders a species eligible for the selection of longevity supporting genes in evolution?

Rather than a small number of specific "old age genes" that, just by being present as wild or mutated alleles confer extreme species longevity, it seems that certain combinations of genes involved in choreographing the relevant processes of life-long metabolic performance, are important in determining the velocity of aging. In order to be selected, these genetic "choreographers" need to confer fitness advantage to the animals at a young age (see [START_REF] Williams | Pleitropy, natural selection, and the evolution of senescence[END_REF][START_REF] Zwaan | The evolutionary genetics of ageing and longevity[END_REF]. Along this line of thinking, the "gene cluster hypothesis of aging" has been put forward by G. [START_REF] Barja | The gene cluster hypothesis of aging and longevity[END_REF], who proposes central homeotic regulator genes (transcription factors like hox genes) to coordinate and optimize lifetime gene transcription for an optimal exploitation of environmental resources and adaptation to environmental constraints (such as times of low food availability in nature). Bivalves are of low complexity in their organizational hierarchies and ontogeny when compared to insects or mammals. This might make it easier for them to rapidly adjust their metabolism in order to meet abruptly changing environmental conditions. The regulatory cascades involved in these adaptive changes may be shorter and less complex, and could potentially contribute to the understanding of the genetic basis of longevity in higher organisms.

Lower complexity and high adaptability may, moreover, render long lived bivalves more tolerant of non-lethal deleterious mutations, acting late in life and resulting in a graded loss of individual fitness that accelerates aging [START_REF] Dall | Mutation Rates: does Complexity Matter[END_REF]. This may mean a higher probability of individuals surviving to ages closer to MLSP, once predation pressure (and other impact immediately killing the animals) has been ruled out. It may also make possible to sample individuals closer to maximum age in bivalve populations. In agreement with this, large infaunal bivalves with heavy shells, which experience relatively low pressure from predation, have the highest recorded MLSPs. They have presumably optimized their genomic life history program for increased longevity with undiminished or even increasing reproductive output at old age [START_REF] Williams | Natural Selection, the Costs of Reproduction, and a Refinement of Lack's Principle[END_REF]. Important candidate traits include late maturation, low (less than yearly) reproductive frequency, self-programmed hypometabolism, as well as mitochondrial oxyconformity and low basal ROS formation rates.

Tumour formation would range among "late acting deleterious mutations" and has not yet been recognized as a major cause of death in aged bivalves. Blood cancer, as well as different malignant neoplasms have been identified in bivalves and are listed by [START_REF] Cheng | Noninfectious diseases of marine molluscs[END_REF] for several species. Experiments to induce tumours in bivalves using carcinogenic hydrocarbons generally fail, but application of aromatic amines can cause basophilic cell neoplasms and hematopoetic cancer (200-400 ppm diemthylnitrosamines causes cancer in painters shell Unio pictorum, [START_REF] Khudolei | Development of tumors in Unio pictorum bivalve mollusks under the influence of N-nitroso compounds[END_REF], as these compounds are being converted to mutagens in bivalve digestive glands. The effect of tumour formation on animal life expectancy is unclear. Thus, mass mortality in the hard shell clam Mercenaria spp. could not be related to germ cell tumours (germinoma) occurring massively in this species. Instead, increased tumour incidence is detected during times of rapid environmental change [START_REF] Hesselmann | Gonadal neoplasms in hard shell clams Mercenaria spp. from the Indian River, Florida: Occurrence, prevalence, and histopathology[END_REF]. Germinal tumours were also seasonally increased and more abundant at warm summer water temperatures. Hard shell clams from Florida generally displayed higher tumour incidence than clams from colder Rhode Island waters [START_REF] Hesselmann | Gonadal neoplasms in hard shell clams Mercenaria spp. from the Indian River, Florida: Occurrence, prevalence, and histopathology[END_REF]. Thus, higher susceptibility to malignant neoplasmic diseases may be involved in shortening population specific maximum lifespan in bivalves with a geographical distribution between different climate zones. Tumour promoters discussed in the literature are infestations by herpes-type viruses which are more common at warmer water temperatures. On the other hand, there might also be a general predisposition through certain proto-oncogenes (genes that by mutation are converted into tumour inducing oncogenes) in warm water bivalves. Information on proto-oncogene signalling cascades in molluscan species is largely missing, however, Degnan and collaborators confirmed conservation of the ets-multigene family, homolog to the human oncogenes, throughout nearly all marine metazoan phyla [START_REF] Degnan | The ets multigene family is conserved throughout the metazoa[END_REF], including molluscs and sponges. The proteins encoded by these ets proto-oncogenes are involved in the regulation of multicellular development and cell proliferation, but their specific function in protostome metazoans, or their role in tumour formation, are far from clear. However, higher tumour incidence in warmer waters underlines temperature is a prime factor in control of bivalve life expectancy.

Another interesting question is whether minor variations in maximum lifespans between different populations of one species within a climatic zone can have a genetic or genomic basis. These variations are reported from the long lived pearl clam, which in the Russian Arctic may attain population Amax between 190 (Varzuga) and 114 (Keret) (Ziuganov et al 2000: Tab.1). In our investigation of Arctica islandica, the most conspicuous discrepancy occurred between 3 populations with the same habitat temperature range (0 and 10°C) but from different locations and with very different Amax of 370 (Iceland), 93 (North Norway) and 53 (White Sea) (Begum and Basova unpubl. data). Is this due to environmental effects that limit lifespan in one environment and not in the other? This would be in line with the symmorphosis concept proposed by [START_REF] Weibel | The concept of symmorphosis: a testable hypothesis of structure-function relationship[END_REF], which states that biological systems are designed to meet, but not exceed, their natural requirements. Pleiotropic tradeoffs are largely a function of environmental forcing of the budgeting of energy between fitness and fecundity during species lifetime. Minor variations of MLSP between populations by the 1.3 to 2 fold therefore seem to be primarily a consequence of environmental adaptations and ecological adjustments see also [START_REF] Barja | The gene cluster hypothesis of aging and longevity[END_REF].

Heterozygosity (heterosis) may be the link between the genetic and adaptive physiological determinators of population MLSP. It is an important criterion for animal fitness, as heterozygous alleles for functional proteins theoretically increase adaptability. Bivalves with multiple allozyme heterozygosity display lower metabolic rates [START_REF] Koehn | A genetic/physiological explanation for differential growth rate among individuals of the American oyster, Crassostrea virginica (Gmelin)[END_REF] and lower rates of protein turnover, which enables them to invest more energy into growth, reproduction and presumably predator avoidance [START_REF] Hawkins | Protein turnover, physiological energetics and heterozygosity in the blue mussel, Mytilus edulis: the basis of variable age-specific growth[END_REF][START_REF] Myrand | Selection against blue mussel (Mytilus edulis L.) homozygotes under various stressful conditions[END_REF].

Meanwhile, an extensive body of literature documents better resistance to stress to be due to elevate levels of heterozygosity in bivalves, gastropods and fish (cited in [START_REF] Myrand | Selection against blue mussel (Mytilus edulis L.) homozygotes under various stressful conditions[END_REF]. The authors speculate that heterozygous bivalves may be more apt to enter hypometabolic states, but this is not yet backed by experimental evidence. To the contrary, correlation of allozyme heterozygosity with either growth, reproduction, or survival (individual lifespan) is not found in pectinids [START_REF] Volkaert | Allozyme and physiological variation in the scallop Placopecten magellanicus and a general model for the effects of heterozygosity on fitness in marine molluscs[END_REF][START_REF] Bricelj | Resource allocation and population genetics of the bay scallop, Argopecten irradians irradians: effect of age and alloenzyme heterozygosity on reproductive output[END_REF][START_REF] Fevolden | Allozymic variability in the Iceland scallop Chlamys islandica: geographic variation and lack of growth-heterozygosity correlations[END_REF]. This speaks against heterozygote superiority supporting predator avoidance in motile bivalves, and, indeed, from the marine ecologist's point of view, predator avoidance is a behavioural trait and presumably involves a multitude of genetic and physiological traits and cascades. Moreover, hypometabolism is not an option for scallops given their active energy consuming life style. A more radical reduction of genetic variability may be the consequence of important losses of genetic material including key telomeric sequences, possibly occurring in the evolution of the bay scallop, as proposed by [START_REF] Estabrooks | The possible role of telomers in the short life span of the bay scallop, Argopecten irradians irradians (Lamarck1819)[END_REF] and referred above. This could indeed be the radical way of shortening life expectancy in scallops.

In the context of the possible effect of gene flow on allele frequencies and aging in bivalves, a noticeable discrepancy exists between North and South polar regions. Supposing heterozygosity depends on gene flow (which is not entirely true, C. Held pers. communication) and further supposing that longevity is supported by heterozygosity in Arctic and Antarctic sessile molluscs: Can this help in explaining why extremely long lived bivalves such as Arctica islandica, Hiatella arctica and Margaritifera margaritifera are known from Arctic and Subarctic and not from Antarctic regions? The longest lived Antarctic bivalve so far reported in the literature is the infaunal nuculanid Yoldia eightsi which can live well over 50y. This is slightly more than the recorded Amax of the Antarctic mud clam, Laternula elliptica with a certified maximum age of 36y and the Antarctic scallop Adamussium colbecki (>40y). Other invertebrates in the Antarctic have been aged to over 50 years including the sea urchins Sterechinus antarcticus and Sterechinus neumayeri [START_REF] Brey | Growth and production of Sterechinus neumayeri (Echinoidea Echinodermata) at contrasting sites in McMurdo Sound Antarctica[END_REF] and 2 Antarctic brachiopods live over 55 years [START_REF] Peck | Bomb signals in old Antarctic brachiopods[END_REF], but none reach ages over 100 y comparable to the long-lived Arctic molluscs.

The Antarctic circumpolar current forms a barrier that reduces gene flow and genetically isolates the stenothermal Antarctic molluscs [START_REF] Patarnello | A study of enzyme polymorphism in the Antarctic amphipod Paramorea walkeri Stebbing[END_REF]. Many Antarctic species are endemic and have optimized their ecology and physiology at constant low water temperature, with low flexibility and tolerance to environmental fluctuation. Genes/alleles selected during 10-15 millions of years of Antarctic evolution guarantee optimal thermal adaptation. The trade-off for this specialization may be found in the relatively short reproductive lifespan of Antarctic bivalves, between late maturation on the one hand, as development of Antarctic bivalve trochophora larvae takes 3-15 times longer than in temperate species, and moderate longevity on the other [START_REF] Peck | Environmental constraints on life histories in Antarctic ecosystems: tempos, timings and predictability[END_REF].

Data on heterozygosity in Arctic and Antarctic invertebrates are limited: The Iceland scallop, Chlamys islandica, is an Arctic and subarctic species with "exceptional variation at several gene loci" including stress genes like superoxide dismustase and glycolytic genes [START_REF] Fevolden | Allozymic variability in the Iceland scallop Chlamys islandica: geographic variation and lack of growth-heterozygosity correlations[END_REF]). In contrast, 3 species of liparid fish in Spitsbergen showed extremely low allelic heterozygosity [START_REF] Fevolden | Intra-and interspecific allozymic variation in Liparis fabricii and Liparis gibbus (Teleostei, Liparididae) from Spitsbergen waters[END_REF]. This led Fevolden to conclude that Iceland scallops represent an exception of high heterozygosity in cold polar environments. In the Antarctic, [START_REF] Patarnello | A study of enzyme polymorphism in the Antarctic amphipod Paramorea walkeri Stebbing[END_REF] found very low average heterozygosity of 0.6 and 0.7% in 22 investigated gene loci of 2 amphipods, supporting the idea that homozygous "specialists" with higher metabolic efficiency through cold adapted proteins/genes/alleles are selected under Antarctic conditions. However, given the scarcity of data in the Antarctic, as well as the variability in methods, numbers and nature of genes in the available data sets, it seems that any conclusion would be preliminary and, again, opens the field for future research.

Conclusions and further research directions.

Bivalve molluscs are promising models for age research because they allow very clear distinctions between internal and environmental factors that control the velocity of senescence in cells and tissues. Similar ecotypes, or even the same species can be obtained from different climatic backgrounds and aged by hard structure analysis, yielding information on the role of climatic adaptation on population MLSP, and the ecological and physiological strategies that stabilize the population under the given conditions. As different bivalve life time strategies involve early and late onset of maturation with semelparous or iteroparous reproduction, we can analyze the trade-offs in both systems. Some species are able to maintain their population based on very short lifespans, and the genes dispensable in those species following early maturation may be just as informative for ageing studies, as the analysis of genes that are most actively transcribed in old individuals of the longest lived bivalve species.

In the future, a major interest will be to analyse the importance of gene heterozygosity in relation to species adapting to stress, exploiting a niche and extending lifespan. Specifically, we need to understand more about the roles of hypometabolism in bivalve ageing, as it seems that the longest lived species employ intermittent dormancy as a basic behaviour, and that seasonal low food conditions could indeed be life prolonging. Studies must include the analysis of gene transcription before, during and after self-induced and seasonal dormancy, to understand which genes and pathways are important for cellular maintenance. Table 1: Changes of metabolic and oxidative stress parameters over lifetime in marine bivalves from different climatic environments. Data from 1: [START_REF] Hopkins | Age differences and the respiration in muscle tissue of mollusks[END_REF]), 2: [START_REF] Sukhotin | Age-dependence of metabolism in mussels Mytilus edulis (L.) from the White Sea[END_REF]), 3: (Philipp et al. 2005b), 4: (Philipp et al. 2006), 5: (Philipp et al. 2008), 6: (Abele et al. 2008), 7: (Viarengo et al. 1991), 8: (Philipp et al. 2005a), 9: (Sukhotin et al. 2002), 10 (Canesi and Viarengo 1997), 11: (Hole et al. 1995), 12: (Strahl et al. 2007)

Figure 1 :

 1 Figure legendsFigure 1: Principle of ROS control in low PO 2 (left) and high PO 2 (right) strategic lifestyles in bivalves.

Figure 2 :

 2 Figure 2: Change in respiration rate of isolated mitochondria with age of the bivalves Mya

Figure 3 :

 3 Figure 3: Antioxidant capacity of molluscs (black dots: scallops, black squares: clams, grey

Fig. 2 :

 2 Fig. 2: Change in respiration rate of isolated mitochondria with age of the bivalves Mya

  Fig.3


Posadas Sj

Caz

Largo

De La Gándara

De Miguel

Dr E De

Dr S Posadas

on antioxidants of major organs of aged rats

Keywords: Protective effect of super critical fluid rosemary extract, Rosemarinus oficcinalis Oxidative stress, ageing, SFE rosemary, carnosic acid, Rosmarinus officinalis

Rosemary leaves, "Rosmarinus officinalis", possess a variety of antioxidant, anti-tumoral and anti-inflammatory bioactivities. We hypothesized that rosemary extract could enhance antioxidant defenses and improve antioxidant status in aged rats.

This work evaluates whether supplementing their diet with super critical fluid (SFE) rosemary extract containing 20% antioxidant carnosic acid (CA) reduces oxidative stress in aged rats.

Aged Wistar rats (20 months old) were included in the study. Rats were fed for twelve weeks with a standard kibble (80%) supplemented with turkey breast (20%) containing none or one of two different SFE rosemary concentrations (0.2% and 0.02%). After sacrifice, tissue samples were collected from heart and brain (cortex and hippocampus).

Enzyme activities of catalase (CAT), glutathione pexoxidase (GPX), superoxide dismutase (SOD) and nitric oxide synthase (NOS) were quantitatively analyzed. Lipid peroxidation and levels of reactive oxygen species (ROS) were also determined.

Rosemary decreased lipid peroxidation in both brain tissues. The levels of catalase activities in heart and cortex were decreased in the rosemary-treated groups. The SFE rosemary treated rats presented lower NOS levels in heart and lower ROS levels in hippocampus than the control rats.

Supplementing the diet of aged rats with SFE rosemay extract produced a decrease in antioxidant enzyme activity, lipid peroxidation and ROS levels that was significant for catalase activity in heart and brain, NOS in heart, and LPO and ROS levels in different brain tissues. These observations suggest that the rosemary supplement improved the oxidative stress status in old rats.

Introduction

Oxidative stress results from an imbalance between the cellular production of reactive oxygen species (ROS) and the antioxidant mechanisms that remove them [START_REF] Halliwell | Reactive oxygen species and the central nervous system[END_REF]. Oxidative stress can damage the cell through the oxidization of cellular elements like membrane lipids, proteins and DNA. Numerous studies have reported correlations between age and the accumulation of oxidative damage in cellular macromolecules [START_REF] Floyd | Oxidative stress in brain aging. Implications for therapeutics of neurodegenerative diseases[END_REF][START_REF] Stadtman | Protein oxidation in aging and age-related diseases[END_REF]. For example, aged rats exhibit increased lipid peroxidation (Calabrese et al., 2004, Devi and[START_REF] Devi | Regional responses in antioxidant system to exercise training and dietary Vitamin E in aging rat brain[END_REF][START_REF] Gupta | Age-related elevation of lipid peroxidation products: diminution of superoxide dismutase activity in the central nervous system of rats[END_REF][START_REF] Murray | Evidence that increased hippocampal expression of the cytokine interleukin-1 beta is a common trigger for age-and stress-induced impairments in long-term potentiation[END_REF][START_REF] O'donnell | Dietary antioxidant supplementation reverses age-related neuronal changes[END_REF] and protein oxidation [START_REF] Cini | Studies on lipid peroxidation and protein oxidation in the aging brain[END_REF][START_REF] Forster | Age-related losses of cognitive function and motor skills in mice are associated with oxidative protein damage in the brain[END_REF][START_REF] Sohal | Oxidative damage, mitochondrial oxidant generation and antioxidant defenses during aging and in response to food restriction in the mouse[END_REF] in their brains. Additionally, it has been demonstrated that aged rats have increased oxidative damage in both nuclear and mitochondrial DNA [START_REF] Hamilton | Does oxidative damage to DNA increase with age?[END_REF]. The strong correlation between increased age and oxidative stress and the accumulation of oxidative damage has suggested the oxidative stress hypothesis of ageing [START_REF] Beckman | The free radical theory of aging matures[END_REF]. The increase in agerelated oxidative damage could result from a multitude of factors, including a decrease in antioxidant defenses.

Several antioxidant mechanisms counterbalance the potential deleterious effects of ROS.

Among the enzymatic ROS scavengers are superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) [START_REF] Brigelius-Flohe | Tissue-specific functions of individual glutathione peroxidases[END_REF][START_REF] Halliwell | Drug antioxidant effects. A basis for drug selection?[END_REF]. There are also hydrophilic radical scavengers such as ascorbate (Vitamin C), urate, and glutathione, as well as lipophilic radical scavengers such as tocopherol (Vitamin E), carotenoids and flavonoids. Therefore, dietary supplementation with antioxidants could conceivably protect against the molecular effects of lipid peroxidation, free radicals and ROS and also delay the progress of many chronic diseases [START_REF] Lai | Studies on the antioxidative activities of Hsian-tsao (Mesona procumbens Hemsl) leaf gum[END_REF][START_REF] Gulcin | Screening of antioxidant and antimicrobial activities of anise (Pimpinella anisum L.) seed extracts[END_REF]. Research into the characteristics of traditional herbs has revealed several interesting bioactive compounds. Consequently, compounds (particullarly from natural sources) with potentially antioxidative properties are being sought.

Rosemarinus officinalis leaves possess a variety of bioactive agents, including antioxidants [START_REF] Auroma | An evaluation of the antioxidant and antiviral action of extracts of rosemary and Provençal herbs[END_REF][START_REF] Ramirez | Isolation of functional ingredients from rosemary by preparative-supercritical fluid (Prep-SFC)[END_REF], anti-tumorals [START_REF] Singletary | Inhibition by rosemary and carnosol of 7, 12dimethy (benz-a anthracene (DMBA)-induced rat mammary tumorigenesis and in vivo DMBA-DNA adduct formation[END_REF] and anti-inflammatories [START_REF] Altinier | Characterization of topical antiinflammatory compounds in Rosmarinus officinalis L[END_REF]. The main relevant constituents are composed of vast numbers of polyphenolics, including carnosic acid, carnosol, rosemarinic acid, ursolic acid, etc [START_REF] Ramirez | Isolation of functional ingredients from rosemary by preparative-supercritical fluid (Prep-SFC)[END_REF][START_REF] Richheimer | Antioxidant activity of lipid-soluble phenolic diterpenes from rosemary[END_REF][START_REF] Seronans | Liquid Chromatographicmass spectrometric análisis of supercritical-fluid extracts of rosemary plants[END_REF]. Among these, carnosic acid (a phenolic diterpene compound), and carnosol are the most potent antioxidant constituents (about 90% of antioxidant activity). The disadvantages of CA, however, are its thermal-, photo-, and oxidation-labile physicochemical properties. The problems derived from its physico-chemical properties can be avoided using super critical fluid extraction to produce rosemary extract, which, because of the low temperatures at which it operates, is very suitable for the extraction of thermolabile compounds like antioxidants [START_REF] Ramirez | Isolation of functional ingredients from rosemary by preparative-supercritical fluid (Prep-SFC)[END_REF]. Futhermore, CA has antimicrobial activity [START_REF] Moreno | Antioxidant and antimicrobial activities of rosemary extracts linked to their polyphenol composition[END_REF][START_REF] Oluwatuyi | Antibacterial and resistance modifying activity of Rosmarinus of.cinalis[END_REF], can inhibit lipid absorption in humans [START_REF] Ninomiya | Carnosic acid, a new class of lipid absorption inhibitor from sage[END_REF] and is a free radical scavenger, due to its phenolic skeleton [START_REF] Del Bano | Phenolic diterpenes, .avones, and rosmarinic acid distribution during the development of leaves, .owers, stems, and roots of Rosmarinus of.cinalis. Antioxidant activity[END_REF][START_REF] Masuda | Antioxidant mechanism of carnosic acid: structural identi.cation of two oxidation products[END_REF][START_REF] Masuda | Recovery mechanism of the antioxidant activity from carnosic acid quinone, an oxidized sage and rosemary antioxidant[END_REF]. Nevertheless, little information is available about the dietary value of rosemary extract in regard to its antioxidant activity and possible application in inhibiting oxidative stress in aged individuals.

The present study was designed to determine whether two concentrations (0.2 and 0.02%) of a rosemary extract obtained via the supercritic fluid extraction method [START_REF] Ramirez | Isolation of functional ingredients from rosemary by preparative-supercritical fluid (Prep-SFC)[END_REF] and containing 20% CA (with high free radical scavenging capacity) could enhance antioxidant status in aged rats. We focused our attention on its effects on the activities of catalase (CAT), glutathione peroxidase (GPX) and superoxide dismutase (SOD) as well as on the levels of nitric oxide synthase activity (NOS), ROS and lipid peroxidation.

Material and Methods

Animals

Male Wistar rats between 20 and 21 months old (550-700 g body wt.), were obtained from Harlam (Barcelona, Spain) and acclimated for at least 1 week prior to experimental use. Rats were distributed randomly into three groups of ten animals each. Table I shows the different groups and diets.

Diets

Animals were fed standard kibble (80%) plus turkey breast in pellet form (20%, donated by FRIAL, S A (Madrid, Spain) for one week to accustom the animals to the diet. Afterwards, animals were fed the following twelve weeks with kibble plus the meat alone or supplemented with either 0.2% or 0.02% SFE rosemary extract (see Table I). Frial, SA (Madrid Spain), a Spanish food manufacturer, provided turkey breast pellets that were plain or supplemented with 0.2% or 0.02% SFE rosemary extract. Body weight did not show significant differences between groups at any time during the study.

Sample collection and tissue preparation

At twelve weeks on the diet, rats were killed by decapitation under light anesthesia with isoflurane (Forane ® , at 2%). Heart and brain were quickly removed. Cerebral cortex and hippocampus were isolated [START_REF] Cini | Studies on lipid peroxidation and protein oxidation in the aging brain[END_REF], weighed and rinsed several times in ice-cold ACSF medium. Afterwards all samples were washed in an ice-cold phosphate buffer saline (PBS). The washed samples were immediately immersed in liquid nitrogen and stored at -70º C until biochemical analysis.

On the day of use, the frozen tissue samples were quickly weighed and homogenized 1:10 in ice-cold 50 mM potassium phosphate buffer (pH 7.4) containing 1 mM EDTA and centrifuged at 12000 × g for 15 min at 4º C. The supernatants were separated and used for protein determination, enzyme activity assay, lipid peroxidation and ROS measurement.

Protein assay

The total protein concentrations in the tissue homogenates were measured by Bradford's method (Pierce, BCA Protein Assay kit, USA) using bovine serum albumin as standard.

Catalase activity assay

Catalase activity was spectrophotometrically measured in tissue homogenates as in [START_REF] Cohen | Measurement of catalase activity in tissue extracts[END_REF], following the manufacturer´s protocol (Catalase Assay kit" Cayman ® Chemical, USA). This method uses a formaldehyde solution as standard. The absorbance of standard and samples was read at 540 nm with a plate reader (Biotek Instruments, Inc.

Vermont, USA). Catalase activity was expressed in nmol/min/mg of protein.

SOD activity assay

Total SOD activity (cytosolic and mitochondrial) was determined as in [START_REF] Paoletti | Determination of superoxide dismutase activity by purely chemical system based on NAD (P)H oxidation[END_REF] following the manufacturer´s intructions (Superoxide Dismutase assay kit" Cayman ® Chemical, USA). Supernatants were desalted by passage through a Sephadex G-25 column. The SOD samples were also treated with a mixture of ethanol-chloroform (2:1, v/v) and distilled water to eliminate hemoglobin and red blood cells and then read on plate reader (Biotek Instruments, Inc. Vermont, USA) at 450 nm. Results are expressed as units per mg of protein (U/mg of protein).

GPX activity assay

GPX activity was assayed as in [START_REF] Paglia | Studies on the quantitative and qualitative characterization of erithrocyte glutathione peroxidase[END_REF] following the manufacturer´s instructions (Glutathione Peroxidase assay kit" Cayman ® Chemical, USA). This method is based on the oxidation of NADPH to NAD  , which is accompanied by a decrease in absorbance at 340nm. GPX activity was measured by initiating the reaction with 2.4 mM cumene hydroperoxide. One unit is defined as the amount of enzyme that oxidizes 1 μmol of NADPH per min at 25°C. The absorbance was read every minute at 340 nm using a plate reader (Biotek Instruments, Inc. Vermont, USA) to obtain at least 5 time points. The GPX activity was calculated in nmol/min/mg of protein.

Oxide Nitric Synthase activity

NOS activity was measured in all tissues according to the method described in (Nims R.W., 1995) and following the manufacturer´s instructions in "Nitrate/Nitrite Colorimetric Assay Kit (LDH method)" (Cayman ® Chemical, USA). Prior to assay homogenated samples were filtered using a 30 kDa molecular weight cut-off filter. The sample volume for assay was a maximum of 40l of the filtrate. A nitrate standard curve was included in order to quantitate sample nitrate  nitrite concentrations. The adsorbancies were read at 540 nm using a plate reader (Biotek Instruments, Inc. Vermont, USA). Results are expressed in M units/mg of protein.

Lipid Peroxidation: Determination of malondialdehyde (MDA)

Heart and brain lipid peroxidation was determined by measuring the level of MDA using the Ohkawa technique (Ohkawa, H., 1979) following the manufacturer´s instructions (TBARS assay kit" Cayman ® Chemical, USA). This method is based on the reaction of thiobarbituric acid with malonyl dialdehyde MDA. MDA dilutions ranged between 0 and 50 M were used as standards. Results were measured on a 532 nm plate reader (Biotek Instruments, Inc. Vermont, USA). MDA concentration was expressed as M of MDA/mg of protein.

Assay for Reactive Oxygen Species (ROS assay)

A fluorometric assay [START_REF] Socci | Evidence that oxidative stress is associatted with the pathophysiology of inherited hydrocephalus in the H-Tx rat model[END_REF]

Statistical analyses

All calculations were performed using SPSS 11.0 statistical software. Data from different groups were statistically analysed and compared using analysis of variance (ANOVA) followed by post hoc analysis using the Dunnett and Tukey tests. P-values less than 0.05 were considered statistically significant. Results were expressed as means ± S.D.

Results

Lipid Peroxidation

The results in heart did not show a significant effect from SFE rosemary extract on lipid peroxidation in both groups with a F(2, 29) = 2.63; p0.131. However, the rosemary extract supplement did significantly decrease lipid peroxidation in the cortex and hippocampus with F(2, 29)= 14.31; p0.001 for the former and F(2, 29)= 9.8; p0.001 for the latter. The effect was stronger in the animals treated with 0.2% (p0.001) than in the animals treated with 0.02% rosemary extract (p0.029), in comparison to the controls. The hippocampus only showed significantly decreased peroxidation in the 0.2% rosemary treated rats (p0.001). Rats receiving the 0.02% rosemary supplement did not show a significant decrease (p0.08). Lipid peroxidation levels are represented in a bar diagram for cortex (figure 1A) and hippocampus (figure 1B) and measured as MDA concentration in (M)/ mg of protein.

CAT, GPX and SOD Activity

In heart tissue, ANOVA analysis of data showed significantly decreased CAT activity in rosemary treated animals F(2, 29)=6.815; p0.003. Results for catalase activity are shown in a bar diagram (figure 2A) with catalase activity measured as nmol/min/g of protein.

Differences between groups were at the p0.05 level with the 0.2% rosemary extract and the p0.003 level with the 0.02% rosemary extract. The levels of catalase activity were higher in controls than in either study group. Data also showed a significant effect from SFE rosemary extract on CAT activity in the cortex F(2, 29)=5.56; p0.007. Figure 2B shows the results in the different study groups. The cortical effect was significant with both 0.2% rosemary extract (p0.05) and 0.02% rosemary extract (p0.017). However, ANOVA data did not show a significant effect from rosemary extract on CAT activity in the hippocampus when the three groups were compared F (2, 29)=0.421; p0.663. The ANOVA for SOD activity did not show an effect from rosemary extract on enzyme activity in any of the three tissues: heart, cortex or hippocampus (F (2, 29)=0.940; p0.40, F(2, 29)=0.158; p0.855 and F(2, 29)= 3.56; p0.54, respectively). Table II gives the mean and SD for SOD and GPX, which showed no significant differences.

NOS activity

Heart NOS activity levels were significantly decreased by SFE rosemary extract F(2, 29)=4.4; p0.029. Interestingly, when we compared each group individually against the others we observed a statistical difference between control and rosemary extract at 0.02%. (p0.05). Figure 3 shows a bar diagram for NOS activity in the different study groups. NOS activity is expressed as the concentration of nitrate+nitrite in M units/ g of protein.

However the ANOVA did not show a significant effect from rosemary extract on enzyme activity in hippocampus (F (2, 29)=0.111; p0.89). Table II shows the non-significant values for NOS.

ROS levels

ANOVA data did not show a significant effect from SFE rosemary extract on ROS levels in heart or cortex, (F (2, 29)=2.86; p0.08 for the former and F(2, 29)=0.39; p0.68 for the second tissue). In hippocampus, however ANOVA did show that the rosemary extract significantly decreased ROS levels (F (2, 29)=26.21; p0.001). The effect of 0.2% rosemary extract was (p0.001) stronger than that of 0.02% rosemary extract (p0.002).

The figure 4 bar diagram shows the results for ROS levels in this tissue in the different study groups, expressed as fluorescence units per mg of total protein.

Discussion

The aging process itself exemplifies the cumulative effect of deterioration caused by free radicals in cells and tissues. Although most tissues, including brain and heart, have built-in mechanisms to counteract these radicals, the antioxidant defense is overwhelmed by the aging process, disease aside. The role of enzymatic antioxidants in ageing is controversial. Normal ageing is accompanied by a decline in antioxidant defenses reflected in reduced glutathione in blood and different tissues of animals and humans [START_REF] Muthuswamy | Oxidative stress-mediated macromolecular damage and dwindle in antioxidant status in aged rat brain regions: Role of l-carnitine and DL-alpha-lipoic acid[END_REF]. In fact, supplementing the diet with natural compounds with antioxidant properties has been shown to be beneficial (De la [START_REF] De La Fuente | Antioxidants as modulators of immune function[END_REF].

Nevertheless, it has recently been hypothesized that antioxidants do not seem to control the kinetics of ageing [START_REF] Barja | Free radicals and aging[END_REF]. Many of these antioxidant enzymes are induced by stress, so higher levels would indicate either a better protection or, alternatively, a greater need for antioxidant defenses. In fact, the antioxidant mechanisms are a harmonic system and the interactions among them are complex.

In this work we have analyzed whether two different areas of the brain, namely the cerebral cortex and the hippocampus respond to the effects of rosemary extract in an aged animal model using an extract containing 20% CA and obtained with the supercritical fluid extraction method [START_REF] Ramirez | Isolation of functional ingredients from rosemary by preparative-supercritical fluid (Prep-SFC)[END_REF] at two concentrations (0.2% and 0.02%). Additionally, we also included heart tissue to evaluate the possible effects on cardiovascular oxidative stress status in aged rats.

Lipid peroxidation, a process induced by free radicals, leads to oxidative deterioration of polyunsaturated lipids. Only low levels of lipid peroxides occur in body tissues under physiological conditions. An excessive generation of free radicals leads to peroxidative changes that ultimately result in enhanced lipid peroxidation [START_REF] Rikans | Lipid peroxidation, antioxidant protection and aging[END_REF]. We inferred lipid peroxidation from the level of MDA, which is a secondary product of lipid peroxidation, and is used as an indicator of tissue damage [START_REF] Ohkawa | Assay of lipid peroxides in animal tissue by thiobarbituric acid reaction[END_REF].

Ageing has been reported to be associated with increased disruption of lipid membranes leading to a subsequent formation of peroxide radicals [START_REF] Noguchi | Dynamics of lipid peroxidation and its inhibition by antioxidants[END_REF]. The available literature indicates that the brain has higher levels of lipids and consumes a greater quantity of oxygen than other organs, and in fact some studies have reported an increase in lipid peroxidation with ageing [START_REF] Farooqui | Glutathione and Lipid Peroxidation in the Aging Rats[END_REF][START_REF] Jayakumar | Protective effect of an extract of the oyster mushroom, Pleurotus ostreatus, on antioxidants of major organs of aged rats[END_REF][START_REF] Serrano | Reactive oxygen species and synaptic plasticity in the aging hippocampus[END_REF]. In our results, the level of membrane lipid products in brain was significantly lower in the groups receiving the rosemary extract. The two concentrations of rosemary extract (0.2% and 0.02%) had a significant antioxidant lipid effect in cortex, but, and surprisingly, only the lowest concentration (0.02%) of rosemary extract had a significant effect in hippocampus. These data corroborate findings in earlier investigations where antioxidants and administration of a natural grape seed extract decreased lipid peroxidation in the brain of aged rats [START_REF] Arivazhagan | Effect of dl-α-lipoic acid on neural antioxidants in aged rats[END_REF][START_REF] Balu | Rejuvenation of antioxidant system in central nervous system of aged rats by grape seed extract[END_REF]. The observed reduction in MDA levels in aged rats following the administration of rosemary extract suggest Rosmarinus officinalis, may have an antioxidant effect in the brain.

When we examined heart tissue we did not observe significant differences between the rosemary extract treated and the control groups, although levels of lipid peroxidation were lower in the former. Recent studies report increased MDA levels in the heart of aged rats [START_REF] Jayakumar | Protective effect of an extract of the oyster mushroom, Pleurotus ostreatus, on antioxidants of major organs of aged rats[END_REF][START_REF] Sivonova | Relationship between antioxidant potential and oxidative damage to lipids, proteins and dna in aged rats[END_REF]. Therefore, rosemary has some antioxidant effect in heart tissue, which somehow reverses the lipid peroxidation observed in aged individuals, but it is not enough to be deemed significant in our study.

Living tissues are endowed with innate antioxidant defence mechanisms, including the CAT, SOD and GPX. CAT has been shown to be responsible for the detoxification of significant amounts of H 2 O 2 [START_REF] Cheng | Photoinactivation of catalase[END_REF]. SOD catalyzes the removal of superoxide radical O 2 . -, which would otherwise damage the cell membrane and biological structures. GPX catalyses the reduction of H 2 O 2 to H 2 O and O 2. In general, a reduction in the activity of these enzymes is associated with an accumulation of highly reactive free radicals, and can lead to deleterious effects such as loss of cell membrane integrity and function [START_REF] Reedy | Studies on spice principle as an antioxidant in the inhibition of lipid peroxidation of rat liver microsomes[END_REF][START_REF] Sheela | Antiperoxide effects of S-allyl cystein sulphoxide isolated from Allium sativum Linn and gugulipid in chlosterol diet fed rats[END_REF]. We found diminished levels of CAT activity in brain and heart tissues in both rosemary extract treated groups, and this decrease was statistically significant in heart and cortex but not in the hippocampus. This decrease in the enzymatic activity was significant in heart with both rosemary extract concentrations (0.02%, 0.2%), but only the higher concentration (0.2%) had an effect in cortex. The available information in literature about age-related changes in rodent antioxidant enzymes indicates that catalase activity is increased with ageing and catalase activity changes are more selective in tissues such as heart and brain [START_REF] Calabrese | Increased expression of heat shock proteins in rat brain during aging: relationship with mitochondrial function and glutathione redox state[END_REF]. The lower CAT activity levels observed here in the rosemary-treated animals are related to the effect of the supplement. In agreement with the literature our results could suggest that rosemary extract somehow reverses the effects of aging on catalase activity in heart and cortex, indicating the antioxidant potential of the rosemary extract.

The findings on SOD and GPX activities were not statistically significant, although they did show some reduction in these enzymatic activities in the rosemary treated groups in brain and heart. The available information about the activity and expression of GPX and SOD is contradictory, with decrease, no changes and increases with ageing in different tissues, among them, heart and brain, all being reported [START_REF] Benzi | Age-and peroxidative stress-related modifications of the cerebral enzymatic activities linked to mitochondria and the glutathione system[END_REF][START_REF] Carrillo | The ability of (-)deprenyl to increase superoxide dismutase activities in the rat is tissue and brain region selective[END_REF][START_REF] Doğru-Abbasoğlu | Lipid peroxidation and antioxidant enzymes in livers and brains of aged rats[END_REF][START_REF] Danh | Differential changes in superoxide dismutase activity in brain and liver of old rats and mice[END_REF][START_REF] Gupta | Age-related elevation of lipid peroxidation products: diminution of superoxide dismutase activity in the central nervous system of rats[END_REF][START_REF] Scarpa | Age dependence of the level of the enzymes involved in the protection against active oxygen species in the rat brain[END_REF][START_REF] Warner | Superoxide dismutase, aging and degenerative diseases[END_REF]. In fact, changes in antioxidant enzyme activity need to be carefully interpreted, taking into consideration the facts that these changes are markedly variable depending on sex as well as the organs and brain regions examined and even on the animal model or analytic techniques employed in different studies.

There are two main forms of NOS in the heart, inducible NOS (iNOS) located in the myocytes, and endothelial NOS (eNOS), located in endothelial cells [START_REF] Bolli | Cardioprotective function of inducible nitric oxide synthase and role of nitric oxide in myocardial ischemia and preconditioning: an overview of a decade of research[END_REF]. NOS has a dual cardioprotective role: initiator and mediator. The mechanism by which NOS protects heart tissue has not yet been totally clarified, although a number of different mechanisms have been suggested, the most important being antioxidant action [START_REF] Bolli | Cardioprotective function of inducible nitric oxide synthase and role of nitric oxide in myocardial ischemia and preconditioning: an overview of a decade of research[END_REF]. We could not find significant differences in the levels of the enzyme activity in the brain regions analyzed. Our results found NOS decreases in heart tissue in the groups treated with rosemary extract (0.02%) and these differences with the control group were statistically significant. The available information indicates that aged Wistar rats show increased levels of NOS [START_REF] Cernadas | Expression of constitutive and inducible nitric oxide synthases in the vascular wall of young and aging rats[END_REF][START_REF] Zieman | Upregulation of the nitric oxide-cGMP pathway in aged myocardium: physiological response to l-arginine[END_REF]. Notably, recent studies on the functional role of NOS in cardioprotection report that augmented NO levels are related to heart faliure, whereas NOS decreases levels of NO activity and protects this tissue.

Futhermore, dietary deficiencies of oligoelements like copper can raise NOS levels and there is a clear relationship with the appearance of heart tissue abnormalities and elevated NOS levels [START_REF] Saari | Cardiac nitric oxide synthases are elevated in dietary copper deficiency[END_REF]. Therefore, our results could indicate a protective action by rosemary extract in heart tissue through decreases in NOS activity levels, and subsequently, NO production.

In general, the ageing process is related with an increase in the production of the different reactive oxygen species (Hagen et al., 2002). Our results showed no significant differences in ROS levels between rosemary extract treated groups and the control group in either heart or cortex. However, the group comparison in the hippocampus showed significantly lower ROS levels in this structure with both concentrations of rosemary extract. We therefore suggest that rosemary extract has an antioxidant effect as a free radical scavenger in this organ. This agrees with a growing number of studies showing that natural extracts and phytochemicals have a positive impact on brain ageing through their action on ROS, particulally in the hippocampus [START_REF] Bastianetto | Natural extracts as possible protective agents of brain aging[END_REF].

As far as we know this is the first work that shows antioxidant effects of rosemary extract in aged rats (in vivo). In conclusion, in aged rats rosemary extract consumption decreases cerebral catalase activity, lipid peroxidation and ROS levels, thus protecting the brain, and decreases the activity of catalase and NOS in the heart, protecting this organ. The effects of the two rosemary concentrations were different in the three tissue types since lipid peroxidation, ROS levels and enzymatic activities were different in both treated groups.

Further studies are required to determine the causes behind the distinct effects of the two different rosemary extract concentrations and it remains to be seen whether a similar protective effect can be demonstrated in aged humans. Thus, rosemary extract could be incorporated to the diet as a nutritional supplement, to augment the body´s defenses against oxidative stress.

Legends to the figures. Tables Table I. Different groups, the characteristics of the diet and the meat food composition.

  Animals were individually housed with free access to food and water, and maintained under a normal light-dark cycle in the Experimental Surgery Service of La Paz Hospital. The present study was approved by the Institutional Animal Ethics Committee (IAEC), La Paz Hospital, Madrid, Spain, in accordance with the Spanish law for the protection of experimental animals and other research purposes: RD 1201/2005.

  was used to determine the relative levels of different reactive oxygen species. This assay measures the oxidative conversion of stable 2´, 7´-dichlorodihydrofluorencein diacetate (H 2 DCFDA, Invitrogen, Molecular Probes) to the highly fluorescent 2´, 7´-diclorofluorescein in the presence of sterases, especially hydrogen peroxide. Fractions from tissue homogenates were loaded with 40 l of 1.25 mM 2´, 7´-dichlorodihydrofluorencein diacetate in methanol. A remaining fraction with methanol was used as a blank to control for tissue autofluorescence. All samples were incubated for 15 min in a 32º C water bath. Fluorescence was determined at four time points postincubation (0, 30, 60, 90 min) using a plate reader (Biotek Instruments, Inc. Vermont, USA), at 510 nm excitation, with 528 nm emission. Blank readings were subtracted from loaded sample readings, with values indexed per milligram of total protein. Readings at 60 minutes post incubation were considered optimal.

  GPX activity in heart, cortex or hippocampus was not affected by rosemary extract at either concentration (F (2, 29)=0.477, p0.63; F(2, 29)=0.42; p0.66 and F (2, 29)= 1.00; p0.38 respectively).

Figure 1 .

 1 Figure 1. Bar diagram showing the lipid peroxidation in the different goups in cortex (A) and hippocampus (B).  indicates significant at p0.001 level (control vs 0.2% rosemary extract) and  at p0.03 level (control vs 0.02% rosemary extract). Bar diagram represents the mean and SD values for each group.

Figure 2 .

 2 Figure 2. Bar diagram showing the catalase activity in the different goups in heart (A) and cortex (B).  indicates significant at p0.05 level (control vs 0.2% rosemary extract) and  at p0.017 level (control vs 0.02% rosemary extract). Bar diagram represents the mean and SD values for each group.

Figure 3 .

 3 Figure 3. Bar diagram showing the NOS activity the different goups in heart.  indicates significant at p0.05 level (control vs 0.02% rosemary extract). Bar diagram represents the mean and SD values for each group.

Figure 4 .

 4 Figure 4. Bar diagram showing the ROS levels in the different goups in hippocampus.  indicates significant at p0.001 level (control vs 0.2% rosemary extract) and  at p0.002 level (control vs 0.2% rosemary extract). Bar diagram represents the mean and SD values for each group.

Figure

  Figure(s)

Table II .

 II Statitistical descriptive data of SOD, GPX and NOS activity in heart, cortex and hippocampus. Values are mean  S. D. of ten animals per group. SOD activity is expressed as units per mg of protein. GPX activity is expressed as nmol/min/mg of protein. NOS activity is expressed as M units per mg of protein. Values with different superscripts between groups in each column are significantly different (p0.05).Rosemary 0.02% 31.404.7 48.126.7 15.353.5

		Tissue	Groups	SOD	GPX	NOS
			Control	47.506.3 21.143.8 5.700.9 a
		heart	Rosemary 0.02% 44.856.2 17.752.9 3.870.9 b
			Rosemary 0.2%	41.905.9 18.113.0 5.501.1 a
			Control	35.734.9 74.609.9 11.401.7
		cortex	Rosemary 0.02% 32.874.6 67.758.9 11.101.7
		Groups	Rosemary 0.2%	Extract concentration (%) 31.614.2 68.768.8 10.781.5
	1 Control (n=10)	Control	0 32.675.0 56.707.1 14.292.1
	2 C. A. Ext. 20% (n=10) hippocampus		0.2
	3 C. A. Ext. 20% (n=10) Rosemary 0.2%	0.02 30.004.5 52.447.0	14.93.2
		Functional meat food composition		Values per 100gr
	1	Energy (Kcal)			107
	2	Proteins (g)			19.0
	3	Carbohydrates (g)		1.0
	4	Fat (g)			2.0
	5	Saturated (g)			0.7
	6	Monosaturated (g)		0.8
			(-3+-6)		0.5
	8	Na + (g)			0.5
	9	Ratio (-3/-6)		0.5

Polyunsaturated (g)

Acknowledgments: This work is supported by Grants: Consolider CSA 2007-00063 and Comunity of Madrid 0505-AGR-0553. Sinforiano J. Posadas is a post-doctoral fellow supported by "Juan de la Cierva" program, Ministry of Science (MEC), Madrid, Spain. We thank Dr. J. Matés and Dr. J. J. Merino for their helpful discussion of the manuscript. We thank to Frial, SA (Madrid Spain) for their kind gift of supplemented and regular turkey breast; Frial has given no other support to this study.