Regulation of membrane phospholipids during the adult life of worker honey bee.

Two female castes that are genetically identical are found in honey bees: workers and queens. Adult female honey bees differ in their morphology and behaviors, but the most intriguing difference between the castes is the difference in their longevity. Queens live for years while workers live generally for weeks. The mechanisms that mediate this extraordinary difference in lifespan remain mostly unknown. Both castes share similar developmental stages and are fed liquid food (i.e. a jelly) during development. However, after emergence, workers begin to feed on pollen while queens are fed the same larval food for their entire life. Pollen has a high content of polyunsaturated fatty acids (PUFA) while royal jelly has negligible amounts. The difference in food during adult life leads to drastic changes in membrane phospholipids of female honey bees, and those changes have been proposed as mechanisms that could explain the difference in lifespan. To provide further details on those mechanisms, we characterized the membrane phospholipids of adult workers at seven different ages covering all life-history stages. Our results suggest that the majority of changes in worker membranes occur in the first four days of adult life. Shortly after emergence, workers increase their level of total phospholipids by producing phospholipids that contained saturated (SFA) and monounsaturated fatty acids (MUFA). From the second day, workers start replacing fatty acid chains from those pre-synthesized molecules with PUFA acquired from pollen. After four days, worker membranes are set and appear to be maintained for the rest of adult life, suggesting that damaged PUFA are replaced effectively. Plasmalogen phospholipids increase continuously throughout worker adult life, suggesting that plasmalogen might help to reduce lipid peroxidation in worker membranes. We postulate that the diet-induced increase in PUFA in worker membranes makes them far more prone to lipid-based oxidative damage compared to queens.

The adult lifespan of the female honey bee (Apis mellifera): Metabolic rate, AGE pigment and the effect of dietary fatty acids.

Female honey bees can be queens or workers and although genetically identical, workers have an adult lifespan of weeks while queens can live for years. The mechanisms underlying this extraordinary difference remain unknown. This study examines three potential explanations of the queen-worker lifespan difference. Metabolic rates were similar in age-matched queens and workers and thus are not an explanation. The accumulation of fluorescent AGE pigment has been successfully used as a good measure of cellular senescence in many species. Unlike other animals, AGE pigment level reduced during adult life of queens and workers. This unusual finding suggests female honey bees can either modify, or remove from their body, AGE pigment. Another queen-worker difference is that, as adults, workers eat pollen but queens do not. Pollen is a source of polyunsaturated fatty acids. Its consumption explains the queen-worker difference in membrane fat composition of female adult honey bees which has previously been suggested as a cause of the lifespan difference. We were able to produce "queen-worker" membrane differences in workers by manipulation of diet that did not change worker lifespan and we can, thus, also rule out pollen consumption by workers as an explanation of the dramatic queen-worker lifespan difference.

The under-appreciated fats of life: the two types of polyunsaturated fats.

There are two types of polyunsaturated fatty acids (i.e. fats that contain multiple carbon-carbon double bonds) - omega-6 and omega-3. They are not interconvertible, and they contribute 'double-bonded carbons' to different depths in bilayer membranes, with different effects on membrane processes. This Commentary emphasises the importance of these fats for biological membrane function and examines their evolution and biochemistry. Omega-6 and omega-3 fatty acids are separately essential in the diet of animals, and they pass up the food chain largely from plants, with 'seeds' being a prevalent source of omega-6, and 'leaves' a prevalent source of omega-3. The dietary balance between these fatty acids has a strong influence on membrane composition. Although this aspect of diet has been little investigated outside of the biomedical field, emerging evidence shows it can alter important physiological capacities of animals (e.g. exercise endurance and adiposity), which has implications for activities such as avian migration and hibernation and torpor, as well as significant implications for human health. This Commentary will focus on the separate effects of omega-3 and omega-6 on membrane properties and will emphasise the importance of the balance between these two fatty acids in determining the function of biological membranes; I hope to convince the reader that fats should be considered first and foremost as the basic unit of biological membranes, and secondarily as a means of energy storage.

Honey bee caste lipidomics in relation to life-history stage and the long life of the queen.

Honey bees have evolved a system in which fertilised eggs transit through the same developmental stages but can become either workers or queens. This difference is determined by their diet through development. Whereas workers live for weeks (normally 2-6 weeks), queens can live for years. Unfertilised eggs also develop through the same stages but result in a short-lived male caste (drones). Workers and drones are fed pollen throughout their late larval and adult life stages, while queens are fed exclusively on royal jelly and do not eat pollen. Pollen has a high content of polyunsaturated fatty acids (PUFA) while royal jelly has a negligible amount of PUFA. To investigate the role of dietary PUFA lipids and their oxidation in the longevity difference of honey bees, membrane fatty acid composition of the three castes was characterised at six different life-history stages (larva, pupa, emergent and different adult stages) through mass spectrometry. All castes were found to share a similar membrane phospholipid composition during early larval development. However, at pupation, drones and workers increased their level of PUFA, whilst queens increased their level of monounsaturated fatty acids. After emergence, worker bees further increased their level of PUFA by 5-fold across most phospholipid classes. In contrast, the membrane phospholipids of adult queens remained highly monounsaturated throughout their adult life. We postulate that this diet-induced increase in membrane PUFA results in more oxidative damage and is potentially responsible for the much shorter lifespan of worker bees compared with long-lived queens.

Diet fatty acid profile, membrane composition and lifespan: an experimental study using the blowfly (Calliphora stygia).

The membrane pacemaker theory of ageing proposes that the polyunsaturated fatty acid (PUFA) composition of membrane lipids of a species is an important determinant of its maximum lifespan. We report three experiments using the blowfly Calliphora stygia, where this theory was tested by manipulation of dietary fat profile. Although the fat profile of the larval diet resulted in small alterations of individual membrane fatty acids, it had no effect on the peroxidation index (PI) of membrane lipids and furthermore had no effect on maximum lifespan. Similarly, manipulation of the fat profile of the adult diet resulted in small changes in individual fatty acids, but had no effect on the PI of membrane lipids. There was a small increase in maximum lifespan when adult diet was supplemented with PUFA in form of vegetable oils but no effect when diet was supplemented with pure PUFA. This difference is possibly due to antioxidant content of vegetable oils. The relative refractoriness of membrane PI to dramatic changes in response to diet is similar to the situation in the rat. These results also indicate the blowfly is unable to convert 18-carbon PUFA to more highly polyunsaturated 20- and 22-carbon PUFA.

Polyunsaturated fats, membrane lipids and animal longevity.

Fatty acids are essential for life because they are essential components of cellular membranes. Lower animals can synthesize all four classes of fatty acids from non-lipid sources, but both omega-6 and omega-3 cannot be synthesized de novo by 'higher' animals and are therefore essential components of their diet. The relationship between normal variation in diet fatty acid composition and membrane fatty acid composition is little investigated. Studies in the rat show that, with respect to the general classes of fatty acids (saturated, monounsaturated and polyunsaturated) membrane fatty acid composition is homeostatically regulated despite diet variation. This is not the case for fatty acid composition of storage lipids, which responds to diet variation. Polyunsaturated fatty acids are important determinants of physical and chemical properties of membranes. They are the substrates for lipid peroxidation and it is possible to calculate a peroxidation index (PI) for a particular membrane composition. Membrane PI appears to be homeostatically regulated with respect to diet PI. Membrane fatty acid composition varies among species and membrane PI is inversely correlated to longevity in mammals, birds, bivalve molluscs, honeybees and the nematode Caenorhabditis elegans.

Effect of temperature on the rate of ageing: an experimental study of the blowfly Calliphora stygia.

All organisms age, the rate of which can be measured by demographic analysis of mortality rates. The rate of ageing is thermally sensitive in ectothermic invertebrates and we examined the effects of temperature on both demographic rates of ageing and on cellular senescence in the blowfly, Calliphora stygia. The short lifespan of these flies is advantageous for demographic measurements while their large body size permits individual-based biochemical characterisation. Blowflies maintained at temperatures from 12°C to 34°C had a five to six-fold decrease in maximum and average longevity, respectively. Mortality rates were best described by a two-phase Gompertz relation, which revealed the first-phase of ageing to be much more temperature sensitive than the second stage. Flies held at low temperatures had both a slower first-phase rate of ageing and a delayed onset of second-phase ageing, which significantly extended their longevity compared with those at high temperatures. Blowflies that were transferred from 29°C to 15°C had higher first-phase mortality rates than those of flies held at constant 15°C, but their onset of second-phase ageing was deferred beyond that of flies held constantly at this temperature. The accumulation of fluorescent AGE pigment, a measure of cellular oxidative damage, increased steadily over time in all blowflies, irrespective of the temporal pattern of mortality. Pigment accumulated steadily during periods of 'negligible senescence', as measured by minimal rate of mortality, and the rate of accumulation was significantly affected by temperature. Thus accumulation of AGE pigment is more representative of chronological age than a reflection of biological age or a cause of mortality.

Does the oxidative stress theory of aging explain longevity differences in birds? II. Antioxidant systems and oxidative damage.

The oxidative damage hypothesis of aging posits that the accumulation of oxidative damage is a determinant of an animal species' maximum lifespan potential (MLSP). Recent findings in extremely long-living mammal species such as naked mole-rats challenge this proposition. Among birds, parrots are exceptionally long-living with an average MLSP of 25 years, and with some species living more than 70 years. By contrast, quail are among the shortest living bird species, averaging about 5-fold lower MLSP than parrots. To test if parrots have correspondingly (i) superior antioxidant protection and (ii) lower levels of oxidative damage compared to similar-sized quail, we measured (i) total antioxidant capacity, uric acid and reduced glutathione (GSH) levels, as well as the activities of enzymatic antioxidants (superoxide dismutase, glutathione peroxidase and catalase), and (ii) markers of mitochondrial DNA damage (8-OHdG), protein damage (protein carbonyls) and lipid peroxidation (lipid hydroperoxides and TBARS) in three species of long-living parrots and compared these results to corresponding measures in two species of short-living quails (average MLSP=5.5 years). All birds were fed the same diet to exclude differences in dietary antioxidant levels. Tissue antioxidants and oxidative damage were determined both 'per mg protein' and 'per g tissue'. Only glutathione peroxidase was consistently higher in tissues of the long-living parrots and suggests higher protection against the harmful effects of hydroperoxides, which might be important for parrot longevity. The levels of oxidative damage were mostly statistically indistinguishable between parrots and quails (67%), occasionally higher (25%), but rarely lower (8%) in the parrots. Despite indications of higher protection against some aspects of oxidative stress in the parrots, the pronounced longevity of parrots appears to be independent of their antioxidant mechanisms and their accumulation of oxidative damage.

Fatty acid composition of membrane bilayers: importance of diet polyunsaturated fat balance.

In one of the most extensive analyses to date we show that the balance of diet n-3 and n-6 polyunsaturated fatty acids (PUFA) is the most important determinant of membrane composition in the rat under 'normal' conditions. Young adult male Sprague-Dawley rats were fed one of twelve moderate-fat diets (25% of total energy) for 8weeks. Diets differed only in fatty acid (FA) profiles, with saturate (SFA) content ranging 8-88% of total FAs, monounsaturate (MUFA) 6-65%, total PUFA 4-81%, n-6 PUFA 3-70% and n-3 PUFA 1-70%. Diet PUFA included only essential FAs 18:2n-6 and 18:3n-3. Balance between n-3 and n-6 PUFA is defined as the PUFA balance (n-3 PUFA as % of total PUFA) and ranged 1-86% in the diets. FA composition was measured for brain, heart, liver, skeletal muscle, erythrocytes and plasma phospholipids, as well as adipose tissue and plasma triglycerides. The conformer-regulator model was used (slope=1 indicates membrane composition completely conforming to diet). Extensive changes in diet SFA, MUFA and PUFA had minimal effect on membranes (average slopes 0.01, 0.07, 0.07 respectively), but considerable influence on adipose tissue and plasma triglycerides (average slopes 0.27, 0.53, 0.47 respectively). Diet balance between n-3 and n-6 PUFA had a biphasic influence on membrane composition. When n-3 PUFA<10% of total PUFA, membrane composition completely conformed to diet (average slope 0.95), while diet PUFA balance>10% had little influence (average slope 0.19). The modern human diet has an average PUFA balance ~10% and this will likely have significant health implications.

Metabolic rate and membrane fatty acid composition in birds: a comparison between long-living parrots and short-living fowl.

Both basal metabolic rate (BMR) and maximum lifespan potential (MLSP) vary with body size in mammals and birds and it has been suggested that these are mediated through size-related variation in membrane fatty acid composition. Whereas the physical properties of membrane fatty acids affect the activity of membrane proteins and, indirectly, an animal's BMR, it is the susceptibility of those fatty acids to peroxidation which influence MLSP. Although there is a correlation between body size and MLSP, there is considerable MLSP variation independent of body size. For example, among bird families, Galliformes (fowl) are relatively short-living and Psittaciformes (parrots) are unusually long-living, with some parrot species reaching maximum lifespans of more than 100 years. We determined BMR and tissue phospholipid fatty acid composition in seven tissues from three species of parrots with an average MLSP of 27 years and from two species of quails with an average MLSP of 5.5 years. We also characterised mitochondrial phospholipids in two of these tissues. Neither BMR nor membrane susceptibility to peroxidation corresponded with differences in MLSP among the birds we measured. We did find that (1) all birds had lower n-3 polyunsaturated fatty acid content in mitochondrial membranes compared to those of the corresponding tissue, and that (2) irrespective of reliance on flight for locomotion, both pectoral and leg muscle had an almost identical membrane fatty acid composition in all birds.

Does the oxidative stress theory of aging explain longevity differences in birds? I. Mitochondrial ROS production.

Mitochondrial reactive oxygen species (ROS) production rates are reported to be inversely related to maximum lifespan potential (MLSP) in mammals and also to be higher in short-living mammals compared to short-living birds. The mammal-bird comparison, however, is mainly based on studies of rats and pigeons. To date, there has been no systematic examination of ROS production in birds that differ in MLSP. Here we report a comparison of mitochondrial ROS production in two short-living (quails) and three long-living bird species (parrots) that exhibit, on average, a 5-fold longevity difference. Mitochondrial ROS production was determined both in isolated mitochondria (heart, skeletal muscle and liver) as traditionally done and also in intact erythrocytes. In all four tissues, mitochondrial ROS production was similar in quails and parrots and showed no correspondence with known longevity differences. The lack of a consistent difference between quails and parrots was not due to differences in mitochondrial content as ROS production in relation to oxygen consumption (determined as the free radical leak) showed a similar pattern. These findings cast doubt on the robustness of the oxidative stress theory of aging.

The long life of birds: the rat-pigeon comparison revisited.

The most studied comparison of aging and maximum lifespan potential (MLSP) among endotherms involves the 7-fold longevity difference between rats (MLSP 5y) and pigeons (MLSP 35y). A widely accepted theory explaining MLSP differences between species is the oxidative stress theory, which purports that reactive oxygen species (ROS) produced during mitochondrial respiration damage bio-molecules and eventually lead to the breakdown of regulatory systems and consequent death. Previous rat-pigeon studies compared only aspects of the oxidative stress theory and most concluded that the lower mitochondrial superoxide production of pigeons compared to rats was responsible for their much greater longevity. This conclusion is based mainly on data from one tissue (the heart) using one mitochondrial substrate (succinate). Studies on heart mitochondria using pyruvate as a mitochondrial substrate gave contradictory results. We believe the conclusion that birds produce less mitochondrial superoxide than mammals is unwarranted. We have revisited the rat-pigeon comparison in the most comprehensive manner to date. We have measured superoxide production (by heart, skeletal muscle and liver mitochondria), five different antioxidants in plasma, three tissues and mitochondria, membrane fatty acid composition (in seven tissues and three mitochondria), and biomarkers of oxidative damage. The only substantial and consistent difference that we have observed between rats and pigeons is their membrane fatty acid composition, with rats having membranes that are more susceptible to damage. This suggests that, although there was no difference in superoxide production, there is likely a much greater production of lipid-based ROS in the rat. We conclude that the differences in superoxide production reported previously were due to the arbitrary selection of heart muscle to source mitochondria and the provision of succinate. Had mitochondria been harvested from other tissues or other relevant mitochondrial metabolic substrates been used, then very different conclusions regarding differences in oxidative stress would have been reached.

The ω-3 and ω-6 fats in meals: a proposal for a simple new label.

OBJECTIVE: The ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) are separate essential dietary fatty acids that play a key role in many physiologic processes in higher animals. The content of these PUFAs is relatively well described for many individual food components. Our goal in this study was to analyze the PUFA content of whole meals and produce a simple measurement to estimate the intake of these fatty acids. METHODS: The fatty acid profile and macronutrient composition were determined for a range of fast food, cuisine (restaurant-prepared), and home-prepared whole meals commonly consumed by Australians. RESULTS: Across the different meals there was significant variation in protein (4-fold), fat (13-fold), and carbohydrate (23-fold) contents. With regard to the fatty acid profile, saturated and monounsaturated fatty acids made up approximately 80% of total fatty acids for most meals. The ω-6 PUFAs were substantially more abundant than ω-3 PUFAs for most meals. The balance of dietary ω-3 and ω-6 PUFAs is an important determinant of their metabolic effects within the body, and accordingly we calculated the percentage of the total PUFA comprised of ω-3 PUFAs and referred to this as the PUFA Balance. This parameter showed the greatest variation among the different meals (>45-fold). CONCLUSION: The relative proportions of ω-3 and ω-6 PUFAs vary greatly across meals. PUFA Balance is a useful tool that will allow individuals to more easily monitor and balance their intake of ω-3 and ω-6 fats.

Metabolism and longevity: is there a role for membrane fatty acids?

More than 100 years ago, Max Rubner combined the fact that both metabolic rate and longevity of mammals varies with body size to calculate that "life energy potential" (lifetime energy turnover per kilogram) was relatively constant. This calculation linked longevity to aerobic metabolism which in turn led to the "rate-of-living" and ultimately the "oxidative stress" theories of aging. However, the link between metabolic rate and longevity is imperfect. Although unknown in Rubner's time, one aspect of body composition of mammals also varies with body size, namely the fatty acid composition of membranes. Fatty acids vary dramatically in their susceptibility to peroxidation and the products of lipid peroxidation are very powerful reactive molecules that damage other cellular molecules. The "membrane pacemaker" modification of the "oxidative stress" theory of aging proposes that fatty acid composition of membranes, via its influence on peroxidation of lipids, is an important determinant of lifespan (and a link between metabolism and longevity). The relationship between membrane fatty acid composition and longevity is discussed for (1) mammals of different body size, (2) birds of different body size, (3) mammals and birds that are exceptionally long-living for their size, (4) strains of mice that vary in longevity, (5) calorie-restriction extension of longevity in rodents, (6) differences in longevity between queen and worker honeybees, and (7) variation in longevity among humans. Most of these comparisons support an important role for membrane fatty acid composition in the determination of longevity. It is apparent that membrane composition is regulated for each species. Provided the diet is not deficient in polyunsaturated fat, it has minimal influence on a species' membrane fatty acid composition and likely also on it's maximum longevity. The exceptional longevity of Homo sapiens combined with the limited knowledge of the fatty acid composition of human tissues support the potential importance of mitochondrial membranes in determination of longevity.

Membrane fatty acid composition of rat skeletal muscle is most responsive to the balance of dietary n-3 and n-6 PUFA.

The present study quantifies the relationships between diet fatty acid profile and fatty acid composition of rat skeletal muscle phospholipids. Young adult male Sprague-Dawley rats were fed, for 8 weeks, on one of twelve moderate-fat diets (25 % of total energy) differing only in fatty acid profile. SFA content ranged from 8-88 % of total fatty acids, MUFA 6-65 %, total PUFA 4-81 %, n-6 PUFA 3-70 % and n-3 PUFA 1-70 %. Diet PUFA included only essential fatty acids 18 : 2n-6 and 18 : 3n-3. The balance between n-3 and n-6 PUFA (PUFA balance) in the diet ranged from 1 : 99 to 86 : 14 % n-3 PUFA:n-6 PUFA. The slope of muscle phospholipid composition plotted against diet composition quantifies the response of muscle membrane composition to dietary fat (0, no response; 1, complete conformity with diet). The resulting slopes were 0.02 (SFA), 0.10 (PUFA), 0.11 (MUFA), 0.14 (n-3 PUFA) and 0.23 (n-6 PUFA). The response to PUFA balance was biphasic with a slope of 0.98 below 10 % diet PUFA balance and 0.16 above 10 %. Thus, low diet PUFA balance has greater influence on muscle composition than 18-carbon n-3 or n-6 PUFA individually. Equations provided may allow prediction of muscle composition for other diet studies. Diet PUFA balance dramatically affects muscle 20 : 4n-6 and 22 : 6n-3. This may have significant implications for some disease states in human subjects.

Experimental studies of blowfly (Calliphora stygia) longevity: A little dietary fat is beneficial but too much is detrimental.

This paper is one in a series of experimental studies on the effects of food composition on aging and longevity, using the golden-haired blowfly Calliphora stygia as the animal model. Here we examine how diet fat content affects blowfly life history traits such as longevity, reproduction, feeding rate, body mass, total fat content and membrane fatty acid composition. The highest median and maximum longevity was observed in blowflies fed on low fat diets, while high-fat diets caused more rapid death of the blowflies. A major result was that blowflies feeding on the lowest fat diet had the highest maximal lifespan demonstrating that low levels of diet fat enhanced blowfly lifespan. Diet also influenced gender-specific mortality rates; females lived longer on a high-fat diet, while males lived longer on a low fat diet. Furthermore, we provide data for and explain how blowfly feeding rates, egg production and male harassment affected blowfly longevity. Our results highlight the need for further studies to understand how dietary fats are metabolised and utilised in the golden-haired blowfly.

Metabolic depression during aestivation does not involve remodelling of membrane fatty acids in two Australian frogs.

Changes in membrane lipid composition (membrane remodelling) have been associated with metabolic depression in some aestivating snails but has not been studied in aestivating frogs. This study examined the membrane phospholipid composition of two Australian aestivating frog species Cyclorana alboguttata and Cyclorana australis. The results showed no major membrane remodelling of tissue in either frog species, or in mitochondria of C. alboguttata due to aestivation. Mitochondrial membrane remodelling was not investigated in C. australis. Where investigated in C. alboguttata, total protein and phospholipid content, and citrate synthase (CS) and cytochrome c oxidase (CCO) activities in tissues and mitochondria mostly did not change with aestivation in liver. In skeletal muscle, however, CS and CCO activities, mitochondrial and tissue phospholipids, and mitochondrial protein decreased with aestivation. These decreases in muscle indicate that skeletal muscle mitochondrial content may decrease during aestivation. Na(+)K(+)ATPase activity of both frog species showed no effect of aestivation. In C. alboguttata different fat diets had a major effect on both tissue and mitochondrial phospholipid composition indicating an ability to remodel membrane composition that is not utilised in aestivation. Therefore, changes in lipid composition associated with some aestivating snails do not occur during aestivation in these Australian frogs.

Phospholipid composition of the rat lens is independent of diet.

Dietary fatty acids are known to influence the phospholipid composition of many tissues in the body, with lipid turnover occurring rapidly. The aim of this study was to investigate whether changes in the fatty acid composition of the diet can affect the phospholipid composition of the lens. Male Sprague-Dawley rats were fed three diets with distinct profiles in both essential and non-essential fatty acids. After 8 weeks, lenses and skeletal muscle were removed, and the lenses sectioned into nuclear and cortical regions. In these experiments, the lens cortex was synthesised during the course of the variable lipid diet. Phospholipids were then identified by electrospray ionisation tandem mass spectrometry, and quantified via the use of internal standards. The phospholipid compositions of the nuclear and cortical regions of the lens differed slightly between the two regions, but comparison of the equivalent regions across the diet groups showed remarkable similarity. In contrast, the phospholipid composition of skeletal muscle (medial gastrocnemius) in these rats varied significantly. This study provides the first direct evidence to show that the phospholipid composition of the lens is tightly regulated and thus appears to be independent of diet. As phospholipids determine membrane fluidity and influence the activity and function of integral membrane proteins, regulation of their composition may be important for the function of the lens.

The exceptional longevity of an egg-laying mammal, the short-beaked echidna (Tachyglossus aculeatus) is associated with peroxidation-resistant membrane composition.

The echidna Tachyglossus aculeatus is a monotreme mammal from Australia that is exceptionally long-living. Its documented maximum lifespan of 50 years is 3.7 times that predicted from its body mass. Other exceptionally long-living mammals (naked mole-rats and humans) are known to have peroxidation-resistant membrane composition, raising the question about echidnas. Phospholipids were extracted from skeletal muscle, liver and liver mitochondria of echidnas and fatty acid composition measured. As with other exceptionally long-living mammals, membrane lipids of echidna tissues were found to have a lower content of polyunsaturates and a higher content of monounsaturates than predicted for their body size. The peroxidation index (=peroxidation susceptibility) calculated from this membrane composition was lower-than-expected for their body size, indicating that the cellular membranes of echidnas would be peroxidation-resistant. Additionally when the calculated peroxidation index was plotted against maximum lifespan, the echidna values conformed to the relationship for mammals in general. These findings support the membrane pacemaker theory of aging and emphasise the potential importance of membrane fatty acid composition in aging and in the determination of maximum longevity.