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Experimental Gerontology xxx (2007) xxxxxx www.elsevier.com/locate/expgero

Extended longevity of queen honey bees compared to workers is associated with peroxidation-resistant membranes
Laura Saade Haddad
a

a,1

, Louie Kelbert b, A.J. Hulbert

a,*

Metabolic Research Centre, and School of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia b Kel-Bee Apiaries, Warilla, NSW 2528, Australia Received 23 January 2007; received in revised form 26 February 2007; accepted 27 February 2007

Abstract In the honey bee (Apis mellifera), depending on what they are fed, female eggs become either workers or queens. Although queens and workers share a common genome, the maximum lifespan of queens is an order-of-magnitude longer than workers. The mechanistic basis of this longevity dierence is unknown. In order to test if dierences in membrane composition could be involved we have compared the fatty acid composition of phospholipids of queen and worker honey bees. The cell membranes of both young and old honey bee queens are highly monounsaturated with very low content of polyunsaturates. Newly emerged workers have a similar membrane fatty acid composition to queens but within the rst week of hive life, they increase the polyunsaturate content and decrease the monounsaturate content of their membranes, probably as a result of pollen consumption. This means their membranes likely become more susceptible to lipid peroxidation in this rst week of hive life. The results support the suggestion that membrane composition might be an important factor in the determination of maximum lifespan. Assuming the same slope of the relationship between membrane peroxidation index and maximum lifespan as previously observed for mammal and bird species, we propose that the 3-fold dierence in peroxidation index of phospholipids of queens and workers is large enough to account for the order-of-magnitude dierence in their longevity. 2007 Elsevier Inc. All rights reserved.
Keywords: Monounsaturates; Polyunsaturates; Lipid peroxidation; Lifespan; Longevity; Aging

1. Introduction The social insects have been suggested as good model organisms in which to investigate the mechanisms of ageing because there is considerable variation in lifespan between genetically identical queens and workers (Keller and Genoud, 1997; Keller and Jemielity, 2006). Depending on what they are fed, female eggs of the honey bee (Apis mellifera) become either workers or queens and while worker honey bees have a maximum lifespan of a few months, queen honeybees can live for years (Winston, 1987). Queen honey bees are generally replaced after 12 years in a commercial hive (for productivity reasons) and
Corresponding author. Tel.: +61 2 4221 3437; fax: +61 2 4221 4135. E-mail address: hulbert@uow.edu.au (A.J. Hulbert). 1 Present address: Department of Physiology, Biological Sciences Institute, University of Sao Paulo, Sao Paulo, SP, Brazil. 0531-5565/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2007.02.008
*

have adult lifespans of 25 years (Sammataro and Avitabile, 1998) with the longest recorded lifespan for a honey bee queen being 8 years (Bozina, 1961 cited by Page and Peng, 2001). Worker honey bees spend the rst part of their adult life solely in the hive and commence foraging (thus leaving the hive) after a few weeks. In summer, their adult lifespan is 1538 days (Sammataro and Avitabile, 1998). Worker bees generally die on a foraging trip where they experience a constant probability of death per unit time away from the colony, and most worker bees do not reach senescence (Visscher and Dukas, 1997). However, if prevented from shifting to foraging activity they are recorded as having a maximum lifespan of 75135 days (Haydek, 1963, cited by Amdam and Page, 2005). Over winter, worker bees can also develop into a stress-resistant form (called the diunitus stage) and their maximum adult lifespan is recorded as 140320 days over winter (Sammataro

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and Avitabile, 1998). Thus, the maximum lifespan of queen honey bees is about an order-of-magnitude greater than that of worker honey bees. The mechanistic basis for this very large dierence in lifespan of these female bees is currently not known. The oxidative stress theory, currently the most accepted mechanistic theory of ageing, proposes that normal metabolic activity results in the inevitable production of reactive oxygen species (ROS) which if not inactivated by antioxidant defences result in damage to important nucleic acids, proteins and lipids. This damage accumulates over time resulting in aging and consequently the determination of maximum lifespan (Sohal and Weindruch, 1996; Beckman and Ames, 1998). Queen and worker honey bees have essentially the same mass-specic metabolic rates (Fahrenholz et al., 1992) and therefore rate-of-living dierences are not likely an explanation of the longevity dierence. It might be suggested that the ight activity associated with foraging is responsible for the short lifespan of workers, but this cannot be a complete explanation as the longevity dierence is maintained even when workers are prevented from foraging. Both the expression of antioxidant enzyme genes (Corona et al., 2005) as well as antioxidant enzyme activities (Weirich et al., 2002) are essentially the same in workers and queens and are therefore also not likely an explanation. In Lasius niger, an ant species which also shows an order-of-magnitude longevity dierence between queens and workers, the activity of the antioxidant superoxide dismutase is actually lower in long-living queens than in workers (Parker et al., 2004). The longevity dierence between short-lived summer and long-lived winter worker bees has been suggested to be due to a decreased antioxidant defence (Amdam and Omholt, 2002). Vitellogenin (a 118kDa yolk precursor protein) has been shown to protect worker honey bees from oxidative stress (Seehuus et al., 2006) and involved in the immunosenescence that occurs when worker bees enter the foraging stage (Amdam et al., 2004, 2005). Although vitellogenin is likely involved in the longevity dierences between summer and winter worker bees, as well as the increased longevity when workers are prevented from foraging, whether it can fully explain the extended longevity of queens compared to workers is not certain. It would not appear to be a universal mechanism as, for example, in Caenorhabditis elegans reduced activity of the genes encoding vitellogenin does not shorten lifespan (which is the expectation of reduced antioxidant defence) but instead lengthens lifespan of this nematode (Murphy et al., 2003). Recently a modication of the oxidative stress theory has been proposed which suggests that the fatty acid composition of cell membranes might be an important factor in the determination of a species maximum lifespan (Pamplona et al., 2002; Hulbert, 2005). This modication of the oxidative stress theory of aging highlights (i) the fact that membrane fatty acid composition varies in a systematic manner between species, (ii) that many of the products of

lipid peroxidation are themselves powerful ROS and (iii) that fatty acids dier dramatically in their susceptibility to peroxidative damage, with monounsaturates and saturated fatty acids being resistant to peroxidative damage and only polyunsaturates being susceptible to peroxidation. The more polyunsaturated the fatty acid the more susceptible it is to peroxidation (Holman, 1954). When the fatty acid composition of a membrane is known, its susceptibility to peroxidative damage can be calculated and expressed as a peroxidation index (PI). It has been shown that dierences in the peroxidation index of membranes is inversely related to maximum lifespan of dierent-sized mammals (Pamplona et al., 1998, 1999). It has also been shown that the longer maximum lifespan of birds compared to similar-sized mammals is associated with more peroxidation-resistant membrane composition and that mammals and birds show essentially the same inverse relationship between maximum longevity and peroxidation index (Pamplona et al., 1996; Hulbert, 2003, 2005). The extreme maximum longevity of the naked mole-rat ($28 years) compared to that of mice (34 years) (Buenstein, 2005) has been recently shown to also be associated with dierences in membrane fatty acid composition of tissues from these two similar-sized rodent species (Hulbert et al., 2006a), as has the extended longevity of some wild-derived strains of Mus compared to laboratory strains (Hulbert et al., 2006b). Similarly, caloric-restriction has been shown to result in membrane fatty acid changes in both rats (Laganiere and Yu, 1987) and mice (Faulks et al., 2006) that make the membranes more resistant to peroxidative damage. The role of membrane composition and lipid peroxidation in the determination of a species maximum longevity has been recently reviewed (Hulbert et al., 2007). The present study was carried out to examine if the difference in longevity between queen and worker honey bees (described above) is also associated with dierences in the fatty acid composition of their respective membranes. The results to be presented show this is the case. 2. Materials and methods The bees used in this study were all obtained during DecemberMarch period of the 20052006 austral summer. Young queens ($4 week post eclosion) were purchased from Australian Queen Bee Exporters (Blayney, NSW, Australia). All other bees were taken from hives in the Wollongong area. Old queens ($2 years old) and worker bees from a number of age-classes were selected by one of us (Kelbert a professional apiarist of 27 years experience). The age-classes of worker bees analysed were; (i) newly emerged workers (<12 h post eclosion), (ii) young hive bees ($1 week post eclosion), (iii) older hive bees ($3 weeks post eclosion) and (iv) forager bees ($5 weeks post eclosion). Upon arrival at the University of Wollongong, all bees were immediately killed by immersion in liquid nitrogen and the head, thorax and abdomen regions were

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separated. All bees (apart from young queens) were alive and in transit for about 12 h before killing and obtaining of tissues. Young queens were in transit for 24 h. Unpublished results suggest this has no eect on membrane composition. Total lipid was extracted using ultra-pure grade chloroform:methanol (2:1 v/v) containing butylated hydroxytoluene (0.01% w/v) as an antioxidant. Phospholipids were separated from neutral lipids by solid phase extraction on silicic acid columns (Waters Corp. Milford, MA, USA,). Phospholipids were transmethylated (Lepage and Roy, 1986) and fatty acid methyl esters were separated by gasliquid chromatography on a Shimadzu GC-17A gas chromatograph (Shimadzu corp. Kyoto, Japan) with a fused silica capillary column. Individual fatty acids were identied by comparing each peaks retention time to those of external standards and then expressed as the mol % of total fatty acids. Unsaturation index (UI) is the number of double bonds per 100 acyl chains and is calculated as UI = (% monoenoics) + (2 % dienoics) + (3 % trienoics) + (4 % tetraenoics) + (5 % pentaenoics) + (6 % hexaenoics). Peroxidation index (PI) is an indicator of the susceptibility of phospholipids to peroxidative damage and is calculated as PI = (0.025 % monoenoics) + (1 % dienoics) + (2 % trienoics) + (4 % tetraenoics) + (6 % pentaenoics) + (8 % hexaenoics). Signicant eects were determined by ANOVA conducted using JMP v5.1 statistical package (SAS Institute Inc.). Where ANOVA revealed a signicant eect, Tukeys post hoc test was used to identify signicant dierences. 3. Results The specic fatty acid composition of phospholipids from the head, thorax and abdomen segments of honey bees are presented in Table 1 and trends are illustrated in Fig. 1. As can be seen from Table 1, phospholipids from all three body segments (head, thorax and abdomen) do not dier signicantly in the total percent of unsaturated fatty acids (UFA) between the dierent classes of honey bees analysed, but there are several signicant dierences between honey bee classes in the types of UFA present. The phospholipids (and thus membrane bilayers) of honey bees are highly unsaturated with an average 76% of all phospholipid acyl chains being UFA (averaging 73%, 85% and 71% in phospholipids from the head, thorax and abdomen, respectively). Within the UFA, the dominant type of fatty acyl chains present are the monounsaturates (MUFA), with both n-6 and n-3 polyunsaturates (PUFA) being present in smaller amounts. When averaged over all honey bee classes and body segment categories, MUFA constitute 75% of all UFA (the averages for head, thorax and abdomen being, respectively, 65%, 83% and 78%). These averages however hide major dierences between queen and worker honey bees. There is not a single signif-

icant dierence observed in membrane fatty acyl composition between young hive workers, old hive workers or forager worker bees. However, newly emerged worker bees dier considerably in membrane composition compared to the other worker bees (to be discussed later) and unless specied otherwise, mention of worker honey bees in the remainder of this article refers only to the three older age-classes of worker bees (and not to the newly emerged workers). Membrane bilayers in queen bees are considerably more monounsaturated and less polyunsaturated than those of workers. For example on average, 91% of all UFA in queens are MUFA compared to 62% in worker bees. Correspondingly, an average 9% of UFA are PUFA in queens compared to 38% in workers. The dierences in the acyl composition of phospholipids from all three body segments are relatively small (Table 1) with phospholipids from the head having relatively more PUFA than phospholipids from both the thorax and abdomen. The pattern of variation among the dierent types of honey bees is essentially the same in phospholipids from the head, thorax and abdomen (see Fig. 1) and suggest the mechanisms controlling these dierences aect all cells in the bees body. There is relatively little dierence in the acyl composition of phospholipids between young queens ($4 week post eclosion) and old queens ($2 years post eclosion). The differences are greatest in phospholipids from the head. There are negligible dierences in the acyl composition of abdominal phospholipids from young and old queen bees (see Table 1). In phospholipids from the head and thorax there is a decrease in MUFA content and increase in PUFA content with age in the queen bees, however only the head MUFA changes are statistically signicant. One consistent change with age of queens is in the balance between n-3 PUFA and n-6 PUFA. In the very young queens, n-3 PUFA constitutes 82% of all PUFA (respectively, 80%, 87% and 79% in head, thorax and abdomen), whereas in old queen bees n-3 PUFA are on average 62% of all PUFA (respectively, 55%, 64% and 68% in head, thorax and abdomen). An unexpected nding was that the membrane composition of newly emerged worker bees is intermediate between that observed in queen bees and the older worker bees (see Table 1 and Fig. 1). Indeed, the membrane composition of these emergent workers are generally more similar to queens than to the older workers, in that there are more signicant dierences between emergent and older workers than between emergent workers and queens (Table 1). For example, on average, PUFAs constitute 17% of all UFAs in phospholipids from recently emerged worker bees, compared to 9% in queen bees and 38% in older worker bees. Consequently, on average, MUFAs constitute 83% of UFA in emergent worker bees compared to 91% in queen and only 62% in the older worker honey bees. After we became aware of this dierence between newly emerged and older worker honey bees, on the nal collecting trip we also obtained two worker larvae (estimated age $5 days) for phospholipid fatty acid analysis. As well, on

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Table 1 Fatty acid composition (mol %) of phospholipids from the head, thorax and abdomen of honey bee queens and workers
Queen bees Young Head 16:0 16:1 n-7 17:1 n-7 18:0 18:1 n-9 18:2 n-6 18:3 n-6 18:3 n-3 20:0 20:1 n-9 20:4 n-6 % UFA % MUFA % PUFA % n-6 PUFA % n-3 PUFA n-3(% PUFA) % C20-22 UI PI Thorax 16:0 16:1 n-7 18:0 18:1 n-9 18:2 n-6 18:3 n-6 18:3 n-3 20:0 20:1 n-9 % UFA % MUFA % PUFA % n-6 PUFA % n-3 PUFA n-3 (% PUFA) % C20-22 UI PI Abdomen 16:0 16:1 n-7 18:0 18:1 n-9 18:2 n-6 18:3 n-6 18:3 n-3 20:0 20:1 n-9 % UFA % MUFA % PUFA % n-6 PUFA % n-3 PUFA n-3 (% PUFA) % C20-22 UI PI (n = 4) 9.3 3.3 6.0 0.6a 0.1 0.0a 9.3 2.0 66.9 6.1a 0.7 0.3a 0.8 0.5a 5.0 1.5a 0.2 0.1 0.5 0.2a 0.0 0.0a 80.5 5.4 73.7 6.3a 6.8 2.1a 1.5 0.6a 5.3 1.5a 80.2 2.5a 1.5 0.7ab 94.1 5.3ab 16.0 3.9a (n = 4) 5.2 0.6 7.5 1.1ab 6.4 1.6ab 77.5 1.6a 0.2 0.0a 0.0 0.0 1.1 0.2a 0.3 0.1 0.5 0.2 87.4 2.3 85.7 2.5a 1.7 0.2a 0.2 0.0a 1.5 0.2a 87.4 1.5a 1.4 0.3 91.4 21.7a 7.0 0.8a (n = 4) 6.8 2.1 5.4 0.7a 9.2 0.8 70.6 2.5a 0.3 0.1a 0.9 0.9 3.7 1.6 0.3 0.1 0.5 0.4 82.0 3.5 76.8 2.3a 5.2 2.3a 1.3 0.8a 3.9 1.6ab 78.9 3.3a 1.7 0.8 92.7 7.4 13.3 4.3ab Old (n = 4) 13.7 1.9 3.5 0.8ab 8.1 2.6b 20.9 3.4 32.3 7.7bc 1.6 0.2a 3.1 0.7a 6.3 1.0ab 1.5 0.2 5.3 2.6b 0.7 0.4b 62.1 5.0 49.2 4.0bc 12.9 1.1ab 5.8 0.4ab 7.1 0.7ab 55.2 0.8bc 9.1 2.7a 90.2 6.1b 32.0 1.4ab (n = 4) 5.9 0.8 9.2 1.1a 7.6 0.3ab 67.2 2.4ab 2.2 0.6a 0.2 0.1 5.4 2.1bc 0.8 0.5 0.4 0.1 84.8 1.5 76.8 2.8ab 8.0 2.6a 2.6 0.5a 5.5 2.2bc 63.7 6.8b 1.5 0.4 98.8 5.2ab 16.1 4.7a (n = 4) 10.2 0.4 7.0 0.2b 10.3 0.4 65.3 0.5ab 1.0 0.1a 0.0 0.0 2.9 0.9 0.9 0.3 0.8 0.1 77.1 1.1 73.1 0.5a 4.0 0.9a 1.0 0.1a 3.0 0.9b 67.5 12.4ab 1.8 0.3 84.3 2.8 9.2 1.8a Worker bees Emergent (n = 5) 10.3 2.1 1.7 0.6b 0.0 0.0a 15.1 2.2 52.1 2.6ab 3.2 0.2a 1.2 0.8a 14.2 1.9abc 0.0 0.0 0.4 0.4a 0.0 0.0a 72.9 4.5 54.2 2.3b 18.6 2.4ab 4.4 0.9a 14.2 1.9abc 75.8 3.8ab 0.4 0.4b 106.9 9.3ab 35.4 4.8ab (n = 6) 9.2 1.9 3.7 0.3c 13.2 2.0a 64.2 5.2ab 3.0 0.3a 0.7 0.5 3.0 0.5ab 1.1 0.3 0.0 0.0 75.6 4.5 68.4 5.0bc 7.3 0.7a 4.1 0.8a 3.1 0.4ab 44.8 5.8c 1.9 0.7 87.6 3.4a 14.1 1.5a (n = 6) 10.6 2.1 0.6 0.1c 16.5 3.0 54.9 6.5abc 3.9 1.5a 0.8 0.5 4.1 1.7 1.1 0.4 0.1 0.0 66.1 5.3 56.5 5.8ab 9.6 3.1a 5.4 1.4a 4.2 1.8b 40.0 5.4bc 2.7 0.7 81.8 7.6 17.4 5.4ab Young hive (n = 5) 8.1 2.6 0.5 0.3b 0.00.0a 14.6 1.4 33.0 3.6bc 10.5 1.1b 10.8 4.5ab 20.1 4.6c 0.1 0.1 0.0 0.0a 0.0 0.0a 75.1 4.8 33.7 3.6c 41.4 6.4c 21.2 4.1bc 20.2 4.5bc 47.7 6.2c 0.6 0.6b 147.7 16.5ab 73.7 11.9c (n = 6) 4.7 0.7 5.4 0.6bc 5.9 0.4b 61.8 1.8b 13.4 2.1b 0.0 0.0 7.8 0.6c 0.2 0.1 0.3 0.3 88.8 0.9 67.5 1.9bc 21.3 2.4b 13.4 2.1b 7.9 0.6c 39.3 4.6c 0.7 0.4 118.0 3.4bc 31.0 2.8b (n = 6) 14.4 1.3 0.6 0.2c 17.0 2.8 31.1 4.6d 11.8 1.9b 2.4 1.7 10.3 2.1 1.1 0.3 5.0 3.0 62.7 4.6 37.0 2.9c 25.7 2.1b 14.2 1.0b 11.4 2.0a 42.9 5.2bc 9.3 4.1 108.3 8.0 48.3 7.5c Older hive (n = 6) 11.7 3.2 0.5 0.3b 0.0 0.0a 14.9 4.2 32.5 5.4c 8.2 1.7b 10.4 4.1ab 13.4 3.0bc 2.9 2.8 0.0 0.0a 0.0 0.0a 67.7 10.3 34.7 4.5c 33.1 7.3bc 19.7 4.8bc 13.4 3.0abc 39.5 6.1c 4.4 3.2ab 127.0 22.2ab 60.0 12.6bc (n = 6) 5.3 0.8 3.9 1.1bc 7.3 0.9ab 56.6 2.0b 14.9 1.4b 0.3 0.2 8.1 0.6c 1.5 1.2 0.9 0.9 84.8 2.7 61.4 2.1c 23.4 1.6b 15.2 1.2b 8.2 0.5c 35.3 1.1c 2.8 1.5 117.4 3.7bc 34.3 1.4b (n = 6) 11.3 2.5 0.9 0.3c 15.9 1.9 42.5 4.1cd 16.7 2.0b 0.5 0.5 8.0 1.8 0.9 0.5 0.6 0.5 69.1 4.9 44.0 3.7bc 25.1 2.5b 17.1 1.7b 8.0 1.8ab 30.8 5.5c 2.2 1.2 103.5 7.8 35.2 3.4bc Foragers (n = 7) 6.9 1.3 1.4 1.4b 0.3 0.3a 12.5 1.7 35.7 2.3bc 7.7 0.8b 17.2 2.9b 16.5 0.9bc 0.3 0.3 0.50.5a 0.0 0.0a 79.5 2.9 38.1 1.7c 41.4 2.7c 24.9 2.9c 16.5 0.9bc 40.9 3.8c 0.7 0.5b 154.6 7.7a 76.0 5.2c (n = 7) 5.0 1.3 2.8 0.5c 7.8 2.0ab 60.3 2.1b 15.0 1.2b 0.2 0.2 8.1 0.5c 0.1 0.1 0.0 0.0 86.3 3.6 63.1 2.5bc 23.2 1.5b 15.1 1.1b 8.1 0.5c 35.0 0.9c 0.2 0.1 117.8 5.2c 33.1 1.8b (n = 7) 9.0 2.3 1.2 0.2c 17.8 2.1 47.7 5.1bcd 11.1 2.0b 1.5 1.1 7.0 1.2 0.9 0.5 0.0 0.0 69.7 5.3 48.9 5.1bc 20.7 2.0b 13.7 1.4b 7.0 1.2ab 33.3 4.1c 2.4 0.9 100.1 6.4 31.6 2.6bc NS 0.004 <0.0001 NS <0.0001 <0.0001 0.004 0.004 NS 0.003 0.006 NS <0.0001 <0.0001 <0.0001 0.006 <0.0001 0.02 0.01 0.0001 NS <0.0001 0.03 0.002 <0.0001 NS <0.0001 NS NS NS 0.0002 <0.0001 <0.0001 <0.0001 <0.0001 NS <0.0001 <0.0001 NS <0.0001 NS 0.0001 <0.0001 NS NS NS NS NS <0.0001 <0.0001 <0.0001 0.01 <0.0001 NS NS <0.0001 Signicance of dierence

Fatty acids identied by number of C atoms: number of double bonds and position of terminal double bond.

Values are shown as means SEM. Within each row, values that do not share a common superscript letter are signicantly dierent. Values do not necessarily add up to 100% because only values for the major fatty acids are shown. NS, no signicant dierence; UFA, unsaturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; UI, unsaturation index; PI, peroxidation index.

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Fig. 1. Classes of fatty acid composition of phospholipids from the head thorax and abdomen of honey bee queens and workers. For each circle, clockwise from top: the white sector represents total saturated fatty acids, the light grey sector represents total monounsaturated fatty acids, the dark hatched sector represents n-6 polyunsaturated fatty acids and the black sector represents n-3 polyunsaturated fatty acids. Actual values are presented in Table 1.

this nal collection trip, two forager worker bees still had pollen on their legs. This was removed, the pollen pooled and analysed for the fatty acid composition of total pollen lipids (as opposed to pollen phospholipids). The results of these analyses are presented in Table 2. The acyl composition of worker bee larval phospholipids (Table 2) is similar to that of adult queen bee phospholipids and unlike the composition of phospholipids from the older adult worker bees (see Table 1). In this respect the membrane composition of newly emerged worker bees is intermediate between that of worker larvae and the older (hive and forager) worker bees. The fatty acid composition of pollen lipids (Table 2) is very unlike that of bee phospholipids. Pollen lipids are much more polyunsaturated with a low MUFA content. The fatty acids found in the pollen lipids are one-third saturated fatty acid chains (SFA) and two-thirds UFA. Unlike the situation in bee phospholipids (where MUFA are the predominant unsaturates) in pollen lipids MUFA represent only 12% and PUFA are 88% of all UFA. The PUFA found in pollen lipids consist of approximately equal amounts of n-6 PUFA and n-3 PUFA. While both MUFA and PUFA contribute double bonds to membrane bilayers and thus to the calculation of the unsaturation index (UI) of bee membranes, essentially only PUFA are capable of being damaged by peroxidation and thus PUFA (and not MUFA) contribute to the calculation of the peroxidation index (PI) of bee membranes. The difference in phospholipid PUFA and MUFA content between dierent bee types means that there is considerably less variation in the UI values of dierent bee membranes than in the PI values. For example, the maximum UI is on average only 71% higher than the minimum UI of phospholipids from bee heads. Similarly, the maximum UI calculated for phospholipids from thorax and abdomen of

Table 2 Fatty acid composition (mol %) of phospholipids from honey bee larvae and pollen collected from the forager worker honey bees
Larvae (n = 2) 16:0 16:1 n-7 18:0 18:1 n-9/n-7 18:2 n-6 18:3 n-6 18:3 n-3 20:0 20:1 n-9 22:0 22:1 n-9 24:0 % UFA % MUFA % PUFA % n-6 PUFA % n-3 PUFA n-3 (%PUFA) %C20-22 UI PI 13.6 5.0 1.6 1.0 7.3 1.3 64.9 4.9 1.4 0.1 0.2 0.3 2.8 2.6 0.5 0.3 2.5 2.5 0.3 0.3 1.9 1.9 0.3 0.3 75.7 4.0 71.0 6.2 4.6 2.2 1.6 0.4 3.0 2.7 57.9 29.2 5.5 5.3 84.1 0.8 10.8 4.9 Pollen (n = 1) 23.5 0.3 3.9 7.4 26.6 0.9 31.4 2.8 0.0 2.7 0.0 0.0 66.6 7.7 58.9 27.5 31.4 53.3 5.5 157.7 91.3

Fatty acids identied by number of C atoms: number of double bonds and position of terminal double bond. Values for larvae are shown as means SEM. Values do not necessarily add up to 100% because only values for the major fatty acids are shown. UFA, unsaturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; UI, unsaturation index; PI, peroxidation index.

bees are, respectively, 35% and 32% greater than the minimum UI values. However, the maximum PI values calculated for phospholipids from head, thorax and abdomen are, respectively, 375%, 390% and 425% greater than the minimum PI values.

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The PI values calculated for phospholipids from the head, thorax and abdomens of the dierent bees are illustrated in Fig. 2. As can be seen from this gure, both young and old queen honey bees have phospholipids (and thus membranes) with a low PI value. Phospholipids from newly emerged worker bees have similar PI values to those calculated for queen bee phospholipids, but have signicantly lower PI values than those calculated for older worker bees. There is no signicant dierence in PI values calculated for phospholipids from (young and old) hive and forager worker bees. This means that the membranes of queen honey bees (and newly emerged workers) are more resistant to peroxidation than those from hive and forager worker honey bees. This dierence is due to the fact that the membranes of worker bees are more polyunsaturated and less monounsaturated than those of queen (and newly emerged worker) honey bees.

Although the PUFA content of membranes from larval and newly emerged worker bees is low, there are approximately equal amounts of n-6 and n-3 PUFA. Following eclosion, there is greater increase in the n-6 PUFA than in the n-3 PUFA such that in the membranes of older workers there is approximately two n-6 PUFA chains for every single n-3 PUFA. In young queen bees, n-3 PUFA represent about four-fths of all PUFA, however in old queen bees they represent only a half to two-thirds of all PUFA.

4. Discussion Although long-chain highly polyunsaturated fatty acids are signicant components of vertebrate brains, this appears not to be the case for insects. For example, the fatty acid composition of phospholipids from the heads of Drosophila shows them to have short-chain but lack long-chain PUFA (e.g. Stark et al., 1993). This is also true for honey bees where the short-chained omega-3 linolenic acid (18:3 n-3) is the most polyunsaturated fatty acid found in signicant quantities in the heads of honey bees. Since they are presumably unable synthesise it, the presence of this omega-3 PUFA in the heads of queen bees (at levels less than those measured in the heads of workers) suggest that it is obtained from the secretions that workers feed to queens as this is the only source of food intake by queen bees. The reason why the fatty acid composition of membranes might be important in the determination of longevity is that many of the products of lipid peroxidation are themselves powerful reactive oxygen species and that fatty acids dier dramatically in their susceptibility to lipid peroxidation. It is the single-bonded carbon atoms that are located between double-bonded carbon atoms in fatty acid chains (the bis-allylic atoms) that are most attacked by the metabolism-derived free radicals (Halliwell and Gutteridge, 1999) and consequently it is only PUFA that are signicant substrates for lipid peroxidation. The more polyunsaturated the PUFA the more prone it is to peroxidation and the more lipid-based ROS will be produced. Saturated fatty acids cannot be peroxidised and MUFA are also extremely resistant to peroxidation. Holman (1954) empirically determined the susceptibility of dierent fatty acids to peroxidation and demonstrated that the two 18-carbon polyunsaturates; linolenic acid (18:3 n-3) and linoleic acid (18:2 n-6) are, respectively, 80 and 40 times more prone to lipid peroxidation than the 18-carbon monounsaturated oleic acid (18:1 n-9). By combining these empirically determined peroxidative susceptibilities of individual fatty acid chains with the specic fatty acid composition of membranes, it is possible to calculate a peroxidation index (PI), which gives a quantitative measure of the susceptibility of the membrane to peroxidative damage (see Section 2). The smaller the PI value the more resistant the membrane is to peroxidative damage.

Fig. 2. The peroxidation index for phospholipids from the head, thorax and abdomen of honey bee queens and workers. See Section 2 for calculation of peroxidation index. Error bars represent SEM.

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This approach has been successful in examining the distinctive maximum lifespans of dierent species. For example, the trend for large mammal and bird species to, in general live longer than small mammal and bird species can be related to the body-size dierences in membrane composition in mammals and birds (Hulbert, 2005). Recently, it has been demonstrated that the longest-living rodent species, the naked mole-rat (Heterocephalus glaber) which has a 28 year maximum lifespan compared to the much shorter-living ($3 year) but similar-sized Mus musculus (Buenstein, 2005) has a membrane fatty acid composition with a PI commensurate with its long lifespan (Hulbert et al., 2006a). Similarly within Mus musculus, there are wild-derived strains that show extended longevities compared to lab Mus strains when kept under identical conditions (Miller et al., 2002) and these long-living strains also have a peroxidation-resistant membrane composition (Hulbert et al., 2006b). The current measurements were performed to see if the large longevity dierences between queen and worker honey bees are also associated with membrane composition dierences. The phospholipids (and consequently membranes) of queen honey bees are more monounsaturated and less polyunsaturated than those of worker bees and will thus be considerably more resistant to peroxidative damage. These dierences are manifest in all three body segments (i.e. head, thorax and abdomen). Furthermore queen honey bees maintain membranes that will be peroxidation-resistant until (at least) 2 years old. We found very small dierences in the membrane composition between young ($4 week) and old ($2 year) queen bees. Since in the current study we obtained these queens from dierent sources we cannot ascertain the importance of the small dierences we have observed. What is important however is that although the queens came from dierent sources, there was no signicant dierence in the PI of their membrane phospholipids. An interesting nding was that newly emerged worker bees were more similar in membrane composition to queen bees than older worker bees, and that worker bee larvae were similar to newly emerged workers. The increased membrane PUFA content of worker bees, and consequently their increased susceptibility to peroxidation, are thus due to events that occur after emergence and during the rst week of hive life. During this period there is a 2 3-fold increase in the peroxidative susceptibility of the membranes of worker bees due to a 23-fold increase in membrane PUFA content. For a number of reasons, it is most likely that this increased membrane PUFA content is due to the consumption of pollen. Although higher animals (including insects and thus honey bees) can synthesise both SFA and MUFA de novo from non-lipid sources, they are unable to synthesise PUFA de novo and thus these must be obtained from the diet. As can be seen in Table 2, pollen has high PUFA content (59% of all pollen fatty acids are PUFA) and the two PUFA present in pollen are the predominant PUFA that increase during this rst week of hive

life (Table 1). Furthermore, worker bees begin consuming pollen within the rst few hours after emergence and reach their maximum consumption level when about 5 days old (p54 of Winston, 1987). The products of lipid peroxidation are many and include uorescent pigments and the volatile hydrocarbons, pentane and ethane (produced, respectively, from peroxidation of n-6 PUFA and n-3 PUFA). Young and Tappel (1978) reported that queen honey bees had 37% less uorescent pigment in their thorax than did hive and forager worker bees and that newly emerged workers were intermediate with 13% less pigment than the older worker bees. The uorescent pigment content in the thorax of worker honey bees increases during the rst 4 weeks of adult life (Young and Robinson, 1983). Furthermore, honey bee thorax is reported to also have a low content of vitamin E (an important membrane antioxidant) and the average rate of pentane exhalation by worker bees is similar to that of vitamin E-decient rats (Young and Tappel, 1978). Female larvae less than 3 days old are bipotent and whether they develop into queens or workers depends on the quantity and quality of larval food fed to them by young nurse worker bees. The larval food produced by these nurse worker bees consists of the combined secretions of the mandibular glands and the hypopharyngeal glands which are located in the worker bees head. The food fed to future queens is called royal jelly and is approximately 1:1 mix of hypopharyngeal:mandibular glandular secretions while that fed to future workers is called worker jelly and is an approximately 2:1 mix of hypopharyngeal:mandibular secretions. During the rst week following emergence worker bees gradually convert to consumption of honey and pollen, while a queen continues to be fed royal jelly by worker bees (directly into her mouth) throughout her entire adult life (Sammataro and Avitabile, 1998). Our interpretation of the results obtained in this study suggest that it is what is not in royal jelly that is responsible for the extended longevity of the queen. Royal jelly consists of water (6070%) together with sugars (2040% of dry mass), proteins (1650% of dry mass) and vitamins and minerals with the lipid component being relatively small (315% of dry mass). Most of the lipids are present as short-chain free fatty acids (i.e. not triglycerides, diglycerides or monoglycerides) that are either hydroxylated or dicarboxylic fatty acids (Lercker et al., 1981). These unusual fatty acids appear to be important antimicrobial and antifungal defences (e.g. Melliou and Chinou, 2005) as well as a defence against parasitic mites (Drijfhout et al., 2005). Lercker et al. (1981) found that the majority of these short-chain fatty acids were 810 carbons long and proposed that they were formed from the degradation of the 18-carbon fatty acids that are the dominant fatty acids of pollen (e.g. see Table 2). Interestingly, they reported the almost complete absence of the fatty acids (free or combined) characteristic of pollen in the lipids of royal jelly (Lercker et al., 1981, p918). Thus, we propose that the feeding of royal jelly to the queen throughout her adult life is a

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way of keeping her away from the polyunsaturates in pollen and consequently allow her to maintain peroxidation-resistant membranes throughout her life. Workers, on the other hand, feed directly on pollen stores after they emerge and the composition of their cellular membranes changes, becoming more susceptible to peroxidative damage and consequently results in a shorter adult lifespan compared to the queen. Another way of describing the dierence is to point out that it is what worker bees eat that is responsible for their shortened lifespan. Male honey bees (drones) are also shorter-living than queens and also consume pollen (Sammataro and Avitabile, 1998). We have not measured their membrane fatty acid composition in the current study and do not know of any study with this information. We report here the observation of a correlation between membrane composition and relative longevities of queen and worker honey bees. This correlation is similar to that previously described for endothermic vertebrates (mammals and birds), namely that lower levels of peroxidisable fatty acids in membranes is associated with a longer maximum lifespan. While, observations of correlations, of course, do not prove cause and eect, the presence of a correlation is a necessary condition for a causeeect relationship and the absence of correlation is evidence against a causeeect relationship. Only experimental manipulation of membrane composition, coupled with the observation of correlation between such treatments and an eect on longevity can prove a causeeect relationship. An advantage of the current nding is that experimental testing of a link between membrane composition and longevity is feasible in this system. An obvious question is whether the observed dierence in membrane composition is quantitatively large enough to account the order-of-magnitude longevity dierence between these female bee phenotypes? When the averaged PI of young and old queen honeys is compared to the average PI of young hive, old hive and forager worker bees, it is can be seen that queen honey bees have a PI that is about one-third of the value for worker bees (respectively, 34%, 35% and 29% for head, thorax and abdomen, see Fig. 2). Among mammal and bird species, it has been previously shown that the PI of skeletal muscle phospholipids and liver mitochondrial phospholipids are, respectively, proportional to maximum lifespan to the 0.30 and 0.40 power (Hulbert, 2005). These mathematical relationships allow calculation of the changes in PI associated with changes in species longevity. They show that, in mammals and birds, a 19% decrease in peroxidation susceptibility (PI) of muscle membranes and a 24% decrease in PI of liver mitochondrial phospholipids is associated with every doubling of maximum lifespan. Assuming that the same proportionality between PI and maximum lifespan observed in mammals and birds, is also found in honey bees, it can be calculated that the approximately 3-fold dierence in PI between workers and queens (i.e. $67% decrease) will be associated with a 1640-fold dierence in maximum lifespan. In other words, the membrane composition dier-

ences we have measured in the current study are large enough to potentially explain the maximum longevity difference between queen and worker honey bees (assuming the same proportionality between PI and maximum lifespan as previously observed in mammals and birds). Acknowledgements We thank Sarah Abbott and Emily Slee for their technical assistance. This work was supported by a grant from the Australian Research Council (to A.J.H.) and L.H. was supported by a scholarship from CAPES, Brazil. References
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