EXG5174S0531-5565(98)00078-310.1016/S0531-5565(98)00078-3Elsevier Science Inc.Table 1Means (± standard errors) for longevity and female fecundity indices of the short-lived and long-lived bean weevils lines legendTraitShort-lived lineLong-lived linet-testnmean ± S.E.nmean ± S.E.LongevityFemales9511.58 ± 0.279719.19 ± 0.5412.59aMales9211.61 ± 0.259315.42 ± 0.437.67aFecundityEarly (1–2 days)10123.83 ± 1.1510015.60 ± 1.214.93aLate (≥9 days)1000.36 ± 0.201003.59 ± 0.754.14aTotal10049.57 ± 1.8210048.02 ± 2.341.19legendn = The number of analyzed individuals.ap < 0.001.Table 2Means (± standard errors) for activities (in units/mg proteins) of SOD and catalase in the young (one-day-old) and old (10-day-old) virgin and mated females and males of the short-lived and long-lived lineslegendEnzymeShort-lived lineLong-lived lineYoungOldYoungOldSODFemalesVirgin31.69 ± 3.4822.57 ± 1.3929.59 ± 1.5427.40 ± 2.67Mated26.86 ± 1.2427.59 ± 0.55MalesVirgin31.87 ± 1.6419.14 ± 1.1832.49 ± 2.4223.79 ± 1.65Mated19.52 ± 1.5227.99 ± 3.28Grand Mean31.78 ± 1.9122.02 ± 0.9631.04 ± 1.5126.69 ± 1.09CatalaseFemalesVirgin29.62 ± 0.4229.89 ± 2.7227.43 ± 0.8234.77 ± 3.38Mated35.33 ± 5.9343.91 ± 6.18MalesVirgin8.84 ± 0.298.37 ± 0.998.03 ± 0.507.36 ± 0.15Mated10.99 ± 0.4612.79 ± 2.33Grand Mean19.23 ± 3.6621.15 ± 3.1617.73 ± 3.4434.71 ± 3.96legendSample sizes for each mean are 5. Grand means are calculated on the pooled data of the corresponding groups.Table 3Two-way ANOVA for SOD and catalase activities in the young (one-day-old) females and males originated from the short-lived and long-lived linesSource of variationdfSOD M.S. (×10−3)FCAT M.S. (×10−3)FLine10.320.077.585.24aSex12.750.581409.48974.27bLine × sex11.140.230.130.09Error164.771.47ap < 0.05;bp < 0.001.Table 4Two-way ANOVA for SOD and catalase activities for virgin weevils of different ages (one day old and 10-days old) from short- and long-lived linesSource of variationdfSOD M.S. (×10−3)FCAT M.S. (×10−3)FFemalesLine13.960.631.150.27Age140.866.51a10.862.58Line × age113.332.1212.012.85Error166.284.21MalesLine112.583.279.922.35Age1159.4641.48b5.991.42Line × age19.762.540.000.00Error163.844.22ap < 0.05;bp < 0.001.Table 5Three-way ANOVA for SOD and catalase activities in the old (10-day-old) virgin and mated females and males from the short- and long-lived linesSource of variationdfSOD M.S. (×10−3)FCAT M.S. (×10−3)FLine (A)172.4615.59b13.590.79Sex (B)146.289.96b3070.31179.69bMating status (C)116.193.49126.507.40aA × B114.913.2115.950.93A × C10.030.007.480.43B × C10.140.0333.691.97A × B × C19.842.122.160.13Error324.6517.09ap < 0.05;bp < 0.001.Original PapersActivity of superoxide dismutase and catalase in the bean weevil (acanthoscelides obtectus) selected for postponed senescenceSeslijaDarkaŠešlijaaBlagojevicDuškoBlagojevićaSpasicMihajloSpasićaTucicNikolaTucića*tucic@opennet.orgaDepartment of Evolutionary Biology, Institute for Biological Research, 29 Novembra 142, 11000 Belgrade, Serbia, Yugoslavia*Corresponding authorAbstractRelationship of superoxide dismutase and catalase activities and aging were tested using bean weevil lines selected for postponed senescence. The beetles of different age (young and old) and mating status (virgin and mated) from the extended longevity lines were compared with their counterparts derived from the short-lived lines for activities of SOD and catalase. The old beetles from the long-lived lines had statistically significant higher activity of SOD than their controls. Although we did not find a significant effect of catalase on longevity, beetles originating from both types of lines exhibited an increased catalase activity during mating processes. In addition, we did observe an increased activity of catalase in one-day-old beetles of the short-lived lines relative to the same-aged individuals of the long-lived lines.KeywordsAcanthoscelides obtectusSuperoxide dismutaseCatalaseLongevitySelectionThe identification of underlying mechanisms of aging is regarded to be an elusive task, largely due to the complexity of aging processes. In addition, most of experimental methods approaching this problem have proven to be inadequate for delineation of the physiological mechanisms that determine rates and patterns of aging. For example, the usual methods of pursuing this goal, such as the studies of the temporal correlates of aging in defined cohorts, have not contributed much to the understanding of genetic and physiological mechanisms controlling aging (see review in Finch, 1990; Rose, 1991). The most important drawback to chronological measures of aging is that many age-related physiological changes are not the same as time-dependent changes (see, e.g., Baker, 1976). Also, experimental studies attempting to test causal mechanism of the aging processes by using a single gene mutant or different environmental factors that “accelerate the rate of aging” seem to be inappropriate because under these circumstances organisms could be dying because of novel pathologies rather than an acceleration of normal aging processes (Maynard Smith, 1966; Hutchinson & Rose, 1987). For these reasons, it has been argued (Johnson, 1987; Rose, 1991; Arking, 1995) that organisms undergoing genetically postponed aging represent the best experimental system that would allow study of the processes important in the normal aging situation.Such a model system for the analysis of underlying genetic and physiological mechanisms of aging constitutes lines with genetically postponed aging obtained from selection for survival to, and reproduction at, later ages. The important advantage to the use of these lines is that they have been obtained by the action of selection on allelic variation affecting aging at great many loci within outbred populations of Drosophila melanogaster (Luckinbill et al., 1984; Rose, 1984, Partridge & Flower, 1992) and Acanthoscelides obtectus (Tucić et al., 1996, 1997). In experiments with these holometabolous insect species in which selection on quantitative genetic variation proceeded by using either young or old adults as parents in each generation, it was found that the “old” lines had higher longevity (and delayed fertility) than the “young” ones. Besides, these experiments have successfully corroborated the basic assumption of the general evolutionary theory of aging (Medawar, 1952; Rose, 1991). As pointed out above, the obtained lines are an excellent experimental system for the study of the nonpathological processes of aging.Because short-lived and long-lived lines are derived by imposing selection on natural variants of many genes affecting aging, it is very likely a priori, that different species (e.g., Drosophila vs. Acanthoscelides) or lines of the same species originating from different laboratories have attained specific longevities by various underlying mechanisms. This expectation seems to be fulfilled with lines of D. melanogaster, with extented longevity stemmed out from different laboratories (see Arking, 1995). Accordingly, when dealing with a topic as complex as aging, it is essential to determine empirically mechanism(s) operative in the short-lived and long-lived lines of A. obtectus obtained in our laboratory.As a part of our effort to elucidate actual mechanisms affecting the aging processes in the bean weevils, in the present study we investigated the activities of two enzymes of great interest to gerontologists—superoxid dismutase (SOD), and catalase. These free radical scavenging enzymes are of interest because of SOD’s role in catalyzing the conversion of superoxide radicals to hydrogen peroxide (McCord & Fridovich, 1969), the latter then undergoing conversion to water due to the action of catalase (Deisseroth & Dounce, 1970). Because free radicals act damaging macromolecules within a cell, it has been proposed that they are important factors in aging (Harman, 1956), and that SOD and catalase could play the antiaging role. The most convincing experimental evidence that supports the free radical theory of aging was provided by Orr and Sohal (1994). In their experiments, D. melanogaster transgenic flies carrying three copies of SOD and catalase genes exhibited about a one-third extension of longevity, a lower rate of senescence, and a lower amount of protein oxidative damage compared to diploid controls. However, analyses of these enzymes in D. melanogaster lines with markedly different adult longevities led to contradictory results. For example, Bartosz et al. (1979) and Fleming et al. (1992) detected a positive correlation between longevity and antioxidant capacity, whereas Massie et al. (1975), Niedzwiecki et al. (1992), and Durusov et al. (1995) did not observe such a correlation. Moreover, a comparison of antioxidant defense in several mammalian species did not suggest a clear association between this defense and longevity (Sohal & Orr, 1992).The long-lived D. melanogaster lines selected by Luckinbill et al. (1984) and those by Rose (1984) differ substantially in their physiology, including the antioxidant defense system. The long-lived flies obtained in the former laboratory show a significant increase in the level and activity of both SOD and catalase (and some other enzymatic and nonenzymatic components of the antioxidant defense system) relative to the control flies, but their resistance to starvation and desiccation is low (Arking et al., 1993; Arking, 1995; Dudas & Arking, 1995). However, although more active allele of the SOD gene is associated with postponed aging lines of Rose (1984), it seems that this allele does not directly increase life span or later fecundity (Tyler et al., 1993). Here, however, the long-lived flies display enhanced resistance to starvation, desiccation, ethanol vapor, and heat (Service et al., 1985; Service, 1987; Graves et al., 1992). Thus, in D. melanogaster there is more than one route to extended longevity. The present work is an attempt to obtain further information on the activity of SOD and catalase in aging of Acanthoscelides obtectus, another insect species for which long-lived and short-lived lines produced by the manipulation with reproductive schedules exist.The fact that sexual activity reduces longevity complicates the interpretation of the long-term studies that use artificial manipulation of reproductive schedules. Although the hypothesis that selection for increased longevity acts on genes that regulate short-term mortality risk through the regulation of sexual behavior (Partridge, 1987) has been refuted (Service, 1989; Partridge & Flower, 1992; Tucić et al., 1996), it is possible that elevated sexual activity in the later ages of the long-lived lines is in a positive correlation with the efficient system of antioxidant defense. This could be expected because, in accordance with “rate of living theory” (Pearl, 1928), a higher sexual activity of females and males means that they are more metabolically active, which implies that these individuals produce a higher level of free radicals. To test this hypothesis, activities of the SOD and catalase were analyzed in the virgin and mated females and males originating from the short- and long-lived lines of the bean weevil.1Materials and methods1.1The short- and long-lived bean weevil linesThe lines used in the present study were derived from a population of bean weevil (Acanthoscelides obtectus) maintained in the laboratory from 1986 (Tucić et al., 1996). This population (the “base”) was synthesised by mass mating equal numbers of adults from three local populations of A. obtectus captured in the vicinity of Belgrade (Yug.). The base population was maintained at large size (about 5,000 individuals each generation) on Phaseolus vulgaris, c.v. “gradistanac” seeds for about a 40-day interval. All cultures were maintained in a dark incubator at 30°C and about 70% humidity.The lines used in this study were obtained from later generations of lines on which selection had been imposed for either early (“young” lines, Y) or late-life (“old” lines, O) reproductive success (Tucić et al., 1996, 1997). The four replicate lines per each selection regime were established. The following summarizes the selection procedures used to maintain the Y-selected and O-selected lines (details are given in Tucić et al., 1996).Beetles in the Y-selected lines were allowed to lay eggs at the ages of one to two days after emergence. In each generation about 300–500 individuals were used as the next generation breeders. This treatment should have given rise to beetles with enhanced fitness during early adult life. In the present study we have used “young” lines selected for the 69 generations.In the O-selected lines (55 generations), the beetles were reproduced from 10th day after emergence until death. Prior to that period experimental adults were kept in vials (females and males together) without bean seeds (in the absence of seeds the egg production is low).All the assays described below were done using four-way crosses within each selection regime. These crosses were obtained by crossing the F1s of different pairs of replicate lines within each selection regime, that is, (Y1 × Y2) × (Y3 × Y4), and (O1 × O2) × (O3 × O4), where the subscript numbers refer to the specific replicate lines (thereafter denoted simply as Y and O lines). The outcrossing of replicate lines should have removed any effect of inbreeding depression, and it diminished any epistatic interactions among genes originating during the long-term selection due to Wahlund’s effect or mutation pressures. Also, protocols used to construct four-way crosses (see Tucić et al., 1997 for details) ensured that selected lines passed through two generations of common conditions.1.2Enzyme assaysEnzyme assays were carried out for females and males of different age (1 and 10 days old) and mating status (virgin and mated) from each experimental group. Twenty individuals were homogenized in 0.25 M sucrose, 50 mM Tris, and 0.1 mM EDTA solution, pH 7.4, using a Janke-Kunkel Ka-Werk Ultra-Turrax homogenizer at 0–4°C Homogenates were sonicated according to Takada et al. (1982). After centrifugation (90 min, 10,500g, 4°C), protein content was determined in the supernates (Lowry et al., 1951) used also in enzyme activity assays. Measurements of the five different samples of each enzyme were made for each sex/age/mating status group.1.3Superoxide dismutaseSuperoxide dismutase (SOD, EC 1.15.1.1) activity was measured as described by Misra and Fridovich (1972). The rate of inhibition of epinephrine autoxidation at alkaline pH in the presence of SOD was spectrophotometrically estimated. One unit of SOD activity was defined as the amount of enzyme inhibiting oxidation of epinephrine by 50% under appropriate reaction conditions. The SOD specific activity was expressed as units per mg protein.1.4CatalaseCatalase (EC 1.11.1.6) activity was determined after Beutler (1982). Decomposition of H2O2 was monitored spectrophotometrically, and a unit of catalase activity was defined as 1 μM H2O2 decomposed per minute, assuming a molar extinction coefficient of 62.4 at 230 nm.2ResultsData shown in Table 1 are the mean longevities and three important fecundity indices of females that characterized lines selected for early and late reproduction for 69 and 55 generations, respectively. As may be seen, there are significant direct responses to both selection regimes; females from the Y lines exhibited significantly higher early fecundity than those from the O lines, whereas for the late fecundity, the opposite trend was observed. In addition, we have corroborated our earlier findings (Tucić et al., 1996, 1997) that selection for early and late reproductive effort does not change the total number of eggs laid by females during the whole lifespan. It is important to note that here, as in the earlier generations of selection, the effect of imposed age-specific selection on longevity, cohorts sampled from the O lines have significantly greater mean longevities relative to Y lines.Records of SOD and catalase activities in 1- and 10-day-old virgin and mated females and males originating from the short-lived and long-lived lines are listed in Table 2. To test differences in the activities of the analyzed enzymes among these groups we performed several ANOVAs (data are log transformed).A two-way ANOVA, with lines and sex of one-day-old beetles as the factors (Table 3), revealed significant differences in the catalase activities (but at the 0.05 level), both between the lines (at the 0.05 level; with the activity of the catalase significantly higher in the short-lived than in the long-lived lines) and sex (in both lines catalase activities in females exceeded more than three times those in males; Table 2).The differences observed above in catalase activities between the lines disappeared when virgins of both 1- and 10-day-old beetles were compared (Table 4). Because the data in Table 1 indicate the opposite pattern of age-dependent catalase activity between the sexes, a two-way ANOVA (with line as one factor and age of beetles as the other factor) were done separately for each sex (Table 4). Increased catalase activity in old females relative to the one-day-old females and its opposite trend in males was, however, statistically insignificant. Although the “line effect” was also absent with regard to SOD activity, virgin 10-day-old females and males had significantly lower activity of this enzyme than their one-day-old counterparts (Table 4).The results of three-way ANOVAs on the SOD and catalase activities over line, sex, and mating status of 10-day-old beetles as factors are listed in Table 5. Here we observed interesting differences between the SOD and catalase activities with respect to line and mating status effects. The old beetles sampled from the long-lived lines exhibited significantly higher SOD activity (at the 0.01 level) than the same aged beetles from the short-lived lines. At the same time, significant “line effect” was absent for catalase activity. Although both the SOD and catalase activities were somewhat higher in females and males kept together their whole life (Table 1), a significant difference between virgin and mated beetles has been observed only for catalase (but at the 0.05 level; Table 5). Also here, in contrast to the data for one-day-old beetles, both enzyme activities were significantly lower in males than in females.3DiscussionThe results presented in this study suggest that SOD might play a role in the postponing, and thus controlling, aging in Acanthoscelides obtectus. Although there are no available data on allelic differences at the SOD structural gene between the lines, pattern of differentiation in the SOD activities between short- and long-lived lines (i.e., statistically detectable difference between 10-day-old beetles and the lack of differentiation between young individuals sampled from different lines; Tables 3 and 5) could be attributed to differences in the regulation of structural genes common to both lines. In other words, in both lines we observed a significant age-specific decline in SOD activity (the “age effect” in Table 4), but this decrease was less pronounced in the long-lived lines. In this respect, our lines seem quite different from those obtained by Tyler et al. (1993), who found that more active SOD alleles were associated with postponed aging of Drosophila melanogaster laboratory lines. However, our interpretation is in accordance with the findings of Dudas and Arking (1995) and Burde and Arking (1998). Their data, obtained by gel electrophoresis, did not provide any evidence on the allelic differences at the SOD gene between the short- and long lived Drosophila lines, which, however, exhibited differences at the level of SOD activity.Orr and Sohal (1994) showed that transgenic Drosophila flies carrying extra copies of SOD and catalase exhibited lifespan extension as a consequence of a slower rate of aging (evidenced by the Gompertz parametric analysis), whereas selected long-lived Drosophila lines seemed to live long as a consequence of a decreased initial mortality rate with no change in the aging rate (Arking, 1995; Dudas & Arking, 1995). Our data, based on the Gompertz parametric analyses (Tucić et al., 1996), suggest that the “young” and “old” selection regimes in the bean weevil produce a change both in the timing of mortality (or in the initial mortality rate) and in rate of change of age-specific mortality. Which of these two important aging events in the bean weevil is possibly connected with the observed level of SOD activity remains to be shown.If, as our data suggest, the long-lived lines exhibit elevated level of the SOD activities, then a concordant increase could be expected in the activity of the catalase, which, acting together with SOD provides the primary enzymatic antioxidant defense. Elevated levels of SOD in the absence of a compensatory upregulation of catalase are thought to lead to H2O2 accumulation, which may give rise to cytotoxic effects (Fleming et al., 1992, and references therein). However, we did not detect any deterioration in the life history traits of the long-lived beetles (on the contrary; egg-to-adult viability, e.g., of the long-lived weevils, was higher than that of the short-lived individuals; Tucić et al., 1996), despite the fact that we failed to demonstrate the increase of catalase activity in these lines. However, this failure arose from the lack of a statistically detectable (in our sample sizes) increase of catalase activity in the long-lived lines but not from the entire absence of any increment in the activity of this free radical scavening enzyme. Careful inspection of the data in Table 2 indicates that grand mean (over both mating status and sexes) of 10-day-old beetles for the catalase is about 14% higher in the long-lived than in the short-lived lines. At the same time, the increase in SOD activity of the same age group in the long-lived relative to the short-lived lines was, on average, some 18%. There are two conclusions that could be drawn from these data. First, the observed increase in the SOD activity appears to provide additional protection against oxidative stress and results in an increased lifespan. Fleming et al. (1992) estimated the increase of SOD expression to be up to 35% (without any change of catalase activity), which could result in an increased longevity, and that levels of SOD above those appear to be toxic for Drosophila. Unfortunately, in the absence of transgenic individuals such an estimation is not possible for the bean weevil. Second, and more important from the standpoint of the evolutionary biologists, it seems that imposed selection regimes generate genotypes in which functionally coupled activities of the free radical scavening enzymes are elevated in a balanced way.Our data also raise the possibility that changes in catalase activity may be an important mechanism operating in the mating activities of the bean weevil. Although both the SOD and catalase activities increased in the mated females and males relative to the virgin beetles (Table 2), only in the latter was a statistically detectable increment of activity recorded (Table 5). It seems, therefore, that different aspects of reproductive activity in the bean weevils, such as courtships and other mating activities of females and males, oogenesis, and spermatogenesis, are protected by antioxidant enzymes. This is not unexpected, because neuropeptides involved in the expression of ecdysone during ovarian development (Lagaueux et al., 1977; Goltzene et al., 1978; Zhu et al., 1983) and spermatogenesis (Koolman et al., 1979) are important regulators of the mating activities, fertilization, and production of gametes in insects. Physiological effects of the ecdysone are realized through 20-hydroxyecdysone, which is regulated by the activity of the cytochrome P-450–linked enzymes which in turn, generate free radicals (Hoffman & Hetru, 1983).However, how could one explain increased level of catalase activity in the one-day-old beetles in the short-lived lines relative to the same aged individuals of the long-lived lines? The reason for such a result could be found in our experimental procedures involved in production of the short- and long-lived lines. Namely, our “young” lines (which bring about to the short-lived beetles) are leaving their progenies only during the first two days after emergence. They are, therefore, under strong selection for shortening the period between emergence and peak of reproductive activity in this insect species (about three to four days after emergence; Tucić et al., 1996). As a by-product of such a selection regime, we have genotypes characterized by elevated catalase activity coincident with the period of the “permitted” reproduction. Presently, we do not know what these genotypes are. They could be more active catalase alleles expressed in the early adult life, or the alleles of the other genes involved in the regulation of catalase activity.ReferencesArking 1995R.ArkingAntioxidant genes and other mechanisms involved in the extended longevity of DrosophilaR.G.CutlerL.PackerJ.BertramA.MoriOxidative Stress and Aging1995Birkhauser VerlagBasel123138Arking et al 1993R.ArkingS.P.DudasG.T.BakerGenetic and environmental factors regulating the expression of an extended longevity phenotype in a long lived strain of DrosophilaGenetica911993127142Baker 1976G.T.BakerInsect flight muscleMaturation and senescenceGerontology221976334361Bartosz et al 1979G.BartoszW.LeykoR.FriedSuperoxidase dismutase and life-span of Drosophila melanogasterExperientia35197911931194Beutler 1982E.BeutlerCatalaseE.BeutlerRed Cell Metabolism, A Manual of Biochemical Methods1982Grune and Straton, IncNew York105106Burde and Arking 1998H.R.BurdeR.ArkingImmunological confirmation of elevated levels of CuZn superoxide dismutase protein in an artificially selected long-lived strain of Drosophila melanogasterExp Geront331998227237Deisseroth and Dounce 1970A.DeisserothA.L.DounceCatalasePhysical and chemical properties, mechanism and catalysis and physiological rolePhysiol Rev501970319375Dudas and Arking 1995S.P.DudasR.ArkingA coordinate up-regulation of the antioxidant gene activities is associated with the delayed onset of senescence in a long-lived strain of DrosophilaJ Gerontol Biol Sci501995B117B127Durusov et al 1995M.DurusovN.DirihN.BozcukAge-related activity of catalase in different genotypes of Drosophila melanogasterExp Gerontol3019957786Finch 1990C.E.FinchLongevity, Senescence, and the Genome1990University of Chicago PressChicagoFleming et al 1992J.E.FlemingI.ReveillaudA.NiedzwieckiRole of oxidative stress in Drosophila agingMutat Res2751992267279Goltzene et al 1978F.GoltzeneM.LaugueuxM.CharletJ.A.HoffmannThe follicle cell epithelium of maturing ovaries of Locusta migratoriaA new biosynthetic tissue for ecdysoneHoppe-Seyler’s Z Physiol Chem359197814271434Graves et al 1992J.L.GravesE.C.ToolsonC.JeongL.N.VuM.R.RoseDesiccation, flight, glycogen, and postponed senescence in Drosophila melanogasterPhysiol Zool651992268286Harman 1956D.HarmanAging—A theory based on free radical and radiation chemistryJ Gerontol111956298300Hoffmann and Hetru 1983J.A.HoffmannC.HetruEcdysoneR.G.H.DownerH.LauferEndocrinology of Insects1983Alan R. Liss, IncNew York6588Hutchinson and Rose 1987E.W.HutchinsonM.R.RoseGenetics of aging in insectsRev Biol Res Aging319876270Johnson 1987T.E.JohnsonAging can be genetically dissected into component processes using long-lived lines of Caenorhabditis elegansProc Natl Acad Sci USA84198737773781Koolman et al 1979J.KoolmanK.SchellerB.BodensteinEcdysteroids in the adult male blowfly Calliphora vicinaExperientia351979134135Lagueux et al 1997M.LagueuxM.HirnJ.A.HoffmanEcdysone during ovarian development in Locusta mugratiraInsect Biochem231997109120Lowry et al 1951D.H.LowryN.J.RosebroughA.L.FarrR.J.RandalProtein measurements with folin-phenol reagentJ Biol Chem1931951265275Luckinbill et al 1984L.S.LuckinbillR.ArkingM.J.ClareW.C.CiroccoS.A.BuckSelection for delayed senescence in Drosophila melanogasterEvolution3819849961003Massie et al 1975H.R.MassieM.B.BairdM.M.McMahonLoss of mitochondrial DNA with agingGerontology211975231237Maynard Smith 1966J.Maynard SmithTheories of agingP.L.KrohnTopics in the Biology of Aging1966InterscienceNew York135McCord and Fridovich 1969J.McCordI.FridovichSuperoxidase dismutase. An enzymic function for erythrocupein (hemocupein)J Biol Chem244196960496055Medawar 1952P.B.MedawarAn Unsolved Problem of Biology1952H. K. LewisLondonMisra and Fridovich 1972H.P.MisraI.FridovichThe role of superoxide anion in the autooxidation of epinephrine and a simple assay for superoxide dismutaseJ Biol Chem247197231703175Niedzwiecki et al 1992A.NiedzwieckiL.ReveillaudJ.F.FlemingChanges in superoxyde dismutase and catalase in aging heat shocked DrosophilaFree Radic Res Commun171992355367Orr and Sohal 1994W.C.OrrR.S.SohalExtension of life-span by overexpression of superoxide dismutase and catalase in Drosophila melanogasterScience263199411281130Partridge 1987L.PartridgeIs accelerated senescence a cost of reproduction?Funct Ecol11987317320Partridge and Flower 1992L.K.PartridgeK.FlowerDirect and correlated response to selection on age at reproduction Drosophila melanogasterEvolution4619927691Pearl 1928R.PearlThe Rate of Living1928KnopfNew YorkRose 1991M.R.RoseEvolutionary Biology of Aging1991Oxford University PressNew YorkRose 1984M.R.RoseLaboratory evolution of postponed senescence in Drosophila melanogasterEvolution38198410041010Service 1987P.M.ServicePhysiological mechanisms of increased stress resistance in Drosophila melanogaster selected for postponed senescencePhysiol Zool601987321326Service 1989P.M.ServiceThe effect of mating status on lifespan, egg laying and starvation resistance in Drosophila melanogaster in relation to selection on longevityJ Insect Physiol351989447452Service et al 1985P.M.ServiceE.W.HutchinsonM.D.MackinleyM.R.RoseResistance to environmental stress in Drosophila melanogaster selected for postponed senescencePhysiol Zool581985380389Sohal and Orr 1992R.S.SohalW.C.OrrRelationship between antioxidants, prooxidants, and the aging processAnn NY Acad Sci66319927484Takada et al 1982Y.TakadaT.NogushiM.KayiyamaSuperoxide dismutase in various tissues from rabbits bearing the Vx-2 carcinoma in the maxillary sinusCancer Res42198242334235Tucic et al 1996N.TucićI.GliksmanD.ŠešlijaD.MilanovićS.MikuljanacO.StojkovićLaboratory evolution of longevity in the bean weevil (Acanthoscelides obtectus)J Evol Biol91996485503Tucic et al 1997N.TucićO.StojkovićI.GliksmanD.MilanovićD.ŠešlijaLaboratory evolution of life history traits in the bean weevil (Acanthoscelides obtectus)The effects of density-dependent and age-specific selectionEvolution51199718961909Tyler et al 1993R.H.TylerH.BrarM.SinghA.LatorreJ.L.GravesL.D.MuellerM.R.RoseF.J.AyalaThe effects of superoxidase dismutase alleles on aging in DrosophilaGenetica911993143150Zhu et al 1983X.X.ZhuH.GfellerB.LanzreinEcdysteroids during oogenesis in the ovoviviparous cockroach Nauphoeta cinereaJ Insect Physiol291983225236