juvenile and steroid hormones in drosophila … is a major insect hormone but may ... 20e is an...

I. Introduction

Hormones coordinate diverse develop-mental and physiological processes andregulate the allocation of metabolicresources to different organs and life-history stages (Finch & Rose, 1995). Manyinsects display an amazing amount ofphenotypic variation in their life histo-ries, which is mediated by the effects ofhormones (Dingle & Winchell, 1997;Nijhout, 1994). Juvenile hormone (JH)1and the steroid hormone ecdysone (activeform: 20-hydroxy-ecdysone, 20E) have fas-cinating physiological effects on various

aspects of development and the adult phe-notype. Consequently, the endocrineeffects of a single hormone on multipletraits (“hormonal pleiotropy”) may offerpromising insights into the mechanismsregulating complex phenotypes (Dingle &Winchell, 1997; Ketterson & Nolan, 1992;Zera & Harshman, 2001), including theaging phenotype (Bartke & Lane, 2001;Finch & Rose, 1995; Kenyon, 2001; Tatar,2004; Tatar et al., 2003).

The study of endocrine aspects ofaging is not new. In 1889, Charles-Edouard Brown-Séquard, the “father” ofendocrinology, was one of the first tosuggest that alterations of endocrineglands or hormone metabolism may bedeterminants of human aging (Hayflick,1994). For the case of insects, Meir PaulPener showed more than 30 yearsago that removal of JH extends life spanin three grasshopper species (Pener,1972). While JH and 20E are bestknown for their major roles in pre-adultdevelopment and adult reproduction(Kozlova & Thummel, 2000; Riddiford,1993, 1994; Truman & Riddiford, 2002;

Handbook of the Biology of Aging, Sixth EditionCopyright © 2001 by Academic Press. All rights of reproduction in any form reserved.

407

Chapter 15

Juvenile and Steroid Hormones in Drosophilamelanogaster Longevity

Meng-Ping Tu, Thomas Flatt, and Marc Tatar

1Abbreviations: aa,. abnormal abdomen; CA, corpusallatum, corpora allata; CC, corpus cardiacum,corpora cardiaca; CNS, central nervous system;DAF, dauer formation; dFOXO, Drosophilaforkhead transcription factor FOXO; DILPs,Drosophila insulin-like peptides; EcR, ecdysonereceptor; 20E, 20-hydroxy-ecdysone; IGF, insulin-like growth factor; InR, insulin-like receptor; IPCs,insulin-producing cells; JH, juvenile hormone; JHA,juvenile hormone analog; JHB3, juvenile hormone 3bisepoxide; MET, methoprene-tolerant; MNCs,median neurosecretory cells; USP, ultraspiracle.

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Wyatt & Davey, 1996), these hor-mones also show intriguing effects oninsect longevity. Recent studies haveshown that downregulation of JH or20E can slow senescence (Herman &Tatar, 2001; Simon et al., 2003; Tataret al., 2001a,b). Here we review hor-monal effects on aging in Drosophilamelanogaster (for a review of the genet-ics of aging in Drosophila, see Stearns &Partridge, 2001; also see Ford & Tower,Chapter 15, this volume). Although wewill focus our discussion on Drosophila,a model system with both ample geneticand endocrinological data, we will alsodraw parallels to the endocrine controlof aging in other systems (other insects,the nematode Caenorhabditis elegans,and vertebrates). We shall argue thatstudying endocrine regulation can offerpromising insights into the mechanismsand the evolution of senescence.

II. JH and 20E: Two Major InsectHormones

A. JH

The insect hormone JH is a sesquiter-penoid compound produced by the cor-pora allata (CA), a pair of endocrine glandswith nervous connections to the brain(Nijhout, 1994; Tobe & Stay, 1985). In lar-val Drosophila, the single corpus allatum(CA) makes part of the so-called ringgland, a compound structure consisting ofthe CA and two other endocrine tissues,the prothoracic glands and the corporacardiaca (CC); in adult flies, the CA iscloser to the CC, whereas the prothoraciccells of the ring gland have degenerated(see Bodenstein 1950; Richard et al., 1989;Siegmund & Korge, 2001).

JH is a major insect hormone but mayalso exist in other arthropods and canbe produced by some plant species(Tobe & Bendena, 1999). In mostinsects, JH regulates critical physiologi-cal processes, including metamorphosis

and reproduction (Dubrobsky, 2005;Gilbert et al., 2000; Riddiford, 1993).Insects produce at least eight differenttypes of juvenoids (0, I, II, III, 4-Methyl-JH, JH III-bis-epoxide [JHB3], and thetwo hydroxy-JH’s 8’-OH-JH III and 12’-OH-JH III), the most common typebeing JH III (Darrouzet et al., 1997;Richard et al., 1989; Riddiford, 1994).D. melanogaster produces both JH IIIand JHB3, both of which have endocrinefunction in dipterans (Riddiford, 1993;Yin et al., 1995). Whereas the effects ofJH III are better known, JHB3 appearsto be the major product of the CAin higher dipterans (so-called cyclor-rhaphan flies, including Drosophila andthe housefly Musca domestica), yet itsfunction awaits further study (Richardet al., 1989; Teal & Gomez-Simuta,2002; Yin et al., 1995).

In pre-adult development and metamor-phosis, JH functions as a “status quo” hor-mone, allowing continued growth afterecdysteroid-induced molting (Riddiford,1996). Metamorphosis can only take placewhen the ecdysteroids act in the absenceof JH. During the final larval instar, the JHtiter declines due to a cessation of synthe-sis and increased degradation in thehemolymph and target tissues. Absence ofJH triggers the release of prothoraci-cotropic hormone (PTTH), which in turninduces the secretion of 20E and theonset of metamorphosis (Nijhout, 1994).Although an experimental withdrawal ofJH during development can lead to prema-ture metamorphosis, an excess of JH priorto pupation results in delayed metamor-phosis (Hammock et al., 1990; Nijhout,2003; Riddiford, 1985). Unlike in someother insects, application of exogenous JHdoes not prevent the larval-pupal trans-formation in Drosophila (Riddiford &Ashburner, 1991). However, JH can dis-rupt the metamorphosis of the nervousand muscular system and disturb the nor-mal differentiation of the abdomen in thefly (Restifo & Wilson, 1998; Riddiford &

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Ashburner, 1991). Furthermore, high con-centrations of exogenous JH can prolongdevelopmental time or even inhibit eclo-sion without affecting the larval-pupaltransformation (Riddiford & Ashburner,1991).

In the adult fly, JH is crucial forthe coordination of reproductive matura-tion in both sexes. In females, JH acts onoocyte maturation, including stimulationof vitellogenin synthesis and uptake ofvitellogenin by the ovary (Bownes, 1982,1989; Dubrovsky et al., 2002; Gavin &Williamson, 1976; Postlethwait & Weiser,1973; Shemshedini & Wilson, 1993), andsexual receptivity (Manning, 1966; Ringoet al., 1991). In contrast, much lessis known about the effects of JH on malereproduction. By analogy with otherinsects, JH may affect protein synthesis inthe male accessory glands, sexual matura-tion, courtship behavior, and pheromoneproduction (Bownes, 1982; Cook, 1973;Manning, 1967; Nijhout, 1994; Teal et al.,2000; Wilson et al., 2003). In otherinsects, JH can also affect diapause regula-tion, migratory behavior, wing lengthpolyphenism, horn development in scarabbeetles, seasonal form development, loco-motory behavior, immune function, castedetermination and division of labor insocial hymenopterans and isopterans,and learning and memory (Belgacem &Martin, 2002; Hartfelder, 2000; Nijhout,1994; Riddiford, 1994; Rolff & Siva-Jothy,2002; Teal et al., 2000; Wyatt & Davey,1996). Thus, JH is truly a “master” hor-mone in insects (Hartfelder, 2000;Wheeler & Nijhout, 2003). As we shalldiscuss in this review, JH also has intrigu-ing effects on insect aging.

B. 20E

Steroid hormones, such as ecdysteroids(including ecdysone and its active form20-hydroxyecdysone, 20E), are anotherclass of vital hormones in insects. Inpre-adult flies, 20E is produced in the lar-

val prothoracic gland, which (togetherwith the larval CA and the CC) makespart of the larval ring gland in dipterans(Bodenstein, 1950). In adult female flies,the ovary is the major 20E producingtissue (Chavez et al., 2000; Gäde et al.,1997; Gilbert et al., 2002; Hagedorn,1985; Kozlova & Thummel, 2000), and20E appears to be produced in both ovar-ian follicle and nurse cells (Chavez et al.,2000, Gilbert et al., 2002; Schwartz et al.,1985, 1989). Unfortunately, we knowalmost nothing about the production andmetabolism of 20E in adult maleDrosophila and other insects (Nijhout,1994; Riddiford, 1993); by analogy withother insects, Drosophila males may pro-duce 20E in their testes (Gäde et al.,1997; Hagedorn, 1985). Together with JH,20E is an important regulator of develop-mental transitions and metamorphosis(Dubrovsky, 2005; Kozlova & Thummel,2000). In adults, 20E is well known for itseffects on oogenesis, much like JH, yetother adult functions have remained elu-sive (Dubrovsky, 2005; Kozlova &Thummel, 2000). Similarly, the adultfunction of 20E in male Drosophila ispoorly understood (Riddiford, 1993); 20Emay affect Drosophila spermatogenesis,as it does in other insects (Nijhout, 1994).

C. Interaction Between JH and 20E

Both JH and 20E play major antago-nistic or synergistic roles in regulatingDrosophila development (Dubrovsky,2005; Kozlova & Thummel, 2000, 2003;Riddiford, 1993; Truman & Riddiford,2002; Zhou & Riddiford, 2002). Theinteraction between JH and 20E seems totake place in target tissues such as thefat body (adipose tissue), epithelium, andthe ovary. For example, 20E circulatingin the hemolymph appears to inhibitJH-induced production of vitellogeninin the fat body (Engelmann, 2002; Solleret al., 1999; Stay et al., 1980). However,in some insects, such as the silkworm

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Bombyx mori, 20E can stimulate JH syn-thesis (Gu & Chow, 2003), and JH itselfcan stimulate 20E production in certainimmature lepidopterans and possiblyother insect species (Hiruma et al.,1978). Thus, while 20E and JH appear toco-regulate reproduction, there is anintricate yet not well understood hor-monal feedback between these key hor-mones (Dubrovsky, 2005).

III. Effects of JH and 20E onDrosophila Aging

A. JH and Agings

As we will discuss below, there is nowincreasing evidence showing that JH is akey regulator of aging in several insectspecies, including Drosophila (see alsoTatar, 2004; Tatar et al., 2003).

1. JH and the Abnormal AbdomenSyndrome

One of the first indications of an effectof JH on dipteran life history wasfound in Hawaiian Drosophila mercato-rum (DeSalle & Templeton, 1986;Templeton, 1982, 1983; Templeton &Rankin, 1978; Thomas, 1991). In thisspecies, the abnormal abdomen (aa)genotype has a decreased JH esterase(JHE) activity, which may lead to a highJH titer in the hemolymph (Templetonet al., 1993; Thomas, 1991). The aaphenotype has increased developmentaltime, early sexual maturation, increasedfecundity, and decreased longevity amongfemales (Hollocher & Templeton, 1994;Templeton, 1982, 1983; Templeton &Rankin, 1978; but see Thomas, 1991).In males, developmental time is notaffected, whereas sexual maturationis delayed, mating success decreased,and longevity increased (Hollocher &Templeton, 1994). Thus, the aageno-type affects male and female lifehistory differently.

Although aa females seem to be long-lived (Templeton, 1982, 1983), the effectsof aa on life span and other life-historytraits may depend on nutrient conditions(Thomas, 1991). Contrary to the findingsof Templeton (1982, 1983), both malesand females of aa genotypes generallyexhibited greater longevity than non-aagenotypes when reared on various con-centrations of dry yeast in the foodmedium (Thomas, 1991). This life-spanextension was observed for all yeast con-centrations, except for the concentrationused by Templeton (1982, 1983), whichreduced life span. Thus, nutrition mayaffect D. melanogaster life span andother life-history traits through changesin JH signaling (see section V).

In addition, reproduction appears not totrade off with survival in these long-livedaa genotypes because females showed bothgreater fecundity and longevity than non-aa females (Thomas, 1991). This contra-dicts many experiments in Drosophila thatshow that life-span extension is typicallyaccompanied by reduced reproduction (fora review, see Stearns & Partridge, 2001).Thus, the study by Thomas (1991) adds toa growing number of examples suggestingthat the tradeoff between reproduction andlife span can be uncoupled under somecircumstances (Barnes & Partridge, 2003;Good & Tatar, 2001; Hwangbo et al., 2004;Leroi, 2001; Marden et al., 2003; Tu &Tatar, 2003).

While the various life-history effectsobserved in D. mercatorum may indeed beproximally controlled by JH, a direct prooffor such an effect is lacking. Whether theaa genotype has an increased JH titerremains to be determined using a directJH titer assay rather than measuringthe turnover rate of degradation enzymes(Thomas, 1991). Yet, despite the uncer-tainty surrounding the pleiotropy of the aagenotype and the role of JH in the aa syn-drome, it is interesting to note that aagenotypes differ remarkably from wildtypeflies in both life span and JH metabolism,


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suggesting that JH may be a proximatedeterminant of life span.

2. JH, Reproductive Diapause, andSenescence Plasticity

Many adult insects use token cues to ini-tiate diapause in response to seasonablypredictable stressful or harsh environmen-tal conditions. Diapause is a hormonallymediated state of reduced metabolism,developmental arrest, increased stressresistance, and altered behavior (Nijhout1994; Tatar & Yin, 2001); the develop-mental arrest associated with diapauseis reflected in an arrest of oogenesis,male accessory gland synthesis, and mat-ing (“reproductive diapause”). In manyinsects, JH is proximately involved in reg-ulating diapause (Nijhout, 1994; Tatar,2004; Tatar & Yin, 2001).

JH controls reproductive diapause ininsects as variable as butterflies (Danausplexippus; Herman & Tatar, 2001; Tatar,2004; Tatar & Yin, 2001), several grasshop-per species (Pener, 1972; Tatar & Yin,2001), and several species of Drosophila(D. macroptera and D. grisea: Kambysellis& Heed, 1974; D. melanogaster: Saunderset al., 1989; Tatar & Yin, 2001; Tatar et al.,2001a).

For example, several temperate-zone species of Drosophila, includingD. melanogaster, D. triauraria, D. lit-toralis, and the cave-dwelling speciesD. grisea and D. macroptera, are knownto over-winter as diapausing adults(Kambysellis & Heed, 1974; Saunderset al., 1989; Tatar, 2004; Tatar & Yin,2001). As shown by Tatar and colleagues(2001a), traits specific for diapause in D.melanogaster (arrest of oogenesis, resist-ance to exogenous stress, negligiblesenescence during diapause) are con-trolled by JH (Tatar & Yin, 2001). JHmay thus be a key mediator of senes-cence plasticity and the tradeoffbetween reproduction and longevity.

Diapausing females downregulateJH synthesis in response to shorterday length and cool temperatures.Consequently, females enter ovarianarrest and show reduced age-specific mor-tality as compared to non-diapausingfemale cohorts that were started synchro-nously with the diapausing cohort. Thediapause phenotype can be rescued byapplication of the synthetic JH metho-prene; this treatment terminates ovarianarrest, makes flies more sensitive tooxidative stress, and reduces post-dia-pause longevity (Tatar et al., 2001b).Methoprene is a JH analog (JHA) that ischemically more stable and much morepotent than JH itself (see Wilson, 2004,and references therein). Although highdoses of methoprene, a commonly usedinsecticide, can be toxic to insects, insectphysiologists have confirmed in numer-ous reports that methoprene behaves as afaithful mimic of JH action in insects ingeneral and in Drosophila in particular,both in vivo and in vitro (e.g., Riddiford& Ashburner, 1991; Wilson, 2004, andreferences therein; but also see Zera,2004).

In C. elegans, larval diapause (for-mation of so-called dauer larvae) isunder the control of the insulin signalingpathway, which signals through aninsulin-like receptor encoded by thedauerformation 2 gene (daf-2), thehomolog of the Drosophila insulin-likereceptor (InR) locus. Mutations in daf-2cause dramatic life-span extension(Dorman et al., 1995; Kenyon et al.,1993; Kenyon, 2001). Interestingly, in D.melanogaster, mutant InR genotypes livelonger and exhibit small and immatureovaries, very similar to those observed indiapausing wildtype Drosophila (Tataret al., 2001b). This suggests that flies inreproductive diapause “phenocopy” thephenotype of InR mutants. Thus, theremay exist an analogy between diapauseand insulin signaling in C. elegans andD. melanogaster.


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3. JH in Mutants of the Insulin SignalingPathway

Several D. melanogaster mutant geno-types of InR and chico (encoding theinsulin-receptor substrate) are long-lived(Clancy et al., 2001; Tatar et al., 2001b;Tu et al., 2002a). For example, a heteroal-lelic InR mutant (InRp5545/InRE19) pro-duces dwarf females with extended lifespan up to 85 percent (Tatar et al., 2001b).Similarly, homozygous mutants of chicoare sterile and very long-lived dwarfs,whereas heterozygous mutants of chicoexhibit normal body size, reduced fecun-dity, and extended life span (Clancy et al.,2001; Tu et al., 2002a).

However, not all InR mutant allelesincrease longevity: since InR gene is ahighly pleiotropic locus, some allelesmay have deleterious developmentaleffects carrying over to adults and coun-terbalancing the positive effects on aging(Tatar et al., 2001b). Furthermore, unlikein C. elegans, hypomorphic insulin sig-naling mutants in Drosophila may havedifferent effects on life span in malesand females (Tu et al., 2002a). WhileDrosophila mutants of InR and chicoextend female longevity by 36 to 85 per-cent, the same alleles do not seem to pro-duce an extension of mean longevity inmales (Clancy et al., 2001; Tatar et al.,2001b). However, males of InR heteroal-lelic mutants have an increased lifeexpectancy measured at age 20 days(Tatar et al., 2001b), and the chico1 muta-tion extends male longevity but has age-independent effects on adult mortalitythat counteract the strong impact of slowaging on life expectancy seen in chicomutant females (Tu et al., 2002a).Similarly, reducing insulin signaling byexperimental ablation of insulin-produc-ing cells (IPCs) reduces age-dependentmortality, yet this effect is masked atyoung ages due to a high age-independentrisk of death (Wessells et al., 2004; butsee Broughton et al., 2005).

Interestingly, the extended life-span phe-notype of some insulin signaling mutantsis likely to be caused by JH deficiency. JHsynthesis is negligible in InR dwarfs (Tataret al., 2001b), and homozygous chicomutants are also JH deficient (Tu et al.,2005). Although InR mutant females areinfertile with non-vitellogenic ovaries, eggdevelopment can be restored by applica-tion of methoprene. Furthermore, treat-ment with methoprene restores wildtypelongevity to the long-lived InR mutants.Thus, JH deficiency, resulting from muta-tion in the insulin signaling pathway,may retard senescence, possibly throughhormone-mediated effects on adult repro-duction, physiology, and somatic mainte-nance. Consequently, Tatar and colleagues(2001b) suggest that infertility may not bethe direct cause of slowed aging but thatJH may simply control both fertility andlongevity; JH may therefore be a key regu-lator for both traits. However, JH synthesisis also known to be reduced in a homozy-gous InR mutant genotype with normallife span; thus, the lack of JH may not besufficient to extend life span under all cir-cumstances (Tatar et al., 2001b). Similarly,whether short-lived insulin signalingmutants have upregulated JH is currentlyunknown.

4. Effects of Manipulating JH on Life Span

Using mutants to examine the effectsof JH on life span may be problematicbecause mutants can exhibit unspecificpleiotropic effects that are unrelated tochanges in JH signaling. This problemmay be overcome by examining theeffects of applying exogenous JH or JHAssuch as methoprene. Treating wildtypeflies with methropene increases earlyfecundity but decreases longevity andstress resistance (Flatt & Kawecki, inpreparation; Salmon et al., 2001, Tataret al., 2001a,b). For example, methoprenetreatment of diapausing D. melanogaster


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restores vitellogenesis and egg produc-tion yet increases demographic senes-cence (Tatar et al., 2001a). Similarly, asdiscussed above (section III.A.3), Tatarand colleagues (2001b) found that thesterility phenotype of InR mutants canbe rescued by methoprene treatment,which restores egg development andreduces life expectancy to that of wild-type flies, whereas methoprene treat-ment of InR wildtype controls did notincrease adult mortality.

Application of commonly used JHinhibitors, such as precocene (Wilsonet al., 1983) or fluvastatin (Debernardet al., 1994), can reduce or inhibit JH syn-thesis in the CA and may thus be used tostudy the effects of JH deficiency upon lifespan. However, these inhibitors alsoappear to have unspecific and toxic effects(e.g., Debernard et al., 1994; Zera, 2004,and references therein). For example, highdoses of fluvastatin kill locusts, whereassurgical removal of CA is not lethal(Debernard et al., 1994). Thus, the useful-ness of JH inhibitors for manipulating lifespan through changes in JH signaling isquestionable. A different approach is toselect wildtype flies for resistance totoxic doses of methoprene (T. Flatt &T. J. Kawecki, unpublished results). Fliesthat evolve specific insensitivity to JH,either by constitutive upregulation of JHesterases or by reduced JH binding, mayexhibit increased longevity because JHdeficiency is known to slow aging.Interestingly, flies selected for metho-prene resistance rapidly evolved bothmethoprene- and JH III-resistance andshowed extended life span (T. Flatt & T. J.Kawecki, unpublished results). However,the underlying mechanisms for this life-span extension are unknown and may notbe JH-related.

B. 20E and Aging

Although we know far less about theeffects of steroid hormones on life span

than about JH effects on aging, it nowseems clear that 20E is a second candi-date regulator of Drosophila aging (alsosee Simon et al., 2003; Tatar, 2004; Tataret al., 2003).

1. Long-Lived Insulin Signaling MutantsHave Reduced 20E Titers

In adult insects, ecdysteroids are made inthe ovaries and the testes (Chavez et al.,2000; Gäde et al., 1997; Gilbert et al.,2002; Hagedorn, 1985). Tu and colleagues(2002b) measured ecdysteroid synthesisin isolated ovaries of InR mutants invitro and found that ovarian ecdysteroidsynthesis of mutant females was reducedas compared to wildtype. How 20E defi-ciency affects aging in InR mutants iscurrently not understood; 20E may affectlife span by serving as a pro-aging hor-monal signal or by regulating the rela-tionship between reproduction and aging(Tatar, 2004; Tatar et al., 2004; Tu et al.,2002b). Clearly, it would be interestingto see whether and how treatment of20E-deficient InR mutants with 20Eaffects aging. In addition, because 20E isa major product of the Drosophila ovaryand plays a pervasive role in femalereproduction, we may speculate that thesex-specific effects of insulin signalingon aging seen in the fly may be related todifferences in 20E signaling betweenmales and females.

2. Mutations in the Ecdysone ReceptorExtend Life Span

Simon and colleagues (2003) demonstratethat flies heterozygous for mutations ofthe ecdysone receptor (EcR) gene exhibitincreased life span and stress resistancewithout decreases in reproduction oractivity. Although almost nothing isknown about the production, metabolism,and role of 20E in adult male Drosophila,it is interesting to note that mutations inEcR extended life span in both males and


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females, suggesting that 20E signalingaffects aging in both sexes. Furthermore, amutant involved in the 20E biosynthesispathway (DTS-3) displays the same phe-notype; this phenotype can be rescued bythe application of 20E (Simon et al., 2003).These results are consistent with reducedpost-eclosion levels of ecdysteroids inlong-lived females from a selection experi-ment for life-span extension (Harshman,1999). Thus, the few examples at handclearly suggest that 20E deficiency slowsaging.

C. Interaction Effects of JH and 20Eon Aging

Interactions between JH and 20E maynot be restricted to pre-adult develop-ment, metamorphosis, and reproduction(Dubrovsky, 2005), but may also extend tothe aging phenotype. In mosquitoes, appli-cation of bovine insulin acts directly inthe ovary to regulate ecdysteroid synthesis(Riehle & Brown, 1999); insulin is alsoknown to regulate germ-line stem cellproliferation in D. melanogaster ovaries(Drummond-Barbosa & Spradling, 2001).As discussed above (sections II.C andIII.B.1), the ovaries of the JH-deficient InRmutants produce little ecdysteroids (Tuet al., 2002b), and 20E can, depending onthe species, inhibit or stimulate JH syn-thesis (Gu & Chow, 2003; Soller et al.,1999). Thus, 20E may be a gonad-derivedsignal through which insulin and JH affectinsect aging (Tatar, 2004; Tatar et al.,2003). In addition, reproductive diapauseand diapause senescence may not beexclusively controlled by JH. Applicationof JH III or JHB3 to abdomens of diapaus-ing female flies can restore vitellogenesis(Saunders et al., 1989). However, terminat-ing diapause by warming flies from 11 °Cto 25 °C results in a significant increase inthe synthesis of ecdysteroids, but not JH(Saunders et al., 1989). Furthermore, theinjection of 20E can also elicit vitellogene-sis and terminate diapause (Richard et al.,

1998, 2001), as has been observed with JH.This suggests that JH and 20E may inter-act in affecting diapause and levels of age-specific mortality during diapause.Interestingly, although JH and 20E oftenhave antagonistic effects on the same traitor process, both hormones seem to havepositive effects on female reproductionand negative effects on female life span.However, whether and how 20E inter-acts with JH to affect male life historyremains unknown. Clearly, the interactiveeffects of these hormones on aging awaitfurther study.

IV. Candidate Genes AffectingLife Span Through JH and 20E

SignalingA. Insulin Signaling Affects Both JH

and 20E

The insulin/IGF (insulin-like growth fac-tor) signaling pathway has profoundeffects on aging in a variety of organ-isms, such as the nematode C. elegans,Drosophila, and rodents, and is sus-pected to have similar effects in humans(Kenyon, 2001; Partridge & Gems, 2002;Tatar, 2004; Tatar et al., 2003). Studies ofmutants of InR, chico, and EcR suggestthat JH and 20E may be secondary pro-aging signals downstream of insulin/IGF(see section III). As discussed above,long-lived mutants of InR are bothJH and 20E-deficient, suggesting thatinsulin signaling is a major regulator ofthese secondary hormones. Indeed, thestimulation of ecdysteroid synthesis byinsulin signaling is well known in manyinsects (Graf et al., 1997; Hagedorn,1985; Nagasawa, 1992; Riehle & Brown,1999). For example, mosquitoes synthe-size ovarian ecdysteroids after a bloodmeal, and this synthesis depends oninsulin signaling. Sugar-fed mosquitofemales do not produce ecdysteroids, butapplication of exogenous bovine insulincan stimulate ovarian ecdysteroid


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synthesis (Riehle & Brown, 1999). Buthow does insulin signaling regulate JHand 20E synthesis?

In response to environmental or inter-nal stimuli such as nutrition, insulin-pro-ducing cells (IPCs, belonging to the classof median neurosecretory cells, MNCs)located in the pars intercerebralis of thebrain produce seven different Drosophilainsulin-like peptides (DILPs; Brogioloet al., 2001; Broughton et al., 2005).These DILPs are then released into theprotocerebrum, at the CC, and into thehemolymph (Ikeya et al., 2002; Rulifsonet al., 2002). Little is known aboutthe effects of individual DILPs, butthe expression of the genes dilp3 anddilp5 seems to be regulated by nutrition,and overexpression of dilp1–7 promotesgrowth (Hwangbo et al., 2004; Ikeya etal., 2002; Rulifson et al., 2002). Insulinsignaling may regulate JH and 20E syn-thesis in at least two not mutually exclu-sive ways (Tatar, 2004). First, circulatingDILPs in the hemolymph activate insulinsignaling by binding to the InR receptorsin the target tissues and hence stimulategrowth in these tissues. For example, InRmutants are dwarfs with both reduced JHsynthesis and corpus allatum (CA) size;insulin may thus affect JH synthesisby affecting CA development and growth(Tatar et al., 2001b; Tu et al., 2005).Similarly, InR mutants have an approxi-mately 50 percent reduction of wildtypeovariole number; mutants may thereforeproduce less 20E because of a reducednumber of ovarian follicle cells synthesiz-ing 20E (Tu & Tatar, 2003; Tu et al.,2002b;). Second, JH and 20E synthesismay be indirectly modulated by insulinsignaling. For example, because JH syn-thesis is regulated by neuropeptides (Tobe& Bendena, 1999), DILPs may indirectlyregulate the production of JH by affectingthese neuropeptides. In chico mutants, JHsynthesis relative to CA size is dispropor-tionately reduced, suggesting that insulinsignaling can regulate adult JH synthesis

independent of affecting CA developmentand growth (Tu et al., 2005). The insulinsignaling–dependent regulation of JH and20E may also be supported by the obser-vation that ablation of IPCs can extendlife span in Drosophila (Broughton et al.,2005; Wessells et al., 2004). However, itis currently unknown whether this effectoccurs through the downregulation ofJH or 20E. Clearly, future work needs totest whether ablation of IPCs or downreg-ulation of DILP expression reduces JHand/or 20E. Similarly, reducing insulinsignaling by transgenically overexpress-ing dPTEN (encoding a phosphatase andtensin homolog protein that antagonizesinsulin signaling) and dFOXO (a fork-head transcription factor downstream ofinsulin signaling whose activity is inhib-ited by insulin signaling) in the head fatbody of the fly can slow aging (Hwangboet al., 2004), yet it remains to be testedwhether these genes regulate longevitythrough effects on JH or 20E production.Figure 15.1 shows an integrated model ofthe endocrine regulation of Drosophilaaging.

Although JH and 20E are not producedby C. elegans and rodents, the existence ofsecondary pro-aging hormones in theseorganisms has been postulated (Tataret al., 2003; also see Gill et al., 2004). Forexample, in rodents, insulin signaling inthe hypothalamus regulates the pituitarygland, which secretes secondary hormonessuch as thyroid-stimulating hormone(TSH), follicle-stimulating hormone (FSH),growth hormone (GH), and luteinizinghormone (LH); TSH in turn regulates thethyroid gland to produce the thyroxin hor-mones T3 and T4. The pituitary may beseen as the mammalian equivalent of theCC and CA, and thyroxin has been tenta-tively suggested to share cellular functionswith JH (Davey, 2000). Remarkably, a newstudy supports the idea that thyroxin maybe a pro-aging hormone like JH (Vergaraet al., 2004). Long-lived Snell dwarf mice(mice homozygous for the Pit1 mutation)


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Figure 15.1 Integrated model for the endocrine regulation of aging, based on studies in D. melanogaster(see Hwangbo et al., 2004; Tatar, 2004; Tatar et al., 2003) External cues like nutrition stimulate insulin-producing cells (IPCs) to secrete insulin-like peptides (DILPs), which bind and activate the insulin-likereceptor at the target tissues (InR in D. melanogaster, DAF-2 in C. elegans). Ligand binding at InR in turninduces the insulin/IGF-1 signaling cascade in cells of the CNS and other tissues, such as the fat body (pri-mary insulin signaling). Induction of insulin/IGF-1 signaling suppresses a forkhead transcription factordownstream of insulin signaling (dFOXO in D. melanogaster, DAF-16 in C. elegans) required for life-spanextension by slowed insulin signaling. Activation of this transcription factor (or inactivation of InR orablation of IPCs) extends life span (Broughton et al., 2005; Hwangbo et al., 2004; Kenyon et al., 1993; Tataret al., 2001b; Wessells et al., 2004). Insulin signaling also has secondary effects: (1) insulin signaling affectsinsulin production by participating in endocrine and paracrine regulatory feedback circuits to regulateDILP transcription (Hwangbo et al., 2004) and (2) insulin signaling affects the production of secondaryaging regulatory signals such as JH from the CA or 20E from the gonads (Simon et al., 2003; Tatar, 2004;Tatar et al., 2001b, 2003; Tu et al., 2002b;). In Drosophila and other insects, these secondary endocrine sig-nals (unknown hormones in C. elegans, but see Gill et al., 2004) suppress life-span extension (as well asstress resistance, immunity, and somatic maintenance) and upregulate gonad activity and reproduction(Tatar, 2004; Tatar et al., 2003). Note that external cues may also directly affect the production of JH and20E. Thus, insulin signaling may either directly affect aging (through dFOXO) or indirectly (through sec-ondary pro-aging hormones). See text for further details.

have multiple hormonal defects, butwhether these deficiencies are causallyresponsible for the slow aging phenotypehas remained unclear (Tatar et al., 2003).

Vergara and colleagues (2004) show thattreatment with the thyroxine hormone T4restores the reduced senescence phenotypein the long-lived Snell mice. Although it

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cannot be ruled out that the T4 treatmentresults in a toxic effect reducing life span(Vergara et al., 2004), the restoration of theanti-aging effects by T4 is highly reminis-cent of the life-span reduction seen inlong-lived JH-deficient flies when treatedwith methoprene.

B. Genes Involved in JH Signaling

The genetics of JH signaling are currentlynot well understood (Dubrovsky, 2005).Moreover, the life-span effects of mostDrosophila genes involved in JH signal-ing are unknown. Yet, despite our lim-ited understanding, there exist severalinteresting candidate genes implicated inJH signaling that may affect the agingphenotype.

A major reason limiting our under-standing of JH signaling is the unknownnature of the JH receptor (Dubrovsky,2005; Gilbert et al., 2000; Henrich &Brown, 1995; Jones & Sharp, 1997;Truman & Riddiford, 2002). Recent evi-dence suggests that ultraspiracle (usp),encoding a retinoid X receptor, may bea JH receptor because JH is closelyrelated to retinoic acid (RA) and USPprotein can bind JH (Gilbert et al.,2000; Jones & Sharp, 1997; Truman &Riddford, 2002). Although USP doesnot show high-affinity binding to JH(Jones & Sharp, 1997), it forms a het-erodimer with the ecdysone receptor(EcR) (Gilbert et al., 2002; Truman &Riddiford, 2002), which is itself knownto have effects on Drosophila life span(Simon et al., 2003). Thus, given thecommon interactions between JH and20E and given that mutations in EcR orJH deficiency extend life span, ultraspir-acle appears to be a promising candidategene affecting aging. Yet, while usp prob-ably plays a functional role in JH signal-ing, the low binding affinity for JH is notconsistent with usp encoding a JH recep-tor (Dubrovsky, 2005).

Another candidate gene for the JHreceptor is the X-linked gene Methoprene-

tolerant (Met), encoding a 85 kD high-affinity JH binding protein essential fortransducing JH signals (Ashok et al., 1998;Pursley et al., 2000; Restifo & Wilson,1998; Shemshedini & Wilson, 1990;Wilson et al., 2003). Met mutant flies pro-duce JH in normal amounts but are upto 100 times less sensitive to JH III andmethoprene than Met� flies (Pursleyet al., 2000; Shemshedini & Wilson, 1990;Shemshedini et al., 1990). In adults, theMet gene has important effects on life-history traits, such as developmentaltime, onset of reproduction, and age-specific fecundity (Flatt & Kawecki,2004; Minkoff & Wilson, 1992; Wilson &Ashok, 1998; Wilson et al., 2003). Giventhese pleiotropic life-history effects ofMet, presumably mediated by JH signal-ing, one may expect that this locus alsoaffects life span. However, inconsistentwith the notion of a JH receptor, Met nullmutants are completely viable. Thus, theexact role of Met in JH signaling remainsunclear (Dubrovsky, 2005).

Mutations of the apterous (ap) generesult in sterile females due to the devel-opment of non-vitellogenic ovaries(Ringo et al., 1991). Mutant males arebehaviorally sterile, spend less timecourting, and are less likely to performsome elements of courtship behaviorthan age-matched wildtype males, yetthey have fertile gametes. Adult mutantflies are JH deficient (Altatraz et al.1991), and application of JH or metho-prene to newly eclosed mutant femalesresults in vitellogenic oocytes(Postlethwait & Weiser 1973), suggestingthat the development of oocytes is pro-foundly affected by JH. A recent studyclearly supports the notion that apterousis involved in JH signaling; in apterousmutants, the levels of two JH-induciblegenes (Dubrovsky et al. 2002), JhI-21 andminidiscs (mdn), are strongly reduced,and methoprene treatment can rescuethis defect. Thus, since apterous mutantsare JH-deficient, like long-lived mutantsof InR and chico, apterous may be an


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interesting candidate gene affectingaging.

Flies with a mutation in the cricklet(clt) gene have reduced yolk protein syn-thesis, larval fat bodies persisting intothe adult stage, and an arrested oocytedevelopment in the pre-vitellogenic stage(Shirras & Bownes, 1989). Methoprene hasno effect on the fat body synthesis of yolkand vitellogenesis in the mutants; ovariantransplant experiments suggest thatfemales have sufficient JH concentrationsto promote oogenesis (Shirras & Bownes,1989). This indicates that the gene mayencode a protein downstream of JH syn-thesis, such as a receptor or transcriptionfactor, which is nonfunctional in themutants (Shirras & Bownes, 1989).However, as for many other genes affect-ing JH signaling, the potential effects ofclt on aging await further study.

In female Drosophila, the post-matingresponse consists of increased egg depo-sition and reduced receptivity to malesand is regulated by sex peptide (SP), con-tained in the male seminal fluid andtransferred to the female upon mating.SP is known to stimulate JH synthesisin the mature CA (Moshitzky et al.,1996). Consequently, JH appears to bea downstream component in the SPresponse cascade, causing the progres-sion of vitellogenic oocytes after matingor SP application (Moshitzky et al.,1996; Soller et al., 1999). Remarkably,work by Geiger-Thornsberry & Mackay(2004) shows that the sex peptide locus(Acp70, accessory protein 70) harborsgenetic variation for life span. Thus, thesex peptide gene is another promisingcandidate gene for aging studies, in par-ticular since it may affect life spanthrough its effects on female JH signal-ing (Flatt, 2004).

Biogenic amines, such as dopamine,octopamine, and serotonin, are known toaffect JH synthesis in insects (Lafon-Cazal & Baehr, 1988; Rachinsky, 1994;Roeder, 1999). For example, octopamine

stimulates JH production in locusts (Lafon-Cazal & Baehr, 1988), and both octopamineand serotonin promote JH synthesis inhoneybees (Rachinsky, 1994). In D.melanogaster and D. virilis, dopamineseems to stimulate synthesis of JH inimmature females but inhibits its syn-thesis in mature, intensely reproducingfemales; similarly, octopamine appears toblock JH production (Chentsova et al.2002; Grutenko et al., 2001; Rauschenbachet al., 2002). Two recent experiments sug-gest that biogenic amines are importantdeterminants of life span. Sze and col-leagues (2000) found that a serotonin-syn-thesis mutant in C. elegans exhibitsextended reproductive life span, and DeLuca and colleagues (2003) report that thegene coding for the enzyme dopa decar-boxylase (DDC), required for the final cat-alytic step in the synthesis of dopamineand serotonin, harbors significant amountsof variation for Drosophila longevity. Thus,although C. elegans is not known to pro-duce JH, these results suggest that biogenicamine metabolism may affect the levels ofsecondary endocrine hormone with effectson aging. However, it remains to be deter-mined whether ddc mutants in Drosophilaexhibit variation in JH signaling that may,at least partially, account for the variationin life span attributed to this locus.

Table 15.1 summarizes information onsome Drosophila genes involved in JHsignaling; these genes may be promisingcandidate genes affecting aging.

C. Genes Involved in 20E Signaling

In contrast to JH, we have a fairly goodunderstanding of the molecular mecha-nisms of both ecdysteroid synthesisand 20E action, particularly at theonset of metamorphosis (Dubrovsky,2005). For example, 20E binds to theecdysone receptor (EcR), which formsa heterodimer receptor complex withUSP (Gilbert et al., 2002; Truman &Riddiford, 2002), and recent work


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Table 15.1Examples of Drosophila Genes Involved in JH Signaling. Because JH is known to have major effects on insect

life span, these genes represent candidate genes for aging. Further information on each of these and furthercandidate genes, including references, can be found on the flybase Website at: http://flybase.bio.indiana.edu;

this database is searchable using gene names, flybase accession numbers, or keywords.

Gene Function Mutant Phenotype and Flybase Biological Processes Accession

Acp70 (sex peptide; Male product contained Female mating FBgn0003034accessory gland in seminal fluid; has defective; injection of peptide 70A) hormone activity; Acp70A stimulates

negatively regulates JH synthesis in the female receptivity and female, increases egg post-mating response production and mimics

the effects of mating; genetic variation for life span detected as a function of this gene

adp (adipose) Involved in Mutations result in FBgn0000057carbohydrate and hypertrophied adult lipid metabolism fat body with enlarged

lipid vesicles and hypertrophied female corpora allata; mutants are viable, starvation resistant, male and female fertile, yet egg hatchability is reduced and eclosion delayed

ap (apterous) Product exhibits Mutations affect halteres, FBgn0000099zinc ion binding and muscle development, is involved in neuroanatomy, neurogenesis ovarian development,

oogenesis, and female receptivity; some mutants are JH-deficient; JH application can restore vitellogenesis and positively affects

ovarian maturationclt (cricklet) Carboxylesterase activity Defective in yolk protein FBgn0000326

synthesis, histolysis of the larval fat body, vitellogenesis, and synthesis of larval serum protein 2;gene may encode a protein essential for mediating JH signaling in target tissues

e (ebony) Product exhibits Mutations affect wing FBgn0000527beta-alanyl-dopamine and adult cuticle; synthase activity, mutants are locomotor involved in cuticle rhythm and body color pigmentation defective; 1-day-old

mutant females show a significantly lower JH-hydrolyzing activity as compared to wildtype

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Table 15.1 (Cont’d)


Fas2 (fasciclin 2) Involved in neuronal Mutations affect the FBgn0000635cell recognition and embryonic neurons and organ looping and 13 other tissues; symmetry mutants are larval

recessive lethal and have embryonic neuroanatomical defects; the spin mutant allele affects neurosecretory cells innervating the corpus allatum, male genitalia rotation, and interacts with the Met locus

fs(2)B (female sterile (2) Female reproduction; Recessive female sterile; FBgn0000949Bridges) also affects endocrine cells of the

function corpus-allatum-corpus-cardiacum (CA/CC) complex of homozygous mutant females can probably not release hormone product and undergo degenerative changes; enlarged abdominal fat cells

ibx (icebox) Encodes a product Various mutant FBgn0041750involved in female phenotypes, including courtship behavior female mating defective,

female courtship defective, yet fertile and viable; treatment with JH analog methoprene results in more mating in homozygous females

InR (insulin-like Insulin-like receptor; Heterozygous mutants FBgn0013984receptor) binds Drosophila are dwarf, female sterile,

insulin-like peptides and long-lived; some mutants have lowered JH and 20E production

jhamt (JH acid JH acid Unknown FBgn0028841methyltransferase) methyltransferase

activity involved in JH biosynthesis

Jhbp-30, 63, and 80 JH binding activity Unknown FBgn0013282; (JH binding in larval fat FBgn0013283; proteins – 30kD, body cell nuclei FBgn001328463kD, and 80kD)

Jhe (JH esterase) JH esterase activity Unknown FBgn0010052involved in JH catabolism

Jheh1, Jheh2, and JH epoxide hydrolase Unknown FBgn0010053;Jheh3 (JH epoxide activity involved in hydrolases 1, 2, 3) JH catabolism;

maybe involved in FBgn0034405; defense response FBgn0034406

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JhI-1and JhI-26 Unknown; Unknown FBgn0028426; (JH-inducible gene expression FBgn0028424proteins 1 and 26) induced by JHJhI-21 (JH-inducible L-amino acid Unknown FBgn0028425protein 21) transporter activity;

amino acid metabolism?l(2)gl (lethal (2) Product with myosin Mutations affect FBgn0002121

giant larvae) binding, involved in the dorsal neurotransmitter mesothoracic disc, secretion the larval brain, and

21 other tissues; recessive lethal and tumorigenic; wildtype allele important for onset of vitellogenesis and oocyte growth, follicle cell migration and organization, and germline cell viability; some mutants have reduced size of the larval ring gland (consisting of the corpora cardiaca, the corpora allata, and the prothoracic gland), presumably resulting in endocrine deficiency

Methoprene-tolerant Encodes a product with Loss-of-function alleles FBgn0002723(Met 5 Rst(1)JH, JH binding, a putative have reduced numbers Resistance to JH) JH receptor; involved in of stage S8 to 9 and

regulation of stage S10 to 14 oocytes, transcription are methoprene and

JH III resistant, viable, but have reduced female fertility; Met interacts with Br and Fas2

mama (maternal ? Mutants show lipid FBgn0000988metaphase arrest) accumulation,

hypertrophied corpora allata, are viable, recessive female sterile, maternal effect recessive lethal, and reduced male fertile

Mdh1 (malate L-malate dehydrogenase Unknown; response of FBgn0002699dehydrogenase 1) activity Mdh1 to JH depends on

ecdysteroids; during interecdysial period of the last instar Mdh1rapidly responds to JH by increasing activity

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Rbp9 (RNA binding RNA binding, Loss-of-function FBgn0010263protein 9) involved in egg mutations affect

chamber formation the ovariole, the cystocyte, and the egg chamber and are female sterile; cells of the corpus-allatum corpus-cardiacum complex of homozygous mutant females can probably not release their hormone products and undergo degenerative changes

Tbh (tyramine ? Tyramine-beta Loss-of-function FBgn0010329hydroxylase) hydroxylase activity mutations are viable,

involved in behavioral male fertile and response to ethanol female sterile; under

normal conditions, young mutant females have a higher JH-hydrolyzing activity than wildtype

usp (ultrapiracle) Product with Mutations affect the FBgn0003964ligand-dependent embryonic/larval nuclear receptor anterior spiracle, activity; forms the imaginal discs, heterodimer with the embryonic ecdysone receptor; larval midgut, and are binds JH with recessive lethal, low affinity; has hypoactive and touch cell-autonomous role in sensitivity defective; controlling neuronal mutations show a remodeling range of imaginal

disc phenotypesVha44 Hydrogen-exporting Unknown FBgn0020611

(vacuolar H[�] ATPase activity, ATPase) phosphorylative

mechanism, involved in JH biosynthesis

Yp 1, 2, and 3 Structural molecule Mutations conditionally FBgn0004045; (yolk proteins 1, 2, activity involved in affect egg production FBgn0005391; and 3) vitellogenesis and the adult fat body FBgn0004047

and are dominant femalesterile; the JH analogmethoprene upregulates yolk proteins

confirms the importance of this het-erodimer complex for Drosophiladevelopment (Hall & Thummel, 1998;Hodin & Riddiford, 1998; Dubrovsky,

2005). When bound to the receptor, 20Einduces a number of early responsegenesm such as Broad (Br), a geneessential for transducing 20E signals

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during metamorphic development oflarval and imaginal tissues (Restifo &Wilson, 1998; Zhou & Riddiford, 2002),as well as E74 and E75, which seem tobe required for oogenesis (Dubrovsky,2005; Kozlova & Thummel, 2000). Incontrast to the genetic control of JHsynthesis, the field has recently wit-nessed major progress in identifyingcritical genes required for 20E synthe-sis, involving key enzymes encoded bygenes such as defective in the avoid-ance of repellents (dare), disembodied(dib), ghost (gho), phantom (phm),shade (shd), shadow (sad), spook (spo),

and Start1 (Buszczak et al., 1999;Chavez et al., 2000; Gilbert et al., 2002;Petryk et al., 2003; Roth et al., 2004;Warren et al., 2004). Because thesegenes affect 20E synthesis and because20E deficiency is known to slow aging,some of these genes may be interestingcandidate genes affecting aging.

Table 15.2 summarizes informa-tion on some fly genes known to beinvolved in 20E signaling; our futureunderstanding of the endocrine regula-tion of aging may be improved by study-ing the effects of these genes on theaging phenotype.


Table 15.2Examples of Drosophila Genes Involved in 20E Signaling. Because 20E affects insect aging, these genes

may represent candidate genes for aging. Further information on each of these and other candidate genescan be found at: http://flybase.bio.indiana.edu ; this data base is searchable using gene names, flybase

accession numbers, or keywords.


EcR (ecdysone receptor) 20E receptor Constitutive FBgn0000546heterozygous mutants extend life span; follicle cell expression of dominant negative for EcR results in female sterility; germline clones have arrested mid-oogenesis

E74 and E75 Orphan receptors; Germline clones have FBgn0000567; (ecdysone-induced transcription factors arrested mid-oogenesis, FBgn0000568proteins 74 and 75) required for mediating degenerate

20E response egg chambers; some mutants have low 20E titers

Br (broad) Transcription factor; Various mutant FBgn0000210a major 20E phenotypes, including inducible gene; also developmental arrest, interacts with the metathoracic tarsal Met locus segment formation,

optic lobe formation, effects on adult brain, and reduced transcription rate or stability of the small heat shock protein mRNAs

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dare (defective in Adrenodoxin reductase Various mutant FBgn0015582the avoidance required for ecdysteroid phenotypes; blocked of repellents) synthesis ecdysteroid synthesis;

abnormal response to olfactory stimuli, degenerate nervous system; germ-line clones arrest oogenesis at stage 8/9

dib (disembodied) Mitochondrial Low ecdysteroid FBgn0000449cytochrome P450 synthesis and various required for other mutant hydroxylation in phenotypes, including ecdysteroid no differentiation of the biosynthesis cuticle or the

head skeletongho (ghost) ?; involved in Mutants have

ecdysteroid undifferentiated cuticle FBgn0001106biosynthesis pathway due to defect in 20E

signaling but normal embryonic ecdysteroid titers

InR (insulin-like Insulin-like receptor, Heterozygous FBgn0013984receptor) Ibinding Drosophila mutants are dwarf,

insulin-like peptides female sterile, and long-lived; some mutants have lowered JH and 20E production

phm (phantom) ? Mutations affect the FBgn0004959embryonic cuticle, the embryonic head, and oogenesis; embryonic recessive lethal; mutants display a posterior contraction and poorly differentiated cuticle

sad (shadow) Mitochondrial Mutants have low larval FBgn0003312cytochrome P450 ecdysteroid titers; required for no differentiation of the hydroxylation in cuticle or the head ecdysteroid synthesis skeleton

shd (shade) P450 enzyme, Mutants have FBgn0003388converting ecdysone no differentiation of to 20E cuticle or head skeleton;

ovarian enzyme activity required for female fertility; low embryonic 20E production

spo (spook) ?, transporter for Mutants with low FBgn0003486shuttling embryonic ecdysteroid

titers; no differentiation of the cuticle or the head skeleton

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D. Interactions between JH and 20ESignaling

As discussed above (section IV.B), theinteractive effects of JH and 20E are sup-ported by the finding that the ecdysonereceptor (EcR), affecting fly life span,dimerizes with the USP, which is a can-didate receptor for JH (Dubrovsky, 2005;Truman & Riddiford, 2002). The poten-tial importance of this receptor complexfor aging is underscored by the sug-gestion that the molecular chaperonesHsp70 and Hsp90 and the histonedeacetylases Sin3A/Rpd3 interact withEcR/USP (Arbeitmann & Hogness, 2000;Tsai et al., 1999). These genes areknown to affect life span: overexpres-sion of chaperones extends longevity inD. melanogaster and C. elegans (Tataret al., 1997; Yokoyama et al., 2002), andmutation of the gene encoding Rpd3increases the life span of yeast andD. melanogaster (Kim et al., 1999;Rogina et al., 2003). It is thus interestingto speculate that the aging effects ofEcR, and potentially those of theEcR/USP complex, may be mediated bythese genes.

Interestingly, two 20E-induced tran-scription factors, Br and E75, seem to beintimately involved in the cross-talkbetween JH and 20E signaling (Dubrovsky,2005). JH and 20E are known to regulatethe expression of Br (Dubrovsky, 2005, andreferences therein), and preliminary datasuggest that Br interacts epistatically withthe putative JH receptor gene Met (Restifo& Wilson, 1998). In view of this interac-tion, it would be interesting to study theeffects of Br on aging. The E75 gene can beactivated by both JH and 20E, and the iso-form E75A is the first transcription factorwhose expression is known to be directlyinduced by JH (Dubrovsky, 2005, and ref-erences therein). Thus, E75 may be animportant mediator of the interactionbetween JH and 20E (Dubrovsky, 2005),and it would be important to examinewhether this locus affects aging.

V. Hormones, Nutrition, and Life Span

A. Nutrition Regulates JH and 20ESynthesis

In insects, nutritional status is wellknown to affect both developmentand reproduction. Proper nutritionprovides an organism with the energyrequired for development, growth,reproduction, and somatic maintenance.In some insects, insufficient nutritionsuppresses egg development by inhibit-ing CA or ovarian function, as seen inmosquitoes and some higher dipterans(Wheeler, 1996). For example, vitellogen-esis in the mosquito Aedes aegyptidepends on interactions among JH, 20E,and other endocrine factors. These hor-mones, however, are released only after ablood meal (Dhadialla & Raikhel, 1994).A protein-rich diet is also necessary toinitiate JH and 20E synthesis in otherhigher flies, such as the house fly M.domestica (Adams & Gerst, 1991, 1992)and the black blow fly Phormia regina(Liu et al., 1988; Yin & Stoffolano, 1990;Yin et al., 1990; Zou et al., 1989).

In D. melanogaster, yeast appears tobe a major stimulus in activating theendocrine system. For example, DILP pro-duction by the IPCs is activated uponyeast feeding in adult flies that wereyeast-deprived as third instar larvae (Tu &Tatar, 2003). DILPs may be required forJH and 20E synthesis because reducedinsulin signaling, as observed in InR andchico mutants, is known to result in JHand 20E deficiency. This hypothesis maybe supported by the observation thatJH synthesis is elevated upon adult yeastfeeding in flies that were yeast-deprived asthird instar larvae as compared to controlflies yeast-starved both during third larvalinstar and adulthood (Tu & Tatar, 2003).Thus, these results suggest that JH pro-duction is directly regulated by adult feed-ing, not larval feeding. 20E also respondsto nutrition in the fruit fly. For instance,


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flies fed on a diet consisting only of sugarand water produce much less ecdysteroidsat both 24 hours and 48 hours of adult-hood than yeast-fed control flies, whereasre-feeding female yeast after a 24-houryeast starvation period induces a highovarian ecdysteroid production (Schwartzet al., 1985). The above results thereforesuggest that the effects of dietary manipu-lation on life span may be mediated bynutrition-induced changes of pro-aginghormones such as JH and 20E.

B. Nutrient Sensing Pathways AffectAging Through Hormones

An adequate physiological response tonutrient levels is a key determinantof survival and somatic maintenance. InDrosophila, responses to nutrition aremediated by highly conserved nutrientsensing pathways, such as the insulin/IGFsignaling pathway and the target ofrapamycin (TOR) pathway, both of whichare major growth regulators (Chen et al.,1996; Ikeya et al., 2002; Oldham et al.,2000).

For the case of insulin signaling, thegene expression of dilp3 and dilp3, but notdilp2, is regulated by nutrient availabil-ity, and overexpressing dilp1–7 promotesgrowth (Ikeya et al., 2002). Interestingly,recent work shows intimate connectionsbetween DILP expression, insulin signal-ing, and the fat body, which presumablyrepresents a major nutrient sensing tissuein the fly. The fat body is an important tar-get for both JH and 20E, a major organ forthe synthesis of vitellogenins and antibac-terial peptides, and also has essentialnutrient storage functions.

In C. elegans, activation of the gene daf-16, a forkhead transcription factor down-stream of insulin signaling, is required forthe life-span extension induced by reducedinsulin signaling (Kenyon et al., 1993).Interestingly, in adult Drosophila, limitedactivation of dFOXO, the homolog ofC. elegans daf-16, in the head (pericere-

bral) fat body uniquely reduces expres-sion of neuronally synthesized DILP2 (butnot that of DILP3 and DILP5), repressesendogenous insulin-dependent signaling inthe abdominal fat body, and extends lifespan (Hwangbo et al., 2004). These non-autonomous and systemic effects suggestthat the adult head fat body is a majorendocrine site (Hwangbo et al., 2004).Furthermore, these studies indicate thatDILPs 2, 3, and 5 may have different phys-iological functions (Hwangbo et al., 2004;Ikeya et al., 2002). DILP2 responds to long-term downregulation of insulin signaling,whereas DILP3 and DILP5 do not. In con-trast, DILP3 and DILP5 respond to acutestarvation, whereas DILP2 does not. Thus,DILP3 and DILP5 may mediate the short-term response to changes in nutrient lev-els, whereas DILP2 may adjust a fly’s lifehistory in response to sustained periodsof reduced insulin signaling. Clearly, itwould be very informative to examinethe effects of individual DILPs on agingand on the production of JH and 20E indifferent developmental stages and underdifferent nutritional conditions (also seeFigure 15.1).

In addition to insulin signaling, theTOR and the slimfast pathways are majornutrition sensing pathways regulatinggrowth through the fat body (Colombaniet al., 2003). The involvement of thefat body in insulin, slimfast, and TOR sig-naling and its response to nutrition chal-lenge suggest that this tissue is importantfor both development and the regulationof aging. It is thus conceivable that down-regulating the slimfast and TOR pathwaysin the fat body may increase longevity.Indeed, recent work demonstrates thatconstitutive suppression of the dTORpathway, either ubiquitously or in the fatbody only, can extend life span (Kapahiet al., 2004). This life-span extensiondepends on nutritional conditions, sug-gesting a possible link between the TORpathway and dietary restriction (Kapahiet al., 2004). For example, in yeast


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(Saccharomyces cerevisiae), the TORpathway mediates cell growth in responseto nutrient availability, in part by inducingribosomal protein gene expression throughhistone acetylation (Rohde & Cardenas,2003). Thus, although the longevity exten-sion by downregulation of TOR may relateto caloric restriction, the underlyingmechanisms remain unclear. Interestingly,the dTOR and slimfast pathways areknown to interact with the insulin signal-ing pathway (Colombani et al., 2003;Hafen, 2004; Oldham & Hafen, 2003), butwhether and how dTOR and slimfastaffect hormones such as JH and 20Eremains unknown.

C. Connection Between CaloricRestriction, Hormones, and Life Span

To date, caloric restriction has been oneof the most effective interventionsextending life span in model organisms,including yeast, nematodes, flies, androdents (Kenyon, 2001; Masoro, 2000; butsee Carey et al., 2002). Similarly, as dis-cussed above, nutrition has major effectson the production of hormones (DILPs,JH, and 20E) that are intimately involvedin the regulation of aging. It is thus inter-esting to speculate that the longevityeffects of caloric restriction may be medi-ated by hormones.

In Drosophila, dietary restriction affectsboth reproduction and age-specific mortal-ity (Good & Tatar, 2001). For instance, lowadult nutrition induces an arrest in earlystem-cell proliferation and alters the fre-quency of cell death at two pre-vitel-logenic checkpoints; this response requiresintact insulin signaling (Drummond-Barbosa & Spradling, 2001). Similarly,caloric (or dietary) restriction can dramati-cally extend fly life span (Chapman &Partridge, 1996; Chippindale et al., 1993).In contrast, complete yeast starvation ofadult flies shortens life span (Chippindaleet al., 1993; Good & Tatar, 2001; Tu &Tatar, 2003), suggesting that yeast is a

crucial dietary component for survival andsomatic maintenance.

Yeast restriction during late develop-ment may silence insulin signalingthroughout metamorphosis into adult-hood, and thereby extend life span. Totest this hypothesis, Tu and Tatar (2003)studied aging in adult flies that wereyeast-deprived as third instar larvae. Asexpected, adult flies from yeast-deprivedlarvae phenocopied insulin signalingmutants by exhibiting prolonged devel-opmental time, small body size, reducedovariole number, and reduced fecundity(Tu & Tatar, 2003). Furthermore, yeastdeprivation reduced insulin signaling:adult flies from yeast deprived larvaehad reduced numbers of insulin-positivevesicles. However, unlike constitu-tive insulin signaling mutants of InR orchico, adults from yeast-deprived larvaedid not exhibit decreased age-specificmortality. Interestingly, yeast feedingincreased both insulin-like peptide andJH levels in adult flies from yeast-deprived larvae as compared to flies thatwere yeast-deprived throughout boththeir larval and adult life (Tu & Tatar,2003). This suggests that adult insulinand JH are regulated by adult nutritionalstate and that slowed aging specificallyrequires reduced insulin signaling or JHdeficiency in the adult.

A better understanding of the interplaybetween nutrition and hormones inaffecting aging is likely to come fromgenetic analysis. For example, recent find-ings show that mutations in the geneencoding the Rpd3 histone deacetylase,likely to be involved in caloric restriction,promote life span (Rogina et al., 2003).Interestingly, the histone deacetylasesSin3A/Rpd3 interact with the EcR/USPcomplex (Tsai et al., 1999), suggestingthat they may respond to JH and 20E sig-naling. Furthermore, in yeast, caloricrestriction extends life span by activatingSir2, a member of the sirtuin familyof NAD�-dependent protein deacetylases.


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In C. elegans, a homolog of Sir2 appears toact in the insulin signaling pathwayupstream of DAF-16; overexpression ofthe Sir2 gene extends worm life span in adaf-16 dependent manner (Tissenbaum &Guarente, 2001). Sir2 can also be activatedby several sirtuin-activating compounds(STACs) found in plants. For example, thenatural compound resveratrol, found inred wine, activates sirtuins in both C. ele-gans and Drosophila and extends bothworm and fly life span (Wood et al., 2004).The life-span extension induced by resver-atrol seems to be independent of caloricrestriction: resveratrol does not increaselife span in calorically restricted long-lived worms and flies, suggesting thatresveratrol affects life span through amechanism related to caloric restriction(Wood et al., 2004). It is noteworthy thatthe structure of resveratrol is somewhatsimilar to that of JH or 20E, with multiplesix-carbon rings and long carbon chainbranches, and it will be interesting todetermine whether and how JH and 20Esignal through a sirtuin pathway to regu-late aging under starvation conditions.However, in summary, our current under-standing of how nutrition and hormonalsignaling interact in affecting life spanremains very limited.

VI. Hormonal Effects on StressResistance and Immunity

A. Hormones and Stress Resistance

Upregulation of stress resistance is thoughtto be one of the major ways for organismsto regulate senescence (Jazwinski, 1996;Johnson et al., 1996; Lithgow, 1996).During the aging process, molecular chap-erones such as heat-shock proteins arethought to combat stress-related senescentdysfunction. For example, transgenicDrosophila with extra copies of the heatshock protein gene hsp70 show increasedlife span upon heat shock induction (Tataret al., 1997). Similarly, long-lived InR

mutants or transgenic flies overexpress-ing dFOXO have elevated resistance toparaquat, a commonly used free-radicalreagent (Hwangbo et al., 2004; Tatar et al.,2001b). Since InR mutants have low JHand 20E synthesis, it is possible that JHand 20E negatively regulate stress responsein flies. For example, high JH and 20E lev-els may increase reproduction at the costof decreased stress resistance and short-ened life span. This model is indeed sup-ported by recent experiments. For example,Salmon and colleagues (2001) found thatmethoprene application increased repro-duction in female fruit flies yet decreasedstress resistance, measured as the suscepti-bility to starvation and oxidative stress.Another good example comes from bury-ing beetles (Nicrophorus spp.), in whichstarvation stress decreases both the JH titerand fecundity, whereas treatment with JHor a JH analog reduces starvation resist-ance (Trumbo & Robinson, 2004).

B. Hormonal Effects on Immunity

The optimal function of the immune sys-tem is of crucial importance for survivaland somatic maintenance (Arlt &Hewison, 2004). For example, there aremany well-known links between longevityand immune-response genes in mammals(Flurkey et al., 2001), such as genes of themajor histocompatibility complex (MHC,see review by Ginaldi & Sternberg, 2003).In contrast to mammals, insects such asDrosophila do not possess adaptive immu-nity but exhibit innate immunity to com-bat microbial infections (Hoffmann, 2003;Tzou et al., 2002).

How hormones in general modulateimmune function in insects is not wellunderstood, but recent studies suggest thatJH regulates immunity (Rantala et al.2003; Rolff & Siva-Jothy, 2002). In themealworm beetle (Tenebrio molitor)immunity (phenoloxidase levels) isreduced by mating activity, and this trade-off seems to be regulated by JH (Rolff &


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Siva-Jothy, 2002). Application of the JHinhibitor fluvastatin increases immuneactivity; thus, JH specifically downregu-lates immune function (Rolff & Siva-Jothy,2002). Similarly, Rantala and colleagues(2003), using the same species, have shownthat the tradeoff between immune func-tion and sexual advertisement (i.e.,pheromone production) is mediated by JH.Thus, JH, a major gonadotropic hormone,has negative effects on immune function.This observation is interesting in view ofthe fact that reproductive hormones in ver-tebrates can often have negative effects onthe immune system, as is the case fortestosterone (e.g., Casto et al., 2001).

To initiate an improved understandingof hormonal effects on immunity, our lab-oratory has recently begun to explore theeffects of JH on primary immune responsegenes in Drosophila (M. Tatar, unpub-lished). In this preliminary experiment,flies were yeast-starved for 5 days to lowertheir endogenous JH titer and to synchro-nize their physiology. Subsequently, the JHanalog methoprene was topically appliedto individual flies, using ethanol-treatedflies as control. RNA transcript levels fromthese two groups were then analyzed usingAffymetrix gene chips (two replicate chipsper group). From these data, with theFatiGO software (Al-Shahrour et al., 2004),we find that genes with functions forresponse to biotic stimuli are relativelyenriched by JH treatment. This gene ontol-ogy category includes genes involved inresponse to microbial infection, starvation,and oxidative stress. Our set of JH respon-sive genes (criterion: at least two-foldchange in gene expression as compared tothe untreated control) consisted of 270probe sets (160 downregulated and 110upregulated, of a total 14,009 sets with6,142 annotated). Noticeably, for this set,12.9 percent of the genes showing changesin gene expression belong to the category“response to biotic stimuli.”

To test whether different categoricalresponses represent the biological effect

of JH as distinct from a chance observa-tion, we compared the observed represen-tation within gene ontology categoriesto the expected representation assuminga process of random sampling. Genesinvolved in “monosaccharide metabo-lism” were significantly over-represented(3.91 percent observed relative to1.21 percent expected from the annotatedgenome; Fisher’s Exact Test, P � 0.0032).As well, the categories “response topest/pathogen/parasite” and “response tobiotic stimuli” were enriched relative tochance expectation (respectively: 3.91percent versus 1.25 percent, P � 0.0040;13.04 percent versus 7.55 percent, P �0.0051). Genes involved in response tobiotic stimuli (including parasites andpathogens) are thus significantly over-represented, suggesting that JH influ-ences the state of adult defense response,including immune function.

Among the genes that show a responseto biotic stimuli, is there any bias towardupregulation versus downregulation? Forinstance, among “cell organization andbiogenesis” genes, 16.84 percent wereupregulated relative to 4.41 percentdownregulated (Fisher’s Exact Test, P �0.0024). In contrast, among genes in the“biotic response” category, 17.65 percentwere downregulated compared to only6.32 percent in the upregulated set(Fisher’s Exact Test, P � 0.0159). Thus,although JH stimulates the expression ofsome of these genes, these data overallsuggest that JH functions to suppressmany aspects of cellular and systemicstress response (see Table 15.3).

VII. Conclusions

Here we have reviewed the effects of hor-mones on fly aging. The hormonal mech-anisms affecting aging are manifest bothas genetic polymorphisms (as seen inendocrine deficient mutants) and as phe-notypic/physiological plasticity (as seen


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Table 15.3JH-Induced Drosophila Genes Involved in Defense or Immune Response. The table provides informationon genes involved in defense, stress, or immune response whose expression is either upregulated or down-regulated by application of the JH analog methoprene (M. Tatar, unpublished data). Further information on

each of these genes can be found at: http:/ /flybase.bio.indiana.edu.

Gene Name Function in Reaction to Downregulated or Flybase AccessionBiological Stimulus Upregulated

Dorsal-related Defense and immune Up FBgn0011274immunity factor response, response to

fungiTurandot M Humoral defense Up FBgn0031701

mechanismTetraspanin 96F B-cell mediated Up FBgn0027865

immunityTraf3 Defense response Up FBgn0030748CG6662 Defense response Up FBgn0035907Tetraspanin 74F Defense response Up FBgn0036769CG6435 Defense response, Down FBgn0034165

defense response to bacteria

CG7627 Defense response, Down FBgn0032026response to toxin

JH expoxide Defense response, Down FBgn0034405hydrolase 2 response to toxin

Drosocin Defense response to Down FBgn0010388gram-positive and -negative bacteria

Immune induced Defense response Down FBgn0034328molecule 23

PHGPx Defense response, Down FBgn0035438response to toxin

Ejaculatory bulb Response to virus Down FBgn0011695protein III

CG12780 Gram-negative Down FBgn0033301bacterial binding, defense response

Metchnikowin Antibacterial and Down FBgn0014865antifungal humoral response

CG1702 Defense response, Down FBgn0031117response to toxin

CG6426 Defense response to Down FBgn0034162bacteria

CG5397 Defense response Down FBgn0031327CG13422 Defense response to Down FBgn0034511

gram-negative bacteriaCG10307 Defense response Down FBgn0034655Transferrin 1 Defense response Down FBgn002235518 wheeler Antibacterial Down FBgn0004364

humoral response, immune response

Diptericin B Antibacterial Down FBgn0034407humoral response

Hemolectin Defense response Down FBgn0029167Diptericin Defense response to Down FBgn0004240

gram-negative bacteria

(continues)

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in reproductive diapause and senescenceplasticity). These sources of variation arelikely to involve common endocrine reg-ulatory mechanisms.

As in worms and rodents, reducedinsulin signaling can slow aging in the fly(see Figure 15.1). Downstream of insulinsignaling, fly aging seems to be regulatedby secondary hormones, such as thesesquiterpenoid JH and the steroid 20E.Several lines of evidence indicate that bothJH and 20E deficiency can dramaticallyslow aging while increasing stress resist-ance or immune function. Insects such asDrosophila may use JH and 20E to adap-tively coordinate (and, if necessary, tradeoff) the expression of the “reproductivefunction” versus the “survival function”in response to environmental cues such astemperature or nutrition. Yet, although insome cases JH and 20E co-affect life spanand reproduction, endocrine interventionstypically slow aging without causing costsin reproduction. The conditional uncou-pling of reproduction and survival willthus require us to rethink classical modelsfor the evolution of senescence, includingWilliams’ (1957) antagonistic pleiotropyhypothesis and the concept of senescencetradeoffs caused by costs of reproduction.Recent work on the Drosophila fat bodyhas also advanced our understanding ofthe tissue specificity of endocrine effectson life span. The fat body appears toregulate aging both autonomously andnon-autonomously by affecting insulin sig-

naling and probably secondary hormonessuch as JH and 20E.

Despite these insights into theendocrine control of aging in Drosophilaand other insects, there remain manyunresolved and difficult questions. Forinstance, we need to know how generalthe effects of JH and 20E upon aging are:Does JH or 20E deficiency invariablyextend life span, or is this a conditionaleffect, for instance depending on species,nutrition, temperature, and sex? Do bothJH and 20E have identical effects onlife span, or do they differ and how?Answering these questions will dependon suitable tools for manipulating JH and20E signaling. For example, do syntheticJH and 20E inhibitors or inhibitory neu-ropeptides such as allatostatins extendlife span? Does overexpression of JH- or20E-degrading enzymes in transgenicallyengineered insects slow aging? Similarly,the molecular details of JH and, to alesser extent, 20E signaling remain rela-tively poorly understood. This raisesmany difficult questions: What is themolecular nature of the JH receptor, andhow does it modulate aging? Do most, ifnot all, JH and 20E signaling genes affectaging? Through which downstream genesdo the JH and 20E signaling pathwaysregulate aging? How exactly does insulinsignaling affect JH and 20E signaling?What is the nature of the secondaryhormones mediating the effects of fatbody insulin signaling on whole-animal


Gene Name Function in Reaction to Downregulated or Flybase AccessionBiological Stimulus Upregulated

CG1681 Defense response Down FBgn0030484CG2736 Defense response Down FBgn0035090CG8336 Defense response Down FBgn0036020

takeout Behavioral response Down FBgn0039298to starvation

CG18522 Defense response; Down FBgn0038347reactive oxygen species metabolism

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life span? How do caloric restriction andhormones interact at the molecular levelto affect longevity? We can make furtherobservations and ask related questions.For example, JH and 20E seem to modu-late evolutionarily relevant tradeoff rela-tionships between fitness components.How, mechanistically, do JH and 20Emodulate these tradeoffs between repro-duction and survival, stress resistance,and somatic maintenance? Are theendocrine loci affecting these tradeoffsgenetically variable and subject to natu-ral selection in wild populations? Finally,we may ask: Do other insect hormonesthan JH and 20E have major effects onthe aging phenotype? What are the func-tional equivalents of JH and 20E in otherorganisms and how do they affect aging?

These are but a few of the most press-ing questions to be addressed in the nearfuture. To address them, we will needto combine molecular and evolutionarygenetics, endocrinology, nutritional physi-ology, and biodemography. We have manyhypotheses, patterns, and models, but lit-tle solid data. Understanding the geneticand endocrine regulation of aging remainsa fundamental, yet promising, challengefor modern molecular biogerontology.

Acknowledgements

We thank Kyung-Jin Min for helpful discus-sions, James Cypser for help with the JH genechip study, and Faye Lemieux and the othermembers of the Tatar Lab for producing manyof the data we have summarized. This workwas supported by the American Federationfor Aging Research (MPT); the Swiss NationalScience Foundation, the Roche ResearchFoundation, and the Swiss Study Foundation(TF); the National Institute of Health; and theEllison Medical Foundation (MT).

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