BACKGROUND
juvenile hormone  plays a central role in insect development where it interacts with ecdysone to maintain larval status quo between molts  <cit> .
in adults, jh takes on different functions.
from work with a variety of insects, there is consensus that jh produced in adult corpora allata modulates ovarian maturation, in part through its regulation of yolk protein uptake while ecdysone derived from the follicle cells induces yolk protein synthesis in fat bodies  <cit> .
jh in the adult also affects learning, migration, diapause and innate immunity  <cit> .
in the monarch butterfly, some grasshoppers and the firebug pyrrhocoris apterus, surgical allatectomy extends lifespan, suggesting that jh is a pro-aging hormone  <cit> .
drosophila melanogaster produces three forms of juvenile hormone: methyl farnesoate , jhiii and bisepoxide jhiii   <cit> .
jhbiii is unique to dipterans and is the most abundant jh in flies.
in other insects, exogenous jh or juvenile hormone analogs , such as methoprene, induce supernumerary juvenile or pupal stages and block metamorphosis, but drosophila larvae treated with jh analogs still initiate metamorphosis  <cit> , although high doses of jha produce a mosaic adult where the abdomen is covered with pupal cuticle  <cit> .
the high dosage required to induce this phenotype, and an artifact produced by a common genetic marker , have complicated our ability to study the roles of jh in drosophila development  <cit> .
even less is known about the role of jh in the drosophila adult, in part because treating wildtype adults with jh analogs produces few obvious phenotypes and because surgical allatectomy is not practical.
one potential role for jh in adult drosophila became apparent in studies of fly insulin-like receptor mutants.
drosophila insulin-like receptor  mutants have ovaries similar to those seen in wildtype flies during reproductive diapause  <cit> .
drosophila reproductive diapause is maintained in part by reduced jh  <cit> , and insulin-like signaling mutants were found to be jh deficient  <cit> .
inr mutants are long-lived, and jha treatment restores lifespan toward that of wildtype flies.
together these observations motivated the question of our current study: is jh deficiency sufficient to extend lifespan in drosophila, and if so, by what mechanism?
jh production in drosophila might be experimentally reduced in several ways.
a classic genetic approach used the apterous mutant, which regulates development in wing, muscle and axons, and neuroendocrine control of juvenile hormone as well as other neuropeptides  <cit> .
more recently, the p{gal4} insertion  was found to be expressed in larval corpora allata <cit>  and riddiford et al.
<cit>  used aug <dig> to drive uas-grim to induce apoptosis in larval corpora allata .
allatectomized larvae produced small pupae that died at head eversion and showed many photoreceptor development defects.
liu et al.
<cit>  used this genotype to reveal how the jh-interacting proteins met  and gce  regulate larval fat body programmed cell death.
to study adult traits, gruntenko et al.
<cit>  drove ca apoptosis with a combination of uas-reaper and uas-hid.
unlike aug21 > uas-grim, this genotype produced viable adults and showed impaired jh metabolism, decreased resistance to heat stress, and reduced female fecundity.
this genetically mediated ca-ablation also increased the level of dopamine in young females, accompanied by decreases in the activity of enzymes responsible for both the synthesis and degradation of this amine  <cit> .
here, we modified these approaches to study how aging is affected by allatectomy that reduces endogenous jh in adult drosophila.
we used aug <dig> to over-express nuclear inhibitor of protein phosphatase type  <dig>   <cit>  and thus eliminate the ca of newly eclosed adults.
from these adults and their coisogenic controls, we measured lifespan and tested potential mechanisms by which reduced jh could contribute to longevity assurance.
allatectomy reduced egg production as expected, and fecundity was partially restored by treating adults with exogenous jha.
reduced jh extended lifespan in fertile flies and, importantly, it also did so in adults made sterile by the dominant ovod <dig> mutation, which inhibits egg maturation  <cit> .
these data suggest that reduced jh extends lifespan by mechanisms independent of direct physiological trade-offs with egg production.
allatectomy extended lifespan robustly in flies fed different concentrations of yeast, eliminating a potential explanation based on mechanisms of dietary restriction.
finally, we analyzed gene expression profiles from allatectomized adults when fertile and when ovod <dig> sterile, and from the intersection of these data identified soma-related genes through which jh may potentially modulate longevity.
RESULTS
corpora allata knockout
jh is normally abundant in females, at least up to age  <dig> days  <cit> .
here, the genotype aug21-gal <dig>  uas-gfp >uas-nipp <dig>  produced adults within  <dig> to  <dig> hours post-eclosion with small to non-existent corpora allata and limited levels of jh as measured by gas chromatography–mass spectrometry.
females of wildtype and parental genotypes contained between  <dig> and  <dig> pg jh per gram body mass while cako females had less than  <dig> pg/g .
cako reduces fecundity and impedes adult fat body development
cako strongly reduced egg production.
when measured from caged cohorts, average daily fecundity of control genotypes peaked at  <dig> to  <dig> eggs per female, while cako females laid less than  <dig> eggs per day .
fecundity was also measured from individually maintained females .
from eclosion to age  <dig> days, wildtype females produced two-fold more eggs than cako.
notably, the variance in total egg production was two-fold greater in cako, suggesting there were underlying differences in the efficiency of corpora allata knockout.
thus, when measured from a collection of caged adults, the net sample of cako females may yield some eggs because a few females retain sufficient levels of jh.
individually held cako females show that some individuals indeed produce nearly wildtype quantities of eggs while some produce very few eggs.
in the cases where eggs are produced, these females may have initiated these eggs before the ca-ablation was complete, retained enough residual jh after the ablation-period to mature some eggs, or have the capacity to produce eggs in the absence of jh.
we cannot distinguish between these alternatives because measuring jh levels requires destructive samples comprised of many individuals.
to test if reduced fecundity was caused specifically by the loss of jh in allatectomized females we measured daily fecundity in adults exposed to methoprene .
as previously reported  <cit> , jha did not affect egg production in wildtype females but jha increased egg production in allatectomized females .
notably, complete rescue of daily fecundity was statistically achieved only after seven days of jha treatment, suggesting that the loss of jh in cako impairs some aspect of post-eclosion ontogeny that takes some time to restore.
in many insects, including drosophila, jh modulates fecundity at least in part because the hormone is required to induce yolk proteins uptake into oocytes  <cit> , while ecdysone produced from egg follicles induces yolk protein mrna expressed in the fat body  <cit> .
we found yolk protein mrna  was reduced in cako females while treatment of these flies with jha partially restored their yolk protein expression .
likewise, wildtype females had mature ovarioles that contained previtellogenic egg chambers at stages  <dig> to  <dig>  one or two yolk-containing egg chambers  and a mature egg .
in contrast, ovarioles from cako females contained only previtellogenic egg chambers, or they were ‘discontinuous’ because they contained previtellogenic chambers with one mature egg.
jha treatment increased the proportion of mature ovarioles in cako females but did not affect the distribution of ovariole types in wildtype females.
together these data suggest that cako females have reduced fecundity because they have limited yolk protein.
the discontinuous quality of cako ovarioles may arise because newly eclosed females possess enough jh at eclosion to initiate one mature egg but with allatectomy they cannot sustain vitellogenesis.
adult fat body is a major site of yolk protein synthesis.
at emergence, drosophila adults contain larval fat body  <cit> .
this larval fat body undergoes histolysis in  <dig> to  <dig> hours in wildtype adults, while adult fat body develops from progenitor cells.
postlethwait and jones  <cit>  recognized that jh plays a role in larval fat histolysis.
here, we evaluated fat body status in 15-day-old females.
among  <dig> wildtype females all but one exclusively contained adult fat body.
among  <dig> cako females,  <dig> retained some larval fat body while  <dig> contained only adult fat body.
on the other hand,  <dig> of  <dig> cako females treated with jha contained only adult fat body.
cako adults also expressed mrna from genes that are characteristic of larval fat body, including larval serum protein  <dig>  and fat body protein  <dig>  .
these larval-associated mrna were repressed when cako females were treated with jha soon after eclosion.
the impaired fecundity and low expression of yolk protein mrna of cako adults may arise in part because they delay full development of adult fat bodies.
treating cako adults with jha induces the transition from larval to adult fat body and may thus facilitate yolk protein production and fecundity.
cako extends lifespan independent of egg production
surgical allatectomy extends lifespan in desert grasshoppers, monarch butterflies and the firebug pyrrhocoris apterus <cit> .
in each case, life expectancy of adults treated with jh or jha is reduced to that of sham-operated adults.
while these results are robust, little is known about the mechanisms by which jh affects lifespan.
drosophila provides a tractable system to study potential mechanism, and cako offers a useful platform to describe how loss of jh modulates adult drosophila lifespan.
the aug <dig> gfp and nipp <dig> stocks were introgressed into two wildtype backgrounds.
survival of female offspring with the aug <dig> gfp; nipp <dig>  genotype were then compared to otherwise coisogenic parental genotypes and wildtype stocks .
in the w <dig> background, cako increased adult survival relative to all genetic controls  by consistently reducing age-specific adult mortality .
female longevity was likewise extended when cako was introgressed into the white-dahomey  wildtype background .
although the role of jh in males is generally enigmatic, cako was also sufficient to extend male lifespan .
to establish whether loss of jh upon allatectomy is sufficient to extend lifespan we exposed control and cako adult females to the jha methoprene .
longevity was extended in cako adults exposed to vehicle relative to similarly treated wildtype controls.
survival of cako flies exposed to jha was reduced to that of wildtype flies with or without jha.
as seen with surgically allatectomized insects, the longevity benefit from genetically ablated ca in drosophila is reversed by jha.
the observed gain in lifespan appears to be specifically caused by the loss of jh in cako adults.
longevity is extended in many animals when fecundity is experimentally repressed, presumably by preventing physiological trade-offs between survival and egg production  <cit> .
reduced egg production is also a feature of insect reproductive diapause, a state induced in part by reduced jh synthesis  <cit> .
reproductive diapause slows aging in drosophila melanogaster as it does in the endemic d. triauraria  and d. littoralis   <cit> .
eliminating reproductive trade-offs may account for the increased longevity of cako.
to test this hypothesis we backcrossed ovod <dig> into w <dig> and generated ovod1; aug <dig>  gfp.
ovod <dig> dominantly represses egg maturation by autonomously blocking oocyte progression at stages  <dig> and  <dig>  <cit> .
this mutation has been used previously to demonstrate that drosophila longevity assurance produced by dietary restriction is not caused by reduction in egg maturation  <cit> .
importantly, our data show that cako arrests oogenesis at the pre-vitellogenic check-point of stages  <dig> to  <dig>  <cit> .
both ovod <dig> and cako; ovod <dig> females are sterile because they arrest oocytes at stages  <dig> to 3; the oocyte arrest of ovod <dig> supersedes the later stage arrest of cako.
in this way, both genotypes similarly avoid survival costs of reproduction associated specifically with egg production.
all the same, sterile cako; ovod <dig> females remained long-lived relative to sterile ovod <dig> controls .
avoiding costs of egg production does not fully account for the mechanism by which cako extends lifespan.
cako independent of reproduction modulates somatically related genes
these results suggest that jh controls aging to some extent because it directly affects mechanisms of somatic survival.
dietary restriction is one process that may somatically control drosophila longevity independent of egg production  <cit> .
to test if reduced jh and dietary restriction mediate lifespan through common mechanisms, we measured lifespan of adults maintained on five diets ranging from  <dig> % to 16% yeast.
median lifespan was increased in both wildtype and cako adults when the quantity of dietary yeast was reduced.
the lifespan of cako was greater than that of wildtype at every diet level , and the magnitude of mortality differences between these genotypes was similar across all diets .
dietary restriction and cako thus appear to affect longevity through independent mechanisms.
extended longevity in drosophila is often associated with resistance to exogenous stress  <cit> .
resistance to heat and oxidative stress is also a feature of drosophila reproductive diapause, and this trait is blunted when adults are treated with jha  <cit> .
gruntenko et al.
<cit>  found allatectomized females, but not males, were resistant to heat stress.
here we see that cako extended survival in females exposed to h2o <dig> and this capacity was reduced when adults were simultaneously treated with jha .
cako, however, did not increase survival when flies were starved, although starvation survival was reduced when cako and wildtype females were treated with jha .
cako thus appears to extend lifespan directly through effects upon somatic physiology, stress resistance or homeostasis rather than indirectly by avoiding costs of egg production.
to identify somatic processes affected by jh independent of egg production, we compared gene expression profiles of cako females to expression profiles of wildtype controls, and we compared cako; ovod <dig> females to sterile ovod <dig>  controls.
these overlapping sets revealed  <dig> genes with changes correlated to longevity assurance but independent of reproductive state .
the genes are categorized into two groups.
group  <dig> includes transcripts that increase in cako and reflect genes that are normally repressed by jh in wildtype flies  .
the induction of these genes is positively correlated with longevity extension; these genes thus contain candidates that confer longevity assurance.
group  <dig> includes transcripts that decrease in cako and thus reflect genes that are otherwise induced in animals with intact corpora allata  .
group  <dig> genes reflect genes that are associated with pro-aging processes.
besides representing different individual genes, these groups also differ in their biological functions represented through gene ontology  where genes induced by jh involve processes of protein digestion or catabolism while those repressed by jh are statistically associated with oxidative reduction and substrate transport.
to validate this analysis for a sample of genes, we conducted real time quantitative pcr from cako and wildtype females in both sterile  and fertile genetic backgrounds, and when fertile flies were treated with jha .
from group  <dig>  we tested obp99b.
mrna of obp99b was increased by allatectomy in both reproductive states , and this expression was repressed when females were treated with jha .
obp99b is an odorant binding protein expressed in antennae and in fat body, and its expression was previously identified as jh-responsive in allatectomized larvae  <cit> .
some odorant receptor proteins can modulate drosophila aging; mutants of orb83b extend lifespan and enhance stress resistance  <cit> .
obp99b was also independently identified as a candidate gene to affect longevity in a mapping analysis of wildtype drosophila  <cit> .
among genes induced by jh and associated with a pro-aging state , we confirmed the expected mrna responses for jon25bii and ana .
the gene anachronism  encodes a secreted glycoprotein that participates in neuroblast development, neuromuscular function and wing development  <cit> .
jon25bii is a predicted endopeptidase expressed abundantly in the midgut and is associated with drosophila immune and xenobiotic responses  <cit> .
jon25bii and ana have been identified as factors expressed in a fly model of mitochondrial disease  <cit> ; they may contribute to aging by facilitating mitochondria functions that reduce survival  <cit> .
besides factors in these overlap groups, several genes of special interest changed in response to cako but only in one reproductive condition .
kruppel homolog  <dig>  is a jh-regulated zinc-finger transcription factor where it functions in processes such as metamorphosis, neurogenesis and foraging behavior  <cit> .
in our arrays and confirmatory qpcr, mrna for kr-h <dig> decreased in cako females of the fertile background exclusively, and this response was reversed by jha treatment .
the gene takeout is a small peptide with sequence similarity to manduca sexta juvenile hormone binding protein  <cit> , although functional studies in drosophila have yet to confirm jh binding for the takeout protein.
bauer et al.
<cit>  report that takeout is up-regulated in experimental conditions that extend drosophila lifespan, and transgenic over-expression of takeout is sufficient to extend lifespan.
the longevity benefit of takeout overexpression is repressed when flies are treated with jha , and jha increases takeout mrna in wildtype flies  <cit> .
in our analysis, however, , mrna of takeout does not differ among wildtype and cako, but it is strongly elevated in ovod <dig>  and this difference is eliminated in allatectomized ovod <dig> females.
we find no effect of jha treatment upon takeout mrna abundance in either wildtype or cako genotypes.
the regulation of takeout by jh appears to be complex and must depend on uncontrolled factors that differ among these studies.
ubiquitously expressed mutations of the insulin/igf signaling system in drosophila extend lifespan and reduce the production of jh from adult corpora allata <cit> .
on the other hand, an inverse interaction between jh and insulin signaling was seen in tribolium castaneum where reduced jh promoted expression of insulin-like peptides  <cit> .
here we see that eliminating adult jh in drosophila does not affect abundance of dilp <dig> or dilp <dig> mrna .
abundance of dilp <dig> mrna decreases with cako in the sterile ovod <dig> background, but not in fertile flies .
in contrast, dilp <dig> mrna declines strongly in cako adults in the fertile background, while dilp <dig> mrna is already low in all sterile ovod <dig> females.
dilp <dig> is predominantly expressed in adult fat body unlike neuron-expressed dilp <dig>  dilp <dig> and dilp <dig> <cit> .
overall, these data suggest that the impact of jh on aging is distal to longevity-regulatory dilp <dig>  while jh contributes to the expression of dilp <dig>  perhaps through its control of adult fat body.
CONCLUSIONS
juvenile hormone is an integrator of insect development, physiology and life history.
in adult insects jh modulates alternative states of active reproduction versus dispersal, reproductive diapause and torpor  <cit> .
jh supports the reproductive state in part by inducing fatty acid oxidation  <cit> , and yolk protein uptake  <cit> .
in contrast, jh is reduced during adult reproductive diapause or torpor.
such adults can be migratory  <cit> , express lipogenic enzymes, accumulate lipids and triglycerides  <cit> , resist exogenous stress, and live a relatively long time  <cit> .
many features of these syndromes appear in drosophila, where adults in reproductive diapause have reduced titers of jh and increased triglycerides, glycogen, stress resistance and lifespan  <cit> .
our current work establishes that reduced jh is sufficient to extend drosophila lifespan.
allatectomy increases drosophila lifespan and this survival benefit is reduced by treatment with jha.
although allatectomy also reduces fecundity in a jha sensitive manner, the longevity of allatectomized adults is still expressed in otherwise sterile females.
at a physiological level, jh appears to independently modulate egg production and survival.
under some ecological conditions a trade-off may occur between these fitness traits, and in this way selection would favor juvenile hormone-mediated pleiotropy between reproduction and aging.
hodkova  <cit>  makes a similar argument for pyrrhocoris apterus where surgical allatectomy extends lifespan in both fertile and surgically sterilized females.
our microarray analysis demonstrates there are at least some direct somatic physiological targets of jh that may be independent of its effects upon reproduction.
this gene set provides a platform for hypotheses on how jh may mechanistically control longevity assurance.

 juvenile hormone plays a central role in insect development where it interacts with ecdysone to maintain larval status quo between molts. in adults, jh takes on different functions. from work with a variety of insects, there is consensus that jh produced in adult corpora allata modulates ovarian maturation, in part through its regulation of yolk protein uptake while ecdysone derived from the follicle cells induces yolk protein synthesis in fat bodies. jh in the adult also affects learning, migration, diapause and innate immunity. in the monarch butterfly, some grasshoppers and the firebug pyrrhocoris apterus, surgical allatectomy extends lifespan, suggesting that jh is a pro-aging hormone. drosophila melanogaster produces three forms of juvenile hormone: methyl farnesoate, jhiii and bisepoxide jhiii. jhbiii is unique to dipterans and is the most abundant jh in flies. in other insects, exogenous jh or juvenile hormone analogs, such as methoprene, induce supernumerary juvenile or pupal stages and block metamorphosis, but drosophila larvae treated with jh analogs still initiate metamorphosis, although high doses of jha produce a mosaic adult where the abdomen is covered with pupal cuticle. the high dosage required to induce this phenotype, and an artifact produced by a common genetic marker, have complicated our ability to study the roles of jh in drosophila development. even less is known about the role of jh in the drosophila adult, in part because treating wildtype adults with jh analogs produces few obvious phenotypes and because surgical allatectomy is not practical. one potential role for jh in adult drosophila became apparent in studies of fly insulin-like receptor mutants. drosophila insulin-like receptor mutants have ovaries similar to those seen in wildtype flies during reproductive diapause. drosophila reproductive diapause is maintained in part by reduced jh, and insulin-like signaling mutants were found to be jh deficient. inr mutants are long-lived, and jha treatment restores lifespan toward that of wildtype flies. together these observations motivated the question of our current study: is jh deficiency sufficient to extend lifespan in drosophila, and if so, by what mechanism? jh production in drosophila might be experimentally reduced in several ways. a classic genetic approach used the apterous mutant, which regulates development in wing, muscle and axons, and neuroendocrine control of juvenile hormone as well as other neuropeptides. more recently, the p{gal4} insertion was found to be expressed in larval corpora allata and riddiford et al. used aug to drive uas-grim to induce apoptosis in larval corpora allata. allatectomized larvae produced small pupae that died at head eversion and showed many photoreceptor development defects. liu et al. used this genotype to reveal how the jh-interacting proteins met and gce regulate larval fat body programmed cell death. to study adult traits, gruntenko et al. drove ca apoptosis with a combination of uas-reaper and uas-hid. unlike aug21 > uas-grim, this genotype produced viable adults and showed impaired jh metabolism, decreased resistance to heat stress, and reduced female fecundity. this genetically mediated ca-ablation also increased the level of dopamine in young females, accompanied by decreases in the activity of enzymes responsible for both the synthesis and degradation of this amine. here, we modified these approaches to study how aging is affected by allatectomy that reduces endogenous jh in adult drosophila. we used aug to over-express nuclear inhibitor of protein phosphatase type and thus eliminate the ca of newly eclosed adults. from these adults and their coisogenic controls, we measured lifespan and tested potential mechanisms by which reduced jh could contribute to longevity assurance. allatectomy reduced egg production as expected, and fecundity was partially restored by treating adults with exogenous jha. reduced jh extended lifespan in fertile flies and, importantly, it also did so in adults made sterile by the dominant ovod mutation, which inhibits egg maturation. these data suggest that reduced jh extends lifespan by mechanisms independent of direct physiological trade-offs with egg production. allatectomy extended lifespan robustly in flies fed different concentrations of yeast, eliminating a potential explanation based on mechanisms of dietary restriction. finally, we analyzed gene expression profiles from allatectomized adults when fertile and when ovod sterile, and from the intersection of these data identified soma-related genes through which jh may potentially modulate longevity.  corpora allata knockout jh is normally abundant in females, at least up to age days. here, the genotype aug21-gal uas-gfp >uas-nipp produced adults within to hours post-eclosion with small to non-existent corpora allata and limited levels of jh as measured by gas chromatography–mass spectrometry. females of wildtype and parental genotypes contained between and pg jh per gram body mass while cako females had less than pg/g. cako reduces fecundity and impedes adult fat body development cako strongly reduced egg production. when measured from caged cohorts, average daily fecundity of control genotypes peaked at to eggs per female, while cako females laid less than eggs per day. fecundity was also measured from individually maintained females. from eclosion to age days, wildtype females produced two-fold more eggs than cako. notably, the variance in total egg production was two-fold greater in cako, suggesting there were underlying differences in the efficiency of corpora allata knockout. thus, when measured from a collection of caged adults, the net sample of cako females may yield some eggs because a few females retain sufficient levels of jh. individually held cako females show that some individuals indeed produce nearly wildtype quantities of eggs while some produce very few eggs. in the cases where eggs are produced, these females may have initiated these eggs before the ca-ablation was complete, retained enough residual jh after the ablation-period to mature some eggs, or have the capacity to produce eggs in the absence of jh. we cannot distinguish between these alternatives because measuring jh levels requires destructive samples comprised of many individuals. to test if reduced fecundity was caused specifically by the loss of jh in allatectomized females we measured daily fecundity in adults exposed to methoprene. as previously reported, jha did not affect egg production in wildtype females but jha increased egg production in allatectomized females. notably, complete rescue of daily fecundity was statistically achieved only after seven days of jha treatment, suggesting that the loss of jh in cako impairs some aspect of post-eclosion ontogeny that takes some time to restore. in many insects, including drosophila, jh modulates fecundity at least in part because the hormone is required to induce yolk proteins uptake into oocytes, while ecdysone produced from egg follicles induces yolk protein mrna expressed in the fat body. we found yolk protein mrna was reduced in cako females while treatment of these flies with jha partially restored their yolk protein expression. likewise, wildtype females had mature ovarioles that contained previtellogenic egg chambers at stages to one or two yolk-containing egg chambers and a mature egg. in contrast, ovarioles from cako females contained only previtellogenic egg chambers, or they were ‘discontinuous’ because they contained previtellogenic chambers with one mature egg. jha treatment increased the proportion of mature ovarioles in cako females but did not affect the distribution of ovariole types in wildtype females. together these data suggest that cako females have reduced fecundity because they have limited yolk protein. the discontinuous quality of cako ovarioles may arise because newly eclosed females possess enough jh at eclosion to initiate one mature egg but with allatectomy they cannot sustain vitellogenesis. adult fat body is a major site of yolk protein synthesis. at emergence, drosophila adults contain larval fat body. this larval fat body undergoes histolysis in to hours in wildtype adults, while adult fat body develops from progenitor cells. postlethwait and jones recognized that jh plays a role in larval fat histolysis. here, we evaluated fat body status in 15-day-old females. among wildtype females all but one exclusively contained adult fat body. among cako females, retained some larval fat body while contained only adult fat body. on the other hand, of cako females treated with jha contained only adult fat body. cako adults also expressed mrna from genes that are characteristic of larval fat body, including larval serum protein and fat body protein. these larval-associated mrna were repressed when cako females were treated with jha soon after eclosion. the impaired fecundity and low expression of yolk protein mrna of cako adults may arise in part because they delay full development of adult fat bodies. treating cako adults with jha induces the transition from larval to adult fat body and may thus facilitate yolk protein production and fecundity. cako extends lifespan independent of egg production surgical allatectomy extends lifespan in desert grasshoppers, monarch butterflies and the firebug pyrrhocoris apterus. in each case, life expectancy of adults treated with jh or jha is reduced to that of sham-operated adults. while these results are robust, little is known about the mechanisms by which jh affects lifespan. drosophila provides a tractable system to study potential mechanism, and cako offers a useful platform to describe how loss of jh modulates adult drosophila lifespan. the aug gfp and nipp stocks were introgressed into two wildtype backgrounds. survival of female offspring with the aug gfp; nipp genotype were then compared to otherwise coisogenic parental genotypes and wildtype stocks. in the w background, cako increased adult survival relative to all genetic controls by consistently reducing age-specific adult mortality. female longevity was likewise extended when cako was introgressed into the white-dahomey wildtype background. although the role of jh in males is generally enigmatic, cako was also sufficient to extend male lifespan. to establish whether loss of jh upon allatectomy is sufficient to extend lifespan we exposed control and cako adult females to the jha methoprene. longevity was extended in cako adults exposed to vehicle relative to similarly treated wildtype controls. survival of cako flies exposed to jha was reduced to that of wildtype flies with or without jha. as seen with surgically allatectomized insects, the longevity benefit from genetically ablated ca in drosophila is reversed by jha. the observed gain in lifespan appears to be specifically caused by the loss of jh in cako adults. longevity is extended in many animals when fecundity is experimentally repressed, presumably by preventing physiological trade-offs between survival and egg production. reduced egg production is also a feature of insect reproductive diapause, a state induced in part by reduced jh synthesis. reproductive diapause slows aging in drosophila melanogaster as it does in the endemic d. triauraria and d. littoralis. eliminating reproductive trade-offs may account for the increased longevity of cako. to test this hypothesis we backcrossed ovod into w and generated ovod1; aug gfp. ovod dominantly represses egg maturation by autonomously blocking oocyte progression at stages and. this mutation has been used previously to demonstrate that drosophila longevity assurance produced by dietary restriction is not caused by reduction in egg maturation. importantly, our data show that cako arrests oogenesis at the pre-vitellogenic check-point of stages to. both ovod and cako; ovod females are sterile because they arrest oocytes at stages to 3; the oocyte arrest of ovod supersedes the later stage arrest of cako. in this way, both genotypes similarly avoid survival costs of reproduction associated specifically with egg production. all the same, sterile cako; ovod females remained long-lived relative to sterile ovod controls. avoiding costs of egg production does not fully account for the mechanism by which cako extends lifespan. cako independent of reproduction modulates somatically related genes these results suggest that jh controls aging to some extent because it directly affects mechanisms of somatic survival. dietary restriction is one process that may somatically control drosophila longevity independent of egg production. to test if reduced jh and dietary restriction mediate lifespan through common mechanisms, we measured lifespan of adults maintained on five diets ranging from % to 16% yeast. median lifespan was increased in both wildtype and cako adults when the quantity of dietary yeast was reduced. the lifespan of cako was greater than that of wildtype at every diet level, and the magnitude of mortality differences between these genotypes was similar across all diets. dietary restriction and cako thus appear to affect longevity through independent mechanisms. extended longevity in drosophila is often associated with resistance to exogenous stress. resistance to heat and oxidative stress is also a feature of drosophila reproductive diapause, and this trait is blunted when adults are treated with jha. gruntenko et al. found allatectomized females, but not males, were resistant to heat stress. here we see that cako extended survival in females exposed to h2o and this capacity was reduced when adults were simultaneously treated with jha. cako, however, did not increase survival when flies were starved, although starvation survival was reduced when cako and wildtype females were treated with jha. cako thus appears to extend lifespan directly through effects upon somatic physiology, stress resistance or homeostasis rather than indirectly by avoiding costs of egg production. to identify somatic processes affected by jh independent of egg production, we compared gene expression profiles of cako females to expression profiles of wildtype controls, and we compared cako; ovod females to sterile ovod controls. these overlapping sets revealed genes with changes correlated to longevity assurance but independent of reproductive state. the genes are categorized into two groups. group includes transcripts that increase in cako and reflect genes that are normally repressed by jh in wildtype flies. the induction of these genes is positively correlated with longevity extension; these genes thus contain candidates that confer longevity assurance. group includes transcripts that decrease in cako and thus reflect genes that are otherwise induced in animals with intact corpora allata. group genes reflect genes that are associated with pro-aging processes. besides representing different individual genes, these groups also differ in their biological functions represented through gene ontology where genes induced by jh involve processes of protein digestion or catabolism while those repressed by jh are statistically associated with oxidative reduction and substrate transport. to validate this analysis for a sample of genes, we conducted real time quantitative pcr from cako and wildtype females in both sterile and fertile genetic backgrounds, and when fertile flies were treated with jha. from group we tested obp99b. mrna of obp99b was increased by allatectomy in both reproductive states, and this expression was repressed when females were treated with jha. obp99b is an odorant binding protein expressed in antennae and in fat body, and its expression was previously identified as jh-responsive in allatectomized larvae. some odorant receptor proteins can modulate drosophila aging; mutants of orb83b extend lifespan and enhance stress resistance. obp99b was also independently identified as a candidate gene to affect longevity in a mapping analysis of wildtype drosophila. among genes induced by jh and associated with a pro-aging state, we confirmed the expected mrna responses for jon25bii and ana. the gene anachronism encodes a secreted glycoprotein that participates in neuroblast development, neuromuscular function and wing development. jon25bii is a predicted endopeptidase expressed abundantly in the midgut and is associated with drosophila immune and xenobiotic responses. jon25bii and ana have been identified as factors expressed in a fly model of mitochondrial disease; they may contribute to aging by facilitating mitochondria functions that reduce survival. besides factors in these overlap groups, several genes of special interest changed in response to cako but only in one reproductive condition. kruppel homolog is a jh-regulated zinc-finger transcription factor where it functions in processes such as metamorphosis, neurogenesis and foraging behavior. in our arrays and confirmatory qpcr, mrna for kr-h decreased in cako females of the fertile background exclusively, and this response was reversed by jha treatment. the gene takeout is a small peptide with sequence similarity to manduca sexta juvenile hormone binding protein, although functional studies in drosophila have yet to confirm jh binding for the takeout protein. bauer et al. report that takeout is up-regulated in experimental conditions that extend drosophila lifespan, and transgenic over-expression of takeout is sufficient to extend lifespan. the longevity benefit of takeout overexpression is repressed when flies are treated with jha, and jha increases takeout mrna in wildtype flies. in our analysis, however,, mrna of takeout does not differ among wildtype and cako, but it is strongly elevated in ovod and this difference is eliminated in allatectomized ovod females. we find no effect of jha treatment upon takeout mrna abundance in either wildtype or cako genotypes. the regulation of takeout by jh appears to be complex and must depend on uncontrolled factors that differ among these studies. ubiquitously expressed mutations of the insulin/igf signaling system in drosophila extend lifespan and reduce the production of jh from adult corpora allata. on the other hand, an inverse interaction between jh and insulin signaling was seen in tribolium castaneum where reduced jh promoted expression of insulin-like peptides. here we see that eliminating adult jh in drosophila does not affect abundance of dilp or dilp mrna. abundance of dilp mrna decreases with cako in the sterile ovod background, but not in fertile flies. in contrast, dilp mrna declines strongly in cako adults in the fertile background, while dilp mrna is already low in all sterile ovod females. dilp is predominantly expressed in adult fat body unlike neuron-expressed dilp dilp and dilp. overall, these data suggest that the impact of jh on aging is distal to longevity-regulatory dilp while jh contributes to the expression of dilp perhaps through its control of adult fat body.  juvenile hormone is an integrator of insect development, physiology and life history. in adult insects jh modulates alternative states of active reproduction versus dispersal, reproductive diapause and torpor. jh supports the reproductive state in part by inducing fatty acid oxidation, and yolk protein uptake. in contrast, jh is reduced during adult reproductive diapause or torpor. such adults can be migratory, express lipogenic enzymes, accumulate lipids and triglycerides, resist exogenous stress, and live a relatively long time. many features of these syndromes appear in drosophila, where adults in reproductive diapause have reduced titers of jh and increased triglycerides, glycogen, stress resistance and lifespan. our current work establishes that reduced jh is sufficient to extend drosophila lifespan. allatectomy increases drosophila lifespan and this survival benefit is reduced by treatment with jha. although allatectomy also reduces fecundity in a jha sensitive manner, the longevity of allatectomized adults is still expressed in otherwise sterile females. at a physiological level, jh appears to independently modulate egg production and survival. under some ecological conditions a trade-off may occur between these fitness traits, and in this way selection would favor juvenile hormone-mediated pleiotropy between reproduction and aging. hodkova makes a similar argument for pyrrhocoris apterus where surgical allatectomy extends lifespan in both fertile and surgically sterilized females. our microarray analysis demonstrates there are at least some direct somatic physiological targets of jh that may be independent of its effects upon reproduction. this gene set provides a platform for hypotheses on how jh may mechanistically control longevity assurance. 
nor does the longevity benefit appear to function through mechanisms by which dietary restriction extends longevity. dietary restriction further extended the lifespan of knockout females. in an analysis of expression profiles of knockout females in fertile and sterile backgrounds, about genes changed in response to loss of juvenile hormone independent of reproductive state. we identify transcripts that change in response to juvenile hormone independent of reproductive state and suggest these represent somatically expressed genes that could modulate how juvenile hormone controls persistence and longevity. knockout females remained relatively long-lived even when crossed into a genotype that blocked all egg production. our data do not support a mechanism for juvenile hormone control of longevity simply based on reducing the physiological costs of egg production. in contrast, little is known about how juvenile hormone affects adult drosophila melanogaster. here we adapt a genetic approach to knock out the corpora allata in adult drosophila melanogaster and characterize adult life history phenotypes produced by reduction of juvenile hormone. reduced juvenile hormone alone is sufficient to extend the lifespan of drosophila melanogaster. previous work suggests that insulin signaling may modulate drosophila aging in part through its impact on juvenile hormone titer, but no data yet address whether reduction of juvenile hormone is sufficient to control drosophila life span. juvenile hormone has been demonstrated to control adult lifespan in a number of non-model insects where surgical removal of the corpora allata eliminates the hormone’s source. with this system we test potential explanations for how juvenile hormone modulates aging. treating these adults with the juvenile hormone analog methoprene restored all traits toward wildtype. reduced juvenile hormone limits reproduction by inhibiting the production of yolked eggs, and this may arise because juvenile hormone is required for the post-eclosion development of the vitellogenin-producing adult fat body. corpora allata knockout adults had greatly reduced fecundity, inhibited oogenesis, impaired adult fat body development and extended lifespan.
 juvenile hormone has been demonstrated to control adult lifespan in a number of non-model insects where surgical removal of the corpora allata eliminates the hormone’s source. in contrast, little is known about how juvenile hormone affects adult drosophila melanogaster. previous work suggests that insulin signaling may modulate drosophila aging in part through its impact on juvenile hormone titer, but no data yet address whether reduction of juvenile hormone is sufficient to control drosophila life span. here we adapt a genetic approach to knock out the corpora allata in adult drosophila melanogaster and characterize adult life history phenotypes produced by reduction of juvenile hormone. with this system we test potential explanations for how juvenile hormone modulates aging.  a tissue specific driver inducing an inhibitor of a protein phosphatase was used to ablate the corpora allata while permitting normal development of adult flies. corpora allata knockout adults had greatly reduced fecundity, inhibited oogenesis, impaired adult fat body development and extended lifespan. treating these adults with the juvenile hormone analog methoprene restored all traits toward wildtype. knockout females remained relatively long-lived even when crossed into a genotype that blocked all egg production. dietary restriction further extended the lifespan of knockout females. in an analysis of expression profiles of knockout females in fertile and sterile backgrounds, about genes changed in response to loss of juvenile hormone independent of reproductive state.  reduced juvenile hormone alone is sufficient to extend the lifespan of drosophila melanogaster. reduced juvenile hormone limits reproduction by inhibiting the production of yolked eggs, and this may arise because juvenile hormone is required for the post-eclosion development of the vitellogenin-producing adult fat body. our data do not support a mechanism for juvenile hormone control of longevity simply based on reducing the physiological costs of egg production. nor does the longevity benefit appear to function through mechanisms by which dietary restriction extends longevity. we identify transcripts that change in response to juvenile hormone independent of reproductive state and suggest these represent somatically expressed genes that could modulate how juvenile hormone controls persistence and longevity. juvenile hormonedrosophilalifespanfecundityfat bodygene expression 