Lifespan and Metabolism
During the last 15 years, the insulin/IGF-1/daf-2 signaling pathway has been shown to be the most potent regulator of lifespan in C. elegans. Signaling from DAF-2 is mediated through the AGE-1 phosphatidylinositol 3-kinase (PI3K), PDK-1, and AKT-1/2 kinases, to antagonize DAF-16, orthologous to human FoxO, a forkhead transcription factor. The function of this pathway in mediating metabolism and aging is conserved in C. elegans, Drosophila, and mammals. Because so much of the insulin signaling pathway is conserved, the new components we discover in C. elegans will have broad relevance to mammalian insulin signaling and longevity control.
Genetic analysis in C. elegans continues to identify components of the pathway that are likely to reveal human variation in insulin-like signaling, with medical significance for diabetes and the understanding of how insulin signaling and analogous hormonal pathways couple chronological age to many late onset diseases. Using RNAi to screen for defects in daf-2 pathway mediated longevity regulation, we identified a comprehensive genetic network necessary for the longevity response to low daf-2 insulin/IGF1 signaling (9). Similarly, our proteomic analysis of insulin signaling components has identified other new and unstudied candidate genes to act in insulin signaling. The use of RNAi screens and proteomics in C. elegans is opportune for two reasons: first, the tissues where insulin signaling is key for metabolic control has changed dramatically over the past decade. No longer is an exploration of insulin signaling only in the liver or muscle or even pancreas definitive. Neural and adipose centers of insulin signaling have emerged. We have identified new protein components of insulin signaling from whole animal extracts, and our RNAi screens are done in the whole animal, so insulin signaling across tissues is surveyed. This is unlike mammalian insulin signaling functional genomics which may assay for insulin responses in tissue culture, but not in the physiological context of a whole organism. Aging and diabetes may be more physiological and endocrine, not easily modeled in cell culture. In this way, the C. elegans insulin signaling genetic system is better model system for human aging and human diabetes than human cell culture.
Gene activities that mediate the longevity regulation by C. elegans insulin-like signaling. Samuelson, A. V. , Carr, C. E., and G. Ruvkun. 2007. Gene activities that mediate increased lifespan of C. elegans insulin-like signaling mutants. Genes and Development 21:2976-94.
Genetic and RNA interference screens for lifespan regulatory genes have revealed that the daf-2 insulin-like signaling pathway plays a major role in C. elegans metablism and longevity. This pathway converges on the DAF-16 transcription factor to regulate aging by controlling the expression of a large number of genes. We conducted a genome-wide RNA interference screen to identify genes necessary for daf-2 mutants to survive nearly twice as long as wild type, and identified approximately 200 gene inactivations that shorten daf-2 lifespan to near that of wild type but have much more minor impact when inactivated in wild type. Of the gene inactivations identified in our screen, most enriched are genes annotated to mediate vesicle sorting. For example, compared to loss of daf-16, inactivation of Y65B4A.3 caused the greatest suppression of daf-2 phenotypes. Y65B4A.3 is homologous to human charged multivesicular body protein 6 and the myristolyated subunit of yeast ESCRT-III, the endosomal sorting complex required for transport of transmembrane proteins into the multivesicular body pathway to the lysosomal/vacuolar lumen. Sixteen other endocytosis/vesicular trafficking related genes also suppressed daf-2. One of the responses to a decline in daf-2 is dramatic upregulation of the DAF-16 target gene sod-3, a manganese superoxide dismutase. Thirty-four gene inactivations suppressed the daf-2-dependent induction of sod-3 expression, including smk-1/ protein phosphatase regulatory subunit, mag-1/exon junction complex component, F28D1.9/fatty acid transporter, and cua-1/cation transporter.
Proteomic exploration of the insulin signaling pathway (not yet published)
In the absence of insulin-like signaling, DAF-16/FoxO binds to the promoters of numerous metabolic and stress response genes and can either activate or repress their transcription by RNA Pol II. Such transcriptional outputs cause enhanced stress resistance and a change to fat storage metabolism, and enhanced longevity. Using a proteomic approach, we have identified dozens of candidate protein interactors with insulin-signaling components. We constructed functional translational fusions to epitope tags for the transcription factor FOXO/DAF-16 and one of its activators, SMK-1. FOXO/DAF-16 mediates the transcriptional output of insulin/IGF signaling. SMK-1 is predicted to be a regulatory subunit of protein phosphatase 4 and has genetically been shown to promote DAF-16 activity. We integrated the fusion protein constructs into the C. elegans genome and to study DAF-16/FOXO in variable states of activation, we used three different strain backgrounds: wild type, daf-2(e1370) a strong kinase domain mutant with low insulin like signaling, and daf-18(mg198)/PTEN phosphatase null allele with constitutively high insulin like signaling. Epitope-tagged DAF-16/FOXO from each strain grown in large quantity (tens of liters of growth medium and tens of ml of packed worms) was purified by immunoprecipitation from each of these strains. Co-purifying potential physical interactors of the fusion proteins were identified by mass spectrometry (LC-MS/MS).
In order to determine the background of these purifications, parallel control purifications from worms lacking the fusion proteins were performed. Many of the FOXO/DAF-16 binding partners have chromatin or stress response related annotations. To test their function in daf-2 regulation of lifespan, we inactivated each corresponding gene by RNAi and surveyed phenotypes expected for either enhancement or loss of DAF-16/FOXO activity. We tested each gene inactivation in wild type animals for an extension of lifespan, enhanced resistance to heat or oxidative stress or inappropriate expression of the DAF-16/FOXO-activated gene sod-3. Conversely, we tested in daf-2/insulin receptor mutant animals for suppression of extended lifespan, suppression of resistance to heat or oxidative stress or an inability to overexpress sod-3. Of 89 binding partners tested, 65% showed significant lifespan or stress resistance phenotypes in at least one and 32% in multiple assays. The genes identified by this screen are highly enriched for acting in the longevity pathway: 18 % of the DAF-16/FoxO binding partners are required for lifespan extension in daf-2(e1370), while a genome-wide screen for the same phenotype had a discovery rate near 1%.
Lifespan regulation by evolutionarily conserved genes essential for viability Curran, S. P. and G. Ruvkun. 2007. PloS Genetics 3(4):e56
We have discovered that other arrest points induced by gene inactivations of core cellular components such as the ribosome or mitochondrion or cytoskeleton may be as highly regulated and as key to lifespan regulation as the insulin regulated dauer arrest point. In common with the dauer arrest point, these other arrest points are induced by environmental inputs, for example starvation or natural products that target conserved cellular components such as the mitochondrion or the ribosome. Our analysis suggests that the integrity of these core cellular components are assessed, either in cells that tend to be the most exposed to the environment, or in all cells, and that a signaling pathway to endocrine control of development and reproduction may operate.
To reveal this essential pathway signaling system, we screened the 2700 gene inactivations that cause larval arrest or reproductive arrest in C. elegans for increased adult lifespan by initiating the gene knockdown once the animal had reached adulthood, thus bypassing any developmental abnormalities. We identified 64 genes that can extend lifespan when inactivated post-developmentally. More than 90% of the genes we identified were conserved from yeast to humans. Our yield of 64 gene inactivations out of 2700 tested (~2.4%) is a four-fold increased yield than the previous 89 gene inactivations out of 16,000 screened (~0.6%), and a higher proportion of the gene inactivations cause large increases in longevity.
To classify the pathways represented by these new genes, we performed secondary assays: DAF-16 localization, sod-3 expression, arrested larval survival, suppression of polyglutamine aggregation, and aberrant fat metabolism and clustered the genes by the phenotypes observed. Our analysis placed some of these longevity genes within the insulin-signaling pathway, while others were independent of this pathway.
Stress resistance may contribute directly to extended longevity, and is co-regulated with longevity. We have observed a panel of a dozen stress-responsive GFP fusions in strains experiencing gene inactivations corresponding to a library of ~200 lifespan-extending RNAi clones. We tested a heat induced GFP fusion, hsp-16::GFP, an ER stress induced gene, hsp-4::GFP, and two mitochondrial stress induced genes, hsp-6::GFP and gst-4::GFP. Most of the longevity inducing gene inactivations activated one or more stress reporter genes and the pattern of stress reporter activation was characteristic for ribomsomal inactivation for example compared to mitochondrial inactivation.
The integrity of these core cellular components may be assessed in cells that tend to be the most exposed to the environment, and a signaling pathway to endocrine control of development and reproduction may operate. For example, we have found that there are sentinel signaling cells: the intestine where microbes are first encountered in an intimate way and sensory neurons that might “probe” the antibiotic environment before the rest of the cells of the animal are inhibited. We used a collection of strains in which gene knockdowns can be accomplished within a single, restricted tissue using the same technique of feeding animals bacteria expressing dsRNA against target genes. The strain backgrounds used for these experiments are fully defective for RNAi because they lack the necessary gene (either rde-1 or sid-1). Only the tissue(s) in which gene function is inactivated by RNAi of essential genes are those in which the tissue-specific promoters rescue rde-1 or sid-1 expression is rescued, in neurons or intestine or muscle. In addition, we used a mutant strain proficient for RNAi in the germline but not in somatic tissue (rrf-1) due to differing mechanisms of RNAi in these tissue types. Inactivation of core cell components in each tissue tested – the hypodermis, the intestine, the gonad and body wall muscle – is competent to trigger similar responses to essential gene inactivation as inactivation of these genes in all tissues. This suggests the existence of an endocrine system for response to essential gene inactivation. Similar endocrine outputs from mitochondrial gene inactivation have been found by the Dillin lab (12).
The developmental arrests induced by these gene inactivations and the longevity induction may be a “programmed” response to a deficiency in a key function, and active signaling programs may mediate the arrest point, as a sort of “developmental checkpoint”. Such an arrest program interpretation depends on two key attributes: 1. The arrest should be reversible. We have observed that when drug is removed in many cases, the arrest is reversible. 2. There should be mutations that disable the arrest program. This is also established below.
Stress decoupled mutants shorten the lifespan of insulin signaling and other long lived mutants (not published)
A common theme to many of the cellular components that induce increased longevity when inactivated is that many are targets of antibiotics produced by a range of fungi and microbes that nematodes encounter in the environment. We hypothesize that as a larvae or adult enters an environment with an antibiotic, there may be signaling pathways that detect, for example, antibiotic-induced ribosomal deficiency to trigger cessation of reproductive developmental trajectory, arrest at a particular developmental point, as well as longevity enhancing pathways and behavioral aversion programs to allow the arrested animal to escape and survive long enough to reanimate reproduction and be alive for that joyous experience. The induced stress adaptation and survival pathways would ensure that the animal could escape the antibiotic and resume reproductive development in a less toxic environment or feeding on less toxic microbes. Inhibition of translation by RNAi of translation factors may mimic the ribosomal function deficiency induced by antibiotics in the normal C. elegans ecosystem, and trigger the physiological response of developmental arrest and cessation of reproduction and initiation of longevity programs in the arrested larvae or adults. Exposure of C. elegans to many of these drugs cause similar developmental arrest and a modest increase in lifespan, but the drugs are not as potent as the gene inactivation by RNAi. This may be because drug detoxification is highly evolved —-they induce a variety of detoxification pathways that may be more effective on the small molecule drugs than on gene inactivation by RNAi.
The same stress reporter genes activated by the longevity inducing gene inactivations are also induced by the drugs. Tunicamycin is a natural product the bacterium Streptomyces Iysosuperficus; it inhibits ER N-linked glycosylation and strongly induces HSP-4, a component of the ER unfolded protein response. Antimycin is a natural product Streptomyces and inhibits the mitochondrial electon transport complex 3 by binding the cytochrome b subunit and induces hsp-6, a component of the mitochondrial unfold protein response.
We screened for new gene inactivations that cause a failure to induce the ER or mitochondrial or other stress GFP fusion genes under drug or stressed conditions—-these gene inactivations may cause animals to be “blind” to mitochondrial or ribosomal dysfunction, and therefore not induce these GFP fusion genes. We then asked if a failure to induce these genes reflects a general decoupling of the surveillance of the ribosome for example to the induction of longevity normally induced by essential gene inactivations. In this way, we will discern the signaling pathway, both within cells and between cells for decrements in core cellular functions.
So far from these pilot screens, we have identified 30 gene inactivations that fail to activate the drug induced reporter genes. If the cytoprotective pathways used as stress reporters in the studies above are normally induced by mutants or gene inactivations that confer increased longevity and are part of the program for increased longevity, a decoupling of their induction might shorten the lifespan of long lived mutants more than wild type. We tested whether the 30 gene inactivations with defects in cytoprotective gene induction also abrogated the increase in lifespan induced by mitochondrial dysfunction, reduced feeding, or disruption of insulin signaling. In these experiments, 12 of 30 gene inactivations tested abrogate 2/3 or more of the lifespan extension observed in eat-2, isp-1 and/or daf-2 mutants. While dcp-66, pas-3 and arf-3 exert their largest suppression of lifespan in isp-1, inactivation of cpf-2, wnk-1 and nekl-2 are most potent in the eat-2 mutant. phi-50, ima-3, gob-1, ufd-1, let-70, and elt-2 are critical to lifespan extension in both the isp-1 and eat-2 mutants. Two of these, phi-50 and ima-3, also reduce the lifespan of daf-2 mutants by more than 2/3. These gene inactivations are analogous to the ceh-23 suppression of mitochondrial mutant lifespan increase (16).
A soma-to-germline transformation phenotype in long-lived C. elegans mutants (Curran et al, Nature 459: 1079-84)
Genomic instability of somatic cells is a hallmark of increased age among most organisms. Protection of the germline on the other hand is an evolutionarily conserved trait. We uncovered a soma-to-germline transfomation phenotype among Caenorhabditis elegans longevity mutants. Mutations that inhibit insulin-like signaling cause misexpression of a germline restricted gene, pgl-1::gfp in the intestine and hypodermis. Mutants in the synMuvB class of genes are Eri and display somatic misexpression of the normally germline-limited P-granule component PGL-1. We monitored a PGL-1::GFP protein fusion in daf-2 or age-1 mutant strains. pie-1 and pgl-1 are exclusively expressed in the germline of wild type animals. Decreased insulin-signaling caused strong misexpression of PGL-1:GFP in hypodermal and intestinal somatic tissues of dauers and late larval stage animals. By qPCR, we could verify strong up-regulation of the P granule components pgl-1, pgl-2, and pgl-3 in the soma of daf-2 and age-1 mutants. This misexpression of germline components was strongly suppressed by mutations in the FoxO transcription factor DAF-16, the transcriptional output that is repressed by the insulin-like signaling pathway.
The misexpression of germline markers suggests that the somatic cells of an insulin-signaling mutant are more germline-like. Germline-transformed somatic cells, like germ cells, may engage additional protective pathways that prevent or slow genomic destabilization; the effect of which could cause an increased ability to respond to stress and extend lifespan. We tested the ability of daf-2 mutants to protect somatic tissues from genomic instability by feeding a sublibrary of RNAi clones previously shown to induced somatic nuclear DNA mutation. RNAi targeting rpa-2, srxa-6/dnJ-25, and F49E12.6 causes an increased rate of somatic mutation. These gene inactivations cause nuclear DNA damage as measured by the somatic expression of a normally out-of-frame lacZ transgene. Expression is induced following a nucleotide insertion/deletion event that places the lacZ gene in-frame. After feeding these RNAi clones, X-gal staining of animals carrying this transgene in the daf-2(e1368) background displayed reduced lacZ expression in somatic cells compared to animals harboring the transgene alone, suggesting a reduction in genomic instability in the somatic cells of the insulin-like signaling mutants.
If the misexpression of germline genes in somatic tissues contributes to the longevity phenotype of insulin signaling mutants then removal of the genes by RNAi should suppress the longevity phenotype. To test this we used RNAi to ablate the misexpressed germline transcripts in daf-2 mutant animals. Depletion of these misexpressed germline genes in the daf-2 mutant reduced the lifespan of these normally long-lived animals but not wild type animals. The enhanced RNAi phenotype of synMuvB mutations is suppressed by loss of function mutations in chromatin remodeling ATPase, isw-1 and the SET domain containing protein, mes-4. Similarly, RNAi towards isw-1 and mes-4 could partially suppress the enhanced longevity of daf-2(e1368).
We tested whether any of the other genes identified in our post-developmental RNAi screen to extend lifespan also displayed somatic cell to germline cell fate transformations by tracking the expression of endogenous PGL-1. PGL-1 is normally restricted to the germline where it forms perinuclear punctate structures around mitotic and meiotic germcells. RNAi clones targeting two components of the cytosolic chaperonin complex cct-4 and cct-6 also caused somatic expression of PGL-1. The somatic PGL-1 protein localized to punctate perinuclear rings in hypodermal cells and also produced intestinal cytoplasmic granules.
Germ cells employ protective mechanisms to ensure genetic integrity. The soma-to-germline transformation provides a potential mechanism for the observation that long-lived mutants exhibit resistance to genotoxic stress. We hypothesize that increased genomic stability in somatic cells may be an important pathway facilitating lifespan extension. It will be interesting to investigate if this is a broadly conserved mechanism given that protection of the germline is a shared trait across species.
We have discovered that the daf-2 insulin/IGF1 pathway also regulates the intensity of RNAi in C. elegans. Long-lived mutants in daf-2 and age-1 are enhanced for RNAi and misexpress the normally germline restricted gene P granule RNAi components in the intestine and hypodermis. Mutants in the synMuvB class of genes are also enhanced for RNAi (Eri) and display somatic misexpression of the normally germline-limited P-granule components. The misexpression of germline markers suggests that the somatic cells of an insulin-signaling mutant are more germline-like. Germline-transformed somatic cells, like germ cells, engage additional protective pathways that prevent or slow genomic destabilization to cause an increased ability to respond to stress and extend lifespan. The germline RNAi components that we study are universal to animals. The genes we will discover in this proposal that mediate the regulation of the P granule components are likely to be universal to all animals and to act in aging pathways of humans as well.
Our genetic analysis of aging in the reproductive system of C. elegans is thematically related to the regulation of germ cell pathways in somatic cells. We have found many gene inactivations affect reproductive senescence, some of which also affect organismal aging, some of which do not. Because the reproductive system in mammals is tightly coupled to an endocrine system that assesses nutritional and stress status, our genetic analysis of this system in C. elegans is likely to reveal previously unknown endocrine axes and signals.
Regulation of RNAi by the daf-2 insulin-like signaling pathway (published as Wang, D. and G. Ruvkun. 2004. The insulin pathway regulates RNAi in C. elegans. Cold Spring Harbor Symposium on Quantitative Biology, 69th edition, 69: 429-433.).
C.elegans strains with decreased insulin-like signaling have a more intense RNAi response than wild type. Such regulation of RNAi by this stress and longevity signaling pathway suggests a role in response to pathogens such as viruses. For example, mutants lacking age-1 show enhanced response to RNAi. age-1(mg305) responds to RNAi of lin-1 with a multiple vulva phenotype in 96% of the animals vs 0% on wild-type animals. Similarly, other RNAi of a range of histone genes is enhanced in age-1 strains. Northern analysis shows that after feeding his-44 dsRNA, his-44 mRNA level is significantly decreased in age-1(mg305), whereas no change is observed in wild type. A daf-2; daf-16 double mutant or an age-1; daf-16 double mutant do not show enhanced RNAi. These data suggest that insulin-like signaling normally inhibits RNAi via the DAF-16 transcriptional cascade. Dauer-constitutive mutants in other two pathways, daf-7/ TGF-b and daf-11/ guanylate cyclase are either unaffected or very weakly enhanced, in contrast to the daf-2 pathway mutations. Our model for this enhanced RNAi is that is the misexpression of germline specific RNAi factors that confers the enhanced somatic RNAi in insulin signaling mutants.
Lin-35/retinoblastoma regulation of somatic/germ line specification (Wang et al, Nature 436: 593-597 and unpublished date since that paper being written up now)
The Retinoblastoma (Rb) protein functions as a crucial transcriptional repressor in tumor suppression. In C. elegans, genes encoding Rb (lin-35), components of the core complex and the recruited chromatin factors belong to the synthetic multivulva B (synMuv B) gene class, which repress transcription during vulval development. A whole genome RNAi screen for synMuv suppressors identified mostly chromatin factors7, suggesting that, as in other organisms, the biological roles of lin-35/Rb in C. elegans are primarily chromatin regulation.
Mutations in lin-35/Rb lead to soma-to-germline transformation, similar to the daf-2 insulin signaling mutants. As in the case of the daf-2 mutant, lin-35 mutant worms misexpress the germline specific protein PGL-1 in somatic tissues8. Unlike daf-2 mutant animals, these ectopically expressed PGL-1 proteins form perinuclear granules, reminiscent of its natural P-granule localization in the germline9, indicating that additional germline specific genes may also be misexpressed. lin-35 mutant worms also misexpress germline-enriched genes in somatic cells. Therefore, loss of lin-35/Rb induces a germline state in the soma. PGL-1 misexpression in lin-35 and other synMuvB mutants can be suppressed by inactivating several Muv suppressing chromatin factors7, 13, 14 (mes-4, isw-1 and mrg-1). However, not all Muv suppressing-chromatin factors are PGL-1 suppressing7, suggesting that preventing soma-to-germline transformation is different from preventing Muv formation.
The added germline expression in the soma of lin-35 mutants likely contributes to some of their organismal phenotypes. This is particularly likely for the enhanced RNAi phenotype observed in lin-35 mutants, since many genes functioning in RNAi or related processes are predominantly expressed in the germline15. Indeed, several germline-enriched Argonaute genes are significantly misexpressed in the soma of these worms.
To examine the extent of germline transformation, we compared two microarray analyses performed on lin-35 mutant worms at the first larval stage (L1)10, 11. L1 worms contain a very minimum germline and thus mostly represent expression profiles in the somatic tissues. This analysis identified 295 genes that are commonly upregulated in both arrays. Among them, 141 genes (~50%) were normally germline-enriched as they were depleted in mutant worms lacking a germline15. Thus, germline genes are preferentially upregulated in lin-35 mutant worms, suggesting an extensive germline transformation in the soma.
To verify such a germline transformation in the soma, we measured germline-specific transcripts in the soma of lin-35 mutants. This was achieved by performing the experiments in a glp-4 mutant, which are defective in germline proliferation so only expression in somatic cells is monitored in whole animal RNA preps. We focused on 9 genes that are involved in small RNA function, including genes that encode germline-enriched RNAi factors and components of the germline-restricted P-granules. Real-time RT-PCR on isolated total RNA confirmed the upregulation for all 9 genes. This is shown Table 1.
We further characterized the function of the heterochromatin factors via a biochemical approach. We created transgenic lines that express GFP-tagged LIN-61/L3MBT and immunopurified LIN-61::GFP containing protein complex using anti-GFP antibody. Copurifying proteins were identified by mass spectrometry. The only synMuv B proteins that copurify were LIN-13 and HPL-2/HP1, known synMuvB genes, suggesting the existence of a separate complex formed by synMuv B heterochromatin proteins.
The extensive misexpression of germline genes suggests that germline transformation may involve the activation of one or more master regulators that trigger subsequent cascades of misexpression. Inactivation of mes-4, encoding a histone H3K36 dimethyltransferase21, suppress multiple phenotypes of lin-35 mutants, including Muv and PGL-1 misexpression. We found that the somatic misexpression of 4 of the 9 genes in the pilot set depends on mes-4 (Table 1, last 4 columns), showing that it is a master regulator.
Genome-wide RNAi screening for late reproductivity genes (not yet published)
There is a correlation between late fertility and longevity in many species. Both daf-2 mutants with decreased insulin like signaling and tph-1 mutants deficient in serotonin signaling that act upstream of daf-2 cause late fertility. We developed a simple genetic selection for late reproduction mutants. We use the sqt-3 (e2117) temperature sensitive collagen mutant to kill off the early clutch of embryos laid at the non permissive temperature of 25 degrees. sqt-3 encodes a cuticle collagen, the temperature sensitive allele (e2117) of which is embryonic lethal at 26℃. sqt-3 (e2117) mutants cease progeny production completely and undergo reproductive senescence after day 4 of adulthood at 26℃. To screen for mutants with delayed reproductive senescence, sqt-3 (e2117) mutant animals were fed a library of RNAi clones to test each of 20,000 gene inactivations, were grown at 26℃ for 5 days, during which all F3 progeny are lethal due to the collagen mutation. This lethality is not reversible by lowering the temperature. After the mostly post reproductive 5 day old adults are shifted to 15 degrees, wild-type reproductively senescenced worms will not produce any progeny, while the mutants or gene inactivations with late reproductivity still generate progeny. The background that we must distinguish are gene inactivations that induce greater production of sperm, which naturally limit brood size, and gene inactivations that generally slow down metabolism and development, essentially making the rate of living during the 5 day senescence period a shorter period in biological time. Our intitial controls for this are to measure brood size relative to control; those strains with a larger than normal brood size are suspected to have increased sperm production. This could also be due to increased lifespan of sperm, not an uninteresting topic. Our control for slower metabolism is to record the developmental time from L1 to adult; those gene inactivations with normal developmental time, and normal brood size are our top candidates. We have screened the entire genome based on this experimental design. Totally, 32 genes have been identified from these primary screens. Most but not all of these hits do not affect growth rate or brood size. The hit rate is around 1%. One recent paper from the Murphy lab found that the daf-4 TGF beta pathway receptor affects C. elegans reproductive lifespan 155. We did retrieve daf-3 in our screen, endorsing this finding. But many of our hits are much better than daf-3, so we believe that our analysis will be more thorough.