BioMed Central Page 1 of 13 (page number not for citation purposes) BMC Developmental Biology Open Access Research article Age- and calorie-independent life span extension from dietary restriction by bacterial deprivation in Caenorhabditis elegans Erica D Smith*1,2, Tammi L Kaeberlein1, Brynn T Lydum1, Jennifer Sager1, K Linnea Welton2, Brian K Kennedy2 and Matt Kaeberlein*1 Address: 1Department of Pathology, University of Washington, Seattle, WA 98195, USA and 2Department of Biochemistry, University of Washington, Seattle, WA 98195 USA Email: Erica D Smith* - ericas4@u.washington.edu Tammi L Kaeberlein - tkaeber@yahoo.com Brynn T Lydum - btlydum@u.washington.edu Jennifer Sager - jnnys@u.washington.edu K Linnea Welton - linnea.welton@gmail.com Brian K Kennedy - bkenn@u.washington.edu Matt Kaeberlein* - kaeber@u.washington.edu * Corresponding authors Abstract Background: Dietary restriction (DR) increases life span and delays age-associated disease in many organisms. The mechanism by which DR enhances longevity is not well understood. Results: Using bacterial food deprivation as a means of DR in C. elegans, we show that transient DR confers long-term benefits including stress resistance and increased longevity. Consistent with studies in the fruit fly and in mice, we demonstrate that DR also enhances survival when initiated late in life. DR by bacterial food deprivation significantly increases life span in worms when initiated as late as 24 days of adulthood, an age at which greater than 50% of the cohort have died. These survival benefits are, at least partially, independent of food consumption, as control fed animals are no longer consuming bacterial food at this advanced age. Animals separated from the bacterial lawn by a barrier of solid agar have a life span intermediate between control fed and food restricted animals. Thus, we find that life span extension from bacterial deprivation can be partially suppressed by a diffusible component of the bacterial food source, suggesting a calorie-independent mechanism for life span extension by dietary restriction. Conclusion: Based on these findings, we propose that dietary restriction by bacterial deprivation increases longevity in C. elegans by a combination of reduced food consumption and decreased food sensing. Background Dietary restriction (DR), also referred to as calorie restric- tion, is an intervention that extends life span and delays the onset of age-related phenotypes in nearly all eukaryo- tic organisms in which it has been tested [1]. Simplisti- cally, it is defined as a significant reduction in dietary intake in the absence of malnutrition. Many different approaches can be used to achieve DR. In mice and rats for example, life span extension is observed by either reduc- ing the amount of food consumed daily (compared to an ad libitum control group) or by imposing an intermittent fasting regimen [2,3]. In addition to simply reducing the amount of food intake, the effects of altering dietary composition has also been examined in different organisms. Methionine-restricted Published: 5 May 2008 BMC Developmental Biology 2008, 8:49 doi:10.1186/1471-213X-8-49 Received: 26 September 2007 Accepted: 5 May 2008 This article is available from: http://www.biomedcentral.com/1471-213X/8/49 �� 2008 Smith et al licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

BMC Developmental Biology 2008, 8:49 http://www.biomedcentral.com/1471-213X/8/49 Page 2 of 13 (page number not for citation purposes) mice [4] and rats [5] have an extended life span, suggest- ing that the nutritional composition of the diet can influ- ence longevity in mammals. In fruit flies, reducing either the yeast extract or sugar composition of the food supply extends life span, although yeast extract appears to have the greatest impact on longevity [6]. Restriction of amino acids also increases life span in flies [7]. In yeast, reducing either the amount of glucose or amino acids in the growth media increases replicative life span [8-10]. Multiple methods for DR have been used in C. elegans, as in other model organisms [11]. One commonly used method in C. elegans is a genetic model, mutation of eat- 2. The eat mutants were originally identified in a screen for defects in feeding behavior ��� they have reduced food con- sumption due to pharyngeal pumping defects [12,13]. Several alleles of eat-2 (and other eat mutants) increase life span, with longevity generally correlating with the degree to which pumping rate is decreased [14]. Life span extension from mutation of eat-2 is independent of the FOXO-family transcription factor DAF-16 and is additive with a longevity-enhancing allele of the insulin/IGF-1-like receptor daf-2 [14]. This has led to the generally accepted model that DR acts to modulate longevity in a genetic pathway distinct from insulin/IGF-1-like signaling (IIS) [15,16]. As with any genetic model of DR, however, eat mutants are not suitable for certain studies. The relative activity associated with different eat-2 alleles has not been completely characterized, so it is unclear whether life span extension from DR is maximized in studies using these alleles. In addition, eat mutants are food restricted from hatching, and DR during development may have second- ary effects on adult physiology that are not fully under- stood. DR by reducing food availability also increases life span in C. elegans. The standard method for culturing C. elegans in the laboratory is to maintain the nematodes on the sur- face of nematode growth medium (NGM) nutrient agar with E. coli OP50 as the food source. DR on NGM nutrient agar has been achieved by reducing the amount of pep- tone in the media so as to limit bacterial growth [17] or by reducing the amount of live bacteria present on the sur- face of the NGM nutrient agar [18]. Two methods of DR have also been described using non-standard, liquid- based growth conditions: bacterial dilution in S basal medium and axenic growth [19-23]. Both liquid-based methods increase life span relative to animals fed a diet of E. coli OP50 in S basal, and axenic growth behaves simi- larly to mutation of eat-2 in epistasis experiments with IIS [24]. Nonetheless, neither method is widely used for aging studies. This may be due to the potential differences in growth under standard conditions on an agar surface versus growth in liquid culture. Axenic growth is reported to cause delayed development and poor growth [22,25], and feeding animals E. coli in S basal shortens life span rel- ative to growth on NGM agar [19]. In addition, DR during development has been shown to be sub-optimal, since animals switched to DR just before adulthood are longer- lived than those maintained on DR from hatching [26]. Recently, two groups independently reported an novel reduced bacterial feeding DR protocol carried out under standard conditions [27,28]. By measuring adult life span as a function of E. coli food concentration, both studies determined that complete removal of bacterial food early in adulthood (BD, bacterial food deprivation also referred to as dietary restriction through food deprivation or dietary deprivation) optimally increases median and maximum life span. BD differs from previous studies describing DR by axenic growth in the culture method (solid versus liquid media) and the time of initiation (reproductively mature adults versus hatchlings)[27]. Although bacterial food is completely absent during adulthood in the BD regimen, BD-treated animals do not suffer from malnutrition (as evidenced by their increased life span and stress resistance [27,28]), thus meeting the commonly accepted definition for DR. Life span exten- sion from BD has been recently validated in multiple wild-derived C. elegans strains, as well as in a second closely related nematode species C. remanei [29]. Like mutation of eat-2 or axenic growth [14,24], BD appears to modulate longevity by a mechanism distinct from reduced IIS [27,28]. While BD results in a more robust life span increase than mutation of eat-2, eat-2 mutants subjected to BD are not longer-lived than wild type animals on BD [27,28]. Taken together, these obser- vations indicate that BD and mutation of eat-2 are likely to increase life span by similar or overlapping mecha- nisms. Thus, BD represents a simple plate-based method for DR that maximizes longevity from food restriction under standard nematode growth conditions and does not require additional genetic manipulation. In order to better characterize the mechanism by which BD slows aging in C. elegans, we determined the effect of BD on survival as a function of the age at which BD is ini- tiated. Previously, our group and an independent study [27,28] reported that BD increases life span in C. elegans when initiated at the last stage of larval development (L4) through day 11 of adulthood. Here, we expand this anal- ysis to more advanced ages, after which mortality has become significant in the population. Similar to prior observations in Drosophila [30] and in mice [31], we find that BD enhances survival even when imposed late in life. Transient exposure to BD confers a life span benefit that correlates with stress resistance, as measured by thermo- tolerance. We further demonstrate that age-independent life span extension in response to BD is not solely due to