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Defining Wild-Type Life Span in Caenorhabditis elegans

^a The Galton Laboratory, Department of Biology, University College London, England
^b Molecular Biology Program and Division of Biological Sciences, University of Missouri–Columbia, USA

David Gems, The Galton Laboratory, Department of Biology, University College London, 4 Stephenson Way, London NW1 2HE, England E-mail: dgems{at}galton.ucl.ac.uk.

John A. Faulkner, PhD

	Abstract

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The nematode Caenorhabditis elegans reproduces predominantly as a self-fertilizing hermaphrodite, and this drives laboratory populations to be homozygous at all genetic loci. Passaging of stocks can lead to fixation of spontaneous mutations, especially when the latter do not result in a selective disadvantage under laboratory conditions. Life span may be such a trait, since a comparison of six wild-type N2 lines derived from a common ancestor (but maintained separately in several laboratories) revealed four variants with median adult life spans ranging from 12.0 ± 0.8 to 17.0 ± 0.6 days at 20°C. Fertility was also reduced in the two shortest-lived strains. We determined which life span most closely corresponds to that of the authentic wild type by two means. Firstly, N2 hermaphrodites were compared with seven C. elegans wild isolates. The latter were found to resemble only the longest-lived N2 strain. Comparison of male life spans of six lines also revealed additional strain variation. Secondly, life spans of F1 progeny issuing from crosses between N2 variants showed that short life spans were recessive, indicating that they result from loss-of-function mutations. We infer that the longest-lived N2 variant best resembles the original N2 isolate. This is the N2 male stock currently distributed by the Caenorhabditis Genetics Center.

DIFFERENCES in maximum life span between animal species are genetically determined ⁽¹⁾. The mechanisms by which the genome determines the rate of aging are unknown, and are currently the subject of intense investigation. One recent and fruitful approach has been the study of mutations that slow or delay aging in model organisms such as the nematode Caenorhabditis elegans (reviewed in ref. ⁽²⁾,⁽³⁾,⁽⁴⁾).

A potential confounding variable in studies of the genetics of aging in model organisms is the existence of genetic variation among standard laboratory reference stocks. For example, estimates of the life span of hermaphrodites of the C. elegans wild-type laboratory strain, Bristol (N2), vary over almost a twofold range, with median life spans ranging from 11.8 days ⁽⁵⁾ to 20 days ⁽⁶⁾, when grown on agar plates at 20°C with Escherichia coli. Such differences have been attributed to environmental variation, because growth conditions are known to affect life span ⁽⁷⁾.

The N2 strain ⁽⁸⁾, originally isolated from mushroom compost ⁽⁹⁾, has been used by laboratories around the world as the wild-type C. elegans for nearly 30 years. To determine possible genetic differences between N2 stocks from different laboratories, we measured life spans of six stocks from several sources. These include two male stocks (composed of males and hermaphrodites), which are normally maintained separately from hermaphrodite stocks to provide a supply of males for genetic crosses. Males arise spontaneously at a frequency of 0.1% in hermaphrodite populations by meiotic nondisjunction of the X chromosome ⁽¹⁰⁾, creating XO males that can be propagated by mating with hermaphrodites. We identified four genetically distinct variants with median adult hermaphrodite life spans ranging from 12–17 days. It then became necessary to define the wild-type life span for C. elegans. Our analysis suggests that the longest-lived of the four strains best resembles the ancestral wild-type N2. This strain, the N2 male stock currently distributed by the Caenorhabditis Genetics Center (CGC, University of Minnesota, Minneapolis) is significantly longer-lived than the CGC hermaphrodite stock. We recommend that all future studies of life span use the CGC male stock as a starting point.

	Methods

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Media and strains.
DRM (male stock) and DRH (hermaphrodite stock) were obtained from J. Hodgkin and D.L. Baillie, respectively, in 1974, at the Medical Research Council Laboratory of Molecular Biology (LMB), Cambridge, UK. After passaging on agar plates, these were brought to the University of Missouri–Columbia (MU) in 1975 by D.L. Riddle, frozen in liquid nitrogen, and were distributed through the Caenorhabditis Genetics Center (CGC) when it was located at MU from 1979–1992. The male and hermaphrodite stocks distributed by the current CGC (University of Minnesota) are continuous with DRM and DRH, respectively. JW is a stock obtained by D.L. Riddle from J.G. White at the LMB in Cambridge, UK, in 1975, which had been previously maintained for several years separately from the DR stock. The JW stock was stored in liquid nitrogen from 1975 until its use in this work. The S. Ward laboratory N2 strain (here designated BA) was kindly provided by W.A. Van Voorhies (University of Arizona, Tucson) in 1995. Non-N2 wild-type isolates were AB1 and AB2 (isolated and frozen in 1983), and CB4555, CB4854, CB4855, CB4857, CB4932, and RC301 (stocks obtained from those frozen by the CGC in 1991). The provenances and idiosyncrasies of the non-N2 strains are described by Hodgkin and Doniach ⁽¹¹⁾.

Animals were maintained at 20°C in 60-mm Petri dishes containing 12 ml NG agar. These were seeded with E. coli OP50 as a food source ^(8)(12). To ensure that all N2 strains obtained from outside sources were not grown with variant bacterial strains, all were grown from eggs which had been sterilized by exposure to hypochlorite bleach ⁽¹²⁾.

Analysis of genetic dominance.
To obtain populations of F1 hermaphrodites from crosses between N2 strains, approximately 10 males were crossed with 3 hermaphrodites. Animals were transferred to fresh plates after 24 hours to exclude self-progeny produced before mating had occurred. After 24 hours laying eggs, hermaphrodites were removed, and when progeny had developed to the L4 stage, the sex ratio of animals on a sample section of the agar was scored. This was achieved by first excising 1-cm² sections from each plate and transferring each to a fresh plate. In all cases the sex ratio was approximately 1:1, indicating that the great majority of the hermaphrodites were the result of crossing rather than selfing. L4 hermaphrodites were then picked for life span assays from the plates from which the 1-cm² sections had been taken.

Life span analysis.
Hermaphrodite life span assays were initiated using approximately 20–50 L4 larvae. Animals were transferred daily to fresh plates during the egg-laying stage, and less frequently thereafter. Males were maintained in isolation throughout life, because male-male interactions greatly reduce life span ⁽¹³⁾. Animals that died as a consequence of internal hatching of larvae, or that crawled up the wall of the Petri dish, were not included in the life span measurements. (In the case of solitary males, most of the starting population suffered premature death as a result of the latter behavior.) Animals were scored as dead on the basis of absence of any movement and failure to respond to a gentle poke in the head region with a platinum wire. Life span is expressed as the period from the late L4 stage until death, and as weighted means of median or maximum life spans from measurements from several trials. Maximum life span was measured as the last day on which a live worm was observed. Calculations of weighted means of life spans, pairwise comparisons and correlation analyses were carried out using the statistical analysis program JMP version 3 (SAS Institute, Inc., Cary, NC).

	Results

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Genetic Variation Between N2 Lines
We compared the life span and fertility of hermaphrodites of six N2 stocks from several sources. These included the D.L. Riddle laboratory N2 male and hermaphrodite stocks, here designated DRM and DRH, respectively. The current Caenorhabditis Genetics Center (CGC) male and hermaphrodite N2 stocks (here designated CGCM and CGCH, respectively) are derived from DRM and DRH, respectively (see Methods), and no differences in hermaphrodite life span were detected between the respective DR and CGC strains (data for CGC strains not shown). Therefore, the CGC strains were taken to be identical to their DR counterparts. The line designated BA was obtained from the laboratory of S. Ward. The stock designated JW was for several years maintained separately from the stock from which the DR and CGC lines were derived (see Methods).

Although BA and JW hermaphrodites appeared vigorous and healthy when young, they were significantly shorter lived than in DRM and DRH, apart from maximum life span of BA (Fig. 1, Table 1 , Table 2 ). That median but not maximum life span is reduced in BA suggests that this strain contains defects that reduce viability in mid- and late-adulthood (Fig. 1). The maximum life span of BA is significantly greater than that of JW (Tukey-Kramer test, see Table 2 ), indicating that these strains may not be identical.

View larger version (24K): [in this window] [in a new window]   Figure 1. Survival of hermaphrodites of six N2 stocks from several sources at 20°C. CGCM and CGCH are male and hermaphrodite stocks distributed by the Caenorhabditis Genetics Center (University of Minnesota), and are continuous with DRM and DRH, respectively. Note that it is hermaphrodite life span that is being measured in each case.

View larger version (31K): [in this window] [in a new window]   Figure 2. Variation of unmated hermaphrodite brood size distributions between three N2 variants at 20°C.

Which variant, if any, should be treated as wild type? Two approaches were taken to establish which N2 variant most closely resembles the original wild-type Bristol isolate. Firstly, we compared N2 hermaphrodite life spans to those of other wild isolates, to exclude any N2 variants whose life span was significantly different from the species-typical wild-type life span. Secondly, we examined the genetic dominance of the variant N2 life span traits.

Comparison of N2 Lines With Other C. elegans Wild-Type Strains
In seven wild isolates, median hermaphrodite life span was found to range from 14.1 ± 2.6 to 17.8 ± 0.6 days, and maximum life span from (Table 1 ). No significant differences in life span were observed (one-way analysis of variance [ANOVA]). Life span values from all seven strains were summed and compared to those of DRM, DRH, BA and JW (Table 2 ). Median life span of BA, and both median and maximum life span of JW, were significantly less than that of the wild isolates. We compared the median life spans of DRM, DRH, and the wild isolates, adjusted to a normal distribution, and found that DRH was significantly shorter lived than the wild isolates and DRM ; Student's t test), whereas DRM was not shorter lived than the wild isolates The maximum life span did not differ between DRH, DRM and the wild isolates (data not shown). These data indicate that DRM is the best approximation of wild type, whereas DRH, BA and JW are short-lived derivatives.

A limited analysis of male life span confirmed strain differences between DRM and JW, and revealed additional variation among the wild isolates (Table 3 ). Males were maintained in isolation, because male-male interactions reduce life span ⁽¹³⁾. DRM male life span was significantly greater than that of JW, indicating that N2 strain variation also affects male life span (Table 3 ). Overall, the life spans of AB2, CB4855, and RC301 males were consistently lower than those of DRM and AB1 males. For example, in single pairwise comparisons, AB2, CB4855, and RC301 median male life spans were significantly less than that of DRM (Student's t test, respectively).

BA/JW hybrids were significantly shorter lived than DRM and DRH, but not different from BA and JW parents. This lack of genetic complementation suggests that BA and JW contain the same life-shortening mutations, perhaps reflecting common ancestry. Maximum BA/JW life span was significantly less than that of BA (Student's t test. p = .02), suggesting that JW may contain a dominant mutation that shortens maximum life span, at least in a BA/JW hybrid background. Taken together, the five hybrids (excluding JW/BA) were slightly shorter lived than the wild isolates (, for median and maximum life span, respectively; Student's t test), but neither group differed significantly in life span from DRM.

	Discussion

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These results demonstrate the existence of four genetic variants of the C. elegans laboratory wild-type, Bristol N2 strain. Using C. elegans as a biological model, it is important to use a strain that is wild type rather than mutant as a starting point for investigations. Until now, N2 has been defined as wild type as a matter of convention. The variation between N2 lines identified here raises the question of which one better corresponds to wild type. We used two approaches to establish which N2 line best resembles wild type: comparison with other wild isolates of C. elegans, and the presumed dominance of the wild-type life span in F1 hermaphrodites from crosses between variants. Identifying the wild-type N2 strain by comparison with wild isolates is based on the supposition that life span in C. elegans as a species is relatively invariant. The similarity between the hermaphrodite life spans of the seven wild isolate strains, originating from Australia, Europe, and North America, supports this view, as does a study of five other wild isolates maintained in liquid culture ⁽¹⁶⁾.

By contrast, a limited study of life span in males revealed a significant variation in life span among the wild isolates (Table 3 ). This is consistent with previous work by Johnson and Hutchinson ⁽¹⁶⁾. Interestingly, AB2, CB4855, and RC301 males, which are shorter lived, deposit a mating plug on the hermaphrodite during mating, whereas N2 and AB1 males, which are longer lived, do not ⁽¹¹⁾. Possibly the capacity to produce mating plugs is associated with a reduction in male longevity. If so, this would be an example of a trade-off between fitness traits, in which mating plug deposition enhances reproductive success ⁽¹⁷⁾, but limits longevity.

Accumulation of spontaneous deleterious mutations in laboratory stocks has been observed in the Bergerac strain of C. elegans, which was found to exhibit a high frequency of transposon Tc1 transposition ^(18)(19), and in the "wild-type" laboratory strain of Caenorhabditis briggsae, which was found to harbor X-linked recessive mutations affecting movement, chemotaxis and dauer larva formation, and autosomal recessive mutations affecting male tail development ⁽²⁰⁾. These stocks were maintained as growing laboratory cultures for more than 30 years.

The observed variation in life span among N2 lines could have resulted from the accumulation of mildly deleterious loss-of-function mutations. This view is supported by the recessive life span traits of BA and JW. Rosenbluth, Cuddeford, and Baillie ⁽²¹⁾ estimated the frequency of spontaneous lethal mutations in C. elegans to be about 0.2–0.3% per animal per generation. Park and Horvitz ⁽²²⁾ estimated that loss-of-function mutations in up to 50% of C. elegans genes may result in no detectable phenotype.

Mutants with reduced life span are essentially not detectable in the context of routine stock maintenance, and they may suffer from no selective disadvantage. It remains possible that the short life span and reduced brood sizes observed are the result of adaptation to laboratory culture conditions, and that these traits are linked to some unidentified fitness advantage. It might seem unlikely that reduced fertility would be an advantage, unless it were coupled with increased early reproduction. C. elegans hermaphrodites that produce and store more sperm are delayed in the switch to oocyte production. Although their reproductive potential is greater (because the number of stored sperm limits the production of self-progeny) the onset of reproduction is later, resulting in a selective disadvantage ⁽²³⁾. However, a simpler explanation is that genetic stocks are generally passaged with relatively few individuals (a practice that varies among investigators), making the "founder effect" possible. Populations founded by one or a few individuals can by chance be homozygous for a deleterious trait. Crossing with males should promote heterozygosity and reduce the probability that mutations become fixed in this way. Furthermore, the presence of males may convey a reproductive advantage to hermaphrodites with greater longevity because their fecundity is not limited by their own sperm production. Perhaps such factors account for the difference between DR male and hermaphrodite stocks.

In conclusion, three shorter-lived variants of the wild-type C. elegans strain N2 are the result of mutation accumulation that occurred in the absence of outcrossing. The existence of short-lived mutant N2 variants is likely to have contributed to the variation in previously reported life span estimates, for example, that of Van Voorhies ⁽⁵⁾, which employed the short-lived strain BA. We recommend that future studies using C. elegans as a model treat the Caenorhabditis Genetics Center N2 male stock as the wild type. Over the years, the DR and CGC stocks have been periodically refreshed from frozen stocks rather than continuously cultivated. The former is a common practice in many C. elegans laboratories, and should minimize the accumulation of genetic variation. Genetic variation among N2 lines is a potential confounding variable in the study of the effects of single gene mutations on aging in C. elegans. This may be avoided by multiple backcrossing of all mutations into the same N2 background prior to use. Use of the CGC N2 male stock is recommended, because it is possible that the pathology of aging in the short-lived variants of N2 is distinct from that of the putative wild-type CGC N2 male stock. Thus, mutations affecting the rate of aging may produce different effects in the N2 variants.

	Acknowledgments

 
We thank Jason Kennington and David Clancy for statistical assistance, and Patrice Albert for helpful discussion. This work was funded by a University of Missouri Molecular Biology Fellowship, and a Royal Society University Research Fellowship, and BBSRC grant 31/SAG09982 to D.G. and by DHHS grants GM60151 and AG12689 to D.L.R. Some nematode strains used in this study were provided by the Caenorhabditis Genetics Center, which is funded by the DHHS National Center for Research Resources.

Received January 24, 2000

Accepted February 22, 2000

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