variation in caenorhabditis elegans dauer larva formation

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Develop. Growth Differ. (2003) 45, 389–396 Variation in Caenorhabditis elegans dauer larva formation Mark E. Viney,* Michael P. Gardner and Joseph A. Jackson School of Biological Sciences, University of Bristol, Woodland Road, Bristol, BS8 1UG, UK Dauer larvae of Caenorhabditis elegans are formed when young larvae experience conditions of low food availability and high conspecific population density; non-dauer, third stage larvae are formed in conditions of plenty. This developmental response to environmental conditions is an example of phenotypic plasticity; that is, an environmentally induced change in phenotype and, as such, a manifestation of a genotype–environment interaction. Extensive variation was found in reaction norms of phenotypic plasticity of dauer formation among wild lines of C. elegans. Recombinant-inbred lines were constructed from parental lines with very different reaction norms of dauer formation. These recombinant-inbred lines had a wide range of reaction norms, of a range greater than that set by the parental lines. The natural variation in reaction norms of dauer formation in C. elegans is, presumably, an adaptation to enhance fitness under the lines’ different natural prevailing conditions. The genetic basis of this variation, as well as its phenotypic consequences, are now ripe for further investigation. Key words: Caenorhabditis elegans, dauer larva, phenotypic plasticity, reaction norm. Introduction The nematode Caenorhabditis elegans has a devel- opmental choice in its life cycle which is modulated by its environmental conditions. In the ‘normal’ life cycle, adult hermaphrodites lay eggs which hatch to release first stage larvae (L1s), which molt through three further larval stages (L2s–L4s) before finally molting into adults. In the alternative developmental pathway, larvae develop into an ‘alternative’ L3s stage, known as a dauer larva. Dauer larvae are arrested, long- lived, environmentally resistant forms which have a morphology, physiology and behavior distinct from other larval stages (Riddle & Albert 1997). The choice between development into ‘normal’, non-dauer L3s and dauer larvae is modulated by food availability, dauer pheromone concentration and temperature. Dauer pheromone is produced constitutively by all worms, and its concentration is used by worms as a measure of conspecific population density. A high pheromone : food ratio favors the formation of dauer larvae, whereas the opposite conditions favor normal, non-dauer development (Riddle & Albert 1997). Thus, dauer larvae preferentially develop at times of environ- mental stress and their environmental resistance and longevity are, presumably, an adaptation to surviving these unfavorable conditions. There has been extensive genetic and molecular analysis of the signal transduction pathway by which developing larvae make and enact the dauer or non- dauer developmental choice (Riddle & Albert 1997). This has provided a good understanding of these processes; however, essentially nothing is known about whether there is natural variation in C. elegans dauer larva formation and the form of any such variation. The development of larvae into dauer or non-dauer L3s is an example of phenotypic plasticity; that is, an environmentally induced change in phenotype (Schlichting & Pigliucci 1998). The nature, form or extent of any phenotypically plastic response may vary between different genotypes. For example, for C. elegans, consider the dauer larva formation of two different (wild-type) genotypes in the same environ- mental conditions: one genotype may produce a very large number of dauer larvae, whereas the other may produce only a very small number. Both genotypes of worms are able to develop into dauer larvae, but the magnitude of the response is different between the two genotypes. Thus, here, the different genotypes have different types or forms of phenotypic plasticity of dauer larva formation. The form of plasticity of a phenotype is formally known as its reaction norm (Scheiner 1993; Pigliucci 2001). For any pheno- typically plastic response, different reaction norms have evolved and are maintained by selection and *Author to whom all correspondence should be addressed. Email: [email protected] Received 17 March 2003; revised 2 June 2003; accepted 3 June 2003.

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Page 1: Variation in Caenorhabditis elegans dauer larva formation

Develop. Growth Differ.

(2003)

45

, 389–396

Variation in

Caenorhabditis elegans

dauer larva formation

Mark E. Viney,* Michael P. Gardner and Joseph A. Jackson

School of Biological Sciences, University of Bristol, Woodland Road, Bristol, BS8 1UG, UK

Dauer larvae of

Caenorhabditis elegans

are formed when young larvae experience conditions of low foodavailability and high conspecific population density; non-dauer, third stage larvae are formed in conditions ofplenty. This developmental response to environmental conditions is an example of phenotypic plasticity; that is,an environmentally induced change in phenotype and, as such, a manifestation of a genotype–environmentinteraction. Extensive variation was found in reaction norms of phenotypic plasticity of dauer formation amongwild lines of

C. elegans

. Recombinant-inbred lines were constructed from parental lines with very differentreaction norms of dauer formation. These recombinant-inbred lines had a wide range of reaction norms, of arange greater than that set by the parental lines. The natural variation in reaction norms of dauer formation in

C. elegans

is, presumably, an adaptation to enhance fitness under the lines’ different natural prevailingconditions. The genetic basis of this variation, as well as its phenotypic consequences, are now ripe for furtherinvestigation.

Key words:

Caenorhabditis elegans

, dauer larva, phenotypic plasticity, reaction norm.

Introduction

The nematode

Caenorhabditis elegans

has a devel-opmental choice in its life cycle which is modulated byits environmental conditions. In the ‘normal’ life cycle,adult hermaphrodites lay eggs which hatch to releasefirst stage larvae (L1s), which molt through threefurther larval stages (L2s–L4s) before finally moltinginto adults. In the alternative developmental pathway,larvae develop into an ‘alternative’ L3s stage, knownas a dauer larva. Dauer larvae are arrested, long-lived, environmentally resistant forms which have amorphology, physiology and behavior distinct fromother larval stages (Riddle & Albert 1997). The choicebetween development into ‘normal’, non-dauer L3sand dauer larvae is modulated by food availability,dauer pheromone concentration and temperature.Dauer pheromone is produced constitutively by allworms, and its concentration is used by worms asa measure of conspecific population density. A highpheromone : food ratio favors the formation of dauerlarvae, whereas the opposite conditions favor normal,non-dauer development (Riddle & Albert 1997). Thus,dauer larvae preferentially develop at times of environ-mental stress and their environmental resistance and

longevity are, presumably, an adaptation to survivingthese unfavorable conditions.

There has been extensive genetic and molecularanalysis of the signal transduction pathway by whichdeveloping larvae make and enact the dauer or non-dauer developmental choice (Riddle & Albert 1997).This has provided a good understanding of theseprocesses; however, essentially nothing is knownabout whether there is natural variation in

C. elegans

dauer larva formation and the form of any suchvariation.

The development of larvae into dauer or non-dauerL3s is an example of phenotypic plasticity; that is,an environmentally induced change in phenotype(Schlichting & Pigliucci 1998). The nature, form orextent of any phenotypically plastic response mayvary between different genotypes. For example, for

C. elegans

, consider the dauer larva formation of twodifferent (wild-type) genotypes in the same environ-mental conditions: one genotype may produce a verylarge number of dauer larvae, whereas the other mayproduce only a very small number. Both genotypes ofworms are able to develop into dauer larvae, but themagnitude of the response is different between thetwo genotypes. Thus, here, the different genotypeshave different types or forms of phenotypic plasticityof dauer larva formation. The form of plasticity of aphenotype is formally known as its reaction norm(Scheiner 1993; Pigliucci 2001). For any pheno-typically plastic response, different reaction normshave evolved and are maintained by selection and

*Author to whom all correspondence should be addressed.Email: [email protected] 17 March 2003; revised 2 June 2003; accepted

3 June 2003.

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390 M. E. Viney

et al.

are therefore, presumably, adaptations to the likelyenvironment of that genotype. Thus, for example,reaction norms of dauer and non-dauer developmentof

C. elegans

may vary between worms from differentgeographical locations, because different reactionnorms of dauer formation are more fit in differentenvironments.

The way in which selection acts on reaction normshas been the subject of much debate and, in essence,this debate has attempted to determine whetherselection acts directly on reaction norms per se orwhether selection acts on the mean value of a pheno-type in a particular environment, which thereby affectsthe reaction norm (Via

et al

. 1995). Two concepts forthe genetic basis of plasticity can be distinguished: (i)allelic sensitivity, in which different alleles havedifferent phenotypic effects in different environments;and (ii) gene regulation, in which regulatory locicontrol or affect the expression of other loci in differentenvironments. These two views are not mutually exclu-sive (Via

et al

. 1995). The genetic and molecular basisof natural variation in reaction norms of a plasticphenotype for any organism is not known (Pigliucci2001). Determining this is important to be able tounderstand and predict its response to selection andevolution. Considering further the example of two

C. elegans

genotypes, each with different reactionnorms of dauer formation, the question raised is: Arethe different reaction norms due to any, or all of,variation in environmental sensation, variation ininformation transduction, variation in the execution ofthe dauer development program (e.g. morphogen-esis) or variation in other, as yet unknown, controlsacting on dauer development?

Here we have searched for, and found, variation inreaction norms of dauer formation in a range of wildlines of

C. elegans

. We have also determined thedistribution of these reaction norms in recombinant-inbred lines of

C. elegans

. Understanding the eco-logical significance of this variation, as well as otherphenotypic trade-offs, is now possible. The geneticand genome resources available with

C. elegans

presents a particularly good prospect for understand-ing the genetic and molecular basis of this variation inreaction norms of this phenotypically plastic trait.

Materials and Methods

Worms

The dauer formation phenotype of eight wild lines(CB4932, CB4555, CB4853, CB4856, N2, TR389,AB2 and DR1350) (Table 1), which represent distinctlines from a range of different geographic locations,was examined. All lines were obtained from the

Caenorhabditis

Genetics Center (Minneapolis, MN,USA), and maintained using standard methods(Sulston & Hodgkin 1988).

To begin to investigate the genetic basis ofvariation in reaction norms of dauer development,we generated recombinant-inbred lines (RIL) usingtwo parental strains (N2 and DR1350) which hadvery different reaction norms. To generate theseN2

DR1350 RIL, individual hermaphrodite L4s ofDR1350 were mated with a number of N2 males.Individual F

1

progeny resulting from each matinggroup were maintained on separate plates.N2

DR1350 cross-progeny were identified asthose which segregated both bordering and solitaryphenotypes (De Bono & Bargmann 1998) in the F

2

generation. DR1350 has a bordering phenotype; N2has a solitary phenotype. Bordering and solitaryphenotypes are most readily seen as populationphenotypes and to do this, individual F

2

progenywere maintained on plates until a population of wormshad developed, at which time the phenotype wasdetermined from plate observations. To propagateand inbreed the RIL, randomly selected individual F

2

progeny of the F

1

worms were transferred to separateplates and allowed to self-fertilize; individual F

3

wormsfrom each F

2

plate were transferred to separate platesand allowed to self fertilize; etc. (Johnson & Wood1982). This process was repeated for 30 generations,at which point the lines were cryopreserved. Forty RILwere generated in this way.

Dauer formation assays

The preparation of dauer pheromone and dauerformation assays were carried out essentially asdescribed by Golden and Riddle (1984a), with theexception that the preparations of diluted

Escherichiacoli

OP50 food used in the assays was made in wateronly. Dauer pheromone was prepared from the super-natant of liquid cultures of N2. One batch was used forall assays of the RIL and one batch was used for all ofthe other assays, and thus the quantities of dauerpheromone used between these two batches, and the

Table 1.

The origin and Tc1 pattern of eight wild lines of

Caenorhabditis elegans

(Hodgkin & Doniach 1997)

Line Location Tc1

CB4932 UK L14CB4555 CA, USA H6CB4853 CA, USA L3N2 UK L1CB4856 HI, USA L9TR389 WI, USA L1AB2 Australia L8DR1350 CA, USA L3

Page 3: Variation in Caenorhabditis elegans dauer larva formation

C. elegans

dauer larva formation 391

assays in which they were used, were not compar-able. The quantity of dauer pheromone and of foodwas varied in different assays, as described below. Allassays were carried out at 25

C. For each worm line,each set of food and pheromone conditions wastriplicated within an assay, and each assay was itselftriplicated, unless otherwise stated. For the analysis ofthe 40 RIL, these observations were made in 10separate assays (assays 1 and 2 = 12 RIL; assays 3and 4 = four RIL; assays 5–10 = eight RIL each) withN2 and DR1350 controls in each assay. Each set ofconditions was duplicated (assay 1 and 2) or triplic-ated (assays 3–10) for each RIL and control line ineach assay. The assays were completed in five timeblocks (time 1 = assays 1 and 2; time 2 = assays 3and 4; time 3 = assays 5 and 6; time 4 = assays 7 and8; time 5 = assays 9 and 10) and strains were nestedwithin assay.

Statistical analyses

To determine the effect of the environmental con-ditions (dauer pheromone and food concentration)and worm line or RIL on the proportion of dauer larvaethat developed, generalized linear models were con-structed, with a binomial error structure with a logisticlink function in which the number of eggs that devel-oped into dauer larvae was the response variable. Insuch models where the ratio of the residual scaleddeviance to the residual degrees of freedom wasfound to be greater than one (indicating overdis-persion of the data), this ratio was used as the esti-mated scale of the model. Each term of the model wasadded sequentially to the model and an analysis ofdeviance table constructed. The test statistic usedwas

2

if the scale was fixed and F if the scale was

estimated. The concentration of pheromone and foodwas included in the analysis as integer values. Thisanalysis was undertaken using GLMStat version 5.3(Ken Beath, Newcastle, NSW, Australia). Note,

ASSAY

was also included as a term in all relevant analyses.The dauer formation assays of all 40 RIL were

undertaken in a series of assays conducted atdifferent times. To take account of this in the dataanalysis, both

ASSAY

and

TIME

were included as factorssuch that

LINE

was nested within

ASSAY

, which was itselfnested within

TIME

. The nested analysis was doneusing the statistical package JMP3.2.2 (SAS InstituteInc., Cary, NC, USA). The arcsine transformed pro-portion of dauer larvae was the response variable, andthis variable was weighted by the total number oflarvae in the assay.

LINE

and

ASSAY

were randomeffects in the model and JMP synthesized denomin-ators for each effect to be tested using the expectedmean squares table. In the nested analysis, someeffects were not tested with respect to the residualerror. For instance,

LINE

[

ASSAY

,

TIME

] was tested using

PHEROMONE

LINE

[

ASSAY

,

TIME

] mean square as testdenominator.

For analysis of the RIL, the difference in the propor-tion of dauer larvae that formed at the two (10 and40 µL) concentrations of pheromone was calculatedas: (mean proportion within an assay at 40 µL) –(mean proportion within an assay at 10 µL). Thus, foreach RIL there were two data that were analyzed, onefrom each assay. The arcsine transformed differencesin proportion of dauer larvae were the response vari-able.

LINE

and

ASSAY

were each nested within

TIME

. Weused a probability level of

P

< 0.01 as significant forall of these analyses.

Results

Dauer formation in eight wild lines of

Caenorhabditis elegans

The mean proportion of dauer larvae that developedfor eight wild lines of

C. elegans

(Table 1) in thepresence of two concentrations of dauer pheromone(25 and 75 µL per 2 mL plate) with 1.25% w/w food isshown in Figure 1. Analysis of these data show thatthere were significant effects of

PHEROMONE

(

F

= 81.53,

P

< 0.0001) and

LINE

(

F

= 22.96,

P

< 0.001) on theproportion of dauer larvae that developed.

Extensive variation in

Caenorhabditis elegans

dauer formation

Following this analysis, a subset of lines (N2, AB2,DR1350 and CB4932) were chosen for more detailed

Fig. 1.

Dauer formation of eight wild lines of

Caenorhabditiselegans

at two concentrations of pheromone (µL per 2 mL plate)with 1.25% w/w food. The values shown are the overall propor-tion of dauer larvae that developed in all three assays combined.The reaction norm of dauer formation are the slopes of the lines.

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392 M. E. Viney

et al.

analysis. These four lines represent the range of dauerformation phenotypes seen in the eight lines (aboveand Fig. 1). The proportion of dauer larvae formed bythese four lines in the presence of three concen-trations of pheromone (25, 75 and 125 µL per 2 mLplate) at two dilutions of food (2.5 and 1.25% w/w) isshown in Figure 2. The development of dauer larvaewas affected by food and pheromone (

FOOD

:

F

=14.26,

P

= 0.0002;

PHEROMONE

:

F

= 128.4,

P

< 0.0001)and the lines varied in their dauer formation (

LINE

:

F

= 308.9,

P

< 0.0001). In addition, the four linesdiffered in their response to changes in food and inpheromone concentrations (

PHEROMONE

LINE

:

F

=2.98,

P

= 0.009;

FOOD

LINE

:

F

= 16.17,

P

< 0.0001),

and thus differed in their reaction norms of dauerformation with respect to changes in these environ-mental (food and pheromone) conditions. The twoenvironmental conditions did not interact (

FOOD

PHEROMONE

:

F

= 3.30,

P

= 0.0397;

FOOD

PHEROMONE

LINE

:

F

= 1.56,

P

= 0.1635), whichsuggests that the reaction norms with respect topheromone and with respect to food are independent.There was substantial interassay variation; significanteffects:

ASSAY

:

F

= 45.41,

P < 0.0001; ASSAY � LINE:F = 11.65, P < 0.0001; ASSAY � FOOD: F = 5.36,P = 0.0057; ASSAY � FOOD � LINE: F = 3.25, P = 0.005;and ASSAY � FOOD � PHEROMONE: F = 3.40, P = 0.0108;non-significant effects: ASSAY � PHEROMONE: F = 1.79,P = 0.1334.

Dauer formation in recombinant inbred lines

The dauer formation of the 40 RIL were measuredunder two concentrations of pheromone (10 and 40 µLper 2 mL plate) and one dilution of food (2.5% w/w).We analyzed these RIL for their response to phero-mone rather than to food, because controlling differentconcentrations of pheromone is more robust thancontrolling concentrations of food.

Analysis of the data for the 40 RIL (excluding the N2and DR1350 controls) show that dauer developmentwas affected by pheromone and that the lines variedin their development (PHEROMONE: F = 1148.24,P < 0.0001; LINE[ASSAY, TIME]: F = 1.77, P = 0.0090).The lines differed in their response to changes inpheromone (PHEROMONE � LINE[ASSAY, TIME]: F = 9.73,P < 0.0001) and thus differed in their reaction normsof dauer formation with respect to pheromone. There

Fig. 2. Dauer formation of four lines (N2, AB2, DR1350 andCB4932) of Caenorhabditis elegans at three concentrations ofpheromone (µL per 2 mL plate) at two dilutions of food (—,1.25% w/w; - - -, 2.5% w/w). The values shown are the overallproportion of dauer larvae that developed in all three assayscombined. The reaction norm of dauer formation are the slopesof the lines.

Fig. 3. A frequency distributionof the proportion of dauer larvaeformed at two 10 (�), and 40 µL(�) per 2 mL plate (concen-trations of pheromone) and thedifference in proportion ( ) ofdauer larvae formed at theseconcentrations of pheromone for40 N2 � DR1350 recombinant-inbred lines (RIL). The values forN2 and DR1350 (shown in paren-theses) are the mean difference inproportion of the within assaymeans from the 10 assays usedfor analysis of the RIL. The meanproportion of dauer larvae formedwith 10 and 40 µL of pheromonefor N2 are 8.2 and 47.42 andfor DR1350 are 0.89 and 23.60,respectively.

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C. elegans dauer larva formation 393

was substantial variation between different assaysand times at which assays were performed;significant effects: TIME: F = 13.37, P = 0.0060;PHEROMONE � TIME: F = 30.11, P = 0.0007; non-significant effects: ASSAY[TIME]: F = 0.72, P = 0.6620;PHEROMONE � ASSAY[TIME]: F = 0.29, P = 0.9197. Thiseffect of TIME was due to period 5 (mean dauerformation: 23.4, 20.5, 22.3, 20.2 and 11.1 for TIME

periods 1–5, respectively); if TIME period 5 wasexcluded from the analysis then TIME was no longersignificant (TIME: F = 3.02, P = 0.148) and the otherterms, including TIME, remained non-significant(ASSAY[TIME]: F = 0.62, P = 0.654; PHEROMONE �ASSAY[TIME]: F = 0.39, P = 0.815) and all other effectsremained significant (PHEROMONE � TIME: F = 31.17,P = 0.0021; PHEROMONE: F = 799.47, P < 0.0001;LINE[ASSAY, TIME]: F = 2.16, P = 0.0023; PHEROMONE �LINE[ASSAY, TIME]: F = 7.43, P < 0.0001).

The reaction norm of dauer formation can beexpressed as the difference in the proportion of dauerlarvae formed at two concentrations of pheromone. Afrequency distribution of this difference in proportion(as well as the dauer formation at each pheromoneconcentration) for the 40 RIL is shown in Figure 3. Thisshows that the reaction norm of dauer formation withrespect to pheromone concentration varied exten-sively in the RIL and over a range greater than thatencompassed by the parental, N2 and DR1350 lines.The lines differed in their differences in proportion(LINE[TIME]: F = 10.66, P < 0.0001), which is in agree-ment with the other analysis of these data (above). Inthis analysis, there was no detectable variationbetween assays or times at which assays were carriedout (ASSAY[TIME]: F = 1.82, P = 0.134; TIME: F = 3.46,P = 0.0179). The lines varied in their dauer formation

at the high (40 µL per 2 mL plate) pheromone concen-tration (LINE[TIME]: F = 12.75, P < 0.0001) and therewere no ASSAY or TIME effects (ASSAY[TIME]: F = 3.42,P = 0.013; TIME: F = 1.25, P = 0.308). The lines did notvary in their dauer formation at the low (10 µL per 2 mLplate) pheromone concentration (LINE[TIME]: F = 1.80,P < 0.0430) and there were no ASSAY or TIME effects(ASSAY[TIME]: F = 0.483, P = 0.787; TIME: F = 16.90,P = 0.0002).

For the 40 RIL, we correlated the proportion of dauerlarvae formed at each pheromone concentrationagainst each other and against the difference inproportion. There was a significant rank correlationbetween the proportion of dauer larvae formed at thehigh (40 µL per 2 mL plate) pheromone concentrationand the difference in proportion (r = 0.9214,P < 0.0001), but no other significant correlations(Fig. 4). This suggests that the extensive variation inthe RIL reaction norms of dauer formation is largelydue to variation in their dauer formation at high phero-mone concentrations and not due to variation in theirdauer formation at low pheromone concentrations.

Fig. 4. A scatter plot of the proportion of dauer larvae formedat a high concentration (40 µL per 2 mL plate) of pheromone andthe difference in proportion of larvae formed at two concen-trations of pheromone for 40 N2 � DR1350 recombinant-inbredlines (RIL), with a best-fit regression line.

Fig. 5. A scatter plot of the difference between a recombinant-inbred line (RIL) and (a) N2 and (b) DR1350 in the proportion ofdauer larvae formed at low (10 µL per 2 mL plate) and high(40 µL per 2 mL plate) concentrations of pheromone.

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394 M. E. Viney et al.

To further understand how the RIL differed from theparental lines, we compared the dauer formation ofeach RIL at both pheromone concentrations to that ofN2 and DR1350 (Fig. 5). This shows that comparedwith N2, the RIL varied in their dauer formation at bothpheromone concentrations in all possible ways(Fig. 5a). The majority of lines (bottom left-hand quad-rant) formed less dauer larvae at both pheromoneconcentrations; a substantial number (top left-handquadrant) formed more dauer larvae at high phero-mone concentrations, but less at low concentrations;while the remaining lines formed more dauer larvae atlow pheromone concentrations (right-hand quadrants)with an approximately equal mix of responses (greaterand lesser) to high pheromone concentrations. Incontrast, compared with DR1350, most of the RILformed more dauer larvae at low pheromone concen-trations, with an approximately equal mix of responses(greater and lesser) to high pheromone concen-trations (Fig. 5b).

The 40 RIL had a range of bordering or solitaryphenotypes (De Bono & Bargmann 1998). To deter-mine whether there was any association between thisphenotype and the dauer formation phenotype in theRIL, the bordering or solitary phenotype was scoredfor the 40 RIL by plate observation. Of the 40 RIL, 16had a bordering phenotype, 21 had a solitary pheno-type and three were ambiguous, and thereforeexcluded from further analysis. The mean dauer form-ation at each pheromone concentration for thosestrains that had bordering and solitary phenotypeswas 4.6 and 5.5% (10 µL pheromone per 2 mL plate)and 32.6 and 33.3% (40 µL pheromone per 2 mLplate), respectively. These means are not significantlydifferent at either pheromone concentration (10 µL:F = 0.18, P = 0.67; 40 µL: F = 0.01, P = 0.921) andneither was there a difference in the 40–10 µL differ-ence in proportion (F = 0.002, P = 0.968). Thus, thereis no effect of the bordering phenotype on dauerformation.

Discussion

We examined dauer formation in a range of wild linesof C. elegans under a range of dauer inducing con-ditions. We showed that there is extensive variationbetween wild lines in their propensity for the formationof dauer larvae under these assay conditions. Somelines (e.g. N2) readily form dauer larvae whereasothers (e.g. AB2 and DR1350) do so much less readilyor barely at all (e.g. CB4932). Lines with such lowlevels of dauer larva formation may be due to naturalvariation in genes known by mutagenesis analysis tocause a dauer-defective phenotype (Riddle & Albert

1997), or due to natural variation in other, as yetuncharacterized, loci. N2 is the ‘standard’ wild-typeline used for virtually all genetic analyses. In this study,N2 most readily formed dauer larvae, and the possi-bility should be considered that this is a consequenceof its long history of laboratory culture.

In all assays, dauer formation was affected bypheromone and/or food concentration per se, aswould be expected (Riddle & Albert 1997). However,in addition, examination of a subset of lines showedthat the lines also varied in their reaction norm ofdauer formation with respect to food and pheromoneconditions. Analysis of the RIL also showed furtherextensive variation in their reaction norms of dauerpheromone (Fig. 3) over a range greater than thatencompassed by the parental lines. The RIL varied intheir dauer formation in response to different phero-mone concentrations in all possible ways compared toboth parental lines (Fig. 5). Thus, among the RIL therewere individual lines that had greater or lesser dauerformation at either pheromone concentration com-pared to both parental lines. However, the correlationof dauer formation at a high pheromone concentrationwith the reaction norm of dauer formation (the differ-ence in proportion; Fig. 4) in the RIL shows that mostof the variation in this reaction norm is brought aboutby lines increasing their dauer formation response tohigh concentrations of pheromone.

The occurrence of progeny with more extremephenotypes than their parents is known as trans-gressive segregation, and this has been reported inC. elegans for other life-history traits (Johnson 1987).Transgressive segregation suggests that there aremultiple loci affecting the trait in question, in this casethe reaction norm of dauer development. For thedifference in proportion of dauer larvae formed for theRIL (Fig. 3), the range (D) and variance (Vp) is 66.98and 354.6, respectively. This phenotypic distributioncan be used to estimate the minimum number (k) ofquantitative trait loci (QTL) underlying this phenotypefrom Taylor’s modification of Wright’s formula (k = D2/4Vp) (van Swinderen et al. 1997) which in this casesuggests that there are three QTL underlying this trait.The mean phenotypic plasticity of dauer formation ofthe RIL was midway between that of the two parentallines, suggesting that no selection occurred duringthe construction of the RIL: such selection has beenreported in other studies of C. elegans (Shook et al.1996).

In our analyses, there was persistent, substantialvariation between different assays in dauer formationof the same line under the same dauer pheromoneand food conditions. For example, in the analysis offour wild lines for three concentrations of pheromone

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C. elegans dauer larva formation 395

and two concentrations of food, ASSAY was a signifi-cant single term effect as well as in two, two-way andtwo, three-way interactions. Despite continual refine-ment of our dauer assay procedures, this inter-assayvariation persisted. Our observations of inter-assayvariation also suggest that accurate definition of dauerformation phenotypes of mutant alleles and compari-son of these between different laboratories is likely tobe difficult.

The range of environmental conditions in which wemeasured dauer formation appears to include thestrongest dauer inducing conditions possible. Analy-sis of dauer formation of four C. elegans lines withthree concentrations of pheromone (Fig. 2) shows thatdauer formation is not linear over this range. Rather,dauer formation is asymptotic with increasing phero-mone concentrations, as has been observed by otherworkers (Golden & Riddle 1984b; Ailion & Thomas2000). One can envisage that such an asymptoticresponse may be due to saturation of the sensoryprocess involved in the initiation of dauer formationand/or in the subsequent transduction of that inform-ation. Overall, in all of our analyses, the greatest levelof dauer development was 87%. We did attempt togenerate still greater dauer formation, but wereunsuccessful. Thus, in assays at 25�C, if increasinglygreater concentrations of dauer pheromone wereused, worms left the agar and did not develop further;and if decreasingly smaller amounts of food werepresent, larvae did not develop beyond first or secondstage larvae (data not shown). In addition, it ispossible that a greater level of dauer formation couldbe induced at higher temperatures. Analysis ofC. elegans strains and mutants has shown that dauerformation assays undertaken at 27�C (near thethermal limit of C. elegans) greatly increases dauerformation with up to 100% dauer formation (Ailion &Thomas 2000). Thus, analysis of the wild lines(Table 1) or the RIL under the same food and phero-mone conditions, but at 27�C, are likely to generatedifferent reaction norms of dauer formation.

Until relatively recently, there has been little con-sideration of inter-isolate variation in C. elegans,although there is growing interest with this and othernematodes (Delattre & Félix 2001; Sommer et al.2001). Natural isolates of C. elegans have beendescribed with respect to variation in their Tc1insertion patterns and variation in the formation ofcopulatory plugs (Hodgkin & Doniach 1997), life spanand other life-history traits (Johnson & Hutchinson1993). The variation between C. elegans lines in theirbordering or solitary phenotypes has been investi-gated and has been found to be due to a poly-morphism in a neuropeptide receptor-like molecule

(De Bono & Bargmann 1998). Different isolates ofC. elegans vary in their adult body size and thegenetics of this is beginning to be investigated (Knightet al. 2001). The distribution of single nucleotide poly-morphisms between different C. elegans lines hasshown extensive variation, although with no generaldiscernible pattern or correlation with the geograph-ical origin of different lines (Koch et al. 2000). Theavailability of these polymorphisms should now allowthe rapid mapping of the phenotypic plasticity ofdauer formation (Wicks et al. 2001).

We have shown that lines of C. elegans vary in theirreaction norm of dauer formation. One can envisagea number of ways in which such variation could occur.Thus, lines may differ in their sensitivity of theirsensation and reception of the extrinsic cues (foodand pheromone concentrations) used to determinewhether or not to develop into a dauer larva . Alterna-tively, this sensation may not vary between lines butrather some aspect of the transduction of that inform-ation may vary, thereby bringing about quantitativelyand/or qualitatively different signals to induce dauerdevelopment. Further still, lines may vary in the ‘effici-ency’ of the phenotypic expression of the dauer ornon-dauer development for the same stimulatorysignal. Variation between worm lines in any or all ofthese steps in dauer formation could vary and be thebasis by which lines vary in their reaction norm ofdauer formation. Molecular identification of the locithat underlie this variation in reaction norm will resolvethis question as well as more generally explain thegenetic architecture underlying these phenomena. Animportant question that will be addressed by suchstudies is whether the genes that control the reactionnorm of dauer formation are genes within or withoutthe known (from mutagenesis analyses) dauerdevelopment pathway itself. The complete genomesequence of C. elegans is known and is predicted tocontain approximately 19 000 genes (The C. elegansSequencing Consortium 1998). Up to half of these areuncharacterized, and systematic screening has foundno apparent knock-out phenotype for more than 75%of genes (Fraser et al. 2000). It is therefore possiblethat some of these as yet uncharacterized genesmay be involved in variation in reaction norms ofC. elegans dauer development.

Acknowledgements

We thank Simon Harvey for initial discussions whichled to the start of this work and the Leverhulme Trustfor two pilot project grants. Strains were suppliedby the Caenorhabditis Genetics Center which is

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396 M. E. Viney et al.

supported by the National Institutes of Health,National Center for Research Resources, USA.

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