Arresting development arrests aging in the nematode Caenorhabditis elegans

Mechanisms of Ageing and Development, 28 (1984) 23-40 Elsevier Scientific Publishers Ireland Ltd.

ARRESTING DEVELOPMENT CAENORHABDITIS E L E G A N S

ARRESTS

AGING

23

IN

THE

NEMATODE

THOMAS E. JOHNSONa'*, DAVID H. MITCHELLb'**, SUSAN KLINEa, REBECCA KEMALb and JOHN FOY b aMolecular, Cellular and Developmental Biology and Institute for Behavioral Genetics, University of Colorado, Boulder, CO 80309 and bDepartment of Cell Physiology, Boston Biomedical Research Institute, 20 Staniford Street, Boston, MA 02114 (U.S.A.) (Received April 3rd, 1984)

SUMMARY Larval development of the nematode, Caenorhabditis elegans, can be arrested by either of two different treatments: (1) complete starvation, or (2) growth in a partially defined culture medium (axenic medium) of strains adapted to bacterial growth. The developmental arrest is complete under total starvation and the starved populations live about 10 days. The developmental block is incomplete in axenic medium; most animals mature but maturation takes 10 times longer than normal. If developmentally arrested cultures are returned to growth on E. coli, both the completely starved and the axenically arrested cultures mature at normal rates. Life-span is prolonged by I day for each day of complete starvation; life-span is prolonged by 0.7 days for each day of axenic arrest. These results suggest that aging and development are closely coupled in this system. The results are discussed in terms of previous observations on nutritional deprivation in other invertebrates and caloric restriction in mammals and are interpreted in light of theoretical models of senescence.

Key words: Developmental program; Programmed senescence; Control of senescence; Caloric restriction

INTRODUCTION Caenorhabditis elegans is a non-parasitic, bacterial-feeding nematode about 1 mm in length. It is currently being used in a number of studies throughout the world as a model *To whom all correspondence and reprint requests should be sent: Dr. Thomas E. Johnson, Molecular Biology and Biochemistry, University of California, lrvine, CA 92717, U.S.A. (714) 856-6867 **Current address: Sensor Diagnostics, 751 Greetree Rd, Pacific Palisades, CA 90272 (U.S.A.) 0047-6374/84/$03.00 Printed and Published in Ireland

© 1984 Elsevier Scientific Publishers Ireland Ltd

24 developmental system. Over the last 6 years this species has also been chosen for use in a number of studies of aging. The organism can be grown with simple microbiological techniques [1] and has a generation time of 3.5 days at 20°C [2]. Life-span is between 12 and 30 days depending upon the exact conditions of maintenance [3] and is about 19 days under the monoxenic conditions (growth on bacteria alone) used for some of these experiments [4] and about 30 days in a partially defined culture medium (axenic conditions [5] ). C. elegans is genetically well defined. Genetic analysis is greatly facilitated by its self-fertilizing, hermaphroditic mode of reproduction, which makes the isolation of mutants easy, and by the occurrence of spontaneous males, which can be used to construct stocks and map new mutations [6]. We have previously shown that it is possible to obtain strains that are longer-lived than wild-type using standard selective breeding approaches [4 and Johnson, unpublished observations]. In this study we show that environmental manipulations, i.e. transient starvation and development arrest in axenic medium, also have significant effects on the length of life. These treatments result in the extension of both mean and maximum life-spans of treated cultures and block larval development as well. Klass [3] has shown that length of life and fecundity are influenced by bacterial concentration. He showed that the mean life-span is maximized at concentrations of 10 ~ bacteria/ml, while maximum fecundity is obtained at 10 9 bacteria/ml. In previous studies the effects of axenic growth were compared to growth on bacteria [5]. In general, axenic cultures promote prolonged times of development and longer life-spans than is typical in bacterially maintained cultures. In this paper, we show that both development and senescence are arrested by two transient starvation protocols. We interpret this observation to mean that aging is closely coupled to development in the nematode, C. elegans.

MATERIAL AND METHODS

Stocks and media Stocks were maintained as described by Brenner [1]. The axenic medium was that of Rothstein [7] as modified by Gandhi et al. [5]. Stocks used for this study include the Brenner wild type strain, N2, obtained from Dr. D. Hirsh, and an N2 derivative strain, N2A, isolated in the laboratory of Dr. D.R. Sanadi after prolonged culture in axenic medium. N2A carries a recessive, sex-linked mutation enabling it to grow well in axenic medium (Mitchell et al., in preparation). Survival analyses for experiments in monoxenic culture were as described by Johnson and Wood [4] ; axenic growth and survival techniques are described by Gandhi et al., [5]. Experiments involving complete starvation were carried out by Johnson and Kline in the laboratory of W.B. Wood; axenic starvation experiments were performed by Mitchell,

Kemal and Foy.

25 Starvation protocol Complete starvation was achieved by harvesting monoxenically grown adults using the egg isolation technique of Emmons et al. [8]. Eggs were allowed to hatch overnight in S buffer [1] without cholesterol and with no added E. coli. Those stocks starved at the time of hatch were kept in this medium. Stocks starved at later times in larval development were first fed E. coli by growth of the cultures on nematode growth medium (NGM) preseeded with a lawn of E. coli strain 0P50 [1]. At 24 or 48 h larvae were washed free of this medium using S buffer. Larvae were washed by centrifugation (2000 rev/min for 30 sec) three times or more with S buffer until bacteria were fewer than 106/ ml in the wash. Starved larvae were maintained in S basal free of cholesterol and E. coli until refeeding. Cultures were refed E. coli strain 0P50 at 109/ml and survival was analyzed. Axenic arrest protocol Axenic cultures were harvested from NGM preseeded with E. coli as described by Brenner [1]. Eggs were suspended in distilled water and agitated for 1 rain in a vortex mixer to dislodge E. coli coating the eggs. Eggs were then washed five times in distilled water to remove E. coli in suspension. Axenization was performed as described by Gandhi et al. [5]. The eggs were treated with glutaraldehyde (10% [v/v] for 30 min) at room temperature to kill any remaining/:: coli and any hatched larvae or adults remaining in the preparation. The eggs were washed 3 times in distilled water to remove glutaraldehyde. About 50 eggs were then placed back on NGM pre-seeded with E. coli to serve as a nonarrested control population. The remaining eggs were distributed into 24-well COSTAR cluster dishes containing 1 ml of axenic medium at a density of several hundred eggs per well and allowed to hatch overnight. The next day hatched worms were distributed into fresh axenic medium at 3 0 - 4 0 worms/well and kept in this medium for the indicated times. Size measurements were made at the indicated times by observing 20 worms in each well. Cultures were monitored for the appearance of rare escapees from arrest (see Results); these were removed before maturation. At the indicated times, axenically arrested worms were removed from axenic medium, transferred to individual segments of quartered Petri dishes containing NGM and a spot of E. coli, and allowed to mature. Twelve to 36 h after maturing, all adults were transferred to a new NGM plate with E. coli and containing 30 #M FUdR [Mitchell et al., submitted] to prevent further reproduction and to prevent death of adults from intrauterine hatching of young. The next day worms were transferred again to fresh FUdR medium. Egg production, quality of movement and general appearance were then monitored until death as described below. This FUdR treatment at the time of the adult molt results in normal aging, lifespan and adult characteristics [Mitchell et al., submitted]. Fertility was taken to be the sum of the number of larvae on NGM and the number of larvae and fertilized but unhatched eggs on FUdR medium. As a control, eggs from strain N2A were axenized in parallel with N2 eggs. After hatching, individual worms were placed in separate COSTAR wells in axenic medium and allowed to mature. At 12-36 h after maturation, worms were transferred to axenic

26 medium containing 25 /aM FUdR [5] to prevent further reproduction and intrauterine hatching of young. The next day, worms were transferred to fresh axenic medium containing 25 /aM FUdR and observed until death. All observations and manipulations were performed in a laminar flow hood under sterile conditions.

Aging indices Aging indices associate an arbitrary numerical value with three traits that change clearly and reproducibly with age: (1) reproductive state; (2) degree and quality of movement; and (3) general appearance. For each trait "1" is defined as a state characteristic of young adults and "5" as a state characteristic of very old adults (Table I). To determine these indices four or more worms were assayed weekly beginning 1-2 days after maturity. Size measures and statistics Size measurements were made with a Wild M5A or a Bausch and Lomb StereoZoom 7 stereodissecting microscope equipped with an ocular micrometer. Bacterial concentrations were determined using a Petroff-Hauser counter. Best fit analysis was performed using either of two survival statistics previously described [4]. Raw data were incremented by periods of 1 day and the resulting data compared to the unstarved control. The increment yielding the highest P values is taken as the best fit of the starved population to the control. RESULTS

Developmental arrest by complete starvation Eggs from the N2 strain ofC. elegans were harvested as described and allowed to hatch in the absence of an exogenous food source (starvation). Individual first larval stage worms were periodically monitored for growth; .no significant increase in the mean length of the population was observed over a 12-day period of starvation (Fig. I). Populations of second larval stage animals were also isolated 24 h after egg isolation and subjected to starvation conditions. Again there was no significant increase in size during 11 days of starvation (Fig. 1). In both instances arrested worms continued to move vigorously for many days. Animals isolated at 48 h (fourth larval stage) also showed no increase in body length after starvation but in contrast to earlier times of block, animals survived starvation only for periods of about 2 days, dying from matricidal hatching (hatching of fertile eggs within the body of the mother). Starvation at 48 h could therefore not be monitored for more than 2 days. Developmental arrest in axenic medium Newly axenized first stage larval populations from the N2 strain showed almost complete blockage of growth and maturation for periods up to 4 weeks in Rothstein's. modified axenic medium [5], in a manner apparently similar to completely starved worms (Fig. 2 and Table If). These worms continued to move vigorously for many days.

Less opaque. Pigment spots may be more prominent.

May be somewhat transparent in places. May look grainy; not smooth-looking. May include prominent pigment spots.

Clearly transparent in places. May look disordered inside, including large vacuolate-looking regions. Pigment spots may be dark.

Many parts of body look transparent. May include very dark localized pigment spots. May look shrunken or flabby or both. May have lost internal structure especially in intestine which may look partly hydrolyzed. May be highly vacuolated or grainy.

Moving whole body, but clearly slower. Still moving constantly and spontaneously or nearly so.

Moving whole body fairly strongly, but not all the time. Still moving spontaneously. Or moving, but quite slowly with non sinusoid or exaggerated sinusoid shape. Moving head only, most of the time, with body immobile. Whole body may move, especially if touched but very slowly and with great difficulty. Non-sinusoidal. Immobile head and/or body or very slight movement of head or body, for >10 sec. Usually moves only if touched.

Laying many oocytes, few or no eggs. Eggs or ooeytes visible intern',ally even if not being laid.

Gonad not clearly atrophied, but no eggs or oocytes visible internally.

Gonad clearly atrophied; often dumb-bell shaped, granular or condensedlooking.

Gonad very clearly atrophied; or extruded from vulva.

Opaque, "full", solid, smooth. Relatively pale, inconspicuous or smoothly distributed pigment spots. Well-ordered internal structure.

Rapid, spontaneous, continuous whole body sinusoidal movements.

Laying fertilized eggs

General appearance

1

-

Movement

Sexually immature

Reproductive state

Characteristics corresponding to aging index value

0

Aging index value

AGING INDICES

TABLE I

tO ---d

2.8 BODY LENGTHS 16

14

12

I0

08

06 04

02

0

I 5

0

I0

TIME (DAYS) Fig. 1. Shown are mean body lengths -+ S.D. after complete starvation at different times, followed by refeeding: control, no starvation ( . - n - . ) ; starved at the time o f hatching (..zx..), at 24 h after egg isolation ( - • - ) and at 48 h after egg isolation ( - = ) . Refeeding o f starved cultures is indicated by arrows at days 4, 6, 9, and 12: growth after feeding of cultures starved at hatch ( -- o .... ) or at 24 h (o).

BODY LENGTHS 14

12

•

I0

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/ ,

I I I

E 08 E

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I

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04

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I /

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I

I

I

]o

20

30

TiME (Days) Fig. 2. Shown are mean body length after axenic arrest for different lengths of time, followed by transfer to NGM seeded with E. coll. Return to bacterial growth is indicated by arrows at days 7, 15, 21 and 28: axenic starvation ( c - ) , bacterial growth ( - e - -).

T A B L E I1

100%

-+ 0.4 -* 0.4 ± 0.3 ± 0.4

3.0 ± 0.5

33.0 ± 12

2.5 17.5 23.0 30.0

Juvenile period (days ± S.D.)

± ± ± ±

9 7 6 7

28 ± 6

33 ± 12

26 20 t7 20

Adult period (days ± S.D.)

-

-

2.5 2.5 2.2 2.4

± 0.4 +- 0.4 ± 0.3 ± 0.6

Time to maturity after release from arrest {days ± S.D.)

-

few eggs

211 -+ 52 165 ± 4 6 99 ± 4 0

Fertility {progeny per Adult ± S.D.)

0

97 68 -

Fraction arrested (~}

aAfter longer p e r i o d s of s t a r v a t i o n p h e n o t y p i c a l l y a b n o r m a l i n d i v i d u a l s were o b s e r v e d (see text). These w o r m s were n o t i n c l u d e d in the a n a l y s e s p r e s e n t e d in this table.

N2A control ( N = 19)

Whole Lifespan (N = 20)

75%

100% 72% 34% 20%

35) 34) 32) 30)

0 15 21 28

(N= {N = (N= {N =

% Healthy a adults

Days arrested

CHARAC'FERISTICS OF AXENICALLY ARRESTED WORMS

30 The slow development of N2 was in sharp contrast to strain N2A [5] which, when grown in the same medium, matured in 3 days (Table I1). Axenically arrested N2 larvae are not dauer larvae. Dauer larvae are an alternative third larval stage in C. elegans, in which development is arrested by adverse environmental conditions, particularly a lack of nutrients [9]. Unlike dauer larvae, axenically arrested worms show pharyngeal pumping and are killed by exposure to a 1% sodium dodecyl sulfate. Most N2 larvae eventually mature in axenic medium, but at rates 10 times slower than under growth in bacteria. The time needed for maturation of N2 worms in axenic medium varies greatly from worm to worm. The vast majority of worms remain small and sexually immature for more than 2 weeks (Table II, fraction arrested). However, even in cultures a few days old, rate individuals "escape" arrest and begin to grow rapidly. After 15 days, the fraction of worms escaping arrest each day increases markedly until, by 33 days of age, the majority of N2 worms have reached adulthood. More than 75% of arrested N2 worms eventually mature if left in axenic medium.

Life-span of cultures in developmental arrest Not surprisingly, complete starvation of Ll's from strain N2 severely reduced mean life-span. Starved populations survived for about 10 days and then began dying quickly with kinetics quite different from that associated with normal aging (Cuccaro and Johnson, unpublished). Axenic arrest of Ll's from strain N2 resulted in markedly different survival kinetics. Arrested larvae did not die prematurely, and most larvae eventually matured, as already described. In fact, the lifespan of N2 worms that spend their entire life in axenic medium is doubled compared to either strain N2 on monoxenic medium or strain N2A in axenic medium, with a life-span approximately equally divided between a much elongated larval period and a normal or slightly lengthened period of adulthood (Table II). Reversal of developmental arrest by refeeding Upon refeeding of the starved cultures, normal rates of larval development resumed, as monitored by increase in mean length of larvae (Fig. 1). Normal rates of development were also observed when axenically arrested N2 larvae were returned to monoxenic growth conditions (Fig. 2). The time to maturity after refeeding remained almost constant (Table II) in axenically starved cultures even up to 28 days. However, longer periods of axenic arrest yielded populations whose adult length was less than that of control populations (Fig. 2). A similar reduction in adult length may be seen in cultures that were again fed after 12 days of complete starvation (Fig. 1). Worms that were axenically arrested for 7 or fewer days appeared normal when released from axenic arrest and allowed to mature, as judged by their morphology, behavior and fertility. However, populations that were arrested for 2 - 4 weeks exhibited increasing numbers of individuals that appeared morphologically abnormal by the time they reached adulthood [see Table I1, % healthy adults]. The most common abnormalities were:

31

SURVIVAL after ARREST by STARVATION ~.~.~~~,

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AGE (DAYS) Fig. 3. These are survival curves of populations of worms which underwent starvation and ret'eeding: control, unstarved ( - u - ) . (A) Animals were starved at the time of hatch and refed at 4 days ( - ' - ) , 6 days ( A _ ) , 9 days ( - c - ) or 12 days ( - o - ) . (B) Animals were starved at 24 h after egg isolation and refed at 4 days (_A_), at 6 days ( - ~ - ) , at 9 days ( - o - ) , and at 12 days ( - e - ) . (C) Animals were starved 48 h after egg isolation and refed at 2 days after egg isolation ( - - c . - - ) ,

32

TABLE Ill BEST FIT OF LIFE-SPAN OF COMPLETELY ARRESTED WITH CONTROL POPULATION

Length of arrest

Mean life-span (days)

None 2 days 4 days 6 days 8 days 10 days

21.7 21.5 23.8 25.4 26.1 32.6

Life-span to obtain best fit a (incremen t)

Probability of similarity

0 +3 +4 +6 + 11

0.62 0.72 0.77 0.64 0.70

days days days days days

aBest-fit was obtained by incrementing the control population in steps of 1 day. Survival curves were compared using the Log-rank test implemented on a CDC Cyber 172 [4]. TABLE IV LIFE-SPANS OF DEVELOPMENTALLY ARRESTED POPULATIONS

Treatment

Complete starvation Experiment A

Complete starvation Experiment B

Treatment begins (days)

Treatment ends {days)

Mean life-span (days +-S.E.)

Population size

Control 0 0 0 0 0 0

not stopped 2 4 6 8 10

21.2 10.9 21.5 23.8 25.4 26.1 32.6

-+ 0.7 -+ 0.1 ~- 0.6 -+ 0.8 -~ 0.7 -+ 1.1 + 1.6

81 50 44 19 39 29 14

2

4 6 9 12 4 6 9 12 4

18,6 25.9 27,4 29,2 32,4 23.5 24,8 28.2 31.4 23.0

-+ 0.6 a'b'c -+ 0.7 a +- 0.9 a +- 0.7 a -+ 0.7 a -+ 0.7 b -+ 0,6 b +- 0.7 b +- 0 . 7 b -+ 0.6 c

77 90 87 95 79 81 86 86 79 40

Control 0 0 0

15 21 28

29.0 37.0 40.0 50.0

+- 1d -+ 1d ± 1d +- 1d

35 34 32 30

Control 0 0 0 0 1 1 1 1

Axenic arrest

aData bData CData dData

for for for for

survival populations survival populations survival populations survival populations

of of of of

Fig. Fig. Fig. Fig.

3A. 3B. 3C. 4.

33 1. An abnormal bulge at the anus, and a rapid decrease in body diameter posterior to the anus ("choptail"); 2. Extruded gonads from the vulva; 3. Slow or irregularly moving animals; 4. Premature death (often associated with matricidal hatching), defined as death within 2 days after maturity. Even worms that lacked overt abnormalities may have lowered total brood size (Table II) or short average adult length. Aging and senescence o f refed cultures

The survival curves of populations of worms that experienced total starvation were normally shaped (Fig. 3). Note that these data are representative (especially for 10 or more days of treatment) of only those animals surviving the starvation protocol. An analysis of these survival curves was carried out by a best fit procedure in which the survival curve of the control starved population was compared to that of the non-starved populations in increments of 1 day (Table Ill). The best fit was almost equal to the total amount o f time the population was starved. Indeed, correlations of mean life-spans of the starved populations (Table IV) with time of starvation were significant for all three experimental populations (Table V). There was an essentially linear regression o f mean life-span with time of starvation, with the slope of the regression almost equal to one (Fig. 5 and Table V), suggesting that in all cases of complete starvation an extra day of life was obtained for each day the culture was starved. Similar results are seen after axe'nic arrest. Again, survival curves were normally shaped (Fig. 4) and life-span was significantly extended by the experimental treatment (Table IV). Note that these data are representative only of arrested animals which were morphologically normal after release from axenic arrest. There is a tendency for the length of the adult phase to be somewhat decreased with longer times o f starvation. A linear regression of the time of axenic starvation on mean life-span of the starved cultures suggested that there was about 0.7 days of additional life for every 1 day of starvation (Table V).

TABLE V LINEAR REGRESSION OF OBSERVED MEAN LIFE-SPAN ON LENGTH OF ARREST Experiment and number

7~'meof starvation

1 2 3 4

at hatch at hatch 24 h at hatch

Starvation medium

S basal "S basal S basal Axenic

Number of points

Regression (slope +-95% confidence interval)

Correlation

6 5 5 4

1.0 ± 0.8 1.1 -+0.9 1.1 -+0.9 0.7 -+ 1.3

0.93 a 0.97 a 0.99 a 0.96 b

aprobability of not significantly differing from 0. P < 0.01. bprobability of not significantly differing from 0, P < 0.05.

34

SURVIVAL after AXENIC ARREST I00

80 z

\\

> 60 C,"

ONoS,orvo,,on

O'3 Z 0

• 0-1 Day Axemc Medium A..~

40

0-7 Day 0 0-14 Day

{_)

~

20

• 0-21 Day

I

I

I

I

I0

15

20

25

30

35

40

45

50

AGE (DAYS) Fig. 4. S h o w n are survival curves o f populations undergoing a x e n i c arrest at the time o f hatch f o l l o w e d by refeeding with bacteria: control, unstarved ( n ); refed at 1 day (__A_), 7 days ( ," ), 14 days ( :7 ) , 2 1 d a y s ( - e ).

The effects of axenic arrest on physiological aging were consistent with the observed extensions of life-spans. A set of three aging indices (Table I) showed reproducible and internally consistent changes during aging of a control population (Fig. 6A). For clarity of presentation in Fig. 6B, these indices were averaged and plotted together with the day 0, control. Averaged indices for axenically arrested populations (Fig. 6B) showed that REGRESSION of L I F E S P A N

on TIME

of S T A R V A T I O N 32 _

/

30 _

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z 26 Q,_ 03 w u.. 24 z

22

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LENGTH of STARVATION (DAYS)

Fig. 5. Linear regression analysis of mean lifespan of starved and refed populations of worms on length of starvation period: starvation at time of hatch (data not shown; . , . e. , . ), starvation at time of hatch (data as shown in Fig. 3A;

s

) and at 24 h a f t e r egg isolation (data of Fig. 3B; - _z~_ _).

35

AGING I N D I C E S 5

A

4

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0 - - - - 0 Reproduction t1-----.tl Appearance A - - A Movement

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hl --I

> 2 x i.i_i f-h z I

• DAY0 o DAY15

-

• DAY 21 DAY 28

1 l0

I 20

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AGE (Days)

Fig. 6. Displayed are aging indices used for scoring physiological age of cultures undergoing axenic arrest and refeeding with bacteria. (A) Aging indices for unstarved control: reproduction ( - - ) , appearance ( . . . e . . . ), and movement ( - ~ - ) . (B) Average combined aging indices of populations starved and refed at day 0 (o), day 15 (v), day 21 (A), and at day 28 (:0.

physiological aging is arrested b y the axenic arrest regimen. As judged by their fertility, m o v e m e n t , and general appearance - all described b y the single, averaged aging index experimental worms scored as markedly younger than controls. These data, like those for life-span, showed that axenic arrest o f development caused arrest o f aging.

36 DISCUSSION Both development and senescence are arrested by starvation We have shown that two different conditions which arrest larval development of the nematode, C. elegans, also arrest senescence: either complete starvation (Fig. 1 and 3) or growth in a semi-defined axenic medium (Figs. 2, 4 and 6). In complete starvation there is a 1-day increase in total life-span for each day that the culture is starved (Fig. 5, Table V). In other words, aging is completely blocked and there is no effect on the length of the adult life-span. Similar findings are seen in cultures starved at three different points throughout the larval development period. Axenic arrest yields results which are similar but not identical to those seen in the complete starvation protocols; there is an increase in the total length of life of about 0.7 days for every day of axenic starvation (Table V); that is, aging appears to be partially blocked. By several other criteria, as well, axenic arrest of development appears to be incomplete: worms do develop, although slowly, in axenic conditions; they can spontaneously escape arrest and develop rapidly; and, when released from axenic arrest by transfer to a bacterial food source, worms often develop abnormally, which is consistent with the notion that limited developmental processes proceed during axenic arrest and that unbalanced growth may therefore occur. Thus, axenic arrest is likely to be more complex than arrest by complete starvation. On the other hand, axenic arrest, unlike complete starvation, may be caused by a specific and limited set of missing nutrients. This view is backed up by the observation that single gene mutations can confer the ability to grow well in axenic media (Mitchell et al., in preparation). Another interesting finding can be seen in those animals which underwent complete starvation at 48 h after hatch (Fig. 1). This time corresponds to completion of about 80% of the developmental period. Even though further increase in length is blocked, these cultures complete development and become sexually mature adults, producing eggs which hatch within the mother, leading to her death. These morphological changes are dramatic

and can be detected with a dissecting microscope. Some further development may also occur in animals arrested by starvation at earlier times in development, but this development is not readily detectable with a dissecting microscope. Studies of ongoing somatic cell lineages in starved larval cultures, using Nomarski optics, could determine how quickly developmental arrest follows starvation. There may be a critical size or stage at which the larva is committed to complete development and cannot be arrested by a starvation block. The ability of most worms to "escape" axenic arrest and develop rapidly after a point in larval development corresponding to about 15 days of axenic arrest also suggests a critical stage after which the worm is committed to complete larval development. This time point corresponds to the stage at which developing larvae become insensitive to growth arrest induced by 400 gM FUdR [10] and may correspond with a point at which the somatic cell lineages of the worm are largely complete [11,12]. Potential problems The arrest regimens are not without deleterious effects. Prolonged arrest by either

37 regimen leads to smaller adult length. Periods of axenic arrest greater than 2 weeks lead to decreases in brood size and to several types of morphological abnormalities (Table 1I). Moreover, completely starved larvae only survive about 10 days if not transferred to growth medium (Table IV and Cuccaro and Johnson, unpublished observations). Under both arrest protocols, some animals do not survive longer periods of arrest. It could therefore be argued that selection for long-lived survivors could be responsible for the increased mean life-spans. Three lines of reasoning argue against this interpretation. First, most animals survive both protocols for periods up to a week; axenic arrest results in little mortality, developmental abnormality, or escape from arrest for 2 weeks. Second, not only mean life-span but also maximum life-spans show corresponding increases after arrest (Figs. 3 and 4); since a rare sub-population with greatly increased maximum life-spans is not detected in unstarved, control populations, it is unlikely that such a subpopulation exists in these experiments. Third, the shapes of survival curves can be compared with each other and differences detected statistically; when we compared survival curves of control and experimental populations, we found that the time (days) added to the control to obtain a best-fit of the two curves closely corresponded to the length of the starvation period (Table Ill). Moreover, the shape of the survival curves are the same for most of the data, showing only a longer plateau phase; in particular, the mortality rates, corrected for length of arrest, are quite similar for control and experimental populations. (Figs. 3A,B, and Fig. 4: 1,7, 14 days starvation). This is consistent with the notion that aging and mortality proceed at a normal rate after the period of arrest. It is inconsistent with the selection model which predicts higher rates of mortality in older populations. In two cases maximum life-span has not been extended and higher mortality rates are detected (e.g. Fig. 3C and Fig. 4, 28 days). In these two groups we cannot exclude the possibility that worms surviving the experimental treatments are not representative of the entire population.

Results with other organisms Comfort [13] reviews early studies of starvation effects on life-span in several invertebrate systems; starvation increases the life-span of ticks dramatically. In Drosophila starvation-induced delay of pupation has no effect on length of pupation or adult lifespan [14]. Similarly, Ingle et al. [15] showed that in Daphnia, starvation slows development and increases the length of life in proportion to the length of starvation. Turkish hamsters undergo hibernation characterized by a metabolic decrease and display increased life-spans in direct proportion to their periods of hibernation [16]. Our observations on C. elegans can also be related to experiments of growth retardation in rodents. One of the major mechanisms by which caloric restriction (see review [17] ) has been thought to extend life-span is through the extension of the growth period and a corresponding prolongation of total life-span [ 18,19]. From the more detailed studies of a number of physiological systems in caloric-restricted rodents, it is clear that dietary restriction causes a large number of changes and probably has major effects other than merely prolonging the immature stage of development [20-22]. Food restriction (as opposed to complete starvation) has been imposed, in C. elegans, by lowering bacterial concentra-

38 tion and results in prolongation of life [3,23]. The effects of food restriction on rates of development have not been determined. A re development and senescence coordinately controlled?

The data presented show that at two points in larval development a complete developmental arrest stops the aging processes. This could be because the same rate-determining process governs both development and senescence or because the process of senescence does not begin till the developmental processes are completed. These results should be viewed in light of observations by Klass and Hirsh [23] who showed similar extensions of life-span when larval development was arrested by entry of larvae into the dauer larval state. Since the dauer is a specialized third larval stage [9] which has a 50% survival time in excess of 60 days [23], it was not immediately obvious that the results seen with worms in the dauer state would apply to worms arrested in the normal developmental sequence. Our studies show that the dauer state is not a necessary prerequisite for the extension of life by arrest of development, although we were only able to extend total life-span by 12--20 days as compared to the 60 day extensions observed by Klass and Hirsh. They point out that the absence of any observable decrease in adult life-span despite extended periods in the dauer state indicates that agents truly external to the organism must not play a significant role in determining life-span of the organism and therefore rules out mechanisms of senescence which are completely dependent on the physical environment. Yeargers [24] observed that dauer larvae are resistant to high levels of 3,-irradiation and suggested..."that the dormant dauer state might be a period of active repair". The radiation resistance of dauer larvae is under study in our laboratory. Preliminary data suggest that the dauer is no more or less resistant to radiation than are normal third stage larvae (Johnson and Etebar, unpublished observations). Anderson [25] reported a fourfold increase in the specific activity of superoxide dismutase in dauer larvae despite a 50% decrease in the rate of oxygen consumption [26]. He suggested that this increased ability to capture superoxide radicals might account for the long life-span and radiation resistance of the dauer. Klass and Hirsh ascribe the relative immortality of the dauer to the fact that the dauer is "dormant" and suggest that normal aging is an innate process whose rate is directly related to the metabolic rate. However, dauer larvae are metabolically active as shown by the fact that oxygen consumption is on the order of 50% of that observed in adults [26] and by the fact that dauer larvae move rapidly and spontaneously for many days after entering the dauer state. These observations suggest that the complete arrest of aging in dauers is not due to a complete suspension of metabolic processes. Similarly, under complete starvation larvae continue to move vigorously again suggesting that the observed lifespan extension is not due to a complete arrest of metabolic processes. Our observations suggest a simple alternative explanation for the extended life-span of arrested larvae and of the dauer: the normal senescent processes follow or are otherwise coupled to the developmental program. By this model, dauers would live longer because

39 the dauer state is distinct from the normal senescent program. Our developmental coupling hypothesis makes strong and testable predictions: first, halting development in other ways should halt senescence; and second, starvation after the completion o f development might affect senescence differently from starvation before such completion. We have not been able to test these predictions using the starvation regimen described herein because this regimen delays egg lay in adults resulting in the hatch of unlaid eggs within the bodies of their mothers, thereby killing them. It should be possible to test this model using temperature sensitive mutants which block egg development thereby preventing hatching o f the eggs within the body of the mother or by blocking hatch with 30/aM F U d R (Mitchell and Santelli, in preparation). Alternatively, it is now possible to transfer N2 larvae and adults of various ages from bacterial growth medium to axenic medium (Mitchell, unpublished observations). Preliminary experiments suggest that N2 worms, axenized as adults, may live only slightly longer than untreated controls. A closer examination of these processes should yield a more complete understanding of the coupling of the developmental and the senescence processes. ACKNOWLEDGEMENTS This research was supported by Public Health Research Grants Nos. HD-11762 to W.B. Wood, AG-01236 to T.E. Johnson, and AG00952 to D.H.M.; and by NSF Grant No. PCM 8208652 to T.E.J. Thanks to Cynthia Verdecia and Patrice Cuccaro for help in preparing the manuscript. REFERENCES 1 S. Brenner, The genetics of Caenorhabditis elegans. Genetics, 77 (1974) 71 94. 2 L. Byerly, R.C. Cassada and R.L. Russell, The life cycle of the nematode Caenorhabditis elegans 1. Wild-type growth and reproduction. Dev. BioL, 51 (1976) 23-33. 3 M.R. Klass, Aging in the nematode Caenorhabditis elegans: Major biological and environmental factors influencing life span. Mech. Ageing Dev., 6 (1977) 413-429. 4 T.E. Johnson and W.B. Wood, Genetic analysis of the life-span in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA, 79 (1982) 6603-6607, 5 S. Gandhi, J. Santelli, D.H. Mitchell, J.W. Stiles and D.R. Sanadi, A simple method for maintaining large, aging populations of Caenorhabditis elegans. Mech. Ageing Dev., 12 ~1~80) 137-150. 6 R.K. Herman and H.R. Horvitz, Genetic analysis of Caenorhabditis elegans. In B.M. Zuckerman (ed.), Nematodes as Biological Models, Vol. 1, Academic Press, New York, 1980, pp. 227 261. 7 M. Rothstein, Practical methods for the axenic culture of the free-living nematodes, Turbatrix aeeti and Caenohabditis briggsae. Comp. Biochem. Physiol., 49B (1974) 669-678. 8 S.W. Emmons, M.R. Klass and D. Hirsh, Analysis of the constancy of DNA sequences during development and evolution of the nematode Caenorhabditis elegans. Proc. Natl. Acad. Sci. LISA, 76 (1979) 1333-1337. 9 R.C. Cassada and R.L. Russell, The dauerlarva, a post-embryonic developmental variant of the nematode Caenorhabditis elegans. Dev. Biol., 46 (1977) 326 342. 10 D.H. Mitchell, J.W. Stiles, J. Santelli and D.R. Sanadi, Synchronous growth and aging of Caenorhabditis elegans in the presence of Fluorodeoxyuridine. J. Gerontol., 34 (1979) 28 36. 11 J.E. Sulston and H.R. Horvitz, Post-embryonic cell lineages of the nematode, Caenorhabditis elegans. Dev. Biol., 56 (1977) 110-156.

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