Influence of preimaginal environment on fecundity and ageing in Drosophila melanogaster hybrids—III. Developmental speed and life-span

Exp. Geront. Vol. 6, pp. 427-445. Pergamon Press 1971. Printed in Great Britain

INFLUENCE OF PREIMAGINAL ENVIRONMENT ON F E C U N D I T Y A N D A G E I N G I N DROSOPHILA MELANOGASTER H Y B R I D S - - I I I . DEVELOPMENTAL SPEED AND LIFE-SPAN F. A. LINTS and C. V. LINTS Laboratoire de G~n~tique, Facult~ des Sciences Agronomiques, Universit~ de Louvain, Kardinaal Mercierlaan, 92, 3030 Heverlee, Belgium

(Received 3 August 1971)

INTRODUCTION BROtmLY speaking there are two major theories about the mechanisms of ageing. The supporters of the first theory argue that random cell damage is chiefly responsible for the events characteristic of ageing and which culminate in death. Cell damage could be supposed to be mediated either by mutation or through errors in protein biosynthesis. So far experiments to test the random errors theory of ageing, involving exposure of organisms to unnaturally large or even small amounts of agents such as X-rays and mutagenic agents (for reviews see Clark, 1964; Welch, 1967) have been controversial and inconclusive; it was even recently stated that, in Drosophila at least, such radiations studies had nothing to do with the ageing phenomenon itself (Atlan, Miquel and Binnard, 1969). Lewis and Holliday (1970) working with Neurosporahave shown that in some senescent mutants death could result either from errors in protein synthesis or from a mutation rate increasing sharply before death. Their work however does not necessarily have a direct bearing on the mechanism of ageing in higher organisms, devoid of adult cell division. A second theory of ageing proposes that the process is closely linked to development and is therefore, at least partially, under genetical control. As Muller (1963) has argued senescence and death appear as the final stages in the process of development. It may then further be argued that the study of longevity becomes a particular case or rather the prolongation of studies in the genetics of development. It is still too soon to decide between those theories. In any case it is extremely difficult to demonstrate that they are mutually exclusive (Bullough, 1967). Indeed if the frequency of errors---accident which disrupts the structure of genes or chromosomes, changes in enzymes concerned with DNA synthesis, errors in protein biosynthesis, for instance--is not random, but is, in the words of Comfort (1968) a built-in consequence of differentiation--without being therefore and necessarily under a direct genetical control--then the apparent contradiction between both theories disappears. To demonstrate that ageing and longevity are one of the facets of the genetically and environmentally controlled process of development requires first that the handling, through the environment, of the process of development results in concomitant and consistant variations of the diverse manifestations of ageing and essentially of life-span itself: and secondly that genes which regulate development in one or the other way also 427

428

F. A. LINTS AND C, V. LINTS

regulate the manifestations of senescence and determine life-span. In this paper, and as a first step, we will try to show that variations in life-span are indeed closely and precisely linked to the variations in the process of development provoked by diverse environmental factors. If molecular information loss, which, as we have seen, may occur at different levels, is the time keeper of ageing and if that information loss is a part of the development programme, that implies that the only likely way of prolonging life-span must be through the stretching of the development considered as a whole. In testing a programmed senescence theory of ageing, Drosophila, insects in general, and other poikilotherms are particularly suitable. Indeed, unlike homeotherms, the developmental homeostatic mechanisms of poikilotherms are not very strictly regulated. For instance, by manipulating the conditions in which embryonic and larval life is spent the duration of development, which we will define as the time between fertilization and the emergence of the imago, can be considerably modified. Holometabolous insects, like Drosophila, present another remarkable phenomenon: it is now clear (Bozcuk, 1970) that their tissues show no mitoses after emergence of the imago, which means that the death of the organism must to a certain extent coincide with cellular death. With regard to other organisms Drosophila presents a third important advantage: one may study populations showing an identical genotype, viz. highly inbred lines or F1 hybrid populations obtained after crossing highly inbred lines. Clark (1964) has emphasized that in longevity studies the use of hybrid populations is more interesting than the use of highly inbred lines; indeed in such hybrid populations, which, as for other traits, exhibit heterosis for longevity (Clarke and Maynard Smith, 1955 ; Lints, 1961, 1962), death is less likely to be due to the presence of deleterious genes in the homozygous state and therefore to defective processes manifested in the presenescent period of life. It may furthermore be noted that the phenotypic variance of any quantitative trait is, in such hybrids, entirely of environmental origin; this must later permit an estimate of the relative importance of the genetic and environmental components of the variance of life-span. In other words, by using, in the present experiment, F1 hybrid Drosophilait is possible to study the physiology of development of a given genotype as opposed to the genetics of the physiology of development. We attempted to prolong the duration of development of Drosophila melanogaster hybrids by manipulating in various ways the preimaginal environment. We succeeded in prolonging it both by increasing, at constant temperature, the preimaginal population density, and by decreasing, at constant population density, the developmental temperature. In the first case the result was a decrease in size and a significant increase in imaginal life-span (Lints and IAnts, 1969b); in the second case it resulted in an increase in size and a significant prolongation of the imaginal life-span (Lints and Lints, in press). We therefore assumed a simple relation between life-span and duration of development (Lints and Lints, 1971). However a more careful examination of the data revealed afterwards that that simple relation was indeed too simple and that a more precise--and probably more pertinent--relation could be established between adult life-span and a variable which includes duration of development and a function of size. M A T E R I A L AND M E T H O D S A detailed account of the material and methods used in this series of experiments has been given elsewhere (Lints and Lints, 1969b and in press). It suffices here to remind

DEVELOPMENTALSPEEDAND LIFE-SPAN

429

that in a first experiment DrosophilamelanogasterFI hybrids were grown at the constant temperature of 25°C and at preimaginal population densities of 3, 7, 15, 30, 60, 120, 240 and 480 eggs per standard vial, and that, in a second experiment, hybrids developed at a constant population density of 30 eggs per standard vial at the temperatures of 16, 19, 22, 25, 28 and 31°C. In both experiments the emerged adults, isolated by pairs, were observed, at 25°C, during the whole life-time. The first experiment will be called the "density", the second the "temperature" experiment.

RESULTS AND DISCUSSION

The experimental data pertaining to the density and temperature experiments have been given in two previous papers (Lints and Lints, loc. cit.). Each reciprocal hybrid being considered separately those data related to the following items: duration of

26-

AGA AAG Z .~

2O-

oGA ~AG

9

"TEMPERATURE.

~DENSITY.

10,

Z O n, 14' r~

10-

3

7

I

'

15 !

30 ~

|0 ~

120 t

4~0

240 ~

!

LOG. P R E I M A G I N A L POPULATION D E N S I T Y

,~ PREIMA61NAL

FIG. | .

,;

,'~

2~

,'~

TEMPERATURE

Duration of development, in days, in function of the preimaginal temperature (lower abscissa) and the log. of the preimaginal population density (upper abscissa).

430

F. A. LINTS AND C. V. LINTS

development, imaginal size, total fecundity, mean daily egg-production and longevity. It is unnecessary to reconsider then here as such, as we are essentially interested in the comparative study of both sets of results. We will first consider the relationship between duration of development and imaginal size and try to show how precisely the variations of those variables depend on each other; their definite degree of dependence being reflected in what will be defined as the developmental speed. We will secondly try to understand in how far total fecundity and mean daily egg production depend on genotype and environment and how they are related to the other traits considered. Finally we will show how the imaginal life-span of Drosophila melanogaster depends on the developmental speed.

1. The relations between duration of development and imaginalsize : The developmental speed Figure 1 shows how the duration of development varies with the variations in preimaginal temperature or population density. Duration of development is defined as the time between the fertilization--which, due to the particular egg collecting technique used, coincides with the moment of egg-laying--and the emergence of the imago.

O

1,20,

~k

~.10'

100

0.90

~z

~_

"TEMPERATU'E"

oG~

X

"

0.50-

0 .3 i L06.

~ , PREIMASINAL ~ 32 PR'EIMAGINAL

15 ,

310

, 28

~0 ~

POPULATION , 24

120 ~

240

DENSITY . 20

~

4CO i

I ~6

TEMPERATURE

~;~. 2. ~horacic size, in rams, in f ~ c t i o , of the pre~aginM t e m p e r a t e (lower ~bscissa) ~ d the log. of the p r e i m a ~ a l population densiw (u~per abscissa).

DEVELOPMENTAL SPEED AND LIFE-SPAN

431

Figure 2 shows how the imaginal size--in fact the thoracic size measured according to the technique devised by Robertson and Reeve (1952)--evolves with the same variations in the environment. Several points emerge from those data. First, under the respective influences of the variations in temperature or in population density the relations between duration of development and size are completely reversed. Indeed the correlation coefficient between those two variables equals 0.915 (n = 12; P < 0.001) for the temperature data and --0.947 (n = 16; P < 0.001) for the density data. Secondly, the duration of development of flies developed at a constant population density increases, between 31 ° and 16°C, from a little less than 8 days to almost 27 days, a difference of 19 days. In contrast with that large increase, flies developed at 25°C and at densities varying between 3 and 480 eggs per vial present durations of development which increase from 8 to less than 16 days, a difference of only 8 days (Fig. 1). Thirdly, with regard to the same variations in the environmental conditions, the size of the temperature flies varies from 0.96 to 1.06 mm--roughly a 15 per cent increase in volume--whilst the density flies varies from 1.10 to 0.80 m m - - a more than 50 per cent decrease in volume--(Fig. 2). Thus on the one side--temperature flies--a large increase in duration is accompanied by a small increase in size, and on the other side~-density flies--a small increase in duration is accompanied by a large decrease in size. One must therefore try to see if the apparently inconsistent relations between those two variables are indeed incoherent, in other words if the two variables vary independently from each other, or if they cannot be shown to obey a definite rule. We define the developmental speed or the speed of growth as the ratio between the cube of size~in mm3--and the duration of development in days. Ideally that developmental speed should have been calculated as the ratio of weight on duration of development; unfortunately while thoracic size appears in time as a very constant character, the weight of female Drosophila was shown to vary to a great extent in a very short time (Gruwez and Lints, unpublished). When the growth rate of the temperature and density flies is plotted as a function of the variations in preimaginal environment--as in Figs. 1 and 2--one obtains two straight lines which perfectly coincide, except at the highest rates, viz. at high temperature and at low population density (Fig. 3). From the graph we infer the following. First: independently of the very different experimental conditions used, the variations in developmental rate appear to follow the same rule. Indeed that rate increases regularly and in an identical way---except at the highest rates--in function of the increase in preimaginal temperature and of the decrease in preimaginal population density. Secondly: identical variations in rate of growth may be obtained through large variations in size accompanied by small variations in duration of development, or by small variations in size accompanied by large variations in duration. In other words a given developmental rate, as it was defined, may involve flies of very different sizes and/or very different durations of development. Thirdly: at the highest developmental speeds, which as we shall see are detrimental for the emergence of adults, the linearity of the increase disappears. Concerning the temperature flies, at high temperature the speed of growth diminishes because, although the imaginal size still diminishes (Fig. 2) the duration of development can be reduced no further (Fig. 1). Extrapolating the curve gives a zero rate around 32-33°C, which is indeed the upper lethal temperature for Drosophila. With regard to the density flies, at low preimaginal population density the speed of growth still increases, but slightly: indeed size moderately D

432

F . A . LINTS AND C. V. LINTS

0,150.

~

&,A{~TEMPERATURE" AGA oGA • AG"DENSITY"

o

Q 0.100'

.< Z

o 0,050

\ \ 3 , LOG,

7

15

30

60

120

240 i

PREIMAGINAL

3;

,~,

PREI M A G I N A L

POPULATION

2',

480 ,

DENSITY

,'~

,'~

TEMPERATURE

FzG. 3. Developmental speed, in mm3/days, in function of the preimaginal temperature (lower abscissa) and the log. of the preimaginal population density (upper abscissa). The stippled line is an extrapolation. increases (Fig. 2) whilst duration is somewhat reduced, remaining however a little longer than the shortest observed for the temperature flies (Fig. 1). Fourthly: extrapolating the curve at the low rates and supposing that the variations in rate will remain linear gives a zero rate at a developmental temperature of 10°C and at a population density of around 2000 eggs per vial. What does that developmental rate represent? Obviously it is the result of the opposite effects of anabolism and catabolism. Indeed in the case of development occurring at increasing population densities--but at constant temperature the duration of development increases relatively little and size diminishes considerably, which finally results in a slackening developmental rate. It means that the duration of development cannot be extended beyond a certain point simply because the reaction speed of the different biosyntheses can only be slowed down to a small extent, the speed of enzyme synthesis, or the rate of production of the different hormones which control morphogenesis being probably under a rather strict temperature control: the anabolie processes must therefore take place at a more or less constant speed. But, due to the more and more intense crowding in the population, which probably results in a more and more difficult search for food, and thus in a larger energy expenditure, the catabolic processes become greater

DEVELOPMENTAL SPEED AND LIFE-SPAN

433

•nd arc paid at the expense of s~zc. The duration of the entire phenomenon of morphogenesis being regulated to a certain extent, at high densities, the final result is therefore a small fly with a low developmental speed. In the case of development occurring at different decreasing prcimaginal temperat u r e s - a n d at constant population dcnsity--s~zc increases somewhat whilst duration is considerably prolonged which, as in the first case, results in a slackening developmental speed. The considerable prolongation of the duration must occur because the reaction speeds of the hiosynthcscs arc slowed down in function of the decrease in temperature. In the long run the total production of the anabolic processes must bc greater. This resembles a situation described by Church and Robc~tson (1955). They found, in selected lines of Drosophila--where variations may bc duc in part to genetic factors, whilst ours arc entirely environmental in origin--that the duration of development is negatively correlated with the rate of synthesis of DNA but positively with the absolute DNA content in the adult. As regards to the catabolic processes their sum, on the average and as the larval activities remain probably more or less constant, probably does not v a ~ v c ~ much from one temperature to the other. At low temperature therefore the final result is a large fly with a low developmental rate. It could also be supposed that the final large size observed at low temperature is duc to the fact that the larvae arc permitted a longer feeding period. This could bc partially true. However wc believe that the final size is, as in the case of the density flies, the net result of the anabolic and catabolic processes. Indeed Gray (1925) and Spaas and Hcuts (1958), working with different species or intcrspccific hybrids of Salmo, have shown that eggs developed at low temperature result in larger alevins than eggs developed at high temperature. The food reserves contained in the egg being of course identical the observed variations in size must bc duc to a changing balance between anabolism and catabolism. Concluding: in a large array of environmental conditions the apparently inconsistent and independent variations in both duration of development and imaginal size in fact closely depend on each other. Their dependence expresses itself under the form of developmental rate. Wc will scc that the biological meaning of that variable is great. Indeed the imaginal life-span of flies developed in the environments considered does not depend on the size of the imago, nor on the extent, separately considered, of the anaholic and catabolic processes in the prcimaginal life, nor on the duration of development; it precisely depends on the developmental speed.

2. Fecundity: Laying period, mean daily egg-production, total fecundity In Drosophila fecundity remains one of the less known quantitative traits. There are probably numerous reasons to explain this gap. A definite method of measurement, and consequently a precise definition of fecundity, has never been unanimously accepted, since a good method of measurement requires daily and tedious countings extended over a period of two or three months. We are however relatively well informed about the influence on fecundity of genetical or contemporaneous environmental factors. Less is known of the magnitude of the influence of parental factors, i.e. the influence of the physiological and morphological characters of the parents, whatever the origins of the variations in those characters. Completely ignored is the magnitude of the influence of the preimaginal environment (for review, see Lints and Lints, 1969a). The total number of eggs laid by a female during her life-time is of course only a crude approximation of fecundity. Equal number of eggs may be laid in a short time if

434

F. A. LINTS AND C. V. LINTS

the daily egg-production is high, in a longer time if it is low. We therefore will consider three items: the laying period, the mean daily egg-production, i.e. the total eggproduction divided by the number of days of egg-laying and finally the total fecundity itself. The laying period is always and very strictly correlated with the imaginal life-span (see Results and Discussion, Section 3 : Imaginal life-span). T A B L E 1.

CORRELATIONS BETWEEN MEAN DAILY EGG-PRODUCTION AND SIZE~ DURATION OF DEVELOPMENT, DEVELOPMENTAL SPEED AND LONGEVITY

Size "Temperature" "Den s i t y " "Temperature and Density" * P

Duration of development

Developmental speed

0"390 -0"884* -0"276

0" 559 § 0" 908 * 0"432~:

Longevity

-0"501 0"902* 0"4285

0"709~ -0-888* -0"325

n 12 16 28

< 0-001.

J'0"001 < P < 0-01. $0"01 < P < 0-02. §0"02 < P < 0-05. 64. 60"

•

/

56.

/ 52-

•/ . /

* •

• ..

o 48' = ~-

/

~ 44. =¢

:/

.40. •

~ 3~

~/

39.

~

~8

•

/

.~" •

/ ~

•

•

/

o.

,

/

/

•

•

/ /'%

//

• / ~-/ ~ / // ~

//

/

//

~/./'

24

~

~,¢•

j"

/ ¢¢~ ~

/ ~

20

~

~

~

16

,~

4'a s'o s'2 5~

5g

5~

~b ~'2 o?

g~ ~

SIZE(ARBIXRABYUNITS)

FIG. 4. Regressions between mean daily egg-production (total fecundity divided by the number of days of egg-laying) and imaginal size. • . . . . Data and regression of the experiment realized at constant preimaginal population density and at different preimaginal temperatures :"temperature" data. • . . . . . . Data and regression of the experiment realized at constant preimaginal temperature and at different preimaginal population densities: "density" data.

435

DEVELOPMENTAL SP]Z~D AND L I I ~ - S P A N

Concerning mean daily egg-production different correlations have been calculated between that variable and the other traits measured, viz. size, duration of development, developmental speed and longevity. Calculations have been made on the one side for the "temperature" and "density" data separately considered, on the other side for the pooled data (Table 1). Numerous correlations are significant, but only in the case of the mean daily egg-production size relations are the three correlation coefficients significantwat least at the 5 per cent level--and are the signs of the coefficients identical. The mean daily egg-production thus apparently varies with the size of the imago: it increases with the increase in imaginal size (Fig. 4). The coefficients of correlation are respectively equal to 0.559 (n = 12; 0.02 < P < 0.05) for the temperature data, 0.908 (n = 18; P < 0-001) for the density data and 0.436 (n = 28; 0.001 < P < 0.01) for the pooled data. Yet in that last case the sum of squares of the deviations from the regression equal 3,317 out of a total sum of squares of 4,097, whilst it only equals 365 out of a total of 2,081 for the density data and whilst it rises to 411 out of a total of 598 in the case of the temperature data. 64605652:~

4844 40 36 32. 28. 24eAG

~,AG

20" 16•

:i

~

~

~'o ~

~o =,io 4~o

LOG, PREIMAGINAL POPULATION DENSITY

~"~

2~

~,~

2~,

~

-~.,

PREIMAGINAL TIEMPERATORE

Fro. 5. Mean daily egg-production, at 25°C, in function of the preimaginal population density and temperature. Figure 5 shows how the mean daily egg-production varies as a function of the preimaginal population density and temperature. Its comparison with Fig. 1 (size in function of the preimaginal environment) shows two points. The first is that the mean daily egg-production varies very regularly with the variations in size provoked by the preimaginal population density. The second is that this is not true in the case of the

436

F. A. LINTS AND C. V. LINTS

temperature data; indeed in that case a slight optimum for daily egg-production apparently occurs for flies with the lowest size, i.e. grown at 31 ° and 28°C; the daily eggproduction however does not increase for flies which have a size larger than the one attained at 25°C, i.e. the 22 °, 19 ° and 16°C imagos. The relation between mean daily egg-production and size, though significant, is thus not entirely satisfying and certainly does not cover the entire reality. As yet however we have no better explanation for the evidence. On the average the mean total number of eggs laid during life time by both reciprocal hybrids decreased markedly from one experiment to the other. It was shown (Lints and Lints, in press) that the decrease was highly significant. About 1 yr separates both experiments. This means about 25 generations of inbreeding in the lines crossed to obtain the hybrids. It is probable that the quite large discrepancy between both experiments is due to the effects of genetic drift in the one or the other, or in both of the inbred lines used. The important fact however is that, in a given experiment, the mean total fecundity--measured in a constant environment, at 25°C--remains constant for a wide range of preimaginal environmental conditions. Indeed a Tukey test applied to the "density" data indicates that the Abeele ? × Gabarros ~ (AG) hybrids, have, when grown at the preimaginal population density of 480 eggs per vial, a total fecundity which is significantly inferior to that found for the hybrids grown in the other densities and that the same applies for the Gabarros ? × Abeele ~ (GA) hybrids gro~vn at the densities of 480 and 240 eggs per vial ; the same test shows that, at the 5 per cent level, all the other data may be considered as statistically non-different (Fig. 6). Furthermore

~o0-

~ 2000¢:~ =~ ¢.~ ~_ 1500-

0 GA ~ AG

1000

~/

~ AG

500

~

~

1'5 ~0

(~0 12'0 2'~0 4~0

LOG. PREIMAGINAL POPULATION DENSITY

.3'1

.2'5

2'5

2'2

1~)

1'6'c

PREIMAGINAL TEMPERATURE

FIc. 6. Total fecundity, at 25°C, in function of the preimaginal population density and temperature.

DEVELOPMENTAL SPEED AND LIFE-SPAN

437

the same test applied to the temperature data shows that the GA and AG hybrids, when grown at 31 ° and 28°C, have fecundities inferior to the ones observed for the other developmental temperatures and that those last ones, at the 5 per cent level, do not differ significantly from each other (Fig. 6). The total fecundity of a given genotype therefore appears, within large limits of size, duration of development and developmental speed provoked by a large array of environments, as a particularly stable trait. To realize how large indeed those limits are, one may, for instance, underline the fact that the AG hybrids, developed at the preimaginal density of 240 eggs per vial, have a body volume which is almost 50 per cent inferior to the volume of the imago developed at the density of 30 eggs per vial; notwithstanding that diameter decrease in size the total fecundity of both those groups of imagos is equal: their laying period however, as well as their mean daily egg-production are very different (Lints and Lints, 1969b).

3. Imaginal life-span Since Loeb and Northrop (1917) showed that the duration of larval, pupal and adult life is negatively correlated with temperature, innumerable examples have been given of the fact that the total life-span of insects in general can be increased by keeping any or all of the stages from egg to imago at low temperature (references on that matter may be found in the excellent Bibliography of Drosophila published by Muller, 1939 and Herskowitz, 1952, 1958, 1963, 1969). This however only relates to the direct influence of environmental temperature. Concerning the indirect influence of temperature on imaginal life-span it is now demonstrated that the temperature at which the preimaginal, i.e. the embryonic and/or postembryonic development of Drosophila takes place affects the duration of subsequent imaginal life. This was demonstrated by experiments with Drosophila melanogaster which show that raising the preimaginal temperature to 28°C for instance shortens the adult life-span at various other temperatures, whereas lowering the developmental temperature, to 18°C for instance, has the opposite effect (Alpatov and Pearl, 1929; Lints, 1963b; Lints and Lints, 1971; Burcombe and Hollingsworth, 1970). With regard to the indirect influence of preimaginal population density there is still, as noted by Comfort (1964), some disagreement whether delayed growth of larvae, which is one of the effects of crowding, leads to an increase in imaginal life-span. That disagreement essentially relies upon the experiments of Northrop, 1917; Loeb and Northrop, 1917) who found no such increase in imagos reared from retarded larvae. The growth of those larvae was delayed by keeping them for various periods on a yeastless medium. However there are probably no good reasons for comparing the effects of larval underfeeding or feeding with toxic substances and the effects of crowding in presence of large and sufficient amounts of food. These effects are essentially different and underfeeding or feeding with toxic substances, during the embryonic or postembryonic stages, must normally lead, when essential metabolites are absent or destroyed, to a reduction of the imaginal life-span. Whatever it is, it is now clearly demonstrated that increasing the larval population density may considerably prolong the imaginal life-span (Miller and Thomas, 1958; Lints, 1963a and b; Lints and Lints, 1969b). Thus variations in preimaginal population density and temperature were shown to influence to a large extent the imaginal life-span of a given genotype measured in one precise environment. For instance, at 25°C, the life-spans of hybrids grown at 7 or 480 eggs per vial are respectively of 39.5 ± 5.23 and 66.3 4- 4.08 days, an increase of more

438

F. A. LINTS AND C. V. LINTS

than 60 per cent; the life-spans, at 25°C, of hybrids grown at 31 ° and 16°C are respectively of 43.7 4- 4.3 and 66.7 :t: 1.8 days, an increase of more than 50 per cent. Different relations between life-span and the other traits measured have been looked at. The correlation coefficients between size and life-span equal 0.691 (0.001 < P < 0.01; n = 12) for the temperature data, but -- 0.819 (P < 0.001; n = 16) for the density data. The opposite signs between both those significant coefficients point to the fact that in any case size in itself plays no direct role in the determinism of life-span by Drosophila (Fig. 7).

~

66•

•

• •

\

64-

i "\'\

• 62-

// / /

"\ ~ /

i

/)X, i 60-

~, .

~.

~ /~ /

X.

/ /

58"

A

/

~ ,~t/

•

k

X

/

• • k

56-

"~ , o'~

54-

• ,

~.

•

52

% ~, "k

50

~. N oN

48.

3'5

" 4'0

~'0

dO LIFE+SPAN(DAYS)

7~

FIO. 7. Regressions b e t w e e n t h o r a c i c s i z e - - i n a r b i t r a r y units, 60 u n i t s b e i n g equal to 1 m m - - a n d longevity in days. T e m p e r a t u r e ( A , - - - ) a n d density ( • , - . . . . . ) data are separately considered.

The signs of the coefficients of correlation calculated between duration of development and life-span are identical for the temperature and density data, separate or pooled (Table 2). They are 0.514 (n = 28; P < 0.001) for the pooled data, 0.769 (n = 16; P < 0.001)for the density data and 0.593 (n = 12; P < 0-05)for the temperature data. From the pooled data a curvilinear regression line of the form Y = 9.5 ÷ 5.9X -0.16X 2 (R -----0.72; n = 28; P < 0.001) is significantly superior to the linear regression (F251 = 13.2; P = 0.001) (Fig. 8) (Lints and Lints, 1971). However these relations are not entirely satisfying. Indeed the sum of squares of the deviations from the regression are relatively high in comparison with the sum of squares of the deviations due to the regression (Table 2). Other relations were accordingly sought. For instance, Fig. 9 shows the relations which exist between life-span and the ratio of size (expressed in mms) and duration of development (expressed in days). Table 2 shows how the sum of squares of the deviation from regression diminishes considerably with regard to the same value calculated for the regression duration--life-span.

439

DEVELOPMENTAL SPEED AND LIFE-SPAN • 26, 24,

~ 20

I¢ ~. 18

¢ ~ 16. om14" i,-

~2-

g

10

1~" ~,.,~.~.~.~.~.~.~.o~ •

• ~

•

8.

~'5

~b

s

s

•o

~, •

2'0

8'0

LIFE-SPAN (DAYS)

rb

FIG. 8. Regressions between duration of development, in days, and imaginal life-span, in days. • " T e m p e r a t u r e " data • " D e n s i t y " data. '---Regression for the temperature data. ...... Regression for the density data. ~ Regression for the pooled data. T h e curvilinear regression for the pooled data is significantly superior to the linear regression for the same data.

T A B L E 2. RELATIONS BETWERN LIFE-SPAN AND DURATION OF DEVELOPMENT~ THE RATIO SIZE/DURATION OF DEVELOPMENT AND DEVELOPMENTAL SPEED FORTHE "TEMPERATLrRE' ~AND " D E N S I T Y " DATA, SEPARATE AND POOLED. COEFFICIENT OF CORRELATION AND SIGNIFICANCE (7), ORIGIN AND SAMPLE COEFFICIENT OF THE REGRESSION ( a AND b), SUM OF SQUARES OF THE DEVIATIONS DUE TO THE REGRESSION ( b ~ x y ) AND SUM OF SQUARES OF THE DEVIATION FROM THE REGRESSION (~ff~ - - b ~ y )

Duration of development

t° d t°+d

Size/duration

t° d

D e v e l o p m e n t a l speed

t° d

t°+d

t°+d

r

a

0"593§ 0"769* 0"514t

43.41 19.95 43.02

- 0"703t - 0.828* -0"758*

70.34 80-79 74.71

- 0.718~ - 0.852* -0.801 *

72"57 71"85 71.90

" T e m p e r a t u r e " : n = 12 " D e n s i t y " : n = 16 " T e m p e r a t u r e + D e n s i t y " : n = 28

b

bY,x y

0.73 3"34 0"88

277.4 706"4 525.4

509.1 500.2 1478-9

-2.99 -4.40 -3.57

388.8 828.0 1146.9

397"7 378.6 857.5

-193"31 -179.30 -182.45

410.1 876.8 1285.9

376.4 329.7 718.4

* P < 0"001. "1"0"001 < P < 0-01. ~ 0 ' 0 1 < P < 0"02. § 0"02 < P < 0"05.

Y.y~ -- b Z x y

440

F.A. LINTS AND C. V. LINTS

•

i / / /~1!

3-

il / •

=/

~/////~

• /////~///'~

,U.: //•o

•

• ~N 8 ~

• ./ i

a'~

,

4b

o ,,,~'.

1 •

5b

7'0

6b

U~-S~A~(~S)

FIG. 9. Regressions between the ratio size/duration of development and longevity. Symbolism as in Fig. 8. 0.03-

o/~ •

/4

•

/~

.

°

~,

0~07-

~. ~ 0~09-

,

~ 0,11 0.

i/y'• IZI

=~

0.13

• •

~5

"

~

~b

i

°

i II/'j~lll i ~ I~• . , ~ ., /~'

~'o

6~ i~s~ u~-s~

r~

FIG. 10. Regressions between developmental speed and longevity. Symbolism as in Fig. 8.

The nearest relationship however is between life-span and developmental speed (Fig. 10; Table 2). In that case the parameters of the regression lines--for the data pooled and separate~therefore almost coincide, and finally the sum of squares of the deviations from the regression is minimal.

DEVELOPMENTAL SPEED AND LIFE-SPAN

441

Different conclusions emerge from the precise relationship disclosed between the imaginal life-span of a given genotype and elements which are completely determined before the emergence of the imago. First of all the ubiquity of that rdation must be confirmed with other genotypes and controlled or rather refined over a wide range of imaginal environments. It is dear also that claims about the relationships between lifespan and other quantitative traits, as size and duration of development, must be accepted with caution when those traits are separately considered. But the important fact is the following. The respective influences of preimaginal and imaginal environments on the determinism of life-span should be clearly separated. Theories of ageing have been suggested which depend on the study of the influence of temperature, temperature variations, thermic shocks and radiation treatments during adult life-span and which absolutely do not take into account the possible influence of preimaginal environment. It now appears that both those influences may have extremely different effects. CONCLUSION The study of development, fecundity and life-span of Drosophila melanogaster hybrids grown in a large range of environments and observed during the whole imaginal life in the same environment has yielded some indications bearing to the determinisms of fecundity and longevity. Concerning fecundity, it appears that that variable may be divided into three components: the mean daily egg-production, the total fecundity and the laying period which is invariably linked with life-span. The three components are function of different determinisms. Total fecundity essentially depends on the genotype, whilst mean daily egg-production is largely determined by the size of the imago, whatever the environmental conditions responsible of that size. Those results permit to have doubts about the validity of a number of observations--and conclusions--regarding fecundity. Particularly doubtful are claims about a possible relation between fecundity and longevity. Numerous authors have indeed measured fecundity only for a few days after emergence. That method is based on a series of evidences provided by Gowen and Johnson (1946). These authors however worked with different species or races of Drosophila and their evidence concerning inter- or intraspecific differences, for a large part genetically determined, now appear to have no bearing on intraspecific differences provoked essentially by the environment. Clearly the possible influence of diverse variables on the life-span of a given genotype of Drosophila melanogastercould be ruled out by the present experiments. Imaginal life-span does not depend on the size of the imago. The inverse relationship between those two variables under the respective influences of variations in preimaginal temperature and population density demonstrates it without ambiguity. Life-span also does not depend on the speed of the anabolic processes in the preimaginal life. If it did depend on it, and if one admits that the speed of the anabolic processes, or at least the inferior limit of it, essentially depends on the temperature, then all the flies grown at 25°C, at different population densities, should have longevities differing from each other much less than what was observed. The magnitude of the catabolic processes in the preimaginal life also does not determine the imaginal life-span. If it did and if one admits the hypothesis invoked to explain the very reduced size and the not too much prolonged duration of development of flies grown at 25°C at a very high population

442

F. A. LINTS AND C. V. LINTS

density, the life-span of those last flies should be shorter than that of flies grown at the same temperature and at low population density: this is not so. Duration of development also does not determine life-span, at least not solely. Indeed the longest mean longevities observed in the temperature and density flies are almost equal, viz. 66.7 ± 1-8 days (AG16°c) and 66.3 -4-4.1 days (GA480); in fact the durations of development of those flies are the longest observed in both the temperature and the density experiments; they differ however very much being respectively equal to 27.0 ± 0.18 days in the temperature experiment and to 14.6 ± 0.08 days in the density experiment. Life-span of a given genotype in a given environment finally depends on the developmental speed, i.e. the productivity of the genotype per unit of time, or the resultant, due to environmental circumstances, of anabolism and catabolism. As said before it is now demonstrated that tissues of holometabolous insects show no mitoses after the emergence of the imago (Bozcuk, 1970). The death of the organism must therefore, to a certain extent, coincide with the cellular death. On the other hand it is known, from studies carried out in higher organisms, that, when a cell enters the ageing pathway, it has a life expectancy that is characteristic of the tissue. When the mitotic rate of the tissue is high, as in the intestinal lining, the life expectancy of the functional cells is only a few days (Lesher, Fry and Kohn, 1961); when the mitotic rate is moderate, as in epidermis, the life expectancy of the functional cells may be a few weeks (Scott and Ekel, 1963); and when the mitotic rate is low, as in liver, the life expectancy of the functional cells may exceed one year (McDonald, 1961). Artificial prolongation of the epidermal cell life was obtained by Bullough and Ebling (1952) in conditions of stress which provoked a decreased mitotic rate; that result was extended to liver cells by McDonald (1961). We have, as yet, no direct evidence concerning the mitotic rate in temperature and density flies. However, some indirect evidence exists. It is indeed known from previous experiments that the decrease in size obtained by increasing larval density or developmental temperature--variations in size which are respectively accompanied by a longer or a shorter duration of development--follows from a decrease in both cell size and cell number (Deleour and Lints, 1967). Two non-exclusive hypotheses may be put forward to explain the influence on adult life-span of the variations in developmental speed. In a first hypothesis it would be assumed that the preimaginal environment partially determines the adult life-span through an interference with subcellular constituents which must pass the metamorphosis and yet remain intact and coded in the one or the other way. It is maybe not unreasonable to postulate that organisms whose metabolic machinery is adapted, during development, to work at a slow speed, whose anabolism-catabolism ratio results in a slow developmental speed, proceed to work slowly during their adult life. Perhaps, as supposed by Burcombe and Hollingsworth (1970) to explain the influence of preimaginal temperature on adult life-span, does the developmental speed affect the type of molecule that is synthesized and the rates at which these molecules carry out metabolism; in other words the types and the working capacities of molecules could be different when synthesized at different speeds. This hypothesis resembles Medvedev's delayed maturity hypothesis. Medvedev (1966) assumes that the lengthening of the adult life-span may be obtained by stretching the development programme. The programme of our slowly developing flies being, on the organismic, cellular and subcellular level and under the influence of various environmental factors, more slowly run during the developmental period, it could be argued that slower running speed is maintained during adult life. The action of destruc-

DEVELOPMENTALSPEEDAND LIFE-SPAN

443

tion of the cell at the appropriate time (Bullough, 1967), as defined by the programme, would then come later in time. In a second hypothesis developmental speed could be associated with mitotic division rate. W h e n the growth speed is accelerated or decelerated it may, as a first approximation, be admitted that the same happens for the mitotic division rate. One actually knows very well that anarchic and rapid cell division is generally associated with an abnormally large number of chromosomal rearrangements. It could then be argued that a slower mitotic division rate could be accompanied by a reduced n u m b e r of errors of replication of the hereditary material. This should then be reflected in later life in a lower number of errors of synthesis which could justify a later, programmed or unprogrammed, death of the cell and of the organism.

REFERENCES ALPATOV,W. W. and Pma~L, R. (1929) Am. Nat. 63, 37. ATLAS, H., MIQUEL, J. and BINN~mD, R. (1969) 3t. Gerentol. 24, 1. BOZCUK, N. (1970) Phil. thesis of the Univ. Sussex. BULLOUGH,W. S. (1967) The Evolution of Differentiation. Academic Press, New York. BULLOUGH,W. S. and EBLIN~, F. J. (1952)~. Anat. 86, 29. BlmCOMBE, J. V. and HOLLINOSWORTH,M. J. (1970) Gerentologia 16, 172. CHURCH, R. B. and ROBERt'SON,F. W. (1966) ~enet. Res. 7, 383. CLARK, A. M. (1964) Adv. gerent. Res. 1, 207. CLARKE,J. M. and MAY~IAROSMITH,J. (1955)j~. Genet. 53, 172. COMI~ORT,A. (1964) Ageing: The Biology of Senescence. Routledge and Kegan. COMFORT, A. (1968) Nature, Lond. 217, 320. DELCOUR, J. and LINTS, F. A. (1967) Genetica 37, 543. Gowm% J. W. and JOHNSON, L. E. (1946) Am. Nat. 80, 149. GRAY, J. (1926)j~. exp. Biol. 6, 125. HERSKOWlTZ,I. H. Bibliography on the Genetics of Drosophila. (1952) Part II : Famham Royal. (1958) Part III: Indiana University Press. (1963) Part IV: McGraw-Hill. (1969) Part V: McMillan. LESHER, S., FRY, R. J. M. and KOHN, H. I. (1961) Lab. Invest. 10, 291. LEWIS, C. M. and HOLLIDAY,R. (1970) Nature, Lend. 228, 877. LISTS, F. A. (1961) Genetica 32, 177. LINTS, F. A. (1962) Acta biotheor. 16, 1. LINTS, F. A. (1963a) Nature, Lend. 197, 1128. LINTS, F. A. (1963b) Bull. Biol. France-Bdgique 97, 605. LINTS, F. A. and LINTS, C. V. (1969a) Exp. Gerent. 4, 81. Liars, F. A. and LINTS, C. V. (1969b) Exp. Gerent. 4, 231. LINTS, F. A. and LINTS, C. V. (1971) Nature, Lend. 229, 86. LINTS, F. A. and LINTS, C. V. Exp. Geront. 6, 417. LOEB, J. and NORTHROP,J. H. (1917)ft. biol. Chem. 32, 103. McDONALD, R. A. (1961) Archs intern. Med. 107, 335. MEDVEDEV,Z. A. (1966) Protein Biosynthesis. Oliver and Boyd. MILLER, R. S. and THOMAS,J. L. (1958) Ecology 39, 118. MULLER, H. J. (1939) Bibliography on the Genetics of Drosophila. Oliver and Boyd. MULLER, H. J. (1963) In Cellular Basis and Actiology of Late ,~omatic Effects of Ionizing Radiation. Academic Press, New York. NORTHROP, J. (1917)~. blol. Chem. 32, 123. ROBERTSON,F. W. and RimvE, E. C. R. (1952)~. Genet. 50, 414. SCOTT, E. J. van and EKEL, T. M. (1963) Archs Derm. 88, 373. SPAAS, J. T. and HEUTS, M. J. (1958) Hydrobiologia 12, 1. WELCH, J. P. (1967) Adv. gerent. Res. 2, 1.

F. A. LINTS AND C. V. LINTS S u m m a r y - - M e a n daily egg-production, total fecundity and imaginal life-span of Drosophila melanogaster hybrids, grown in various preimaginal environments, viz. six temperatures and eight population densities, were measured at 25°C. Duration of development and size at emergence were also recorded. Duration of development and size of hybrids grown at various temperatures are positively correlated. T h e correlation between those traits is negative for hybrids grown at various population densities. Very different in magnitude and even opposed, the variations of both those traits depend however on each other; this is testified by the identical behaviours of the variations in developmental speed as a function of both preimaginal temperature and population density. Developmental speed is defined as the ratio of the cube of size on duration of development. Total fecundity essentially depends on genotype, and only at extreme values upon the preimaginal environment. Mean daily egg-production, on the contrary, depends on size at emergence, whatever the conditions responsible for that size. Imaginal life-span, which, for a given genotype and in a given imaginal environment, may pass from the simple to more than the double, strictly depends on the developmental speed. Two hypotheses are put forward to explain those data. The first appeals to Medvedev's delayed maturity hypothesis of ageing and the second one links developmental speed to mitotic division rate. R 6 s u m 6 - - L a moyenne de ponte journalibre, la f6condit6 totale et la long6vit6 imaginale d'hybrides de Drosophila melanogaster, qui s'dtaient ddvelopp6s dans u n grand nombre de milieux pr6imaginaux diff6rents, ont 6t6 mesurdes h 25°C. La dur6e de d6veloppement et la taille h l'6mergence de ces hybrides ont 6galement 6t6 6tudi6es. Les milieux choisis comprenaient six temp6ratures et huit densit6s de population pr6imaginales. Lorsque des hybrides croissent ~ diff6rentes temp6ratures pr6imaginales les caract~res dur6e de d6veloppement et taille sont corr616s positivement, tandis que ces m~mes caractbres le sont n6gativement lorsque ces hybrides se d6veloppent h diffdrentes densit6s de population pr6imaginales. M~me oppos6es et de grandeur fort diff6rente les variations de ces deux caractbres d6pendent clairement l'une de l'autre ainsi qu'en t6moignent les allures identiques des variations de la vitesse de d6veloppement en fonction tant de la temp6rature que de la densit6 de population pr6imaginale. La vitesse de d6veloppement est d6finie comme 6tant 6gale au rapport entre le cube de la taille et la durde de d6veloppement. I1 a 6t6 montr6 dgalement que la f6condit6 totale d6pendait essentiellement du g6notype et fort peu, sinon aux valeurs extremes, des conditions du milieu pr6imaginal. La moyenne de ponte journalibre, par contre, d6pend de la taille h l'emergence quelles que soient d'ailleurs les conditions de milieu responsables de cette taille. La dur6e de vie imaginale d ' u n g6notype donn~ /~ une temp6rature pr~cise-dur6e de vie qui peut varier du simple au plus du double--d6pend strictement de la vitesse de d6veloppement. Deux hypotheses sont avanc6es pour expliquer ces r6sultats. L'tme fair appel, en la modifiant quelque peu, ~ l'hypoth~se de la maturit6 diff6r6e de Medvedev; l'autre lie la vitesse de d6veloppement aux taux de division mitotique.

Zusammerdasstmg--Die mittlere tiigliche Eiproduktion, die Gesamffruchtbarkeit und die imaginale Lebensdauer yon Hybriden von Drosophila melanogaster, welche in verschiedenen pr~iimaginalen Bedingungen (sechs Temperaturen und acht Populationsdichten) gezogen wurden, wurden bei 25 ° bestimmt. Die Dauer der Entwicklungsphase u n d die Gr6Be beim Schliipfen wurden ebenfalls bestimmt. Dauer der Entwicklungsphase und Gr613e der Hybriden, welche bei verschiedenen Temperaturen gezogen werden, sind positiv korreliert. Die Korrelation dieser

445

DEVELOPMENTAL SPEED AND LIFE-SPAN

Eigenschaften ist negativ bei Hybriden, welche bei versehiedenen Populationsdichten gezogen werden. Obwohl sehr verschieden in der Gr613e u n d sogar gegengerichtet, h~ngen die Variationen Beider Eigenschaften jedoch voneinander ab; dies wird belegt durch das identische Verhalten der Variationen in der Entwicklungsgeschwindigkeit als Funktion von priiimaginaler Temperatur u n d Populationsdichte. Die Entwicklungsgeschwindigkeit wird definiert als das Verh~iltnis der 3. Potenz der Gr613e zur Entwicklungsdauer. Die Gesamtfruchtbarkeit h~ngt im wesentlichen vom GenotTp ab, n u r bei Extremwerten v o n d e r pr~iimaginalen Umgebung. Die mittlere t~gliche Eiproduktion h~ingt jedoch im Gegensatz dazu yon der Gr613e beim Schliipfen ab, wobei die Gr613e bestimmenden Faktoren noch often sind. Die imaginale Lebensdauer, welche bei bestimmtem Genotyp und bestimmter Imaginalumgebung bis auf das Doppelte zunehmen kann, h~ngt streng von der Entwicklungsgeschwindigkeitab. Zwei Hypothesen zur Erkliirung der Befunde werden gegeben. Die erste lehnt an Medvedev's Altershypothese der verz6gerten Reifung an, die zweite verbindet die Entwicklungsgeschwindigkeit mit der Mitoserate. P e 3 ~ M e ~ C p c ~ eme~memm~ m~menoc~ocTs, o6ma~ nno~oB~rocTs a n p o ~ o ~ sao~s ~ s3po~ r a 6 p ~ o s Drosophila melanog~ter, s ~ e ~ Bp ~ ~ ~ ~ c ~ , a ~e~HpH m~TH T e ~ e p a ~ n B B~b~ n o ~ HO~ ~OTHO~, ~Mep~ ~ n 25 ° ~ . T a ~ e ~ ~ c ~ ~e~H~Tb p~B~ ~ p~Mep ~ B ~ . ~ e ~ b p~B~ n p~Mep ~ 6 p ~ o ~ , ~ p ~ c ~ np~ p ~ ~ pampa, o6aap~r nono~Te~ ~op~. H o p ~ MC~ 3T~ np~3~aK~ oTp~aTe~n~ y ~6p~oe, B~p~e~ ~ff p ~ J m ~ ~OT~ffX no~n~ .

XOTff ~ p ~

~OT~OnOnO~, no~e~ ~pn~ ~ nO~O~O~ pa3Mepa K ~

e

O ~ O ~ 3 T ~ ~ H 3 H a K O B C ~ H O O T ~ a ~ T C f f nO ~ o~B3a~o3aB~c~. O~ 3TOM C B ~ C T ~ B y ~ T

~ ~opo~n p ~ ,

~OTH~T~. ~ p~B~.

~opo~b

~

~

p~B~

n ~ ~ n o ~ O~e~eTCff

C

~ ~ O~KOB~

Te~epaT~bI

OTHO~CM

Ky~a

~ ~O~OB~b 3 a B f f c ~ B ~HOBHOM OT r e ~ o T ~ a ~ TO~KO ~H K p ~ 3Ha~e~X~T ~ H ~ H O ~ C~. C ~p~o~ CTOpO~I, C ~ e~e~eBffa~ ~ e H ~ 3 a B f f C ~ OT p~Mepa n p n Bb~eTe~ H ~ 3 a B ~ O OT y C ~ O B ~ ~ ~ Ha 3TOT p a 3 M e p . ~pO~O~e~a~Tb ~ 0 ~ ~ ~ H O ~ np~eM ona ff~o~cff

~3~ B3poc~ Ha~KO~ ~ ~oro ~Ho~a C ~ e ~ MO~CT nOB~CffT~ff OT ~ o ~ o r o ~o ~ o r o B ~poro~ 3 a ~ o ~ OT c K O p ~ T ~ p ~ B ~ .

H B

cpoKa,

~ o 6 ~ c n e ~ 3T~ ~ 6 ~ n~O~C~ ~ r ~ O T C 3 ~ : n e p B ~ 6~npyeTCff aa ~OTe~ M e ~ e ~ , c o r n a c a o KOTOpO~ ~ a ~ e ~eT 3~ep~a 3~n~, a BTOp~ C B ~ ~opo~b p~B~ Co c K O p ~ T M m ~ o ~ K o r o ~ene~.