Respiration in Drosophila—III influence of preimaginal environment on respiration and ageing in Drosophila melanogaster hybrids

Exp. Geront. Vol. 4. pp. 81-94. Pergamon Press1969. Printed in G r u t Britain

RESPIRATION IN DROSOPHILA--III INFLUENCE OF PREIMAGINAL ENVIRONMENT ON RESPIRATION AND AGEING IN DROSOPHILA MELANOGASTER HYBRIDS F. A. LINT$ and C. V. LINT$ F. A. Janssens Memorial Laboratory for Genetics, Agricultural Institute of the University, Parc d'Arenberg, Heverlee-Louvain, Belgium

(Received 25 November 1968) INTRODUCTION RATEof oxygen consumption, fecundity and other biometrie traits of different populations of Drosophila melanogaster imagos were measured at 25°C from emergence to death. The populations studied included a wild type Gabarros, an inbred line Gabarros, and reciprocal hybrids obtained by pairing Gabarros and Abeele inbred lines. It was shown that in precise environmental conditions the mean respiration rate of a given genotype as well as the regression of its respiration rate with age are largely independent of the expression of other biometric traits. More precisely it was shown that when the data relating to the three populations studied are pooled together there exists no relation between the mean respiration rate and the other quantitative traits measured, viz. size, weight and longevity; particularly there exists no relation between the decreases in respiration rate and in fecundity with age (Lints and Lints, 1968). As a next step it then appears interesting to see to what extent the respiration rate and the evolution of that rate with age are determined by genotype and environment respectively. It is well known that environment influences to a large extent the expression of diverse biometric traits in insects. More specifically preimaginal environmental conditions may to a certain degree determine traits as size, duration of development and longevity (Lints and Lints, 1965, 1968). This paper reports experiments in which preimaginal stages of Drosophila melanogaster hybrids--reciprocal crosses between Abeele and Gabarros inbred lines--were cultured at a fixed temperature (25°C) under two different degrees of larval crowding; duration of development, size and life span were determined for those different genotype-environment combinations, while measurements of fecundity and respiration rate were carried out at 25°C throughout life, on individual females isolated with a male. A further paper will describe similar experiments in which the preimaginal stages are cultured under a fixed degree of larval crowding at three different temperatures. M A T E R I A L AND M E T H O D S The Ila~ generation of the Gabarros4 inbred line of the Gabarros strain (Spain)and the It62 generation of the Abeele inbred line (Belgium) were reciprocally crossed in order to obtain the hybrids Gabarros 4~ × Abeele ~ (GA) and Abeele ~ × Gabarros 4c~ (AG). A report on the previous history of the Abeele and Gabarros strains, from 1959 on, was 81

82

F. A. LINTS AND C. V. LINTS

published earlier (Lints and Lints, 1965). The eggs required for experiment were collected in the following way. Virgin females and males were kept in separate bottles. The day before the experiment, they were put together, transferred to a fresh medium and fed with a large amount of live yeast. Eggs laid by young flies aged 3-9 days were collected on an agar-agar gelled acetic alcohol medium during a 4-hr period (cf. Heuts, 1956; Lints, 1960; Parsons, 1964; Lints, Lints and Zeuthen, 1967). They were then redistributed in lots of 30 (30 replicates) or 240 (6 replicates) in standard culture tubes in which the surface area of the medium was 5.5 cm% and the total weight of the food 10.0 4- 0.1 g. The medium was the classical agar-agar-sugar syrup-cornmeal-baker's yeast mixture. The culture temperature, 25°C, was regulated to a tenth of a degree. Upon emergence only those females having a modal duration of development--9 and 14 days respectively at densities 30 and 240 eggs per tube--were kept for measurement of thorax size, which was realized according to the method of Robertson and Reeve (1952). Only individuals of modal size were retained for the subsequent measurements. Oxygen consumption measurements were carried out according to the method described by Gregg and Lints (1967). Since the organisms are not starved but immobile during the experiment, these measurements reflect the energy needed to maintain a steady-state and to sustain a limited growth, i.e. to fuel the chemical reactions involved in maintaining respiration itself and in synthetizing materials for cellular repair, growth or reproduction; in experiments performed on insects under these conditions only a negligible amount of energy is used for physical purposes. When followed throughout the whole life span of the imago the respiration measurements will then furnish estimates of the relative "rates of living" of the different genotype-preimaginal environment combinations studied. For the four genotype-preimaginal environment combinations here considered 355 determinations of oxygen consumption were made, of which 132 had to be discarded for various technical reasons. Measurements were made at 25°C, from a few days after emergence up to the death of the imagos. Concurrently, fecundity, fertility and life span were recorded for a parallel group of females also possessing modal durations of development and size. For each genotypepreimaginal environment combination studied, twenty females, each with a male, were kept in individual tubes, daily renewed. Dead males were replaced, as life span was taken into account only in case of the females. The daily egg-production of ten of these females was noted, the daily brood retained and the number of emergences counted in due time.

RESULTS

Figures 1-4 show the results of this series of measurements. Table I gives the mean oxygen consumption rates, and the mean size, weight and life span for each of the four genotype-preimaginal environment combinations studied. Table 2 presents various data relating to fecundity and fertility. Table 3 gives the correlations found between rates of oxygen consumption and age, and the parameters and significance of the regressions of oxygen consumption on age. The main features of the results may be enumerated as follows: (1) Between emergence and death the two reciprocal hybrids have almost identical respiration rates at each of the larval population densities used (see Table 1). Expressed in/zl O~/mg fresh weight/hr, the respiration rates are 3-69 q- 0-11 and 3.40 -4- 0-10 for

RESPIRATION IN DRO$OPHILA--III

100.6

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days FIC. 1. Relation between rate of oxygen consumption, age and fecundity in imagos of the Gabarros 4 inbred ~ × Abeele inbred c~hybrid reared at a density of 30 eggs/5 "7 cm z (GAs0). Abscissa: age in days. Right ordinate: oxygen consumption in tzl O2/mg fresh wt./hr (solid circles). Left ordinate: total number of eggs laid per day by ten females (open circles).

TABLE

1.

RATES OF OXYGEN CONSUMPTION PREIMAGINAL

ENVIRONMENT

Oxygen consumption (~I O~/mg fresh weight/hour) G ~ A 3 30 A~ Gc~ 30 G~ Ac~ 240 AS G 3 240

3.694-0-11 3.40 .4- 0.10 3-66 .4- 0.12 3-51 .4- 0.10

AND OTHER QUANTITATIVE COMBINATIONS

Thoracic size (arbitrary units) 41.1 41 "5 34"8 35-5

±0.10 .4- 0.08 .4- 0.12 .4- 0.14

OF

CHARACTERS IN FOUR GENOTYPE-

Drosophila melanogagter Weight (mg) 1.31 1"33 0.88 0.92

.4-0.02 .4- 0.02 .4- 0.01 q- 0.02

Life span (days) 46.4.4-2.5 43.7 -4- 3.0 58.8 .4- 3-2 59.1 .4- 2 . 4

T h e thoracic size is expressed in arbitrary units, 1 m m being equal to 40 units ; it is the modal size of individuals with a modal duration of development. T h e weight, given in mg, expresses the mean weight of all the individuals used to measure rate of oxygen consumption, weight being recorded before oxygen uptake. Life span expresses the mean duration of imaginal life from records over twenty individuals.

84

F. A. LINTS AND C. V. LINT$

J ,m o

g,

100 90, 80 5 70 60 50 40. 30 20 10,

01

FzG. 2. As in Fig. 1, in imagos of the Abeele inbred ~ × Gabarros 4 inbred 3 hybrid, reared at a density of 30 eggs/5.7 cm~(AGs0).

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FIG. 3. As in Fig. 1, in imagos of the Gabarros 4 inbred ~ × Abeele inbred of c~ hybrid, reared at a density of 240 eggs/5"7 c m 2 ( G A 2 4 0 ) .

85

RESPIRATION IN DROSOPHILA~III

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days FIo. 4. As in Fig. 1, in imagos of the Abeele inbred ~ × Gabarros 4 inbred c2hybrid, reared at a density of 240 eggs/5.7 cm2(AGz40). GA and AG30, 3"66 -b 0.12 and 3"51 4- 0.10 for GA and AG240. Tested by means of the analysis of covariance, none of the differences observed is significant. (2) The average respiration rates of the hybrid imagos do not depend on the preimaginal population density (Table 1). (3) Respiration rate decreases with age in both hybrids when larval population density was 30 individuals per tube (Table 3). The regression coefficients are higher in AG than in GA hybrids, but not significantly. There is no relation between respiration rate and age for either hybrid at population density 240 larvae per tube. (4) The imaginal life span of both reciprocal hybrids is very significantly increased when the preimaginal population density is raised from 30 to 240 eggs per standard tube. Life span is prolonged by more than 26 per cent in the GA hybrids (58.8 -4- 3.2 vs. 46"4 4- 2.5 days), and by more than 35 per cent in the AG (59.1 4- 2.4 vs. 43"7 4- 3.0 days) (Table 1). (5) At the higher larval population density for both reciprocal hybrids size is greatly reduced and modal duration of development considerably prolonged (see Table 1 and Material and Methods section). Yet in the case of the AG hybrid the mean total fecundity is not reduced; in fact it increases a little though not significantly (AGa0:1853 4- 230; AG94o: 2021 4- 75 eggs; t = 0-9; P: n.s.) (see Table 2). However in the GA hybrid the mean total fecundity decreases slightly when larval density per tube is raised from 30 to 240 (GAa0:2227 4- 100; GA240:1876 4- 116 eggs; t = 3.2; P < 0.001). (6) The maximal daily egg production is attained much later at preimaginal rearing densities of 240 eggs per tube as opposed to only 30 eggs per tube, namely, 13.1 4- 2.2 days for GAz40 vs. 6.4 4- 0.9 days for GAa0 (t = 2.8; 0.01 < P < 0-02) and 10.4 4- 0"9 days for AG240vs. 4.2 4- 0-2 days for AGso(t = 6.4;P < 0.001) (see Table 2). In addition

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F. A. LINTS AND C. V. LINTS

TABLE 2. FECUNDITY, FERTILITY AND RELATED TRAITS FOR FOUR GENOTYPE-PREIMAGINAL ENVIRONMENT COMBINATIONS OF Drosophila melanogaster Fecundity (total n u m b e r of eggs laid)

G? A3 A~ G3 G~ A~ A? G3

30 30 240 240

2227 1853 1876 2021

± 100 4- 230 -4- 116 4- 75

Fertility M a x i m a l daily L a y i n g p e r i o d M e a n daily ( n u m b e r of egg p r o d u c t i o n ( n u m b e r of days) egg p r o d u c t i o n eggs h a t c h i n g ) a t t a i n e d o n day:

1846 1568 1602 1736

i 102 4- 190 :L 80 4- 93

6"4 4.2 13-1 10.4

i 0.9 4- 0.2 4- 2.2 4- 0.9

43.5 35.2 46-8 50-2

-4- 3"1 4- 3.5 4- 4.5 4- 2"8

50-44- 3"2 56-7 4- 3"9 37.2 4- 2.5 38"6 4- 2"2

T h e figures for fecundity, fertility a n d related traits are m e a n s f r o m t e n individuals.

TABLE 3.

RELATIONS BETWEEN OXYGEN CONSUMPTION AND AGE FOR FOUR GENOTYPE-PREIMAGINAL ENVIRONMENT COMBINATIONS OF Drosophila melanogaster

r02 cons.--age

G? Ad' A~ Gd' G~ A3 A~ G3

30 30 240 240

- 0"375* - 0'648t -- 0" 152 n.s. - 0" 154 n.s.

Regression equation Y = 4.25 - 0"019X Y = 4"23 - 0"031X n.s. n.s.

95% confidence interval for a y 4"47--4"04 4-40--4-06 ---

t-value for t h e difference b e t w e e n regression coefficients

1 "41 n.s. ° ----

~fP < 0.001. * 0 . 0 0 1 < P < 0.01. n.s. non significant. o with A~ G3 30

mean daily egg production is much smaller after higher rearing density, namely, 37.2 -42.5 eggs per day for GA240 vs. 50.4 4- 3.2 for GA30 (t = 3.2; P < 0.001) and 38.6 42.2 eggs per day for AG240 vs. 56.7 4- 3-9 for AG30 (t = 4.2; P < 0.001). Finally, the mean egg-laying period is prolonged when the larvae are crowded (see Table 2), highly significantly in the case of the A G hybrid (50.2 4- 2-8 days for 240 vs. 35.2 4- 3.5 days for 30 eggs per tube (t = 3.4; P < 0.001), non significantly in the G A hybrid (46-8 44-5 days for 240 vs. 43.5 4- 3.1 days for 30 eggs per tube (t = 0.6; P : n.s.). (7) No relation emerges between rate of oxygen consumption and the other quantitative traits measured, when all four genotype-environment combinations are considered together. Specifically, the observations do not reveal any relation between the age function of respiration rate and fecundity respectively; the data also do not reveal any correlation between mean rate of oxygen consumption and life span (Tables 1 and 2, Figs. 1-4). D I S C U S S I O N

I t is well known that environment influences to a large extent diverse biometric traits in insects. One should, however, distinguish between contemporaneous and antecedent environmental influences. Contemporaneous influences of the environment have been recognized since Loeb and Northrop (1917) showed that the duration of larval, pupal and

RESPIRATION IN DROSOPHILA--III

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adult life is negatively correlated with temperature and Alpatov and Pearl (1929) demonstrated that size depends upon temperature in a similar way. More recently investigators have been concerned with effects of "density" (number of larvae per unit surface or/and volume: larval crowding; number of imagos per unit surface or/and volume: population density), but here a clear picture does not emerge from the results of biometric analysis, essentially because at high population densities underfeeding can begin to play a role in metabolism (cf. Bakker, 1961). It is certainly difficult to establish a borderline between effects due to larval crowding and, those due to underfeeding, but in spite of this complication it now seems generally accepted that increasing larval density diminishes the size of the larvae and consequently the size of the imago at emergence and prolongs duration of larval and/or pupal life in Drosophila (Sang, 1949). Thus reversed relations between size and duration of development under the respective influences of temperature and of larval density must be emphasized (Lints, 1963 ; Lints and Lints, 1965). Besides the contemporaneous responses of organisms to environmental influence there are subsequent effects resulting from antecedent environmental influences. Thus the temperature at which embryonic and postembryonic development of Drosophila takes place is known to affect the duration of subsequent life. This was established by Alpatov and Pearl (1929) who showed that raising the temperature (to 28°C) during development shortens adult life span at various temperatures, whereas lowering the developmental temperature (to 18°C) has the opposite effect. Recently these findings have been confirmed (Lints, 1963). Subsequent effects of larval crowding, in the absence of underfeeding, on quantitative traits of the imago are less clear. It was demonstrated however in Drosophila melanogaster,that, within certain limits, and depending to a large extent on genotype, an increase in larval population density prolongs the duration of imaginal life (Miller and Thomas, 1958; Lints, 1963). It was claimed for Drosophilapersimilis that crowding the larvae does not alter the average daily egg production measured over a period of only 30 days, at 16°C (Spiess, 1958). However, these results are contrary to the views of Sang (1949) who counted numbers of eggs produced over 9 days only, and was led to the conclusion that in Drosophila melanogaster fecundity varies in proportion to body size under the influence of crowding and underfeeding. Thus for a given genotype and within a given imaginal environment, it appears possible to alter life span and other quantitative traits through manipulation of the preimaginal environment, e.g. by crowding the larvae or varying their temperature of incubation. Reciprocal hybrids, reared at larval population densities of 30 and 240 individuals per standard tube, have equivalent respiration rates measured as/zl O2/mg fresh weight/ hour in the period from emergence until death of the imagos. This does not mean of course that a maternal or cytoplasmic influence would not become obvious if the hybrids were tested in other environments. The relativity of the expression of maternal or cytoplasmic influences to environment has been demonstrated repeatedly (Lints, 1960, 1962). More important, however, in relation to the problem of the influence of the preimaginal environment on quantitative imaginal traits is the fact that the mean rates of oxygen consumption are essentially the same within each pair of fixed genotype: altered preimaginal environment combinations. In contrast to this similarity flies developed from larvae cultured at the higher density of 240 eggs per tube live longer and have smaller weights (and sizes) than flies emerged from larval cultures of density 30 per tube. Now if total oxygen consumption,per unit of weight and per individual are calculated for each strain

88

F.A. LINTS AND C. V. LINT$

and larval culture density from the data of Table 1, the following figures are obtained for GA30, AG30, GAz4o and AG240 respectively: (i) total oxygen consumption in ~l/mg fresh weight/life: 4.1, 3.6, 5.1 and 4"9; (ii) total oxygen consumption in/~1/ individual/life: 5"3, 4"7, 4"5 and 4.6. The figures in the second set closely resemble one another but, in the absence of a possibility to calculate their standard errors, it is difficult to ascertain whether they do or do not differ significantly. To test this definitively, the rates of oxygen consumption of a series of individuals should be measured throughout life and compared with the life spans of those same individuals; technically, however, this is very difficult to accomplish for the observed flies generally die accidentally, following the measurement of oxygen consumption (cf. Gregg and Lints, 1967). Expressed in microlitres per milligram per hour and calculated from the data of the whole life the mean respiration rate thus appears as strongly determined by the genotype, and as rather independent on the antecedent environment. This is not the case for other biometric traits as longevity and certain characteristics of the laying, the expression of which is largely dependent on the (contemporaneous and) antecedent environment. That explains of course why no relations between mean respiration rate and other biometric traits measured have been disclosed. At population densities of 30 eggs per tube, the GA and AG reciprocal hybrids both show a definite negative regression of rate of oxygen consumption on age; the decrease is steep and relatively constant judged from the scatter of the experimental points around the calculated regression line. Curiously enough, at the higher larval density of 240 eggs per tube neither hybrid shows any significant correlation of this type; hence here the rate of ageing, as based on the change in rate of oxygen consumption with age, is very different from that observed in adults developed from larvae kept under less crowded conditions. Recently, Comfort (1968) restated the widely held view that ageing is due to cellular information loss, probably in determined cells, at the molecular level. Furthermore, he submits that "if information-loss is the time-keeper of ageing, there are by consensus two hypotheses to explain its occurence: (a) that it is caused by the random accumulation of chemical noise in the cellular information system, and is itself unprogrammed; (b) that it is a built-in consequence of the differentiation caused by the switching-off of synthetic processes which cannot be switched on again without loss of differentiation, or to the evolutionary accumulation of late-acting deleterious genes" and proposes that "the only likely way of prolonging vigour will prove to be through stretching the development programme as a whole". Now in the present study on hybrid Drosophila genotypes, crowding the larvae has the dual effect of prolonging the period of larval and/or pupal development, and of lengthening the duration of adult life, the extended imaginal life span being characterized by a more or less constant rate of oxygen consumption. Furthermore, when development takes place at the higher larval population density, the maximal daily egg production is attained later in both hybrids, while the laying period for its part is extended very significantly in the AG hybrid, non significantly in the GA hybrid. These data appear to be in favour of the "delayed maturity hypothesis" of Medvedev (1967, cited by Comfort, 1968). However it is not clear why there is no detectable decrease in rate of oxygen consumption with age, not even at later stages of adult life, in the flies developed from larvae cultured under crowded conditions, but it is possible that more data or a more refined technique would reveal the existence of such a correlation. In any case, a simple explanation invoking an unwitting selection, at the end of the experimental period, of relatively long-lived individuals with high metabolic rate,

RESPIRATION IN DROSOPHILA--III

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can most probably be ruled out. Indeed the experiments were carried out on FI hybrids, and in order to reduce even more an eventual residual genetic variance (cf. Petit, 1953; Lints and Lints, 1965) a double selection was realized in the imagos obtained, by choosing from the largest daily emergence (modal duration of development) the individuals with a m o d a l size.

It must be remarked that these results are indirectly at variance with the data of Clarke and Maynard Smith (1966) on changes in rate of protein synthesis with age in Drosophila subobscura, as were our previous findings on respiration in diverse genotypes (cf. Discussion in Lints and Lints, 1968). They are even more incompatible with recent observations of Tribe (1966) who found a positive correlation between rate of oxygen consumption and age in Calliphoraerythrocephala. However Tribe performed his experiments on grouped individuals using the Warburg technique and it has been clearly shown that discrepancies in such oxygen consumption studies can be due to differences in grouping (Chen, 1951). Moreover, it is not the first time that contradictions arise when Drosophila is compared with other insects; conflicting results have also been obtained concerning the influence of preimaginal temperatures on the span of imaginal life in Drosophila melanogaster (Alpatov and Pearl, 1929; Lints, 1963) and Mormoniella vitripennis (Clark and Kidwell, 1967) respectively. No relationship is apparent either between life span and fecundity (total number of eggs laid) or between life span and fertility (total number of hatched imagos) when mean fecundities, fertilities and life spans are compared for all of the genotype-environment combinations studied (see Tables 1 and 2). This is at variance with the claim of Maynard Smith (1958) that a longer adult life span is correlated with a reduced egg production; however, he investigated mated, virgin and ovariless Drosophila subobscura females and his data are not directly comparable with the present findings. Now when the individual data from the four genotype-environment combinations are pooled together, and the relation between life span and fecundity examined, then a highly significant positive correlation is disclosed between total number of eggs laid and life span (r = 0-42; d.f. : 38; 0.001 < P < 0-01). Clark and Smith (1967) treated their data from two species of Bracon, B. serinopae and B. hebitor, in the same manner but found no interdependence between fecundity and life span in that instance. Thus it becomes more and more clear that there is in all likelihood in Drosophila and certainly in insects, no generally-valid correlation between the kind of quantitative characters under discussion here. A precise knowledge about conditions in preimaginal environment, a complete description of the manner in which measurements have been made, and a detailed analysis in as large as possible an array of imaginal environments are all absolutely necessary to establish unequivocally the authenticity of so-called classical relationships. Table 4 may serve to underline this viewpoint. Finally another point, relevant to the relations between weight and rate of oxygen consumption, or rather between the variation of both those factors, must be discussed. It is generally accepted that in mammals--the familiar mouse--elephant plot--the oxygen consumption per unit of weight decreases with increasing body weight, or differently said that the increase in metabolism is not directly proportional to the increase in total body weight. In insects a good deal of effort has been devoted to trials in finding a general rule which should apply to the relations between the variation in oxygen metabolism and weight. On log. log coordinates and on a scale large enough to span several orders of magnitude of weight, a plot of respiratory rate against body weight

90

F. A. LINTS AND C. V. LINTS TABLE 4. ENVIRONMENTALINFLUENCES ON VARIOUS QUANTITATIVETRAITS Preimaginal treatment

Temperature 7

Larval density (with low or no 7 underfeeding) (3)

Quality or quantity of "~ food

Size and weight

Effects on duration of adult preimaginal life span life

~

"~

"~ (1)

"~

7

7

Fecundity

? (4)

References

LOEB and NORTHROP, 1917 ALPATOVand PEARL, 1929 ALPATOV, 1932 LINTS, 1963 DAVID and CLAVEL, 1967 (2) ROBERTSON and SANG, 1944 SANG, 1949, 1950 SPIESS, 1958 MILLER and THOMAS, 1958 MISHIMA, 1962 LINTS, 1963 (5), 1968 BHALLAand SOKAL, 1964 (6) ALPATOV, 1932 ROBERTSON and SANG, 1944 SANG, 1950 BAggER, 1961 LINTS, 1962

Note: Not all of the relations between preimaginal treatments and effects on the various quantitative traits here considered are studied in each of the references cited. (1) The contrary was shown to be true in Mormoniella vitripennis (Clark and Kidwell, 1967). (2) These authors have clearly shown that the relations between environmental conditions (temperature) and the quantitative traits here emphasized are valid only in a "normal" temperature range, ,~ 17-29°C, which is in fact the temperature range usually utilized in Drosophila experiments. Outside this range the correlations may be altered or even reversed. (3) Certain discrepancies in the existing literature could be due to the fact that some of the larval densities experimented with are outside the range where there is a linear correlation between larval density and the quantitative trait measured, as exemplified in the work of David and Clavel (1967) on temperature effects. Undercrowding effects have been recorded by Sang (1950) and Mishima (1962). (4) The relation between fecundity and larval population density is not clear. Spiess (1958) claimed that raising the population density of Drosophila persimilis larvae to 500 larvae/19 cm a (which corresponds more or less to the "high" density of 240 eggs/5-7 cm ~ of the present experiments) did not affect the fecundity of the resulting imagos. His claim, however, was based on an average fecundity at 16°C measured over 30 days only. Robertson and Sang (1944) and Sang (1950) contended that fecundity was roughly proportional to size, but made observations for 9 days only. As it has been shown that flies developed from larvae cultured at high population densities may live and lay eggs longer than those from less crowded larvae, it is obvious that daily egg-production records extending over the whole adult life are badly needed. (5) T h e effect of change in larval population density on life span in Drosophila melanogaster imagos was shown to be strongly influenced by the genotype. (6) In Musca domestica.

RESPIRATION IN DROSOPHILA--III

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generally yields a straight line. The slope of the lines corresponds to a given power of weight varying between 0.75 and 1 (reviews in yon Bertalanffy, 1951 ; Keister and Buck, 1964). However when a narrower scale of weight is used, of the order of magnitude of intraspecific variations, the experimental data in some cases reveal an extremely slight, in some cases even negative increase of oxygen consumption with growth in size (Zeuthen, 1952). Now it is shown that within a species, different genotypes respire amounts of oxygen which bear no relation to their weights or sizes. For instance, the "wild" genotype of the Gabarros strain, with a mean weight of 1.37 + 0.02 mg is found to respire 2.84 4- 0.10/A O2/mg fresh wt./hr, while the corresponding figures for the Fl15 generation of the genotype inbred Gabarros 4 line are 1.18 -4- 0.02 mg and 3.04 + 0.10/zl O2/mg fresh wt./hr (Lints and Lints, 1968), and for the hybrid Gabarros 4 inbred ~ × Abeele inbred ~ are 1.31 + 0.02 mg and 3.69 -4- 0.11/zl O2/mg fresh wt./hr (Table 1). Furthermore in the present study the adult weights of two reciprocal hybrids vary in response to a difference in the preimaginal environment, while the mean rates of consumption of oxygen remain unchanged. It is concluded that at a given temperature the mean rate of oxygen consumption per unit of weight is determined essentially by the genotype and is much less under the influence of the preimaginal environment than other quantitative traits. This also demonstrates the fact that respiratory control can be only tenuously related causally to body weight or size per se. Furthermore it is shown that, in function of age, respiratory rate evolves or not, depending on the antecedent environment. Those last data have to be related to recent findings on the evolution with age of flight muscles sarcosomes size and number (a review in Tribe, 1966); they also may be referred to data on decline with age of ATPase activity in female Musca domestica (Rockstein and Gutfreund, 1961 ; Clark and Rockstein, 1964) and with observations on cytochrome C oxidase activity in the giant mitochondria of ageing house flies (Clark and Rockstein, 1964; Rockstein, 1967). Considering these points it must be admitted that the respiratory control must now be sought for and located at a subcellular or enzymatic level and even further analysed at a genetical level. Acknowledgements--We are indebted to Mr. M. Ringel6 and Mr. R. Stiers for valuable technical

help. Many thanks are due to Prof. Dr. M. J. Heuts for some stimulating discussions on the topic. We wish to express our sincere gratitude to Prof. Dr. Erik Zeuthen for his continuous interest in our work and for a critical but friendly reading of the manuscript. REFERENCES ALPATOV, W. W. (1932).,~. exp. Zool. 63, 407. ALPATOV, W. W. and PEARL, R. (1929) Amer. Nat. 63, 37.

B^KKER, K. (1961) Arch. nderl. Zool. 14, 200. BHALLA,S. C. and SOKAL,R. C. (1964) Evolution 18, 312. CHEN, P. S. (1951) Z . f . ind. Abst. Vet. Lehre 84, 38. CLARK,A. M. and KIOWELL,R. N. (1967) Exp. Geront. 2, 79. CLARK,A. M. and ROCKSTEIN,M. (1964) Ageing in insects, In Physiology of Insecta. Academic Press, New York. CL~mK,A. M. and SMITH,R. E. (1967) Ann. entom. Soc. America 60, 903. CLaarcE, J. M. and MAWraXDSMITH, J. (1966) Nature, Lond. 209, 627. COMFORT,A. (1968) Nature, Lond. 217, 320. DAVID, J. and CLAWL, M. F. (1967) J. insect PhysioL 13, 717. GaEGC, J. H. and Lxrers, F. A. (1967) C.R. Tray. Lab. Carlsberg 36, 25. H~UTS, M. J. (1956) Agricultura 4, 343.

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KEISTER,M. and BucK, J. (1964) Aging in insects, In Physiology oflnsecta. Academic Press, New York. LINTS, C. V., LINTS, F. A. and ZEtrrHEN, E. (1967) C.R. Tray. Lab. Carlsberg 36, 35. LINTS, F. A. (1960) Genetica 31, 188. LINTS, F. A. (1962) Acta Biotheoretica 16, 1. LINTS, F. A. (1963) Bull. biol. France Belgique 97, 605. Lncrs, F. A. and LINTS, C. V. (1965) Genetica 36, 183. LINTS, F. A. and LmTS, C. V. (1968) Exp. Geront. 3, 341. LOEB, J. and NORTHROe, J. H. (1917) 07. biol. Chem. 32, 103. MAYNARD SMITH, J. (1958) J . exp. Biol. 35, 832. MEDWDEV, Z . A . (1967) Protein Biosynthesis. Oliver and Boyd. MmLER, R. S. and THOMAS, J. L. (1958) Ecology 39, 118. MISHIMA, J. (1962)jTap. 07. Ecology 12, 235. P~SONS, P. A. (1964) Quart. Rev. Biol. 39, 258. PETIT, C. (1963) Arm. de G~n~tique 6, 29. ROBm~TSON, F. W. and R ~ w , E. C. R. (1952) 07. Gen. 50, 416. ROBERTSON, F. W. and SANa, J. H. (1944) Proc. Roy. Soc. B. 132, 258. ROCKSTEIN, M. (1967) Syrnp. Soc. cell. Biol. 21, 337. ROCKS~IN, M. and GUTFm~UND, D. E. (1961) Science 133, 1476. SANa, J. H. (1949) Physiol. Zool. 22, 183. S~u'qo, J. H. (1950) Biol. Rev. 25, 188. SPIESS, E. B. (1958) Evolution 12, 234. TRIBE, IV[. A. (1966)07. insect Physiol. 12, 1577. VON BEaTALar,rFt~'Z, L. (1951) tlmer. Nat. 85, 111. ZEU'rHEN, E. (1952) Quart. Rev. Biol. 28, 1.

S u m m a r y - - R a t e of oxygen consumption, daily, mean and total fecundity, fertility and adult life span of Drosophila melanogaster imagos were measured, at 25°C, from emergence up to the death. Reciprocal hybrids, from the cross between two highly inbred lines, Gabarros 4 and Abeele, produced at preimaginal densities of 30 and 240 eggs per standard tube of 5"7 cm 2 of surface, were used. T h e respiration measurements were carried out according to the technique of Gregg and Lints; all the measurements were realized on individual females; age, size and weight were taken into account. Increase of the preimaginal eggs (and larvae) density reduces the size of the emerging adults, prolongs the duration of development and lengthens the adult life span; the total fecundity do not appear to vary considerably; the mean fecundity, however, is much smaller, the egg-laying period is prolonged and the maximal daily egg production is attained later. Between emergence and death the two reciprocal hybrids have almost identical mean respiration rates at both the population (eggs and larvae) densities studied. Respiration rate decreases with age in both hybrids when larval population density is 30 eggs per tube. There is no relation between respiration rate and age for either hybrid at population density of 240 eggs per tube. N o relation emerges between rate of oxygen consumption and the other quantitative traits measured when all four genotype--environment combinations are considered together. Specifically the observations do not reveal any correlation between mean rate of oxygen consumption and mean daily egg-production, or between mean rat e of oxygen consumption and life span. T h e results are discussed in relation with different ageing and respiratory control theories.

RESPIRATION IN DROSOPHILA--III

R e s u m ~ - - - L e taux de consommation d'oxyg~ne, la f~condit6 journalitre, moyenne et totale, la fertilit~ et la long~vit6 d'imagos de Drosophila melanogaster ont ~t6 mesurts, h 25°C, de l'tmergence ~ la mort. Des hybrides rtciproques, issus du croisement de deux ligntes hautement consanguines, Gabarros 4 et Abeele ont ~t6 produits ~ des densit~s pr~imaginales de 30 et 240 oeufs par milieu de culture standard d'une surface de 5-7 crn 2. La technique de mesure de la respiration est celle de Gregg et Lints; toutes les mesures ont 6t6 rtalistes sur des individus femelles isol~s et il a ~t~ tenu compte de l'age, du poids et de la taille. L'accroissement de la densit6 de culture pr~imaginale r~duit la taille des imagos, prolonge la durte de dtveloppement et la dur~e de vie; la ftcondit6 totale ne semble gutre vari~e, la ftcondit6 moyenne par contre est beaucoup plus faible, la p~riode de ponte est prolongte et le maximum de la ponte joumali~re est atteint plus tardivement. Le taux moyen de la respiration, mesur6 de l'tmergence a la mort, est identique pour les deux hybrides r~ciproques aux deux densit~s (d'oeufs et de larves) utilistes. Le taux de respiration d~croit r~guli~rement au cours de la vie pour des individus ~lev~s h faible densitt; il est par contre constant pour des hybrides obtenus ~ haute densitC Lorsque les donntes relatives aux quatre combinaisons gtnotype---milieu sont groupies, on ne trouve pas de relation entre le taux de consommation d'oxygtne et les autres caract~res quantitatifs ~tudi~s. En particulier les donn~es ne rtv~lent aucune corrtlation entre le taux moyen de respiration et la ponte joumali~re, ni entre ce taux et la long~vit& Les rtsultats sont discutts par rapport ~ diff~rentes theories du vieillissement et du mtcanisme de contr61e de la respiration.

Sauerstoffverbrauch, die t~igliche, mittlere und die Gesamteierproduktion, die Fertilit~it u n d die Lebensdauer von Drosophila melanogaster-Imagines wurde bei 25°C vom Schliipfen bis zum Tode gemessen. Es wurden reziproke Hybride der Kreuzung zwischen zwei stark ingezfichteten Linien --Gabarros 4 u n d Abeele--verwendet, die mit einer priiimaginalen Dichte yon 30 u n d 240 Eiem pro Standardrthrchen yon 5,7 cm ~ Oberfl~iche gezogen wurden. Die Atmungsmessungen wurden nach der Methode yon Gregg und Lints durchgeffihrt. Alle Messungen wurden an einzelnen Weibchen vorgenommen. Alter, Gr6Be u n d Gewicht wurden beriicksichtigt. Die Erh6hung der pr~iimaginalen Eier-[und Larven-]dichte vermindert die GrtBe der schliipfenden Adulten, verl~ingert die Entwicklungsphase u n d die Lebensdauer. Die Gesamteierproduktion scheint sich nicht betr~ichtlich zu ver~indem. Die mittlere Eierproduktion ist jedoch sehr viel kleiner. Die Legeperiode wird verl~ngert, und die htchsten t~iglichen Legeraten werden erst sp~iter erreicht. Zwischen Schliapfen und T o d haben die beiden Hybriden fast identische mittlere AtmungsgrtBen bei beiden untersuchten Dichten [Eier u n d Larven]. Die AtmungsgrtBe n i m m t bei beiden Hybriden mit dem Alter ab, wenn die Larvendichte 30 Eier pro R6hrchen betriigt. Bei keinem der beiden Hybriden besteht eine Beziehung zwischen AtmungsgrtBe u n d Alter bei einer Eierdichte yon 240 pro Rthrchen. Zwischen Sauerstoffverbrauch u n d den anderen gemessenen quantitativen Merkmalen ergibt sich keine Beziehung, wenn alle vier Kombinationen von Genotyp u n d Umgebung zusammen betrachtet werden. Insbesondere liiBt sich keine Korrelation zwischen dem mittleren Sauerstoffverbrauch und der mittleren t~iglichen Eierproduktion oder zwischen dem mittleren Sauerstoffverbrauch u n d der Lebensdauer finden. Die Ergebnisse werden hinsichtlich der verschiedenen Alters- u n d Atmungskontrolltheorien diskutiert. Zusammenfassung--Der

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