Studies in ageing—VII Integration of genotypes, homeostasis, and the expression of ageing processes in Drosophila

Exp. Geront. Vol. 2, pp. 241-248. Pergamon Press 1967. Printed in Great Britain

INTEGRATION EXPRESSION

STUDIES IN AGEING--VII OF GENOTYPES, HOMEOSTASIS, AND THE OF AGEING PROCESSES IN DROSOPHILA K. C. SONDHI

Department of Zoology and Physiology, Rutgers University, Newark, New Jersey

(Received 1 August 1967) INTRODUCTION Ix EARLIERcommunications (Sondhi, 1964, 1967a), the role of developmental processes in determining the adult physiology of Drosphila was considered. Inbred and outbred populations of Drosophila melanogaster were allowed to undergo embryonic and postembryonic development at a lower temperature, the effects of which were studied by examining the adult life span and fecundity at the normal temperature. The results of these experiments showed genotypic differences for responsiveness to a changed environment. Inbred lines revealed an undiminished adult life span and fecundity or a drastic reduction in both the physiological parameters. Outbred populations, on the other hand, showed an increase in the adult longevity or fecundity. Most of the changes observed in inbred and outbred populations were considered in terms of homeostatic mechanisms which bring about homeostatic adjustment within a certain range of environmental variation (see Sondhi, 1966, 1967b). The realized gains in the adult life span or in the egg production of outbred populations were explained as due to adaptive responses. The nature of results obtained on longevity and fecundity led to the conclusion that the expression of both characters is determined by inter-acting, compensatory systems, which come into being during development and which continue operating during the adult life. Since prolongation of the adult life span or an increase in the egg production was only observed in outbred populations, similar experiments were designed to study the differences in responsiveness of heterozygous populations with changed genetic backgrounds. M A T E R I A L S AND M E T H O D S The Samarkand inbred line, which had been initially inbred for over 200 generations, was kept in mass-cultures for over 30 generations, and afterwards in sib-matings for approximately 25 generations. The Samarhand strain was reciprocally crossed with another Swedish-b 8 strain of Drosophila melanogaster that had been kept without interruption in sib-matings for over 70 generations. For brevity, the cross between Swedish-b s females and Samarkand males will be abbreviated as Hy, and that between Samarkand females and Swedish-b s males, as hY. Parental populations were kept, in pairs, in food vials for 3-4 days after emergence at 25 4- 1.00°C, thereafter each population was divided at random into two groups. One group was permitted to lay eggs at 25 i 1.00°C, and the other, at 16 ± 1.00°C. The offspring consisted of 4-7 day old fertilized females 241

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allowed to lay eggs individually over 24-hr periods in half-pint milk bottles containing a medium of maize meal, agar and molasses, to which a suspension of living yeast was added. Individuals emerging within 3-7 days of the eclosion period were collected, in pairs, in vials fitted with "dual purpose plastic stoppers" containing the food medium. T h e two sets of each population, one that had been kept at the normal temperature, N, and the other, at a lower temperature, L, were kept on the same shelf of the incubator at 25 1.00°C. T h e number of eggs laid by each female was recorded each day during the life span. T h e life tables for hybrid populations were prepared by recording the number of deaths daily after eclosion. T h e technical procedures employed for handling Drosophila populations, for measuring the adult life span and female fecundity have been described previously (Sondhi, 1965, 1966, 1967a). For convenience, the first set of H y and hY populations that had been kept during the pre-adult life at temperature L and during the adult life at N temperature will be referred to as : H y ( L N ) and hY(LN), respectively ; the two populations of the second set, which had been kept during all phases of life at N temperature, will be designated as: H y ( N N ) and hY(NN), correspondingly. RESULTS Table 1 shows the average adult life span of experimental and control populations. Survival curves of males and females in H y ( L N ) , Hy(NN), hY(LN) and hY(NN) populations are displayed in Fig. 1. It is interesting to note that the average expectation T A B L E 1.

M E A N SURVIVAL TIMES I N DAYS OF EXPERIMENTAL

Population LN Hy (males) Hy (females) Sexes combined hY (males) bY (females) Sexes combined

(LN)

AND CONTROL

Average adult life span ( ± standard error) NN difference

48' 57 :+_ 2' 32 (66) 56.96 :k 1 '85 (60) 52-57 ± 1-54 (126)

73- 91 ± 1 •30 (72) 78.76 ± 1"86 (81) 76.45 ± 1'47 (153)

66.34 ± 1.55 (78) 65.02 :k_ 1 "42 (79) 65"68 ± 1.03 (157)

78.36 ± 1-17 (83) 74"26 ~ 1 '61 (88) 76"25 ± 1'02 (171)

(NN)

POPULATIONS

P

25.34

< 0' 01

21 "80

: 0"01

23'88

< 0"01

12.02

< 0.01

9'24

< 0'01

10'57

< 0"01

Figures in brackets represent the nunber of individuals employed in outbred populations. of adult life of the H y ( N N ) and hY(NN) populations is approximately the same ( P = > 0.50). In the two populations, although the adult longevity of H y ( N N ) females does not differ appreciably from that of females in the hY(NN) group ( P = > 0.05), considerable differences exist in the average imaginal life span of males in the H y ( N N ) and hY(NN) groups. T h e expectation of adult life of hY(NN) males is significantly greater than ( P = < 0.02) that of males in the H y ( N N ) population. A drastic reduction in the adult longevity of both males and females is observed in the H y ( L N ) and h Y ( L N ) groups

STUDIES IN AGEING--VII

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compared with the control populations, Hy(NN) and hY(NN), respectively. It is interesting to note that the Hy(LN) and hY(LN) populations show differences in responsiveness to a changed developmental environment. The reduction in the average adult survival of Hy(LN) group is significantly greater than (P -- < 0.01) that in the hY(LN) population. Similar differences exist for the adult survival between males (P = < 0.01) and females (P = < 0.01) in the two populations, Hy(LN) and hY(LN).

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Table 2 shows the average fecundity of the experimental and control populations. The egg production of Hy(LN) and hY(LN) populations is approximately the same (P = > 0.03). Similarly, no appreciable difference (P = > 0.04) is observed between the fecundity of Hy(NN) and hY(NN) hybrid populations. However, the average egg production of Hy(LN) or of hY(LN) population undergoes a significant decrease (P = < 0.01) compared with that in the controls, Hy(NN) and hY(NN), respectively. TABLE 2.

T H E T O T A L N U M B E R OF EGGS L A I D PER F E M A L E I N E X P E R I M E N T A L

(LN)

AND CONTROL

(NN)

POPULATIONS

Population Hy hY

Average total fecundity LN NN 497.06 ± 21.98 (60) 529.70 ± 19.61 (78)

925.16 ± 29.98 (74) 962.00 ± 24.21 (85)

P < 0-01 < 0.01

Figures in brackets represent the number of individuals employed in outbred populations. DISCUSSION The results of the present experiments reveal that first generation, reciprocal hybrid populations differ in several respects under both controlled and experimental conditions. The mechanisms underlying the observed differences, as well as similarities, will be discussed. The average adult longevity or fecundity of outbred, control populations, Hy(NN) and hY(NN), is approximately the same. Considerable differences exist, however, in the expectation of adult life of Hy(NN) and hY(NN) males, the latter being longer than the former. An explanation of this difference could be sought in terms of chromosomal differences in the two sexes. In reciprocal hybrid populations, under normal conditions, and probably in the present case, differences in males can arise because of differences in the sex-chromosomes, the other chromosomal complements being equal to both populations. Thus, a male in the Hy(NN) population will have an X-chromosome from the Swedish-b s female and a Y-chromosome from the Samarkand male, whereas a reverse situation will occur in individuals of the hY(NN) population; that is, males will have the X-chromosomes from Samarkand females and the Y-chromosomes from Swedish-b 8 males. Since the Y-chromosome in Drosophila melanogaster contributes very little in terms of the chromosomal genes (Morgan, 1926; Bridges and Brehme, 1944), much of the observed differences in the two reciprocal hybrid populations can be attributed to the X-chromosome differences. Therefore, an increase in the span of adult life of hY(NN) males appears largely to be due to the presence of the X-chromosomes from Samarkand females and, possibly, to their interactions with the remaining chromosomes. An examination of the Hy(LN) and hY(LN) populations shows that the adult longevity in the former is more adversely affected than in the latter because of an environmental change during development. Differences in the adult survival of first generation hybrids, obtained by reciprocally crossing two strains, may arise due to some kind of maternal effect. However, further work is required to justify its presence in the experimental populations.

STUDIES IN AGEING---VII

245

A comparison of the expectation of adult life of the two sexes in the treated and untreated populations reveals (Table 1) that in both hybrids the survival of males is more adversely affected compared with females. These results suggest the presence of a sex-difference for the adult life span in the genotypes studied, females being able to survive a temperature change better than males. However, such a difference may or may not be present in the adult populations with different genetic or environmental backgrounds (see Sondhi, 1967a). The major interest of the findings lies in their showing that outbred populations, in contrast to the earlier hybrids that were obtained by crossing strains on which inbreeding was practised without interruption (Sondhi, 1964, 1967a), undergo a decline in the fitness. The small differences in the fitness of outbred populations involving strains with different breeding backgrounds can be attributed to inequalities of environment, for the external environment to which the strains were exposed under the experimental or controlled conditions was not very different. Thus, a reduction in the fitness of the present experimental populations appears in a large measure to be the consequence of changes in the genetic backgrounds (refer to materials and methods section) of the parental strains. This apparently affects the genetic make-up of hybrids so that fitness is reduced in a changed environment. Since the adult longevity or fecundity of Hy(NN) and hY(NN) hybrids shows a striking increase compared with the earlier Hy(NN) populations, a reduced fitness of the present populations cannot be the result of a loss of heterozygosity. The observed differences, on the contrary, appear to be produced despite an increase in heterozygosity because of a change in the breeding procedure of one of the parental strains partly contributing to the genetic constitution of the present outbred populations. This suggests that the experimental populations, which were capable of responding adaptively earlier, even in the presence of increased heterozygosity, have undergone adverse effects on the fitness. A decline in the fitness of a population does not involve the absence of homeostatic mechanisms. The presence of homeostatic mechanisms in the experimental populations with a reduced fitness is demonstrated by the fact that both reciprocal hybrids show an approximately constant fecundity in the presence of significant differences in the adult longevity. Therefore, the inability of the genotypes to regulate effectively in the changed environment can serve as a satisfactory explanation of the present results. And this means that, besides heterozygosity, the nature of integration of genotypes must be important for eliciting homeostatic or adaptive responses contributory to the fitness of a population under changed conditions of environment (excellent discussions on related topics will be found in numerous volumes of Cold Spring Harbour Symposia on Quantitative Biology). Acknowledgements--The present study was completed at the Department of Laboratories, Newark Beth Israel Hospital, Newark, New Jersey, during the tenure of a Rutgers Research Council faculty fellowship. Supported by a research grant GB-3219 from the National Science Foundation, USPHS Biomedical Sciences grant FR- 7059, and a Research Project grant from Rutgers Research Council. The expert technical assistance received from Francine Fainman, Dorothy Rapp, Leo Masciulli and Harvey Striar is gratefully acknowledged.

246

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REFERENCES

BRIDGES,C. B. a n d BREHME, K. S. (1944) Carnegie Inst. Wash. Pub 552, l. MORG.~.N, T . H. (1926) The Theory of the Gene. N e w H a v e n : Yale U n i v e r s i t y Press. SONDHt, K. C. (1964) Am. Zool. 4, 286. SoNrmi, K. C. (1965) Life Sei. 4, 57. SONDHI, K. C. ( 1 9 6 6 ) J . exp. Zool. 162, 89. SONDHI, K. C. (1967a) Proc. natn. Acad. Sci. U.S.A. in press. SONDHI, K. C. (1967b) Exp. Gerontol. 2, 233. Summary--Experiments were p e r f o r m e d on first g e n e r a t i o n h y b r i d populations, o b t a i n e d b y reciprocally crossing two Dros@hila melanogaster strains, one i n b r e d c o n t i n u o u s l y , a n d the other, before a n d after keeping p a r e n t s in m a s s - c u l t u r e s for s o m e generations. P a r e n t a l p o p u l a t i o n s were divided at r a n d o m into two groups. O n e g r o u p was p e r m i t t e d to lay eggs at 16 : 1.00cC, a n d the other, at 25 ~ I'00~C. T h e a d u l t longevity a n d the female fecundity, w h i c h p r o v i d e some m e a s u r e of fitness in the treated a n d u n t r e a t e d populations, were m e a s u r e d at 25 _ 1"00~C. W h i l e no appreciable differences were o b s e r v e d in the physiological p a r a m e t e r s m e a s u r e d in the c o m b i n e d sexes, the average a d u l t life span of males in one h y b r i d was c o n s i d e r a b l y increased c o m p a r e d with t h a t of males in the o t h e r population. It is suffgested t h a t the o b s e r v e d differences in the longevity are due t o t h e X - c h r o m o s o m e differences and, possibly, to t h e i r interactions w i t h the o t h e r c h r o m o s o m e s . O n the o t h e r h a n d , the m e a n adult longevity of males in the treated p o p u l a t i o n s was more adversely affected t h a n t h a t of females, indicating a sex difference for surviving, as adults, a t e m p e r a t u r e c h a n g e i n t r o d u c e d d u r i n g d e v e l o p m e n t . Because significant differences in the average adult life span, a l t h o u g h not in the average egg p r o d u c t i o n , were f o u n d in the two hybrids, the presence of some kind of m a t e r n a l effect is indicated. A c o m p a r i s o n of the e x p e r i m e n t a l p o p u l a t i o n s revealed a drastic r e d u c t i o n in the fitness c o m p a r e d w i t h the controls. Evidence is p r e s e n t e d to show t h a t this is due to the inability of h o m e o s t a t i c m e c h a n i s m s to regulate effectively in a c h a n g e d e n v i r o n m e n t a n d that, besides heterozygosity, the n a t u r e of i n t e g r a t i o n of genotypes is essential for eliciting h o m e o s t a t i c or adaptive responses c o n t r i b u t o r y to the fitness o f a population. R6sum6--On a fait des expdriences sur des p o p u l a t i o n s h y b r i d e s de p r e m i 6 r e gdn6ration, o b t e n u e s p a r c r o i s e m e n t rdciproque de deux races de Drosopkila melanogaster, d o n t l ' u n e avait 6td 6lev~e c o n t i n f i m e n t en c o n s a n g u i n i t d et l'autre a v a n t et apr6s avoir laiss6 les p a r e n t s dans des cultures en masse p e n d a n t q u e l q u e s g~ndrations. Les p o p u l a t i o n s P1 o n t &6 rdparties an h a s a r d sur deux groupes. Au p r e m i e r groupe, on a p e r m i s de p o n d r e des oeufs 'a 16 - 1,00°C, au second groupe, ',i 25 i: 1,00°C. L a longdvit6 des adultes et la f & o n d i t 6 des femelles qui d o n n e n t u n e certaine m e s u r e de sant6 p h y s i q u e dans les p o p u l a t i o n s trait6es et n o n trait~es, o n t dtd ddtermindes ~ 25 t 1,00:C. T a n d i s q u ' a u c u n e diff6rence appr4ciable n ' a p u &re observ~e e n t r e les param & r e s physiologiques mesurds dans les deux sexes pris ensemble, la durde de vie adulte m o y e n n e des mfiles d ' u n des h y b r i d e s &ait prolongde c o n s i d d r a b l e m e n t par r a p p o r t aux mfiles de l'autre population. O n s u p p o s e que les diffdrences du longdvitd observdes sont dries fi des diffdrences dans les c h r o m o s o m e s X et, 6 v e n t u e l l e m e n t , ~ leur interactions avec d ' a u t r e s c h r o m o s o m e s . D ' a u t r e part, ladur&, m o y e n n e de la vie adulte des re'ales &ait afl'ectSe plus d d f a v o r a b l e m e n t dans les p o p u l a t i o n s traitdes que celle des femelles, ce qui p o r t e ~ croire qu'il y ait une diffdrence e n t r e les sexes en ce qui c o n c e r n e la survie, au stade adulte, ?~ u n c h a n g e m e n t de t e m p d r a t u r e i n t r o d u i t au cours du d ~ v e l o p p e m e n t . E t a n t d o n n d que des diff6rences significatives dans la durde m o y e n n e de la vie adulte, mais pas dans la p r o d u c t i o n m o y e n n e d'oeufs, ont 6td trouvdes chez les deux hybrides, u n certain effet m a t e r n e l est probable.

STUDIES IN AGEING--VII La c o m p a r a i s o n des populations d ' e x p d r i e n c e rdvdla une rdduction spectaculaire de la sant6 p h y s i q u e par r a p p o r t aux tdmoins. Des faits sont prdsentds t e n d a n t fi d d m o n t r e r que ceci est dfi ~ l'impossibilitd p o u r les m d c a n i s m e s d'homdostasie, d ' e f f e c t u e r la r f g u l a t i o n de fagon efficace dans u n milieu modifi6 et que, ~t part l'hdtdrozygotie, la nature de l'intdgration des g6notypes est essentielle p o u r le d d c l e n c h e m e n t de rfiactions h o m 6 o s t a t i q u e s ou adaptatives c o n t r i b u a n t ~ la santd p h y s i q u e de la population.

Zusammenfassung--Die V e r s u c h e w u r d e n mit H y b r i d e n p o p u l a t i o n e n der ersten G e n e r a t i o n durchgefiJhrt. Zwei Stiimme yon Drosophila melanogaster w u r d e n s y m m e t r i s c h gekreuzt, der eine war ein kontinuierlich ingez(ichteter S t a m m , der a n d e r e s t a m m t e aus einer tiber einige G e n e r a t i o n e n geftihrten Grol3kultur. Die E l t e r n p o p u l a t i o n e n w u r d e n o h n e Auslese in zwei G r u p p e n geteilt. Eine G r u p p e w u r d e z u m Eierlegen bei 16 m 1,00"C, die andere bei 25 :: 1 , 0 0 C gehalten. Die L e b e n s d a u e r der e r w a c h s e n e n T i e r e u n d die F r u c h t b a r k e i t der \ V e i b c h e n w u r d e n als Mal3 der L e b e n s k r a f t in der b e h a n d e l t e n u n d der nicht b e h a n d e l t e n Population bei 25 -. 1,00°C b e s t i m m t . W i i h r e n d bei den k o m b i n i e r t e n G e s c h l e c h t e r n keine wesentlichen U n t e r s c h i e d e in den physiologischen P a r a m e t e r n g e m e s s e n w u r d e n , war die d u r c h s c h n i t t l i c h e L e b e n s d a u e r der M i i n n c h e n einer H y b r i d e n p o p u l a t i o n betrachtlich gegeniiber der der M i i n n c h e n in der a n d e r e n Population erh6ht. Die b e o b a c h t e t e n U n t e r s c h i e d e in der L e b e n s d a u e r sind v e r m u t l i c h d u t c h X - C h r o m o s o m e n u n t e r s c h i e d e zu erkl~iren, m 6 g l i c h e r w e i s e auch d u r c h W e c h s e l w i r k u n g mit den a n d e r e n C h r o m o somen. A u f der a n d e r e n Seite war die L e b e n s d a u e r der M i i n n c h e n aus der b e h a n d e l t e n P o p u l a t i o n s c h w e r e r beeintriichtigt als die der W e i b c h e n , ein H i n w e i s auf G e s c h l e c h t s u n t e r s c h i e d e der e r w a c h s e n e n T i e r e in l~lberleben einer T e m p e r a t u r iinderung wiihrend der E n t w i c k l u n g s p h a s e . Da bei den H.vbriden signifikante U n t e r s c h i e d e in der L e b e n s d a u e r , n i c h t aber in der d u r c h s c h n i t t l i c h e n Eierp r o d u k t i o n g e f u n d e n w u r d e n , ist w a h r s c h e i n l i c h ein m a t e r n e l l e r Faktor beteiligt. Ein Vergleich der V e r s u c h s p o p u l a t i o n e n mit den K o n t r o l l g r u p p e n zeigte eine starke R e d u z i e r u n g der Lebenskraft. Es w e r d e n H i n w e i s e gegeben, dab dies auf die U n f a h i g k e i t h o m f o s t a t i s c h e r M e c h a n i s m e n in der wirksamen Regulation bei ver~inderter U m g e b u n g zuriickzufiihren ist. N e b e n der H o m o z y g o t i e ist die K e n n t n i s der Integrationsart der G e n o t y p e n n o t w e n d i g ftir die Aufkl~irung h o m 6 o s t atischer o d e r adaptativer Reaktionen, welche zu der L e b e n s k r a f t einer Population beitragen.

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