UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Environmental and genetic influences on the life span of adult Drosophila melanogaster Richter, Murray Dean 1986

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1986_A6_7 R55.pdf [ 5.42MB ]
Metadata
JSON: 831-1.0096796.json
JSON-LD: 831-1.0096796-ld.json
RDF/XML (Pretty): 831-1.0096796-rdf.xml
RDF/JSON: 831-1.0096796-rdf.json
Turtle: 831-1.0096796-turtle.txt
N-Triples: 831-1.0096796-rdf-ntriples.txt
Original Record: 831-1.0096796-source.json
Full Text
831-1.0096796-fulltext.txt
Citation
831-1.0096796.ris

Full Text

ENVIRONMENTAL AND GENETIC INFLUENCES ON THE LIFE SPAN OF ADULT DROSOPHILA MELANOGASTER by MURRAY DEAN RICHTER B.Sc, The University of British Columbia, 1979 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Zoology) We accept this thesis as conforming to the required standard October 1986 ©Murray Dean Richter In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date Oc T o f f ^ ^ IJ } 1^ 8Q ii ABSTRACT The onset of senescence and the rate at which organisms age are influenced by both extrinsic and intrinsic factors. Extrinsic factors include temperature, humidity and diet. Intrinsic factors arise within an organism as a result of normal metabolism. An example is the production of free radicals, highly reactive compounds that usually arise as byproducts of normal oxidative functions. Another less commonly considered instrinsic factor is the genetic makeup of an organism. Not only do different species have different characteristic life spans, but different strains within a species also have different characteristic life spans. This study examines how environmental and genetic differences affect the adult life span of Drosophila melanogaster. Drosophila was chosen because of its well characterized genetics, the ease with which it can be cultured and its life span measured, and the ability to control environmental conditions, particularly temperature. The study used three different approaches. The first approach looked at the influence of preimaginal development on adult longevity. The duration of development of melanogaster was altered using developmental temperature shifts. Both a wild-type strain and a mutant strain that displays a longer developmental period at 29°C than at 22°C were examined. No correlation was found between developmental duration and adult life span in either strain. The second approach examined the differences in adult longevity that existed between a highly inbred laboratory strain and four strains recently isolated from the wild. This was done to assess the suitability of highly inbred strains for studying aging. The laboratory strain showed much less variation in life span than did any of the recently isolated strains. The third approach looked at the possible roles of DNA damage accumulation and DNA repair in controlling life span in Drosophila. Mutagen-sensitive (mus) mutants representing strains that are potentially defective in DNA repair were examined to see if their life spans were altered relative to control strains. Little difference was found between mus strains and the controls. Where differences did exist, it was not clear that they were a direct result of defective repair in the adult. The results of each approach are discussed with respect to current knowledge of the processes controlling aging and senescence in Drosophila and other organisms. iv TABLE OF CONTENTS Page ABSTRACT ii LIST OF TABLES . v LIST OF FIGURES vii ACKNOWLEDGEMENTS x GENERAL INTRODUCTION 1 CHAPTER ONE 5 Introduction . . . . . . . . . . . . . 5 Materials and Methods . . . . . . . . . . . 7 Results . 9 Discussion . . . . . . . . . . . . 5 6 CHAPTER TWO 61 Introduction . . . . . . . . . . . . . 61 Materials and Methods . . . . . 6 3 Results . . . . . . . . . . . . . . 64 Discussion . . . . . . . . . . . . . 86 CHAPTER THREE 94 Introduction . . . . . . . . . . . . . 94 Materials and Methods . . . . . . . . . . . 97 Results 102 Discussion . . . . . . . . . . . .161 SUMMARY 168 REFERENCES . . 170 LIST OF TABLES TABLE 1. The effects of different culture temperatures and combinations of culture temperatures on the duration of preimaginal development in Oregon-R TABLE 2. The effects of different culture temperatures and combinations of culture temperatures on the duration of preimaginal development in strain 957 TABLE 3. Oregon-R adult longevity at 29°C TABLE 4. Oregon-R adult longevity at 22°C TABLE 5. Strain 957 male adult longevity at 29°C TABLE 6. Strain 957 female adult longevity at 29°C TABLE 7. Strain 957 male adult longevity at 22°C TABLE 8. Strain 957 female adult longevity at 22°C TABLE 9. Adult life spans of wild strains at 29°C TABLE 10.Analysis of variance based on data of Table 9 TABLE 11.Adult life spans of wild strains at 22°C TABLE 12.Analysis of variance based on data of Table 11 TABLE 13.Adult life span of Oregon-R TABLE 14.Mean life spans of non-irradiated b p_r cn/b p_I cn. A-l /A-1. D-132/D-132. G-92/G-92 and Canton-S males from 25 krad and 10 krad experiments . . . . TABLE 15.Mean life spans of non-irradiated b p_r cn/b p_r cn, A-l /A-1. D-132/D-132. G-92/G-92 and Canton-S females from 25 krad and 10 krad experiments . . . . TABLE 16.Mean life spans of non-irradiated b p_r cn/CvO , A-l/CvO. D-132/CvO and G-92/CvO males from 25 krad and 10 krad experiments . . . . . . . TABLE 17.Mean life spans of non-irradiated b p_r cn/CvO. A-l/CvO. D-132/CvO and G-92/CvO females from 25 krad and 10 krad experiments . . . . . TABLE 18.Mean life spans of b_ EI cn/b pr c n , A-l /A-1. D-132/D-132 and Canton-S males exposed to 25 krads of gamma radiation . . . . TABLE 19.Mean life spans of b p_r cn/b pr cn, A-l /A-1. D-132/D-132 and Canton-S females exposed to 25 krads of gamma radiation . . . . TABLE 20.Mean life spans of b p_r cn /CvO. A-l/CvO and D-132/CvO males exposed to 25 krads of gamma radiation . . . . . TABLE 21.Mean life spans of b p_i cn /CvO. A-l/CvO and D-132/CvO females exposed to 25 krads of gamma radiation . . . . . TABLE 22.Mean life spans of b p_r cn / b pr c n , A-l/A-1. D-132/D-132. G-92/G-92 and Canton-S males exposed to 10 krads of gamma radiation . . . . . . . TABLE 23.Mean life spans of b p_r cn / b pr c n , A-l/A-1. D-132/D-132. G-92/G-92 and Canton-S females exposed to 10 krads of gamma radiation . . . . . . TABLE 24.Mean life spans of b pr cn /CvO. A-l/CvO. D-132/CvO and G-92/CvO males exposed to 10 krads of gamma radiation TABLE 25.Mean life spans of b p_r cn /CvO. A-l/CyO, D-132/CvO and G-92/CvO females exposed to 10 krads of gamma radiation . . . . vii LIST OF FIGURES Page FIGURE 1. Relationship between total duration of development and duration of development at 17°C or 29°C for Oregon-R . . 1 3 FIGURE 2. Relationship between total duration of development and duration of development at 17°C or 29°C for strain 957 . 1 7 FIGURE 3. Survival curves of Oregon-R males maintained at 29°C . 2 1 FIGURE 4. Survival curves of Oregon-R females maintained at 29°C . 2 3 FIGURE 5. Relationship between mean life span and duration of development for Oregon-R at 29°C 25 FIGURE 6. Survival curves of Oregon-R males maintained at 22°C . 2 8 FIGURE 7. Survival curves of Oregon-R females maintained at 22°C . . . 3 0 FIGURE 8. Relationship between mean life span and duration of development for Oregon-R at 22°C . . . . . . . 33 FIGURE 9. Survival curves of strain 957 males maintained at 29°C . . . 3 7 FIGURE lO.Survival curves of strain 957 females maintained at 29°C . . 3 9 FIGURE 11.Relationship between mean life span and duration of development for 957 males at 29°C 42 FIGURE 12.Relationship between mean life span and duration of development for 957 females at 29°C . . . . . . . 44 FIGURE 13.Survival curves of strain 957 males maintained at 22°C . 4 8 FIGURE 14.Survival curves of strain 957 females maintained at 22°C . . 5 0 FIGURE 15.Relationship between mean life span and duration of development for 257 males at 22°C 52 FIGURE 16.Relationship between mean life span and duration of development for 957 females at 22°C . . . . . . . 54 FIGURE H.Survival curves of strain D adults maintained at 29°C . 6 8 FIGURE 18.Survival curves of strain E adults maintained at 29°C . . 7 0 FIGURE 19.Survival curves of strain F adults maintained at 29°C . 7 2 FIGURE 20.Survival curves of strain G adults maintained at 29°C . . 7 4 viii FIGURE 21.Survival curves of strain D adults maintained at 22°C . 7 9 FIGURE 22.Survival curves of strain E adults maintained at 22°C . .81 FIGURE 23.Survival curves of strain F adults maintained at 22°C . . 8 3 FIGURE 24.Survival curves of strain G adults maintained at 22°C . . . 8 5 FIGURE 25.Survival curves of Oregon-R adults maintained at 29°C . 8 9 FIGURE 26.Survival curves of Oregon-R adults maintained at 22°C . 9 1 FIGURE 27.Mating and separation scheme for experiments comparing the effects of 0 krads and 25 krads on the strains b px cn/b pr cn, b p_I cn/CvO. A-l/A-1. A-l/CvO. D-132/D-132 and D-132/CvO 101 FIGURE 28.Survival curves of non-irradiated b p_r cn/b pr cn, A-l/A-1. D-132/D-132. G-92/G-92 and Canton-S males maintained at 22°C 104 FIGURE 29.Survival curves of non-irradiated b pr cn/b pr cn. A-l/A-1. D-132/D-132. G-92/G-92and Canton-S males maintained at 29°C 106 FIGURE 30.Survival curves of non-irradiated b pr cn/b pr cn, A-l/A-1. D-132/D-132. G-92/G-92 and Canton-S females maintained at 22°C 110 FIGURE 31.Survival curves of non-irradiated b pr cn/b p_r cn, A-l/A-1. D-132/D-132. G-92/G-92 and Canton-S females maintained at 29°C 112 FIGURE 32.Survival curves of non-irradiated b pr cn/CvO. A-l/CvO. D-132/CvO and G-92/CvO males maintained at 22°C 115 FIGURE 33.Survival curves of non-irradiated b p_r cn/CvO. A-l/CvO. D-132/CvO and G-92/CvO males maintained at 29°C 117 FIGURE 34.Survival curves of non-irradiated b p_I cn/CvO. A-l/CvO. P-132/CvO and G-92/CvO females maintained at 22°C . 121 FIGURE 35.Survival curves of non-irradiated b_ PT cn/CvO. A-l/CvO. D-132/CvO and G-92/CvO females maintained at 29°C 123 FIGURE 36.Survival curves of pr cn/b pr cn, A-l/A-1. D-132/D-132 and Canton-S males exposed to 25 krads of gamma radiation .127 FIGURE 37.Survival curves of b_ p_I cn/b pr cji, A-l /A-1. D-132/D-132 and Canton-S females exposed to 25 krads of gamma radiation FIGURE 38.Survival curves of b p_I cn/CvO. A-l/CvO. and D-132/CvO males exposed to 25 krads of gamma radiation FIGURE 39.Survival curves of b pr cn/CvO. A-l/CvO. and D-132/CvO females exposed to 25 krads of gamma radiation FIGURE 40.Survival curves of fe pr cn/b pr cn, A-l /A-1. D-132/D-132. G-92/G-92 and Canton-S males exposed to 10 krads of gamma radiation and maintained at 22°C . . . . FIGURE 41.Survival curves of b pr cn/b pr cn, A-l /A-1. D-132/D-132. G-92/G-92 and Canton-S males exposed to 10 krads of gamma radiation and maintained at 29°C . . . . FIGURE 42.Survival curves of b pr cn/b pr cn, A-l /A-1. D-132/D-132. G-92/G-92 and Canton-S females exposed to 10 krads of gamma radiation and maintained at 22°C . . . . FIGURE 43.Survival curves of b pr cn/b pr cn. A-l /A-1. D-132/D-132. G-92/G-92 and Canton-S females exposed to 10 krads of gamma radiation and maintained at 29°C . . . . FIGURE 44.Survival curves of b pr cn/CvO. A-l/CvO. D-132/CvO. and G-92/CvO males exposed to 10 krads of gamma radiation and maintained at 22°C . . . . FIGURE 45.Survival curves of b pr cn/CvO. A-l/CvO. D-132/CvO. and G-92/CvO males exposed to 10 krads of gamma radiation and maintained at 29°C . . . . FIGURE 46.Survival curves of b pi cn/CvO. A-l/CvO. D-132/CvO. and G-92/CvO females exposed to 10 krads of gamma radiation and maintained at 22°C . . . . FIGURE 47.Survival curves of b pr cn/CvO. A-l/CvO. D-132/CvO.and G-92/CvO females exposed to 10 krads of gamma radiation and maintained at 29°C . . . . X ACKNOWLEDGEMENTS I offer my most sincere thanks to: Dr. Thomas Grigliatti for his guidance, encouragement and helpful criticism; Dr. Donald Sinclair for his support, ideas and critical reading of this thesis; Dr. Robert Devlin for his many helpful suggestions and good discussions; Mr. Daryl Henderson for the use of his stocks in Chapter Three (and for turning over the odd fly for me); Ms. Kathy Kafer and Mr. Ian Rolston for collecting much of the data in Chapter Two; The members of Room #3447 for their friendship; and Cathy without whose love and patience I would never have been able to complete this work. GENERAL INTRODUCTION As many societies experience an increase in the proportion of elderly in the population, the need to understand the basic processes involved in aging becomes more important. Medicine has successfully prolonged the average human life span, but has had much less success in concomitantly prolonging human health. Therefore, gerontological research at both the applied and the basic levels has expanded rapidly over the past 20 years. Unfortunately, the nebulous nature of the aging process has lead to difficulties in attacking the problem experimentally. Simplistically, aging can be defined as the process of growing old. It begins at fertilization, ends at death and does not describe a particular period in an organism's life span. Senescence, on the other hand, describes the deteriorative processes that occur in later life following reproductive maturity of the organism. These two terms are often used interchangeably, but it is an examination of senescence that is of more concern in this thesis. Several theories of aging have been proposed. The theories can be grouped into two broad categories (for reviews, see Lamb, 1977, Lints, 1978, and Strehler, 1977). In one category are theories proposing that stochastic events are responsible for the changes that occur during senescence. According to this view, aging is caused by "wear and tear" changes that accumulate at both the cellular level and the tissue level. In metazoans, death occurs when the extent of cumulative damage and resulting cell and tissue death cause failure of a vital organ. Some of the theories in this category are the error-catastrophe or protein error hypothesis (Orgel, 1963), the somatic mutation theory (Curtis, 1963) and the free radical theory (Harman, 1982). Theories in the other category postulate that aging and death are a normal part of the entire process of development and, as such, are programmed events. Programmed death is 2 known to occur at the cellular level in many organisms. For example, during the period of morphogenesis in insects, a large amount of death occurs in preimaginal tissue to allow formation of adult structures. At the level of the whole organism, programmed death is viewed as an adaptive trait for a species whereby old individuals in a population are eliminated so that younger ones may thrive. However, a clear link has yet to be established between programmed senescence at the cellular level and at the level of the whole organism (Kirkwood, 1977 and 1984). Several model systems have been developed for use in gerontological research to better understand the aging process. The most commonly used organisms in this research are laboratory rodents. Both rats and mice represent systems that should be similar to human aging. An advantage to using these animals is that it is now relatively easy to obtain genetically well-defined strains. However, there are many disadvantages in using rodents. They are long-lived compared to other model systems and are usually available only in low numbers. In addition, various factors that have significant effects on longevity (e.g., disease, diet and environmental conditions) are less easily controlled in rodents than in other model systems. Cell cultures are also commonly used to study aging. Hayflick and Moorhead (1961) have demonstrated that cell cultures established from a variety of normal human tissues have limited division potential. Hayflick (1965) proposed that the limited replicative ability was due to cellular senescence. An inverse correlation between donor age and doubling potential has been demonstrated in human cell cultures and there is evidence that a direct relationship exists between doubling potential and species life span (Hayflick, 1984). However, the inability of this model system to explain how senescence progresses in non-dividing cells (e.g., nervous tissue) has limited the general applicability of the results. In addition, the apparent immortality of transformed cell lines supports the argument that the limited division potential of cells from normal tissues is more a reflection of differentiation than senescence 3 (Bell et aL, 1978). The influence of culture conditions on culture life span also indicates that cell culture has somewhat limited potential as a general model system for aging (Rothstein, 1982). Invertebrates are also used extensively in aging research. Protozoa represent a unique model system for eukaryotes because clonal and colonial aging, as well as cellular aging, can be studied simultaneously (Smith-Sonneborn, 1981 and 1984). Of special interest is the ability to alter clonal life span in Paramecium using physical and chemical agents with known modes of action (Smith-Sonneborn, 1979; Smith-Sonneborn gt aL, 1983). The nematode Caenorhabditis elegans is also being used increasingly in aging research. This is due to the excellent characterization of the organism at the molecular, morphological and genetic levels (Johnson, 1983 and 1984). Like Paramecium. C elegans is used to test compounds that can alter life span (Zuckerman, 1983). The invertebrate most commonly used as a model system for aging is Drosophila  melanogaster. Lamb (1978), Lints (1978) and Rockstein and Miquel (1973) all provide comprehensive reviews of the literature of Drosophila pertaining to the study of senescence. No organism is better suited to a genetic analysis of the factors controlling aging. The postmitotic nature of the somatic tissue in the adult makes Drosophila an excellent model system for the changes seen in mammalian tissues composed of non-dividing cells (Miquel et aJL, 1979). Furthermore, behavioral (Leffelaar and Grigliatti, 1984b), anatomical and cytological (Miquel et al , 1979) characterizations during the adult life span have provided markers by which changes that occur during aging may be followed. Gerontological research in Drosophila has aimed at resolving whether the life span of adult flies is determined by programmed events (i.e., aging in the adult represents the final stages of development) or whether it is wear and tear existing in adults that eventually leads to death. Lints (1978) argues that the ability to alter adult life span by altering preimaginal growth rate is evidence for a type of programming that begins even before adult life. He 4 further contends that such a programming functions at many levels and is not necessarily genetically determined. Miquel et a_L (1976), on the other hand, argue that it is principally the metabolic rate of Drosophila that determines its life span. This is based on numerous studies that have examined adult longevity at different temperatures and under different dietary conditions (Lamb, 1978). This thesis attempts to resolve some of the factors controlling aging in Drosophila. Three areas were explored. The first chapter describes experiments that examine the effects of altering the duration of preimaginal development on adult longevity, using different developmental temperatures to change the duration of development. Furthermore, a comparison was made between a wild-type strain and a strain displaying an unusual extension of the length of the developmental period in response to temperature. The second chapter describes an investigation of the pattern of age-related death in a highly inbred wild-type stock compared to the patterns seen in four lines of flies recently isolated from the wild. The third chapter describes experiments designed to investigate the possible role of DNA repair in controlling life span, using mutants carrying putative lesions in DNA repair systems. 5 CHAPTER ONE INTRODUCTION The fact that the life span of poikilotherms is dependent, to some extent, on temperature is generally taken to be supportive of Pearl's "rate of living" theory of aging (Pearl, 1928). The theory essentially argues that while an organism ages, vital substances are used up or, alternatively, deleterious substances are accumulated. The rate of usage or accumulation is dependent on environmental temperature for poikilotherms. Thus, the higher the temperature, the more rapid the rate of consumption or accumulation and, consequently, the shorter the life span of the organism. For Drosophila melanogaster. the temperature coefficient for adult longevity, Q 1 0 , is 2 to 3 (Lamb, 1978; Leffelaar and Grigliatti, 1984b). That is, a two- to three-fold reduction in life span occurs with every 10°C rise in temperature. This relationship holds within the normal physiological limits for Drosophila. Environmental conditions during the preimaginal stages of Drosophila development have been observed to affect adult longevity. Alpatov and Pearl (1929) monitored the adult life spans of both sexes of a wild-type Drosophila strain that had been cultured at either 18°C or 28°C during preimaginal development. They found that at three different adult temperatures, the flies that developed at the lower temperature lived longer. They also found that the 18°C- cultured flies took longer to develop, as expected, and had a larger adult body size. Burcombe and Hollingsworth (1970) obtained similar results in a study of adult longevity in the first generation hybrid progeny of two highly inbred wild-type laboratory strains. Hybrids, developed at either 15°C or 30°C, had similar adult life spans when the adults were maintained at 15°C. However, when life span was measured at 30°C, flies reared at 15°C lived longer than those reared at 30°C. Consistent with Alpatov and Pearl's study, the flies raised at the lower temperature had a longer duration of development and a larger body mass. 6 Lints and Lints (1969 and 1971a) also examined the relationship between preimaginal development and adult longevity in Drosophila melanogaster hybrids. In the first experiment, all hybrid progeny were cultured at 25°C, but preimaginal population densities ranged from a low of 3 to a high of 480 eggs/vial (Lints and Lints, 1969). Analysis was restricted to females possessing both a modal duration of development and a modal size for each population density. They found that with increased culture density, the duration of development increased, whereas the size of the adult flies decreased. Adult life span also increased with increasing population density. In their second experiment, Lints and Lints (1971a) maintained a constant preimaginal density and varied the developmental temperature between 17°C and 31°C. They found high negative correlations between developmental temperature and duration of development and between developmental temperature and adult body size. They also observed that adult life span exhibited a similar negative correlation with preimaginal culture temperature. The important finding in both studies is that as the duration of development increased, so did the adult life span. Both Burcombe and Hollingsworth (1970) and Lints and Lints (1971a) have argued that adult life span is somehow determined by the length of the developmental period in Drosophila. More importantly, Lints and Lints (1971b) have argued that the key relationship is between growth rate and adult longevity and that duration of preimaginal growth is more or less a reflection, albeit a negative one, of growth rate. However, experiments with Tribolium castaneum are not consistent with this hypothesis. When Lints and Soliman (1977) compared the growth rates of eight wild strains of Tribolium with the mean adult life span, they found a positive correlation. Therefore, the exact relationship between adult life span and the duration of development and/or growth rate remains to be resolved. If adult life span is, in fact, positively correlated with the length of preimaginal development in Drosophila. it should be possible to determine the developmental interval during which adult longevity is determined. This interval could be as long as the entire 7 preimaginal period or as short as a discrete stage within the period. If adult longevity is set by the developmental rate during a discrete interval, then a series of temperature shifts during development should identify this period. Temperature-shift studies during development have proven useful in identifying the interval during which a temperature-sensitive gene product is required in Drosophila (Suzuki, 1970). However, none of the previous Drosophila studies have examined how stage-specific delays in preimaginal development affect adult longevity. The experiments reported here use a series of temperature shifts during development to alter the length of the preimaginal period. Adult life spans were then measured to determine whether or not the longevity of flies was affected. In this way, the relationship between the duration of preimaginal growth and the life span of adult Drosophila may be better understood. MATERIALS AND METHODS Stocks and Culture Conditions Two strains of Drosophila melanogaster were used in these experiments. Oregon-R, a highly inbred wild-type strain, was used as the control. The other strain, 957. is derived from Oregon-R and carries an X-chromosome linked mutation. This mutation results in a temperature-sensitive (t.s.) delayed development phenotype. At 22°C, the duration of preimaginal development of 957 is 16 to 19 days, similar to that of Oregon-R. At 29°C, preimaginal development of this mutant is extended to over 25 days whereas that of Oregon-R is only of 10 days duration. Cultures of 957 maintained at 29°C for longer than 26 to 27 days show complete pupal lethality. Both strains were maintained at 22 + 1°C and 50-60% relative humidity in quarter-pint bottles containing 24 ml of medium. The food used was a standard agar^ cornmeal-yeast medium containing 2.4 g/1 of methyl-p-hydroxybenzoate to inhibit mold growth and 50 mg/1 8 of either streptomycin or tetracycline as bacterial inhibitors. A 14 hour light-10 hour dark cycle was used for both culture maintenance and longevity analyses. Alteration of Duration of Development Male and female adults (3 to 5 days post-eclosion) were placed in empty culture bottles and 5 cm-diameter petri plates containing 12 ml of medium were attached to the bottles. Females were allowed to lay eggs for 10 hours at 22°C. Embryos were collected from the plates and placed in 2.5 cm-diameter x 9.5 cm shell vials each containing 6 ml of medium. Density of the cultures was maintained at 50 to 60 eggs in a vial. 162 vials were set up for Oregon-R and 272 for strain 957. The vials were kept at 22°C for 20 hours, the time at which the ratio of embryos to first instar larvae is approximately 1:1. Cultures were then placed at different temperatures as follows: 50 vials of Oregon-R at 29°C, 100 vials at 17°C and 12 vials at 22°C; 135 vials of £57 at 29°C, 122 vials at 17°C and 15 vials at 22°C. In the "shift-up" or "U" series of temperature shifts during development, 5 vials from each strain were shifted from 17°C to 22°C at various times during preimaginal growth. The cultures were then allowed to complete development at 22°C. The duration of development at 17°C and the most common developmental stage at the time of the shift were recorded. Some cultures were allowed to complete development at 17°C. The "shift-down" or "D" series of shifts were performed in a similar manner except that 4 vials of Oregon-R and 5 vials of 957 were shifted from 29°C to 22°C at various times and then the cultures were allowed to complete preimaginal growth at 22°C. Some of the Oregon-R cultures completed development at 29°C. Due to the lethal effect of 957. none of these cultures yielded adults at 29°C. For both strains, some cultures were kept at 22°C during the entire developmental period. Measurement of Adult Life Span Adult life span was measured at 22+1 °C and 29+l°C. In most cases, only the flies that emerged on the day of maximal eclosion for any one shift were used for determining adult longevity. However, it was necessary to make a second collection for some cultures on the following day due to low numbers of emerging adults. Flies from any one developmental shift were examined at only one temperature. Flies from the subsequent shift were examined at the other temperature. This was continued in an alternating manner for all shifts. Flies that emerged from Oregon-R cultures maintained at 22°C or 29°C throughout development were collected several times with the day of peak eclosion being recorded. Due to developmental asynchrony in Oregon-R cultures maintained through to eclosion at 17°C, only one collection was made. 957 flies that were cultured continuously at 22°C or 17°C were handled in a similar manner. For all cultures, newly eclosed flies were collected over 24 hour periods. Males and females were separated and place no more than 10 flies to a vial. The flies were then divided into two groups. One group was placed at 22°C and the other group was placed at 29°C. Transfers to vials with fresh medium were performed every 3 days at both temperatures. RESULTS Environmental and Genetic Alterations of the Duration of Preimaginal Development The length of the developmental period of both strains used in this study was altered by varying the amount of time preimaginal cultures were maintained at different temperatures. Table 1 summarizes the effects of culturing Oregon-R at 17°C, 22°C and 29°C for different periods of time on the total duration of development (i.e. from egg laying to day of maximal eclosion). As expected, the total duration of development increased with the amount of time maintained at lower temperatures. Cultures maintained at 29°C for progressively longer periods in the D-series had shortened developmental periods while those 10 TABLE 1 The effects of different culture temperatures and combinations of culture temperatures on the duration of preimaginal development in Oregon-R. Shift or Duration of Development Stage Total Developmental Development at Time of Shift Duration of Temperature (Days) at Development 17°C 29°C (Days) 29°C 0 10 10 D-9 0 8.6 Pupae (^ 50% Pigmented) 11 D-8 0 7.7 Pupae 12 D-7 0 6.8 Pupae (few 3rd instar) 12 D-6 0 5.8 3rd instar and pupae 13 D-5 0 4.8 3rd instar 13 D-4 0 3.8 3rd instar 14 D-3 0 2.8 2nd and 3rd instar 14 D-2 0 1.8 2nd instar 15 D-l 0 0.8 1st instar 15 22°C 0 0 15 U-l 1.2 0 2nd instar (few 1st instar) 17-18 U-2 2.1 0 2nd instar 18 U-3 3.1 0 2nd instar 18 U-4 4.1 0 2nd instar 19 U-5 5.1 0 2nd instar (few 3rd instar) 20 U-6 6.0 0 3rd instar (few 2nd instar) 20 U-7 6.9 0 3rd instar 21 U-8 8.0 0 3rd instar 22 U-9 9.0 0 3rd instar 21-22 U-10 10.0 0 3rd instar 21-22 U- l l 11.1 0 3rd instar 21-22 U-12 12.2 0 3rd instar (few pupae) 22 U-13 13.1 0 3rd instar and pupae 22-23 U-14 15.2 0 Pupae (few 3rd instar) 23-24 U-15 16.1 0 Pupae 24-25 U-16 18.1 0 Pupae (pigmented) 24-25 U-17 21.1 0 Pupae (eyes apparent) 26 U-18 23.2 0 Pupae (eyes and wings apparent) 26-27 17°C 29 0 29 11 maintained at 17°C for progressively longer periods in the U-series had extended developmental periods. This situation is typical of poikilotherms. In addition, no stage appeared particularly sensitive to the alterations in temperature. Figure 1 shows that the relationship between total duration of development and duration of development at either 17°C or 29°C was linear. The correlation coefficient (r) for the U-series (including 17°C and 22°C) is 0.977 and for the D-series (including 29°C and 22°C) is -0.979. Both of these values are highly significant (p<0.001). Table 2 summarizes the results of temperature shifts performed on strain 957. The U-series of shifts altered the duration of development in a manner similar to the U-series for Oregon-R. Figure 2 shows a regression plot for 957 similar to that for Oregon-R in Figure 1. The correlation coefficient for the U-series (including 17°C and 22°C) is 0.978 (p<0.001). Based on a Student's t-test, the regression coefficient (slope) for the 957 U-series (b=0.443) is not significantly different from the regression coefficient for the Oregon-R U-series (b=0.422) at 0.20 <p<0.50. In contrast, the D-series for 957 was markedly different from the D-series for Oregon-R. Table 2 and Figure 2 show that the total duration of development increased as the amount of time that 957 cultures were maintained at 29°C increased. Table 2 also shows that third instar larvae and, to a lesser extent, pupae are more sensitive to the delay caused by 29°C than embryos and first and second instars. This is evident in Figure 2 where it can be seen that cultures maintained at 29°C longer than 19 to 20 days showed a rapid increase in duration of development. However, the overall relationship is approximately linear (r=0.974 for D-series including 22°C, p<0.001). Figure 2 also shows that culturing strain 957 at 29°C was more effective in delaying development than culturing at 17°C, especially for later shifts. Note that the regression coefficient for the D-series (b=0.711) is significantly greater than the regression coefficient for the U-series (b=0.443) at p<0.0005. 12 FIGURE 1 Relationship Between Total Duration of Development and Duration of Development at 17°C or 29°C for Oregon-R. r = correlation coefficient b = regression coefficient + 95% confidence limits a = y - intercept U - series of shifts O r = 0.977 b 0.422 + 0.045 a 17.1 D - series of shifts % r -0.979 b -0.507 ± 0.079 a 15.5 13 3 0 • D CO £ 2 5 Q o . o UJ Q_ O o o o 2 0 ^ o p. LU > LU Q 1 5 U_ O z g cc ZD Q o o • • • 1 I I I I 1 1 0 5 10 15 2 0 2 5 3 0 DURATION OF D E V E L O P M E N T (DAYS) AT 17°C OR 29°C 14 TABLE 2 The Effects of Different Culture Temperatures and Combinations of Culture Temperatures on the Duration of Preimaginal Development in Strain 957. Shift or Developmental Temperature Duration of Development (Days) at 17°C 29°C Development Stage at Time of Shift Total Duration of Development (Days) D-25 0 24.9 60% 3rd instar : 40% pupae (many pupae dead) 36-37 D-24 0 23.8 75% 3rd instar : 25% pupae (some pupae dead) 35-36 D-23 0 22.8 80% 3rd instar : 20% pupae 34-35 D-22 0 21.8 90% 3rd instar : 10% pupae 33 D-21 0 21.3 3rd instar (few pupae) 32 D-20 0 20.3 3rd instar (few pupae) 31 D-19 0 19.2 3rd instar 28-29 D-18 0 18.3 3rd instar 27-28 D-17 0 16.3 3rd instar 26 D-16 0 15.3 3rd instar 26 D-15 0 14.3 3rd instar 25-26 D-14 0 13.2 3rd instar 24 D-13 0 12.1 3rd instar 25 D-12 0 11.2 3rd instar 24-25 D-l l 0 10.0 3rd instar (very few 2nd instar) 24-25 D-10 0 9.9 3rd instar (few 2nd instar) 24 D-9 0 9.0 50% 3rd instar : 50% 2nd instar 24 D-8 0 8.1 2nd instar (few 3rd instar) 22 D-7 0 7.0 2nd instar 21 D-6 0 6.1 2nd instar 21 D-5 0 5.0' 2nd instar 20 D-4 0 4.2 2nd instar (few 1st instar) 19 D-3 0 3.1 1st instar 19 D-2 0 2.1 1st instar 19 D-l 0 1.1 embryos and 1st instar 19 22°C 0 0 17-18 U-l 1.0 0 1st instar 18 U-2 2.0 0 1st instar 20 U-3 3.0 0 1st instar (very few 2nd instar) 19 15 TABLE 2 (CONT'D.) Shift or Duration of Development Stage Total Developmental Development at Time of Shift Duration of Temperature (Days) at Development 17°C 29°C (Days) U-4 4.0 0 2nd instar 19 U-5 5.0 0 2nd instar 21-22 U-6 6.0 0 2nd instar 22 U-7 6.8 0 " 2nd instar 22 U-8 7.8 0 2nd instar 22 U-9 8.9 0 3rd instar 23 U-10 9 8 0 3rd instar 22-23 U- l l 10.9 0 3rd instar 23 U-12 12.0 0 3rd instar 24-25 U-13 13.0 0 3rd instar 24-25 U-14 14.0 0 3rd instar (few pupae) 24-25 U-15 16.1 0 3rd instar (few pupae) 26 U-16 17.0 0 50% 3rd instar : 50% pupae 27 U-17 18.0 0 70% pupae : 30% 3rd instar 27 U-18 19.0 0 90% pupae : 10% 3rd instar 27 U-19 21.0 0 Pupae 27-28 U-20 24.1 0 Pupae (eyes apparent) 31 U-21 26.0 0 Pupae (eyes and wings apparent) 28-29 U-22 28.0 0 Pupae (few closed) 30 17°C 30-31.0 0 30-31 16 FIGURE 2 Relationship Between Total Duration of Development and Duration of Development at 17°C 29°C for Strain 957. U-series o r = 0.978 b = 0.443 + 0.042 a = 18.5 D-series • r b a = 0.974 = 0.711 + 0.070 = 16.6 17 4 0 - | 35H 30H 25H oo.o o oo>»' 16H 10 o — r ~ 15 " T " 5 — r ~ 10 2 0 2 5 DURATION OF D E V E L O P M E N T (DAYS) AT 17°C OR 29°C 30 3 5 18 Relationship between duration of development and adult longevity Oregon-R Table 3 summarizes the results of altering the duration of development on adult longevity at 29°C for Oregon-R. The table shows that there is little or no correlation between the length of preimaginal growth and mean life span at this temperature. This is true for both males and females. Sample survival curves for Oregon-R at 29°C are presented in Figures 3 and 4. There is a large amount of variation evident in the curves, but there is no consistent pattern of shifting of the curves to the right as duration of development increased. Moreover, whereas Table 3 indicates there is a fair amount of variation in mean life span, the survival curves show a great deal of consistency with respect to maximum life span. As a statistical test of the relationship between Oregon-R adult longevity and duration of development, a regression analysis of mean life span on duration of development was performed. For this analysis, the standard deviation was not taken into account even though this did not make use of all the information and is generally not recommended (Zar, 1984). However, the difference in sample sizes between groups would have weighted some mean life spans in the regression much more heavily than others. The results of the analysis are shown in Figure 5. The slopes of the regression lines for both males and females are slightly negative, indicating an inverse relationship between duration of development and mean life span. However, the 95% confidence intervals on the lines show that neither slope is significantly different from 0. The correlation coefficients are also not significant (r m a l e s = -0.290, 0.20<p<0.50; r f e m a l e e = -0.167, p>0.50). Table 4 summarizes the 22°C results for Oregon-R. There is much more variation in mean life span at 22°C than there is at 29°C. This is also apparent in the 22°C survival curves shown in Figures 6 and 7. For males, the extremes for mean life span (89.9 days for D-7 versus 47.0 days for 17°C) are reflected in the survival curves (see Figure 6), but no 19 TABLE 3 Oregon-R Adult Longevity at 29°C A. Males B. Females Shift or Total Duration Mean Life Standard Sample Developmental of Development Span in Deviation Size Temperature (Days) Days in Days 29°C D-8 D-6 D-4 D-2 22°C U-l U-3 U-5 U-7 U-9 U - l l U-13 U-15 U-17 17°C 10 12 13 14 15 15 17-18 18 20 21 21-22 21- 22 22- 23 24-25 26 29 35.7 28.9 30.8 30.4 28.2 33.0 33.6 31.8 32.7 32.1 33.6 30.6 26.1 33.6 31.1 26.4 5.3 5.5 4.9 5.1 5.5 7.2 4.6 5.6 4.4 5.5 5.7 4.7 4.7 5.2 6.9 5.8 117 50 49 56 60 109 50 47 48 70 69 59 25 28 32 47 B. 29°C D-8 D-6 D-4 D-2 22°C U-l U-3 U-5 U-7 U-9 U - l l U-13 U-15 U-17 17°C 10 12 13 14 15 15 17-18 18 20 21 21-22 21- 22 22- 23 24-25 26 29 36.0 39.5 37.7 37.1 37.4 35.6 37.1 37.0 40.9 41.8 33.9 37.5 37.1 37.2 38.5 32.9 7.4 8.3 7.0 9.6 10.4 7.8 9.2 7.6 2.7 4.3 9.3 11.5 10.9 9.9 10.9 6.4 119 61 62 56 57 157 77 63 33 33 33 53 23 23 43 45 20 FIGURE 3 Survival Curves of Oregon-R Males Maintained at 29°C 22 FIGURE 4 Survival Curves of Oregon-R Females Maintained at 29°C a - 29°C — — — — — D-8 D-4 22°C b - 22°C U-3 U-9 U-15 17°C % SURVIVING % SURVIV ING 24 FIGURE 5 Relationship Between Mean Life Span and Duration of Development for Oregon-R at 29°C. Females O r = -0.167 b - -0.070 ± 0.235 Males • r = -0.290 b= -0.145 + 0.274 50 45H O o CO CN co Q z 35 CO LU o ° o Ob O Q 30i z < LU 25H 20 n— 25 "T - | 1 - I 5 10 15 20 DURATION OF D E V E L O P M E N T (DAYS) 26 TABLE 4 Oregon-R Adult Longevity at 22°C A. Males B. Females Shift or Total Duration Mean Life Standard Sample Developmental of Development Span in Deviation Size Temperature (Days) Days in Days A. 29°C 10 67.2 18.7 116 D-9 11 64.3 23.7 59 D-7 12 89.9 20.9 65 D-5 13 81.6 17.7 59 D-3 14 77.2 13.8 79 D-l 15 69.3 14.0 40 22°C 15 78.5 20.2 124 U-2 18 74.1 14.6 68 U-4 19 73.6 12.1 68 U-6 20 88.6 19.1 60 U-8 22 74.1 17.1 40 U-10 21-22 70.9 14.2 60 U-12 22 73.2 10.2 23 U-14 23-24 77.6 29.8 29 U-16 24-25 64.6 16.8 40 U-18 26-27 76.2 27.8 37 17°C 29 47.0 26.4 38 B. 29°C 10 85.0 16.7 155 D-9 11 84.3 29.1 59 D-7 12 90.2 29.4 61 D-5 13 89.5 31.7 53 D-3 14 88.5 30.2 57 D-l 15 90.3 26.8 46 22°C 15 91.5 20.1 138 U-2 18 91.7 22.0 51 U-4 19 85.4 22.2 38 U-6 20 94.9 13.3 9 U-8 22 82.7 24.2 38 U-10 21-22 88.8 22.3 42 U-12 22 92.5 25.4 26 U-14 23-24 72.5 31.0 48 U-16 24-25 70.8 27.1 39 U-18 26-27 65.2 28.3 48 17°C 29 76.1 20.0 47 27 FIGURE 6 Survival Curves of Oregon-R Males Maintained at 22°C. a - 29°C — — _ — D-7 D-3 22°C b - 22°C U _ 4  U-10 U-16 17°C 29 FIGURE 7 Survival Curves of Oregon-R Females Maintained at 22°C. a - 29°C D-7 D-3 22°c b - 22°C T j _ 4  U-10 U-16 17°C 31 correlation is evident between duration of development and longevity. The female results are much less variable than the male results both for mean life spans and for survival curves (see Figure 7). Here too, there is no correlation between the duration of development and longevity. Figure 8 shows the regression analysis on 22°C results. As was the case at 29°C, the regression lines have negative slopes. The slope of the male regression line is not significantly different from 0 within the 95% confidence limits and the correlation coefficient is not significant (r = -0.359. 0.10<p<0.20). However, the female regression line does have a significant slope and the correlation coefficient is also significant (r=-0.600, 0.01<p<0.02), unlike the situation at 29°C. Females in groups U-14, U-16, and U-18 had much lower mean life spans than the others at 22°C and their grouping towards one end of the regression produces the significant correlation. The results from 22°C and 29°C indicate that females live slightly longer than males at both temperatures. The mean life span for all females at 29°C was 37.3 days and for all males was 31.2 days, the two values differing by a factor of 1.20. The corresponding values at 22°C were 84.7 days from females and 73.4 days for males, the factor here being 1.15 which is very similar to the 29°C value. Therefore, it does not appear that the adult longevity of one sex is affected more by temperature then the longevity of the other sex. Strain 952 The effects of delaying development on life span were also examined in strain 957. Tables 5 and 6 summarize the 29°C results for males and females, respectively. There appears to be no relationship between life span and duration of development for either sex. On the other hand, the survival curves shown in Figures 9 and 10 reflect substantial variability of longevity for both sexes. This variability is more pronounced in the D-series curves. As was the case with Oregon-R at 29°C, the 9_5_7 survival curves show no consistent 32 FIGURE 8 Relationship Between Mean Life Span and Duration of Development for Oregon-R at 22°C Females o r = -0.600 b = -0.905 ± 0.664 Males • r = -0.359 b = -0.622 ±0.891 33 1 0 0 - 1 o 9 0 H o CM CM 5 8 0 ^ GO Q 70H s co LU O o o 60H z < 5 0 H 4 0 5 10 15 2 0 2 5 DURATION OF D E V E L O P M E N T (DAYS) 30 34 TABLE 5 Strain 957 Male Adult Longevity at 29°C Shift or Total Duration Mean Life Standard Sample Developmental of Development Span in Deviation Size Temperature (Days) Days in Days D-24 35-36 28.4 6.6 14 D-22 33 33.2 7.0 15 D-20 31 35.2 8.0 21 D-18 27-28 38.8 5.0 43 D-16 26 40.8 5.2 45 D-14 24 43.3 5.0 43 D-12 24-25 41.4 2.6 53 D-10 24 26.1 7.3 39 D-8 22 30.2 7.9 60 D-6 21 34.1 4.9 46 D-4 19 27.3 8.7 47 D-2 19 27.2 7.5 70 22°C 17-18 35.4 3.2 199 U-2 20 32.6 3.0 75 U-4 19 33.8 3.6 49 U-6 22 31.4 2.5 59 U-8 22 31.4 3.7 68 U-10 22-23 31.2 3.2 49 U-12 24-25 38.3 3.8 60 U-14 24-25 36.1 5.9 30 U-16 27 35.9 5.7 50 U-18 27 36.4 5.1 69 U-20 31 31.6 5.4 57 U-22 30 33.9 4.9 72 17°C 31 31.9 6.8 116 35 TABLE 6 Strain 95J7 Female Adult Longevity at 29°C Shift or Total Duration Mean Life Standard Sample Developmental of Development Span in Deviation Size Temperature (Days) Days in Days D-24 35-36 28.7 7.3 49 D-22 33 37.0 7.2 26 D-20 31 37.6 6.0 14 D-18 27-28 39.2 6.1 33 D-16 26 44.0 4.3 36 D-14 24 44.1 5.8 53 D-12 24-25 41.7 4.2 65 D-10 24 32.0 5.4 33 D-8 22 35.4 3.8 41 D-6 21 31.5 5.7 50 D-4 19 30.8 7.0 60 D-2 19 31.7 6.5 47 22°C 17-18 31.9 4.8 193 U-2 20 31.8 3.5 77 U-4 19 33.5 3.8 58 U-6 22 31.8 3.0 60 U-8 22 30.7 4.2 57 U-10 22-23 31.2 2.2 48 U-12 24-25 37.1 3.7 64 U-14 24-25 37.5 2.9 39 U-16 27 32.9 6.2 55 U-18 27 35.8 4.3 68 U-20 31 31.8 5.0 56 U-22 30 31.3 3.7 68 17°C 31 31.0 3.9 142 36 FIGURE 9 Survival Curves of Strain 95J7 Males Maintained at 29°C. a - D-24 D-18 D-12 D-6 _ _ _ _ _ _ 22°C b - 22°C U-6 U-12 U-18 17°C 38 FIGURE 10 Survival Curves of Strain 957 Females Maintained at 29°C a - D-24 D-18 D-6 _ _ _ _ _ _ 22°C b - 22°C U-6 U-I2 U-18 40 pattern in shifting as the length of development increased. The regression analysis of mean life span at 29°C on duration of development is shown in Figure 11 for males and in Figure 12 for females. Both sexes have a slightly positive correlation between mean life span and length of preimaginal growth for the D-series although neither correlation is significant ( r m a l e g = 0.095, p»0.50; r f e m a l e s = 0.164, p>0.50). The regressions for the U-series are clearly not significant ( r m a l e s = -0.023, p»0.50; r f e m a l e s = -0.006, p>»0.50). The large variability in longevity for the D-series flies is well-illustrated in the regression analysis plots. It can be seen that the overall maximum and minimum life spans at 29°C for both sexes fall within the D-series. The plots also show that beyond shift D-10, a strongly negative relationship exists between duration of development and mean life span. Tables 7 and 8 summarize the 22°C results for strain 957. These results are similar to the 29°C results in that no correlation is apparent between mean life span and duration of development. Figures 13 and 14 show some of the survival curves for 22°C. Once again, the D-series curves show a higher degree of variability than curves shown from the U-series. Except for the survival curve representing D-23 males in Figure 13A, there appears to be less variability in the 22°C curves than in the 29°C curves. This is very clear when examining the U-series. The situation is opposite to that observed with Oregon-R and was not expected as there is generally more variation in adult longevity at lower temperatures. Especially consistent at 22°C are the maximum life spans for both sexes in the U-series. Almost all the curves show that the final deaths occurred between 115 and 120 days. The 22°C regression plots for 957 are shown in Figures 15 and 16 for males and females, respectively. The regression lines for the D-series have negative slopes indicating a negative correlation between mean life span and length of development. While the female regression is not significant (r = -0.485, 0.01 <p<0.20), the male regression is significant as indicated by the 95% confidence limits on the slope and the correlation coefficient (r = 41 FIGURE 11 Relationship Between Mean Life Span and Duration of Development for 957 Males at 29°C U-series + 17°C + 22°C O r = -0.023 b = -0.012 ±0.349 D-series + 22°C « r = 0.095 b = 0.100 + 0.696 42 5CH 2 0 ^ — , , , , r 10 15 2 0 2 5 3 0 3 5 4 0 DURATION OF D E V E L O P M E N T (DAYS) 43 FIGURE 12 Relationship Between Mean Life Span and Duration of Development for 957 Females at 29°C U-series + 17°C + 22°C O r = -0.006 b = -0.003 ± 0.346 D-series + 22°C « r = 0.164 b = 0.154 + 0.614 44 5 0 - i 1 I 1 1 1 1 1 10 15 2 0 2 5 3 0 3 5 4 0 DURATION OF D E V E L O P M E N T (DAYS) TABLE 7 45 Strain 957 Male Adult Longevity at 22°C Shift or Total Duration Mean Life Standard Sample Developmental of Development Span in Deviation Size Temperature (Days) Days in Days D-25 36-37 59.7 3.2 3 D-23 34-35 56.5 23.4 28 D-21 32 69.8 34.3 16 D-19 28-29 82.7 26.1 22 D-17 26 92.1 27.9 37 D-15 25-26 94.6 21.3 68 D-13 25 88.1 36.8 35 D-l l 24-25 83.0 30.0 30 D-9 24 83.9 21.7 37 D-7 21 85.1 20.4 50 D-5 20 81.9 22.8 50 D-3 19 83.8 26.3 80 D-l 19 87.0 21.3 46 22°C 17-18 82.2 19.6 184 U-l 18 83.3 21.8 48 U-3 19 81.1 20.5 50 U-5 21-22 81.1 20.5 50 U-7 22 76.3 26.8 50 U-9 23 81.9 18.9 59 U- l l 23 84.9 18.1 50 U-13 24-25 86.3 18.4 41 U-15 26 81.8 25.7 66 U-17 27 83.1 22.0 47 U-19 27-28 87.9 18.1 66 U-21 28-29 81.6 22.9 59 17°C 31 76.0 17.1 123 46 TABLE 8 Strain 9J2 Female Adult Longevity at 22°C Shift or Total Duration Mean Life Standard Sample Developmental of Development Span in Deviation Size Temperature (Days) Days in Days D-25 36-37 61.7 25.9 22 D-23 34-35 75.8 29.0 58 D-21 32 77.5 36.1 26 D-19 28-29 78.1 19.5 16 D-17 26 98.6 27.2 37 D-15 25-26 87.0 31.3 43 D-13 25 92.1 20.6 35 D-l l 24-25 89.3 28.6 59 D-9 24 82.6 27.2 34 D-7 21 83.5 30.0 56 D-5 20 79.0 26.1 72 D-3 19 78.6 27.7 72 D-l -19 80.3 25.9 46 22°C 17-18 87.8 21.9 193 U-l 18 78.6 23.1 90 U-3 19 82.9 24.8 59 U-5 21-22 85.2 24.3 50 U-7 22 86.2 18.0 59 U-9 23 79.9 18.4 55 U- l l 23 85.2 20.7 49 U-13 24-25 92.3 23.0 57 U-15 26 85.5 25.8 86 U-17 27 84.6 22.2 55 U-19 27-28 83.9 24.0 68 U-21 28-29 79.0 25.7 82 17°C 31 81.7 17.3 104 47 FIGURE 13 Survival Curves of Strain 9J57 Males Maintained at 22°C. a - D-23 D-17 D-5 22°C b - 22°C U-5 U - l l U-17 49 FIGURE 14 Survival Curves of Strain 957 Females Maintained at 22°C a - D-23 D-17 D- l l D-5 22°C b - 22°C U-5 U - l l U-17 51 FIGURE 15 Relationship Between Mean Life Span and Duration of Development for 957 Males at 22°C U-series + 17°C + 22°C O r = -0.068 b = -0.055 + 0.531 D-series + 22°C # r = -0.721 b = -0.1.359 + 0.821 52 53 FIGURE 16 Relationship Between Mean Life Span and Duration of Development for 957 Females at 22°C U-series + 17°C + 22°C O r = -0.111 b = -0.101 ± 0.596 D-series + 22°C • r = -0.485 b = -0.720 + 0.816 t 54 1 0 0 - ) CJ o CN CM t j CO $ Q Z co LU z < LU 5 90H 80H 7 0 60H 50H 4 0 15 3 5 — 1 4 0 10 2 0 2 5 3 0 DURATION OF D E V E L O P M E N T (DAYS) 55 -0.721, 0.002<p<0.005). This is due to the low mean life spans obtained for D-23 and D-25 males. A regression based on the D-series that excludes D-23 and D-25 is not significant. The U-series regressions are not significant ( r m a l e B = -0.068, p»0.50; r f e m a ] e s = -0.111, p>0.50), as was the case at 29°C. While the results obtained for 957 indicate a much larger degree of variability for the D-series than for the U-series at both temperatures, the overall mean life spans within each series are very similar: (a) 29°C, males D-series + 22°C, mean life span = 34.0 days. U-series + 17°C + 22°C, mean life span = 33.8 days. (b) 29°C, females D-series + 22°C, mean life span = 35.8 days. U-series + 17°C + 22°C, mean life span = 32.9 days. (c) 22°C, males D-series + 22°C, mean life span = 80.7 days. U-series + 17°C + 22°C, mean life span 82.1 = days. (a) 22°C, females D-series + 22°C, mean life span = 82.3 days. U-series + 17°C + 22°C, mean life span = 84.1 days. In addition, females and males live approximately the same length of time at both temperatures. The mean life span for all females at 29°C was 34.5 days and for all males at 29°C was 33.8 days. These values differ by a factor of 1.02. At 22°C, the mean life span for all females was 83.0 days and for all females was 81.4 days, again differing by a factor of 1.02. Therefore, the situation for strain 957 is similar to the wild-type strain, Oregon-R, in that the sexes do not respond differently to changes in the temperature at which adults are maintained. DISCUSSION 56 The results of this study show that there is no influence of the duration of preimaginal development on adult longevity. Neither the wild-type strain, Oregon-R, nor the mutant strain 957. showed consistent alterations in life span at either 22°C or 29°C when development was progressively delayed. Both the general observations made on the survival curves and the regression analyses of mean life span on duration of development support these conclusions. It is not surprising that the Oregon-R strain showed no difference between 22°C and 29°C, in terms of the response of adult longevity to different developmental treatments. Leffelaar and Grigliatti (1984b) have shown that 22°C and 29°C survival curves for both Oregon-R males and Oregon-R females are coincident when adjusted for the difference in the rate of living at the two temperatures. In the same study, reproducible patterns of age-correlated behavioral changes were observed to be very similar at the two temperatures. Strain 957 has a phenotype of delayed preimaginal development when cultured at 29°C. Although the main reason for looking at adult longevity in 9_5_7 was to see how the delayed development phenotype affected life span, it was also of interest to know if the mutation acted in the adult. A comparison of Oregoh-R and 957 overall mean life spans for each sex indicates that the two strains do not differ markedly in their longevity at either 22°C or 29°C. This is also supported by the survival curves of the two strains. Thus, the mutation does not extend adult life span and, consequently, it does not appear that the gene product is necessary in adult Drosophila. Examination of the survival curves shows that there is more variation in male longevity than in female longevity. This is true for both strains at both adult temperatures. In addition, male life span was somewhat shorter than female life span, although the difference was very small in strain 957. Leffelaar and Grigliatti (1984a) found similar differences 57 between males and females when examining Oregon-R and five different temperature-sensitive adult-lethal mutants. The difference between male and female longevity varies from strain to strain in Drosophila (Lamb, 1978) and even may depend on the mating status of both females (Maynard Smith, 1958; Rose and Charlesworth, 1981a) and males (Partridge and Farquhar, 1981). In the experiment reported here, flies were collected every 24 hours which is not frequent enough to ensure virginity at either 22°C or 29°C (Ashburner and Thompson, 1978). Therefore, neither the mating status of the flies nor its effect on longevity could be assessed. Previous studies of the effects of developmental conditions on adult longevity in Drosophila have utilized two approaches. The first approach has involved culturing the preimaginal stages at different temperatures (Alpatov and Pearl, 1929; Burcombe and Hollingworth, 1970; Lints and Lints, 1971a). The second approach has involved the use of different culture densities, the duration development increasing as the preimaginal culture density increases (Lints and Lints, 1969). In contrast with the present work, all of these studies have suggested that a positive correlation does exist between the duration of development and adult life span in Drosophila. However, several important differences exist between these previous reports and the work reported here. One difference is that hybrid strains are the ones most commonly used to study aging in D. melanogaster. Lints and Lints (1971b) have argued that inbred lines such as those examined in the present study should not be used to examine normal aging and senescence. They argue that death in inbred strains could be due to a high frequency of "deleterious" genes existing in a homozygous state. Furthermore, they argue that these genes could act in the "presenescent" period of life (i.e., the period prior to sexual maturity and peak fecundity), causing "presenescent" death. This is based on the well documented fact that hybrid strains of Drosophila melanogaster display heterosis for adult longevity (Bozcuk 1978 and 1981; Lamb, 1978). Therefore, the highly inbred nature of some strains does have life-58 shortening effects. However, there is no a Priori reason to believe that genes whose expression act to decrease adult longevity, are only acting in a "presenescent" manner. A more likely situation is that such gene expression is part of the whole pattern of senescence. Rose (1983a and 1983b) has criticized the use of hybrid strains and stated that hybridization of highly inbred strains creates "genetic" and "evolutionary disequilibrium." That is, in looking at hybrids, one may be studying aging in a genetically unstable situation. A difference between this study and that of Lints and Lints (1971a) lies in the parameters used in correlation with adult life span. In the latter report, the effects of preimaginal temperature on adult life span were examined in females produced by crossing two wild-type strains of Drosophila. Cultures were maintained at six different temperatures throughout development. Upon eclosion, females of a modal size and a modal duration of development from each preimaginal environment were set at 15°C and their life spans were measured. A significant negative correlation was found between developmental temperature and mean life span. As duration of development normally increases with decreasing developmental temperature, Lints and Lints argue that there is a positive correlation between duration of development and mean life span. However, examination of their data reveals that when duration of development is compared directly to mean adult life span, the correlation is only marginally significant at the 5% level. The range of temperatures over which cultures were maintained by Lints and Lints (1971a) also differs from the present study. The highest culture temperature used by Lints and Lints was 31°C. This temperature usually increases the duration of all developmental stages (i.e., embryo, larva and pupa) relative to 28°C (Ashburn and Thompson, 1978) which was the next highest temperature used for culturing. In addition, culture mortality usually shows a rapid increase with temperature above 30°C (Ashburner and Thompson, 1978). Thus, the very short life span of females developed at 31°C in Lints and Lints' study may be more representative of a situation of physiological stress, rather than shortened development. If 59 the mean life spans of 31°C-developed flies is ignored, then the correlation is no longer significant. Furthermore, Cohet (1975) has observed that flies developed at temperatures below 17°C have shorter life spans than flies developed above 17°C. Therefore, a significant correlation between developmental temperature and mean life span is not necessarily indicative of a similar correlation between duration of development and mean life span. It is important to note that previous studies have usually reported some measure of the rate of growth during the preimaginal stages. For example, Lints and Lints (1971b) have observed that growth rate (i.e., size of newly eclosed adults/duration of development) is more closely correlated with life span than is duration of development. Typically, the growth rate-mean life span correlation is negative (Lamb, 1978), indicating that a more rapid growth rate results in shorter lived flies. To explain this, Lints and Lints (1971b) argue that slowing the growth rate slows the expression of developmental programs. They argue further that senescence is part of normal development and, consequently, must be a genetically programmed event, an idea originally proposed by Muller (1963). By slowing the expression of the developmental program the entire program is "preset" to unfold at a slower rate, thus delaying the onset of senescence. Lints and Lints (1971b) also postulate that slowing the rate of growth at lower temperatures also slows the mitotic cell division rate, a slower mitotic rate during development would result in fewer spontaneous errors occurring during DNA replication and during RNA and protein synthesis. The reduced number of errors in DNA would be reflected later in life as a delayed time of cell and organismal death. The latter explanation is very similar to both the somatic-mutation theory of aging and the protein-error (error-catastrophe) theory of aging. Interestingly, Lints (1978) has claimed that there is little evidence to support either of these theories and that the evidence that does exist is inconclusive. In the present study, the size of flies was not measured, so no estimate of growth rate was obtained. If growth rate was slowed as duration of development increased in either 60 Oregon-R or strain 957. it is highly unlikely that a significant correlation between growth rate and adult longevity would have been observed, for example, there was more than a two-fold difference in the duration of development of 957 files when comparing cultures developed at 22°C to cultures that were involved in the later D-series shifts. Yet, no change in the size of flies was noticed. In fact, for both strains and under all developmental conditions, no apparent change occurred in body size. Therefore, it seems reasonable to assume that growth rate was probably slowed as the duration of development increased. With no consistent pattern of change being observed in mean adult life span, the present study does not support the idea of programmed senescence proposed by Lints and Lints (1971b). The lack of a substantial difference in the life spans of the wild-type strain (Oregon-R) and the mutant strain (957) indicates that while the mutation delays development at 29°C, it does not delay senescence in the adult. This provides further support for the argument that delaying preimaginal development does not extend adult longevity. However, this does not imply that all mutations that affect development have no affect on life span. Leffelaar and Grigliatti (1984a) have described several mutant strains that show preimaginal death when developed at 29°C. When cultured at 22°C, preimaginal death does not occur, but if the adults are then shifted to 29°C the life spans of these strains are altered. Unfortunately, the exact nature of the defects caused by these mutations is not understood. Further characterization of these strains should provide more information on the mechanisms involved in the control of aging and death in Drosophila. One approach that may prove useful in gaining a better understanding of the genetic control of senescence is to use mutant strains that have already been characterized as being defective in cellular maintenance functions. One such class of mutants in D. melanogaster that could be studied are the mutagen-sensitive (mus) mutants. Many of these strains are known to be defective in various DNA repair pathways. By using such strains, it can be asked if and how their adult life spans are affected. The effects of mus mutations on adult longevity will be examined in Chapter Three. CHAPTER TWO INTRODUCTION 61 When a phenomenon as complex as aging is studied in the laboratory, two questions arise about the general applicability of the conclusions. First, do the environmental conditions under which the experiment is performed (e.g., temperature, humidity, food, population density) reflect the natural environment? The laboratory situation is seldom similar to an organism's natural environment, but in order to obtain accurate and repeatable results it is necessary to standardize environmental variables. This is especially important when comparisons are made between different lines or strains of the organism. Second, are the strains being studied typical of non-laboratory strains? For a genetic analysis, this question is very important to consider. Laboratory strains of Drosophila melanogaster are often highly inbred. As a consequence, these strains probably have a much lower genetic variability than those in nature. Thus, it is possible that the use of highly inbred laboratory strains does not accurately reflect the situation in the wild. One way of partially alleviating this problem is to use lines that have been recently isolated from the wild. Presumably, the genetic variability that occurs in nature would be characteristic of such strains. Investigations of population-based theories of aging in Drosophila often make use of strains that have high genetic variability. Rose (1983a and 1984) and Rose and Charlesworth (1981b) argue that the use of these strains allows examination of how the pattern of senescence evolves in a species such as Drosophila melanogaster. Their contention is that genes which influence the fitness of the organism act in a pleiotropic manner, affecting characteristics like fertility and fecundity early in adult life and senescence at later ages. By selecting for a particular pattern of early life-history characteristics, they suggest that it is possible to change later life-history characteristics that may be seemingly unrelated. Attempts to select for alterations in patterns of life-history (e.g., increased life span) 62 have been performed using highly inbred Drosophila strains. For example, Lints §1 ah. (1979) attempted to select lines that showed increased longevity from a laboratory wild-type stock, Oregon-R. This was done by retaining the offspring from flies that were in the 25% longest-lived portion of the population maintained as single male-female pairs. The selection procedure, repeated over eight generations, produced no significant change in life span between experimental and unselected controls. The reason for the inability to select for long-lived lines was probably that the Oregon-R strain had low genetic variability resulting from inbreeding in the laboratory over several hundred generations. Luckinbill gj aL (1984) and Rose and Charlesworth (1981b) used strains that had been bred to maximize genetic variability. While they did not select directly for adult life span, selection schemes for early and late reproduction used in both studies yielded short and long lived lines, respectively. It is of interest to know if and how strains recently isolated from the wild differ in their pattern of life span from highly inbred wild-type Drosophila stocks that have been maintained in the laboratory for many years. Although wild strains may better represent natural aging, laboratory strains may be preferable for genetic analyses of aging because of their well characterized genetic background. To clarify these issues, the adult life span of four PJ. melanogaster lines that were isolated from the wild and maintained in the lab for approximately one year before testing were examined. Two different adult temperatures and two different preimaginal growth' temperatures were used in this study. The results were compared to the life span of a wild-type stock, Oregon-R, that has been cultured in the laboratory for about 60 years. In this way, the use of highly inbred strains to study aging in a laboratory environment can be assessed. 63 MATERIALS AND METHODS Stocks and Culture Conditions Five Drosophila melanogaster strains were examined in this study. The wild-type Oregon-R stock has been inbred in the laboratory over a 50 to 60 year period. The other four strains were isolated from the wild one year before studying their adult longevity. Initially, eight non-virgin Drosophila females were isolated from the Vancouver area and brought into the laboratory. The progeny of four of these females led to established lines (designated strains D, E, F and G) that were concluded to be melanogaster based on reciprocal crosses to Oregon-R and two other highly inbred EL melanogaster wild-type strains, Canton-S and Samarkand. All stocks were maintained under the same conditions as those described in Chapter One. Determination of Adult Life Span Parents of the flies whose longevity was to be measured were allowed to oviposit in bottles over a 12 hour period at 22°C. For the wild strains, half of the cultures remained at 22°C to complete development. The other half were transferred to 29°C where they were kept until eclosion. The Oregon-R strain was cultured only at 29°C during preimaginal growth. When rapid eclosion began, bottles were cleared and held for 24 hours. Flies were then collected, separated by sex and placed no more than 10 flies to a 2.5 cm diameter x 9.5 cm vial containing 6 ml of medium. Half of the flies from each culture were placed at 22°C and half were placed at 29°C. A second collection was made from the following 24 hour period for the wild strains and Oregon-R. These flies were handled in the same way. For Oregon-R, a third collection was made from which all flies were kept at 22°C. Flies were transferred to vials containing fresh food on a 2 day-2 day-3 day interval. The number of flies still alive and their age were recorded at each transfer. This was 64 continued until all flies were dead in the sample, the determination of mean life span and the plotting of survival curves are described in Chapter One. RESULTS Wild Strain Adult Longevity The adult life span of the four wild strains was determined at both 22°C and 29°C following development at either of these temperatures. Each strain was tested twice using 40 to 60 individuals from each sex per test. Mean life spans at 29°C are shown in Table 9. The results are based on the combined data of the two tests. Table 10 summarizes the results of the analyses of variance based on the data in Table 9. Strains D, F and G showed no significant difference (p=0.05) in life span between flies developed at 29°C and those developed at 22°C for either sex. Only strain E males and females lived significantly longer at 29° following 29°C development as compared to 22°C development. Sex differences in adult life span varied from strain to strain. For example, a significant difference between male and female life span was observed in strain D following development at 29°C, in strain F following development at 22°C and in strain G following development at 29°C. However, all other differences were not statistically significant. The 29°C survival curves for the wild strains are shown in Figures 17 through 20. Strains F and G display more or less "rectangular" survival curves. These curves are characteristic of populations in which the force of mortality (i.e. rate of death divided by number of individuals alive at the beginning of a specific interval) increases with age (Lamb, 1978). Strain D males raised at 22°C and strain D females raised at either temperature also have rectangular curves. Strain E flies raised at 29°C exhibit curves that are somewhat less rectangular. The curves for this strain following development at 22°C are almost diagonal. This type of curve is typical of populations showing little or no change in the force of 65 TABLE 9 Adult Life Spans of Wild Strains at 22°C. Sex Strain Developmental Mean Standard Temperature Life Span Deivation (°C) (DAYS) (DAYS) Sample Coefficient Size of Variation (%) 29 22 40.0 42.3 14.2 13.5 85 87 35.5 31.9 Male 29 22 29 22 46.9 33.3 42.7 43.9 13.2 20.9 7.7 12.6 97 100 108 104 28.1 62.8 18.0 28.7 29 22 45.3 44.7 10.5 13.6 114 109 23.2 30.4 D 29 22 48.9 46.2 13.8 13.8 103 90 28.2 29.9 Female 29 22 29 22 47.6 39.5 45.3 48.1 15.9 21.1 13.7 12.6 93 80 85 98 33.4 53.4 30.2 26.2 29 22 48.4 47.7 11.5 14.2 99 100 23.8 29.8 66 TABLE 10 Analysis of Variance Based on Data of Table 9. Strain D Based on Developmental Temperature Sex F Level of Significance Male 1.19 0.10<p<0.25 Female 1.84 0.10<p<0.25 Based on Sex Developmental Temperature (°C) 29 22 18.9 3.61 Level of Significance p<0.0005 0.05<p<0.10 Male 29.6 p«0.0005 Female 8.26 0.0025<p<0.005 29 22 0.109 3.88 p»0.25 0.05<p<0.10 Male 0.706 p>0.25 Female 2.07 0.10<p<0.25 29 22 2.78 5.61 0.05<p<0.10 0.01 p>0.025 Male 0.137 p»0.25 Female 0.146 p»0.25 29 22 4.23 2.43 0.025<p<0.05 0.10<p<0.25 67 FIGURE 17 Survival Curves of Strain D Adults Maintained at 29°C. Development Temperatures = 22°C { { Development Temperature = 29°C { { — -a - Males b - Females 69 FIGURE 18 Survival Curves of Strain E Adults Maintained at 29°C. Development Temperatures = 22°C { { Development Temperature = 29°C { { — • a - Males b - Females % SURVIVING o 71 FIGURE 19 Survival Curves of Strain F Adults Maintained at 29°C. Development Temperatures = 22°C { { Development Temperature = 29°C { { a - Males b - Females 73 FIGURE 20 Survival Curves of Strain G Adults Maintained at 29°C. Development Temperatures = 22°C { { Development Temperature = 29°C { { a - Males b - Females % SURVIVING % SURVIVING 75 mortality (Lamb, 1978). The mean life spans of the wild strains at 22°C are listed in Table 11 and the results of the corresponding analyses of variance are summarized in Table 12. With the exceptions of strain G males and strain D females, there was a significant difference in life span at 22°C when a comparison is made between flies raised at this temperature and flies raised at 29°C. Flies developed at 29°C lived longer than their 22°C-raised counterparts. (However, note that strain F males lived longer when developed at 22°C.) These results show that raising the strains at the two different temperatures had more striking effects on adult life span at 22°C than at 29°C. More marked sex differences were also apparent when life span was measured at 22°C. Only Strain F flies developed at 29°C and strain G flies developed at either temperature showed no statistically significant difference between the sexes at 22°C. Where differences in adult life span do exist between the sexes at 22°C, they are not necessarily consistent with the differences that existed at 29°C. For example, strain F females were longer lived than their male counterparts at 29°C, but at 22°C the females were shorter lived. In general, the wild strains showed much more variation in their life spans at 22°C than they did at 29°C. The coefficients of variation for life span listed in Tables 9 and 11 are the best indicators of this temperature difference. The average coefficient of variation at 22°C is 40.0% while at 29°C the average is 32.8%. The 22°C survival curves shown in Figures 21 through 24 also illustrate the larger amount of variation at 22°C. The decline in the percentage of flies surviving was more gradual at 22°C than at 29°C. Only the curves for strains F and G, shown in Figures 23 and 24, respectively, strongly resemble the rectangular shape. In addition, the decline in the 22°C populations relative to the maximum life spans at this temperature occurred much earlier than it did at 29°C. This is best illustrated in the curves for strains D and E (Figures 21 and 22) where the shape of the curves is almost diagonal, indicating constant death rates in these strains at 22°C. 76 TABLE 11 Adult Life Spans of Wild Strains at 22°C. Sex Strain Developmental Temperature (°C) Mean Standard Sample Coefficient Life Span Deviation Size of Variation (DAYS) (DAYS) (%) D 29 22 69.7 59.3 23.8 29.9 98 96 34.1 50.4 Male 29 22 29 22 93.8 78.2 79.4 96.0 30.0 41.7 25.4 26.4 91 89 96 102 32.0 53.3 32.0 27.5 29 22 78.7 72.3 21.7 28.9 111 102 27.6 40.0 D 29 22 77.5 75.4 28.3 30.4 101 96 36.5 40.3 Female 29 22 29 22 77.9 63.0 87.7 77.9 28.9 32.7 32.5 32.1 96 80 82 99 37.1 51.9 37.1 41.2 29 22 79.6 68.4 30.5 28.7 99 97 38.3 42.0 77 TABLE 12 Analysis of Variance Based on Data of Table 11. Strain D Based on Developmental Temperature Sex F Level of Significance Male 7.20 0.005<p<0.01 Female 0.252 p>0.25 Based on Sex Developmental Temperature (°C) 29 22 F Level of Significance 4.42 13.7 0.25<p<0.05 p<0.0005 Male 8.33 0.025<p<0.005 Female 10.3 0.001<p<0.0025 29 22 13.6 6.84 p>0.0005 0.005<p<0.01 Male 20.3 p>0.0005 Female 4.13 0.025<p<0.05 29 22 36.5 19.1 0.05<p<0.10 p<0.0005 Male 3.37 0.05<p<0.10 Female 7.00 0.005<p<0.01 29 22 0.062 0.192 p»>0.025 p»0.25 78 FIGURE 21 Survival Curves of Strain D Adults Maintained at 22°C. Development Temperatures = 22°C { { Development Temperature = 29°C { a - Males b - Females 80 FIGURE 22 Survival Curves of Strain E Adults Maintained at 22°C. Development Temperatures = 22°C { { Development Temperature = 29°C { { a - Males b - Females % SURVIVING 00 82 FIGURE 23 Survival Curves of Strain F Adults Maintained at 22°C. Development Temperatures = 22°C { { Development Temperature = 29°C { { a - Males b - Females 84 FIGURE 24 Survival Curves of Strain G Adults Maintained at 22°C. Development Temperatures = 22°C { { Development Temperature = 29°C { { a - Males b - Females 86 Oreeon-R Adult Longevity The life span of the highly inbred wild-type stock, Oregon-R, was determined at 22°C and 29°C. Only 29°C was used as the preimaginal culture temperature because the adult life span of Oregon-R is not dependent on the developmental temperature within normal physiological limits (see Chapter One). The results are shown in Table 13. The most interesting aspect of the results was that no significant difference in mean life span existed between males and females at 22°C (F=1.650, 0.10<p<1.25), but at 29°C, the difference was highly significant (F= 104.2, p«<0.0005). The difference at 29°C may be due to very low variations in life span, especially in females. The survival curves for Oregon-R, shown in Figures 25 and 26, are highly rectangular. Death of almost all the flies occurred over a very short period of time at both 22°C and 29°C. This is particularly clear when the Oregon-R curves are compared to the wild strain curves. Although the maximum life span of Oregon-R was generally lower than that of the wild strains, the major decline in the population was considerably delayed in Oregon-R relative to the wild strains. The coefficients of variation for Oregon-R provide the best measure of the low variability of the Oregon-R results. At 22°C, the overall coefficient of variation is 21.8% and, at 29°C, it is 18.5%. These values are almost half the corresponding values for the wild strains. DISCUSSION The results of the present study indicate that D^  melanogaster strains recently isolated from the wild display more variation in life span than highly inbred strains when life span is monitored under laboratory conditions. The high degree of variability in the wild strains was apparent in flies maintained at either 22°C or 29°C. In addition, there is no consistent effect of developmental temperature on adult life span. This supports the findings in Chapter 87 TABLE 13 Adult Life Span of Oregon-R. Sex Adult Mean Standard Sample Coefficient Temperature Life Span Deviation Size of Variation (°C) (Days) (Days) (%) 29 38.5 7.8 188 20.3 Male 22 95.7 21.3 137 22.3 29 45.5 6.6 255 14.5 Female 22 92.7 19.8 175 21.4 FIGURE 25 Survival Curves of Oregon-R Adults Maintained at 29°C. a - Males b - Females 90 FIGURE 26 Survival Curves of Oregon-R Adults Maintained at 22°C. a - Males b - Females 92 One that adult longevity is not dependent on the duration of preimaginal growth. The large amount of variability in the adult longevity of the wild strains may reflect a highly heterogeneous genetic background. However, several of the 22°C survival curves show an early plateau followed by a gradual decline in the population. This plateau may be due to inbreeding over one year of laboratory culture. Studies of these strains following even longer periods of laboratory culture should then produce survival curves more similar to those of Oregon-R. Alternatively, the plateau may simply represent the amount of time needed to ensure the presence of subsequent generations prior to the onset of senescence. Under this situation, the survival curves should not change unless selection pressures in the lab cause changes in fertility, fecundity and the pattern of reproduction. The possibility that reduced genetic variation (as a result of inbreeding) could lead to alterations in the pattern of adult survival led Rose (1983a) to argue that the study of the evolution of senescence in Drosophila requires the use of strains that have a high degree of genetic variability. He contends that selection (as it applies to species survival) has occurred in a manner such that genes that act in a beneficial way early in adult life may later produce deleterious effects. Such a selection system can only be demonstrated using strains maintained in a way that their genetic variability remains high. Rose has termed this system "antagonistic pleiotropy." Previous studies supporting this theory (Rose and Charlesworth, 1981a and 1981b) have shown that adult life span is, in fact, reduced when selection for peak reproduction early in adult life is applied and that selection for a pattern of later peak reproduction is accompanied by an increase in life span. Such a system of aging may occur in the wild. Once reproduction is ensured, senescence can occur. If reproduction is delayed then senescence must also be delayed in order to ensure species survival. However, it is unlikely that such a system exists in highly inbred strains in the laboratory. Oregon-R reproduces in more than adequate numbers within the first two weeks of adult life at 22°C. Yet this study shows that no significant decline 93 in the population occurs until approximately 12 weeks at 22°C. Therefore, the difference in the pattern of death between Oregon-R and the wild strains may reflect the evolution of different systems of aging between the two environments rather than merely a difference in genetic variability. It is possible that, under conditions such as those that occur in the wild, death may be unrelated to age. Factors such as predation and nutritional fluctuations may be much more important in determining how long an organism lives. For example, Cannon (1966) has observed that when the highly inbred Oregon-R strain is placed under conditions designed to mimic those in the wild, it displays a more variable life span. It was also observed that strains carrying several mutations in an Oregon-R background have survival patterns similar to those of Oregon-R when they too are placed under wild conditions. Yet under laboratory conditions such as those described in this study, the life-shortening effects of even single mutations have been well documented (Lamb, 1978). It is not likely that the observations of Cannon (1966) can be explained simply by a rapid and sudden increase in genetic variation in the inbred stocks. Therefore, it is clear that, under laboratory conditions, a direct identification of the genetic factors involved in aging would be difficult when wild strains such as those used in the present study are examined. A genetic dissection of aging would be next to impossible to perform using strains with a high degree of variability in both their genetic backgrounds and their life spans. Even highly inbred strains such as Oregon-R display a certain amount of shifting in their survival curves each time the strain is tested. However, the relatively narrow range of age over which death occurs in Oregon-R makes life span a more precisely defined phenotype in this strain than it is in the wild strains. Thus, it appears that a genetic dissection of aging in Drosophila melanogaster is best performed using established wild-type strains with well-characterized genetic backgrounds. 94 CHAPTER THREE INTRODUCTION The efficiencies of cellular repair systems may determine, to a great extent, the rates at which physical and chemical lesions accumulate in cells. The accumulation of DNA damage could result in aberrant macromolecular synthesis, cellular dysfunction and, eventually, cell death. Therefore, the efficiency of DNA repair may play a role in determining when a cell dies. When cell death reaches a critical point in metazoans, tissue and organ death are likely to occur. These processes would ultimately lead to death of the organism. Therefore, a correlation may exist between the rate of accumulation of DNA damage, as it is determined by DNA repair, and species longevity (Hart et a_L, 1979; Hart and Modak, 1980). In fact, some researchers feel that DNA damage is the most basic and most direct cause of senescence (for an extensive review of this area, see Gensler and Bernstein, 1981). Most studies of the relationship between DNA repair and life span have been performed using mammalian cell culture. Hart and Setlow (1974) studied excision repair activity in primary fibroblast cultures established from seven different mammalian species. They measured the rate and the extent of 3H-thymidine incorporation into DNA following ultraviolet irradiation of cells. A linear relationship was found between the logarithm of species maximum life span and the amount of unscheduled DNA synthesis (i.e., repair synthesis). This relationship was not due to a higher level of ultraviolet-induced dimers in the DNA of longer-lived species because these workers also found the number of dimers per given length of DNA was the same in all cultures. Similar results were obtained by Francis et ah (1981) using fibroblast cultures established from 21 different mammalian species. Using an assay system involving the measurement of the molecular weight of photolysed DNA that had incorporated 5-bromodeoxyuridine during repair, they found a positive linear relationship between average species life span and the number of DNA excision repair sites measured 20-95 22 hours following ultraviolet treatment of the cultures. Hall et aK (1984) have shown a correlation between maximum species life span and ultraviolet-induced excision repair in both primary fibroblast and lymphocyte cell cultures established from several different primates. In humans, fibroblast cultures established from individuals suffering from progeria (a genetic disease with many symptoms of premature aging) have a decreased ability to repair single-stranded DNA breaks induced by gamma rays (Brown §i aL, 1978). However, the claim that progeria represents a disease caused by a DNA repair defect has not been substantiated (Brown and Wisniewski, 1983). An association between DNA repair and longevity has also been observed in non-mammalian organisms. For example, Smith-Sonneborn (1979) found that the clonal aging of Paramecium tetraurelia was dependent on the accumulation of DNA damage. Ultraviolet irradiation of cultures maintained in the dark reduced clonal life span. In addition, the dose required to reduce the mean clonal life span decreased as the fission age at the time of irradiation increased. Exposure to visible light prior to irradiation produced the same effects. However, when photorepair was allowed to occur following the ultraviolet treatment, clonal life span returned to or exceeded control values. Hence, it appears that although ultraviolet irradiation induced sufficient damage to cause premature death of cell lines, it also induced repair systems following ultraviolet treatment. It is possible that photorepair removed most of the damage caused by irradiation and the induced repair systems were then able to repair damage that had accumulated during normal senescence. Thus, the clonal life span was extended. Novobiocin, an antibiotic known to inhibit both regular DNA synthesis and DNA synthesis that occurs during repair, was also used to examine the effects of ultraviolet light on clonal life span (Smith-Sonneborn gj. aL, 1983 and 1984). Treatment with novobiocin alone did not reduce the fission number. However, the reduction normally caused by ultraviolet-irradiation was enhanced when combined with the drug. Photorepair again reversed the effects, although not to the extent that was observed following ultraviolet treatment alone. 96 Not all studies have supported the idea of the relationship between species longevity and DNA repair ability. For example, Kato §1 aL (1980) examined the amount of unscheduled DNA synthesis (measured as the incorporation of 3H-thymidine) that occurred in cell cultures following exposure to ultraviolet light. They found no correlation between maximum life span and the extent of this synthesis in cultures established from 34 species of mammals. However, these workers were not consistent in their choice of tissue source. Cell cultures were variously established from adult tissues such as lung, epidermis or kidney, or from embryonic fibroblasts. It is possible that different tissues can show different levels of repair even within the same organism (Hall £t aL, 1984). The study was also not consistent in the use of primary cultures. Woodhead et aL (1980) found no significant difference in the amount of excision repair ability (measured as the number of pyrimidine dimers sensitive to endonuclease treatment 24 hours after ultraviolet irradiation) between cell cultures derived from three cold-blooded vertebrates: the Amazon Molly (life span = three years); the rainbow trout (life span = eight years); the box turtle (life span as high as 118 years). Unfortunately, this study also was not consistent in terms of tissue source and use of primary cell cultures. Therefore, the results of both studies are questionable. The study of DNA repair as it relates to senescence has not been pursued in Drosophila. The reason for this is that most cells in adult Drosophila do not undergo mitosis, thus precluding the establishment of primary cell cultures from the adult. On the other hand, DNA repair during preimaginal development has been studied extensively in melanogaster. This is particularly true from a genetic perspective. About 50 mutagen-sensitive (mus) mutants have been isolated. Mus loci have been identified on the X-chromosome and on both of the major autosomes. In addition, the ability to culture embryonic cells from Drosophila has allowed biochemical characterization of many of these mutants. Several different mus mutants have been associated with deficiencies in one or more repair processes including excision repair, postreplication repair and photorepair, as well as DNA synthesis (Boyd §_1 aL, 97 1980 and 1983). The experiments reported here examine the effects of three mus mutations on adult longevity. The three mutations were isolated in a screen for second chromosome mus mutants by Henderson §t aL (in press). It is assumed that all three mutations represent lesions in some aspect of DNA repair because most mus mutants examined to date appear to be repair defective. The effects of ionizing radiation on the longevity of the mutants was also assessed. While more specific DNA damaging agents exist, ionizing radiation was used for the following reasons: (a) the dose administered can be easily controlled; (b) although chemical agents are more specific, previous experiments utilizing such agents have produced ambiguous results (Graf and Wurgler, 1979); (c) ultraviolet irradiation may cause death by damaging superficial structures such as the eyes (Atlan et aL, 1969b) rather than by damage to DNA specifically; (d) the evidence that does exist for the role of DNA repair in the aging process in adult insects comes from studies utilizing low doses of neutron radiation in Tribolium (Ducoff, 1976) and low doses of ionizing radiation in Drosophila (Baxter and Blair, 1969). MATERIALS AND METHODS Stocks and Culture Conditions One of the control strains of Drosophila melanogaster used in this study carries the mutations black (bj, purple (pr) and cinnabar (cn) on the second chromosome. The b_ pr cn chromosome is maintained over the multiply inverted second chromosome Curly of Oster (CvO). CvO carries the dominant mutation curly wings (Cv) and the recessive markers p_r and £n. Since C_y_ acts as a recessive lethal mutation, the balanced stock produces b_ p_r cn/b pr cn. homozygotes and b_ pr cn/CvO heterozygotes. Other strains that were studied carried one of three mutations that had been induced on 98 the b pr cn chromosome. These mutations, designated A - l . D-l32 and G-92. confer hypersensitivity to killing by the mutagen methyl methane sulfonate (MMS) when it is administered to embryos homozygous for any one of the three mutations. A - l and D-l32 are temperature-sensitive mutations with hypersensitivity occurring at 29°C, but not at 22°C. Tentative results indicate that A- l and D-l32 homozygotes are also hypersensitive to gamma radiation, but the temperature sensitivity here has not been clearly established. Homozygotes for G-92 die when cultured at 29°C during development even in the absence of MMS. The hypersensitivity to the mutagen is displayed at 22°C. Females homozygous for G-92 are completely sterile. All three mutations fall under the classification of mus mutations and may represent lesions in DNA repair systems or other systems of DNA metabolism. For simplicity, the b pr cn chromosomes carrying each of the three mus mutations will be abbreviated using only the mutation designation. For example, A - l b pr cn/A-1 b pr cn individuals will be designated A-l/A-1 while A- l b pr cn/CvO individuals will be designated A-l/CvO. This will also apply to D-l 32 and G-92. A wild type Drosophila strain, Canton-S, was also examined. Canton-S cannot be used directly as a control strain because it has a different genetic background than the b pr cn/CvO stock. However, it was of interest to know if the observed effects of gamma radiation on life span in the other strains were peculiar to their genetic backgrounds. All stocks were maintained under the same conditions as those described in Chapter One. 0 Krad vs 25 Krad Experiment In this experiment, the effects of a 25 krad dose of gamma radiation were examined in flies homozygous or heterozygous for the b pr cn, A- l and D-l32 chromosomes (G-92 was not subjected to this analysis). Virgin b pr cn/CvO. A -1 /CvO and D-I32/Cv0 females were mated to the corresponding heterozygous males and allowed to lay eggs in culture bottles. All 99 cultures were kept at 22°C during the entire developmental period. When the rate of eclosion began to increase rapidly, the bottles were cleared and newly eclosed adults were collected over periods of 12 hours or less. The sexes were separated immediately in order to ensure that all flies being studied were virgins. Flies were then further separated according to the scheme in Figure 27 and placed no more than 10 flies in a vial (2.5 cm diameter x 9.5 cm containing 6 ml of medium). Flies whose life spans were to be measured at 29°C were shifted to the higher temperature immediately. Flies were irradiated at about 24 hours post-eclosion. Any flies that died prior to the treatment were not counted as part of the original population. Samples were exposed in their culture vials to a 6 0Co source at a dose rate of approximately 1,000 rads/minute. Immediately following irradiation, flies were transferred to vials containing fresh food. No flies died during the irradiation period in any experiment. Untreated flies were also transferred to vials with fresh food one day post-eclosion. The duration of life span was determined using the procedures described in Chapter Two. 0 Krads vs 10 Krad Experiment A second experiment was carried out to examine the effects of a 10 krad dose of gamma radiation. Flies homozygous and heterozygous for G-92 were included in this experiment along with the strains used in the 25 krad study (i.e., b_ EI cn/b EI CD., E EI cn/C_y0, A-l /A-1. A-1/Cv0. D-132/D-I32 and D-132/CvQ). The collection of flies and their irradiation was performed in a manner similar to that of the previous experiment. However, the parents of flies that were examined were a mixture of homozygotes and heterozygotes, unlike the 25 krad experiment where all parents used were heterozygotes. In addition, separate groups of control i> EI cn/b EI £0. and b. EI cn/CvO flies were used in comparisons 100 FIGURE 27 Mating and separation scheme for experiment comparing the effects of 0 krads and 25 krads on the strains p_ p_I £n/i> PI cn, b pr cn/CvO. A-l /A-1. A-l/CvO. D-132/D-132 and P-132/Cv0. N.B. Following separation of males and females, and of homozygotes and heterozygotes, flies were divided into approximately equal sized samples for the two temperatures and, subsequently, for the irradiated and non-irradiated samples. b pr c n / C y O <f (or A - 1 / C v O c f or D - 1 3 2 / C v O c f ) X b pr c n / C v O 9 (or A J / C y O 9 o r D - 1 3 2 / C v O 9 ) c f b pr c n / b p i c n b pr c n / C v O b pr c n / b pr QD b pr c n / C y O 22°C 29°C 22°C 29°C A 1 Ni A 1 N l A 1 Nl A 1 N l 22°C 29°C 22°C 29°C I = Irradiated A A A A I N l I N l I N l I N l Nl = N o n - i r r a d i a t e d 102 with A - l and D-132 flies and with G-92 flies. Canton-S Wild-type Canton flies were collected and handled in a manner identical to the other experiments. Doses of 0, 10 and 25 krads were used for this strain as well. RESULTS Non-irradiated flies The life spans of adult Drosophila that were not irradiated were examined to determine the differences in longevity that occur naturally between the strains used in this study. Both sexes were studied at 22°C and 29°C, the permissive and restrictive temperatures respectively for the musts mutations A - l . D-132 and G-92. Homozygotes The 22°C survival curves of untreated homozygous males are shown in Figure 28. The curves have been separated according to the experiments with which they coincide. Figure 29 shows the 29°C survival curves. The figures show that b p_I cn/b pr cn control males lived longer than either A-l/A-1 or D-132/D-132 males at both 22°C and 29°C in the 25 krad experiment. However, the differences were much less in the 10 krad experiment. At 22°C, there is virtually no difference between the curves for the three strains. The mean life spans listed in Table 14 show that the mean life span of b pr cn/b pr cn at 22°C decreased by 10 days from the 24 krad to the 10 krad experiment. At 29°C, b pr cn/b pj_ cn males showed little change in mean life span, but A-l/A-1 males had an increase in the 10 krad experiment. D-132/D-132 males showed an increase in mean life span in the 10 krad experiment at 22°C, but no change at 29°C. 103 FIGURE 28 Survival Curves of Non-irradiated fe p_r c_n/fe pr cn, A-l /A-1. D-132/D-132. G-92/G-92 and Canton-S Males Maintained at 22°C. a 25 krad experiment fe pr cn/b pr cn A-l/A-1  D-132/D-132  b - 10 krad experiment fe pr cn/b pr cn* A-l/A-1  D-132/D-132  c 10 krad experiment fe EI cn/b EI cn* G-92/G-92 _ _ _ _ _ _ Canton-S *As mentioned in the Materials and Methods, separate groups of fe p_r cn/fe pjr _ flies were used in comparisons with A-l/A-1 and P-132/D-132 and with G-92/G-92. This applies to both sexes at both 22°C and 29°C, and to heterozygotes as well. 105 FIGURE 29 Survival Curves of Non-irradiated p_ pr cn/b. _ cn, A-l /A-1. D-132/D-132. G-92/G-92 and Canton-S Males Maintained at 29°C. a 25 krad experiment b pr cn/b p_r cn A-l/A-1  P-132/D-132  b - 10 krad experiment b pr cn/b pr cn • •— A-l/A-1  D-132/D-132  c - 10 krad experiment b_ pr cn/b pr cn G-92/G-92 Canton-S 107 TABLE 14 Mean Life Spans of Non-irradiated b_ p_r £n/fi pr c_n, A-l /A-1. D-132/D-132. G-92/G-92 and Canton-S Males from 25 krad and 10 krad Experiments Temperature Experiment Strain Standard Sample Mean Deviation Size Life Span of Mean % Difference from b pr cn/b p_r cn 25 krad b pr cn/b. PI cn A-l/A-1  D-132/D-132 63 99.4 60- 87.6 65 77.4 25.2 15.6 14.0 -11.9 -22.1 22°C E EI £n/fe EI SB 60 89.2 10 krad A-l/A-1 50 85.6 D-132/D-I32 49 85.2 22.6 14.8 13.5 4.0 4.5 b pr cn/b pr cn 48 94.3 10 krad G-92/G-92 60 51.4 15.8 17.8 -45.5 Canton-S 159 99.9 19.1 b- EI cn/E EI Sn 64 44.7 25 krad A-l/A-1 60 39.4 D-132/D-I32 62 40.0 8.9 3.8 4.7 •11.9 •10.5 29°C E EI cn/E EI cn 60 45.1 10 krad A-l/A-1 48 44.3 D-132/D-132 49 40.6 8.7 8.4 11.5 • 1.8 •10.0 E EI cn/b pr cn 50 39.0 10 krad G-92/G-92 60 26.4 9.4 8.3 -33.0 Canton-S 70 45.5 7.7 10S G-92/G-92 males were clearly shorter lived than their fe pr cn/b _ c_n counterparts at both temperatures. This is obvious from both the survival curves and mean life spans. On the other hand, Canton-S males have a life span very similar to that of fe pr c_rt/fe p_r _ males. Figures 30 and 31 show the survival curves at 22°C and 29°C, respectively, of non-irradiated homozygous females. Table 15 lists the corresponding mean life spans. At 29°C, b pr cn/b pr cn females were longer lived than either A-l/A-1 or D-132/D-132 females in both the 25 krad and 10 krad experiments. However, the situation at 22°C was not as clear. In the 25 krad experiment, fe pr cn/b pr c_n and A-l/A-1 had very similar life spans. In the 10 krad experiment, A-l/A-1 females showed a drastic reduction in mean life span and the corresponding survival curve in Figure 30b is indicative of a bimodal distribution for age at death within the A-l/A-1 sample. If the earliest deaths (i.e., prior to 20 days of age) are ignored for A-l/A-1 females at 22°C in the 10 krad experiment, then the survival curve is similar to the one obtained in the 25 krad experiment (see Figure 30a). fe pr cn/fe pr cn females that were used as controls for G-92/G-92 females also showed a bimodal survival curve at 22°C (see Figure 30c) though not to be the extent seen with A-1/A-l. Like their male siblings, G-92/G-92 females were much shorter lived than b pr cn/b pr cn females at both temperatures. Canton-S females had life spans very similar to those of b pr cn/fe pr c_n females as was the situation in males. Heterozygotes To determine whether or not any of the three mus,'8 mutations act in a dominant manner, the heterozygous strains fe pr cn/CvO. A-l/CvO. D-132/CvO and G-92/Cv0 were also examined. The survival curves of non-irradiated males are shown in Figures 32 (22°C) and 33 (29°C). The mean life spans of these samples are summarized in Table 16. It is apparent that no clear pattern exists with respect to the adult longevity of these males. For example, 109 FIGURE 30 Survival Curves of Non-irradiated p_ p_r £n/b_ pr cn, A-l /A-1. D-132/D-132. G-92/G-92 and Canton-S Females Maintained at 22°C. a 25 krad experiment b pr cn/b pr cn A-l/A-1  D-132/D-132  b - 10 krad experiment b pr cn/b pr cn Azl/A-X D-132/D-132  c 10 krad experiment b_ pr cn/b pr cn G-92/G-92 Canton-S 1 0 0 A G E (DAYS) AT 22°C I l l FIGURE 31 Survival Curves of Non-irradiated b_ pjr cn/b_ pr £H, A-l /A-1. D-132/D-132. G-92/G-92 and Canton-S Females and Maintained at 29°C. a 25 krad experiment E pr cn/p pr en • A-l/A-1  D-132/D-132  b 10 krad experiment b_ pr cn/b pr gn — A-l/A-1  D-132/D-132  c - 10 krad experiment b_ EI cn/b EI cn G-92/G-92 Canton-S 1 0 0 o z > > QZ CO 112 10 T 2 0 T 3 0 r 4 0 5 0 A G E (DAYS) AT 2 9 ° C 0 10 2 0 3 0 4 0 6 0 6 0 7 0 A G E (DAYS) AT 2 9 ° C 113 TABLE 15 Mean Life Spans of Non-irradiated b pr cn/b pr cn, A-l /A-1. D-132/D-132. G-92/G-92 and Canton-S Females from 25 krad and 10 krad Experiments Temperature Experiment Strain Standard Sample Mean Deviation Size Life Span of Mean % Difference from b pr cn/b pr cn b pr cn/b pr cn 60 91.8 25 krad A-l/A-1 60 88.1 D-132/D-132 69 82.0 18.8 16.5 14.4 - 4.0 - 1 0 . 7 22°C b pr cn/b pr cn 60 83.2 10 krad A-l/A-1 50 52.8 D-132/D-132 44 74.2 25.9 40.4 23.0 -36.5 -10.8 b pr cn/b pr cn 50 85.9 10 krad G-92/G-92 60 36.4 20.1 14.5 -57.6 Canton-S 142 101.6 27.2 b pr cn/b pr cn 59 49.9 25 krad A-l/A-1 60 40.5 D-132/D-132 62 38.9 8.4 3.9 8.2 - 18 .8 -22 .0 29°C b pr cn/b pr cn 59 48.3 10 krad A-l/A-1 50 36.5 D-132/D-132 45 36.2 12.2 14.6 15.4 -24.4 -25.1 b pr cn/b pr cn 49 36.0 10 krad G-92/G-92 60 19.1 18.7 6.2 - 46 .9 Canton-S 60 46.6 6.9 114 FIGURE 32 Survival Curves of Non-irradiated b pr cn/CvO. A-l/CvO. D-132/CvO and G-92/Cv0 Males Maintained at 22°C. a - 25 krad experiment b pr cn/CvO  A-l/CvO D-132/CvO  b - 10 krad experiment b pr cn/CvO  A_/C_vO D-132/CvO  c 10 krad experiment b pr cn/CvO  G-92/CvO 1 0 0 AGE (DAYS) AT 22°C 1 0 0 116 FIGURE 33 Survival Curves of Non-irradiated fe pi cn/CvO. A-l/CvO. D-132/CvO and G-92/CvO Males Maintained at 29°C. a - 25 krad experiment b pr cn/CvO A-l/CvO D-132/CvO b 10 krad experiment fe pr cn/CvO A-l/CvO D-132/CvO c - 10 krad experiment fe pr cn/CvO  G-92/CvO TABLE 16 118 Mean Life Spans of Non-irradiated p pr cn/CvO. A-l/CvO. D-132/CvO and G-92/CvO Males from 25 krad and 10 krad Experiments Temperature Experiment Strain Standard Sample Mean Deviation Size Life Span of Mean % Difference from b pr cn/CvO b pr cn/CvO 60, 108.9 25 krad A-l/CvO 59 106.3 D-132/CvO 58 88.7 17.2 22.1 16.6 - 2.4 -18.5 22°C 10 krad p pr cn/CvO  A-l/CvO  D-132/CvO 59 59 60 99.5 89.1 102.6 16.6 18.1 15.3 -10.5 + 3.1 10 krad b pr cn/CvO  G-92/CvO 49 59 113.6 105.9 11.6 20.8 6.8 b_ pr cn/£vO 59 50.2 25 krad A-l/CvO 55 46.9 D-132/CvO 58 42.9 3.3 11.3 8.7 - 6.6 -14.5 29°C b pr cn/CvO 60 50.9 10 krad A-l/CvO 60 55.1 D-132/CvO 59 47.8 6.3 4.2 5.7 + 8.3 - 6.1 b_ pr cn/CvO 10 krad G-92/CvO 50 59 51.8 51.9 3.4 6.8 + 0.2 119 fe pr cn/CvO and A-l/CvQ males had very similar life spans at 22°C in the 25 krad experiment whereas P-132/CvO males were clearly shorter lived. However, fe pr cn/CvO and D-132/CvO males had similar life spans at 22°C in the 10 krad experiment and A- l /CvO was the shorter lived strain. At 29°C, D-132/CvO males were the shortest lived in both experiments. In the experiment where the life spans of fe pr cn/CvO and G-92/CvO males were compared, little difference exists between these two strains. This was especially true at 29°C where it can be seen that the mean life spans are almost identical and the survival curves are nearly coincident (see Figure 33c). The results obtained with heterozygous females that were not irradiated are more consistent than the results seen with their male counterparts. Figures 34 and 35 show that fe pr cn/CvO females were the longest lived in all comparisons. This is particularly clear at 22°C. Furthermore, Table 17 shows that A-l/CvO. D-132/CvO and G-92/CvO females had mean life spans that were at least 12% lower than those of fe pr cn/CvO females at 22°C and at least 9% lower at 29°C. Finally, it is important to note that A-l/CvO females did not display a bimodal survival curve at 22°C in the 10 krad experiment, unlike their A-l/A-1 sibling females. Thus, it appears that none of the mus'* mutations act in a dominant manner when adult longevity is examined. In addition, A-l/A-1 and D-132/D-132 flies did have shorter life spans than fe pr c_n/b pr cn flies, but the difference is often only marginal. Only strain G-92/G-92 had a clearly reduced adult life span in both sexes and at both 22°C and 29°C. Comparison of Flies Exposed to 25 Krads To assess whether or not DNA repair occurs in adult Drosophila and, if it does, how it affects adult longevity, flies were exposed to 25 krads of gamma radiation at one day of adult age. If repair in the adult does influence life span, then it is possible that the homozygous mus'1 strains may be more sensitive to the radiation treatment than fe pr cn/b pr 120 FIGURE 34 Survival Curves of Non-irradiated b_ pr cn/CvO. A-l/CvO. D-132/CvO and G-92/CvO Females Maintained at 22°C. a 25 krad experiment p pr cn/CvO  A-l/CvO  D-132/CvO  b 10 krad experiment b pr cn/CvO  A-l/CvO  D-132/CvO  c - 10 krad experiment b pr cn/CvO  G-92/CvO 122 FIGURE 35 Survival Curves of Non-irradiated b_ pr cn/CyO. A-l/CvO. D-132/CvO and G-92/CvO Females Maintained at 29°C. a - 25 krad experiment b pr cn/CvO _ A-l/CvO  D-132/CvO  b 10 krad experiment b pr cn/CvO  A-l/CvO  D-132/CvO  c - 10 krad experiment p pr cn/CvO  G-92/CvO 124 TABLE 17 Mean Life Spans of Non-irradiated b pr cn/CvO. A-l/CvO. D-132/CvO and G-92/CvO Females from 25 krad and 10 krad Experiments Standard Sample Mean Deviation % Difference from Temperature Experiment Strain Size Life Span of Mean b pr cn/CvO b pr cn/CvO 59 118.1 16.3 25 krad A-l/CvO 59 103.5 8.7 -12.4 D-132/CvO 57 99.2 15.4 -16.0 b pr cn/CvO 60 105.9 14.1 10 krad A-1/CvO 60 81.0 20.8 -23.5 22°C D-132/CvO 59 81.2 23.3 -23.3 b pr cn/CvO 49 111.4 23.9 10 krad G-92/CvO 60 93.7 16.9 -15.9 b pr cn/CvO 59 53.2 2.2 25 krad A-l/CvO 60 47.0 8.2 -11.7 D-132/CvO 58 46.5 4.0 -12.6 b pr cn/CvO 59 52.5 10.1 10 krad A-1/CvO 59 44.8 15.5 -14.7 29°C D-132/CvO 58 47.6 11.4 - 9.3 b pr cn/CvO 50 54.1 7.4 10 krad G-92/CvO 59 48.3 10.7 -10.7 125 cn flies or heterozygous flies. Homozygotes The survival curves of b pr cn/b pr cn, A-l/A-1. D-132/P-132 and Canton-S males exposed to 25 krads are shown in Figure 36. The corresponding mean life spans are summarized in Table 18. It is clear from both the survival curves and the mean life span data that the 25 krad treatment reduces adult longevity in all strains. D-132/D-132 males showed the smallest reduction with a 14.5% decrease in mean life span at 22°C and a 24% decrease at 29°C relative to non-irradiated D-132/D-132 males (See Table 14). The other strains showed reductions of 28% to 29% at 22°C and 31% to 36% at 29°C. The radiation treatment did not cause any significant increase in the difference between the life span of p pr cn/b pr cn males and either A-l/A-1 or D-132/D-132 males. In fact, the radiation treatment usually caused a slight decrease in this difference. Therefore, neither A-l/A-1 nor D-132/D-132 males are more sensitive to radiation than b pr cn/b pr cn males. The survival curves of homozygous females exposed to 25 krads (shown in Figure 37) reveal that females were much more sensitive to the treatment than males. This is particularly clear in p pr cn/b pr cn. Table 19 shows that these females had a 60% reduction in mean life span at 22°C following irradiation, double the reduction in males. At 29°C, the reduction was 47.1% in p pr cn/b pr cn females. The other strains also showed a larger reduction in the mean life spans of females than males as a result of irradiation. The only exception to this occurred in Canton-S flies at 29°C (36.3% reduction for males compared to 32.8% reduction for females). As was the case with males, neither A-l/A-1 nor P-132/D-132 females appeared to be more sensitive to the radiation treatment than b_ pr cn/b pr cn females. At 22°C, the irradiated musu females actually had higher mean life spans than their b_ pr cn/b pr cn counterparts. 126 FIGURE 36 Survival Curves of b EI cn/b pjr cn, A-l /A-1. D-132/D-132 and Canton-S Males Exposed to 25 krads of Gamma Radiation. b pr cn/b pr cn AM/AjJ. D-132/D-132  Canton-S a - 22°C b - 29°C 128 TABLE 18 Mean Life Spans of p px cn/p pr £n, A-l/A-1. D-132/D-132. and Canton-S Males Exposed to 25 krad of Gamma Radiation. % Difference Standard % Difference From Non-Sample Mean Deviation From irradiated Temperature Strain Size Life Span of Mean t_ pr cn/p pr cn Counterparts p pr cn/b pr cn 65 70.4 17.5 -29.2 A-l/A-1 60 63.0 14.9 -10.6 -28.1 22°C D-132/D-132 67 66.2 8.2 - 6.0 -14.5 Canton-S 69 71.5 7.4 -28.4 p pr cn/b pr cn 64 30.8 6.5 -31.1 A-l/A-1 59 26.4 2.7 -14.3 -33.0 29°C D-132/D-132 70 30.4 5.9 - 1.3 -24.0 Canton-S 69 29.0 4.2 -36.3 129 FIGURE 37 Survival Curves of b pr cn/b. pr cn, A-l /A-1. P-132/D-132 and Canton-S Females Exposed to 25 krads of Gamma Radiation. b pr cn/b pr cn A-l/A-1  D-132/D-132  Canton-S a - 22°C b - 29°C T A B L E 19 131 Mean Life Spans of p pr c_n/b pr cn, A-l/A-1. D-132/D-132. and Canton-S Females Exposed to 25 krad of Gamma Radiation. % Difference Standard % Difference From Non-Sample Mean Deviation From irradiated Temperature Strain Size Life Span of Mean p pr cn/b pr cn Counterparts b pr cn/b pr cn 60 36.7 12.8 -60.0 A-l /A-I 60 41.4 16.1 +12.8 -53.0 22°C D-132/D-132 64 45.0 14.9 +22.8 -45.1 Canton-S 60 59.8 22.9 -41.1 b pr cn/b pr cn 60 26.4 10.4 -47.1 A-l/A-1 60 24.8 7.0 - 6.2 -38.8 29°C D-132/D-132 64 24.4 8.2 - 7.6 -37.3 Canton-S 60 31.3 10.1 -32.8 132 Heterozygotes Heterozygous flies were also exposed to radiation to examine their response to the treatment. The survival curves for b pr cn/CvO. A-l/CvO and D-132/CvO males are shown in Figure 38. Mean life spans are listed in Table 20. Very little difference exists between the three strains at both temperatures. However, it is clear that the 25 krad treatment does reduce life span. This varies between 15.6% (D-132/CvO at 22°C) and 39.4% (b pr cn/CvO at 29°C). The survival curves for heterozygous females are shown in Figure 39 and mean life span data is shown in Table 21. Once again, females were more sensitive to the radiation treatment than males. In addition, the reductions in mean life span for heterozygous females were greater than those seen in homozygous females. Comparisons between the strains reveals that irradiated b pr cn/CvO females were much shorter lived than irradiated D- 132/CvO females at 22°C. However, other strain differences were between 8% and 10%. It is clear that the 25 krad treatment causes a large reduction in the adult longevity of all strains at both temperatures. Females are more sensitive than males at this radiation level. However, the mus'8 strains are not more sensitive to radiation (as measured by adult survival) than the repair-proficient strains. Comparison of Flies Exposed to 10 Krads Due to the very large reduction in adult longevity caused by 25 krads of gamma radiation, a lower dose of 10 krads was also used. It was possible that a lower dose may Cause no reduction in the life span of repair-proficient strains, but produce some effect in musts strains. 133 FIGURE 38 Survival Curves of fi EI cn/£yj), A-l/CvO and D-132/CyO Males Exposed to 25 krads of Gamma Radiation. b pr cn/CyO A-l/CvO  D-132/CvO  a - 22°C b - 29°C 135 T A B L E 20 Mean Life Spans of p pr cn/C__, A-1/CvO and D-132/CvO Males Exposed to 25 krad of Gamma Radiation. % Difference Standard % Difference From Non-Sample Mean Deviation From irradiated Temperature Strain Size Life Span of Mean p pr cn/CvO Counterparts p pr cn/CvO 60 74.9 11.9 -31.2 A-l/CvO 60 72.5 14.2 - 3.2 -31.8 22°C D-132/CvO 59 74.6 9.6 - 0.4 -15.6 b pi cn/Cvo 60 30.4 3.2 -39.4 A-l/CvO 60 30.0 4.8 - 1.3 -36.0 29°C D-I32/Cv0 60 32.1 4.6 + 5.6 -25.2 136 FIGURE 39 Survival Curves of b pr cn/CvO. A-l/CvO and D-132/CvO Females Exposed to 25 krads of Gamma Radiation. b pr cn/CvO — A-l/CvO  D-132/CvO  a - 22°C b - 29°C 138 TABLE 21 Mean Life Spans of p pr gn/CyQ, A-l/CvO and D-132/CvO Females Exposed to 25 krad of Gamma Radiation. Temperature Strain Standard Sample Mean Deviation Size Life Span of Mean % Difference % Difference From Non-From irradiated b pr cn/CvO Counterparts b_ pr £n/CyO 59 30.8 11.3 -73.9 A-l/CvO 59 33.9 10.6 +10.0 -67.2 22°C D-132/CvO 60 39.6 10.3 +28.6 -60.1 29°C P pr cn/Cvo A-l/CvO  D-132/CvO 59 26.0 8.8 -51.1 60 23.9 7.5 - 8.2 -49.1 59 28.6 8.0 +10.0 -38.5 139 Homozygotes The survival curves of pr cn/fi p_r c_n, A-l/A-1. D-132/D-132. G-92/G-92. and Canton-S males exposed to 10 krads of radiation are shown in Figures 40 (22°C) and 41 (29°C). The curves for b pr cn/b pr cn and D-132/D-132 are very similar at both 22°C and 29°C. In addition, their mean life spans differ by less than 5% (see Table 22). The radiation treatment appears to have reduced life span very little for these two strains and for Canton-S. On the other hand, A-l/A-1 males were shorter lived than either b pr cn/b pr cn or D-132/D-132. At 29°C, A-l/A-1 males had a mean life span 22% lower than that of irradiated b pr cn/b pr cn males and 32.1% lower than that of non-irradiated A-l/A-1 males. Irradiated G-92/G-92 males had much shorter life spans than other irradiated males at both temperatures, but showed virtually no reduction in life span as a result of the radiation treatment. Thus, this level of radiation appears to caus e a significant reduction in male adult longevity only in strain A-l/A-1 and this reduction is very clear only at 29°C. Figures 42 (22°C) and 43 (29°C) show the survival curves for b pr cn/b Pi cn, A-l/A-1. D-132/D-132. G-92/G-92. and Canton-S females exposed to 10 krads of radiation. Mean life spans are listed in Table 23. No large reduction in female longevity occurred in b pr cn/b pr cn, D-132/D-132. G-92/G-92 and Canton-S flies as a result of the radiation treatment. However, A-l/A-1 females appeared to be very sensitive at 29°C, similar to their A-l/A-1 male siblings. It is important to point out that the unusual shape of the survival curves for these females makes it difficult to assess if, in fact, the reduction in mean life span at 29°C was a result of the radiation treatment. It is possible that the large amount of variation in age at death, even in non-irradiated A-l/A-1 females in the 10 krad experiment could produce what appears to be a reduction in life span due to irradiation. 140 FIGURE 40 Survival Curves of b pr cn/b. pr cn, A-l /A-1. D-132/P-132. G-92/G-92 and Canton-S Males Exposed to 10 krads of Gamma Radiation and Maintained at 22°C. a - b_ pr cn/b EI cn A-l/A-1  D-132/D-132 ----b - b pr cn/b pr cn G-92/G-92 _ _ _ _ _ _ Canton-S 142 FIGURE 41 Survival Curves of b_ pr cn/b. pr cj\, A-l /A-1. D-132/D-I32. G-92/G-92 and Canton-S males Exposed to 10 krads of Gamma Radiation and Maintained at 29°C. a - p pr cn/b pr cn A _ 7 A _ D-132/D-132 b b pr cn/b pr cn G-92/G-92 Canton-S % SURVIVING % SURVIV ING 144 TABLE 22 Mean Life Spans of p pr cn/b pr cn, A-l /A-1. D-132/D-132. G-92/G-92 and Canton-S Males Exposed to 10 krad of Gamma Radiation. % Difference Standard % Difference From Non-Sample Mean Deviation From irradiated Temperature Strain Size Life Span of Mean fi pr cn/b pr cn Counterparts b_ pr cn/b pr cn 58 81.7 , 18.4 - 8.4 A-l/A-1 58 70.3 22.6 -14.0 -17.9 22°C D-132/D-132 60 78.0 15.7 - 4.5 - 8.5 b_ pr cn / b pr c n 50 91.3 20.3 - 3.2 G-92/G-92 60 51.3 14.9 -43.8 - 0.2 Canton-S 69 97.1 10.4 - 2.8 b pr cn / b pr cn 60 . 38.6 10.7 -14.4 A-l/A-1 58 30.1 11.6 -22.0 -32.1 29°C D-132/D-I32 60 38.3 7.5 - 0.8 - 5.7 b pr cn/p pr c_n 50 41.2 8.4 + 5.6 G-92/G-92 60 26.4 7.9 -43.8 0 Canton-S 70 40.8 6.5 -12.2 145 FIGURE 42 Survival Curves of b_ pr cn/b pr cn, A-l /A-1. P-132/D-132. G-92/G-92 and Canton-S Females Exposed to 10 krads of Gamma Radiation and Maintained at 22°C. a - b pr cn/b pr cn A-l/A-1  D-132/D-132  b b pr cn/b pr cn G-92/G-92 Canton-S 147 FIGURE 43 Survival Curves of b pr cn/b pr cn, A-l /A-1. D-132/P-132. G-92/G-92 and Canton-S Females Exposed to 10 krads of Gamma Radiation and Maintained at 29°C. a - b pr cn/b pr cn — A-l/A-1  D-132/P-132  b b pr cn/b pr cn G-92/G-92 Canton-S 149 TABLE 23 Mean Life Spans of p pr c_n/p pr cn, A - l / A - L D-132/D-132. G-92/G-92 and Canton-S Females Exposed to 10 krad of Gamma Radiation. % Difference Standard % Difference From Non-Sample Mean Deviation From irradiated Temperature Strain Size Life Span of Mean b pr cn/b pr cn Counterparts fi Pi cn/b, pr c_n 60 78.6 24.4 - 5.5 A-l/A-1 59 54.8 34.3 -30.3 + 3.8 D-132/D-132 60 76.7 • 12.3 - 2.4 + 3.4 b pr cn/b pr cn 47 80.0 25.1 - 6.9 G-92/G-92 60 34.3 15.5 -57.1 - 5.8 Canton-S 69 103.2 23.4 + 1.6 b pr cn/b pr cn 60 43.7 12.5 - 9.5 A-l/A-1 60 23.9 14.3 -45.3 -34.5 29°C D-132/D-132 60 38.7 8.7 -11.4 + 6.9 b pi cn/b pr £n 50 42.4 14.3 +17.8 G-92/G-92 60 17.6 5.4 -58.5 - 7.9 Canton-S 70 47.0 7.4 - — + 0.9 Heterozygotes Figures 44 and 45 show the 22°C and 29°C survival curves, respectively, of p pr cn/CvO. A - l /CvO. D-132/CvO and G-92/CvO males that were exposed to 10 krads of radiation. Little difference existed between the strains. However, the values for mean life span listed in Table 24 indicate that somewhat large reductions in life span occurred in b pr cn/CvO males that were examined concommitantly with G-92/CvO males as a result of the radiation treatment. In addition, large reductions in life span occurred at 29°C in both G-92/CvO and A- l /CvO males. This was unusual because the homozygous sibling males of G-92/CvO males showed virtually no change in life span following irradiation at the same level. The survival curves for heterozygous females exposed to 10 krads of radiation are shown in Figures 46 (22°C) and 47 (29°C). The corresponding mean life spans are listed in Table 25. The results are somewhat different than the results for males. For example, b pr cn/CvO females showed no major reduction in adult life span at either 22°C or 29°C following irradiation. In addition, A-l/CvO females were much shorter lived than both b pr cn/CvO and D-132/CvO females following irradiation, unlike their heterozygous male siblings. Finally, G-92/CvO females were much shorter lived than b pr cn/CvO females at 29°C, opposite to the situation in males. In summary, the 10 krad treatment did not reduce the adult life span of homozygotes except for A- l /A-I males and females at 29°C. As mentioned previously, there is difficulty in assessing whether or not the reduction is due to irradiation because of the unusual bimodal survival curves. In heterozygotes, the situation is difficult to interpret because of the unexpected reductions in life span seen particularly in b pr cn/CvO. A- l /CvO and 92/CvO. 151 FIGURE 44 Survival Curves of b_ p_r cn/CvO. A-l/CvO. D-132/CvO and G-92/CvO Males Exposed to 10 krads of Gamma Radiation and Maintained at 22°C. a - b pr cn/CvO  A-l/CvO D-132/CvO  b - b pr cn/CvO  % SURVIVING % SURVIV ING 153 FIGURE 45 Survival Curves of_ pr cn/CvO. A-l/CvO. D-I32/Cv0 and G-92/CvO Males exposed to 10 krads of Gamma Radiation and Maintained at 29°C. a - b pr cn/CvO A-l/CvO  D-132/CvO b b pr cn/CvO  G-92/CvO 1 0 0 o z > > DC CO 154 ~r 10 2 0 3 0 4 0 5 0 A G E (DAYS) AT 29°C o z > > oc CO 1 0 0 80H 60H 40H 2 0 H A G E (DAYS) AT 29°C 155 TABLE 24 Mean Life Spans of p pr cji/r_0, A-I/CvO. D-132/CvO and G-92/CvO Males Exposed to 10 krad of Gamma Radiation. Temperature Strain Standard Sample Mean Deviation Size Life Span of Mean % Difference From p pr cn/CyO % Difference From Non-irradiated Counterparts fi pr cn/CvO 59 92.8 . 6.8 - 6.7 A-l/CvO 60 85.5 19.8 - 7.9 - 4.0 22°C D-132/CvQ 60 91.1 19.3 - 1.8 -11.2 bprcji/CxO 50 93.4 8.8 -17.8 G-92/CvO 59 99.2 14.2 + 6.2 - 6.3 fipra_/£y0 60 44.9 3.3 -11.8 A-1/Cv0 60 43.1 8.0 - 4.0 -21.8 29°C D-132/CvQ 59 43.1 3.5 - 4.0 - 9.8 b pr cn/CvO 50 40.9 7.0 -21.0 G-92/CvO 60 42.9 4.5 + 4.9 -17.3 156 FIGURE 46 Survival Curves of b_ pr cn/CvO. A- l /CvO. D-132/CvO and G-92/CvO Females exposed to 10 krads of Gamma Radiation and Maintained at 22°C. a - p pr cn/CvO A-l/CvO  D-132/CvO  b p Pi cn/CvO  G-92/CvO 158 FIGURE 47 Survival Curves of p pr cn/CvO. A-l/CvO. D-132/CyO and G-92/CvO Females exposed to 10 krads of Gamma Radiation and Maintained at 29°C. a - b pr cn/CvO  A-l/CvO D-132/CvO b b pr cn/CvO  G-92/CvO 0 10 20 30 40 50 60 70 A G E (DAYS) AT 29°C 160 TABLE 25 Mean Life Spans of p pr cn/CvO. A-l/CvO. D-132/CvO and G-92/CvO Females Exposed to 10 krads of Gamma Radiation. % Difference Temperature 22°C Standard % Difference From Non-Sample Mean Deviation From irradiated s Strain Size Life Span of Mean b pr cn/CyO Counterparts b pr cn/CvO 60 99.8 20.6 - 5.8 A-l/CvO 60 66.1 28.1 -33.8 -18.4 D-132/Cv0 60 95.8 14.1 - 4.0 +18.0 b pr cn/CvO 49 104.4 15.6 - 6.3 G-92/CvO 59 93.8 19.5 -10.2 + 0.1 b pr cn/CvO 60 53.3 3.6 + 1.5 A-l/CvO 60 38.0 12.7 -28.7 -15.2 29°C D-132/CvO 60 45.6 9.0 -14.4 - 4.2 b pr cn/CvO 50 51.7 7.4 - 4.4 G-92/CvO 60 35.8 7.1 -30.8 -25.9 161 DISCUSSION The results of this study show that non-irradiated adults homozygous for any one of the three mus1' mutations examined here were shorter lived than p pr _/b_ pr en adults. In no instance did the mean life spans of A-l/A-1. D-132/D-132 or G-92/G-92 adults exceed that of the p pr c_r_/p pr c_n adults used in the same experiment. However, the difference between non-irradiated homozygous mutants and non-irradiated controls varied considerably. Reductions in mean life spans of the mutant strains ranged from 1.8% (A-l/A-1 males at 29°C in 10 krad experiment, Table 14) to 57.6% (G-92/G-92 females at 22°C, Table 15), relative to the mean life spans of the p pr cn/b pr cn control strain. An unexpected finding was the generally reduced longevity of untreated A-l /CvO. D-132/CvO and G-92/CvO flies, relative to p pr cn/CvO flies. In only 3 out of 20 comparisons did non-irradiated individuals heterozygous for one of the three mutations have mean life spans equal to or greater than that of the corresponding group of non-irradiated b pr cn/CvO heterozygotes. Such a finding is difficult to explain. Preliminary characterizations of the mutagen sensitivity conferred by these mutations have not indicated that any one is a dominant or a semi-dominant mutation (D. Henderson, personal communication). However, direct tests for dominance have not been performed. The 25 krad dose of gamma radiation administered to flies homozygous or heterozygous for the i> pr cn, A- l or D-132 chromosome clearly had a life shortening effect. The average reduction in mean life span (relative to non-irradiated flies of the same genotype) was 28% for males and 52% for females. A similar large reduction in adult longevity also occurred in Canton-S flies, although the difference between the response in males and the response in females was lower in the wild-type. The results of the experiment in which flies were exposed to a lower radiation dose of 162 10 krads are more difficult to interpret than those of the first experiment. All males showed a reduction in mean life span when irradiated. This reduction ranged from 4% to 32%. A-1/A-l males were the most sensitive at both temperatures (i.e. they showed the largest percent difference between non-irradiated and 10 krad-irradiated males). Female response to radiation varied from an 18% increase (D-132/CvO females at 22°C) to a 35% decrease (A-l/A-i females at 29°C) in mean life span. It is of interest to note that irradiated D-132/CvO females exhibited a 4% decrease in mean life span at 29°C whereas irradiated A-l/A-1 females at 22°C showed a 4% increase. Unlike the 25 krad treatment, the response of males and the response of females to the 10 krad treatment did not differ significantly. This was also the case with G-92/G-92 flies where neither sex showed a significant reduction of the mean life span when exposed to 10 krads of gamma radiation. In light of the difficulty in interpreting the results of the 10 krad experiment, it is important to point out that survival curves obtained from several A-l/A-1 and A- l /CvO samples in this experiment are unusual in that they deviate from the standard rectangular-shaped curves. The appearance of bimodal survival curves for these strains might be explained by the manner in which the experiments were performed. Only heterozygous parents were used in the 25 krad experiment whereas both homozygous and heterozygous parents were used in the 10 krad experiment. Thus, it is possible that A-l/A-1 females could exert a maternal effect on their progeny. In order to affect the offspring as adults, the maternal effect would have to act on cells whose fate is determined early in development and in structures that are maintained throughout development into the adult stage. One example of such a tissue is the central nervous system in Drosophila. To test whether or not a maternal effect an adult longevity does exist, progeny of the same genotype from both homozygous and heterozygous mothers would have to be compared. The differential response between males and females to the 25 krad dose of radiation is similar to that observed by Giess and Planel (1977) after irradiating four-day-old Oregon-R 163 flies at doses of 25, 50 or 75 krads. Females lived longer than males in non-irradiated cultures whereas males were the longer lived sex at each level of radiation. An interesting finding in their work was that the radiation-induced life span shortening in females did not vary with the dose. Males, on the other hand, showed a decreasing mean life span with the increase in dose. Giess (1980) extended this investigation by using additional doses of 15 and 20 krads and by comparing flies irradiated on the first, fourth or eighth day of adult life. Older females showed a smaller reduction in mean life span than younger females, but only after doses of 20 or 25 krads. Males were still less radio-sensitive than females, but the pattern of radio-sensitivity in males did not change with age. Giess posits that the radiation-induced response depends on the physiological maturity of adult Drosophila. That is, the age-related change seen in females with regard to their response to radiation is due to physiological maturation. He further contends that the maturation occurs more rapidly in males and may bring with it more efficient repair. Thus, the reduction in life span following irradiation may not represent an acceleration of the processes involved in natural senescence (Atlan e_t ah, 1979a; Miquel §i ah, 1972), but the radiation responses may expose some of the changes that occur during normal aging. In contrast to the findings of Giess and co-workers, results of other studies indicate that males are more sensitive to the life-shortening effects of ionizing radiation. For example, Gartner (1973a) compared non-irradiated Canton-S flies to those exposed to 33, 66 and 93 krads of gamma radiation. At each dose, males showed a proportionally larger reduction in their mean life span than females. However, several differences exist between Gartner's procedures and the procedures of Giess and Planel (1977) and Giess (1980). Different wild-type strains were used and different culture temperatures were also employed. It is possible that the response to radiation may be strain specific and, to some extent, temperature-sensitive. Both possibilities are supported by the present findings. The studies also differed in dose-rate at which radiation was administered. Gartner used a ^ Co source delivering 164 9,000 rads/minute whereas the source used by Giess and Planel delivered 1,000 rads/minute. It has been suggested by Lamb (1978) that flies may exhibit differential responses to different dose rates, even if the amount of total radiation is the same. The results of the present study are in better agreement with the work of Giess and Planel, at least in the comparison of non-irradiated and 25 krad-irradiated flies. The use of mus mutants such as those involved in this study may be useful for testing Giess' speculation. It is probable that if repair ability in somatic tissue increases with physiological maturation, repair ability may also increase in gonadal tissue. Graf and Wurgler (1978) have found that there is a possible maternal effect for DNA repair. Embryos derived from females homozygous for a mus mutation may have lower repair efficiency than embryos derived from females carrying the wild-type allele for the mus locus. Therefore, it is possible that if repair efficiency increases with physiological maturation, the probability that homozygous mus embryos would survive mutagen treatment should increase with the age of repair-proficient mothers. Such a possibility could be tested using the somatic repair assay developed by Henderson et aL (in press). Studies on the life-shortening effects of ionizing radiation in Drosophila have not determined if the reduction in life span is due solely to DNA damage. For example, ionizing radiation causes an acceleration of the cytological changes seen in normal aging (e.g. lipo-pigment accumulation, mitochondrial enlargement) but also produces some uncharacteristic changes especially at doses above 40 krads (Gartner, 1973b; Miquel et _ , 1972). This is especially important to consider in light of the results from the present study. These results suggest that it may be difficult to assess the causes of death in experiments where doses of greater than 25 krads are used because of the potential for widespread damage. Damage to other cellular components such as the cell membrane and mitochondria could also contribute to cell deaih. Irraaiation of Drosophila at low doses (i.e. less than 20 krads) has, in general, not been 165 observed to reduce adult longevity (Gartner, 1984). Some studies have actually reported increases in life span, especially at doses of 10 krads or less (reviewed by Lamb, 1978). Moreover, it has been reported that doses as high as 40 krads increase both mean and maximum life span in the adult housefly, Musca domestica (Allen and Sohal, 1982). The present study indicates that a 10 krad dose causes very little or no reduction of adult Drosophila life span in the repair-proficient strains p pr cn/b pr c_n, b_ pr cn/CvO. D-132/CvO. and G-92/CvO and Canton-S. Even strains that are potentially repair-deficient, D-132/D-132 and G-92/G-92. showed no appreciable reduction in longevity. Only individuals bearing A- l (i.e. heterozygotes and homozygotes) showed significantly reduced life spans in the 10 krad experiment. All mus mutations that have been biochemically characterized appear to represent lesions in various DNA repair pathways (Boyd £_ _ , 1983). Prior to the experiments reported here, only one mus mutation, mei-41. has been examined for its effects on longevity (Miquel §_ §J_, 1983). The life span of males carrying mei-41 was compared to control males carrying the X-chromosome on which the mutation was originally induced. Three strains of wild-type males were also examined. Metabolic activity, measured as ul 02 consumption/mg fly/24 hours, and mating ability were also determined for the five lines of males (it is not clear whether these two parameters were averaged over several determinations during the life span or whether single measurements were made at one time during the life span). The results showed that mei-41 males had the lowest values for all three parameters. When the wild-type flies and control flies were considered separately, there was an inverse relationship between mean life span and metabolic activity. Therefore, it did not appear that the shorter life span of mei-41 was due to a higher metabolic rate. The authors concluded that mei-41 males lacked some form of "inherent vitality" and that the early death of the mutants was not representative of accelerated aging. While the mus strains in the present study were also used to establish whether or not 166 repair-deficient mutations alter life span, it is important to know to what extent, if any, mus* genes remain functional in the adult. In other words, the reductions in adult life span observed in homozygous mus strains in these experiments could be due to deficiencies in DNA metabolism during development rather than reflections of accelerated senescent processes as they occur in the adult. The irradiation of adult Drosophila is one method of determining if the products of mus* genes are functioning in the adult soma. Flies deficient for repair functions in the adult should be more radiation-sensitive than repair-proficient flies. On the other hand, if repair processes are not essential in the adult, then mus flies should not be more radiation-sensitive than control flies. However, the fact that mus homozygotes at 29°C were not more sensitive to 25 krads of gamma radiation than homozygotes at 22°C or p pr cn/b pr c_n controls at either temperature does not preclude the possibility that DNA repair functions occur in adults. Under the conditions used here and with the genetic background of the strains studied, it is possible that any differences in DNA repair that exist between strains are negated by the high dose. Furthermore, levels of radiation of this magnitude have been reported to reduce wild-type adult longevity anywhere from 5% to 65% (Giess, 1980). It is possible that 25 krads may cause other extensive cellular damage (i.e. other than DNA damage) that would not be influenced by DNA repair enzymes and yet could lead to death. The 10 krad dose revealed that A-l/A-1 flies appeared to be more radiation-sensitive than all other strains at 29°C. Therefore, there is some indication that repair may be functional in the adult and may influence how rapidly Drosophila ages. Further characterization of this mutant is necessary, however, to determine if it is actually defective in DNA repair in adults. Biochemical characterization of DNA repair may be difficult due to the post-mitotic nature of adult somatic tissue in Drosophila. However, recent experiments with the nematode Turbatrix aceti should prove useful. Like Drosophila. the adult is composed of non-dividing cells except for germinal tissue. Excision repair capacity shows a decrease with 167 increasing age following either ultraviolet irradiation (Targovnik g_t aL, 1984) or X-irradiation (Targovnik ei aL, 1985). If it does appear that DNA damage accumulation and the efficiency of repair processes are involved in normal aging, then mutants that cause an acceleration of damage accumulation should show signs of accelerated aging. Behavioral tests such as those used by Leffelaar and Grigliatti (1984b) should prove useful in assessing these mutants. The normal age-related changes in cell morphology such as cytoplasmic organelle loss and age pigment accumulation (Miquel g_t aL, 1979) could also be examined. These changes correlate well with behavioral changes such as decline in negative geotaxis. At present, there are about 50 loci in Drosophila that have been identified by mutagen sensitivity (Boyd et aL, 1983; Henderson §_ aL, in press). Mutants at several of these loci show some degree of sensitivity to X-rays. This provides a group of mutants that could also be examined for alterations in adult longevity. In addition, acute exposure to gamma rays could be performed at later ages in the adult to see if changes occur in the response to radiation. Chronic exposure to lower doses may also prove insightful in determining whether or not repair occurs in adults. 168 SUMMARY Previous studies on aging and senescence in Drosophila melanogaster have typically examined the role of environmental conditions in the determination of adult life span. Other studies have often compared different strains in terms of how long the adults live. However, no systematic attempt has been made to examine the interaction between genotype and the environment in Drosophila. This study looked at this interaction and approached the problem from three different directions. The first approach examined how alterations in the duration of preimaginal development affect adult longevity. Previous studies have suggested that a positive correlation exists between the duration of development and adult life span. In the experiments reported here, a series of temperature shifts during the preimaginal period were used to alter developmental duration and then adult life span was determined at two temperatures. No correlation between the length of the developmental period and life span was found in either the wild-type strain Oregon-R or the temperature-sensitive delayed development strain 957. In addition, 957 displayed no extended life span at 29°C relative to Oregon-R. Therefore, it does not seem likely that the duration of development is a determining factor in controlling senescence in Drosophila. Furthermore, these findings to not support theories which propose that the onset of senescence and death are programmed events. The second approach was a comparison of adult life span between a highly inbred laboratory strain and strains recently isolated from the wild. At both 22°C and 29°C, the recently isolated strains displayed a fair degree of variability in life span within samples whereas the highly inbred strain showed a much narrower range over which most death occurred. If a genetic characterization of aging is to be carried out (especially by mutational dissection) under laboratory conditions, low variability in the life span of control stocks is desirable. Otherwise, the isolation of short or long lived strains becomes difficult 169 because of background variation. Therefore, it is preferable to use well characterized highly inbred wild-type strains when performing an analysis in the laboratory even though the pattern of death may be far removed from that occuring in nature. The third approach assessed the possible role of DNA repair in determining adult life span in Drosophila melanogaster. Of three potentially repair-defective strains that were examined, only one was clearly shorter lived than the control strain. As the adults of this strain were not more sensitive to the life-shortening effects of radiation than was the control strain, the reduced life span cannot be definitely attributed to defective repair in the adult. Another strain appeared to be more sensitive than controls at 29°C to a lower radiation level, but an unusual bimodal pattern of death within both irradiated and non-irradiated mutant samples makes the interpretation of these results difficult. Thus, no direct evidence exists to support the role of DNA repair in adult Drosophila as a factor in aging. 170 REFERENCES Allen, R.G. and R.S. Sohal (1982). Life-lengthening effects of gamma-radiation on the adult housefly, Musca domestica. Mech. Ageing Dev. 20: 369-375. Alpatov, W.W. and R. Pearl (1929). Experimental studies on the duration of life - XII. Influence of temperature during the larval period and adult life on the duraiton of the life of the imago of Drosophila melanogaster. Amer. Naturalist 63:37-67. Ashburner, M. and J.N. Thompson, Jr. (1978). The laboratory culture of Drosophila. In M. Ashburner and T.R.F. Wright (eds.): "The Genetics and Biology of Drosophila. Vol. 2a." Academic Press, London, pp. 1-109. Atlan, H., J. Miquel and R. Binnard (1969a). Differences between radiation-induced life shortening and natural aging in Drosophila melanogaster. J. Geront. 24:1-4. Atlan, H., J. Miquel and G. Welch (1969b). Life-shortening effects of ultraviolet radiation on Drosophila melanogaster imagoes. Pros. Inf. Serv. 44:88. Baxter, R.C. and H.A. Blair (1969). Recovery and overrecovery from acute radiation injury as a function of age in Prosophila. Radiat. Res. 39:345-360. Bell, E., L.F. Marek, P.S. Levinstone, C. Merrill, S. Sher, LT. Young and M. Eden (1978). Loss of division potential in vitro: aging or differentiation? Science 202:1158-1163. Boyd, J.B., P.V. Harris, C.J. Osgood and K.E. Smith (1980). Biochemical characterization of repair-deficient mutants of Prosophila. In W.M. Genoroso, M.P. Shelby and F.J. de Serres (eds.): "PNA Repair and Mutagenesis in Eukaryotes." Plenum Press, New York, pp. 209-221. Boyd, J.B., P.V. Harris, J.M. Presley and M. Narachi (1983). Prosophila melanogaster: a model eukaryote for the study of PNA repair. In E.C. Friedberg and B.A. Bridges (eds.): "Cellular Responses to PNA Pamage." Alan R. Liss, New York, pp. 107-123. Bozcuk, A.N. (1978). The effect of some genotypes on the longevity of adult Prosophila. Exp.  Geront. 13:279-285. Bozcuk, A.N. (1981). Genetics of longevity in Prosophila - V. The specific and hybridised effects of rolled, sepia, ebony and eyeless autosomal mutants. Exp. Geront. 16:415-427. Brown, W.T., J.B. Little, J. Epstein and J.R. Williams (1978). PNA repair defect in progeric - cells. In P. Bergsma and P.E. Harrison (eds.): "Genetic Effects on Aging. Birth Pefects: Original Article Series, Vol. 14." Alan R. Liss, New York, pp. 417-430. Brown, W.T. and H.M. Wisniewski (1983). Genetics of human aging. In M. Rothstein (ed.).: "Review of Biological Research in Aging, Vol. 1." Alan R. Liss, New York, pp. 81-99. Burcombe, J.V. and M.J. Hollingsworth (1970). The relationship between developmental temperature and longevity in Prosophila. Gerontologia 16:172-181. 171 Cannon, G.B. (1966). Competition, viability and longevity in experimental populations. Evolution 20:117-131. Cohet, Y. (1975). Epigenetic influences on the lifespan of the Drosophila: Existence of an optimal growth temperature for adult longevity. Exp. Geront. 1_Q_: 181 -184. Curtis, H.J. (1963). Biological mechanisms underlying the aging process. Science. 141:686-694. Ducoff, H.S. (1976). Radiation-induced increase in lifespan of insects: Implications for theories of mammalian aging and radiosensitivity. In: "Biological and Environmental Effects of Low-Level Radiation, Vol. 1." IAEA, Vienna, pp. 103-109. Francis, A.A., W.H. Lee and J.D. Regan (1981). The relationship of DNA excision repair of ultraviolet-induced lesions to the maximum life span of mammals. Mech. Ageing Dev. 16:181-189. Gartner, L.P. (1973a). Radiation-induced life span shortening in Drosophila. Gerontologia 19:295-302. Gartner, L.P. (1973b). Ultrastructural examination of ageing and radiation-induced life-span shortening in adult Drosophila melanogaster. Int. J. Radiat. Biol. 23:23-39. Gartner, L.P. (1984). The effects of 4000 and 8000 R of X-irradiation on Drosophila life span. Pros. Inf. Serv. 60:107-108. Gensler, H.L. and H. Bernstein (1981). DNA damage as the primary cause of aging. Quart. Rev.  Biol. 56:279-303. Giess, M.C. (1980). Differences between natural ageing and radio-induced shortening of the life expectancy in Drosophila melanogaster. Gerontology 26:301-310. Giess, M.C. and H. Planel (1977). Influence of sex on the radiation-induced life span modifications in Drosophila melanogaster. Gerontology 23:325-333. Graf, U. and F.E. Wurgler (1978). Mutagen-sensitive mutants in Drosophila: Relative MMS sensitivity and maternal effects. Mutation Res. 52:381-394. Graf, U. and F.E. Wurgler (1979). Lack of correlation between MMS-toxicity in larvae and in adults of mutagen-sensitive mutants of Drosophila melanogaster. Environ. Mut. L239-248. Hall, K.Y., R.W. Hart, A.K. Benirschke and R.L. Walford (1984). Correlation between ultraviolet-induced DNA repair in primate lymphocytes and fibroblasts and species maximum achievable life span. Mech. Ageing Dev. 24:163-173. Harman, D. (1982). The free-radical theory of aging. In W.A. Pryor (ed.): "Free Radicals in Biology, Vol. V." Academic Press, New York, pp. 255-275. Hart, R.W . SM D'Ambrosio and K.J. Ng (1979). Longevity, stability and DNA repair. Mech.  Ageing Dev. 9:203-223. 172 Hart, R.W. and S.P. Modak (1980). Aging and changes in genetic information. In K. Oota, R. Makinodan, M. Iriki and L.S. Baker (eds.): "Aging Phenomena: Relationships among Different Levels of Organization." Plenum Press, New York, pp. 123-137. Hart, R.W. and R.B. Setlow (1974). Correlation between deoxyribonucleic acid excision-repair and life-span in a number of mammalian species. Proc. Natl. Acad. Sci. USA 71:2169-2173 Hayflick, L. (1965). The limited in vitro lifetime of human diploid cell strains. Exp. Cell Res. 3_7:614-636. Hayflick, L. (1984). Intracellular determinants of cell aging. Mech. Ageing Dev. 28:177-185. Hayflick, L. and P.S. Moorhead (1961). The serial cultivation of human diploid cell strains. EXP. Cell Res. 25:585-621. Henderson, D., D.A. Bailey, D.A.R. Sinclair and T.A. Grigliatti. The isolation and characterization of second chromosome mutagen-sensitive mutants in Drosophila  melanogaster. Mutation Res, (in press). Johnson, T.E. (1983). Aging in Caenorhabditis elegans. In M. Rothstein (ed.): "Review of Biological Research in Aging, Vol. 1." Alan R. Liss, New York, pp. 37-49. Johnson, T.E. (1984). Analysis of the biological basis of aging in the nematode, with special emphasis on Caenorhabditis elegans. In D.H. Mitchell and T.E. Johnson (eds.): "Invertebrate Models in Aging Research." CRC Press, Boca Raton, pp. 59-93. Kato, H., M. Harada, K. Tsuchiya and K. Moriwaki (1980). Absence of correlation between DNA repair in ultraviolet irradiated mammalian cells and life span of the donor species. Japan. J. Genet. 55:99-108. Kirkwood, T.B.L. (1977). Evolution of ageing. Nature 270:301-304. Kirkwood, T.B.L. (1984). Towards a unified theory of cellular ageing. In H.W. Sauer (ed.).: "Monographs in Developmental Biology, Vol. 17: Cellular Ageing." S. Karger, Basel, pp. 9-20. Lamb, M.J. (1977). "Biology of Ageing." Blackie, London. Lamb, M.J. (1978). Ageing. In M. Ashburner and T.R.F. Wright (eds.): "The Genetics and Biology of Drosophila, Vol. 2c." Academic Press, London, pp. 43-104. Leffelaar, D. and T.A. Grigliatti (1984a). A mutation in Drosophila that appears to accelerate .' aging. Dev. Genet. 4:199-210. Leffelaar, D. and T. Grigliatti (1984b). Age-dependent behavior loss in adult Drosophila  melanogaster. Dev. Genet. 4:211-227. Lints, F.A. (1978). "Interdisciplinary Topics in Gerontology, Vol. 14: Genetics and Ageing." S. Karger, Basel. 173 Lints, F.A. and CV. Lints (1969). Influence of preimaginal environment on fecundity and ageing in Drosophila melanogaster hybrids - I. Preimaginal population density. Exp.  Geront. 4:231-244. Lints, F.A. and CV. Lints (1971a). Influence of preimaginal environment on fecundity and ageing in Drosophila melanogaster hybrids - II. Preimaginal temperature. Exp. Geront. 6:417-426. Lints, F.A. and CV. Lints (1971b). Influence of preimaginal environment on fecundity and ageing in Drosophila melanogaster hybrids - III. Developmental speed and life-span. Exp. Geront. 6:427-445. Lints, F.A. and M.H. Soliman (1977). Growth rate and longevity in Drosophila melanogaster and Tribolium castaneum. Nature 266:624-625. Lints, F.A., J. Stoll, G. Gruwez and CV. Lints (1979). An attempt to select for increased longevity in Drosophila melanogaster. Gerontology 25:192-204. Luckinbill, L.S., R. Arking, M.J. Clarke, W.C Cirocco and S.A. Buck (1984). Selection for delayed senescence in Drosophila melanogaster. Evolution 3_8:996-1003. Maynard Smith, J. (1958). The effects of temperature and of egg-laying on the longevity of Drosophila subobscura. J. Exp. Biol. 3.5:832-842. Miquel, J., K.G. Bensch, D.E. Philpott and H. Atlan (1972). Natural aging and radiation-induced life shortening in Drosophila melanogaster. Mech. Ageing Dev. 1:71-97. Miquel, J., R. Binnard and J.E. Fleming (1983). Role of metabolic rate and DNA-repair in Drosophila aging: Implications for the mitochondrial mutation theory of aging. Exp.  Geront. 18:167-171. Miquel, J., A.C. Economos, K.G. Bensch, H. Atlan and J.E. Johnson, Jr. (1979). Review of cell aging in Drosophila and mouse. Age 2:78-88. Miquel, J., P.R. Lundgren, K.G. Bensch and H. Atlan (1976). Effects of temperature on the lifespan, vitality and fine structure of Drosophila melanogaster. Mech. Ageing Dev. £347-370. Muller, H.J. (1963). Mechanisms of life-span shortening. In R.J.C. Harris (ed.): "Cellular Basis and Aetiology of Late Somatic Effects of Ionizing Radiation." Academic Press, New York, pp. 235-245. Orgel, L.E. (1963). The maintenance of the accuracy of protein synthesis and its relevance to ageing. Proc. Natl. Acad. Sci. USA 49:517-521. Partridge, L. and M. Farquhar (1981). Sexual activity reduces lifespan of male fruitflies. Nature 294:580-582. Pearl, R. (1928). "The Rate of Living." University of London Press, London. Rockstein, M. and J. Miquel (1973). Aging in insects. In M. Rockstein (ed.): "The Physiology of Insecta, Second Edition." Academic Press, New York, pp. 371-478. 174 Rose, M.R. (1983a). Evolution of aging. In M. Rothstein (ed.): "Review of Biological Research in Aging, Vol. I." Alan R. Liss, New York, pp. 19-24. Rose, M.R. (1983b). Theories of life-history evolution. Amer. Zool. 23_:15-23. Rose, M.R. (1984). Laboratory evolution of postponed senescence in Drosophila melanogaster. Evolution 3_8:1004-1010. Rose, M.R. and B. Charlesworth (1981a). Genetics of life history in Drosophila melanogaster. I. Sib analysis of adult females. Genetics 9.7:173-186. Rose, M.R. and B. Charlesworth (1981b). Genetics of life history in Drosophila melanogaster. II. Exploratory selection experiments. Genetics 97:187-196. Rothstein, M. (1982). "Biochemical Approaches to Aging." Academic Press, New York. Smith-Sonneborn, J. (1979). DNA repair and longevity assurance in Paramecium tetraurelia. Science 203:1115-1117. Smith-Sonneborn, J. (1981). Genetics and aging in protozoa. Int. Rev. Cvtol. 73:319-354. Smith-Sonneborn, J. (1984). Protozoan Aging. In D.H. Mitchell and T.E. Johnson (eds.): "Invertebrate Models in Aging Research." CRC Press, Boca Raton, pp. 1-14. Smith-Sonneborn, J., P.D. Lipetz and R.E. Stephens (1983). Paramecium bioassay of longevity modulating agents. In W. Regelson and F.M. Sinex (eds.): "Intervention in the Aging Process, Part B: Basic Research and Preclinical Screening." Alan R. Liss, New York, pp. 253-273. Smith-Sonneborn, J., P.D. Lipetz and R.E. Stephens (1984). Novobiocin inhibition of dark repair and longevity in Paramecium. In A. Collins, C.S. Downes and R.T. Johnson (eds.): "DNA Repair and Its Inhibition." IRL Press, Oxford, pp. 309-318. Strehler, B. (1977). "Time, Cells and Aging: Second Edition." Academic Press, New York. Suzuki, D.T. (1970). Temperature-sensitive mutations in Drosophila melanogaster. Science 170:695-706. Targovnik, H.S., S.E. Locher and P.V. Hariharan (1985). Age associated alteration in DNA damage and repair capacity in Turbatrix aceti exposed to ionizing radiation. Int. J.  Radiat. Biol. 47:255-260. Targovnik, H.S. S.E. Locher, T.F. Hart and P.V. Hariharan (1984). Age-related changes in the excision reapir capacity of Turbatrix aceti. Mech. Ageing Dev. 27:73-81. Woodland, A.D., R.B. Setlow and E. Grist (1980). DNA repair and longevity in three species of cold-blooded vertebrates. Exp. Geront. _:301-304. Zar, J.H. (1984). "Biostatistical Analysis, Second Edition." Prentice-Hall, Englewood Cliffs, N.J. 175 Zuckerman, B.M. (1983). The free-living nematode Caenorhabditis elegans as a rapid screen for compounds to retard aging. In W. Regelson and R.M. Sinex (eds.): "Intervention in the Aging Process, Part B: Basic Research and Preclinical Screening." Alan R. Liss, New York, pp. 275-285. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0096796/manifest

Comment

Related Items