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Longevity, behaviour, and mapping of three temperature sensitive adult lethal alleles of Drosophila melanogaster Hansen, Beverley Nina 1991

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LONGEVITY, BEHAVIOUR, AND MAPPING OF THREE TEMPERATURE SENSITIVE A D U L T L E T H A L A L L E L E S OF DROSOPHILA MELANOGASTER by BEVERLEY NINA HANSEN B.Sc.(Honours), The University of Saskatchewan, Regina, 1970 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF T H E REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Zoology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 1991 © Beverley Nina Hansen, 1991. 7 ' In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of 7no1onv The University of British Columbia Vancouver, Canada Date October 1991 DE-6 (2/88) ABSTRACT The rate at which an organism ages, as well as the onset of senescence are determined by many factors. Different species, as well as different strains of the same species, have characteristic lifespans. This study is an investigation of three strains of Drosophila melanogaster, to test their influences on ageing and senescence. Temperature-sensitive putative allele mutants adl-16tsl, adl-16ts2, and adl-16flrdI, and wild-type Oregon-R (control) Drosophila melanogaster were examined for patterns of age dependent behaviour loss over the course of their adult lifespans. Longevity, geotaxis, phototaxis, and motor activity were examined at both the permissive temperature, 22°C, and the restrictive temperature, 2 9 ° C . adl-16tsl displayed a longevity and behaviour loss pattern similar to the wild-type strain at 22°C. At 29°C, longevity was significantly reduced compared to wild-type, and the pattern of age-dependent behaviour loss was compressed into a shorter time frame. This pattern of behaviour loss was consistent with that expected from a mutation which increases the rate of ageing (Leffelaar and Grigliatti, 1984). The lifespan of adl-16ts2 at 2 2 ° C was reduced, when compared with Oregon-R, but the behaviour loss pattern was similar. At 29° C the flies died rapidly, with almost total, immediate, behaviour loss. Survival curves at 22°C, 25°C, and 29°C adjusted for the rate of living were coincident. Flies of the type adl-16flrdI, isolated in a separate screen as flight-reduced, demonstrated differences from adl-16tsl and adl-16ts2 in longevity curve shape as well as behaviour. Lifespan was reduced at both 22°C and 29°C, and although behaviour differed slightly in young flies, the behaviour loss pattern was similar to that of adl-16tsl. The order of severity of effects of the restrictive temperature from the least affected allele to the most affected was adl-16flrdI, adl-16tsl, and adl-16ts2. At 22°C, hybrid flies of type adl-16tslladl-16ts2 and adl-16ts2ladl-Ififlrdl exhibited lifespans comparable to Oregon-R, the latter being longer lived than either parental strain. The hybrid strain adl-16tslIadl-16flrdIwas demonstrated to have longevity intermediate between parent types, and reduced with respect to Oregon-R. Geotactic behaviour was reduced in all three hybrids at 2 2 ° C , but phototaxis and motor activity were similar to that of wild-type flies. At 29°C all hybrid strains could be said to demonstrate intermediate lifespan, between females of the generating parent strains. Complementation did not occur, and thus all mutants under study were confirmed to be alleles. Behaviour was reduced in adl-16tslladl-16ts2 and adl-16ts2ladl-l6flrd at 29°C, but not in adl-I6tslladl-16flrd. Flies of type Deficiency/mutant were found to have greatly reduced lifespans at both 22°C and 29°C. The order of severity of effects was Dfladl-16tsl, Df/adl-16ts2, and Dfladl-16flrdI, with the last being the most severely affected, with deformities noted at both temperatures. This result confirms the cytological location of these mutants relative to their genetically determined position. The focus of action of the mutant gene adl-16ts2 was mapped two ways by means of gynandromorph analyses. The drop-dead behaviour was mapped against a background map constructed for this study. This behaviour appears to map to the ventral thoracic region, and most likely involves presumptive mesodermal tissue. The paralysis behaviour noted in these flies at 2 9 ° C was then mapped separately for each leg. Three foci were found. A l l three appear to map to presumptive nervous tissue. Involvement of nerve and muscle tissues would not be surprising, considering the behaviours noted earlier. The discussion involves speculation as to the precise function of all three adl-16ts genes. V TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS v LIST OF TABLES v i LIST OF FIGURES ix ACKNOWLEDGEMENTS xiii GENERAL INTRODUCTION 1 CHAPTER ONE Introduction 14 Materials and Methods 17 Results 21 Discussion 91 CHAPTER TWO Introduction 98 Materials and Methods 101 Results 105 Discussion 135 CHAPTER THREE Introduction 140 Materials and Methods 141 Results 143 Discussion 163 CHAPTER FOUR Introduction 168 Materials and Methods 174 Results 180 Discussion 197 SUMMARY DISCUSSION 208 REFERENCES 212 APPENDICES 222 vi LIST OF TABLES Page TABLE 1-1 Repetitions of Survival and Behaviour Tests. 21 TABLE 1-2 Longevity of Oregon-R Adults Maintained at 22°C 40 or 29°C Posteclosion. TABLE 1-3 Conversion Factors for Differences in Rate of Living of 41 Oregon-R at 22°C or 29°C Posteclosion. TABLE 1-4 Longevity of adl-16tsl Adults Maintained at 22°C or 29°C 52 Posteclosion. TABLE 1-5 Longevity of adl-16ts 1 Adults Maintained at 22°C or 29°C 53 Posteclosion TABLE 1-6 Longevity of adl-16ts2 Adults Maintained at 22°C or 29°C 66 Posteclosion. TABLE 1-7 Conversion Factors for Differences in Rate of Living of 67 adl-16ts2 at 22°C and 29°C. TABLE 1-8 Longevity of adl-16ts2 Males Maintained at 22°C or 29°C 67 Posteclosion. TABLE 1-9 Longevity of adl-16flrdIAdults Maintained at 22°C or 29°C 78 Posteclosion TABLE 1-10 Conversion Factors for Differences in Rate of Living of 79 adl-16flrdIm 22°C and 29°C. TABLE 1-11 Longevity of Adult Hybrid Females Maintained at 22°C 88 Posteclosion. TABLE 1-12 Longevity of Adult Hybrid Females Maintained at 29°C 89 Posteclosion. TABLE 2-1 Behaviour Loss with age in Oregon-R Males at 22°C 120 and at 29°C. TABLE 2-2 Behaviour Loss with age in Oregon-R Females at 22°C 121 and at 29°C. TABLE 2-3 Behaviour Loss with age in adl-16tsl Males at 22°C 122 and at 29°C. TABLE 2-4 Behaviour Loss with age in adl-16tsl Females at 22°C and at 29°C. TABLE 2-5 Behaviour Loss with age in adl-16ts2 Males at 22°C and at 29°C. TABLE 2-6 Behaviour Loss with age in adl-16ts2 Females at 22°C and at 29° C. TABLE 2-7 Behaviour Loss with age in adl-16flrdI Males at 22°C and at 29°C. TABLE 2-8 Behaviour Loss with age in adl-16flrdI Females at 22°C and at 29°C. TABLE 2-9 Behaviour Loss with age in adl-16tsl I adl-16ts2 Hybrid Females at 22°C and at 29°C. TABLE 2-10 Behaviour Loss with age in adl-16tslladl-16flrdI Hybrid Females at 22°C and at 29°C. TABLE 2-11 Behaviour Loss with age in adl-16ts2ladl-16flrdI Hybrid Females at 22°C and at 29°C. TABLE 2-12 Maximal Behaviour in all strains Tested at 22°C and 29°C TABLE 2-13 Total Behaviour Loss of Mutant Strains Relative to D-100 and Oregon-R. TABLE 3-1 Viability Ratios. TABLE 3-2 Progeny of Deficiency Crosses at 22°C. TABLE 3-3 adl-16tsl/Deficiency Compared with adl-16tsl Homozygotes and Hemizygotes at 22°C. TABLE 3-4 adl-16ts21 Deficiency Compared with adl-16ts2 Homozygotes and Hemizygotes at 22°C TABLE 3-5 adl-16flrdIi'Deficiency Compared with adl-16flrdI Homozygotes and Hemizygotes. TABLE 3-6 adl-16tsl/Deficiency Compared with adl-16tsl Homozygotes and Hemizygotes at 29°C. TABLE 3-7 adl-16ts2/Deficiency Compared with adl-16ts2 Homozygotes and Hemizygotes. vii i Page TABLE 3-8 adl-16flrdIlDeficiency Compared with adl-16flrdI 158 Homozygotes and Hemizygotes. TABLE 3-9 Conversion Factors for Differences in Rate of Living of 159 Dflmutant at 22°C and 29°C. TABLE 4-1 Progeny Genotypes from Ring-X Chromosome Female 181 Crossed with adl-16ts2 Males. TABLE 4-2 Drop-dead Behaviour Mapped to 9 Landmarks. 187 TABLE 4-3 Distances Between Landmarks. 185 TABLE 4-4 Percent Maleness of the Nine Structural Landmarks Used. 187 TABLE 4-5 Preliminary Mosaic Mapping of Drop-dead Focus. 188 Head, Thorax, and Abdomen. TABLE 4-6 Analysis of Four Landmarks for Domineering Foci. 193 TABLE 4-7 Number of Legs Mutant, and Probability of Early Death. 194 TABLE 4-8 Map Data for Paralysis of Legs 1, 2, and 3. 195 IX FIGURE 1-1 FIGURE 1-2 FIGURE 1-3 FIGURE 1-4 FIGURE 1-5 FIGURE 1-6 FIGURE 1-7 FIGURE 1-8 FIGURE 1-9 FIGURE 1-10 FIGURE 1-11 FIGURE 1-12 FIGURE 1-13 FIGURE 1-14 LIST OF FIGURES Oregon-R Males Survival Curve at 22°C. Oregon-R Males Survival Curve at 29°C. Oregon-R Males Survival Curves at 22°C and 29°C Adjusted for Rate of Living Oregon-R Females Survival Curve at 22°C Oregon-R Females Survival Curve at 29°C Oregon-R Females Survival Curves at 22°C and 29°C Adjusted for Rate of Living Oregon-R Males and Females Survival Curves at 22°C Oregon-R Males and Females Survival Curves at 29°C Oregon-R Males and Females Survival Curves at 22°C and 29°C Adjusted for Rate of Living Oregon-R Males and Females Survival Curves at 22°C and 29°C adl-16tsl Males Survival Curve at 22°C adl-16tsl Males Survival Curve at 29°C adl-16tsl Males Survival Curves at 22°C and 29°C Adjusted for Rate of Living adl-16tsl Males Survival Curves at 22°C and 29°C Adjusted for adl-16tsl Rate of Living Page 30 31 32 33 34 35 36 37 38 39 44 45 46 47 FIGURE 1-15 FIGURE 1-16 FIGURE 1-17 FIGURE 1-18 FIGURE 1-19 FIGURE 1-20 FIGURE 1-21 FIGURE 1-22 FIGURE 1-23 FIGURE 1-24 FIGURE 1-25 FIGURE 1-26 FIGURE 1-27 FIGURE 1-28 adl-16tsl Females Survival Curve at 22°C adl-16tsl Females Survival Curve at 29°C adl-16tsl Fe males Survival Curves at 22°C and 29°C Adjusted for Rate of Living adl-16tsl Females Survival Curves at 22°C and 29°C Adjusted for adl-16tsl Rate of Living adl-16ts2 Males Survival Curve at 22°C adl-16ts2 Males Survival Curve at 29°C adl-16ts2 Males Survival Curves at 22°C and 29°C Adjusted for adl-16ts2 Rate of Living adl-16ts2 Males Survival Curves at 22°C and 29°C Adjusted for Rate of Living adl-16ts2 Males Survival Curve at 25°C adl-16ts2 Males Survival Curves at 22°C, 25°C and 29°C adl-16ts2 Females Survival Curve at 22°C adl-16is2 Females Survival Curve at 29°C adl-16ts2 Females Survival Curves at 22°C and 29°C Adjusted for adl-16ts2 Rate of Living adl-16ts2 Females Survival Curves at 22°C and 29°C Adjusted for Rate of Living xi FIGURE 1-29 FIGURE 1-30 FIGURE 1-31 FIGURE 1-32 FIGURE 1-33 FIGURE 1-34 FIGURE 1-35 FIGURE 1-36 FIGURE 1-37 FIGURE 1-38 FIGURE 1-39 FIGURE 1-40 adl-16flrdI Males Survival Curve at 22°C adl-16flrdI Males Survival Curve at 29°C adl-16flrdI Males Survival Curves at 22°C and 29°C Adjusted for adl-16f^rdI Rate of Living adl-16flrdI Males Survival Curves at 22°C and 29°C Adjusted for Rate of Living adl-16flrdI Females Survival Curve at 22°C adl-16flrdI¥e males Survival Curve at 29°C adV-irj/'^ Females Survival Curves at 22°C and 29°C Adjusted for adl-16f^rdI Rate of Living adl-16flrdI Females Survival Curves at 22°C and 29°C Adjusted for Rate of Living adl-16ts Hybrid Females Survival Curves at 22°C adl-16ts2/adl-16flrdI Hybrid Females adl-16ts2, adl-16flrdlHomozygotes Survival Curves at 29°C adl-16tslladl-16ts2 Hybrid Females adl-16tsl, adl-16ts2 Homozygotes Survival Curves at 29°C adl-16tsl/adl-16flrdI Hybrid Females adl-16tsl, adl-16flrdIHomozygotes Survival Curves at 29°C Page 70 71 72 73 74 75 76 77 82 83 84 85 FIGURE 1-41 adl-16ts Hybrid Females Survival Curves at 29°C 86 x i i Page FIGURE 1-42 adl-16ts Homozygous Females 87 Survival Curves at 29°C FIGURE 2-1 Geotactic Response of Oregon-R Males (5cm) 108 FIGURE 2-2 Geotactic Response of Oregon-R Females (5cm) 109 FIGURE 2-3 Geotactic Response of Oregon-R Males (10cm) 110 FIGURE 2-4 Geotactic Response of Oregon-R Females (10cm) 111 FIGURE 2-5 Geotactic Response of Oregon-R Males (15cm) 112 FIGURE 2-6 Geotactic Response of Oregon-R Females (15cm) 113 FIGURE 2-7 Phototactic Response of Oregon-R Males 115 FIGURE 2-8 Phototactic Response of Oregon-R Females 116 FIGURE 2-9 Motor Activity of of Oregon-R Males 118 FIGURE 2-10 Motor Activity of of Oregon-R Females 119 FIGURE 3-1 Diagram of Wing of adl-16flrdI/Deficiency 145 FIGURE 3-2 Comparison of Genetic and Cytological Location 152 of aciZ-id" Gene FIGURE 3-3 Deficiency/adl-16ts Mutants 161 Survival Curves at 22°C FIGURE 3-4 Deficiency I adl-16ts Mutants 162 Survival Curves at 29°C FIGURE 4-1 Percent Mosaics Alive or Paralysed 183 FIGURE 4-2 Background Fate Map Diagram 186 FIGURE 4-3 Fate Map of Drop-Dead Behaviour 190 FIGURE 4-4 Map Showing Expected Position of Presumptive 191 Neural and Mesodermal Tissue Superimposed on Figure 4-3 FIGURE 4-5 Fate Map of Paralysis of Legs 196 ACKNOWLEDGEMENTS Notwithstanding many difficulties in the accomplishment of this project, I wish to thank the following people in direct proportion to the extent of their help, encouragement, and support. Bent, Trent, Shane, Garth and Rianna Hansen; Kathleen Fitzpatrick, Vet. Lloyd; Alex Holm, Jim Kettlewell, Kim Sykes; Lois Campbell; Dr. Don Sinclair, Dr. Jim Berger and, last but not least, Dr. Thomas Grigliatti. 1 General Introduction A centuries old obsession with eternal youth has brought us no closer to solving the mystery of ageing. It remains a mystery, and no less compelling. There is no consensus on the causes of ageing, nor a single theory that explains the physiological changes associated with it. The etiology of ageing is considered to be extremely complex. As population demographics change, with the concomitant increase in the proportion of older people in society, gerontological research is becoming more important. Diseases such as progeria, and Alzheimer's, as well as other degenerative disorders in man, are active areas of research, and, since the amount of experimentation involving humans is obviously limited, other species afford an excellent substitute to test various theories. Micro-organisms, and insects, especially Drosophila, with its well known genetics, are frequently used as model systems to elucidate difficult or complex problems such as ageing. Ageing is the change in structure and function, occuring naturally over time, which increases the probability of the death of an organism (Coni, Davison, and Webster, 1984). Accidents, or diseases are usually not considered part of normal ageing. Rockstein and Miquel (1973), who have been actively involved in the study of ageing for many years, define it as "the sum total of those time-dependent, reproducible changes both in structure and/or function for a given organism, species, or strain, during its total lifespan." In general, lifespan can be divided into three phases: juvenile, reproductive, and senescent. For Drosophila, the juvenile stages include the egg, larva, and pupa; the reproductive phase is represented by the first, and larger portion of the adult lifespan; and senescence occurs just prior to death, and involves the deteriorative changes which occur towards the end of life. Many theories have been proposed to explain the causes of senescence and death. Minot (1907), Muller (1963), and more recently Timeras (1978), and Lints (1978), postulated that ageing and death are continuous with morphogenesis, and are simply the final result of differentiation, meaning that they are events programmed into the genome. By this, it is interpreted that specific physiological events act in such a way that ageing occurs, and death is inevitable. Timeras proposed an ontogenetic program or "master plan", regulated by neuroendocrine controls located in the brain. It is known that in vertebrates the hypothalamus controls the secretion of a number of hormones and neurotransmitters. In man, for example, it controls the release of hormones responsible for puberty, ovulation, menstruation, and menopause. In the case of the Pacific salmon, shortly after spawning, a huge release of hormones precedes death. While it can be argued that there is an evolutionary advantage for programmed death, (i.e. genetic control), ageing is certainly not the only means by which this can be accomplished. More evidence exists to support programmed death than programmed ageing (Collatz and Sohal, 1986). The lifespan characteristic for each species provides evidence for programmed death and suggests some type of genetically determined biological clock. Time-dependent changes seen in mammals, for example presbyopia, or the greying of hair, which occur with ageing, also seem to indicate genetic determination. Although there is some evidence to support the genetic basis of ageing and senescence, other intrinsic metabolic factors may be involved. Examples are randomly occurring errors in normal biochemical pathways and cellular events, such as accumulation of faulty proteins (protein error hypothesis; Orgel, 1963), and aberrant membranes or somatic mutations (Curtis, 1963). Changes of this type are generally known as "wear and tear", and occur at both the cellular and tissue level. The incidence of such mutations and errors increases with age, as aberrent products accumulate, necessary products diminish, and vital organs cease to function, eventually causing death. This theory is supported by some evidence that free radicals, the by-products of oxygen metabolism, accumulate with age, and damage cell membranes (Harman, 1982). A third hypothesis of ageing posits that it results from extrinsic factors, such as bacterial or viral infections, radiation, toxins, or temperature. It is widely accepted that metabolic rate is directly temperature dependent. This is especially true for poikilothermic animals, such as insects. Coping with environmental abuse, such as habitat destruction, not only leads to death of the individual, but also death of the species. At the present time there is great concern about this in relation to the destruction of rainforests and the increase in ultraviolet radiation exposure due to the reduction of the ozone layer. Two paradigms related to the three theories of ageing explained above and specifically directed at poikilotherms are the threshhold theory of ageing, and the rate of living hypothesis. Clark and Maynard Smith (1961 a and b) proposed the threshhold theory of ageing which argues that ageing is independent of temperature, but vitality decreases with time and at a certain threshhold dying begins within a population. This will occur more rapidly at a higher temperature, because the threshhold is higher. It may also be strain or species specific. On the other hand, the "rate of living" hypothesis proposed by Alpatov and Pearl in 1929, states that at a higher temperature poikilotherms are more active, and thus have a higher rate of living. They propose that faster ageing is due to a limited amount of vitality, which is exhausted more rapidly at higher temperatures, leading to a shortened lifespan. Much conflicting data has been published on these two hypotheses. Miquel et al. (1976), and also Lamb (1978), appear to support the rate of living hypothesis within the 18-25°C temperature range, but there is no unequivocal support for either theory (Lamb, 1978). It is extremely unlikely that any one theory can explain ageing, rather, it seems logical that ageing is due to the complex interaction of many mechanisms. For example, phenotype is well known to be the result of both genotype and environment. Wright and Davidson, (1980) rejected a simplistic view of ageing, and reviews by Rockstein and Miquel (1973), Lints (1978), and Collatz and Sohal (1986), all attest to the complex nature, and the lack of any satisfactory explanation of ageing and senescence. To test any of the above theories and hypotheses associated with ageing, ways to quantify and measure it objectively are needed. Although lifespan is relatively simple to measure, the other characteristics of ageing need to be quantified so that the biological or physiological state of an organism can be correlated with its chronological age. This is difficult, because experimental manipulation of organisms frequently alters lifespan, and organisms such as Drosophila, maintained under laboratory conditions for generations, often have abnormal lifespans. Nevertheless, genetic alterations which modify the biological or physiological age of an organism to one "younger" or "older" than expected, could be said to influence ageing. Regardless of the complexity of the situation we may, however, examine one or two factors at a time. The environmental effect of temperature has been extremely useful in the study of gene expression especially in poikilotherms. It has been used as a tool to test reactions in living organisms without destroying their "normal" functions. As early as 1891, temperature was associated with change in pattern of phenotype in insects. Merrifield demonstrated in 1893 that temperature shocks administered to lepidoptera pupae caused changes in the pattern and intensity of wing markings. Temperature is frequently used to demonstrate conditional mutations such as those above which exhibit normal phenotypes in one environment (permissive), and produce abnormal phenotypes in another (restrictive). Temperature sensitive (ts) mutations are just one type of conditional mutation (Suzuki, 1970). The earliest report of temperature sensitivity in Drosophila, genetically the most extensively characterized insect, was the reduplicated legs mutation (rdp), reported in 1915 by Hoge. She found rdp to be cold sensitive, with high penetrance at low temperatures. She also demonstrated by using temperature-shift studies, that the larval stage required the restrictive temperature for expression of this mutation. Since that time, a great many temperature sensitive insects have been discovered, and temperature sensitivity in Drosophila has been extensively reported by Suzuki and other researchers. Drosophila is also an excellent organism to use as a tool for investigating ageing because it has a short lifespan, is easy to keep in large numbers, and is well suited for genetic analysis. Little or no cell division occurs in Drosophila larvae, except for imaginal disc tissue (Bodenstein, 1950), and no cell division occurs in adults where cells are post-mitotic except for reproductive tissue. Routine replacement of ageing cells is eliminated, making Drosophila an ideal model organism for ageing of post-mitotic mammalian tissue, such as nervous tissue (Miquel et al. 1979). In microorganisms, temperature sensitivity has been shown to result from missense mutations, which alter single amino acids in proteins (Whittmann and Whittmann-Liebold, 1966). These mutations cause loss of biological function at restrictive temperatures by altering the thermostability of tertiary and quaternary protein structure (Jockusch, 1966; Guthrie et al., 1969). Lethal mutations can be maintained at the permissive temperature and the cause of lethality examined under restrictive conditions. To study the time of activity of any temperature sensitive genes, cultures may be shifted between permissive and restrictive temperatures. Tarasoff and Suzuki, (1970), Poodry, Hall and Suzuki, (1973), and Homyk and Grigliatti, (1983) have all demonstrated defects in Drosophila caused by exposure of mutant juvenile flies (e.g. shibiretsl) to restrictive temperatures during development. Times at which adult specific temperature sensitive genes and their products are active might be identified by similar methods. If certain genes are turned on and off in the adult fly, they might be associated with the programming of ageing and death. Unfortunately there are few bio-markers of ageing in adult Drosophila. Anatomical changes such as tattered wings are frequently seen in older flies, but this is not universal, and may be a product of environment. Various tissue specific morphological changes with age have been documented, for example those in the nervous system and muscle tissue demonstrated by Miquel in 1971. Pigment granules and other biochemical changes associated with ageing may be used to quantify ageing, but are time consuming to perform, and require sacrifice of the organism. Although changes in activity of enzymes such as catalase and peroxidase with age have been demonstrated (Burcombe et al., 1972), (Armstrong et al., 1978), and (Nicolosi et al, 1973), these changes are small, and therefore might not be easy to demonstrate or use as reliable markers of physiological age. More recently Fleming et al. (1986), examined over 100 polypeptides by electrophoresis in flies aged 10, 28, and 44 days old. They found that although the "qualitative pattern of gene expression is identical in young and old flies, profound quantitative changes occur in the expression of proteins during the Drosophila lifespan". Among the polypeptides evident there were, however, seven relatively abundant polypeptides expressed in 28 day old flies, which were not detected in 10 and 44 day old flies. No suggestion as to the role of the products of these particular genes was made, but generalizations about the effects of the composition changes of proteins present were given. A different approach, that of age-related behaviour changes of Drosophila, might provide useful bio-markers, since many observed behaviours are innate, and therefore genetically programmed. The sacrifice of flies to test for age is also eliminated. Behaviour is most visible in the reproductive phase of Drosophila and may serve as a useful bio-marker of age if it can be shown to be a reproducable phenotype. Elens (1972), and Elens et al. (1971), recorded differences in phototaxis of flies 5 and 30 days old. Frequently researchers report two or three different ages for various behaviour tests, but rarely has behaviour been tested over the lifespan of Drosophila. In 1972 Miquel et al. measured geotaxis and mating behaviour in wild-type and irradiated flies. They later related temperature and ageing with changes in viability, geotaxis, & mating behaviour during adult lifespan of flies at 18°C, 2 1 ° C , and 27 °C (1976). Economos et al. (1979), tested mating behaviour in male D. melanogaster and correlated decreased viability of flies with the age at which they become impotent. They demonstrated that mating behaviour in male flies can be quantified over lifespan and is reliable as a bio-marker of age. Of importance for the mosaic study in this thesis, age is related to fertility or maternal age effect. A higher number of gynandromorph flies are produced by older female flies (Hall, Gelbart, and Kankel, 1976). Since it is well established that the age of flies affects their behaviour, and testing behaviour does not require sacrifice of flies, the idea that behaviour is a reliable and quantifiable parameter of adult physiological age, as reported by Leffelaar and Grigliatti (1984), warrants further testing. They examined the lifespan and patterns of behaviour loss of five temperature sensitive X-linked adult lethal mutants of Drosophila. One of these, adl-16tsl< appears to accelerate the normal ageing process. The ability to perform normal phototactic responses is lost first, and this is followed by a loss of geotactic response, during the reproductive phase of adult populations. Motor ability is not lost until well into the death phase of the population, the time period during which most of the population dies. Since the onset of senescence is often not clear, loss of behavioural fitness might function as a suitable bio-marker of physiological age especially during the reproductive phase, where physiological or anatomical bio-markers of age are absent. This thesis continues and expands the studies reported by Leffelaar and Grigliatti. The first premise that will be examined is that ageing is genetically programmed, and is therefore part of the developmental process. Point mutations in single genes, which increase or decrease the rate of ageing, would support this hypothesis. Three temperature-sensitive adult-lethal mutations were used, all of which were found to be alleles of the adl-16 gene in Drosophila melanogaster. Al l three alleles, adl-16tsl, adl-16ts2, and adl-16flrai were isolated from Oregon-R wild-type strain as ethyl methanesulfonate-induced X-linked point mutations in stwo separate screens, adl-16flraI was reported by Homyk and Sheppard in 1977, and adl-16tsl and adl-16ts2 were isolated by Schellenbarger and Cross in 1979 (Homyk et al, 1986). At the permissive temperature (22°C) , all the above mutants have fairly normal lifespans and behaviour. At the non-permissive temperature ( 2 9 ° C ) , all die prematurely (adl-16ts2, adl-16tsl, and adlflraI\ in order of effects from most severe to least affected). The phenotype of these alleles suggests that each is defective in a single vital gene, the function of which is necessary for normal lifespan. As well as being adult-lethal, all three mutants die as embryos or larval instars if exposed to the restrictive temperature from the deposition of the egg (Homyk et al., 1986). Also, the effect of shifting mutants to the restrictive temperature at any age leads to their demise (personal observation). In most cases premature death of flies will have nothing to do with the ageing process, but rather will be due to the elimination of some required biochemical function. But do these ts mutations affect the ageing process, or do they cause the loss of some vital function at 2 9 ° C ? If a mutation affects the rate at which ageing occurs, one would expect the same pattern of behaviour-loss or the loss of any set of bio-markers, at 22°C as at 2 9 ° C , except that at 2 9 ° C , this pattern would be compressed into a shorter time frame. This particular pattern should also be present in the control flies, Oregon-R, but should reveal a greater compression factor in mutant flies. Conversely, if the behaviour-loss patterns of a ts mutant and Oregon-R are different at 29°C as compared to 22°C, then we might conclude that this gene is not directly involved in the ageing process. The alleles named above have been examined in four ways. The longevity parameters and survival curves of each strain, and hybrids between strains, were determined for males and females at both the permissive temperature, 2 2 ° C , and the restrictive temperature, 2 9 ° C , and compared with that of wild-type Oregon-R flies. This experiment is described in Chapter 1. The second chapter describes and examines age related behaviour loss patterns for all types of flies examined in Chapter 1. Behaviours tested were geotaxis, phototaxis, and motor ability. Questions under examination here are: Can a quantifiable and repeatable behaviour pattern be demonstrated? Is there a specific pattern of age-dependent behaviour loss? Can the pattern of behaviour loss be used as a bio-marker of the ageing process? Is the pattern of behaviour-loss consistent with a mutation which accelerates the ageing process or is adl-16tsl simply a mutation which disrupts an essential function, but which has little or nothing to do with normal ageing? Chapter three examines the allelic relationship of the three genes under study, as well as their physical location on the X-chromosome. Mutant flies were crossed with flies deficient in the band 13F to 14B region, Df( 1 )sd72b26/FM y B wa, thought to be located in "the same region of the X-chromosome as the genes of interest, to find out if the genes could be uncovered, and to see what effect a single female dose of gene product would have on lifespan and behaviour. Hemizygotes were examined for changes in phenotype, anatomy, and longevity. An examination of the fertility of the alleles of adl-16ts /deficiency hybrids was also performed. In chapter four, the question of what type of tissue is affected by the adl-16ts2 m u t a t i o n is explored by the mosaic analysis of gynandromorphs of ln(l)wvC/ adl-16ts2. It was hypothesized that this mutation, which causes early death and paralysis at the restrictive temperature, might map to presumptive nervous or mesodermal tissue. From previously published data (Leffelaar and Grigliatti, 1984), it was expected that the severity of expression of the genes under study compared with the control would be: Or-R < adl-16tsl < adl-16 t s 2 with respect to lifespan, and behaviour. Where adl-16flraI would fit into this sequence was unknown prior to this study. Although this mutant was isolated because of reduction in flight ability, other adult behaviours had not previously been tested. The behaviour of females of adl-16ts2, and the hybrid females of the above alleles were not tested prior to this study. CHAPTER 1 INTRODUCTION While it is interesting to estimate the contribution of genotype versus environment on the longevity of an organism, a far more complex issue involves the interaction of these two factors in determining ageing and ultimate lifespan. The longevity of specific populations is predictable, and if it is under predominantly genetic influence, then it should be possible to select for increases or decreases in length of life. If however, intrinsic factors other than genotype, or extrinsic factors exert a greater effect, then genetic selection would not produce significant changes in longevity. Various attempts have been made to increase longevity and delay senescence in Drosophila melanogaster, for example those by Lints et al. (1979), and Clare and Luckinbill (1984). The latter demonstrate quite clearly that it is possible to select for either increased or decreased lifespan (Collatz and Sohal, 1986). The inference which can be made from this is that lifespan is under genetic control, a posit which has proved elusive to supporting documentation. The presence of homozygous mutant genes results in decreased lifespan as a rule (Arking and Clare, 1986). The alleles involved in this study decrease lifespan at the restrictive temperature. A decrease in lifespan does not necessarily support the position that ageing is under genetic control, but might do so if a pattern of behaviour-loss or other bio-markers can be demonstrated to accelerate at the same rate as the survival of the strain decreases. Such may be the case with the mutant adl-16tsl. To test the hypothesis that adl-16tsl accelerates ageing it is necessary to demonstrate that the lifespan and behaviour parameters such as those cited by Leffelaar and Grigliatti (1984), can be replicated. To this end, the experiments involving Oregon-R, (the control strain), and adl-16tsl, were repeated. In addition to this, adl-16ts2, and adl-16flrdI, alleles of adl-16tsl were tested, and the results scrutinized for any similarities of behaviour or lifespan. The expectations for adl-16flrdI were that since it was isolated in a separate screen to detect adult behaviour mutants with an emphasis on flight, its phenotype with respect to behaviour might differ from the other two alleles. The pleiotrophic nature and differences between different alleles of temperature-sensitive mutants has been reported previously (Homyk and Grigliatti, 1983) and will be discussed more fully in the next chapter. Temperatures of 2 2 ° C and 29°C were chosen for these experiments because these temperatures had been used to isolate the mutants. In addition 2 9 ° C , the restrictive temperature for the adl-16ts mutants, has no adverse effects on development, behaviour and vitality of the control strain Oregon-R (Leffelaar and Grigliatti, 1984). It is well established that different strains of adult lethal mutants have characteristic lifespans. In order to test this specific longevity with respect to the alleles of adl-16ts, and before behaviour tests were performed, it was necessary to establish survival patterns for the mutants under study. This chapter examines the survival curves for the three mutants listed above, and for their hybrids. Ultimately it is hoped that the combination of these results with those of behaviour, and other tests will generate some useful information about these phenotypes, regardless of whether that information is related to ageing. MATERIALS AND METHODS Stocks Oregon-R, a highly inbred wild-type laboratory strain of Drosophila melanogaster, was used as a control for the following experiments. Mutant stocks involved were: adl-16tsl, adl-16ts2, and adl-16flrdI, which are temperature-sensitive strains derived from Oregon-R after E M S treatment. The mutant flies exhibit relatively normal lifespans at 2 2 ° C , but die prematurely at 2 9 ° C . In some previously published literature adl-16 t s l , adl-16ts2, and adl-16flraI have been given other names, so a table listing these is shown below (Homyk et al., 1984). NAMES OF MUTANTS Current Name Former Name Isolation Name l(l)adl-16^1 S6-1 DC836 l(l)adl-16ts2 S6-2 DC359 l(l)adl-16flrdI S6-Flrd f l r d l 1 Reciprocal crosses between flies of these three mutant alleles were made to test hybrids. Virgin females for these crosses were obtained by clearing expanded stock bottles, and collecting females within the first 8 hours after eclosion. These flies were then mated with males of the same age. The following crosses were made: 1) adl-16tsl female 2) adl-16ts2 female 3) adl-16tsl female 4) ad/-7<5»2 female 5) adl-ltflrd! female 6) adl-16flrdI female <8) adl-16ts2 male <8> adl-16tsl male <g> adl-16flrdImule. <8> adl-16flrdI male <8> adl-16tsl male <8> adl-16ts2 male (adl-l6tslladl-16ts2) (adl-16ts2ladl-16^1) (adl-16*sl/adl-16flrdi) (adl-16^2/adl-16/^1) (adl-16flrdIladl-16tsl) (adl-l6f^dlladl-16^2) Progeny from these crosses were used in the survival and behaviour tests outlined below. For hybrids, where flies tested were all female, reciprocal crosses were made and tested for both behaviour and longevity to observe maternal effect (if any). Culture Conditions Standard cornmeal agar medium was used as food. The food composition was as follows: 1,000 g H2O, 76 g cornmeal, 7 g agar, 63 g dextrose, 31 g sucrose, 32 g dry brewers yeast, 8.7 g NaK tartrate, 0.54 g CaCl2, 2.4 g Tegosept, 50 mg ampicillin, and 10 mg streptomycin. A l l cultures were maintained at a constant day-night cycle (14 hours light, 10 hours dark) at either 22°C + 1°C, or 29°C ± 0 . 5 ° C , and 50-60% relative humidity. Stocks were expanded in quarter-pint bottles containing 24 ml of medium, then 20-30 pairs of males and females 1-3 days old were placed in bottles containing fresh medium. Sufficient bottles were set up to produce over 500 progeny of the same age. For most of the experiments 30 bottles were required. After 3-5 days, the adult flies were discarded and the eggs allowed to develop at 22°C. The parents used to produce progeny for experimental purposes were thus at the peak of their reproductive period to avoid the possible Lansing effect of parental age on longevity and behaviour (see Lamb, 1978). After clearing the bottles in the evening, newly eclosed adults were collected over a twelve hour period, separated by sex, and placed into 8-dram shell vials containing 6 ml of the above food. Each vial contained either 10 males or 10 females. This ensured that all flies used in experiments were virgins, of the same age, and in uncrowded conditions. Vials were selected at random, numbered, and grouped in sets of five vials (50 flies). Half the vials were used for experiments at 2 2 ° C ± 1 ° C , and the other half incubated at 2 9 ° C ± 0 . 5 ° C . A set of 50 Oregon-R flies of each sex was set up on the same day to be used as controls for each experiment. Adult lifespan was measured at 2 2 ° C ± 1 ° C and 29°C±0 .5°C . For the majority of tests, only those flies which emerged between 8 a.m. and 8 p.m. on the day of maximal eclosion were used to determine adult longevity. Some tests required the use of flies from two consecutive days, but this was avoided wherever possible. One set of flies was tested for survival only, but thereafter, additional sets of flies were tested for both survival and behaviour concurrently since no differences were noted in the survival of behaviour-tested and untested flies in the first set of behaviour trials (see Chapter 2). In those groups kept at 22°C, flies were transferred to vials with fresh medium every 2-4 days; for flies maintained at 29°C, transfers were made every 2-3 days. Flies which escaped, or were crushed during transfer were subtracted from the total possible flies for each test. Bacterial contamination of vials occurred from time to time, especially with adl-16flrdI. These vials were discarded, and the number of flies subtracted from the totals. To obtain survival curves, the number of living flies per vial was counted daily for the mutants adl-16tsl and adl-16ts2 and all hybrids at 29°C, and at each transfer for adl-16 f l r d I at 29°C and Oregon-R at both 22°C arid 29°C. The total number of flies present was summed. Flies were considered dead when they made no response to a gentle prod with a paint brush. This same brush was used to transfer flies that were living but not very mobile. Wherever possible a minimum of 200 flies (N) of each sex was used to determine longevity for a given population. The number of flies present at the beginning of each experiment is quoted, although the survival percentage and other parameters were adjusted to allow for removal of flies which escaped or died accidently. Sample sizes were smaller for some trials, and have been quoted individually. Controls of Oregon-R and both mutant parent types were used as necessary. Each experiment was repeated at least three times. Table 1-1 lists the tests made for survival and behaviour (discussed in the next chapter). In total, over 20,000 flies were tested for survival and behaviour. 21 Table 1-1 REPETITIONS OF SURVIVAL AND BEHAVIOUR TESTS 22°C 22°C 29°C 29°C Strain males females males females 1. Oregon-R 7 7 7 7 2. adl-16tsl 4 4 4 4 3. adl-16ts2 4 4 4 4 4. a d l - ^ d 1 6 6 6 6 5. adl-16tsl/adl-16ts2 3 3 6. adl-16tsl/adl-16flrdi 4 4 7. adl-16ts2/adl-16 f l rdl 3 3 8. adl-lGts^Oregon-R 1 1 9. adl-16 t s 2 /Oregon-R 1 1 10. adl-16 f l rdi/Oregon-R 1 1 11. adl-16 t s 2 1*25°C Total Experiments Performed 21 34 22 34 Number of flies per test, N=200 or greater, unless otherwise stated in the text. RESULTS Survival Curves In general four phases can be seen in any one survival curve. During the first phase, or reproductive phase (RP), flies were fertile, and most deaths were accidental. It corresponds to the first portion of the graph where few flies die, consequently it may be horizontal. The second phase revealed the onset of death, death phase onset, ( D P o n s e t ) of the population. If few flies died, this change is gradual, but if many did there is quite a sharp downturn in the graph. The onset of death may be classed as early (E), middle (M), late (L), or very late (V), representing commencement on days 0-5, 6-10, 11-18, and in excess of 18 respectively at 2 9 ° C . The figure quoted for D P onset in the Tables represents the day that the death phase onset began, averaged over the populations for that particular experiment. The next phase, called the death phase, (DP), represents the time during which most of the population died If death occured over a relatively short period of time, it would appear that it had been programmed, and the survival curve tended towards vertical. The end of the death phase occurred when the number of flies dying per day decreased significantly. The first day (average) on which this was noted has been used as the death phase end, (DP e n d) in Tables. The final phase of the graph, called the final death phase, (FDP), represents the few individuals who died later. This may occur because of the particular combination of genes present, or simply the fact that as the other flies died, less and less crowded conditions were present. Usually the final death phase represents under 5% of the population. Survival curves have been divided into four categories. Comparisons can then be made between graphs produced from data collected. At 29°C, curves with a distinct onset and end of death phase, the entire death phase occupying under 12 days are called type A curves, or rectangular curves. A population with this type of curve clearly manifests senescence. When the graph appears more rounded, with less distinct onset of the death phase, a death phase lasting for 12-18 days and therefore being less steep, this is called a type B curve. Most organisms manifest this type of survival curve (Rockstein and Miquel, 1973). If the onset of the death phase is unclear, and appears to merge with the reproductive phase in a long steep downward trend with mild slope lasting 18-24 days, the survival curve is said to be type C. In the final type of curve, type D, the onset of death, a death phase lasting in excess of 24 days, and death phase end are impossible to distinguish. The latter two types of curves reveal a fairly constant overall mortality, where no real senescence is exhibited and death could even be considered to be a random event (Rockstein and Miquel, 1973). The survival curves presented below are those from one experiment unless otherwise noted. An attempt has been made to classify them according to type, and make inferences about senescence. Conversion factors (defined below) used on graphs between temperatures reflect those for that particular experiment so that coincident graphs can be produced. Pooled data was used to calculate the figures given in tables, so these results represent averages for a large number of flies over a minimum of three trials each. Composite survival curves showing both sexes and/or temperatures are given to clarify comparisons. Data quoted in tables has been included to enable comparisons to be made regarding the average parameters of survival for each strain of flies tested. The figures were calculated as follows: Lifespan Mean (L S Y) is the average age of flies at death. Since flies maintained at 22°C were examined every 2-4 days, and adl -16 f l r d I and Oregon-R were examined every 2-3 days at 2 9 ° C , rather than estimating the death pattern during these time intervals, flies were considered to have died on the day that the culture was examined. Al l data were calculated in this manner, meaning that there will be a slight increase in values for several parameters, but the data are internally consistent so valid comparisons can be made between data. The average L S X for each trial was found, and then these means for replicate experiments were averaged. Death Phase Mean (DPvJ The average age at death of individuals dying between the death phase onset and end was calculated. The day of death was assumed to be the day of examination of the culture and as explained above, this will cause a slight increase in values obtained. This calculation usually involved in excess of 80% of the flies in any one test. Means from separate replicate tests were then averaged. Death Phase Onset (DPonset). For each trial, the day of onset of death of the population was recorded. These values were averaged for replicate experiments to obtain the mean. Death Phase End (DP P nd). This average was calculated in a similar manner, except that the day that the death phase ended was used in the calculation. D-20. D-50. D-95 . and D-100 represent the days posteclosion when 20, 50, 95, or 100% of the population had died. Average figures are quoted in the tables. The Slope of the Death Phase ( D P s i o p e ) represents the rate at which death occurs; the higher this figure, the more rapid the death rate. This figure was calculated using the death phase onset and end as start and end-points. The percentage of flies alive at the time of D P end was subtracted from that of the DP0nset and this figure was divided by the number of days between DP onset and end. Average D P slopes are given and represent average percentage of flies dying per day. Conversion Factors Since the lifespan of flies is lower at higher temperature, conversion factors for the rate of living were calculated. Values for L S x , DPx, DPonset, D P e n d , D-20, D-50, D-95, and D-100 for Oregon-R at 22°C for males or females were divided by the corresponding values from 29°C experiments. This yielded a set of conversion factors for each sex. Each set of figures was averaged to obtain the general conversion factors to be used for comparison of survival of Oregon-R flies of each sex at each temperature. For Oregon-R females, a factor of 2.32 was found, and for males a factor of 2.49 was obtained between the temperatures 22°C and 29°C. This means that at 29°C, one day of life for a female fly is equivalent to 2.32 days at 22°C, and one day at 29°C for a male fly is equivalent to 2.49 days at 22°C. These conversion figures have been used subsequently in this thesis to compare lifespans of Oregon-R flies with each other and the lifespans of mutant flies. They were also used to convert survival curves between 2 2 ° C and 2 9 ° C for all strains tested. Individual conversion factors were also calculated for each mutant, and used in producing the coincident graphs shown below. The only graphics program available to me at the time of writing this thesis did not permit the use of two X axes, so the number of days at 2 9 ° C was multiplied by the appropriate male or female conversion factor, so that comparative graphs could be drawn. The second abscissa (not depicted) is understood to apply to the 29°C flies. It is unfortunate that the graphics program used did not have the facility to show the second X axis, but conversion factors are given with each graph so that comparisons can be made. OregonrR Adult Longevity. Since Oregon-R was used as a control stock for all the following experiments, great care was exercised in examining its longevity and behaviour because this would provide a hallmark against which differences observed in the test strains would be compared. At 22°G the mean lifespan of females exceeded that of males by 5.9 days, and in general lifespan parameters were more consistent over all results calculated, which can be seen from the low figure for the standard deviations in Table 1-2. The average lifespan of males and females differed by a factor of 1.06. There was less variation in all lifespan parameters at 29°C than at 2 2 ° C , and female lifespan exceeded that of males by 4.9 days, lifespans differing by a factor of 1.11. The lower variation at 2 9 ° C could be expected from the fact that the environment of the 29°C incubator with respect to both temperature and humidity was much more uniform than that of the laboratory, which acted as the 22°C chamber. The values 1.06 and 1.11 obtained by comparing male and female lifespans at the two temperatures used are very similar, therefore it does not seem that the longevity of the adult of one sex is more affected by temperature than the other in Oregon-R. Figures 1-1 and 1-2 show survival curves for Oregon-R males at 22°C and 2 9 ° C respectively. Although survival was recorded every 2-4 days, only every second or third point is shown on the 22°C survival curves to prevent obscuring the graphs. This was done for all 22°C graphs to follow. Figure 1-3 demonstrates that when the graph at 29°C is adjusted to compensate for the rate of living by multiplication with the conversion factor 2.5 (the value for this experiment), the two graphs are extremely similar, and could be said to coincide. Figures 1-4 and 1-5 are the survival curves for female flies at 22°C and 2 9 ° C respectively. Figure 1-6 shows the coincident graph for flies at 22°C and 29°C after the 29°C figures have been adjusted for the rate of living by a factor of 2.3. Figures 1-7 and 1-8 show the separate survival curves for males and females of Oregon-R at 22°C, and 29°C respectively. Figure 1-9 contains graphs of males and females at 22°C as well as males and females at 29°C adjusted for the rate of living. This set of graphs may be difficult to see. What is important is that the 22°C and 2 9 ° C graphs are essentially coincidental for each sex, and that the general shape is consistent; thereby demonstrating that temperature seems to have a similar effect on both male and female flies. Figure 1-10 depicts the above graphs without symbols, and with the scale for graphs at 29°C unadjusted for the rate of living, permitting comparison of the original data. A l l four curves have their own individual characteristics, but are quite similar in shape. In general, few flies died during the reproductive phase of the lifespan, and when the death phase was reached, most flies died within a few days. The curves are sigmoidal in shape, (type A), with a distinct reproductive phase and death phases including a very late (V) onset of death, which might be suggestive of genetic programming. In general, the degree of variability was low, which means that Oregon-R can be used as a control stock for this type of experiment. The stability of Oregon-R is also fortunate because the mutant alleles were derived from Oregon-R stock, and its genetics and behaviour have been extensively studied, and are well characterized. Graphs showing males or females at both experimental temperatures are shown so that comparisons can be made concerning^the effects of temperature. Once the conversion factor is used to expand the days at 2 9 ° C to compensate for the higher rate of living at the higher temperature, it can be seen that the curves parallel one another (Figures 1-3, 1-6 and 1-9), meaning that a temperature of 29°C is not deleterious to these flies. Al l graphs are results from single experiments. Average results are calculated for the replicates of experiments, and these numbers are shown in Tables 1-2 and 1-3 along with calculations for standard deviations. Figure 1-1 OREGON-R MALES Survival Curve at 22°C Males (22°C) N=200 Age (Days) at 22°C Figure 1-2 OREGON-R MALES Survival Curve at 29°C Males (29°C) N=250 0 10 20 30 40 50 60 Age (Days) at 29°C 32 Figure 1-3 OREGON-R MALES Survival Curves at 22°C and 29°C Males (22°C) N=200 Males (29°C) N=250 Age (Days) at 22°C Note: 29°C days have been adjusted by Conversion Factor 2.5 33 Figure 1-4 OREGON-R FEMALES Survival Curve at 22°C Females (22°C) N=200 Age (Days) at 22°C 34 Figure 1-5 Age (Days) at 29°C 35 Figure 1-6 OREGON-R FEMALES Survival Curves at 22°C and 29°C co > 120 100 --•a— Females (22°C) N=200 -•— Females (29°C) N=200 100 200 Age (Days) at 22°C Note: 29°C days have been adjusted by Conversion Factor 2.3 36 Figure 1.-7 OREGON-R MALES AND FEMALES Survival Curves at 22°C Males (22°C) N=200 Females (22°C) N=200 Age (Days) at 22°C Figure 1-8 38 Figure 1-9 OREGON-R MALES AND FEMALES Survival Curves at 22°C and 29°C 120 100 Co" 80 -60 -40 -20 -- 0 — Males (22°C) N=200 •+— Males (29°C) N=250 -•— Females (22°C) N=200 •+— Females (29°C) N=200 1 00 Age (Days) at 22°C 200 Note: 29°C days have been adjusted by Conversion Factor 2.5 (males), and 2.3 (females). Figure 1-10 Table 1-2 LONGEVITY OF OREGON-R ADULTS MAINTAINED A T 22°C or 29°C POSTECLOSION Males 22°C ( X ± SD) Females 22°C ( X ± SD) Males 29°C (X ± SD) Females 29°C (X ± SD) L S X 97.3 ± 3.3 103.2 ± 0 . 3 41.6 ± 0 . 9 46.2 ± 1.7 D P X 105.1 ± 0 . 6 109.7 ± 0 . 5 42.8 ± 1.0 47.5 ± 1.4 DP onset 80 ± 3 . 5 89.5 ± 5 . 5 36 ± 1.0 37.3 ± 2 . 3 D-20 79 ± 6 . 2 94.5 ± 0 . 5 35 ± 3.2 42.3 ± 2 . 4 D-50 103.3 ± 3 . 1 108.5 ± 1.5 42.3 ± 1.5 47 ± 1.7 D-95 126 ± 1.0 127.7 ± 1.5 49.7 ± 0 . 3 53.3 ± 1.5 DP end 123.6 ± 4.4 124.5 ± 1.5 48 ± 1.5 53 ± 1.2 D-100 142 ± 1.5 138.5 ± 0.5 51.7 ± 0 . 7 58.6 ± 1.4 DPslope 1.8 ± 0 . 2 2.13 ± 0 . 1 6.9 ± 0 . 2 5.7 ± 0 . 5 The wild-type strain, Oregon-R was used as a control for all experiments. DP slope is given as % death per day. Al l other data are given in days ± standard deviation, obtained from three separate experiments, n=3 (total # of flies = 1440 males, 1620 females). L S X : Life-span mean; D P X : death phase mean; DPonset and DPend: onset and end of death phase, respectively; D-20, 50, 95, 100: time at which 20, 50, 95, and 100 % of the population had died, respectively. Table 1-3 CONVERSION FACTORS FOR DIFFERENCES IN RATE OF LIVING OF OREGON-R at 22°C and 29°C Males Females L S X 2.3 2.2 DP X 2.5 2.3 DPonset 2.2 2.4 D-20 2.3 2.2 D-50 2.4 2.3 D-95 2.5 2.4 DPend 2.6 2.3 D-100 2.7 2.4 Average 2 . 4 9 ± 0 . 1 7 2 . 3 2 ± 0 . 0 8 Each number represents the ratio of the respective 2 2 ° C and 2 9 ° C values for longevity. One 29°C day for females = 2.32 days at 22°C. One 29°C day for males = 2.49 days at 22°C. adl-16tsl Longevity The earliest result to be noted with this mutant was an obvious change in phenotype at the restrictive temperature. Within two days swollen abdomens were noted in both male and female flies. The proportion of flies with abdomenal distension, the onset of this phenomenon, and its expression were seen to be greater in the female than in the male fly, but by day three almost all flies exhibited this phenomenon, and in some cases abdomens were so swollen that it became difficult to aspirate flies for motor experiments. Swollen abdomens were noted to be filled either with gas or fluid. By day five, swelling in the abdomens of the males diminished, the flies appeared weak and began to walk in wobbly circles (mostly anticlockwise). Approximately one third of the males were affected by day six. On day seven, the same behaviour was noted in females, along with a reduction in swelling of the abdomen. This behaviour preceded death by approximately one day. By day three, almost all flies also had permanently extended probosci. I later related the extension of the proboscis to paralysis, and thought that adl-16tsl flies were unable to retract the organ at 2 9 ° C . I also propose that the impairment of the proboscis has an effect on the ability of these flies to absorb food and water. It was noted that in dead flies the proboscis was in the retracted position. Swollen abdomens and extended probosci were also observed in adl-1 6 t s l flies maintained at 2 2 ° C , the earliest being on day 22 posteclosion in a female fly. At the time of onset of the death phase many male and female flies were noted to exhibit swollen abdomens, as well as excessive grooming, but the phenomenon was not universal, as it was at 2 9 ° C . The proportion of flies affected increased to approximately 20% toward the end of the death phase. It should be noted that swollen abdomens and extended probosci were also seen on occasion in populations of Oregon-R flies but in extremely low frequency. As can be seen from Figures 1-11 and 1-15, the survival curves of adl-16tsl males and females respectively at 22°C, and also Table 1-4, the lifespan parameters of this mutant fly were reduced minimally compared with that of Oregon-R. The onset of the death phase was not as sharp, and the slope of the death phase was slightly increased. Overall the graphs for survival of this mutant fly were quite similar to those of wild-type. At 2 9 ° C , however, the lifespan of adl-16tsl was greatly reduced. In fact lifespan was reduced almost exactly five-fold relative to Oregon-R. Figures 1-12 and 1-16 show typical survival curves for males and females at this temperature: Figures 1-13 and 1-17 show survival curves for males at 22°C and 29°C, and females at 22°C and 29°C respectively. The 29° curves in these graphs have been converted by the factors for Oregon-R rate of living differences between these temperatures. Male days at 2 9 ° C have been multiplied by 2.5, and female days at 29°C have been increased by a factor of 2.3. The deleterious effect of the restrictive temperature on lifespan can be clearly seen. The survival curves for adl-16tsl at 29°C were of type A with death phase onset in the middle range (M) at 2 9 ° C again indicating the influence of senescence. To test whether an accelerated ageing effect could be demonstrated, the days at 29°C were increased by a factor of 10.2 for males and 11.6 for females, the conversion factors calculated for adl-16tsl, producing the coincident graphs depicted in Figures 1-14 and 1-18. Figure 1-11 Age (Days) at 22°C Figure 1-12 adl-16ts1 Males Survival Curve at 29°C Males (29°C) N=200 0 2 4 6 8 10 12 Age (Days) at 29°C 46 Figure 1-13 120 100 > adl-16 ts1 Males Survival Curves at 22°C and 29°C - G — Males (22°C) N=140 — Males (29°C)N=140 Age (Days) at 22°C Note: 29°C days have been adjusted by Conversion Factor 2.5 Figure 1-14 adl-16 ts1 Males Survival Curves at 22°C and 29°C 120 — Males (22°C) N=140 -*— Males (29°C) N=140 0 20 40 60 80 100 120 Age (Days) at 22°C Note: 29°C days have been adjusted by Conversion Factor 10.2. 48 Figure 1-15 adl-16 ts1 Females Survival Curve at 22°C 120 20 40 60 Age (Days) at 22°G Females (22°C) N=200 120 Figure 1-16 adl-16 ts1 Females Survival Curve at 29°C 49 120 Females (29°C)N=200 50 Figure 1-17 adl-16 ts1 Females Survival Curves at 22°C and 29°C 120 100 co > CO — Females (22°C) N=200 •+— Females (29°C) N=200 20 40 60 80 100 120 Age (Days) at 22°C Note: 29°C days have been adjusted by Conversion Factor 2.3 51 Figure 1-18 adl-16 ts1 Females Survival Curves at 22°C and 29°C 120 — Females (22°C) N=200 -•— Females (29°C) N=200 o Survival (Days) at 22°C 1 o o 200 Note: 29°C days have been adjusted by Conversion Factor 11.6 Table 1-4 LONGEVITY OF adl-16^ A D U L T S MAINTAINED A T 22°C OR 29°C POSTECLOSION Males 22°C CX ± SD) Females 22°C (X ± SD) Males 29°C (X ± SD) Females 29°C (X ± SD) L S X 80.8 ± 0.7 99.2 ± 0.8 8.5 ± 0.7 9.1 + 0.3 DP X 83.1 ± 2.3 101.2 ± 0.9 8.3 ± 0.6 9.1 ± 0.2 DPonset 49.7 ± 1.3 54.0 ± 4.0 4.2 ± 0.4 4.3 ± 0.9 D-20 66.7 ± 3.9 87.5 ± 2.5 6.4 ± 0.7 7.4 ± 0.1 D-50 81.3 ± 2.4 102 ± 1.0 8.1 ± 1.0 9.3 ± 0.4 D-95 102.7 ± 4.2 114.5 ± 0.5 10.1 ± 1.0 11.1 ± 0.9 DPend 101 ± 3.7 114 ± 1.0 10 ± 0.7 11 ± 0.0 D-100 114.7 ± 1.3 119 ± 1.0 11.5 ± 0.9 12.7 ± 0.9 PD slope 1.87 ± 0.1 3.29 ± 0.6 22.2 ± 0.7 22.7 ± 4.1 Data are given in days ± standard deviation, obtained from three separate experiments n=3 (total# of flies = 1240 males, 1426 females). L S X : Life-span mean; D P X : death phase mean; DPonset and DPend' onset and end of death phase, respectively; D-20, 50, 95, 100: time at which 20, 50, 95, and 100 % of the population had died, respectively. D P s i 0 p e is given as % death per day. Table 1-5 CONVERSION FACTORS FOR DIFFERENCES IN R A T E OF LIVING OF adl-W*1 A T 22°C AND 29°C Males Females L S X 9.4 10.9 PD X 10.0 11.2 DPonset 11.7 12.6 D-20 10.3 11.9 D-50 10.0 10.9 D-95 10.2 10.3 DPend 10.1 10.4 D-100 10.0 9.4 Average 1 0 . 2 ± 0 . 6 1 0 . 9 ± 0 . 9 Each number represents the ratio of the respective 2 2 ° C and 2 9 ° C values for longevity. One day at 29°C is equivalent to 10.2 days at 22°C for males, and 10.9 days at 22°C for females. adl-16ts2 Longevity Most debilitating effects on these flies at 29°C were obvious within 24 hours. After one day of exposure to this temperature few flies could stand. A few managed to climb a little, and a few to walk shakily, but the majority were found lying on their sides, or more frequently on their wings, with their legs folded and severely impaired. A variety of small twitches of head, feet, and abdomen or small wing movements confirmed life in each fly. When touched with a brush, they would kick, but could not stand even if placed right side up after they had been found prostrate. It was recognized that these flies would die quickly, since they would be without nourishment. Swollen abdomens and extended probosci were not noted. At 2 2 ° C the above changes were not noted. The only general observations were that immediately prior to the death phase many males appeared to be preoccupied with rubbing themselves with their legs, and several females exhibited swollen abdomens. One test of survival of male flies at 25°C was made towards the end of all the experiments, to see if a less debilitating effect than that at 29°C could be obtained for use in behaviour tests. Since no other tests were made at this temperature, little is available to compare , it with. Hovever the preliminary indication is that this temperature would be useful for experiments with this mutant, so the survival curve is shown in Figures 1-23 and 1-24. A comparison between flies at 2 2 ° C , 2 5 ° C and 2 9 ° C can be seen in Table 1-8. The following observations were made. On day 1, the flies were very active, but by day 2 they had become unco-ordinated, twitchy and bump sensitive, and mostly remained on the bottom of the vial. By day 7 many were unable to stand or walk. By examining Figures 1-19 and 1-25, as well as Table 1-6 it can be seen that at 22°C mean lifespan for adl-16ts2 is shorter than either adl-16tsl or Oregon-R. Average female lifespan of 72.4 days exceeded that of average male lifespan (64.9) by 7.5 days. The mean of the death phase was lowered by similar amounts. The onset of the death phase was earlier and more rapid as was the rate of death, shown by the increase in slope of the death phase. In general, even at 22°C, adl-16ts2 dislpay slightly reduced viability compared with Oregon-R. Figures 1-20, 1-26 and Table 6 clearly show that at 29°C, changes in all parameters for adl-16ts2 were enormous. Increased temperature has a totally debilitating effect on these flies. Survival curves were all type A with early onset of death (E). The slope of these curves was extremely high indicating that death was rapid at this temperature. In fact lifespan was reduced approximately ten-fold relative to Oregon-R. Figures 1-22 and 1-28 show the comparative effects of temperatures 22°C and 29°C on adl-1 <5"2. The 29°C days have been converted for the mutant flies by (Oregon-R) factors 2.5 for males and 2.3 for females. However when the conversion factors determined for adl-16ts2 were used, 23.2 for females and 23.5 for males, the coincident graphs in Figures 1-21 and 1-27 can be seen. If the conversion factor 3.5 is applied to the days at 29°C for these flies, the curves at 25°C and 29°C also become coincident. Thus it could be argued that this mutation also accelerates lifespan, albeit at a faster rate than adl-16tsl. If the behaviour parameters show the same pattern, possibly this mutation could also be said to accelerate ageing. In general, the conversion factors for rate of living adjustments in this mutant were very consistent (see Table 1-7), and the variability of results shown by the standard deviations was low. 56 Figure 1-19 Age (Days) at 22°C Figure 1-20 adl-16 ts2 MALES Survival Curve at 29°C 40 H 20 H Males 29°C N=200 Age (Days) 58 Figure 1-21 adl-16 ts2 MALES Survival Curves at 22°C or 29°C 120 -*>— Males (22°C) N=200 — Males (29°C) N=200 20 40 60 80 100 120 Age (days) at 22°C Note: 29°C days were adjusted by Conversion Factor 23.5 59 Figure 1-22 adl-16 ts2 Males Survival Curves at 22°C and 29°C 120 Males (22°C) N=200 Males (29°C) N=200 r 80 1 00 40 60 Age (Days) at 22°C Note: 29°C days were adjusted by Conversion Factor 2.5 Figure 1-23 Age (Days) at 25°C Figure 1-24 adl-16 ts2 Males Survival Curves at 22°C, 25°C and 29°C 120 T 100 -80 -CO •| 60 3 CO 40 -Males (29°C) N=200 Males (22°C) N=200 Males (25°C) N=100 20 -—i— 20 40 60 80 100 Age (Days) at 25°C Figure 1-25 Figure 1-26 64 Figure 1-27 adl-16ts2 FEMALES Survival Curves at 22°C or 29°C 120 -s— Females (22°C) N=200 -•— Females (29°C) N=200 20 40 60 80 100 120 Age (Days) at 22°C Note: 29°C days were adjusted by Conversion Factor 23.15 Figure 1-28 Note: 29°C days were adjusted by Conversion Factor 2.32 Table 1-6 LONGEVITY OF adl-16ts2 A D U L T S MAINTAINED A T 22°C OR 29°C POSTECLOSION Males Females Males Females 22°C 22°C 29°C 29°C (X + SD( (X ± SD) (X ± SD) (X ± SD) L S X 64.9 ± 7.5 74.2 ± 11.9 2.9 ± 0.1 3.2 ± 0.2 DP X 67.0 ± 6.7 76.9 ± 12.9 2.9 ± 0.1 3.2 ± 0.2 DPonset 50.3 ± 2.2 53.7 ± 8.9 2.0 ± 0.0 2.0 ± 0.0 D-20 53.0 ± 7.5 54.7 ± 12.3 2.2 ± 0.5 2.5 ± 0.4 D-50 65.0 ± 6.7 72.3 ± 10.1 2.7 ± 0.4 2.9 ± 0.3 D-95 84.3 ± 8.4 89.3 ± 11.9 3.6 ± 0.4 4.0 ± 0.3 DPend 80.3 ± 7.9 88.0 ± 10.7 3.5 ± 0.5 4.0 + 0.0 D-100 93.7 ± 7.6 95.3 + 10.2 3.8 + 0.4 4.7 ± 0.5 DPslope 2.6 ± 0.4 2.9 ± 0.78 48.6 ± 2.4 45.9 ± 1.6 Data are given in days ± standard deviation, obtained from three separate experiments n=3 (total # of flies = 1146 males, 1130 females). L S X : Life-span mean; D P X : death phase mean; D P o n s e t and DPend: onset and end of death phase, respectively; D-20, 50, 95, 100: time at which 20, 50, 95, and 100 % of the population had died, respectively. DP s i 0 p e , the rate of death of flies per day. Table 1-7 CONVERSION FACTORS FOR DIFFERENCES IN R A T E OF LIVING OF adl-16ts2 at 22°C and 29°C Males Females L S X 22.1 23 DP X 22.8 23.8.0 DPonset 23.5 26.8 D-20 23.8 21.6 D-50 24.5 24.9 D-95 23.8 22.6 DPend 22.9 22.0 D-100 24.9 20.1 Average 23.5+0.9 2 3 . 1 ± 2 . 0 Each number represents the ratio of 22°C and 29°C values for longevity. Table 1-8 LONGEVITY OF adl-16 t s 2 M A L E S MAINTAINED A T 22°C, 25°C OR 29°C POSTECLOSION. Temperature 22°C 25°C 29°C L S X 64.9 9.7 2.9 DP X 67.0 9.8 2.9 DPonset 47.0 7.0 2.0 D-20 53.0 8.0 2.2 D-50 65.0 9.3 2.6 D-95 84.3 10.9 3.6 DPend 80.3 11.0 3.5 D-100 93.7 12.0 3.8 DPsloDe 2.6 19.5 48.6 Data are given in days ± standarc deviation, obtained from one experiment at 25°C, total # of flies = 100 males, L S X : Life-span mean; D P X : death phase mean; DPo nset and D P e n d : onset and end of death phase, respectively; D-20, 50, 95, 100 : time at which 20, 50, 95, and 100 % of the population had died, respectively. D P s i 0 p e » the rate of death of flies per day. adl-16flrdI Longevity Of all the strains used in these studies, adl-16flrdI proved most troublesome and variable. At 22°C, many black pupae were seen in stock bottles, indicating a lowered viability even at this temperature. Vials of flies frequently became contaminated with bacteria, and had to be discarded. Whether the remaining flies were affected is unknown, but since other strains of flies tested did not show this level of contamination, I conclude that these flies may be susceptible to disease, and have contaminated their environment, rather than environmental bacteria affecting them. Flies were slower in movement generally, and tended to hop rather than fly. As early as day 10 after eclosion several females exhibited swollen abdomens and proboscis extension to a small degree. The number of individuals affected increased slowly throughout lifespan. At 2 9 ° C , swollen abdomens were noted within 24 hours of exposure to increased temperature. By the second day over 50% of flies exhibited this trait, which continued and increased both in number of flies affected and severity. Proboscis extension corresponded with abdomen extension. The abdomens became hugely swollen, and almost transparent. It was also noted that at this temperature some flies held their wings up at an unusual angle. At both temperatures there was a great deal of variation of experimental results with this stock, which explains the larger number of replicate experiments. It seemed that whenever I repeated the experiments a different result was obtained. However when these results were averaged, and analysed, a clear picture was seen, and was surprisingly consistent with respect to conversion factors (see Table 1-9). Table 1-8 and the survival curves in Figures 1-29 and 1-33 at 22°C, show the death phase onset is variable and slow, tending to merge with the death phase. Graphs are mostly of type C, with a slope close to of 2.1 for females, and 1.9 for males. Males lived slightly longer than females, as can be seen from the means for lifespan and death phase. Lifespan means for males and females were, 54.4 and 53.7 days respectively, and the means of the death phase were 58.27 days for males and 55.6 days for females. Lifespan was near half that of Oregon-R. By examining the survival curves in Figures 1-30 and 1-34 and Table 1-8 at 2 9 ° C , it can be seen that the death phase onset was intermediate (M), and the survival curve Type A. Female lifespan exceeded that of males by 3.47 days, and death phase by 3.75 days on average respectively. The conversion factors used to adjust the rate of living between 22°C and 29°C were consistently around 5 for males, and 4 for females, see Figures 1-31 and 1-35. The high figures for standard deviation reflect the extreme variability of the stock, as do the survival curves. The fact that the survival curves adjusted for rate of living do not coincide is also not surprising. Averaged curves obviously would have shown a closer fit, but would not have demonstrated the variable nature of the survival curves for this mutant as effectively as those presented, which are from one set of data. Figure 1-29 adl-16 flrdl Males Survival Curve at 22°C Males (22°C) N=200 Age (Days) at 22°C Figure 1-30 Age (Days) at 29°C 72 Figure 1-31 adl-16 flrdl Males Survival Curves at 22°C and 29°C CO > 3 CO 120 100 - Q — Males (22°C) N=200 — Males (29°C) N=200 T 0 20 40 60 80 100 120 Age (Days) at 22°C Note: 29°C days were adjusted by Conversion Factor 5. Figure 1-32 adl-16ts flrdl Survival Curves at 22°C and 29°C 120 Males (22°C) N=200 Males (29°C) N=200 Age (Days) at 22°C Note: 29°C days were adjusted by Conversion Factor 2.5. Figure 1-33 Figure 1-34 Figure 1-35 77 Figure 1-36 adl-16 ts flrd 1 FEMALES Survival Curves at 22°C and 29°C ra > 3 CO 120 100 Female (22°C) N=200 • — Females (29°C) N=200 T 0 20 40 60 Age (Days) at 22°C Note: 29°C days were adjusted by Conversion Factor 2.3. Table 1-9 LONGEVITY OF adl-16flrdI ADULTS MAINTAINED A T 22°C OR 29°C POSTECLOSION Males 22°C (X ± SD) Females 22°C (X ± SD) Males 29°C (X ± SD) Females 29°C (X ± SD) L S X 54.4 ± 10.6 53.67 ± 1 6 . 3 10.8 ± 1.0 14.3 ± 1.6 DPx 58.3 ± 11.0 55.58 ± 1 7 . 7 10.9 ± 1.2 14.7 ± 1.8 DPonset 35.7 ± 8.2 32.75 ± 2 2 . 7 5.5 ± 2.1 8.8 ± 2.3 D-20 35.7 ± 12.4 35.5 ± 20.9 7.6 ± 2.5 8.9 ± 1.9 D-50 54.7 ± 11.6 49.0 ± 21.6 11.2 ± 2.4 13.3 ± 2.2 D-95 76.0 ± 6.4 83.5 ± 4.7 15.5 ± 2.4 18.1 ± 1.9 DPend 74.7 ± 9.4 85.0 ± 4.9 17.8 ± 2.3 19.3 ± 3.7 D-100 83.3 ± 3.1 88.5 ± 5.9 18.0 ± 1.2 22.5 ± 1.8 DPsloDe 2.1 ± 0.3 1.91 ± 1.0 9.5 ± 1.3 9.0 ± 2.2 Data are given in days ± standard deviation, obtained from three separate experiments n=4 (total# of flies = 1400 males, 1600 females). L S X : Life-span mean; D P X : death phase mean; DP o nset and DPend^ onset and end of death phase, respectively; D-20, 50, 95, 100: time at which 20, 50, 95, and 100 % of the population had died, respectively. DP s i 0 pe is given as % death per day. Table 1-10 CONVERSION FACTORS FOR DIFFERENCES IN R A T E OF LIVING OF adl-16ftrdi at 22°C and 29°C Males Females L S X 5.0 3.8 DP X 5.3 3.8 DPonset 6.5 3.7 D-20 4.7 4.0 D-50 4.9 3.7 D-95 4.9 4.6 Dpend 4.2 4.4 D-100 4.6 3.9 Average 5.0 ± 0.6 4.0 ± 0.3 Each number represents the ratio of the respective 22°C and 29°C values for longevity. Longevity of Hybrids Between Strains After examining hybrids of all possible combinations of mutant alleles, it was found that there was no significant difference in either survival or behaviour patterns for reciprocal crosses. Values for X 2 fell between 0.006 and 0.05, meaning that differences such as those found in the results could be expected to occur in more than 50% of similar experiments. Maternal effect was therefore excluded as a possibility. Only one set of survival curves, and behaviour patterns (see Chapter 2) are presented here. Data have been pooled for the statistical analyses in Table 1-11 and Table 1-12. The controls used in these experiments have also been pooled, and are used for comparison with hybrid data. These data are presented in Chapter 3 with deficiency data. It is interesting to note how similar this data is to that quoted earlier. Flies in the latter sample were not tested for behaviour. Therefore, testing for ability to perform defined behaviours apparently has no adverse (nor beneficial) effect on longevity. At 22°C the survival of the hybrid adl-16tsl I adl-16ts2 was almost identical to that of the adl-16tsl homozygote, which, in turn was very similar to Oregon-R , as expected. The hybrid of adl-16tsl/adl-16flrdI was intermediate between females of the parent strains. This is not surprising, because adl-16 flrdI lifespan is quite reduced with respect to Oregon-R and adl-16tsl. An unexpected result was that the hybrid adl-16ts2ladl-16flrdI had the greatest longevity of all. Since females of the parent types had the shortest lifespans, it was expected that this hybrid would also have its lifespan curtailed. The result may be due to heterosis or gene interaction, the two being difficult to distinguish. Since survival curves of all homozygotes of these alleles have been presented earlier, one graph only, Figure 1-37 depicts the survival curves of all three hybrids of adl-16ts at 2 2 ° C . Relevent data is presented in Table 1-11. At 2 9 ° C the survival curve of adl-16ts2ladl-16flrdI was almost exactly midway between those of homozygous females of the parent types (see Figure 1-38). The distances between curves were calculated at D-20, D-50 and D-95 and averaged. The hybrid flies were 50.3% longer lived than adl-16ts2, and had 49.7% shorter lives than adl-16flrdI. For adl-16tslladl-16ts2, the lifespan at 29°C was also intermediate. Comparison of the D-20, D-50, and D-95 values yielded differences of 52.3% from adl-16ts2, and 47.3% from adl-16tsl (see Figure 1-39). Such values would be expected from hybrids of hypomorphs lacking complementation, and so these mutants were confirmed to be alleles. The lifespan of adl-16tslladl-16flrdI was unusual relative to the two parent types. Between D-20 and D-50, it was reduced even compared with adl-16tsl, the shorter lived parental strain. Between D-50 and D-95, the survival curves cross, and the latter part of the lifespan of this hybrid follows more closely that of the longer lived parent strain, adl-16flrdI, but it could be said to be intermediate for this portion of lifespan. The survival curve is shown in Figure 1-40. The thought occurs that the change in the curve is about the time adl-16tsl flies die (10-12 days), so it might be possible that the corresponding gene product is non functional after that. Figure 1-41 shows all hybrids of adl-16ts, and 1-42 depicts the homozygote females for comparison. Figure 1-37 adl-16 ts HYBRID FEMALES Survival Curves at 22°C adl-16ts2/adl-16flrdl N=200 adl-16 ts1/adl-16 ts2 N=200 adl-16ts1/adl-16flrdl N=382 0 10 20 30 40 50 60 70 80 90 100110120130 Age (Days) at 22°C 83 Figure 1-38 adl-16 ts2 /adl-16 flrdl HYBRIDS adl-16 ts2, adl-16 flrdl HOMOZYGOTES Survival Curves at 29°C Age (Days) at 29°C 8 4 Figure 1-39 adl-16 ts1/adl-16 ts2 HYBRIDS adl-16 ts1, adl-16 ts2 HOMOZYGOTES Survival Curves at 29°C adl-16 ts1/adl-16 ts2 N=600 adl-16 ts1 Homozygote N=559 adl-16 ts2 Homozygotes N=525 0 2 4 6 8 10 12 14 Age (Days) at 29°C 85 Figure 1-40 adl-16 ts1/adl-16 flrdl HYBRIDS adl-16 ts1, adl-16 flrdl HOMOZYGOTES Survival Curves at 29°C 0 2 4 6 8 10 12 14 16 18 20 22 24 Survival (Days) at 29°C 86 Figure 1-41 Age (Days) at 29°C 87 Figure 1-42 120 -i 100 -(0 > CO adl-16 ts HOMOZYGOTE FEMALES Survival Curves at 29°C adl-16ts1 Homozygotes N=559 • — adl-16 ts2 Homozygotes N=525 adl-16 flrd I Homozygotes N=600 4 6 8 10 12 14 16 18 20 22 24 Age (Days) at 29°C 88 Table 1-11 LONGEVITY OF ADULT HYBRID FEMALES MAINTAINED A T 22°C POSTECLOSION. HYBRID \adl-16tslladl-16flrdI adl-16ts2ladl-16flrdI adl-16^1! adl-16^2 Number 382 200 200 L S X 81.9 110.8 99.23 DP X 86.1 112.4 102.7 DPonset 6 1 80 60 D-20 70.5 103 88 D-50 84.5 112 103 D-95 94 122 119 DPend 96 122 119 D-100 101.5 129 127 DPslope 2.5 2.1 1.5 Data are given in days obtained by pooling the results from separate experiments. L S X : Life-span mean; D P X : death phase mean; DP o n set and DP e nd: onset and end of death phase, respectively; D-20, 50, 95, 100: time at which 20, 50, 95, and 100 % of the population had died, respectively. D P s i 0 p e is given as % death per day. Table 1-12 LONGEVITY OF ADULT HYBRID FEMALES MAINTAINED A T 29°C POSTECLOSION. HYBRID adl-16tsl/adl-16flr<11 adl-16^2/adl-16f^di adl-16^ 1 adl-16^2 Number 888 641 600 L S X 9.3 7.5 6.5 DP X 9.3 7.2 6.4 DPonset 1 4 3 D-20 6.7 5,3 4.8 D-50 8.7 7.0 6.2 D-95 15.3 10.7 7.7 DPend 15 10 8 D-100 22.0 14.0 10.0 DPslope 6.7 15.3 19.2 Data are given in days was obtained from several separate experiments. L S X : Life-span mean; D P X : death phase mean; DP o n set and DPend: onset and end of death phase, respectively; D-20, 50, 95, 100: time at which 20, 50, 95, and 100 % of the population had died, respectively. D P s i 0 p e is given as % death per day. Longevity of adl-16ts /Oregon-R One set of experiments with each of the mutant alleles heterozygous with wild-type was completed early in this research. At the time, I thought that only confirmation that the adl-16ts alleles were recessive was necessary. It was noted that longevity of the heterozygote was slightly greater than that of the wild-type, Oregon-R. The small extension of life present was assumed to be due to heterosis, and the alleles under study were thenceforth considered recessive. DISCUSSION The temperature coefficient for Drosophila melanogaster longevity, Q i O , is 2 to 3 ( Lamb, 1978; Leffelaar and Grigliatti, 1984 ). This means that for every 10 degree rise in temperature, there is a two-to three- fold reduction in lifespan. This relationship holds true for temperatures of 22°C and 29°C which are within normal physiological limits for Drosophila and was confirmed in these experiments where a 7C° increase in temperature yielded conversion factors of 2.3 for females and 2.5 for males. These results are in an acceptable range. In general male flies in any one sample, and overall, did not live as long as their female counterparts, except in the case of adl-16flrdIat 2 2 ° C . This was true for both temperatures used in these sets of experiments. Also it does not seem that the longevity of the adult of one sex is more affected by temperature than the other. This was demonstrated by the close values obtained for conversion factors for rate of living for any one strain. This result confirms that of Leffelaar and Griggliatti (1984), who demonstrated that survival curves of Oregon-R males and females and adl-16tsl are sex-specific and coincident when adjusted for the rate of living at the two temperatures used. It also extends their study by demonstrating that adl-16ts2 has sex-specific coincident survival curves. It is questionable that the premise includes adl-16flrdI. A temperature of 2 9 ° C was not detrimental to Oregon-R, but it certainly was to all the mutant strains and hybrids studied. Even at 22°C some effects of temperature could be seen on the alleles of adl-16ts. For the following comparisons death phase means were used rather than lifespan means because they eliminate the exceptional survivors in the final death phase, and the few unexplained deaths in the reproductive phase and thus are more reliable for comparative purposes. For adl-16^ females at 22°C, the D P x ( O r e g o n - R ) / D P x ( a d / - 7 t f » 7 ) = 1.1, that is, the ratio of the death phase means is 1.1 ± 0.04 for females which represents 89-96% of Oregon-R lifespan. For males the corresponding calculation yields a ratio of 1.26 ± 0 . 0 5 meaning that male lifespan is 76-83% that of Oregon-R. The male lifespan was reduced more than that of females, but both are essentially the same as for Oregon-R. Using the same calculations, adl-16ts2 female lifespan is 66-74% that of Oregon-R, and male lifespan is 60-68%. Thus male and female lifespans were reduced by about the same amount relative to their wild-type counterparts. Therefore even at 2 2 ° C , they displayed slightly reduced viabilities. When flies and larvae of all ages were shifted to 29°C, all died, leading me to believe that the temperature sensitivity of these flies is continuous. In mutant strains shifted to 29°C within 12 hours of egg deposition, larvae survive to the 2nd-3rd larval instar. This suggests that, even during development, death is not immediate. In the case of adl-16flrdI, for females the lifespan was 47-54% of Oregon-R, and for males it was 50-59%. Thus these flies lived about half as long as their wild-type counterparts at 2 2 ° C . Both sexes had lifespans reduced by approximately the same amount. It is known that the lethal phase in this mutant occurs in the second or third larval stage (Homyk et al., 1986), but observations regarding many pupae which do not eclose in stock maintained at 22°C, suggest that viability throughout lifespan may be reduced, both at 2 2 ° C and at 2 9 ° C . The simple experiment of shifting these flies in a bottle containing larvae and flies of all ages to the restrictive temperature, resulting in death of flies or larvae within a few days, caused me to think that temperature sensitivity is continuous at 2 9 ° C . Further experiments are needed to confirm the nature of the temperature sensitivity of all three alleles studied here. It is well known that heterosis increases lifespan in general (Lamb, 1978), and the mutant flies used were produced from Oregon-R stock, many generations earlier. Comparatively speaking, these flies were relatively homozygous except for the mutant character under study. Lints and Lints (1979), argue that inbred lines may have a high frequency of deleterious genes existing in a homozygous state, and therefore should not be used to study normal ageing and senescence, since these genes could act to shorten lifespan. However all the flies used in this study are of similar background, which means that the background genome has been standardized and so the effects of each mutant should be easier to compare. After assessing the possibility of a maternal effect associated with the alleles under study, by means of reciprocal crosses between the mutant types and examining hybrids for behaviour and longevity characteristics, I concluded that there was no maternal effect discernable. At 2 2 ° C , for hybrids between adl-16tsl and adl-16ts2 viz, adl-16tslladl-16ts2 females, the lifespan was 92-97% that of Oregon-R females. Since adl-16tsl homozygous females exhibit close to wild-type lifespan, but adl-16ts2 homozygous females have reduced lifespan, this suggests that adl-16tsl is a hypomorph. In the case of hybrids between adl-16ts2 and adl-16flrdI female lifespan was 92-108% that of wild-type females suggesting that adl-16flrdI is also a hypomorph. This would imply a series for the lethal phene, with alleles varying in strength as follows: adl-16ts2 > adl-16tsl > adl-16flrdI. Hybrids of adl-16tsl and adl-16flrdIpresented a different picture. Females of type adl-16tsl/adl- 16flrdIlived only 70-80% as long as their wild-type counterparts. It should be recalled, however that adl-16flrdI had the shortest lifespan of the alleles at 2 2 ° C . The lifespan for the hybrid was intermediate between that for the homozygotes of the parent types, which was the expected result. Another way of analyzing these hybrids would be to compare lifespan with females from each parent stock. When this was determined at 22°C, it was found that: a) adl-16ts2 I adl-16flraI hybrid flies lived longer that either parent stock. Compared with homozygous adl-16flraI the hybrids lived twice as long (1 .98± 0.56), and relative to adl-16ts2 homozygotes lifespan was almost half as long again(1.4 ± 0 . 1 1 ) . b) adl-16tsl I adl-16flraI were found to be intermediate between homozygous parent types adl-16tsl and adl-16flraI , with no overlap in the length of the lifespans. c) adl-16tsI I adl-16ts2 hybrid females were slightly longer lived than either parent type, adl-16tsl and adl-16ts2 In the case of those hybrids that lived longer than either parent type, it is interesting to speculate whether this difference is due to heterosis, resulting from mating strains which have been separated for many generations, or whether it is due to some type of gene interaction. At 29°C Oregon-R flies of either sex lived close to five times as long as adl-16tsl flies. In the case of adl-16ts2 an approximately ten-fold reduction in lifespan relative to Oregon-R was seen. The lifespan of adl-16flraI was around 30% that of wild-type, that is, a 1.7-fold reduction in longevity was seen at 29°C. Overall then, it can be said that adl-16ts2 was the most temperature sensitive, adl-16tsl was intermediate, and adl- 16flrdIwas least affected. For hybrids at 29°C, reduction of lifespan was clear. In the case of adl- 16tsl I adl-16ts2 the lifespan was approximately midway between that of females of the parent types. This represents a 7-fold reduction relative to Oregon-R. For adl-16ts2l adl-16flraI the hybrid lifespan was again intermediate between females of the parent strains, with about a 5.7-fold reduction in lifespan relative to Oregon-R. In general, intermediate lifespans could be said to be dosage related, half the normal quantity of each allele product being present. Although adl-16tslladl-16flraI was the longest lived of the hybrid flies at 2 9 ° C , as expected from a simple hypomorphic series, the survival curve was not intermediate between females of the two parent types. These flies demonstrated reduced viability over either parent at 29°C until approximately 10-12 days old, at which time the survival behaviour resembled the longer lived parent more closely, with an extremely long death phase end. Overall there was about a 3.5-fold reduction in longevity relative to Oregon-R. It is not surprising that the two most severe of the mutant strains together produce the most severely affected hybrid, nor that the most and least affected parent types generate an intermediate effect. What is surprising is that the two least affected parent types produce a hybrid which is still severely affected, paralleling the other two hybrids, but overall closer to the moderately affected parent type. That the order differs at 22°C and 29°C is also surprising. adl-16tsll adl-16flrdI has the longest lifespan at 29°C and the shortest at 22°C. Gould there possibly be some relation to the time of action of these mutations? Is it possible that the protein product of adl-16tsl is non-existent or non-functional after 10-12 days at 29°C? The overall conclusion that can be drawn from the above is, nevertheless that these mutants do not complement, and may thus be considered alleles. They are recessive to wild-type, and express their phenotype at the restrictive temperature, 2 9 ° C , albeit with some effects even at the permissive temperature. There may also be some interesting gene interaction between alleles. Now that survival parameters are available for these strains of flies, it would be interesting to examine other known aspects of ageing such as pigment accumulation, organelle loss, or histological changes associated with ageing. Such comparisons might very well correlate with age related behaviour changes to be discussed in the next chapter. C H A P T E R 2  INTRODUCTION While it is well known that longevity is influenced by behaviour in Drosophila, for example the life shortening effect of mating for male flies, and mating and fecundity for females, (Lints and Soliman, 1988), and a wealth of information is available about mutants with fascinating behaviour e.g. savoir-faire, dunce, fruitless, stuck, easily shocked, tko, Ether-a-gogo, stoned, to name just a few esoteric ones, might it be possible to use normal behaviours to elucidate the controlling factors of lifespan? Insight into age-related changes is provided by longitudinal studies of the relationship of behaviour to ageing. Few studies exist which examine behaviour in ageing flies, as most reported behaviours are those of young flies (Lints and Soliman, 1988). A small subset of the above examine the genetics of behaviour in relation to ageing. Age-related behaviour loss was used as a bio-marker of age in the study by Leffelaar and Griggliatti (1984), Loss of geotaxis, phototaxis and motor abilities were shown to follow a set pattern in wild-type flies at both 2 2 ° C and 2 9 ° C . Furthermore, a temperature sensitive mutant, adl-16tsl was shown to have a pattern of behaviour loss identical to that of wild-type at both temperatures. What was extremely interesting was that at 29°C not only was the longevity decreased, the pattern of behaviour loss was also compressed into a shorter time period, proportional to the reduction in lifespan. Several other mutants tested did not behave this way. It is thus possible that adl-16tsl may be a mutation which accelerates normal ageing at the restrictive temperature. The results of testing this mutation and its other alleles adl-16ts2 and adl-16flrdI as well as hybrids between these alleles are presented in this chapter. The specific behaviours described and measured in Chapter 2 are geotaxis, phototaxis and motor activity. Other behaviours such as mating behaviour, and flight have been extensively researched, and it would be interesting to see the results of such an analysis added to the age related behaviour loss studies for the alleles presented here. Nevertheless, it was desirable at this time to test the repeatability of the aforementioned studies, and extend them to include the other alleles of adl-16tsl-Phototaxis has been studied since 1905 when Carpenter noted the response of Drosophila to light, gravity and mechanical stimulation (Carpenter, 1905). It is a complex behavioural response involving absorption of light by eye pigments, neural transmission of impulses to the central nervous system, and integration there with other signals, followed by the response that the fly walks (or flies) towards the light source in the case of Drosophila melanogaster. Such a behavioural trait is of importance for survival, and thus has evolutionary significance. It is affected by various environmental factors such as humidity, temperature, mechanical stimulation, and 100 density. Thus experimental design is extremely important when comparisons between results are made, and the experimental design here attempted to standardize those parameters (see Materials and Methods). Since differences have been noted between different strains of flies, a genetic basis for phototaxis has been proposed (Benzer, 1967). Benzer found great differences in six wild strains of Drosophila by his countercurrent distribution method of separating flies. In the same study he also examined various mutants and also mutilated flies, with legs or wings removed. Phototaxis is under genetic control, with the X-chromosome implicated (Grossfield, 1978). Age and sex of flies also affect results. Thus by including sex in the controlled parameters of the experiments, the two parameters of interest: strain and ageing, have been isolated for study. The second behaviour examined was geotaxis, which is defined as a directed movement in relation to gravity. The sensory input and analysis of stimuli by the organism is not well understood, but the antennae are thought to be involved (Grossfield, 1978). The trait is known to be under polygenic control (Lints and Soliman, 1988). Finally, locomotor activity was studied, motor activity being defined as general activity such as walking or flying. It should be noted that this ability has a strong influence on the previous behaviours, since flies unable to walk will not be able to respond phototactically, or geotactically, in the test chambers used (flying excluded). There is difficulty in distinguishing spontaneous motor activity from activity induced by manipulation of the flies. In all of the experiments performed, manipulation of flies immediately preceded measurement of behaviour, so that some of this behaviour could be ascribed to a "startle" or "escape" response. Nevertheless, this does not preclude conclusions being drawn about ageing since all the tests were performed in the same manner, and were conducted at regular intervals throughout the entire lifespan of each strain. MATERIALS AND METHODS The stocks used for the behaviour tests are shown in Table 1-1 in Chapter 1. For all behaviour tests flies eclosing within the same 12 hour period were separated by sex, placed in vials containing ten flies of one sex, and the vials were divided at random, half for maintenance at 22°C, and the remainder placed in the 29°C incubator as soon as was practicable. Controls of Oregon-R were set up on the same day. For most tests a minimum number of 200 flies was used. Behaviour Tests Geotactic, phototactic, and motor behaviour of all strains listed in Table 1-1 were measured. Behaviour tests were performed at the temperature of maintenance of flies, either 22°C or 29°C. Males and females of each strain, as well as hybrid females, were tested separately, so that any sexual dimorphism could be noted. Tests were performed each day for flies at 29°C, and once every 3-5 days 102 at 22°C, for temperature sensitive flies, to coincide with the time at which they were transferred to fresh containers and food. Oregon-R control flies and adl-16flrdI were tested every second day at 2 9 ° C , and every 3-5 days at 2 2 ° C . Tests were performed between 10:00 a.m. and 6:00 p.m. Although the test intervals were short time periods, these had proved sufficient to demonstrate behaviour loss in other studies. The behaviour of each strain of flies was tested throughout its lifespan. For each strain of flies except hybrids, at least three sets of data were obtained and analysed (see Table 1-1). 1. Geotaxis Drosophila respond to gravity. When disturbed, they exhibit negative geotaxis by crawling up the side of a vial. The geotaxis chamber used was a glass tube of 3 mm thickness, 30 cm long and 17 mm in internal diameter, with one end open, and the base covered by fine nylon mesh (as used to cover carbon dioxide anaesthetizers). A scale in centimeters was drawn on the side of the tube with a blue marking pen. To test geotaxis, flies from one vial were admitted to the chamber through a plastic funnel, which was then stoppered with cotton batting. Flies in the column were knocked to the bottom of the chamber by tapping it on a foam pad. The column was then held vertically on the desk, and the trial time started. After a 20 second time interval the number of flies in each 5 cm section of the vial were counted and recorded for sections 0-5 cm, 5-10 cm, 10-15 cm, and above 15 cm. This test was repeated five times in succession, then a second vial of flies was tested, so that there were 100 fly 103 trials per experiment. Vials were chosen at random, and each sex and strain of flies were tested. A result of all flies reaching the section above 15 cm was considered 100%. Since no strains of flies including Oregon-R reached 100%, the maximum behaviour of a population was used to calculate 50% behaviour, meaning 50% of the maximum possible behaviour for that population, (e.g. if a population had 86% as its maximum behaviour level, then its expected 50% behaviour was 43%.) In preliminary tests it was noted, after the column broke and was replaced, that the cleanliness and newness of glass had a great effect on the geotactic behaviour of flies. Flies were also noted to climb much more readily in the well used vials of the laboratory, than in the relatively new unscratched geotaxis testing chamber. To minimize these effects, three columns were made, and were used at random throughout the experiments. 2. Phototaxis This test was performed immediately following the geotaxis test using the same tube of flies set horizontally, with an opaque black velveteen cloth placed over the 0-15 cm area, and a 60 watt light shining horizontally at the 15-30 cm end. For each test, the cover was placed on the column, and then the flies were knocked to the bottom of the chamber as before. The tube was then set horizontally, and gently rolled back and forth about once every second in a 5 cm area. Drosophila melanogaster are positively phototactic, and this behaviour was quantified by counting the greatest number of flies in the lighted end of the tube at any one time during a 30 second time interval. Five trials were recorded for each set of flies, then this test was repeated using a second vial of flies (i.e. 100 fly trials per strain and sex of flies). 100% was defined as all flies above the 15 cm mark within the time limit. Since the populations of 100 flies never exhibited 100% behaviour, (i.e. 100 flies in the 15-30 cm lighted area in the five trials), maximum behaviour for each strain was compared using average percentage scores achieved, and behaviour loss was compared in relation to these scores, for example, a 50% behaviour loss for a strain of flies with a maximum behaviour of 60% would then be a demonstrated behaviour of 30%. 3. Motor activity For this test flies were aspirated from their vials into a glass tube modified into a pipette, (see diagram). Each fly was blown into the motor activity chamber. This chamber is made of plexiglass, and is 14.5 cm in diameter, and 0.6 cm deep, with a small hole in the top. It was placed on fresh 1 cm graph paper (Campus No. 4157), which acted as the bottom of the chamber. Each fly was observed for 10 seconds, and the number of lines crossed counted and recorded, moving the disc on the graph paper so that the flies did not come to rest at the side. Flies were added one by one until all from one vial were in the chamber and had been tested. Flies were then anaesthetized with carbon dioxide so that they could be removed from the chamber, and placed in a fresh vial. Vials containing these flies were marked, and the flies were not used in survival studies, or other behaviour tests to avoid possible effects of gas on viability or behaviour. One set of 50 vials was kept separate for motor tests. 20 flies were tested for each trial. The mean activity per fly was then calculated. The absolute maximum level of activity was a value of 38 lines crossed in ten seconds by Oregon-R males at 2 9 ° C . It was defined as 100% for motor activity. For comparisons between strains and drawing graphs, percentaged values have been used. To obtain the 50% activity for any strain, the maximum activity for that strain was divided by 2. The absolute numbers of lines crossed provided a good comparison between types of flies even though the distance covered was not possible to calculate in centimeters. Minimum behaviour for each test was defined as a score of zero for the test, and the corresponding age of the flies was the first day on which behaviour was absent. Tests continued even after behaviour was recorded as zero, to allow for any fluctuations in the final point. In the case of phototaxis in particular, an initial reading of zero was often followed by a few very low behaviours before the final zero was obtained. A score sheet for behaviour is included as Appendix i. RESULTS To be able to compare behaviour loss at two different temperatures, it was necessary to determine that the restrictive temperature of 29°C (for temperature sensitive strains) had no deleterious effects on wild-type flies compared with that of the permissive temperature. This was shown in Chapter 1, where coincident graphs for survival were drawn after conversion factors for the rate of living were calculated and used. Conversion factors of 2.5 and 2.3 for males and females respectively of Oregon-R were used to adjust 29°C graphs for all flies so that the deleterious affect of temperature on the mutant strains as well as their hybrids could be demonstrated. To compare the patterns of behaviour loss to longevity, the percentage of flies displaying a given behaviour was plotted against the age of the population in days at 22°C. Since the graphics program used did not permit the use of two axes, the number of days at 29°C were multiplied by the appropriate male or female conversion factor, so that the graphs could be drawn. The second abscissa (not depicted) is understood to apply to the 29°C flies. When the same conversion factors were applied to behaviour loss patterns, it was possible to compare results obtained at each temperature for each strain. The curves for behaviour loss for Oregon-R are shown in Figures 2-1, through 2-10. Each curve represents the results from one experiment. Many more data points were obtained, but only those sufficient to demonstrate the curves are shown, so that data points do not clutter or obscure the graphs. Although behaviour curves for Oregon-R males and females only are shown, data for the other strains was quite similar, and has been presented in the tables for comparison. Geotactic response of wild-type males and females has been depicted in Figures 2-1 to 2-6. Each value represents the percentage of individuals that climbed to the height shown on the graph (5, 10 or 15 em) within the test interval. Phototactic response of Oregon-R flies has been shown in Figures 2-7 and 2-8, and motor activity is depicted in Figures 2-9 and 2-10. D I A G R A M OF PIPRTTF. 108 Figure 2-1 Geotactic Response of Oregon-R Males at 22°C or 29°C to Height 5 cm. Age (Days) at 22°C Note: 29°C days have been multiplied by the factor 2.5 to adjust for the rate of living. Figure 2-2 E o LO o CD o > O ) cu 100 -i Geotactic Response of Oregon-R Females at 22°C or 29°C to Height 5 cm. Females at 22°C Females at 29°C Age (Days) at 22°C Note: 29°C days have been multiplied by the factor 2.3 to adjust for the rate of living. Figure 2-3 2. E o o o © <D > "To CD 0> 100 -i Geotactic Response of Oregon-R Males at 22°C or 29°C to Height 10 cm. - Males at 22°c « — Males at 29°C 40 60 Age (Days) at 22°C 1 oo Note: 29°C days have been multiplied by the factor 2.5 to adjust for the rate of living. ; Note: The two curves for males at 29°C, involve a computer derived curve of best fit (without symbols), and actual experimental results (with diamond shaped symbols). Figure 2-4 E o o 8 o > CB 8> 100 n Geotactic Response of Oregon-R Females at 22°C or 29°C to Height 10 cm. - Females at 22°C * — Females at 29°C Age (Days) at 22°C Note: 29°C days have been multiplied by the factor 2.3 to adjust for the rate of living. Figure 2-5 Geotactic Response of Oregon-R Males at 22°C or 29°C to Height 15 cm. Males at 22°C Males at 29°C 1 oo Age (Days) at 22°C Note: 29°C days have been multiplied by the factor 2.5 to adjust the rate of living. Figure 2-6 Geotactic Response of Oregon-R Females at 22°C or 29°C to Height 15 cm. 100 -i s 0 10 20 30 40 50 60 Age (Days) at 22°C Note: 29°C days have been multiplied by the factor 2.3 to adjust for the rate of living. It is clear that geotactic behaviour in Oregon-R reaches a maximum within a few days after eclosion, and that flies maintained at 29°C have a higher level of geotactic activity than those at 22°C. Within a few days, geotaxis begins to decrease quite rapidly, and is lost in the order geotaxis 15 cm, 10 cm, then 5 cm. In general, male flies exhibit greater geotaxis than do females. These generalizations hold true for all the strains tested. The shape of the two curves at 22°C or 29°C, although similar, is not congruent. From the fact that a higher level of geotactic behaviour was seen at 29°C, it could be concluded that this temperature does not have a negative impact on that behaviour. Geotactic behaviour in strains other than Oregon-R can be seen in the tables below. In the case of adl-16flrdI as well as all three hybrids, geotaxis at maximum was reduced relative to the other strains at 22°C, which behaved similarly to Oregon-R. At 2 9 ° C the geotactic behaviour of adl-16ts2 was virtually unmeasurable, the flies became paralysed early, usually within the first 24 hours. Only geotaxis at 5 cm was measurable in adl-16tsl/adl-16ts2, and adl-16ts2ladl-16flrdIas flies did not reach the 10 cm mark. adl-16tsl and adl-16tslIadl-16flrdI behaviour were similar to Oregon-R, but geotactic behaviour of adl-16flrdI was reduced in comparison with both these strains as well as Oregon-R. Phototactic behaviour is shown in the following two figures: Figure 2-7 Phototactic Behaviour of Oregon-R Males at 22°C or 29°C. Age (Days) at 22°C Note: 29°C days have been multiplied by the factor 2.5 to adjust for the rate of living. Figure 2-8 CO o o 0_ CD > O Q . 100 n Phototactic Behaviour of Oregon-R Females at 22°C or 29°C Age (Days) at 22°C - Females at 22°C • — Females at 29°C 120 Note: 29°C days have been multiplied by the factor 2.3 to adjust for the rate of living. From the above two graphs, it can be determined that phototactic behaviour was greater at 29°C than at 22°C. The behaviour curves were not coincident but were of similar shape. In the case of males, the behaviour was greater in young flies, but overall there was not much difference in the time of complete loss of phototaxis for each sex. For all of the strains tested, this behaviour was lost after geotaxis 5cm, at 2 2 ° C . At the time of 50% behaviour loss, occasionally phototaxis level was greater than motor activity on the scales used, but it was always greater than geotaxis 10cm. There did not seem to be much reduction of phototactic activity in adl-16ts2 at 2 2 ° C , despite the fact that these flies had vermilion eyes, a factor which is said to reduce phototaxis (Grossfield, 1975). At 29°C for some strains, total loss of phototaxis was observed at a very similar time to geotaxis 5cm, but in most strains it was lost later. At the time that 50% behaviour was displayed, there was much more variability between the strains with regard to phototaxis, several showing a level below that for geotaxis 5cm It should be noted that although the 29°C incubator was lighted, this illumination did not equal that of the laboratory in either intensity or duration, the lights of the latter often being left on for extended times when people were working late or early. This may have affected the results obtained. Figure 2-9 Motor Activity of Oregon-R Males at 22°C or 29°C 100 n Age (Days) at 22°C Note: 29°C days have been multiplied by the factor 2.5 to adjust for the rate of living. Tests were performed at the temperature of maintenance of the flies. Figure 2-10 Motor Activity of Oregon-R Females at 22°C or 29°C Age (Days) at 22°C Note: 29°C days have been multiplied by the factor 2.3 to adjust for the rate of living. Tests were performed at the temperature of maintenance of the flies. Motor activity as evidenced by walking was not totally lost in any strain at either 22°C or 29°C until near the end of life. Clearly it was the last behaviour to be lost, and often this loss was many days after all other measured behaviour had been lost even at 2 9 ° C . Since walking is necessary for expression of geotaxis and phototaxis this is not surprising. For adl-16ts2 and adl-16flrdI at the time of 50% behaviour loss, less motor activity than phototaxis was shown, which may be an example of motor activity reduction being more rapid, and phototaxis decline less rapid early in life. Nevertheless motor activity was retained much longer. By examining the following Tables 2-1 to 2-10, all of the above parameters may be examined in more detail. Space precludes the inclusion of graphic representation for all behaviours of each separate sex and strain examined. Table 2-1 Behaviour Loss with Age in Oregon-R Males maintained at 22°C Maximum Activity 50% Activity Minimum Activity % Day % Day % Day Geotaxis-15 70.7 1-6 35.3 14.3 0 57 Geotaxis-10 88 1-6 44 14.8 0 69.5 Geotaxis-5 96.7 1-7 48.3 32.3 0 80 Phototaxis 69.7 2-5 34.9 48.3 0 114 Motor 33.2 2-8 16.6 42.0 0 126.5 Behaviour Loss with Age in Oregon-R Males maintained at 29°C Maximum Activity 50% Activity Minimum Activity % Day % Day % Day Geotaxis-15 90.5 1-5 45.5 13.5 0 36 Geotaxis-10 99 1-5 49.0 22.0 0 41 Geotaxis-5 100 2-7 50 26.5 0 43.5 Phototaxis 94 7-10 47 29 o 44.6 Motor 34.5 7-13 17.3 36 0 50.5 Table 2-2 Behaviour Loss with Age in Oregon-R Females maintained at 22°C Maximum Activity 50% Activity Minimum Activity % Day % Day % Day Geotaxis-15 58.3 1-5 29.2 7.0 0 54 Geotaxis-10 75.5 1-5 37.5 15.0 0 69.5 Geotaxis-5 91.7 1-6 45.8 25.5 6 73.5 Phototaxis 74.3 5-10 37.2 37.5 0 109.5 Motor 33.5 8-10 16.7 57.5 0 120 Behaviour Loss with Age in Oregon-R Females maintained at 29°C Maximum Activity 50% Activity Minimum Activity % Day % Day % Day Geotaxis-15 89.7 1-3 44.8 20.0 0 37 Geotaxis-10 95 1-2 47.5 20.8 0 39 Geotaxis-5 98 7-10 49 26 0 45.5 Phototaxis 87.7 3-10 43.9 28.8 0 48 Motor 35.5 9-13 17.7 30.5 0 53.5 By examining Tables 2-1 and 2-2 for Oregon-R and the Tables 2-2 and 2-3 which follow, the great similarity of Oregon-R and adl-16tsl can easily be seen. Despite the fact that all parameters are compressed into one fifth the time at 29°C for adl-16tsl, behaviour loss except for phototaxis at 50% is extremely consistent. Table 2-3 Behaviour Loss with Age in adl-16tsl Males maintained at 22°C Maximum Activity 50% Activity Minimum Activity % Day % Day % Day Geotaxis-15 72.5 1-4 36 4.5 0 24.5 Geotaxis-10 84.5 1-5 42.3 4.3 0 58 Geotaxis-5 92.5 1-7 46.3 21.3 0 6 1 Phototaxis 78 1-8 39 31.5 0 69 Motor 30.1 3-10 15.1 28.8 0 92.5 Behaviour Loss with Age in adl-16tsl Males maintained at 29°C Maximum Activity 50% Activity Minimum Activity % Day % Day % Day Geotaxis-15 84 42 2.3 0 4.5 Geotaxis-10 94.5 1 47.5 2.8 0 5.5 Geotaxis-5 97.5 1 48.8 3.3 0 6 Phototaxis 81.5 1 40.8 2.3 0 6 Motor 29.5 1 14.8 3.8 0 7.5 123 Table 2-4 Behaviour Loss with Age in adl-16tsl Females maintained at 22°C Maximum Activity 50% Activity Minimum Activity % Day % Day % Day Geotaxis-15 69 1-6 34.6 3.3 0 24.5 Geotaxis-10 90 1-7 45 6.5 0 40 Geotaxis-5 96.5 1-8 48.3 13 0 62 Phototaxis 84 1-7 42 29.8 0 77 Motor 28.5 1-10 14.3 41.3 0 101 Behaviour Loss with Age in adl-16tsl Females maintained at 29°C Maximum Activity 50% Activity Minimum Activity % Day % Day % Day Geotaxis-15 80.5 40.3 2 0 3.5 Geotaxis-10 92.5 1 46.2 2 0 4.5 Geotaxis-5 95.5 1 47.8 2.8 0 5.5 Phototaxis 86.5 1 43.3 2.3 0 5.5 Motor 29.5 1 14.8 3.5 ..........ffl»*wvmwmW o 6.0 By examining the following Tables 2-5 and 2-6 for adl-16ts2 it can clearly be seen that there is virtually no measurable behaviour at 2 9 ° C . This mutant demonstrated almost total debilitation within 24 hours. It is thus suggested that behaviour tests at a temperature of 25 °C may yield some results which could be used for comparative purposes, because it seems likely from the small amount of evidence available that this allele as well as adl-16tsl may also accelerate the rate of ageing, but at an even faster rate. Table 2-5 Behaviour Loss with Age in adl-16ts2 Males maintained at 22°C Maximum Activity 50% Activity Minimum Activity % Day % Day % Day Geotaxis-15 82.5 1 41.3 9.3 0 15.5 Geotaxis-10 93.1 1-3 46.5 11.5 0 22.5 Geotaxis-5 99 1-5 49.5 13.5 0 36.5 Phototaxis 70 4-10 35 24 6 47.5 Motor 23.6 4-10 11.8 18.3 0 72 Behaviour Loss with Age in adl-16ts2 Males maintained at 29°C Maximum Activity 50% Activity Minimum Activity % Day % Day % Day Geotaxis-15 0 1 0 0 1 Geotaxis-10 0 1 0 1 0 1 Geotaxis-5 3 1 1.5 1.1 0 1.3 Phototaxis 0.9 1 0.5 1.3 0 1.7 Motor 0.8 1 0.4 1.3 0 1.7 Table 2-6 Behaviour Loss with Age in adl-16ts2 Females maintained at 22°C Maximum Activity 50% Activity Minimum Activity % Day % Day % Day Geotaxis-15 52 1 26 4.7 0 13.5 Geotaxis-10 8 1 1 40.5 7 b 1 8 Geotaxis-5 95.5 1-6 47.5 12.5 0 27.5 Phototaxis 70.5 4-10 30 19.5 0 50 Motor 25.1 4-10 12.5 15.5 o 77 Behaviour Loss with Age in adl-16ts2 Females maintained at 29°C Maximum Activity 50% Activity Minimum Activity % Day % Day % Day Geotaxis-15 0 1 0 1 0 1 Geotaxis-10 0 1 0 1 0 1 Geotaxis-5 2.3 1 1.2 1.2 0 1.3 Phototaxis 0.8 1 0.4 1.2 0 1.3 Motor 1.2 1 .9,6 1.3 0 1.7 In the case of adl-16flraI it can be seen that all parameters for behaviour were reduced at 22°C (as well as the lifespan mentioned in Chapter 1). Geotaxis at 15 cm was reduced four-fold, as was phototaxis, and motor activity was reduced by approximately one third. This leaves less room for further reductions at 2 9 ° C , but at that temperature geotaxis of all levels was reduced by 20%. The fact that phototaxis and motor activity were not further reduced is interesting, because climbing is not necessary for these behaviours. Of all alleles this strain demonstrated the least behaviour at 22°C. At 29°C it was intermediate between the other two strains in terms of reduction of behaviours. Table 2-7 Behaviour Loss with Age in adl- 16flrdlMales maintained at 22°C Maximum Activity 50% Activity j Minimum J Activity % Day % Day % Day. Geotaxis-15 17 1-6 8.5 6 0 1 4 Geotaxis-10 55.3 1-10 27.7 9.2 0 34 Geotaxis-5 83 1-10 41.5 16.2 0 55 Phototaxis 17.3 9-14 8.7 23.7 o 59.3 Motor 20.8 6-9 10.4 26.3 0 78 Behaviour Loss with Age in adl-16flraI Males maintained at 29°C Maximum Activity 50% Activity Minimum Activity % Day % Day % Day Geotaxis-15 6 1-2 3 2 0 2.7 Geotaxis-10 31.7 1-2 15.8 2.8 0 3.7 Geotaxis-5 64.7 1-2 32.3 3.3 0 8 Phototaxis 1 6 2-4 7.8 7.2 0 11.7 Motor 20 1-4 10.1 5.2 0 16.7 Table 2-8 Behaviour Loss with Age in adl-16flrdIFemales maintained at 22°C Maximum Activity 50% Activity Minimum Activity % Day % Day % Day Geotaxis-15 18.7 5 9.3 7 0 9 Geotaxis-10 40.7 5-17 20.3 18.3 0 27 Geotaxis-5 94.5 5-14 47.3 24 0 61.3 Phototaxis 16.3 1-14 8.2 44.5 0 66 Motor 21.7 1-14 10.8 37.7 0 79 Behaviour Loss with Age in adl-16flrdIFemales maintained at 29°C Maximum Activity 50% Activity Minimum Activity % Day % Day % Day Geotaxis-15 1 1 0.5 1.5 0 2 Geotaxis-10 13.7 1-2 6.8 2.8 0 4.3 Geotaxis-5 47.3 1-2 23.7 3.3 0 10 Phototaxis 15.3 2-4 7.7 8.5 0 11.7 Motor 18.5 1-2 9.2 3.3 0 19.3 The hybrid presented in Table 2-9, adl-16tslladl-16ts2 demonstrated reduced geotaxis at 2 2 ° C , although phototaxis and motor activity appear normal. Thus 2 2 ° C may not be a totally permissive temperature for this strain. At 2 9 ° C all behaviours were further reduced by approximately 20%. In adl- 16tsl/adl- 16ts2 all behaviours were intermediate between those of parent strains at the restrictive temperature. For adl-l6ts2ladl-16flrdI behaviour was somewhat reduced at 2 2 ° C , but of the hybrids it was the least affected at this temperature (see Table 2-11). However a reduction in behaviour was seen in the results for geotaxis, the lifespan having been comparable to that of Oregon-R at that temperature. Phototaxis and motor ability were normal at 22°C. It is interesting that geotaxis, phototaxis and motor ability are less severely affected in the hybrid adl-16ts2ladl-16flraI than in either parent strain at 29°C. The hybrid adl- 16tslladl- 16flraIwas the most interesting of all (see Table 2-10). At 2 2 ° C , geotaxis was reduced to intermediate compared with the other two hybrids, but phototaxis as well as motor activity were normal. In other words all three hybrids had similar behaviour type at 22°C, demonstrating a reduction in levels of geotaxis. In the case of this hybrid lifespan was reduced at 22°C, and was intermediate between females of the generating lines. At 2 9 ° C a surprising result was found. A l l behaviours tested for this hybrid resulted in the high normal range. It may be recalled that this hybrid was the longest lived of the hybrids at 29°C. It could be argued that this combination of alleles provides some protection against the deleterious effects of temperature, at least as far as behaviour is concerned. It would appear that the presence of the allele adl-16flrdI had a moderating effect on the severity of expression of the other two alleles at the restrictive temperature. Table 2-9 Behaviour Loss with Age in adl-16tslladl-16ts2 Hvbrid Females maintained at 22°C Maximum 50% Activity Minimum Activity Activity % Day % Day % Day Geotaxis-15 18.5 6.5 9.3 1 1 0 23 Geotaxis-10 3 1 6.5 15.5 12 0 23 Geotaxis-5 48 6.5 24.0 22.5 0 55 Phototaxis 72.5 6.5 36.3 26.8 0 96 Motor 30.732 11.5 16.0 32.5 0 102 Behaviour Loss with Ase in adl-16tslladl-16ts2 Hvbrid Females maintained at 29°C Maximum. 50% Activity Minimum Activity Activity % Day % Day % Day Geotaxis-15 0 0 0 Geotaxis-10 13 6.5 1.5 0 2 Geotaxis-5 35 1 17.5 1.5 0 2 Phototaxis 54 1 27 1.5 0 3 Motor 16.7 1 8.3 1.5 6 4 Table 2-10 Behaviour Loss with Age in adl-16ts 11 adl-16flraI Hybrid Females maintained at 22°C Maximum Activity 50% Activity Minimum Activity % Day % Day % Day Geotaxis-15 34.7 7 17.3 18 0 47.3 Geotaxis-10 5 7 10 28.5 1 23.2 b 54.7 Geotaxis-5 70 10 35.0 34.2 0 72 Phototaxis 87.3 5.3 42 33.2 0 90 Motor 37.0 7.3 18.5 51.8 0 100.3 Behaviour Loss with Aee in adl-16tsl 1 adl-16flraI Hvbrid Females maintained at 29°C Maximum Activity 50% Activity Minimum Activity % Day % Day % Day Geotaxis-15 92 1 46 1.5 0 3 Geotaxis-10 98.5 1 49.3 2.0 0 4 Geotaxis-5 99.5 1 49.8 2.5 0 5 Phototaxis 87.5 1 43.8 2.0 0 5.5 Motor 29.3 1 14.6 2.5 0 9.5 Table 2-11 Behaviour Loss with Age in adl-16flrdIladl-16ts2 Hvbrid Females maintained at 22°C Maximum Activity 50% Activity Minimum Activity % Day % Day % Day Geotaxis-15 43.5 5.5 21.8 12.5 0 35.5 Geotaxis-10 65 5.5 32.5 15.8 6 52.5 Geotaxis-5 82 5.5 41.0 19.8 0 78 Phototaxis 82 9.5 41.0 38.5 6 102.5 Motor 38.7 8 19.1 68.8 0 122.5 Behaviour Loss with Age in adl-16flrdIladl-16ts2 Hvbrid Females maintained at 29°C Maximum Activity ' 50% Activity Minimum Activity % Day % Day % Day Geotaxis-15 43 1.5 21.5 2.3 0 3 Geotaxis-10 60.5 1.5 30.3 2.3 0 3 Geotaxis-5 72 1.5 36 2.3 0 3 Phototaxis 68 1.5 34 2.3 0 3.5 Motor 23.7 1.5 11.7 2.3 0 8.5 So that a comparison between strains can be made more easily, the following table is presented showing maximal behaviour for each strain for all behaviours tested. Table 2-12 Maximal Behaviour in Al l Strains Tested at 22°C Strain of Flies Tested Geotaxis 15 cm Geotaxis 10 cm Geotaxis 5 cm Photo-taxis Motor Activity Oregon-R Males 70.7 88 96.7 69.7 33.2 Oregon-R Females 58.3 75.5 91.7 74.3 33.5 adl-16 t s l Males 72.5 84.5 92.5 78 30 adl-16 t s l Females 69 90 96.5 84 28.5 adl-16* 2 Males 82.5 93 99 70 23.6 adl-16 t s 2 Females 52 81 95.5 70.5 25.1 adl- 16Ardl Males 17 55 83 17.3 20.8 a d l - 1 6 f l r d I Females 18.7 40.7 94.5 16.3 21.7 a d l - 1 6 t s l / a d l - 1 6 t s 2 18.5 3 1 48 72.5 32 adl-16tsl /adl-16 f l r di 34 57 7 0 87.3 37 a d l - 1 6 t s 2 / a d l - 1 6 « ' - d i 43.5 65 82 82 38.7 Maximal Behaviour in Al l Strains Tested at 29°C Strain of Flies Tested Geotaxis 15 cm Geotaxis 10 cm Geotaxis 5 cm Photo-taxis Motor Activity Oregon-R Males 90.5 99 100 94 34.5 Oregon-R Females 89.7 95 98 87.7 35.5 adl-16tsl Males 8 4 94.5 97.5 81.5 29.5 adl-16 t s l Females 80.5 92.5 95.5 86.5 29.5 adl-16 t s 2 Males 0 0 3 0.9 0.8 adl-16 t s 2 Females 0 0 2.3 0.8 1.2 a d l - 1 6 f l r d I Males 6 31.7 64.7 16 20 a d l - 1 6 f l r d I Females 1 13.7 47.3 15.3 18.5 adl-16tsVadl-16ts2 0 13 35 54 16.7 adl -16ts l /adl -16 f l r d I 92 98.5 99.5 87.5 29.3 a d l - 1 6 t s 2 / a d l - 1 6 f l r d I 43 6 0 72 68 23.7 Table 2-12 examines behaviour of each strain at its maximal level at the beginning of adult life. From this table it can clearly be seen that the only strain which resembles Oregon-R is adl-16tsl. In fact the parameters of behaviour for this strain are very similar to Oregon-R at both the permissive and restrictive temperatures, except that at the restrictive temperature, all are accelerated. By inspecting the Tables 2-1 to 2-4, it can be seen that at 22°C the two strains were quite similar. What is even more interesting is the similarity of these strains at 29°C. The premise related to ageing for this mutant is that behaviour loss at 2 9 ° C follows a similar pattern to that at 22°C, but at an accelerated rate. The following Table (Table 2-13) has been prepared to demonstrate this effect at 2 9 ° C over the total lifespan. Total behaviour loss has been divided by the value D-100 for each strain, and then the strains have been compared to Oregon-R (i.e. Total loss in Oregon-R/D-100 Oregon-R divided by Total loss in mutant/D-100 mutant). Table 2-13 Total Behaviour Loss of Mutant Strains Relative to D-100 and Oregon-R at 29°C Strain of Flies Tested Geo-taxis 15 cm Geo-taxis 10 cm Geo-taxis 5 cm Photo-taxis Motor Act iv-ity A v e r -age and S.D. adl-16^ 1 Males 1.84 1.65 1.62 1.65 1.51 1 . 6 6 ± . l l adl -16 t s l Females 2.25 1.91 1.81 1.91 1.94 1 . 9 6 ± . 1 5 adl-16ts2 Males - - 2.47 1.91 2.18 2 . 1 9 ± . 2 3 adl-16 t s 2 Females - - 2.79 2.93 2.52 2 . 7 5 ± . 1 7 adl-16 f l r<H Males 4.67 3.76 1.91 1.32 1.05 2.5411.4 a d l - 1 6 f l r d I Females 7.0 3.52 1.77 1.58 1.06 2.9912.2 adl-16tsl/adl-16ts2 - 3.35 3.90 2.73 2.28 3.071.61 adl -16ts l /ad l -16 f l r d I 4.50 3.72 3.39 3.28 2.11 3.41.77 a d l - 1 6 t s 2 / a d l - 1 6 « r d i 3.0 1.49 3.71 3.28 1.49 2.591.93 For adl-16tsl for all behaviour parameters averaged at the time of their loss relative to the lifespan of this mutant at 2 9 ° C , males differed by 6% and females differed by 8% from Oregon-R. In addition to this, there was very little variation in any of the total loss parameters relative to Oregon-R. adl-16ts2 for those parameters which could be measured, differed by 11% for males, and 6% for females from Oregon-R. Thus it is likely that this mutant also resembles Oregon-R in terms of behaviour loss patterns, but this loss is extremely accelerated. A measurement at 2 5 ° C might prove interesting. By comparison, adl-16flraI differed greatly in behaviour loss patterns from Oregon-R, the males by 56%, and the females by 73%. Differences were from 20% to 36% for the three hybrids of adl-16ts. DISCUSSION From the results of the behaviour tests for each strain, it is clear that behavioural responses could serve as bio-markers of age in Drosophila. Not only did each strain demonstrate a unique pattern of behaviour at the two temperatures used in the experiments, but behaviour loss was consistent and measurable. Furthermore, in spite of the small differences in experimental design used, the results are are similar to those obtained by Leffelaar and Grigliatti (1984). In the experiment measuring geotaxis, Leffelaar and Grigliatti used two empty glass vials connected together at their open ends by a piece of tape. Five and fifteen cm intervals were marked on the side of the vials. In my experiments, a cylinder was used, so that no crack at the half-way point would be available for flies to use in climbing. Also three 5 cm intervals were tested, which would not have been possible with tape obscuring the view. The bother of re-taping at each experiment was also eliminated. New glass was used in either experiment, but the diameter of their two vails was not given. The base of the chamber differed, for theirs, it was glass, for these experiments it was nylon mesh glued to the outside of the glass. It was thought that this would minimize any damage to flies when they were knocked to the bottom of the chamber. For the experiment testing phototaxis, a 60 watt lamp was used in these experiments, and 10 cm of space was available in the lighted area. The other experiments used a 6 volt lamp, and only 5 cm was available in the lighted area. For the experiment for motor activity, the grid of squares on the paper base differed. For my experiments it was 1 cm graph paper with tenths marked. They used paper with squares measuring 1.25 cm per side. Their tests for motor behaviour were performed once a week at 22°C for males only. My motor tests were performed at the temperature of maintenance of the flies, 22°C and 2 9 ° C , on a more frequent basis to correspond with turning over of flies and for both sexes. Thus despite the many differences in experimental design, very comparable results were obtained. The independent verification of results, supports the idea that behaviour makes an excellent bio-marker of age. Although behavioural levels varied from day to day, the continual trend in behaviour loss was apparent in populations of flies after the time at which maximal behaviour was seen. The total behaviour loss pattern was consistently geotaxis (all levels), phototaxis, and finally motor activity for flies of all strains and each sex. Of all the behaviours measured, phototaxis varied the most, and might not be as good a bio-marker of age at the 50% point as the other two behaviours. The behaviour loss pattern in the second half of adult life was extremely consistent. At 2 2 ° C populations of flies reached maximum behaviour within a few days after eclosion. This high level of behaviour was maintained for up to ten days. At 29°C maximal behaviour was usually seen within three days. On these graphs adjusted for rate of living, it would appear that there is a slight delay in the appearance of maximal behaviour. This was possibly due to the flies acclimating fully to this temperature. Previous to this, larvae and pupae had been maintained at 22°C. After the maximum was achieved at either temperature, there was a decline in demonstrated behaviours until total loss of behaviour occured. In these experiments, phototaxis was lost after geotaxis 5 cm, not before, as in the experiments by Leffelaar and Grigliatti, but the design of the experiments differ. The shape of the behaviour loss curves is similar, and the days on which total behaviour loss occurred agree rather well. The reduction in geotaxis reported by Miquel et al., (1976) at 27°C was not observed. Rather, flies of wild-type and adl-16tsl demonstrated an increased behaviour at the higher temperature. The pattern of behaviour loss at 22°C was similar in both males and females, and was consistent for each strain over several trials. There was also a good agreement between behaviour loss and chronological age of the population. Loss of geotaxis 15, 10, and 5 cm occured in order, usually beginning in the second third of life and ending in the last third. Phototaxis was lost next, after approximately 80% of total lifespan. Finally motor behaviour was lost, but not until well into the final death phase. The same pattern was found for adl-16tsJ at 2 9 ° C . adl-16ts2 was too severely debilitated by this temperature for any definite conclusions to be made, but at 22°C, the pattern of behaviour loss was as that for Oregon-R and adl-16tsl. In the case of adl-16ts2, the chromosome was also marked with scute, (sc), cross-veinless, (cv), vermilion, (v), and forked, (f). The mutations scute, and cross-veinless cause wing beat frequency reduction. Vermilion as well as forked flies have been noted to have normal mating behaviour, but the mutation vermilion has been reported to reduce phototaxis (Fingerman, 1952). No reduction of phototaxis was noted in these experiments, and no other impediments were found because of these markers, but it would have been better to use an unmarked chromosome for adl-16ts2. The analysis of hybrids of the temperature-sensitive alleles studied was extremely interesting. Not only was reduced behaviour demonstrated at 2 9 ° C , as expected, but at 2 2 ° C a reduction in geotaxis was also seen. The most surprising result was that of adl-16tslladl-16flrdI which revealed a behaviour loss pattern similar to that of adl-16tslat 2 9 ° C , but a lifespan intermediate between that for females of the two generating strains at 22°C. The other two hybrids demonstrated reduced behaviour at 2 9 ° C as expected. This may represent the first longitudinal study of behaviour in alleles as well as their hybrids. It is certainly the first such study for temperature sensitive mutant alleles throughout adult life. Because of the consistency and repeatability of results, behaviour loss could thus be a useful tool to identify mutations (as in the case of hybrids at 22°C) , or to define age for experimentation. Finding mutations which alter the pattern of ageing will assist in understanding what controls it. The possibility that adl-16tsl and adl-16ts2 might be such mutations is encourageing. The remainder of this thesis deals with the location of these alleles as well as adl-16 flrdl o n the X-chromosome, so that they might be cloned for further study; and location of the type of tissue affected by the gene product produced. CHAPTER 3 INTRODUCTION In general, deletions in chromosomes cause death of the organism if homozygous. In heterozygotes, they permit phenotypic expression of recessive genes as pseudodominance, the genes being present in single dose. This allows the cytological localization of genes, so that a correlation can be made between the genetic map, based on linkage studies, and the cytological map inferred by the presence of deficiency loops visible in polytene chromosomes, combined with pseudodominance. Furthermore, a single dose of gene product in the deficiency female can then be compared with the action of the same gene in male flies which have a single dose of gene product, but with dosage compensation. Inferences may then be made as to the mode of gene action in the mutant allele; whether amorph, hypomorph, neomorph, or hypermorph. An X-chromosome with a deletion which uncovered shits, known to be very close to adl-16ts alleles, (the positions are 52.2 and 52.9 respectively mapped by usual recombinant methods), Df(l)sd72b26 which uncovers bands 13F1-14B1, was available in this laboratory and given to me by kind favour of Don Sinclair. If the alleles for adl-16ts lie in the area corresponding to the deficiency, their location and function might be further elucidated by studying them in combination with this deficiency chromosome. This would enable comparisons of homozygotes, with hemizygotes in either males or females. The three strains of flies hybrid for the deficiency and each adl-16ts mutant allele were examined for temperature sensitivity, fertility, longevity, behaviour, and the presence of any visible phenotypical changes. Some of these tests were quite preliminary, and should be continued by another researcher, because these early results indicate some interesting possibilities. MATERIALS AND METHODS Male flies of all three mutant alleles of adl-16ts were mated with Df( 1 )sd72b26IFM y B wa virgin females. Thirteen replicates of each cross were made. To optimize the number of progeny, all vials were kept and examined after each transfer (every 2-4 days). The Deficiency stock was also crossed with Oregon-R. The following crosses were made: 1) Df(l )sd72b26IFM y B ® FM y B w« IY (stocks) 2) Df(l )sd72b26IFM y B w« ® Oregon-RIY (viability ratio) 3) Df(l )sd72l>26IFM y B wa ® adl-16**1IY 4) Df(l )sd72b26/FM y B wa adl-16**2 IY 5) Df(l )sd72b26/FM y B w« ® adl-16flrdIIY For simplicity, Df(l)sd72b2^IFM y B wa will be called D//FM7. Cross #1 was necessary to maintain the deficiency chromosome over a balancer with the visible markers yellow, white-apricot, and Bar. FM7IFM7 is female sterile, due to sn *2, and DflY is lethal. Cross #2 was used to compare viability of Oregon-R with the deficiency chromosome, i.e. Dflwild compared with FM7lwild should give a 1:1 ratio. If this was not the case, then viability in Dflmutant might be lowered due to the action of the Df chromosome, which must be eliminated as a possibility before viability decisions regarding Dflmutant could be made. Crosses #3-5 involved the mutant alleles under study. The following progeny types were expected: DflY Dflmutant FM7lmutant FM7IY dies normal eye Wide Bar eye white Bar eye yellow Fertility of Deficiency/Mutant at 22°C A test of the fertility of Df I mutant flies was tried at 22°C. Flies which eclosed from the test crosses (#3-5 above) were mated with the FM7IY males which eclosed from the same crosses, and the progeny scored. Since no attempt had been made to separate the females as virgins, this was appropriate. The few mutant 10 males present were assumed to be sterile. These flies were maintained at 22°C. Controls used were female flies of the each mutant stock (20 males and females of each strain) and females of type FM?'I'mutant (30 of each strain) Viability and Development at 29°C To test whether the Df I mutant could develop to adult flies at 29°C, virgin females of type DflFM7 were mated to males of each of the mutant strains. The female flies were removed after 4 days. Eight vials were maintained at 2 2 ° C , and 5 vials were shifted to the restrictive temperature. Thirteen replicates were made of each of the following crosses. Controls used were male and female mutants of all three types (30), as well as DflFM7 flies (30). The following crosses were made: a) DflFM7 ® adl-16^/Y b) DflFMl ® adl-16^2iY c) Df/FM7 <8> adl-16flrdl/Y Twenty white pre-pupal larvae of adl-16ts2 were shifted to 2 9 ° C . Ten such small experiments were performed. Heat pulses of two hours, four hours, six hours or continuous heat were applied. One vial of 20 pre-pupal larvae was left at 2 2 ° C . The effects of temperature on development of the adult are noted below. RESULTS General 22°C For Df(l)sd72b26/adl-16tsl swollen abdomens were visible by day three, and by day 7, proboscis extension was present, and abdomens were very swollen. Flies walked jerkily or weakly during the last 10 days of life. Light colored wings showed posterior wing vein variability (missing or incomplete) Df( 1 )sd72b26/adl-16ts2 showed very swollen abdomens, as well as proboscis extension. Walking problems were seen within a week; the gait was jerky and shaky. Wings were light colored, and cross veins were deformed or missing. This was unexpected, because although the chromosome with the mutant allele was marked with sc, cv, v, and/ , the Df chromosome was c v + . Df( 1 )sd72b26ladl-16flraI all held their wings up. These wings appeared dark colored and crinkled, and frequent abnormalities such as extra folds and bristles and/or hinge parts missing were noted on examination (see Figure 3-1 and photograph 3-1). The wing deformities were reminiscent of those seen on engrailed (Lawrence and Morata, 1976; Kornberg, 1981), or Minute (Sinclair, 1984). The flies walked as if on stilts, probably caused by the fact that they had deformed, sometimes flattened, legs and feet with tarsi and claws missing or abnormal. The body appeared very dark, and was missing some hairs. 1 4 5 Figure 3-1  D I A G R A M O F W I N G O F Df/adl-16fl£dl Part of wing missing General characteristics a) Wings seemed more opaque, darker, as if wrinkled, or more hairs present. Some hairs were different in shape. b) Wing size and shape differed. c) Parts of the wing were often missing, especially bristles. d) Posterior crossvein was frequently missing or deformed. e) The body of the fly was dark. f) legs and feet were deformed, with claws and pads often missing. Legs seemed fragile and weak. Normal W i n p Photograph 3-1  PHOTOGRAPH OF WING OF Df/adl-16flidi 146 The viability ratio of Oregon-RIFM7 to Oregon-R/Df was very close to 1:1 (86:83) meaning that the deficiency chromosome did not reduce viability, and so any reductions in viability seen with Df/mutant would be due to the effects of the mutant. The number of flies of type Df/mutant was divided by the number of flies of type FM?'/mutant to obtain the viability ratio for each strain. The ratios in Table 3-1 were found. Table 3-1  VIABILITY RATIOS 22°C 29°C Df/mutant:Mutant/FM7 Percent Percent adl-16tsl 140/195 72% 0% adl-16ts2 135/260 52% 0% adl-16flrcil 137/227 60% 0% X2Values were 39.56 for Dfladl-16^2, 22.26 for Df/adl-l6f^rdl, and 9.02 for adl-16tsl. Thus the Df/mutant eclosed at a significantly reduced frequency relative to FM7/mutant. At 2 2 ° C viability was thus significantly reduced in all three alleles over deficiency, adl-16ts2 causing the greatest reduction in viability. Once eclosed, though, flies of type adl-16flrdI died the most rapidly. At 29°C no Df/mutant flies developed to the imago. This represents a highly significant reduction in viability! 148 Table 3-2 PROGENY OF DEFICIENCY CROSSES A T 22°C Genotype Number Phenotype at 22°C Dfl adl-16"1 140 Swollen abdomen. Proboscis extension Wing abnormalities. Problems walking FM7ladl-16tsl 195 Normal wings, normal behaviour FM7IY 101 Normal wings, normal behaviour DflY (adl-16^110) 15 Al l had to be adl-16 t s l , tested as such. Dfladl-16ts2 135 Abnormal crossveins. Swollen abdomen Proboscis extension. Problems walking FM7ladl-16ts2 260 Normal wings, normal behaviour FM7IY 183 Normal wings, normal behaviour DflY (adl-16ts2/0) 2 Crossveinless, vermilion, forked, had to be adl-16 t s 2 and tested to be such. Dfladl-16flrdi 137 Wings up. Wings dark and abnormal. Eclosed late. Died quickly. Problems walking, deformed legs and feet. FM7I adl-16flr<U 227 Normal wings, normal behaviour FM7IY 107 Normal wings, normal behaviour DFIY (adl-16flrdll0) 1 Normal wings, lived 67 days at 22°C. Total 1503 The presence of adl-16tslIO non-disjunction flies surprised me, but they must have been of this type, because DflY does not usually survive, and these flies when tested at 29°C , died as expected for mutant flies of the appropriate genotype e.g. adl-16tsl 10 died in 5-8 days. Furthermore, the two from the cross containing adl-16ts2 were crossveinless vermilion and forked (the markers for the chromosome containing the mutant gene). The 15 from adl-16tsl parent type, were all from one vial. 149 Fertility of Deficiency/mutant at 22°C Df/adl-16tsl was fertile, producing 15 female and 10 male offspring. Whether the females were D//FM7 or F M71 adl-16tsl was not determined. The males were adl-16tslIY Dfladl-16ts2 was also fertile, producing the following offspring: 9 FM7IDf females, 26 FM7/adl-16ts2 females and 23 adl-16™2IY males. No flies of type Df/Y were produced from either of these crosses. The females could be distinguished in this case by vermilion eyes being present in FM7ladl-16ts2. Dfladl-16flrdI was not fertile. No mating was seen, no eggs were present, and no larvae were seen even though these flies of this genotype were repeatedly tested and examined. Over 90% of the control flies of type mutantlFM7 and adl-16tsl&2 were alive at the end of the above experiments i.e. 90% lived in excess of 60 days. Fifty percent of control stock of type adl-16flrdI were alive on day 50. General Observations at 29°C. Df/adl-16tsl died rapidly at 29°C. By day 2 swollen abdomens and proboscis extension were noted, and by day 4 all flies were dead. Flies of type Df/adl- 16ts2 were paralysed after 24 hours, and dead by day three. Al l Df/adl- 16flrdlheld their wings up, and died extremely rapidly at 29 °C. Most were dead within 24 hours, and the remainder were dead by day 2. Controls of type FM7/mutant lived in excess of 30 days at 29°C, and flies of the three mutant alleles died according to their expected lifespans. Viability and Development at 29°C No flies of type Dflmutant developed at 29°C. Although larvae were seen in all vials, no pupae developed. This was expected because the temperature sensitive lethal phases are embryo to second larval instar for adl-16tsl, and larval instar 2-3 for both adl-16ts2, and adl-16flrdl (Homyk et al, 1986). From the cross DflFM7 with adl-16ts]/Y, 13 FM7IY males and 16 FM7ladl-16"1 females were produced. From the cross DflFM7 with adl-16ts2, 11 FM7IY males and 14 FM7ladl-16ts2 females eclosed. In the case of adl-16flraI crossed with DflFM7 lower numbers of progeny were produced, 4 FM7/Y, and 6 FM7/adl-16f^di. In total, 28 flies of type FM7IY eclosed, in addition there were 16 adl-16"1 IFM7, 14 adl-16"2IFM7 and 6 adl-16flraI/FM7 which eclosed. No flies of type DflY survived. From the pre-pupal heat pulse tests of adl-16ts2, it was found that after a heat pulse of two hours 39/60 adults eclosed, after four hours, 22/40 eclosed, after six hours 13/20 eclosed. Of those exposed to continuous heat, 7/80 eclosed alive, two died immediately, a further 21/80 flies were parity eclosed. In each case (except for continuous heat where some early pupal death was seen) the remainder appeared fully developed to the red-eye stage but died before eclosing. Some of these flies were removed from the pupal case, and all flies were examined for eye scars as seen in shits, but none were found. The 20 pre-pupal larvae left at 2 2 ° C developed normally. It appears that shifting adl-16ts2 to the restrictive temperature is detrimental at all stages of life. Comparison of Homozygous and Hemizygous Flies In order to compare homozygous flies of each allele type with hemizygous flies either male or Df/mutant, and heterozygous flies, the following six Tables (3-3 to 3-8) are given. Data given in the tables is pooled data from deficiency crosses, and the controls used with each cross. The lifespan parameters for mutant strains were calculated separately for Chapter 3, but are quite similar to those in Chapter 1, where many flies had been tested for behavioural responses. This provides support for the position that behaviour tests had little effect on longevity. Table 3-9 shows conversion factors for the rate of living between results for Df/mutant at 22°C and 29°C. It can be clearly seen that the position of all three alleles tested was confirmed to correspond to the position of the Deficiency. Thus the genetically determined position, 52.9, and the cytological position, Bands 13Fi to 14Bi correspond. The adl-16ts gene is located between parats at 53.9 and shits at 52.2 on the X-chromosome (see Figure 3-2). Figure 3-2 COMPARISON OF GENETIC AND CYTOLOGICAL LOCATION OF adl-16^ GENE Cytological map adapted from Handbook of Genetics, Ed. , R .C. King, 1975. sJL sk SX>*\ ' < 1 u So &.f SI 5-2 £3 X - C\\ ycv^ o S c T A B L E 3-3 adl- 16&Lt'Deficiency Compared with fl<j/-76^-Homozygote and Hemizygote at 22°C adl-16"1/ Deficiency adl-16"1/ adl-16"1 adl-16"1/ Y adl-16"1/ adl-16"2 adl-16"1/ adl-16flrdi L S X 48.9 91.8 80.8 99.2 81.9 DP X 47.1 101.0 83.1 102.7 86.1 DPonset 32 86.3 56.3 60 61 D-20 40 87.3 67 88 70.5 D-50 46 102 8 1 103 84.5 D-95 59 115 102 119 94 DPend 60 110 101 119 96 D-100 65 120 115 127 101.5 DPslope 3.4 2.9 1.9 1.5 2.5 154 T A B L E 3-4 adl-16^-1 Deficiency Compared with a(j/-7f3&2-Homozygote and Hemizygote at 22°C adl!6ts2/ Deficiency adl-16^2/ adl-16ts2 adl-16ts2l Y adl-16"2/ adl-16tsl adl-16"2/ adl-16flrdI L S X 30.5 74 65 99.2 111 DP X 27.0 77 67 102.7 111 PJP0nset 13 58 50.7 60 80 D-20 20.5 55 52.3 88 103 D-50 30 76 65 103 112 D-95 45 90 84 119 122 DPend 4 1 87 80.3 119 122 D-100 48 95 94 127 129 DPslope 2.9 2.9 2.6 1.5 2.1 155 T A B L E 3-5 adl-168*411 Deficiency Compared with a<i/-76ff£<&-Homozy gote and Hemizygote at 22°C > adl-16f^rdI /Deficiency adl-16flr<H Iadlll6flrdl adl-16flrdI IY adl-16flrdI ladl-16tsl adl-16"2 1adl-16flrdI L S X 4.1 53 54 81.9 111 DP X 4.0 59 58 86.1 112 DPonset 2.5 33 37 6 1 80 D-20 2.9 36 36 70.5 103 D-50 3.3 47 " " ' — • " 55 84.5 112 D-95 4.7 86 76 94 122 DPend 4 89 77 96 122 D-100 6.5 92 83 101.5 129 DPslope 61.1 2.0 2.0 2.5 2.1 0 156 T A B L E 3-6 adl-'Deficiency Compared with aci/-76^-Homozygote and Hemizygote at 29°C adl-16"1/ Deficiency adl-16"1/ adl-16"1 adl-16"1/ Y adl-16"1/ adl-16"2 adl-16"1/ adl-16^1 L S X 3.7 9.1 8.6 6.5 9.7 DP X 3.65 9.3 8.4 6.4 9.3 DPonset 2 7.3 5.3 3 1 D-20 2.8 7.4 6.5 4.8 6.7 D-50 3.25 9.3 8.1 6.2 8.7 D-95 4.4 11.1 10 7.7 15.3 DPend 4 10.3 10 8 15 D-100 5.5 12.7 11.5 10 22 DPslope 46.5 22.7 19.8 19.2 6.7 157 T A B L E 3-7 adl-16^2-/Deficiency Compared with adl-7r5£s2-Homozygote and Hemizygote at 29°C adl-16"2/ Deficiency adl-16"2/ adl-16"2 adl-16"2/ Y adl-16"2/ adl-16"1 adl-16"2/ adl-16ftrdi L S X 2.3 3.3 2.9 6.5 7.5 DP X 2.2 3.3 2.9 6.4 7.2 DPonset 1.0 1.3 1.7 -3 4 D-20 1.35 2 2.2 4.8 5.3 D-50 1.75 2.5 6.2 7.0 D-95 2.65 3.6 3.3 7.7 10.7 DPend 2.5 3.7 3.3 8 10 D-100 3.5 4.3 3.7 10 14 DPslope 90 45.3 48.2 19.2 15.3 1 5 8 T A B L E 3 - 8 adl-16fkdLlDeficiency Compared with arf/-/6fffl2LHomozvgote and Hemizygote at 29°C adl-16flrdl IDeficieny adl-16flrdI ladl-16flrdI adl-16flrdl IY adl-16flrdI /adl-16"1 adl-16"2/ adl-16flrdi L S X 1 . 1 5 1 4 . 3 1 1 . 3 9 . 7 7 . 5 D P X 1 1 4 . 7 1 1 . 7 9 . 3 7 . 2 D P o n s e t . 5 9 . 8 7 . 3 1 4 D - 2 0 0 . 5 8 . 9 8 . 8 6 . 7 5 . 3 D - 5 0 0 . 6 1 3 . 3 1 1 2 8 . 7 7 D - 9 5 1 . 5 5 1 8 . 9 1 6 1 5 . 3 1 0 . 7 D P e n d 1 1 8 1 5 . 7 1 5 1 0 D - 1 0 0 2 2 3 . 8 2 0 2 2 1 4 D P s l o D e 1 1 0 9 . 0 1 0 6 . 7 1 5 . 3 Table 3-9 CONVERSION FACTORS FOR DIFFERENCES IN R A T E OF LIVING OF Df/MUTANT at 22°C and 29°C Dfladl-I6tsl Df/adl-16ts2 Dfladl-16flr<U L S X 13.2 13.7 3.6 DP X 12.9 12.3 1 4.0 DPonset 16.0 13 5.0 D-20 14.3 14.8 5.8 D-50 14.2 17.4 5.5 D-95 13.4 17.0 2.9 DPend 15 17.0 4.0 D-100 11.8 13.9 3.3 Average 1 3 . 9 ± 1 . 2 1 4 . 9 ± 1 . 9 4 . 3 ± 1 . 0 Each number represents the ratio of the respective 2 2 ° C and 2 9 ° C values for longevity. One 29°C day for Df/adl-16 t s l = 13.9 days at 22°C. One 29°C day for adl-16 t s 2 = 14.9 days at 22°C. One 29°C day for Df/adl-16 f l r d I = 4.2 days at 22°C. Bearing in mind that the survival of all mutants/Deficiency were severely reduced to begin with, the following compares survival curves of each strain of Df/mutant at the two temperatures used. Figures used in Chapter 1 were used to make these comparisons. In the case of adl-16tsl the conversion factor was 10.9 for females, and 10.2 for males. In the hemizygote (Deficiency/adl- 16tsl) it was 13.9. For adl-16ts2, the conversion factors were 23.1 for females, and 23.5 for males, and for the Df hybrid it was 14.9. This represents a lower degree of compression, but the Df/adl-16ts2 l i fespan was very reduced at 22°C. For adl-16flrdI conversion factors of 3.9 and 5.0 were found in stock flies, but a factor of 4.3 for Dfladl-16flrdI. Of all the alleles, adl-16flrdI was the most (drastically) affected by being in the hemizygous state. Although the parent strain (adl-16flrdI) tolerated increase of temperature better than either adl-16tsl o r 2 , the hemizygoteIDeficiency died exceedingly rapidly at both temperatures. It should also be pointed out that this strain had a lifespan reduced by approximately 50% relative to Oregon-R even at 2 2 ° C , and so the smaller conversion factors reflect quite drastic curtailment of lifespan. Nevertheless the effects of dosage compensation can be clearly seen in the males of the parent stock, which are also hemizygotes for these X-linked genes, but in all respects are very similar to homozygous females. Figure 3-3 DEFICIENCY/adl-16 MUTANTS Survival Curves at 29°C Age (Days) DISCUSSION Complementation data between the three putative alleles of adl-16ts seems to indicate that the three are all hypomorphic alleles of one locus, if hybrid data alone are considered. From this data adl-16ts2 would appear to be the most severely affected allele, adl-16tsl next, and adl-16flrdI the least affected allele with respect to the temperature change from the permissive to restrictive temperature. In round figures, the differences were 10-fold, 5-fold, and 3.3-fold reductions in lifespan. Survival curves for hybrids are intermediate between pairs of alleles, and so are viabilities. This is exactly as expected if each allele is hypomorphic and the hybrids produce intermediate quantities of the gene product from that locus. However the deficiency data produce some surprising, and at first glance, inexplicable results. Dfladl-16tsl and Dfl' adl-16ts2 each have viability curves which would be expected of hypomorphic alleles. Each shows a more severely curtailed lifespan over the deficiency than does the corresponding homozygote of the allele, that for Dfladl-16tsl being longer than that for Dfladl-16ts2. This is precisely what would be predicted, since a hypomorph in combination with an amorph (deficiency), should produce less product or product activity than a double dose of the hypomorph. The problem with accepting this hypothesis is that it does not include adl-16flrdIwhich behaves differently. This allele was the least affected at at 2 9 ° C but the Df/adl-16flrdIresulted in the most reduced lifespan of all at both temperatures. Although not surprising at 22°C, because this allele has the shortest lifespan at that temperature, it was expected that at 29°C Df/adl-16flraI would show the greatest longevity of the three, based on the assumption that the less severe alleles should also show less severe effects in combination with the Deficiency. This not being the case, it became necessary to re-examine the previous assumptions regarding the allelism and the types of mutations of these three loci. Several possibilities were briefly considered and discarded. For instance, adl-16flraI cannot be a hypermorph, since in combination with a deficiency, it would be expected to yield a lifespan closer to the wild-type than would a homozygote. Another possibility might be the site of mutation (hence the type of mutation) differing in these alleles. For instance, adl-16tsl&2 which were more similar in all tests may be caused by a change in only one amino acid in the protein of the gene product, leading to different thermolability, which causes the differences in results. Most E M S induced mutations are of this type, as mentioned earlier. Mutations, though, may also occur in the promoter or terminator areas near genes, and thus alter transcription or translation, e.g. the case of Sgs-4 mutations where several underproducers of Sgs-4 are caused by deletions or insertions upstream from the 5' end of the structural gene. In some cases the Sgs-4 gene product is reduced by 50-100-fold (hobo insert) and four different transcripts are produced (Suzuki et al., 1986). Thus the control of a structural gene may be involved in the case of adl-16 f l r d I - or of course, the reverse is also possible. Another possibility, that of a second site mutation near adl-16flrdI could not explain these results since some effect would be observed in the homozygote. The possibility does exist that a second site mutation exists which is highly dosage sensitive, that is, it is also hypomorphic and when adl- 16flrdI is homozygous the reduction in the second site product is not sufficient to produce a discernable effect. However, over a deficiency, the gene product drops below a threshhold level (50% for instance) and a drastic effect is observed. This is possible. One could also argue that the aci/- /6'/ ' r ^ / product itself is necessary in certain threshhold amounts, but then one would expect similar results from the other two alleles, which is not the case. Yet another explanation may be that adl-16flrdI is not in fact allelic with the other two mutants, but is actually a separate locus, the product of which may fall below a certain level without severe consequences. The following discussion examines the data presented and questions whether this hypothesis is reasonable. adl-16flrdI has shown a number of differences from the other two (perhaps) alleles. For example, the lifespan data were much more variable than those of the other mutants, and the shape of the viability curves is quite different, tending more to the diagonal as lifespan continues. To demonstrate this the graphs can be plotted differently. If the age (days) of final death is plotted on the abscissa, and temperature on the ordinate, we find that the homozygotes of adl-16tsl&2 have similar slope, and are to the right of adl-16flrdI which differs in two ways. First the slope is much greater, and second, this curve clearly crosses the other two. A similar graph results with the Dflmutant, but with Df/adl-16flrdI shifted to lie to the left of the other two hybrid curves. It is this shift in position which is difficult to explain. The regular survival curve of adl-16flrdI has a less clearly defined death phase region and the curve here resembles somewhat the type of curve that might be expected when a certain proportion of the death occurring is not age-related but is random as in drop-dead where the probability of death is constant with a half life of about 2 days (Benzer, 1971; Hotta and Benzer, 1972). In the case of drop-dead temperature has little effect on death rate, but clearly it affects adl-16flrdI. Perhaps corresponding to the diagonal portion of the curve, there is a surplus or deficit in some necessary product, which crosses a threshhold (maybe 50% of gene product), at which time death occurs. This would account for the sudden lethal effect when adl-16flrdI is combined with a deficiency for the region. adl-16flrdI also showed some developmental defects in combination with the deficiency, which were not observed in the other putative alleles. Again this seems strange, that the least severe in a series of alleles produced more developmental defects than the more severe alleles. The thought occurs that this gene may be a structural gene and the other two regulatory, if, indeed they do represent different loci. If adl-16flrdI and adl-16tsl&2 are really separate loci, then how could their apparent failure to complement be explained? One explanation is that of strong interaction between the loci, which have an interdependant function, such that heterozygotes of any two of them will cause a reduction in viability. I strongly suspect that the region from shits to parats may represent a structural gene family related to nerve function. There seem to be many similarities in the phenotypes these genes produce. Much other evidence in the papers written about these genes points to nerve involvement (Grigliatti et al., 1972, 1973; Suzuki et al., 1971; Poodry et al., 1973; Ganetzky, 1984). In very simple terms, adl-16ts2 is the most temperature sensitive, while adl-16flrdI is the most dose sensitive. Perhaps these are merely examples of the pleiotropy of these temperature sensitive mutants, and no further explanation is needed. It is interesting however, to speculate which tissue these genes affect. With clear nerve involvement, this could be the central or peripheral nervous system, the junctions between nerves and muscles, (the affector organs for movement), or muscular tissue. Mosaic analysis, the results of which are discussed in Chapter 4, has been used to attempt to resolve this issue. C H A P T E R 4 INTRODUCTION Classical genetic recombination methods allow precise determination of the location of a gene on a chromosome. The position of alleles can be confirmed by complementation tests, as well as mapping the gene of interest over a deficiency chromosome, to test the putative mutant position. As has been noted in the previous chapter, inferences can then be made concerning the mode of action of the genes, whether amorphic, hypomorphic, hypermorphic or neomorphic. However it is more difficult to to identify the focus of the genetic alteration; the anatomical site at which a gene exerts its primary effect. A method which is used to locate the focus of action of a gene is called focus mapping. It involves the use of gynandromorphs, first reported in D. melanogaster by T .H. Morgan in 1914. Early studies of these flies led to a realization that they would provide powerful tools for studying development (Sturtevant, 1929). Since then, both the mosaics and methods to use them have evolved considerably. For temperature sensitive behavioural mutations, such as adl-16ts2, the focus might be a particular region of the central nervous system, a single cell, or even a distant organ, the malfunction of which influences behaviour only indirectly. Behavioural responses may be disrupted by altering the ability of the individual to detect stimuli, (defective sensory receptors) to transmit and process the information, (peripheral sensory, or central nervous system defects), or to adequately respond to the stimulus, (altered motor neurons, neuromuscular junctions, or muscular system). Indeed, any or all of the above may be involved, as single, or pleiotropic effects. Temperature sensitive lethal genes would be expected to encompass an even wider range of possibilities. Several other adl mutants have been shown to have pleiotropic effects (Homyk et al., 1983). The mosaic technique has proven to be a precise method for pinpointing foci involved. The analysis of behavioural mutants of Drosophila melanogaster has been greatly aided by the use of gynandromorphs. Gynandromorphs are mosaic individuals which are part wild-type (XX) and part mutant (XO). They are most easily generated by using unstable ring-X chromosomes, designated Xr, which are frequently lost very early in the development of a fruitfly (Patterson, 1933; Hall, Gelbart, and Kankel, 1976). The resultant individual is composed of some cells bearing a full complement of chromosomes, and others which lack the Xr chromosome (an X X r / X O gynander). If the normal X chromosomes bears the mutant gene, then this gene will be expressed in X O tissues, but not in the cells containing the ring chromosome, which are wild-type for the gene of interest, and for marker genes. In order for this technique to be effective, the mutant gene must be fully penetrant, that is, expressed in all flies which carry two copies (or one unpaired copy), of the recessive mutation; and autonomous, i.e. expressed only in those tissues bearing the mutant gene. A mutation in which the gene is either partly penetrant, or produces a diffusible product which can travel from tissue to tissue, as in the case of the vermilion eye color mutant, would yield vague results at best (Hotta and Benzer, 1970). The orientation of the boundary between normal and mutant tissues depends on events occurring early in development. In Drosophila, the first nuclear division spindle is oriented arbitrarily in space (Parks, 1936). The first nine nuclear divisions occur in a syncytium-like cluster without membranes being laid down. Within this cluster, very little mixing apparently occurs in that the relative positions of the nuclei during the syncytial divisions and their migration to the cell surface tend to be maintained. After the nuclei from these nine divisions migrate to the surface of the egg three more divisions occur. Only then do cell membranes appear, and a blastoderm one cell thick is formed (Sonnenblick, 1965). Since the ring chromosome is lost at the first or second nuclear division, the resultant clones of X X r and X O nuclei each populate half the blastoderm. Since there is little mixing, the boundary is a closed figure, girdling the egg surface; its arrangement in each mosaic is different, depending on the orientation of the first spindle. Once the blastoderm is formed, the surface site occupied by a cell largely decides its fate (Geigy, 1931; Howland and Child, 1935; Hathaway and Selman, 1961; Chan and Gehring, 1971). Calculations based on correlation between wild-type cuticle (female), and mutant cuticle (male), can be used to construct a two-dimensional fate map of the blastoderm surface indicating the relative positions of presumptive adult structures. Garcia-Bellido and Merriam (1969) used 379 mosaics of D. simulans to construct a self consistent fate map for adult surface structures. Hotta and Benzer, (1974) have demonstrated that it is possible to "locate the anatomical site of abnormalities affecting behaviour" by constructing embryonic fate maps. Most importantly a mutant behavioural, or drop-dead ts focus can be located on such a map. Homyk, in 1977 mapped ten recessive behavioural mutants, of which at least two were temperature sensitive. From the location of such foci, the corresponding adult structures which are affected can be deduced and examined. Thus it may be possible to determine the type of tissue in which the adl-16 gene exerts its primary effect. If the non-ring chromosome in a gynandromorph is a marked chromosome, bearing the gene of interest flanked by markers then, tissue containing this gene can be inferred by the presence of the markers. Because loss of the ring-X chromosome usually occurs during the first division of the zygote nucleus of a female embryo (Lifschytz and Falk, 1969), this would give rise to two clones of nuclei, one with a single marked X chromosome bearing the mutant gene, which is thus expressed in male tissue, and the other with two X chromosomes, resulting in phenotypically wild female cells. Hall, Gelbart and Kankel (1976), argue that the ring-X is lost at the second nuclear division, producing two of the four daughter cells as X O , and the other two as XXr . Either way, gynandromorphs are generated in which approximately half the tissue is expected to express the mutant gene and the remainder of tissue to be wild-type. A correlation between the expression of the mutant gene in some body parts, and the mutant behaviour of the fly as a whole, should pinpoint those parts which must be mutant for mutant behaviour to be expressed, as well as body parts which appear not to influence behaviour. Temperature sensitive lethality would be demonstrated in the same manner, with the extra step of placing examined flies at the restrictive temperature, and recording lifespan. If cells destined to form two body structures are located close to each other, then the chance that they are genetically different in a mosaic is small. The further apart any two blastoderm sites are, the greater is the probability that the randomly oriented boundary in a mosaic blastula will pass between them. The probability that this will occur is directly proportional to the distance between them. To determine that distance, many mosaics are examined. Fate mapping is analogous to the construction of the one-dimensional map of the order of genes on a chromosome based on the frequencies of crossing over between the genes, with the difference that the fate map in two dimensions represents a three-dimensional blastoderm. Using mosaic individuals, a map of surface landmarks (external body parts on adult or larval flies) can be constructed by determining the distance between them. If the mosaic dividing line falls between two ipsilateral sites on the blastoderm (each being the primordium of a corresponding surface landmark), they will be of opposite genotype. The distance between the two sites is given as the total proportion of flies in which the landmarks in question are of different sex, multiplied by 100 to give a percentage of the distance between them. One map unit, or one sturt, (named in honour of Sturtevant) is defined as 1% probability that the mosaic boundary will fall between two structures. The distance of a third site from the original two may then be measured in the same way, and a map drawn by triangulation. Extension of this process to other landmarks, measured two by two, leads to construction of a two dimensional map that corresponds to the relative positions of the sites on the blastula surface giving rise to these adult landmarks. Sturtevant (1929) using D. simulans established the first fate map showing the embryonic relationships of precursor cells for the imaginal tergites and sternites. 703 gynandromorphs were used by Hotta and Benzer in 1972 to produce a fate map of Drosophila melanogaster. In a fly that is mosaic for behaviour, that character can be scored as mutant or normal, the same way that a visible part of the body can be scored. Recessive behavioural genes, combined with marker genes on the X chromosome, and viewed in mosaics can show whether the behaviour exhibited by the mosaic fly corresponds to the same or a different genotype as a particular surface landmark. If one is mutant, and the other normal, it may be concluded that, in that individual, the mosaic boundary on the blastula passed between the primordial site for the surface landmark and the one for the behavioural focus. By observing many mosaics, the distance can be obtained, in sturts, from the external landmark to the behavioural focus. This focus can then be added to the previously established map. Genetic mosaics have been used in many studies to determine the tissues which were required to be wild-type in order to produce wild-type behaviour, and also to determine the focus of various behaviour mutants. For example, Hall (1979) used genetic mosaics to derive a courtship pathway for male Drosophila. Not only was he able to distinguish a number of steps in the courtship pathway and their relative order (flies defective at one stage would not progress to subsequent stages), but also the tissues which apparently directed each behaviour. In some cases the brain was the key structure, other steps required thoracic tissue to be normal, still others depended on the genotype of the genital regions. Hotta and Benzer (1972) determined the foci for a number of behaviour mutants including wingsup and Hyperkinetic. Flanagan (1977) determined the primary focus for the lethal mutant doomed. MATERIALS AND METHODS The allele I decided to map is adl-16ts2. The reason is that it is the most severe of those under study; adl-16ts2 flies showing paralysis in less than 24 hours at 29°C, followed by death a few days later. As a consequence, faster preparation of the necessary recombinant-X chromosome was possible. Also a simple paralysis and/or death type response would be easy to score in mosaics. Since mosaics have reduced viability, it would be more difficult to score a mutant with a longer lifespan. In order to map the focus of adl-16ts2, using In(l)wvC as the ring-X chromosome, classical recombination methods were employed to genetically link the chitin markers for yellow body color (y), white eyes (w), and forked bristles (f36a) onto the chromosome containing adl-16ts2. Cuticular areas containing all three of these markers were assumed to carry adl-16ts2, since they flanked the gene of interest which was present in the marked stock. Stock flies containing adl-16ts2 carried markers for scute bristles, crossveinless wings, vermilion eyes and forked bristles, i.e. sc cv v adl-16ts2f. It was decided to use the markers yellow (body), white eye, and extremely forked bristles, to correspond with ring-X stock available, and to avoid vermilion, which is not autonomous, for scoring mosaics. Autonomy in Drosophila is defined by the phenotypic reflection of a tissue's genotype in spite of being surrounded by cells of a different genotype. A strain of yellow, f o r k e d 3 6 3 was obtained from Kathy McElwain from the University of Utah. Yellow, white flies were available in our laboratory. The following crosses were made: 1) yf36a/yf36a <g> sc cv v adl-16^2 f/Y 2) yf36a/sc c v v adl-16^2 f <g> yf36a/Y Among progeny, sc cv v (?adl-16ts2 )f36aIY recombinants might contain the gene of interest. These were crossed as below, then the male was removed after 5 days and tested at 29°C. If paralysis and death occurred as expected, those lines were continued. 3) sc cv v (?adl-16"2)f36aIY ® sc cv v adl-16"2f /sc cv v adl-16ts2f Self-cross the FI A)sc cv v (?adl-16ts2> f36"lsc cv v adl-16"2f® sc cv v (?adl-16ts2)f36a Five days after the cross was made the male was tested at 29°C. If adl-16ts2 was present, this line was continued and sc cv v adl-Ifits2f36a males and females expanded. The line was tested to check that the gene of interest was present. 5) sc cv v adl-16"2f36a/Y <g> y w ly w 6) sc cv v adl-16ts2f36aly w <8> y w IY Among the progeny, recombinants of the type y w (?adl-16ts2)f36al Y were isolated and crossed with y wly w females. After mating for 5 days the males were tested at 29°C, and if found to be adl-16"2> the line was continued. 7) y w (?adl-16ts2)f36a ® y w ly w 8) yw(?adl-16ts2)f36aly w ® y w (?adl-16"2)f3^IY Lines of y w (?adl-16ts2)f36a males and females from the above cross were tested at 29°C. Those containing adl-16ts2 were expanded and used for the mosaic cross. Originally, I intended to use the lab stock of In( 1 )wvClywspl <8> ywspl/w + Y However it had probably stabilized, because it produced insufficient mosaics even after selection, outcrossing, and other attempts to increase number of mosaics. Therefore it was not used. Although sc cv v adl-16ts2 f 3 6 a and y v adl-16ts2f36a marked chromosomes were prepared, it was decided not to use a marked chromosome containing vermilion because eye color is wild-type in a gynandromorph mosaic for wild-type and vermilion tissue because of non-autonomy of vermilion (Lindsley and Grell, 1968). A y2 cho2 adl-16ts2 f chromosome was also constructed but I found the mutant y2 difficult to differentiate from wild-type color - especially in smaller patches of mosaic tissue, so this prepared chromosome was not used. The female parents in the mosaic generating cross had the unstable r ing-X chromosome, R(1 )wvC maintained over a balancer chromosome: In( 1 )wvCI In( 1 )dl49, y w lzs 1(2) <8> A 49 y w lz/ y+Y Obtained from Jeff Hall of Brandeis University (with grateful thanks). Virgin ring-X females were crossed to y w adl-16ts2 f36a/Y males, or to control males of y w fi6aI Y, and the progeny scored. Care was taken to score all progeny from each cross, to avoid missing thoracic mosaics, which tend to eclose last (Hall, Gelbart and Kankel, 1976). This produced five progeny types listed in table 4-1. Mutations for yellow, forked, and white are known to express their phenotype autonomously. Mosaics of In(l)wvC,+ + + +/y w adl2 f 3 6 a were examined and the exact area of yellow, white, forked tissue recorded on a detailed diagram of the fly, (see Appendices ii and iii). 233 mosaic flies raised continuously at 2 2 ° C , were collected and their cuticular phenotypes examined. A diagram was made of each fly, with the wild-type tissue coded red, and the mutant tissue coded yellow. These flies were kept at 22° in vials for one to three days, during which scoring was performed, and behaviour noted. They were then shifted to 2 9 ° C . Thereafter, their behaviour and survival were monitored daily. Survivors were transferred to fresh medium at two day intervals. Transferring these flies within a 24 hour period after eclosion, and scoring them all in that time period, proved impossible. This problem was circumvented by testing a bottle of adl-16ts2 mixed age flies and larvae at 29°C. The bottle was shifted from 22°C to 29°C, and it was noted that flies of all ages died equally quickly. This confirmed a casual earlier observation that adult adl-16ts2 of any age are equally quickly affected by the restrictive temperature. 100 non-mosaic ring-X females were shifted to 2 9 ° C , transferred to fresh vials every second day and observed for 32 days, as controls. Of the 233 mosaic flies collected, 225 were used in the mosaic analysis. This represents 450 fly sides used in the calculations for mapping. Eight flies were not included in the calculations because of abnormalities such as a missing leg, which would have made them impossible to score accurately, or which might have effected their behaviour or lifespan. The resulting mosaic data were analyzed according to the procedure of Hotta and Benzer (1974), Kankel and Hall (1976), and Homyk (1977). The usual calculation formula is shown below: Distance between = # of mosaics in which structures differ vino blastoderm cells total # of mosaics scored A B (sturts) = # of times A and B are different x 100 total times A and B are scored To produce a mosaic map using the sturt as defined above, a 50% maleness is assumed, but rarely obtained. The calculations are based on the premise that approximately half of the average fly will be male, and the other half, female. To test whether a 50% maleness was present in the data obtained the maleness average was calculated for each structure as follows: maleness average = # of times a given structure is male # of times same structure was scored If the maleness value falls below 50%, this indicates that the ring-X chromosome was lost in divisions later that the first or second, and is compensated for by considering the boundary line in a mosaic to fall between different genotypes, rather than different sexes. In this case, the calculation for sturt distances listed above would be modified to accommodate a less than 50% maleness, by using a unit called the sturtoid. A B (sturtoids) = # of times A and B are different genotype # of times A is cf + # of times B is o" RESULTS Five progeny types were produced by the cross In(l)wvCl In(l)dl49, y w lzs ® A 49 y w Iz I y+ Y. They are listed in Table 4-1, with numbers of flies scored. Of the In(l) wvC/y w adl-16ts2 f36a flies, 233/988 or 23.58% showed external mosaicism. This is a high percentage which is only obtained if selection of the strain for mosaics has been maximized (Hall, Gelbart, and Kankel, 1976). It was noted early in the experiment that there was an extremely high correlation between male (mutant) tissue and paralysis at 2 9 ° C for any mobile body part. In fact after paralysis was noted, re-examination of a fly revealed a tiny patch of mutant tissue in a few cases. Table 4-1 Cross: In(l) w^Cnn(l)dl49 y w Iz* ® y w adl-16ts2f36a/Y females males Progeny Genotype Number Percent I n ( l ) w v C / y w a d l - 1 6 t s 2 f 3 6 a mosaic females 233 7.9% I n ( l ) W v C / y w adl-16ts2f3 6a non-mosaic females, (controls) 755 26.29% In(l)dl 49y w lz s/y w adl-16 t s 2 yellow white females 1534 52.04% y w adl-16ts2f3 6a yellow white forked (XO) males 89 3.02% I n ( l ) W v C / Y Ring-X chromosome males 317 10.75% Total 2948 100 Of the 100 control mosaics placed at 29°C, 93% lived beyond day 10. No behavoural abnormalities were observed; in particular no paralysis. On day 20, 76% of controls were still alive, with no paralysis visible, on day 32, 56% of the controls were alive, and some abnormal behaviour was noted. Since the adl-16ts2 m u t a n t expresses its phenotype prior to day 4, which was used as a cut-off for observation of both lethality (drop-dead), and paralysis phenotypes, clearly the mutant flies die before their control counterparts. As can be seen in the survival curve in Figure 4-1 for mosaics containing adl-16 t s 2 , 74.7% (168/225) had died by day 4, and 85.3% by day 10. The curve is reminiscent of that shown for adl-2 by Homyk et al., in 1986. A l l mosaic flies were carefully monitored for their entire lifespan, and all behaviour abnormalities recorded. It was noted early in the scoring of mosaics, that tissue containing the allele of interest at 29°C became paralysed within 24 hours. This assisted as a double check for scoring, since in a few instances early in the experiment, after paralysis was noted, the part involved was re-examined and almost invariably a small patch of mutant tissue was found. Body parts involved were: wings, which were held at unusual angles, and useless for flight; probosci, which were extended, pointing towards the wild-type side if half was involved; eyes, which caused the fly to walk in circles, or climb in a helical pattern; abdomen, dragged on the medium; and legs, which were paralysed. If several legs were affected, the fly could not stand, even though attempts were made continually. FIGURE 4-1  % Mosaics Alive or Paralyzed 120 Days at 29°C Survival and Leg Paralysis of adl-16 t s 2 mosaics at 29°C. MAPPING OF adl-16"2 The drop-dead behaviour and the paralysis of the legs of the fly were mapped separately. The first analysis resulted in the construction of a fate map for nine external surface markers on the adult fly: the third antennal segment (Ant.), proboscis (Pro.), ocellar bristle (Oc.B.), humerus (Hum), presutural bristle (P.St.), Coxae 1, 2, and 3 (Co. l , 2, or 3), and the third abdominal sternite (Stn.3). Abbreviations for these structures are given in parentheses. Then drop-dead behaviour of adl-16ts2 was mapped against this background. Finally the paralysis of each leg was mapped separately against the leg and its three closest external markers. BACKGROUND MAP Table 4-2 shows the results of the fate map analysis for the nine body structures scored. The distances observed are comparable, though not identical to those obtained by Hotta and Benzer (1972). The map obtained from these values is shown in Figure 4-1. As expected, it is similar to their background map. A l l behaviours studied are mapped against this background. 185 Table 4-3 Distances Between Landmarks (to nearest Sturt) Midline Ant Oc.Br Pro. [Hum. P.St. Co.l Co.2 Co.3 Stn.3 14 Ant. 1 1 Oc.Br. 8 9 Pro. 15 19 1 3 Hum. 25 29 15 P.St. 3 1 34 22 18 18 Co.l 27 28 21 22 12 21 Co.2 33 38 30 28 17 14 17 Co.3 37 40 32 26 16 19 1 1 20 Stn.3 47 49 39 [30 25 32 25 20 The validity of the analysis depends upon the assumption that all parts are equally likely to be effected. To address this assumption, The degree of maleness of each landmark used was calculated as follows. maleness average = # of times a given structure is male # of times same structure was scored (see Table 4-4). It was found that the ring-X stock from Brandeis used in this experiment In(l) w v C / I n ( l ) dl49,y w lz s gave 49.93 % maleness over 9 external structural landmarks involving 4050 fly sides examined. Values ranged from 45.5% to 54%. These values are shown in Table 4-4. They are all close to the expected 50%, thus justifying the use of an analysis similar to that used by Hotta and Benzer, and so the unit used for calculations was the sturt. Figure 4-2 Background Fate Map Diagram. Scale: 1 Sturt = 2 mm. Distances between structures are given in Table 4-3. 187 Table 4-2 Drop-dead Behaviour Mapped to 9  Landmarks Landmark Drop-dead Oc. Br. 29 Ant. 25 Pro. 26 Pst. 1 9 Hu. 25 Co.l 17 Co.2 19 Co.3 20 Stn.3 30 Note: Numbers refer to distances in sturts between landmarks and a presumed drop-dead focus. T A B L E 4-4 PERCENT MALENESS OF THE NINE STRUCTURAL LANDMARKS USED Landmark # scored #male % male exp# male X 2 ( 0 - E ) 2 / E Antenna 450 207.5 41.6 225 0.16 Proboscis 450 229 50.8 225 0.17 Oc.Bristle 450 214 47.5 225 0.54 Pre.St.Br. 450 233 51.7 225 0.28 Coxa 1 450 232 51.5 225 0.22 Coxa 2 450 243 54.0 225 1.44 Coxa 3 450 241.5 53.6 225 1 1.21 Humerus 450 219 48.7 225 0.16 Stn. 3 450 205 45.5 225 1.76 Total 4050 2024 449.4 625 5.86 To know if this is less than expected at error probability of 5%, 8 degrees of freedom gives a X2=15.507, which is above 5.86. The X 2 value falls between the 50 and 90% level of probability so this level of deviation could be expected 50-90% of the time. D R O P - D E A D FOCUS It is useful to first determine whether the drop-dead focus is nearest to the head, thorax, or abdomen. Mosaics which had either entirely mutant or entirely wild-type heads were examined. Of the 51 mosaics in which the heads were completely mutant, 47 showed mutant {drop-dead) behaviour, and four showed wild-type behaviour. Of the 58 flies whose heads were entirely wild-type, 24 showed mutant behaviour and 34 wild-type. This gives a total of 28 mosaics in which the genotype of the head as a whole, and the behaviour differ. The map distance calculated for these data is 26 sturts. The same analysis was performed for the thorax and the abdomen, giving map distances of 16 and 33 sturts, respectively (see Table 4-5). Table 4-5 Preliminary Mosaic Mapping of drop-dead Focus.  Head. Thorax, and Abdomen. Major Division Genotype (N) Behaviour Number Head (109) 28/109=25.68 Sturts Mutant (51) Mutant 4 7 Wild Type 4 Wild Type (58) Mutant 24 Wild Type 34 Thorax (51) 8/51 = 15.68 Sturts Mutant (29) Mutant 25 Wild Type 4 Wild Type (22) Mutant 4 Wild Type 1 8 Abdomen (67) 22/67=32.83 Sturts Mutant (21) Mutant 17 Wild Type 4 Wild Type (46) Mutant 18 Wild Type 28 Table 4-5 shows the results of the analysis of the drop-dead behaviour of the adl-16ts2 mutant. The drop-dead focus is shown in Figure 4-3. This behaviour appears to map in the ventral thoracic region, in a vaguely defined position which most likely represents mesoderm. Figure 4-4 shows the expected positions of presumptive mesodermal and nervous tissue (dotted lines), superimposed on Figure 4-3. <r Figure 4-3 Fate Map of drop-dead Behaviour. Scale: 1 Sturt = 2 mm. Distances'between structures are given in Table 4-3. v Figure 4-4 Map Showing Expected Positions of Presumptive Neural and Mesodermal Tissue Superimposed on Figure 4-3 / / PP3~ presumptive V^J nervous - - - \ > j Presumptive mesodermal tissue. DOMINEERING OR SUBMISSIVE FOCUS In flies which were roughly bilaterally mosaic, it was noted that if one side or the other of the fly was mutant, that fly would die. Of 14 approximately bilateral mutants, 11 died in less than five days, the remainder died prior to 10 days. This is reminiscent of the data for &dl-2ts presented by Homyk et al. (1986). Thus, there existed the possibility that there were two behavioural foci, only one of which must be mutant to kill the individual. This is the definition of a domineering focus as outlined by Hotta and Benzer (1972). Accordingly, behaviour was analysed using their approach for domineering foci. For each landmark (the four closest to the presumed thoracic focus were used: the three coxae and the presutural bristle) the distance from the landmark to the focus, the distance from the landmark to the midline, and the distance between foci were calculated. According to Hotta and Benzer, the distance between foci should be the same, if the proposal that the two foci have a domineering relationship is correct. This is because the distance between foci should be the same, no matter which landmark is used as a reference point. Table 4-6 shows that for each of the four landmarks used, the distance between foci is quite different. Thus although it initially appeared that there were two domineering foci, this was not the case. Table 4-6 Analysis of Four Landmarks for Domineering Foci (After Hotta and Benzer Distance to Focus Distance to Midline Distance Between Foci P.St. 20 1 8 39 Coxa 1 19 1 8 3 1 Coxa 2 21 21 28 Coxa 3 23 17 3 1 Note: Distances are given in sturts. If there are not two domineering foci, then what explanation could support the observation that when the legs on one side or the other of the fly were mutant, that fly usually died? A closer examination of the data showed that even in mosaics which were not divided bilaterally, death occurred in nearly all individuals with three or more legs mutant, whether or not they were on the same side. Thus the number of flies with 0, 1, 2, 3, 4, 5, or 6, legs mutant were counted and the proportion of each class which died determined. The results of this analysis can be seen in Table 4-7. It is clear from these data that a fly having 4 or more legs mutant has only about a 5% chance of surviving past 4 days (the cut-off point), and with 5 or 6 legs mutant, virtually no chance of survival. On the other hand, even with no legs affected, an individual still had a 15% chance of dying. See Table 4-7 Table 4-7 Number of Legs Mutant, and Probability of Early Death # Legs Affected Early Death Non-Mutant Total % Died Early 0 4 23 27 15% 1 4 13 17 24% 2 1 1 12 23 48% 3 45 14 59 76% 4 17 1 18 94% 5 15 0 15 100% 6 65 66 99% MULTIPLE FOCUS This led to the hypothesis that adl-16ts2 shows early death due to paralysis and subsequent starving or the cessation of other vital body functions due to internal paralysis. It was predicted that flies with a mutant proboscis might not be able to eat, so probosci were re-examined in the mosaic flies. Only two flies were found with no mutant legs, and a fully mutant proboscis. In the first of these, the entire head was mutant. It died in under four days. The second fly lived nine days, but some normal tissue was observed on the head. Of four flies with partly mutant proboscis, accompanied by wild-type legs, two showed drop-dead behaviour and two lived over 24 days. This is a rather small sample, and no conclusion can be drawn from the observations with respect to the involvement of probosci as a cause for death. It will be recalled, however that flies of type adl-16"1 lived several days with extended probosci (Chapter 1). During examination of the flies it was noted that the paralysis of each leg was largely independent of the other legs (like the mutant hyperkinetic, in which shaking of each leg was independent of that of the other legs; Hotta and Benzer, 1972), and like the mutants adl-lts], rex, and sesE, analysed by Homyk et al. (1986), therefore it was decided to map the paralysis of each leg separately. If the paralysis is independent in each leg, a separate focus should be obtained for each. Table 4-8 shows the results of the maps. Table 4-8 Map Data for Paralysis of Legs 1. 2. and 3. Distance to Nearest Sturt Paralysis Coxa 1 Coxa 2 Coxa 3 P.St. Leg 1 6 1 1 20 14 Leg 2 1 2 8 1 3 16 Leg 3 1 9 14 7 1 9 The four nearest landmarks to each focus were used. Fig 4-5 includes the focus obtained for each leg. For legs 1 and 3, the paralytic foci lie just ventral and posterior to the primordial region of coxas 1 and 3 , respectively. The focus for leg 2 is ventral and slightly anterior to the primordial region for coxa 2. The foci become slightly less distinct towards the posterior, i.e. the focus for leg 1 is quite sharp, that for leg 2 slightly less distinct, and that for leg 3 relatively diffuse. Nevertheless even the background map of Hotta and Benzer shows sizeable areas for mapping of legs. In all cases, however, there is an apparent neural focus (ventral thorax), possibly corresponding to the thoracic ganglia for each leg. Fate Map of Paralysis of Legs Note: Leg 1 (PI), Leg 2 (P2), Leg 3 (P3) DISCUSSION Complete debilitating paralysis of flies of type adl-16ts2 occurred when adults of any age were placed at 29°C indicating a continuous requirement for the normal gene product in the adult (personal observation). The fact that larvae of these flies die as second or third larval instars indicates that this product is also required for normal development. The product of this gene may represent a protein important for the normal metabolism in the fly. To determine the tissues affected by the expression of the adl-16ts2 gene, mosaic analysis was completed. It was hypothesized that nervous or muscular tissue was compromised at the restrictive temperature. The map distances used are not exactly additive. This is to be expected because, when the distance between two points is large, the probability becomes considerable that a boundary, having once intersected the line connecting the two sites, may cross back again, so that both sites end up with the same genotype. Large distances thus tend to appear shorter than their true value, in analogy to the effect of double crossing over in genetic mapping. The true distance between widely separated points is most accurately obtained by the summation of small intervals. The probability of a cleavage plane bisecting two points on the three-dimensional surface of the sphere refers to the angle between two points from the centre, or the arc distance between two points on the surface. This arc distance is curved. The shorter it is, the closer the straight line obtained as sturt distance approximates it. Another source of distortion of the map is caused by the fact that the convex egg surface is represented on two dimensionally. The overall orientation of the map with respect to antero-posterior and dorso-ventral axes can be decided from other embryological information. The maps represent the best attempt by hand to fit all obtained distances into a two-dimensional array, bearing in mind as well that the egg is an oval shape, rather than a circle. The distances estimated by triangulation agree fairly well with the distances obtained mathematically where long distances are concerned. In cases where two or more distances could not be entirely reconciled, the shorter distances were taken as the most likely to be correct. The fate map analyses have yielded a focus for the premature death of adl-16ts2 in the ventral thorax, in an area which most likely represents mesodermal tissue. The paralytic effect of the mutant maps separately to three foci most likely located in nervous tissue, and possibly corresponding to the thoracic ganglia for each leg. The question thus arises, are there two sets of foci? and if so which of them represents the primary defect for the mutation? If two sets of foci are involved, then it must also be assumed that paralysis and lethality cosegregate. If there is only one type of focus, then is the second focus an artefact, or an indirect result of the first focus? The argument for one focus, is as follows: There is a possibility that there are two sets of foci: one where the mutant acts to cause death, and another set for the individual paralysis of each limb. This seems unlikely, because of those mosaics which died early, (in less than 4 days), virtually all showed paralysis (those which did not show paralysis, died before paralysis could be scored), which argues against separate foci for paralysis and death. If the idea of separate foci is correct, then how can the differing map locations of early death and paralysis be explained? I initially believed this to be an artefact based upon the hypothesis that the mutation acts to paralyse whatever part of the fly is mutant, and that death results when a large enough proportion of the fly is mutant. This idea arose because of the observation that the male tissue, in almost all cases, appeared to be paralyzed. For example affected wings were observed sticking out at abnormal angles and these were never moved back into a more normal position. Affected probosci were found to be permanently extended. Also some partly mutant probosci were bent towards the affected region. Flies with one mutant eye walked in circles, with the wild-type eye towards the centre, indicating that the other eye was defective (Benzer, 1971). Flies with affected abdomens were unable to hold up the abdomen in a normal position, rather they dragged it along. Unfortunately it was often not possible to score the presence or absence of paralysis in these structures because mosaics which died early were frequently found on their backs, held to the moist medium by surface tension. It may be recalled that this is the most typical pose of adl-16ts2 flies at the restrictive temperature. However it was possible to test the paralysis of the legs when the flies were found in this position. It was simply necessary to prod each leg very gently with a soft brush and determine whether or not the leg moved in response. If the artefact hypothesis - that paralysis of the mutant, and subsequent starvation or cessation of other body functions is what actually kills the flies, - is correct, then the drop-dead behaviour ought to map equally distantly from all body parts. This distance should correspond roughly to the proportion of the fly which must be mutant in order for it to die. This would assume that all parts have an equal likelihood of being affected, and that each part is equally effective in contributing to the death of the organism. The first assumption presents no problem. Many studies (Flanagan, 1977; Garcia-Bellido and Merriam, 1969; Hall, Gelbart and Kankel, 1976; Homyk, 1977; Hall, 1978;) have utilized gynandromorph analyses and in all cases the results have been shown to be valid. Confirmation of damaged tissues of the expected type has supported the tissue of choice for the focus in their studies. Such studies can only be valid if all cells in the fly have an equal chance of being mutant. However the second assumption seems questionable. Simply by considering the effect on the fly of a paralyzed humerus, for example, compared with a paralyzed proboscis one can see that some parts of the fly are much more vital than others, and that paralysis of one might cause starvation, while that of another might have a slight effect, if any, on viability. This made it necessary to modify the artefact hypothesis somewhat; adding that certain parts of the fly were more critical than others in determining whether or not the individual would die. Paralysis of the legs was chosen for scoring both because the ability to walk is directly related to survival, and because the legs were easy to score for paralysis in all of the mosaics. If it had been possible to score sufficient probosci separately, they would have been included in the analysis. The precedent of such analyses by the researchers mentioned above, makes this analysis all the more plausible. The analysis revealed that the greater the number of limbs affected, the greater the likelihood that the individual would die. Flies with four limbs affected had a very slim chance of living beyond 4 days, and with more than 4 legs affected, had none. That flies with no mutant limbs had a 15% chance of early death indicates that other body structures also contribute to the early death. The proboscis is the likeliest candidate, although internal structures may also be involved, for example nerve ganglia or the digestive system. Pearl and Parker, (1924) determined the longevity of flies deprived of food and or moisture. They found that at 25° C flies deprived of food and moisture lived for 2-3 days, and those deprived only of food lived approximately 4 days. At 2 9 ° C adl-16ts2 flies become paralysed within 24 hours and live 1-4 days, which corresponds quite well to the results of Pearl and Parker. It thus seems reasonable to conclude that the adl-16ts2 flies also starve to death, and this accounts for the fate mapping results. The artefact hypothesis thus posits that paralysis related to nervous tissue is the primary focus of the gene, and death is a secondary effect resulting from paralysis. The problem with accepting this hypothesis is to reconcile these data and assumptions with the graph in Figure 4-1, which clearly shows different curves for paralysis and survival. If, in fact there are two sets of foci (the second hypothesis), then both mesoderm and neural tissue are involved in the expression of the adl-16ts2 gene. It is not at all unusual for temperature sensitive adult lethal mutants to demonstrate pleiotropic effects including lethality as in the temperature-sensitive mutations for flight (Homyk and Grigliatti, 1983). Nor is it exceptional that two foci be found, one for lethality, the other(s) for a behavioural component of phenotype. Examples are the Hyperkinetic1 jumping action mapping to the brain, while life-shortening maps to the anterior thoracic ganglion (Trout and Kaplan, 1981), and leg shaking maps to a separate focus for each leg (Hotta and Benzer, 1972). There was also an independent mosaic death focus in this strain which mapped in the region of the wing (Trout and Kaplan, 1981). Homyk et al. (1986), found a pair of neural foci, each of which appeared to include three separate foci for leg paralysis in sesE. For both adl-ltsl and rex they found separate mesodermal foci for each leg, and because of the difference in survival curves for mortality and paralysis, they inferred that a separate focus for lethality existed, but they were not able to determine it because death of the mosaics was still occurring when the study ended. The graph in Figure 4-1 concerning percentage of flies alive, and percentage paralyzed for my data, shows two completely different patterns. It is extremely similar to those found by Homyk et al. (1986) for adl-1tsl and rex. This supports the hypothesis that the early death and paralysis foci are separate in adl-16ts2. If this is so, then the focus for early death mapping to mesoderm would be expected to be a domineering focus, as explained in the results. Since the data for distances between foci are not the same, this presents a problem in accepting this hypothesis. Early in the scoring process of the experiment, it was noted that flies with four limbs affected (those four limbs often being adjacent) died rapidly. Some flies with only dorsal thoracic tissue affected survived, so I thought at that time that the focus would map to the mid ventral ganglion, as a single submissive focus. However too many bilateral mosaics died too quickly to support this assumption, so the interpretation of their being a pair of domineering foci seems logical considering the data. There is no problem accepting the three foci for paralysis of legs in neural tissue for either of the above hypotheses. These foci correspond closely to the position of the presumptive legs, which would indicate that each leg is under the control of a separate noninteracting focus. These foci most likely are located in the neural tissue of the thoracic ganglia. In fact paralysis of legs was noted to correlate almost 100% with the presence of mutant leg tissue (cuticle markers). Whether these foci represent the primary focus is more difficult to say. Lethality and paralysis seem to co-segregate in adl-16"2. The fact that the adl-16ts2 mutant may map to either neural or mesodermal tissue or both, is interesting because a great many genes involved in nervous system function are located near the adl-16ts2 site (52.9) on the X chromosome. These include: bang-sensitive at 47.2 (Grigliatti et al, 1973), Ether-a-gogo at 50.0 (King, 1975), shibirets located at 52.2 (Grigliatti et al., 1973), para" at 53.9 (Suzuki et al., 1971), stress sensitive (Homyk et al., (1980, 1986), and stoned at 66.3 (Grigliatti et al., 1973). The shi" locus is of particular interest in regard to adl-16"2, because preliminary complementation data (Sinclair, personal communication), suggest either that adl-16" is allelic with shi"' or that strong interactions take place between the two loci. Interactions between loci with related function are well known, and two nervous system mutants, para", (on chromosome 1, see above) and nap" ( 2nd chromosome), provide a good example of this. Both are temperature sensitive mutants, with reversible paralysis observed at the restrictive temperature. Studies have shown both to be associated with sodium channel function, (Ganetzky, 1984). Double mutants are lethal at the permissive temperature, and, even heterozygotes of para"/ + show some lethality in a nap" background. It was suggested that the two loci each code for one of the subunits required for the pores of the sodium channels. I suggest that adl-16" and shits alleles may interact in a similar way, that is, that the two loci each perform a strongly interdependent function, or that the two are allelic. Kelly demonstrated that two alleles of shits (2 a n a 6K are involved in the production of some component of the regenerative sodium channel, using tetrodotoxin, which specifically blocks this channel of the action potential mechanism, and furthermore that this blockage was temperature related (Kelly, 1974). Kosaka and Ikeda (1983) have related shitsl to the reversible blockage of membrane retrieval and endocytosis involving the labyrinthine channels and coated pits and vesicles of Garland cells. Attempts are being made to clone shits , and it is apparently involved in microtubule function, which explains a great number of the developmental defects caused by the mutant, (Poodry et al., 1973). I would suggest that similar developmental studies be performed on all the adl-16ts alleles so that they can be better compared with those of shits. Developmental studies such as have been completed for shits including histological examination of eggs and larvae developing at the restrictive temperature are indicated for adl-16ts alleles and might explain whether mesodermal and nervous tissue develop normally, since these mutants die at larval stages 2-3 at the restrictive temperature. Shits may code for one structural protien, adl-16ts, perhaps another. Alternatively, either or both could code for enzymes involved in the assembly or disassembly of microtubules, with one having a more crucial developmental role than the other. Since the adl-16ts2 locus appears to compress age-related behaviour loss, it is even possible that its product is regulatory rather than structural, and acts to control the timing of certain events, rather than the events themselves. Both shits (Sinclair, personal communication) and adlts mutants are uncovered by the same deficiency, and when shits has been cloned, certain probes and restriction sites will be available, and I therefore suggest that a walk (strenuous hike) through the region from sh its to parats might be very useful, (with the possibility of isolation of a gene product), in elucidating function. If shits and the adl-16ts are alleles, then the nature of each defect might then be determined. In the meantime, to confirm or eliminate the possibility that shits and adl-16tsl ,2,&flrdi a r e alleles, detailed fine structure mapping would help to clarify the relationship between the many loci which are found in that region. This interesting area certainly deserves further investigation. The relationship of the three adl-16ts mutants under study to ageing, seems unclear. While adl-16tsl has been found to accelerate age-related behaviour loss, adl-16ts2 dies too early at the restrictive temperature to determine such loss, although at 2 2 ° C , the pattern of behaviour loss is somewhat like that of adl-16tsl- Most adlts mutants exhibit distinct pleiotropic phenotypes, so it is not surprising that there are many differences observed in these alleles including adl-16flrd. During adult life, all normal metabolic reactions must occur, and a few processes of behaviour and reproduction change. Mutations affecting essential processes are likely to shorten the lifespan of the fly, if not kill it, so the distinction between cessation of normal function due to mutation and normal gene regulation causing ageing is difficult to separate. However, the connection of the locus of adl-16ts2 to neural or mesodermal tissue in no way disqualifies this mutant as one involved in the ageing process. As pointed out in the introduction, nervous system and muscle cell degeneration is a well documented age-related change, and the onset of damage correlates well with the observation of behaviour loss (Miquel, 1971). In fact, in a mutant which alters behaviour, it would be strange if the these tissues were not involved, although it is not impossible. SUMMARY DISCUSSION An enormous amount of research data exists on Drosophila melanogaster. Studies have concerned longevity, behaviour, histology, biochemistry, and genetics and other esoteric topics. Much evidence has been amassed, but rarely have two or more of the above subjects been considered at a time. To date, no one study has examined a group of alleles, with respect to longevity, behaviour, gene location and tissue of gene action over the lifespan of the flies involved. This study has attempted to do that. In the first chapter, it was demonstrated that lifespan is predictable under controlled conditions, and that it is strain and sex specific for these mutant alleles and their hybrids as well as wild-type flies. This provided a basis upon which behaviour was tested in the second chapter. Behaviours geotaxis, phototaxis and motor activity were found to be lost in the consistent order that they were listed in the previous sentence, and furthermore, adl-16tsl compared with Oregon-R at either the permissive or restrictive temperature demonstrated a relationship with the wild-type strain which could be said to be related to ageing. Whether it is related to ageing, or merely parallels it remains to be tested further. Since behaviours were consistent, and quantifiable, and there was a definite pattern of behaviour loss for each strain examined, these parameters could be used as bio-markers of ageing in Drosophila. Lifespans of hybrids between the three alleles adl-16tsl, adl-16ts2, and adl-16flrdIwere found to be intermadiate between those of females of the generating parent strains at 2 9 ° C , confirming their allelic nature. The three mutants were located within the bands 13Fi to 14Bi, of the X-chromosome since all were uncovered by the deficiency Df(l)sd72b26. Hybrids between mutants and deficiency had greatly reduced viability and lifespan, as well as some anatomical deformities. It would thus be interesting to test the genetic map location of 52.9 by fine mapping these alleles. There are many similarities between the adl-16ts alleles and shits or parats which are located on either side of them, so hybrid studies between these alleles might prove interesting. The mosaic analysis of adl-16ts2 demonstrated two types of focus. The drop-dead behaviour mapped to one diffuse focus which appeared to be in an area of presumptive mesoderm, while three foci were found for paralysis close to each set of legs, in an area most likely to be presumptive nervous tissue. An examination of neural tissue of the thoracic ganglia is thus suggested. It would be interesting to test the eyes of flies of adl-16ts2 at 29°C to see if the E R G pattern is different at the restrictive temperature. Such an analysis might point to either a neural deficit or a deficit in the receptor cells which transmit impulses to the neurons. Although allelic, flies of type adl-16flraIdemonstrated many differences from the other two alleles, in lifespan, behaviour, and also as a hybrid with the deficiency. It was the shortest lived at 2 2 ° C , but the longest at 29°C. The effects of the deficiency as a hybrid with this allele were the most severe at both temperatures. When combined with other alleles in hybrids, there was a very noticeable effect of the presence of this allele. Since this mutant was isolated in a screen for mutations affecting flight, it would be interesting to test this behaviour more extensively in all three alleles over their lifespan. Flight ability might then be added to the other possible bio-markers of age. Additional behaviours which could be tested are mating behaviour as well as fertility and fecundity. For the allele adl-16ts2, testing at 25°C might give better results for behaviour tests, because behaviour is lost so early at 2 9 ° C . If the experiments were repeated at this temperature comparisons could be made between alleles including adl-16ts2. Prior to this, it would be advisable to remove the markers sc, cv, v, and f, to avoid any possible interference in experimental results. If behaviour and longevity studies were to be repeated it would be interesting to include some alleles of shits and parats. Other suggestions for additional research include temperature shift studies, to determine if temperature-sensitive periods exist. Preliminary testing of the alleles indicated that the restrictive temperature effect was continuous when flies were shifted to it at any age. Histological examination of the alleles both at 22°C and at 2 9 ° C might elucidate the problem manifested at the restrictive temperature. This could be tried in mosaics of adl-16ts2 in particular, to demonstrate differences in affected and normal tissue. Such a study could encompass flies of different ages, to examine the question of a genetic basis of ageing in adl-16tsl in particular. If, in fact adl-16tsl or adl-16ts2 do have a role in the ageing process, then further testing will support this idea. This research, although it confirms the results obtained previously, does not explain the nature of the gene product for adl-16ts, or how it works, nor could it be expected to do so. 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