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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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L O N G E V I T Y , BEHAVIOUR, A N D MAPPING O F T H R E E T E M P E R A T U R E SENSITIVE A D U L T L E T H A L A L L E L E S OF DROSOPHILA MELANOGASTER  by  B E V E R L E Y NINA HANSEN B.Sc.(Honours), The University of Saskatchewan,  Regina,  1970  A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T O F T H E REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in  T H E F A C U L T Y OF G R A D U A T E STUDIES (Zoology) We  accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH COLUMBIA October 1991 ©  Beverley Nina Hansen, 1991.  7 '  In  presenting this  degree at the  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make it  agree that permission for extensive  scholarly purposes may be her  for  It  is  granted  by the  understood  that  head of copying  my 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  DE-6 (2/88)  October  1991  ABSTRACT The  rate  at  which  an  organism  ages,  as  well  senescence are determined by many factors. well  as  different  lifespans.  strains  the  same  the  Different  species,  have  adl-16f ,  putative  and  lrdI  melanogaster  allele  wild-type  were  mutants  geotaxis, phototaxis,  tsl  Oregon-R  examined  as  Drosophila  for  adl-16 ,  tsl  ts2  (control)  patterns  of  Drosophila  age  temperature,  dependent Longevity,  and motor activity were examined  at both the  2 2 ° C , and the restrictive temperature,  compared to  behaviour loss  was  At 2 9 ° C ,  wild-type,  longevity  and the  compressed  into  pattern  a shorter  was of  time  significantly age-dependent  frame.  pattern of behaviour loss was consistent with that expected mutation which increases the rate of ageing 1984).  29°C.  displayed a longevity and behaviour loss pattern similar to  the wild-type strain at 2 2 ° C . reduced  species,  adl-16 ,  behaviour loss over the course of their adult lifespans.  adl-16  of  to test their influences on ageing and senescence.  Temperature-sensitive  permissive  onset  characteristic  This study is an investigation of three strains of  melanogaster,  and  of  as  The lifespan  of  adl-16  at  ts2  This from a  (Leffelaar and Grigliatti,  22°C  was  reduced,  when  compared with Oregon-R, but the behaviour loss pattern was similar. At  29° C  the  flies  behaviour loss.  died  rapidly,  with  in  a  total,  immediate,  Survival curves at 2 2 ° C , 2 5 ° C , and 2 9 ° C adjusted for  the rate of living were coincident. isolated  almost  separate  screen  Flies of the type as  flight-reduced,  adl-16f , lrdI  demonstrated  differences  from adl-16  well as behaviour. although  and adl-16  tsl  in longevity  ts2  curve shape as  Lifespan was reduced at both 2 2 ° C and 2 9 ° C , and  behaviour differed  slightly  in young  loss pattern was similar to that of adl-16 .  flies,  the  behaviour  The order of severity of  tsl  effects of the restrictive temperature from the least affected the most affected was adl-16f , lrdI  adl-16 ,  and  tsl  At 2 2 ° C , hybrid flies of type adl-16 ladl-16 tsl  Ififlrdl exhibited longer  lived  lifespans  16 Iadl-16f was tsl  between  parental  parent  types,  and  Geotactic behaviour was phototaxis  to  reduced  adl-16 ladlts2  the latter being  The hybrid  have  longevity  with  respect  lifespan,  between  to  Oregon-R.  reduced in all three hybrids at 2 2 ° C ,  females  Complementation did not confirmed  16 ladl-16  to  be  of  the  generating  occur, and thus  alleles. ts2  flies.  strains.  under  reduced  in  at 2 9 ° C , but not in  lrd  but  intermediate  parent  all mutants  Behaviour was  and adl-16 ladl-l6f  ts2  adl-  intermediate  all hybrid strains could be said to demonstrate  tsl  strain  and motor activity were similar to that of wild-type  At 2 9 ° C  were  ts2  and  ts2  strain.  demonstrated  lrdI  adl-16 .  comparable to Oregon-R,  than either  allele to  study adladl-  I6tslladl-16fl . rd  Flies of type Deficiency/mutant lifespans at both 2 2 ° C and 2 9 ° C . Dfladl-16 , tsl  most  severely  Df/adl-16 , ts2  affected,  were found to have greatly reduced The order of severity of effects was  and Dfladl-16f , lrdI  with deformities  with the last being the  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-16 ways  by means  behaviour this  The  gynandromorph analyses.  was mapped against  study. This  region,  of  likely  involves  behaviour noted  presumptive  in these  flies  tissues  would  behaviours noted earlier.  not  mesodermal  tissue.  at 2 9 ° C  was then  Three foci were found.  A l l three  appear to map to presumptive nervous tissue. muscle  drop-dead  a background map constructed for  mapped separately for each leg.  and  The  behaviour appears to map to the ventral thoracic  and most  paralysis  was mapped two  ts2  be  Involvement of nerve  surprising,  considering  the  The discussion involves speculation as to  the precise function of all three adl-16  ts  genes.  V  T A B L E OF CONTENTS Page ABSTRACT  ii  TABLE OF CONTENTS  v  LIST OF TABLES  vi  LIST OF FIGURES  ix  ACKNOWLEDGEMENTS GENERAL INTRODUCTION  xiii 1  CHAPTER ONE Introduction Materials and Methods Results Discussion  14 17 21 91  CHAPTER TWO Introduction Materials and Methods Results Discussion  98 101 105 135  CHAPTER THREE Introduction Materials and Methods Results Discussion  140 141 143 163  CHAPTER FOUR Introduction Materials and Methods Results Discussion  168 174 180 197  SUMMARY DISCUSSION  208  REFERENCES  212  APPENDICES  222  vi  LIST OF T A B L E S Page TABLE 1-1  Repetitions of Survival and Behaviour Tests.  21  TABLE 1-2  Longevity of Oregon-R Adults Maintained at 22°C or 29°C Posteclosion.  40  TABLE 1-3  Conversion Factors for Differences in Rate of Living of Oregon-R at 22°C or 29°C Posteclosion.  41  TABLE 1-4  Longevity of adl-16 Posteclosion.  Adults Maintained at 22°C or 29°C  52  TABLE 1-5  Longevity of adl-16 Posteclosion  Adults Maintained at 22°C or 29°C  53  TABLE 1-6  Longevity of adl-16 Posteclosion.  Adults Maintained at 22°C or 29°C  66  TABLE 1-7  Conversion Factors for Differences in Rate of Living of adl-16 at 22°C and 29°C.  67  TABLE 1-8  Longevity of adl-16 Posteclosion.  67  TABLE 1-9  Longevity of adl-16f Adults Posteclosion  tsl  ts  ts2  1  ts2  ts2  lrdI  Males Maintained at 22°C or 29°C  Maintained at 22°C or 29°C  78  TABLE 1-10 Conversion Factors for Differences in Rate of Living of adl-16f m 22°C and 29°C.  79  TABLE 1-11 Longevity of Adult Hybrid Females Maintained at 22°C Posteclosion.  88  TABLE 1-12 Longevity of Adult Hybrid Females Maintained at 29°C Posteclosion.  89  lrdI  TABLE 2-1  Behaviour Loss with age in Oregon-R Males at 22°C and at 29°C.  120  T A B L E 2-2  Behaviour Loss with age in Oregon-R Females at 22°C and at 29°C.  121  TABLE 2-3  Behaviour Loss with age in adl-16 and at 29°C.  122  tsl  Males at 22°C  TABLE 2-4  Behaviour Loss with age in adl-16 and at 29°C.  tsl  Females at 22°C  TABLE 2-5  Behaviour Loss with age in adl-16 and at 29°C.  ts2  Males at 22°C  TABLE 2-6  Behaviour Loss with age in adl-16 and at 29° C.  TABLE 2-7  Behaviour Loss with age in adl-16f and at 29°C.  TABLE 2-8  Behaviour Loss with age in adl-16f and at 29°C.  TABLE 2-9  Behaviour Loss with age in adl-16 Females at 22°C and at 29°C.  ts2  Females at 22°C  Males at 22°C  lrdI  Females at 22°C  lrdI  tsl  I adl-16  Hybrid  ts2  TABLE 2-10 Behaviour Loss with age in adl-16 ladl-16f Females at 22°C and at 29°C. tsl  Hybrid  lrdI  TABLE 2-11 Behaviour Loss with age in adl-16 ladl-16f Females at 22°C and at 29°C. ts2  Hybrid  lrdI  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-16 /Deficiency Compared with adl-16 Homozygotes and Hemizygotes at 22°C.  TABLE 3-4  adl-16 1 Deficiency Compared with adl-16 Homozygotes and Hemizygotes at 22°C  TABLE 3-5  adl-16f i'Deficiency Compared with Homozygotes and Hemizygotes.  TABLE 3-6  adl-16 /Deficiency Compared with adl-16 Homozygotes and Hemizygotes at 29°C.  TABLE 3-7  adl-16 /Deficiency Compared with Homozygotes and Hemizygotes.  tsl  ts2  lrdI  tsl  ts2  adl-16f  lrdI  tsl  ts2  tsl  adl-16  ts2  vii i Page TABLE 3-8  adl-16f lDeficiency Compared with adl-16f Homozygotes and Hemizygotes.  158  TABLE 3-9  Conversion Factors for Differences in Rate of Living of Dflmutant at 22°C and 29°C.  159  TABLE 4-1  Progeny Genotypes from Ring-X Crossed with adl-16 Males.  181  lrdI  lrdI  Chromosome Female  ts2  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. Head, Thorax, and Abdomen.  188  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  LIST OF FIGURES  Page FIGURE 1-1  Oregon-R Males Survival Curve at 22°C.  30  FIGURE 1-2  Oregon-R Males Survival Curve at 29°C.  31  FIGURE 1-3  Oregon-R Males Survival Curves at 22°C and 29°C Adjusted for Rate of Living  32  FIGURE 1-4  Oregon-R Females Survival Curve at 22°C  33  FIGURE 1-5  Oregon-R Females Survival Curve at 29°C  34  FIGURE 1-6  Oregon-R Females Survival Curves at 22°C and 29°C Adjusted for Rate of Living  35  FIGURE 1-7  Oregon-R Males and Females Survival Curves at 22°C  36  FIGURE 1-8  Oregon-R Males and Females Survival Curves at 29°C  37  FIGURE 1-9  Oregon-R Males and Females Survival Curves at 22°C and 29°C Adjusted for Rate of Living  38  FIGURE 1-10  Oregon-R Males and Females Survival Curves at 22°C and 29°C  39  FIGURE 1-11  adl-16  Males Survival Curve at 22°C  44  FIGURE 1-12  adl-16  Males Survival Curve at 29°C  45  FIGURE 1-13  adl-16  Males Survival Curves at 22°C and 29°C Adjusted for Rate of Living  46  FIGURE 1-14  adl-16  Males Survival Curves at 22°C and 29°C Adjusted for adl-16 Rate of Living  47  tsl  tsl  tsl  tsl  tsl  Females Survival Curve at 22°C  FIGURE 1-15  adl-16  FIGURE 1-16  adl-16 l  FIGURE 1-17  adl-16  FIGURE 1-18  Females Survival Curves at 22°C and 29°C Adjusted for adl-16 Rate of Living adl-16 Males Survival Curve at 22°C  tsl  Females Survival Curve at 29°C ts  Fe males Survival Curves at 22°C and 29°C Adjusted for Rate of Living tsl  adl-16 l ts  tsl  FIGURE 1-19  ts2  Males Survival Curve at 29°C  FIGURE 1-20  adl-16  FIGURE 1-21  adl-16  ts2  Males Survival Curves at 22°C and 29°C Adjusted for adl-16 Rate of Living ts2  ts2  Males Survival Curves at 22°C and 29°C Adjusted for Rate of Living  FIGURE 1-22  adl-16  FIGURE 1-23  adl-16  FIGURE 1-24  adl-16  FIGURE 1-25  adl-16  FIGURE 1-26  adl-16  FIGURE 1-27  Females Survival Curves at 22°C and 29°C Adjusted for adl-16 Rate of Living adl-16 Females Survival Curves at 22°C and 29°C Adjusted for Rate of Living  ts2  Males Survival Curve at 25°C ts2  Males Survival Curves at 22°C, 25°C and 29°C ts2  Females Survival Curve at 22°C ts2  Females Survival Curve at 29°C  adl-16  is2  ts2  ts2  FIGURE 1-28  ts2  xi  Page FIGURE 1-29  adl-16f  lrdI  Males Survival Curve at 22°C  70  FIGURE 1-30  adl-16f  lrdI  Males  71  FIGURE 1-31  adl-16f  lrdI  Males  72  Survival Curve at 29°C  Survival Curves at 22°C and 29°C Adjusted for adl-16f^ Rate of Living rdI  73  FIGURE 1-32  adl-16f  FIGURE 1-33  adl-16f  FIGURE 1-34  adl-16f ¥e  males Survival Curve at 29°C  75  FIGURE 1-35  adV-irj/'^Females Survival Curves at 22°C and 29°C Adjusted for adl-16f^ Rate of Living  76  adl-16f  77  lrdI  Males  Survival Curves at 22°C and 29°C Adjusted for Rate of Living lrdI  Females  Survival Curve at 22°C lrdI  74  rdI  FIGURE 1-36 FIGURE 1-37 FIGURE 1-38  lrdI  Females  Survival Curves at 22°C and 29°C Adjusted for Rate of Living adl-16 Hybrid Females Survival Curves at 22°C ts  82  adl-16 /adl-16f Hybrid Females lrdl adl-16 , adl-16f Homozygotes  83  ts2  lrdI  ts2  Survival Curves at 29°C  FIGURE 1-39  adl-16 ladl-16 adl-16 , adl-16  Hybrid Females Homozygotes Survival Curves at 29°C  84  FIGURE 1-40  adl-16 /adl-16f Hybrid Females lrdI adl-16 , adl-16f Homozygotes  85  Hybrid Females Survival Curves at 29°C  86  tsl  ts2  tsl  tsl  ts2  lrdI  tsl  Survival Curves at 29°C  FIGURE 1-41  adl-16  ts  xi i  Page FIGURE 1-42  adl-16  ts  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  FIGURE 2-9  Motor Activity of of Oregon-R Males  118  FIGURE 2-10 FIGURE 3-1  Motor Activity of of Oregon-R Females Diagram of Wing of adl-16f /Deficiency  119 145  FIGURE 3-2  Comparison of Genetic and Cytological Location of aciZ-id" Gene  152  FIGURE 3-3  Deficiency/adl-16  Mutants Survival Curves at 22°C  161  FIGURE 3-4  Deficiency I adl-16  Mutants Survival Curves at 29°C  162  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 Neural and Mesodermal Tissue Superimposed on Figure 4-3  191  FIGURE 4-5  Fate Map of Paralysis of Legs  196  lrdI  ts  ts  116  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. compelling. single it.  It remains a mystery, and no less  There is no consensus on the causes of ageing, nor a  theory that explains the physiological changes associated  with  The etiology of ageing is considered to be extremely complex.  population  demographics  change,  with  the  concomitant  increase  As in  the proportion of older people in society, gerontological research is becoming  more  important.  Alzheimer's,  as  active  of research, and, since  areas  involving  well  humans  is  as  Diseases  other  such  degenerative  obviously  limited,  insects,  Drosophila,  used  disorders  other  to test various theories.  frequently  progeria,  the amount of  excellent substitute especially  as  with its  in  and  man, are  experimentation  species  afford  an  Micro-organisms, and  well known genetics, are  as model systems to elucidate  difficult or complex  problems such as ageing.  Ageing is over  time,  the change which  in structure and function, occuring naturally  increases  the  probability  organism (Coni, Davison, and Webster, 1984). are usually Miquel ageing  not considered  (1973),  who  have  part of normal been  for many years, define  dependent,  actively  of  the  death  of  an  Accidents, or diseases ageing.  involved  Rockstein and in the  study  of  it as "the sum total of those time-  reproducible changes  both  in  structure  and/or  function  for a given organism, species, or strain, during its total lifespan." general,  lifespan  can  be  divided  reproductive,  and senescent.  include  egg,  the  larva,  into  three  For Drosophila,  and  pupa;  the  phases:  In  juvenile,  the juvenile  reproductive  stages  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  senescence recently  have  and  been  death.  Minot  Timeras (1978),  and death are continuous final  result  of  proposed (1907),  and Lints  to  explain  Muller  (1978), postulated  programmed into the genome.  meaning  causes  (1963),  with morphogenesis,  differentiation,  the  and that  more ageing  and are simply  that  they  are  of  the  events  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. it  controls  the  release  of  hormones  ovulation, menstruation, and menopause.  In man, for example,  responsible  for  puberty,  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 Sohal,  1986).  evidence  The lifespan characteristic for each species provides  for  genetically  (Collatz and  programmed  determined  death  biological  seen in mammals, for example  and  suggests  clock.  some  type  Time-dependent  presbyopia, or the  of  changes  greying of hair,  which occur with ageing, also seem to indicate genetic determination.  Although ageing  there  and  is  some evidence  senescence,  involved.  Examples  biochemical pathways faulty proteins  other are  to  support the  intrinsic  randomly  occurring  known  of  factors  may  be  errors  in  normal  and cellular events, such as accumulation of  (protein error hypothesis;  generally  basis  metabolic  Orgel,  membranes or somatic mutations (Curtis, 1963). are  genetic  as  "wear  and tear",  1963), and aberrant Changes of this type  and occur  at  both  the  cellular and tissue level.  The incidence of such mutations and errors  increases  aberrent  products  with  age,  as  diminish, and vital  causing death.  products  organs  cease  accumulate, to  function,  This theory is supported by some evidence  necessary eventually that free  radicals, the by-products of oxygen metabolism, accumulate with age, and damage cell membranes (Harman, 1982).  A  third  factors,  hypothesis such  temperature. temperature  as  of  ageing  posits  bacterial or viral  It is widely dependent.  animals, such as insects.  accepted  This  is  that it results  infections,  from extrinsic  radiation, toxins,  or  that metabolic rate is directly  especially  true for poikilothermic  Coping with environmental abuse, such as  habitat destruction, not only leads to death of the individual, but also  death of the about  this  species.  in  At the present  relation  to  the  time there is  destruction  of  great concern  rainforests  and  the  increase in ultraviolet radiation exposure due to the reduction of the ozone layer.  Two  paradigms  above  related  and specifically  theory  of  Maynard ageing  ageing,  to  the  ageing  explained  directed at poikilotherms are the  threshhold  and the  Smith (1961  which argues  three  rate  of  theories  living  of  hypothesis.  Clark  a and b) proposed the threshhold theory that ageing  is independent  and of  of temperature, but  vitality decreases with time and at a certain threshhold dying begins within  a population.  This  will  occur  more  temperature, because the threshhold is higher. or species specific. proposed  in  1929,  of  a higher  It may also be strain  states that  temperature poikilotherms are more active,  amount  at  On the other hand, the "rate of living" hypothesis  by Alpatov and Pearl  rate of living.  rapidly  and thus  at a higher  have  a higher  They propose that faster ageing is due to a limited  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.  and  also  hypothesis  Lamb  (1978),  within the  appear  18-25°C  to  support  temperature  the  range,  rate  of  but there  (1976), living 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  ageing,  ways  Although  the  to  above  quantify  lifespan  is  theories  and hypotheses associated  and measure  relatively  it  objectively  simple  to  are  measure,  with  needed.  the  other  characteristics of ageing need to be quantified so that the biological or physiological chronological  state of  age.  an organism can  This  manipulation of organisms such as Drosophila, generations, alterations  often  is  have  which modify  difficult,  frequently  maintained  be  because  alters  under  correlated  lifespan,  laboratory  abnormal lifespans.  with  its  experimental and organisms conditions  Nevertheless,  the biological or physiological  for  genetic  age  of an  organism to one "younger" or "older" than expected, could be said to influence  ageing.  Regardless  of  the  complexity  of  the  situation  examine one or two factors at a time. temperature  has  been  extremely  test reactions functions.  however,  in  the  study  of  gene  It has been used as a tool to  in living organisms without  destroying  their "normal"  As early as 1891, temperature was associated with change  in pattern of phenotype in insects. that  may,  The environmental effect of  useful  expression especially in poikilotherms.  we  temperature  shocks  Merrifield demonstrated in  administered  to  lepidoptera pupae  1893  caused  changes in the pattern and intensity of wing markings. is  frequently  those above  used  to  demonstrate  which exhibit  (permissive),  and  (restrictive).  normal  produce  conditional mutations phenotypes  abnormal  Temperature sensitive  Temperature  in one  such  as  environment  phenotypes  in  another  (ts) mutations are just one type  of conditional mutation (Suzuki, 1970).  The  earliest  report  genetically  the  reduplicated found  rdp  of  most  temperature extensively  legs mutation (rdp), to  be  temperatures.  cold  She  also  sensitive,  sensitivity  in  characterized  Drosophila,  insect,  was  reported in 1915 by Hoge. with  demonstrated  high by  penetrance  using  at  the She low  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  been discovered, and temperature sensitivity in Drosophila extensively  reported by Suzuki and other researchers.  also an excellent  have  has been  Drosophila  is  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. Drosophila  Little or no cell division occurs in  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 (Jockusch,  of  1966;  tertiary  Guthrie  and  quaternary  et al., 1969).  Lethal  protein  structure  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  demonstrated  Grigliatti,  (1983)  have  all  Drosophila caused by exposure shibire ) to restrictive temperatures tsl  which adult specific  of  mutant  defects  juvenile  during development.  in  flies  (e.g.  Times at  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  Drosophila.  are  few  bio-markers  Anatomical changes  of such  ageing as  in  tattered  adult wings  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.  are  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  peroxidase 1972),  with  in age  (Armstrong  activity have  et al.,  of  been  enzymes  such  demonstrated  as  catalase  (Burcombe  1978), and (Nicolosi et al,  1973),  and  et  al.,  these  changes are small, and therefore might not be easy to demonstrate or use as reliable markers of physiological age. et al. flies  More recently Fleming  (1986), examined over 100 polypeptides by electrophoresis in 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  proteins during the Drosophila evident  there  were,  occur  lifespan".  however,  in  the  expression  of  Among the polypeptides  seven  relatively  abundant  polypeptides expressed in 28 day old flies, which were not detected in  10 and 44 day old flies.  products  of  these  particular  No suggestion genes  was  as to the role of the  made,  but  generalizations  about the effects of the composition changes of proteins present were given.  A  different  Drosophila,  approach, that  of  age-related  behaviour  changes  of  might provide useful bio-markers, since many observed  behaviours are innate, and therefore  genetically  sacrifice of flies to test for age is also eliminated. visible in the reproductive phase of Drosophila  programmed.  The  Behaviour is most and may serve as a  useful bio-marker of age if it can be shown to be a reproducable  phenotype.  Elens  differences  (1972),  in phototaxis  and  Elens  of flies  et  5 and 30  researchers report two or three different tests,  but  rarely has  Drosophila. behaviour  in  behaviour  al.  been  days  old.  recorded Frequently  ages for various behaviour  tested  In 1972 Miquel  et al.  wild-type  irradiated flies.  and  (1971),  over  measured  the  lifespan  geotaxis They  of  and mating later  related  temperature and ageing with changes in viability, geotaxis, & mating behaviour during adult lifespan  of flies at 1 8 ° C , 2 1 ° C , and 27 ° C  (1976).  Economos  et  melanogaster at which  al.  (1979),  tested  mating  behaviour  in  male  D.  and correlated decreased viability of flies with the age  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. number of gynandromorph flies  A higher  are produced by older female  flies  (Hall, Gelbart, and Kankel, 1976).  Since  it  is well  established  behaviour, and testing  that  the  age  of  flies  affects  behaviour does not require sacrifice  their  of  flies,  the idea that behaviour is a reliable and quantifiable parameter of adult  physiological  age,  as  reported  (1984), warrants further testing. patterns  of  behaviour  loss  of  accelerate  the  normal ageing  Leffelaar  They examined five  adult lethal mutants of Drosophila. to  by  temperature  the  and  Grigliatti  lifespan  and  sensitive  X-linked  One of these, adl-16 <  appears  tsl  process.  The ability to perform  normal phototactic responses loss  of geotactic  populations. of  the  during  the reproductive phase  of adult  Motor ability is not lost until well into the death phase  population,  population dies. of  response,  is lost first, and this is followed by a  the  period  during  which  most  of  the  Since the onset of senescence is often not clear, loss  behavioural fitness  physiological  time  age  might function as  especially  during  the  a suitable  bio-marker of  reproductive phase,  where  physiological or anatomical bio-markers of age are absent.  This thesis continues and expands the studies reported by Leffelaar and Grigliatti. is  The first premise that will be examined is that ageing  genetically  developmental increase  or  programmed, process.  Point  decrease  hypothesis.  Three  and  the  is  therefore  mutations  rate  of  part  in  single  ageing,  would  temperature-sensitive  of  genes,  which  support  adult-lethal  the  this  mutations  were used, all of which were found to be alleles of the adl-16 gene in Drosophila  melanogaster.  adl-16fl i  were isolated  ra  methanesulfonate-induced screens, and  adl-16f tsl  tsl  from  X-linked  and adl-16  the  fairly  permissive normal  temperature adlf \ lraI  were  ts2  Cross in 1979 (Homyk et al,  At  Oregon-R  wild-type  point mutations  adl-16 , ts2  strain as in stwo  and ethyl  separate  was reported by Homyk and Sheppard in 1977,  lraI  adl-16  A l l three alleles, adl-16 ,  (29°C),  by  Schellenbarger and  1986).  temperature  lifespans  isolated  and  (22°C),  all the above mutants have  behaviour.  At  the  all die prematurely (adl-16 ,  non-permissive adl-16 ,  and  in order of effects from most severe to least affected).  The  ts2  tsl  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  of the egg (Homyk et al., 1986). to  the  restrictive  (personal  temperature  deposition  Also, the effect of shifting mutants at  any  age  leads  to  their  demise  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 29°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 2 2 ° C  pattern  would  be  as at 2 9 ° C ,  compressed  into  a  except that at 2 9 ° C ,  shorter  time  frame.  this This  particular pattern should also be present in the control flies, OregonR, but should reveal  a greater compression  factor in mutant  flies.  Conversely, if the behaviour-loss patterns of a ts mutant and OregonR are different at 2 9 ° C as compared to 2 2 ° 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,  29°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  loss patterns for all types of flies examined in Chapter 1. tested were geotaxis, phototaxis, and motor ability. examination here are: pattern  be  dependent used  as  Is  behaviour loss? a bio-marker of consistent  there  a  Questions under  specific  Can the pattern the  ageing  with  ageing process or is adl-16 essential  Behaviours  Can a quantifiable and repeatable  demonstrated?  behaviour-loss  behaviour  a  of  process?  mutation  behaviour  pattern  of  age-  behaviour loss  be  Is  of  which  the  pattern  accelerates  the  simply a mutation which disrupts an  tsl  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 )sd  72b26  /FM  y B w, a  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 dose  of  gene  Hemizygotes longevity. /deficiency  product  would  have  what effect on  lifespan  a single and  female  behaviour.  were examined for changes in phenotype, anatomy, and A n examination of the fertility of the alleles of hybrids was also performed.  adl-16  ts  In chapter four, the question of what type of tissue is affected by the adl-16  ts2  m u t a t i o n is  gynandromorphs this  mutation,  restrictive  ln(l)w / vC  which  causes  temperature,  mesodermal  might  by  the  adl-16 . death  map  analysis  It was hypothesized  ts2  early  mosaic  to  and  paralysis  presumptive  nervous  expected  published data (Leffelaar and Grigliatti,  that  the  severity  of  expression  of  the  that the or  with respect to lifespan, and behaviour.  1984), it  genes under  study compared with the control would be: Or-R < adl-16 t s 2  at  of  tissue.  From previously was  of  explored  < adl-16  tsl  Where adl-16f  lraI  fit into this sequence was unknown prior to this study.  would  Although this  mutant was isolated because of reduction in flight ability, other adult behaviours  had  not  females of adl-16 , ts2  previously  been  tested.  The  behaviour  and the hybrid females of the above  were not tested prior to this study.  of  alleles  CHAPTER 1 INTRODUCTION While  it  is  versus  environment on the  complex  interesting  issue  to  involves  estimate  the  longevity  the  contribution of  genotype  of an organism, a far more  interaction  of  determining ageing and ultimate lifespan.  these  two  factors  The longevity of  in  specific  populations is predictable, and if it is under predominantly genetic influence, decreases genotype,  then  it  should  be  in length of life. or extrinsic  possible  to  select  for  increases  or  If however, intrinsic factors other than  factors  exert  a greater effect,  then  genetic  selection would not produce significant changes in longevity.  Various  attempts  have  been  senescence in Drosophila al.  made to increase longevity  melanogaster,  for example those by Lints et  (1979), and Clare and Luckinbill (1984). The latter  quite  clearly that it is  possible  to  select  for either  decreased lifespan (Collatz and Sohal, 1986). can posit  demonstrate increased or  The inference which  be made from this is that lifespan is under genetic which has proved elusive  presence  of homozygous  lifespan  lifespan does  not  at  the  control, a  to supporting documentation.  mutant genes results in decreased  as a rule (Arking and Clare, 1986). decrease  and delay  lifespan  The alleles involved in this study  restrictive  necessarily  The  temperature.  support the  position  A  decrease  that  ageing  in 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. with the mutant  To  test  the  necessary  Such may be the case  adl-16 . tsl  hypothesis  to  that  demonstrate  adl-16 that  tsl  the  accelerates lifespan  ageing  and  it  is  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-16 ,  were repeated.  tsl  16 , ts2  and adl-16f , lrdI  alleles of adl-16  tsl  In addition to this,  adl-  were tested, and the results  scrutinized for any similarities of behaviour or lifespan.  The  expectations  for adl-16f  were that since it was isolated in a  lrdI  separate screen to detect adult behaviour mutants with an emphasis on  flight, its phenotype with respect to behaviour might differ from  the  other  between reported  two  alleles.  The pleiotrophic nature  different alleles previously  and  differences  of temperature-sensitive mutants has  (Homyk  and  Grigliatti,  1983)  and  will  been be  discussed more fully in the next chapter.  Temperatures of 2 2 ° C  and 2 9 ° C  were chosen for these experiments  because these temperatures had been used to isolate the mutants. addition 2 9 ° C ,  the restrictive temperature for the adl-16  ts  In  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-16 , ts  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 A N D METHODS  Stocks Oregon-R, a highly inbred wild-type laboratory strain of  Drosophila  melanogaster,  was used as a control for the following experiments.  Mutant  involved were:  stocks  adl-16 ,  which are temperature-sensitive EMS  The  adl-16 , ts2  and  adl-16f ,  strains derived from Oregon-R  lrdI  after  treatment.  mutant flies exhibit relatively normal lifespans at 2 2 ° C , but die  prematurely 16  tsl  t s l  at 2 9 ° C .  , adl-16ts2,  In some  and adl-16f  lraI  previously published literature  adl-  have been given other names, so a  table listing these is shown below (Homyk et al., 1984). N A M E S OF M U T A N T S Current Name  Former Name  l(l)adl-16^ l(l)adl-16ts2 l(l)adl-16f  S6-1 S6-2 S6-Flrd  1  lrdI  Reciprocal crosses between made to test hybrids. by clearing  flies  Isolation Name DC836 DC359 flrdl 1  of these three mutant alleles  were  Virgin females for these crosses were obtained  expanded stock bottles, and collecting females within the  first 8 hours after eclosion. of the same age.  These flies were then mated with males  The following crosses were made:  (adl-l6 ladl-16 ) (adl-16 ladl-16^1)  1)  adl-16  tsl  female  <8) adl-16  ts2  male  2)  adl-16  ts2  female  <8> adl-16  tsl  male  3)  adl-16  tsl  female  <g>  4)  ad/-7<5»2 female  <8> adl-16fl  5)  adl-ltflrd!  female  <8> adl-16  tsl  male  (adl-16fl ladl-16tsl)  6)  adl-16fl  female  <8> adl-16  ts2  male  (adl-l6f^ ladl-16^ )  rdI  tsl  ts2  (adl-16*sl/adl-16fl i)  adl-16f mule.  rd  lrdI  male  rdI  ts2  (adl-16^ /adl-16/^1) 2  rdI  dl  2  Progeny from these crosses were used in the survival and behaviour tests outlined below. reciprocal  crosses  For hybrids, where flies tested were all female,  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,  streptomycin.  2.4  g  Tegosept,  50  mg  ampicillin,  and  10  mg  A l l cultures were maintained at a constant day-night  cycle (14 hours light, 10 hours dark) at either 2 2 ° C + 1°C, or 2 9 ° C ± 0 . 5 ° C , and 50-60% relative humidity.  Stocks  were  expanded in quarter-pint bottles  containing 24  medium, then 20-30 pairs of males and females placed in bottles  containing fresh medium.  1-3  experiments 30 bottles were required.  days old were  Sufficient bottles  set up to produce over 500 progeny of the same age.  ml of  were  For most of the  After 3-5 days, the adult flies  were discarded and the eggs allowed to develop at 2 2 ° 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  were  collected  over a twelve  bottles  in  the  evening,  newly  eclosed  adults  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. in  sets  of  Vials were selected five  vials  (50  at random, numbered, and grouped  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 2 9 ° 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  differences  were  noted  and  in  the  behaviour survival  concurrently of  since  behaviour-tested  no and  untested flies in the first set of behaviour trials (see Chapter 2).  In  those groups kept at 2 2 ° C , flies were transferred to vials with fresh medium every 2-4 days; for flies maintained at 2 9 ° C , transfers were made every 2-3 days.  Flies which escaped, or were crushed during  transfer were subtracted from the total possible Bacterial  contamination  of  vials  occurred  flies  from  for each test. time  to  time,  especially  with  adl-16f .  These vials were discarded, and the  lrdI  number of flies subtracted from the totals.  To  obtain survival curves, the number of living flies  counted daily for the mutants adl-16  and adl-16  tsl  at 2 9 ° C , and at each transfer for a d l - 1 6 both 2 2 ° C arid 2 9 ° C .  flrdI  per vial  was  and all hybrids  ts2  at 2 9 ° C  and Oregon-R at  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. of  Wherever possible a minimum  200 flies (N) of each sex was used to determine longevity for a  given population. each  experiment  The number of flies present at the beginning of is  quoted,  although  the  survival percentage  other parameters were adjusted to allow for removal of flies escaped  or died accidently.  Sample sizes were  trials, and have been quoted individually.  which  smaller for some  Controls of Oregon-R and  both mutant parent types were used as necessary. was repeated at least three times.  and  Each experiment  Table 1-1 lists the tests made for  survival and behaviour (discussed in the next chapter). 20,000 flies were tested for survival and behaviour.  In total, over  21  Table  1-1  REPETITIONS OF SURVIVAL A N D BEHAVIOUR TESTS 22°C males  Strain 1. 2. 3. 4. 5.  Oregon-R adl-16tsl adl-16ts adl-^d adl-16tsl/adl-16ts  22°C females  7 4 4 6  2  1  7 4 4 6 3 4 3 1 1 1  2  6. adl-16tsl/adl-16flrdi 7. adl-16ts2/adl-16 dl 8. adl-lGts^Oregon-R 9. adl-16 /Oregon-R 10. adl-16 di/Oregon-R flr  ts2  flr  11. a d l - 1 6 Total Experiments Performed ts2  21  34  29°C males  29°C females  7 4 4 6  1*25°C 22  7 4 4 6 3 4 3 1 1 1 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. the first phase, or reproductive most deaths were accidental. the graph where few The (DP but  flies  phase  (RP), flies were fertile, and  It corresponds to the first portion of  die, consequently it may be horizontal.  second phase revealed the onset of death, death phase o n s e t  ) of the population.  During  onset,  If few flies died, this change is gradual,  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, and  in excess of  18 respectively at 2 9 ° C .  11-18,  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  during which most of the population died relatively  short period of time,  the  time  If death occured over a  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, ( D P d ) in Tables. en  The final phase of the graph, called the final represents  the  few  individuals who  died  death phase,  later.  This  may  (FDP), occur  because of the particular combination of genes present, or simply the fact that as the other flies were present.  died, less and less crowded conditions  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  2 9 ° 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. manifests  senescence.  A population with this  type  of curve clearly  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 to be type C .  days, the survival curve is said  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. fairly  constant  overall  The latter two types of curves reveal a 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. according  to  type,  Conversion  factors  temperatures  reflect  coincident calculate  graphs the  A n attempt has been made to classify them and  make  (defined those can  figures  be  inferences  below)  for  that  used  in  on  senescence.  graphs  between  particular experiment  produced.  given  about  Pooled  tables,  so  data  these  so  was  results  that  used  to  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 2 2 ° C  were examined every 2-4  adl-16  and  examined every 2-3  Oregon-R  estimating  were  days, and  days at 2 9 ° C ,  flrdI  rather than  the death pattern during these time intervals, flies  were  considered to have died on the day that the culture was  examined.  A l l data were calculated in this manner, meaning that there will be a slight  increase  internally  in values  consistent  so  for several valid  data.  The average  means  for replicate experiments  Death  Phase  comparisons  L S for each X  Mean  parameters,  trial  was  can  but the be  data are  made  between  found, and then these  were averaged.  (DPvJ The average  age at death of individuals  dying between the death phase onset and end was calculated. day  of  death  was  assumed  to  be the  day  of  examination  The 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. then  Means from separate replicate tests were  averaged.  Death Phase Onset (DP set). on  of  the  population  was  For each trial, the day of onset of death  recorded. These  replicate experiments to obtain the mean.  values  were  averaged  for  Death Phase End ( D P n d ) . P  This average was calculated in a similar manner, except that the day that the death phase ended was used in the calculation.  D - 2 0 . D - 5 0 . D - 9 5 . and D-100  represent the days posteclosion  20, 50, 95, or 100% of the population had died.  when  Average figures are  quoted in the tables.  The Slope of the Death Phase ( D P i s  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 DP nset and this figure was 0  divided by the number of days between DP onset and end.  Average  D P slopes are given and represent average percentage of flies per  dying  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 , D P x ,  DPonset, D P d , D-20, D-50, D-95, and D-100 for Oregon-R at 2 2 ° C for e n  males  or females  were  2 9 ° C experiments. sex.  Each  set  divided  by the corresponding values  from  This yielded a set of conversion factors for each 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. of  2.32  was  found,  and for males  For Oregon-R females, a factor a factor of 2.49  between the temperatures 2 2 ° C and 2 9 ° C .  was obtained  This means that at 2 9 ° C ,  one day of life for a female fly is equivalent to 2.32 days at 2 2 ° C , and one day at 2 9 ° C for a male fly is equivalent to 2.49 days at 2 2 ° C . These conversion figures have been used subsequently in this thesis to  compare lifespans  of  lifespans of mutant flies. curves  between  22°C  Oregon-R  flies  with each  other  and the  They were also used to convert survival  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 29°C  was  factor,  multiplied by the appropriate male or female  so  that  comparative  graphs  could  be  drawn.  conversion The  second  abscissa (not depicted) is understood to apply to the 2 9 ° 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 experiments,  used as a control stock  for all the  following  great care was exercised in examining its longevity and  behaviour  because  this  would  provide  a  hallmark against  differences  observed in the test strains would be compared.  which  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 2 9 ° C than at 2 2 ° C , and female lifespan exceeded that of  males by 4.9 days, lifespans  lower  variation at 2 9 ° C  differing by a factor of 1.11.  could be expected  from the fact  that  The the  environment of the 2 9 ° C incubator with respect to both temperature and  humidity was  much more uniform than that of the laboratory,  which acted as the 2 2 ° 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 2 2 ° C and  29°C  respectively.  Although survival was recorded every  2-4  days, only every second or third point is shown on the 2 2 ° C survival curves to prevent obscuring the graphs. graphs to follow.  Figure  1-3  This was done for all 2 2 ° C  demonstrates  that when the graph at  2 9 ° 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 2 2 ° C and  29°C  respectively.  Figure  1-6  shows the coincident graph for  flies at 2 2 ° C and 2 9 ° C after the 2 9 ° 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 2 2 ° C , and 2 9 ° C respectively.  Figure 1-9 contains graphs of males and females at 2 2 ° C as well as males and females at 2 9 ° C adjusted for the rate of living.  This set of  graphs may be difficult to see.  What is important is that the 2 2 ° C  and  coincidental for each sex,  the  29°C  graphs are essentially  general  shape  is  consistent;  thereby  and that  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 2 9 ° C unadjusted for the rate of living, permitting comparison of the original data.  All  four curves  quite  similar  have  in  their  shape.  reproductive phase  own In  individual characteristics,  general,  of the lifespan,  few  shape,  (type  A ) , with  died  a distinct  of  genetic  reproductive phase  variability was  programming.  low, which means  In  the was  The curves are sigmoidal  phases including a very late (V) onset of death, suggestive  during  and when the death phase  reached, most flies died within a few days. in  flies  but are  and death  which might  general,  the  degree  be of  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 OregonR  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. at 2 9 ° C  Once the conversion factor is used to expand the days  to compensate  temperature,  it  can  be  for the higher rate of living at the higher seen  that  the  curves  parallel one  another  (Figures 1-3, 1-6 and 1-9), meaning that a temperature of 2 9 ° C is not deleterious  to these flies.  All  graphs are results from single experiments.  calculated for the replicates of experiments, shown  in Tables 1-2  deviations.  and 1-3  Average results are  and these numbers are  along with calculations for standard  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  Age (Days) at 29°C  40  50  60  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: 2 9 ° 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 120 100 -  co >  Age (Days) at 22°C  100  -•a—  Females (22°C) N=200  -•—  Females (29°C) N=200  200  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 -  -0—  Males (22°C) N=200  •+—  Males (29°C) N=250  -•—  Females (22°C) N=200  •+—  Females (29°C) N=200  20 1 00  Age (Days) at 22°C  200  Note: 2 9 ° 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)  LS  X  97.3 ± 3.3  103.2 ± 0 . 3  41.6 ± 0 . 9  46.2 ± 1.7  DP  X  105.1 ± 0 . 6  109.7 ± 0 . 5  42.8 ± 1.0  47.5 ± 1.4  80 ± 3 . 5  89.5 ± 5 . 5  36 ± 1.0  37.3 ± 2 . 3  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  123.6 ± 4.4  124.5 ± 1.5  48 ± 1.5  53 ± 1.2  142 ± 1.5  138.5 ± 0.5  51.7 ± 0 . 7  58.6 ± 1.4  1.8 ± 0 . 2  2.13 ± 0 . 1  6.9 ± 0 . 2  5.7 ± 0 . 5  DP onset D-20  DP end D-100 DPslope The  wild-type  experiments. are  given  separate females).  strain,  Oregon-R  was  used  as  a  control  DP slope is given as % death per day. in  days  experiments,  ±  standard  n=3  (total  deviation, #  of  flies  1440  all  A l l other data  obtained =  for  from  three  males,  1620  L S : Life-span mean; D P : death phase mean; DPonset and X  X  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 R A T E OF LIVING OF OREGON-R at 22°C and 2 9 ° C Males Females LS DP  2.2 2.3  2.3 2.5 2.2 2.3 2.4  X  X  2.5 2.6 2.7  2.4 2.2 2.3 2.4 2.3 2.4  2.49±0.17  2.32±0.08  DPonset D-20 D-50 D-95 DPend D-100 Average  Each number represents the ratio of the respective 2 2 ° C values for longevity.  and 2 9 ° C  One 2 9 ° C day for females = 2.32 days at 2 2 ° C .  One 2 9 ° C day for males = 2.49 days at 2 2 ° C .  adl-16 The  tsl  Longevity  earliest result to be noted with this  mutant was  change in phenotype at the restrictive temperature.  an obvious  Within two days  swollen abdomens were noted in both male and female flies.  The  proportion  this  of  phenomenon, female  flies  with  and its  than in the  abdomenal distension,  expression male  fly,  were  seen  but by day  to  the  onset  be  greater  three  almost  of in all  the flies  exhibited this  phenomenon, and in some  swollen  it  that  became  difficult  to  cases abdomens aspirate  flies  were  for  so  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). were affected  by day six.  Approximately one third of the males 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,  probosci. and  almost  flies  also  had  permanently  extended  I later related the extension of the proboscis to paralysis,  thought that adl-16  29°C.  all  flies were unable to retract the organ at  tsl  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 16  t s l  flies  maintained  at  posteclosion in a female fly.  22°C,  the  earliest  being  on  day  adl22  At the time of onset of the death phase  many male and female flies were noted to exhibit swollen abdomens, as  well  universal,  as  excessive  as  it  was  grooming, at  29°C.  but  the  phenomenon  The proportion of  flies  was  not  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  adl-16  tsl  and 1-15,  the survival curves of  males and females respectively at 2 2 ° C , and also Table  the lifespan parameters  1-4,  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-16  was greatly reduced.  tsl  In  fact lifespan was reduced almost exactly five-fold relative to OregonR.  Figures 1-12 and 1-16 show typical survival curves for males and  females  at this temperature:  Figures 1-13  and 1-17  curves for males at 2 2 ° C and 2 9 ° C , and females respectively. by  The 2 9 ° curves in these graphs have been converted  temperatures. Male days at 2 9 ° C female days at 2 9 ° C effect  clearly seen. A  at 2 2 ° C and 2 9 ° C  the factors for Oregon-R rate of living differences  deleterious  show survival  between these  have been multiplied by 2.5, and  have been increased by a factor of 2.3.  The  of the restrictive temperature on lifespan can be  The survival curves for adl-16  with death phase  tsl  at 2 9 ° C were of type  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 2 9 ° C were increased by a factor of 10.2 for males and 11.6  for females,  the  conversion  factors  calculated  for  producing the coincident graphs depicted in Figures 1-14  adl-16 , tsl  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  Age (Days) at 29°C  6  8  10  12  46  Figure  1-13 adl-16 ts1 Males Survival Curves at 22°C and 29°C  120 100  >  -G—  Males (22°C) N=140  —  Males (29°C)N=140  Age (Days) at 22°C Note: 2 9 ° 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  0  20  40  60  80  100  —  Males (22°C) N=140  -*—  Males (29°C) N=140  120  Age (Days) at 22°C  Note: 2 9 ° C days have been adjusted by Conversion Factor 10.2.  48  Figure  1-15 adl-16 ts1 Females Survival Curve at 22°C  120  Females (22°C) N=200  20  40  60  Age (Days) at 22°G  120  49  Figure  1-16  adl-16 ts1 Females Survival Curve at 29°C 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  20  40 60 Age (Days) at 22°C  80  100  Females (22°C) N=200 Females (29°C) N=200  120  Note: 2 9 ° 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  o  Survival (Days) at 22°C 1 o o  —  Females (22°C) N=200  -•—  Females (29°C) N=200  200  Note: 2 9 ° C days have been adjusted by Conversion Factor 11.6  Table  1-4  L O N G E V I T Y OF adl-16^ A D U L T S MAINTAINED A T 22°C OR 29°C POSTECLOSION Males 22°C CX ± SD) LS  X  DP  X  80.8 83.1 49.7 66.7  DPonset D-20 D-50 D-95  ± 0.7 ± 2.3 ± 1.3 ± 3.9  81.3 102.7 101 114.7 1.87  DPend D-100 PD slope  ± ± ± ± ±  2.4 4.2 3.7 1.3 0.1  Females 22°C (X ± SD) 99.2 101.2 54.0 87.5 102 114.5 114 119 3.29  ± ± ± ± ± ± ± ± ±  0.8 0.9 4.0 2.5 1.0 0.5 1.0 1.0 0.6  Males 29°C (X ± SD) 8.5 8.3 4.2 6.4 8.1 10.1 10 11.5 22.2  Females 29°C (X ± SD)  0.7 0.6 0.4 0.7  9.1 9.1 4.3 7.4  + ± ± ±  0.3 0.2 0.9 0.1  ± 1.0 ± 1.0 ± 0.7 ± 0.9 ± 0.7  9.3 11.1 11 12.7 22.7  ± ± ± ± ±  0.4 0.9 0.0 0.9 4.1  ± ± ± ±  Data are given in days ±  standard deviation, obtained from  three  separate  (total#  1426  females).  experiments  n=3  of  flies  =  1240  males,  L S : Life-span mean; D P : death phase mean; DPonset and X  X  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 i p s  0  e  is given as % death per day.  Table  1-5  CONVERSION FACTORS FOR DIFFERENCES IN R A T E OF LIVING O F adl-W* A T 22°C A N D 29°C 1  LS PD  X  X  DPonset D-20 D-50 D-95 DPend D-100 Average Each number represents values for longevity.  Males  Females  9.4 10.0 11.7 10.3 10.0 10.2 10.1 10.0 10.2±0.6  10.9 11.2 12.6 11.9 10.9 10.3 10.4 9.4 10.9±0.9  the ratio of the respective  22°C  and  29°C  One day at 2 9 ° C is equivalent to 10.2 days at  2 2 ° C for males, and 10.9 days at 2 2 ° C for females.  adl-16  ts2  Longevity  Most debilitating effects on these flies at 2 9 ° C were obvious within 24 hours.  After one day of exposure to this temperature few  could stand.  A few  managed to climb a little, and a few  flies  to walk  shakily, but the majority were found lying on their sides, or more frequently impaired.  on  their  wings,  with  their  legs  folded  and  severely  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  nourishment.  would  Swollen  die  quickly, since  abdomens  they  and extended  would  be  probosci  without  were  not  noted.  At  22°C  the  above  observations males  changes  were  were that immediately  appeared  to  be  preoccupied  not  noted.  The only  general  prior to the death phase with  rubbing  themselves  their legs, and several females exhibited swollen abdomens.  many with  One test  of survival of male flies at 2 5 ° C was made towards the end of all the experiments, to see if a less debilitating effect than that at 2 9 ° C could be obtained for use in behaviour tests. made  at  this  temperature,  little  is  Since no other tests were  available  to  compare , it  with.  Hovever the preliminary indication is that this temperature would be useful  for experiments  with  shown  in Figures  and 1-24.  22°C,  25°C  1-23  and 2 9 ° C  this  can be  mutant,  so  the  survival curve  is  A comparison between flies  at  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 2 2 ° C mean lifespan for adl-16 adl-16  tsl  or Oregon-R.  Average  ts2  female  is shorter than either lifespan  of  exceeded that of average male lifespan (64.9) by 7.5 days. of the death phase was lowered by similar amounts. death phase  was  earlier and more rapid as was  72.4  days  The mean  The onset of the the rate of death,  shown by the increase in slope of the death phase. at 2 2 ° C , adl-16  dislpay  ts2  slightly  In general, even  reduced viability compared with  Oregon-R. Figures 1-20,  1-26 and Table 6 clearly show that at 2 9 ° C , changes in  all parameters for adl-16  ts2  were enormous.  Increased temperature  has a totally debilitating effect on these flies. all type A with early onset of death (E). was  extremely  temperature.  high  indicating  In fact  lifespan  that  was  Survival curves were  The slope of these curves death  was  rapid  at  this  reduced approximately ten-fold  relative to Oregon-R. Figures 1-22  and 1-28  show the comparative effects of temperatures  2 2 ° C and 2 9 ° C on adl-1 <5"2. the mutant flies females. 16  ts2  The 2 9 ° C days have been converted for  by (Oregon-R) factors 2.5  for males  and 2.3  However when the conversion factors determined for  for adl-  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 2 9 ° C for these flies, the curves at 2 5 ° C and 2 9 ° C also become coincident. this mutation also adl-16 . tsl  If  accelerates  the  Thus it could be argued that  lifespan, albeit at a faster rate than  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  Males 29°C N=200 40 H 20 H  Age (Days)  58  Figure  1-21 adl-16 ts2 MALES Survival Curves at 22°C or 29°C  120  20 40 60 Age (days) at 22°C  80  100  -*>—  Males (22°C) N=200  —  Males (29°C) N=200  120  Note: 2 9 ° 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  40 Age (Days) at 22°C  60  r  80  1 00  Note: 2 9 ° 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 Males (29°C) N=200  CO  •| 60  Males (22°C) N=200  3 CO  Males (25°C) N=100  40 20 -  —i— 20  40  Age (Days) at 25°C  60  80  100  Figure  1-25  Figure  1-26  64  Figure  1-27 adl-16ts2 FEMALES Survival Curves at 22°C or 29°C  120  20  40  60  80  100  -s—  Females (22°C) N=200  -•—  Females (29°C) N=200  120  Age (Days) at 22°C Note: 2 9 ° C days were adjusted by Conversion Factor 23.15  Figure  1-28  Note: 2 9 ° C days were adjusted by Conversion Factor 2.32  Table  1-6  L O N G E V I T Y OF adl-16 A D U L T S MAINTAINED A T 22°C OR 29°C POSTECLOSION ts2  Males 22°C (X + SD( LS DP  64.9 67.0 50.3 53.0 65.0 84.3 80.3 93.7 2.6  X  X  DPonset D-20 D-50 D-95 DPend D-100 DPslope  ± ± ± ± ± ± ± ± ±  7.5 6.7 2.2 7.5 6.7 8.4 7.9 7.6 0.4  Data are given in days ± separate females).  experiments  n=3  Females 22°C (X ± SD) 74.2 76.9 53.7 54.7 72.3 89.3 88.0 95.3 2.9  ± 11.9 ± 12.9 ± 8.9 ± 12.3 ± 10.1 ± 11.9 ± 10.7 + 10.2 ± 0.78  Males 29°C (X ± SD) 2.9 2.9 2.0 2.2 2.7 3.6 3.5 3.8 48.6  ± ± ± ± ± ± ± + ±  0.1 0.1 0.0 0.5 0.4 0.4 0.5 0.4 2.4  Females 29°C (X ± SD) 3.2 3.2 2.0 2.5 2.9 4.0 4.0 4.7 45.9  0.2 0.2 0.0 0.4 0.3 0.3 0.0 0.5 1.6  standard deviation, obtained from (total  #  of  flies  =  1146  males,  L S : Life-span mean; D P : death phase mean; D P X  ± ± ± ± ± ± + ± ±  X  o n s e t  three 1130 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 i p , the rate of death of flies per day. s  0  e  Table  1-7  CONVERSION FACTORS FOR DIFFERENCES IN R A T E O F LIVING O F adl-16 at 22°C and 2 9 ° C ts2  LS  X  DP  X  DPonset D-20 D-50  Males  Females  22.1 22.8 23.5 23.8 24.5 23.8 22.9  23 23.8.0 26.8 21.6 24.9  22.6 22.0 DPend 20.1 D-100 24.9 23.5+0.9 23.1±2.0 Average Each number represents the ratio of 2 2 ° C and 2 9 ° C values for longevity. D-95  Table  1-8  L O N G E V I T Y O F adl-16 M A L E S MAINTAINED A T 22°C, 25°C OR 29°C POSTECLOSION. ts2  Temperature  22°C  25°C  29°C  64.9 9.7 2.9 67.0 9.8 2.9 47.0 2.0 7.0 DPonset 53.0 2.2 D-20 8.0 2.6 D-50 65.0 9.3 3.6 D-95 84.3 10.9 80.3 11.0 3.5 DPend 93.7 12.0 3.8 D-100 2.6 48.6 19.5 DPsloDe Data are given in days ± standarc deviation, obtained from one experiment at 2 5 ° C , total # of flies = 100 males, L S : Life-span mean; D P : death phase mean; DP set and D P 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 i p » the rate of death of flies per day. LS DP  X  X  X  X  on  e n  s  0  e  adl-16f  Longevity  lrdI  Of  all the strains used in these studies,  troublesome and variable.  adl-16f  proved  lrdI  most  At 2 2 ° 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  unknown, but since  the  remaining  flies  were  affected  other strains of flies tested did not show  level of contamination, I conclude that these flies may be to  disease,  and have  environmental  contaminated  bacteria  affecting  their  environment,  them.  Flies  10  after  eclosion  and  proboscis  extension  individuals affected At 2 9 ° C , to  several to  increased  females a  small  slowly  this  susceptible rather than  were  slower  movement generally, and tended to hop rather than fly. day  is  in  As early as  exhibited  swollen  degree.  The  abdomens number  of  throughout lifespan.  swollen abdomens were noted within 24 hours of exposure  increased  temperature.  By the  second  day over  50%  of  flies  exhibited this trait, which continued and increased both in number of flies  affected  abdomen  and severity.  extension.  almost transparent.  The  Proboscis extension abdomens  became  corresponded with  hugely  swollen,  and  It was also noted that at this temperature some  flies held their wings up at an unusual angle.  At  both  temperatures  experimental number  of  results replicate  there with  was  this  a  great  stock,  experiments.  deal  which It  of  explains  seemed  that  variation the  of  larger  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  factors (see Table  with  respect  to  conversion  1-9).  Table 1-8 and the survival curves in Figures 1-29 and 1-33  at 2 2 ° 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. than females, phase. days  Males lived slightly longer  as can be seen from the means for lifespan and death  Lifespan means for males and females respectively,  and the  means  of  the  were, 54.4  death phase  days for males and 55.6 days for females.  and 53.7  were  58.27  Lifespan was near half  that of Oregon-R.  By  examining  Table  1-8  the  at 2 9 ° C ,  survival curves  in Figures  1-30  on  1-34  and  it can be seen that the death phase onset was  intermediate (M), and the survival curve Type A . exceeded  and  Female lifespan  that of males by 3.47 days, and death phase by 3.75  average respectively.  days  The conversion factors used to adjust the  rate of living between 2 2 ° C and 2 9 ° C were consistently around 5 for males,  and 4 for females,  see  figures  for standard deviation reflect  stock, as do the survival curves.  Figures  1-31  and 1-35.  the extreme  The high  variability of the  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  120  100  -Q—  CO  > 3  —  CO  T  0  20  40 60 Age (Days) at 22°C  80  100  120  Note: 2 9 ° C days were adjusted by Conversion Factor 5.  Males (22°C) N=200 Males (29°C) N=200  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: 2 9 ° 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  120  100  ra >  Female (22°C) N=200  3 CO  • —  T  0  20 40 Age (Days) at 22°C  60  Note: 2 9 ° C days were adjusted by Conversion Factor 2.3.  Females (29°C) N=200  Table 1-9 L O N G E V I T Y O F adl-16fl A D U L T S MAINTAINED A T 22°C OR 29°C POSTECLOSION rdI  Males 22°C (X ± SD) LS  X  DPx  DPonset D-20 D-50 D-95 DPend D-100 DPsloDe  54.4 58.3 35.7 35.7  ± ± ± ±  54.7 76.0 74.7 83.3 2.1  ± ± ± ± ±  Females 22°C (X ± SD)  10.6 53.67 ± 1 6 . 3 11.0 55.58 ± 1 7 . 7 8.2 32.75 ± 2 2 . 7 12.4 35.5 ± 20.9 11.6 49.0 ± 21.6 6.4 83.5 ± 4.7 9.4 85.0 ± 4.9 3.1 88.5 ± 5.9 0.3 1.91 ± 1.0  Males 29°C (X ± SD)  Females 29°C (X ± SD)  10.8 10.9 5.5 7.6 11.2 15.5 17.8 18.0 9.5  14.3 14.7 8.8 8.9 13.3 18.1 19.3 22.5 9.0  ± ± ± ± ± ± ± ± ±  1.0 1.2 2.1 2.5 2.4 2.4 2.3 1.2 1.3  ± ± ± ± ± ± ± ± ±  1.6 1.8 2.3 1.9 2.2 1.9 3.7 1.8 2.2  Data are given in days ± standard deviation, obtained from  three  separate  1600  females).  experiments  n=4  (total#  of  flies  =  1400 males,  L S : Life-span mean; D P : death phase mean; DP set and X  X  on  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 i p e is given as % death per day. s  0  Table  1-10  CONVERSION FACTORS FOR DIFFERENCES IN R A T E O F LIVING O F adl-16ft di at 22°C and 2 9 ° C r  Males LS DP  Females  3.8 5.0 5.3 3.8 6.5 3.7 DPonset 4.0 D-20 4.7 4.9 3.7 D-50 4.9 4.6 D-95 Dpend 4.4 4.2 D-100 4.6 3.9 Average 5.0 ± 0.6 4.0 ± 0.3 Each number represents the ratio of the respective 2 2 ° C and 2 9 ° C values for longevity. X  X  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. fell between 0.006 and 0.05,  meaning that differences  Values for X  2  such as those  found in the results could be expected to occur in more than 50% of similar experiments. possibility. (see  Maternal  effect  Chapter 2) are presented here.  these  therefore  excluded as a  Only one set of survival curves, and behaviour patterns  statistical analyses in Table 1-11 in  was  experiments  have  comparison with hybrid data.  also  Data have been pooled for the  and Table 1-12. been  pooled,  The controls used and  are  used  for  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  behaviours  apparently has  no  for  ability  adverse  to  (nor  perform  beneficial)  defined  effect  on  longevity. At 2 2 ° C the survival of the hybrid adl-16 identical to that of the adl-16  tsl  I adl-16  was  ts2  homozygote, which, in turn was very  tsl  similar to Oregon-R , as expected.  The hybrid of  adl-16 /adl-16f tsl  was intermediate between females of the parent strains. surprising, because adl-16 fl  rdI  to Oregon-R and adl-16 . tsl  adl-16 ladl-16f ts2  of  lrdI  This is not  lifespan is quite reduced with respect  An unexpected result was that the hybrid  had the greatest longevity of all.  lrdI  almost  Since females  the parent types had the shortest lifespans, it was expected that  this hybrid would also have its lifespan curtailed.  The result may be  due  being  to  heterosis  distinguish.  or  gene  interaction, the  two  difficult  Since survival curves of all homozygotes of these alleles  have been presented earlier, one graph only, Figure 1-37 survival curves of all three hybrids of adl-16 data is presented in Table  At  29°C  to  the  survival  depicts the  at 2 2 ° C .  ts  Relevent  1-11.  curve of adl-16 ladl-16f ts2  was  lrdI  almost  exactly midway between those of homozygous females of the parent types  (see  Figure  1-38).  The  distances  between  calculated at D-20, D-50 and D-95 and averaged. were 50.3% longer lived than adl-16 , ts2  than  adl-16f . lrdI  curves  The hybrid  were flies  and had 49.7% shorter lives  For adl-16 ladl-16 , tsl  the lifespan at 2 9 ° C was also intermediate.  ts2  Comparison of the D-20, D-50, and D-95 values yielded differences of 52.3% from adl-16 ,  and 47.3% from adl-16  ts2  (see Figure 1-39).  tsl  Such values would be expected from hybrids of hypomorphs lacking complementation, and so these mutants were confirmed to be alleles.  The lifespan of adl-16 ladl-16f tsl  parent  types.  Between  compared with adl-16 , tsl  lrdI  was unusual relative to the two  D-20 and D-50, it  was reduced  the shorter lived parental strain.  even  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-16f , lrdI  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-16  tsl  flies  die (10-12 days), so it might be possible  corresponding gene product is non functional after that. shows all hybrids of adl-16 , ts  females  for comparison.  and 1-42 depicts  that the  Figure 1-41  the homozygote  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  84  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  Age (Days) at 29°C  10  12  14  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  Survival (Days) at 29°C  16  18  20  22  24  86  Figure  1-41  Age (Days) at 2 9 ° C  87  Figure  120  1-42  -i  adl-16 ts HOMOZYGOTE FEMALES Survival Curves at 29°C  100 -  adl-16ts1 Homozygotes N=559 (0  • —  >  adl-16 ts2 Homozygotes N=525 adl-16 flrd I Homozygotes N=600  CO  4  6  8  10  12  Age (Days) at 29°C  14  16  18  20  22  24  88  Table 1-11 LONGEVITY OF A D U L T HYBRID F E M A L E S MAINTAINED A T 22°C POSTECLOSION. HYBRID \adl-16 ladl-16fl tsl  Number LS X  DP  X  DPonset D-20 D-50 D-95 DPend D-100 DPslope  rdI  382 81.9 86.1 6 1 70.5 84.5 94 96 101.5 2.5  adl-16 ladl-16f ts2  lrdI  200 110.8 112.4 80 103 112 122 122 129 2.1  adl-16^ ! adl-16^ 1  200 99.23 102.7 60 88 103 119 119 127 1.5  Data are given in days obtained by pooling the results from separate experiments. and  L S : Life-span mean; D P : death phase mean; D P s e t X  X  on  DP nd: onset and end of death phase, respectively; D-20, 50, 95, e  100: time at which 20, 50, 95, and 100 % of the population had died, respectively.  DP i p s  0  e  is given as % death per day.  2  Table 1-12 LONGEVITY OF A D U L T HYBRID FEMALES MAINTAINED A T 29°C POSTECLOSION. HYBRID  adl-16 /adl-16f < tsl  lr  Number LS X  DPonset D-20 D-50 D-95 DPend D-100 DPslope Data  are given  experiments. and  adl-16^ /adl-16f^di 2  in  adl-16^ 1 adl-16^  641 7.5 7.2 4  888 9.3 9.3 1 6.7 8.7 15.3 15 22.0 6.7  X  DP  11  600 6.5 6.4 3 4.8 6.2 7.7 8 10.0 19.2  5,3 7.0 10.7 10 14.0 15.3 days  was  obtained  from  several  separate  L S : Life-span mean; D P : death phase mean; D P s e t X  X  on  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 i p s  0  e  is given as % death per day.  2  Longevity of adl-16  ts  /Oregon-R  One set of experiments with each of the mutant alleles heterozygous with wild-type was completed early in this research. thought  that  recessive  only  confirmation  was necessary.  It  that  the adl-16  was noted  ts  that  At the time, I alleles  longevity  were  of the  heterozygote was slightly greater than that of the wild-type, OregonR.  The small extension of life present was assumed to be due to  heterosis, recessive.  and the alleles under study were  thenceforth considered  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 twoto three- fold reduction in lifespan.  This relationship holds true for  temperatures of 2 2 ° C and 2 9 ° C which are within normal physiological limits for Drosophila  and was confirmed in these experiments where  a 7 C ° 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 22°C.  This  experiments.  was  adl-16f at lrdI  true for both temperatures used in these sets of  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  Leffelaar  living and  for any  one  Griggliatti  strain.  (1984),  This  who  result  demonstrated  curves of Oregon-R males and females and adl-16 and  coincident  temperatures that adl-16  ts2  when  used. has  adjusted It  also  for  the  extends  sex-specific  rate  of  living  lrdI  that  of  survival  sex-specific at  the  two  by demonstrating  survival  adl-16f .  that  are  tsl  their study  coincident  questionable that the premise includes  confirms  curves.  It  is  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  2 2 ° C some effects of temperature could be seen on the alleles of adl16 .  For the following comparisons death phase means were used  ts  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 2 2 ° C , the D P ( O r e g o n - R ) / D P ( a d / - 7 t f » ) = 1.1, 7  x  x  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  corresponding calculation yields a ratio of 1.26 ± 0 . 0 5 male lifespan is 76-83% that of Oregon-R. reduced more than that of females,  meaning  the that  The male lifespan was  but both are essentially  the same  as for Oregon-R. Using the same calculations, adl-16 of  female lifespan is 66-74% that  ts2  Oregon-R, and male lifespan is 60-68%.  lifespans  Thus male and female  were reduced by about the same amount relative  wild-type  counterparts.  slightly reduced viabilities.  Therefore  even  at 2 2 ° C ,  they  to their displayed  When flies and larvae of all ages were  shifted to 2 9 ° C , all died, leading me to believe that the temperature sensitivity 29°C 3rd  of these flies is continuous.  In mutant strains shifted to  within 12 hours of egg deposition, larvae survive to the 2ndlarval  instar.  This  death is not immediate.  suggests  that,  even  during  development,  In the case of adl-16f ,  for females the lifespan  lrdI  Oregon-R, and for males it was 50-59%.  was  Thus these flies lived about  half as long as their wild-type counterparts at 2 2 ° C . lifespans  Both sexes had  reduced by approximately the same amount.  that the lethal phase  1986), but observations  throughout lifespan The simple  or third  regarding many  pupae which do not eclose in stock maintained at 2 2 ° C ,  29°C.  It is known  in this mutant occurs in the second  larval stage (Homyk et al.,  viability  47-54% of  suggest that  may be reduced, both at 2 2 ° C  experiment  of  shifting  these flies  and at  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  experiments  are needed  sensitivity to  is  continuous  confirm the  nature  at 2 9 ° C . of  the  Further  temperature  sensitivity of all three alleles studied here.  It is well known that heterosis increases 1978),  and  the  mutant  stock, many generations were relatively study.  flies  used  earlier.  homozygous  were produced from  mutant character under  Lints and Lints (1979), argue that inbred lines may have a  therefore  should  not  be  genes existing used  to  in a homozygous  study  normal  the  means  flies that  used the  in this  study  are of  background genome has  state,  ageing  senescence, since these genes could act to shorten lifespan. all  Oregon-R  Comparatively speaking, these flies  except for the  high frequency of deleterious and  lifespan in general (Lamb,  and  However  similar background, which been  standardized and  the effects of each mutant should be easier to compare.  so  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  characteristics,  I  concluded  hybrids for behaviour that  there  and longevity  was no maternal  effect  discernable.  At  22°C,  for hybrids between  16 ladl-16 tsl  females. type  females,  ts2  Since adl-16  lifespan,  adl-16  homozygous  tsl  but adl-16  between  and adl-16  viz, adl-  ts2  the lifespan was 92-97% that of Oregon-R  adl-16  ts2  females exhibit close to wild-  homozygous  ts2  lifespan, this suggests that adl-16 hybrids  tsl  females  have  is a hypomorph.  tsl  and adl-16f  lrdI  female  reduced  In the case of  lifespan  108% that of wild-type females suggesting that adl-16f  is also a  lrdI  hypomorph.  was 92-  This would imply a series for the lethal phene,  alleles varying in strength as follows:  adl-16  ts2  with  > adl-16  tsl  > adl-  a different  picture.  16fl . rdI  Hybrids of adl-16  tsl  and adl-16f presented lrdI  Females of type adl-16 /adl-  16f lived  their wild-type  It should be recalled, however  tsl  adl-16f  had the shortest lifespan  lrdI  lifespan  counterparts.  lrdI  for the hybrid  homozygotes  only 70-80% as long as  of the alleles at 2 2 ° C .  was intermediate  between  that  that The  for the  of the parent types, which was the expected result.  Another  way of analyzing  these  hybrids would  be to compare  lifespan  with  each  parent  When  females  from  stock.  this was  determined at 2 2 ° C , it was found that:  a) adl-16 stock.  I adl-16f  ts2  Compared  hybrid flies lived longer that either parent  lraI  with  twice as long ( 1 . 9 8 ±  homozygous  adl-16f  0.56), and relative to adl-16  lifespan was almost half as long again(1.4 b) adl-16  I adl-16f  tsl  homozygous  the  lraI  were  lraI  parent types  adl-16  hybrids lived homozygotes  ts2  ±0.11).  found to be intermediate tsl  and adl-16f  lraI  between  , with no overlap  in the length of the lifespans. c) adl-16  I adl-16  tsI  ts2  hybrid  than either parent type, adl-16  In  tsl  females and  were  adl-16  slightly  longer lived  ts2  the case of those hybrids that lived longer than either parent  type, it is interesting to speculate whether this difference is due to heterosis, for  resulting from mating strains which have been  many generations,  or whether it is due to some  separated  type of gene  interaction.  At 2 9 ° C Oregon-R flies of either sex lived close to five times as long as adl-16  flies.  tsl  In the case of adl-16  ts2  an approximately ten-fold  reduction in lifespan relative to Oregon-R was seen. adl-16f  lraI  was  around 30% that of wild-type, that is, a  reduction in longevity was seen at 2 9 ° C . that adl-16  ts2  The lifespan of  was the most  intermediate, and adl- 16f was lrdI  1.7-fold  Overall then, it can be said  temperature sensitive, least affected.  adl-16  tsl  was  For hybrids at 2 9 ° C , reduction of lifespan was clear. adl- 16 that  tsl  I adl-16  the lifespan was approximately midway  ts2  of females  of the parent  reduction relative to Oregon-R.  types.  This  For adl-16 l  lifespan  was  again  intermediate  strains,  with  about  a 5.7-fold  Oregon-R.  In the case of  adl-16f  ts2  between reduction  represents  females  a  7-fold  the hybrid  lraI  of  in lifespan  In general, intermediate lifespans  between  the parent relative  to  could be said to be  dosage related, half the normal quantity of each allele product being present.  Although adl-16 ladl-16f tsl  flies  at 2 9 ° C ,  survival  curve  parent types.  was the longest lived of the hybrid  lraI  as expected  from  a simple hypomorphic series, the  was not intermediate  between  females  of the two  These flies demonstrated reduced viability over either  parent at 2 9 ° 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  most and least affected parent types generate  hybrid,  nor that the  an intermediate  effect.  What is surprising is that the two least affected parent types produce a hybrid which is still severely hybrids,  affected,  paralleling the other two  but overall closer to the moderately affected  That the order differs at 2 2 ° C and 2 9 ° C is also surprising.  parent type. adl-16 l tsl  adl-16f  has the longest lifespan at 2 9 ° C and the shortest at 2 2 ° C .  lrdI  Gould there possibly be some relation to the time of action of these mutations? Is it possible that the protein product of adl-16  tsl  is non-  existent or non-functional after 10-12 days at 2 9 ° C ?  The  overall  nevertheless  conclusion  can be drawn  from  phenotype  They are recessive  at the restrictive  temperature,  some interesting gene interaction between  and may thus  29°C,  albeit  with  There may also be  alleles.  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 associated with  is,  to wild-type, and express  some effects even at the permissive temperature.  Now  the above  that these mutants do not complement,  be considered alleles. their  that  with ageing.  age related  chapter.  changes  Such comparisons might very well correlate  behaviour changes  to be discussed  in the next  CHAPTER 2 INTRODUCTION While it is well known that longevity is influenced by behaviour in Drosophila, flies,  for example the life shortening effect of mating for male  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,  shocked, tko, Ether-a-gogo, might  it  be  controlling  possible  factors  to  of  dunce, fruitless,  stuck, easily  stoned, to name just a few esoteric ones, use  lifespan?  normal  behaviours  Insight  into  to  elucidate  age-related  the  changes  is  provided by longitudinal studies of the relationship of behaviour to ageing. most  Few studies exist which examine behaviour in ageing flies, as reported  behaviours  Soliman, 1988).  are  those  of  young  flies  (Lints  and  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  flies at both 2 2 ° C  and 2 9 ° C .  mutant,  was  adl-16  identical  to  tsl  that  of  shown  wild-type  Furthermore, a temperature sensitive to have a pattern of behaviour loss at  both  the pattern of behaviour loss was also compressed into a proportional  to  the  reduction  Several other mutants tested did not behave this way.  the  was  decreased,  period,  not only was  What  interesting  time  that at 2 9 ° C  temperatures.  extremely  shorter  was  a set pattern in wild-type  in  longevity  lifespan.  It  is  thus  accelerates  possible  that  adl-16  may  tsl  be  a  normal ageing at the restrictive temperature.  of testing this mutation and its other alleles adl-16 as  well  mutation  as  hybrids  between  these  alleles  are  The results  and  ts2  which  adl-16f  lrdI  presented  in  this  chapter. The  specific  behaviours described and measured  geotaxis, phototaxis  and motor activity.  in Chapter 2 are  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  alleles of  adl-16 -  Phototaxis  has  studies,  and extend  them  to include the  other  tsl  been  studied since  response of Drosophila (Carpenter,  1905).  1905  when  Carpenter noted  the  to light, gravity and mechanical stimulation  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 behavioural  trait  evolutionary  significance.  factors  as  such  is  of  importance It is  for  affected  melanogaster. survival,  and  Such a thus  has  by various environmental  humidity, temperature, mechanical stimulation, and  100  density.  Thus  comparisons here  experimental  between results  attempted  to  design  is  extremely  important  when  are made, and the experimental  standardize  those parameters  (see  design  Materials and  Methods).  Since differences a genetic  basis  have been noted between different strains of for phototaxis  Benzer found great differences his  has  been  study  he  flies,  with  legs  also examined or wings  (Benzer,  1967).  in six wild strains of Drosophila  countercurrent distribution method  same  proposed  flies,  of  various  removed.  separating  mutants  flies.  by  In  the  and also mutilated  Phototaxis  is  under  genetic  control, with the X-chromosome implicated (Grossfield, 1978). and sex  of flies also affect  controlled  parameters  of  results.  the  Thus by including sex  experiments,  the  two  Age in the  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 as general activity such as walking or flying.  defined  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 induced  in distinguishing spontaneous  motor activity from activity  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 A N D METHODS  The  stocks used for the behaviour tests are shown in Table  Chapter 1.  For all behaviour tests flies eclosing within the same 12  hour period were flies  1-1 in  separated by sex, placed in vials containing ten  of one sex, and the vials were  divided at random,  half for  maintenance at 2 2 ° C , and the remainder placed in the 2 9 ° C incubator as soon as was practicable. same day.  Controls of Oregon-R were set up on the  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 2 2 ° C or 2 9 ° C . females  of  separately,  each  strain, as  well  so that any sexual  as  hybrid  females,  Males and were  dimorphism could be noted.  tested Tests  were performed each day for flies at 2 9 ° C , and once every 3-5 days  102  at 2 2 ° C , for temperature sensitive flies, to coincide with the time at which they were transferred to fresh containers and food. control flies and  and adl-16f  lrdI  were tested every second day at  29°C,  Tests were performed between  10:00  every 3-5 days at 2 2 ° C .  a.m.  and 6:00  p.m.  Oregon-R  Although  the  test intervals were  short time  periods, these had proved sufficient to demonstrate behaviour loss in other  studies.  The behaviour of  throughout its lifespan.  each  strain of  For each strain of flies  flies  was  except  tested  hybrids, at  least three sets of data were obtained and analysed (see Table  1-1).  1. Geotaxis  Drosophila negative  respond to  gravity.  When  disturbed, they  geotaxis by crawling up the side of a vial.  exhibit  The geotaxis  chamber used was a glass tube of 3 mm thickness, 30 cm long and 17 mm by  in internal diameter, with one end open, and the base covered 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. time interval the number of flies  After a 20 second  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. and strain of flies  Vials were chosen at random, and each  were tested.  A result of all flies  section above 15 cm was considered 100%. including  Oregon-R  reached  100%,  the  sex  reaching the  Since no strains of flies 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  behaviour was  maximum behaviour level, then its  expected  50%  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  using the same tube of flies velveteen  following  the  geotaxis  test  set horizontally, with an opaque black  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 bottom of the chamber as before.  were knocked to the  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). within  the  time  100% was defined as all flies above the 15 cm mark 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  compared in relation to these scores, for example,  was  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). motor activity chamber.  Each fly was blown into the  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. seconds,  and the  number of  lines  Each fly was observed for 10 crossed  counted  and recorded,  moving the disc on the graph paper so that the flies did not come to rest at the side. were  in  the  anaesthetized  Flies were added one by one until all from one vial chamber  with  and  had  carbon dioxide  been so  tested.  that  they  from the chamber, and placed in a fresh vial.  Flies  were  could be  then  removed  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.  flies were tested for each trial. calculated.  The mean activity per fly was then  The absolute maximum level of activity was a value of 38  lines crossed in ten seconds by Oregon-R males defined as 100% for motor activity. and  20  at 2 9 ° C .  It was  For comparisons between strains  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. good  The absolute numbers of lines crossed provided a  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 was obtained.  by a few  very low behaviours before the final  zero  A score sheet for behaviour is included as Appendix i.  RESULTS To it  be able to compare behaviour loss at two different temperatures, was  necessary  to  determine  that  the  restrictive  temperature  of  2 9 ° 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  calculated and used.  factors  for  the  rate  of  living  were  Conversion factors of 2.5 and 2.3 for males and  females respectively of Oregon-R were used to adjust 2 9 ° 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  percentage  the  patterns  of flies  of  behaviour  loss  to  longevity,  displaying a given behaviour was plotted  the age of the population in days at 2 2 ° C .  the  against  Since the graphics program  used did not permit the use of two axes, the number of days at 2 9 ° 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 2 9 ° C flies.  When the patterns,  same it  temperature Oregon-R  was for  are  conversion factors possible each  shown  to  strain.  were  compare  applied to behaviour loss results  The curves  in Figures 2-1,  obtained, but only those sufficient  at  each  behaviour loss  through 2-10.  represents the results from one experiment. were  for  obtained  Each  for  curve  Many more data points  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 shown, data for the other strains was presented in the tables for comparison.  quite  only are  similar, and has  been  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: 2 9 ° C days have been multiplied by the factor 2.5 to adjust for the rate of living.  Figure  2-2  100  -i  Geotactic Response of Oregon-R Females at 22°C or 29°C to Height 5 cm.  E o  LO  Females at 22°C  o CD  Females at 29°C  o  > O)  cu  Age (Days) at 22°C  Note: 2 9 ° C days have been multiplied by the factor 2.3 to adjust for the rate of living.  Figure  2-3  100  Geotactic Response of Oregon-R Males at 22°C or 29°C to Height 10 cm. -i  2. E o  o  o ©  -  Males at 22°c  «—  Males at 29°C  <D >  "To  CD 0>  40  60  1 oo  Age (Days) at 22°C  Note: 2 9 ° C days have been multiplied by the factor 2.5 to adjust for the rate of living. ; Note: The two curves for males at 2 9 ° C , involve a computer derived curve of best fit (without symbols), and actual experimental results (with diamond shaped symbols).  Figure 2-4  100 n  Geotactic Response of Oregon-R Females at 22°C or 29°C to Height 10 cm.  E o o  8 o  -  Females at 22°C  * —  Females at 29°C  > CB  8>  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  0  s  10  20  30  40  50  60  Age (Days) at 22°C  Note: 2 9 ° 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 2 9 ° C  have a higher level of geotactic activity than those at 2 2 ° 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.  exhibit greater geotaxis than do females. true for all the strains tested.  In general, male  flies  These generalizations hold  The shape of the two curves at 2 2 ° C or  2 9 ° C , although similar, is not congruent.  From the fact that a higher  level of geotactic behaviour was seen at 2 9 ° C , it could be concluded  that  this  temperature  behaviour.  does  have  a  negative  impact  on  that  Geotactic behaviour in strains other than Oregon-R can be  seen in the tables below. three  not  hybrids, geotaxis  In the case of adl-16f  as well as all  lrdI  at  maximum was  reduced  relative  to  the  other strains at 2 2 ° C , which behaved similarly to Oregon-R.  At  29°C  the  unmeasurable, first  24  cm mark.  the  hours.  16 /adl-16 , tsl  geotactic  ts2  flies Only  behaviour became  of  geotaxis at 5 cm was ts2  tsl  and adl-16 Iadl-16f  lrdI  virtually within  measurable  flies  lrdI  tsl  was  ts2  paralysed early, usually  and adl-16 ladl-16f as  adl-16  adl-16  in  the adl-  did not reach the 10 behaviour were similar  to Oregon-R, but geotactic behaviour of adl-16f  lrdI  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  Phototactic Behaviour of Oregon-R Females at 22°C or 29°C  100 n  CO  o o  0_  CD >  -  Females at 22°C  • —  Females at 29°C  O Q.  120 Age (Days) at 22°C  Note: 2 9 ° 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 2 9 ° C than at 2 2 ° 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  geotaxis  5cm,  at  the  strains  22°C.  At  tested, this the  time  behaviour was of  50%  lost  after  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-16  ts2  at  22°C,  despite the fact that these flies  had vermilion eyes, a factor  which is said to reduce phototaxis (Grossfield, 1975). At 2 9 ° 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.  was  At the time that 50% behaviour was  displayed, there  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 2 9 ° 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  results  obtained.  late  or early.  This  may have  affected  the  Figure  2-9  Motor Activity of Oregon-R Males at 22°C or 29°C 100 n  Age (Days) at 22°C  Note: 2 9 ° 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: 2 9 ° 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 2 2 ° C or 2 9 ° 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-16  ts2  and adl-16f  lrdI  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,  parameters may be examined in more detail. inclusion separate  of sex  graphic  representation  for  all  all of the  above  Space precludes behaviours  of  the each  and strain examined.  Table  2-1  Behaviour Loss with Age in Oregon-R Males maintained at 2 2 ° C  Geotaxis-15 Geotaxis-10 Geotaxis-5 Phototaxis Motor  Maximum Activity % Day 70.7 1-6 88 1-6 96.7 1-7 69.7 2-5 33.2 2-8  50% Activity  % 35.3 44 48.3 34.9 16.6  Day 14.3 14.8 32.3 48.3 42.0  Minimum Activity % Day 57 0 69.5 0 0 80 114 0 0 126.5  Behaviour Loss with Age in Oregon-R Males maintained at 2 9 ° C  Geotaxis-15 Geotaxis-10 Geotaxis-5 Phototaxis Motor  Maximum Activity % Day 90.5 1-5 99 1-5 100 2-7 94 7-10 34.5 7-13  50% Activity  % 45.5 49.0 50 47 17.3  Day 13.5 22.0 26.5 29 36  Minimum Activity % Day 0 36 41 0 0 43.5 44.6 o 0 50.5  Table 2-2 Behaviour Loss with Age in Oregon-R Females maintained at 2 2 ° C  Geotaxis-15 Geotaxis-10 Geotaxis-5 Phototaxis Motor  Maximum Activity % Day 58.3 1-5 75.5 1-5 1-6 91.7 5-10 74.3 8-10 33.5  50% Activity  % 29.2 37.5 45.8 37.2 16.7  Day 7.0 15.0 25.5 37.5 57.5  Minimum Activity Day % 54 0 69.5 0  6 0 0  73.5 109.5 120  Behaviour Loss with Age in Oregon-R Females maintained at 2 9 ° C  Geotaxis-15 Geotaxis-10 Geotaxis-5 Phototaxis Motor  By  Maximum Activity % Day 89.7 1-3 95 1-2 98 7-10 87.7 3-10 35.5 9-13  50% Activity  % 44.8 47.5 49 43.9 17.7  Day 20.0 20.8 26 28.8 30.5  Minimum Activity % Day 0 37 39 0 45.5 0 0 48 53.5 0  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 can  easily  be seen.  Despite  the fact  that  adl-16  tsl  all parameters are  compressed into one fifth the time at 2 9 ° C for adl-16 , tsl  loss except for phototaxis at 50% is extremely consistent.  behaviour  Table 2-3 Behaviour Loss with Age in adl-16  Geotaxis-15 Geotaxis-10 Geotaxis-5 Phototaxis Motor  Maximum Activity % Day 1-4 72.5 84.5 1-5 92.5 1-7 78 1-8 30.1 3-10  Geotaxis-15 Geotaxis-10 Geotaxis-5 Phototaxis Motor  Males maintained at 2 2 ° C  50% Activity % 36 42.3 46.3 39 15.1  Behaviour Loss with Age in adl-16 Maximum Activity % Day 84 1 94.5 1 97.5 1 81.5 1 29.5  tsl  tsl  Day 4.5 4.3 21.3 31.5 28.8  Males maintained at 2 9 ° C  50% Activity % 42 47.5 48.8 40.8 14.8  Minimum Activity % Day 0 24.5 58 0 6 1 0 69 0 92.5 0  Day 2.3 2.8 3.3 2.3 3.8  Minimum Activity % Day 0 4.5 0 5.5 0 6 6 0 0 7.5  123  Table 2-4 Behaviour Loss with Age in adl-16  Geotaxis-15 Geotaxis-10 Geotaxis-5 Phototaxis Motor  Maximum Activity % Day 1-6 69 1-7 90 96.5 1-8 84 1-7 28.5 1-10  tsl  50% Activity  %  Geotaxis-15 Geotaxis-10 Geotaxis-5 Phototaxis Motor  By  29.5  1  Day 3.3 6.5 13 29.8 41.3  34.6 45 48.3 42 14.3  Behaviour Loss with Age in adl-16 Maximum Activity % Day 80.5 1 92.5 1 95.5 1 86.5  Females maintained at 2 2 ° C  tsl  Minimum Activity Day % 0 24.5 0 40 62 0 77 0 101 0  Females maintained at 2 9 ° C  50% Activity  %  Day 2  40.3 46.2 47.8 43.3 14.8  2 2.8 2.3 3.5 ..........ffl»*wvmwmW  Minimum Activity % Day 3.5 0 0 4.5 0 5.5 0 5.5 6.0 o  examining the following Tables 2-5 and 2-6 for adl-16  ts2  it can  clearly  be seen that there is virtually no measurable behaviour at  29°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-16  tsl  rate of ageing, but at an even faster rate.  may also accelerate the  Table 2-5 Behaviour Loss with Age in adl-16  Geotaxis-15 Geotaxis-10 Geotaxis-5 Phototaxis Motor  Maximum Activity Day % 1 82.5 93.1 1-3 99 1-5 70 4-10 4-10 23.6  ts2  50% Activity  % 41.3 46.5 49.5 35 11.8  Behaviour Loss with Age in adl-16 Maximum Activity  % Geotaxis-15 Geotaxis-10 Geotaxis-5 Phototaxis Motor  0 0 3 0.9 0.8  Males maintained at 2 2 ° C  ts2  Day 9.3 11.5 13.5 24 18.3  % 0 0 1.5 0.5 0.4  % 0 0 0  6 0  Day 15.5 22.5 36.5 47.5 72  Males maintained at 2 9 ° C  50% Activity Day 1 1 1 1 1  Minimum Activity  Minimum Activity  Day  %  1 1.1 1.3 1.3  0 0 0 0 0  Day 1 1 1.3 1.7 1.7  Table 2-6 Behaviour Loss with Age in adl-16  ts2  Maximum Activity Day % 52 1 1 8 1 1-6 95.5 70.5 4-10 4-10 25.1  Geotaxis-15 Geotaxis-10 Geotaxis-5 Phototaxis Motor  %  Day 4.7 7 12.5 19.5 15.5  26 40.5 47.5 30 12.5 ts2  Maximum Activity % Day 1 0 1 0 1 2.3 1 0.8 1 1.2  In the case of adl-16f  Minimum Activity Day % 0 13.5 b 1 8 27.5 0 0 50 77 o  50% Activity  Behaviour Loss with Age in adl-16  Geotaxis-15 Geotaxis-10 Geotaxis-5 Phototaxis Motor  Females maintained at 2 2 ° C  Females maintained at 2 9 ° C Minimum Activity Day % 1 0 1 0 0 1.3 0 1.3 0 1.7  50% Activity  %  Day 1 1 1.2 1.2 1.3  0 0 1.2 0.4  .9,6  it can be seen that all parameters for  lraI  behaviour were reduced at 2 2 ° C (as well as the lifespan mentioned in Chapter  1).  phototaxis, third.  Geotaxis and motor  at 15 cm was reduced activity  was reduced  four-fold, as was  by approximately one  This leaves less room for further reductions at 2 9 ° C , but at  that temperature geotaxis of all levels was reduced by 20%. that  phototaxis  interesting,  and motor  activity  were  not further  because climbing is not necessary  The fact  reduced  is  for these behaviours.  Of all alleles this strain demonstrated the least behaviour at 2 2 ° C .  At  2 9 ° C it was intermediate between the other two strains in terms of reduction of behaviours.  Table 2-7 Behaviour Loss with Age in adl- 16f Males  maintained at 2 2 ° C  lrdl  Geotaxis-15 Geotaxis-10 Geotaxis-5 Phototaxis Motor  Maximum Activity % Day 17 1-6 1-10 55.3 83 1-10 9-14 17.3 6-9 20.8  50% Activity  Geotaxis-15 Geotaxis-10 Geotaxis-5 Phototaxis Motor  Minimum Activity  %  Day  %  8.5 27.7 41.5 8.7 10.4  6 9.2 16.2 23.7 26.3  0 0 0  Behaviour Loss with Age in adl-16f Maximum Activity % Day 1-2 6 1-2 31.7 1-2 64.7 2-4 1 6 1-4 20  j J  lraI  3 15.8 32.3 7.8 10.1  0  Males maintained at 2 9 ° C  50% Activity  %  o  Day. 1 4 34 55 59.3 78  Day 2 2.8 3.3 7.2 5.2  Minimum Activity % Day 2.7 0 0 3.7 0 8 0 11.7 0 16.7  Table 2-8 Behaviour Loss with Age in adl-16fl Females  maintained at 2 2 ° C  rdI  Maximum Activity % Day 18.7 5 40.7 5-17 5-14 94.5 1-14 16.3 1-14 21.7  Geotaxis-15 Geotaxis-10 Geotaxis-5 Phototaxis Motor  Day 7 18.3 24 44.5 37.7  % 9.3 20.3 47.3 8.2 10.8  Behaviour Loss with Age in adl-16f Females  maintained at 2 9 ° C  lrdI  Maximum Activity Day % 1 1 1-2 13.7 1-2 47.3 2-4 15.3 1-2 18.5  Geotaxis-15 Geotaxis-10 Geotaxis-5 Phototaxis Motor  Minimum Activity % Day 9 0 0 27 0 61.3 0 66 0 79  50% Activity  Minimum Activity Day % 2 0 0 4.3 0 10 0 11.7 0 19.3  50% Activity % 0.5 6.8 23.7 7.7 9.2  Day 1.5 2.8 3.3 8.5 3.3  The hybrid presented in Table 2-9, adl-16 ladl-16 tsl  reduced appear  geotaxis normal.  temperature  at 2 2 ° C , Thus  for this  although  22°C  strain.  reduced by approximately 20%. were intermediate temperature.  phototaxis  may not be At 2 9 ° C  a  demonstrated  ts2  and motor totally  all behaviours  In adl- 16 /adl- 16 tsl  ts2  activity  permissive were  further  all behaviours  between those of parent strains at the restrictive  For adl-l6 ladl-16f ts2  but  behaviour was somewhat  lrdI  of the hybrids it was the least affected  Table 2-11).  at this temperature (see  However a reduction in behaviour was seen in the  results for geotaxis, Oregon-R  reduced at 2 2 ° C ,  the lifespan having been comparable to that of  at that temperature.  normal at 2 2 ° C .  Phototaxis  and motor ability  were  It is interesting that geotaxis, phototaxis and motor  ability are less severely affected  in the hybrid  adl-16 ladl-16f ts2  lraI  than in either parent strain at 2 9 ° C .  The  hybrid adl- 16 ladl-  16f was  tsl  Table  2-10).  compared  lraI  At 2 2 ° C ,  with  geotaxis  to  intermediate  the other two hybrids, but phototaxis  as well as  motor activity were normal.  and  was reduced  In other words all three hybrids had  similar behaviour type at 2 2 ° C , of geotaxis.  the most interesting of all (see  demonstrating a reduction in levels  In the case of this hybrid lifespan was reduced at 2 2 ° C ,  was intermediate between females  29°C  a surprising result was found.  of the generating lines.  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 2 9 ° C . argued  that  this  combination  against  the deleterious  behaviour is concerned. allele  adl-16f  lrdI  effects  of alleles  provides  of temperature,  It could be  some protection  at least  as far as  It would appear that the presence  had a moderating  At  effect  on the  of the  severity  expression of the other two alleles at the restrictive temperature.  of  Table 2-9 Behaviour Loss with Age in adl-16 ladl-16 maintained at 2 2 ° C tsl  Geotaxis-15 Geotaxis-10 Geotaxis-5 Phototaxis Motor  Maximum Activity Day % 18.5 6.5 3 1 6.5 48 6.5 72.5 6.5 30.732 11.5  ts2  50% Activity % 9.3 15.5 24.0 36.3 16.0  Day 1 1 12 22.5 26.8 32.5  Behaviour Loss with Ase in adl-16 ladl-16 maintained at 2 9 ° C tsl  Geotaxis-15 Geotaxis-10 Geotaxis-5 Phototaxis Motor  Maximum. Activity Day % 0 13 1 35 54 1 1 16.7  ts2  50% Activity %  Day  0 6.5 17.5 27 8.3  1.5 1.5 1.5 1.5  Hvbrid  Females  Minimum Activity % Day 0 23 0 23 55 0 0 96 102 0 Hvbrid  Females  Minimum Activity % Day 0 2 0 2 0 3 0 4 6  Table 2-10 Behaviour Loss with Age in adl-16 1 adl-16f maintained at 2 2 ° C ts  Geotaxis-15 Geotaxis-10 Geotaxis-5 Phototaxis Motor  Maximum Activity % Day 34.7 7 57 10 70 10 87.3 5.3 37.0 7.3  1  lraI  50% Activity % 17.3 28.5 1 35.0 42 18.5  Day 18 23.2 34.2 33.2 51.8  Behaviour Loss with Aee in adl-16 1 adl-16f maintained at 2 9 ° C tsl  Geotaxis-15 Geotaxis-10 Geotaxis-5 Phototaxis Motor  Maximum Activity % Day 92 1 98.5 1 99.5 1 1 87.5 29.3 1  lraI  50% Activity % 46 49.3 49.8 43.8 14.6  Day 1.5 2.0 2.5 2.0 2.5  Hybrid  Females  Minimum Activity % Day 47.3 0 54.7 b 72 0 90 0 100.3 0 Hvbrid Females  Minimum Activity % Day 0 3 4 0 0 0 0  5 5.5 9.5  Table  2-11  Behaviour Loss with Age in adl-16f ladl-16 maintained at 2 2 ° C lrdI  Geotaxis-15 Geotaxis-10 Geotaxis-5 Phototaxis Motor  Maximum Activity % Day 43.5 5.5 65 5.5 82 5.5 82 9.5 38.7 8  50% Activity  % 21.8 32.5 41.0 41.0 19.1  Day 12.5 15.8 19.8 38.5 68.8  Behaviour Loss with Age in adl-16f ladl-16 maintained at 2 9 ° C lrdI  Geotaxis-15 Geotaxis-10 Geotaxis-5 Phototaxis Motor  Maximum Activity ' % Day 43 1.5 60.5 1.5 72 1.5 68 1.5 23.7 1.5  table  is  presented  ts2  50% Activity  % 21.5 30.3 36 34 11.7  So that a comparison between following  ts2  Day 2.3 2.3 2.3 2.3 2.3  Hvbrid  Females  Minimum Activity Day % 0 35.5 6 52.5 0 78 6 102.5 0 122.5 Hvbrid  Females  Minimum Activity % Day 0 3 0 3 0 3 0 3.5 0 8.5  strains can be made more easily, the showing  strain for all behaviours tested.  maximal behaviour for each  Table  2-12  Maximal Behaviour in A l l Strains Tested at 2 2 ° C Strain of Flies Tested Oregon-R Males Oregon-R Females adl-16 Males adl-16 Females adl-16* Males adl-16 Females adl- 16Ardl M a l e s tsl  tsl  2  ts2  adl-16 Females adl-16 /adl-16 adl-16tsl/adl-16 di adl-16ts2/adl-16«'-di f l r d I  t s l  t s 2  flr  Geotaxis 15 cm  Geotaxis 10 cm  Geotaxis 5 cm  70.7 58.3 72.5 69 82.5 52 17 18.7 18.5 34 43.5  88 75.5 84.5 90 93 81 55 40.7 3 1 57 65  96.7 91.7 92.5 96.5 99 95.5 83 94.5 48 70 82  Photo- Motor taxis Activity 69.7 74.3 78 84 70 70.5 17.3 16.3 72.5 87.3 82  33.2 33.5 30 28.5 23.6 25.1 20.8 21.7 32 37 38.7  Maximal Behaviour in A l l Strains Tested at 2 9 ° C Strain of Flies Tested Oregon-R Males Oregon-R Females adl-16tsl M a l e s adl-16 Females adl-16 Males adl-16 Females adl-16 Males adl-16 Females adl-16tsVadl-16ts2 tsl  ts2  ts2  f l r d I  f l r d I  adl-16tsl/adl-16 adl-16 /adl-16 t s 2  flrdI  f l r d I  Geotaxis 15 cm  Geotaxis 10 cm  Geotaxis 5 cm  90.5 89.7 84 80.5 0 0 6 1 0 92 43  99 95 94.5 92.5 0 0 31.7 13.7 13 98.5 60  100 98 97.5 95.5 3 2.3 64.7 47.3 35 99.5 72  Photo- Motor Activity taxis 94 87.7 81.5 86.5 0.9 0.8 16 15.3 54 87.5 68  34.5 35.5 29.5 29.5 0.8 1.2 20 18.5 16.7 29.3 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-16 .  In fact the  tsl  parameters of behaviour for this strain are very similar to Oregon-R at both the permissive the  and restrictive temperatures,  restrictive temperature, all are accelerated.  Tables 2-1 quite  except that at  By inspecting  the  to 2-4, it can be seen that at 2 2 ° C the two strains were  similar.  What is even  these strains at 2 9 ° C .  more interesting  is  the  similarity of  The premise related to ageing for this mutant  is that behaviour loss at 2 9 ° C 2 2 ° C , but at an accelerated rate.  follows  a similar pattern to that at  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 2 9 ° C Strain of Flies Tested  Geotaxis 15 cm  Geotaxis 10 cm  Geotaxis 5 cm  Photo- Motor taxis Activity  Average and S.D.  adl-16^ M a l e s  1.84  1.65  1.62  1.65  1.51  1.66±.ll  adl-16  2.25  1.91  1.81  1.91  1.94  1.96±.15  -  -  2.47  1.91  2.18  2.19±.23  2.79  2.93  2.52  2.75±.17  adl-16 <H M a l e s  4.67  3.76  1.91  1.32  1.05  2.5411.4  adl-16 Females adl-16tsl/adl-16ts2  7.0  3.52  1.77  1.58  1.06  2.9912.2  -  3.35  3.90  2.73  2.28  3.071.61  adl-16tsl/adl-16 adl-16ts2/ dl-16«rdi  4.50  3.72  3.39  3.28  2.11  3.41.77  3.0  1.49  3.71  3.28  1.49  2.591.93  1  tsl  Females  adl-16ts2 M a l e s adl-16  ts2  Females  flr  f l r d I  flrdI  a  For adl-16 their loss differed  for all behaviour parameters averaged at the time of  tsl  relative  to  the  lifespan  by 6% and females  of  this  mutant at 2 9 ° C ,  differed by 8% from  males  Oregon-R.  In  addition to this, there was very little variation in any of the total loss parameters  relative  to  Oregon-R.  adl-16  ts2  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  25°C  might  prove  interesting. By  comparison, adl-16f  from  lraI  Oregon-R,  the  differed greatly in behaviour loss patterns  males  by  56%,  and  the  females  Differences were from 20% to 36% for the three hybrids of  by  73%.  adl-16 . ts  DISCUSSION From the results of the behaviour tests for each strain, it is clear that behavioural  responses  Drosophila. behaviour  could  serve  as  bio-markers  of  age  in  Not only did each strain demonstrate a unique pattern of at  the  two  temperatures  used  in  the  behaviour loss was consistent and measurable.  experiments,  but  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  two empty glass vials connected together  and Grigliatti used  at their open ends by a  piece of tape.  Five and fifteen cm intervals were marked on the side  of  In my experiments, a cylinder was used, so that no  the vials.  crack at the half-way point would be available for flies climbing.  to use in  Also three 5 cm intervals were tested, which would not  have been possible with tape obscuring the view. taping at each experiment was also eliminated.  The bother of reNew glass was used  in either experiment, but the diameter of their two vails was given.  The base of the chamber differed, for theirs, it was glass, for  these experiments glass.  not  it was  nylon mesh glued to the outside  of the  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. it was 1 cm graph paper with tenths marked. squares measuring 1.25 cm per side.  For my experiments They used paper with  Their tests for motor behaviour  were performed once a week at 2 2 ° C for males only.  M y motor tests  were performed at the temperature of maintenance of the flies, and  22°C  2 9 ° C , on a more frequent basis to correspond with turning over  of flies and for both sexes.  Thus  despite  the  many  comparable results results,  supports  marker of age.  differences  were obtained. the  idea  that  in  experimental  The independent  behaviour  makes  design,  very  verification of  an excellent  bio-  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. behaviour  loss  pattern  was  consistently  geotaxis  (all  The total 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  22°C  populations  of flies reached maximum behaviour within a  few days after eclosion. for  up to ten days.  within three days.  This high level of behaviour was maintained  At 2 9 ° C  maximal behaviour was  usually seen  On these graphs adjusted for rate of living, it  would  appear  that  there  maximal behaviour. fully  to this  temperature.  there  a  slight  delay  in  the  appearance  was  Previous to this, larvae and pupae had After the maximum was achieved at either  a decline  in demonstrated  behaviours  until  total loss of behaviour occured.  In these experiments, phototaxis  lost  before,  after  geotaxis  of  This was possibly due to the flies acclimating  been maintained at 2 2 ° C . temperature,  is  5  cm,  not  as  in  the  experiments  Leffelaar and Grigliatti, but the design of the experiments differ.  was by 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 2 7 ° C was not observed. Rather, flies of wild-type and adl-16  tsl  demonstrated  an  increased  behaviour at the higher temperature.  The pattern of behaviour loss at 2 2 ° 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. lifespan.  Phototaxis was lost next, after approximately 80% of total Finally motor behaviour was lost, but not until well into the  final death phase. adl-16  ts2  was  too  The same pattern was found for adl-16 severely  debilitated  by this  tsJ  temperature  at 2 9 ° C . for any  definite conclusions to be made, but at 2 2 ° C , the pattern of behaviour loss was as that for Oregon-R and  adl-16 . tsl  In the case of adl-16 ,  the chromosome was also marked with scute,  ts2  (sc),  cross-veinless,  (cv),  the  mutation  vermilion  reported to reduce phototaxis  (Fingerman, 1952).  phototaxis  these  was  impediments  noted  were  in  found because  beat  The  Vermilion as well as forked flies have been noted to have but  wing  (f).  reduction.  behaviour,  cause  forked,  scute,  mating  cross-veinless  and  mutations  normal  and  vermilion, (v),  these  and  markers, but  have been better to use an unmarked chromosome for  The  analysis  was  of hybrids of the temperature-sensitive  extremely  demonstrated  interesting.  at  29°C,  geotaxis was also seen. 16 ladl-16f tsl  as  Not  only  expected,  but  was at  has  been  No reduction of  experiments,  of  frequency  other  it  would  adl-16 . ts2  alleles studied  reduced  22°C  no  behaviour  a reduction in  The most surprising result was that of  adl-  which revealed a behaviour loss pattern similar to  lrdI  that of adl-16 at  2 9 ° C , but a lifespan intermediate between that for  tsl  females of the two generating strains at 2 2 ° C .  The other two hybrids  demonstrated  expected.  reduced behaviour at 2 9 ° C  as  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 2 2 ° C ) ,  mutations  which  or to define age for experimentation.  alter  the  understanding what controls it.  pattern  of  ageing  will  Finding assist  The possibility that adl-16  tsl  in and  adl-16  might be such mutations is encourageing.  ts2  The remainder  of this thesis deals with the location of these alleles as well as  adl-  16 flrdl  for  o  n  the  X-chromosome, so  that  they  might  be  cloned  further study; and location of the type of tissue affected by the product produced.  gene  CHAPTER 3 INTRODUCTION  In general, deletions in chromosomes cause death of the organism if homozygous. recessive  In heterozygotes,  genes  single dose.  as  they permit phenotypic expression of  pseudodominance,  the  genes  being  present  in  This allows the cytological localization of genes, so that a  correlation can be made between the genetic map, based on linkage studies,  and  deficiency  the  loops  pseudodominance.  cytological visible  map  in polytene  inferred  by  the  chromosomes,  presence  combined  of with  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. of  gene action  neomorph,  in the  Inferences may then be made as to the mode mutant allele;  whether  amorph, hypomorph,  or hypermorph.  An X-chromosome with a deletion which uncovered be very close to respectively which  adl-16  ts  alleles,  (the  positions  mapped by usual recombinant methods),  uncovers  bands  13F1-14B1, was  ts  ts  are 52.2  known to and  Df(l)sd  If the alleles for adl-  lie in the area corresponding to the deficiency, their location and  function  might  be  combination  with this  comparisons  of  females.  further  elucidated  deficiency  homozygotes,  by  chromosome.  with hemizygotes  studying This  them  would  in either  in  enable  males  52.9  72b26  available in this laboratory  and given to me by kind favour of Don Sinclair. 16  shi ,  or  The  three strains of flies hybrid for the deficiency and each  mutant  allele  were  examined  for  temperature  sensitivity,  adl-16  ts  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 some interesting  these early results  indicate  possibilities.  MATERIALS AND METHODS  Male flies Df( 1 )sd  of all three mutant alleles of adl-16  IFM  y B w virgin females.  72b26  kept  and  Deficiency  with  To optimize the number of progeny, all vials were  examined stock  mated  Thirteen replicates of each  a  cross were made.  were  ts  after  was  also  each  transfer  crossed  (every  2-4  with Oregon-R.  days).  The  The following  crosses were made: IFM  y B  ®  FM y B w« IY (stocks)  IFM  y B w«  ®  Oregon-RIY  Df(l )sd7 > IFM  y B w  ®  adl-16** IY  4)  Df(l )sd b26/FM  y B w  5)  Df(l )sd  y B w«  1)  Df(l )sd  72b26  2)  Df(l )sd  72b26  3)  2l  26  72  a  1  adl-16** IY  a  /FM  72b26  For simplicity, Df(l)sd ^IFM 72b2  (viability ratio)  2  ®  adl-16f IY lrdI  y B w will be called a  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  was  #2  used  to  compare  viability  deficiency chromosome, i.e. Dflwild give a 1:1 ratio.  of  Oregon-R with  compared with FM7lwild  If this was not the case, then viability in  should  Dflmutant  might be lowered due to the action of the Df chromosome, must  be  eliminated  regarding Dflmutant Crosses #3-5  as  a  possibility  before  viability  DflY dies  which decisions  could be made.  involved the mutant alleles under study.  progeny types were  the  The following  expected:  Dflmutant normal eye  FM7lmutant Wide Bar eye  FM7IY white Bar eye yellow  Fertility of Deficiency/Mutant at 2 2 ° C A test of the fertility of Df I mutant which eclosed the FM7IY  flies was tried at 2 2 ° C .  from the test crosses (#3-5  males  progeny scored.  which eclosed  from the  were  mated with  same crosses, and the  Since no attempt had been made to separate  females as virgins, this was appropriate. present were assumed to be sterile. 22°C.  above)  Flies  The few  mutant 10  males  These flies were maintained at  Controls used were female flies of the each mutant stock  males and females of each strain) and females of type (30 of each strain)  the  (20  FM?'I'mutant  Viability and Development at 2 9 ° C  To test whether the Df I mutant  could develop to adult flies at 2 9 ° C ,  virgin females of type DflFM7  were mated to males of each of the  mutant strains. vials  were  The female flies were removed after 4 days.  maintained at 2 2 ° C ,  restrictive temperature. the following crosses.  and 5  vials  ®  adl-16^/Y  b)  DflFMl  ®  adl-16^2iY  c)  Df/FM7  <8>  adl-16flrdl/Y  flies (30).  The following  white  pre-pupal larvae of adl-16  such small experiments  were shifted to  ts2  were performed.  Heat pulses  29°C.  of  hours, four hours, six hours or continuous heat were applied. vial  of  the  were made:  DflFM7  Ten  to  Controls used were male and female mutants  a)  Twenty  shifted  Thirteen replicates were made of each of  of all three types (30), as well as DflFM7 crosses  were  Eight  20  pre-pupal  larvae was  left  at  22°C.  The effects  two One of  temperature on development of the adult are noted below. RESULTS General 2 2 ° C For Df(l)sd  /adl-16  72b26  tsl  swollen  abdomens were  visible  by day  three, and by day 7, proboscis extension was present, and abdomens  were very swollen. days  of  life.  Light  variability (missing  Df( 1 )sd  72b26  Flies walked jerkily or weakly during the last 10  /adl-16  or  colored  proboscis extension.  wing  vein  very swollen  abdomens,  as well  as  Wings were light colored, and cross veins  or missing.  the chromosome  posterior  Walking problems were seen within a week; the  gait was jerky and shaky. were deformed  showed  incomplete)  showed  ts2  wings  This was  with the mutant allele  unexpected,  because  although  was marked with sc, cv, v,  a n d / , the D f chromosome was c v . +  Df( 1 )sd  ladl-16f  72b26  all held their wings up.  lraI  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 deformities  (see  Figure  3-1  and  photograph  3-1).  were reminiscent of those seen on engrailed  The  wing  (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, missing  sometimes flattened,  or  abnormal.  missing some hairs.  The  legs and feet with tarsi and claws body  appeared  very  dark,  and  was  14 5 Figure DIAGRAM  3-1  O F W I N G O F Df/adl-16fl£dl  Part of wing missing  General characteristics a) b) c) d) e) f)  Wings seemed more opaque, darker, as if wrinkled, or more hairs present. Some hairs were different in shape. Wing size and shape differed. Parts of the wing were often missing, especially bristles. Posterior crossvein was frequently missing or deformed. The body of the fly was dark. legs and feet were deformed, with claws and pads often missing. Legs seemed fragile and weak. Normal  Winp  146  Photograph  3-1  P H O T O G R A P H O F WING OF Df/adl-16flidi  The viability ratio of Oregon-RIFM7 1:1  to Oregon-R/Df  (86:83) meaning that the deficiency  viability,  and so  was very close to  chromosome did not reduce  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 flies of type FM?'/mutant  was divided by the number of  to obtain the viability ratio for each strain.  The ratios in Table 3-1 were found.  Table  3-1  VIABILITY RATIOS 29°C  22°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% X2 alues were 39.56 for Dfladl-16^ , 22.26 for Df/adl-l6f^ , and 9.02 for adl-16 . Thus the Df/mutant eclosed at a significantly reduced frequency relative to FM7/mutant. 2  rdl  V  tsl  At  22°C  viability was thus significantly reduced in all three alleles  over deficiency,  adl-16  ts2  causing the greatest reduction in viability.  Once eclosed, though, flies of type adl-16f  lrdI  At 2 9 ° C no Df/mutant  died the most rapidly.  flies developed to the imago.  highly significant reduction in viability!  This represents a  148  Table 3-2 PROGENY OF DEFICIENCY CROSSES A T 22°C Number  Genotype Dfl  adl-16"  140  1  FM7ladl-16 FM7IY DflY (adl-16^ 10)  195 101 15  Dfladl-16  135  FM7ladl-16ts2  260 183 2  tsl  1  ts2  FM7IY DflY  (adl-16 /0) ts2  Phenotype at 2 2 ° C Swollen abdomen. Proboscis extension Wing abnormalities. Problems walking Normal wings, normal behaviour Normal wings, normal behaviour A l l had to be a d l - 1 6 , tested as such. tsl  Abnormal crossveins. Swollen abdomen Proboscis extension. Problems walking Normal wings, normal behaviour Normal wings, normal behaviour Crossveinless, vermilion, forked, had to be adl-16 and tested to be such. ts2  Dfladl-16flrdi  137  FM7I adl-16fl <U FM7IY DFIY (adl16flrdll0)  227 107 1  Total  1503  r  The  presence  of adl-16 IO tsl  Wings up. Wings dark and abnormal. Eclosed late. Died quickly. Problems walking, deformed legs and feet. Normal wings, normal behaviour Normal wings, normal behaviour Normal wings, lived 67 days at 2 2 ° C .  non-disjunction flies  they must have been of this type, because DflY survive, and these flies when tested at 2 9 ° C  surprised me, but does  tsl  crossveinless vermilion and forked (the markers for the  were all from one vial.  The 15 from adl-16  tsl  for  10 died in 5-8  Furthermore, the two from the cross containing adl-16  containing the mutant gene).  usually  , died as expected  mutant flies of the appropriate genotype e.g. adl-16 days.  not  ts2  were  chromosome parent  type,  149 Fertility of Deficiency/mutant at 2 2 ° C  Df/adl-16  was fertile, producing 15 female and 10 male offspring.  tsl  Whether  the  females  were  determined.  The males were  Dfladl-16  was  ts2  FM7IDf No The  D//FM7 tsl  also fertile, producing the following offspring:  flies of type Df/Y  lrdI  females and 23 adl-16™ IY  ts2  2  males.  were produced from either of these crosses.  FM7ladl-16 . ts2  was not fertile.  No mating was seen, no eggs were  present, and no larvae were seen even genotype  9  could be distinguished in this case by vermilion eyes  being present in  Dfladl-16f  was not  tsl  adl-16 IY  females, 26 FM7/adl-16  females  or F M71 adl-16  were repeatedly tested  though these flies  and examined.  Over 90% of the control flies of type mutantlFM7  and  were alive at the end of the above experiments i.e. excess of 60 days.  of this  adl-16  tsl&2  90% lived in  Fifty percent of control stock of type  adl-16f  lrdI  were alive on day 50.  General Observations at 2 9 ° C . Df/adl-16  tsl  died rapidly at 2 9 ° C .  By day 2 swollen abdomens and  proboscis extension were noted, and by day 4 all flies Flies of type Df/adl- 16  ts2  day three.  were dead.  were paralysed after 24 hours, and dead by  A l l Df/adl- 16f held lrdl  their wings up, and died extremely  rapidly  at  29  °C.  Most  were  remainder were dead by day 2.  dead  within  24  hours,  and  Controls of type FM7/mutant  the  lived  in excess of 30 days at 2 9 ° C , and flies of the three mutant alleles died according to their expected lifespans.  Viability and Development at 2 9 ° C  No flies of type Dflmutant developed at 2 9 ° C .  Although larvae were  seen in all vials, no pupae developed. This was expected because the temperature  sensitive  instar for adl-16 , (Homyk  16 /Y, produced. and  14  et al,  1986).  males  FM7ladl-16  and 6  embryo  and 16 FM7ladl-16"  with adl-16 ,  In the case of  1  IFM7,  to  were males lraI  of  eclosed,  two  hours 39/60  after  continuous  six  heat,  14  adl-16" IFM7 2  adults eclosed,  hours  13/20  7/80  eclosed  FM7IY  survived.  it was found that  ts2  exposed  adl-  adl-16f  No flies of type DflY  From the pre-pupal heat pulse tests of adl-16 ,  22/40  with  In total, 28 flies of type  which eclosed.  lraI  hours,  and adl-  lower numbers of progeny were produced, 4  FM7/adl-16f^di.  pulse  larval  11 FM7IY  ts2  females eclosed.  ts2  and 6 adl-16f /FM7  a heat  second  females  1  eclosed, in addition there were 16 adl-16"  after  to  From the cross DflFM7  From the cross DflFM7  crossed with DflFM7 FM7/Y,  are  ts2  13 FM7IY  ts]  phases  and larval instar 2-3 for both adl-16 ,  tsl  16flrdl  lethal  eclosed.  four  Of those  alive,  immediately, a further 21/80 flies were parity eclosed.  after  two  died  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  shi ,  but  22°C  none  were  developed  found.  normally.  The It  20  appears  pre-pupal that  larvae  shifting  left  adl-16  at to  ts2  ts  the  restrictive temperature is detrimental at all stages of life.  Comparison of Homozygous and Hemizygous Flies  In  order to  hemizygous  compare flies  homozygous  either male  the following six Tables (3-3  flies  each  allele  type  or Df/mutant, and heterozygous to 3-8)  tables is pooled data from deficiency with each cross.  of  The lifespan  are given.  with flies,  Data given in the  crosses, and the controls  used  parameters for mutant strains were  calculated separately for Chapter 3, but are quite similar to those in Chapter  1,  responses.  where This  tests had little  many provides  effect  flies  had  been  support for  on longevity.  the  tested  for  position  Table 3-9  behavioural  that  shows  behaviour conversion  factors for the rate of living between results for Df/mutant at 2 2 ° C and 2 9 ° 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  position,  determined  position,  Bands  13Fi to 14Bi correspond.  para  at 53.9 and shi  2).  ts  ts  52.9,  and the  The adl-16  ts  cytological  gene is located between  at 52.2 on the X-chromosome (see  Figure 3-  Figure  3-2  COMPARISON O F GENETIC A N D C Y T O L O G I C A L L O C A T I O N O F adl-16^ GENE Cytological map adapted from Handbook of Genetics, E d . , R . C . King, 1975.  sJL '  So  <  sk  1  &.f SI  SX>*\  u  5-2 X - C\\ ycv^ o S c  £3  T A B L E 3-3  adl- 16&Lt'Deficiency Compared with  fl<j/-76^-Homozygote  and Hemizygote at 2 2 ° C  adl-16" /  adl-16" / 1  adl-16" /  adl-16" /  adl-16" /  Deficiency  adl-16"  1  Y  adl-16"  adl-16fl di  1  1  1  2  1  r  48.9  91.8  80.8  99.2  81.9  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  LS  X  DP  X  154  T A B L E 3-4  adl-16^-1 Deficiency  Compared with a(j/-7f3& -Homozygote 2  and Hemizygote at 2 2 ° C  adl!6ts2/  adl-16^ /  adl-16 l  adl-16" /  adl-16" /  Deficiency  adl-16  Y  adl-16  adl-16f  2  ts2  ts2  2  tsl  2  lrdI  30.5  74  65  99.2  111  27.0  77  67  102.7  111  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  LS  X  DP  X  PJP nset 0  155  T A B L E 3-5  adl-168*411 Deficiency  Compared with a<i/-76ff£<&-Homozy gote  and Hemizygote at 2 2 ° C  >  adl-16f^  adl-16fl <H r  adl-16f  adl-16fl  /Deficiency  Iadlll6f  lrdl  IY  ladl-16  rdI  lrdI  rdI  tsl  adl-16"  2  1adl-16f  LS  X  4.1  53  54  81.9  111  DP  X  4.0  59  58  86.1  112  DPonset  2.5  33  37  61  80  D-20  2.9  36  70.5  103  36 " "  '  —  •  "  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  61.1  2.0  2.0  2.5  2.1  DPslope  0  lrdI  156  T A B L E 3-6  adl-'Deficiency  Compared with aci/-76^-Homozygote and Hemizygote at 2 9 ° C  adl-16" /  adl-16" / 1  adl-16" /  adl-16" /  adl-16" /  Deficiency  adl-16"  1  Y  adl-16"  adl-16^1  1  LS  1  1  2  1  3.7  9.1  8.6  6.5  9.7  3.65  9.3  8.4  6.4  9.3  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  46.5  22.7  19.8  19.2  6.7  X  DP  X  DPonset  DPslope  157  T A B L E 3-7  adl-16^2-/Deficiency  Compared with  adl-7r5£s -Homozygote 2  and Hemizygote at 2 9 ° C  adl-16" /  adl-16" / 2  adl-16" /  adl-16" /  adl-16" /  Deficiency  adl-16"  2  Y  adl-16"  adl-16ftrdi  2  2  2  LS  X  2.3  3.3  2.9  6.5  DP  X  2.2  3.3  2.9  6.4  2  1  7.5 7.2 -  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  DPonset  158  T A B L E  adl-16fkdLlDeficiency  3-8  Compared with arf/-/6fffl2LHomozvgote  and Hemizygote at 2 9 ° C  L S D P  X  X  DPonset D-20  adl-16flrdl  adl-16f  IDeficieny  ladl-16f  lrdI  lrdI  adl-16flrdl  adl-16fl  IY  /adl-16"  rdI  1  adl-16" / 2  adl-16flrdi  1.15  14.3  11.3  9.7  7.5  1  14.7  11.7  9.3  7.2  .5  9.8  7.3  1  4  0.5  8.9  8.8  6.7  5.3  8.7  7  15.3  10.7  1  D - 5 0  0.6  13.3  1 2  D-95  1.55  18.9  16  DPend  1  D - 1 0 0  2  23.8  110  9.0  DPsloDe  1 8  15.7  1 5  10  2 0  2 2  1 4  10  6.7  15.3  Table  3-9  CONVERSION FACTORS FOR DIFFERENCES IN R A T E OF LIVING OF Df/MUTANT at 22°C and 2 9 ° C Dfladl-16fl <U Dfladl-I6 Df/adl-16 tsl  LS DP  13.2 12.9 16.0 14.3 14.2 13.4 15 11.8 13.9±1.2  X  X  DPonset D-20 D-50 D-95 DPend D-100 Average  r  ts2  13.7 12.3 13 14.8 17.4 17.0 17.0 13.9 14.9±1.9  3.6 4.0 5.0 5.8 5.5 2.9 4.0 3.3 4.3±1.0  1  Each number represents the ratio of the respective 2 2 ° C values for longevity. 22°C. for  One 2 9 ° C day for Df/adl-16  One 2 9 ° C day for adl-16  Df/adl-16  ts2  and 2 9 ° C  tsl  = 13.9 days at  = 14.9 days at 2 2 ° C .  One 2 9 ° C day  = 4.2 days at 2 2 ° C .  flrdI  Bearing in mind that the survival of all mutants/Deficiency severely  reduced to  begin  with,  curves of each strain of Df/mutant  the  following  compares  were survival  at the two temperatures used.  Figures used in Chapter 1 were used to make these comparisons. the case of adl-16 10.2 for males. For adl-16 , ts2  tsl  the conversion factor was 10.9 for females, and  In the hemizygote (Deficiency/adl-  of  16 ) tsl  it was 13.9.  the conversion factors were 23.1 for females, and 23.5  for males, and for the Df hybrid it was 14.9. degree  In  compression,  but  the  Df/adl-16  ts2  This represents a lower l i f e s p a n was  very  reduced at 2 2 ° C .  For adl-16f  conversion factors of 3.9  lrdI  and 5.0  were found in stock flies, but a factor of 4.3 for Dfladl-16f .  Of all  lrdI  the alleles, adl-16f  was the most (drastically) affected by being in  lrdI  the  hemizygous  tolerated  state.  increase  of  Although the temperature  the hemizygoteIDeficiency temperatures.  better  died  parent  strain  than either  exceedingly  (adl-16f ) lrdI  adl-16  rapidly  tsl  at  and so  the  curtailment of  Nevertheless in  the  these  males  2  ,  both  smaller  conversion  factors  reflect  quite  even at drastic  lifespan.  the effects of dosage compensation can be clearly seen of the parent stock,  X-linked  homozygous  r  It should also be pointed out that this strain had a  lifespan reduced by approximately 50% relative to Oregon-R 22°C,  o  genes,  females.  but  in  all  which are also hemizygotes respects  are  very  similar  for to  Figure 3-3  DEFICIENCY/adl-16 MUTANTS Survival Curves at 29°C  Age (Days)  DISCUSSION  Complementation data between the three putative alleles of  adl-16  seems to indicate that the three are all hypomorphic alleles of locus, if hybrid data alone are considered.  From this data  would appear to be the most severely affected and  adl-16f  the  lrdI  temperature  change  In round figures,  least  from  affected  the  permissive  the differences  reductions in lifespan.  allele  were  with  expected  is  allele  the  and  3.3-fold  Survival curves for hybrids are intermediate and so are viabilities.  each  to  temperature.  10-fold, 5-fold,  between pairs of alleles, if  ts2  next,  tsl  respect  to restrictive  one  adl-16  allele, adl-16  ts  hypomorphic and  This is exactly the  as  hybrids produce  intermediate quantities of the gene product from that locus.  However the  deficiency  data produce some surprising, and at first  glance, inexplicable results. viability  curves  which  Dfladl-16  would  Each  shows a more severely  than  does  Dfladl-16  tsl  the  corresponding  tsl  and Dfl' adl-16  be expected  of  curtailed lifespan homozygote  of  being longer than that for Dfladl-16 . ts2  ts2  each have  hypomorphic over the the  allele,  alleles.  deficiency that  for  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-16f which  behaves  lrdI  affected  at at 2 9 ° C  reduced  lifespan  differently.  This  allele  was  but the Df/adl-16f resulted  in  lrdI  of  all  at  both  the  temperatures.  least  the  most  Although  not  surprising at 2 2 ° C , because this allele has the shortest lifespan at that temperature, it was expected that at 2 9 ° C Df/adl-16f  would  lraI  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  previous  case,  assumptions  mutations  of  considered  these  and  hypermorph, expected  the  it  regarding  three  loci.  discarded.  since  became  For  the  necessary  to  allelism  and  Several  to yield a lifespan  closer  the  possibilities  instance,  in combination with  re-examine  of  briefly  cannot be a  lraI  a deficiency,  to the wild-type  types  were  adl-16f  the  it would  be  than would a  homozygote.  Another possibility might be the site of mutation (hence the type of mutation) differing in these alleles.  For instance, adl-16  which  tsl&2  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 thermolability, which causes the differences  in results.  induced mutations are of this type, as mentioned earlier. though,  may  different  Most E M S Mutations,  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 gene.  or insertions  upstream from  the  5' end of the structural  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 a d l - 1 6  flrdI  - or of course, the reverse is also possible.  Another possibility, that of a second site mutation near  adl-16f  lrdI  could not explain these results since some effect would be observed in the homozygote.  The possibility does exist that a second  mutation exists which is highly dosage hypomorphic the  second  effect.  and when  adl- 16f  lrdI  is  sensitive,  that is, it is  homozygous  site product is not sufficient  site also  the reduction in  to produce a discernable  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 a c i / - / 6 ' / ' ^ p r o d u c t r  /  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-16f  lrdI  with the product  other two of  which  consequences. and questions  adl-16f  lrdI  mutants, but is may fall  below  is not in fact allelic  actually a separate a certain level  locus,  without  the  severe  The following discussion examines the data presented whether this hypothesis is reasonable.  has shown a number of differences  (perhaps) alleles.  from the other two  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. differently.  To demonstrate this the graphs can be plotted  If the age (days) of final death is plotted on the abscissa,  and temperature on the ordinate, we find that the homozygotes adl-16  have similar slope, and are to the right of  tsl&2  which differs in two ways.  First the  slope  of  adl-16f  lrdI  is much greater, and  second, this curve clearly crosses the other two.  A similar graph  results with the Dflmutant,  shifted to lie to  but with Df/adl-16f  the left of the other two hybrid curves. which is difficult to explain. has  lrdI  It is this shift in position  The regular survival curve of  adl-16f  lrdI  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 but clearly it affects  temperature has little effect on death rate,  adl-16f .  Perhaps corresponding to  lrdI  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. sudden lethal effect when adl-16f  lrdI  for the region.  adl-16f  lrdI  also  showed  This would account for the is combined with a deficiency 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. structural  gene  and the  represent different If adl-16f  The thought occurs that this gene may be a two  regulatory, if,  indeed  they  do  loci.  and adl-16  lrdI  other  are really separate loci, then how could  tsl&2  their apparent failure to complement be explained?  One explanation  is  which  that  of  strong  interaction  between  the  loci,  interdependant function, such that heterozygotes will cause a reduction in viability. from shi nerve  to para  ts  ts  function.  have  of any two of them  I strongly suspect that the region  may represent a structural gene family related to There  seem  phenotypes these genes produce.  to  be  many  similarities  in  1972,  the  Much other evidence in the papers  written about these genes points to nerve involvement (Grigliatti al.,  an  1973; Suzuki et al., 1971; Poodry et al.,  et  1973;  Ganetzky,  temperature  sensitive,  1984).  In very simple terms, adl-16 while  adl-16f  lrdI  merely  examples  is of  mutants,  and no  however,  to speculate  organs  the for  the  most  is  the  most  dose sensitive.  pleiotropy  further explanation  nerve involvement, system,  the  ts2  of is  these  Perhaps these are temperature  needed.  It  which tissue these genes affect.  is  sensitive interesting  With clear  this could be the central or peripheral nervous  junctions movement),  between  nerves  and  or muscular tissue.  muscles,  (the  affector  Mosaic analysis,  the  results of which are discussed in Chapter 4, has been used to attempt to resolve this issue.  CHAPTER 4 INTRODUCTION  Classical genetic recombination methods  allow precise  of the location of a gene on a chromosome.  determination  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. more difficult to to identify the focus  However it is  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 reported in of  mapping. D.  It involves the use of gynandromorphs, first  melanogaster  by T . H . Morgan in 1914.  Early studies  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-16 ,  the focus might be a particular region of the central nervous a single influences  cell,  or even  a distant  organ, the  behaviour only indirectly.  malfunction of  ts2  system, which  Behavioural responses may be  disrupted by altering the ability of the individual to detect stimuli,  (defective  sensory  receptors)  to  transmit  and  process  information, (peripheral sensory, or central nervous system or to  adequately  respond to the  stimulus, (altered  neuromuscular junctions, or muscular system). the  above  may  be  Temperature sensitive  involved,  as  single,  Indeed, any or all of or  pleiotropic to  Several other adl  been shown to have pleiotropic effects  defects),  motor neurons,  lethal genes would be expected  an even wider range of possibilities.  the  (Homyk  effects.  encompass  mutants have  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  ( X X ) and part  mosaic  individuals which  mutant (XO).  are  part  wild-type  They are most easily generated by using unstable ring-  X chromosomes, designated X r , which are frequently lost very early in the development of a fruitfly (Patterson, 1933; Kankel,  1976).  Hall, Gelbart, and  The resultant individual is composed of some  cells  bearing a full complement of chromosomes, and others which lack the  X r chromosome  chromosomes expressed  bears  (an the  XXr/XO mutant  gynander). gene,  then  in X O tissues, but not in the cells  If this  the  normal  gene  will  be  containing the ring  chromosome, which are wild-type for the gene of interest, marker genes.  X  and for  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.  penetrant,  A  mutation  or produces  in  which  a diffusible  the  gene  is  either  partly  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. the  first  nuclear  division  spindle  is  oriented  In  Drosophila,  arbitrarily  in  space  (Parks, 1936). The first nine nuclear divisions occur in a syncytiumlike cluster without membranes being laid down.  Within this cluster,  very little mixing apparently occurs in that the relative positions the nuclei during the syncytial divisions cell surface tend to be maintained. divisions occur.  migrate  to  the  surface  of  and their migration to the  After the nuclei from these nine of  Only then do cell membranes  the  egg  three  more  divisions  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  orientation of the first spindle.  is  different,  depending  on  Once the blastoderm is formed, the  surface site occupied by a cell largely decides its fate (Geigy, Howland and Child, Gehring,  1935;  Hathaway and Selman,  1961;  1931;  Chan and  1971).  Calculations based on correlation between wild-type cuticle and  the  mutant  cuticle  (male),  can  be  used  to  construct  (female), a  two-  dimensional relative  fate  map  positions  of  of  the  blastoderm  presumptive  adult  surface  indicating  structures.  Garcia-Bellido  and Merriam (1969) used 379 mosaics of D. simulans self  consistent  fate  map for adult  surface  fate  drop-dead  ts focus can be located on such a map.  were  ten  recessive  temperature  corresponding  Most importantly a mutant  behavioural mutants,  sensitive.  adult structures  and examined.  the  non-ring  the  of  to "locate  the  behavioural, or Homyk, in 1977  which at least  location  of  which are affected  Thus it may be possible  tissue in which the adl-16  If  From  Hotta and  behaviour" by constructing  embryonic  mapped  maps.  to construct a  structures.  Benzer, (1974) have demonstrated that it is possible anatomical site of abnormalities affecting  the  two  such foci, can  be  the  deduced  to determine the type of  gene exerts its primary effect.  chromosome  in  a  gynandromorph is  chromosome, bearing the gene of interest  a marked  flanked by markers then,  tissue containing this gene can be inferred by the presence  of the  markers.  occurs  Because  loss  of  the ring-X  chromosome  usually  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 X X r . 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, behaviour.  as well as body parts which appear not to Temperature sensitive lethality  would  be  influence  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. that  distance,  analogous  to  many the  mosaics  construction  are of  examined. the  Fate  one-dimensional  To determine mapping map of  is 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  triangulation. measured  two  in  the  Extension by two,  map that corresponds  same of  way,  this  and to  map  drawn  by  other  landmarks,  leads to construction of a two  dimensional  to the relative  process  a  positions  of the  sites on the  blastula surface giving rise to these adult landmarks.  Sturtevant showing  (1929) using D. the  embryonic  simulans  relationships  imaginal tergites and sternites. Hotta and Benzer in 1972  703  established the first fate map of  precursor  cells  for  the  gynandromorphs were used by  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 tissues  have been used in many studies  which were  wild-type  required  to  behaviour, and also  be  to determine the  wild-type in order to produce  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 to subsequent  stages), but also the tissues which apparently directed  each behaviour. other  steps  at one stage would not progress  In some  required  cases the brain  thoracic  tissue  to  was be  normal,  depended on the genotype of the genital regions. (1972)  determined  including  wingsup  the  foci  for  a  number of  and Hyperkinetic.  the key structure, still  others  Hotta and Benzer behaviour  mutants  Flanagan (1977) determined  the primary focus for the lethal mutant  doomed.  MATERIALS A N D METHODS The allele I decided to map is adl-16 .  The reason is that it is the  ts2  most severe of those under study; adl-16  ts2  flies  showing paralysis in  less than 24 hours at 2 9 ° C , followed by death a few days later. consequence,  faster  preparation  chromosome was possible. response  of  the  necessary  As a  recombinant-X  Also a simple paralysis and/or death type  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-16 ,  using  ts2  chromosome,  classical  recombination  In(l)w  as the ring-X  vC  methods  were  employed  link the chitin markers for yellow body color (y),  genetically  eyes (w), and forked bristles (f )  onto the chromosome  36a  adl-16 .  to  white  containing  Cuticular areas containing all three of these markers were  ts2  assumed to carry adl-16 ,  since they flanked the gene of  ts2  interest  which was present in the marked stock.  Stock flies  containing adl-16  crossveinless wings, adl-16 f.  and extremely  available, scoring  and  to  mosaics.  phenotypic  Utah.  3 6 3  bristles,  vermilion eyes and forked bristles, i.e. sc cv v white  forked bristles, to correspond with ring-X avoid  vermilion, which  Autonomy  reflection  of  a  in  is  not  Drosophila  tissue's  surrounded by cells of a different forked  scute  It was decided to use the markers yellow (body),  ts2  eye,  carried markers for  ts2  genotype.  autonomous, is  genotype  stock  in  defined spite  of  A strain of  by  yellow,  was obtained from Kathy McElwain from the University of  Yellow, white  flies  2)  f36a/ f36a  y  the  being  were available  in our laboratory. The  following crosses were made: 1)  for  <g>  y  yf36a/  sc  adl-16^ f 2  c  v  v  <g>  sc cv v adl-16^ f/Y 2  yf /Y 36a  Among  progeny,  sc cv v (?adl-16  contain the gene of interest.  )f IY  ts2  recombinants  36a  might  These were crossed as below, then the  male was removed after 5 days and tested at 2 9 ° C .  If paralysis and  death occurred as expected, those lines were continued. 3)  sc cv v (?adl-16" )f IY 2  ®  36a  sc cv v adl-16" f /sc cv v adl-16 f 2  ts2  Self-cross the FI A)sc cv v (?adl-16 > f "lsc ts2  cv v adl-16" f®  36  2  sc cv v  (?adl-16 )f ts2  Five days after the cross was made the male was tested at 2 9 ° C . adl-16  ts2  was  present,  this  line  was  Ifits2f36a males and females expanded.  36a  If  continued and sc cv v adlThe line was tested to check  that the gene of interest was present. 5)  sc cv v adl-16" f36a/Y  6)  sc cv v adl-16 f ly  <g>  2  ts2  w  36a  y w ly w  <8>  y w IY  Among the progeny, recombinants of the type y w (?adl-16 )f l ts2  were isolated and crossed with y wly w females.  36a  Y  After mating for 5  days the males were tested at 2 9 ° C , and if found to be adl-16" > the 2  line was continued. 7)  y w (?adl-16 )f  8)  yw(?adl-16 )f ly  ts2  Lines of y w  ts2  36a  w  36a  (?adl-16 )f ts2  cross were tested at 2 9 ° C .  ®  y w ly w  ®  y w  (?adl-16" )f ^IY 2  males and females  36a  Those containing adl-16  and used for the mosaic cross.  ts2  from  3  the above  were expanded  Originally, I intended to use the lab stock of In( 1 )w lywspl <8> vC  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  v adl-16 f  sc  chromosomes  cv  v adl-16  ts2  f  3 6 a  and y  were prepared, it was  ts2  marked  36a  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 y  2  adl-16 y  2  cho  2  f chromosome was also constructed but I found the mutant  ts2  difficult  to  differentiate  smaller patches  from  of mosaic tissue,  wild-type  color  -  especially  in  so this prepared chromosome was  not used.  The female parents in the mosaic generating cross had the unstable ring-X  chromosome,  chromosome:  R(1 )w  vC  maintained  over  a  balancer  In( 1 )w CI In( 1 )dl49, y w lz 1(2) <8> A 49 y w lz/ y+Y v  s  Obtained from Jeff Hall of Brandeis University (with grateful thanks). Virgin ring-X females were crossed to y w adl-16 to control males of y w  fi I 6a  ts2  f /Y  males, or  36a  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,  autonomously.  and white Mosaics  of  are known to In(l)w ,+ vC  express  + + +/y  w  their adl f 2  phenotype 3 6 a  were  examined and the exact area of yellow, white, forked tissue recorded  on  a detailed diagram of the fly, (see  mosaic  flies  raised continuously  cuticular phenotypes with  the  yellow.  examined.  wild-type  Appendices ii and iii).  at 2 2 ° C ,  were collected  A diagram was  tissue coded  red, and the  233  and their  made of each mutant tissue  fly,  coded  These flies were kept at 2 2 ° in vials for one to three days,  during  which  scoring was  performed, and behaviour noted.  were then shifted to 2 9 ° C .  They  Thereafter, their behaviour and survival  were monitored daily.  Survivors were transferred to fresh medium  at two  Transferring  day  intervals.  these flies  within  a 24  hour  period after eclosion, and scoring them all in that time period, proved impossible. adl-16  This problem was circumvented by testing  a bottle  mixed age flies and larvae at 2 9 ° C . The bottle was  ts2  of  shifted  from 2 2 ° C to 2 9 ° C , and it was noted that flies of all ages died equally quickly. This confirmed a casual earlier observation that adult 16  of  ts2  any  age  temperature. transferred days, the  equally  quickly  affected  by  the  restrictive  100 non-mosaic ring-X females were shifted to  fresh  as controls. mosaic  are  vials  every  second  day  adl-  to  and observed  29°C, for  32  Of the 233 mosaic flies collected, 225 were used in  analysis.  This  mapping.  represents  in  the  included  in  the  because of abnormalities such as a missing them  might  have  effected  impossible  their  to  behaviour  score or  not  used  calculations  made  were  sides  for  have  flies  fly  calculations  would  Eight  450  leg,  accurately,  lifespan.  The  which  or which 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 blastoderm cells  =  # of mosaics in which structures differ v i n o total # of mosaics scored  A B (sturts) =  # of times A and B are different total times A and B are scored  x 100  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 male, and the other half, female. was  present  in  the  data  fly will  be  To test whether a 50% maleness  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 case,  the  different  genotypes,  calculation  for  rather  sturt  than  distances  different listed  sexes.  above  In  would  this 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)w l  In(l)dl49,  vC  y w lz  s  ® A 49 y w Iz I y  numbers of flies scored. 233/988  or 23.58%  percentage mosaics  which  Y. They are listed in Table 4-1,  +  Of the In(l)  showed  is  only  w /y vC  external obtained  w adl-16  mosaicism. if  selection  ts2  This of  f  36a  is  with flies, a high  the  strain  for  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) any  mobile  body  part.  In  fact  tissue and paralysis at 2 9 ° C after  paralysis  was  noted,  for re-  examination of a fly revealed a tiny patch of mutant tissue in a few cases.  Table Cross: In(l)  Progeny  4-1  w^Cn (l)dl49 y w Iz* ® y w adl-16 f /Y females males ts2  Genotype  Number  v C  t s 2  y  7.9%  755  26.29%  1534  52.04%  89  3.02%  317  10.75%  2948  100  3 6 a  w  non-mosaic females, (controls) In(l)dl 49y w lz /y w a d l - 1 6 yellow white females y w adl-16ts2f3 6a s  ts2  yellow white forked (XO) males In(l) vC/Y Ring-X chromosome males Total  Percent  233  In(l)w /y w adl-16 f mosaic females In(l) vC/ adl-16ts2f3 6a W  36a  n  W  Of the 100 control mosaics placed at 2 9 ° C , 93% lived beyond day 10. No  behavoural  paralysis.  abnormalities  On day 20,  76%  were  observed;  of controls  were  in  particular  no  still alive, with no  paralysis visible, on day 32, 56% of the controls were alive, and some abnormal  behaviour  was  noted.  Since  the  adl-16  ts2  mutant  expresses its phenotype prior to day 4, which was used as a cut-off for  observation  of  both  As can be seen in the survival curve in Figure 4-1 for  and 85.3% by day 10. adl-2  by Homyk et al.,  ts2  die  before  their  paralysis  counterparts.  adl-16 ,  flies  and  clearly  containing  mutant  (drop-dead),  phenotypes,  mosaics  the  lethality  control  74.7% (168/225) had died by day 4,  The curve is reminiscent of that shown for 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 2 9 ° C became paralysed within 24 hours.  This  assisted as a double check for scoring, since in a few instances early in the experiment, re-examined was  found.  after paralysis was  and almost  noted, the part involved  invariably a small patch of  Body parts involved were: wings,  mutant  was  tissue  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, attempts were made  the fly could not stand, even though  continually.  FIGURE 4-1 % Mosaics Alive or Paralyzed  120  Days at 29°C  Survival and Leg Paralysis of adl-16  MAPPING O F adl-16"  The  drop-dead  were  mapped  ts2  mosaics at 2 9 ° C .  2  behaviour and the paralysis of the legs of the separately.  The  first  analysis  resulted  in  fly 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,  Abbreviations  2,  or 3),  and the  for these structures  third  abdominal  are given  sternite  in parentheses.  (Stn.3). Then  drop-dead  behaviour  background.  of  adl-16  ts2  was  mapped  against  this  Finally the paralysis of each leg was mapped separately  against the leg and its three closest external markers.  BACKGROUND MAP  Table 4-2 body  shows the results of the fate map analysis  structures  scored.  The  distances  observed  are  for the  nine  comparable,  though not identical to those obtained by Hotta and Benzer (1972). The  map obtained  expected,  it  is  from these values is  similar to  studied are mapped against  their  shown  in Figure 4-1.  background map.  this background.  As  A l l behaviours  185  Table 4-3 Distances Between Landmarks (to nearest Sturt) Midline 14 1 1 9 13  Ant. Oc.Br. Pro. Hum. P.St.  Co.l Co.2 Co.3 Stn.3  18 21 17 20  The  Ant  Oc.Br Pro. [Hum.  P.St.  8 15 25 31 27 33 37 47  19 29 34 28 38 40 49  12  15 22 21 30 32 39  18 22 28 26 [30  17 16 25  Co.l  Co.2  Co.3  14 19 32  11 25  20  Stn.3  validity of the analysis depends upon the assumption that all  parts are equally likely to be effected. The  degree  of maleness  To address this assumption,  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  experiment In(l) over  9  external  the  w  v C  ring-X  /In(l)  structural  stock  from  dl49,y w l z  s  Brandeis used gave  in  this  49.93 % maleness  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  Drop-dead  Table 4-2 Behaviour Mapped to 9 Landmarks Drop-dead  Landmark  29 Oc. Br. 25 Ant. 26 Pro. 19 Pst. 25 Hu. 17 Co.l Co.2 19 20 Co.3 30 Stn.3 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 T H E NINE STRUCTURAL L A N D M A R K S USED Landmark Antenna Proboscis Oc.Bristle Pre.St.Br. Coxa 1 Coxa 2 Coxa 3 Humerus Stn. 3 Total  # scored 450 450 450 450 450 450 450 450 450 4050  #male  % male  207.5 229 214 233 232 243 241.5 219 205 2024  exp# male  41.6 50.8 47.5 51.7 51.5 54.0 53.6 48.7 45.5 449.4  X (0-E) /E 2  225 225 225 225 225 225 225 225 225 625  1  2  0.16 0.17 0.54 0.28 0.22 1.44 1.21 0.16 1.76 5.86  To know if this is less than expected  at error probability of 5%, 8  degrees of freedom gives a X =15.507,  which is above 5.86.  2  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 to the head, thorax, or abdomen. mutant  or entirely  wild-type  mosaics  in which  the heads  mutant  {drop-dead)  behaviour.  Mosaics which had either entirely  heads  were  behaviour,  mutant,  and four  O f the 51 47  showed  showed  wild-type  O f the 58 flies whose heads were entirely wild-type, 24  in which  behaviour differ. sturts.  examined.  were completely  showed mutant behaviour and 34 wild-type. mosaics  focus is nearest  This gives a total of 28  the genotype of the head as a whole,  and the  The map distance calculated for these data is 26  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 Head. Thorax, and Abdomen. Major Division  Genotype (N)  Behaviour  Head (109)  Mutant (51)  28/109=25.68  Wild Type (58)  Mutant Wild Type Mutant Wild Type Mutant Wild Type Mutant Wild Type Mutant Wild Type Mutant Wild Type  Thorax  Sturts  (51)  8/51 = 15.68  Sturts  Mutant (29) Wild Type (22)  Abdomen  (67)  Mutant (21)  22/67=32.83  Sturts  Wild Type (46)  Focus.  Number 47 4 24 34 25 4 4 1 8 17 4 18 28  Table  4-5  shows  the  results  behaviour of the adl-16ts2 Figure 4-3.  mesodermal Figure  4-3.  the  analysis  of  mutant. The drop-dead  the  drop-dead  focus is shown in  This behaviour appears to map in the ventral thoracic  region, in a vaguely mesoderm.  of  defined  Figure 4-4 and  position which most  shows the expected  nervous  tissue  (dotted  likely  positions lines),  represents  of presumptive  superimposed  on  <r  Figure 4-3  Fate Map of  drop-dead  Scale: 1 Sturt = 2 mm. Distances'between  Behaviour.  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. approximately bilateral mutants,  11 died in less than five  remainder died prior to 10 days. &dl-2  ts  presented  possibility  by  days,  the  This is reminiscent of the data for  Homyk et al. (1986).  Thus, there existed the  that there were two behavioural foci, only one of which  must be mutant to kill  the individual.  domineering  focus  Accordingly,  behaviour  domineering presumed  Of 14  as  foci.  was  focus  Hotta  landmark  and  using  their  approach  the  from the landmark to the focus,  the  three  closest  for  and  the  four  (1972).  the  used:  (the  Benzer  to  were  the distance  by  analysed  For each  thoracic  presutural bristle)  outlined  This is the definition of a  coxae  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 have  a domineering  relationship  is  correct.  This is  foci  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.  although it initially appeared that there were two domineering this was not the case.  Thus foci,  Table 4-6 Analysis of Four Landmarks for Domineering Foci (After Hotta and Benzer Distance to Midline Distance Between Foci Distance to Focus 39 31 28 31  18 20 18 19 Coxa 1 21 21 Coxa 2 17 23 Coxa 3 Note: Distances are given in sturts. P.St.  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? of  the data showed  bilaterally,  death  that even  in mosaics  A closer examination  which were  not divided  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 1 2  4 4 11  3 4 5 6  45 17 15 65  23 13 12 14  27 17 23 59 18 15 66  15% 24% 48% 76% 94% 100% 99%  1 0  MULTIPLE FOCUS  This led to the hypothesis paralysis  and  subsequent  that adl-16ts2 starving  or the  body functions due to internal paralysis.  shows early death due to cessation  of  other  vital  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. entire head was mutant.  In the first of these, the  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 cause for death. 16"  1  with respect  to the involvement  of probosci as a  It will be recalled, however that flies of type  lived several days with extended probosci (Chapter 1).  adl-  During examination of the flies it was noted that the paralysis of each leg  was  largely  hyperkinetic,  independent  of  the  other  legs  (like  the  mutant  in which shaking of each leg was independent of that of  the other legs; Hotta and Benzer, 1972), and like the mutants l ,  adl-  rex, and sesE, analysed by Homyk et al. (1986), therefore it was  ts]  decided to map the paralysis of each leg separately.  If the paralysis  is independent in each leg, a separate focus should be each.  obtained for  Table 4-8 shows the results of the maps. Table 4-8 Map Data for Paralysis of Legs 1. 2. and 3.  Paralysis Leg 1 Leg 2 Leg 3  Distance to Nearest Sturt Coxa 1 Coxa 2 Coxa 3  P.St.  1 1 8 14  14 16 19  6 12 19  20 13 7  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  Nevertheless  less distinct,  and that for leg  3 relatively  diffuse.  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-16  occurred  ts2  when adults of any age were placed at 2 9 ° C indicating a continuous requirement  for  observation).  the  normal  gene  product in  the  adult  (personal  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  important for the normal metabolism in the fly. tissues affected analysis  was  by the  expression  completed.  It  To determine the  of the adl-16  was  a protein  hypothesized  gene,  ts2  that  mosaic  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  bisecting two points on the three-dimensional surface of the  plane 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  antero-posterior  and dorso-ventral axes can be decided from  to  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  is  egg  estimated  an  by  obtained  oval  shape,  triangulation  mathematically  rather than agree  where  fairly  long  a circle.  The  distances  well  the  distances  distances  with 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-16  ts2  represents  in the  ventral thorax, in an area which  mesodermal  tissue.  The paralytic effect  of  most the  likely 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? foci  If two sets of  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,  paralysis of each limb.  and  another  set  for  the  individual  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 mutant. tissue,  when  a large enough  This idea arose because  proportion  of  wings  angles and  these were never moved back into a more normal position. probosci were found to be permanently extended.  centre,  1971).  in  indicating that  Unfortunately  a  Flies with  walked in circles, with the wild-type eye  Flies with affected  abdomen  Affected  Also some partly  mutant probosci were bent towards the affected region.  the  normal  it was  the  other  eye  was  defective  towards (Benzer,  abdomens were unable to hold up the  position,  often  is  For example  were observed sticking out at abnormal  one mutant eye  fly  of the observation that the male  in almost all cases, appeared to be paralyzed.  affected  the  rather  not possible  they to  dragged  score  the  it  along.  presence  or  absence of paralysis in these structures because mosaics which died early  were  frequently  found  medium by surface tension. typical  pose  of  adl-16  ts2  on  their  backs,  held  to  the  moist  It may be recalled that this is the most 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  subsequent  hypothesis  -  that  paralysis  of  the  mutant,  starvation or cessation of other body functions  actually kills the flies,  - is correct, then the drop-dead  ought to map equally distantly from all body parts.  and  is  what  behaviour 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, effective  in contributing to the death  assumption  no  Hall, Gelbart and Kankel,  1976;  Homyk,  1977;  utilized gynandromorph analyses  and  all  cases  the  results  have  studies  The first  Garcia-Bellido and Merriam, 1969; 1978;) have  Many  organism.  1977;  Hall,  problem.  of the  (Flanagan,  in  presents  and that each part is equally  been  shown  Confirmation of damaged tissues of the expected the tissue of choice for the focus in their studies.  to  be  valid.  type has supported 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 some parts of  the  fly  are much more vital than others,  that  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 modify  the  artefact  hypothesis  somewhat;  to  adding that certain parts  of the fly were more critical than others in determining whether or not the individual would die. scoring  both  because  the  Paralysis of the legs was chosen for  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,  would  precedent  they of  such  have  been  analyses  by  included in the  the  researchers  analysis.  mentioned  The 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 the  likeliest  candidate,  although  internal  structures  may  also  involved, for example nerve ganglia or the digestive system. and Parker,  (1924) determined  food and or moisture.  approximately  paralysed quite  well  reasonable and  this  hypothesis  within to  24  the  longevity  flies  Pearl  deprived of  4  days, and those deprived only of  days.  hours results  At  29°C  for  the  adl-16  flies  ts2  and live  1-4  days,  of  and  Parker.  Pearl  to conclude that the adl-16 accounts  of  be  They found that at 2 5 ° C flies deprived of food  and moisture lived for 2-3 lived  the  is  fate  ts2  which It  food  become  corresponds thus  seems  flies also starve to death,  mapping  results.  The  artefact  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-16 adult  ts2  lethal  gene. mutants  It is not at all unusual for temperature sensitive to  demonstrate  pleiotropic  lethality as in the temperature-sensitive and Grigliatti, 1983).  effects  including  mutations for flight (Homyk  Nor is it exceptional that two foci be found, one  for lethality, the other(s) for a behavioural component of phenotype. Examples are the Hyperkinetic  jumping action mapping to the brain,  1  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. mesodermal  foci  survival  curves  separate  focus  For both adl-l  tsl  for each for for  leg,  and rex  and because  mortality and paralysis, lethality  existed,  but  of  they found separate the  they  they  difference  inferred  were  not  that able  in a to  determine it because death of the mosaics was still occurring when the study ended.  The graph in Figure 4-1 percentage  concerning percentage  paralyzed for my data, shows two  patterns.  It is extremely  (1986) for adl-1  If this is so, then the focus  results.  al.  that the  adl-16 . ts2  to be a domineering focus,  as explained in the  Since the data for distances between foci are not the same,  scoring process limbs affected Some flies  this hypothesis.  (those four limbs often  being adjacent)  with only dorsal thoracic tissue affected  at that time that the focus  died  Early in the  of the experiment, it was noted that flies with four  too  quickly  to  died rapidly. survived, so I  would map to the mid ventral  ganglion, as a single submissive focus. mosaics  different  for early death mapping to mesoderm  this presents a problem in accepting  thought  completely  This supports the hypothesis  early death and paralysis foci are separate in  would be expected  alive, and  similar to those found by Homyk et  and rex.  tsl  of flies  However too many bilateral  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  correspond closely would  indicate  that  noninteracting focus.  of  the  to the position each  leg  is  above of  the  hypotheses.  These  presumptive  under the  control  of  legs, a  foci which  separate  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  difficult to say.  is more  Lethality and paralysis seem to co-segregate in  adl-  16" . 2  The fact that the adl-16  ts2  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 site (52.9) on the X chromosome. 47.2  (Grigliatti  shibire  ts  et al, 1973),  adl-16  These include: bang-sensitive  Ether-a-gogo  at 50.0  located at 52.2 (Grigliatti et al., 1973), para"  (King,  ts2  at  1975),  at 53.9 (Suzuki  et al., 1971), stress sensitive (Homyk et al., (1980, 1986), and stoned at 66.3 (Grigliatti et al., 1973). in regard to adl-16" ,  because  2  (Sinclair,  personal  allelic with shi"'  The shi" locus is of particular interest preliminary complementation  communication),  suggest  either  that  data  adl-16"  is  or that strong interactions take place between the  two loci.  Interactions between loci with related function are well known, and two nervous system and nap" are  mutants, para",  (on chromosome  1, see  above)  ( 2nd chromosome), provide a good example of this.  temperature  sensitive  mutants,  with  reversible  Both  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 shi  alleles may interact in a similar way, that is, that the two  ts  loci each perform a strongly interdependent function, or that the two are allelic.  Kelly demonstrated that two alleles of shi the  production  of  some  component  of  ts  (  2  a  n  the  a  6  K  are involved in  regenerative  sodium  channel, using tetrodotoxin, which specifically blocks this channel of the was  action potential temperature  have related shi and endocytosis  mechanism, and furthermore that this  related  (Kelly,  1974).  Kosaka  blockage  and Ikeda (1983)  to the reversible blockage of membrane retrieval  tsl  involving the labyrinthine channels  and coated  pits  and vesicles of Garland cells.  Attempts are being made to clone shi in  microtubule  function,  which  ts  , and it is apparently involved  explains  a  great  number  of  the  developmental defects caused by the mutant, (Poodry et al., 1973). I would  suggest that  all the adl-16  ts  those of shi . ts  for shi  developing alleles  alleles  so  that  they  can  studies be performed on be  better  compared  Developmental studies such as have been  including  ts  similar developmental  histological  at the restrictive  and might  of  eggs  completed  and  temperature are indicated for  ts  develop normally, since these mutants die at larval stages 2-3  at the  adl-16 , ts  for  Shi  ts  perhaps another.  enzymes  microtubules,  involved with  one  mesodermal  adl-16  tissue  temperature.  whether  larvae  and nervous  restrictive  explain  examination  with  may code  for one  structural protien,  Alternatively, either or both could code in  having  the  assembly  a more  crucial  or  disassembly  developmental  of role  than the other. Since the adl-16  locus  ts2  related  behaviour  loss,  it  is  even  appears to compress  possible  that  its  age-  product  regulatory rather than structural, and acts to control the  is  timing of  certain events, rather than the events themselves.  Both shi  ts  (Sinclair, personal communication)  uncovered by the same deficiency,  and adl  ts  and when shi  mutants are  has been cloned,  ts  certain probes and restriction sites will be available, and I therefore suggest that a walk (strenuous para  hike) through the region from sh i  ts  might be very useful,  ts  (with the possibility  gene product), in elucidating function.  If shi  ts  of isolation  of a  and the adl-16  are  ts  alleles, then the nature of each defect might then be determined. the meantime,  to confirm or eliminate  adl-16  ,2,&flrdi  help  clarify  tsl  to  a  r  e  the  alleles,  detailed  relationship  found in that region.  the possibility that shi  ts  fine  to  In and  structure mapping would  between the  many loci  which  are  This interesting area certainly deserves further  investigation.  The relationship of the three adl-16 seems unclear. While adl-16 behaviour  loss,  adl-16  mutants  under study to  ageing,  has been found to accelerate  tsl  related  ts  ts2  dies too  early at the  age-  restrictive  temperature to determine such loss, although at 2 2 ° C , the pattern of behaviour loss is somewhat like that of adl-16 -  Most adl  tsl  exhibit there  pleiotropic phenotypes,  are many differences  16f .  so  it is  not  mutants  surprising that  observed in these alleles including  adl-  During adult life, all normal metabolic reactions must occur,  lrd  and  distinct  ts  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  of  adl-16  gene  regulation  causing ageing is difficult to separate.  However,  the  mesodermal  connection tissue  in  of  no  the way  involved in the ageing process. nervous  system and muscle  locus  disqualifies  this  to neural or  ts2  mutant  as  one  As pointed out in the introduction,  cell degeneration  age-related change, and the onset of  is a well  documented  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.  S U M M A R Y DISCUSSION An  enormous  amount  melanogaster.  of  research  Studies  have  data  exists  concerned  on  Drosophila  longevity,  behaviour,  histology, biochemistry, and genetics and other esoteric topics. evidence  has  been  amassed,  but rarely  have  above subjects been considered at a time. examined  a group of  alleles,  two  or more  Much of  the  To date, no one study has  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 Oregon-R  sentence, at  demonstrated  either  and  furthermore,  the  a relationship  permissive with the  be said to be related to ageing.  adl-16 or  tsl  compared  restrictive  wild-type  with  temperature  strain which could  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-16 ,  adl-16 ,  tsl  and  adl-16f were  females  of  the  allelic nature. to  found  lrdI  14Bi, of  deficiency had  be  intermadiate  parent strains  between  at 2 9 ° C ,  of  confirming their  the  X-chromosome  Df(l)sd  72b26  .  since  all  were  uncovered  Hybrids between mutants  reduced  viability  deformities.  and  It would  lifespan,  thus  be  as  and  interesting  similarities  between  the  adl-16  as to  the  some  test  the  There are  alleles and shi  ts  by  deficiency  well  genetic map location of 52.9 by fine mapping these alleles. many  those  The three mutants were located within the bands 13Fi  greatly  anatomical  generating  to  ts2  ts  or  para  ts  which are located on either side of them, so hybrid studies between these alleles might prove interesting.  The  mosaic analysis of adl-16  The  drop-dead  behaviour  demonstrated  ts2  mapped  to  one  two types of diffuse  focus  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. tissue  of  the  thoracic  ganglia  is  A n examination of neural  thus  suggested.  interesting to test the eyes of flies of adl-16 ERG  pattern is  analysis  different  at the  might point to either  restrictive  It  at 2 9 ° C  ts2  temperature.  a neural deficit  would  be  to see if the Such an  or a deficit  in the  receptor cells which transmit impulses to the neurons.  Although differences  allelic, from  flies the  of  type  other two  adl-16f demonstrated lraI  alleles,  also as a hybrid with the deficiency.  in lifespan,  many behaviour, and  It was the shortest lived at  2 2 ° C , but the longest at 2 9 ° C . hybrid with this  allele  When  with  combined  The effects of the deficiency  were the most other  alleles  severe at both  in hybrids, there  noticeable effect of the presence of this allele. isolated  in  a  screen  for  mutations  as a  temperatures. was  a very  Since this mutant was  affecting  flight,  it  would  be  interesting to test this behaviour more extensively in all three alleles over their lifespan. possible  Flight ability might then be added to the other  bio-markers of age.  Additional behaviours which could be  tested are mating behaviour as well as fertility and fecundity.  For the allele adl-16 ,  testing at 2 5 ° C might give better results for  ts2  behaviour tests, because behaviour is lost so early at 2 9 ° C .  If the  experiments  could  were  repeated  at  this  temperature  be made between alleles including adl-16 .  comparisons  Prior to this, it would  ts2  be advisable to remove the markers sc, cv, v, and f, to avoid any possible longevity  interference studies  in  experimental  were to  include some alleles of shi  Other  be  repeated  and  para .  it  to  Preliminary  determine testing  temperature effect any age.  if  of was  the  If  would  be  behaviour  and  interesting  to  ts  suggestions for additional research  studies,  29°C  ts  results.  include  temperature  temperature-sensitive alleles  continuous  indicated  when  periods  that  the  shift exist.  restrictive  flies were shifted  to it at  Histological examination of the alleles both at 2 2 ° C and at  might  temperature.  elucidate This  the  could  particular, to demonstrate  problem be  tried  differences  manifested  at  the  in  of  adl-16  mosaics  in affected  restrictive ts2  in  and normal tissue.  Such a study could encompass flies of different ages, to examine the question of a genetic basis of ageing in adl-16 If, in fact adl-16  or adl-16  tsl  tsl  in particular.  do have a role in the ageing process,  ts2  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-16 , ts  expected disruption proves Whether question.  to do  so.  of  some  nor  In fact premature death may be due to required  disproves  ageing  is  or how it works, nor could it be  that  biochemical longevity  genetically  is  function  which  genetically  programmed remains  the  neither  controlled. a tantilizing  Until the reason for the deleterious effects of temperature  on the alleles of adl-16  ts  is known, as well as their normal role, their  part in ageing, if any, will remain unsolved.  REFERENCES  Alpatov, W . W . and R. Pearl (1929). Experimental studies on the duration of life. XII. Influence of temperature during the larval period and adult life on the duration of life of the imago of Amer. Nat. 63: 37-67.  Drosophila melanogaster.  Arking, R and M . 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Cell 19: 133-141.  Appendix i (see pi05 )  222  fKotor  "1  A 1 i  <3  3  '¥  ¥  IL  rt  /  f  it  2.  1  */  3  AO  V  3  2 ft S)ec<L  ii  1  i  z. 3  3  ¥  b rt  it  2 3  AD  \  3  Appendix i i i  Z24  


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