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Isolation and study of two mutants affecting motor activity in Drosophila melanogaster. Williamson, Rodney, L.M. 1971

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THE ISOLATION AND STUDY OF TWO MUTANTS AFFECTING MOTOR ACTIVITY I N DROSOPHILA MELANOGASTER  by Rodney L. M. W i l l i a m s o n B.Sc., U n i v e r s i t y  of B r i t i s h  Columbia, 1968  A' THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  Genetics in  the Department o f Zoology  We a c c e p t t h i s t h e s i s as conforming required standard  t o the  THE UNIVERSITY OF BRITISH COLUMBIA May, 1971  In  presenting  this  an a d v a n c e d  degree  the L i b r a r y  shall  I  f u r t h e r agree  for  scholarly  by h i s of  this  written  thesis at  the U n i v e r s i t y  make  permission.  Department  of  of  it  by  shall  requirements  for  I agree  r e f e r e n c e and copying of  this  that  not  copying  or  for  that  study. thesis  t h e Head o f my D e p a r t m e n t  is understood  financial gain  the  B r i t i s h Columbia,  for extensive  p u r p o s e s may be g r a n t e d  for  fulfilment of  it freely available  that permission  representatives, thesis  in p a r t i a l  or  publication  be a l l o w e d w i t h o u t my  ii ABSTRACT Mitants  o f P r o s o p h i l a melanogaster which a r e p a r a l y s e d by  exposure t o one temperature, b u t r e c o v e r m o b i l i t y a t another  temperature  may a i d i n t h e i n v e s t i g a t i o n o f t h e n e u r a l and muscular components which govern motor a c t i v i t y .  With the help of a mechanical screening  method, a r e c e s s i v e s e x - l i n k e d t e m p e r a t u r e - s e n s i t i v e p a r a l y t i c mutant (para^  s  -53°9) was d i s c o v e r e d among t h e progeny o f e t h y l methanet«?  s u l p h o n a t e - t r e a t e d males and attached-X females,  para  flies  which  had been r a i s e d a t 22°C were p a r a l y s e d w i t h i n 5 seconds a f t e r t r a n s f e r 29°C, b u t q u i c k l y r e g a i n e d m o b i l i t y when r e t u r n e d t o 2 2 ° C  to  l e f t a t 29°C f o r p r o l o n g e d mobility.  When  periods, the f l i e s gradually regained  Further studies i n d i c a t e d t h a t t h e time r e q u i r e d f o r  r e c o v e r y f o l l o w i n g an i n c r e a s e i n temperature was d i r e c t l y r e l a t e d t o the magnitude and r a t e o f t h e temperature r i s e . Temperature-sensitive p a r a l y s i s was seen o n l y i n a d u l t f l i e s . The  abnormal movements which a r e c h a r a c t e r i s t i c o f t h e b e h a v i o u r a l  mutants Hk stopped  , Hk  , Sh , when  l i n k e d t o para  i n males,were q u i c k l y  and s t a r t e d by temperature s h i f t s from 22°C t o 29°C and 29°C t o  o 22 C, r e s p e c t i v e l y .  The p o s s i b l e s i g n i f i c a n c e o f t h e s e o b s e r v a t i o n s i s  discussed. p a r a ^ / M ( T ) 0 females e x h i b i t e d t e m p e r a t u r e - s e n s i t i v e s  paralysis.  The p o s s i b i l i t y t h a t t h e chromosome b e a r i n g t h e M(l)0 mutation might ts a l s o c a r r y a d e l e t i o n or mutant a l l e l e o f p a r a  has n o t y e t been  investigated. A s e x - l i n k e d dominant mutation which caused abnormal movements o f t h e head and appendages under ether a n a e s t h e s i a as w e l l as shuddering  iii movements i n unetherised f l i e s was also discovered. was called Shuddering (Shu - 55«1).  The mutation  The shuddering movements could  be temporarily suppressed by feeding the flies media containing LiCl but not NaCl,  NH4.CI  or KC1.  The evidence presented i n this and other studies suggests that ts the effects of para  and Shu mutations upon motor activity are  mediated through their effects upon the nervous system,,  iv TABLE OF CONTENTS Page Introduction  1  Methods and M a t e r i a l s  ^  Results  24  Discussion  ^  Summary  60  Bibliography  6  2  V  LIST OF TABLES TABLE  Page  1„  Frequency of spontaneous shuddering i n Shu/FM6 f l i e s after different salt treatments.  2„  Total numbers of f l i e s tested for shuddering induced by mechanical stimulation after different salt treatments„  28  3«  Estimated number of f l i e s screened for sex-linked . recessive and autosomal dominant temperaturesensitive paralysiso  31  4„  Types of movements which may be seen during recovery from temperature induced paralysis,,  36  5.  Recombination data for para mutants„  6.  Reisolation of single mutation chromosomes by recombination of double mutants„  2  ^  ts  and different Neuro  kk ^5  LIST OF FIGURES Figure  Page  1,  Type I-a screening device for isolating paralysed flies,  5  2,  Type I-b screening device for isolating paralysed flies.  ?  3o  Type II screening device for isolating paralysed flies.  9  4,  End views of the type II screening device during the isolation of paralysed f l i e s ,  10  5o  Procedure for detection of a dominant temperature sensitive paralytic mutant.  12  6,  Procedure for detection of a sex-linked recessive or autosomal dominant temperature-sensitive paralytic mutant.  13  7,  Twin water-heated chambers for comparing behaviour at constant temperatures.  18  8.  A semi-immersible chamber for observing behaviour during temperature changes.  19  9.  Protocol for generating and testing Neuro para^ /Y males,  10,  Reduction of shuddering of Shu/FM6 f l i e s subjected to mechanical stimulation,  11,  Recovery of motor competence with time after increases i n temperature.  12,  The number of para f l i e s , preconditioned at 22°G (solid line) and 17°C (dashes) which were able to stand after being transferred to 29.5°G.  13,  The number of para f l i e s able to stand when temperatures were changed from (22.2°-22.4°C) to (27.0 -27o6°C) within (a) 14 minutes and (b) 6 minutes. Solid line - number of f l i e s able to. stand; dashes - temperature,  s  ts  2 2  2  9  38  ts  o  ts 14,  The number of para f l i e s able to stand when temperatures were changed from (21.6°-22,0 C) to (28,5°-29,5°C) within (a) 24 minutes (b) 6 minutes. Solid line - number of f l i e s standing; dashes temperature.  41  V1X  Figure 15.  Page Genetic locat: Genetic locations X chromosome.  5  ts o f HK.para  and Sh  on t h e  43  viii ACKNOWLEDGEMENT I am very grateful to Dr, David T. Suzuki for providing me with a most interesting research problem, and for his continued guidance, faith and help. I am very grateful to Dr. Leonie K. Piternick for her enthusiastic encouragement and help in the early stages of this work. It has been a great pleasure to work with Mr. Thomas G r i g l i a t t i who shared equally in the task of finding and characterising the para^ mutant. s  I would like to thank my father, Dr. Bruce Williamson for bringing lithium ion therapy to my attention and providing me with his professional view of i t s efficacy in the treatment of manicdepression and related disorders. I wish to thank a l l the members of Dr, Suzuki's laboratory whose help and encouragement have made this work both possible and enjoyable.  1 INTRODUCTION Comparative neuroanatomy was founded in the Alexandrian schools of medicine in 300 B.C.  Since that time, knowledge of the nervous  systems of numerous animals has been enlarged through many disciplines. The current interest in neurobiology represents a renewed awareness of the diversity of nervous systems which can now be analysed with a large assortment of technical and biological tools.  In view of the  dramatic success of genetic analyses of microbial systems, many investigators now wonder whether the mysteries of the nervous system may also be amenable to a similar strategy. Although the effects of single mutations upon behaviour have been studied for some time (McEwen, 1918), an organised pursuit of single mutations which affect a specific nervous function was f i r s t launched in Drosophila melanogaster by Benzer many nonphototactic mutants.  (I967), His search has yielded  Those which are characterisable by  abnormalities in their electroretinograms have been assigned to five cistrons of the X chromosome by genetic mapping and complementation tests (Hotta and Benzer,  1970),  Brenner (1970) pointed out that the morphogenesis of a nervous system probably requires extensive genetic regulation.  Mutations  affecting regulation are therefore likely to be found among behavioural mutants. Accordingly, Brenner has conducted a search for nonspecified behavioural deviants using a Nematode which possesses 50 axons and a ganglion comprised of 20 neurons.  350 mutants have been characterised  and 20 functional groups have been defined.  Electron microscopic  studies of some of the mutants have revealed alterations of the nerve  2  circuitry.  Unfortunately, the species has not been accessible to  electrophysiological probes. Whiting (1932) was able to determine that the expression of sex specific behaviour in the wasp Habrobracon juglandis depended upon the genetic composition of the head, by studying gynandromorphs. Genetic mosaics have also been analysed by Ikeda and Kaplan (1970b) and Hotta and Benzer (1970) to determine the regional specificity and autonomy of behavioural mutants of Drosophila.  In the latter studies, the  frequency of genetic mosaics was increased by using the somatically vC unstable ring chromosome In(l)w  (Ronton, 1955) or the mutation  claret-nondisjunctional (Lewis and Gencarella, 1952). The genetic manipulations made possible by the variety of chromosome aberrations and mutations in Drosophila melanogaster. together with the accumulated knowledge of the organism, sustain i t s usefulness as a tool for genetic study.  Furthermore, the neuro-  physiology of the organism is now accessible to intracellular electrical recording techniques (Ikeda and Kaplan, 1970a).  The use of  mutations to probe the adult motor nervous system was initiated by Kaplan and Trout (1969) and Ikeda and Kaplan (1970a,b).  The existing  knowldge of this system and its relationship to muscular physiology make i t a promising area for molecular investigation. The f i r s t step towards a genetic study of neural and muscular function is the isolation of mutants.  Paralysis is both a logical and  a feasible mutant phenotype to look for.  The extensive documentation  of conditional mutations whose phenotypes are temperature-dependent (Suzuki, 1970) suggested a search for temperature-dependent  paralysis,  A mutation which allows mobility at one temperature, but is reversibly  3 paralysed by another provides a basis for large scale screening methods for i t s detection. If the f l y recovers mobility after i t i s returned to the "permissive" temperature, the mutation may be perpetuated as a stock. By screening for temperature-sensitive paralysis, individuals exhibiting various kinds of debilitated motor activity might also be detected.  With the recovery of each behavioural mutant, the  probability that one of them w i l l be accessible to an existing means of investigation increases. The same rationale applies to the paral y t i c phenotype i t s e l f .  From a variety of defects which cause  paralysis, a new access to the neuromuscular system may be found.  METHODS AND MATERIALS General Procedures. Complete descriptions of the mutations affecting behaviour w i l l be given;  other stocks used routinely have been described by  Lindsley andGraLl (1968). Unless specified, the medium was the standard mixture used at the California Institute of Technology. In experiments requiring media containing various molarities of LiCl, NH^.C1, KC1 and NaCl the appropriate salt solution was diluted ten fold with standard media and blended with a Sorvall Omnimixer. Crosses were made i n shell vials or quarter and half pint milk bottles.  The standard laboratory temperature was 21.5 - 1°C«  Screening devices for recovery of paralysed f l i e s . Theoretically a paralysed individual should be easy to find within a group of active f l i e s .  Unfortunately, a caged population of normal  f l i e s usually includes many which stay perfectly s t i l l at any one moment. Because each f l y goes through its own cycles of activity and inactivity, the membership of the motionless segment of the population is constantly changing.  For this reason, direct attempts to determine  which motionless f l y was actually paralysed proved to be impractical. Two types of apparatus were designed to overcome this problem. Considerable success in separating mobile from immobile f l i e s was achieved by placing the f l i e s in an empty shell v i a l which was capped with a paper funnel leading upward into another shell v i a l containing medium (Figure 1). The f l i e s , driven by their tendency to move upward and the attraction of the medium, climbed into the upper  FIGURE 1 Type I-a screening device for isolating paralysed f l i e s . The device consists of an upper and lower v i a l facing mouth to mouth separated by an upwardly directed paper funnel. The upper v i a l contains media. An active f l y (A) climbs into the upper v i a l . After many f l i e s have l e f t the lower v i a l , the paralysed f l y (P) i s more easily noticed among the diminished population.  6 vial.  The cone was a f a i r l y efficient valve which prevented migrants  from returning.  Immobile individuals were selected from the diminished  population of the lower v i a l . the type I-a screener.  This assembly w i l l be referred to as  A modification of this design is shown in  Figure 2, Vials positioned above and below funnels cut out of -§•" plexiglass were held in place by foam rubber cushions.  This device  (type I-b) permitted easy handling of the vials and provided an unobstructed view of the f l i e s . When a large population of flies is confined to a small space, the f l i e s tend to cluster, their activity seems aimless and their antigeotactic behaviour diminishes.  Consequently, the type I designs  imposed a limit of a few hundred f l i e s per v i a l for optimal screening efficiency, Benzer (19^7) found that nonphototactic mutants could be selected by counter current distribution from a population of several hundred f l i e s placed in an 18 x 150 mm test tube.  However, each group of flies  to be tested must be put through several operations and tubes, A second limitation of the type I device was the dependence on a constant behaviour pattern, namely migration through the funnel into the upper v i a l .  Many f l i e s were capable of climbing, but simply  remained in the lower v i a l .  Some showed no antigeotactic tendency  while other f l i e s stopped inside the funnel at i t s lower edge. The efficiency of the screener was somewhat improved by selecting for a strain of f l i e s characterised by i t s ability to reach the upper v i a l quickly. Mutants were induced and selected from this stock which w i l l be referred to as F,C, for fast climbers.  However, heterogeneous  behaviour was never eliminated. Consequently, a more efficient  7  i  FIGURE 2  Type I-b screening device f o r i s o l a t i n g paralysed f l i e s . The device works on the same p r i n c i p l e as the type I-a. In t h i s case, the upper and lower v i a l s are pushed a g a i n s t funnels of p l e x i g l a s s with foam rubber cushions.  8 separating device was needed. The type I I screener (Figures 3 and 4) i s b a s i c a l l y an elongated box with a diagonal b a r r i e r (b) extending the length of the box and a drawer (d) f i t t i n g into the acute angle between the b a r r i e r and the bottom of the box.  The diagrams of the end view of the box, shown i n  Figure 4, i l l u s t r a t e the sequence of operations used to i s o l a t e an immobilised i n d i v i d u a l (P) from a large population.  Rotation of the  box clockwise 90° and then counterclockwise 90° ( i , e , , from p o s i t i o n 1 to 2 to 3) causes P to f a l l i n t o the drawer.  The a c t i v i t y of the  population i s greatly increased by introducing a small quantity of vinegar and detergent solution onto the l e f t side of the b a r r i e r (3)» A l l f l i e s which are not inside the drawer can be eliminated by r o t a t i n g the box 90° and shaking the active f l i e s i n t o the solution (4), F i n a l l y , P can be selected from among the small number of individuals which remain i n the drawer ( 5 ) , The design minimises the deleterious effects of crowding, as w e l l as the number of p h y s i c a l obstacles which tend to i n h i b i t free movement. Six  thousand  f l i e s at a time could be r e a d i l y screened i n the type I I  box shown i n Figure 3« Screening procedure f o r dominant temperature-sensitive mutants. The e f f i c i e n c y of the type I screener depended upon the speed with which a large proportion of the f l i e s would enter the upper v i a l . In t h i s respect, Oregon-R was better suited to the apparatus Samarkand.  than  The e f f i c i e n c y of separation was further improved by  occasionally regenerating the stock with f l i e s which had reached the upper v i a l most quickly.  9 FIGURE 3 Type I I s c r e e n i n g d e v i c e f o r i s o l a t i n g p a r a l y s e d f l i e s . dimensions 6" x 6" x 1 8 " (^-" p l e x i g l a s s ) .  Vinegar Feed P i p e F l y Entrance Vinegar I n l e t  Drawer Stop V e n t i l a t i o n Hole Diagonal 3 a r r i e r  Outside  10 FIGURE 4 End views of the type I I screening device during the i s o l a t i o n of paralysed f l i e s . Steps 1 to 5 represent successive stages i n the procedure. The l a b e l b r e f e r s t o a diagonal b a r r i e r ; d i s a drawer P i s an immobilised f l y . The hatched area represents a vinegar and detergent s o l u t i o n . The operation of t h i s device i s explained on page 8 .  11 F.C, Oregon-R males were l e f t f o r 24 hours i n h a l f p i n t m i l k bottles  containing  two d i s c s  o f Whatman No. 1 f i l t e r paper dampened  w i t h 1 ml o f 0.025M e t h y l methanesulphonate (EMS) i n a 1$ s u c r o s e solution  (Lewis and Bacher, 1968).  The mutagen t r e a t e d  males were t h e n  mated w i t h F.C. Oregon-R females i n q u a r t e r and h a l f p i n t m i l k b o t t l e s (10  t o 15 p a i r s p e r b o t t l e ) . e v e r y 3 t o 8 days.  bottles  These f l i e s were t r a n s f e r r e d t o f r e s h Eggs were never c o l l e c t e d beyond 16 days  a f t e r treatment. The  progeny were r a i s e d a t 22°C,  After  e c l o s i o n , t h e y were  p l a c e d i n t o a t y p e I or type II s c r e e n e r which had been prewarmed t o o 29 C„ for  o A f t e r 10 minutes t o 2 hours a t 29 C, t h e p o p u l a t i o n was s c r e e n e d  immobile f l i e s .  Each immobile f l y which r e c o v e r e d m o b i l i t y a t  o 22  was t h e n c r o s s e d t o 3 F.C. Oregon-R f l i e s  progeny o f t h e f e r t i l e  in a shell vial.  The  i n d i v i d u a l s were a g a i n r a i s e d a t 22°C and then  tested  f o r the inheritance  vial.  This  o f p a r a l y s i s by d i r e c t t r a n s f e r t o a 29°C  procedure i s o u t l i n e d i n F i g u r e  S c r e e n i n g procedure f o r s e x - l i n k e d m u t a t i o n s. A search f o r sex-linked  recessive  5. and autosomal dominant  mutants was a l s o conducted ( F i g u r e  6).  EMS-treated Oregon-R males were c r o s s e d t o F.C. females c a r r y i n g an attached-X  chromosome  This  p e r m i t t e d t h e r e c o v e r y o f male  progeny c a r r y i n g mutagenised p a t e r n a l X chromosomes. c a r r i e d out i n a t y p e II a p p a r a t u s .  A l l s c r e e n i n g was  The s e l e c t e d males and females  were i n d i v i d u a l l y c r o s s e d t o F.C, XX/Y females and F.C, Oregon-R males, respectively.  T h e i r progeny were t e s t e d f o r i n h e r i t e d temperature-  s e n s i t i v e p a r a l y s i s as d e s c r i b e d above.  12  FIGURE 5 Procedure for detection of a dominant temperature-sensitive paralytic mutant. FoC.  0regon-R^(0 025M E K 3 ) o  x  F  0  C . Oregon-R  22°C  F i adults shifted from 2 2 ° C to 2 9 ° C Immobile f l i e s selected with a type I or type II screener  Flies selected are returned to 22 C  Flies recovering mobility at 22 C are mated  A  F.C, 22°C  Oregon-Ror  F . C . Oregon-R 22°C  Tested for paralysis induced by a 2 2 ° C to 2 9 ° C shift Tested for recovery by returning f l i e s to 22 C  13  FIGURE 6 Procedure for detection of a sex-linked recessive or autosomal dominant temperature-sensitive paralytic mutant. F.C. 0regon-R^/(0.025M EMS)  x  F C. f\/Y,+ 8  ^  22°C F i adults shifted from 22°C to 29°C Immobile f l i e s selected with a type II screener  • o Immobile f l i e s returned to 22 C  Flies which recover are mated  <5*xF.C. XX/Y,+ 22°C  or  F.C. Oregon-R^ 22°C O  a  Tested for paralysis induced by a 22 C to 29 C shift Tested for recovery by returning f l i e s to 22 C  14 Recovery  and g e n e t i c mapping o f a s h u d d e r i n g mutant.  In the s c r e e n f o r dominant t e m p e r a t u r e - s e n s i t i v e p a r a l y s i s u s i n g a type I-b d e v i c e , a female which o c c a s i o n a l l y shuddered was i n the lower v i a l . l i n k e d dominant;  The b e h a v i o u r was  found t o be i n h e r i t e d as a sex-  c o n s e q u e n t l y , t h e mutant chromosome was  t h e m u l t i p l y i n v e r t e d chromosome,  noticed  b a l a n c e d over  FM6,  While a n a e s t h e t i s e d under e t h e r , the shuddering f l i e s  scissored  t h e i r wings and shook t h e i r l e g s i n q u i c k v i g o r o u s spasms.  In o r d e r  t o l o c a l i s e t h e g e n e t i c s i t e r e s p o n s i b l e f o r the b e h a v i o u r under e t h e r , females were c r o s s e d t o f l i e s  c a r r y i n g t h e f o l l o w i n g markers  by t h e i r symbols and g e n e t i c l o c a t i o n ) ;  y e l l o w - y_  (followed  (0.0), c r o s s v e i n l e s s -  cv (13.7), v e r m i l i o n - v (33.0), f o r k e d - f (56,7), c a r n a t i o n - car  (62,5).  Heterozygous  progeny were s c o r e d .  females were t e s t c r o s s e d and a l l male and  female  The shuddering males were extremely weak and  u n c o o r d i n a t e d and s u r v i v e d o n l y f o r one t o t h r e e days under good c u l t u r e conditions.  I n o r d e r t o reduce the s e v e r e e f f e c t s o f crowding  c o m p e t i t i o n on t h e s e f l i e s ,  s i n g l e females were mated t o t h r e e males i n  s h e l l v i a l s and were s e r i a l l y t r a n s f e r r e d t o new 10  and  v i a l s each day f o r  days b e f o r e b e i n g d i s c a r d e d . T h i s mutant w i l l h e r e a f t e r be r e f e r r e d t o as Shuddering  or Shu,  The e f f e c t s o f monovalent c a t i o n s on shuddering b e h a v i o u r . The p o s s i b i l i t y t h a t l i t h i u m i o n s might b e h a v i o u r was  suppress  shuddering  suggested by t h e i r t h e r a p e u t i c e f f e c t s upon nervous  d i s o r d e r s i n guinea p i g s and man  (Cade,  1949).  The s i m i l a r i t i e s between  shuddering and t h e s e d i s o r d e r s w i l l be d i s c u s s e d l a t e r . were a l s o t e s t e d because  Ammonium i o n s  the aberrant l e g shaking of n e u r o l o g i c a l  15 mutants of Drosophila under ether anaesthesia was reversibly suppressed by ammonia (Kaplan, personal communication). Four pairs of Shu/FM6 f l i e s were placed in quarter pint bottles (1 pair per bottle) containing various concentrations of LiCl, KCi, NaCl or NH^Cl, and twelve pairs were placed on standard media.  The  number of jerks and shudders produced by each pair of f l i e s during one minute was recorded daily.  Shudders lasting for about three seconds  or more were counted as two shudders.  Prior to each observation, the  bottle cap was replaced with a transparent plastic petri plate. Following this disruptive manipulation, the bottle was l e f t undisturbed. Under these conditions, f l i e s treated with 0,32 M to 0,35 M LiCl ceased shuddering after three and four days of treatment; mortality rate was high.  however, the  Consequently, a second series of experiments  was performed to test the capacity of LiCl to suppress shuddering induced by mechanical stimulation, and to determine whether or not the f l i e s would continue to live, i f they were returned to standard media at c r i t i c a l times. Groups of 5 to 40 Shu/FM6 f l i e s were placed in quarter pint milk bottles on standard media and media containing LiCl, NaCl and NH/j,Cl at 0,2M,  0.32M and 0.33M concentrations. Every 12 or 24 hours, f l i e s in  each bottle were shaken down repeatedly.  By one and a half days of age,  Shu/FM6 f l i e s kept on standard media would inevitably give a shuddering response to this type of mechanical stimulation. were recorded:  Two types of responses  "no shuddering" or "slight shuddering".  The latter  class was defined by a few slight jerks seen within a group.  Two  groups of 40 f l i e s were transferred from 0.32M LiCl to standard media after three days.  The rest of the f l i e s were returned to standard media  16 a f t e r s i x days or at various e a r l i e r times when the f i r s t deaths occurred i n each b o t t l e . Bottles of media which were not used immediately were stored i n p l a s t i c bags to reduce desiccation and consequent changes i n s a l t concentration.  The f l i e s were usually transferred to new media every  three days i n order to minimise exposure of the f l i e s to desiccated and microbially infected media.  V i r g i n females were used i n most  experiments t o obviate changes i n the behaviour of adults on media which had become softened and dampened by developing larvae.  In  cultures containing many larvae, Oregon-R f l i e s walked slowly with a s l i g h t side to side wobble.  The same behaviour was accentuated i n  Shu/FM6 f l i e s i n which the wobble frequently became a more vigorous shudder.  Observation of a temperature-sensitive p a r a l y t i c mutant. A male whose paralysis was temperature-dependent was from an  mother using a type I I screening device.  isolated  Upon crossing the  male to XX/Y females, a l l male offspring showed temperature-sensitive paralysis.  Females heterozygous f o r the mutation and FM6 were  unaffected by temperature, thereby showing the mutation to be recessive. Homozygous females were also paralysed at 29°C. Males carrying the mutation were crossed to y cv v f car and t h e i r o F-j_ daughters were testcrossed at 22 C„ the  The F  2  progeny were scored f o r  v i s i b l e markers and the males were screened f o r paralysis at 29°C,  The mutation was c a l l e d p a r a l y t i c temperature-sensitive and designated ts as para  .  Preliminary observations of the effects of temperature upon the  17 behaviour of para  ts  were made by simply transferring the f l i e s to  v i a l s which had been preconditioned to various  temperatures.  In order to examine behaviour at constant temperatures, the f l i e s were placed into twin observation chambers which were b u i l t into a transparent temperature-controlled water bath (Figure 7)*»  The temperature  i n the chambers was maintained by constantly exchanging water between the observation bath and a Blue M constant temperature bath.  The  temperature was monitored with a Yellow Springs Instrument Company general purpose thermistor probe and registered on a Rustrak recorder,, ts Generally 5 t o 10 para  and an equal number of Oregon-R f l i e s were  observed over a two hour period following transfer of the f l i e s t o the observation chamber.  Most experiments required one person t o monitor  behaviour continuously while another recorded the changes i n behaviour with temperature  and time.  A second type of chamber was constructed i n order t o observe the f l i e s while the temperature was being changed (Figure 8 ) . The f l i e s were placed i n the bottom of three chambers which were formed by stacking four 3.5cm p l a s t i c p e t r i plates on top of each other. sealed together with p l a s t i c i n s u l a t i o n tape.  The chambers were  A 1mm a i r hole was made i n  each of the upper three plates, each hole having a staggered p o s i t i o n , with respect to i t s v e r t i c a l axis, i n r e l a t i o n t o the other two. A thermistor wrapped i n i n s u l a t i o n tape above the sensing r e s i s t o r was seated snugly i n t o the lower chamber.  By p a r t i a l l y submerging t h i s  assembly i n a water bath, the temperature  of the bottom chamber was  quickly altered and equilibrated within the variations of the bath i t s e l f . * This apparatus was designed and b u i l t by Tom G r i g l i a t t i .  18  FIGURE  Twin w a t e r - h e a t e d temneratures.  7  chambers f o r c o m p a r i n g b e h a v i o u r  at constant  phermistor  Observation  chambers  19 FIGURE  8  A s e m i - i m m e r s i b l e chamber f o r o b s e r v i n g b e h a v i o u r d u r i n g changes.  temperature  20 The q u i c k e s t temperature changes were produced by moving t h e chamber from one b a t h t o a n o t h e r .  More g r a d u a l temperature i n c r e a s e s were  a c h i e v e d by immersing t h e chamber i n a Haake F,S, c o n s t a n t temperature b a t h and r a i s i n g t h e b a t h temperature  electrically.  O b s e r v a t i o n s were made t h r o u g h a d i s s e c t i n g microscope w i t h subs t a g e i l l u m i n a t i o n by l i g h t r e f l e c t e d from t h e bottom o f t h e b a t h . Overhead  light  c o u l d n o t be used because t h e r e f l e c t i o n s obscured t h e ts  view, and t h e heat r a d i a t i o n was  s u f f i c i e n t l y high t o immobilise para  flies„ Because t h e f l i e s were a n a e s t h e t i s e d w i t h e t h e r i n o r d e r t o p l a c e them i n t h e chamber, and because t h e chamber temperature was  altered  by  h a n d l i n g d u r i n g i t s assembly, t h e f l i e s were m a i n t a i n e d f o r an hour a t o 22  C f o l l o w i n g t h e i r r e c o v e r y from t h e a n a e s t h e t i c ,  I n t e r a c t i o n s between p a r a  ts  and n e u r o l o g i c a l m u t a t i o n s .  IP Under e t h e r a n a e s t h e s i a , t h e s e x - l i n k e d mutants (30.9 ±  0  6 ) , Hyperkinetic  2 1  - HK  2 T  ( 3 0 . 4 + . 7 ) and S h a k e r  ( 5 8 , 2 1 , 6 ) shake t h e i r l e g s v i g o r o u s l y and r a p i d l y 1969).  Hyperkinetic 5  - Sh  5  These tremulous f l i e s  " n e u r o l o g i c a l mutants" by Kaplan and T r o u t ( 1 9 & 9 ) g e n e r a l i s e d by t h e a b b r e v i a t i o n Ikeda and Kaplan ( 1 9 7 0 a ) IP r a p i d l e g s h a k i n g o f HK  a m  -HK  5  (Kaplan and T r o u t ,  I n t h e case o f S h . t h e l e g movements a r e accompanied  r e p e a t e d s c i s s o r i n g o f t h e wings.  ip  by  were c a l l e d  * w i l l hereafter  be  Neuro.  demonstrated t h a t t h e c h a r a c t e r i s t i c  o r i g i n a t e d as impulse b u r s t s i n t h e t h r e e  p a i r e d motor areas o f t h e t h o r a c i c g a n g l i o n .  The r h y t h m i c b u r s t s were  t r u l y endogenous and r e c e i v e d no c o n t r i b u t i o n s from t h e c e p h a l i c , s e n s o r y or neuromuscular systems.  S t u d i e s o f b i l a t e r a l t h o r a c i c mosaics  21 containing HK /+ * and HK /0 tissues (Ikeda and Kaplan, 1970b) 1P  1P  IP  IP /0 and HK  indicated that the electrophysiology of HK  /+ tissues and  the associated behaviour was b i l a t e r a l l y autonomous within the thorax and also independent of the r e s t of the body.  The interactions of  Neuro and para"*-' mutations could be best analysed i n Neuro para^ /Y males, / ts 5  5  Single Neuro +/+ para  females were placed with wild type males  for 7 days at 22°C and then discarded (Figure 9, step l ) .  The male  progeny were scored f o r temperature-sensitive paralysis at 29°C and either-induced leg shaking at 22°C and 29°C The phenotypes were ts compared with Neuro, para and Oregon-R stocks. Newly a r i s i n g ts 0  phenotypes which exhibited characteristics of both Neuro and para were designated Neuro para^ /Y. The genetic i n t e g r i t y of the phenotype S  was tested by crossing each male t o females carrying attached-X chromosomes and scoring f o r the same phenotype i n male progeny (Figure 9, step 2 ) .  The double mutation condition of the chromosomes  could be v e r i f i e d by r e - i s o l a t i n g the s i n g l e mutations by recombination. Therefore, single Neuro para^ /++ females derived from each of the S  o r i g i n a l putative recombinant double mutant males were crossed t o Oregon-R males (step 4),  The male progeny were then scored f o r the  ts occurrence of para and Neuro behaviour separately. ts Interaction of para  and Minutes.  The p o s s i b i l i t y that the mutation M(l)0. located at 56.6, might be a deletion which would span the p a r a l y t i c region, prompted a t e s t f o r ts pseudodominance of para when heterozygous with M(l)0. A p a r t i a l  t<?  paralysis d i s t i n c t from that of para * For a l l p r a c t i c a l purposes HK  homozygotes was observed i n  can be regarded as recessive.  22 FIGURE  9  Protocol f o r generating and testing Neuro p a r a / Y males ts  Steps 1.  ++/l/x  + para /Neuro + ^ ts  Select putative  2.  |  3o  k.  + +/+  Neuro p a r a ^ / Y t f  Neuro para  x  + +/Yd* x  1  ts /Y &  Neuro p a r a / + + |»  Score f o r recovery of Neuro+ZY^  7  tpara^/Y^  t s  23 M(1)0 f / p a r a ^ females, but not i n f / p a r a ^ controls. s  s  This suggested  that Minute mutations i n general might a f f e c t the expression of para^ . s  ts Male para  f l i e s were crossed to single females of the following  genotypes:  M(l)0 f/FM6, M(2)e /SM5;  e l(3)e;  S  M(3)w /ln(3R)C. e l ( 3 ) e . B  on two separate occasions.  M(2)S /SM5; 7  M(3)w *Vln(3R)C. 12  Five matings of each type were made  The progeny were  raised to 22°C,  transferred d i r e c t l y into 29°C v i a l s and observed f o r any change i n behaviour.  2h  RESULTS  Shuddering:  phenotype and map p o s i t i o n  There were no obvious and Shu/+ f l i e s  b e h a v i o u r a l d i f f e r e n c e s between Shu/FM6  i n e i t h e r t h e •'conscious" or a n a e s t h e t i s e d s t a t e s .  S i n c e t h e Shuddering mutation was c o n v e n i e n t l y m a i n t a i n e d females,  they were t h e main s u b j e c t s o f study,  A f o u r day o l d Shu/FM6  f l y shuddered on an average o f 8 times a minute, a l t h o u g h v a r i e d from l e s s t h a n 1 t o more t h a n 50 times  i n Shu/FM6  a minute.  individuals The shuddering  phenotype was produced by a r a p i d sequence o f l e g j e r k s which caused t h e f l y t o lunge i n v a r i o u s d i r e c t i o n s . for  The r e s u l t a n t "shudder" l a s t e d  a f r a c t i o n o f a second t o about f o u r seconds, and v a r i e d i n  i n t e n s i t y from a s l i g h t j e r k t o a shudder which was s t r o n g enough t o throw t h e f l y on i t s back. it  Although  shuddering  occurred  spontaneously,  c o u l d a l s o be e l i c i t e d by g e n t l y moving a b o t t l e c o n t a i n i n g Shu/FM6  f l i e s which had p r e v i o u s l y been l e f t  undisturbed.  D r o s o p h i l a can o f t e n be induced t o f l y when they a r e l i f t e d  from  a s u r f a c e by means o f a t o o t h p i c k g l u e d t o t h e d o r s a l s i d e o f t h e thorax.  Under t h i s t e t h e r e d f l i g h t c o n d i t i o n , Shu/FM6 f l i e s v i b r a t e d  t h e i r legs continuously.  I n r a r e i n s t a n c e s , Oregon-R f l i e s  also  v i b r a t e d t h e i r l e g s , b u t t h i s movement was i n e v i t a b l y stopped  during  flight. When Shu/FM6 f l i e s were a n a e s t h e t i s e d w i t h e t h e r , they remained s t i l l f o r o n l y a b r i e f p e r i o d b e f o r e shaking t h e i r l e g s v i g o r o u s l y and rapidly.  The shaking was generated  femurs remained r e l a t i v e l y s t i l l .  i n t h e t i b i a e and t a r s a e w h i l e t h e The i n t e n s i t y o f t h e shaking was  25 spasmodic,  u n l i k e t h e continuous tremor  of the legs i n tethered flight,,  E v e n t u a l l y wing s c i s s o r i n g began, accompanied b y movements o f t h e h a l t e r e s , antennae and head.  These l a t e r movements d i d n o t n o r m a l l y  b e g i n s i m u l t a n e o u s l y , b u t t h e y were o f t e n observed t o be i n synchrony w i t h one another.  Very s i m i l a r movements were seen i n e t h e r i s e d  which were d e c a p i t a t e d .  When t h e s e h e a d l e s s f l i e s  e t h e r , t h e y r i g h t e d themselves  and t h e n shuddered  flies  r e c o v e r e d from occasionally,  a l t h o u g h t h e s h u d d e r i n g seemed i n f r e q u e n t when compared w i t h whole  flies.  None o f t h e s e p a t t e r n s o f b e h a v i o u r was observed i n whole or d e c a p i t a t e d FM6 or Oregon-R  flies.  Shu/Y males were extremely shaky. backs f r e q u e n t l y .  They were thrown onto  their  Under e t h e r t h e y shook t h e i r l e g s more v i g o r o u s l y  than the heterozygotes.  When t h e y were a b l e t o walk, t h e males u s u a l l y  dragged t h e i r wings which were s h r i v e l l e d and h e l d l a t e r a l l y ,  drooping  towards t h e t i p s . Shuddering mapped a t 55.1 - 0.4*.  T h i s p o s i t i o n was c a l c u l a t e d  by t h e r e l a t i v e p o s i t i o n o f t h e mutation observed between v and f and p l a c e d i n t o t h e book d i s t a n c e . is  The book v a l u e f o r t h e v t o f d i s t a n c e  23.7 u n i t s whereas t h e observed d i s t a n c e was 19.6 u n i t s .  b e h a v i o u r o f recombinants  The  a f t e r r e c o v e r y from e t h e r i n d i c a t e d t h a t  s h u d d e r i n g , l e g s h a k i n g and wing s c i s s o r i n g were n o t s e p a r a b l e by c r o s s i n g over and t h e r e f o r e were p h e n o t y p i c m a n i f e s t a t i o n s o f t h e same mutation.  E f f e c t s o f monovalent c a t i o n s on shuddering b e h a v i o u r T a b l e 1 summarises t h e r e s u l t s o f two experiments which t e s t e d  * The  95$ c o n f i d e n c e l i m i t s were c a l c u l a t e d , b y t h e method o f Stevens (19^2)  TABLE 1 Frequency of spontaneous shuddering i n Shu/FM6 f l i e s a f t e r d i f f e r e n t s a l t treatments. Frequency i s expressed as shudders per f l y per minute. Where death has occurred, the survivors are presented i n brackets as a f r a c t i o n of the o r i g i n a l number of f l i e s . Experiment 1 Age i n days Days on treatment Molarity 0.0 Salt /0.12 [0.18 L i C l J o . 24 /0.30 \ 0,36  Experiment 2  0-2  1-3  2-4  0  1  2  7.1  13.1  12,4  4,4 7.3 7.0 6.1(7/8) 5.5(7/8)  7.5 12.5 11.5 5.6(7/8) 2.2(5/8)  11,4 9.1 17.8 9.7(7/8) 0.0(5/8)  0.32 0.33 0,34 0.35 0.36  1-1 1/2 1  2-2 1/2  3-3 1/2  4-4 1/2  2  3  4  4.7  6.8  8.1  0.4(7/8) 0.5(6/8) 0.0(7/8) 0.1(6/8) 0.8  0.5(6/8) 5.3(6/8) 0.0(4/8) 1.0(5/8) 3.2  0.0(6/8) 0.0(6/8) 0.0(4/8) 0.0(5/8) 1.6  0,0(6/8) 1.0(2/8) (0/8) 0.0(4/8) 0,0(5/8)  Molarity 0.0 2  /0,12 10.18 NaCl "S0.24 J0„30 \0.36  9.7 10,1 7.9 9.9 12,3  15.8 14,9 7.9 13.5 12.4  13.0 9.9 12,1 15.3 12.3(6/8)  0.32 0.33 0.34 0.35 0.35  1.6 4,0 2,6 1.5 3.2  4.5 7.7 8,8 4.3 6.0  6,3 8.8 10.3 10.1 9.3  8.9 8.8 11.1 10,9 9.0  /0,12 0,18 N%C1 { 0.24 0.30 \0.36  8,8 3.5 12.4 12.1 9.8  9.4 14.9 7.9 13.5 12.4  0.8 5.5 7.3 14,5 4.0  0.32 0.33 0.34 0.35 0.36  3.7 1.1 4.3 2,6 2.6  12.6 6.9 5.0 2.8 8,6  6.8 5.9 4.9 3.6 1.8  9.0 9.0 3.9 3.8 5.8  0.32 0.33 0.34 0.35 0.36  5.3 3.2 3.6 1.7 1.0  2.1 3.1 0.8 0.4 0.9  0.5 3.1 5.9 3.^ 2.3  4.4 5.0(7/8) 5.8 2.0 1.9  / KCl  \ \  27 the e f f e c t s o f v a r i o u s s a l t s on spontaneous Shu/FM6  flies.  the f l i e s  The experiments  shuddering i n u n d i s t u r b e d  d i f f e r e d w i t h r e s p e c t t o t h e ages o f  and t h e times a t which o b s e r v a t i o n s were made r e l a t i v e t o t h e  beginning of treatments.  The shuddering f r e q u e n c y o f f l i e s  s t a n d a r d media and N a C l media i n c r e a s e d w i t h time. seen i n f l i e s placed on NH^Cl and KC1.  p l a c e d on  No c l e a r t r e n d s were  0„32M t o 0,35M L i C l  seemed t o  suppress shuddering a f t e r t h r e e or f o u r days o f t r e a t m e n t , however many f l i e s  died during t h i s  time,  A second s e t o f experiments  t e s t e d t h e c a p a c i t y o f L i C l t o suppress  s h u d d e r i n g i n d u c e d by m e c h a n i c a l s t i m u l a t i o n and attempted t o i n c r e a s e s u r v i v a l by r e t u r n i n g t h e f l i e s times.  The number o f f l i e s  t o s t a n d a r d media a t v a r i o u s c r i t i c a l  p l a c e d on each treatment t o g e t h e r w i t h t h e  number which s u b s e q u e n t l y d i e d i s p r e s e n t e d i n T a b l e 2, of  t h e t r e a t m e n t s which suppressed s h u d d e r i n g a r e p r e s e n t e d i n F i g u r e  A l t h o u g h many f l i e s survivors  d i e d on  (102 out o f 131)  0.32M and 0.33M L i C l ,  a f t e r t h r e e days o f treatment w i t h  Shu/FM6  flies  03 .2M or 0.33M L i C l .  series  would be shuddering a t an average r a t e o f 7 times a minute.  flies  on  0.33M  NaCl.  The  conditions,  o f an age comparable t o t h e t r e a t e d  was a l s o suppressed i n a group  The  s u p p r e s s i o n o f shudder-  p r e v i o u s d a t a i n d i c a t e d t h a t even under l e s s d i s t u r b i n g untreated  10.  t h e number o f  d i d n o t change from day 6 t o day 13.  t e s t s c l e a r l y i n d i c a t e d a r e d u c t i o n or complete ing  The r e s u l t s  Shuddering  o f 5 f l i e s p l a c e d on 0.2M L i C l and 5  Shuddering  c o u l d n o t be e l i c i t e d from t h e l a t t e r  group when t h e y were f i r s t p l a c e d on t h e t r e a t e d media.  This  s i v e b e h a v i o u r was o c c a s i o n a l l y seen i n v e r y young f l i e s . was e a s i l y e l i c i t e d on subsequent  days.  nonrespon-  Shuddering  Therefore, the i n i t i a l  o b s e r v a t i o n p r o b a b l y d i d n o t r e f l e c t an e f f e c t o f t h e NaCl,  28 TABLE 2 T o t a l numbers of f l i e s tested f o r shuddering induced by mechanical stimulation a f t e r d i f f e r e n t s a l t treatments.  0.32M  0.33M  66 ( 1 5 died)  6 5 (14 died)  0.2M 25  LiCl  5 ( 1 died)  NaCl  10  NH^Cl Standard Media  45  5  5  10  10  FIGURE 10 Reduction of shuddering i n Shu/FM6 f l i e s subjected to mechanical stimulation. White squares indicate s l i g h t shuddering, black squares no shuddering. Arrows indicate duration of treatment. Survivors are presented as a f r a c t i o n of the o r i g i n a l sample s i z e , M and N s i g n i f y observations made at midnight and noon, r e s p e c t i v e l y . Time i n days Age of f l i e s  # Observations per 24 hours  Survivors/ f l i e s tested  Treatment  0  1  ..  2  3  M  M  0-1  1  29/40  ,32M L i C l  0-1  1  30/40  ,33M L i C l  1/2-1  2  4/6  .32M L i C l  1/2-1  2  4/5  .33M L i C l  1-11/2  2  9/10  ,32M L i C l  1 - 1 1/2  2  8/10  .33M L i C l  1 - 1 1/2  2  9/10  ,32M L i C l  M N M • • •  1 - 1 1/2  2  9/10  ,33M L i C l  M N M • % •  1/2-1  2  5/5  .2 M L i C l  1/2-1  2  5/5  ,33M NaCl  M  M  N M N • • M N M N  • • • •  •  M N M  M • .  N •  6  5  »"  n  HMD • • •  4  N M  • •  M •  •  M •  N M N M  m  mm  N •  M a  M N UP  t  30 L e t h a r g i c behaviour and a f i n e tremor which a r e symptoms t o x i c l e v e l s o f l i t h i u m i n r a t s and men  (Schou, 1958,  1959),  of were  •oe:casi«nal.]^ seen i n Shu/FM6 i n d i v i d u a l s and were u s u a l l y f o l l o w e d by death.  However, most f l i e s i n which shuddering had been suppressed  showed n e i t h e r o f t h e s e symptoms.  I n f a c t , t h e y appeared t o be more  a c t i v e t h a n u n t r e a t e d Shu/FM6 f l i e s  o f t h e same age.  ts I s o l a t i o n of p a r a An e s t i m a t e o f t h e number o f f l i e s screened f o r s e x - l i n k e d r e c e s s i v e or autosomal dominant t e m p e r a t u r e - s e n s i t i v e p a r a l y s i s  was  o b t a i n e d by c o u n t i n g t h e number o f f l i e s i n one or two b o t t l e s out o f every t h i r t y s c r e e n e d .  The r e s u l t s a r e summarised i n T a b l e  Of t h e e s t i m a t e d 250,000 f l i e s 29°C,  200  screened, 293  o f t h e s e were found t o be dead.  3°  were i m m o b i l i s e d a t  I t i s n o t known whether  d e a t h o c c u r r e d b e f o r e , d u r i n g or a f t e r t h e s c r e e n i n g p r o c e s s . 93 was  f l i e s which r e c o v e r e d m o b i l i t y a t 22°C, 34 were f e r t i l e .  Of t h e One f l y  found t o t r a n s m i t h e r i t a b l e t e m p e r a t u r e - s e n s i t i v e p a r a l y s i s .  s e x - l i n k e d mutation, p a r a ^ . mapped a t 53<>9 ( S u z u k i e t a l , , s  I t i s thought t h a t a much l a r g e r number of f i i e s was  The  1971).  screened f o r  dominant t e m p e r a t u r e - s e n s i t i v e p a r a l y s i s , a l t h o u g h e s t i m a t e s o f t h i s number were n o t made. may  The chances  o f f i n d i n g t h e dominant  mutation  have been a l t e r e d by t h e use o f two types of s c r e e n i n g d e v i c e s  r a t h e r t h a n one.  For t h e s e r e a s o n s , a v a l i d comparison  of t h e two s c r e e n i n g procedures  of t h e success  cannot be made. ts  E f f e c t s o f d i f f e r e n t temperatures o At 22  on p a r a  flies  ts C, p a r a  flies  e x h i b i t e d normal w a l k i n g , c l i m b i n g and  31 TABLE 3  E s t i m a t e d number o f f l i e s s c r e e n e d f o r s e x - l i n k e d r e c e s s i v e and autosomal dominant t e m p e r a t u r e - s e n s i t i v e p a r a l y s i s .  Type o f f l i e s  Number  Total f l i e s  screened  250,000  Immobilised  a t 29°C  293 200  Dead Recovered m o b i l i t y a t 22°C  93  F e r t i l e a t 22°C Temperature-sensitive  p a r a l y t i c mutations  1  32 flying abilities.  When shaken  i n t o v i a l s which had been p r e h e a t e d t o  o 29 C, they became p a r a l y s e d w i t h i n 5 seconds.  Upon r e t u r n i n g t o 22  t h e y r e g a i n e d v e r y a c t i v e m o b i l i t y w i t h i n 2 or 3 seconds.  0  C,  Paralysis  and  r e c o v e r y c o u l d be induced r e p e a t e d l y i n t h i s way w i t h no apparent harm to did to  o ts When kept a t 29 C f o r p r o l o n g e d p e r i o d s , p a r a flies  the f l i e s .  not remain p a r a l y s e d . their feet.  At s t i l l  A f t e r 30 minutes, most f l i e s were a b l e t o get  l a t e r t i m e s , they were a b l e t o walk and c l i m b .  However, t h e s e f l i e s never e x h i b i t e d t h e same degree o f c o o r d i n a t i o n and a c t i v i t y t h a t they had shown a t 22  o  C,  At any time, a s h i f t t o 22  would r e s u l t i n an immediate r e c o v e r y o f w i l d type  W  C  activity.  The mutation d i d n o t appear t o a f f e c t v i s c e r a l muscles. s h i f t from 22 C t o 29  o  After a  C, h e a r t pumping c o u l d s t i l l be seen t h r o u g h t h e t<5  d o r s a l abdominal  c u t i c l e of para  flies.  a 22°C t o a 29°C D r o s o p h i l a R i n g e r s s o l u t i o n the p a r a ^  s  A f t e r being t r a n s f e r r e d  from  ( E p h r u s s i and Beadle, 1936),  e j a c u l a t o r y duct a l s o continued t o p u l s a t e .  None o f t h e l a r v a l i n s t a r s was  a f f e c t e d by t r a n s f e r from a 22°C  c u l t u r e t o D r o s o p h i l a R i n g e r s s o l u t i o n s kept a t 29°C, comparative study o f t h e development o f p a r a ^ / Y s  and  However, a X^,p_ara^f /Y +  s i b l i n g s i n d i c a t e d t h a t the number o f s u c c e s s f u l e c l o s i o n s by t h e males was  normal when pupae r a i s e d a t 29°C were s h i f t e d t o 22°C p r i o r t o  e c l o s i o n , but g r e a t l y reduced when pupae were m a i n t a i n e d a t 29°C ( S u z u k i et a l , , 1971)„ The b e h a v i o u r a l responses t o i n c r e a s e d temperature were s t u d i e d i n f l i e s which had been r a i s e d a t 22°C by shaking f l i e s from 22°C v i a l s i n t o s e p a r a t e water-heated b e h a v i o u r were made on 20  chambers.  Observations o f changes i n  o f each sex of p a r a  t s  one degree i n t e r v a l s over a 22°G t o 35°C range.  and Oregon-R f l i e s a t Behaviour was  monitored  33 continuously for the f i r s t hour and then once every 10 minutes over the second hour. For purposes of general comparison,  s p e c i f i c types of behaviour  were c l a s s i f i e d i n the following order of increased motor competence; p a r a l y s i s , leg shaking, s e l f - r i g h t i n g to a standing position, walking, Figure 11 i s a graph of these motor a c t i v i t i e s ,  climbing, f l y i n g .  exhibited by at least 50$ of the f l i e s , p l o t t e d on an a r b i t r a r y scale against time.  Recovery rates were f a r from uniform within a group;  i n i t i a t i o n of the same a c t i v i t i e s being separated by as much as 50 minutes i n d i f f e r e n t f l i e s .  Although the average mobility c l e a r l y  improved with time, the recovery of each f l y was not always progressive. F l i e s which had demonstrated good walking and climbing a b i l i t i e s would occasionally revert to a very weak stance f o r several minutes.  Even  within a single f l y , recovery did not appear t o be uniformly progressive. Often one or two legs on one side of the body would move while the legs on the opposite side remained s t i l l .  Differences i n the extension of  the legs frequently caused the f l y t o l i s t while standing and p i t c h and r o l l while walking. temperatures  The d e b i l i t a t i n g effects of progressively higher  were manifested i n both the extent of the i n i t i a l response  and the length of time taken t o recover further a c t i v i t y at various temperatures,  para  ts  f l i e s were noticeably d e b i l i t a t e d by s h i f t s to  the 26°C t o 28°C range. o paralysed.  At higher temperatures  they were completely  ts  At 29 C, para  f l i e s were able to climb after 70 minutes;  o at 31 C, 105 minutes were needed t o regain the same a c t i v i t y .  After  two hours at any temperature up to 33°C, the f l i e s were able to recover mobility upon returning t o 22°C„  However, a two hour exposure to 34° C  resulted i n 90$ of the f l i e s dying.  Oregon-R f l i e s were unaffected by  34 FIGURE 11  Recovery  o f motor competence w i t h time a f t e r i n c r e a s e s i n temperature.  FLYING  PARALYSIS  60 TIME (MINUTES)  90  120  35 r a n g i n g from 22°C t o 37°C,  two hour exposures t o temperatures A list  o f the types o f movements observed d u r i n g r e c o v e r y a t  i m m o b i l i s i n g temperatures T a b l e 4,  i s p r e s e n t e d i n r o u g h l y c h r o n o l o g i c a l order i n  A l l o f the movements l i s t e d were r a r e l y seen i n a s i n g l e f l y .  However, they were crude i n d i c a t o r s o f l e v e l s o f r e c o v e r y , o A l t h o u g h 22  C had been chosen as t h e p e r m i s s i v e temperature f o r  s c r e e n i n g and most s h i f t experiments, i t was that para  "t s  f l i e s r a i s e d a t 17  t r a n s f e r r e d t o 22°C a few seconds.  0  o C became  subsequently discovered  slow or immobile when  Upon r e t u r n i n g t o 17°C,  t h e y became a c t i v e w i t h i n  Thus i t appeared t h a t the p a r a  t h e f l y s e n s i t i v e t o temperature  ts  mutation had r e n d e r e d  i n c r e a s e s over a t l e a s t a 17°C  to  o 29  C range. The experiments which f o l l o w e d t h e d i s c o v e r y o f 22°C s e n s i t i v i t y  were d e s i g n e d t o t e s t a simple model which had been used t o e x p l a i n  ts p a r a l y s i s and r e c o v e r y phenomena.  For h e u r i s t i c purposes, p a r a  might be assumed t o produce an enzyme whose c a t a l y t i c e f f i c i e n c y an i n v e r s e f u n c t i o n o f temperature temperature)  ( i , e , , Km  was  i s a d i r e c t f u n c t i o n of  and whose r e a c t i o n p r o d u c t p e r m i t t e d m o b i l i t y o f the f l y  when t h e p r o d u c t was  formed a t a s u f f i c i e n t r a t e .  Given equal s u b s t r a t e  c o n c e n t r a t i o n s , the r a t e o f p r o d u c t f o r m a t i o n would d i m i n i s h a t h i g h temperatures  and motor a c t i v i t y would consequently d i m i n i s h a l s o .  However, t h e reduced e f f i c i e n c y o f t h e enzyme would a l s o cause t h e s u b s t r a t e c o n c e n t r a t i o n t o i n c r e a s e i f t h e s u b s t r a t e were s u p p l i e d t o t h e enzyme a t a s u f f i c i e n t r a t e .  I f t h e s u b s t r a t e l e v e l then became  h i g h enough t o overcome the i n c r e a s e d Km o f t h e enzyme, p r o d u c t f o r m a t i o n might a g a i n r e a c h a r a t e which would permit m o b i l i t y .  This hypothesis  i s p r e s e n t e d as an example o f a b a s i c concept, namely a "damming up"  36 TABLE 4  Types o f movement which may be seen d u r i n g r e c o v e r y from temperaturei n d u c e d p a r a l y s i s . The l i s t i s i n r o u g h l y c h r o n o l o g i c a l o r d e r ,  1,  P a r a l y s i s (legs  2,  Extension  3,  Tarsal twitching.  4,  Extension  5,  Slow movements o f the wings l a t e r a l l y  6,  V e n t r a l f l e x i n g o f the p o s t e r i o r abdomen. genitalia.  7,  Leg  8,  Righting.  9,  Walking,  and  Preening,  11.  Climbing. Flying.  c o n t r a c t i o n of the  c o n t r a c t i o n of the  Excretion.  extension  10.  12.  and  contracted), femurs.  tibias, or d o r s a l l y and v e n t r a l l y , Movements of  Head nodding.  f o l l o w e d by pawing movements.  the  37 effect.  The a c c u m u l a t i o n o f a n e u r a l t r a n s m i t t o r might have f i t t h e  o b s e r v a t i o n s as w e l l , i f t h i s p r o c e s s were more c l e a r l y  understood.  I f t h e n o t i o n o f damming up s u b s t r a t e were c o r r e c t , f l i e s c o n d i t i o n e d at  17°C  to  t a k e l o n g e r t o r e c o v e r m o b i l i t y a t 29°C t h a n f l i e s  w i t h a r e l a t i v e l y low s u b s t r a t e c o n c e n t r a t i o n , would be  A c c o r d i n g l y , 44 p a r a ^ at  s  flies  22°C were d i v i d e d i n t o two  w h i l e t h e o t h e r was  (0  c o n d i t i o n e d t o 22°C.  t o 3 days o f age) which had been r a i s e d  e q u a l groups.  p l a c e d a t 17°C  One group was  f o r t h r e e days.  left  groups  O  f l i e s were t h e n s i m u l t a n e o u s l y t r a n s f e r r e d t o 29.5  para  d e t a i l e d comparison two  a t 22°C,  22°C and 17°C  4- c  of  expected  groups  o f 19  b a s i c parameters  was  made o f two groups  C.  A  of 3 f l i e s w h i l e the remaining  f l i e s were observed more c a s u a l l y f o r the r e c o v e r y o f o f m o b i l i t y , such as s t a n d i n g , w a l k i n g and c l i m b i n g .  By a l l measured c r i t e r i a , 17°C-conditioned f l i e s r e c o v e r e d more s l o w l y than f l i e s  c o n d i t i o n e d t o 22°C.  The d i f f e r e n c e i s i l l u s t r a t e d by t h e  graph shown i n F i g u r e 12 i n which t h e numbers o f f l i e s i s p l o t t e d against time. approach  able t o stand  Only a f t e r more t h a n an hour do the two  curves  each o t h e r .  A l t h o u g h t h e s e experiments  s u p p o r t the " s u b s t r a t e a c c u m u l a t i o n "  model, t h e y a l s o suggest t h a t t h e time t a k e n f o r r e c o v e r y i s i n v e r s e l y r e l a t e d t o t h e temperature  shock.*  While temperature  i n t u i t i v e l y u n d e r s t a n d a b l e term, f o r purposes d e f i n e d here as i n c r e a s e i n body temperature  shock i s an  o f c l a r i t y i t s h a l l be per u n i t o f time.  Thus i t s  magnitude a t any i n s t a n t i s a d i r e c t f u n c t i o n o f the d i f f e r e n c e i n temperature between t h e f l y ' s body and i t s environment.  * I am g r a t e f u l t o Dr. Hans S t i c h f o r r a i s i n g t h i s i m p o r t a n t p o i n t .  38 FIGURE  12  The number of p a r a f l i e s , preconditioned at 22°C ( s o l i d l i n e ) and 17°C (dashes) which were able t o stand a f t e r being transferred t o 29.5°C t s  22 20  Time (Minutes)  39 I f the temperature were r a i s e d at d i f f e r e n t rates over the same i n t e r v a l , and w i t h i n a short enough  time to induce p a r a l y s i s , the rates  of recovery should d i s t i n g u i s h between the v a l i d i t i e s of the two arguments.  According t o the "shock hypothesis", the f a s t e r r i s e i n  temperature should prolong recovery.  On the other hand, the quicker  a r r i v a l at the high temperature should cause the substrate t o accumulate at i t s maximum r a t e from an e a r l i e r time, thus shortening the time needed f o r recovery by the "accumulation hypothesis". The e f f e c t of temperature shock was studied i n four groups of ts para  f l i e s , each group being comprised of 5 males and 5 females, 0  t o 2 days o l d .  Observations of the number of f l i e s able to stand  were made once a minute during and f o l l o w i n g each temperature r i s e . When the temperature was r a i s e d from ( 2 2 , 2 ° - 22.4°C) t o ( 2 7 , 0 ° - 27,6°C) w i t h i n a 14 (Figure 13 a) and 6 minute (Figure 13b) i n t e r v a l , the response was d r a m a t i c a l l y d i f f e r e n t .  The same e f f e c t s can be seen i n  Figures 14a and 14b, although t h i s comparison may not be v a l i d since the f a s t e r temperature r i s e was a l s o greater by one degree, t s  These experiments suggest that para  f l i e s are s e n s i t i v e not only  to temperature, but a l s o t o temperature shock.  The e f f e c t of shock was  seen i n the f l y ' s i n i t i a l response and i n i t s r a t e of recovery.  The  r e s u l t s c o n t r a d i c t a "substrate accumulation hypothesis", ts The responses of para  f l i e s i n the above experiments strongly  suggest that the monitored temperatures were underestimates of the a c t u a l chamber temperatures.  The source of e r r o r was probably heat loss  from the thermistor r e s i s t o r by conduction along 1 cm,of probe w i r e . Fortunately the s i g n i f i c a n c e of these experiments i s not changed by this error.  40  FIGURE 13 The number of p a r a f l i e s able to stand when temperatures were ehan from (22.2°- 22,4°C) to (27.0° - 27.6°C) within (a) 14 minutes, (b) 6 minutes. S o l i d l i n e - number of f l i e s able t o stand; dashes temperature. u s  Z o  z < 0  20 —I— Time  (Minutes)  40  60  80  I(0  OT UJ u. o  (b)  28°  cz Hi  CO  2.T  z  v. 25 •«««. *  01  24'  22  A  41 FIGURE 14 .ts The number o f p a r a f l i e s a b l e t o s t a n d when temperatures were changed from (21.6° - 22 cT~to (28.5° - 29.5°C) w i t h i n (a) 24 minutes, (b) 6 minutes. S o l i d l i n e - number o f f l i e s s t a n d i n g ; dashes temperature. J  u  a z <  I  0  20  to  Time  I (b)  29°.  f-  40  60  co  80  100 -f—  120  (Minutes)  u. o  ' ^  ce  i l i  jo  26°J / 27  9 8  26*1  7  2S  6 5  5  4  2,4° 23? 22°  co  A  r\  3 2 I 10  Ul CO 2  z  I n t e r a c t i o n s between p a r a P u t a t i v e Neuro p a r a  ts  and n e u r o l o g i c a l mutants  ts /  /Y males e x h i b i t i n g t h e c h a r a c t e r i s t i c s o f ts b o t h mutations were r e c o v e r e d from Neuro/para females. The s t a n d a r d can be seen i n F i g u r e 1 5 ,  p o s i t i o n s o f t h e Neuro mutations  The number  ts / of  HK p a r a  /Y recombinants  was l e s s than expected, w h i l e t h e number o f  p a r a ^ S h / Y males agreed w e l l w i t h t h e o r e t i c a l r e c o m b i n a t i o n d i s t a n c e s 5  (Table 5 ) .  These d i s c r e p a n c i e s p r o b a b l y r e f l e c t d i f f e r e n c e s i n s u r v i v a l  under crowded c o n d i t i o n s . The double mutant n a t u r e o f t h e p h e n o t y p i c a l l y s e l e c t e d was  c o n f i r m e d i n a l l b u t two c a s e s .  recombinants  In both cases, t h e exceptions  r e s u l t e d from c l a s s i f i c a t i o n o f HK /Y males as H K 1P  1 P  para /Y. t s  Each  double mutant was v e r i f i e d by r e i s o l a t i n g s i n g l e mutant chromosomes by r e c o m b i n a t i o n ( T a b l e 6), The response o f Neuro p a r a / Y t s  males t o a 2 2 ° C t o 2 9 ° C s h i f t was  a q u i c k c e s s a t i o n o f a l l movement whether induced by e t h e r a n a e s t h e t i s a t i o n or n o t . at  A l l movements c o u l d be r e s t o r e d a t 2 2 ° C and stopped a g a i n  2 9 ° C w i t h i n a few seconds, HK  29°C,  /Y f l i e s were a l s o somewhat s e n s i t i v e t o s h i f t s from 2 2 ° C t o E t h e r - i n d u c e d l e g s h a k i n g would cease w i t h i n 1 5 seconds t o a  minute o f exposure t o 2 9 ° C ,  U n l i k e t h e l e g s o f HK"*" para^ /Y, t h e s e l e g s P  began t o shake a g a i n a f t e r about a minute.  S  Leg s h a k i n g c o u l d a l s o be  r e i n i t i a t e d b y r e t u r n i n g t h e HK /Y f l i e s t o 2 2 ° C , b u t c o u l d n o t be 1P  stopped a g a i n by r e t u r n i n g them t o 2 9 ° C ,  IP  t h a t t h e s e n s i t i v i t y o f HK  n o t always, be demonstrated. from t h a t o f HK n onr e p e a t a b l e .  para /Y t s  I t s h o u l d a l s o be  /Y f l i e s t o temperature  emphasised  c o u l d u s u a l l y , but 1P  I n s h o r t , t h e response o f HK  /Y  differed  by b e i n g slow, u n r e l i a b l e , u n s u s t a i n e d and  43  FIGURE 15  ts G e n e t i c l o c a t i o n s o f Hk  5  para  5  and Sh_  on t h e X chromosome.  pord-53.9 iJK-30.5  I  v-33.0  -56.7  TABLE 5 Recombination data f o r para  Maternal parent + para /HK + ts  tS  + para  ts  # female progeny  1P  lrj 21  ,  /HK  +  V  and d i f f e r e n t  5 2 3  421  Neuro mutants, Male progeny recovered  Non-crossovers HK'IP  Crossovers 2 6 1  +  para  2T HK'  +  +  oara  +  SJr  t s  t s  Crossing over  +  102  HK  176  +  108  HK  +  1P  para  4 9  t s  +  2T  para  (23.4)  1 7 o 9  ( 2 3 . 9 )  11  4 5  t s  14,2  1 7  -3-  para  ts  , +/+  5  Sir n  1  5 9 0  para  * f i g u r e i n bracket i s standard d i s t a n c e .  ts ^ +  1 5 4  1 1 6  +  para  ts  +  4  5 SIT  5 e  3 o 2  (3.8)  1  TABLE 6 Reisolation  o f s i n g l e mutation  chromosomes by r e c o m b i n a t i o n  o f double  mutants.  Male progeny r e c o v e r e d Maternal parent  HK para /++ 1 P  t s  # female progeny  911  Non-crossovers  +  HK para /++ 2 T  t s  2,261  HK  +  2 T  +  para  t s  Sh /++ 5  706  +  para +  *  para  f i g u r e i n bracket i s standard d i s t a n c e .  t s  268  489  t s  7  Sh +  Crossovers  210  HjT^para^.  3  5  $ Crossing over  HT^ +  HK  92  + para  +  2 T  t s  62  224  5  +  Para  193  +  Sh£  18  314  para  +  7  t s  t s  24.4 (23.4)  23.5 (23.4)  152  5.1  (3.8)  46 The behaviour 22°C  o f HK /Y and H K p a r a / Y f l i e s were s i m i l a r a t 1P  1 P  t s  Both showed a k i n e t o g e n i c response  0  (jumping i n response t o hand  movements) and b o t h shook t h e i r l e g s v i g o r o u s l y under e t h e r a n a e s t h e s i a , HK  para  /Y f l i e s  movements a t 22°C„  d i d not show a k i n e t o g e n i c response t o hand  Under e t h e r , l e g shaking was  v e r y subdued when  2T / compared w i t h HK the tarsae.  IT and i n many c a s e s , was  A t 22°C, S]£ p a r a  shaking seen i n Sh^/Y did  not  mutants under e t h e r a n a e s t h e s i a , b u t the wings  The v i g o r o u s l e g shaking o f HK ts were r e s t o r e d when t h e p a r a s  of  /Y males e x h i b i t e d t h e c h a r a c t e r i s t i c l e g  scissor,  Neuro p a r a ^  reduced t o a r a p i d tremor  chromosomes by  2T  /Y and t h e wing s c i s s o r i n g o f  mutation was  5  ShPJj  removed from t h e r e s p e c t i v e  recombination,  ts I n t e r a c t i o n between p a r a Of t h e Minutes  Minutes  t e s t e d i n combination w i t h p a r a  i n t e r a c t e d t o produce shift,  and  M(l)0 f/para^  t s  . only M(l)0  a change i n b e h a v i o u r f o l l o w i n g a 22°C t o 29°C s  females assumed s t a t i o n a r y p o s i t i o n s on t h e  floor  and w a l l s o f t h e o b s e r v a t i o n chamber f o r 3 minutes or more w h i l e FM6/Y and FM6/para  c o n t r o l s remained  homozygotes, M ( l ) 0  f/para^  s  highly active.  U n l i k e para  f l i e s were a b l e t o remain  a f t e r t h e r i s e i n temperature,  w a l k i n g and c l i m b i n g a c t i v i t y took about two hours. f l i e s , m o b i l i t y c o u l d be q u i c k l y and t o 22°C,  feet  although the stances of the f l i e s  weak and t h e f i r s t movements were extremely slow.  the f l i e s  on t h e i r  seemed  The r e c o v e r y o f normal ts As w i t h p a r a  c o m p l e t e l y r e s t o r e d by  returning  47 DISCUSSION  The  s e a r c h f o r i n h e r i t e d t e m p e r a t u r e - s e n s i t i v e p a r a l y s i s has l e d t o  t h e d i s c o v e r y o f two s e x - l i n k e d para  mutations  -(53.9) and Shu - ( 5 5 . 1 ) .  t s  which a f f e c t motor a c t i v i t y ;  A cross of S h u / p a r a  t S  females by p a r a ^ / Y s  males f a i l e d t o y i e l d w i l d types o r double mutant recombinants 2,256 progeny.  T h e r e f o r e , the Shu and p a r a  t s  mutations  among  may be much  c l o s e r than f i r s t estimates had i n d i c a t e d . ts A t h i r d mutation, M(1)0 - 5 6 . 5 ) , appeared t o produce  t o i n t e r a c t with para ts an a l t e r e d p a r a l y t i c phenotype i n p a r a /M(1)0 f e m a l e s . ts  However, a d i r e c t comparison y e t been made. studied  Furthermore,  o f such females w i t h p a r a  the M ( l ) Q - b e a r i n g chromosome was n o t  g e n e t i c a l l y or c y t o l o g i c a l l y .  pre-existing  f l i e s has n o t  Thus, i t i s n o t known whether a  para a l l e l e or d e l e t i o n o f the para" " l o c u s e x i s t s on t h e 1  chromosome. U n t i l further  relationships  i n f o r m a t i o n i s o b t a i n e d on t h e g e n e t i c and f u n c t i o n a l ts  o f the Shu and p a r a  shuddering and p a r a l y s i s  mutations, an u n d e r s t a n d i n g o f  can o n l y be i n f e r r e d by p h e n o t y p i c  symptoms  and t h e a l t e r a t i o n o f t h e i r e x p r e s s i o n by g e n e t i c , c h e m i c a l and p h y s i c a l manipulation. Shu/FM6 f l i e s a r e s i m i l a r t o the homozygous n e u r o l o g i c a l  IP HK  2T 5 , HK and S h ,  mutants,  i n t h a t t h e y a l l e x h i b i t abnormally v i g o r o u s and  r a p i d l e g shaking under e t h e r a n a e s t h e s i a .  IP  The b e h a v i o u r o f HK  /+  2T and HK  /+ females was found t o be v e r y d i f f i c u l t  e i t h e r Canton-S o r Oregon-R w i l d t y p e f e m a l e s .  to distinguish  The l e g shaking o f  Sh /+ was subdued compared w i t h the homozygous females, but not 5  suppressed.  from  Comparisons were drawn between t h e homozygous  completely  neurological  48  mutants ( H K , H K 1P  2T  and Sh ) 5  phenotypic closeness;  and Shu/FM6 females because of t h e i r  a l b e i t the genetic comparison i s i n c o n s i s t a n t .  The movements of Shu/FM6 and S h  5  f l i e s under ether are s i m i l a r i n  t h a t they both s c i s s o r t h e i r wings and halteres as w e l l as nodding t h e i r heads and extending t h e i r antennae.  However, when Shu/FM6 and S h  were e t h e r i s e d simultaneously and examined together, S h  5  5  f l i e s were  found t o begin shaking t h e i r legs much sooner than Shu/FM6 f l i e s d i d . The wing s c i s s o r i n g of S h  5  f l i e s involved shorter, quicker movements.  was also noted that the leg shaking movements of S h  5  It  f l i e s involved a l l  segments of l e g while the movements of Shu/FM6 f l i e s were mainly confined t o the t i b i a and t a r s u s .  Thus, S h  5  and Shu/FM6 were pheno-  typically distinguishable. 5 Sh , HK  IP  2T and HK  f l i e s jump i n response t o the v i s u a l stimulus  of a hand movement or t o mechanical a g i t a t i o n (Kaplan and Trout, 1969). However, Shu/FM6 f l i e s responded only to mechanical s t i m u l a t i o n and d i d so by shuddering while standing i n one spot.  I n d i v i d u a l Shu/FM6 females  continued t o shudder p e r i o d i c a l l y , even when l e f t f o r long periods i n undisturbed i s o l a t i o n , IP The pattern of r a p i d l e g shaking i n HK  f l i e s has been demonstrated  to r e s u l t from r a p i d bursts of neural a c t i v i t y (Ikeda and Kaplan, 1970a), The f a c t that the movements of the wings, h a l t e r e s , head and antennae of S h  5  f l i e s and Shu/FM6 females were u s u a l l y i n phase with one another  a l s o suggests they were e l i c i t e d by a common neurogenic a c t i v i t y rather than independent myogenic impulses. The p o s s i b i l i t y that l i t h i u m might suppress the shuddering behaviour of Shu/FM6 f l i e s was suggested by i t s therapeutic e f f e c t s on analogous disorders i n guinea p i g and man (Cade, 1949),  The analogies drawn  49 between f l i e s and man were crude.  Y e t the v a r i e t y o f b e h a v i o u r a l  d i s o r d e r s which t h i s i o n had been shown t o suppress suggested t h a t i t a c t e d upon a common f a c t o r which might even be found i n f l i e s . Cade (1949) d i s c o v e r e d t h a t l i t h i u m s a l t s were a b l e t o suppress t h e s e v e r e c o n v u l s a n t e f f e c t o f i n t r a p e r i t o n e a l urea i n j e c t i o n s upon guinea pigs.  He a l s o d i s c o v e r e d t h a t t h e same treatment seemed t o e l i m i n a t e  t h e e x p r e s s i o n o f c h r o n i c and r e c u r r e n t mania i n man. Van der Velde (1970) subsequent patients.  According t o  s t u d i e s have i n v o l v e d more t h a n 39000  C r i t i c a l evidence f o r t h e s u c c e s s f u l treatment o f mania has  been s u p p l i e d by Maggs (I963).  S t r o n g evidence f o r i t s p r o p h y l a c t i c  a c t i o n a g a i n s t t h e r e c u r r e n c e o f d e p r e s s i o n i n manic d e p r e s s i v e psychoses et  has a l s o been p r e s e n t e d (Baasturp and Schou, 1967;  a l , , 1969)o  Shou (1959; 1963) noted t h a t u n l i k e o t h e r psycho-  t h e r a p e u t i c drugs, l i t h i u m d i d n o t suppress normal mental, or  Goodwin  physical activity.  emotional  Thus, t h e i o n seemed t o a c t upon t h e d i s o r d e r  per se. The s i m i l a r i t y between t h e shuddering b e h a v i o u r o f Shu/FM6 f l i e s and u r e a - i n d u c e d c l o n i c c o n v u l s i o n s i n guinea p i g s i s fair!Ly o b v i o u s . Both a r e sudden, v i g o r o u s , u n c o n t r o l l e d motor a c t i v i t i e s .  The syndromes  of r e c u r r e n t mania and manic d e p r e s s i o n can a l s o be compared w i t h t h e s h u d d e r i n g o f Shu/FM6 i n t h a t they a r e b o t h c h a r a c t e r i s e d by r i s e s and falls  i n motor a c t i v i t y  ( K r a e p e l i n , 1906;  A r i e t y , 1959); a l b e i t t h e  r i s e i n a c t i v i t y o f t h e f l y i s more sudden and u n c o n t r o l l e d . rises  In man t h e  i n motor a c t i v i t y a r e accompanied by a l o s s o f c o n t r o l over t h e  l a r g e r e p i t o i r e o f b e h a v i o u r which i s c h a r a c t e r i s t i c o f h i s s p e c i e s . Thus he becomes v e r b o s e , i n t r u s i v e and o c c a s i o n a l l y d e s t r u c t i v e ( K r a e p e l i n , 1906; A r i e t y , 1959).  Manic and d e p r e s s e d s t a t e s may  occur  50 repeatedly  ( L u n d q u i s t , 1945)  environmental stress Shu/FM6 f l i e s  and o f t e n cannot be a t t r i b u t e d t o  (Hudgens e t a l . , 1967)=  The shuddering o f  i s a l s o r e c u r r e n t and sponeous a l t h o u g h t h e shuddering  response can a l s o be e l i c i t e d by m e c h a n i c a l  stimulation.  Among the c h l o r i d e s a l t s t e s t e d , L i C l demonstrated c a p a c i t y t o supress b o t h i n d u c e d and spontaneous Shu/FM6 f l i e s .  However, i t was  them.  shuddering a c t i v i t y i n  n o t p o s s i b l e t o s u s t a i n doses  would supress t h e i n c r e a s e d nervous killing  a unique  a c t i v i t y of older f l i e s  which  without  I t i s i n t e r e s t i n g t o note t h a t o l d e r p e o p l e a r e a l s o l e s s  l i k e l y t o respond t o l i t h i u m t h e r a p y (Van der V e l d e , 1970). The observed t o x i c i t y o f l i t h i u m i n f l i e s suggests t h a t the s u p r e s s i o n o f shuddering might be a p a t h o l o g i c a l p r e l u d e t o death. T h i s was  r u l e d out by the s u r v i v a l o f l i t h i u m - t r e a t e d f l i e s which  ceased t o shudder  f o r one or more days, when t h e y were p l a c e d on  s t a n d a r d medium.  Most f l i e s  e x h i b i t e d w i l d type l e v e l s  had  i n which shuddering had been suppressed o f a c t i v i t y and showed no t o x i c symptoms.  The attempt t o suppress shuddering i n a l l o f t h e t r e a t e d  flies  p r o b a b l y r e q u i r e d t h e use o f s a l t c o n c e n t r a t i o n s which exceeded t o l e r a n c e o f some i n d i v i d u a l s i n t h e sample.  the  The uptake, e x c r e t i o n  and  and s e n s i t i v i t i e s t o l i t h i u m have been found t o d i f f e r among i n d i v i d u a l s i n b o t h man  and animals  (Schou, 1958,  1959;  q u e n t l y , d u r i n g l i t h i u m t h e r a p y i n man,  Weischer,  the dosage must be  w h i l e b l o o d l e v e l s and b e h a v i o u r a r e monitored was  1969).  Conse-  controlled  (Schou, 1959).  Shuddering  suppressed i n a group o f f i v e f l i e s t r e a t e d w i t h a r e l a t i v e l y  dose o f L i C l  (0.2M) ( F i g u r e 10)  which had no e f f e c t on s u r v i v a l .  However, h i g h e r c o n c e n t r a t i o n s would have exceeded and may  have k i l l e d t h e f l i e s .  low  the t h e r a p e u t i c l e v e l  51 L i t h i u m has  a l s o been found t o supress i n d u c e d a g g r e s s i o n i n r a t s ,  mice and golden hamsters as w e l l as t h e n a t u r a l a g g r e s s i o n o f male Siamese f i g h t i n g f i s h t h e s e treatments  (Weischer,  d i d not suppress  1969;  Sheard,  1970).  Once a g a i n ,  normal l e v e l s o f a c t i v i t y .  experiments were p r o b a b l y suggested  These  by a g g r e s s i v e b e h a v i o u r which i s  o f t e n d i s p l a y e d i n t h e manic s t a t e .  At t h i s time i t does not seem  p r o f i t a b l e t o examine t h e v a l i d i t y of t h i s  comparison or t h e a n a l o g i e s  which have been drawn between f l i e s and man.  On t h e o t h e r hand, the  a e t i o l o g i c a l s i g n i f i c a n c e o f t h e s e experiments cannot be i g n o r e d . r e s u l t s suggest  t h a t among the mechanisms which g i v e r i s e t o t h e s e  v a r i o u s forms o f behaviour may  The  i n such w i d e l y s e p a r a t e d organisms, t h e r e  be one which i s common t o them a l l .  I t follows that future  i n v e s t i g a t i o n s i n t o the causes of shuddering  behaviour  i n Drosophila  might b e n e f i t from i n f o r m a t i o n c o n c e r n i n g the e f f e c t s o f l i t h i u m manic d e p r e s s i v e b i o c h e m i s t r y i n man  on  and r e l a t e d b i o c h e m i s t r y i n other  animals. B e f o r e r e v i e w i n g the b i o c h e m i s t r y o f t h e s e psychoses, important  t o d i s t i n g u i s h a t l e a s t two  inherited disorders.  i t is Manic  d e p r e s s i o n appears t o be t r a n s m i t t e d as a s e x - l i n k e d , dominant  mutation a  w i t h p a r t i a l penetrance.  I t s l i n k a g e w i t h c o l o u r b l i n d n e s s and t h e  b l o o d group has been demonstrated ( R e i c h e t a l . , 1969; Tanna,  I969).  whereas  6ji  Manic d e p r e s s i v e males have f a t h e r s who  o f t h e i r mothers a r e a f f e c t e d  Winokur a r e not  (Winokur e t a l . ,  Xg  and affected  I969).  First  degree r e l a t i v e s o f manic d e p r e s s i v e probands o f t e n e x p e r i e n c e  only  depression.  relatives  Another d i s o r d e r i n which b o t h probands and t h e i r  show o n l y d e p r e s s i o n appears not t o be s e x - l i n k e d (Winokur and  1965;  Winokur et a l . ,  1969).  Pitts,  52 L i t h i u m has been shown t o a c t a g a i n s t mania and manic W h i l e l i t h i u m seems t o r e l i e v e some types (Baastrup  and Schou, 196?;  of r e c u r r e n t  Gooodwin e t a l . , 1969)  depression  I t s u s e as a g e n e r a l  treatment o f d e p r e s s e d p a t i e n t s has been q u e s t i o n e d The d i s c o v e r y , by Coppen e t a l . ,  depression.  1970).  (Van d e r V e l d e ,  (1965) t h a t l i t h i u m t r e a t m e n t  caused a drop i n i n t r a c e l l u l a r sodium, gained  s i g n i f i c a n c e i n view o f  the l a t e r d i s c o v e r y t h a t i n t r a c e l l u l a r sodium was r e l a t e d t o mood i n the f o l l o w i n g way: (Coppen e t a l . ,  Normal  1966),  Na  +  <  Depressive  Na  +  <  Manic  Na  +  I t i s l i k e l y t h a t t h e change i n i n t r a c e l l u l a r  sodium c o n c e n t r a t i o n occurs  i n nerve c e l l s as w e l l as other t i s s u e s .  L i t h i u m has been shown t o compete s u c e s s f u l l y w i t h sodium f o r t h e " e a r l y " i n f l u x channels i n t h e f o r m a t i o n Chandler,  1965; C o l e , I968).  o f a c t i o n p o t e n t i a l s (Meves and  A t t h e same t i m e , t h e a c t i v e removal o f  l i t h i u m from t h e c e l l occurs more s l o w l y t h a n sodium ( A r a k i e t a l . . 1 9 6 5 ) . The l i n k i n g nonadrenaline,  o f d e p r e s s i o n t o low l e v e l s o f s e r a t o n i n and  and o f mania t o h i g h l e v e l s o f t h e s e b i o g e n i c amines has  l a r g e l y been i n f e r r e d from t h e e f f e c t s o f r e s e r p i n e , imipramine and t h e manoamine o x i d a s e Davis,  1965;  i n h i b i t o r s on mood and body b i o c h e m i s t r y  Schildkraut,  (Bunney and  1965).  L i t h i u m i o n s have been shown t o a c c e l e r a t e t h e uptake o f n o r a d r e n a l i n e i n t o preparations  o f synaptosomes i n v i t r o  Uptake o f s e r a t o n i n i n t o t h e r e d b l o o d c e l l s  (Colburn e t a l . ,  o f manic  p a t i e n t s i s a l s o i n c r e a s e d b y l i t h i u m (Murphey e t a l . ,  1967).  deppressive  1970).  Katz e t a l . ,  (1968) d i s c o v e r e d t h a t l i t h i u m seemed t o reduce t h e r a t e a t which noradrenaline brain slices.  and s e r a t o n i n were r e l e a s e d from  electrically-stimulated  These r e s u l t s may i n d i c a t e t h a t l i t h i u m i n c r e a s e s t h e  53 e f f i c i e n c y o f t h e r e u p t a k e mechanism f o r b i o g e n i c amines, Schandberg e t a l , , (196?) found t h a t l i t h i u m a c c e l e r a t e d n o r e p i n e p h i n e c a t a b o l i s m w i t h i n a few hours o f treatment. B l i s s and A i l i o n ,  However,  (1970) m a i n t a i n e d t h a t a f t e r two weeks o f l i t h i u m  treatment no change i n n o r a d r e n a l i n e  metabolism c o u l d be d e t e c t e d .  I t i s known t h a t i n t r a c e l l u l a r l e v e l s o f adenosine monophosphate (cyclic-AMP)  3'5 -cyclic 9  r i s e i n response t o t h e catecholamines and  s e r a t o n i n i n many t a r g e t t i s s u e s ( S u t h e r l a n d , not t o o s u r p r i s i n g t o f i n d t h a t cyclic-AMP  1968),  Therefore,  i t is  i s excreted a t higher  than  normal r a t e s i n manic p a t i e n t s and lower t h a n normal r a t e s i n depressed patients  ( P a u l e t a l , , 1971a).  The t r a n s i t i o n from the depressed t o  t h e manic s t a t e i s a l s o marked by a t r a n s i t o r y i n c r e a s e i n cyclic-AMP e x c r e t i o n which i s even g r e a t e r than t h a t o f the manic s t a t e e t a l . , 1971b),  (Paul  These authors have a l s o shown t h a t l e v e l s o f cyclic-AMP  f o l l o w the d i r e c t i o n o f c l i n i c a l change brought about by l i t h i u m . cyclic-AMP  Thus  dropped i n p a t i e n t s r e c o v e r i n g from mania and r o s e i n  p a t i e n t s r e c o v e r i n g from d e p r e s s i o n  ( P a u l e t a l , . 1971a),  T h i s f i n d i n g tends t o broaden r a t h e r t h a n c o n f i n e t h e problem, s i n c e cyclic-AMP e t a l . , 1968). i s involved — production chemical  i s a common i n t e r m e d i a t e  i n hormal c o n t r o l  I n v e s t i g a t i o n s o f two other systems i n which g l y c o l y c i s (Sutherland  (Sutherland  cyclic-AMP  and Robinson, 1966) and s t e r o i d  e t a l . , I968) —  have n o t y e t r e v e a l e d c l e a r  r e l a t i o n s h i p s w i t h manic d e p r e s s i v e  R u b i n , 1967; Van der Velde,  (Sutherland,  disorders  (Coppen, 1967;  1969; Heninger and M e u l l e r , 1970),  Thus f a r , the mechanism upon which l i t h i u m operates t o produce i t s e f f e c t has n o t been d i s c o v e r e d . the b i o c h e m i s t r y  To t h i s end i t may be u s e f u l t o compare  o f manic d e p r e s s i v e  p a t i e n t s w i t h t h a t o f Shuddering  54  flies. Whereas t h e p a t t e r n s o f b e h a v i o u r seen i n Shuddering f l i e s  suggested  a nervous d i s o r d e r , t h e temperature-induced c e s s a t i o n o f a l l behaviour ts patterns i n para the  effect.  flies  c o u l d have any number o f p o s s i b l e bases f o r  The r e q u i s i t e i n s i g h t i n t o t h e t i s s u e s p e c i f i c i t y o f t h e  mutation was  g a i n e d by examining g e n e t i c mosaic i n d i v i d u a l s  from y p a r a ^ / l n ( l ) w ^ . - H - zygotes (Suzuki et a l . , 1971). vC S  V  s o m a t i c a l l y u n s t a b l e r i n g X chromosome, l n ( l ) w produced  (Hinton,  derived  Loss of the  1955)  i n d i v i d u a l s which c a r r i e d b o t h y para^ /++ and y p a r a ^ / 0 s  T i s s u e d e r i v e d from X/0  cells  cells.  s  c o u l d be r e c o g n i z e d e x t e r n a l l y by t h e  e x p r e s s i o n o f the r e c e s s i v e c u t i c u l a r mutation, y_. I t was  d i s c o v e r e d t h a t f l i e s w i t h y e l l o w l e g s and w i l d t y p e b o d i e s  were a b l e t o move t h e i r l e g s back and f o r t h a t h i g h temperature, b u t were u n a b l e t o c o o r d i n a t e or p o s i t i o n t h e i r l e g s p r o p e r l y .  F l i e s with  y e l l o w t h o r a c e s c o n t r a c t e d t h e i r l e g s i n response t o a temperature  rise,  whereas f l i e s w i t h y e l l o w heads assumed a normal s t a n c e but were u n a b l e to walk. F l i e s w i t h y e l l o w abdomens were u n a f f e c t e d by temperature. B i l a t e r a l , mosaic f l i e s moved t h e i r w i l d t y p e l e g s a t h i g h temperature, b u t were u n a b l e t o move t h e i r y e l l o w l e g s . The r e s u l t s o f t h e s e ts experiments  indicate  t h a t the p a r a  mutation p r o b a b l y has a d i r e c t  e f f e c t upon the nervous system which i s n o t mediated through a f r e e l y circulating factor.  Furthermore, the presence o f the mutation i n  d i f f e r e n t p a r t s o f t h e nervous system appears t o l e a d t o b e h a v i o u r a l l y distinct  consequences.  The o b s e r v a t i o n t h a t t h e r e c o v e r y o f m o b i l i t y i n ts  d i f f e r e n t p a r t s o f the body o f p a r a seems t o occur a t d i f f e r e n t r a t e s may In  flies  a t h i g h temperature  often  a l s o i n d i c a t e t h e gene's autonomy.  a d d i t i o n t o w a l k i n g and c l i m b i n g a c t i v i t i e s , t e t h e r e d f l i g h t  was  55 a l s o found t o be s e n s i t i v e t o temperature  change (Suzuki e t a l  F l i g h t c o u l d be q u i c k l y i n i t i a t e d or stopped by exposure  M  1971)»  t o 22°C and  o 29 C, r e s p e c t i v e l y . IP The f a c t t h a t t h e temperature characteristic of para^  S  s e n s i t i v i t y o f HK  impulses b u r s t from t h e t h o r a c i c g a n g l i o n .  o f HK  para  /y i s  r a t h e r t h a n H K ^ suggests t h a t p a r a ^  or i n d i r e c t l y supresses t h e i n i t i a t i o n or subsequent  2T  ts  s  directly  conduction of  The temperature-dependence  ts para  /Y males supports  this contention since the expression of  2T t h e HK  a l l e l e was n o t s u b j e c t t o temperature  change.  Some o f t h e mosaic f l i e s who had mutant t i s s u e around  one eye were  observed t o f o l l o w a h e l i c a l p a t h a t 29°C when c l i m b i n g up t h e i n s i d e surface of a cylinder  ( S u z u k i e t a l . , 1971).  S i n c e t h e mutant eye was  f a c i n g upward d u r i n g t h e climb i t was thought t h a t v i s i o n i n t h a t eye might be i m p a i r e d . response and 29°C.  These mosaic i n d i v i d u a l s showed a normal optomotor  ( i . e . , t h e y t u r n e d i n t h e d i r e c t i o n o f moving s t r i p e s ) a t 22°C When p a i n t , which had been shown t o e f f e c t i v e l y shut out  l i g h t , was used t o cover t h e w i l d type eye and o c e l l i , t h e f l i e s  still  showed a normal optomotor response a t 22°G, b u t behaved ambiguously a t 29°C.  Normal changes i n p o t e n t i a l o f t h e s u r f a c e o f t h e eye i n response ts  t o s t i m u l a t i o n by l i g h t  (Hotta and Benzer,  19&9)  were r e c o r d e d f o r p a r a  a t b o t h 22°C and 3 0 ° C * S t u d i e s o f m i c r o b i a l systems have i n d i c a t e d t h a t a  temperature-  induced l o s s o f f u n c t i o n may be caused by t h e t h e r m a l i n s t a b i l i t y o f a p a r t i c u l a r molecule.  The i n s t a b i l i t y can be caused by a s i n g l e amino a c i d  * The e l e c t r o r e t n a g r a m s were o b t a i n e d f o r us through t h e kindness o f change p r o t e iand n (Jockusch, 1964, 1966) or a s i n g l e base change Drs. w Yi ot sh hi i nk iaHotta Seymour Benzer.  56 w i t h i n a t r a n s f e r RNA  1970), The c a p a c i t y o f some  s p e c i e s (Smith e t a l „,  mutant molecules t o f u n c t i o n n o r m a l l y depends upon t h e temperature a t the time of t h e i r temperatures  s y n t h e s i s and assembly,  but does n o t depend on  1964;  (Edgar and L i e l a u s i s ,  f u n c t i o n i n g o f o t h e r mutant molecules i s subsequent  S a d l e r and N o v i c k ,  1964),  The  temperature-dependent  t o syntheses (Maas and D a v i s , 1952;  and N o v i c k ,  subsequent  I g a r a s h i , 1969;  Sadler  1965). ts  The speed o f response o f p a r a the  possibility  f l i e s t o temperature  o f much macromolecular  change p r e c l u d e s  s y n t h e s i s and suggests t h a t  a l t e r a t i o n s i n b e h a v i o u r a r e mediated t h r o u g h a p r e - e x i s t i n g thermol a b i l e product. the  A l t h o u g h mutant p r o t e i n s have o f t e n been s i g h t e d as  b a s i s o f temperature s e n s i t i v i t y ,  the nature of the thermolabile  ts f a c t o r i n para  flies  The s e n s i t i v i t y  cannot be assumed.  o f a mutant f u n c t i o n t o a p a r t i c u l a r  temperature  change i s o f t e n an e x a g g e r a t i o n o f t h e w i l d t y p e s e n s i t i v i t y t o t h e same change,  Guthrie et a l . ,  of r i b o s o m a l assembly sensitivity activity  (1969) a t t r i b u t e d the enhanced r e c o v e r y  d e f e c t s among c o l d - s e n s i t i v e mutants t o t h e normal  of t h i s process t o c o l d .  ( I g a r a s h i , 1969)  The s e n s i t i v i t y  and t h e h a l f l i f e  enzyme (Horowitz and F l i n g ,  1952)  of catalase  o f pantothenate  synthesising  t o i n c r e a s e d temperature i s g r o s s l y  exaggerated i n t h e mutant c o n d i t i o n ,  O'Donnovan and Ingraham  (I965)  d i s c o v e r e d t h a t a mutant p h o s p h o r i b o s y l ATP p y r o p h o s p h o r a l a s e i n E, which had a g r e a t l y i n c r e a s e d s e n s i t i v i t y histidine. at  The a d d i t i o n a l s e n s i t i v i t y  low temperatures was  t o feedback i n h i b i t i o n  o f t h e mutant enzyme t o  coli  by inhibition  about the same as t h a t o f t h e w i l d type enzyme,  but the consequences t o t h e growth o f t h e mutant were much g r e a t e r . The p r o c e s s o f s c r e e n i n g f o r h e a t - s e n s i t i v e mutants may t e n d t o  57 s e l e c t systems which a r e a l r e a d y somewhat s e n s i t i v e t o heat p r i o r t o mutation. viewed  I f t h e phenomenon o f t e m p e r a t u r e - s e n s i t i v e p a r a l y s i s i s  i n t h i s way,  t h e demonstrated  system t o i n c r e a s e d temperatures Taylor  s e n s i t i v i t y of the c e n t r a l  acquires a s p e c i a l i n t e r e s t .  Kerkut and  (1958) observed t h a t the maximum impulse f r e q u e n c y e m i t t e d by an  i s o l a t e d t h o r a c i c or abdominal temperature  cockroach ganglion occurred a t the  a t which t h e a n i m a l had been p r e c o n d i t i o n e d and f e l l o f f  g r a d u a l l y above and below t h a t temperature. by changing t h e temperature  These r e a d i n g s were o b t a i n e d  o f t h e g a n g l i o n s l o w l y and p e r m i t t i n g time  f o r the frequency t o reach a steady s t a t e .  A r a p i d temperature i n c r e a s e  o f l6°C caused impulses t o cease almost immediately. seconds  nervous  A f t e r eleven  o f i n a c t i v i t y , t h e impulses suddenly resumed a t a h i g h e r  frequency than before.  The same e f f e c t was  i n c r a y f i s h abdomenal g a n g l i a and a 3°C (Kerkut and T a y l o r , temperature  1958).  a c h i e v e d by a 13°C  increase  increase i n slug pedal ganglia  In a l l t h e s e systems, q u i c k drops i n  i n d u c e d t r a n s i e n t i n c r e a s e s i n impulse f r e q u e n c y .  These  t r a n s i e n t responses t o temperature were c h a r a c t e r i s t i c o f some, b u t n o t all  c e n t r a l nervous  elements  (Kerkut and T a y l o r ,  1958).  Nevertheless,  the speed o f r e a c t i o n o f t h e g a n g l i a which c o n t r o l m o b i l i t y t o c o l d ts and heat shock was  very reminiscent of para  behaviour.  The b i o c h e m i c a l l i m i t s which d e f i n e an organism's range as w e l l as i t s means o f a c c l i m a t i s i n g t o a new been e x t e n s i v e l y s t u d i e d . and Somero  temperature  temperature  have  I n t h e i r r e v i e w o f t h i s s u b j e c t , Hochachka  (1971) have s t r e s s e d t h e importance o f enzyme s u b s t r a t e  a f f i n i t y , as i m p l i e d by low Km v a l u e s , i n d e t e r m i n i n g t h e range immediately e x p l o i t a b l e temperatures.  A d a p t a t i o n t o a new  of  range  may  r e q u i r e de novo s y n t h e s i s o f an enzyme whose l o w e s t Km v a l u e s extend  58 over t h e new range.  Thus, when Rainbow t r o u t were t r a n s f e r r e d from  t o 2°C t h e y were i m m o b i l i z e d . was  accompanied  V?°C  A f t e r a few weeks, a r e t u r n o f m o b i l i t y  by t h e appearance o f a new low temperature a c e t y l -  c h o l e n e s t e r a s e and t h e d i s a p p e a r a n c e o f a h i g h temperature form (Baldwin and Hochachka,  1970),  I t appears t h a t t h i s i s a s i m p l e  example o f a common phenomenon (Hochachka and Somero, 1971) < > Long term a d a p t a t i o n i s a l s o accompanied  by i o n i c  changes  ( H e i n i c h e and Houston, 19&5) a^d a l t e r a t i o n s i n t h e p h o s p h o l i p i d s p e c i e s of t h e c e n t r a l nervous system (Roots, 1968),  Preliminary tests of short  ts and l o n g term a c c l i m a t i o n o f p a r a  f l i e s t o h i g h temperature suggested  the e x i s t e n c e o f s h o r t and l o n g term mechanisms f o r r e g a i n i n g (Suzuki e t a l , ,  1971),  mobility  The s h o r t term advantage appears t o be q u i c k l y  l o s t a f t e r a b r i e f s h i f t down f o l l o w e d by a r e t u r n t o h i g h temperature. The l o n g term g a i n i s n o t s o e a s i l y l o s t and may, t h e r e f o r e , be accomp l i s h e d by an a c c l i m a t i o n p r o c e s s such as de novo s y n t h e s i s o f a new ts f a c t o r t o r e p l a c e or a i d the para  factor.  An organism's i n s e n s i t i v i t y t o temperature need n o t always on t h e i n s e n s i t i v i t y o f systems w i t h i n i t .  Hochachka  and Samero  depend  (1971)  have named f i v e mechanisms which a r e p r o b a b l y r e q u i r e d t o b a l a n c e t h e s e n s i t i v i t y o f f r u c t o s e d i p h o s p h a t a s e t o AMP i n h i b i t i o n a t low temperatures.  By m u t a t i o n t h i s k i n d o f b a l a n c e c o u l d e a s i l y be u p s e t  (O'Donnovan and Ingraham,  I965).  The p o s i t i o n s and n a t u r e o f t h e t e m p e r a t u r e - s e n s i t i v e elements o f ts the para  nervous system a r e y e t unknown and must await t h e r e s u l t s o f  * electrophysiological investigation. The d i s c o v e r y o f t h e d r a m a t i c r i s e i n a c e t y l c h o l e n e s t e r a s e and These s t u d i e s a r e b e i n g conducted by Dr, Kazuo Ikeda,  59 choleneacetyltransferase a c t i v i t i e s  i n D r o s o p h i l a melanogaster d u r i n g  a d u l t metamorphosis (Dewhurst e t a l , , 1970)  provides a p l a u s i b l e  explanation f o r the a d u l t - s p e c i f i c f u n c t i o n of the para  mutation.  However, the a c t i v i t i e s o f t h e s e enzymes were not found t o be s e n s i t i v e i n para  ts  flies,  T u n n i c l i f f et a l .  temperature-  *  (1970) d i s c o v e r e d t h a t dopamine c o n c e n t r a t i o n s  a r e below normal i n an a c t i v e s t r a i n o f D r o s o p h i l a melanogaster above normal i n an i n a c t i v e s t r a i n . noradrenaline.  The  o p p o s i t e was  Thus, n o r a d r e n a l i n e has become one  and  true for  o f the  candidates  ts for  b i o c h e m i c a l study i n b o t h p a r a  and Shuddering  flies.  However, i f  a l t e r e d l e v e l s o f n o r a d r e n a l i n e were d i s c o v e r e d i n t h e s e f l i e s , problem o f p r i m a r y The  cause would s t i l l  i n v e s t i g a t i o n o f primary  the  remain.  causes o f b e h a v i o u r a l d e f e c t s i s b e s t  c a r r i e d out i n s i n g l e mutant systems.  The  s e l e c t i v e recovery of s i n g l e  gene mutants a f f e c t i n g t h e motor system i s f e a s i b l e and has, i n t h i s i n s t a n c e , y i e l d e d two  mutants o f g e n e r a l n e u r o l o g i c a l i n t e r e s t .  T h i s b i o c h e m i c a l a n a l y s i s was  conducted  by Dr„  Linda  Hall,  6o  SUMMARY  W i t h the i n t e n t i o n of r e c o v e r i n g mutations a f f e c t i n g n e u r a l and/or muscular systems, a s e a r c h f o r a t e m p e r a t u r e - s e n s i t i v e mutant among D r o s o p h i l a melanogaster a d u l t s was  conducted.  Mechanical  d e v i c e s f o r t h e s e p a r a t i o n o f a c t i v e from i m m o b i l i s e d constructed.  Two  screening  f l i e s were  mating schemes were u s e d t o r e c o v e r chromosomes from  males t r e a t e d w i t h e t h y l methanesulphonate.  I n one scheme the t r e a t e d  males were c r o s s e d w i t h w i l d t y p e females so t h a t o n l y dominant mutations would be d e t e c t e d i n the F]_ g e n e r a t i o n .  In t h e second scheme, the males  were c r o s s e d t o attached-X (XX/Y) females, of  thus p e r m i t t i n g t h e d e t e c t i o n  s e x - l i n k e d r e c e s s i v e or autosomal dominant mutations.  g e n e r a t i o n s were r a i s e d t o adulthood i n d i v i d u a l s which were i m m o b i l i s e d From approximately  a t 22°C and t h e n screened  250,000 progeny of XX/Y  as p a r a ^  s  for  a t 29°C, females,  e x h i b i t e d h e r i t a b l e t e m p e r a t u r e - s e n s i t i v e p a r a l y s i s was m u t a t i o n (symbolised  The F]_  one male which recovered.  The  f o r p a r a l y t i c - t e m p e r a t u r e - s e n s i t i v e ) was  found t o be r e c e s s i v e and s e x - l i n k e d , mapping g e n e t i c a l l y a t 53«9o para^  s  f l i e s which had been r a i s e d a t 22°C were p a r a l y s e d w i t h i n  5 seconds o f exposure t o 29°C and r e c o v e r e d f u l l m o b i l i t y w i t h i n a seconds o f exposure t o 22°G,  S e n s i t i v i t y t o temperature change  few  was  detected only i n adult f l i e s . Prolonged  exposure of p a r a  recovery of m o b i l i t y .  t s  f l i e s t o 29°C r e s u l t e d i n a  gradual  I n g e n e r a l , the l e n g t h o f the r e c o v e r y time  d i r e c t l y r e l a t e d t o the magnitude and r a t e o f the temperature r i s e .  was  61 The movements o f e t h e r i s e d and n o n - e t h e r i s e d HK HK^para^ /Y 5  IP  para  t  and para^ Sh~VY males were s u b j e c t t o t h e same s  /Y, temperature-  ts s e n s i t i v e p a r a l y s i s that para IP leg  s h a k i n g o f HK  by temperature  flies  exhibited.  The o b s e r v a t i o n t h a t  ts para  /Y males was r a p i d l y s u p r e s s e d and r e i n i t i a t e d ts  change suggests t h a t p a r a  may have d i r e c t l y or  i n d i r e c t l y s u p r e s s e d t h e i n i t i a t i o n or subsequent  c o n d u c t i o n o f impulse  b u r s t s which have been shown t o cause t h e c h a r a c t e r i s t i c l e g shaking o f HK^  flies. In  t h e s e a r c h f o r a dominant mutation  causing temperature-sensitive  p a r a l y s i s a f l y c a r r y i n g a dominant mutation which caused shuddering was d i s c o v e r e d .  periodic  The s e x - l i n k e d m u t a t i o n , c a l l e d Shuddering  (Shu), mapped g e n e t i c a l l y a t 55.1.  I t was p o s s i b l e t o suppress t h e  shuddering b e h a v i o r by f e e d i n g t h e f l i e s media c o n t a i n i n g L i C l b u t n o t NaCl, K C l , or NH^Cl.  The s i g n i f i c a n c e o f t h e s e o b s e r v a t i o n s  has been  c o n s i d e r e d i n t h e l i g h t o f o t h e r e f f e c t s o f l i t h i u m i o n s upon t h e b i o c h e m i s t r y , p h y s i o l o g y and b e h a v i o u r o f man and o t h e r a n i m a l s . Under e t h e r a n a e s t h e s i a Shu f l i e s vigorously.  shook t h e i r l e g s r a p i d l y and  Abnormal movements o f t h e wings, h a l t e r e s , head and  antennae were a l s o observed.  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