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The establishment of the dosage-mortality curve for an aphid : a problem in methodology. Pearson, William Daniel 1968

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THE ESTABLISHMENT OF THE DOSAGE-MORTALITY CURVE FOR AN APHID: A PROBLEM IN METHODOLOGY by WILLIAM DANIEL PEARSON B.S:.'A., University of B r i t i s h Columbia, 1965  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Division of PLANT SCIENCE We accept t h i s thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA' April, 1 9 6 8  In p r e s e n t i n g  this thesis  in p a r t i a l  f u l f i l m e n t o f the requirements f o r an  advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y  a v a i l a b l e f o r r e f e r e n c e and study.  agree that p e r m i s s i o n f o r e x t e n s i v e  I further  copying.of t h i s t h e s i s f o r s c h o l a r l y  purposes may be g r a n t e d by the Head o f my Department o r by h i s representatives.  I t i s understood that c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r  financial  gain  Department nf  s h a l l not be a l l o w e d w i t h o u t my w r i t t e n  Pjt&^jf  Ss&*je*4<s4JLs  The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada  Date  Hfi^cA  /£>, /96>8  permission.  I  ABSTRACT A r e l i a b l e technique for establishing the dosage: mortality curve for an aphid i s described.  Standardized  conditions of rearing, c o l l e c t i n g , treating and posttreatment holding are combined with application of measured droplets of malathion i n acetone solution to individual aphids.  The importance of each of these  factors i s discussed.  The L D ^ Q of malathion to the pea  aphid i s 23.6 nanograms per aphid.  The 95$ f i d u c i a l  l i m i t s about t h i s estimate are 18.4 and 3 1 . 4 nanograms per aphid.  The slope (± S,E.,n = 7) o'f .the." log-do sage:  probit-mortality l i n e i s 5 .5 - 0 . 4 .  The mathematical  and b i o l o g i c a l c r i t e r i a used to evaluate the technique, and the possible application of the technique to select i o n and studies of insecticide resistant clonal l i n e s of aphids are also discussed. Exposure of aphids on treated glass surfaces, and spraying with solutions or emulsions are also i n v e s t i gated and are discussed.  These methods are unsatisfac-  tory for investigation of the genetics and toxicology of insecticide resistance i n aphid populations.  i i TABLE OP CONTENTS ABSTRACT  i  TABLE OP CONTENTS  i  L I S T OP TABLES  i  L I S T OP FIGURES  i  i  i  v  ACKNOWLEDGEMENTS  v i  INTRODUCTION  1  GENERAL MATERIALS AND METHODS  6  The I n s e c t i c i d e  6  The A p h i d  6  Analysis  9  o f M o r t a l i t y Data  10  EXPERIMENTS Exposure t o Treated Surfaces  10  Group T o p i c a l  Application: Malathion Solutions  13  Group T o p i c a l  A p p l i c a t i o n : Malathion Emulsions  19  Individual Topical  35  Application  RESULTS  39  DISCUSSION  45  Evaluation  45  o f t h e Technique  S i g n i f i c a n c e o f t h e Dosage-Mortality. Curve  57  S e l e c t i o n f o r R e s i s t a n c e W i t h i n a Pea Aphid Clone  58  The P o s s i b i l i t y o f R e s i s t a n c e a Laboratory Clone  60  Within  SUMMARY  65  LITERATURE CITED  66  APPENDIX  70  iii L I S T OF TABLES Table I  II  III  IV  V  VI  VII  VIII  IX  " M o r t a l i t y o f l a s t nymphal i n s t a r and a d u l t A* P i s u m e x p o s e d f o r 15 m i n u t e s o n g l a s s p l a t e s sprayed w i t h malathion I n acetone solutions.  Page  12  M o r t a l i t y o f l a s t nymphal i n s t a r and a d u l t A* P i s u m s p r a y e d w i t h m a l a t h i o n i n 50$ e t h a n o l s o l u t i o n s t h e n h e l d on t r e a t e d s u r f a c e f o r 30 m i n u t e s i n a c l o s e d d i s h .  15  P e r c e n t m o r t a l i t y i n 4 s e t s o f 9-day o l d A. p i s u m s p r a y e d w i t h v a r i o u s m a l a t h i o n c o n c e n t r a t i o n s i n 50$ e t h a n o l a n d l e f t o n t h e t r e a t e d s u r f a c e f o r 30 m i n u t e s .  17  P e r c e n t m o r t a l i t y i n 3 s e t s o f 9-day o l d A. p i s u m s p r a y e d w i t h v a r i o u s m a l a t h i o n c o n c e n t r a t i o n s i n 50$ e t h a n o l a n d l e f t on t h e t r e a t e d s u r f a c e f o r 30 m i n u t e s .  18  P e r c e n t m o r t a l i t y i n 12 s e t s o f 9-day o l d A. p i s u m s p r a y e d w i t h m a l a t h i o n emulsions o f v a r i o u s c o n c e n t r a t i o n s t h e n l e f t on t h e t r e a t e d s u r f a c e f o r 30 m i n u t e s .  21  M o r t a l i t i e s o f 9-day o l d A. p i s u m s p r a y e d w i t h malathion emulsions o f v a r i o u s concent r a t i o n s u s i n g a w e t t e r in. t h e d i s t i l l e d water c a r r i e r .  24  P e r c e n t m o r t a l i t y o f 9-day o l d A. p i s u m r e a r e d under constant c o n d i t i o n s f o r t h r e e g e n e r a t i o n s , s p r a y e d w i t h m a l a t h i o n emulsions containing a wetter. A p h i d s l e f t on t h e t r e a t e d s u r f a c e f o r 30 m i n u t e s . M o r t a l i t y a s s e s s e d a t 48 h o u r s .  28  P e r c e n t m o r t a l i t y o f 9-day o l d A. p i s u m , reared under constant c o n d i t i o n s f o r t h r e e g e n e r a t i o n s , s p r a y e d w i t h m a l a t h i o n emulsions containing a wetter. Aphids l e f t on t h e t r e a t e d s u r f a c e f o r 30 m i n u t e s t h e n t r a n s f e r r e d t o e x c i s e d p l a n t s I n Hoagland's f o r 48 h o u r s b e f o r e m o r t a l i t y a s s e s s m e n t .  30  C o r r e l a t i o n between age o f e m u l s i o n ( i n d a y s ) and L C ^ Q o b t a i n e d by p r o b i t a n a l y s i s .  32  iv Table X  XI  XII  XIII  Page P e r c e n t m o r t a l i t y o f 9-day o l d A. p i s u m r e a r e d under constant c o n d i t i o n s f o r t h r e e g e n e r a t i o n s , s p r a y e d w i t h m a l a t h i o n emulsions c o n t a i n i n g a w e t t e r . Aphids l e f t on -the t r e a t e d s u r f a c e f o r 30 m i n u t e s t h e n t r a n s f e r r e d t o l i v i n g p l a n t s f o r 43 h o u r s b e f o r e m o r t a l i t y assessment.  34  P e r c e n t m o r t a l i t y o f 9-day o l d A. p i s u m t o w h i c h 0.5 m i c r o l i t e r d r o p s o f m a l a t h i o n i n a c e t o n e s o l u t i o n were a p p l i e d on t h e abdominal dorsum w i t h a s y r i n g e m i c r o - b u r e t . A p h i d s h e l d on s o l i d s u r f a c e f o r t r e a t m e n t , t h e n t r a n s f e r r e d t o l i v e p l a n t s f o r 48 h o u r s p r i o r t o m o r t a l i t y assessment,  37  P e r c e n t m o r t a l i t y o f 9 - d a y o l d A. p i s u m t o w h i c h 0.5 m i c r o l i t e r d r o p s o f m a l a t h i o n i n a c e t o n e s o l u t i o n v/ere a p p l i e d on t h e abdominal dorsum w i t h a s y r i n g e m i c r o b u r e t . A p h i d s h e l d on a 1 2 - s t r a n d p e r cm s a r a n screen f o r treatment, then t r a n s f e r r e d t o l i v e p l a n t s f o r 48 h o u r s b e f o r e m o r t a l i t y assessment. C a l c u l a t i o n o f t h e upper f i d u c i a l l i m i t a b o u t t h e l d - p l i n e o f maximum t o x i c i t y o f m a l a t h i o n t o _A. p i s u m .  40  73  V  L I S T OF FIGURES Figure 1  Page The t w o c e n t r a l l o g - d o s a g e : p r o b i t - m o r t a l i t y l i n e s , o b t a i n e d by t h e method o f r e l a t i v e p o t e n c y , d e s c r i b e t h e maximum a n d minimum t o x i c i t y o f malathion t o Acyrthosiphum pisum (Harris). The t w o o u t e r c u r v e s a r e t h e 9 5 $ f i d u c i a l l i m i t s (see Appendix) about t h e b a n d f o r m e d b y t h e two c e n t r a l l i n e s ; t h e s e outer curves i n d i c a t e the area w i t h i n which the t r u e l d - p l i n e f o r the clone i n expected to f a l l  4 l  vi A0KN0WLEIX1EMENTS I am g r a t e f u l t o Dr. H. R. MacCarthy who s u p e r v i s e d t h i s work, p r o v i d e d o f f i c e and l a b o r a t o r y space a t t h e Canada Department o f A g r i c u l t u r e Research S t a t i o n , and o f f e r e d many h e l p f u l suggestions d u r i n g t h e p r e p a r a t i o n o f t h e manuscript.  I a l s o wish t o express my s i n c e r e  a p p r e c i a t i o n t o Dr. G. G. E. Scudder, Dr. G. W.  Eaton,  Dr. D, G. F i n l a y s o n , and Dr. A. J . Renney f o r t h e i r constructive c r i t i c i s m of the t h e s i s . My s p e c i a l thanks a r e extended t o Dr. W. T. Cram, f o r a host o f items t o o numerous t o d e t a i l .  Thanks a r e  a l s o extended t o Mr. B. D. F r a z e r and Dr. A. R. Forbes f o r much h e l p f u l d i s c u s s i o n d u r i n g t h e course o f t h e work.  I a l s o wish t o thank my w i f e , Verna, and my  mother, Mrs, Dorothy Pearson, f o r much h e l p and u n f a i l i n g support d u r i n g t h e course o f t h e study and p r e p a r a t i o n o f the t h e s i s .  The support and encouragement o f my good  f r i e n d s and f e l l o w students, p a r t i c u l a r l y Mrs. K. G.* G. B a r r e t t and Mr. R. D. King, a r e a l s o g r a t e f u l l y acknowledged.  1 INTRODUCTION The  members o f t h e A p h i d i d a e  are very important  to agri-  c u l t u r e b e c a u s e o f t h e v a s t amount o f damage t h e y c a u s e t o crops.  Reductions  i n v i g o r and i n q u a l i t y and q u a n t i t y o f  t h e c r o p c a n be a t t r i b u t e d d i r e c t l y t o a p h i d excretion. and  f e e d i n g and  More s e r i o u s e f f e c t s a r e a t t r i b u t a b l e t o t o x i n s  v i r u s e s i n j e c t e d i n t o p l a n t t i s s u e s by f e e d i n g o r p r o b i n g  aphids.  Aphids  form t h e l a r g e s t group o f p l a n t - v i r u s v e c t o r s ;  i n i 9 6 0 , w i t h 87$ o f t h e A p h i d i d a e  remaining  t o be t e s t e d  w i t h v i r u s , 173  s p e c i e s had been found  t o be v e c t o r s  Day  1962).  production o f alate  and Eastop,  forms throughout  The c o n t i n u o u s  t h e summer, a n d t h e i r p o o r a b i l i t y  c r i m i n a t e between h o s t and non-host s p e c i e s w i t h o u t the t i s s u e s ,  (Kennedy,  contribute greatly to their  to disprobing  e f f i c i e n c y as  vectors. F o r t u n a t e l y , most s p e c i e s o f e c o n o m i c i m p o r t a n c e a r e c o n t r o l l a b l e by t h e u s e o f r e s i s t a n t p l a n t v a r i e t i e s o r by efficient  c o n t a c t , fumigant  there i s l i t t l e or toxic  o r systemic  insecticides.  d a n g e r o f damage t o c r o p s by v i r u s  e f f e c t s o f aphids,  insecticidal  couraged because t h e r e a r e adverse  The  e f f e c t s on p r e d a t o r s a n d  (Forbes,  success o f t h e Aphididae  infection  controls are dis-  p a r a s i t e s which g e n e r a l l y a r e able themselves l a t i o n s b e l o w an e c o n o m i c l e v e l  Where  t o h o l d popu-  1962).  a s a group can be a t t r i b u t e d  to t h e evolution, o f a s p e c i a l i z e d c y c l e which i s c l o s e l y adapted t o t h e annual  c y c l e s o f i t s host p l a n t s .  features o f the aphid cycles are:  The m a i n  a thelytokous, or solely  2  female-produqing,  spring and summer phase during which one  or many generations occur l i v i n g usually on one or more species of herbaceous plantsj and a sexual f a l l generation, usually on a woody plant, which allows for gene segregation and recombination, thereby retaining the evolutionary p o t e n t i a l i t y present i n a normal population.  The summer  phase, aided by v i v i p a r i t y , allows for rapid population i n crease and ensures that the reproducing population at the end of the phase w i l l consist almost entirely of individuals which were best adapted to the conditions prevalent during the phase. ing  These characteristics are advantageous i n evolv-  forms suitable to changing environments and in. the race  to exploit new ecological niches or situations.  Some species  l i v i n g i n mild climates have secondarily lost the sexual f a l l generations and now reproduce entirely by theiytoky. In these species the genetic constitution i s extremely stable (Soumalainen,  19^2j  White,  1945).  Nevertheless, several  independent instances of the evolution, of insecticide r e s i s t ance in the Aphididae during the past decade have given reason to doubt t h i s presumed s t a b i l i t y .  In two of these  cases, the population was d e f i n i t e l y thelytokous.  No males  of the parathion-resistant spotted a l f a l f a aphid, Therioaphis maculata (Buckton), have been reported from west of the Rocky Mountains where resistance developed  (Stern,  1962).  A thelytokous population of the strawberry aphid, Chaetosiphon f r a g a e f o l i i (Cockerell), resistant to the cyclodiene insecticide endosulfan, has also been reported (Shanks,  1967)•  3 Three o t h e r oases o f aphid r e s i s t a n c e t o organophosphates have been r e p o r t e d .  Two o f t h e s e w e r e g r e e n h o u s e p o p u l a t i o n s  o f t h e g r e e n p e a c h a p h i d , Myzus p e r s i c a e ( S u l z e r ) ,  (Baerecke,  1962j Dunn a n d Kempton, 1966), w h e r e s e x u a l r e p r o d u c t i o n i s u n l i k e l y t o h a v e o c c u r r e d ; t h e t h i r d was a p a r a t h i o n - r e s i s t ant p o p u l a t i o n o f t h e walnut  a p h i d , Chromaphis  (Kltb.) discovered i n northern California 1954),  et a l , ,  i n the area  Males o f t h i s  ( E s s i g , 1955).  .luglandicola  (Michelbacher,  species a r e thought  t o occur  Mechanisms whereby t h e s e  instances  o f r e s i s t a n c e h a v e e v o l v e d a r e unknown, a n d t h e r e i s t h u s a n interest  i n them f r o m b o t h t h e a c a d e m i c a n d t h e a p p l i e d  j o i n t s o f view. L i t t l e p r e c i s e t c x i c o l o g i c a l w o r k h a s been done on a p h i d s . Stern had  (1962), who e x p o s e d a p h i d s on f o l i a g e b o u q u e t s w h i c h  been dipped  i n emulsified insecticides, reported  dosage-mortality  curves.  reliable  However, t h e c u r v e s o b t a i n e d b y  Dunn a n d Kempton (1966), who e x p o s e d a p h i d s o n l e a v e s w i t h I n s e c t i c i d e emulsions,  were n o t s a t i s f a c t o r y .  h o l d e r a n d D I c k e (1966) o b t a i n e d r e l i a b l e c u r v e s  sprayed  Burk-  f o r other  s m a l l i n s e c t s by u s i n g s t a n d a r d i z e d g l a s s and composition surfaces sprayed w i t h i n s e c t i c i d e emulsions ders.  The P o t t e r s p r a y t o w e r  ( P o t t e r , 1952) was u s e d by  P o t t e r a n d G i l l h a m (1958), Y u l e apply i n s e c t i c i d e all  o r w e t t a b l e pow-  (1964), a n d P a t w a (1965) t o  solutions d i r e c t l y t o aphids.  These workers  reported r e l i a b l e dosage-mortality  curves.  The  i s s e e n i n Dunn a n d  r e s u l t o f inadequate  technique  K e m p t o n ' s (1966) r e p o r t o f t h e i r a t t e m p t  to trace the decline  4 o f demeton-methyl r e s i s t a n c e i n a c l o n e of the green peach aphid.  T h e s e w o r k e r s w e r e n o t a b l e t o make v a l i d  between g e n e r a t i o n s to determine  test.  s u s c e p t i b l e clone appeared t o  develop  f i v e - f o l d r e s i s t a n c e as compared w i t h an  earlier  T h i s apparent  aphid's  c o n c e n t r a t i o n s were used  m e d i a n l e t h a l t i m e s ; when t h e same c o n c e n t r a -  t i o n s were used t h e f o u r and  since different  comparisons  r e s i s t a n c e c o u l d be an a r t i f a c t  of  the  b e h a v i o r ; t h o s e t h a t were a c t i v e would a c q u i r e a  l e t h a l dose within, a short p e r i o d . w o r k e r s w e r e a b l e t o d r a w was when s e l e c t i o n p r e s s u r e was  The  only conclusion these  that the r e s i s t a n c e declined  removed.  The  present  study  was  p r o m p t e d by t h e i n c r e a s i n g l y c l e a r n e e d f o r a p r e c i s e t e c h nique  f o r e s t a b l i s h i n g dosage-mortality curves A dosage-mortality  f o r aphids.  curve i s a l i n e d e s c r i b i n g the  r e l a t i o n s h i p b e t w e e n t h e e f f e c t o f a t o x i c a n t and of a population. Pechner law  t h e members  T h i s r e l a t i o n s h i p u s u a l l y f i t s t h e Weber-  ( H o s k i n s and  C r a i g , 19^2), w h i c h s t a t e s t h a t  change i n magnitude o r i n t e n s i t y o f a b i o l o g i c a l response p r o p o r t i o n a l not t o t h e change i n s t i m u l u s but t o l o g a r i t h m o f t h i s change. of responses  c a n be n o r m a l i z e d  t i o n of dosages.  is  the  distribution  by a l o g a r i t h m i c t r a n s f o r m a -  A further straightening of t h i s  curve, which i s d i f f i c u l t son,  I t follows that the  the  t o use  sigmoidal  f o r i n t e r p o l a t i o n or  compari-  i s o b t a i n e d by t r a n s f o r m i n g m o r t a l i t y f r o m p e r c e n t a g e  p r o b i t s , which a r e coded s t a n d a r d d e v i a t i o n u n i t s . straightened curve mortality line,  and  i s properly c a l l e d the log  to  The  dosage-probit  h e r e a f t e r i s r e f e r r e d t o as t h e l d - p  line.  5  The essential characteristics of t h i s l i n e are I t s position and slope, and . t h e i r standard deviations. Replications employing .precise methods w i l l give Id-p l i n e s with both positions and slopes r e l a t i v e l y constant, and small standard deviations. The values derived from the Id-p l i n e serve as reference points f o r future observations of the aphid population. They must therefore be r e l i a b l e and repeatable. The computed L C ^ Q values are estimates of the concentrations which w i l l k i l l 50 percent of the tested populations and indicate the median t o x i c i t y of the Insecticide to 'individual aphids.  The 95 percent f i d u c i a l l i m i t s about  these estimates indicate the range within which the true L C ^ Q may be expected to f a l l , and give an indication of the confidence which may be placed i n the estimate (Hosklns and Craig,  1962},  The slope of the ld-p l i n e i s a more useful value since i t indicates the variance of response to the insecticide i n the tested population. The standard deviation of the slope (referred to as SD^ i n tables) i s a measure of variation attributable to the method alone. Only when a technique produces repeatable results can investigation begin on the mechanisms whereby resistance has evolved.  6  GENERAL MATERIALS AND The  METHODS  insecticide The  i n s e c t i c i d e u s e d v/as s e c o n d a r y s t a n d a r d  malathion  1  (0,0-dimethyl S - ( l , 2 - d i c a r b e t h o x y e t h y l )  phosphoro d i t h i o a t e )  o f 95$  acetone s o l u t i o n s ,  purity.  The  w e r e s t o r e d a t -8  pure malathion,  C when n o t  and  i n use.  A l c o h o l i c s o l u t i o n s and  e m u l s i b l e f o r m u l a t i o n s were s t o r e d a t room t e m p e r a t u r e , glassware  was  washed w i t h sodium d i c h r o m a t e - s u l p h u r i c  s o l u t i o n , r i n s e d w i t h d i s t i l l e d water, then agent grade acetone before Malathion i s an  was  acid  rinsed with re-  use.  chosen as the t e s t i n s e c t i c i d e because i t  organophosphate, the c l a s s of I n s e c t i c i d e i n v o l v e d i n  most i n s t a n c e s o f a p h i d r e s i s t a n c e t o i n s e c t i c i d e s , b e c a u s e o f i t s low  mammalian t o x i c i t y .  and  I f selection for  r e s i s t a n c e w i t h i n t h e c l o n e were s u c c e s s f u l , p a t t e r n s c r o s s r e s i s t a n c e t o o t h e r o r g a n o p h o s p h a t e s and  whereby the r e s i s t a n c e had  of  carbamates  w o u l d p r o v i d e a n o t h e r means o f i n v e s t i g a t i n g t h e  The  All  mechanisms  evolved.  Aphid C o l o n i e s o f t h e pea  aphid, Acyrthosiphon  pisum  (Harris),  u s e d t h r o u g h o u t t h i s i n v e s t i g a t i o n , w e r e r e a r e d on  broad  bean, V i c i a  These  f a b a L.,  v a r i e t y E x h i b i t i o n Long Pod.  w e r e grown i n a p o t t i n g m i x t u r e moss and  find  sand.  No  of equal p a r t s of  sphagnum  a d d i t i o n a l n u t r i e n t s were added.  The  use o f p l a n t s w h i l e they were young r e d u c e d t h e  L.  O b t a i n e d f r o m C y a n a m i d o f Canada L i m i t e d , 1 C i t y D r i v e , R e x d a l e , Ont.  possibility View  7 o f v a r i a t i o n between  p l a n t s s i n c e t h e y were s t i l l  n u t r i e n t s f r o m t h e endosperm  drawing  o f t h e s e e d when t h e y w e r e  dis-  carded. The a p h i d s w e r e d r a w n f r o m a c l o n e e s t a b l i s h e d o n b r o a d bean  i n 1965 a t t h e Canada D e p a r t m e n t  S t a t i o n , V a n c o u v e r , B. C.  o f A g r i c u l t u r e Research  The l a b o r a t o r y c o l o n y , f r o m w h i c h  t h i s c l o n e was d e r i v e d , o r i g i n a t e d f r o m a c o l l e c t i o n o f a p t e r o u s v i r g i n o p a r a e f o u n d on a l f a l f a , growing a t the U n i v e r s i t y o f B r i t i s h  Medicago  s a t i v a L.  C o l u m b i a i n 1963.  C o l o n i e s w e r e r e a r e d i n i n s e c t - p r o o f c a g e s 30.5 cm w i d e x 45.7 cm h i g h x 44.5 cm d e e p .  The f r o n t , b a c k a n d f l o o r  o f t h e cages were o f sanded, u n p a i n t e d f i r plywood.  The  s i d e s were o f c l e a r p o l y e t h e l e n e f i l m , and t h e t o p o f c l e a r Kodapak . front.  A c c e s s t o t h e c a g e was b y a 23x38 cm d o o r a t t h e  The d o o r was h e l d i n p l a c e by a b a c k i n g o f l i g h t  p l y w o o d , f a c e d w i t h 1,3  cm w i d e foam p l a s t i c w e a t h e r  p i n g , on t h e i n s i d e o f t h e d o o r .  fir  strip-  F o u r t u r n b u t t o n s on t h e  o u t s i d e made t h e d o o r e s c a p e - p r o o f . V e n t i l a t i o n was p r o v i d e d by a f o r c e d a i r s y s t e m d e l i v e r i n g f i l t e r e d a i r a t t h e r a t e o f 0.17  m3 p e r m i n u t e t h r o u g h  a s c r e e n e d 4.5 cm h o l e a t t h e b a c k o f t h e c a g e .  There were  t h r e e 4.5 crn e x h a u s t h o l e s i n t h e d o o r o f t h e c a g e s c r e e n e d w i t h n y l o n o r g a n d y , 40 s t r a n d s p e r cm.  A f o u r t h 4.5 cm  h o l e i n t h e d o o r was u s e d f o r w a t e r i n g t h e p l a n t s . closed this 2.  A cork  hole.  A transparent p l a s t i c sheeting obtained from Bumaby O r c h i d s , B u m a b y , B. C.  8  The t e m p e r a t u r e i n s i d e t h e cage was m a i n t a i n e d a t 2 1 . 7 - 2C, and t h e vapor p r e s s u r e d e f i c i t a t 12.5 ± 3 . 5 millibars.  S i x t e e n hours i l l u m i n a t i o n p e r day was p r o v i d e d  by s i x 2.4-m c o o l w h i t e f l u o r e s c e n t t u b e s p l a c e d 35 cm above the  soil  surface i n the pots.  The l i g h t i n t e n s i t y ranged  from 2100 t o 5000 l u x , from t h e s o i l  surface t o the c e i l i n g  of t h e cage. The a p h i d s used i n t h e f i r s t p a r t o f t h e s e i n v e s t i g a t i o n s were mature nymphs and a d u l t s c o l l e c t e d a s t h e y were r e q u i r e d from c o l o n i z e d p l a n t s . to  No s p e c i a l e f f o r t was made  m a i n t a i n optimum o r u n i f o r m c o n d i t i o n s d u r i n g t h e p r e v i o u s  generations. For the  c o l l e c t i n g , t h e i n f e s t e d p l a n t s were p l a c e d o v e r  c o l l e c t i o n d i s h and tapped s h a r p l y w i t h a b r u s h t o d i s -  lodge t h e a p h i d s .  A vacuum p e n c i l  ( G l a s s , 19^5) was t h e n  used t o t r a n s f e r t h e r e q u i r e d number o f a p h i d s t o p e t r i p l a t e s i n which t h e y were h e l d u n t i l t r e a t e d . A P o t t e r s p r a y tower ( P o t t e r , 1952) was used f o r e x p e r i ments I n v o l v i n g spray a p p l i c a t i o n s t o s u r f a c e s o r a p h i d s . U n l e s s o t h e r w i s e s t a t e s , one-ml a l i q u o t s o f m a t e r i a l were a p p l i e d w i t h t h e tower o p e r a t i n g a t a p r e s s u r e o f 15 cm mercury.  There was no t e m p e r a t u r e c o n t r o l i n t h e tower but  normal room t e m p e r a t u r e s o f 2 2 , 0 C t o 2 6 . 5 C were common. T r e a t e d a p h i d s were caged on young bean p l a n t s and kept under 16 hours p e r day o f a r t i f i c i a l l i g h t a t 21.7 £ 2C and 12.5 £ 3.5 m i l l i b a r s vapor p r e s s u r e d e f i c i t f o r t h e d u r a t i o n of  the post-treatment holding period.  Q  A n a l y s i s o f M o r t a l i t y Data The r e s u l t s were a n a l y s e d by an IBM 7044 computer, u s i n g two programs.  These programs showed by standard maxi-  mum l i k e l i h o o d methods o f p r o b i t the r e g r e s s i o n o f p r o b i t  a n a l y s i s (Finney,  response on l o g dosage.  l i n e program^ computed a l i n e  f o r each r e p l i c a t e .  1962) A single The  second program^" a l s o computed a l i n e f o r each r e p l i c a t e , t h e n t e s t e d the l i n e s f o r p a r a l l e l i s m and computed the common r e g r e s s i o n , from the pooled r e s u l t s .  3.  Adapted by P. M. Morse and E . A. Reimer, S t a t i s t i c a l Research S e r v i c e , C. D. A . from the o r i g i n a l program by M. J . Garber. Users l i b r a r y No. 162O-O6.O.O93.  4.  Taken from the program by R. J . Daum and C. G i v e n s , U. S . D. A . Users l i b r a r y No. l 6 2 0 - 0 6 , 0 . 0 8 5 .  10  EXPERIMENTS Exposure on a treated surface To establish the appropriate concentration range f o r the malathion, clean glass plates were sprayed with solutions of 0 , 10, 100, and 1000 parts per million (ppm) i n acetone. Twenty aphids were confined continuously on each of these plates by Fluon^-coated Kodapak cylinders, 1.8 cm high x 8 cm diameter.  On the plate sprayed with acetone only most  of the aphids were s t i l l a l i v e after 16 hours, but a l l aphids i n the other treatments were dead. However, the behavior of the aphids exposed to the 100 ppm deposit i n d i cated that they had picked up enough malathion after 90 minutes on the surface to affect t h e i r coordination.  This  loss of coordination was preceded by a period of extreme excitement which appeared 15 minutes after the aphids were placed on the surface. The length of time the aphids were withheld from t h e i r normal environment on the plant influenced the rate of mortality.  Since the aphid's response to the insecticide  should be Isolated from confounding factors (Hoskins and Craig, 1 9 6 2 ) , the length of the exposure was reduced to as short a period as possible. In. an attempt to establish an appropriate exposure time, glass plates were sprayed with a solution of 100 ppm of malathion i n acetone and aphids were confined on these 5.  A polytetrafluoroethylene dispersion manufactured by Imperial Chemical Industries Ltd.  11  surfaces for periods of 5, 10, 20 and 40 minutes.  The  aphids were then removed to holding cages for 24 hours before mortality was assessed.  The holding cage consisted  of a piece of butyrate^ tubing, 5-cm diameter x 11-cm  high,  with a ring of Fluon. at the top to prevent escapes. was mounted on a 5-cm  This  square p l a s t i c pot containing a  young bean plant. The 40 minute exposure period resulted in JO percent mortality but the others had no apparent effect.  This  exposure period was probably too long, A series of higher concentrations, 125, 250, 500 and 1000 ppm were next tested with a constant exposure period of 15 minutes.  The slopes and  LC^QS  of the ld-p lines  from these t e s t s were erratic (Table I ) , and revealed inconsistency i n aphid contamination by the insecticide.  Busvine  and Barnes (1947), using naturally c r y s t a l l i n e organochlorine insecticides dissolved i n acetone, state that t h i s method i s not satisfactory since "the evaporation of the v o l a t i l e solvent leads to irregular sizes and distribution, of the crystals, with consequent erratic r e s u l t s " .  Mala-  thion Is naturally a viscous l i q u i d ( S p i l l e r , 1 9 6 l ) , and does not form c r y s t a l s .  Spraying a surface with malathion  dissolved in a small amount of acetone i s comparable to ultra-low volume application in s t i l l a i r . Most of the 6.  A seamless transparent display tubing obtained from P l a s t i c Bottle Sales Ltd., Weston, Ontario.  12  TABLE I , Mortality of last nymphal instar and adult A, pisum exposed f o r 15 minutes on glass plates sprayed with malathion in acetone solutions. Malathion ppm  1  0 125 250 500  11 21 11 0  22 23 9 47  18 11 27 19  1000  39  27  19  Stat1stics LCcg (PPm) F i d u c i a l Limits  Percent mortality i n replicate 2  3  a  Slope (b) SD ±  b  1121.12  n.s.  9.67 93.48  40,413.56  n.s.  0.81 1.94  0.00  n.s. -0.23 2.08  a. S t a t i s t i c s f o r a l l tables, except Table VI, obtained by probit analysis, n.s. Regression not s i g n i f i c a n t (p = . 0 5 ) . F i d u c i a l l i m i t s not computed.  13  acetone evaporates before i t reaches the target, and the malathion forms a deposit of very small, evenly dispersed particles.  The greatest factor i n these erratic results i s  probably the a c t i v i t y of the aphids.  Those individuals  that move around a great deal come i n contact with, and are contaminated by, more insecticide. (1958),  Gast  (1959)  Nevertheless, Ebeling  and Burkholder and Dicke  (1966)  have  reported good results with other insects using similar methods. Group Topical Application:  Malathion Solutions.  Following the f a i l u r e of exposure of aphids on a treated surface, spraying them d i r e c t l y with acetone-malathion  solu-  tions by placing them into the Potter tower i n the bottom of a p e t r i dish was t r i e d .  At the highest concentrations of  500 and 1000 ppm, the aphids showed great excitement 15 to 20 minutes after they had been sprayed; after 24 hours, mortali t y in. the control treatments did not d i f f e r from that i n the highest treatments.  This suggested a f a i l u r e of effect-  ive contact between the insecticide and the aphids. In order to examine the spray pattern and deposit formed by the acetone:malathion  solution, 9-cm f i l t e r paper  c i r c l e s v/ere sprayed with 1, 2 and 3 ml of acetone containing neutral red dye.  The deposit formed by 1 and 2 ml of solu-  tion was even but barely v i s i b l e . deposit.  Three ml formed a good  These deposits were compared with that formed by  1 ml of 50$ ethanol solution containing neutral red, which  14  produced a somewhat better deposit than three ml of acetone, demonstrating  that the atomized acetone evaporated before i t  reached the target. When the malathionracetone  solutions  were sprayed, much of the atomized malathion was carried away i n the exhaust from the tower.  Also, since the aphids  were not anesthetized, the p e t r i plate had to be removed from the tower quickly, and there was no opportunity for small droplets s t i l l i n the tower to settle onto the aphids. Simplicity i s one of the prime objectives i n the development of a bioassay technique and the best way to achieve a simple relationship between insecticide dosage and mortali t y i s to use as few a u x i l i a r y chemicals as possible (Hoskins and Craig,  1962).  Since malathion i s soluble i n  ethanol ( S p i l l e r , 1961) and small quantities of ethanol are not toxic to the aphid, the acetone solvent-carrier was replaced with 50$ ethanol. To establish the effective concentration range with the 50$ ethanol solvent-carrier, three sets of aphids were sprayed with concentrations of 25, .100, 250, and 1000 ppm malathion.  The three sets were used to compare the effects  of removing the aphids from the treated surface  immediately  and of holding them on the surface for 30 minutes i n closed, or i n ventilated, p e t r i dishes.  There were no readily dis-  cernible differences between the three conditions, and since the 30-minute exposure period i n a closed p e t r i dish was the simplest method, t h i s was chosen. Five concentrations i n the range indicated by the  15  TABLE I I , Mortality of last nymphal instar and adult A. pisum sprayed with malathion i n 50$ ethanol solutions then held on the treated surface for 30 minutes i n a closed dish. Malathion ppm 0 12.5 25.0 40.0 50.0 80.0 100,0 120,0 160.0 200.0  1  Percent mortality i n t e s t : 2  3  4  15 0 26  15 10 10  0  5  —  —  0 0  0 0  0  0  60 75  20 100  5  __  0  25  -75  —  —  —  —  100  100  -—  Statistics LC50 (ppm) Fiducial Limits Upper Lower  b.b  Slope (b) ^b  b.b,  ±s  80.2  cL  127.56  e cl »  n.s.  99.01 56,40 6.32 1.91  cL . cl  1  37.78 46.33  n.s.  Regression not significant (p = .05}. F i d u c i a l l i m i t s not computed.  a.a.  Analysis abandoned. Pooling of dosage classes resulted in loss of degrees of freedom.  b.b.  See text.  16  previous t e s t , 12.5 to 200 ppm malathion, were used with the method established.  The two ld-p l i n e s computed from  the four data sets did not agree (Table I I ) ; i t was also evident that the concentration range was s t i l l too wide. Log-dosage probit l i n e s could not be obtained for tests 1 and 3- In test 1, the computations for probit analysis exceeded the capacity of the computer, i . e . , there were more than 42 s i g n i f i c a n t figures in one of the quantities in the c a l c u l a t i o n . In test 3, the pooling of dosage classes resulted in the loss of degrees of freedom for the calculations, and the analysis was abandoned. Some of the variation between tests up to t h i s point probably arose from the different ages and consequent physiological conditions of the aphids when treated.  To  eliminate t h i s uncertainty, only 9-day old aphids were used for a l l future t e s t s . To obtain aphids of a constant age, plants were infested with reproducing adults, which v/ere then removed  24  hours l a t e r '(Kenten,  1955).  After  9  days  the nymphs deposited had a l l reached maturity and were collected. The results of the next two tests, using only 9-day old aphids, were erratic but somewhat less so. I I I and IV).  (Tables  In the f i r s t series of tests (Table I I I ) ,  mortality was assessed at 48 hours.  This was done i n an  effort to reduce the d i f f i c u l t y , and probability, of error in the dead:live decision.  At 24 hours t h i s decision was  17 TABLE I I I . sprayed  Percent  m o r t a l i t y i n 4 s e t s o f 9~day o l d A. p i s u m  with various malathion  i n 50$ e t h a n o l  concentrations  l e f t on t h e t r e a t e d s u r f a c e f o r 30 m i n u t e s .  and  Malathion. ppm 0 80 100 120  Percent  1  2 19  15 0 40 10 25 70  140 160  mortality i n replicate  0 55 50  30  40  3  "4  39 15 15 20 50 100  0 69 55  45  50 95  Statistics LC (ppm) Fiducial Limits Upper Lower 5 0  Slope (b) ±SL\ n.s.  154.8 167.7 147.0  515.7 n.s.  143.7 n.s.  1.08 1.86  79.59 124.41  24.95 8.47  Regression not s i g n i f i c a n t not computed.  (p = . 0 5 ) .  Fiducial  73.7 n.s.  1.57 2.65 limits  18  TABLE I V . sprayed and  Percent  m o r t a l i t y i n 3 s e t s o f 9-day o l d A. p i s u m  with various malathion  left  concentrations  i n 50% e t h a n o l  on t h e t r e a t e d s u r f a c e f o r 30 m i n u t e s .  Malathion ppm  1  0 100 120 140 160 180  0 0 35 50 70 95  Percent 2  mortality i n replicate  0 0 10" 4o 95 90  3  5 5 40 80 100 95  Statistics L 0  5  (PPm)  0  . F i d u c i a l .Lim.it s Upper Lower Slope (b) ±SD b  138.27  142.15  127.38  146.65 130.04  148.90 135.32  134.59 II8.69  17.40 2.93  14.79 2.76  12.06 2.03  19  d i f f i c u l t ; many aphids were s t i l l a l i v e and capable of coordinated movement, but were very excited, shrunken, and appeared to be dying.  The diminished size of the aphids  appeared to be an effect of the alcohol, because those treated i n the check were also affected.  Since t h i s shrunken  excited condition v/as s t i l l present at 48 hours and the d i f f i c u l t y of the dead-live decision was not reduced, the 24-hour post-treatment holding period v/as used in the second series of tests (Table IV). Erratic mortality and Id-p l i n e s obtained from the four replicates shown in Table I I I indicate irregular contact between, the insecticide and the aphid.  To remedy t h i s , a  wetting agent was added to the solutions.  The v/etter,  7  Triton X - 1 0 0 , reduces the contact angle between the solution and the aphid's c u t i c l e . After the wetter had been added, and the solution shaken vigorously for f i v e minutes, small globules of undissolved material could be seen in the solution.  Since these  may have been undissolved malathion the use of 50$ ethanol as a solvent-carrier v/as Group Topical Application:  discontinued. Malathion Emulsions.  The f a i l u r e of simple insecticide-solvent systems led to attempts to apply the insecticide in the emulsified state. For the solvent, the meta isomer of xylene v/as chosen, since malathion. i s readily soluble i n i t , and i t has the high 7. Triton X-100, a surface active agent, i s a r a l k y l polyether alcohol, manufactured by Rohm & Haas.  20  b o i l i n g point of 139 0 with low vapor pressure.  Thus the  solvent would not evaporate either when the emulsion was sprayed, or i f the two phases of the emulsion separated during storage. A stock 10% emulsible concentrate was made by dissolvo  ing 1.000 g malathion and 0.5 ml of an emulsifier i n m-xylene to make 10 ml of solution.  Emulsions of the  desired concentration v/ere made by d i l u t i o n of the stock concentrate with d i s t i l l e d water.  When the emulsion was  applied the water was repelled by the waxy layer of the e p i c u t l c l e , leaving the xylene containing malathion to spread over the c u t i c l e . Since the dead:live count at 24 hours was subject to error due to the d i f f i c u l t y of the decision and the 48-hour period may have introduced adverse effects from the barely adequate cage size, a 30-hour holding period -was t r i e d . Twelve replicates were carried out over a four-day period.  The response of the aphids, and the s t a t i s t i c s com-  puted from the data, were again erratic (Table V). In replicate 6, new, unwashed p e t r i plates v/ere used. The factory washing process apparently l e f t a detergent-like deposit on the glass.  In these plates, the spray deposit  formed a continuous f i l m on the glass, and insecticide contamination of the aphids was very effective; there was 100$ mortality i n a l l treatments except the lowest.  This  8. Triton B - 1956, a wetting, spreading, sticking and emulsifying agent, i s modified phthalic glycerol alkyd resin, manufactured by Rohm & Haas.  T A B L E V.  P e r c e n t m o r t a l i t y i n 12 s e t s o f 9-day o l d A. p i s u m s p r a y e d w i t h m a l a t h i o n  sions o f various concentration Malathion ppm  t h e n l e f t on t h e t r e a t e d s u r f a c e  emul-  f o r 30 m i n u t e s .  Percent m o r t a l i t y i n r e p l i c a t e  0 80 100 120 140 160  1  2  35 45 47 35 65 85  10 45 16 55 60 90  3  4 0 10 15 16 80 95  5 15 5 25 85 95  5 5 15 25 15 90 100  7  6 5  90 100 100 100 100  0  15 25 0 50 100  8 0  0 15 0 0 90  0  9  10  11  10 25 35 25 95  20 45 0 25 55 95  15 0 15 60 100  12 0  0 5 11 5 35 80  Statistics  LC50 (ppm) Fiducial Upper Lower  144.2 Limits 158.5 111.9  17.57 7.78  119.8 n.s.  a . a . a . a . 1 3 1 . 2 a . a . 132.9 n.s. n.s.  135.4 121.6  5.44 a . a . 3.28  Slope  (b)  n.s.  Regression  a.a.  A n a l y s i s abandoned.  ±sr>b  a . a . 129.3  not s i g n i f i c a n t  a.a.  Fiducial  7.51 a . a . 4.77  6.70 3-91  27.60 9.38  146.1 166.1 134.9  148.1 128.0  21.28 a . a . a . a . 4.48 (p = . 0 5 ) .  l4l.3  a.a.  8.86 1.97  l i m i t s n o t computed.  P o o l i n g o f dosage c l a s s e s r e s u l t e d i n l o s s o f degrees o f freedom.  22  indicates f a i l u r e of contact between the aphids arid the insecticide in. the other replicates, which i s also indicated by the wide variations in the slopes of the ld-p lines computed from the replicates.  There i s a specific ld-p l i n e  slope for each insecticide:insect combination;  i f the  applied dosage bears a constant r e l a t i o n to the amount of insecticide actually picked up by the aphid, the method of application has no effect on the slope of the ld-p l i n e (Hoskins and Craig, 1 9 6 2 ) .  With t h i s formulation, the r e l a -  tion was not constant. The high mortality i n replicate 6, Table V,  suggested  a return to the use of the more e f f i c i e n t wetting agent, Triton X-100.  To determine the appropriate amount, emulsions  containing the wetter i n the d i s t i l l e d water carrier were made up.  Beginning at 150 ppm Triton X-100, the concentra-  tion was increased in 100 ppm increments.  These were sprayed  onto glass plates, and the character of the deposits examined under a dissecting microscope.  At 150 and 250  ppm,  the spray formed discrete droplets, but t h e i r contact angle was low.  At 650 to 850 ppm the deposits were even and the  plates were thoroughly wetted. The variance of a sample mean from a binomial d i s t r i b u tion i s estimated by s- = pq/n, where p represents the proportion of successes, i . e . , dead insects, q i s equal to ( l - p), and n represents the number of observations in. a sample.  This d e f i n i t i o n indicates that between-replicate  variation i n mortality resulting from a dosage may  be  23  reduced by increasing n.  I t was decided, therefore, to  increase the number of aphids i n treatments to 5 0 . F i f t y aphids required a larger cage, since the one used previously v/as no more than adequate for the maintenance of 20 aphids over a 24-hour holding period.  One new caging  method t r i e d contained an excised broad bean leaf in a 5.4 cm x 2.1 cm high Stender dish.  A preliminary test had  shown t h i s to be an adequate cage for a 24-hour period. The stock solution of 10$ malathion i n xylene containing 5$ emulsifier v/as diluted with d i s t i l l e d water containing 750 ppm wetter. These emulsions were shaken vigorously before application.  Two replicates of s i x treatments each  were sprayed with a series of concentrations.  The aphids  v/ere kept on the treated surface for 30 minutes i n a closed p e t r i dish, then brushed into the Stender dish.  An un-  sprayed control of 50 aphids v/as also included.  Mortality  was assessed 24 hours l a t e r . The v/etter v/as an extremely effective adjuvant (Table The emulsion did not evaporate during the 30 minute .  VI).  exposure period; some aphids had to be brushed roughly out of the p e t r i dish, others appeared to have drowned-. Mort a l i t y v/as high in a l l treatments except the unsprayed check. The extreme mortality in t h i s t r i a l might have been caused by any number of factors.  The wetter i t s e l f may  have been toxic at the rate employed, or i t may have drowned the aphids by so reducing surface tension that the l i q u i d  24  TABLE V I . malathion  Mortalities emulsions  Unsprayed 0.0 3.1 6.2 12.5 25.0 50.0  with  of various concentrations using a  i n the d i s t i l l e d water Malathion ppm  o f 9 - d a y o l d A. p i s u m s p r a y e d  wetter  carrier. Number d e a d o u t o f 50 p e r - . 1 2 3 41  25  44 41  44 46  43 48  50 50  50 50  treatment  25  blocked the spiracles and entered the tracheae.  An extreme-  l y damp condition during the 24-hour holding period, caused by lack of v e n t i l a t i o n , was probably the largest factor cont r i b u t i n g to the high mortality.  In replicate one, with  3.1 ppm malathion, the Stender used for holding the aphids had a small chip i n the ground glass l i p .  This would permit  the r e l a t i v e humidity to equalize with that i n the holding room, and would permit a i r exchange.  Ventilation was not  possible i n the unsprayed control either, but the aphids were not wet when placed into the dish. have acted as a fumigant.  Malathion may also  I t was apparent that t h i s method  of caging was not satisfactory. Yule (1964) and Patwa (1965) used a similar technique with the pea aphid and the potato aphid, Macrosiphum euphorbiae (Thomas). Using a Potter spray tower, they applied 5 ml aliquots of synergised pyrethrum solutions containing a wetter to aphids held i n a p e t r i half.  After  spraying, the aphids were immediately removed to a clean p e t r i plate and held without food for 24 hours before mort a l i t y assessment.  Yule reported the L C ^ Q as 150 ppm  pyrethrins, with 95$ confidence that the true value f e l l between 96 and 305  ppm.  A broad bean plant, excised under water so as not to interrupt water conduction, was used i n the next cage.  The  floor of t h i s cage was formed by a Mason jar l i d glued into a Mason jar ring 9«6-cm i  n  diameter.  The stem of the plant  v/as held in a 0.8-cm hole i n the floor by a c o l l a r of  26  modelling clay, and protruded into a water reservoir below. The body of the cage was formed by a Kodapak cylinder, 9-5-cm diameter x 18-cm high, f i t t e d inside the Mason jar ring. The top of the cage was nylon organdy. Since t h i s cage provided suitable conditions, the holding period was increased to 48 hours.  This increased the  precision of measurement of the effect of the malathion by reducing the chance of error in the dead:live decision. To reduce the p o s s i b i l i t y of the wetter entering into • the reaction between the aphid and the malathion, new emulsions v/ere made with the concentration of the wetter reduced to 250 ppm.  E a r l i e r tests had shown that t h i s  concentration reduced the contact angle between a glass surface and the emulsion. next 11 tests.  These emulsions were used i n the  A saran screen, 12 strands per cm, v/as used  to hold the aphids i n the p e t r i dish during the 30-minute exposure period. This screen allowed the deposit to evaporate to dryness, and the aphids could be removed gently from the plate at the end of the exposure period. In a further effort to reduce variation between r e p l i cates, i t was decided that the so-called grandmother effect should be taken into account.  The s u s c e p t i b i l i t y of the  aphid may be conditioned by such factors as:  temperature,  photoperiod, vapor pressure d e f i c i t , crowding, and the water relations and nutrient status of the host plants. Ideally these conditions should be constant not only from replicate to replicate for the treated generation, but also  27 f o r t h e two p r e v i o u s g e n e r a t i o n s i n t h e i r p a r e n t a l  lines.  T h e s e new t e c h n i q u e s r e m e d i e d many o f t h e s h o r t c o m i n g s o f former methods.  Low m o r t a l i t y  (Table V I I ) i n d i c a t e d that treatment holding  conditions  w i l t e d and c h l o r o t i c .  treatments  t h e treatment procedure and p o s t were n o t d e t r i m e n t a l  A t 48 h o u r s t h e e x c i s e d  aphids.'  the  i n the control  plants  to the  h a d become  slightly  The s u r v i v i n g a p n i d s , e s p e c i a l l y i n  c o n t r o l and lower c o n c e n t r a t i o n  treatments,  s h r u n k e n b u t f e d a n d showed no s i g n s  were  o f the excitement  symptomatic o f malathion i n t o x i c a t i o n . N e v e r t h e l e s s , t h e r e s u l t s were d i s a p p o i n t i n g . variation great  i n the slopes  The  o f t h e l d - p l i n e s v/as s t i l l t o o  (Table V l l ) , i n d i c a t i n g e i t h e r inconstancy i n contact  between, t h e a p h i d s a n d t h e m a l a t h i o n  (Hoskins and C r a i g ,  1962),  some e x t r a n e o u s f a c t o r .  or i r r e g u l a r interaction with  As a c h e c k on t h e c o n d i t i o n s treatment holding  p r o v i d e d by t h e p o s t -  cages, 48-hour s u r v i v o r s  ments w e r e t r a n s f e r r e d t o f r e s h p l a n t s while  o t h e r s were l e f t  condition  o f some t r e a t -  i n holding  on t h e o r i g i n a l p l a n t s .  The  o f t h e p l a n t s , a n d m o r t a l i t y among t h e a p h i d s  were a s s e s s e d a f t e r an a d d i t i o n a l 48-hour  period.  The a p h i d s w h i c h w e r e t r a n s f e r r e d t o f r e s h expanded t o t h e i r normal s i z e , and t h e r e  plants  v/as l i t t l e  tional  mortality after the additional period.  plants  v/ere s l i g h t l y w i l t e d a n d c h l o r o t i c .  addi-  These  Where t h e  a p h i d s h a d n o t been t r a n s f e r r e d t o f r e s h p l a n t s an  cages,  a d d i t i o n a l 60$ m o r t a l i t y among t h e s u r v i v o r s .  t h e r e v/as These  TABLE V I I .  P e r c e n t m o r t a l i t y o f 9~day o l d A. p i s u m , r e a r e d u n d e r c o n s t a n t c o n d i t i o n s  f o r three generations, sprayed w i t h malathion on t h e t r e a t e d Malathion ppm 0 20 30 40 50 60 80  s u r f a c e f o r 30 m i n u t e s .  1 6 16  2 0 2  16  24  —  —  M o r t a l i t y a s s e s s e d a t 48  94  92 92  57.36  46.48  45.47  53.27  Fiducial Upper Lower  Limits n.s.  n.s.  48.88 42.18  n.s.  S l o p e (b) -feSDb  7.85  3.57 1.53  7.37  7.21 2.29  Aphids  hours.  8 2  --  88  —  containing a wetter.  P e r c e n t tn o r t a l i t y i n t e s t : 4 6 7 5 2 0 0 0 — 0 0 6 12 2 2 20 40 0 17 — 48 57 100 70 50 32 98 98 98 98  3 2 4 10 31  —  —  emulsions  —  2 0 16 52 26  9 0  --0  14 44 34 86  Statistics LCCJQ  ppm  3.48  n.s.  Regression not s i g n i f i c a n t  a.a.  A n a l y s i s abandoned. freedom.  0.84  (p = . 0 5 ) .  a. a.  50.65  ci  0  9. 0  53.34 47.96 a. a.  Fiducial  9.26 1.18  cl •  9.  Q  90.06  59.15  n.s.  n.s.  4.06 3.43  6.93 1.93  l i m i t s n o t computed.  P o o l i n g o f dose c l a s s e s r e s u l t e d i n l o s s o f degrees o f  left  29 plants  were s e v e r e l y  w i l t e d , and t h e r e  spots i n the leaves.  period.  o f t h e h o s t p l a n t s , w h i c h may  mortality during  To g u a r d a g a i n s t  this possibility,  ( H o a g l a n d a n d A r n o n , 1950)  attrialso  48-hour  holding  the plant  main-  the i n i t i a l  t e n a n c e medium was c h a n g e d t o H o a g l a n d ' s c o m p l e t e solution  necrotic  T h i s a d d i t i o n a l m o r t a l i t y was  buted t o the condition have i n f l u e n c e d  were l a r g e  nutrient  since the e a r l i e s t  indi-  c a t i o n o f h o s t d e g e n e r a t i o n was a g e n e r a l c h l o r o s i s . Three r e p l i c a t e s were r u n , s i m i l a r i n a l l r e s p e c t s t e s t s 6 t o 9 i n Table V I I except t h a t was u s e d a s t h e p l a n t  ment h o l d i n g The  Hoagland s s o l u t i o n 1  :  m a i n t e n a n c e medium.  was u s e d t o c h e c k t h e c o n d i t i o n s  p r o v i d e d by t h e p o s t - t r e a t -  cages a l s o .  the d e t e r i o r a t i o n o f the host p l a n t s .  h o u r s t h e p l a n t s were t u r g i d , b u t s m a l l appeared i n t h e leaves. were s e v e r e l y enlarged. the  The same t e s t  u s e o f H o a g l a n d ' s s o l u t i o n was u n s u c c e s s f u l  preventing  to  After.the  added p e r i o d ,  w i l t e d and t h e n e c r o t i c  T h e r e was s t i l l  necrotic  s p o t s were  in 48  After  spots had the plants greatly  an a d d i t i o n a l 60$ m o r t a l i t y  among  s u r v i v i n g a p h i d s , w h i c h d i d n o t o c c u r i f they were  ferred to fresh The fairly  LC^os  plants. computed f r o m t h e s e t h r e e  r e p l i c a t e s agreed  c l o s e l y but the v a r i a t i o n s In slopes  l i n e s was t o o g r e a t  (Table V I I I ) .  of the ld-p  The s l o p e s  given i n  T a b l e s V I I a n d V I I I i n d i c a t e an i n t e r a c t i o n w i t h trolled there  trans-  variable.  On e x a m i n a t i o n o f t h e L C E J Q S  a p p e a r e d t o be a g e n e r a l d e c l i n e  an u n c o n -  obtained  i n the t o x i c i t y o f  30  TABLE V I I I .  Percent  under constant malathion  m o r t a l i t y o f 9 - d a y o l d A. p i s u m ,  conditions f o r three generations,  emulsions  containing a wetter,  reared  sprayed  Aphids l e f t  with  on t h e  s u r f a c e f o r 30 m i n u t e s t h e n t r a n s f e r r e d t o e x c i s e d  treated  p l a n t s i n H o a g l a n d ' s f o r 48 h o u r s b e f o r e m o r t a l i t y Malathion ppm 0 30 40  50 60 80  Percent 1  0 0 0  assessment.  mortality i n replicate  2  3  2 2  16  0 0 2  66  22 44 64  8 18 60  LC 0 Fiducial Limits Upper Lower  72.17  67.65  75.40  77.43 68.32  76.27 62.09  83.49 70.26  Slope (b) +SDb  10.32 1.42  5.46 0.84  8.07 1.19  4 24  Statistics 5  31 the emulsion from t e s t VIII.  1 o f Table V I It o t e s t 2 o f Table  The same e m u l s i o n s w e r e u s e d f o r a l l  these  tests.  I n t h e e m u l s i o n t h e m a l a t h i o n was d i s s o l v e d I n x y l e n e , w h i c h was t h u s d i s p e r s e d a s s m a l l d r o p l e t s t h r o u g h o u t t h e d i s t i l l e d water:wetter c a r r i e r . was  I n t h e o r y each  droplet  i s o l a t e d f r o m t h e c a r r i e r b y an. e n v e l o p i n g f i l m o f t h e  emulsifier.  I t a p p e a r s t h a t t h e m a l a t h i o n was h y d r o l y s e d  even t h o u g h  i t was d i s s o l v e d i n x y l e n e , w h i c h i s i n s o l u b l e  i n w a t e r , and p r o t e c t e d b y t h e f i l m o f e m u l s i f i e r .  This  ran c o u n t e r t o t h e r e p o r t o f Y o s t , F r e d e r i c k and M I g r d i c h i a n (1955), who f o u n d no s i g n i f i c a n t  m a l a t h i o n - x y l e n e e m u l s i o n s i n w a t e r a f t e r seven storage.  T h e r e v/as a s i g n i f i c a n t  was  months  i n c r e a s e i n LC50 w i t h i n -  c r e a s e d age o f t h e e m u l s i o n s used h e r e The  i n 1$  loss of toxicity  (Table X I ) .  basic design o f the post-treatment holding  cage  appropriate t o the aphid's requirements, but t h e rapid  d e t e r i o r a t i o n o f t h e h o s t p l a n t s n e c e s s i t a t e d a change t o p l a n t s growing  i n 10.3 x 10.3-cm s q u a r e p o t s .  cage t o t h e l i v e p l a n t , a base o f u n p a i n t e d f i r  To a d a p t t h e plywood,  w i t h a s l o t c u t t o accommodate t h e p l a n t , was f i t t e d t h e r i m o f t h e p o t , on t h e s o i l . a s t r i p o f masking  inside  The s l o t was s e a l e d  with  tape and a c o l l a r o f m o d e l l i n g c l a y  around t h e base o f t h e p l a n t .  The b o d y o f t h e c a g e was  f i x e d i n t o a c i r c u l a r g r o o v e , 9«5-cm i n d i a m e t e r , a l s o c u t i n t o t h e plywood  base, by s t r i p s o f masking  w a l l o f t h e cage t o t h e s i d e s o f t h e p o t .  tape from t h e These cages  were  32  TABLE I X .  C o r r e l a t i o n between age o f emulsion  LCtjQ o b t a i n e d by p r o b i t  analysis. LC50  (days) 2 3 4 5 7 11 12 13 14  (ppm)  57.4 46.5 45.5 53.3 50.6 90.1 59.2 72.2 67.6 r = .683 * F  * S i g n i f i c a n t at p = . 0 5 .  (1,7)  = 6.1316  ( i n days) and  33  also tested by leaving the aphids on the plants for an addit i o n a l 48-hour period. The emulsions used in the next three tests (Table X) were made within. 20 minutes of being applied. There was no additional mortality in the added period after the i n i t i a l 48 hours.  This fact, and the absence of  mortality in the control treatments attested to the adequacy of the post-treatment holding method. When using the Potter spray tower, there i s no certainty that each aphid w i l l be contacted by the same quantity of insecticide, nor that a constant amount of material w i l l be deposited on the target.  An aphid may be contacted by  a single large droplet, or by a small droplet; the 30-minute exposure period tends t o equalize t h i s d i s p a r i t y , but cannot r e c t i f y i t e n t i r e l y .  Differences In the a c t i v i t y of  individual aphids while on the treated surface also affect t h e i r contact with the toxicant.  The t o t a l material  deposited on the target varies d i r e c t l y with the amount deposited on the walls of the tower, which in turn i s determined by the l e v e l at which turbulence i s i n i t i a t e d . Since the factors causing turbulence are not known, i t cannot be controlled (Potter,  1952;.  Dosage i s the controllable variable and the precision of i t s measurement i s of prime importance.  Imperfections  in the Potter tower do not allow of the required precision in the measurement of dosage and are reflected in the v a r i ation in the slopes and high standard deviations.  34  TABLE X.  Percent  constant  conditions for three generations,  t h i o n emulsions treated  m o r t a l i t y o f 9-day o l d A. p i s u m , r e a r e d u n d e r  containing a wetter.  sprayed w i t h mala-  Aphids  left  on t h e  s u r f a c e f o r 30 m i n u t e s t h e n t r a n s f e r r e d t o l i v i n g  p l a n t s f o r 48 h o u r s b e f o r e m o r t a l i t y Percent  Malathion ppm  1  0 40 50 60 70 80 90 100  assessed. mortality i n test 2  0  0  0 26 92 74 68 100  0 54 24 44 14 94  3 0 12 12 22 36 86 — ——  Statistics LC50 (ppm) Fiducial Slope (b) n.s.  Limits  67.78 n.s.  84.93 n.s.  69.50 n.s.  9.96 4.34  5.19 3.86  7.04 2.77  Regression, not s i g n i f i c a n t not computed.  (p = . 0 5 ) .  Fiducial limits  35  Topical Application to Individuals. The next step i n refining the technique was t o p i c a l application of measured dosages to individuals.  The need  for t h i s refinement was demonstrated by the inadequacy of the Potter tower. Acetone was chosen as the solvent-carrier, because i t has high vapor pressure and evaporates rapidly.  While t h i s  characteristic i s disadvantageous i n spraying, i t i s advantageous when the material i s placed d i r e c t l y on the aphid. A stock 10,000 ppm (one percent) solution of malathion i n acetone was made by weighing 0.500 g of toxicant into a clean, dry, 50-ml volumetric flask, and d i l u t i n g with acetone.  Solutions of the desired concentration were then  made by d i l u t i n g calculated amounts of the stock solution with acetone. To apply the insecticide a Yale B-D Leur 0.25-ml glass hypodermic syringe f i t t e d with a No. 26 square-end, rightangled needle was clamped into the holder of a modified Micro-Metric SB-2^ syringe micro-buret.  The drive spindle  of t h i s applicator had been f i t t e d with a plywood disc 19-cm  i n diameter, i n which 20 equally spaced cogs had been  cut.  A pawl mounted from the applicator base permitted the  operator to devote h i s entire attention to the t i p of the needle and the aphids during operation.  By turning the  plywood disc from one cog to the next the plunger was 9.  Manufactured by Micro-Metric Instrument Company, Cleveland, Ohio.  36  advanced .002 inches, and 0.514 ^ul^of solution was delivered. This droplet of insecticide was transferred to the aphid by touching the t i p of the needle to the dorsum of the aphid's abdomen. The insecticide spread at once to cover the entire abdomen. During the f i r s t t r i a l s the aphids v/ere held for treatment i n glass p e t r i plates which were divided into four compartments by PIuon-coated Kodapak s t r i p s . compartment held five aphids.  Each  The appropriate dosage range  was established by a logarithmic series of dosages ranging from 6 2 . 5 to 1000 ppm (31.25 to 500 ng per aphid).  Forty-  eight hours a f t e r treatment there was 68 and 96 percent mortality at 62.5 and 125 ppm respectively, but at higher dosages mortality was v i r t u a l l y complete.  Since these con-  centrations approximated those used with the Potter tower Table VII, VIII), a similar series of concentrations was used for the next four tests (Table X l ) . Different means of holding the aphids for treatment were t r i e d .  In Test 1, the glass p e t r i dish was divided  into four compartments.  In these, the uneven bottom of the  plate allowed some aphids to move under the barriers from one compartment to another, with the p o s s i b i l i t y of an aphid escaping treatment or being treated twice.  In Test 2  the concentration range was reduced so that more points could be established i n the central portion of the l i n e . For t h i s t r i a l the glass p e t r i dish was divided Into three arenas by Fluon-coated Kodapak cylinders 1.3-cm high.  Each  37  TABLE X I . 0.5  P e r c e n t m o r t a l i t y o f 9 - d a y o l d A. p i s u m t o w h i c h  m i c r o l i t e r drops o f malathion i n acetone s o l u t i o n  were  a p p l i e d on t h e a b d o m i n a l d o r s u m w i t h a s y r i n g e m i c r o - b u r e t . A p h i d s h e l d on s o l i d to  surface f o r treatment, then transferred  l i v e p l a n t s f o r 48 h o u r s p r i o r t o m o r t a l i t y Malathion ppm n g / A p h i d  0 20 30 40 50 60 70 80 100  0 10 15 20 25 30 35  40  50  1  assessment.  Percent mortality i n test 2 3  0 4 33 84 —  92 94  2  4  0  0 —  —  2 16 38 32 62  26 24  64  70 80 92 90  mm  — —  _  8 32 54 80 86 80  Statistics LD50 ( n g / A p h i d ) Fiducial Limit s Upper Lower S l o p e (b) ±SDb  22.29  32.41  21.72  23.33  24.41  20.07  35.36 30.17  26.56 14.89  25.10 21.44  5.30 O.56  5.52 O.78  5.58 1.11  4.94 0.59  38  arena held f i v e aphids.  Low mortality in t h i s t r i a l may  have been the result of insecticide transfer from the aphids to the glass, as some aphids were seen to touch t h e i r abdomens to the glass before the acetone had evaporated. No residue was v i s i b l e on the glass. To check on the p o s s i b i l i t y of insecticide loss the aphids were held i n new p l a s t i c p e t r i plates, which were readily scarred by acetone, for the next two tests (Nos. 3 and 4 ) . Numerous scars, of varying size, i n the p l a s t i c after Test 4 indicated that many aphids had lost various amounts of the material applied.  This loss was probably  the largest factor contributing to the decreased mortality at high dosages, and the variations between tests i n Table XI.  Except for the high standard deviation of the slope  in Test 3, the s t a t i s t i c s computed from these data were very uniform. To reduce the p o s s i b i l i t y of transfer of insecticide from the aphid to the surface on which i t was standing, the treatment surface was changed to 12 strand per cm saran screen.  The number of aphids confined i n the arenas was  changed from 5 i n each to 4 in one, 5 i n another, and 6 i n the t h i r d .  This enabled the operator to discriminate  between arenas, thereby reducing the chance of confusion over which aphids had been treated and which had not.  39  RESULTS At t h i s stage i t was concluded that a suitable technique for establishing the dosage-mortality curve for the pea aphid had been evolved.  Consideration of five major  factors v/as shown to be Important i n establishing t h i s curve.  The major factors were:  the formulation of the  insecticide, and the methods of rearing, c o l l e c t i n g , treating, and post-treatment holding. The repeatability of the results obtained by use of t h i s technique i s demonstrated by the homogeneity of the  LD50  a n d  slope estimates from  replicate to replicate i n Table XII. Two Id-p l i n e s , given in Figure 1, representing the maximum and minimum t o x i c i t y of malathion to the pea aphid, also i l l u s t r a t e t h i s repeatability.  The procedures of the technique evolved are  detailed i n the following paragraphs. The Insecticide Formulation A solution of malathion in acetone v/as found to be best for t o p i c a l application to the pea aphid. ppm  A  10,000  (one percent) stock malathion solution was made by  weighing  0.500  g of toxicant, i . e . , 0.5208 g of the  96$  malathion, into a 50-ml volumetric flask and d i l u t i n g with reagent grade acetone.  One ml of t h i s solution v/as then  diluted to 100 ml to make a 100 ppm solution.  Calculated  amounts of t h i s 100 ppm solution were then further diluted to make 20 ml aliquots of test solutions containing 30, 40, 50,  60, 70 and 80 ppm malathion.  TABLE X I I . Percent mortality of 9 - d a y old A. pisum to which 0.5 m i c r o l i t e r drops of malathion i n acetone solution were applied on theabdominal dorsum with a syringe microburet.  Aphids held on a 12-strand per cm saran screen .for treatment, then transferred  to l i v e plants f o r 48hours before mortality assessment Malathion ppm ng/Aphid 0 30 40 50 60 70 80 100  0 15 20 25 30 35 40 50  1  2  2 4 36 60 72 84 86 96  2 O 44 66 82 82 88 100  0  Percent mortality i n replicate 3  4  5  6  7  7 3 20 76 70 76 92 82  4 18 42 78 70 92 74 96  0 6 38 54 80 90 90 94  0 4 52 75 66 86 84 87  2 6 36 78 74 86 88 96  Statistics (ng/Aphid) Fiduc i a l Limit s Upper Lower  LD50  Slope (b) ±SD b  24.54  23.01  25.69  22.08  23.58  22,91  23.16  26.21 22.88  24.50 21 .39  27.75 23.40  24.01 19.85  25.07 22.02  24.77 20.86  24.71 21.49  5.86 0.58  O .24 0 .62  5.13 0.64  4.51 0.53  6.13 O.58  4.59 0.50  5.97 0,60  4l FIGURE 1. mortality potency,  The  two  lines,  central log-dosage:probitr.  o b t a i n e d by t h e method o f r e l a t i v e  d e s c r i b e t h e maximum and  minimum  of malathion t o Acyrthosiphon pisum two  o u t e r c u r v e s a r e t h e 95$  toxicity  (Harris).  fiducial limits  A p p e n d i x I ) a b o u t t h e b a n d f o r m e d by t h e two lines;  these outer curves i n d i c a t e the area  which the t r u e ld-p l i n e to  fall.  The (see central  within  f o r the clone i s expected  1  J  DOSAGE  I  L J  I  nanograms/aphid  |  I I I  42 Rearing C o l o n i e s o f t h e pea a p h i d , A c y r t h o s i p h o n pisum  (Harris),  used f o r t h i s t e c h n i q u e were r e a r e d i n t h e ca.ges, and under t h e p h y s i c a l e n v i r o n m e n t a l c o n d i t i o n s d e s c r i b e d on Pages 7 and 8.  The c o l o n i e s were m a i n t a i n e d under t h e s e optimum  c o n d i t i o n s f o r t h r e e g e n e r a t i o n s b e f o r e t h e a p h i d s were treated. A s i m p l e method was used t o p r o v i d e u n i f o r m l y l a r g e , h e a l t h y , a p t e r o u s a p h i d s which had not been s u b j e c t e d t o crowding o r drought.  C o l o n i e s were i n i t i a t e d by p l a c i n g 20  n i n e - d a y o l d a p h i d s i n each o f two p o t s c o n t a i n i n g 12 2-8 cm plants.  2.5-cm  Esca/pes were p r e v e n t e d by a F l u o n - c o a t e d  h i g h Kodapak r i n g f i t t e d s n u g l y I n s i d e t h e r i m o f t h e p o t . Twenty pea a p h i d s o f t h i s age produced about 220 nymphs ( F r a z e r , p e r s o n a l communication) d u r i n g 24 h o u r s ,  after  which t h e a d u l t s were removed. Collecting Aphids were c o l l e c t e d when t h e y were 9 - 0 . 5  days o l d .  For c o l l e c t i o n , t h e cage v/as removed g e n t l y from t h e f l o o r t o a bench.  Aphids. not on t h e p l a n t s i n t h e cage were  removed and d e s t r o y e d .  S i n g l e p l a n t s were c u t o f f a t t h e  s o i l l i n e and h e l d over a p y r e x d i s h , t h e i n n e r s i d e s o f which were c o a t e d w i t h F l u o n . g e n t l y brushed  Apterous a d u l t a p h i d s were  from t h e p l a n t i n t o t h i s c o l l e c t i n g  w i t h a c l e a n No. 2 s q u i r r e l h a i r b r u s h .  dish  C a s t s and nymphs  were removed from t h e d i s h and from t h e a d u l t s .  Sub-samples,  43  i.e.,  the aphids collected from one or two plants, were  evenly distributed among the eight p e t r i dishes used to hold them prior to treatment.  Sub-samples were taken u n t i l  enough aphids to furnish 50 for each of seven treatments and a control had been collected.  Aphids used in control  treatments were weighed before being treated as a check on uniformity and to gauge the effects of handling. The aphids were then transferred singly from the holding dish with a vacuum pencil to 10-cm arenas/, the floors of which were of 12-strand per cm saran. screen.  Each arena  was subdivided into three compartments by Fluon-coated Kodapak cylinders, 4-cm diameter x 1.3-cm high.  Fifteen  aphids were placed In each arena, four i n one compartment, five i n another and s i x i n the t h i r d . Treating A Yale B-D Leur 0.25-ml glass hypodermic syringe with the needle i n place was f i l l e d with the appropriate insecticide solution,  A No. 26 needle, 3.8-cm long, with  a 90° bend 2-cm from the t i p , vias used.  The beveled point  of the needle had been ground with an oilstone u n t i l no trace of the bevel remained.  The syringe was cleared of  a i r bubbles by Inverting i t and advancing the plunger. The f i l l e d syringe was clamped into the holder of the modified Micro-Metric SB-2 syringe micro-buret.  The plunger  was advanced, delivering 0 . 5 jul of insecticide which was transferred by touching the t i p of the needle to the dorsum of an aphid's abdomen. The time was recorded at.the  44 beginning and end o f t h e treatment p e r i o d .  A different  random sequence o f treatments was used f o r each  replicate.  Post-treatment h o l d i n g A f t e r treatment the aphids were brushed g e n t l y i n t o the h o l d i n g cage d e s c r i b e d on Page 3 1 .  During the 48-hour  h o l d i n g p e r i o d the p l a n t was s u p p l i e d with adequate water. F o r t y - e i g h t hours a f t e r treatment  the r e s u l t s were assessed  by counting t h e aphids and c l a s s i f y i n g them as l i v i n g or dead.  Any aphid capable o f c o o r d i n a t e d movement was  c l a s s i f i e d as l i v i n g .  45  DISCUSSION E v a l u a t i o n of the Technique The c r i t e r i a  used f o r e v a l u a t i n g a treatment method  vary w i t h the purpose f o r which i t  i s intended,  A field  survey method must be r a p i d and simple enough to be p e r formed s a t i s f a c t o r i l y by i n e x p e r i e n c e d p e r s o n n e l . should employ e a s i l y p o r t a b l e ,  s t a n d a r d i z e d equipment and  m a t e r i a l s t h a t may be prepared i n q u a n t i t y Practicability technique.  It  In advance.  i s the b a s i s f o r e v a l u a t i n g t h i s type  A technique f o r I n s e c t i c i d e  screening or  r e s i s t a n c e s t u d i e s i s judged by d i f f e r e n t These are p r i m a r i l y  of  criteria.  concerned with the accuracy and p r e -  c i s i o n o f the r e s u l t s ; p r a c t i c a l c o n s i d e r a t i o n s o f cost and experience of p e r s o n n e l are secondary. Hoskins and C r a i g ( 1 9 6 2 ) i n d i s c u s s i n g b i o a s s a y s set out ten c r i t e r i a which should be s a t i s f i e d by a t r e a t ment procedure i f applicability.  i s to have g e n e r a l and s p e c i f i c  These concern two g e n e r a l c o n s i d e r a t i o n s :  the mathematical the r e l i a b i l i t y  it  C h i - s q u a r e which g i v e s an I n d i c a t i o n o f the computed l d - p l i n e ;  c a l r e l a t i o n between the I n s e c t i c i d e caused by i t .  The ten c r i t e r i a  are:  constant r e l a t i o n  2.  p r e c i s e measurement o f dosage;  4,  normality of  o f dose to dosage;  evaluation of environment;  practi-  and the response  1.  3, q u a n t i t i a t i v e  and the  effect;  of  46  5 . constancy of environment; 6. s e n s i t i v i t y to variation; 7. reproducibility of results; 8. wide a p p l i c a b i l i t y ; 9. representativity of population; 10, s i m p l i c i t y and rapidity. Application of these c r i t e r i a to the method developed here are discussed i n descending order of importance, 1, Constant Relation of Dose to Dosage Dosage must be accurately reflected by the response over the range of the dosage, and i t must bear a constant r e l a t i o n to the dose, I.e., the insecticide which actually reaches the site of action.  Verifying the second part of  t h i s c r i t e r i o n would need a complex study in I t s e l f , For example, malathion acts as a cholinesterase i n h i b i t o r , but there i s some doubt as to the existence of a cholinesterase system i n many insects (O'Brien, 1966}. less, malathion i s an effective insecticide.  Neverthe-  I have  assumed here that a cholinesterase system exists in the Aphididae and that there i s a constant relation between the dosage and the dose, within the l i m i t s of p r a c t i c a b i l ity.  In at lease one case, the second assumption has been  shown to be unfounded.  Bridges (1957), applying the  pyrethrum derivative a l l e t h r i n t o p i c a l l y to the adult house f l y , Musca domestlca Linnaeus, showed that 95$ of a low dose of 1 jjg had been absorbed after 24 hours but  47 o n l y 47$ o f a h i g h dose o f 12 j j g . The r e l a t i o n between t h e a p p l i e d dosage and t h e dose i s measured i n d i r e c t l y by the d e v i a t i o n o f from t h e normal response d i s t r i b u t i o n .  active  responses  In p r o b i t  analysis  t h e s e d e v i a t i o n s a r e expressed as the s t a n d a r d d e v i a t i o n o f t h e s l o p e and t h e 95$ f i d u c i a l l i m i t s about the comp u t e d l e t h a l dose v a l u e s  (Table X l l ) .  When t o p i c a l  a p p l i c a t i o n s a r e made i n d i v i d u a l l y b o t h o f t h e s e a r e s m a l l , i n d i c a t i n g c l o s e agreement  quantities  w i t h normal response  d i s t r i b u t i o n , and hence w i t h t h e r e q u i r e d c o n s t a n t t i o n between a p p l i e d dosage and a c t i v e 2 . P r e c i s e Measurement o f  dose.  Dosage  S i n c e dosage i s the independent v a r i a b l e , measured p r e c i s e l y .  rela-  i t must be  H o s k i n s and C r a i g (1962) s t a t e  that  e r r o r s I n t h i s measurement a r e common, and t h a t i n v e s t i g a t o r s who a r e i m p r e c i s e g e n e r a l l y a t t r i b u t e t h e r e s u l t i n g l a r g e s t a n d a r d d e v i a t i o n s and wide f i d u c i a l l i m i t s t o biological variation.  Among t h e few I n v e s t i g a t o r s who  r e p o r t t h e i r methods and r e s u l t s i n s u f f i c i e n t d e t a i l t o p e r m i t a c r i t i c a l e v a l u a t i o n , many, such as (1962),  Dittrich  Patwa (1965}, P o t t e r and G i l l h a m (l95§h and  Y u l e (1964),  computed l d - p l i n e s from t h e averaged  o f a number o f r e p l i c a t e s .  This procedure Is  statistically  unsound u n l e s s p r e v i o u s a n a l y s i s has shown t h a t t h e ances o f b a t c h e s from t e s t t o t e s t  exposed t o t h e same dosage a r e (Finney,  1962).  results  vari-  homogeneous  48  The method developed i n this study employed two devices to increase confidence i n the precision of dosage measurement:  to prevent a buildup of a malathion residue,  the t i p of the applicator needle was wiped frequently with a piece of absorbent tissue, and to reduce the possib i l i t y of loss of insecticide before the solvent had evaporated, the aphids were held on a saran screen during treatment. 3 . Quantitative Evaluation of Effect The response must be expressed quantitatively. Pradhan  ( 1 9 4 9 )  abandoned his study with the chrysanthemum  aphid, Macrosiphoniella sanbourni G i l l . , because of d i f f i c u l t i e s i n providing a suitable post-treatment environment and i n making the dead:live decision necessary to the quantal assessment of response.  These d i f f i c u l t i e s were  also encountered i n t h i s study; however, when a suitable environment was provided, and the holding period Increased to 48 hours, the dead:live decision could be made with certainty.  There was seldom doubt as to the present and  future condition of an aphid. Errors i n quantal assessment would probably tend to be biased toward the death response at higher dosages and more random at lower dosages.  This would result i n an  increased slope and larger standard deviation.  49 4.  N o r m a l i t y o f Environment The t r e a t m e n t p r o c e d u r e must b e c a r r i e d o u t i n a n  environment  as n e a r l y normal as p o s s i b l e , w i t h i n t h e l i m i t a -  tions of controlled conditions.  Fulfilling this  condition  ensures t h a t t r e a t e d i n s e c t s remain r e p r e s e n t a t i v e o f t h e i r s p e c i e s and t h a t m o r t a l i t y i n t h e c o n t r o l t r e a t m e n t s i s h e l d t o a minimum.  Y u l e (1964) a n d P a t w a (1965),  investigat-  ing t h e t o x i c i t y o f pyrethrum f o r m u l a t i o n s t o t h e pea aphid a n d t h e p o t a t o a p h i d p r o v i d e d no f o o d d u r i n g t h e i r h o l d i n g p e r i o d p r i o r t o m o r t a l i t y assessment. cedure might  24-hour  This pro-  i n t r o d u c e an. e r r o r b y i n c r e a s i n g m o r t a l i t y a t  low dosages where i n t o x i c a t i o n  i s l i k e l y t o be r e v e r s i b l e .  T h i s would d e c r e a s e t h e s l o p e o f t h e l d - p l i n e , g i v i n g an overestimate of the variance of s u s c e p t i b i l i t y  i n the  population. Environmental conditions at a l l aphid's l i f e environment.  times during the  should approximate those o f t h e aphid's normal In t h e procedure developed here, t h e only  p e r i o d d u r i n g w h i c h t h e a p h i d s were s u b j e c t e d t o abnormal c o n d i t i o n s was d u r i n g t h e a p p l i c a t i o n o f t h e i n s e c t i c i d e . The  l a p s e d t i m e between removal from t h e h o s t p l a n t and  I n t r o d u c t i o n t o t h e h o l d i n g c a g e was 3.5 i" 0.5 h o u r s . D u r i n g t h i s p e r i o d t h e a p h i d s were n o t s u b j e c t e d t o unnece s s a r y e n v i r o n m e n t a l shocks, such as t e m p e r a t u r e  changes.  50 5.  Constancy o f Environment Since t h e a c t i v i t i e s o f i n s e c t s a r e a f f e c t e d by  environmental bioassay.  c o n d i t i o n s t h e s e must b e c o n s i d e r e d i n a  Aphids  are particularly sensitive to their  e n v i r o n m e n t , w h i c h may b e r e f l e c t e d morph, o f t h e i n s e c t .  even i n t h e f o r m , o r  Photoperiod, temperature,  and popu-  l a t i o n d e n s i t y h a v e b e e n shown t o be I n s t r u m e n t a l i n morph d e t e r m i n a t i o n i n t h e v e t c h a p h i d , Megoura v i c i a e  Buckton,  ( L e e s , 1959); t h e cowpea a p h i d , A p h i s . c r a c c i v o r a K o c h , (Johnson,  19^5,  1966b); a n d t h e c a b b a g e a p h i d ,  brassicae  ( L i n n a e u s ) , (Lamb a n d W h i t e , 1966).  Brevicoryne The c o n d i -  t i o n o f t h e h o s t p l a n t a l s o a f f e c t s morph d e t e r m i n a t i o n i n t h e cowpea a p h i d determined  (Johnson,  1966a).  The morph, w h i c h i s  w h i l e t h e a p h i d i s a n embryo i n i t s m o t h e r ' s  ovariole,  i s determined  by t h e maternal  physiology  (Lees,  1961), w h i c h i s i n t u r n i n f l u e n c e d by t h e e n v i r o n m e n t a l c o n d i t i o n s t o which t h e mother i s s u b j e c t e d . s t u d i e s by U i c h a n c o  (1924) on t h e embryogeny a n d p o s t -  n a t a l development o f t h e aphid Dactynotus tanaceti  (Linnaeus)  while t h e parent  Cytological  (= M a c r o s i p h u m )  h a v e shown t h a t o v u l a t i o n b e g i n s  i sstill  i n t h e embryonic s t a t e .  Lees  (1961) s t a t e s t h a t t h e s e x o f t h e embryo i s d e t e r m i n e d t h i s t i m e , and t h a t t h e s e x r a t i o temperature  at  can be m o d i f i e d by t h e  i n t h e grandmother's environment.  Other  environmental  f a c t o r s determine  whether o r not males  be p r o d u c e d .  The e n v i r o n m e n t a l  factors controlling  will this  51  so-called grandmother effect may act d i r e c t l y on the developing oocyte or, more probably, by modifying the grandmother s physiological state. 1  The mother's or grandmother's  physiological state may also influence s u s c e p t i b i l i t y to an insecticide in a daughter or granddaughter. The environment may have a direct effect In the relation between the malathion and the aphid. Young and Roussel (1958) and Rai, _et a l . (1956) found a positive correlation between temperature and the t o x i c i t y of malathion to the b o l l weevil, Anthonomus grandis Boheman, and the house f l y . To avoid introducing errors caused by interactions between the environment and the independent variable i n t h i s study, conditions of l i g h t , temperature, vapor pressure d e f i c i t , host plant condition, and population density were held constant from replicate to replicate and from generation to generation.  To avoid the possi-  b i l i t y of an interaction of treatment time with a daily rhythm of s u s c e p t i b i l i t y , as shown i n the b o l l weevil by Cole and Adkisson (1964) and the two-spotted spider mite, Tetranychus urticae (Koch), by Fisher (1967), the order of treatments was randomized i n each replicate. 6. S e n s i t i v i t y to Variation The bioassay must be s u f f i c i e n t l y sensitive to serve i t s purpose.  This procedure was developed for studies of  s u s c e p t i b i l i t y so that s e n s i t i v i t y here refers to changes  52 in the effect of the insecticide. (1956) d e f i n e d The  first,  stand  H o s k i n s and Gordon  two t y p e s o f d e c r e a s e d  vigor tolerance,  i s an added a b i l i t y t o w i t h -  a toxicant, usually derived  improved n u t r i t i o n  from such f a c t o r s a s  o r extra weight.  i n d i c a t e d by an i n c r e a s e  susceptibility.  i nthe  T h i s would be w i t h no  LD50  significant  c o n c u r r e n t change i n t h e s l o p e o f t h e l d - p l i n e .  The  second, t r u e r e s i s t a n c e , i s a l s o t h e added a b i l i t y t o withstand  a t o x i c a n t , but i t i sacquired  from those i n d i v i d u a l s that at dosages s u f f i c i e n t but  not sufficient  by a n i n c r e a s e  i nthe  survive a toxicant  to k i l l  to kill LD50  slope o f t h e ld-p l i n e .  t h e whole. a n <  ^  genetic  a  small  c o n c u r r e n t change i n t h e s t u d i e s on  parthenogenesis,  To d e t e c t  such changes I n  and t o t r a c e t h e r i s e o f t o l e r a n c e o r  r e s i s t a n c e , t h e method must b e v e r y Hoskins and C r a i g evaluating a bioassay  sensitive.  (1962) a d v o c a t e t w o q u a n t i t i e s f o r technique:  of p r o b i t s about t h e l d - p l i n e , appropriateness  i s indicated  changes a r e expected t o be  ( S u o m a l a i n e n , 1962).  susceptibility,  This  In susceptibility  evolutionary  applied  a part o f the population,  i n s e c t s which reproduce by apomictic any  by i n h e r i t a n c e  t h e standard Sy.x,  deviation  which i n d i c a t e s t h e  o f t h e technique f o r determining  In s u s c e p t i b i l i t y ;  and t h e standard  changes  deviation of log-  dosages about t h e l d - p l i n e / X x . y , which i s a p p r o p r i a t e for  e v a l u a t i n g t h e s u i t a b i l i t y o f methods f o r  residue  53  determination or insecticide screening. In a sensitive test, both of these quantities are small.  For comparison  between insects or insecticides the ratios Sy.x/LD50 and yVx.y/LD50  are more appropriate, since the magnitude of  the standard deviations are partly determined by the units of dosage. Hoskins and Craig explain the derivation of these r a t i o s , but I have been unable to obtain the ratios given i n t h e i r Table I (pp  4 5 2 - 4 5 3 ) ,  either by working  through the data given In. t h e i r table, or by reanalysing the o r i g i n a l data from four of t h e i r sources and applying the formulae they set forth.  Unfortunately, they do not  give a reference to a more detailed account of t h e i r method. The standard deviation of the slope of the ld-p l i n e , with the 9 5 $ f i d u c i a l l i m i t s about the ID^Q, indicate the precision of the bioassay procedure.  These quantities,  taken from the pooled results of the seven replicates shown i n Table XII, are presented graphically i n Figure 1. With the clone used here and the bioassay procedure described, any subsequent test that produced an ld-p l i n e f a l l i n g outside the small c r i t i c a l area i n the figure would represent a significant deviation from t h i s baseline. The r e s t r i c t e d size of t h i s area shows that the bioassay procedure i s of sufficient precision to detect the very small changes expected when selecting f o r insecticide resistance within a clone of a parthenogenetic insect.  54 7. R e p r o d u c i b i l i t y o f r e s u l t s Some v a r i a t i o n b e t w e e n t e s t s i s u n a v o i d a b l e different  i n d i v i d u a l a p h i d s must be  When t h e t e s t s a r e generations  so  used f o r each  a r e t e s t e d , a l a r g e r v a r i a t i o n can  from the  test.  separated i n time that d i f f e r e n t  R e p r o d u c i b i l i t y of bioassay different  since  be  results, therefore,  r e p r o d u c i b i l i t y t o be  expected.  i s quite  expected i n a  p h y s i c a l or chemical a n a l y s i s i n which the materials be  defined  with great  Analysis of (page 40}  accuracy.  seven, r e p l i c a t e s a s g i v e n  shows t h a t a c e t o n e s o l u t i o n s o f  applied t o p i c a l l y to ducible results.  This  the homogeneity of the  ( F i g u r e 1,  the  opposite  estimates  standard  page 4 l ,  and  f r o m one  l i n e s , and  there  Wide  the limits,  replicate slopes  Table X I I ) . s e v e n r e p l i c a t e s by are  no  (p=.05) f r o m p a r a l l e l i s m b e t w e e n  a r e no  significant; deviations The  t h i s a n a l y s i s i s that the hypothesis drawn f r o m t h e  repro-  d e v i a t i o n s of the  data from the  the potency of the malathion.  8.  fiducial  method o f r e l a t i v e p o t e n c y shows t h a t t h e r e  significant deviations the  LD^Q  95$  XII  malathion  s t a t e m e n t i s s u p p o r t e d by  low  An. a n a l y s i s o f t h e the  i n Table  i n d i v i d u a l a p h i d s gave t r u l y  n a r r o w d o s a g e r a n g e i n c l u d e d by t h e  t o t h e n e x t , and  can.  same p o p u l a t i o n  conclusion that the  c a n n o t be  in  drawn from samples were  rejected.  applicability  T o p i c a l a p p l i c a t i o n has  been used s u c c e s s f u l l y w i t h  55  many species of other families i n the past (Busvine,  1957).  With minor modifications to suit the individual requirements of a species, the procedure developed here i s suitable for laboratory work with many species of aphids.  Syringes  with a smaller bore diameter, delivering very small dropl e t s , could be used for treating smaller aphids. rearing, collecting and post-treatment  The  holding methods  can easily be adapted to the individual requirements of a species.  Where insects are able to move about and feed  following treatment, they should be provided with the opportunity to do so. Acetone i s probably the most satisfactory solvent for most of the organic insecticides.  Most are soluble In I t  to a high degree (Metcalf, F l i n t and Metcalf, 1962) so that the wide range of concentrations necessary i n studies of s u s c e p t i b i l i t y and resistance are obtainable.  The use  of a single adjuvant, acting as both solvent and c a r r i e r , reduces the r i s k of interactions which might complicate the relationship between the Insect and insecticide.  The  use of acetone permits v a l i d comparisons to be made between the many insecticides which are soluble i n i t , 9. Representativity of population This c r i t e r i o n i s d i f f i c u l t , i f not impossible, to meet i n a laboratory procedure.  The baseline obtained can-  not be representative of the population because i t applies only to those Individuals in the natural population which  56  have had the same experience of temperature, vapor pressure d e f i c i t , photoperiod, population density, and n u t r i t i o n a l conditions, and have the same age distribution as the tested individuals.  In order to make v a l i d comparisons between,  populations i t would be necessary to i n i t i a t e colonies i n the laboratory and subject them to the same conditions for three generations before determining the ld-p l i n e . Hewlett and Gostick (1955), l  n  t h e i r experiments with  the flour beetle, Trlbolium castaneum Herbst, could not s i g n i f i c a n t l y reduce the variation from batch to batch at a dosage by increasing t h e i r sample size from 50 to 100. Busvine (1957) states that i t i s rarely advantageous to use more than 50 insects per treatment, and sets 15 as the lower l i m i t i f any degree of precision i s desired. The method developed here could be used with any sample sizes between these l i m i t s , but i t would always be best to use the largest number possible, since s t a t i s t i c s computed from large samples tend to give more precise estimates of population, parameters than do those from small samples. 10, Simplicity and r a p i d i t y . The method used here must be judged as a tool f o r research since i t employs equipment best suited to the laboratory, and i t s s e n s i t i v i t y and precision s a t i s f i e s the needs of research i n the genetics and physiology of insecticide resistance.  The performance of the procedures  require no more than patience and a steady hand, nevertheless  57  i t i s time consuming and the results do not come rapidly. Significance of the Dosage-Mortality Curve The average weight of the aphids used for determination of the seven ld-p lines of Table XII was (seven samples of  50  aphids each).  mg  4.110.11  The average  LDEJQ  com-  puted was 47.15 ppm, the equivalent of 23.6 ng per aphid, or 5.8 ug per g of body weight.  This value indicates high  t o x i c i t y but comparisons with other insecticides against the pea aphid, or for malathion against other insects, have not been possible because no reference which gives the necessary information has -.-been found. The slope of the ld-p l i n e Is r e l a t i v e l y steep, i n d i cating that there i s l i t t l e variation i n response o f the aphids to the Insecticide.  The probability of the aphids  being able to discriminate between dosages decreases as the range of dosages i s narrowed.  Even though there are  deviations from the computed ld-p l i n e s , these are not t r u l y aberrant since the dosages used to establish the l i n e varied within very narrow l i m i t s .  The steep slope,  and the lack of consistent or major deviation from the ld-p l i n e give no indication of the presence of a preadapted resistance mechanism i n the clone; there appears to be l i t t l e chance of a resistant population, developing even after repeated selection with the i n s e c t i c i d e .  58  Selection for Resistance Within a Pea Aphid Clone. The Insecticide In most cases where an aphid has developed resistance to an insecticide an organophosphate has been the selecting agent (Baerecke, 1 9 6 2 ; Dunn and Kempton, 1 9 6 6 ; Stern, 1 9 6 2 ; and Michelbacher, et a l , 1 9 5 4 ) .  One exception i s r e s i s t -  ance to the cyclodiene insecticide endosulfan, reported by Shanks  (1967).  Since resistance to organophosphates i s  more common, and insecticides of t h i s class are most often recommended for aphid control, an organophosphate was best for an attempt to select for resistance. Malathion was the insecticide chosen, largely because of i t s low mammalian t o x i c i t y (oral t o x i c i t y to the male white r a t ,  LD50  1375  mg/kg of body weight), and consequently reduced r i s k i n handling. Rearing Endomeiosis i s a type of meiosis during which the normal f i r s t meiotic division i s replaced by chromosome pairing, chiasma formation, and separation, without spindle formation or dissolution of the nuclear membrane (Cognetti, 1961).  Boschetti and P a g l i a i  (1964;  found that endomeiosis  was a regular occurrence In the rose aphid, Macrosiphum rosae (Linnaeus), reared at 1 8 C; but at 2 5 C i t v/as reduced and at 2 8 C i t v/as v i r t u a l l y absent.  Since chiasma forma-  t i o n may promote some genetic v a r i a b i l i t y during the  59 theiyiokous  generations,  e n d o m e i o s i s w o u l d be  favored  r e a r i n g a p h i d s a t a t e m p e r a t u r e o f 20  c or l e s s .  would a l l o w  recessive  gene t h a t  f o r the  expression  resistance the  attained.  i m p r o v e m e n t w o u l d be  rearing  This  d i d a r i s e by p r o v i d i n g a means t h r o u g h w h i c h  homozygous s t a t e m i g h t be No  o f any  by  required  i n any  of the  other  procedures.  Collecting No  b e n e f i c i a l changes are  p r o c e d u r e , as d e s c r i b e d The  r e a r i n g and  p a g e s 42  on  from the  and  described  They a l s o r e d u c e t h e  a p h i d s w h i c h have o p e r a t i v e selected  collecting  43.  c o l l e c t i n g procedures  e n s u r e homogeneous s a m p l e s . excluding  envisaged i n the  resistance  risk  of  factors  sample.  Treating During the tion  should  be  course of s e l e c t i o n , treated.  every second genera-  This pressure  i s equivalent  t h a t o f i n t e n s i v e s e l e c t i o n s i n c e t r e a t m e n t o f an a l s o exposes to the tains.  I f every generation  r i s k of k i l l i n g tion). line, part  i n s e c t i c i d e the  To  a l l the  i s treated there  the  colony  •These p l a n t s and  should  colonies  be  i s an  (personal  be  selected  i s treated, a  u s e d t o c o l o n i z e new  should  increased  observa-  danger of l o s i n g the  even when e v e r y s e c o n d g e n e r a t i o n of the  adult  embryos t h a t I t c o n -  aphids treated  guard against  to  discarded  only  small  plants. after  60  i t i s certain that there are reproducing survivors among the individuals treated with the selection dosage. Although the computations of probit analysis include weighting factors for different sized samples, greater confidence can be placed in data derived from equal sized samples, and these should be employed i f possible, both from treatment to treatment and replicate to replicate. Each time the selection procedure i s carried out, enough treatments should be used that an ld-p l i n e can be computed.  This l i n e gives the dosage required to maintain  selection pressure at a specific l e v e l .  A comparison ld-p  l i n e should be computed for the unselected control colony at the same time, using the same number of aphids per treatment and the same order; during the photoperiod the comparat i v e treatments should be as close in time as possible. The l i n e computed for the unselected, control clone provides the only v a l i d measurement of change in the selected clone.  The series of lines obtained from the control clone  w i l l expose any changes which may occur in a clone not subjected to the selection pressure of malathion. Post-treatment Holding No modification of the suggested procedure seems necessary. The P o s s i b i l i t y of Resistance i n a Laboratory Clone of the Pea Aphid. The source of insecticide resistance l i e s i n the  61  genetic v a r i a b i l i t y of the species. Resistance arises when a gene mutation affects an alteration i n the structure of a molecule or the functioning of a system.  Resistance may be  conferred by a change in any one of the processes of penet r a t i o n , d i s t r i b u t i o n , storage, intoxication, detoxication, or excretion, plus any changes i n the s i t e of action or the aphid's behavior In r e l a t i o n to the insecticide.  Each of  these components may be controlled by one or several gene pairs.  Thus, the chances of resistance arising vary  d i r e c t l y with the number of components i n the intoxication process and.the specific frequency of mutation at the l o c i controlling these components, and inversely with the number of genes controlling each of the separate components. Selection for insecticide resistance i n generally agreed to be exerted on preadapted genes existing at low frequencies i n the population (Crow,  1957).  While Insecti-  cides other than chemosterHants are not mutagenic, the p o s s i b i l i t y of post-adaptation cannot be excluded.  There  i s a good chance of perpetuating mutations conferring resistance when these arise _de novo i n the germ l i n e of aphids exposed to sub-lethal dosages.  Selective, sub-lethal  dosages in the f i e l d result from incomplete spray coverage, p a r t i a l degradation, of insecticide deposits, and insect escapes by migration out of treated areas.  In the labora-  tory they result from controlled dosage l e v e l s . When a gene for resistance occurs and i s expressed i n  62  thelytokously reproducing aphid population, i t s spread within the population w i l l be rapid.  The rapidity results  from the concentrating power of clonal reproduction; however, at the end of the thelytokous portion of the aphid's cycle, migration w i l l ensure that not a l l of the sexually reproducing population w i l l be derived from resistant clones.  Bisexual reproduction might thus actually  impede the r i s e and spread of resistance by inter-breeding resistant and susceptible migrant aphids and so d i l u t i n g the genes for resistance. Resistance to organophosphate insecticides i s generally l a b i l e (Perry,  1964),  indicating that i t i s either d e t r i -  mental i n some manner, or that the mutated genes conferring i t are unstable.  The l a t t e r explanation seems to be the  more reasonable since organophosphate resistance has been observed to decline i n laboratory clones free from competition (Dunn and Kempton,  1966).  This i n s t a b i l i t y , and the  fact that few aphid species have developed resistance to organophosphates, imply that mutations conferring r e s i s t ance are infrequent and that when they do occur there i s a strong homeostatic tendency.  I t follows that the fre-  quency of genes for resistance to organophosphates i s low in the population, although the potential for mutation to the resistant forms exists.  In a laboratory study, carried  out on a very small portion of the population, I t Is impossible that the t o t a l gene pool of the species w i l l be  63  represented.  By e x p o s i n g t h e c l o n e  t o a mutagenic  such as i o n i z i n g r a d i a t i o n o r u l t r a - v i o l e t the p o t e n t i a l i t y o f t h e species the  clone  agent,  l i g h t , more o f  m i g h t be e x p r e s s e d , a n d  w o u l d be more t r u l y r e p r e s e n t a t i v e  o f the total  population. S t u d y o f t h e mechanisms o f o r g a n o p h o s p h a t e  resistance  i s h a n d i c a p p e d by l a c k o f f u n d a m e n t a l knowledge i n t h e major a r e a s o f g e n e t i o n and t o x i c o l o g y . and  The H o m o p t e r a ,  H e t e r o p t e r a , u n l i k e o t h e r a n i m a l s a n d p l a n t s , do n o t  h a v e c e n t r i c chromosomes, b u t s p i n d l e  f i b e r s , which con-  verge on t h e p o l e s and a r e e l a b o r a t e d  along the e n t i r e  poleward surface 1948).  o f e a c h chromosome  (Hughes-Schrader,  The e f f e c t s o f t h i s s i t u a t i o n , p a r t i c u l a r l y on  formation igated.  o f chlasmata during  meiosls,  I f endomeiosis as reported  have n o t been  by C o g n e t t i  Invest-  (1961)  occurs throughout t h e Aphididae, i n v e s t i g a t i o n o f t h e e f f e c t s may be o b s c u r e d b y t h e o b l i g a t o r y i n t e r p o s i t i o n of thelytokous could  generations,  during  o c c u r , between t h e s e x u a l In the area o f toxicology,  which f u r t h e r  portions  chiasmata  of the life-cycle.  n e i t h e r t h e mode, n o r t h e  s i t e , o f a c t i o n o f organophosphate i n s e c t i c i d e s h a s been d e f i n i t e l y established..-..  I t i s known t h a t  these  insecti-  cides  i n h i b i t b o t h i n s e c t a n d mammalian c h o l i n e s t e r a s e , b u t  there  i s still  zyme a n d t h a t certain that  uncertainty  a s t o t h e f u n c t i o n o f t h i s en-  of acetylcholine  ( O ' B r i e n , 1966).  I ti s  a c h o l i n e r g i c system does not o p e r a t e a t t h e  64 neuro-muscular j u n c t i o n  i n insects  ( O ' B r i e n , 1 9 6 6 ) , but  t h i s does not d i s p r o v e t h e o p e r a t i o n o f such a system a t other s i t e s i n the insect.  Inhibition of  cholinesterase  i n homogenates o f i n s e c t head and t h o r a x does not n e c e s s a r i l y prove t h a t a c h o l i n e r g i c insect.  system e x i s t s i n t h e l i v i n g  More i n v e s t i g a t i o n must be added t o t h e enormous  amount a l r e a d y done i n t h i s f i e l d .  65 SUMMARY 1.  T h r e e methods o f e x p o s i n g  aphids  to contact  insecti-  c i d e s were i n v e s t i g a t e d i n o r d e r t o f i n d a t e c h n i q u e f o r e s t a b l i s h i n g the dosage-mortality curve Acyrthosiphon 2.  pisum  Repeatable  (Harris). s t a t i s t i c s w e r e o b t a i n e d when m e a s u r e d  droplets o f malathion aphids.  f o r t h e pea a p h i d ,  were a p p l i e d t o p i c a l l y t o i n d i v i d u a l  T h e s e s t a t i s t i c s v/ere o f t h e h i g h  precision  r e q u i r e d f o r r e s e a r c h i n t o x i c o l o g y and g e n e t i c s . 3.  The s t a t i s t i c s p r o d u c e d by e x p o s u r e on a t r e a t e d  g l a s s s u r f a c e , and d i r e c t or emulsions,  spraying with malathion s o l u t i o n s  v/ere n e i t h e r r e p e a t a b l e n o r o f t h e r e q u i r e d  precision. 4. curve,  For the establishment environmental  of the dosage-mortality  c o n d i t i o n s s h o u l d be c o n s t a n t a n d f a v o r '  able t o t h e aphids throughout  the periods of rearing,  c o l l e c t i n g , t r e a t i n g and p o s t - t r e a t m e n t  holding.  The  aphids  o f t h e two g e n e r a t i o n s p r e v i o u s t o t h e t r e a t e d g e n e r a t i o n s h o u l d a l s o be r e a r e d i n t h e same f a v o r a b l e e n v i r o n m e n t .  66  LITERATURE  CITED  B a e r e c k e , M. L. (1962), R e s i s t e n z v o n Myzus p e r s i c a e ( S u l z . ) g e g e n E605 u n d M e t a s y s t o x . Z. P f l a n z e n k r a n k h u Pflanzenschutz. 69: 453-461. B o z c h e t t i , M. A. a n d P a g l i a i , A. M. ( 1 9 6 4 ) . L'azione d e l l a temperaturea s u l l o v o g e n e s i parthenogenetica d i Macrosiphum r o s a e (Homoptera: A p h i d i d a e ) . C a r y o l o g i a 17: 203-218. 1  B r i d g e s , P. M. ( 1 9 5 7 ) . A b s o r p t i o n a n d m e t a b o l i s m o f (-^C) A l l e t h r i n b y t h e a d u l t h o u s e f l y , Musca d o m e s t i c a L. B i o c h e m . J . 6 6 : 3 1 6 - 3 2 0 . B u r k h o l d e r , ¥. E. a n d D i c k e , R. J . (1966). The t o x i c i t y o f malathion and f e n t h i o n t o dermestid l a r v a e as I n f l u e n c e d by v a r i o u s s u r f a c e s . J . E c o n . E n t o m o l . 5 9 : 253-254, B u s v i n e , J . R. ( 1 9 5 7 ) . A C r i t i c a l Review o f t h e Techniques for Testing Insecticides. Commonwealth I n s t i t u t e E n t o m o l o g y , L o n d o n . 208 p . B u s v i n e , J . R. a n d B a r n e s , S. ( 1 9 4 7 ) . Observations on m o r t a l i t y among i n s e c t s e x p o s e d t o d r y i n s e c t i c i d a l films. B u l l , Entomol. Research 38: 81-90. C o g n e t t i , G. ( 1 9 6 1 ) . Endomeiosis i n parthenogenetic of aphids, E x p e r i e n t i a 17: 168-169.  lines  C o l e , C. L . a n d A d k i s s o n , P. L . ( 1 9 6 4 ) . D a i l y rhythm i n t h e s u s c e p t i b i l i t y o f an i n s e c t t o a t o x i c agent. Science 144: 1 1 4 8 - 1 1 4 9 . Crow, J . P. (1957}. G e n e t i c s o f i n s e c t r e s i s t a n c e t o c h e m i c a l s , Ann. Rev. Entomol. 2 : 227-246. D i t t r i c h , V. ( 1 9 6 2 ) . A comparative study o f t o x i l o g i c a l t e s t methods on a p o p u l a t i o n o f t h e t w o - s p o t t e d s p i d e r mite, Tetranychus t e l a r i u s . J . Econ. Entomol. 55: 6 4 4 - 6 4 8 . Dunn, J . A. a n d Kempton, D. P. (1966). N o n - s t a b l e r e s i s t a n c e t o d e m e t o n - m e t h y l i n a s t r a i n o f Myzus p e r s i c a e . Entomol. E x p t l . Appl. 9:67-73. E b e l i n g , W. ( 1 9 5 8 ) . Vinegar f l y control C a l i f . Agr. 12(6): 12-14.  treatments.  E s s i g , E. 0 . ( 1 9 5 8 ) , I n s e c t s and M i t e s o f Western North America. M a c m i l l a n , New Y o r k . 1050 p .  67  Finney, D, J , (1962). Probit Analysis. 2nd Ed. Univ. Press, Cambridge, England. 318 p.  Cambridge  Fisher, R. W, (1967). Diel periodicity i n s e n s i t i v i t y of Tetranychus urticae (Acarina: Tetranychidae) to d i c o f o l . Can. Entomologist 99: 281-284. Forbes, A. R. (1962). Aphid populations and t h e i r damage to oats i n B r i t i s h Columbia. Can. J. S c i . 42:660-666. Gast, R. T. (1959). Laboratory studies of residual contact effect of 28 insecticides on Japanese beetle adults. J. Econ. Entomol. 52: 9 - 1 2 . Glass, E. H, (I965}. Vacuum-pencil technique for manipulating small Lepidopterous larvae. J . Econ. Entomol. 58:  785-786.  Hewlett, P. S. and Gostick, K. G. (1955). The loss of weight of pyrethrin-treated flour beetles, Tribolium casteneum (Herbst), and i t s application to bioassay. Ann. Appl. B i o l . 4 3 : 213-236. Hoagland, D, R. and Arnon, D. I. ( 1 9 5 0 ) . The water culture method f o r growing plants without s o i l . C a l i f , Agric. Exptl. Sta. Circ. 347. 32 p. Hoskins, W. M. and Craig, R. (1962). Uses of bioassay i n entomology, Ann. Rev. Entomol. 7: 4 3 7 - 4 6 4 . Hoskins, W, M. and Gordon, H. T. (1956). Arthropod r e s i s t ance to chemicals. Ann. Rev, Entomol. 1 : 89-122. Hughes-Schrader, S, (1948). Cytology of coccids (Coccoidea: Homoptera). Advances i n Genet, 2 : 1 2 7 - 2 0 3 . Johnson, B. (1966a). Wing polymorphism i n aphids I I I . The influence of the host plant. Entomol. Exptl. Appl. 9:  213-222.  Johnson, B. (1966b). Wing polymorphism i n aphids VI. The effect of temperature and photoperiod. Entomol, Exptl. Appl. 9: 301-313. Johnson, B. (1965). Wing polymorphism i n aphids I I . Interaction between aphids. Entomol. Exptl. Appl. 8: 4 9 - 6 4 . Kennedy, J . S,, Day, M. F. and Eastop, V. A. (1962). A conspectus of aphids as vectors of plant viruses. Commonwealth Institute Entomology, London. 114 p.  68 Kenten, J . ( 1 9 5 5 ) . The e f f e c t o f p h o t o p e r i o d and temperature on r e p r o d u c t i o n i n Acyrthosiphon pisum ( H a r r i s ) and on the forms produced. B u l l . Entomol. Research. 46a 599-624. Lamb, K. P. and White, D. (1966). E f f e c t o f temperature, s t a r v a t i o n and crowding on p r o d u c t i o n o f a l a t e young by the cabbage aphid, (Brevicoryne b r a s s i c a e ) . Entomol. E x p t l . A p p l . 9 : 179-184". Lees, A. D. ( 1 9 6 1 ) . C l o n a l polymorphism i n a p h i d s . p. 6 8 - 7 9 , i n J . S . Kennedy, (Ed.) Insect polymorphism. Symposium No. I, Roy. Entomol. Soc. Lond. Lees, A. D. ( 1 9 5 9 ) . The r o l e o f photoperiod and temperature i n the d e t e r m i n a t i o n o f parthenogenetic and sexual forms In the a p h i d Megoura v i c i a e Buckton - I The i n f l u e n c e o f these f a c t o r s on apterous v i r g i n o p a r a e and t h e i r progeny. J . I n s . P h y s i o l . 3: 92-117. M e t c a l f , C. L . , F l i n t , W. P. and M e t c a l f , R. L . (1962). D e s t r u c t i v e and u s e f u l i n s e c t s . 4 t h Ed. McGraw-Hill, Toronto. IO87 p. Michelbacher, A, E., Fulmer, 0 . J . , C a s s i l , C. C. and Davis, C. S, ( 1 9 5 4 ) . Walnut aphid r e s i s t a n c e to parat h i o n i n northern C a l i f o r n i a . J . Econ. Entomol.  47:  366-367.  Morse, P. M, and B i c k l e , A. ( 1 9 6 7 ) . The combination o f estimates from s i m i l a r experiments, a l l o w i n g f o r i n t e r experiment v a r i a t i o n . J . Am. S t a t . Assoc. 6 2 : 241-250. O'Brien, R. D.  (1966).  Ann. Rev. Entomol.  Mode o f a c t i o n o f i n s e c t i c i d e s .  1 1 : 369-402.  Patwa, N. ( 1 9 6 5 ) . F a c t o r s i n f l u e n c i n g the t o x i c i t y o f • aqueous f o r m u l a t i o n s o f pyrethrum a g a i n s t an aphid, Macrosiphum euphorbiae (Thomas). Pyrethrum P o s t .  8 (1):  6-7,  19.  P e r r y , A. S. ( 1 9 6 4 ) . The p h y s i o l o g y o f i n s e c t i c i d e r e s i s t ance by i n s e c t s , i n R o c k s t e i n , M. (Ed.), P h y s i o l o g y o f the I n s e c t s , volume 3 . Academic Press, New York. P o t t e r , C. ( 1 9 5 2 ) , An improved l a b o r a t o r y apparatus f o r a p p l y i n g d i r e c t sprays and s u r f a c e f i l m s , with data on the e l e c t r o s t a t i c charge on atomized spray f l u i d s . Ann. A p p l . B i o l . 39: 1-28. P o t t e r , C. and Gillham, E. M. ( 1 9 5 8 ) . E f f e c t o f host p l a n t on the r e s i s t a n c e o f Ac,yrthosiphon pisum ( H a r r i s ) t o i n s e c t i c i d e s . B u l l . Entomol. Research. 48: 3 1 7 - 3 2 2 .  69  Pradhan, S. ( 1 9 4 9 ) . Studies on the t o x i c i t y of Insecticide films: 'I - Preliminary investigations on the concent r a t i o n , time, mortality r e l a t i o n . B u l l , Entomol, Research. 4 0 : 1 - 2 5 . Rai, L., A f i f i , S. A, E,, Fryer, H, C. and Roan, C. C. ( 1 9 5 6 ) . The effect of different temperatures and piperonyl butoxide on the action of malathion on susceptible and DDT-resistant strains of houseflies. J . Econ. Entomol. 49:  307-309.  Shanks, C, H, ( 1 9 6 7 ) . Resistance of the strawberry aphid to endosulfan In southwestern Washington. J . Econ, Entomol. 60:  968-970.  S p i l l e r , D. ( 1 9 6 1 ) . A digest of available information on the insecticide malathion. Advances i n Pest Control Research. 4 : 2 4 9 - 3 3 5 . Stern, V. M. ( 1 9 6 2 ) , Increased resistance to organophosphorus insecticides in. the parthenogenetic spotted a l f a l f a aphid, Therio.aphis maculata, i n C a l i f o r n i a , J . Econ. Entomol. 5 5 : 9 0 0 - 9 0 ^ Suomalainen, E, ( 1 9 6 2 ) . Significance of parthenogenesis i n the evolution of insects. Ann. Rev. Entomol. 7 : 3 4 9 - 3 6 6 . Uichanco, L. B. ( 1 9 2 4 ) . Studies on the embryogeny and postnatal development of the Aphididae with special reference to the history of the "symbiotic organ" or "mycetom". Philippine J. S c i . 2 4 : 143-247. White, M. J. D. ( 1 9 4 5 ) . Animal' cytology and evolution. 1st Ed. Cambridge Univ. Press, Cambridge, England. 3 7 5 p. Yost, J. F., Frederick, J. B."and Migrdichian, V. ( 1 9 5 5 ) . S t a b i l i t y , compatability, and technological data on malathion formulations. Agr, Chem. 1 0 ( 1 0 ) : 42-44, ;  105,  107.  Young, M. R. and Roussel, J . S. ( 1 9 5 8 ) , The effects of temperature on the efficiency of insecticides applied t o p i c a l l y to b o l l weevils d i f f e r i n g i n s u s c e p t i b i l i t y to chlorinated hydrocarbon insecticides. J. Econ. Entomol. 5 1 : 9 3 - 1 0 0 . Yule, W. N. ( 1 9 6 4 ) . Aqueous formulations of pyrethrum for controlling phytophagous Arthropoda - an evaluation using bioassay techniques. Ann. Appl. B i o l . 5 3 : 1 5 - 2 8 .  70 APPENDIX Finney  (1962) g i v e s m e t h o d s o f d e f i n i n g t h e f i d u c i a l  l i m i t s about a s i n g l e l d - p l i n e and about t h e d i f f e r e n c e between  t h e mean d o s a g e s o f two l d - p l i n e s .  However, no  method o f d e f i n i n g t h e f i d u c i a l l i m i t s a b o u t a s e r i e s o f replicates i sgiven  1 0  .  F o r t h i s p u r p o s e I have  combined  two a n a l o g o u s e q u a t i o n s g i v e n by F i n n e y . The q u a n t i t i e s r e q u i r e d f o r t h e c a l c u l a t i o n o f f i d u cial  limits are: Snw  J  w h e r e V- i s t h e v a r i a n c e o f t h e w e i g h t e d mean w o r k i n g p r o b i t a n d Snw i s t h e summation of  o f t h e p r o d u c t s o f t h e number ( n )  i n s e c t s i n e a c h b a t c h a n d t h e w e i g h t (w) a t t a c h e d t o e a c h  observation; "Vb -  1 = _1_ Snw(x-x)2 sSx  w h e r e Vb i s t h e v a r i a n c e o f t h e s l o p e a n d S n w ( x - x ) ^ i s t h e summation  o f t h e squared d e v i a t i o n s  a b o u t t h e w e i g h t e d mean o f t h e d o s a g e V  Y * b V  +  v  (b) o f t h e l d - p l i n e  y( -^) x  ( x ) ; and  2  where Vy i s t h e v a r i a n c e o f t h e expected p r o b i t Y f o r any dosage, x.  Proximate f i d u c i a l l i m i t s t o the l d - p l i n e are  10. I n a r e c e n t a r t i c l e , M o r s e a n d B i c k l e (19^7) have c o n s i d e r e d t h e problem o f combining e s t i m a t e s from a s e r i e s o f s i m i l a r experiments,  71 then given  by FL s Y i  where s  Y  i s the square  s t y  r o o t o f Vy,  t i s Student's  k-2  t with  degrees o f freedom f o r t h e chosen l e v e l o f p r o b a b i l i t y ; k i s t h e number o f d o s a g e s t e s t e d . significant,  Where h e t e r o g e n e i t y  1 1  is  the value of t corresponding to that l e v e l  of  p r o b a b i l i t y i s used. Exact  f i d u c i a l l i m i t s a b o u t x, t h e d o s a g e g i v i n g a  whose p r o b i t i s Y ,  a r e o b t a i n e d by  kill  s o l v i n g an e q u a t i o n w h i c h  g i v e s t h e v a l u e o f x f o r w h i c h Y has a s e l e c t e d f i d u c i a l limit.  T h i s equation, i s  x +  (x-x) ±  _ S _  1-g  * /(l-g)Ofe) bCl-gjV  w h e r e g = t ^ / b ^ S S x , and ly.  +  (V )(x~x)  2  b  o t h e r symbols a r e as d e f i n e d p r e v i o u s -  Where t h e h e t e r o g e n e i t y i s s i g n i f i c a n t l y g r e a t e r  one,  g and t h e e x p r e s s i o n w i t h i n t h e s q u a r e  c r e a s e d by t h e h e t e r o g e n e i t y f a c t o r , In potency,  r o o t must be i n -  X^/k-2.  h i s s e c t i o n on t h e d e t e r m i n a t i o n o f t h e i . e . , the r a t i o  m a t e s o f b, V , D  g, and  these equations.  relative  of e q u a l l y e f f e c t i v e dosages, o f a  number o f p o i s o n s , o r o f one F i n n e y g i v e s an a n a l a g o u s  than  p o i s o n on  several occasions,  set of equations.  Pooled  esti-  the h e t e r o g e n e i t y f a c t o r are used i n  However, t h e e q u a t i o n f o r t h e  fiducial  11. H e t e r o g e n e i t y i s a measure o f t h e d e v i a t i o n s o f . o b s e r v e d from e x p e c t e d numbers, S i g n i f i c a n c e o f h e t e r o g e n e i t y i s j u d g e d by c o m p a r i s o n o f a C h i - s q u a r e d v a l u e - o (SPxy)2 (k-2)~ ^ s£T w i t h t a b u l a t e d Chi-square values. S  l  V  6  n  ^  v2  X  S  S S  v  72  l i m i t s i s appropriate only for the l i m i t about the d i f f e r ence between the mean dosage of two ld-p l i n e s . The equation used for setting the f i d u c i a l l i m i t s about the band formed by the ld-p lines of the most and least potent replicates (Figure l ) i s given i n Table XIII. In t h i s equation, the pooled estimates of b, V^, and g are used and Vy-^ + Vyg i s the sum of the variances of the weighted mean working probits for the least and most potent r e p l i cates.  Since heterogeneity i s s i g n i f i c a n t , g and the expres-  sion, inside the square root are increased by the heterogeneity factor. As an example, the calculation of the maximum l i m i t of t o x i c i t y , i . e . the minimum l i m i t of dosage, i s set out in. Table X I I I .  TABLE X I I I .  Calculation of the upper f i d u c i a l l i m i t about the ld-p l i n e of maximum  t o x i c i t y of malathion to A. pisum.  x A  +  x = 1.7682  a = -4.5253  g =  Het.=  .0214  1% B  -'  {X  X)  ?  W=SJ  C  J  b = 5.5478  3.3084  ( ( 1 _ s )  ( v  v-  4 y- =  ?i  +  D  V  .0146  +  5 10 30 50 70 90 95  Y ! 3.3551 3.7148 4.4756 5.0000 5.5244 6.2816 6.6429  1  ^  E  ( x  b  .0476  =  (35)  =  2.032  " ~ ^ < ->  F  J fo . K i l l  v  x)  Het  G  H  K  A2 1.3543 1.4198 1.5563 1.6508 1.7453 1.8818 1.9473  c  Bpj  G  -0.3639 -0.2984 -O.1619 -0.0647 +0.0271 +O.1636 +0.2291  1.3463 1.4133 1.5528 1.6494 1.7459 1.8854 1.9523  .1324 .0890 .0262 .0042 .0007 .0268 .0525  5  FG+E .0206 .0185 .0155 .0145 .0143 .0156 .0168  K .06815 .06121 .05128 .04797 .04731 .05161 .05558  /K" .0985 .0933 .0854 .0826 .0820 .0857 .0889  J-D/K" 1.2478 1.3200 1.4674 1.5668 I.6639 1.7997 1.8634  1 . Expected probit corresponding to % K i l l . Taken from tables i n Finney ( 1 9 6 2 ) . 2 . Capital l e t t e r s refer to the corresponding underlined portions of the equation. 3 . Position of the ld-p l i n e , expressed as the l o g of the dosage in. parts per million, 1 0  

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