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The genetic control of riboflavin in Tribolium confusum Weber, Joseph 1966

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THE GENETIC CONTROL OF RIBOFLAVIN IN TRIBOLIUM CONFUSUM by JOSEPH WEBER B.Sc., University of B r i t i s h Columbia, 196*+  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department, of Poultry Science  We accept this thesis as conforming  to the  required standard  THE UNIVERSITY OF BRITISH COLUMBIA July, 1 9 6 6  In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h Columbia,, I agree that 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 reference and study.  I f u r t h e r agree that permission., f o r extensive  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 granted by the Head o f my Department or by h i s representatives.  I t i s understood that copying  or 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 f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission.  Department of  p n 1 t r y S r i Pnrn  P  The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada Date__  1 s t August,  1966  ii ABSTRACT The i d e n t i t y o f a y e l l o w f l u o r e s c e n t s u b s t a n c e isolated  f r o m T r i b o l i u m c o n f u s u m , w i t h r i b o f l a v i n was  shown by t h e i n d e p e n d e n t methods o f c h r o m a t o g r a p h y and e l e c t r o p h o r e s i s . products  spectrophotometry, Two  decomposition  o f r i b o f l a v i n a s w e l l a s two c o n j u g a t e d  FMN a n d FAD, w e r e i d e n t i f i e d .  The g e n e t i c  flavines  control of  r i b o f l a v i n a n d i t s r e l a t i o n s h i p t o some m u t a n t e y e c o l o r genes i n t h e p l e i o t r o p i c investigated  h i e r a r c h y o f gene e f f e c t s was  i n a s e r i e s o f e x p e r i m e n t s i n v o l v i n g more  t h a n 8000 T r i b o l i u m f r o m 11 g e n e t i c a l l y d i f f e r e n t All  developmental stages  o f i n d i v i d u a l s homozygous  r e c e s s i v e p e a r l eye c o l o r gene, £ but  lines.  r  fora  ( f o r m e r l y c a l l e d p_,  renamed) w e r e a s s o c i a t e d w i t h a s i g n i f i c a n t , h e r i t a b l e  reduction i n r i b o f l a v i n content  i n t h e body.  r e d u c t i o n was p r i n c i p a l l y i n t h e M a l p i g h i a n In a l l of the other  lines  This tubules.  t e s t e d t h e v i t a m i n was  accumulated i n the tubules  i n r e l a t i v e l y high  concentrations.  The n o r m a l a l l e l e o f t h i s gene was f o u n d t o be dominant w i t h r e s p e c t  t o t h e r i b o f l a v i n phene.  t h e r e s i d u a l g e n o t y p e o f p." b y means o f two 1  incompletely Changing  generations  of r e c u r r e n t s e l e c t i o n d i d not d e t e c t a b l y a l t e r i t s expression.  Two a l l e l e s  o f p e a r l (p_ and p_ ) w e r e s  p h e n o t y p i c a l l y i d e n t i c a l t o p , except a t the l e v e l o f r  t h e r i b o f l a v i n phene. p_  r  Crosses  w e r e shown t o c o m p l e m e n t .  r i b o f l a v i n content t u b u l e s w e r e shown. / I*  of these a l l e l e s Genetic  with  differencesi n  and t h e p i g m e n t a t i o n o f M a l p i g h i a n Individuals of the pearl  phenotype  s  ( £ ) £ ) £ )  always  had c o l o r l e s s M a l p i g h i a n t u b u l e s .  S e x u a l d i m o r p h i s m f o r r i b o f l a v i n c o n t e n t was o b s e r v e d i n all  lines  tested.  The q u a n t i t a t i v e p a r a m e t e r s  of  v a r i a t i o n o f t h e r i b o f l a v i n phene w e r e e x a m i n e d a n d i t s h e r i t a b i l i t y was f o u n d parisons.  t o be h i g h by two d i f f e r e n t com-  TABLE OF CONTENTS INTRODUCTION REVIEW OF THE LITERATURE MATERIALS AND METHODS RESULTS AND DISCUSSION PART I . CHEMICAL ANALYSIS A. P r e l i m i n a r y o b s e r v a t i o n s B. I s o l a t i o n , p u r i f i c a t i o n a n d characteri ation o f spot 5 1. I d e n t i f i c a t i o n b y s p e c t r o p h o t o m e t r y 2. I d e n t i f i c a t i o n b y means o f chromatography 3. I d e n t i f i c a t i o n by means o f electrophoresis DISCUSSION z  PART I I . GENETIC ANALYSIS A. A n a l y s i s o f p e a r l B. A n a l y s i s o f £ l o d C. T e s t f o r a l l e l i s m b e t w e e n r j a n d p_ D. The s y n t h e s i s o f a d o u b l e m u t a n t ; p e a r l , ruby spot E. P a r t i a l a n a l y s i s o f a p o s s i b l e t h i r d a l l e l e ; p, F. D i s t r i b u t i o n a n d d e v e l o p m e n t a l v a r i a t i o n of r i b o f l a v i n 1. D i s t r i b u t i o n o f r i b o f l a v i n 2. D e v e l o p m e n t a l v a r i a t i o n o f r i b o f l a v i n G. G e n e t i c d i f f e r e n c e s i n r i b o f l a v i n c o n c e n t r a t i o n and c o l o r a t i o n o f Malpighian tubules H. E s t i m a t i o n o f h e r i t a b i l i t y o f t h e riboflavin trait DISCUSSION r  3  s  CONCLUSIONS AND SUMMARY LITERATURE CITED  V  L I S T OF TABLES TABLE I.  PAGE Chromatographic d i s t r i b u t i o n and  their R  values  f  of substances  i n W.T.  adult 15  T r i b o l i u m confusum II.  V i s u a l estimation of the yellow fluorescent s u b s t a n c e s i n 11  111.  Mean R^. v a l u e s  lines  o f sample and s t a n d a r d i n  various chromatographic IV.  Rf values  of reference  V. The d i s t r i b u t i o n Fg VI.  compounds a n d s a m p l e s  Comparison of t o t a l  riboflavin  and  +/+  with the  a n d t h e W.T.  of r i b o f l a v i n i n the F  F2 p r o g e n y f r o m t h e c r o s s p / p ,  The d i s t r i b u t i o n and  30  1  32  l o d / l o d x +/+ IX.  28  of recurrently +  the heterozygotes  x  (ug/mg f r e s h  s e l e c t e d p_' f r o m p'/p' x V ,  The d i s t r i b u t i o n  22  o f r i b o f l a v i n i n t h e F-j_ a n d  w e i g h t o f two g e n e r a t i o n s  VII.  21  solvents  progeny from the cross p V p '  parents,  17  o f T. c o n f u s u m  backcross  p/p,lod/lod  o f r i b o f l a v i n i n t h e F^ (BC^) p r o g e n y o f p / p r  r  x 35  VI  TABLE X.  PAGE The d i s t r i b u t i o n F  o f r i b o f l a v i n i n t h e F^,  a n d b a c k c r o s s (BC-^) p r o g e n y  2  of p / p r  r  36  x p/p,rus/rus XI.  The d i s t r i b u t i o n F  of r i b o f l a v i n i n the F ,  and b a c k c r o s s ( B C i ) progeny  2  of p /p r  r  x p/p,dre/dre,cas/cas XII.  The F-^ and F  2  progeny  ^7  from p'/p  and t h e d i s t r i b u t i o n  1  x rus/rus  of r i b o f l a v i n i n the  F3 p r o g e n y f r o m t h e s e l f o f t h e F XIII.  The p h e n o t y p e the  and d i s t r i b u t i o n  progeny  rus/rus" x p /p r  rus/rus  2  of r i b o f l a v i n of  from t h e c r o s s e s " p / p , r  r  r  (!•) and " p / p , r u s / r u s " r  r  x p / p , r u s / r u s (2.) XIV.  hi  D i s t r i b u t i o n o f r i b o f l a v i n i n ug i n t h e pupae a n d a d u l t s o f W.T.  XV.  39  p_ and p." l i n e s s  1  h5  D e v e l o p m e n t a l v a r i a t i o n o f r i b o f l a v i n i n W.T., p_  s  and i ) l i n e s r  m e a s u r e d i n ug/mg f r e s h w e i g h t  ^-8  vii TABLE XVI.  PAGE The means o f r i b o f l a v i n c o n c e n t r a t i o n i n ug/mg f r e s h w e i g h t f o r m a l e s a n d f e m a l e s o f 11  lines  (their  a v e r a g e c o m p a r e d by-  D u n c a n ' s New M u l t i p l e Range T e s t ) a n d t h e color of Malpighian XVII.  52  tubules  Analysis of variance of t o t a l  riboflavin  i n ug/mg f r e s h w e i g h t i n 10 l i n e s  o f T.  confusum XVIII.  5V  C o r r e l a t i o n o f 10 d a y a d u l t w e i g h t  with  r i b o f l a v i n c o n c e n t r a t i o n i n t h e p_ l o d 56  marked s t o c k XIX.  Means o f t o t a l r i b o f l a v i n  i n ug/mg f r e s h w e i g h t  of the progeny from the mating o f three dams w i t h i n e a c h o f t h r e e s i r e s XX.  A n a l y s i s o f v a r i a n c e by sex o f t h e t o t a l riboflavin  i n ug/mg f r e s h w e i g h t i n t h e  p / p , l o d / l o d marked l i n e XXI  59  60  A n a l y s i s o f v a r i a n c e a n d e x p e c t e d mean s q u a r e s of t o t a l r i b o f l a v i n  i n ug/mg f r e s h w e i g h t  i n t h e p / p , l o d / l o d marked l i n e  6l  viii  TABLE XXII.  PAGE Components  of variance estimated  a n a l y s i s of variance of tfbtal  from the riboflavin 63  i n t h e p / p , l o d / l o d marked l i n e XXIII.  Estimates of h e r i t a b i l i t i e s e r r o r s of the r i b o f l a v i n p / p , l o d / l o d marked l i n e  and trait  standard i n the ' 65  ix  L I S T OF FIGURES  FIGURE 1.  PAGE Ultraviolet  a b s o r p t i o n s p e c t r u m o f pure  riboflavin  and r i b o f l a v i n  extract, at  pH 7 2.  Paper  19  electropherogram of f l a v i n -  mononucleotide dinucleotide  (FMN),  flavin-adenine-  (FAD), r i b o f l a v i n ,  a  m i x t u r e o f t h e s e , and w h o l e a n i m a l s o f different 3.  Developmental  variation  Xi ", p_ a n d W.T. 1  s  developmental last  2k  genotypes  instar  of r i b o f l a v i n i n  males and f e m a l e s .  The  s t a g e s a r e : a, eggs; b,  larvae; c, e a r l y ,  d, l a t e  pupae;  e, d a y o l d , f , 10 d a y o l d , g , 30 d a y o l d adults  t+9  X  ACKNOWLEDGEMENTS The  w r i t e r wishes  t o express h i s g r a t e f u l  a p p r e c i a t i o n t o h i s a d v i s o r , D r . C.W. R o b e r t s , f o r his  encouraging  a s s i s t a n c e and c r i t i c i s m  the course o f t h i s  study.  throughout  He i s a l s o i n d e b t e d t o t h e  members o f h i s c o m m i t t e e who c r i t i c a l l y r e a d  this  manuscript. T h a n k s a r e a l s o d u e D r . W.D. a v a i l a b l e the apparatus used  K i t t s who made  i n f l u o r o m e t r y and  spectrophotometry. A p p r e c i a t i o n i s a l s o extended  to fellow  graduate  s t u d e n t s M r . D.C. C r o b e r a n d M i s s C H . C h i f o r assistance with the s t a t i s t i c a l  a n a l y s e s and t h e  many h e l p f u l d i s c u s s i o n s on t h e s u b j e c t o f t h i s F i n a n c i a l a s s i s t a n c e p r o v i d e d by a P o u l t r y Graduate  Research A s s i s t a n t s h i p i s g r a t e f u l l y  S i n c e r e a p p r e c i a t i o n i s a l s o extended for  her preparation of the manuscript.  thesis. Science  acknowledged.  t o h i s v/ife  INTRODUCTION In recent  y e a r s T r i b o l i u m confusum has i n c r e a s i n g l y  been u s e d f o r p o p u l a t i o n  and q u a n t i t a t i v e  genetic  studies.  T h r o u g h t h e s e s t u d i e s a l a r g e number o f  mutations  have b e e n f o u n d a n d c a t e g o r i z e d .  Stocks of  these mutants a r e r e a d i l y a v a i l a b l e throughout and  the United  States,  h o w e v e r , o n l y a f e w have b e e n  investigated i n detail. affect  A number o f m u t a t i o n s w h i c h  t h e p i g m e n t a t i o n o f t h e eye have been  Preliminary  Canada  found.  i n v e s t i g a t i o n s by t h e a u t h o r u s i n g  these  eye m u t a n t s l e d t o t h e d e m o n s t r a t i o n o f q u a l i t a t i v e and  quantitative differences i n a yellow  fluorescent  p i g m e n t t h a t was s u b s e q u e n t l y i d e n t i f i e d a s r i b o f l a v i n . Since  t h i s p i g m e n t i s a v i t a m i n and e s s e n t i a l f o r t h e  s u r v i v a l o f T. c o n f u s u m , i t s i n v e s t i g a t i o n m i g h t c l a r i f y the nature o f the genetic t h o s e genes r e s p o n s i b l e and  i n t e r r e l a t i o n s h i p s between  for riboflavin  t h o s e genes a s s o c i a t e d W o r k e r s have o b s e r v e d  with  the genetics  Drosophila  differences  w i t h eye pigment d i f f e r e n c e s . the a s s o c i a t i o n of r i b o f l a v i n  of pteridines p a r t i c u l a r l y i n  and-Ephestia, yet the genetics  of r i b o f l a v i n  2  has r e c e i v e d v e r y l i t t l e this  t h e s i s was  the genetic  direct attention.  concerned w i t h attempts to define  c o n t r o l of t h i s pigment, w i t h the  d e s c r i p t i o n o f the changes during  Therefore  i n i t s concentration  d e v e l o p m e n t , and w i t h q u a n t i t a t i v e s t r a i n  d i f f e r e n c e s of t h i s  phene.  REVIEW OF M u t a t i o n s are  THE  usually  LITERATURE  classified  according  t o t h e i r most c o n s p i c u o u s e f f e c t s .  In the  eye  directly visible  c o l o r genes, i n i t i a l l y  pigments can  be  observed.  recognizable features by  an  o n l y the As  are  case  a r u l e these e a s i l y  p r e c e d e d and  accompanied  additional  set  o f c h a r a c t e r s w h i c h become  a p p a r e n t o n l y by  the  use  of s p e c i a l methods. T h e  t o t a l i t y of a l l phenes, i n w h i c h a mutant from a "normal" or constitutes  the  Such p l e i o t r o p y  "standard"  or w i l d  "pleiotropic pattern  ( H a d o r n , 195*0 o f  the  respective  of  color  1951;  Hadorn,  1956;  Flavlnes  are  1955).  i n Drosophila  the  Hubby,  isolated with  have been c o n f u s e d  (1956)  from the  Mitchell,  1962).  pteridines,  e a r l i e r l i t e r a t u r e t h e s e two  Viscontini  pteridines  often  1961;  and  concerning  o f g e n e s ( H a d o r n and  Ziegler,  bio-  morphological  h a v e p r o v i d e d much i n f o r m a t i o n  o f compounds may  with  genes, studied  pleiotropic action  i n some o f  factor.  of genie a c t i o n , c o n d i t i o n s  phenes.  the  (W.T.),  manifestation"  gene o r  p h y s i o l o g i c a l , as w e l l as  Ephestia,  deviates  type  c h e m i c a l and Eye  of  and  classes  ( V i s c o n t i n i , et a l . ,  i s o l a t e d r i b o f l a v i n along  h e a d s o f D r o s o p h i l a and  Ephestia.  A close and  riboflavin  r e l a t i o n s h i p e x i s t s between p t e r i n s (Ziegler,  1961).  B o t h have t h e  p y r i r n i d i n e - p y r a z i n e n u c l e u s a n d s h a r e p o s s i b l y some common s t e p s i n b i o s y n t h e s i s  (Weygand a n d W a l d s c h m i d t ,  1955). The  d i s a p p e a r a n c e o f r i b o f l a v i n f r o m t h e eggs  of i n s e c t s and t h e c o i n c i d e n t fluorescent  appearance o f other  compounds w h i c h seem t o h a v e t h e p r o p e r t i e s  of p t e r i d i n e s also  suggests the p o s s i b l e b i o l o g i c a l  d e r i v a t i o n o f p t e r i d i n e s from f l a v i n e s (Bodine and Fitzgerald,  19*+7j  Burgess,  f l a v i n e s as p r o s t h e t i c has  19^9).  The f u n c t i o n o f  groups o f s e v e r a l  been w e l l e s t a b l i s h e d .  dehydrogenases  Whether t h e y have a n a d d i t i o n a l  f u n c t i o n i n i n s e c t s , which t h e i r accumulation i n such l a r g e q u a n t i t i e s m i g h t suggest,,, i s n o t known  (Gilmour,  1961). U s i n g t h e b a c t e r i o l o g i c a l a s s a y , C a s p a r i and Blomstrand deposited  (1958)  out t h a t t h e pigment  i n r o d l e t s I n the outer layer of the t e s t i s  sheath of Ephestia W.T.  pointed  was p a r t l y r i b o f l a v i n .  riboflavin gradually  appears during  l a r v a l i n s t a r , reaches a plateau and  d i s a p p e a r s i n t h e l a t e pupa.  suggested that  I nthe the l a s t  i n t h e e a r l y pupa Subsequently,  they  r i b o f l a v i n m i g h t t h e n be t r a n s f e r r e d  to the Malpighian tubules since the rate of disappearance f r o m t h e t e s t i s was e x a c t l y b a l a n c e d by i t s a p p e a r a n c e i n the Malpighian tubules.  I n a d d i t i o n they demonstrated  t h a t gene a , w h i c h b l o c k s t h e o x i d a t i o n o f t r y p t o p h a n t o k y n u r e n l n , causes t h e appearance  of considerably  h i g h e r amounts o f r i b o f l a v i n i n t h e t e s t i s  sheaths,  b u t t h e r i b o f l a v i n seems t o d i s a p p e a r somextfhat t h a n i n t h e W.T.  The p l e i o t r o p i c  earlier  action of this  gene,  includes not only tryptophan metabolism of p r o t e i n s , but a l s o m e t a b o l i c p r o c e s s e s such as t h e e f f e c t on p t e r i n s a n d r i b o f l a v i n w h i c h c a n n o t be e x p l a i n e d i n d e t a i l a t t h e moment ( Z i e g l e r ,  1961).  I n mutant w e i s s - a u g i g  (wa), w h i c h i s c h a r a c t e r i z e d by t h e s u p p r e s s i o n o f many f l u o r e s c e n t  s u b s t a n c e s (Hadorn and Kuhn,  K u h n a n d B e r g , 1956) the t e s t i s  t h e appearance  s h e a t h s was i n h i b i t e d  19535  of riboflavin i n  at a l ltimes.  The  t o t a l amount o f r i b o f l a v i n i n t h e s e i n d i v i d u a l s (wa) was  r e d u c e d c o m p a r e d t o a i n d i v i d u a l s b y t h e amount  present i n a testes. I n a d d i t i o n t o e f f e c t s due t o s i n g l e C a s p a r i a n d B l o m s t r a n d (1958) d e m o n s t r a t e d  genes, significant  s t r a i n d i f f e r e n c e s i n t h e amount o f r i b o f l a v i n a n d the p a t t e r n of i t s ' disappearance i n Ephestia. E y e c o l o r g e n e s w h i c h a f f e c t t h e amount o f  6 riboflavin  have a l s o been found i n D r o s o p h i l a .  (1951)  H a d o r n and M i t c h e l l ' s r i b o f l a v i n and  s p o t F l h/5  three other substances a l l of  h a v e b e e n shown t o be p t e r i d i n e s . missing  contained  This spot  i n t h e m u t a n t s b r o w n (bw)  and w h i t e  R i b o f l a v i n as w e l l as t h e o t h e r s u b s t a n c e s lacking are  which was (w).  are  i n a n i m a l s homozygous f o r t h e s e g e n e s .  many o t h e r c o l o r m u t a n t s i n w h i c h t h e  o f F l +/5  There  intensity  i s i n c r e a s e d ( c l o t , c l ; s e p i a , sje; s e p i a o i d ,  1  2  2  s e d ; r o s y , ryr  and a l s o e b o n y , 1; c l a r e t ,  ca;  prune,  2 P  n  ) or reduced  g a r n e t , g. ; 2  (white-apricot, w  a  white-eosin, w ; e  p i n k - p e a c h , p_ ; l i g h t , I t ; c a r n a t i o n , c a r ; p  p u r p l e , p_r) as c o m p a r e d t o t h e w i l d t y p e Mitchell, In  1951;  H a d o r n and S c h w i n c k ,  and  1956).  some o f t h e s e c a s e s i t i s known t h a t a y e l l o w  p t e r i d i n e i s accumulated;  but the l i t e r a t u r e  c l a r i f y w h e t h e r t h e amount o f r i b o f l a v i n a l s o a f f e c t e d by t h e s e T a i r a and Nawa  u s i n g the  t h a t m u t a n t se o f D.  showed s i g n i f i c a n t l y  "riboflavin-like" isoxanthopterin  present  not was  bacteriological  c o n t a i n s much more f l a v i n t h a n v e r m i l i o n  (1961)  does  genes.  (1958),  method o f a s s a y , f o u n d  Hubby  (Hadorn  melanogaster  (v) or  bw.  higher l e v e l s of a  pigment i n the t e s t i s o f  ( l i x ) c o m p a r e d t o ry_  and  light Oregon-R.  7 E v i d e n c e f o r the r i b o f l a v i n among t h e the  domestic fowl.  obtained  a highly  incidence and  genetic  higher  c o n t r o l of  f o r m s has  Bernier  and  melanization.  segregation  B o u c h e r and  B u s s e t a l . , 1959;  i n chicks  of these be  from a  in  (1959;  his co-workers  Cowan e t a l . ,  found p h y s i o l o g i c a l c h a r a c t e r i s t i c s a s s o c i a t e d  Their  gene c a u s i n g  riboflavin  d a t a showed t h a t  deficiency  free r i b o f l a v i n  was  r e t a i n e d more e f f e c t i v e l y i n t h e n o r m a l  (Rd-)  t h a n i n the  whereas,  recessive  ( r d r d ) l a y i n g hen,  r i b o f l a v i n r e t e n t i o n i n n o n - l a y e r s was  independent  of the  was  r d gene.  The  more shown t o be  n o r m a l a l l e l e Rd,  incompletely  Although Tribolium i n e c o l o g i c a l , and ( M c D o n a l d and 196 fa, b; 196*+ a , the  b)  has  extensively  genetics  been used  more r e c e n t l y i n g e n e t i c a l  C r e n s h a w and  there  further-  dominant.  P e e r , I96O; S c h l a g e r ,  1965;  L  with  (E)  alleles.  involved  Cowan e t a l . , 196l;  with a recessive (rd).  the  of r i b o f l a v i n d e f i c i e n c y between b l a c k  i n v o l v i n g the  a)  (195*+)  significant difference i n  T h e y s u g g e s t e d t h a t r i b o f l a v i n may  1961  been found i n  Cooney  n o n - b l a c k ( e ) embryos as w e l l as  cross  free  Lerner,  a p p e a r s t o be  no  of p h y s i o l o g i c a l  1963; 196^;  Dawson, Sokoloff,  literature traits.  studies  concerned  MATERIALS AND METHODS A l l c u l t u r e s , p a r e n t s a n d progeny were r e a r e d o f 32° C ,  i n a c o n s t a n t environment humidity.  a n d 72%> r e l a t i v e ( N o . 30)  The medium was p r e p a r e d f r o m s i f t e d  M e o t a w h o l e wheat f l o u r m i x e d w i t h 10% T o r u l a y e a s t (Doty, 1965).  (60° C. f o r  S t e r i l i z a t i o n o f the f l o u r  2k h o u r s ) was p e r f o r m e d p r i o r  t o adding the y e a s t , since  t h e l a t t e r was f o u n d t o c l u m p u p o n  heating.  V i r g i n f e m a l e s were o b t a i n e d f o r a l l  matings  by s e x i n g i n t h e p u p a l s t a g e a n d c h e c k i n g t h e phenotype  upon e c l o s i o n .  M a l e s were sexed  similarly,  w i t h t h e e x c e p t i o n o f some w h i c h w e r e r e m o v e d  from  s t o c k c u l t u r e s i n t h e a d u l t stage and sexed by t h e method o f P f a d t ( 1 9 6 2 ) .  Progeny were u s u a l l y  sexed  i n t h e p u p a l s t a g e , h o w e v e r , some w e r e s e x e d a s adults.  E t h e r i z a t i o n was e m p l o y e d f o r i m m o b i l i z a t i o n .  S m a l l m a t i n g s were c a r r i e d  o u t i n 20 m l . c r e a m e r s ,  l a r g e r ones i n 110 m l . c e l l u l o s e n i t r a t e All  tubes.  o f t h e s t o c k s were o b t a i n e d f r o m t h e  Tribolium Stock Center, U n i v e r s i t y o f C a l i f o r n i a , Berkeley.  T h e y w e r e m a i n t a i n e d i n mass b r e e d i n g  370 m l . c o n t a i n e r s .  A l l c r o s s e s were r e c i p r o c a l and  at  least  i n duplicate.  In general crosses  made by m a t i n g f i v e m a l e s  to f i v e  were  females.  In  a c c o r d a n c e w i t h T r i b o l i u m c o n v e n t i o n males a r e put first  i n the d e s c r i p t i o n  o f t h e s t o c k s u s e d i s as pearl  ocular  from the ommatidia  appearing  gene i s r e f e r r e d pearl" to  description  eye  eliminates  i n a bicolored  (Graham,  t o i n the t e x t  (p_*) and w i l l  the  but not from the  diaphragm r e s u l t i n g  spectacled  The  follows:  (p_), a n a u t o s o m a l r e c e s s i v e , pigment  pearl  of a cross.  1957)*  as  or This  "Graham's  s u b s e q u e n t l y be  redesignated  p_ . r  (p_ ), s p o n t a n e o u s i n t h e B e r k e l e y s  strain;  allelic  (Sokoloff,  synthetic  to pearl described  1963).  by Graham  p.'and p_ a r e p h e n o t y p i c a l l y s  indistinquishable. light  o c u l a r diaphragm  (lod),  i s not s t r i c t l y  m u t a t i o n b u t i t c a n be o b s e r v e d when eye mutants  s u c h as p e a r l  (which m o d i f i e s  pigment  o f the ommatidia)  o f m e l a n i n pigment an e n d o s k e l e t a l  synthesis  diaphragm, to  support  Since the diaphragm i s not  by genes a f f e c t i n g  ommatidia,  color  lod i s  the  s t r u c t u r e which helps  t h e compound e y e . affected  from the o c u l a r  eye  the black  are present,  an autosomal r e c e s s i v e which b l o c k s  an  the c o l o r  of the  i t a p p e a r s as a d a r k band u n d e r t h e  10 c o l o r l e s s or l i g h t l y pigmented marginal ommatidia, g i v i n g the b e e t l e a appearance ( S o k o l o f f ,  spectacled  1962).  ruby spot ( r u s ) , produces a p i n k i s h eye  either i n  the l a t e pupa or i n the t e n e r a l a d u l t , but aging the  eye darkens, l e a v i n g only a  reddish  spot which can be i d e n t i f i e d by c a s t i n g shadow on the b e e t l e ' s  eye  not  a  as i t l i e s on  stage of a d i s s e c t i n g microscope.  with  The  the  spot i s  s t a t i o n a r y f o r , i f the b e e t l e i s r o t a t e d ,  i t moves i n the d i r e c t i o n opposite movement of the b e e t l e .  This  to  the  indicated  that,  i n r e a l i t y , the ommatidia are a l l dark red but  s p e c i a l intense  detect  the pigment ( S o k o l o f f ,  d i r t y p e a r l eye during  lighting  i s required 196l).  (dp_e), i s d i s t i n q u i s h e d  a b r i e f p e r i o d i n the  c y c l e , i n the pupa and  to  from rus  developmental  sometimes i n the  t e n e r a l a d u l t the compound eyes appear uniformly  p i n k i s h i n rus whereas i n dpe  the  eyes o f the l a t e pupa or e a r l y imago appear p e a r l - l i k e except that the c e n t r a l ommatidia appear d i r t y .  When f u l l y c o l o r e d  the eyes of  the imago become dark r e d , perhaps darker than rus., somewhat resembling the  chestnut  eye m u t a t i o n eye  spot  i n T.  castaneum ( S o k o l o f f ,  (es), i s a sex-linked recessive characterized  by a c l e a r  s p o t i n t h e c e n t e r o f t h e eye  c a n be r e a d i l y  to  identify  as t h e i n s e c t a g e s , however i t n e v e r completely  which  s e e n i n young a d u l t s , b u t  becomes i n c r e a s i n g l y d i f f i c u l t  red  196*+).  (McDonald,  disappears  1961).  ( r ) , i s a sex-linked recessive producing  a  light-  r e d a r e a i n t h e eye c o m p a r a b l e i n t h e e a r l y s t a g e s w i t h p e a r l ; w i t h age reduced  the spot  becomes  i n s i z e i n t e r m e d i a t e b e t w e e n p_ and  es  (Ho, 1962). p e a r l , l i g h t o c u l a r d i a p h r a g m (p.,lod); p e a r l , spot  (p_,rus); p e a r l , droopy e l y t r a ,  abdominal s t e r n i t e s  ruby  creased  (p_,dre,cas.), s t o c k s w e r e  obtained from the B e r k e l e y  stock center  along  w i t h the above mutant s t o c k s . CFI-1,  i s an i n b r e d l i n e , M-3 g e n e r a t i o n s o f eye  f r e e ( a s f a r as i s known)  c o l o r mutations.  t h e W.T. CFI-7,  and  b r o t h e r - s i s t e r mated f o r  This l i n e i s used  control.  i s a l s o an i n b r e d l i n e , o b t a i n e d b a s e p o p u l a t i o n as mated f o r ^3 were k e p t  as  same  C F I - 1 , and b r o t h e r - s i s t e r  generations.  B o t h CFI-1  as random m a t i n g c l o s e d  s i n c e t h e i r a r r i v a l from B e r k e l e y generations).  from the  and  CFI-7  populations (about  10  12 A m o d i f i c a t i o n o f the procedure by H a d o r n a n d M i t c h e l l  described  (1951) was u s e d f o r most  of the chromatographic separations. t o g r a m s w h i c h w e r e t o be m e a s u r e d  A l l chroma-  fluorometrically  were p r e p a r e d from b e e t l e s t e n days a f t e r The b e e t l e s were b o i l e d  eclosion.  f o r two m i n u t e s i n d i s t i l l e d  w a t e r i n o r d e r t o c o a g u l a t e p r o t e i n s and t o improve the subsequent  chromatographic r e s o l u t i o n o f  fluorescent substances.  The b o i l i n g was done  d i r e c t l y w i t h t h e l a r v a e and t h e pupae, b u t t h e a d u l t s were immersed b r i e f l y to b o i l i n g .  i n 95$ e t h a n o l p r i o r  E x c e s s w a t e r was r e m o v e d f r o m t h e  b o i l e d a n i m a l s by b l o t t i n g  them o n f i l t e r  paper.  T h e y were t h e n t r a n s f e r r e d i m m e d i a t e l y o n t o Whatman No. 1 . p a p e r .  B e e t l e s w e r e f i r m l y mashed o n t o t h e  p a p e r w i t h a g l a s s r o d a n d a l l o w e d t o d r y a t room temperature i n the dark. removed f r o m t h e paper  R e s i d u a l t i s s u e s were n o t  since the author's pre-  l i m i n a r y experiments demonstrated e x o s k e l t o n does n o t i n t e r f e r e w i t h  that the chitinous chromatography.  I n t e r n a l organs were d i s s e c t e d i n an i n s e c t Ringer  ( P a t t o n a n d C r a i g , 1939) a n d t r a n s f e r r e d  w i t h as l i t t l e  adhering l i q u i d as p o s s i b l e onto t h e  chromatography  paper.  13 The d r i e d chromatography  sheets were r o l l e d  i n t o c y l i n d e r s , s t a p l e d and p l a c e d u p r i g h t i n t o a pyrex tank c o n t a i n i n g the developing solvent.  Unless  o t h e r w i s e s t a t e d a l l experiments were c a r r i e d out i n the a s c e n d i n g d i r e c t i o n i n d a r k w i t h t e r t - b u t a n o l , p y r i d i n e and water (10:3:7) as the s o l v e n t  (Caspari  and B l o m s t r a n d , 1958). When the s o l v e n t f r o n t reached 1^ cm. (about f i v e hours) the c y l i n d e r s were removed and d r i e d i n a darkened fume hood. Developed chromatograms were observed by means of  two u l t r a v i o l e t lamps w i t h p r i n c i p l e e m i s s i o n  at  253 and 366 m i l l i m i c r o n s r e s p e c t i v e l y .  cut  Spots were  out and e x t r a c t e d o v e r n i g h t i n f o u r m l . o f  double d i s t i l l e d water a t room temperature i n a d a r k room.  The samples were r e a d i n a Turner  p h o t o f l u o r o m e t e r , Model 111, u s i n g V36 k7B  and 2A)  and 570 ( f i l t e r 23A)  (filters  m i l l i m i c r o n s as  p r i m a r y and secondary wavelengths and w i t h arrangement 30X  (AOAC, i960).  For q u a n t i t a t i o n  a s t a n d a r d c u r v e was p r e p a r e d from pure u s i n g t h e above methods.  slit  riboflavin  RESULTS AND  PART I .  A.  CHEMICAL ANALYSIS  Preliminary The  W.T.  DISCUSSION  observations  r e s u l t s of paper chromatography o f the  i s shown i n T a b l e I .  and f o u r n o n - f l u o r e s c e n t chromatograms.  In total eight fluorescent s p o t s a p p e a r e d on t h e  The y e l l o w f l u o r e s c e n t s p o t s  a n d 5 a p p e a r e d on a l l t h e c h r o m a t o g r a m s . s e l d o m a p p e a r e d ; when i t was 2 w i t h the i d e n t i c a l R  spot  absent.  The v i o l e t  f  present  yellow  invariably  f l u o r e s c e n t s p o t s k- and 6 v a r i e d  considerably i n i n t e n s i t y of fluorescence. 7 a p p e a r e d w e l l d e f i n e d on f r e s h  red  spot  but  g r a d u a l l y changed  few d a y s .  The  was  still  wet.  when i l l u m i n a t e d w i t h t h e 253 lamp;  i t a l s o disappeared  chromatogram. a n d was never  The b r i c k chromatograms,  t o a d i r t y brown c o l o r a f t e r  f l u o r e s c e n t spot  the chromatogram  2  Spot 3  the  v a l u e was  1,  mu  8 was  only v i s i b l e  principle  emission the  w i t h the solvent f r o n t  e i t h e r brown c o l o r e d o r b l u e  f l u o r e s e e n t , but  both. The r e s u l t s o b t a i n e d  while  Spot 9 weakly absorbed  shortly after drying  S p o t 10 m i g r a t e d  a  by v i s u a l e s t i m a t i o n o f  TABLE I CHROMATOGRAPHIC DISTRIBUTION OF SUBSTANCES AND THEIR R VALUES I N W.T.  Spot No.  ADULT TRIBOLIUM CONFUSUM  visible color  Fluorescent color  R  f  J.  intensity  1  -  Yellow  .05  medium  2  Yellow  Yellow  .27  strong  Violet  .27  medium*  Violet  .50  variable  Yellow  .60  strong  Violet  .6>+  variable  -  • 72  weak  Blue  .81  trace  Absorbs**  .91  trace  Blue***  .97  medium  k  -  5  Yellow  3  6  -  7  Red  8 9 10  Brown  * S e l d o m a p p e a r s ; i n i t sp r e s e n c e t h e y e l l o w ** Maximum a b s o r b a n c e a t 253  spot i s u s u a l l y  mu.  *** T h i s s p o t i s e i t h e r b r o w n , o r b l u e a n d f l u o r e s c e n t .  absent.  16. f l u o r e s c e n c e of the y e l l o w f l u o r e s c e n t 5 i n 11  and  Table I I .  intensely yellow fluorescent  shows c o n s i d e r a b l e v i s u a l v a r i a t i o n eye c o l o r m u t a n t s and  literature 19^9;  for similar  1962;  good i n d i c a t i o n  B. spot 1.  be  Pecakova,  196*+)  and  gave a  riboflavin.  p u r i f i c a t i o n and  characterization  5 I d e n t i f i c a t i o n by  electrophoretic the substance  of  spectrophotometry  spectrophotometry,  and c h r o m a t o g r a p h i c  i n s p o t 5,  on t h a t o f S c h m i d t  identification,  s u s p e c t e d t o be  e x t r a c t e d and p u r i f i e d  based  Comparison  Schmidt  et a l . ,  t h a t i t might  For the purpose  was  chromatographed,  s o l v e n t s ( H a i s and  Harkness  Isolation,  different  spot w i t h those i n the  C a s p a r i and B l o m s t r a n d , 1958;  Viscontini,  of  were  5  Although  never p r e s e n t i n t h i s mutant.  t h e R^ v a l u e s o f t h i s  spot  between  the i n b r e d l i n e s .  a l a r g e number o f p_' i n d i v i d u a l s  of  2  l i n e s o f T. c o n f u s u m a r e shown i n The  s p o t 5 was  s p o t s 1,  riboflavin,  by a m o d i f i e d method  and V i s c o n t i n i  (1962).  TABLE I I VISUAL ESTIMATION OF THE YELLOW FLUORESCENT SUBSTANCES I N 1 1 LINES OF T. CONFUSUM  Lines P cas  P lod P us  o5  +  +  +  +  +  +  + +  +  +  +  2  .1+8  ++  +  ++  +  +  +  + + + +  +  +  5  .60  +  +++  +++  + + _ + +  +  ++  ++  +  68  68  68  68  68  20  d  Spot  R  1  #  f  number chromatographed*  r  e  + +  68  r  * E q u a l numbers o f males and females  p  s  p' es- r  dpe r u s C F I - 1 C F I - 7  +  3 7 5 68 68 68  18  A b o u t one gm. p o r t i o n s o f b e e t l e s w e r e homogenised i n an i c e - c o o l e d P o t t e r - t y p e t i s s u e homogeniser. by  A 95%> e t h a n o l  evaporation  e x t r a c t i o n was f o l l o w e d  o f t h e excess a l c o h o l , and chromato-  g r a p h y o n Whatman No. 1 p a p e r i n t e r t - b u t a n o l : p y r i d i n e : water  (10:3:7)•  The f a s t e r m o v i n g  f r a c t i o n was e l u t e d i n d i s t i l l e d w a t e r a n d concentrated  b y vacuum e v a p o r a t i o n  chromatographed i n n-butanol: C+:l:l).  a t 60°  C,  then  a c e t i c a c i d : water  The f a s t e r f r a c t i o n was e l u t e d a g a i n a n d  n e u t r a l i s e d i n an i c e b a t h .  The s o l v e n t was e v a p o r a t e d  u n d e r vacuum a n d t h e r e s i d u e r e d i s s o l v e d i n a s m a l l amount o f w a t e r . S a l t s w e r e removed b y s u b s e q u e n t c h r o m a t o g r a p h y i n water. the  Following  e l u t i o n the concentration of  s o l u t e was b r o u g h t t o e q u a l i t y b y d i l u t i o n  that o f a s i m i l a r l y prepared Using  pure sample o f r i b o f l a v i n .  a U n i c a m (SP.500) s p e c t r o p h o t o m e t e r , a n u l t r a -  v i o l e t absorption curve The  with  i d e n t i t y of standard  was p r e p a r e d  (Figure  and sample a p p e a r s  1). evident.  FIGURE 1.  ULTRAVIOLET ABSORPTION-SPECTRUM  R I B O F L A V I N AND-RIBOFLAVIN EXTRACT, AT pH 7  OF PURE  20  2.  Identification  by means of chromatography  Whole beetle squashes were made on two d i f f e r e n t grades of f i l t e r paper and chromatographed i n five different  solvents using pure r i b o f l a v i n  with each run for comparison. results  The solvents and the  for each f i l t e r paper are shown i n Table I I I .  Each value was the mean of ten or more determinations; The migration properties of sample and standard appear nearly i d e n t i c a l under a l l conditions. The use. of decomposition products for the i d e n t i f i c a t i o n of r i b o f l a v i n has frequently been employed i n the past (Yagi, 1957: C e r l e t t i and S i l i p r a n d i , 1955). lumiflavin  Using the methods of Yagi (1957)  and lumichrome were prepared by photolysis  of pure r i b o f l a v i n as well as the Tribolium extract. These, along with flavin-mononucleotide (FMN), flavln-adenine-dinucleotide (FAD) and squashes of whole beetles were chromatographed on Whatman No. 1. f i l t e r paper using n-butanol: acetic acid: water (*+:l:5) for solvent. Table IV. The  The results are shown i n  values of reference nucleotides  and decomposition products agree well with those of the corresponding substance i n the extract and whole squashes of beetles.  TABLE I I I MEAN R  VALUES OF SAMPLE AND STANDARD CHROMATOGRAPHIC  SOLVENTS  paper  spot  A  B  Whatman No. 1  spot 5  .32  .60  riboflavin  .31  spot 5 riboflavin  Whatman No. h  * A  n-propanol; IN.  acetic acid  I N VARIOUS  solvents* C  D  E  .37  .25  .32  .6^-  .36  .25  .33  • 39  ,6h  M  .27  • 37  .hi  .66  .h6  .27  .39  (3:1) (10:3 :7)  B  tert-butanol;  p y r i d i n e ; water  C  n - p r o p a n o l ; 1% aqueous ammnonia  D  n-butanol; acetic  a c i d ; water  (*+:l: 1)  E  n-butanol; acetic  a c i d ; water  (*+:l: 5)  (2: 1)  22  TABLE IV R  f  VALUES OF REFERENCE COMPOUNDS AND SAMPLES  compound  reference cpd.  Tribolium extract  whole squashes  color of fluorescence  FAD  .05  -  .05  yellow  FMN  .13  -  .12  yellow  riboflavin  .32  .32  • 31  yellow  lumiflavin  .1+6  .1+6  -  yellow  lumichrome  .63  .63  -  blue  23 J.  I d e n t i f i c a t i o n b y means o f e l e c t r o p h o r e s i s  The method o f Y a g i electrophoretic  (1957)  was u s e d f o r t h e  separation of flavines.  squashes o f d i f f e r e n t  genotypes were r u n c o n c u r r e n t l y  w i t h FMN, FAD r i b o f l a v i n a n d a m i x t u r e P h o s p h a t e b u f f e r o f pH 8,  standards.  was u s e d w i t h c o n s t a n t giving  ^00 v o l t s .  f o u n d most s u i t a b l e Satisfactory  2.  c u r r e n t a t 2.M+  Whatman No. 3MM f o r supporting  of these a n d .05  The r e s u l t s  M  mA/cm.  p a p e r was medium.  s e p a r a t i o n s were o b t a i n e d d u r i n g  two h o u r r u n . in Figure  Sample  a r e shown  a  diagrammatically  riboflavin  FMN  FAD  FAD FMN  +/+  p'/p'  p /p S  S  p/p: d r e / d r e : c a s / c a s  riboflavin FIGURE 2.  PAPER ELECTROPHEROGRAM  OF FLAVINE-MONONUCLEOTIDE ( F M N ) ,  FLAVIN-ADENINE-DINUCLEOTIDE ( F A D ) , R I B O F L A V I N , A MIXTURE AND WHOLE ANIMALS OF DIFFERENT  GENOTYPES.  OF THESE,  DISCUSSION The i d e n t i t y o f t h e y e l l o w s p o t 5)  i s o l a t e d from T r i b o l i u m  fluorescent  substance,  confusum a d u l t s ,  with  r i b o f l a v i n was shown by t h e i n d e p e n d e n t methods o f s p e c t r o p h o t o m e t r y , c h r o m a t o g r a p h y and  electrophoresis.  I n a d d i t i o n , t h e d e c o m p o s i t i o n p r o d u c t s o f s a m p l e and s t a n d a r d r i b o f l a v i n w e r e a l s o shown t o be i d e n t i c a l . a p p e a r s t h a t none o f t h e f l u o r e s c e n t  spots obtained  It in  chromatography ( T a b l e I and T a b l e I I ) were d e c o m p o s i t i o n products of r i b o f l a v i n . values are  Therefore, the r i b o f l a v i n  subsequently obtained  by t h e f o r e g o i n g  assumed t o be r e p r e s e n t a t i v e  of r i b o f l a v i n FMN  method  of the a c t u a l  amount  present.  and FAD w e r e a l s o i d e n t i f i e d  v a l u e s and e l e c t r o p h o r e t i c m i g r a t i o n  b y t h e i r Re-  i n order to  d i s t i n g u i s h them f r o m f r e e r i b o f l a v i n a n d t o o b s e r v e any  p o s s i b l e v a r i a t i o n i n t h e i r l e v e l s due t o  v a r i a t i o n i n the l e v e l of free r i b o f l a v i n .  Such  c o n c o m i t a n t v a r i a t i o n m i g h t be e x p e c t e d o n t h e o r e t i c a l g r o u n d s s i n c e FMN and FAD d e r i v e the molecules from f r e e FMN (Figure that  the f l a v i n part  of  riboflavin.  and FAD a r e p r e s e n t i n a l l s i x g e n o t y p e s  2).  Visual inspection indicated  (Table I I )  f r e e r i b o f l a v i n was p r e s e n t i n v a r y i n g  quantities  26 in a l l lines by v i s u a l the  e x c e p t JD  observation  1  i n w h i c h i t was  p e a r l and p_  o f JD_' ( S o k o l o f f , 1963)  detectable  under u l t r a v i o l e t l i g h t .  eyes o f the f i v e mutant genotypes  phenotypically  not  s  tested  are  has b e e n d e s i g n a t e d  i t was  of i n t e r e s t  a b e r r a t i o n on t h e p a r t o f t h e JD'  stock.  Since  an  to note  allele this  PART I I .  A.  GENETIC ANALYSIS  A n a l y s i s of p e a r l I n the p r e v i o u s  g r a p h y and  s e c t i o n i t was  shown by  chromato-  e l e c t r o p h o r e s i s t h a t t h e p_' m u t a n t l i n e  had  no v i s i b l e amount o f r i b o f l a v i n , w h e r e a s t h e p_ l o d , p_ r u s , and  p_ d r e c a s m u t a n t l i n e s  v i s i b l e q u a n t i t i e s (Table The  t h e p.  1  was  I I . , and  q u e s t i o n a r o s e , was  h e r i t a b l e , and gene.  i f s o , was To  possessed Figure  clearly 2).  this riboflavinless  phene  i t dependent or independent  answer t h i s q u e s t i o n  of  the f o l l o w i n g cross  made:  P  p'/p*  x  F-L The  a b s e n c e o f r i b o f l a v i n i n t h e F^  W.T. o f 3=1  and  The  phene s h o u l d  t o p e a r l ) w o u l d be o n l y be  characters are  showing the presence and  F2  progeny  or  are  F-^ p r o g e n y w e r e a l l p h e n o t y p i c a l l y  possessed r i b o f l a v i n .  (W.T.  +/+  selfed  r e s u l t s of t h i s c r o s s  shown i n T a b l e V.  x  present  I n the F2 expected.  progeny a The  riboflavinless  i n p e a r l s , i f these  a s s o c i a t e d w i t h the  same g e n e .  of the progeny i n Table V i n d i c a t e d the r e s u l t s  ratio  two  Analysis are  TABLE V THE DISTRIBUTION OF R I B O F L A V I N I N THE F  AND F  2  PROGENY*  FROM THE CROSS p'/p* x +/+  generation  phenotype  males  females  total  number chromatographed  F-L  wild  317  ^09  726  IOO  -  -  -  -  wild  653  66*+  1317  100  pearl  1^3  l6h  307  307  pearl F  2  number w i t h riboflavin  100  100  * Progeny from r e c i p r o c a l c r o s s e s were p o o l e d as t h e y were n o t a p p r e c i a b l y  different.  CO  29 significantly different (x  2  =  32.18,  unexpected  d.f.  =1,  s i n c e Graham  f r o m t h e e x p e c t e d 3-1 P =  .005).  (1957)  H o w e v e r , t h i s was n o t  and Park  (1937)  a l o s s o f f i t n e s s o f t h e p e a r l homozygotes. 300 p r o g e n y  showed  With over  t e s t e d , t h e r i b o f l a v i n l e s s phene was a l w a y s  a s s o c i a t e d w i t h t h e p_' g e n e . was  ratio  The p o s s i b i l i t y o f l i n k a g e  somewhat r e d u c e d by t h e f a c t t h a t t h e r e w e r e no w i l d  t y p e s w h i c h l a c k e d r i b o f l a v i n , n o r were t h e r e any p e a r l s w h i c h had r i b o f l a v i n . t o have p r o d u c e d was  as l i t t l e  R e c o m b i n a t i o n w o u l d be e x p e c t e d  such r e c i p r o c a l phenotypes  i f linkage  a s .'3 • -  S i n c e p. a n d p_ l p d have b e e n s e p a r a t e b r e e d i n g 1  p o p u l a t i o n s presumably it  s i n c e t h e y were f i r s t  discovered,  was c o n s i d e r e d p o s s i b l e t h a t t h e a c c u m u l a t i o n o f  d i f f e r e n t m o d i f i e r backgrounds  c o u l d have c a u s e d t h e  observed d i f f e r e n c e i n the r i b o f l a v i n t r a i t .  To c h e c k  t h i s a l t e r n a t i v e , r e c u r r e n t s e l e c t i o n f o r p e a r l was p r a c t i c e d f o r two g e n e r a t i o n s i n t h e c r o s s p ' / p x 1  Twenty progeny were chromatographed  +/+.  from each g e n e r a t i o n  and t h e r i b o f l a v i n was m e a s u r e d f l u o r o m e t r l c a l l y .  These  v a l u e s a r e compared i n T a b l e V I w i t h t h o s e o b t a i n e d f r o m t h e p_' b a s e p o p u l a t i o n (P-j_), a n d F-^ h e t e r o z y g o t e s ( p ' / ) +  and t h e W.T.  TABLE V I COMPARISON OF TOTAL R I B O F L A V I N  (ug/mg FRESH WEIGHT)  OF TWO GENERATIONS OF RECURRENTLY SELECTED p_« FROM p ' / p ' x +/+,  WITH THE PARENTS,  THE HETEROZYGOTES AND THE  genotype  sex  l  p'/p'  male female  .0050 .0060  .0005  l  P'/+  male female  .091 .063  .00!+ .0009  2  P'/P'  male female  .0057 .0062  .0006  P'/P'  male female  .0060 .0059  .0008  male female  .110 .078  .004.004-  generation  P  F  F  W.T.*  * Ten o b s e r v a t i o n s  per sex  riboflavin ug/mg f r e s h weight  +  standard error  .OOO^r  .0004-  .0004-  31 The  v a l u e s o b t a i n e d f o r p j homozygotes must be  viewed w i t h some c a u t i o n as these measurements were made a t the l i m i t s o f s e n s i t i v i t y o f t h e f l u o r o m e t e r . The  results  show t h a t changing o f t h e r e s i d u a l  does n o t d e t e c t a b l y a l t e r t h e a p p a r e n t l y c o n d i t i o n o f p'/p' i n d i v i d u a l s .  genotype  riboflavinless  The W.T. h e t e r o z y g o t e  p V } shows t h a t a t t h e l e v e l o f t h e r i b o f l a v i n phene, +  the  normal a l l e l e i s i n c o m p l e t e l y dominant t o p. . 1  B.  A n a l y s i s o f p_ l o d . p_ l o d , a l t h o u g h p h e n o t y p i c a l l y s i m i l a r t o p',  has  r i b o f l a v i n (Table I I , F i g u r e 2).  S i n c e t h e p_ and p_'  genes a r e supposedly i d e n t i c a l a l l e l e s ( S o k o l o f f , 196^ the  i n t e g r i t y of the p o s i t i v e  )  r i b o f l a v i n phenotype o f  the p_ l o d mutants was t e s t e d .  The f o l l o w i n g  c r o s s was  made t o t e s t i f a r i b o f l a v i n l e s s p_ l o d mutant c o u l d be obtained: p/p  lod/lod  F-L The  results  the F  selfed  o f t h i s c r o s s showing t h e presence o r absence  of r i b o f l a v i n i n t h e ^ VII.  x +/+  and F  2  progeny a r e shown i n T a b l e  The F-j_ progeny were a l l W.T. and had r i b o f l a v i n . 2  t h e expected p h e n o t y p i c r a t i o o f W.T. t o p e a r l ,  In to pearl  TABLE V I I THE DISTRIBUTION OF RIBOFLAVIN I N THE F  1  PROGENY* FROM THE CROSS p/p l o d / l o d  generation  F  phenotype  males  females  total  wild  202  189  391  wild  163  167  330  3^  56  8  10  pearl pearl, lod  AND F x ++  e x p e c t e d number ratio chromatographed  -  number w i t h riboflavin  50  50  12  100  100  90  3  90  90  18  1  18  18  * P r o g e n y f r o m r e c i p r o c a l c r o s s e s were p o o l e d a s t h e y w e r e n o t a p p r e c i a b l y  different.  IV)  33 l i g h t o c u l a r diaphragm i s  12:3:1  and + / ; l o d / l o d g e n o t y p e s  are indistinguishable  +  t h e W.T.  The d e v i a t i o n f r o m t h e e x p e c t e d  significant all  since the p/+;lod/lod  ( x  2  =  3.79,  d.f. =  2,  0.1  ratio.P  from was n o t  0.25),  and  phenotypes were p o s i t i v e f o r r i b o f l a v i n . F r o m t h e s e r e s u l t s i t c a n t e n t a t i v e l y be  concluded  t h a t t h e p e a r l gene i n t h e p_ l o d s t o c k may  n o t be i d e n t i c a l t o t h e p e a r l gene i n t h e p_" s t o c k a s suggested  by S o k o l o f f  A c k e r m a n (1966  ).  (196*+  ) a n d more r e c e n t l y b y  Therefore, i t i s suggested  apparently riboflavinless p  1  that the  be r e - d e s i g n a t e d "_p_ . 11  3^ C.  Test  f o r a l l e l i s m b e t w e e n p_ a n d p_ r  The d i f f e r e n t i a l  r e s p o n s e o f t h e two p e a r l g e n e s  r a i s e d t h e q u e s t i o n , what i s t h e r e l a t i o n s h i p between them i f t h e y a r e n o t a l l e l e s . To a n s w e r t h i s q u e s t i o n , t h e f o l l o w i n g c r o s s e s w e r e made; 1.  P  1  F  x  p /p r  r  x a.  p/p,lod/lod selfed  b. c r o s s e d w i t h 2.  3«  P-j_ p / p r  r  x  p /p r  p/p, r u s / r u s  F-^  a s i n c r o s s 1. a . a n d b.  Pi  P /p  Fj_  as i n c r o s s 1. a . and b.  r  r  x  r  p/p,dre/dre,cas/cas  The r e s u l t s a n d a n a l y s i s o f t h e s e c r o s s e s a r e shown.in Tables  I X , X and X I .  The F-^ p r o g e n y f r o m a l l  t h r e e c r o s s e s were w i l d t y p e and p o s i t i v e f o r r i b o f l a v i n . T h e r e f o r e , p_ a n d p_ a r e n o t t r u e a l l e l e s . r  and  backcross  phenotypic  ratios deviate  None o f t h e F  2  significantly  from expectations a p p l i c a b l e i n the case of s e g r e g a t i o n at a single locus. and  Hence i t may be c o n c l u d e d  t h a t p_  p_ a r e e i t h e r c l o s e l y l i n k e d l o c i w i t h s i m i l a r  f u n c t i o n s , or h e t e r o a l l e l e s capable  r  genetic  (as d e s c r i b e d f o r D r o s o p h i l a  TABLE I X THE DISTRIBUTION OF RIBOFLAVIN I N THE F , 1  PROGENY OF p / p r  cross P /P x p/p,lod/lod r  r  p /P)l°d/ selfed r  +  F  l  F  2  riboflavin present absent  wild  100  wild  pearl,lod wild  r  r  BC  X  pearl reciprocal  BC  1  95** 99  2  AND BACKCROSS (BCj_)  x p/p,lod/lod  phenotype  pearl  P?/P * p /p,lod/+  r  F  total  expected 2 ratio X  -  1899*  -  _  19*+  8  h5  39  3^ 4-1  159  6  13 11  10 15  ^9  2  171  1  172  1  186  1  178  1  8487  -  wild  98 88  pearl  -  82 90  86 92  <P  <  -  -  -  0.721  .5  .75  0.003  .9  .95  0.176  .5  .75  * T o t a l p r o g e n y , o u t o f w h i c h a sample o f 100 was a s s a y e d f o r r i b o f l a v i n . ** The t o p v a l u e r e f e r s t o m a l e s , t h e b o t t o m t o f d m a l e s .  TABLE X THE DISTRIBUTION OF RIBOFLAVIN IN THE PROGENY OF p / p r  r  r  p'/p,rus/+ selfed  F  F  i  wild  100  2  non-pearl** 206*** 21k  r  pVp,rus/+  reciprocal  B C  1  BC-L  -  2  AND BACKCROSS ( B C )  p/p,rus/rus  phenotype  total  expected ratio  -  777*  H-20  1  105 101  97 92  202 193  1  wild  60 71  _  131  1  pearl  -  59 63  122  1  wild  103 106  209  1  pearl  -  213  1  pearl***  pJ7p X  x  riboflavin p r e s e n t absent  cross P /P X p/p,rus/rUs  r  F  98 115  2 X  -  -  0.77  .25  .5  0.320 .5  .75  O.O38 • 75  .9  * T o t a l progeny, out o f which a sample o f 100 was assayed f o r r i b o f l a v i n ** W i l d t y p e s and r u s homozygotes. *** The t o p v a l u e r e f e r s t o males, t h e bottom t o f e m a l e s .  TABLE X I THE D I S T R I B U T I O N OF R I B O F L A V I N I N THE F , ±  PROGENY OF p / p r  r  x  phenotype  riboflavin present absent  p /p x Fj p/p,dre/dre,cas/cas  wild  100  non-pearl**  122*** 110  r  p /p,dre/+,cas/+ selfed r  F  2  pearl  56 6>f  P /p x BC. P /p,dre/+,cas/+ r  r  r  reciprocal  x  BC^  wild  71 68  pearl  -  wild pearl  81 93  -  2  AND BACKCROSS (BCj_)  p/p,dre/dre,cas/cas  cross r  F  —  58 57  65 72  82 87  total  expected ratio x  -  670*  232 ll*f  1  121  1  139  1  137  1  17*+  1  I69  1  * T o t a l p r o g e n y , o u t o f w h i c h a s a m p l e o f 100 was a s s a y e d f o r ** W i l d t y p e s a n d d r e a n d c a s h o m o z y g o t e s . *** The t o p v a l u e r e f e r s t o m a l e s , t h e b o t t o m t o f e m a l e s .  2  < P <  -  -  -  0.021  .75  .9  O.OlM-  .9  .95  0.073  .75  .9  riboflavin  38 b y C a r l s o n , 1959  ) of a p p a r e n t l y complete  complementation  when h e t e r o z y g o u s .  D.  The  s y n t h e s i s o f a double mutant;  p e a r l , ruby spot  I n an experiment u n r e l a t e d t o the s u b j e c t o f t h e s i s , and p r i o r t o t h e r e s u l t s d e s c r i b e d i n t h e  this fore-  g o i n g , t h e f o l l o w i n g c r o s s e s w e r e made t o s y n t h e s i z e d o u b l e m u t a n t p_  r  rji£_ f rom p_  r  p  l  P /p r  r  the  and r u s : x  rus/rus  selfed  S i n c e p_  r  ^2  r u s / r u s progeny were and s e l f e d  F^  p e a r l progeny were  i s epistatic  t o r u s , i t was  f o r r u s h o m o z y g o t e s (F2) f o r p." . 1  I n t h i s way  r  of these crosses.  selected  necessary to  one g e n e r a t i o n p r i o r  all  t o be homozygous f o r p_  selected  p e a r l progeny  rus.  to  select selecting  i n t h e F^  ought  T a b l e X I I . shows t h e  The F-j_ p r o g e n y w e r e d i s c a r d e d  results  after  a f e w d a y s o f m a t i n g , h e n c e no d a t a i s a v a i l a b l e f r o m them. The F  2  p r o g e n y w e r e c o u n t e d b u t n o t s e x e d , h o w e v e r i t was  n o t e d t h a t b o t h sexes were p r e s e n t i n each o f the t h r e e phenotypes. typic  T h i s g e n e r a t i o n gave the u n e x p e c t e d  r a t i o of 9 pearl:  6 r u b y s p o t : 1 W.T.,  pheno-  instead  of  TABLE XII THE F  x  AND F  PROGENY EROM p'/p x rus/rus AND THE DISTRIBUTION 1  2  PROGENY FROM THE SELF OF THE F  OF RIBOFLAVIN IN THE  generation  i  F  F  2  phenotype  wild  males  ruby spot pearl  *  11 73 118  wild pearl  3  females  *  ruby spot  F  0  361 88  * Data unavailable. ** Progeny were not sexed.  373 102  total  *  rus/rus  expected number ratio chromatographed  -  11 73 118  k  73^ 190  -  9 3  20 20  number witl riboflavin  -  -  -  20 20  4-Ct h e e x p e c t e d 9 W.T.:  3 ruby spot: h p e a r l .  e f f o r t t o e x p l a i n the overabundance t h e r u s s t o c k c u l t u r e was  checked  I n an  of p e a r l  progeny  for possible  c o n t a m i n a t i o n w i t h p e a r l , b u t no h o m o z y g o t e s w e r e f o u n d among 209  adults.  However, t h i s does n o t  exclude the p o s s i b i l i t y  o f p e a r l genes e x i s t i n g  heterozygous combination. and t e n f e m a l e p e a r l and chromatographed All  I n t h e F^ p r o g e n y  i n d i v i d u a l s were c h e c k e d .  b e h a v e as p_ r u s w h i c h was S i n c e i n s e c t i o n C. r  produced  phene a p p e a r e d  riboflavin.  I n the next g e n e r a t i o n an  o f t h e r i b o f l a v i n p h e n e , "p_  w i t h p_  t e n male  t o check f o r the presence of  f o u n d t o be p o s i t i v e f o r r i b o f l a v i n . terms  in  ruby spot a d u l t s were  possessed high l e v e l s .  a d d i t i o n a l 100  entirely  A l l were  Apparently i n  r u s " now  r  appeared  to  imported from B e r k e l e y .  i t was  shown t h a t p_ r u s c r o s s e d  a l l W.T.  progeny  i n h a l f of the F  2  o f i n t e r e s t t o knox^ t h e p h e n o t y p e  and  the  riboflavinless  p e a r l progeny, of the  i t was  following  crosses-,  The  1.  "p /p  2.  "p /p r  rus/rus" x r  p /p r  r u s / r u s " x p/p r u s / r u s  r e s u l t s o f t h e s e c r o s s e s a r e shown i n T a b l e Contrary to expectation a l l F  1. w e r e W.T.  progeny  and p o s i t i v e f o r r i b o f l a v i n .  XIII.  from c r o s s  The  progeny  TABLE XIII THE PHENOTYPE AND DISTRIBUTION OF RIBOFLAVIN OF THE F  x  PROGENY* FROM THE CROSSES r  rus/rus x p / p  AND "p /p  r  rus/rus" x p/p rus/rus  r  r  cross  phenotype  1.  wild  (1.)  "p /p  males  r  r  (2.)  number chromatographed  number with riboflavin  females  total  263  50k  100  100  —  _  _  159  159  159  pearl  2.  wild  -  —  pearl  82  77  * Progeny from reciprocal crosses were pooled as they were not appreciably different.  «*2  f r o m c r o s s 2. h o w e v e r , w e r e a l l p e a r l and had  riboflavin.  The u n u s u a l r e s u l t s o f t h e s e e x p e r i m e n t s t h a t t h e r e may  be g e n e t i c f a c t o r s o p e r a t i n g r e m i n i s c e n t  o f a c o n v e r s i o n - t y p e system 1966  indicated  ), which cannot  such as p a r a m u t a t i o n  (Coe,  a t t h e p r e s e n t and from t h e s e  results  a l o n e be e x p l a i n e d .  E.  P a r t i a l analysis of a possible t h i r d a l l e l e ;  (1963)  Sokoloff which W.T.  He f o u n d  at Slough,  , after  s  i n the Berkeley  i t t o be a l l e l i c  d e s c r i b e d b y Graham (1957 designated p  s  r e p o r t e d another p e a r l mutant  occurred spontaneously strain.  p_  )•  synthetic  t o the p e a r l  T h i s new p e a r l  he  the pest i n f e s t a t i o n l a b o r a t o r y  England.  T h i s p e a r l m u t a n t o n l y r e c e n t l y became a v a i l a b l e t o t h i s l a b o r a t o r y , c o n s e q u e n t l y i t s a n a l y s i s was H o w e v e r , i t was  thought  limited  of i n t e r e s t t o i n c l u d e here  what d a t a i s a v a i l a b l e . The eye p h e n o t y p e o f p_  s  f r o m e i t h e r p_  r  o r p_.  i s indistinguishable  The r i b o f l a v i n c o n t e n t o f  day o l d male and female  imagoes were  visually  10  ^3 estimated  f r o m t h e c h r o m a t o g r a m s t o be a b o u t t h e  same a s t h e W.T.  (Table I I . ) .  The f o l l o w i n g  c r o s s e s were made i n o r d e r  to test  p_ a g a i n s t p_ a n d p_ : s  r  C r o s s 1. and as  1.  p /p  s  x  p /p  2.  p /p  s  x  p/p  p r o d u c e d 137  positive  S  s  r  rus/rus  F-^ p r o g e n y ; t h e y were a l l W.T.  f o rriboflavin.  C r o s s 2.  i t o n l y produced three progeny.  that a mite  r  largely  failed  I t i s suspected  i n f e c t i o n may have b e e n t h e c a u s e .  The  t h r e e p r o g e n y , h o w e v e r , were a l l p h e n o t y p i c a l l y p e a r l and  riboflavin  positive.  The r e s u l t s not  homoalleles.  o f c r o s s 1.  show t h a t p  The r e s u l t s  o f c r o s s 2.  p_ a n d J J a r e a l l e l i c , s  (Ackermann,  1966).  r  a n d p_ a r e s  indicate  w h i c h has a l r e a d y b e e n shown  that  F.  Distribution  and d e v e l o p m e n t a l v a r i a t i o n  of  riboflavin 1.  Distribution  of r i b o f l a v i n (1939)j  Based on t h e r e p o r t o f D r i l h o n and B u s n e l who  demonstrated the presence of r i b o f l a v i n  concentrations  i n the Malpighian  tubules  i n high  o f many  t h e pupae and a d u l t s w e r e d i s s e c t e d i n i n s e c t and  the Malpighian  p_ , p_ a n d W.T. r  s  values  Ringer  t u b u l e s were c h r o m a t o g r a p h e d  f r o m t h e r e s t o f t h e body.  The r e s u l t s  obtained  s  I n W.T. and  These  t o weigh.  pupae a n d a d u l t s was i n t h e M a l p i g h i a n the r i b o f l a v i n  content  in  W.T.  tubules.  of the Malpighian  tubules  t h e b o d y i n c r e a s e d p r o p o r t i o n a l l y f r o m pupae t o  a d u l t s , being respectively. content  a b o u t 35% and 15%  of the t o t a l i n both  I n p_ t h e t o t a l i n c r e a s e i n r i b o f l a v i n s  f r o m pupae t o a d u l t s was m o s t l y  i n the Malpighian only differed the Malpighian  tubules.  i n the l e v e l  same.  tubules  due t o i n c r e a s e  Thus t h e two g e n o t y p e s of r i b o f l a v i n present i n  tubules, while the l e v e l  when t h e M a l p i g h i a n the  from  were n o t c o r r e c t e d f o r w e i g h t as t h e M a l p i g h i a n  The g r e a t e s t c o n c e n t r a t i o n o f r i b o f l a v i n p  separately  c o n t r o l are given i n Table XIV.  t u b u l e s were d i f f i c u l t  and  insects,  i n t h e body  a r e r e m o v e d was  essentially  TABLE X I V DISTRIBUTION OF RIBOFLAVIN W.T.  lines  number assayed  source  W.T.  10  Malpighian  p  I N u g * I N THE PUPAE AND ADULTS OF s  tubule  body  E  S  10  Malpighian  tubule  body  10  Malpighian body  AND p_  tubule  r  LINES  pupae + S.E.  a d u l t s + S.E.  .106  .009  .136  .oih  .019  .002  .025  .003  .091  .007  .192  .Olh  .020  .003  .023  .003  .011  .002  .003  .0006  .029  .002  .006  .0006  * T h e s e v a l u e s were n o t c o r r e c t e d f o r w e i g h t  i+6 The p a t t e r n o f d i s t r i b u t i o n t h e p_ and  l i n e was c o m p l e t e l y  p_ . s  of r i b o f l a v i n i n  r e v e r s e d c o m p a r e d t o W.T.  The M a l p i g h i a n t u b u l e s o f t h e pupae  contained  o n l y 2Q% o f t h e t o t a l r i b o f l a v i n a l t h o u g h t h e c o n c e n t ration  i n t h e b o d y was s l i g h t l y h i g h e r c o m p a r e d t o W.T.  and £  I n t h e M a l p i g h i a n t u b u l e s and body o f a d u l t s ,  h o w e v e r , t h e c o n c e n t r a t i o n o f r i b o f l a v i n was to near  2.  reduced  zero.  Developmental v a r i a t i o n  of r i b o f l a v i n  Developmental d i f f e r e n c e s i n r i b o f l a v i n concentration among m u t a n t l i n e s  o f E p h e s t i a h a v e b e e n shown b y  C a s p a r i and Blomstrand  (1958).  Visually  detectable  d i f f e r e n c e s i n t h e amount o f r i b o f l a v i n among m u t a n t lines this  i n a d u l t T r i b o l i u m h a v e a l r e a d y b e e n shown i n thesis  (Table I I . ) .  The f o l l o w i n g methods were u s e d t o e x a m i n e a n d compare  the developmental  stages  o f p_ a n d p_ r  s  t o W.T.  F o u r s a m p l e s e a c h c o n t a i n i n g 50 eggs o n t h e b a s i s o f e q u i v a l e n c e t o a v e r a g e i m a g o n a l body w e i g h t , assayed  forriboflavin.  distinguished after  S i n c e t h e sexes  c a n o n l y be  p u p a t i o n , a mean f o r t h e l a r v a e  was o b t a i n e d f r o m t e n i n d i v i d u a l Separate  were  determinations  determinations.  based on t e n i n d i v i d u a l s  of  h7 e a c h s e x were o b t a i n e d f r o m t h e e a r l y and l a t e and  t h e a d u l t s , 1 d a y , 10 d a y s and 30 d a y s  eclosion. standard  The means o f t h e d e v e l o p m e n t a l e r r o r s a r e g i v e n i n T a b l e XV.  better visualize  the developmental  t h e means a r e g r a p h e d i n F i g u r e The  eggs o f p_ a n d W.T.  pupae  after stages and  I n order t o  and s e x d i f f e r e n c e s  3»  contained very nearly the  s  same amount o f r i b o f l a v i n b u t w e r e  significantly  d i f f e r e n t f r o m t h e eggs o f p_ i n w h i c h r i b o f l a v i n  was  r  v e r y much r e d u c e d .  I n t h e p_ a n d p_ r  s  genotypes  r i b o f l a v i n was a c c u m u l a t e d d u r i n g t h e l a r v a l at  a b o u t t h e same r a t e .  I n t h e W.T.  stages  a slight  reduction  d u r i n g t h e l a r v a l s t a g e was f o l l o w e d b y a r a p i d  rise  which took place during the short time  prior  to  their  interval  pupation. In  the main the pupal  stages o f a l l genotypes  showed a g e n e r a l r e d u c t i o n i n r i b o f l a v i n c o n c e n t r a t i o n . S i n c e t h e T r i b o l i u m do n o t e a t d u r i n g t h i s d e c l i n e agrees  stage,  this  w i t h t h e f i n d i n g s o f F r a e n k e l and B l e w e t t  (194-7) t h a t T r i b o l i u m c a n n o t s y n t h e s i z e r i b o f l a v i n . The  slight  riboflavin  increase i n the p  s  female  pupae  was n o t s i g n i f i c a n t . I n b o t h W.T. of r i b o f l a v i n  than  a n d p_ t h e m a l e s h a d h i g h e r s  the females.  The g r e a t e s t  values sexual  TABLE XV DEVELOPMENTAL VARIATION OF RIBOFLAVIN IN W.T., p_s AND p_r LINES MEASURED IN ug/mg FRESH WEIGHT  lines W.T. eggs* last instar larvae  i  S.E.  p_ ± S.E. s  p  r  ± S.E.  .CM-  .002**  .04,1  .001  .014-  .002  .0^3  .004-  .051  .003  .022  .002 -  e a r l y pupae  .059 .04-7  .004-*** .003  .051  .04-2  .004.004-  .017 .023  .002 .002  l a t e pupae  .054.04-7  .004.004-  .04-8 .CM  .00^ .005  .017 .023  .002 .002  day o l d a d u l t s  .078 .065  .008 .005  .050 .04-5  .004-  .004-  .015 .019  .002 .003  10 day o l d adults  .110 .078  .093 .081  .004.002  .005 .006  .0005 .0004-  30  .131 .101  .004.004.007 .007  .109 .096  .008 .006  .005 .006  .0007 .0007  day o l d adults  * Four d e t e r m i n a t i o n s , each of 50 eggs. ** Standard e r r o r f o r t e n observations each. ***Upper values r e f e r to males, lower ones to females.  ^9  a  b c d e age ( d a y s )  f .  g  FIGURE  3.  DEVELOPMENTAL V A R I A T I O N OF R I B O F L A V I N  p_ ,  AND  V/.T. M A L E S AND- F E M A L E S .  r  p  S  STAGES A R E : a, EGGS; d, L A T E PUPAE; OLD  ADULTS  e, D A Y  b,  LAST  OLD,  THE  DEVELOPMENTAL  INSTAR LARVAE;  f , 10  DAY  IN  c,  O L D , g , 30  EARLYJ DAY  50 d i m o r p h i s m was n o t e d i n t h e 10 .001)  P <  o f W.T.  day o l d a d u l t s  and t h e 30 d a y o l d a d u l t s I n p_  s  significant  o n l y t h e 10  (t =  ( t = 3.03,  day o l d a d u l t s  P^==  .01)  showed  sexual dimorphism f o r r i b o f l a v i n  2.86,  (t =  .02).  P==  The p o s t - p u p a l was  s l i g h t l y delayed  increase  i n the l e v e l  of  i n p_ c o m p a r e d t o li.T, s  riboflavin I n p_ t h e r  downward t r e n d o f t h e p u p a l s t a g e was p r e s e r v e d 10  5.71,  until  days a f t e r e c l o s i o n , f o l l o w i n g t h a t o n l y t r a c e s o f  riboflavin  remained a t a constant  level.  p r e v i o u s l y noted, such low c o n c e n t r a t i o n s  However, as of  riboflavin  were measured a t t h e l i m i t s o f s e n s i t i v i t y o f t h e fluorometer.  Thus a g a i n  the developmental e f f e c t s of  p_ w e r e m a r k e d l y d i f f e r e n t f r o m t h o s e o f p_ r  while two.  s  and  W.T.,  o n l y s m a l l e r d i f f e r e n c e s e x i s t e d between t h e l a t t e r  51 G. and  Genetic  differences i n riboflavin  c o l o r a t i o n of M a l p i g h i a n Genetic  concentration  tubules.  d i f f e r e n c e s f o r c o n c e n t r a t i o n of  i n E p h e s t i a h a v e b e e n shown by C a s p a r i and  (1958)  and  Kikkawa  i n D r o s o p h i l a by B e a d l e  (1953)  Malpighian  (1937)  riboflavin  Blomstrand and  Hubby  a t t r i b u t e d changes i n the c o l o r a t i o n o f  t u b u l e s i n .Bombyx t o t h e p l e i o t r o p i c  o f g e n e s c a u s i n g m u t a n t eye T h i s t h e s i s has  effects  c o l o r pigments.  a l r e a d y shown t h a t  visually  d e t e c t a b l e d i f f e r e n c e s e x i s t e d i n t h e amount o f between mutant l i n e s  (Table I I . ) .  These  d i f f e r e n c e s were q u a n t i t a t e d i n n i n e lines  (EL d r e c a s , EL r u s . p l o d . p_? S  r , dp_e  ) and  i n two  W.T.  a l s o examined.  The  eye 1  c o l o r mutant rus,  ( C F I - 1 and  of the M a l p i g h i a n  Malpighian  tubules.  and W.T.  b l a c k i n r and inbred  The  tubules of non-pearl  r a n g e d f r o m b r o w n i n dpe  and  eye  pigmentation  tubules XVI.  had of  the  c o l o r mutants  es, through  speckled  r u s , to pronounced b l a c k i n the  brown two  lines.  With the e x c e p t i o n of p , r  r a t i o n was  CFI-7).  r e s u l t s are given i n Table  I n d i v i d u a l s of the p e a r l phenotype always colorless Malpighian  riboflavin  visual  EL "5 e s ,  inbred lines  Concurrently the pigmentation was  (1962).  always higher  the r i b o f l a v i n  concent-  i n the males than i n the  52 TABLE X V I THE MEANS* OF R I B O F L A V I N CONCENTRATION I N ug/mg FRESH WEIGHT FOR MALES AND FEMALES OF 11 (THEIR AVERAGE COMPARED BY DUNCAN'S NEW TEST) At©  THE COLOR OF MALPIGHIAN  LINES MULTIPLE RANGE  TUBULES  line  males  females  average  color of Malpighian tubules  p_ d r e c a s  .133  .097  .H5 **  colorless  p_ r u s  .105  .085  .095  b  colorless  p_ l o d  .112  .086  .099  b  colorless  p_  .093  .081  .087  b  colorless  ^ r ***  .005  .006  .006  es  .098  .081  .089  b  brown  rus  .083  .074-  .078  b  speckled  r  .059  .04-7  .053  d  s p e c k l e d brown to black  dpe  .051  .0*+2  ,04-6  d  CFI-1  .110  .078  .09^  black  CFI-7  .077  .054-  ,065  black  s  .  .  a  colorless  c  black  brown  * Means a r e b a s e d on t e n i n d i v i d u a l s p e r s e x . ** A n y two means h a v i n g t h e same l e t t e r a r e n o t s i g n i f i c a n t l y d i f f e r e n t a t P £ ; .05. *** N o t i n c l u d e d i n a n a l y s i s o f v a r i a n c e ; i t s l o w r i b o f l a v i n c o n c e n t r a t i o n has been d e m o n s t r a t e d .  53 females.  The p_ f e m a l e s h a d s l i g h t l y more r i b o f l a v i n r  t h a n t h e males, a l t h o u g h as d i s c u s s e d v a l u e s must be v i e w e d w i t h The  some  earlier,  caution.  r i b o f l a v i n c o n t e n t o f n o n - p e a r l eye c o l o r  m u t a n t s was r o u g h l y p r o p o r t i o n a l  t o the degree o f  p i g m e n t a t i o n o f t h e eyes and t h e M a l p i g h i a n This  these  tubules.  r e l a t i o n s h i p d i d n o t e x i s t i n t h e p e a r l eyed mutant  lines. The  e x t e n t o f v a r i a t i o n between the l i n e s and  between t h e sexes, as w e l l as t h e p o s s i b i l i t y  of a  l i n e by s e x i n t e r a c t i o n was d e t e r m i n e d v i a t h e a n a l y s i s of variance. of variance  The p_ l i n e was e x c l u d e d f r o m t h e a n a l y s i s r  as i t a p p e a r e d t o have no r e l i a b l y  measurable  amounts o f r i b o f l a v i n and i t s s i g n i f i c a n t d i f f e r e n c e the  r e s t o f t h e l i n e s has b e e n d e m o n s t r a t e d .  results of the analysis of variance XVII. lines.  Highly  i s given  significant differences  existed  from  The i n Table between  The d i f f e r e n c e b e t w e e n t h e s e x e s was a l s o  highly  s i g n i f i c a n t , a n d t h e r e was no i n d i c a t i o n o f a l i n e b y sex i n t e r a c t i o n . D u n c a n ' s New M u l t i p l e Range T e s t was u s e d t o demonstrate the extent of l i n e d i f f e r e n c e s . are  shown i n T a b l e X V I .  letter  The r e s u l t s  A n y two means h a v i n g t h e same  are not s i g n i f i c a n t l y d i f f e r e n t a t the p r o b a b i l i t y  l e v e l o f .05.  With the exception  o f t h e p_ d r e c a s l i n e  9+  TABLE X V I I ANALYSIS OF VARIANCE OF TOTAL  RIBOFLAVIN  I N ug/mg FRESH WEIGHT I N 10  L I N E S OF T.  confusum  source of v a r i a n c e  d.f.  mean s q u a r e s  between  lines  9  .00868**  between  sexes  1  .01802**  9  .000M+  i n d i v i d u a l s / l i n e x sex  180  .00031  total  199  l i n e x sex  ** H i g h l y s i g n i f i c a n t  (P  ^.01)  55 t h e means o f t h e - r i b o f l a v i n p o s i t i v e p e a r l l i n e s not  significantly different  from each o t h e r and f r o m  t h e means o f e s , r u s a n d C F I - 1 . to  note  I t was o f i n t e r e s t  t h a t t h e means o f t h e two i n b r e d l i n e s ,  originated  f r o m t h e same p a r e n t s , w e r e  different.  Genetic  were  which  significantly  d i f f e r e n c e s between, as w e l l as  i t f i t h i n h i g h l y i n b r e d l i n e s , have b e e n shown b e f o r e for  red blood c e l l  (Briles,  H.  antigens  i n t h e domestic  fowl  19^9).  Estimation of heritability Prior  to conducting  of the r i b o f l a v i n  trait.  g e n e t i c s t u d i e s on p o p u l a t i o n s  of a species, p a r t i c u l a r l y w i t h respect t o a s i n g l e q u a n t i t a t i v e c h a r a c t e r , i t i s important extent of d i f f e r e n t (Lerner, 1958). individual's weight  sources  Therefore,  of phenotypic  variation  t h e r e l a t i o n s h i p between an  t o t a l r i b o f l a v i n content  was a s s e s s e d .  t o know o f t h e  a n d i t s body  I n a d d i t i o n the h e r i t a b i l i t y of  t h e r i b o f l a v i n t r a i t was u s e d t o d e t e r m i n e  the pro-  p o r t i o n o f t h e t o t a l v a r i a n c e t h a t was a t t r i b u t a b l e t o t h e a v e r a g e e f f e c t s o f t h e genes w h i c h i n f l u e n c e d t h i s trait.  56 The  simple c o r r e l a t i o n  individual  coefficient  ( r ) between  b o d y w e i g h t a n d r i b o f l a v i n c o n t e n t was  a s s e s s e d u s i n g 90 m a l e s a n d 90 f e m a l e s .  The m a l e a n d  female r v a l u e s as w e l l as t h e c o e f f i c i e n t o f determination  o  a r e shown i n T a b l e  XVIII.  TABLE X V I I I CORRELATION OF 10 DAY ADULT WEIGHT WITH R I B O F L A V I N CONCENTRATION I N THE p_ l o d MARKED STOCK  females  males simple c o r r e l a t i o n coefficient  1  coefficient-'- ( r )  • 32**  of determination ( r ) 2  89 d e g r e e s  10%  23^  o f freedom.  ** H i g h l y s i g n i f i c a n t  (P -S .01)  Only a s m a l l percentage o f the v a r i a b i l i t y o f r i b o f l a v i n c o n c e n t r a t i o n c a n be a c c o u n t e d variability females). exists  i n body w e i g h t  ( r  = 10$ i n m a l e s ;  However, a h i g h l y s i g n i f i c a n t  between t h e t r a i t s  f o r by t h e 23$ i n  association  f o r both sexes.  Consequently  57 t h e measurement o f r i b o f l a v i n on a p e r body w e i g h t b a s i s was  n o t i m p e r a t i v e f o r m a l e s , b u t p r o v i d e s more  accurate data i n the case of females. The  p r o g e n y o f p_ l o d (random b r e d f o r o v e r  g e n e r a t i o n s ) were u s e d t o e s t i m a t e t h e of the r i b o f l a v i n t r a i t . mated t o f o u r f e m a l e s . w e r e removed  A f t e r f i v e d a y s , t h e males  f r e s h medium.  t h e f e m a l e s were removed  Mere  W h i l e i t was  number o f s i r e s t e s t e d was  distributed  A f t e r 10  and d i s c a r d e d .  g r o u p s e a c h h a v i n g t h r e e dams successful matings.  heritability  T h i r t e e n males were each  and t h e f e m a l e s w e r e e a c h  into vials containing  36  days  Three  sire  chosen from the  realized  that the  q u i t e l o w , i t was  assumed  that the h e r i t a b i l i t y estimates subsequently obtained w o u l d n o t be u n d u l y a f f e c t e d .  Ten days a f t e r  the progeny were i n d i v i d u a l l y weighed, a n d r i b o f l a v i n was m e a s u r e d  eclosion  chromatographed  by t h e f l u o r o m e t r i c  method.  Ten m a l e and t e n f e m a l e p r o g e n y f r o m e a c h s i r e / d a m mating were a n a l y z e d .  (1956;  a f t e r Becker,  analysis.  The  196*+)  g e n e r a l method o f was  used f o r the  Standard errors of h e r i t a b i l i t y  Cockerham statistical  estimates  w e r e c a l c u l a t e d u s i n g t h e method o f D i c k e r s o n  (i960).  58  The s t a t i s t i c a l model used was as f o l l o w s ; Y  where  ;  ijk  =  u  +  s  i  +  d  i j  +  e  ijk  : t h e r e c o r d o f the k^* progeny o f t h e j ^ * 1  1  dam mated t o t h e i ^ * 1 s i r e ; u : t h e common mean; s^ : the e f f e c t o f t h e i j  t  h  t  h  s i r e ; d.^  dam mated t o t h e 1^  : the e f f e c t of the  s i r e , and ej j  u n e x p l a i n e d e n v i r o n m e n t a l and g e n e t i c  k  : the  deviations  a t t r i b u t a b l e t o t h e k t h progeny from t h e j ^ * mated t o t h e 1^  1  dam  sire.  The average e f f e c t s o f t h e r i b o f l a v i n t r a i t f o r ' t h e s i r e s , t h e dams and t h e sexes,- c a n be a s s e s s e d from t h e means w h i c h a r e p r e s e n t e d i n T a b l e X I X . The means o f t h e male progeny were c o n s i s t e n t l y h i g h e r t h a n t h o s e o f t h e female, progeny.  The average  c o n t r i b u t i o n t o t h e progeny was the same f o r s i r e A and s i r e B.  W i t h t h e e x c e p t i o n o f t h e progeny from  the s i r e A - dam 1 f a m i l y , the progeny o f t h e dams w i t h i n s i r e G had on t h e average t h e l o w e s t r i b o f l a v i n values.  Hence t h e progeny o f s i r e C were a l s o much  lower compared  t o the progeny o f s i r e s A and B.  To determine whether the s e x u a l dimorphism e x h i b i t e d by t h e means was s t a t i s t i c a l l y s i g n i f i c a n t , the a n a l y s i s o f v a r i a n c e was performed (Table X X . ) .  TABLE XIX MEANS OF TOTAL RIBOFLAVIN IN ug/mg FRESH WEIGHT OF THE PROGENY FROM THE MATING OF THREE DAMS WITHIN EACH OF THREE SIRES  sire A dams  sex  1  2  3  sire B  sire mean  1  2  3  sire C  sire mean  1  2  3  sire mean  progeny  M*  .108 .127 .14-7  .127  .111  .115 .13^  .120  .091 .093 .105 .096  mean  F  .072 .098 .096  .089  .085  .108 .096  .096  .062 . 0 7 0 .089  dam mean  .090  .098  .112 .121  sire mean  .108  .074-  .076 . 0 8 1 .097  .111 .115 .108  .085  * M = males, F = females. vn so  60 The  F-test  (P~.0l),  f o r t h e s e x e f f e c t was h i g h l y confirming  t h e above  significant  observations.  TABLE X X ANALYSIS OF VARIANCE BY SEX OF THE TOTAL R I B O F L A V I N I N ug/mg FRESH WEIGHT I N THE p/p  l o d / l o d MARKED L I N E  source o f variance  d.f.  sex  „1  mean s q u a r e s O.O3606**  178  error  0.00061  179  total  ** P ^ . 0 1  Due t o t h e h i g h l y  significant  sexual  h e r i t a b i l i t y was e s t i m a t e d s e p a r a t e l y  f o r each s e x ,  as w e l l a s m a k i n g a c o m b i n e d e s t i m a t e . of variance  dimorphism  The a n a l y s i s  o f t h e male p r o g e n y , f e m a l e p r o g e n y ,  c o m b i n e d s e x e s a s w e l l a s t h e e x p e c t e d mean s q u a r e s are  presented i n Table XXI.  The F - t e s t  dam c o n t r i b u t i o n was h i g h l y  significant  three analyses.  showed t h e i n each o f the  The s i r e c o n t r i b u t i o n was n o t  TABLE X X I ANALYSIS OF VARIANCE AND EXPECTED MEAN SQUARES OF TOTAL RIBOFLAVIN IN ug/mg FRESH WEIGHT IN THE p/p l o d / l o d MARKED LINE  source of variance  mean squares female • progeny combined  d.f.  male progeny  sires  2  0.00785  dams/sires  6  0.00196** 0.00180** 0.00308**  <5 W + K <5 D  0.0004-3  <5 W  individuals/ dams/sires  81 A  total  89  0.00398  0.00034-  e x p e c t e d mean squares'  0.01070  0.00061  <3?W .+ K 2  B  1 When t h e sexes a r e a n a l y z e d s e p a r a t e l y , Kj_ = 10, K 2 = 30. When t h e sexes a r e a n a l y z e d t o g e t h e r , K-j_ = 20, K ** P ^ .01. A F o r t h e combined a n a l y s i s i t i s 171; and B, 179.  2  = 60.  2  2  2  x  2  (5 D + K <S S 2  x  62  s i g n i f i c a n t i n any o f the t h r e e a n a l y s e s .  The  g r e a t amount o f v a r i a t i o n c o n t r i b u t e d by the dams have masked the s i r e The  may  effect.  e s t i m a t e s o f the components o f v a r i a n c e are  presented i n Table X X I I . e s t i m a t e s £ <j£  The  s i r e component, tf£  (additive genetic variance) plus  decreasing f r a c t i o n s of higher order a d d i t i v e x additive epistatic interactions.  I n the absence o f  the l a t t e r e f f e c t s , i+&]r p r o v i d e s an e s t i m a t e o f the a d d i t i v e g e n e t i c v a r i a n c e .  The dam  component <s^  e s t i m a t e s ^6"% + £ d o m i n a n c e v a r i a n c e ) p l u s f r a c t i o n a l higher order e p i s t a t i c terms, plus ( v a r i a n c e due  to m a t e r n a l e f f e c t s ) .  Thus a g a i n  i g n o r i n g the e p i s t a t i c i n t e r a c t i o n s , + and  tj^-v,.  i n c l u d e s 6"%  ,  fc5« ( F a l c o n e r , i960).  F o r each a n a l y s i s the s i r e component of v a r i a n c e ( 6%)  c o n t r i b u t e d 25$  and 71%  i n the male, 13$  i n the female  i n the combined sexes towards the t o t a l v a r i a n c e  f o r the t r a i t .  S i n c e the mean squares from w h i c h  these were e s t i m a t e d were not s i g n i f i c a n t , the percentage v a l u e s i n d i c a t e d d i f f e r e n c e s t h a t were more e a s i l y r e s o l v a b l e i n terms o f the t o t a l v a r i a t i o n i n t h e t h r e e groups o f progeny.  The  high estimate  when the sexes were combined (71$)  was  obtained  p r o b a b l y due  to  TABLE XXII COMPONENTS OF VARIANCE ESTIMATED FROM THE ANALYSIS OF VARIANCE OF TOTAL RIBOFLAVIN IN THE p/p l o d / l o d MARKED  component  is  <SD 2  <5 T 2  LINE  combined value  male progeny value % total  female proeenv % total value  0.000196  25.16  0.000073  13.06  0.000615  71.10  0.000153  19.64-  0.00014-6  26.12  0.000123  14-.  0.0004-30  55.20  0.0003^0  60.82  0.000127  14-. 68  0.000779  100.00  0.000559  100.00  0.000865  100.00  %  total  22  the marked s e x u a l d i m o r p h i s m . dams c o n t r i b u t e d 20$, and  D i f f e r e n c e s among t h e  26$ a n d 1^$  i n t h e male,  combined a n a l y s e s , r e s p e c t i v e l y .  The g r e a t e r  u n i f o r m i t y o f t h e s e components i n d i c a t e d t h a t reliable  estimates  female  fairly  o f h e r i t a b i l i t y may be o b t a i n e d  f r o m them. Since e p i s t a t i c ignored without  i n t e r a c t i o n s c a n u s u a l l y be  i n t r o d u c i n g any s e r i o u s e r r o r ( F a l c o n e r  I960), a n e s t i m a t e o f h e r i t a b i l i t y i n t h e n a r r o w sense from t h e s i r e  component i s g i v e n by  (where &\ i s t h e t o t a l (Cockerham,  1956).  v a r i a n c e , o r ^s"*' » '^. 6  4  A s e c o n d e s t i m a t e , f r o m t h e dam  c o m p o n e n t , k<S /6' ± x>  phenotypic  M-CT^/^  r  s  b i a s e d by d o m i n a n c e a n d m a t e r n a l  e f f e c t s , a l t h o u g h a s d i s c u s s e d a b o v e i t may be more reliable  i n the present  experiment,  due t o t h e g r e a t e r  number o f dams t e s t e d . For each a n a l y s i s the estimates and  their  standard  combined sexes  e r r o r s f o r t h e male, female  a r e given i n Table X X I I I .  the h e r i t a b i l i t y were a n a l y s e d  of h e r i t a b i l i t y  separately, although  dam c o m p o n e n t s : 0.59  Maternal  In general  e s t i m a t e s were h i g h e r when t h e s e x e s t h e combined sex  a n a l y s i s gave more c o m p a r a b l e e s t i m a t e s and  and t h e  a n d 0.57  f o r the sire  respectively.  a n d d o m i n a n c e e f f e c t s i n t h e dam component  TABLE X X I I I ESTIMATES OF H E R I T A B I L I T I E S AND STANDARD ERRORS OF THE RIBOFLAVIN  TRAIT I N THE p/p l o d / l o d MARKED  component  male progeny  sire  1.00*  dam  O.78 + 0.20  s i r e + dam  O.89  * E s t i m a t e s g r e a t e r t h a n 1.00  LINE  h e r i t a b i l l t v ± standard error female progeny combined  + 1.35  + 0.06  were assumed  0.5l  0.59  + 0.34-  +  O.83  1.00 + 0.26  0.57 1 0.4-1  0.77  0.58  + 0.31  t o be e q u a l t o  +  O.63  1.00  ON  66 probably caused the estimate to  be e l e v a t e d .  riboflavin  from t h e female  progeny  The c o n s i s t e n t l y h i g h e r l e v e l s o f  i n t h e m a l e s a n d t h e l o w number o f s i r e s  used provides a p o s s i b l e e x p l a n a t i o n f o r the high heritability obtained  based on t h e s i r e  component w h i c h was  i n t h e a n a l y s i s o f t h e male p r o g e n y .  the h e r i t a b i l i t y  e s t i m a t e s based on t h e s i r e  Since and t h e  dam c o m p o n e n t s c a n o n l y be c o n s i d e r e d e q u a l i n t h e combined a n a l y s i s , i t i s o n l y here t h a t t h e assumption of  (Becker,  cTj«  combining  196*+)  the h e r i t a b i l i t y  was met f o r v a l i d l y estimates  obtained  s e p a r a t e l y f r o m t h e s i r e s a n d t h e dams.  Therefore  from t h e combined a n a l y s i s t h e h e r i t a b i l i t y o f 0 . 5 8 may be t a k e n a s t h e b e s t  estimate  estimate.  Broadly i n t e r p r e t e d the h e r i t a b i l i t y  estimates  i n t h e male p r o g e n y i n d i c a t e d m o s t l y a d d i t i v e v a r i a n c e , whereas t h e h e r i t a b i l i t y female  estimates  genetic i nthe  progeny i n d i c a t e d a d d i t i v e g e n e t i c v a r i a n c e as  w e l l as dominance e f f e c t s and p o s s i b l y m a t e r n a l The  heritability  estimates  effects.  o b t a i n e d from t h e combined  a n a l y s i s w e r e e s s e n t i a l l y t h e same a n d t h e r e was no evidence A valid sexes  f o r dominance e f f e c t s o r m a t e r n a l comparison of the h e r i t a b i l i t i e s  may be d i f f i c u l t  effects.  between t h e  t o i n t e r p r e t on a c c o u n t  extensive sexual dimorphism f o r t h i s  trait.  of the  DISCUSSION I t has  b e e n assumed i n t h i s  thesis that  yellow f l u o r e s c e n t substance separated was  largely riboflavin.  identification and  chromatography  T h i s a s s u m p t i o n was  v i a spectrophotometric,  based  on  chromatographic  e l e c t r o p h o r e t i c methods. All  for  by  the  developmental stages  a r e c e s s i v e p e a r l eye  called  p_, b u t  significant,  o f i n d i v i d u a l s homozygous  c o l o r g e n e , p_  r  (formerly  renamed) w e r e a s s o c i a t e d w i t h  heritable reduction i n riboflavin  i n t h e b o d y and  i n the M a l p i g h i a n  d i f f e r e n c e i n r i b o f l a v i n content  tubules. o f W.T.  t h e c h r o m a t o g r a p h i c method was  t h a t i t may  regarded  be  c r o s s b e t w e e n p_  r  d i s t o r t e d 3>1  ratio  this  e f f e c t was  t h e p_ g e n e , i t may fitness  was  The  be  p_  and W.T. 2  progeny.  This  so  large  suggested r  gene.  observed i n crosses i n v o l v i n g  p o s t u l a t e d t h a t the r e d u c t i o n i n  a s s o c i a t e d w i t h the r i b o f l a v i n l e s s  g e n e s , £ and  adults  gave a g r e a t l y  a s s o c i a t e d w i t h t h e p_ not  r  real.  i n the F  a reduction i n fitness Since  as  content  The  and  e s t a b l i s h e d by  The  a  p_  s  a p p e a r e d t o be  as n o t e d by A c k e r m a n n ( 1 9 6 6 ) .  The  phene.  identical  complementation  alleles  68 d e m o n s t r a t e d b e t w e e n p_ n d p_, a n d b e t w e e n £ r  a n d p_  r  s  a  was an  o n l y s u r p r i s i n g i n s o f a r as i t appeared t o c o n t r a d i c t earlier  t e c h n i c a l note by S o k o l o f f i n T r i b o l i u m  Information be  B u l l e t i n , v o l . 6,  t h a t S o k o l o f f was u s i n g  1963.  o n l y p_ a n d £ , s  unaware o f t h e e x i s t e n c e o f t h e p of d i f f e r e n t  H o w e v e r , i t may  r  line.  environments c o u l d provide  alternative explanation.  being The e f f e c t  a possible  Environment dependent  c o m p l e m e n t a t i o n has been d e m o n s t r a t e d i n m i c r o o r g a n i s m s ( G a r e n a n d G a r e n , 1963; Fincham and S t a d l e r ,  1965; F a n , e t a l . , 1966).  r e a r i n g c o n d i t i o n s o f S o k o l o f f w e r e 30° r e l a t i v e humidity humidity was  i n this  1963;  S c h l e s i n g e r and L e v i n t h a l ,  c o m p a r e d t o 32° investigation.  The  C a n d 70%  C a n d 73%  relative  In addition his  enriched w i t h brewer's y e a s t , r a t h e r than  flour  Torula  yeast. F u n d a m e n t a l l y two d i f f e r e n t t y p e s  o f complementation  may be d i s t i n g u i s h e d ( S c h l e s i n g e r a n d L e v i n t h a l , Firstly,  1963).  i f t h e two m u t a t i o n s a r e i n f u n c t i o n a l l y  d i f f e r e n t regions then they w i l l  o f t h e g e n e t i c map  (inter-cistronic)  a l w a y s complement ( B e n z e r ,  s i t u a t i o n c a n be e x p l a i n e d  1957)•  i f i t i s assumed t h a t  c i s t r o n determines the s t r u c t u r e o f a separate  This each  polypeptide  69 chain.  The "normal" effect would be produced i f each  polypeptide chain was made i n i t s normal configuration by at least one of the two chromosomes.  Complete  complementation would then be expected i f the different polypeptide chains were part of a single enzyme or determined different enzymes i n the same biochemical pathway.  In the second type of complementation  ( i n t r a - c i s t r o n i c ) the two mutations are i n the same c i s t r o n and affect  the same polypeptide chain.  In this  case, the "normal" effect would only be produced beWeen some pairs of mutants.  On the basis of the data presented  here for the p_r gene versus the p_ and p_s genes, i t  is  not possible to d i s t i n g u i s h between i n t e r - and i n t r a c i s t r o n i c complementation.  However, i t was c e r t a i n that  p_r was complementary to p_ and p_s. Brink (1956) demonstrated that regular heritable changes i n the function of a gene can be directed by an allele.  In 1958 he designated this change as a  paramutation.  Paramutation denotes the heritable nature  of the change and the o r i g i n a t i o n through heterozygous association of a sensitive an inducing (paramutagenic)  (paramutable) allele..  recently reviewed by Coe ( 1 9 6 6 ) .  a l l e l e with  This subject was  The r e s u l t s ,  involving  the apparent conversion of p_r to p_ i n the synthesis of  70 the double mutant "p_r rus" are indicative of this type of behaviour.  Time did not permit repeating the lengthy  mating scheme to v e r i f y these r e s u l t s .  While  paramutation has not yet been demonstrated i n animals,  it  i s not unreasonable to assume i t e x i s t s . The relationship between the time of  disappearance  of r i b o f l a v i n i n jj r and the time of deposition of pigments i n the W.T. Malpighian tubules and eyes was of i n t e r e s t .  A relationship was suggested by the fact  that the time during which r i b o f l a v i n disappears corresponds approximately to the period during which pigment synthesis would normally take place.  Furthermore,  mutants p_r, p_ and p_s affect the amount of pigment i n the Malpighian tubules.  The p_r gene reduced the r i b o f l a v i n  content of the tubules, while p_ and p_s did not have the sarnie effect.  A similar s i t u a t i o n exists i n Ephestia  where the gene wa functions i n an analogous manner (Caspari, 1955). The pleiotropic effects of the gene causing the pearl eye and the colorless Malpighian tubules may be resolved by the data of Wolsky and Zamora (i960). They suggested that the dark pigment i n the eyes and Malpighian tubules of Latheticus oryzae, a c l o s e l y related species to Tribolium., are probably ommochromes.  71 The genetic control of the synthesis  of this group  of pigments has been partly documented i n Ephestia (Hadorn and Kuhn, 19535 Caspari, 1955; V i s c o n t i n i , al.,  et  1956; Reisener-Glasewald, 1956; Z i e g l e r , 196l)  and Bombyx (Kikkawa, 1953; Z i e g l e r , 196l).  In  addition i n nearly every case mutants affecting  the  amount of ommochrome pigment have also been shown to affect the amount of r i b o f l a v i n . The p o s s i b i l i t y that r i b o f l a v i n may be involved i n one of the steps leading to ommochrome formation, was "suggested by the experiments of Chareonnet-Harding,  et a l . , (1953) and by Mason (1953) with the r a t .  If  r i b o f l a v i n deficient rats are fed tryptophan, urinary excretion of anthranilic a c i d , kynurenic a c i d , xanthurenic acid and occassionally of kynurenin and 3-hydroxykynurenin i s observed.  These author suggested that r i b o f l a v i n  deficiency may affect the steps leading from kynurenin to 3-hydroxykynurenin. According to a summary by Gilmour (196l), these steps occur i n the chain of reactions leading to ommochrome pigment.  The r i b o f l a v i n  content and the degree of pigmentation of the Malpighian tubules and eyes appeared to be related i n the non-pearl eye color mutant l i n e s .  However i n the p_ versus the p_ r  and p_ l i n e s no such relationship was noted. s  72 The question arises whether the lack of r i b o f l a v i n i n the p_ adults was due to an i n h i b i t i o n r  of i t s formation, to i t s destruction or to an interference with i t s absorption.  N u t r i t i o n a l experiments have  shown that Tribolium confusum cannot survive i n the absence of r i b o f l a v i n , suggesting that they are unable to synthesize this vitamin (Fraenkel and Blewett, 194-7).  The chromatographic pattern of  fluorescent  substances of p r did not contain detectable  amounts  of the usual degradation products, lumiflavin and lumichrome.  Therefore i t was u n l i k e l y that these  products were formed i f r i b o f l a v i n was indeed degraded. However, the p o s s i b i l i t y of b a c t e r i a l degradation of r i b o f l a v i n to non-fluorescent compounds by a symbiotic bacteria,  such as Pseudomonas r i b o f l a v i n u s . so far  not demonstrated i n Tribolium. may exist 196'+).  (Harkness,  This p o s s i b i l i t y would have to be predicated on  an incompatibility system of P. riboflavinus with a l l other strains used. The p o s s i b i l i t y that the lack of r i b o f l a v i n storage i n the Malpighian tubules and the reduced amount of r i b o f l a v i n i n the body of p_ mutants may be due to r  an i n a b i l i t y to r e t a i n this vitamin.  This was suggested  by the findings of Cowan, et a l . , 1964- , 1966, 1966a) i n the domestic fowl.  The concentration-of  free  73 r i b o f l a v i n i n the blood of a mutant s t r a i n of chickens homozygous for r d , f a i l e d to increase as i t did i n normal chickens at the onset of egg production (Maw,  195^;  Boucher et a l . , 1959).  Isotopic  tracer  studies  have shown that free r i b o f l a v i n was absorbed normally from the digestive tract  (Cowan et a l . , 196^). It  thus appeared that the gene conditioned event which prevented accumulation of r i b o f l a v i n i n the blood must take place after  absorption.  The p o s s i b i l i t y that r i b o f l a v i n was  destroyed i n the tissues of the recessive hen was ruled out by subsequent evidence (Cowan et a l . ,  1966)  Recent data (Cowan et a l . , 1966a) showed that i n the mutant laying hen, the recessive gene expressed i t s e l f by a l t e r i n g the renal reabsorptive mechanism for free riboflavin.  As a result of this a l t e r a t i o n , the intact  vitamin was rapidly excreted v i a the urine, hence i t f a i l e d to accumulate i n the blood. In the estimation of h e r i t a b i l i t y of the amount of stored r i b o f l a v i n i n the p_ lod l i n e i t was assumed that the epistatic  contributions to variance were  r e l a t i v e l y unimportant (Falconer, I960).  As far as i t  can be determined the s t r a i n used for the study had been kept as a closed population for a number of years. As Mayr (1963) points out, epistatic  effects would be less  7h important and overdominant effects more important as sources of genetic v a r i a t i o n i n such populations.  In  natural populations which are continually subject to gene flow i n the form of migration, a greater amount of epistasis would interact i n many combinations.  In  closed populations there would be less need for such genes, and i n fact, as Dobzhansky and Spassky (1962) have shown, closed populations can evolve overdominance. The design used for the estimation of h e r i t a b i l i t y i s one of a number of methods available for this (see Falconer, i 9 6 0 ) .  purpose  The h i e r a r c h i c a l design (a  full  and h a l f - s i b relationship) provides a more accurate estimate of h e r i t a b i l i t y than the regression methods.  parent-offspring  In addition an i n d i c a t i o n of the  non-additive genetic variance and maternal effects can be noted. The differences  i n the h e r i t a b i l i t y estimates  based on the combined sexes from that of the separate estimates from the male and female progeny can not be resolved using the available genetic models. the significant differences  In general,  shown between the mutant  l i n e s and especially between the two W.T. inbred l i n e s and the high h e r i t a b i l i t y estimates suggest that the amount of r i b o f l a v i n present i n T. confusum is probably controlled by a small number of genes.  75 Unusual sex ratios were obtained i n many of the crosses i n that more females than males were produced. From a t o t a l of &%5l Tribolium, k-06l were males and ^390 were females.  This was a highly significant  deviation from the expected 1:1 r a t i o ( P ^ .005). There was no i n d i c a t i o n that the pearl eye mutation was a sex-linked character, nor "was there any i n d i c a t i o n that the r i b o f l a v i n l e s s divergent sex r a t i o .  t r a i t was associated with this  Similar sex-ratio differences  (in  pearl eyed stocks) i n favour of females were evident i n the data of Graham (1957) and were also noted i n T. castaneum by Park ( 1 9 3 7 ) .  The p o s s i b i l i t y of genetic  d i s t o r t i o n of the sex r a t i o , as reported for other insects i s not u n l i k e l y (Peacock and Erickson, 1965; Hickey and Craig, 1966).  CONCLUSIONS AND SUMMARY  The i d e n t i t y of a yellow fluorescent  substance  from Tribolium confusum, with r i b o f l a v i n was shown by the independent methods of  spectrophotometry,  chromatography and electrophoresis.  Two decomposition  products of r i b o f l a v i n as well as two conjugated  flavines  FMN and FAD, were i d e n t i f i e d . The genetic control of r i b o f l a v i n and i t s  relation-  ship to some mutant eye color genes i n the p l e i o t r o p i c hierarchy of gene effects was investigated  i n a series  of experiments involving more than 8000 Tribolium from 11 genetically different  lines.  A l l developmental  stages of individuals homozygous for a recessive pearl eye color gene, p_ (formerly c a l l e d p_, but renamed) r  were associated with a s i g n i f i c a n t , r i b o f l a v i n content i n the body.  heritable reduction i n  This reduction was  p r i n c i p a l l y i n the Malpighian tubules.  In a l l of the  other l i n e s tested the vitamin was accumulated i n the• tubules i n r e l a t i v e l y high concentrations.  The normal  a l l e l e of this gene was found to be incompletely dominant with respect to the r i b o f l a v i n phene.  Changing the  residual genotype of p_ by means of two generations r  recurrent selection did not detectably a l t e r i t s  of  expression.  77 Two a l l e l e s  of pearl (p_ and p_ ) were phenotypically s  i d e n t i c a l to p_ , except at the l e v e l of the r i b o f l a v i n r  phene.  Crosses of these a l l e l e s with p_ were shown to r  complement.  The r e s u l t s ,  involving the apparent  conversion of p_ to p_ i n the synthesis of the double r  mutant " p r rus" were indicative of a paramutation-type mechanism. Genetic differences  i n r i b o f l a v i n content and the  pigmentation of Malpighian tubules were shown.  The  r i b o f l a v i n content of non-pearl eye color mutants was roughly proportional to the degree of pigmentation of the eyes and the Malpighian tubules.  Individuals of  the pearl phenotype ( p r , p_ , p_) always had. colorless s  Malpighian tubules.  The depth of pigmentation of adult  eyes i n the mutant l i n e s was roughly proportional to the.degree of pigmentation of the Malpighian tubules. Sexual dimorphism for r i b o f l a v i n content was observed i n a l l l i n e s tested. parameters  The quantitative  of v a r i a t i o n of the r i b o f l a v i n phene were  examined and i t s h e r i t a b i l i t y was found to be high by two different comparisons.  In general an excess of  female progeny from a l l crosses was observed.  LITERATURE CITED Ackermann, M.E. 1 9 6 6 . Personal communication. Department of Genetics, University of C a l i f o r n i a , Berkeley. Association of O f f i c i a l A g r i c u l t u r a l Chemists. p. 658-660. AOAC, Washington 4-, D.C.  I960,  Beadle, G.W. 1937* The inheritance of the color of Malpighian tubes i n Drosophila melanogaster. Amer. Nat., 7 1 : 2 7 7 - 2 7 9 . Becker, W.A. 1964-. Manual of Procedures i n Quantitative Genetics. Washington State U n i v e r s i t y , Pullman, Washington. Benzer, S. 1 9 5 7 . In Symposium on Chemical Basis of Heredity, ed. by W.D. McElroy^and B. Glass, p. 7 0 • Baltimore, The Johns Hopkins Press. Bernier, P . E . and Cooney, T . 195*+. Black down color and r i b o f l a v i n deficiency i n embryos of the domestic fowl. Proceed. World's Poult. Congr., 1 0 : 6 6 - 7 1 . Bodine, J . H . and F i t z g e r a l d , L.R. 194-7. Riboflavin and other fluorescent compounds i n a developing egg (Orthoptera). P h y s i o l . Z o o l . , 2 0 : 14-6-160. Boucher, R . V . , Buss, E . G . and Maw, A . J . G . 1 9 5 9 . Physiological c h a r a c t e r i s t i c s associated with a mutant gene i n chickens that causes a deficiency of r i b o f l a v i n 2 . Blood. Poult. S c i . , 3 8 : 1 1 9 0 . B r i l e s , W.E. 194-9* Heterozygosity of inbred lines of chickens at l o c i effecting the red c e l l antigens. Poult. S c i . , 2 8 : 7 5 9 Brink, R.A. 1 9 5 6 . 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