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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 British Columbia, 196*+ A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department, of Poultry Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July, 1966 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia,, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission., for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of p nn1 t r y S r i Pnr- P The Uni v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada Date__ 1st August, 1966 i i 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 i s o l a t e d from T r i b o l i u m confusum, w i t h r i b o f l a v i n was shown by the independent methods o f s p e c t r o p h o t o m e t r y , chromatography and e l e c t r o p h o r e s i s . Two d e c o m p o s i t i o n p r o d u c t s o f r i b o f l a v i n as w e l l as two c o n j u g a t e d f l a v i n e s FMN and FAD, were i d e n t i f i e d . The g e n e t i c c o n t r o l o f r i b o f l a v i n and i t s r e l a t i o n s h i p t o some mutant eye c o l o r genes i n the p l e i o t r o p i c h i e r a r c h y o f gene e f f e c t s was i n v e s t i g a t e d i n a s e r i e s o f experiments i n v o l v i n g more than 8000 T r i b o l i u m from 11 g e n e t i c a l l y d i f f e r e n t l i n e s . A l l d e v e l o p m e n t a l stages o f i n d i v i d u a l s homozygous f o r a r e c e s s i v e p e a r l eye c o l o r gene, £ r ( f o r m e r l y c a l l e d p_, but renamed) were 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 r e d u c t i o n i n r i b o f l a v i n c o n t e n t i n the body. T h i s r e d u c t i o n was p r i n c i p a l l y i n the M a l p i g h i a n t u b u l e s . I n a l l o f the o t h e r l i n e s t e s t e d the v i t a m i n was accumulated i n the t u b u l e s i n r e l a t i v e l y h i g h c o n c e n t r a t i o n s . The normal a l l e l e o f t h i s gene was found t o be i n c o m p l e t e l y dominant w i t h r e s p e c t t o the r i b o f l a v i n phene. Changing the r e s i d u a l genotype of p.1" by means of two g e n e r a t i o n s o f 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 e x p r e s s i o n . Two a l l e l e s o f p e a r l (p_ and p_s) were 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 r , except a t the l e v e l o f t h e r i b o f l a v i n phene. Cro s s e s o f t h e s e a l l e l e s w i t h p_r were shown t o complement. 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 t e n t and the p i g m e n t a t i o n o f M a l p i g h i a n t u b u l e s were shown. I n d i v i d u a l s o f the p e a r l phenotype / I* 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 dimorphism f o r r i b o f l a v i n c o n t e n t was obser v e d i n a l l l i n e s t e s t e d . The q u a n t i t a t i v e parameters o f v a r i a t i o n o f 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 t o be h i g h by two d i f f e r e n t com-p a r i s o n s . 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 and c h a r a c t e r i z a t i o n o f spot 5 1. I d e n t i f i c a t i o n by 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 by means o f chromatography 3. I d e n t i f i c a t i o n by means o f e l e c t r o p h o r e s i s DISCUSSION 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 between r j r and p_ D. The s y n t h e s i s 3 o f a doub l e mutant; p e a r l , r u b y 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,s F. D i s t r i b u t i o n and 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 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. Developmental 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 M a l p i g h i a n t u b u l e s 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 r i b o f l a v i n t r a i t DISCUSSION CONCLUSIONS AND SUMMARY LITERATURE CITED V LIST OF TABLES TABLE PAGE I. Chromatographic d i s t r i b u t i o n o f substances and t h e i r R f v a l u e s i n W.T. a d u l t T r i b o l i u m confusum 15 I I . V i s u a l e s t i m a t i o n o f the 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 s i n 11 l i n e s of T. confusum 17 111. Mean R^ . v a l u e s o f sample and s t a n d a r d i n v a r i o u s c h r o m a t o g r a p h i c s o l v e n t s 21 IV. Rf v a l u e s o f r e f e r e n c e compounds and samples 22 V. The 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 the F-j_ and Fg progeny from the c r o s s p V p ' x +/+ 28 V I . Comparison o f t o t a l r i b o f l a v i n (ug/mg f r e s h w e i g h t o f two g e n e r a t i o n s o f r e c u r r e n t l y s e l e c t e d p_' from p'/p' x V + , w i t h t h e p a r e n t s , the h e t e r o z y g o t e s and the W.T. 30 V I I . The 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 the F 1 and F2 progeny from the c r o s s p/p, l o d / l o d x +/+ 32 IX. The 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 the F^ and b a c k c r o s s (BC^) progeny o f p r / p r x p / p , l o d / l o d 35 V I TABLE PAGE X. The 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 the F^, F 2 and b a c k c r o s s (BC-^) progeny o f p r / p r x p / p , r u s / r u s 36 X I . The 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 the F , F 2 and b a c k c r o s s ( B C i ) progeny o f p r / p r x p / p , d r e / d r e , c a s / c a s ^7 X I I . The F-^  and F 2 progeny from p'/p 1 x r u s / r u s and t h e 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 the F3 progeny from the s e l f o f the F 2 r u s / r u s 39 X I I I . The phenotype and d i s t r i b u t i o n of r i b o f l a v i n o f the progeny from t h e c r o s s e s " p r / p r , r u s / r u s " x p r / p r (!•) and " p r / p r , r u s / r u s " x p / p , r u s / r u s (2.) hi X I V . 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 the pupae and a d u l t s o f W.T. p_s and p.1" l i n e s h5 XV. Developmental v a r i a t i o n of r i b o f l a v i n i n W.T., p_s and i ) r l i n e s measured i n ug/mg f r e s h w e i g h t -^8 v i i TABLE PAGE X V I . 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 ght f o r males and females o f 11 l i n e s ( t h e i r average compared by-Duncan's New M u l t i p l e Range T e s t ) and t h e c o l o r o f M a l p i g h i a n t u b u l e s 52 X V I I . A n a l y s i s o f v a r i a n c e 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 i n 10 l i n e s o f T. confusum 5V X V I I I . C o r r e l a t i o n o f 10 day a d u l t w e i g h t w i t h 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 marked s t o c k 56 X I X . 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 t h r e e dams w i t h i n each o f t h r e e s i r e s 59 XX. A n a l y s i s o f v a r i a n c e by sex o f the 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 the p / p , l o d / l o d marked l i n e 60 XXI A n a l y s i s o f v a r i a n c e and expected mean squares 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 ght i n the p / p , l o d / l o d marked l i n e 6 l v i i i TABLE PAGE X X I I . Components o f v a r i a n c e e s t i m a t e d from t h e a n a l y s i s o f v a r i a n c e o f t f b t a l r i b o f l a v i n i n the p / p , l o d / l o d marked l i n e 63 X X I I I . E s t i m a t e s o f h e r i t a b i l i t i e s and s t a n d a r d e r r o r s o f the r i b o f l a v i n t r a i t i n the ' p / p , l o d / l o d marked l i n e 65 i x LIST OF FIGURES FIGURE PAGE 1. U l t r a v i o l e t a b s o r p t i o n s p e c t r u m o f pure r i b o f l a v i n and r i b o f l a v i n e x t r a c t , a t pH 7 19 2. Paper e l e c t r o p h e r o g r a m o f f l a v i n -m o n o n u c l e o t i d e (FMN), f l a v i n - a d e n i n e -d i n u c l e o t i d e (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 whole a n i m a l s o f d i f f e r e n t genotypes 2k 3. Developmental v a r i a t i o n o f r i b o f l a v i n i n Xi 1", p_s and W.T. males and f e m a l e s . The d e v e l o p m e n t a l s t a g e s a r e : a, eggs; b, l a s t i n s t a r l a r v a e ; c, e a r l y , d, l a t e pupae; e, day o l d , f , 10 day o l d , g, 30 day o l d a d u l t s 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 , Dr. C.W. R o b e r t s , f o r h i s e n c o u r a g i n g a s s i s t a n c e and c r i t i c i s m t h r o u g h o u t the c o u r s e o f t h i s s t u d y . He i s a l s o i n d e b t e d t o the members o f h i s committee who c r i t i c a l l y r e a d t h i s m a n u s c r i p t . Thanks a r e a l s o due Dr. W.D. K i t t s who made a v a i l a b l e the ap p a r a t u s used i n f l u o r o m e t r y and s p e c t r o p h o t o m e t r y . A p p r e c i a t i o n i s a l s o extended t o f e l l o w g r a d u a t e s t u d e n t s Mr. D.C. Crober and M i s s C H . C h i f o r a s s i s t a n c e w i t h t h e s t a t i s t i c a l a n a l y s e s and the many h e l p f u l d i s c u s s i o n s on the s u b j e c t o f t h i s t h e s 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 S c i e n c e Graduate R e s e a r c h 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 acknowledged. 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 t o h i s v/ife f o r her p r e p a r a t i o n o f the m a n u s c r i p t . INTRODUCTION I n r e c e n t 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 used 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 g e n e t i c s t u d i e s . Through t h e s e s t u d i e s a l a r g e number o f m u t a t i o n s have been found and c a t e g o r i z e d . S t o c k s o f th e s e mutants a r e r e a d i l y a v a i l a b l e t h r o u g h o u t Canada and the U n i t e d S t a t e s , however, o n l y a few have been i n v e s t i g a t e d i n d e t a i l . A number o f m u t a t i o n s w h i c h a f f e c t the p i g m e n t a t i o n o f the eye have been found. P r e l i m i n a r y 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 t h e s e eye mutants l e d t o the 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 q u a n t i t a t i v e d i f f e r e n c e s i n a y e l l o w f l u o r e s c e n t pigment 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 as r i b o f l a v i n . S i n c e t h i s pigment 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. confusum, i t s i n v e s t i g a t i o n might c l a r i f y the n a t u r e o f the g e n e t i c i n t e r r e l a t i o n s h i p s between tho s e genes r e s p o n s i b l e f o r r i b o f l a v i n d i f f e r e n c e s and those genes a s s o c i a t e d w i t h eye pigment d i f f e r e n c e s . Workers have observed the a s s o c i a t i o n o f r i b o f l a v i n w i t h the g e n e t i c s o f p t e r i d i n e s p a r t i c u l a r l y i n D r o s o p h i l a a n d - E p h e s t i a , y e t the g e n e t i c s o f 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 d i r e c t a t t e n t i o n . T h e r e f o r e t h i s t h e s i s was concerned w i t h a t t e m p t s t o d e f i n e t h e g e n e t i c c o n t r o l o f t h i s pigment, w i t h t h e d e s c r i p t i o n o f the changes i n i t s c o n c e n t r a t i o n d u r i n g development, 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 o f t h i s phene. REVIEW OF THE LITERATURE M u t a t i o n s are u s u a l l y c l a s s i f i e d a c c o r d i n g 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 . I n the case o f eye c o l o r genes, i n i t i a l l y o n l y the d i r e c t l y v i s i b l e pigments can be o b s e r v e d . As a r u l e t h e s e e a s i l y r e c o g n i z a b l e f e a t u r e s a r e preceded and accompanied by an a d d i t i o n a l s e t o f c h a r a c t e r s w h i c h become apparent o n l y by the use o f s p e c i a l methods. The t o t a l i t y o f a l l phenes, i n w h i c h a mutant d e v i a t e s from a "normal" or " s t a n d a r d " or w i l d t y p e (W.T.), c o n s t i t u t e s the " p l e i o t r o p i c p a t t e r n o f m a n i f e s t a t i o n " (Hadorn, 195*0 of the r e s p e c t i v e gene or f a c t o r . Such p l e i o t r o p y o f genie a c t i o n , c o n d i t i o n s b i o -c h e m i c a l and p h y s i o l o g i c a l , as w e l l as m o r p h o l o g i c a l phenes. Eye c o l o r genes, s t u d i e d i n D r o s o p h i l a and E p h e s t i a , have p r o v i d e d much i n f o r m a t i o n c o n c e r n i n g the p l e i o t r o p i c a c t i o n o f genes (Hadorn and M i t c h e l l , 1951; Hadorn, 1956; Z i e g l e r , 1961; Hubby, 1962). F l a v l n e s are o f t e n i s o l a t e d w i t h p t e r i d i n e s , and i n some of the e a r l i e r l i t e r a t u r e these two c l a s s e s o f compounds may have been c o n f u s e d ( V i s c o n t i n i , e t a l . , 1955). V i s c o n t i n i (1956) i s o l a t e d r i b o f l a v i n a l o n g w i t h p t e r i d i n e s from the heads o f D r o s o p h i l a and E p h e s t i a . A c l o s e 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 and r i b o f l a v i n ( Z i e g l e r , 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 and share 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 and Waldschmidt, 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 from the eggs o f i n s e c t s and the c o i n c i d e n t appearance o f o t h e r f l u o r e s c e n t compounds w h i c h seem t o have t h e p r o p e r t i e s o f p t e r i d i n e s a l s o s u g gests 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 F i t z g e r a l d , 19*+7j B u r g e s s , 19^9). The f u n c t i o n o f f l a v i n e s as p r o s t h e t i c groups o f s e v e r a l dehydrogenases has been w e l l e s t a b l i s h e d . Whether t h e y have an 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 , w h i c h t h e i r a c c u m u l a t i o n i n such l a r g e q u a n t i t i e s might suggest,,, i s not known ( G i l m o u r , 1961). U s i n g the 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 B l o m s t r a n d (1958) p o i n t e d out t h a t the pigment d e p o s i t e d i n r o d l e t s I n the o u t e r l a y e r o f t h e t e s t i s s h e a t h o f E p h e s t i a was p a r t l y r i b o f l a v i n . I n t h e W.T. r i b o f l a v i n g r a d u a l l y appears d u r i n g the l a s t l a r v a l i n s t a r , reaches a p l a t e a u i n t h e e a r l y pupa and d i s a p p e a r s i n the l a t e pupa. S u b s e q u e n t l y , 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 might t h e n be t r a n s f e r r e d t o the M a l p i g h i a n t u b u l e s s i n c e the r a t e o f d i s a p p e a r a n c e from the 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 appearance i n t he M a l p i g h i a n t u b u l e s . I n a d d i t i o n t h e y 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 the appearance o f c o n s i d e r a b l y h i g h e r amounts o f r i b o f l a v i n i n the t e s t i s s h e a t h s , but 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 e a r l i e r t h a n i n t h e W.T. The p l e i o t r o p i c a c t i o n o f t h i s gene, i n c l u d e s n o t o n l y t r y p t o p h a n m e t a b o l i s m o f 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 and r i b o f l a v i n w h i c h cannot be e x p l a i n e d i n d e t a i l a t the 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 the 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, 19535 Kuhn and B e r g , 1956) the appearance o f r i b o f l a v i n i n the t e s t i s sheaths was i n h i b i t e d a t a l l t i m e s . 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 reduced compared t o a i n d i v i d u a l s by the amount p r e s e n t i n a t e s t e s . 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 genes, C a s p a r i and B l o m s t r a n d (1958) demonstrated s i g n i f i c a n t 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 and t h e p a t t e r n o f i t s ' d i s a p p e a r a n c e i n E p h e s t i a . Eye c o l o r genes w h i c h a f f e c t the amount o f 6 r i b o f l a v i n have a l s o been found i n D r o s o p h i l a . Hadorn and M i t c h e l l ' s (1951) spot F l h/5 c o n t a i n e d r i b o f l a v i n and t h r e e o t h e r s u b s t a n c e s a l l o f w h i c h have been shown t o be p t e r i d i n e s . T h i s spot was m i s s i n g i n t h e mutants brown (bw) and w h i t e (w). R i b o f l a v i n as w e l l as the o t h e r substances a r e l a c k i n g i n a n i m a l s homozygous f o r t h e s e genes. There a r e many o t h e r c o l o r mutants i n w h i c h the i n t e n s i t y o f F l 1+/5 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 , 2 2 sed; r o s y , ryr and a l s o ebony, 1; c l a r e t , c a ; prune, 2 P n ) or reduced ( w h i t e - a p r i c o t , w a w h i t e - e o s i n , w e ; ga r n e t , g.2; p i n k - p e a c h , p_p; l i g h t , I t ; c a r n a t i o n , c a r ; p u r p l e , p_r) as compared t o the w i l d t y pe (Hadorn and M i t c h e l l , 1951; Hadorn and Schwinck, 1956). I n some o f t h e s e cases 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 does not c l a r i f y whether the amount o f r i b o f l a v i n p r e s e n t was a l s o a f f e c t e d by t h e s e genes. T a i r a and Nawa (1958), u s i n g the b a c t e r i o l o g i c a l method of a s s a y , found t h a t mutant se of D. m e l a n o g a s t e r 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 (v) or bw. Hubby (1961) showed s i g n i f i c a n t l y h i g h e r l e v e l s o f a " r i b o f l a v i n - l i k e " pigment i n the t e s t i s o f l i g h t i s o x a n t h o p t e r i n ( l i x ) compared t o ry_ and Oregon-R. 7 E v i d e n c e f o r the g e n e t i c c o n t r o l o f f r e e r i b o f l a v i n among the h i g h e r forms has been found i n t h e domestic f o w l . B e r n i e r and Cooney (195*+) o b t a i n e d a h i g h l y s i g n i f i c a n t d i f f e r e n c e i n t h e i n c i d e n c e o f 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 (E) and n o n - b l a c k (e) embryos as w e l l as i n c h i c k s from a c r o s s i n v o l v i n g the s e g r e g a t i o n o f t h e s e a l l e l e s . They suggested t h a t r i b o f l a v i n may be i n v o l v e d i n m e l a n i z a t i o n . Boucher and h i s co-workers (1959; Buss et a l . , 1959; Cowan e t a l . , 196l; Cowan et a l . , 1961 a) 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 w i t h a r e c e s s i v e gene c a u s i n g r i b o f l a v i n d e f i c i e n c y ( r d ) . T h e i r d a t a showed t h a t f r e e 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 the normal (Rd-) t h a n i n the r e c e s s i v e ( r d r d ) l a y i n g hen, whereas, 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 o f the r d gene. The normal a l l e l e Rd, was f u r t h e r -more shown t o be i n c o m p l e t e l y dominant. A l t h o u g h T r i b o l i u m has e x t e n s i v e l y been used i n e c o l o g i c a l , and more r e c e n t l y i n g e n e t i c a l s t u d i e s (McDonald and P e e r , I96O; S c h l a g e r , 1963; Dawson, 196Lfa, b; 1965; Crenshaw and L e r n e r , 196^; S o k o l o f f , 196*+ a, b) t h e r e appears t o be no l i t e r a t u r e c oncerned w i t h the g e n e t i c s o f p h y s i o l o g i c a l t r a i t s . MATERIALS AND METHODS A l l c u l t u r e s , p a r e n t s and progeny were r e a r e d i n a c o n s t a n t environment o f 32° C , and 72%> r e l a t i v e h u m i d i t y . The medium was pr e p a r e d from s i f t e d (No. 30) Meota whole wheat f l o u r mixed w i t h 10% T o r u l a y e a s t (Doty, 1965). S t e r i l i z a t i o n o f the f l o u r (60° C. f o r 2k h o u r s ) was performed p r i o r t o adding the y e a s t , s i n c e the l a t t e r was found t o clump upon h e a t i n g . V i r g i n females 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 the p u p a l stage and c h e c k i n g t h e phenotype upon e c l o s i o n . Males were sexed s i m i l a r l y , w i t h the e x c e p t i o n o f some wh i c h were removed from s t o c k c u l t u r e s i n t h e a d u l t s tage and sexed by t h e method o f P f a d t (1962). Progeny were u s u a l l y sexed i n the p u p a l s t a g e , however, some were sexed as a d u l t s . E t h e r i z a t i o n was employed f o r i m m o b i l i z a t i o n . S m a l l matings were c a r r i e d out i n 20 ml. creamers, l a r g e r ones i n 110 ml. c e l l u l o s e n i t r a t e t u b e s . A l l o f the s t o c k s were o b t a i n e d from the T r i b o l i u m S t o c k C e n t e r , U n i v e r s i t y o f C a l i f o r n i a , B e r k e l e y . They were 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 a t l e a s t i n d u p l i c a t e . In g e n e r a l c r o s s e s were made by mating f i v e males to f i v e females. In accordance w i t h T r i b o l i u m c o n v e n t i o n males are put f i r s t i n the d e s c r i p t i o n of a c r o s s . The d e s c r i p t i o n o f the stocks used i s as f o l l o w s : p e a r l (p_), an autosomal r e c e s s i v e , e l i m i n a t e s the pigment from the ommatidia but not from the o c u l a r diaphragm r e s u l t i n g i n a b i c o l o r e d or s p e c t a c l e d appearing eye (Graham, 1957)* T h i s gene i s r e f e r r e d to i n the t e x t as "Graham's p e a r l " (p_*) and w i l l subsequently be r e d e s i g n a t e d to p_r . p e a r l (p_s), spontaneous i n the B e r k e l e y s y n t h e t i c s t r a i n ; a l l e l i c to p e a r l d e s c r i b e d by Graham ( S o k o l o f f , 1963) . p.'and p_s are p h e n o t y p i c a l l y i n d i s t i n q u i s h a b l e . l i g h t o c u l a r diaphragm ( l o d ) , i s not s t r i c t l y an eye mutation but i t can be observed when eye c o l o r mutants such as p e a r l (which m o d i f i e s the b l a c k pigment of the ommatidia) are present, l o d i s an autosomal r e c e s s i v e which blocks the s y n t h e s i s of melanin pigment from the o c u l a r diaphragm, an e n d o s k e l e t a l s t r u c t u r e which helps to support the compound eye. Since the diaphragm i s not a f f e c t e d by genes a f f e c t i n g the c o l o r of the ommatidia, i t appears as a dark band under the 10 colorless or l i g h t l y pigmented marginal ommatidia, giving the beetle a spectacled appearance (Sokoloff, 1 9 6 2 ) . ruby spot (rus), produces a pinkish eye either i n the l a t e pupa or i n the teneral adult, but with aging the eye darkens, leaving only a reddish spot which can be i d e n t i f i e d by casting a shadow on the beetle's eye as i t l i e s on the stage of a dissecting microscope. The spot i s not stationary f o r , i f the beetle i s rotated, i t moves i n the d i r e c t i o n opposite to the movement of the beetle. This indicated that, i n r e a l i t y , the ommatidia are a l l dark red but special intense l i g h t i n g i s required to detect the pigment (Sokoloff, 1 9 6 l ) . d i r t y pearl eye (dp_e), i s distinquished from rus during a b r i e f period i n the developmental cycle, i n the pupa and sometimes i n the teneral adult the compound eyes appear uniformly pinkish i n rus whereas i n dpe the eyes of the l a t e pupa or early 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 colored the eyes of the imago become dark red, perhaps darker than rus., somewhat resembling the chestnut eye m u t a t i o n i n T. castaneum ( S o k o l o f f , 196*+). eye spot ( e s ) , i s a s e x - l i n k e d r e c e s s i v e c h a r a c t e r i z e d by a c l e a r spot i n the c e n t e r o f t h e eye w h i c h can be r e a d i l y seen i n young a d u l t s , but becomes i n c r e a s i n g l y d i f f i c u l t t o i d e n t i f y as the i n s e c t ages, however i t never d i s a p p e a r s c o m p l e t e l y (McDonald, 1961). r e d ( r ) , i s a s e x - l i n k e d r e c e s s i v e p r o d u c i n g a l i g h t -r e d a r e a i n the eye comparable i n the 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 the spot becomes reduced i n s i z e i n t e r m e d i a t e between p_ and es (Ho, 1962). p e a r l , l i g h t o c u l a r diaphragm (p.,lod); p e a r l , ruby spot (p_,rus); p e a r l , droopy e l y t r a , c r e a s e d abdominal s t e r n i t e s (p_,dre,cas.), s t o c k s were o b t a i n e d from the B e r k e l e y s t o c k c e n t e r a l o n g w i t h t h e above mutant s t o c k s . C F I - 1 , i s an i n b r e d l i n e , b r o t h e r - s i s t e r mated f o r M-3 g e n e r a t i o n s and f r e e (as f a r as i s known) o f eye c o l o r m u t a t i o n s . T h i s l i n e i s used as t h e W.T. c o n t r o l . C F I - 7 , 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 from the same base p o p u l a t i o n as C F I - 1 , and b r o t h e r - s i s t e r mated f o r ^3 g e n e r a t i o n s . B o t h CFI - 1 and CFI - 7 were k e p t as random mating c l o s e d p o p u l a t i o n s 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 (about 10 g e n e r a t i o n s ) . 12 A m o d i f i c a t i o n o f the pr o c e d u r e d e s c r i b e d by Hadorn and M i t c h e l l (1951) was used f o r most o f the chr o m a t o g r a p h i c s e p a r a t i o n s . A l l chroma-tograms w h i c h were t o be measured f l u o r o m e t r i c a l l y 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 e c l o s i o n . The b e e t l e s were b o i l e d f o r two minutes 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 c h r o m a t o g r a p h i c r e s o l u t i o n o f f l u o r e s c e n t s u b s t a n c e s . 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, but t h e a d u l t s were immersed b r i e f l y i n 95$ e t h a n o l p r i o r t o b o i l i n g . E xcess water was removed from the b o i l e d a n i m a l s by b l o t t i n g them on f i l t e r p aper. They 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 onto Whatman No. 1.paper. B e e t l e s were f i r m l y mashed onto the paper w i t h a g l a s s r o d and a l l o w e d t o d r y a t room t e m p e r a t u r e i n the d a r k . R e s i d u a l t i s s u e s were n ot removed from the paper s i n c e the a u t h o r ' s p r e -l i m i n a r y e x periments demonstrated t h a t the c h i t i n o u s e x o s k e l t o n does not i n t e r f e r e w i t h 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 R i n g e r ( P a t t o n and C r a i g , 1939) and t r a n s f e r r e d w i t h as l i t t l e a d h e r i n g l i q u i d as p o s s i b l e onto the 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 placed 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 so l v e n t . Unless otherwise stated a l l experiments were c a r r i e d out i n the ascending d i r e c t i o n i n dark 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 solvent (Caspari and Blomstrand, 1958). When the solvent 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 emission 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 . Spots were cut out and e x t r a c t e d overnight i n four ml. of double d i s t i l l e d water at room temperature i n a dark room. The samples were read i n a Turner photofluorometer, Model 111, using V36 ( f i l t e r s k7B and 2A) and 570 ( f i l t e r 23A) m i l l i m i c r o n s as primary and secondary wavelengths and w i t h s l i t arrangement 30X (AOAC, i960). For q u a n t i t a t i o n a standard curve was prepared from pure r i b o f l a v i n using the above 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 The r e s u l t s o f paper chromatography o f the W.T. i s shown i n T a b l e I . I n t o t a l e i g h t f l u o r e s c e n t and f o u r n o n - f l u o r e s c e n t s p o t s appeared on the chromatograms. The y e l l o w f l u o r e s c e n t s p o t s 1 , 2 and 5 appeared on a l l t h e chromatograms. Spot 3 seldom appeared; when i t was p r e s e n t t h e y e l l o w spot 2 w i t h the i d e n t i c a l R f v a l u e was i n v a r i a b l y a b s e n t . The v i o l e t 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 c o n s i d e r a b l y i n i n t e n s i t y o f f l u o r e s c e n c e . The b r i c k -r e d spot 7 appeared w e l l d e f i n e d on f r e s h chromatograms, but g r a d u a l l y changed t o a d i r t y brown c o l o r a f t e r a few days. The f l u o r e s c e n t s p o t 8 was o n l y v i s i b l e w h i l e the chromatogram was s t i l l wet. Spot 9 w e a k l y absorbed when i l l u m i n a t e d w i t h t h e 253 mu p r i n c i p l e e m i s s i o n lamp; i t a l s o d i s a p p e a r e d s h o r t l y a f t e r d r y i n g t h e chromatogram. Spot 10 m i g r a t e d w i t h the s o l v e n t f r o n t and was 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 never b o t h . The r e s u l t s o b t a i n e d 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 IN W.T. ADULT TRIBOLIUM CONFUSUM Spot v i s i b l e F l u o r e s c e n t R f i n t e n s i t y No. c o l o r c o l o r J . 1 - Y e l l o w .05 medium 2 Y e l l o w Y e l l o w .27 s t r o n g 3 - V i o l e t .27 medium* k - V i o l e t .50 v a r i a b l e 5 Y e l l o w Y e l l o w .60 s t r o n g 6 - V i o l e t .6>+ v a r i a b l e 7 Red - • 72 weak 8 - B l u e .81 t r a c e 9 - A b s o r b s * * .91 t r a c e 10 Brown B l u e * * * .97 medium * Seldom appears; i n i t s presence the y e l l o w spot i s u s u a l l y a b s e n t . ** Maximum absorbance a t 253 mu. *** T h i s spot i s e i t h e r brown, o r b l u e and f l u o r e s c e n t . 16. f l u o r e s c e n c e o f the y e l l o w f l u o r e s c e n t s p o t s 1, 2 and 5 i n 11 l i n e s o f T. confusum a r e shown i n T a b l e I I . The i n t e n s e l y y e l l o w f l u o r e s c e n t spot 5 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 between d i f f e r e n t eye c o l o r mutants and the i n b r e d l i n e s . A l t h o u g h a l a r g e number o f p_' i n d i v i d u a l s were chromatographed, s p o t 5 was never p r e s e n t i n t h i s mutant. Comparison o f the R^ v a l u e s o f t h i s spot w i t h those i n t h e l i t e r a t u r e f o r s i m i l a r s o l v e n t s ( H a i s and Pecakova, 19^9; C a s p a r i and B l o m s t r a n d , 1958; Schmidt and V i s c o n t i n i , 1962; Harkness e t a l . , 196*+) gave a good i n d i c a t i o n t h a t i t might be r i b o f l a v i n . B. I s o l a t i o n , p u r i f i c a t i o n and c h a r a c t e r i z a t i o n o f spot 5 1. I d e n t i f i c a t i o n by s p e c t r o p h o t o m e t r y F o r the purpose o f s p e c t r o p h o t o m e t r y , e l e c t r o p h o r e t i c and c h r o m a t o g r a p h i c i d e n t i f i c a t i o n , t he substance i n spot 5, s u s p e c t e d t o be r i b o f l a v i n , was e x t r a c t e d and p u r i f i e d by a m o d i f i e d method based on t h a t o f Schmidt and V i s c o n t i n i (1962). TABLE I I VISUAL ESTIMATION OF THE YELLOW FLUORESCENT SUBSTANCES IN 11 LINES OF T. CONFUSUM Lines  P P d r e l o d P Spot R f cas rus p s p' es- r dpe rus C F I - 1 CFI - 7 1 # o 5 + + + + + + + + + + + 2 .1+8 ++ + ++ + + + + + + + + + 5 . 6 0 + + + +++ +++ + + _ + + + + ++ ++ + number 68 68 68 68 3 7 5 68 68 68 68 68 20 chromatographed* * Equal numbers of males and females 18 About one gm. p o r t i o n s o f b e e t l e s were 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. A 95%> e t h a n o l e x t r a c t i o n was f o l l o w e d by e v a p o r a t i o n o f the ex c e s s a l c o h o l , and chromato-graphy on Whatman No. 1 paper 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 moving 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 ter and c o n c e n t r a t e d by vacuum e v a p o r a t i o n a t 60° C , t h e n chromatographed i n n - b u t a n o l : a c e t i c a c i d : water C + : l : l ) . 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 and 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 under vacuum and the 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 were removed by subsequent chromatography i n w a t e r . F o l l o w i n g e l u t i o n the c o n c e n t r a t i o n o f the s o l u t e was brought t o e q u a l i t y by d i l u t i o n w i t h t h a t o f a s i m i l a r l y p r e p a r e d pure sample o f r i b o f l a v i n . U s i n g a Unicam (SP.500) s p e c t r o p h o t o m e t e r , an u l t r a -v i o l e t a b s o r p t i o n c u r v e was p r e p a r e d ( F i g u r e 1). The i d e n t i t y o f s t a n d a r d and sample appears e v i d e n t . FIGURE 1. ULTRAVIOLET ABSORPTION-SPECTRUM OF PURE RIBOFLAVIN AND-RIBOFLAVIN EXTRACT, AT pH 7 20 2. Identification by means of chromatography Whole beetle squashes were made on two different grades of f i l t e r paper and chromatographed in five different solvents using pure riboflavin with each run for comparison. The solvents and the results for each f i l t e r paper are shown in Table III. Each value was the mean of ten or more determinations; The migration properties of sample and standard appear nearly identical under a l l conditions. The use. of decomposition products for the identification of riboflavin has frequently been employed in the past (Yagi, 1957: Cerletti and Siliprandi, 1955). Using the methods of Yagi (1957) lumiflavin and lumichrome were prepared by photolysis of pure riboflavin 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. The results are shown in Table IV. The values of reference nucleotides and decomposition products agree well with those of the corresponding substance in the extract and whole squashes of beetles. TABLE I I I MEAN R VALUES OF SAMPLE AND STANDARD IN VARIOUS CHROMATOGRAPHIC SOLVENTS s o l v e n t s * paper spot A B C D E Whatman spot 5 .32 .60 .37 .25 .32 No. 1 r i b o f l a v i n .31 .6 -^ .36 .25 .33 Whatman spot 5 • 39 ,6h M .27 • 37 No. h r i b o f l a v i n .hi .66 .h6 .27 .39 * A n - p r o p a n o l ; IN. a c e t i c a c i d (3:1) B t e r t - b u t a n o l ; p y r i d i n e ; water (10:3 :7) C n - p r o p a n o l ; 1% aqueous ammnonia (2: 1) D n - b u t a n o l ; a c e t i c a c i d ; water (*+:l: 1) E n - b u t a n o l ; a c e t i c a c i d ; water (*+:l: 5) 22 TABLE IV Rf 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 by 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 (1957) was used f o r the e l e c t r o p h o r e t i c s e p a r a t i o n o f f l a v i n e s . Sample 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 and a m i x t u r e o f t h e s e s t a n d a r d s . Phosphate b u f f e r o f pH 8, and .05 M was used w i t h c o n s t a n t c u r r e n t a t 2.M+ mA/cm. g i v i n g ^00 v o l t s . Whatman No. 3MM paper was found most s u i t a b l e f o r s u p p o r t i n g medium. S a t i s f a c t o r y 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 a two hour r u n . The r e s u l t s a r e shown d i a g r a m m a t i c a l l y i n F i g u r e 2. r i b o f l a v i n FMN FAD FAD +/+ p'/p' p S / p S p/p: d r e / d r e : c a s / c a s FMN r i b o f l a v i n FIGURE 2. PAPER ELECTROPHEROGRAM OF FLAVINE-MONONUCLEOTIDE (FMN), FLAVIN-ADENINE-DINUCLEOTIDE (FAD), RIBOFLAVIN, A MIXTURE OF THESE, AND WHOLE ANIMALS OF DIFFERENT GENOTYPES. DISCUSSION The i d e n t i t y o f the 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 , spot 5) i s o l a t e d from T r i b o l i u m confusum a d u l t s , w i t h r i b o f l a v i n was shown by the independent methods o f s p e c t r o p h o t o m e t r y , chromatography and e l e c t r o p h o r e s i s . I n a d d i t i o n , the d e c o m p o s i t i o n p r o d u c t s o f sample and s t a n d a r d r i b o f l a v i n were a l s o shown t o be i d e n t i c a l . I t appears t h a t none of the f l u o r e s c e n t s p o t s o b t a i n e d i n 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 p r o d u c t s o f r i b o f l a v i n . T h e r e f o r e , t h e r i b o f l a v i n v a l u e s s u b s e q u e n t l y o b t a i n e d by the f o r e g o i n g method a r e assumed to be r e p r e s e n t a t i v e o f the a c t u a l amount o f r i b o f l a v i n p r e s e n t . FMN and FAD were a l s o i d e n t i f i e d by t h e i r Re-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 i n o r d e r t o d i s t i n g u i s h them from f r e e r i b o f l a v i n and to observe 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 o f f r e e 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 might be e x p e c t e d on t h e o r e t i c a l grounds s i n c e FMN and FAD d e r i v e the f l a v i n p a r t o f the m o l e c u l e s from f r e e r i b o f l a v i n . FMN and FAD a r e p r e s e n t i n a l l s i x genotypes ( F i g u r e 2 ) . V i s u a l i n s p e c t i o n i n d i c a t e d (Table I I ) t h a t 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 q u a n t i t i e s 26 i n a l l l i n e s e x cept JD1 i n w h i c h i t was not d e t e c t a b l e by v i s u a l o b s e r v a t i o n under u l t r a v i o l e t l i g h t . S i n c e the eyes o f the f i v e mutant genotypes t e s t e d a r e p h e n o t y p i c a l l y p e a r l and p_s has been d e s i g n a t e d an a l l e l e o f JD_' ( S o k o l o f f , 1963) i t was o f i n t e r e s t t o note t h i s a b e r r a t i o n on the p a r t o f the JD' s t o c k . PART I I . GENETIC ANALYSIS A. A n a l y s i s o f p e a r l I n the p r e v i o u s s e c t i o n i t was shown by chromato-graphy and e l e c t r o p h o r e s i s t h a t the p_' mutant 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 , whereas the p_ l o d , p_ r u s , and p_ d r e cas mutant l i n e s p o s s e s s e d c l e a r l y v i s i b l e q u a n t i t i e s ( T a b l e I I . , and F i g u r e 2 ) . The q u e s t i o n a r o s e , was t h i s r i b o f l a v i n l e s s phene h e r i t a b l e , and i f so, was i t dependent o r independent o f the p.1 gene. To answer 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 was made: P x p'/p* x +/+ F-L s e l f e d The r e s u l t s o f t h i s c r o s s showing the presence o r absence o f r i b o f l a v i n i n the F^ and F2 progeny a r e shown i n T a b l e V. The F-^  progeny were a l l p h e n o t y p i c a l l y W.T. and p o s s e s s e d r i b o f l a v i n . I n the F 2 progeny a r a t i o o f 3=1 (W.T. t o p e a r l ) would be e x p e c t e d . The r i b o f l a v i n l e s s phene s h o u l d o n l y be p r e s e n t i n p e a r l s , i f t h e s e two c h a r a c t e r s a r e a s s o c i a t e d w i t h the same gene. A n a l y s i s o f the progeny i n T a b l e V i n d i c a t e d t h e r e s u l t s a r e TABLE V THE DISTRIBUTION OF RIBOFLAVIN IN THE F AND F 2 PROGENY* FROM THE CROSS p'/p* x +/+ g e n e r a t i o n phenotype males females t o t a l number number w i t h chromatographed r i b o f l a v i n F-L w i l d 317 ^09 726 IOO 100 p e a r l - - - -F 2 w i l d 653 66*+ 1317 100 100 p e a r l 1^ 3 l6h 307 307 * 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 d i f f e r e n t . CO 29 s i g n i f i c a n t l y d i f f e r e n t from t h e e x p e c t e d 3-1 r a t i o ( x 2 = 32.18, d . f . = 1 , P = .005). However, t h i s was n o t unexpected s i n c e Graham (1957) and Park (1937) showed a l o s s o f f i t n e s s o f the p e a r l homozygotes. W i t h over 300 progeny t e s t e d , the r i b o f l a v i n l e s s phene was always a s s o c i a t e d w i t h t h e p_' gene. The p o s s i b i l i t y o f l i n k a g e was somewhat reduced by the f a c t t h a t t h e r e were 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 , nor 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 . R e c o m b i n a t i o n would be ex p e c t e d t o have produced such r e c i p r o c a l phenotypes i f l i n k a g e was as l i t t l e as .'3 • -S i n c e p.1 and p_ l p d have been s e p a r a t e b r e e d i n g p o p u l a t i o n s presumably s i n c e t h e y were f i r s t d i s c o v e r e d , i t was c o n s i d e r e d p o s s i b l e t h a t the 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 caused the observe d 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 check 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 the c r o s s p'/p 1 x +/+. Twenty progeny were chromatographed from each g e n e r a t i o n and the r i b o f l a v i n was measured f l u o r o m e t r l c a l l y . These v a l u e s a re compared i n T a b l e V I w i t h those o b t a i n e d from the p_' base p o p u l a t i o n (P-j_), and F-^  h e t e r o z y g o t e s ( p ' / + ) and the W.T. TABLE V I COMPARISON OF TOTAL RIBOFLAVIN (ug/mg FRESH WEIGHT) OF TWO GENERATIONS OF RECURRENTLY SELECTED p_« FROM p'/p' x +/+, WITH THE PARENTS, THE HETEROZYGOTES AND THE W.T.* g e n e r a t i o n genotype sex r i b o f l a v i n ug/mg f r e s h w e i g h t + s t a n d a r d e r r o r P l p'/p' male female .0050 .0060 .0005 .OOO^r F l P'/+ male female .091 .063 .00!+ .0009 F 2 P'/P' male female .0057 .0062 .0006 .0004-P'/P' male female .0060 .0059 .0008 .0004-- male female .110 .078 .004-.004-* Ten o b s e r v a t i o n s per sex 31 The values obtained f o r pj homozygotes must be viewed w i t h some c a u t i o n as these measurements were made at the l i m i t s of s e n s i t i v i t y of the fluorometer. The r e s u l t s show that changing of the r e s i d u a l genotype does not de t e c t a b l y a l t e r the apparently r i b o f l a v i n l e s s c o n d i t i o n of p'/p' i n d i v i d u a l s . The W.T. heterozygote p V + } shows that at the l e v e l of the r i b o f l a v i n phene, the normal a l l e l e i s incompletely dominant to p.1. B. A n a l y s i s of p_ l o d . p_ l o d , although 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 , Figure 2). Since the p_ and p_' genes are 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 of the p_ l o d mutants was t e s t e d . The f o l l o w i n g cross was made to 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 could be obtained: p/p l o d / l o d x +/+ F-L s e l f e d The r e s u l t s of t h i s cross showing the presence or absence of r i b o f l a v i n i n the ^  and F 2 progeny are shown i n Table V I I . The F-j_ progeny were a l l W.T. and had r i b o f l a v i n . In the F 2 the expected phenotypic r a t i o of W.T. to p e a r l , to p e a r l TABLE V I I THE DISTRIBUTION OF RIBOFLAVIN IN THE F 1 AND F PROGENY* FROM THE CROSS p/p l o d / l o d x ++ g e n e r a t i o n phenotype males females t o t a l e x p e c t e d r a t i o number chromatographed number w i t h r i b o f l a v i n w i l d 202 189 391 - 50 50 F w i l d 163 167 330 12 100 100 p e a r l 3^ 56 90 3 90 90 p e a r l , l o d 8 10 18 1 18 18 * 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 ot a p p r e c i a b l y d i f f e r e n t . IV) 33 l i g h t o c u l a r diaphragm i s 12:3:1 s i n c e t h e p / + ; l o d / l o d and + / + ; l o d / l o d genotypes a re i n d i s t i n g u i s h a b l e from t h e W.T. The d e v i a t i o n from the exp e c t e d ratio.- was not s i g n i f i c a n t ( x 2 = 3.79, d . f . = 2, 0.1 P 0.25), and a l l phenotypes were p o s i t i v e f o r r i b o f l a v i n . From t h e s e r e s u l t s i t can t e n t a t i v e l y be c o n c l u d e d t h a t the p e a r l gene i n the p_ l o d s t o c k may not be i d e n t i c a l t o the p e a r l gene i n the p_" s t o c k as suggested by S o k o l o f f (196*+ ) and more r e c e n t l y by Ackerman (1966 ). T h e r e f o r e , i t i s suggested t h a t t h e a p p a r e n t l y r i b o f l a v i n l e s s p 1 be r e - d e s i g n a t e d "_p_ 1 1. 3^  C. T e s t f o r a l l e l i s m between p_r and p_ The d i f f e r e n t i a l response o f the two p e a r l genes r a i s e d t h e q u e s t i o n , what i s the r e l a t i o n s h i p between them i f they a r e not a l l e l e s . To answer t h i s q u e s t i o n , the f o l l o w i n g c r o s s e s were made; 1. P 1 p r / p r x p / p , l o d / l o d F x a. s e l f e d b. c r o s s e d w i t h p r / p r 2. P-j_ p r / p r x p/p, r u s / r u s F-^  as i n c r o s s 1. a. and b. 3« Pi P r / p r x p / p , d r e / d r e , c a s / c a s Fj_ as i n c r o s s 1. a. and b. The r e s u l t s and 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 Ta b l e s I X , X and X I . The F-^  progeny from a l l t h r e e c r o s s e s were w i l d t ype 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_r and p_ a r e not t r u e a l l e l e s . None o f the F 2 and b a c k c r o s s p h e n o t y p i c r a t i o s d e v i a t e s i g n i f i c a n t l y from e x p e c t a t i o n s a p p l i c a b l e i n t h e case o f s e g r e g a t i o n a t a s i n g l e l o c u s . Hence i t may be c o n c l u d e d t h a t p_r and 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 g e n e t i c f u n c t i o n s , o r h e t e r o a l l e l e s c a p a b l e (as d e s c r i b e d f o r D r o s o p h i l a TABLE IX THE DISTRIBUTION OF RIBOFLAVIN IN THE F1, F 2 AND BACKCROSS (BCj_) PROGENY OF p r / p r x p / p , l o d / l o d c r o s s phenotype r i b o f l a v i n p r e s e n t absent t o t a l e x p e c t e d r a t i o 2 X <P < P r / P r x p / p , l o d / l o d F l w i l d 100 - 1899* - - - -p r/P)l°d/ + s e l f e d F 2 w i l d 9 5 * * 99 _ 19*+ 8 p e a r l 39 h5 3^ 4-1 159 6 0 . 7 2 1 . 5 . 7 5 p e a r l , l o d 13 11 10 15 ^ 9 2 P ? / P r * p r/p,lod/+ BC X w i l d 84-87 171 1 p e a r l - 82 90 172 1 0 .003 . 9 . 9 5 r e c i p r o c a l B C 1 w i l d 98 88 -186 1 p e a r l -86 92 178 1 0 . 1 7 6 . 5 . 7 5 * T o t a l progeny, out of which a sample o f 100 was as s a y e d f o r r i b o f l a v i n . ** The top v a l u e r e f e r s t o males, the bottom to f d m a l e s . TABLE X THE DISTRIBUTION OF RIBOFLAVIN IN THE F 2 AND BACKCROSS ( B C ) PROGENY OF p r / p r x p/p,rus/rus cross phenotype r i b o f l a v i n present absent t o t a l expected r a t i o 2 X P r/P r X p/p,rus/rUs F i w i l d 100 - 777* - - -p'/p,rus/+ s e l f e d F 2 non-pearl** 206*** 21k H-20 1 p e a r l * * * 105 101 97 92 202 193 1 0.77 .25 .5 pJ7pr X pVp,rus/+ B C 1 w i l d 60 71 _ 131 1 p e a r l - 59 63 122 1 0.320 .5 .75 r e c i p r o c a l B C - L w i l d 103 106 -209 1 p e a r l - 98 115 213 1 O.O38 • 75 .9 * T o t a l progeny, out of which a sample of 100 was assayed f o r r i b o f l a v i n ** Wild types and rus homozygotes. *** The top value r e f e r s to males, the bottom to females. TABLE X I THE DISTRIBUTION OF RIBOFLAVIN IN THE F±, F 2 AND BACKCROSS (BCj_) PROGENY OF p r / p r x p / p , d r e / d r e , c a s / c a s c r o s s phenotype r i b o f l a v i n p r e s e n t a b s e n t t o t a l e x p e c t e d r a t i o x 2 < P < p r / p r x F j p / p , d r e / d r e , c a s / c a s w i l d 100 - 670* - - - -p r/p,dre/+,cas/+ F 2 s e l f e d n o n - p e a r l * * 122*** 110 — 232 1 0.021 .75 .9 p e a r l 56 6>f 58 57 l l * f 121 1 P r / p r x BC. P r/p,dre/+,cas/+ x w i l d 71 68 -139 1 p e a r l - 65 72 137 1 O.OlM- .9 .95 r e c i p r o c a l BC^ w i l d 81 93 -17*+ 1 p e a r l -82 87 I69 1 0.073 .75 .9 * T o t a l progeny, out of 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 ** W i l d t y p e s and d r e and cas 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 . 38 by C a r l s o n , 1959 ) o f 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 , r uby 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 i s 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 f o r e -g o i n g , the f o l l o w i n g c r o s s e s were made t o s y n t h e s i z e t h e d o u b l e mutant p_r rji£_ f rom p_r and r u s : p l P r / p r x r u s / r u s s e l f e d ^2 r u s / r u s progeny were s e l e c t e d and s e l f e d F^ p e a r l progeny were s e l e c t e d S i n c e p_r i s e p i s t a t i c t o r u s , i t was n e c e s s a r y t o s e l e c t f o r r u s homozygotes (F2) one g e n e r a t i o n p r i o r t o s e l e c t i n g f o r p.1" . I n t h i s way a l l p e a r l progeny i n the F^ ought t o be homozygous f o r p_r r u s . T a b l e X I I . shows the r e s u l t s o f t h e s e c r o s s e s . The F-j_ progeny were d i s c a r d e d a f t e r a few days o f m a t i n g , hence no d a t a i s a v a i l a b l e from them. The F 2 progeny were counted but not s e x e d , however 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 t h e t h r e e phenotypes. T h i s g e n e r a t i o n gave the unexpected pheno-t y p i c r a t i o o f 9 p e a r l : 6 ruby s p o t : 1 W.T., i n s t e a d o f TABLE XII THE F x AND F 2 PROGENY EROM p'/p1 x rus/rus AND THE DISTRIBUTION OF RIBOFLAVIN IN THE PROGENY FROM THE SELF OF THE F0 rus/rus generation phenotype males females total expected ratio number chromatographed number witl riboflavin F i wild * * * - - -F 2 wild 11 11 9 ruby spot 73 73 3 - -pearl 118 118 k - -F 3 ruby spot 361 373 73^ - 20 20 pearl 88 102 190 - 20 20 * Data unavailable. ** Progeny were not sexed. 4-C-t h e e x p e c t e d 9 W.T.: 3 ruby s p o t : h p e a r l . I n an e f f o r t t o e x p l a i n the overabundance o f p e a r l progeny t h e r u s s t o c k c u l t u r e was checked f o r p o s s i b l e c o n t a m i n a t i o n w i t h p e a r l , but no homozygotes were found among 209 a d u l t s . However, t h i s does not e n t i r e l y e x c l u d e t h e 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 i n h e t e r o z y g o u s c o m b i n a t i o n . I n t h e F^ progeny t e n male and t e n female p e a r l and ruby spot a d u l t s were chromatographed t o check f o r t h e presence o f r i b o f l a v i n . A l l p o s s e s s e d h i g h l e v e l s . I n the n e x t g e n e r a t i o n an a d d i t i o n a l 100 i n d i v i d u a l s were checked. A l l were found t o be p o s i t i v e f o r r i b o f l a v i n . A p p a r e n t l y i n terms o f the r i b o f l a v i n phene, "p_r r u s " now appeared t o behave as p_ r u s w h i c h was i m p o r t e d from B e r k e l e y . S i n c e i n s e c t i o n C. i t was shown t h a t p_ r u s c r o s s e d w i t h p_r produced a l l W.T. progeny and t h e r i b o f l a v i n l e s s phene appeared i n h a l f o f the F 2 p e a r l progeny, i t was o f i n t e r e s t t o knox^ t h e phenotype o f t h e f o l l o w i n g crosses-, 1. "p /p r u s / r u s " x p r/p 2. " p r / p r r u s / r u s " x p/p r u s / r u s The 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 X I I I . C o n t r a r y t o e x p e c t a t i o n a l l F progeny from c r o s s 1. were W.T. and p o s i t i v e f o r r i b o f l a v i n . The progeny TABLE XIII THE PHENOTYPE AND DISTRIBUTION OF RIBOFLAVIN OF THE F x PROGENY* FROM THE CROSSES "p r/p r rus/rus x p r/p r (1.) AND "p r/p r rus/rus" x p/p rus/rus (2.) cross phenotype males females total number chromatographed number with riboflavin 1. wild pearl 263 50k 100 100 2. wild - — — _ _ pearl 82 77 159 159 159 * Progeny from reciprocal crosses were pooled as they were not appreciably different. «*2 from c r o s s 2. however, were a l l p e a r l and had r i b o f l a v i n . The u n u s u a l r e s u l t s o f th e s e e x p e r i m e n t s i n d i c a t e d 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 such as p a r a m u t a t i o n (Coe, 1966 ) , w h i c h cannot a t the p r e s e n t and from t h e s e r e s u l t s a l o n e be e x p l a i n e d . 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_s S o k o l o f f (1963) r e p o r t e d a n o t h e r p e a r l mutant w h i c h o c c u r r e d s p o n t a n e o u s l y i n the B e r k e l e y s y n t h e t i c W.T. s t r a i n . He found i t t o be a l l e l i c t o the p e a r l d e s c r i b e d by Graham (1957 )• T h i s new p e a r l he d e s i g n a t e d p s , a f t e r the p e s t i n f e s t a t i o n l a b o r a t o r y a t S l o u g h , E n g l a n d . T h i s p e a r l mutant 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 l i m i t e d However, i t was thought o f 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 phenotype o f p_s i s i n d i s t i n g u i s h a b l e from e i t h e r p_r or p_. The r i b o f l a v i n c o n t e n t o f 10 day o l d male and female imagoes were v i s u a l l y ^3 e s t i m a t e d from the chromatograms t o be about the same as t h e W.T. ( T a b l e 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 t o t e s t p_s a g a i n s t p_ and p_r : 1. p S/p s x p r / p r 2. p s / p s x p/p r u s / r u s C r o s s 1. produced 137 F-^  progeny; t h e y were a l l W.T. and p o s i t i v e f o r r i b o f l a v i n . C ross 2. l a r g e l y f a i l e d as i t o n l y produced t h r e e progeny. I t i s s u s p e c t e d t h a t a m i t e i n f e c t i o n may have been the cause. The t h r e e progeny, however, were a l l p h e n o t y p i c a l l y p e a r l and r i b o f l a v i n p o s i t i v e . The r e s u l t s o f c r o s s 1. show t h a t p r and p_s a r e not h o m o a l l e l e s . The r e s u l t s o f c r o s s 2. i n d i c a t e t h a t p_ and J J s a r e a l l e l i c , w h i c h has a l r e a d y been shown (Ackermann, 1966). F. D i s t r i b u t i o n and 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 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 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 (1939)j who demonstrated the presence o f r i b o f l a v i n i n h i g h c o n c e n t r a t i o n s i n the M a l p i g h i a n t u b u l e s o f many i n s e c t s , t h e pupae and a d u l t s were d i s s e c t e d i n i n s e c t R i n g e r and the M a l p i g h i a n t u b u l e s were chromatographed s e p a r a t e l y f r om the r e s t o f t h e body. The r e s u l t s o b t a i n e d from p_r, p_s and W.T. c o n t r o l a r e g i v e n i n T a b l e XIV. These v a l u e s were not c o r r e c t e d f o r w e i g h t as the M a l p i g h i a n t u b u l e s were d i f f i c u l t t o weigh. 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 i n W.T. and p s pupae and a d u l t s was i n the M a l p i g h i a n t u b u l e s . I n W.T. the r i b o f l a v i n c o n t e n t o f the M a l p i g h i a n t u b u l e s and t h e body i n c r e a s e d p r o p o r t i o n a l l y from pupae t o a d u l t s , b e i n g about 35% and 15% o f the t o t a l i n b o t h r e s p e c t i v e l y . I n p_s the 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 c o n t e n t from pupae t o a d u l t s was m o s t l y due t o i n c r e a s e i n the M a l p i g h i a n t u b u l e s . Thus the two genotypes o n l y d i f f e r e d i n t h e l e v e l o f r i b o f l a v i n p r e s e n t i n t h e M a l p i g h i a n t u b u l e s , w h i l e the l e v e l i n the body when th e M a l p i g h i a n t u b u l e s a r e removed was e s s e n t i a l l y t h e same. TABLE XIV DISTRIBUTION OF RIBOFLAVIN IN ug* IN THE PUPAE AND ADULTS OF W.T. p s AND p_r LINES lines number assayed source pupae + S.E. a d u l t s + S.E. W.T. 10 M a l p i g h i a n t u b u l e .106 .009 .136 .oih body .019 .002 .025 .003 E S 10 M a l p i g h i a n t u b u l e .091 .007 .192 .Olh body .020 .003 .023 .003 10 M a l p i g h i a n t u b u l e .011 .002 .003 .0006 body .029 .002 .006 .0006 * These v a l u e s were not c o r r e c t e d f o r weight i+6 The p a t t e r n o f 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 the p_ l i n e was c o m p l e t e l y r e v e r s e d compared t o W.T. and p_s. The M a l p i g h i a n t u b u l e s o f t h e pupae c o n t a i n e d o n l y 2Q% o f the 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 co n c e n t -r a t i o n i n the body was s l i g h t l y h i g h e r compared t o W.T. and £ I n the 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 , however, the 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 reduced t o near z e r o . 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 D e v e l o p m e n t a l 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 among mutant l i n e s o f E p h e s t i a have been shown by C a s p a r i and B l o m s t r a n d (1958). V i s u a l l y d e t e c t a b l e 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 mutant l i n e s i n a d u l t T r i b o l i u m have a l r e a d y been shown i n t h i s t h e s i s ( T a b l e I I . ) . The f o l l o w i n g methods were used t o examine and compare the d e v e l o p m e n t a l s t a g e s o f p_r and p_s t o W.T. Four samples each c o n t a i n i n g 50 eggs on the b a s i s o f e q u i v a l e n c e t o average imagonal body w e i g h t , were as s a y e d f o r r i b o f l a v i n . S i n c e the sexes can o n l y be d i s t i n g u i s h e d a f t e r 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 from t e n i n d i v i d u a l d e t e r m i n a t i o n s . S e p a r a t e d e t e r m i n a t i o n s based on t e n i n d i v i d u a l s o f h7 each sex were o b t a i n e d from the e a r l y and l a t e pupae and the a d u l t s , 1 day, 10 days and 30 days a f t e r e c l o s i o n . The means o f the d e v e l o p m e n t a l s t a g e s and t h e i r s t a n d a r d e r r o r s a r e g i v e n i n T a b l e XV. I n o r d e r t o b e t t e r v i s u a l i z e t h e d e v e l o p m e n t a l and sex d i f f e r e n c e s the means are graphed i n F i g u r e 3» The eggs o f p_s and W.T. c o n t a i n e d v e r y n e a r l y t h e same amount o f r i b o f l a v i n but were s i g n i f i c a n t l y d i f f e r e n t from the eggs o f p_r i n w h i c h r i b o f l a v i n was v e r y much reduc e d . I n the p_r and p_s genotypes r i b o f l a v i n was accumulated d u r i n g the l a r v a l s t a g e s a t about the same r a t e . I n the W.T. a s l i g h t r e d u c t i o n d u r i n g the l a r v a l s t a g e was f o l l o w e d by a r a p i d r i s e w h i c h t o o k p l a c e d u r i n g the s h o r t t i m e i n t e r v a l p r i o r t o p u p a t i o n . I n the main the p u p a l s t a g e s 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 the T r i b o l i u m do not eat d u r i n g t h i s s t a g e , t h i s d e c l i n e agrees w i t h the 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 cannot s y n t h e s i z e r i b o f l a v i n . The s l i g h t r i b o f l a v i n i n c r e a s e i n the p s female pupae was not s i g n i f i c a n t . I n b o t h W.T. and p_s the males had h i g h e r v a l u e s o f r i b o f l a v i n t h a n the f e m a l e s . The g r e a t e s t s e x u a l TABLE XV DEVELOPMENTAL VARIATION OF RIBOFLAVIN IN W.T., p_s AND p_r LINES MEASURED IN ug/mg FRESH WEIGHT li n e s W.T. i S.E. p_s ± S.E. p r ± S.E. eggs* .CM- .002** .04,1 .001 .014- .002 l a s t instar .0^3 .004- .051 .003 .022 .002 larvae -early pupae .059 .004-*** .051 .004- .017 .002 .04-7 .003 .04-2 .004- .023 .002 l a t e pupae .054- .004- .04-8 .00^ .017 .002 .04-7 .004- .CM .005 .023 .002 day old adults .078 .008 .050 .004- .015 .002 .065 .005 .04-5 .004- .019 .003 10 day old .110 .004- .093 .004- .005 .0005 adults .078 .004- .081 .002 .006 .0004-30 day old .131 .007 .109 .008 .005 .0007 adults .101 .007 .096 .006 .006 .0007 * Four determinations, each of 50 eggs. ** Standard error for ten observations each. ***Upper values refer to males, lower ones to females. ^9 a b c d e f . g age (days) F I G U R E 3. DEVELOPMENTAL V A R I A T I O N OF R I B O F L A V I N I N p_r, p S AND V/.T. MALES AND- FEMALES. THE DEVELOPMENTAL STAGES ARE: a, EGGS; b, LAST INSTAR LARVAE; c, E A R L Y J d, LAT E PUPAE; e, DAY OLD, f, 10 DAY OLD, g, 30 DAY OLD ADULTS 50 dimorphism was n o t e d i n the 10 day o l d a d u l t s ( t = 5.71, P < .001) and the 30 day o l d a d u l t s ( t = 3.03, P^ == .01) o f W.T. I n p_s o n l y the 10 day o l d a d u l t s showed s i g n i f i c a n t s e x u a l dimorphism f o r r i b o f l a v i n ( t = 2.86, P== .02). The p o s t - p u p a l i n c r e a s e i n t h e l e v e l o f r i b o f l a v i n was s l i g h t l y d e l a y e d i n p_s compared t o li.T, I n p_r t h e downward t r e n d o f the p u p a l stage was p r e s e r v e d u n t i l 10 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 r i b o f l a v i n remained a t a c o n s t a n t l e v e l . However, as p r e v i o u s l y n o t e d , such low c o n c e n t r a t i o n s o f r i b o f l a v i n were measured 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 . Thus a g a i n the d e v e l o p m e n t a l e f f e c t s o f p_r were markedly d i f f e r e n t from t h o s e o f p_s and W.T., w h i l e 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 two. 51 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 M a l p i g h i a n t u b u l e s . G e n e t i c 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 o f r i b o f l a v i n i n E p h e s t i a have been shown by C a s p a r i and B l o m s t r a n d (1958) and i n D r o s o p h i l a by B e a d l e (1937) and Hubby (1962). Kikkawa (1953) 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 M a l p i g h i a n t u b u l e s i n .Bombyx t o the p l e i o t r o p i c e f f e c t s o f genes c a u s i n g mutant eye c o l o r p i g m e n t s . T h i s t h e s i s has a l r e a d y shown t h a t v i s u a l l y 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 the amount o f r i b o f l a v i n between mutant l i n e s ( T a b l e I I . ) . These v i s u a l 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 eye c o l o r mutant l i n e s (EL d r e c a s , EL r u s . p l o d . p_S? EL1"5 e s , r u s , r , dp_e ) and i n two W.T. i n b r e d l i n e s (CFI - 1 and CFI - 7 ) . C o n c u r r e n t l y t h e p i g m e n t a t i o n o f the M a l p i g h i a n t u b u l e s was a l s o examined. The r e s u l t s a r e g i v e n i n T a b l e X V I . I n d i v i d u a l s o f the p e a r l phenotype 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 . The p i g m e n t a t i o n o f t h e M a l p i g h i a n t u b u l e s o f n o n - p e a r l eye c o l o r mutants ranged from brown i n dpe and e s , t h r o u g h s p e c k l e d brown and b l a c k i n r and r u s , t o pronounced b l a c k i n t h e two W.T. i n b r e d l i n e s . W i t h the e x c e p t i o n o f p r , the r i b o f l a v i n c o n c e n t -r a t i o n was always h i g h e r i n the males th a n i n the 52 TABLE XVI THE MEANS* OF RIBOFLAVIN CONCENTRATION IN ug/mg FRESH WEIGHT FOR MALES AND FEMALES OF 11 LINES (THEIR AVERAGE COMPARED BY DUNCAN'S NEW MULTIPLE RANGE TEST) At© THE COLOR OF MALPIGHIAN TUBULES l i n e males females average c o l o r o f M a l p i g h i a n t u b u l e s p_ d re cas .133 .097 . H 5 a * * c o l o r l e s s p_ r u s .105 .085 .095 b c o l o r l e s s p_ l o d .112 .086 .099 b c o l o r l e s s p_s . .093 .081 .087 b c o l o r l e s s ^ r *** .005 .006 .006 c o l o r l e s s es .098 .081 .089 b brown r u s .083 .074- .078 b s p e c k l e d b l a c k r .059 . . 0 4 - 7 .053 d s p e c k l e d brown t o b l a c k dpe .051 .0*+2 , 0 4 - 6 d brown CFI - 1 .110 .078 . 0 9 ^ b l a c k CFI - 7 .077 .054- ,065 c b l a c k * Means a r e based on t e n i n d i v i d u a l s per sex. ** Any two means h a v i n g the 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. *** Not 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 low 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 demonstrated. 53 f e m a l e s . The p_r females had s l i g h t l y more r i b o f l a v i n 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 e a r l i e r , t h e s e v a l u e s must be viewed w i t h some c a u t i o n . The 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 mutants 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 the eyes and the M a l p i g h i a n t u b u l e s . T h i s r e l a t i o n s h i p d i d not e x i s t i n the p e a r l eyed mutant l i n e s . The e x t e n t of v a r i a t i o n between the l i n e s and between t h e s e x e s , as w e l l as the p o s s i b i l i t y of a l i n e by sex i n t e r a c t i o n was det e r m i n e d v i a the a n a l y s i s o f v a r i a n c e . The p_r l i n e was e x c l u d e d from the a n a l y s i s o f v a r i a n c e as i t appeared 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 from the r e s t o f the l i n e s has been demonstrated. The r e s u l t s o f the a n a l y s i s o f v a r i a n c e i s g i v e n i n T a b l e X V I I . H i g h l y s i g n i f i c a n t d i f f e r e n c e s e x i s t e d between l i n e s . The d i f f e r e n c e between the sexes was a l s o h i g h l y s i g n i f i c a n t , and t h e r e was no i n d i c a t i o n o f a l i n e by sex i n t e r a c t i o n . Duncan's New M u l t i p l e Range T e s t was used t o demonstrate the e x t e n t o f l i n e d i f f e r e n c e s . The r e s u l t s a r e shown i n T a b l e X V I . Any two means hav i n g the same l e t t e r a r e 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. W i t h t h e e x c e p t i o n o f the p_ d r e cas l i n e 9+ TABLE X V I I ANALYSIS OF VARIANCE OF TOTAL RIBOFLAVIN IN ug/mg FRESH WEIGHT IN 10 LINES OF T. confusum sou r c e o f v a r i a n c e d . f . mean squares between l i n e s 9 .00868** between sexes 1 .01802** l i n e x sex 9 .000M+ i n d i v i d u a l s / l i n e x sex 180 .00031 t o t a l 199 ** H i g h l y s i g n i f i c a n t (P ^ . 0 1 ) 55 t h e means o f the - 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 were not s i g n i f i c a n t l y d i f f e r e n t from each o t h e r and from the means o f e s , r u s and C F I - 1 . I t was o f i n t e r e s t t o n o te t h a t the means o f the two i n b r e d l i n e s , w h i c h o r i g i n a t e d from the same p a r e n t s , were s i g n i f i c a n t l y d i f f e r e n t . G e n e t i c 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 been shown b e f o r e f o r r e d b l o o d c e l l a n t i g e n s i n t h e domestic f o w l ( B r i l e s , 1 9 ^ 9 ) . 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 of the r i b o f l a v i n t r a i t . P r i o r t o c o n d u c t i n g 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 o f a s p e c i e s , p a r t i c u l a r l y w i t h r e s p e c t 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 i m p o r t a n t t o know o f the e x t e n t o f d i f f e r e n t s o u r c e s o f p h e n o t y p i c v a r i a t i o n ( L e r n e r , 1 9 5 8 ) . T h e r e f o r e , the r e l a t i o n s h i p between an i n d i v i d u a l ' s t o t a l r i b o f l a v i n c o n t e n t and i t s body w e i g h t was a s s e s s e d . I n a d d i t i o n the h e r i t a b i l i t y o f the r i b o f l a v i n t r a i t was used t o dete r m i n e the p r o -p o r t i o n o f the 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 the average e f f e c t s o f the genes w h i c h i n f l u e n c e d t h i s t r a i t . 56 The s i m p l e c o r r e l a t i o n c o e f f i c i e n t ( r ) between i n d i v i d u a l body weight and 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 males and 90 f e m a l e s . The male and female r v a l u e s as w e l l as the c o e f f i c i e n t o f o d e t e r m i n a t i o n a r e shown i n T a b l e X V I I I . TABLE X V I I I CORRELATION OF 10 DAY ADULT WEIGHT WITH RIBOFLAVIN CONCENTRATION IN THE p_ l o d MARKED STOCK males females s i m p l e c o r r e l a t i o n c o e f f i c i e n t - ' - ( r ) • 32** c o e f f i c i e n t o f d e t e r m i n a t i o n ( r 2 ) 10% 23^ 1 89 degrees 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 p e r c e n t a g e 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 can be accounted f o r by t h e v a r i a b i l i t y i n body weight ( r = 10$ i n males; 23$ i n f e m a l e s ) . However, a h i g h l y s i g n i f i c a n t a s s o c i a t i o n e x i s t s between the t r a i t s f o r b o t h s e x e s . C o n s e q u e n t l y 57 t h e measurement o f r i b o f l a v i n on a per body w e i g h t b a s i s was not i m p e r a t i v e f o r males, but p r o v i d e s more a c c u r a t e d a t a i n the case o f f e m a l e s . The progeny o f p_ l o d (random b r e d f o r over 36 g e n e r a t i o n s ) were used t o e s t i m a t e t h e h e r i t a b i l i t y o f the r i b o f l a v i n t r a i t . T h i r t e e n males were each mated t o f o u r f e m a l e s . A f t e r f i v e d a y s , t h e males were removed and the females were each d i s t r i b u t e d i n t o v i a l s c o n t a i n i n g f r e s h medium. A f t e r 10 days the females were removed and d i s c a r d e d . Three s i r e groups each h a v i n g t h r e e dams Mere chosen from the s u c c e s s f u l m a t i n g s . W h i l e i t was r e a l i z e d t h a t the number of s i r e s t e s t e d was q u i t e low, i t was assumed t h a t the h e r i t a b i l i t y e s t i m a t e s s u b s e q u e n t l y o b t a i n e d would not be u n d u l y a f f e c t e d . Ten days a f t e r e c l o s i o n the progeny were i n d i v i d u a l l y weighed, chromatographed and r i b o f l a v i n was measured by t h e f l u o r o m e t r i c method. Ten male and t e n female progeny from each s i r e / d a m mating were a n a l y z e d . The g e n e r a l method o f Cockerham (1956; a f t e r B e c k e r , 196*+) was used f o r the s t a t i s t i c a l a n a l y s i s . S t a n d a r d e r r o r s o f h e r i t a b i l i t y e s t i m a t e s were 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 i j k = u + s i + d i j + e i j k where ; : the record of the k^*1 progeny of the j ^ * 1 dam mated to the i ^ * 1 s i r e ; u : the common mean; s^ : the e f f e c t of the i t h s i r e ; d.^ : the e f f e c t of the j t h dam mated to the 1^ s i r e , and ej j k : the unexplained environmental and genetic d e v i a t i o n s a t t r i b u t a b l e to the k t h progeny from the j ^ * 1 dam mated to the 1^ s i r e . The average e f f e c t s of the r i b o f l a v i n t r a i t for' the s i r e s , the dams and the sexes,- can be assessed from the means which are presented i n Table XIX. The means of the male progeny were c o n s i s t e n t l y higher than those of the female, progeny. The average c o n t r i b u t i o n to the progeny was the same f o r s i r e A and s i r e B. With the exception of the progeny from the s i r e A - dam 1 f a m i l y , the progeny of the dams w i t h i n s i r e G had on the average the lowest r i b o f l a v i n v a l u e s . Hence the progeny of s i r e C were a l s o much lower compared to the progeny of s i r e s A and B. To determine whether the sexual dimorphism e x h i b i t e d by the 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 of variance was performed (Table XX.). 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 sire mean sire B sire mean sire C sire mean dams sex 1 2 3 1 2 3 1 2 3 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 .070 .089 .074-dam mean .090 .112 .121 .098 .111 .115 .076 .081 .097 sire mean .108 .108 .085 * M = males, F = females. vn so 60 The F - t e s t f o r the sex e f f e c t was h i g h l y s i g n i f i c a n t ( P ~ . 0 l ) , c o n f i r m i n g the above o b s e r v a t i o n s . TABLE XX ANALYSIS OF VARIANCE BY SEX OF THE TOTAL RIBOFLAVIN IN ug/mg FRESH WEIGHT IN THE p/p l o d / l o d MARKED LINE s o u r c e o f v a r i a n c e d . f . mean squares sex „ 1 O . O 3 6 0 6 * * e r r o r 178 0.00061 t o t a l 179 ** P ^  . 0 1 Due t o the h i g h l y s i g n i f i c a n t s e x u a l dimorphism 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 as making a combined e s t i m a t e . The a n a l y s i s o f v a r i a n c e o f the male progeny, female progeny, combined sexes as w e l l as t h e e x p e c t e d mean squares a r e p r e s e n t e d i n T a b l e X X I . The F - t e s t showed t h e dam c o n t r i b u t i o n was h i g h l y s i g n i f i c a n t i n each o f the t h r e e a n a l y s e s . The s i r e c o n t r i b u t i o n was not TABLE XXI 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 vari a n c e mean squares male female d.f. progeny • progeny combined expected mean squares' s i r e s dams/sires i n d i v i d u a l s / dams/sires t o t a l 2 6 81 89 A B 0.00785 0.00398 0.01070 <3?W .+ K x (52D + K 2 <S2S 0.00196** 0.00180** 0.00308** <52W + K x <52D 0.0004-3 0.00034- 0.00061 <52W 1 When the sexes are analyzed separately, Kj_ = 10, K 2 = 30. When the sexes are analyzed together, K-j_ = 20, K 2 = 60. ** P ^  .01. A For the combined a n a l y s i s i t i s 171; and B, 179. 62 s i g n i f i c a n t i n any of the three analyses. The great amount of v a r i a t i o n c o n t r i b u t e d by the dams may have masked the s i r e e f f e c t . The estimates of the components of variance are presented i n Table X X I I . The s i r e component, tf£ estimates £ <j£ ( a d d i t i v e 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 a d d i t i v e e p i s t a t i c i n t e r a c t i o n s . In the absence of the l a t t e r e f f e c t s , i+&]r provides an estimate of the a d d i t i v e genetic v a r i a n c e . The dam component <s^ estimates ^6"% + £ d o m i n a n c e variance) plus f r a c t i o n a l higher order e p i s t a t i c terms, plus (variance due to maternal e f f e c t s ) . Thus again 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 , + includes 6"% , tj^-v,. and fc5« (Falconer, i960). For each a n a l y s i s the s i r e component of variance ( 6%) c o n t r i b u t e d 25$ i n the male, 13$ i n the female and 71% i n the combined sexes towards the t o t a l v a riance f o r the t r a i t . Since the mean squares from which these were estimated were not s i g n i f i c a n t , the percentage values i n d i c a t e d d i f f e r e n c e s that were more e a s i l y r e s o l v a b l e i n terms of the t o t a l v a r i a t i o n i n the three groups of progeny. The high estimate obtained when the sexes were combined (71$) was probably due to TABLE XXII COMPONENTS OF VARIANCE ESTIMATED FROM THE ANALYSIS OF VARIANCE OF TOTAL RIBOFLAVIN IN THE p/p lod/lod MARKED LINE component male progeny female proeenv combined value % t o t a l value % t o t a l value % t o t a l i s 0.000196 25.16 0.000073 13.06 0.000615 71.10 <S2D 0.000153 19.64- 0.00014-6 26.12 0.000123 14-. 22 0.0004-30 55.20 0.0003^0 60.82 0.000127 14-. 68 <52T 0.000779 100.00 0.000559 100.00 0.000865 100.00 the marked s e x u a l dimorphism. D i f f e r e n c e s among t h e dams c o n t r i b u t e d 20$, 26$ and 1^ $ i n t h e male, female and 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 f a i r l y r e l i a b l e e s t i m a t e s o f h e r i t a b i l i t y may be o b t a i n e d from them. S i n c e e p i s t a t i c i n t e r a c t i o n s can u s u a l l y be i g n o r e d w i t h o u t 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), an e s t i m a t e o f h e r i t a b i l i t y i n the narrow -sense from the s i r e component i s g i v e n by M - C T ^ / ^ (where &\ i s the t o t a l p h e n o t y p i c v a r i a n c e , o r ^s"*'6»4'^. (Cockerham, 1956). A second e s t i m a t e , from the dam component, k<Sx>/6'r ±s b i a s e d by dominance and m a t e r n a l e f f e c t s , a l t h o u g h as d i s c u s s e d above i t may be more r e l i a b l e i n the p r e s e n t e x p e r i m e n t , due t o t h e g r e a t e r number o f dams t e s t e d . F o r each a n a l y s i s the e s t i m a t e s o f h e r i t a b i l i t y and t h e i r s t a n d a r d e r r o r s f o r the male, female and the combined sexes a r e g i v e n i n T a b l e X X I I I . I n g e n e r a l t h e h e r i t a b i l i t y e s t i m a t e s were h i g h e r when the sexes were a n a l y s e d s e p a r a t e l y , a l t h o u g h the combined sex a n a l y s i s gave more comparable e s t i m a t e s f o r the s i r e and dam components: 0.59 and 0.57 r e s p e c t i v e l y . M a t e r n a l and dominance e f f e c t s i n t h e dam component TABLE X X I I I ESTIMATES OF HERITABILITIES AND STANDARD ERRORS OF THE RIBOFLAVIN TRAIT IN THE p/p l o d / l o d MARKED LINE h e r i t a b i l l t v ± s t a n d a r d e r r o r  component male progeny female progeny combined s i r e 1.00* + 1.35 0 .5 l + 0.34- 0.59 + O.83 dam O.78 + 0.20 1.00 + 0.26 0.57 1 0.4-1 s i r e + dam O.89 + 0.06 0.77 + 0.31 0.58 + O.63 * E s t i m a t e s g r e a t e r than 1.00 were assumed to be e q u a l t o 1.00 ON 66 p r o b a b l y caused the e s t i m a t e from t h e female progeny to be e l e v a t e d . 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 r i b o f l a v i n i n t h e males and the low number o f s i r e s used p r o v i d e s a p o s s i b l e e x p l a n a t i o n f o r the h i g h h e r i t a b i l i t y based on the s i r e component w h i c h was o b t a i n e d i n the a n a l y s i s o f t h e male progeny. S i n c e 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 and the dam components can o n l y be c o n s i d e r e d e q u a l i n the combined a n a l y s i s , i t i s o n l y here t h a t the a s s u m p t i o n o f c T j « ( B e c k e r , 196*+) was met f o r v a l i d l y c ombining the h e r i t a b i l i t y e s t i m a t e s o b t a i n e d s e p a r a t e l y from the s i r e s and t h e dams. T h e r e f o r e from the combined a n a l y s i s the h e r i t a b i l i t y e s t i m a t e o f 0 .58 may be t a k e n as the b e s t e s t i m a t e . B r o a d l y i n t e r p r e t e d the h e r i t a b i l i t y e s t i m a t e s i n the male progeny i n d i c a t e d m o s t l y a d d i t i v e g e n e t i c v a r i a n c e , whereas the h e r i t a b i l i t y e s t i m a t e s i n t h e female 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 e f f e c t s . The h e r i t a b i l i t y e s t i m a t e s o b t a i n e d from t h e combined a n a l y s i s were e s s e n t i a l l y the same and t h e r e was no e v i d e n c e f o r dominance e f f e c t s or m a t e r n a l e f f e c t s . A v a l i d c o m p a r i s o n o f the h e r i t a b i l i t i e s between t h e sexes may be d i f f i c u l t t o i n t e r p r e t on account o f t h e e x t e n s i v e s e x u a l dimorphism f o r t h i s t r a i t . DISCUSSION I t has been assumed i n t h i s t h e s i s t h a t t h e y e l l o w f l u o r e s c e n t substance s e p a r a t e d by chromatography was l a r g e l y r i b o f l a v i n . T h i s a s s u m p t i o n was based on i d e n t i f i c a t i o n v i a s p e c t r o p h o t o m e t r i c , c h r o m a t o g r a p h i c and e l e c t r o p h o r e t i c methods. A l l d e v e l o p m e n t a l st a g e s o f i n d i v i d u a l s homozygous f o r a r e c e s s i v e p e a r l eye c o l o r gene, p_r ( f o r m e r l y c a l l e d p_, but renamed) were 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 r e d u c t i o n i n r i b o f l a v i n c o n t e n t i n the body and i n the M a l p i g h i a n t u b u l e s . The d i f f e r e n c e i n r i b o f l a v i n c o n t e n t o f W.T. and p_r a d u l t s e s t a b l i s h e d by the c h r o m a t o g r a p h i c method was so l a r g e t h a t i t may be r e g a r d e d as r e a l . The c r o s s between p_r and W.T. gave a g r e a t l y d i s t o r t e d 3>1 r a t i o i n the F 2 progeny. T h i s suggested a r e d u c t i o n i n f i t n e s s a s s o c i a t e d w i t h t h e p_r gene. S i n c e t h i s e f f e c t was not observed i n c r o s s e s i n v o l v i n g t h e p_ gene, i t may be 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 f i t n e s s was a s s o c i a t e d w i t h t h e r i b o f l a v i n l e s s phene. The genes, £ and p_s appeared t o be i d e n t i c a l a l l e l e s as n o t e d by Ackermann ( 1 9 6 6 ) . The complementation 68 demonstrated between p_r a n d p_, and between £ r and p_s was 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 an e a r l i e r 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 I n f o r m a t i o n B u l l e t i n , v o l . 6, 1963. However, i t may be t h a t S o k o l o f f was u s i n g o n l y p_ and £ s , b e i n g unaware o f the e x i s t e n c e o f the p r l i n e . The e f f e c t o f d i f f e r e n t environments c o u l d p r o v i d e a p o s s i b l e a l t e r n a t i v e e x p l a n a t i o n . Environment dependent com p l e m e n t a t i o n has been demonstrated i n microorganisms (Garen and Garen, 1963; S c h l e s i n g e r and L e v i n t h a l , 1963; Fincham and S t a d l e r , 1965; Fan, e t a l . , 1966). The 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 were 30° C and 70% r e l a t i v e h u m i d i t y compared t o 32° C and 73% r e l a t i v e h u m i d i t y i n t h i s i n v e s t i g a t i o n . I n a d d i t i o n h i s f l o u r was e n r i c h e d w i t h brewer's y e a s t , r a t h e r t h a n T o r u l a y e a s t . 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 and L e v i n t h a l , 1963). F i r s t l y , i f the 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 r e g i o n s o f the g e n e t i c map ( i n t e r - c i s t r o n i c ) t h e n t h e y w i l l always complement (B e n z e r , 1957)• T h i s s i t u a t i o n can be e x p l a i n e d i f i t i s assumed t h a t each c i s t r o n d e t e r m i n e s t h e s t r u c t u r e o f a s e p a r a t e p o l y p e p t i d e 69 chain. The "normal" effect would be produced i f each polypeptide chain was made in 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 in the same biochemical pathway. In the second type of complementation (intra-cistronic) the two mutations are in the same cistron 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 i s not possible to distinguish between inter- and intra-cistronic complementation. However, i t was certain that p_r was complementary to p_ and p_s. Brink (1956) demonstrated that regular heritable changes in the function of a gene can be directed by an a l l e l e . In 1958 he designated this change as a paramutation. Paramutation denotes the heritable nature of the change and the origination through heterozygous association of a sensitive (paramutable) a l le le with an inducing (paramutagenic) allele.. This subject was recently reviewed by Coe (1966). The results, involving the apparent conversion of p_r to p_ in 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 verify these results . While paramutation has not yet been demonstrated in animals, i t is not unreasonable to assume i t exists. The relationship between the time of disappearance of r iboflavin in jj r and the time of deposition of pigments in the W.T. Malpighian tubules and eyes was of interest. A relationship was suggested by the fact that the time during which riboflavin 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 in the Malpighian tubules. The p_r gene reduced the r iboflavin content of the tubules, while p_ and p_s did not have the sarnie effect. A similar situation exists in Ephestia where the gene wa functions in 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 in the eyes and Malpighian tubules of Latheticus oryzae, a closely related species to Tribolium., are probably ommochromes. 71 The genetic control of the synthesis of this group of pigments has been partly documented in Ephestia (Hadorn and Kuhn, 19535 Caspari, 1955; Viscont ini , et a l . , 1956; Reisener-Glasewald, 1956; Ziegler, 196l) and Bombyx (Kikkawa, 1953; Ziegler, 196l). In addition in nearly every case mutants affecting the amount of ommochrome pigment have also been shown to affect the amount of r ibof lavin. The poss ib i l i ty that r iboflavin may be involved in 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 rat . If riboflavin deficient rats are fed tryptophan, urinary excretion of anthranilic acid, kynurenic acid, xanthurenic acid and occassionally of kynurenin and 3-hydroxykynurenin is observed. These author suggested that r iboflavin deficiency may affect the steps leading from kynurenin to 3-hydroxykynurenin. According to a summary by Gilmour (196l), these steps occur in the chain of reactions leading to ommochrome pigment. The riboflavin content and the degree of pigmentation of the Malpighian tubules and eyes appeared to be related in the non-pearl eye color mutant l ines . However in the p_r versus the p_ and p_s l ines no such relationship was noted. 72 The question arises whether the lack of r iboflavin in the p_r adults was due to an inhibit ion of i t s formation, to i t s destruction or to an interference with i t s absorption. Nutritional experiments have shown that Tribolium confusum cannot survive in the absence of r ibof lavin, 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 unlikely that these products were formed i f r iboflavin was indeed degraded. However, the poss ib i l i ty of bacterial degradation of r iboflavin to non-fluorescent compounds by a symbiotic bacteria, such as Pseudomonas riboflavinus. so far not demonstrated in Tribolium. may exist (Harkness, 196'+). This poss ib i l i ty would have to be predicated on an incompatibility system of P. riboflavinus with a l l other strains used. The poss ib i l i ty that the lack of r ibof lavin storage in the Malpighian tubules and the reduced amount of r iboflavin in the body of p_r mutants may be due to an inab i l i ty to retain this vitamin. This was suggested by the findings of Cowan, et a l . , 1964- , 1966, 1966a) in the domestic fowl. The concentration-of free 73 r iboflavin in the blood of a mutant strain of chickens homozygous for rd, fai led to increase as i t did in normal chickens at the onset of egg production (Maw, 195^ ; Boucher et a l . , 1959). Isotopic tracer studies have shown that free r iboflavin was absorbed normally from the digestive tract (Cowan et a l . , 196^ ). It thus appeared that the gene conditioned event which prevented accumulation of riboflavin in the blood must take place after absorption. The poss ibi l i ty that r iboflavin was destroyed in 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 in the mutant laying hen, the recessive gene expressed i t s e l f by altering the renal reabsorptive mechanism for free r ibof lavin. As a result of this alteration, the intact vitamin was rapidly excreted via the urine, hence i t fai led to accumulate in the blood. In the estimation of her i tab i l i ty of the amount of stored r iboflavin in the p_ lod l ine i t was assumed that the epistatic contributions to variance were relat ively unimportant (Falconer, I960). As far as i t can be determined the strain 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 variation in such populations. In natural populations which are continually subject to gene flow in the form of migration, a greater amount of epistasis would interact in many combinations. In closed populations there would be less need for such genes, and in fact, as Dobzhansky and Spassky (1962) have shown, closed populations can evolve overdominance. The design used for the estimation of her i tab i l i ty is one of a number of methods available for this purpose (see Falconer, i960). The hierarchical design (a f u l l and half-sib relationship) provides a more accurate estimate of her i tab i l i ty than the parent-offspring regression methods. In addition an indication of the non-additive genetic variance and maternal effects can be noted. The differences in the her i tab i l i ty 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. In general, the significant differences shown between the mutant l ines and especially between the two W.T. inbred lines and the high her i tab i l i ty estimates suggest that the amount of r iboflavin present in T. confusum is probably controlled by a small number of genes. 75 Unusual sex ratios were obtained in many of the crosses in that more females than males were produced. From a total of &%5l Tribolium, k-06l were males and ^390 were females. This was a highly significant deviation from the expected 1:1 ratio ( P ^ .005). There was no indication that the pearl eye mutation was a sex-linked character, nor "was there any indication that the riboflavinless t ra i t was associated with this divergent sex rat io . Similar sex-ratio differences (in pearl eyed stocks) in favour of females were evident in the data of Graham (1957) and were also noted in T. castaneum by Park ( 1 9 3 7 ) . The poss ib i l i ty of genetic distortion of the sex rat io , as reported for other insects is not unlikely (Peacock and Erickson, 1965; Hickey and Craig, 1966). CONCLUSIONS AND SUMMARY The identity of a yellow fluorescent substance from Tribolium confusum, with riboflavin was shown by the independent methods of spectrophotometry, chromatography and electrophoresis. Two decomposition products of r ibof lavin as well as two conjugated flavines FMN and FAD, were ident i f ied. The genetic control of r iboflavin and i t s relation-ship to some mutant eye color genes in the pleiotropic hierarchy of gene effects was investigated in a series of experiments involving more than 8000 Tribolium from 11 genetically different l ines . A l l developmental stages of individuals homozygous for a recessive pearl eye color gene, p_r (formerly called p_, but renamed) were associated with a significant, heritable reduction in r iboflavin content in the body. This reduction was principal ly in the Malpighian tubules. In a l l of the other lines tested the vitamin was accumulated in the• tubules in re lat ively high concentrations. The normal a l l e le of this gene was found to be incompletely dominant with respect to the r iboflavin phene. Changing the residual genotype of p_r by means of two generations of recurrent selection did not detectably alter i t s expression. 77 Two al leles of pearl (p_ and p_s) were phenotypically identical to p_r, except at the level of the r iboflavin phene. Crosses of these al le les with p_r were shown to complement. The results , involving the apparent conversion of p_r to p_ in the synthesis of the double mutant " p r rus" were indicative of a paramutation-type mechanism. Genetic differences in r iboflavin content and the pigmentation of Malpighian tubules were shown. The r ibof lavin 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_s, p_) always had. colorless Malpighian tubules. The depth of pigmentation of adult eyes in the mutant l ines was roughly proportional to the.degree of pigmentation of the Malpighian tubules. Sexual dimorphism for r iboflavin content was observed in a l l lines tested. The quantitative parameters of variation of the r iboflavin phene were examined and i t s her i tab i l i ty 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. 1966 . Personal communication. Department of Genetics, University of Cal i fornia , Berkeley. Association of Of f i c i a l Agricultural Chemists. I960, p. 658-660. AOAC, Washington 4-, D.C. Beadle, G.W. 1937* The inheritance of the color of Malpighian tubes in Drosophila melanogaster. Amer. Nat., 7 1 : 2 7 7 - 2 7 9 . Becker, W.A. 1964-. Manual of Procedures in Quantitative  Genetics. Washington State University, Pullman, Washington. Benzer, S. 1 9 5 7 . In Symposium on Chemical Basis of Heredity, ed. by W.D. McElroy^and B. Glass, p. 70• Baltimore, The Johns Hopkins Press. Bernier, P.E. and Cooney, T. 195*+. Black down color and r ibof lavin deficiency in embryos of the domestic fowl. Proceed. World's Poult. Congr., 1 0 : 6 6 - 7 1 . Bodine, J .H . and Fitzgerald, L.R. 194-7. Riboflavin and other fluorescent compounds in a developing egg (Orthoptera). Physiol. Z o o l . , 20 : 14-6-160. Boucher, R .V. , Buss, E.G. and Maw, A . J . G . 1959 . Physiological characteristics associated with a mutant gene in chickens that causes a deficiency of r iboflavin 2 . Blood. Poult. S c i . , 3 8 : 1 1 9 0 . Br i le 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 . , 28 : 759-Brink, R.A. 1956 . A genetic change associated with the R locus in maize which is directed and potentially reversible. Genetics, 4-1: 8 7 2 - 8 8 9 . , 1958 . Paramutation at the R locus in maize. Cold Spr. Harb. Symp. Quant. B i o l . , 23 : 3 7 9 - 3 9 1 . 79 Burgess, L . E . 19*+9. A preliminary quantitative study of pterine pigments in the developing egg of the Grasshopper, Melanoplus d i f ferent ia l i s . Arch. Biochem., 20: 3*+7-355. Buss, E . G . , Boucher, R.V. and Maw, A . J . G . 1959. Physiological characteristics associated with a mutant gene in chickens that causes a deficiency r ibof lavin. 1. Eggs and embryos. Poult. S c i . , 38: 1192. Caspari, E. 1955. On the pigment formation in the testis sheath of Rt and rt of Ephestia ktihniella.• B i o l . Zentr. , 7^:"1^85-602. and Blomstrand, I. 1958. A yellow pigment in the testis of Ephestia: Its development and i t s control by genes. Genetics, *+3: 679-69*+. Carlson, E.A. 1959- Comparative genetics of complex l o c i . Quart. Rev. B i o l . , 3*+: 33-67. Cer le t t i , P. and S i l iprandi , N. 1955• Electrophoretic separation and determination of f lavins. Biochem. Jour., 61: 32>+-328. Charconnet-Harding, F . , Dalgiesh, C.E. and Neuberger, A. 1953. The relation between r iboflavin and tryptophan metabolism, studies in the rat . Biochem. Jour., 53: 513-521. Cockerham, C.C. 1956. Analysis of qualitative gene action. Brookhaven Symposia i n Biology, 9: 53-68. Coe, E . H . , Jr . 1966. The properties, or ig in , and mechanism of conversion-type inheritance at the B locus in maize. Genetics, 53 : 1035-1063. Cowan, J.W., Buss, E.G. and Boucher, R.V. 196l. Physiological characteristics associated with a mutant gene in chickens that causes a deficiency of r iboflavin 3« Total excreta and urine. Abstracts of papers presented at 50th Annual Meeting of the Poult. S c i . , Association, p. 23. , Boucher, R.V. and Buss, E.G. 196la. Physiological characteristics associated with mutant gene in chickens that causes deficiency of r iboflavin h. The effect of estrogen. Ibid. 80 Cowan, J.W., Boucher, R.V. and Buss, E.G. 1964-. Riboflavin u t i l i z a t i o n by a mutant strain of Single Comb White Leghorn Chickens. 2. Absorption of radioactive r iboflavin from the digestive tract. Poult. S c i . , 4-3: 172-174-. , Boucher, R.V. and Buss, E.G. i 9 6 0 . Riboflavin u t i l i z a t i o n by a mutant strain of Single Comb White leghorn Chickens. Poult. S c i . , 4-5: 536-538. , . 1966a. Riboflavin u t i l i z a t i o n by a mutant strain of Single Comb White Leghorn Chickens. Poult. S c i . , 4-5: 538-54-1. Crenshaw, J.W. and Lerner, I.M. 1964-. Productivity of inbred strains of Tribolium confusum and Tribolium castaneum. Ecology, 4-5: 697-705. Dawson, P.S. 1964-a. Genetic homeostasis and developmental rate in Tribolium. Genetics, 51: 873-885. . 1964-b. The use of assortative mating for her i tab i l i ty estimation. Genetics, 4-9: 991-994-. . 1965. Estimation of components of phenotypic variance for developmental rate in Tribolium. Heredity, 20: 4-03-4-17. Dickerson, G.E. i960. Techniques for research in quantitative animal genetics. American Society of Animal Production Publication. Dobzhansky, Th. and Spassky, B. 1962. Genetic dr i f t and natural selection in experimental populations of Drosophila pseudoobscura. Proc. Nat. Acad. S c i . , k8: 1^8-157. Doty, A . E . 1965. An improved dietary supplement for rearing T. confusum. Tribolium Information B u l l . , 8: 73. Drilhon, A. and Busnel, R. 1939* Sur la presence et l a teneur en flavine des tubes de Malpighi des insectes. C R . Acad. S c i . , 208: 839-84-1. Falconer, D.S. i960. Introduction to quantitative  genetics. The Ronald Press Co. , N.Y. p. 3b% 81 Fan, D.P . , Schlesinger, M . J . , Torr iani , A . , Barrett, K.J . and Levinthal, C. 1966. Isolation and characterization of complementation products of Escherichia c o l i alkaline phosphatase. J . Mol. B i o l . , 15.: Finchham, J .R.S. and Stadler, D.R. 1965. Complementation relationships of Neurospora am mutants in relation to their formation of abnormal varieties of glutamate dehydrogenase. Genet. Res., 6: 121-129. Fraenkel, G. and Blewett, M. 19!+7. The importance of fo l ic acid and unidentified members of the vitamin B complex in the nutrit ion of certain insects. Biochem. Jour., *+l: 1+69-1+75. Gilmour, D. 196I. The Biochemistry of Insects. Academic Press, N.Y. Graham, W.M. 1957. Pearl eye in the confused flour beetle, Tribolium confusum Duval (Tenebrionidae) Ent. Mon. Mag., 93: 73-75-Hadorn, E. and Mitchel l , H.K. 195l. Properties of mutants of Drosophila melanogaster and changes during development as revealed by paper chromatography. Proc. Nat. Acad. S c i . , 37: 650-665. and Kuhn, A. 1953- Chromatographysche und fluorometrische Untersuchungen zur biochemischen Polyphanie von Augenfarb-Genen bei Ephestia kuhniella. Z. Naturforsch., 8b: 582-589. . 195*+• Approaches to the study of biochemical and developmental effects of mutations. Caryologia, Suppl. 6: 326-337. . 1956. Patterns of biochemical and developmental pleiotropy. Cold Spr. Harb. Symp. Quant. B i o l . , 21: 363-373. and Schwinck, I. 1956. Fehlen von Isoxanthopterin und Nicht-Autonomie in der Bildune der roten Augen-pigmente bei einer Mutante (rosy2) von Drosophila  melanogaster. Z. Ind., Abst. Vererb., 87: 528-553* Hais, I.M. and Pecakova, L. 191+9« Paper part i t ion chromatography of r iboflavin decomposition products. Nature, 193: 768. 82 Harkness, D.R., Tsai , L and Stadtman, E.R. 1964-. Bacterial degradation of r ibof lavin. V. Stoichiometry of r iboflavin degradation to..* oxamide and other products, oxidation of C labelled intermediates and isolat ion of the Pseudomonad effecting these transformations. Arch. Bioch. Bioph., 108: 323-333. Hickey, A.W. and Craig, G.B. 1966. Genetic distortion of sex ratio in a mosquito Aedes aegypti. Genetics, 53: 1177-1196. Ho, F.K. and Sokoloff, A. 1962. Occurrence of mutations in natural populations of Tribolium. Tribolium Inform. B u l l . , 5- 29-30 Hubby, J .L . 1962. A mutant affecting pteridine metabolism in Drosophila melanogaster. Genetics, 4-7: 109-114-. Kikkawa, H. 1953• Biochemical genetics of Bombyx mor'i (Silkworm). Adv. Genet., 5: 107-14-0. Kuhn, A. and Berg, B. 1956. Uber die Kombination von wa (weissaugig) und biochemica bei Ephestia Kuhniella. Z .-Ind. Abst. Vererb., 87: 335-337. Lerner, I.M. 1958. The Genetic Basis of Selection, p. 298. Wiley, N.Y. McDonald, D.J . and Peer. N.J. i960. Natural selection in experimental populations of Tribolium. I. Preliminary experiments with population cages. Genetics, 4-5: 1317-1333-. I9 6 I . A genetic analysis of the "eyespot" mutation of Tribolium confusum. Jour. Heredity, 52: 261-26^. Mason, M. 1953* The metabolism of•tryptophan in riboflavln-deficient rats. J. B i o l . Chem., 201: 513-518. Maw, A. J .G . 1954-. Inherited r ibof lavin deficiency in chicken eggs. Poult. S c i . , 33 : 216-217. Mayr, E. 1963. Animal Species and Evolution. Harvard Univ. Press, Cambridge. 83 Park, T. 1937. The inheritance of the mutation pearl in the flour beetle Tribolium castaneum (Herbst). Amer. Nat., 71: IH-3-157. Patton, R.L. and Craig, R. 1939* The rate of excretion of certain substances by the larvae of the mealworm, Tenebrio molitor L. Jour. Exp. Zoo l . , 81: l+37-1+57. Peacock, W.J. and Erickson, J . 1965. Segregation distortion and regularly nonfunctional products of spermatogenesis in Drosophila melanogaster. Genetics, 51: 313-328. Pfadt, R.E. (ed.) 1962. Fundamentals of Applied  Entomology. Mac mi 11 an, N.Y. , pp. 6o"8~. Reisener-Glasewald, E . 1956. Uber die Entwicklung des Bestandes an fluoreszierenden Stoffen in den Kopfen von Ephestia kuhniella in Abhangigkeit von ver-schieden Augenfarbgenen. Z. Ind. Abst. Vererb., 87: 668-693. Schlager, G. I963. The ecological genetics of the mutant sooty in populations of Tribolium castaneum. Evolution, 17: 25^ -273. Schlesinger, M.J. and Levinthal, C. 1963. Hybrid protein formation of E. c o l i Alkaline phosphatase leading to in vi tro complementation. J . Mol. B i o l . , 7- 1-12. Schmidt, G.H. and Viscont in i , M. 1962. Fluorescierende Stoffe aus roten Waldameisen der Gattung Formica (Ins. Hym.) Helv. chim. Acta . , h$i 1571-1575. Sokoloff, A. 1961. Section on New Mutants. Tribolium Inform. B u l l . , h: 18. . 1962. Chromosome maps of Tribolium castaneum with suggestions for designation of'chromosome numbers in other tenebrionids. Tribolium Inform. B u l l . , 5: h2-hh. • 1963. Section on New Mutants. Tribolium Inform. B u l l . , 6: 29. . 196*+a. Linkage studies in Tribolium confusum Duval. I. Preliminary linkage studies with autosomal genes. Can. Jour. Genet. Cyt . , 6: 259-270. 8H-Sokoloff, A. 196^ +b. Sex and crossing over in Tribolium  castaneum. Genetics, 50: l+91-^96. Taira, T. and Nawa, S. 1958. No direct metabolic relation between pterines and uric acid, flavines of fo l ic acid in Drosophila melanogaster. Idengaku Zasshi, 33: H-2-H-5. Viscont in i , M . , Schoeller, M . , Loeser, E . , Karrer, P. and Hadorn, E. 1955. Isolierung fluorescierender Stoffe aus Drosophila melanogaster. Helv. chim. Acta, 38: 397-^01. , Kuhn, A . , and Egelhaaf, A. 1956. Isolierung fluorescierender Stoffe aus Ephestia kuhniella. Z. Naturforsch., l i b : 501-50i+7~ Weygand, F. and Waldschmidt, M. 1955. Uber die Bipsynthese des Leukopterins, untersucht mit Cl^-markierten Verbindungen am Kohlweissling. Angew. Chem., 67: 338. Wolsky, A. and Zamora, R. i960. The structure of the pearl eye in Latheticus oryzae. Am. Nat., 9*+: 309-312. Yagi, K. 1957. Dosage des Flavines. B u l l . Soc. Chim., 1957: l5*+3-l550. Ziegler, I. 196l. Genetic aspects of ommochrome and pterin pigments. Adv. in Genet., 10: 3l+9-1+03. 

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