', MUSCLES OF THE DESERT LOCUST - Summit | SFU's...

CYTOCHEMISTRY OF THE DIFFERENTIATING FLIGHT

MUSCLES OF THE DESERT LOCUST ', I

SCHISTOCERCA GREGARIA FORSKAL __L

LYNN CAROL PANAR

B. Sc. , Simon Frase r Universi ty , 1972

A THESIS SUBMITTED I N PARTIAL FULFILLMENT OF

THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

i n t h e Department

Biologica l Sciences

@ LYNN CAROL PANAR 1974

SIMON FRASER UNIVERSITY

August, 1974

A l l r i g h t s reserved. This t h e s i s may not be reproduced i n whole o r i n p a r t , by photocopy o r o t h e r means, without permission of t h e author .

APPROVAL

Name:

Degree:

T i t l e of

Lynn Carol Panar .

Master of Science

Thesis: Cytochemistry of t h e D i f f e r e n t i a t i n g F l i g h t Muscles of t h e Deser t Locust Sch is tocerca g r e g a r i a Forska l

Examining Committee: '

Chairman:

- K. K. Nair

Senior Superv isor

P. B e l t o n .

- - - - T

- - J. M. Webster

PARTIAL COPYRIGHT LICENSE

I hereby g r a n t t o Simon F rase r Univers i ty t h e r i g h t t o lend I

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without my w r i t ten permission.

T i t l e of ~ h e s i s / ~ i s s e r t a t i o n :

Cytochemistry of t h e d i f f e r e n t i a t i n g f l i g h t muscles

of t h e d e s e r t l o c u s t Sch i s toce rca . g rega r i a Forskal .

Author :

( s i g n a t u r e )

Lynn Carol Panar

(name)

(da t e )

ABSTRACT

The purpose of t h i s i n v e s t i g a t i o n was t o s tudy t h e cyto-

chemical changes i n t h e metathoracic median dorsa l l o n g i t u d i n a l

f l i g h t muscle of t h e female d e s e r t l o c u s t , Schis tocerca g regar i a ,

during i t s d i f f e r e n t i a t i o n from t h e las t day of t h e f o u r t h

nymphal i n s t a r , through t h e f i f t h nymphal i n s t a r up t o t h e

e igh th day a f t e r t h e imaginal moult. The parameters examined

were: I. changes accompanying nuclear d i f f e r e n t i a t i o n , with

r e spec t t o : ( 1) DNA content , ( 2 ) t r a n s c r i p t i o n a l a c t i v i t y ,

(3) degree of chromatin condensation and ( 4 ) nuclear a r e a .

11. changes accompanying muscle f i b r e d i f f e r e n t i a t i o n , consider-

ing: (1) RNA content , ( 2 ) p r o t e i n content and (3) cross-

s e c t i o n a l f i b r e a rea .

The r e s u l t s of t h e incorpora t ion of thymidine (methyl 'H)

i n t o muscle n u c l e i show t h a t DNA s y n t h e s i s occurs j u s t p r i o r

t o t h e f i n a l nymphal moult, t h r e e days t h e r e a f t e r and, t o a

l e s s e r degree, f i v e days a f t e r t h e imaginal moult.

Although DNA s y n t h e s i s occurs during d i f f e r e n t i a t i o n

microspectrophotometric a n a l y s i s of Feulgen-stained n u c l e i

shows t h a t po lyp lo id iza t ion i s not a phenomenon a s s o c i a t e d with

t h e d i f f e r e n t i a t i o n of t h e s e muscles. The n u c l e i remain d i p l o i d

throughout t h e developmental per iod.

11 By s t a in ingq ' with 3H-Actinomycin D, followed by autoradfo-

graphy, t h e changes i n t r a n s c r i p t i o n a l a c t i v i t y coinc ident wi th

f l i g h t muscle development were examined. A t t h e beginning of t h e

developmental per iod t h e t r a n s c r i p t i o n a l a c t i v i t y i s r e l a t i v e l y

iii

high and remains so up u n t i l about t h e middle of t h e f i f t h

i n s t a r a f t e r which t ime it d e c l i n e s f a i r l y s t e a d i l y . These

changes i n t r a n s c r i p t i o n a l a c t i v i t y a r e accompanied by changes

i n t h e degree of chromatin condensation. Generally, t h e

pe r iods o f high t r a n s c r i p t i o n a l a c t i v i t y e x h i b i t l e s s condensa-

t i o n of t h e chromatin.

The o r i g i n a l l y high t r a n s c r i p t i o n a l a c t i v i t y i s accompanied

by a s teady o v e r - a l l i n c r e a s e i n t o t a l RNA content during t h e

developmental period; and i n t u r n a l a r g e inc rease i n t o t a l

p r o t e i n content . The c ross - sec t iona l a r e a of t h e muscle f i b r e s

i n c r e a s e s s i g n i f i c a n t l y

t h e f l i g h t muscles.

during t h e growth and d i f f e r e n t i a t i o n

ACKNOWLEDGEMENTS

My s i n c e r e thanks a r e due t o D r . K. K. Nair f o r h i s

cons tan t guidance and encouragement during t h e research f o r and

t h e w r i t i n g of t h i s t h e s i s ; t o D r , P. Belton and D r . J. M.

Webster f o r t h e i r h e l p f u l comments and suggestions; t o M r . Me

Horta f o r r e a r i n g t h e i n s e c t s ; t o D r . Vic Bourne f o r h i s

t e c h n i c a l a s s i s t a n c e ; and t o t h e Nat ional Research Council f o r

t h e i r award of a scholarsh ip .

TABLE OF CONTENTS

Page

Examining Committee Approval . . . . . . . . . . . . . . ii Abst rac t . . . . . . . . . . . . . . . . . . . . . . . . iii Ac know 1 edgement s . . . . . . . . . . . . . . . . . . . . v

T a b l e o f c o n t e n t s . . . . . . . . . . . . . . . . . . . . v i

L i s t of Tables . . . . . . . . . . . . . . . . . . . . . v i i

L i s t of Figures . . . . . . . . . . . . . . . . . . . . . v i i i

In t roduc t ion . . . . . . . . . . . . . . . . . . . . . . 1

Methods and Mate r i a l s . . . . . . . . . . . . . . . . . . 9

. . . . . . . . . . . . . H i s t o l o g i c a l Prepara t ions 9

Microspectrophotometry . . . . . . . . . . . . . . 15

Data Analysis . . . . . . . . . . . . . . . . . . . 16

R e s u l t s * . . . . . . . . . . . . . . . . . . . . . . . . 19

DNA Synthes is . . . . . . . . . . . . . . . . . . . 19 . . . . . . . . . . . . . . . . . . . . BNAContent 19

DNA T r a n s c r i p t i o n a l A c t i v i t y . . . . . . . . . . . 19

Nuclear Area . . . . . . . . . . . . . . . . . . 23

R N A O e e s . . . . . . . . . . . . . . . . . . . . 23

. . . . . . . . . . . . . . . . . . . . . . P r o t e i n 27

. . . . . . . . . . . . Cross-sec t ional F i b r e Area 27

Discussion . . . . . . . . . . . . . . . . . . . . . . . 30

Conclusion . . . . . . . . . . . . . . . . . . . . . . . 58

. . . . . . . . . . . . . . . . . . . . . . . References 39

. . . . . . . . . . . . . . . . . . . . . . . Appendix 1 47

LIST OF TABUS

Page

Table I. Mean e x t i n c t i o n of a Feulgen-stained f l i g h t muscle n u c l e i of S. g regar i a as a func t ion of hydro lys i s i n 3 . 5 N H C l a t 37' C. . . . . 13

Table 11. Regression c o e f f i c i e n t a n a l y s i s of da ta i n F i g . l l . o . . . . . . e . . . e 52

v i i

LIST OF FIGURES

Page

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure 10.

Figure 11.

The metathoracic d o r s a l long i tud ina l f l i g h t muscle of S. g r e g a r i a a t d i f f e r e n t s t a g e s of dev'elopment . . . . . . . . . . . . . . . A matr ix of e x t i n c t i o n values f o r each of t h e p o i n t s measured i n a Feulgen-stained f l i g h t muscle nucleus. . . . . . . . . . . . Thymidine (methyl H 3 ) incorpora t ion i n t o S. - gregar i a f l i g h t muscle nuc le i .

DNA content of S. g r e g a r i a f l i g h t muscle n u c l e i expresse?I as log2 of t o t a l e x t i n c t i o n

Transc r ip t iona l a c t i v i t y of S. g regar i a f l i g h t muscle n u c l e i based on t h e binding of 3~-Actinomycin D t o chromatin, . . . . . . . Rela t ive frequency o f occurrence of p a r t i a l e x t i n c t i o n va lues i n Feulgen- s t a i n e d S. - gregar i a f l i g h t muscle nuc le i . . . . . . . . Area of S. g rega r i a f l i g h t muscle n u c l e i . . - Cytoplasmic RNA c o n t e n t ( t o t a l e x t i n c t i o n ) and i t s concen t ra t ion (mean e x t i n c t i o n ) of S. g regar i a f l i g h t muscle f i b r e s . . . . . . - Cytoplasmic p r o t e i n content t o t a l ex t inc-

i t s c o n c e n t r a t i o n mean ext inc- g r e g a r i a f l i g h t muscle f i b r e s . .

Cross-sec t ional a r e a of S. gregar i a f l i g h t muscle f i b r e s expressed -in terms of a 1 pi s t e p s i z e . . . . . . . . . . . . . . . . . . The r e l a t i o n s h i p between t o t a l e x t i n c t i o n and t h e amount of p r o t e i n s i n s e c t i o n s of f i f t h i n s t a r S. g r e g a r i a f l i g h t muscle . . . -

v i i i

I N T R O D U C T I O N

The i n d i r e c t f l i g h t muscles of most i n s e c t s a r e made up of

l a r g e s i z e d f i b r i l s a r r a n g e d i n t o g i a n t f i b r e s seve ra l hundred pm

i n diameter ( s a c k t o r , 1970) . The f i b r i l s ( m y o f i b r i l s ) a r e d i s -

c r e t e groups of i n t e r d i g i t a t i n g a r r a y s of two s e t s of f i laments ,

t h i c k myosin f i laments , 150 A i n diameter, and t h i n a c t i n f i l a -

ments, 50 2 i n diameter, and t r a n s v e r s e Z-membranes. The f i b r i l s

a r e surrounded by mitochondria, sarcoplasmic reticulum, n u c l e i

and sarcoplasmic sap (~a ruyama, 1965). The f i b r e s a r e charac ter -

i s t i c a l l y invaded by a r i c h t r a c h e o l a r system ( s a c k t o r , 1970).

The f i b r e s a r e arranged around a c e n t r a l t r achea which has

r a d i a t i n g branches ( ~ s h h u r s t , 1967), pene t ra t ing , i n l a r g e

numbers, i n t o t h e i n t e r i o r of t h e f i b r e s (Tiegs, 1955), thus

f a c i l i t a t i n g oxygen t r a n s p o r t by d i f f u s i o n .

The muscle n u c l e i a r e u s u a l l y few i n number and a r e

genera l ly loca ted p e r i p h e r a l l y immediately under t h e sarcolernma

( ~ i e g s , 1955) . The numerous mitochondria a r e s l a b l i k e and very

l a r g e ( s m i t h , 1966) and a r e arranged i n closely-packed elongate

columns, between t h e f i b r i l s ( sack to r , 1970) . The development of i n s e c t f l i g h t muscles i s a complex

process d i f f e r i n g from order t o order and indeed from spec ies

t o spec ies . This i s evident from t h e monumental work of Tiegs

(1955) who examined i n d e t a i l t h e developing f l i g h t muscles of

var ious o r d e r s of i n s e c t s . Among t h e Acr id i idae he has described

t h e postembryonic development of t h e s e muscles i n t h e plague

l o c u s t Chor to ice tes t e r m i n i i ' e ~ a . I n t h e minute f i r s t i n s t a r

nymph, a l l t h e main muscles of t h e a d u l t can be d is t inguished,

i n a very rudimentary condi t ion. During development t h e

f i b r e s both i n c r e a s e i n diameter and i n number. The f i b r e

enlargement u s u a l l y t akes p lace during t h e e a r l y growth per iod

of t h e nymph. I n t h e t h i r d , penul t imate i n s t a r , t h e f i b r e s

begin t o cleave. I n t h e f i n a l i n s t a r , t h e muscle, whose growth

has, till now, merely kept pace with t h a t of t h e nymph, acce le r -

a t e s i t s development i n p repara t ion f o r t h e new func t ion of

f l i g h t . The muscles enlarge up t o t h r e e o r four t imes t h e i r

diameter a t t h e incep t ion of t h e i n s t a r , while t h e nymph

enlarges by about twice,

Bocharova-Messner and Yanchuk ( 1966) observed a similar

p a t t e r n of f l i g h t muscle development i n t h e domestic c r i c k e t ,

Acheta domestics. I n a d d i t i o n they noted t h a t t h e i n t e n s i v e

p repara t ion f o r new d u t i e s a s s o c i a t e d wi th i n t e n s i f i c a t i o n of

energy balance, begins during t h e last t h i r d o f t h e f i n a l nym-

phal i n s t a r ,

Scudder (1971) and Scudder and Hewson (1971) s t u d i e d t h e

f l i g h t muscle development i n t h e Hemipterans Cenocorixa b i f i d a

and Oncopeltus f a s c i a t u s r e spec t ive ly . They found t h a t t h e

dorsolongi tudina l f l i g h t muscles o f t h e f i rs t i n s t a r disappear

and a r e replaced i n l a t e r i n s t a r s by new muscles t h a t func t ion

i n t h e a d u l t . During t h e f i r s t i n s t a r t h e r e a r e four , f u l l y

formed, s t r i a t e d dorsolongi tudina l muscle f i b r e s . During t h e

second f n s t a r , they become surrounded by b a s i p h i l i c c e l l s wi th

l a r g e prominent nucle i . I n t h e t h i r d i n s t a r t h e s e b a s i p h i l f c

c e l l s appear t o have invaded and fragmented t h e o r i g i n a l muscle

f i b r e s . The four th ' i n s t a r i s cha rac te r i zed by t h e predominance

and o rgan iza t ion of t h e b a s i p h i l i c c e l l s i n t o four d i s c r e t e

groups, c l o s e l y packed toge the r . I n t h e f i n a l nymphal i n s t a r

and f o r some time a f t e r t h e imaginal moult t h e s e groups of c e l l s

grow and d i f f e r e n t i a t e i n t o f u n c t i o n a l f l i g h t muscles.

I n recent yea r s cons iderable r e sea rch has been c a r r i e d

out on t h e u l t r a s t r u c t u r e of d i f f e r e n t i a t i n g muscles of i n s e c t s

p a r t i c u l a r l y i n holometabolous spec ies . The development of t h e

f l i g h t muscles has been descr ibed i n Simulium ornatum rant,

1961), Drosophila melanogaster ( Shaf i q , 1963), Antherea pe rny i

( ~ p o t n a and Michejda, 1966), G a l l e r i a melonella (Sahota and

Beckel, 1967) , L u c i l i a cuprina r re gory e t a l . , 1968; P e r i s t i a n i s

and Gregory, 1971) and Cal l iphora erythrocephala ( ~ r o s s l e y ,

l972) , Most of t h e s e s t u d i e s have shown t h a t i n holometaboPous

i n s e c t s t h e l a r v a l muscles degenerate a t pupat ion and t h e

imaginal muscles a r e formed by t h e m u l t i p l i c a t i o n and f u s i o n

of myoblasts i n t o syncyt ia . The s t e p s involved i n t h e formation

of muscle syncyt ia have been s t u d i e d i n depth us ing c u l t u r e d

voluntary s t r i a t e d muscles of v e r t e b r a t e s examined wi th t h e

a i d of t ime-lapse cinematography (capers , 1960; Cooper and

Konigsberg, 1961)~

According t o t h e reviews by Goodman ( 1957) and Murray

(1960) ami tos i s has been impl ica ted i n t h e formation of t h e

syncytium, whereas Cooper and Konigsberg ( 1961) c a s t consider-

a b l e doubt on t h e evidence which had been presented t o support

"ami to t i c r s nuclear d i v i s i o n a s a mechanism of t h e o r i g i n of

m u l t i n u c l e a r i t y of muscle c e l l s . Goodman (1957) and Murray

(1960) had drawn a t t e n t i o n t o de fo rmi t i e s impart ing t o such

n u c l e i a " dumbbell" shape. Cooper and Konigsberg ( 1961) found

t h a t t h i s type of deformity does not l e a d t o d i r e c t d i v i s i o n

of t h e nucleus. The dumbbell shape i s assumed t r a n s i t i o n a l l y ,

t h e nucleus l a t e r resuming i t s e l l i p t i c a l shape.

Capers (1960) noted from h i s observat ions t h a t i n t h e

I course o f r o t a t i o n and migratory a c t i v i t y of t h e nuc le i , a g r e a t

dea l of d i s t o r t i o n commonly occurs. During migrat ion, oval

n u c l e i o f t e n t u r n so t h a t t h e i r long axes a r e perpendicular t o

11 t hose of t h e muscle s t r apss ' . This f r equen t ly r e s u l t s i n t h e

formation o f f o l d s i n t h e nuclear membrane some of which occur

between two n u c l e o l i of t h e same nucleus. Capers ( 1960 )

suggests t h a t t h i s may expla in numerous r e p o r t s t h a t n u c l e i

d iv ide i n t o s e c t o r s each wi th i t s own nucleolus. However, he

found t h a t such d i v i s i o n s a r e not permanent but disapp- Pa r as

soon a s t h e nucleus has completed i t s r o t a t i o n .

Capers (1960) and Cooper and Konigsberg (1961) found t h a t

no mi tos i s , ami tos i s , o r nuclear budding could be observed

during t h e course of muscle development. Cooper and Konigsberg

(1961) noted t h a t d e s p i t e t h e complete absence of m i t o t i c

phenomena i n t h e mul t inuclea te c e l l s , mitoses a r e numerous

among ad jacen t mononucleat ed elements i n c u l t u r e . It seems

un l ike ly , the re fo re , t h a t c u l t u r e cond i t ions p e r s e prevented -- mitos i s . Cooper and Konigsberg (1961) as we l l a s Capers (1960)

5.

repor ted t h a t myoblast f u s i o n was t h e only demonstrable way of

g iv ing r i s e t o mul t inuclea t ion .

This ques t ion seems t o have been resolved as repor ted

i n Fischmanl s (1972) most r ecen t review. Therein he s t a t e s

c l e a r l y t h a t " mult inuclea t ion i n muscle c e l l s r e s u l t s from t h e

cytoplasmic f u s i o n of myogenic c e l l s and not from ami tos i s o r

some o t h e r process involv ing t h e m u l t i p l i c a t i o n of myotube

n u c l e i without cy tokines is . " Evidence f o r t h i s i s obta ined from

f i v e p r i n c i p a l types of experiments, These include: d i r e c t

morphological a n a l y s i s of l i g h t and e l e c t r o n microscopy of

both i n v i t r o and i n vivo systems; q u a n t i t a t i v e DNA measurements - -- by Feulgen microspectrophotometry showing a l l n u c l e i w i t h i n mys-

tubes t o be d ip lo id ; au toradiographic a n a l y s i s of nuclear

s y n t h e s i s u s i n g t r i t i a t e d thymidine as a l a b e l l e d precursor of

DNA which i s found not t o be incorpora ted i n t o t h e nuclear DNA

of myotubes; i n h i b i t o r s t u d i e s i n which DNA s y n t h e s i s i s blocked

by X-rays, n i t rogen mustards, f o l i c a c i d an tagon i s t s , c o l c h i c i n e

e t c . , r e s u l t i n g i n t h e prevent ion of t h e r e p l i c a t i o n of mono-

nuclea ted c e l l s but not myotube formation; and f i n a l l y fus ion

s t u d i e s us ing gene t i c markers.

I n t h e r egenera t ing limb muscles of Pe r ip lane ta americana,

Cowden and Bodenstein (1961) found t h a t when t h e mononucleated

myoblasts reach a s u f f i c i e n t popula t ion dens i ty , l y i n g as t h e y

do i n a space between myotubes, they form i n t e r d i g i t a t i n g chains

and cytoplasmic o u t l i n e s become i n d i s t i n c t . They found no mito-

t i c f i g u r e s even i n t h e mul t inuclea te myotubes. Bhakthan e t a l e --

(1971) while examining t h e r egenera t ion of f l i g h t muscles i n t h e

bark b e e t l e , - I p s paraconfusus (I. - confusus) found t h a t mono-

nuclea ted myoblasts appear and apparen t ly f u s e with o t h e r myo-

b l a s t s t o form mult inucleated c e l l s . From t h e above it may be

concluded t h a t t h e mul t inuclea te cond i t ion of t h e myofibres

of i n s e c t muscles i s achieved by t h e f u s i o n of myoblasts r a t h e r

t h a n by mi tos i s .

Fur ther development o f t h e f l i g h t muscles inc ludes a

cons iderable change i n t h e weight o f t h e muscle. This i s o f

course e s p e c i a l l y no t i ceab le i n t h e hemimetabolous i n s e c t s

where t h e growth and d i f f e r e n t i a t i o n t a k e s p lace over a longer

pe r iod of time. I n t h e d e s e r t l o c u s t Schis tocerca g regar i a ,

t h e f l i g h t muscles grow throughout t h e f o u r t h and f i f t h i n s t a r ,

ceas ing j u s t p r i o r t o and during t h e ecdyses. H i l l and

Goldsworthy (1968) c a l c u l a t e d t h a t t h e f l i g h t muscle inc reases

i n weight by 238% during t h e f o u r t h i n s t a r and by 1624% during

t h e f i f t h i n s t a r . This somatic growth cont inues a f t e r t h e

imaginal moult f o r about e i g h t t o t e n days during which t ime

t h e male experiences a n o v e r a l l 9% inc rease i n dry weight

(walker -- e t a l . , 197'0), and t h e female a 110% i n c r e a s e ill - e t

a l . , 1968). Although t h e s e inc reases i n body weight a r e not - e n t i r e l y due t o growth o f t h e f l i g h t muscles, t h i s probably

c o n s t i t u t e s a l a r g e percentage of it.

These s t r u c t u r a l changes a r e accompanied by biochemical

changes as well . Much of t h e biochemical a n a l y s i s of f l i g h t

muscle development has been c a r r i e d out on var ious Orthopteran

i n s e c t s . BGcher (1965) has done s u b s t a n t i a l work u s i n g Locusta

m i g r a t o r i a . He examined t h c fo l lowing biochemical parameters ;

non- e x t r a c t a b l e p r o t e i n made up p a r t l y by mi tochondr ia l f r a c t i o n s

but c h i e f l y by t h e c o n t r a c t i l e p r o t e i n s ; t h e g lyce ro l - l cphospha te

ox idase r e p r e s e n t i n g t h e c o n s t a n t p r o p o r t i o n complement o f

mi tochondr i a l enzymes; and f i n a l l y f o u r ex t ra -mi tochondr ia1

enzymes involved i n t h e breakdown o f carbohydrate : g lyceraldehyde-

3-phosphate dehydrogenase, g lycerol-1-phosphate dehydrogenase

(GDH) , l a c t o s e dehydrogenase (LDH) and glucose-6-phosphate

dehydrogenase ( G ~ P D H ) . During t h e f i r s t phase of t h e f i f t h

i n s t a r t h e exponen t i a l i n c r e a s e o f a l l parameters i s a lmos t

equa l . During t h e moul t ing i n t e r v a l t h e i n c r e a s e i n LDH

shows a peak, which can p o s s i b l y be a t t r i b u t e d t o t h e growth of

t h e t r a c h e o b l a s t s . Next, t h e a c t i v i t y of GDH i n c r e a s e s by a

f a c t o r of 20 ( ~ r o s e m e r -- e t a l . , 1963) , whereas t h a t o f LDH decrea

s e s by a f a c t o r o f t h r e e . A c t i v i t i e s of G ~ P D H and some o t h e r

enzymes a lmost d i sappear . From about t h e f i f t h t o t h e e i g h t h

day o f imaginal l i f e t h e r e i s a n a lmos t exac t d u p l i c a t i o n of

t h e ex t r ami tochondr i a l enzyme a c t i v i t i e s .

The tremendous i n c r s a s e i n GDH a f t e r t h e l a s t moult i s

due t o n e t enzyme s y n t h e s i s -- de novo, and no t t h e a c t i v a t i o n of

a p r e e x i s t i n g apoenzyme ( ~ r o s e m e r , 1965). Two o t h e r enzymes,

no t mi tochondr ia l , a l s o show a s i g n i f i c a n t i n c r e a s e i n a c t i v i t y

du r ing development. Thesz a r e B-hydroxyacyl-CoA- dehydrogenase

and p- k e t o a c y l t h i c l a s e ( ~ e z n a k k e r s , 1963) . Thi s may be c o r r e l a -

t e d wi th f l i g h t metabolism.

Growth and d i f f e r e n t i a t i o n o f i n s e c t f l i g h t muscles i s

a n o r d e r l y but complex process . It i s undoubtedly c a r e f u l l y

c o n t r o l l e d and guided as a r e most developmental phenomena i n

i n s e c t s . Numerous experiments have been done i n a n at tempt t o

e l u c i d a t e those c o n t r o l mechanisms t h a t a r e r e l a t e d t o somatic

growth i n genera l ( s e e reviews by: Doane, 1973; Gi lbe r t , 1964;

Wigglesworth, 1964). Poels and Beenakkers (1969) and Beenakkers

( 1973) observed t h a t implanta t ion o f a c t i v e corpus a l l a t u m in-

h i b i t e d d i f f e r e n t i a t i o n of f l i g h t muscles i n - L. migra tor ia . From

t h i s they proposed t h a t t h e t r i g g e r f o r d i f f e r e n t i a t i o n of t h e

muscles i s t h e low t i t r e of corpus a l l a tum hormone. The speci-

f i c mechanisms involved i n t h i s process remain t o be e luc ida ted .

From t h e foregoing it i s evident t h a t a number of biochemical

events a s s o c i a t e d wi th muscle d i f f e r e n t i a t i o n i n i n s e c t s a r e

being brought t o l i g h t , However much a l s o s t i l l remains t o be

s tudied . I n an at tempt t o f u r t h e r c l a r i f y some of t h e processes

involved i n d i f f e r e n t i a t i o n of f l i g h t muscles t h e fol lowing s tudy

was undertaken. The o b j e c t i v e s of t h i s study were: I. t o

determine changes accompanying nuclear d i f f e r e n t i a t i o n , wi th

r e spec t to : (1) DNA content , ( 2 ) t r a n s c r i p t i o n a l a c t i v i t y , (3)

degree of chromatin condensation and ( 4 ) nuclear area.. 11. t o

determine t h e changes accompanying muscle f i b r e d i f f e r e n t i a t i o n ,

considering: (1) RNA content , ( 2 ) p r o t e i n content and ( 3 ) cross-

s e c t i o n a l f i b r e a rea .

METHODS AND MATERIALS

The d e s e r t locus ts Schis tocerca g regar i a used i n t h i s

study were r a i s e d on a d i e t o f whole wheat bran and f r e s h g r a s s

( ~ u n t e r - ones, 1956), and rea red under a cons tant l i g h t regime

a t 35 - 38" C and 60 - 7% R. H. Under t h e s e condi t ions t h e

f i f t h i n s t a r nymph moulted t o an a d u l t i n about s i x days.

H i s t o l o g i c a l Preparat ions:

This study was confined t o t h e metathoracic median dorsa l

l o n g i t u d i n a l muscles, Figure 1 i s a p i c t o r i a l r ep resen ta t ion of

the o v e r - a l l change i n s i z e t h a t t h i s p a r t i c u l a r muscle under-

goes during i t s d i f f e r e n t i a t i o n . The changes c h a r a c t e r i z i n g

muscle growth and d i f f e r e n t i a t i o n a r e much exaggerated i n females

as compared with those i n t h e males i ill -- e t a l e , 1968; Walker

e t a l . , 1970) and so only females were used i n t h i s s tudy, -- The f l i g h t muscles begin d i f f e r e n t i a t i n g towards t h e end

of t h e f o u r t h nymphal i n s t a r , and cont inue through t h e f i f t h

nymphal i n s t a r and i n t o about e i g h t days of imaginal l i f e ill e t a l . , 1968). Therefore, t h e fol lowing age groups were chosen -- f o r study: l as t day of t h e f o u r t h nymphal i n s t a r ; f i r s t , t h i r d ,

f i f t h and las t day of t h e f i f t h nymphal i n s t a r ; f i rs t , t h i r d ,

f i f t h and e igh th day a f t e r t h e imaginal moult. Three t o f i v e

i n s e c t s were used f o r each group.

Nucleus:

For s t u d i e s on deoxyribonucleic a c i d (DNA) syn thes i s ,

l o c u s t s were i n j e c t e d under C02 anaes thes ia with 10 p l of

thymidine (methyl H ~ ) ( 2 Ci/rnM, Amersham/Searle, chicago) through

Figure 1. The meta thorac ic dorsal l o n g i t u d i n a l f l i g h t muscle of

female - S. g r e g a r i a a t d i f f e r e n t s t a g e s of development.

These diagrams a r e p i c t o r i a l r e p r e s e n t a t i o n s of a

v e n t r a l view of t h e p a i r of muscles.

A. end of f o u r t h nymphal i n s t a r

B. middle of f i f t h nymphal i n s t a r

C . f ive-day-old a d u l t

M E

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t h e a r t h r o d i a l membrane a t t h e base of t h e second p a i r o f l e g s .

A f t e r i n j e c t i o n , t h e i n s e c t s were r e t u r n e d t o 3 6 & 1 • ‹ C f o r f o u r

hours . The f l i g h t muscles were t h e n d i s s e c t e d out and squashes

were made on g e l a t i n i z e d (0 .5%) s l i d e s u s i n g t h e dry i c e tech-

nique (conger and F a i r c h i l d , 1953) . A f t e r removing t h e cover

s l i p s , t h e s l i d e s were a i r - d r i e d f o r 10 minutes, and f i x e d i n

e thano1 :ace t i c a c i d (3: 1) f o r two hours . A f t e r h y d r a t i o n t h e

s l i d e s were t r e a t e d wi th c o l d t r i c h l o r a c e t i c a c i d (TCA) f o r 15

minutes and washed i n nonrad ioac t ive 0.1% thymidine s o l u t i o n f o r

20 minutes t o remove unbound r a d i o a c t i v e thymidine. The s l i d e s

were a i r - d r i e d and t h e n coa ted w i t h NTB-2 n u c l e a r emulsion

( ~ a s t m a n ~ o d a k ) acco rd ing t o Baserga and Malamud (1969) . Some

s l i d e s were t r e a t e d wi th r i bonuc lease ( ~ ~ a s e ) (sigma Chemical Co.,

St.. Louis, MO. ) , 1 m g p l f o r 1 hour ( ~ e i t c h , 1966) and t h e n

coa t ed w i t h nuc l ea r emulsion. Pr .eparat ions were exposed f o r f o u r

weeks a t 4" C and t h e n processed and s t a i n e d i n methyl green/

pyronin ( ~ e a r s e , 1968). A t l e a s t 500 n u c l e i p e r age group were

examined f o r l a b e l l i n g and from t h e s e da t a t h e percen tage of

l a b e l l e d n u c l e i was c a l c u l a t e d .

To examine changes, i f any, i n t h e t o t a l DNA

con ten t and i t s c o n c e n t r a t i o n du r ing t h e developmental pe r iod ,

e thanol : a c e t i c a c i d (3: 1) f i x e d squashes o f muscles were hydro-

l yzed i n 3.5N hydroch lo r i c a c i d ( H C ~ ) a t 37" C and, 1972) .

To determine t h e optimun h y d r o l y s i s t ime squashes of

f l i g h t muscle of two d i f f e r e n t a g e groups, one-day-old and f i n a l

day f i f t h i n s t a r nymphs, were hydrolyzed i n 3.5N H C 1 f o r 10, 15,

20 and 25 minutes and s t a i n e d i n S c h i f f t s reagent (Basic

Fuchsin, Harleco, ~ h i l a d e l p h i a ) prepared according t o Deitch

(1966). The optimum hydro lys i s t ime was 20 minutes i n both

cases a able I ) . To s t andard ize t h e technique, s l i d e s were

s t a b i l i z e d i n 37' C d i s t i l l e d water before hydrolys is , and were

c h i l l e d a f t e r hydro lys i s i n co ld (4" C ) 1 N H C 1 t o h a l t t h i s

process . The s l i d e s were then s t a i n e d i n S c h i f f l s reagent f o r

one hour and, a f t e r processing, were mounted i n o i l of matching

r e f r a c t i v e index ("D = 1.56, C a r g i l l e , Cedar Grove, N. J. )

Ext inc t ion measurements on 100 n u c l e i per age group were

taken a t 570 nm us ing a Scanning Microscope Photometer (SMP;

Carl Zeiss , Oberkochen, W. ~ e r m a n y ) . These readings were com-

pared wi th those obta ined from s i m i l a r l y f i x e d and s t a i n e d

spermatids ( 1 ~ ) t o determine t h e DNA c l a s s of f l i g h t muscle

nuc le i .

The technique of '' s t a in ing" by 3 ~ - ~ c t i n o m y c i n D ( AMD)

followed by autoradiography (Brachet and Ficq, 1965) was used

t o determine whether t h e t r a n s c r i p t i o n a l a c t i v i t y of t h e

n u c l e i changes during d i f f e r e n t i a t i o n . For t h i s , f l i g h t

muscles were d i s sec ted out and f i x e d i n cold (4" C ) Carnoy

( ~ r a c h e t and Hulin, 1969) f o r four hours. The muscles were

then dehydrated, embedded i n p a r a f f i n and c u t a t 5 p th ick-

ness . The s e c t i o n s were deparaf f in ized , hydrated and t r a n s -

f e r r e d t o a 3 ~ - ~ ~ s o l u t i o n ( 5 $ i / m l , Sp. a c t . 3.8 Ci/rnM,

Schwartz/Mann, Orangeburg, N. Y . ) i n t h e dark. Af te r one hour

t h e s l i d e s were r i n s e d i n d i s t i l l e d water and t r a n s f e r r e d t o a

Mean e x t i n c t i o n of

S. gregar i a a s a -

TABLE I

Feulgen-stained f l i g h t

func t ion of hydro lys i s

37" c

muscle n u c l e i of

i n 3 . 5 N H C 1 a t

-

Hydrolysis Time M.E.'? s.E.* M . E . ~ ? S.E.

To ta l number of n u c l e i measured p e r group was 25.

One-day-old f i f t h i n s t a r nymph.

Last f i f t h i n s t a r

* S.E. = standard e r r o r

Analysis of var iance showed t h a t t h e change i n each age group was s i g n i f i c a n t ( - P < 0.05) . I

nonradioac t ive AMD ( ~a lb iochem, San Diego, ca l i f . ) s o l u t i o n

having a concent ra t ion of 10 - 20 mg/ml f o r one hour. This was

followed by washing i n t a p water f o r 20 hours, a f t e r which t h e

s l i d e s were r i n s e d i n two changes of d i s t i l l e d water and four

changes of 70% ethanol ( ~ b s t e i n , 1969). They were then air-

d r i e d and coated with NTB-2 nuclear emulsion ( ~ a s t m a n ~ o d a k ) .

A f t e r t h r e e days of exposure a t 4 O C, t h e s l i d e s were processed

and s t a i n e d wi th methyl green/pyronin. Using a square g r a t i c u l e

t h e number of s i l v e r g r a i n s pe r g r i d (6.25 pn2) of nuclear a r e a

was determined f o r PO0 n u c l e i per age group,

Cytoplasm:

To examine changes i n t h e r ibonuc le ic a c i d (RNA) and

p r o t e i n l e v e l s during development, f l i g h t muscles were d i s -

sec ted out and f i x e d overnight i n 3% glutaraldehyde i n phosphate

b u f f e r ( p ~ 7 . 4 ) . The t i s s u e was then washed i n two changes of

buf fe r , slowly dehydrated, c l e a r e d and embedded i n p a r a f f i n .

F ive micron s e c t i o n s were c u t , deparaf f i n i z e d , hydrated and

washed i n t h r e e 20-minute changes of t a p water before s t a i n i n g .

Sec t ions f o r RNA a n a l y s i s were s t a i n e d with t h e bas ic

t h i a z i n e dye Azure B ( ~ l l i e d Chemical, Morristown, N. J . ) ,

according t o Flax and Himes (1952). The optimum s t a i n i n g

pe r iod a t 40" C was two hours. The pH of t h e s t a i n i n g s o l u t i o n

was a d j u s t e d t o 4.0 by t h e a d d i t i o n of potassium b i p h t h a l a t e

c r y s t a l s ( ~ e i t c h , 1966).

Sec t ions f o r p r o t e i n a n a l y s i s were s t a i n e d wi th t h e t r i -

phenylmethane dye Coomassie Blue ( ~ d w a r d Gurr Ltd., London,

England). The s t a i n i n g procedure was modified from t h a t de-

s c r i b e d f o r g e l e lec t rophores is . ( s e e Appendix I.) A 1%

stock s o l u t i o n was dissolved i n 12% TCA t o make a 0.1% s t a i n i n g

so lu t ion . S t a i n i n g was done a t 37" C f o r 15 minutes followed

by d e s t a i n i n g i n 7% g l a c i a l a c e t i c a c i d f o r 15 minutes a t room

temperature.

Ex t inc t ion measurements f o r both t h e Azure B and Coomassie

Blue s t a i n e d s e c t i o n s mounted i n o i l ("D = 1.56) were made a t

590 and 595 nm respec t ive ly . The e x t i n c t i o n va lues were de ter -

mined f o r t h e cross-sec t ions o f .100 muscle f i b r e s pe r age group

i n both t h e s e s tud ies .

Microspectrophotometry:

The Feulgen, Azure B and Coomassie Blue s t a i n e d s l i d e s

were examined w i t h t h e SMP. The SMP has a mechanical s t a g e

t h a t e x h i b i t s t h e comb-type movement. According t o Zimmer

( 1 9 7 0 ) ~ t h e f i n e scanning s t a g e can be s h i f t e d a t a r a t e of 60

measuring s t e p s per second, being dr iven by two s tepping motors

which r e c e i v e t h e necessary pu l ses from t h e c o n t r o l u n i t .

Measurements a r e performed i n t h e i n t e r v a l between t h e s t e p s ,

whose l eng th and s i z e of a r e a can be va r i ed (Zimmer, 1970).

The connection of t h i s machine t o a d i g i t a l computer

c r e a t e s p r a c t i c a l l y unl imi ted p o s s i b i l i t i e s f o r t h e d i g i t a l

a n a l y s i s of microscope images. The SMP used i n t h i s study was

on- l ine with a PDP-12 computer ( D i g i t a l Equipment Corporation,

Maynard, Mass.). The program used i s c a l l e d APAMOS (Automatic

Photometric Analysis of Microscopic Objects by scanning) , a

v a r i a t i o n of t h e o r i g i n a l program, TICAS (~axonomic I n t r a c e l l u l a r

Ana ly t i ca l system), descr ibed i n d e t a i l by Weid e t a l , (1968). -- The CRT u n i t a l lows t h e d i sp lay of t h e measured p o i n t s of

var ious ex t inc t ions . I n a d d i t i o n , an e d i t i n g program a l lows t h e

opera to r t o exclude measurements from ad jacen t c e l l s .

A l l measurements were c a r r i ed out with u l t r a f l u a r ob jec-

t i v e (x100, N. A. 1.25) and condenser ( N . A. 0.8) l e n s e s a t 1 .0 p

s t e p s i z e a long t h e X and Y axes. The diameter of t h e measuring

a p e r t u r e was 9.0 p f o r n u c l e i and 1.6 I J ~ f o r f i b r e s .

F igure 2 r ep resen t s a p r i n t - o u t of t h e e x t i n c t i o n va lues

of each of t h e measured p o i n t s of a Feulgen-stained nucleus.

The machine w i l l a l s o p r i n t out t h e da ta as follows:

X= s t e p s Y= l i n e s A= TEb MEk

X r e p r e s e n t s t h e number o f s t e p s on t h e x a x i s and Y t h e number

of l i n e s on t h e y a x i s . A r e p r e s e n t s t h e a r e a a s determined

by t h e number of measured p o i n t s having a t ransmiss ion between

5 and 95%. TE i s t h e t o t a l e x t i n c t i o n . It represen t s t h e t o t a l

of a l l t h e values presented i n f i g u r e 2. ME i s t h e mean

e x t i n c t i o n o r t h e average of a l l t h e s e values.

Data Analysis:

The da ta were analysed u s i n g one way a n a l y s i s of var iance

( ~ u r k h a r d t , 1964).

DNA content was expressed a s log2 of t o t a l e x t i n c t i o n .

This was f o r t h e purpose of determining i f polyploidy i s occur-

r i n g i n t h e developing f l i g h t muscles s i n c e a n inc rease by one

u n i t i n t h e log2 value r e p r e s e n t s a doubling of t h e r e a l value

Figure * 2. A matr ix of e x t i n c t i o n va lues f o r each of t h e p o i n t s

measured i n a Feulgen-stained f l i g h t muscle nucleus.

SFU CYTOLAB PANAR OCT29 73

( ~ i t t w o c h , -- e t a l . , 1966; FOX, 1 9 6 9 ) ~

For determination of t h e r e l a t i v e frequency of occurrence

of e x t i n c t i o n values i n Feulgen-stained nuc le i a computer pro-

gram modified from t h e o r i g i n a l vers ion ( ~ a r t e l s -- e t a l . , 1974)

was used. This program counts t h e frequency of occurrence of

ex t i nc t i on values wi th in t h e range of 0.0 t o 1.8 a t i n t e r v a l s

of 0 .1 f o r each nucleus. From the se a composite histogram f o r

t h e e n t i r e populat ion i s compiled.

RESULTS

DNA Synthesis:

The r e s u l t s from t h e thymidine (methyl H ~ ) i ncorpora t ion

a r e summarized i n f i g u r e 3, Peaks of DNA syn thes i s occur during

t h e four th i n s t a r , j u s t p r i o r t h e f i n a l moult, and

on t h e t h i r d day of t h e f i f t h nymphal i n s t a r . Synthes is de-

c l i n e s r ap id ly and ceases completely p r i o r t o t h e imaginal moult,

There i s one o the r , smaller f i v e days a f t e r t h e imaginal

moult. It i s p e r t i n e n t t o mention here t h a t RNase t reatment had

no e f f e c t on t h e ex ten t of l a b e l l i n g i n t h e s e nucle i .

DNA Content: - ' The da ta from Feulgen microspectrophotometry i n d i c a t e

t h a t no c l e a r - c u t polyploidy i s occurr ing during d i f f e r e n t i a t i o n

(F ig . 4 ) . It seems t h a t t h e f l i g h t muscle n u c l e i a r e d i p l o i d ,

and remain t h u s throughout development.

DNA T r a n s c r i p t i o n a l Act iv i ty : - The t r a n s c r i p t i o n a l a c t i v i t y i s r e l a t i v e l y high t o begin

wi th and remains so, wi th no s i g n i f i c a n t change, up t o t h e f i f t h

day of t h e f i f t h i n s t a r . It t h e n dec l ines s t e a d i l y u n t i l about

t h e t h i r d day of a d u l t l i f e a f t e r which t ime it r i s e s s l i g h t l y

( ~ i g . 5 ) . The o v e r a l l change i s s t a t i s t i c a l l y s i g n i f i c a n t ( P - <

0 ~ 0 5 ) .

The changes i n t r a n s c r i p t i o n a l a c t i v i t y may be expected

t o be coinc ident with changes i n t h e degree of condensation of

t h e chromatin. Transc r ip t iona l ly a c t i v e chromatin would assume

a l e s s condensed s t a t e than t r a n s c r i p t i o n a l l y i n a c t i v e chro-

Figure 3 . ~hymid ine . (methyl H=) incorpora t ion i n t o S. gregar ia - f l i g h t muscle nucle i . Tota l number of nuc le i examined

per age group was a t l e a s t 500.

9 No l abe l l ed nuc le i could be detected i n these

groups.

Figure 4. DNA content of S . gregar i a f l i g h t muscle n u c l e i ex- - pressed a s log2 of t o t a l ex t inc t ion .

A: l a s t day of f o u r t h nymphal i n s t a r ; B, C , D, E:

f i r s t , t h i r d , f i f t h and las t day of f i f t h nymphal

i n s t a r , respect ive ly ; F, G, H, I: f i rs t , t h i r d ,

f i f t h and e ighth day of a d u l t , r e spec t ive ly ; J:

spermatids, To ta l number of n u c l e i measured p e r

age group was 100. 20 spermatid n u c l e i were

measured t o o b t a i n t h e haploid value.

DNA CONTENT AS LOG2 TOTAL EXTINCTION

Figure 5. Transc r ip t iona l a c t i v i t y of S. gregar i a f l i g h t muscle - n u c l e i based on t h e binding of 3~-Actinomycin D t o

chromatin. Each po in t r e p r e s e n t s t h e mean ~t standard

e r r o r of 100 measurements.

matin. This would be r e f l e c t e d by t h e lower percentage of high

dens i ty p o i n t s f o r l e s s condensed chromatin (F ig . 6 ) .

There a r e few high dens i ty p o i n t s i n t h e l a s t day of t h e

four th nymphal i n s t a r but t h e i r frequency inc reases gradual ly

u n t i l t h e f i rs t day a f t e r t h e imaginal moult. The t h i r d day of

a d u l t l i f e i s cha rac te r i zed by a decrease i n t h e frequency

d i s t r i b u t i o n of high dens i ty po in t s . A f u r t h e r inc rease i s

evident on t h e f i f t h day.

Nuclear Area: - The changes i n t h e s i z e o f , t h e n u c l e i over t h e development-

per iod a r e summarized i n f i g u r e The a r e a seen t o

decreas? a t both moults and a l s o a s maturat ion i s achieved.

These changes a r e s t a t i s t i c a l l y s i g n i f i c a n t ( P - < 0.05).

RNA : - Azure B microspectrophotometry c a r r i e d out on cross-

s e c t i o n s of f l i g h t muscle f i b r e s i n d i c a t e s a n o v e r a l l i nc rease

i n t h e t o t a l l e v e l of RNA during development (F ig . 8) . This

t r e n d i s i n t e r r u p t e d only a t t h e t ime of t h e two moults a f t e r

which t h e t o t a l RNA l e v e l cont inues t o increase . I n both cases ,

t h e r i s e resumes immediately a f t e r t h e ecdysis . The apparent

d e c l i n e i n t h e amount of RNA between t h e f i r s t and t h i r d days

a f t e r t h e u l t i m a t e moult i s not s t a t i s t i c a l l y s i g n i f i c a n t ( P - . 0.05).

Other than a s l i g h t i n c r e a s e a t t h e very onse t of

development, t h e concen t ra t ion of RNA dec l ines slowly, but

s t e a d i l y throughout t h i s per iod . Although t h e o v e r a l l change

Figure 6. R e l a t i v e frequency of occurrence of p a r t i a l e x t i n c t i o n

va lues i n Feulgen- s t a i n e d 2. grega.ria f l i g h t muscle

nuc le i .

Figure 7. Area of - S. g r e g a r i a f l i g h t muscle n u c l e i . Each p o i n t

r e p r e s e n t s t h e mean ' s tandard e r r o r o f 100 measure-

ments. A nucleus w i t h a 100 p s t e p s i z e i s equiva-

l e n t t o 76 $ i n a r e a .

(sdap u r d ~ u! pal nseaur se) n u y

Figure 8. Cytoplasmic RNA content ( t o t a l e x t i n c t i o n ) and i t s

concent ra t ion (mean e x t i n c t i o n ) of - S. g regar i a f l i g h t

muscle f i b r e s . Each p o i n t r ep resen t s t h e mean h

standard dev ia t ion of 100 measurements, - t o t a l e x t i n c t i o n

me--- - m mean e x t i n c t i o n

- L

I -

1 -

1 -

1 - -

1 - -

1-

1 - -

1 - - I

3 -

3 - - I

I I I I I I I I 1 1 I I I I I I ,

last clay 1 3 5 last day 1 of 4th of 6th

3 5 8

Inst. L 5 1 h INST AR ~ ~ ~ t a ~ w ADULT b

AGE (DAYS)

a p p e a r s small, a n a l y s i s of v a r i a n c e i n d i c a t e s t h a t it i s s i g -

n i f i c a n t ( P - 4 0.05).

P r o t e i n :

The changes i n t h e amount of t o t a l p r o t e i n i n t h e muscle

f i b r e s fo l lows very c l o s e l y t h o s e changes i n t h e t o t a l RNA

l e v e l s ( F i g s . 8, 9 ) . A s w i th t h e RNA, t h e t o t a l p r o t e i n l e v e l

dec reases s i g n i f i c a n t l y ( P - < 0.05) a t t h e t ime of both t h e

f i n a l nymphal moult and t h e imaginal moult.

The c o n c e n t r a t i o n of p r o t e i n r i s e s a t t h e beginning o f

development and t h e n dec reases slowly and s t e a d i l y throughout

t h e remainder o f t h e growth and d i f f e r e n t i a t i o n p e r i o d ( ~ i g . 9 ) .

C r o s s - s e c t i o n a l F i b r e Area:

The growth and d i f f e r e n t i a t i o n of t h e i n s e c t f l i g h t

muscles i s r e f l e c t e d i n changes o c c u r r i n g i n t h e c r o s s - s e c t i o n a l

a r e a of t h e muscle f i b r e s . Except f o r i n t e r r u p t i o n s a t t h e

t ime o f both t h e moults , t h e c r o s s - s e c t i o n a l a r e a of t h e f i b r e s

i n c r e a s e s r a p i d l y throughout t h e developmental p e r i o d (F ig . 1 0 ) .

During t h e f i f t h nymphal i n s t a r t h e a r e a i n c r e a s e s by 800%

w i t h a subsequent 350% i n c r e a s e i n a r e a du r ing t h e f i r s t e i g h t

days of imaginal l i f e . This s t a t i s t i c a l l y s i g n i f i c a n t ( P <

0.05) i n c r e a s e o f about 12 - fo ld i s no t s u r p r i s i n g i n view of

t h e o v e r a l l i n c r e a s e i n weight that occur s .

Figure 9. Cytoplasmic p r o t e i n content ( t o t a l e x t i n c t i o n ) and

i t s concent ra t ion (mean e x t i n c t i o n ) o f S. g regar i a - f l i g h t muscle f i b r e s . Each po in t r e p r e s e n t s t h e mean

& standard dev ia t ion of 100 measurements.

1- - - - -g t o t a l e x t i n c t i o n

c---------r mean e x t i n c t i o n

.. I l l I I I f I P I I I I I I I

last day of 4th 1 3 3 5 8

5 t h INSTAR Instar

ADU LT-

AGE (DAYS)

Figure 10. Cross-sect ional a r e a of S. g regar i a f l i g h t muscle - f i b r e s expressed i n terms of a 1 pn s t e p s i z e .

Each po in t r ep resen t s t h e mean & standard e r r o r

of 100 measurement

AGE (DAYS)

DISCUSSION

;h t h e r e has been a considera , b l e amount of research

on d i f f e r e n t i a t i o n of s t r i a t e d muscles of v e r t e b r a t e s , l i t t l e i s

known on nuclear d i f f e r e n t i a t i o n of muscles ( s e e reviews by

Fischman, 1972; Holtzer e t a l . , 1972, Goldspink, 1972). -- The a v a i l a b l e data i n d i c a t e t h a t DNA syn thes i s i s seen only i n

t h e mononucleated myoblasts and once a syncytium i s formed by

t h e f u s i o n of t h e s e c e l l s , DNA s y n t h e s i s ceases . The muscle

n u c l e i remain d i p l o i d throughout t h e l i f e of t h e organism.

Information on t h e cytochemistry of i n s e c t muscle n u c l e i

during development and metamorphosis i s a l s o scanty. The

r e s u l t s obtained i n my study show t h a t DNA syn thes i s occurs

during t h e per iod of f l i g h t muscle d i f f e r e n t i a t i o n i n S. gregar ia . - During t h i s t ime . there a r e t h r e e d i s t i n c t peaks of high s y n t h e t i c

a c t i v i t y ( ~ i g . 3 ) .

Krishnakumaran e t a l . (1967) s tud ied t h e p a t t e r n of DNA -- s y n t h e s i s i n d i f f e r e n t t i s s u e s of Lepidopterous i n s e c t s during

growth and metamorphosis. They observed DNA syn thes i s i n

muscle c e l l s not only during t h e l a r v a l i n s t a r s , but a l s o

during pupal-adul t ecdysis . They a l s o not iced l a b e l l i n g of

muscle n u c l e i i n a d u l t cockroaches and mealworms. These

observat ions l e d Krishnakumaran e t a l e (1967) t o hypothesize -- t h a t DNA syn thes i s a t c e r t a i n t imes during development may be

11 f o r e l imina t ion of var ious b iases" thus enabl ing t h e c e l l s t o

be reprogrammed. Although t h i s hypothesis i s a n i n t e r e s t i n g

one i t s v a l i d i t y has y e t t o be e s t ab l i shed .

The f a c t t h a t DNA syn thes i s occurs i n t h e f l i g h t muscles

during d i f f e r e n t i a t i o n r a i s e s p o s s i b i l i t i e s concerning i t s

purpose. Does t h i s i n d i c a t e po lyp lo id iza t ion of n u c l e i o r

t h e i r dup l i ca t ion?

Throughout t h e developmental per iod t h e log2 of t h e t o t a l

e x t i n c t i o n values of t h e f l i g h t muscle n u c l e i remain between

5.0 and 6.0 ( ~ i g . 4 ) . This r ep resen t s a d i p l o i d condi t ion ,

These observat ions conform w i t h those obtained i n v e r t e b r a t e

s k e l e t a l muscles by o t h e r s (Lash e t a l . , 1957; F i r k e t , 1958; -- Basleer , 1962; Cox and Simpson, 1970).

This r u l e s out po lyp lo id iza t ion as t h e reason f o r DNA

syn thes i s . Another p o s s i b i l i t y i s nuclear f i s s i o n . Examina-

t i o n of nuclear a r e a shows t h a t it undergoes s t a t i s t i c a l l y

s i g n i f i c a n t changes over t h e developmental per iod. These

changes ( 20%) do not appear s u b s t a n t i a l enough t o support

t h e p o s s i b i l i t y of f i s s i o n ( ~ i g . 7 ) .

The absence of any m i t o t i c o r a m i t o t i c f i g u r e s as we l l

as a l a c k of po lyp lo id iza t ion i n t h e s e f i b r e s would suggest

a d i f f e r e n t func t ion f o r t h e newly synthesized DNA. From t h e

da ta I have it i s d i f f i c u l t t o e s t a b l i s h t h a t t h i s new DNA

i s metabolic DNA ( ~ e l c , 1972). I n t h e l o c u s t , t h e "precursor9 '

muscle begins syn thes iz ing i t s components immediately a f t e r t h e

moult i n t o t h e f i f t h i n s t a r . This involves t h e s y n t h e s i s of

c e r t a i n molecules l i k e myosin. I n systems which synthes ize

l a r g e q u a n t i t i e s of p r o t e i n s i t w i l l be advantageous t o have

a d d i t i o n a l gene copies f o r t r a n s c r i p t i o n . Pelc (1972) sugges ts

32.

t h a t "owing t o wear and t e a r some copies become unusable and

a r e p e r i o d i c a l l y renewed." I n l i g h t of t h i s it i s tempting

t o suggest t h a t t h e cyc les of DNA syn thes i s i n t h e s e n u c l e i a t

c e r t a i n pe r iods of development r ep resen t t h e syn thes i s of t h e

necessary copies of t h e genome t h a t t r a n s c r i b e f o r s p e c i a l pro-

t e i n s l i k e myosin and c e r t a i n enzyme systems.

DNA t r a n s c r i p t i o n a l a c t i v i t y i s a n e s s e n t i a l , on-going

process i n both developing and mature t i s s u e s . Bonner and

Huang (1963) suggest t h a t h i s tones , bas ic nuclear p r o t e i n s ,

a r e involved i n t h e r e g u l a t i o n of gene expression a t t h e

t r a n s c r i p t i o n a l l e v e l , They surmise t h a t changes i n t h e binding

of h i s t o n e s t o t h e template would make d i f f e r e n t segments of

DNA a v a i l a b l e f o r t r a n s c r i p t i o n . Actinomycin D i s a n a n t i b i o t i c

which binds s p e c i f i c a l l y t o t h e h i s tone- f ree guanosine-containing

s i t e s of t h e DNA molecule ( ~ i i l l e r and Crothers , 1968; Zaj icek

e t a l . , 1970) -- It has been shown conclus ive ly , t h a t t h e binding of AMD

i s dependent upon t h e s t a t e of r e p r e s s i o n of t h e chromatin,

t h a t i s , a n AMD binding p r o f i l e r e f l e c t s changes i n t h e degree

of a s s o c i a t i o n between DNA and chromosomal p r o t e i n s a t d i f f e r e n t

s t a g e s of t h e c e l l cyc le ( ~ r a c h e t and Ficq, 1965; Berlowitz - e t

a l . , 1969; Badr, 1972; Pederson and Robbins, 1972). - Brachet and Malp'oix (1971) s t a t e t h a t AMD binding decreases

when t h e genome i s repressed . This binding decreases towards

t h e f i n a l phase of d i f f e r e n t i a t i o n e s p e c i a l l y i n those c e l l s

t h a t syn thes ize one major p r o t e i n , It has a l s o been shown t h a t

33 *

AMD binding inc reases i n lymphocytes s t imula ted by phytohemag-

g l u t i n i n e (~a rzcynk iewicz e t a l . , 1969; Ringertz e t a l . , 1969; -- -- Ringertz and Bolund, 1969 a, b; R i g l e r e t a l - , 1.969), and i n -- t h e c o l l e t e r i a l gland n u c l e i o f Pe r ip lane ta americana s t imula ted

by juven i l e hormone (Nair and Menon, 1972).

From t h e 3 ~ - ~ ~ study (F'ig. 5) we s e e t h a t i n genera l a s

d i f f e r e n t i a t i o n nears completion t h e o r i g i n a l l y high t r a n s c r i p -

t i o n a l a c t i v i t y dec l ines . The high t r a n s c r i p t i o n a l a c t i v i t y i n

t h e e a r l y s t a g e s i s probably i n d i c a t i v e of increased mRNA pro-

duct ion f o r t h e s y n t h e s i s of s p e c i a l p r o t e i n s such as myosin

and a c t i n . These r e s u l t s support t h e f ind ings of Vogell (1962)

who observed l a r g e amounts of polysomes i n electronmicrographs

11 of I,. migra tor ia precursor muscles'. - The chromatin i n n u c l e i e x i s t s e i t h e r i n d i f fused form

(euchromatin) o r i n a condensed form (heterochromatin) . It i s

be l ieved t h a t euchromatin i s t r a n s c r i p t i o n a l l y a c t i v e , whereas

t h e heterochromatin i s t r a n s c r i p t i o n a l l y i n a c t i v e ( ~ e i f e r e t a l e , -- 1973). If t h i s i s so a n a l y s i s of t h e chromatin p a t t e r n would

g ive some information on t h e gene t i c a c t i v i t y of t h e nucleus.

Frequency d i s t r i b u t i o n of p o i n t s i n a Feulgen-stained nucleus

would show whether t h e r e a r e p o i n t s wi th high e x t i n c t i o n va lues ,

These would rep resen t t h e regions of condensation i n t h e chroma-

t i n .

The r e s u l t s of t h e 3 ~ - ~ ~ study suggest t h a t a frequency

d i s t r i b u t i o n of t h e e x t i n c t i o n va lues of Feulgen-stained f l i g h t

muscle n u c l e i should e x h i b i t more high dens i ty p o i n t s as t h e

age increases . ~ e n e r a l l y , ) t h i s i s t r u e . However, one o t h e r

f a c t o r must a l s o be considered. A change i n t h e phys ica l s t a t e

of t h e chromatin may be i n d i c a t i v e not only of t h e l e v e l of

t r a n s c r i p t i o n a l a c t i v i t y but a l s o of t h e occurrence of DNA

syn thes i s . Indeed, those two age groups wherein no DNA s y n t h e s i s

was de tec ted ( l a s t day of t h e f i f t h nymphal i n s t a r and one-day-

o l d a d u l t ) a l s o have t h e h ighes t percentage of high dens i ty

p o i n t s (Fig. 6 ) . Furthermore, those two age groups showing t h e

h ighes t percentage of thymidine (methyl 'H) incorpora t ion ( l a s t

day of t h e four th nymphal i n s t a r and three-day-old f i f t h i n s t a r

nymph) have t h e lowest percentage of high dens i ty po in t s . There-

f o r e , as expected, where t h e chromatin e x h i b i t s low condensation

and presumably, t h e r e f o r e , high s y n t h e t i c a c t i v i t y , t h e frequency

d i s t r i b u t i o n s show fewer high dens i ty po in t s .

Extensive work has 'been done on f l i g h t muscle development

i n t h e - L. migra tor ia , an i n s e c t c l o s e l y r e l a t e d t o - S. gregar ia .

On t h e b a s i s of f r e s h weight of t h e muscle t h e developmental

process f a l l s i n t o four d i s t i n c t phases (Brosemer -- e t a l . , 1963;

% ~ h e r , 1965). This has been confirmed by u l t r a s t r u c t u r a l and

biochemical s t u d i e s . The f i r s t phase, " l a r v a l growth", com-

p r i s e s most of t h e f i f t h i n s t a r . It i s cha rac te r i zed by a n

inc rease , on a logar i thmic s c a l e , of a l l celJular components.

Although t h e n u c l e i a r e excluded from t h i s ex tens ive growth,

they do remain highly a c t i v e a t t h i s time. The "moulting i n t e r -

val" begins j u s t p r i o r t o t h e imaginal moult and ends a day o r

two a f t e r ecdysis . A t t h i s t ime t h e i n c r e a s e i n weight ceases .

Furthermore, t h e muscles undergo t r a c h e o l e invasion. The "d i f -

f e r m t i a t i o n phase" proceeds until about t h e t h i r d day a f t e r

t h e imaginal moult. It i s dur ing t h i s per iod t h a t growth

resumes coupled wi th d i i f fe ren t ia i ; ion of t h e morphological and

enzymological p a t t e r n s c h a r a c t e r i s t i c of t h e f u n c t i o n a l f l i g h t

I 1 muscle. A f t e r d i f f e r e n t i a t i o n , t h e d u p l i c a t i o n phase" proceeds

u n t l l about t h e e i g h t h day of imaginal l i f e . A t t h i s t ime,

many major components a r e dup l i ca t ed .

An examination o f t h e changcs i n t o t a l RNA and p r o t e i n as

w e l l as i n c r o s s - s e c t i o n a l a r e a of t h e f i b r e s show t h a t t h e

development i n - . S. g r e g a r i a i s very simllar t o t h a t i n - L. m i g r a t o r i a

( ~ i g s . 8, 9, 10).

I n a l l t h r e e of t h e parameters examined t h e " l a r v a l growth

I! A? phase i s c h a r a c t e r i z e d by cons ide rab le i nc reases . The apparen t ?

deci-easz i n f i b r e a r e a dur ing t h e "moul t ing i n t e r v a l " i s no t

s t a t i s t i c a l l y s i g n i f i c a n t . However, t h e r e has obvious ly been

a c e s s a t i o n o f growth. Indeed t o t a l p r o t e i n and t o t a l RNA

l e v e l s d e c l i n e s l i g h t l y a t t h i s t ime. Although t h e y were expected

t o i n c r e a s e dur ing t h e " d i f f e r e n t i a t i o n phase", t h e r ea son they

d i d n o t may be l i n k e d t o t h e t r a n s c r i p t i o n a l a c t i v i t y . The t r a n s -

s c r i p t i o n a l a c t i v i t y i s r e l a t - i ve ly high a t t h e beginning o f . t he

11 l a r v a l growth phase" , but it d e c l i n e s about midway through t h i s

phase and does no t i n c r e a s e a g a i n u n t i l t h e " d i f f e r e n t i a t i o n

phase" has begun. Therefore t h e i n c r o a s e s i n t o t a l RNA and

p r o t e i n c o n t e n t expected a f t e r t h e imaginal moult would not

occur u n t i l a f t e r t h i s s l g n i f i c a n t i n c r e a s e i n t r a n s c r i p t i o n a l

11 a c t i v i t y , And s o t h e d u p l i c a t i o n phase" i s not s u r p r i s i n g l y

a s s o c i a t e d wi th s u b s t a n t Lal i n c r e a s e s i n a l l t h r e e components.

The o v e r a l l i n c r e a s e i n t o t a l RNA con ten t was p r e d i c t a b l e

c o n s i d e r i n g t h e i n i t i a l l y high t r a n s c r i p t i o n a l a c t i v i t y i n t h e

developing f l i g h t muscle n u c l e i . Furthermore, i n view o f t h e

tremendous i n c r e a s e i n s i z e t h a t t h e f l i g h t muscle undergoes

ill and Goldsworthy, 1968; H i l l -- e t a l . , 1968), t h e changes i n

t o t a l p r o t e i n con ten t a r e no t s u r p r i s i n g . And f i n a l l y , w i th

such a s u b s t a n t i a l i n c r e a s e i n p r o t e i n , t h e major c o n s t i t u e n t

o f muscle ( ~ a r u ~ a m a , 1965) , t h e changes i n c r o s s - s e c t i o n a l

muscle f i b r e a r e a a r e t o be expected. Indeed both t h e t o t a l

p r o t e i n con ten t and t h e c r o s s - s e c t i o n a l a r e a i n c r e a s e by a f a c t o r

of 12. Bccher (1965) found a similar i n c r e a s e i n t h e f r e s h

weight o f - L. m i g r a t o r i a f l i g h t muscles du r ing t h e i r development.

The c o n c e n t r a t i o n o f RNA i n t h e f l i g h t muscles dec reases

throughout development wh i l e t h e t o t a l RNA con,tent i n c r e a s e s .

This i s because t h e c r o s s - s e c t i o n a l a r e a o f t h e muscle f i b r e s

i s i n c r e a s i n g a t a h ighe r r a t e t h a n t h e t o t a l RNA. And

so , t h e changes i n t h e c o n c e n t r a t i o n o f p r o t e i n o r RNA i n t h e

developing f l i g h t muscles i s a f u n c t i o n o f t h e changes i n t o t a l

p r o t e i n and RNA and t h e changes i n f i b r e a r e a .

There i s one f i n a l p o i n t t o be d i scussed and t h a t i s t h e

s i g n i f i c a n c e of t h e moul t ing i n t e r v a l . I n - L. m i g r a t o r i a t h e

developing f l i g h t muscles do no t have a t r a c h e o l e system u n t i l

a f t e r t h e f i n a l ecdys i s ( ~ i i c h e r , 1965) . Indeed t h e moul t ing

i n t e r v a l i s c h a r a c t e r i z e d by t h e i n v a g i n a t i o n of t r a c h e o b l a s t s .

Biicher ( 1965)

far under t h e

suggested t h a t development could proceed on ly s o

anae rob ic c o n d i t i o n s t h a t would occur i n the

absence of i n t e r f i b r i l l a r t racheae. Therefore, before develop-

ment can cont inue, t h e t r a c h e o l e system would have t o be

recons t ruc ted . There i s some evidence from t h i s study t h a t t h e

same s i t u a t i o n e x i s t s i n - S. g regar i a . F i r s t l y , t h e r e i s t h i s

c h a r a c t e r i s t i c i n t e r r u p t i o n i n development during t h e moulting

i n t e r v a l ind ica ted by t h e c e s s a t i o n i n t h e inc rease i n t o t a l

RNA and p r o t e i n content and c ross - sec t iona l f i b r e a rea . Secondly,

a t t h i s time, t h e d i s t ance between t h e muscle f i b r e s i s seen t o

i n c r e a s e g r e a t l y which could i n d i c a t e t r a c h e o l a r invagina t ion .

CONCLUSIONS

Cytochemical a n a l y s i s of f l i g h t

S. gregar i a showed t h e following: -- - muscle d i f f e r e n t i a t i o n i n

The f l i g h t muscle n u c l e i remained d ip lo id throughout

t h e per iod of d i f f e r e n t i a t i o n .

Although t h e n u c l e i a r e d ip lo id , c y c l i c a l syn thes i s

of DNA i s seen during d i f f e r e n t i a t i o n . This may

represen t a m p l i f i c a t i o n of c e r t a i n segments of t h e

genome.

The t r a n s c r i p t i o n a l a c t i v i t y of t h e s e n u c l e i i s high

t o begin with but gradual ly dec l ines towards t h e end

of d i f f e r e n t i a t i o n ,

RNA and p r o t e i n content i n c r e a s e during t h e d i f feren-

t i a t i o n per iod by a f a c t o r of four and twelve

respec t ive ly .

The c ross - sec t iona l a r e a of t h e f i b r e s inc rease by

a f a c t o r of 12.

REFERENCES '

A l f e r t , M. and I. I. Geschwind. 1953. A s e l e c t i v e s t a i n i n g method f o r t h e bas ic p r o t e i n s of c e l l nucle i . Proc. Nat. Acad. Sc i . , 39: 991-999.

Ashhurst, D, E. 1967. The f i b r i l l a r f l i g h t muscles of g i a n t water-bugs: an electron-microscope study. J. C e l l Sc i . , 2: 435-444.

Badr, E. A. 1972. Cytophotometric s t u d i e s on h i s t o n e s i n d i f f e r e n t types of c e l l s a s compared t o t h e degree of binding of 3H-actinomycin D t o t h e i r nucle i . Arch. Biol . , 83: 21-27.

Baserga, R. and D. Malamud. 1969. Modern methods i n experi- mental pathology autoradiography: techniques and a p p l i - c a t i o n s . Harper and Row, N. Y . , Evanston and London.

Basleer , R. 1962, Etude l laugmenta t ion du nombre de noyaux dans des bourgeions musculaired c u l t i v e s - i n v i t r o . 2 . anat . Entwicklung. , 123: 184-205.

Bar te l s , P. H, , G. Bahr, W. J e t e r , G. Olson, J. Taylor and G. Weid. 1974. Evaluat ion o f c o r r e l a t i o n a l information i n d i g i t i z e d c e l l images. J. Histochem. Cytochem., 22: 69-79.

Beenakkers, A, M. 1963. Enzyme der det tsaure-oxydat ion i n den flugmuskeln von Locusta migra to r i a wahrend i h r e r entwicklung. Biochem. Z. , 3 3 7 . m 9 .

Beenakkers, A. M. 1973. The in f luence of corpora aLlata on t h e f l i g h t muscle development i n l o c u s t s . J. Endocrinol., 57: 52.

Bhakthan, N. M. , K. K. Nair and J. H. Borden. 1971. Fine s t r u c t u r e of degenerat ing and regenera t ing f l i g h t muslces i n a bark b e e t l e , I p s confusus. 11. Regeneration. Can. J. Z o ~ l . , 4 9 : 8 5 - 8 9 7

Bennett, J. and K. J. Sco t t . 1971. Q u a n t i t a t i v e s t a i n i n g of f r a c t i o n 1 p r o t e i n i n polyacrylamide g e l s us ing Coomassie B r i l l i a n t Blue. Anal. Biochem. , 43: 173-182.

Berlowitz, L., D. P a l l o t t a and C. H, Sibley . 1969. Chromatin and h i s tones : binding of t r i t i a t e d actinomycin D t o heterochromatin i n mealy bugs. Sc i . , 164: 1527-1529.

'1ncludes r e fe rences c i t e d i n body of t e x t a s we l l a s i n t h e appendix.

Bocharova-Messner, 0. M. and K. A. Yanchuk. 1966. Changes i n t h e u l t r a s t r u c t u r e of t h e wing muscles of t h e domestic c r i c k e t ( ~ c h e t a dornestica L. ) i n ontogenesis . Akademiia Nauk U. s.S]R.okladi, ' 170: 657-660.

Bonner, J. and R. C. Huang. 1963, P r o p e r t i e s of ~hromosomal nucleohistone. J. Mol. B io l . , 6:169-174.

Bzotna, M. and J. Michejda. 1966. The u l t r a s t r u c t u r e of f l i g h t muscles i n development of Antherea pernyi Guer. Poznaskie towarzystow pyzyjac io l Nauk Bull . S-. Sciences Biol. Poznan., 7: 85-94.

Brachet, J. 1957. Biochemical Cytology. Academic Press , New York.

Brachet, J. and A. Ficq. 1965. Binding s i t e s of 1 4 ~ - a c t i n o m y c i n D i n amphibian ovocytes and an autoradiography technique f o r t h e d e t e c t i o n of cytoplasmic DNA. Expt l C e l l Res., 38: 153-159.

Brachet, J. and N. Hulin. 1969. Binding of t r i t i a t e d ac t inomycin and c e l l d i f f e r e n t i a t i o n . Nature, 222: 481-482.

Brachet, J. and P. Malpoix. 1971. Macromolecular syn thes i s and nucleocytoplasmic i n t e r a c t i o n s i n e a r l y development, p. 263-316. In: M. Abercombie and J. Brachet (eds . ) . Adv. Morphogenesis Vol. 9. Academic Press , N. Y. and London.

Brosemer, R. W . , W. Vogell and Th. BZicher. 1963. Morphologische und enzymatische muster b e i der entwicklung i n d i r e k t e r flugmuskeln von Locusta migra tor ia . Biochem. Z. , 338: 854-910.

Brosemer, R. W. 1965. The e f f e c t of puromycin and actinomycin D on t h e development of grasshopper f l i g h t muscle g lycerol - phosphate dehydrogenase. Biochim. Biophys . Acta, 99: 388- 399

Biicher, Th. 1965. Formation of t h e s p e c i f i c s t r u c t u r a l and enzymic p a t t e r n of t h e i n s e c t f l i g h t muscle. Biochem. Soc., Lond., 25: 15-28.

Burkhardt, H. 111. 1964. Analysis of variance-PDP-5/8. Decus Program Library, Decus No. 518-9.

Capers, C. R. 1960. Mul t inuclea t ion of s k e l e t a l muscle i n - v i t r o . J. Biophys. Biochem. Cytol. , 7: 559-566.

Conger, A. and A. M. F a i r c h i l d . 1953. Quick-freeze method f o r making smear s l i d e s permanent. S t a i n Tech., 28: 281-289.

Cooper, W. G. and I. R. Konigsberg. 1961. Dynamics of myo- genes is - i n v i t r o . Anat. Rec., 140: 195-206.

Cowden, R. R. and D. Bodenstein. 1961. A cytochemical i n v e s t i - ga t ion of s t r i a t e d muscle d i f f e r e n t i a t i o n i n r egenera t ing limbs of t h e roach, Pe r ip lane ta americana. Embryol., 6: 36-50.

Cox, P. C . and S. B. Simpson. 1970. A microphotometric study of myogenic l i z a r d c e l l s grown i n v i t r o . Devel. Biol . , - 23: 433-443.

Chrambach, A . , R. A. Re i s fe ld , M. Wychoff and J. Zaccari . 1967. A procedure f o r r ap id and s e n s i t i v e s t a i n i n g of p r o t e i n f r a c t i o n a t e d by polyacrylamide g e l e l ec t rophores i s . Anal. Biochem. , 20: 150-154.

Crossley, A. C. 1972. U l t r a s t r u c t u r a l changes during t r a n s i t i o n of l a r v a l t o a d u l t intersegmental muscle a t metamorphosis i n t h e blowfly Cal l iphora erythrocephala . I. Dedi f ferent ia - t i o n and myoblast fus ion . J. Embryol. exp. Morphol., 27: '

43-74.

Darzcynkiewicz, Z . , B. D. G l e d h i l l and N. R. Ringertz . 1969. Changes i n deoxyribonucleoprotein during spermiogenesis i n t h e b u l l . Expt l C e l l Res., 58: 435-438.

Deitch, A. D. 1955. Microspectrophotometric study of t h e bind- i n g of t h e an ion ic dye, Naphthol Yellow S, by t i s s u e s e c t i o n s and by p u r i f i e d p ro te in . Lab. Inves t . , 4:324-351.

Deitch, A. D. 1961. An improved Sakaguchi r e a c t i o n f o r microspectrophotometric use. J. Histochem. Cytochem., 9: 477- 483.

Deitch, A. D. 1966. Cytophotometry of nucle ic a c i d s , p. 327- 354. In: G. L. Weid (ed . ) In t roduc t ion t o Q u a n t i t a t i v e Cytochemistry. Academic Press , New York and London.

Doane, W. 1973. Role of hormones i n i n s e c t development, p , 291-497. In: S. J. Counce and C. H. Waddington ( e d s . ) Developmental Systems: Insec ta Vol. 2. Academic Press , London and New York,

Ebs te in , B. S. 1969. The d i s t r i b u t i o n of DNA wi th in t h e n u c l e o l i of t h e amphibian oocyte a s demonstrated by tri- t i a t e d actinomycin D radioautography. J. C e l l Sc i . , 5: 27-44.

Edwards, F. J. 1969. Development and h i s t o l y s i s of t h e i n d i r e c t f l i g h t muscles i n Dysdzrcus in te rmedius . J. I n s e c t Phys io l . , 15: 1591-1599.

Fand, S. 1972. Environment c o n d i t i o n s f o r op t imal Feulgen h y d r o l y s i s , p. 211-222. In: G. L. Weid and G. F. Bahr ( e d s . ) . I n t r o d u c t i o n t o Q u a n t i t a t i v e Cytochemistry-11. Academic P re s s , N. Y. and London.

Fazekas de S t . Groth, S. , R . G. Webster and A. Datyner. 1963. Two new s t a i n i n g procedures f o r q u a n t i t a t i v e e s t i m a t i o n of p r o t e i n s on e l e c t r o p h o r e t i c s t r i p s . Biochim. Biophys. Acta, 71: 377-391.

Ficq, A. 1955. Etude au to rad iog raph ique du metabolisme des p r o t e i n e s e t des a c i d e s nuc l e iques a u cou r s de l f o o g e n e s e chez l e s b a c t r a c i e n s , E x p t l C e l l Res. , 9: 286-293.

F i r k e t , H. 1958. Researches s u r l a syn these des a c i d e s desocyr ibonuc le iques e t l a p r e p a r a t i o n a l a mi tose dans des c e l l u l l e s c u l t i v e z s i n v i t r o . Arch. Bio l . , 69: 1-166.

Fischman, D. E. 1972. Development of s t r i a t e d muscle, p. 75- 148. In : G. H. Bourne ( e d . ) The . S t r u c t u r e and Funct ion of Muscle Vol. 1. Academic P re s s , N. Y. and London.

F i shbe in , W. N. 1972. Q-uar l t i ta t ive densi tometry of 1-50 g p r o t e i n i n acrylamide gel s l a b s w i th Coomassie blue . '

Anal. Biochem., 46: 388-401.'

Flax, M. H. and M. H. Himes. 1952. Microspectrophotometr ic . a n a l y s i s o f metachromatic s t a i n i n g of n u c l e i c a c i d s . Phys io l . Zool . , 25:297-311.

Fox, D. P. 1969. DNA va lues i n somatic t i s s u e s o f Dermestes ( ~ e r m i s t i d a e : ole opt e r a ) I. Abdominal f a t body and t e s t i s w a l l of t h e a d u l t . Chromosoma ( ~ e r l . ) , 28:445-456.

G i l b e r t , L. I. 1964. P h y s i o l o a o f growth and development: endocr ine a s p e c t s , p. 248-336. In: M. Rocks te in ( e d . ) The Physiology o f I n s e c t a Vol. 1. Academic P re s s , N. Y. and London.

Goldspink, G. 1972. Post embryonic growth and d i f f e r e n t i a t i o n o f s t r i a t e d muscle, p. 179-236. In: G. H. Bourne ( e d . ) The S t r u c t u r e and Funct ion o f Muscle Vol. 1. Academic P r e s s , N. Y. and London.

Gomori, G. 1956. Histoehemi-cal methods f o r prote in-bound su lphydry l and d i s u l p h i d e groups. Q u a r t . J. Microscop. Sc i . , 97: 1-9.

Goodman, G. C. 1957. On t h e r egenera t ion and r e d i f f e r e n t i a t i o n of mammalian s t r i a t e d muscle. J. Morphol., 100: 27-82.

Grant, V. J. 1961. ' Origin and d i f f e r e n t i a t i o n of t h e imaginal i n d i r e c t f l i g h t muscles of Simulium ornatum Meigen ( ~ i ~ t e r a , ~ e m a t o c e r a ) . J. 2001. Soc. ' ~ n d i a , 13: 174-179.

Gregory, D. W . , R. W. Lennie and L. M. B i r t . 1968. An e lec t ron- microscopic study of f l i g h t muscle development i n t h e blow- f l y Luc i l i a cuprina. J, Roy. Microscop. Soc., 88: 151-175.

Herold, R. C. 1965. Development and u l t r a s t r u c t u r a l changes of sarcosomes during honey bee f l i g h t muscle development. Develop. Biol. , 12: 269-286.

H i l l , L. and G. J. Goldsworthy. 1968. Growth, feeding a c t i v i t y and t h e u t i l i z a t i o n of r e se rves i n l a r v a e of Locusta. J. I n s e c t Physiol . , 14: 10854098.

H i l l , L., A. J. Luntz, and P. A. S t e e l e . 1968. The r e l a t i o n - sh ips between somatic growth, ovar ian growth, and feeding a c t i v i t y i n t h e a d u l t d e s e r t l o c u s t . J. I n s e c t Physiol . , 14: 1-20.

Holtzer , H . , H. Weintraub, R. Mayne and B. Mochan. 1972. The c e l l cyc le , c e l l l i neages and c e l l d i f f e r e n t i a t i o n , p. 229-259. In: A. A. Moscona and A. Monroy (eds . ) Current Topics i n Developmental Biology, Vol. 7. Academic Press , N. Y. and London.

Hunter- Jones, P. 1956. I n s t r u c t i o n s f o r Rearing and Breeding Locusts i n t h e Laboratory. Anti-Locust Research Centre, London.

Kei fer , R . , G. Kei fer , R e Salm, R. Rossner and W. S a n d r i t t e r . 1973. A method f o r t h e q u a n t i t a t i v e evalua t ion of eu- and heterochromatin i n i n t erphase n u c l e i us ing cytophotometry and p a t t e r n a n a l y s i s . Be i t r . Path. Bd., 150: 163- 173.

Krishnakumaran, A . , S. J. Berry, H. Oberlander and H. A. Schneiderman. 1967. Nucleic a c i d syn thes i s during i n s e c t development--H. Control of DNA syn thes i s i n t h e cecropia silkworm and o the r s a t u r n i i d moths. J. I n s e c t Phys io l . , 13: 1-57,

Lash, J. W . , H. Hol tzer and H. Swif t . 1957. Regeneration of mature s k e l e t a l muscle. Anat. 'Rec., 128: 679-693.

Maizel, J. V. J r , 1966. Acrylamide-gel electrophorograms by mechanical f r a c t i o n a t i o n : Radioact ive adenovirus p r o t e i n s . Sci . , 151: 988-990.

Maruyama, K. 1965. The biochemistry of t h e c o n t r a c t i l e elements of i n s e c t muscle, p. 451-481. In: M. Rockstein (ed . ) The Physiology of Insec ta . Academic Press , New York.

Mazia, D., P. A. Brewer and M. A l f e r t . 1953. The cytochemical s t a i n i n g and measurement of p r o t e i n with mercuric bromphenol blue. Biol . Bul l . , 104: 57-67.

Meyer, T. S. and B. L. Lamberts. 1965. Use of Coomassie b r i l l i a n t blue R250 f o r t h e e l ec t rophores i s of microgram q u a n t i t i e s of p a r o t i d s a l i v a p r o t e i n s on acrylamide-gel s t r i p s . Biochim. Biophys. Acta, 107: 144-145.

Mittwoch, U., H. Kalmus and W. Webster. 1966. Deoxyribonucleic a c i d values i n d iv id ing and nondividing c e l l s of male and female l a r v a e of t h e honey bee. Nature, 210: 264-266.

Mfiller, W. and D. M. Crothers . 1968. S tudies of t h e binding o f actinomycin and r e l a t e d compounds t o DNA. J. Mol. B io l . , 35: 251-290.

Murray, M. R. 1960, S k e l e t a l muscle t i s s u e i n c u l t u r e , p. 111- 154. In: G. H. Bourne (ed . ) The S t r u c t u r e and Function of Muscle. Academic Press , New York.

Nair, K. K. and M. Menon. 1972. Detect ion of juvenile-hormone- induced gene a c t i v i t y i n t h e c o l l e t e r i a l gland n u c l e i of Pe r ip lane ta by a c t inomycin D s t a i n i n g 1 technique. Experent ia , 28: 577.

Pearse, A. G. E. 1968. Histochemistry: Theore t i ca l and Applied, Vol. 1. J. A. Churchi l l Ltd., London.

Pederson, T. and E. Robbins. 1972. Chromatin s t r u c t u r e and t h e c e l l d i v i s i o n cycle . J. C e l l Biol . , 55:322-327.

Pelc, S. R. 1972. Metabolic DNA i n c i l i a t e d protozoa, s a l i v a r y gland chromosomes and mammalian c e l l s , p. 327-355. In: G. H. Bourne and J. F. D a n i e l l i (eds . ) I n t e r n a t i o n a l Review of Cytology Vol. 32, Academic Press , N. Y. and London.

P e r i s t i a n i s , G. C. and D. W. Gregory. 1971. Early s t a g e s of f l i g h t muscle development i n t h e blowfly L u c i l i a cuprina: A l i g h t and e l e c t r o n microscopic study. J. I n s e c t Physiol . , 17: 1005-1022.

Poels, C. L. M. and A. M. Beenakkers. 1969. The e f f e c t s of corpus a l l a tum implanta t ion on t h e development of f l i g h t muscles and f a t body i n Locusta migra tor ia . Ent. exp. app l . , 12: 312-324.

Rasch, E. and H. Swift . 1960, Microphotometric a n a l y s i s of t h e cytochemical Millon a c t i o n . J. Histochem. Cytochem., 8:4-17.

R ig le r , R. , D. Ki l lander , L. Bolund and N. R. Ringertz. 1969. Cytochemical c h a r a c t e r i z a t i o n of deoxyribonucleoprotein i n ind iv idua l c e l l nuc le i . Expt l Ce l l Res., 55: 215-224.

Ringer tz , N. R. and L. Bolund. 1969a. Actinomycin binding capac i ty of deoxyribonucleoprotein. Biochim. Biophys. Acta, 174:147-154.

11 Ringer tz , N. R. and L. Bolund. 1969b. ~ c t i v a t i o n " of hen ery throc t e deoxyribonucleoprotein. Exptl Ce l l Res. , 55: 205-2y4.

Ringertz , N. R . , 2. Darzcynkiewicz and L. Bolund. 1969. Actinomycin binding p r o p e r t i e s of s t imulated human lymphocytes. Expt l C e l l Res., 56:411-417.

Sacktor , B. 1970. Regulat ion of intermediary metabolism with s p e c i a l r e fe rence t o t h e c o n t r o l mechanism i n i n s e c t f l i g h t muscle, p. 267-347. In: J. W. Beament, J. E. Treherne and V. B. Wigglesworth ( eds . ) Advances i n I n s e c t Physiology Vol. 7. Academic Press , London and New York.

Sahota, T. S. and W. E. Beckel. 1967. The inf luence of epidermis on t h e developing f l i g h t muscles i n Ga l l e r i a mellonel la . Can. J. Zool., 45:421-434.

Scudder, G. G. E. 1971. The postembryonic development of t h e i n d i r e c t f l i g h t muscles i n Cenocorixa b i f i d a ( ~ u n g . ) ( ~ e m i p t e r a : ~ o r i x i d a e ) . Can. J. ~ o o l . ,-87-1398.

Scudder, G. G. E. and R. J. Hewson. 1971. The postembryonic development of t h e i n d i r e c t f l i g h t muscles i n Oncopeltus f a s c i a t u s ( D a l l a s ) ( ~ e m i p t e r a : ~ y g a e d i a ) . Can. J. Zool., 49: 1377-1386.

Shafiq, S. 1963. E lec t ron microscopic s t u d i e s on t h e i n d i r e c t f l i g h t muscles of Drosophila melanogaster 11. Di f fe ren t i a - t i o n of myofibr i ls . J. C e l l Biol. , 17:363-373.

S h i r e r , D. L. 1969~ C u r f i t . Decus Program Library, Decus No. Focal 8-63.

Smith, D. S. 1966, The o rgan iza t ion of f l i g h t muscle f i b r e s i n t h e Odonata. J. Cel l Bio l . , 1: 109-126.

Smith, D. S. 1968, I n s e c t Ce l l s . Oliver and Boyd, Edinburgh.

Tiegs, 0, W. 1955. The f l i g h t muscles of i n s e c t s -- t h e i r anatomy and his tology; with some observat ions on t h e s t r u c t u r e of s t r i a t e d muscle i n general . Ph i l . Trans, Roy. Soc. London, 238: 221-3552.

Vesterberg, 0. 1971. S ta in ing of p r o t e i n zones a f t e r i so- e l e c t r i c focusing polyacrylamide ge l s . Biochim. Biophys. Acta, 243: 345-348.

Vogell, .W. 1962. S. B. Ges. Beford. ges. Naturwiss. Marburg 83/84, 297, a s c i t e d i n Biicher, Th. 1965.

Walker, P. R . , L. H i l l and E. Bailey. 1970. Feeding a c t i v i t y , r e s p i r a t i o n and l i p i d and carbohydrate content of t h e male d e s e r t locus t during a d u l t development. J. I n s e c t Physiol. , 16: 1001-1015.

Weid, G. L., P. H. Bar t e l s , G. F. Bahr, and D. G. Oldf ie ld . 1968. Taxonomic i n t r a - c e l l u l a r a n a l y t i c system (TICAS) f o r c e l l i d e n t i f i c a t i o n . Acta Cytol. , 12: 177-201.

Wigglesworth, V. B. 1964. The hormonal r e g u l a t i o n of growth and reproduct ion i n i n s e c t s , p. 215-226. In: J. W. Beament, J. E. Treherne and V. B. Wigglesworth (eds . ) Advances i n I n s e c t Physiology Vol. 2. Academic Press , New York and London.

Zaj icek, D. , F. J. Swartz and A. D. Floyd. 1970. Ascar is suum and Toxocara canis : Radioautographic d e t e c t i o n of^^ i n F e u l g e n - w i v e muscle nucle i . Expt l P a r a s i t o l . , 27: 516-523.

Zirnrner, H. 1970. Automatic a n a l y s i s of microscopic images. Ze i s s Information, 74:126-132.

47

Appendix I

I n t r o d u c t i o n :

There i s a n u n f o r t u n a t e gap among h i s tochemica l t echniques

f o r q u a n t i t a t i v e p r o t e i n s t a i n s . Gomori (1956) ' ha s desc r ibed

t h e problems a s s o c i a t e d wi th t h o s e p r o t e i n s t a i n s which r e a c t

w i t h t h e su lphydry l and d i s u l p h i d e groups. There a r e a few

s t a i n s which have been uszd s u c c e s s f u l l y i n t h e p a s t f o r p r o t e i n

microspectrophotometry. However, t h e ma jo r i t y of t h e s e a r e f o r

h i s t o n e s . These i nc lude F a s t Green ( ~ l f e r t and Geschwind, 1953) ,

t h e Sakaguchi r e a c t i o n ( ~ e i t c h , 1961) and a Naphthol Yellow S

( ~ e i t c h , 1955) . However, t h e r e a r e problems a s s o c i a t e d w i t h u s e

o f t h e s e techniques . The Sakaguchi r e a c t i o n product i s o f low

i n t e n s i t y ( ~ e i t c h , 1966) and fur thermore i t s development and

p r e s e r v a t i o n a r e t e c h n i c a l l y d i f f i c u l t ( ~ e a r s e , 1968) . a Naphthol

Yellow does no t s t a i n i n t e n s e l y and i s r a t h e r d i f f u s e ( ~ r a c h e t ,

1957)

The commonly used s t a i n f o r g e n e r a l p r o t e i n de t e rmina t ion

i s M i l l o n ' s . However, due t o numerous problems Rasch and Swif t

(1960) found t h a t t h i s t echn ique i s r e a l l y on ly v a l u a b l e i f a n

accu racy of no g r e a t e r t h a n 20% i s d e s i r e d .

Mercuric Bromophenol b lue ( ~ a z i a e t a l . , 1953) i s ano the r -- commonly used p r o t e i n s t a i n . F i cq (1955) found t h a t

t h e s t a i n i n t e n s i t y o f Bromophenol b lue i s s t r o n g l y in f luenced

by t h e presence o r absence o f RNA.

The purpose of t h i s i n v e s t i g a t i o n was t o determine whether

' ~ e f e r e n c e s c i t e d i n t h e appendix a r e l i s t e d i n t h e g e n e r a l r e f e r e n c e l i s t from p. 39.

Coomassie B r i l l i a n t blue could be used f o r t h e es t imat ion of

p r o t e i n s i n t i s s u e s e c t i o n s by cytophotometric methods. It has

been used extens ive ly i n e l ec t rophores i s ( ~ a z e k a s de S t . Groth

e t a l . , 1963; Meyer and Lamberts, 1965; Maizel, 1966; Chrambach -- st a l . , 1967; Bennett and Sco t t , 1971; and Fishbein, 1972). --

Fazekas de S t . Groth e t a l e (1963), Chrambach e t a l . , -- -- (1967) and Fishbein (1972), have shown t h a t t h e i n t e n s i t y of

t h e s t a i n bound t o p r o t e i n s was p ropor t iona l t o t h e concentra-

t i o n of p ro te ins .

Methods and Mater ia ls :

The d o r s a l l o n g i t u d i n a l f l i g h t muscle of a five-day-

maze- o l d f i f t h i n s t a r nymph of - S. g regar i a was f ixed by fib,

s u b s t i t u t i o n ( ~ e a r s e , 1968) and embedded i n p a r a f f i n . Five

micron s e c t i o n s were cu t and placed on quar tz s l i d e s . Some

s e c t i o n s were t r e a t e d with r ibonuclease (sigma Chemical Co.,

S t . Louis, Mo. ) (1 .0 mg/ml) f o r 1 hour a t 37" C ( ~ e i t c h , 1966).

These and t h e c o n t r o l s e c t i o n s were then dehydrated through

grades of e thanol and mounted i n g lyce r ine ( n ~ = 1.459). The

e x t i n c t i o n measurements were made a t 280 nm i n t h e SMP a s

descr ibed on p . 1 5 . The diameter of t h e scanning a p e r t u r e was

1.16 pm and t h e s t e p s i z e was 2 w.

These same s e c t i o n s were t h e n s t a i n e d with Coomassie

Blue (Edward Gurr Ltd. , London, ~ n g l a n d ) a f t e r r e f i x a t i o n i n

12.5% t r i c h l o r o a c e t i c a c i d (TCA) f o r 30 minutes.

Since t h e concen t ra t ion of t h e dye s o l u t i o n does not

appear t o be a c r u c i a l f a c t o r when used between 0.05 and 1.0%

Fishbein, 1972), i n my study t h e s t a i n was made up from a 1% %

s tock so lu t ion , by a 1:20 d i l u t i o n i n 12.5% TCA (~hrambach e t .-

a l . , 1967). These and a l l o t h e r s o l u t i o n s preceding d e s t a i n i n g - were made up with g l a s s d i s t i l l e d water as suggested by Fazekas

de S t . Groth e t a l . (1963). The s e c t i o n s were s t a i n e d f o r one -- hour a t 37" C and des ta ined i n 7% a c e t i c a c i d (Maizel, 1966) f o r

one hour a t room temperature. They were then mounted i n o i l 4

("D = 1.56, Carg i l l e , U. S. A . ) . Ex t inc t ion measurements of t h e s e

s e c t i o n s were taken a t 590 nun us ing t h e same a p e r t u r e and s t e p

s i z e as before,

The da ta were submitted t o " ~ u r f i t " a n a l y s i s , a Decus

program hirer, 1969). This program f i t s weighted o r unweighted

da ta t o a s t r a i g h t l i n e on a Car tes ian , log-log, o r semi-log

graph. It c a l c u l a t e s t h e s lope and t h e i n t e r c e p t of t h e l i n e ,

t h e s tandard e r r o r i n t h e s e values, , t h e r eg ress ion c o e f f i c i e n t

and o t h e r measures of t h e "goodness" of f i t . This was used

t h e r e f o r e t o enable comparison of t h e s lopes us ing t h e s tudent

, t - t e s t .

R e s u l t s and Discussion:

Coomassie Blue i s a triphenylmethane dye which g ives an

except ional ly high colour i n t e n s i t y ( ~ a z e k a s de S t . Groth -- e t a l . ,

1963)

The t o t a l e x t i n c t i o n f o r inc reas ing amounts of p r o t e i n s

i n uns ta ined l o c u s t f l i g h t muscle s e c t i o n s determined by u l t r a -

v i o l e t microspectriophotometry, i s ' p r e s e n t e d i n f i g u r e 1 1 A and B.

Figure 11. The r e l a t i o n s h i p between t o t a l e x t i n c t i o n and t h e

amount of p r o t e i n s i n s e c t i o n s of f i f t h i n s t a r - S.

g regar i a f l i g h t muscle,

A: measured a t 280 nm

B: same a s above a f t e r RNase t reatment f o r one

hour a t 37" C

C: measured a t 590 nm a f t e r s t a i n i n g with Coomassie

blue

D: same a s C preceded by RNase t reatment

N: sample s i z e

r: reg ress ion c o e f f i c i e n t

TOTAL EXTINCTION (590nm ) TOTAL EXTINCTION (280nm)

Figure 1 1 B i s f o r f l i g h t muscle s e c t i o n s previously t r e a t e d

wi th r ibonuclease , Figures 1 1 C and D r ep resen t t h e p r o t e i n

content of t h e same s e c t i o n s a f t e r s t a i n i n g with Coomassie

blue. The s t a t i s t i c a l da ta from t h e s e four graphs i s a l s o

presented i n Table 11.

Although t h e two ways of measuring p r o t e i n content ,

i. e. , u. v. and Coomassie blue microspectrophotom~try, would

be expected t o y i e l d d i f f e r e n t a b s o l u t e r e s u l t s , both t h e

t r e n d s and s lope of t h e r e s u l t i n g l i n e s should be very s i m i l a r .

Table I1 and f i g u r e 11 show t h i s t o be t h e case.

Orences S t a t i s t i c a l a n a l y s i s showed no s i g n i f i c a n t d i f f ,

between any two of t h e s e four popula t ions when r e s u l t i n g

s lopes were compared.

Conclusions:

It appears t h a t Coomassie blue can be used a s a quant i -

t a t i v e s t a i n f o r cytochemical a n a l y s i s o f p ro te ins . Further-

more, t h e presence of RNA does not i n t e r f e r e with t h e Coomassie

Blue s t a i n i n g whereas it does wi th mercuric Bromophenol blue

s t a i n i n g (Ficq , 1955).

It would be d e s i r a b l e t o t e s t t h e in f luence of o t h e r f a c t o r s

l i k e t h e temperature of t h e s t a i n i n g bath and t h e minimum s t a i n -

i n g t ime f o r each t y p e of t i s s u e t o be examined.

TABLE I1

Regression c o e f f i c i e n t a n a l y s i s of da ta i n F ig- 11

Sample N y - i n t e rcep t s lope r

A-D: a s i n Fig. 11

N: sample siz-e

r: r e g r e s s i o n c o e f f i c i e n t

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