', 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

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

my t h e s i s o r d i s s e r t a t i o n ( t h e t i t l e of which i s shown below) t o u s e r s

of t h e Simon F rase r Univers i ty L ib ra ry , and t o make p a r t i a l o r s i n g l e

copies only f o r such use r s o r i n response t o a reques t from the l i b r a r y

of any o t h e r u n i v e r s i t y , o r o the r educa t iona l i n s t i t u t i o n , on i t s own

behal f o r f o r one of i t s u s e r s . I f u r t h e r agree t h a t permission f o r

mu l t ip l e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted

by me or the Dean of Graduate S tudies . It i s understood t h a t copying

o r pub l i ca t ion of t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l no t be allowed

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

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

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

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

LIST OF FIGURES

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)

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

LE 6. GR

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

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

1 - - I

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

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

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

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.

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 ,

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 . ,

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

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

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

Documents

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

The Mechanism of Breathing Air rushing in Intercostal muscles & diaphragm muscles Intercostal muscles & diaphragm muscles Chest Volume Rib Cage Diaphragm.

เรื่องสารเคมีภายในเซลล์ cytochemistry ตอนที่ 1

Le muscle INTRODUCTION. Comparaison des trois types de muscles Muscles squelettiques Muscle cardiaque Muscles lisses Muscles striés Muscles non striés.

MUSCLES .

Lab #15 Muscles. Muscles of the Head and Neck The Axial Muscles Divisions based on location and function: –muscles of head and neck –muscles of vertebral.

FELINE. FACIAL MUSCLES Muscles of the lips and cheeks Muscles of the nose Muscles of the eyelids Muscles of the external ear.

The Cytochemistry of the Amoebocytes and Intestinal ... · 60 Zacks—Cytochemistry of Venus mercenaria OBSERVATIONS Supravital staining. Spreads of atrial muscle stained in i: 10,000

Muscles MUSCLES (“pick things up”)

World History Summer Assignment 2015 (2) · SUMMER ASSIGNMENT FOR WORLD HISTORY ... Gobi Desert Kalahari Desert Sahara Desert Thar Desert Sierra Madre Desert Mojave Desert Namib Desert

Muscles of the Body Healthy Muscles Matter!. Facts About Muscles The body has more than 600 muscles Muscles help you move, lift things, pump blood, and.

Today and Wednesday – muscles! how muscles work – in general muscles that move the mandible abdominal wall muscles anterior and posterior inferior and.

เรื่องสารเคมีภายในเซลล์ cytochemistry ตอนที่ 2

Muscles of the Torso and Limbs Naming Conventions Head and Neck Muscles Trunk Muscles Muscles of the Upper Arm Muscles of the Lower Arm and Hand.

CHAPTER 10 B , MUSCLES AND SKIN - … · CHAPTER 10, Bones, Muscles, and Skin ... Warming up muscles before exercise makes muscles more flexible. c. Muscles never …

Principle Skeletal Muscles 2 Muscles that act on the abdominal wall, muscles used in breathing and muscles that move the pectoral girdle.

Journal of Histochemistry & Cytochemistry

how to build muscles fast 200 muscles recipes

Forelimb Muscles. Extrinsic Muscles Intrinsic Muscles.

Muscles of Mastication Muscles of the Neck Muscles of the Thoracic ...

Muscular System 600 Muscles. 3 types of muscles Smooth Muscles- Which include the muscles of internal organs and blood vessels. These muscles move involuntary.