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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
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
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
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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.
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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
top related