Biosynthesis of hydrocarbons in the American cockroach, Periplaneta americana
by Charles Wayland Conrad
A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY in Chemistry
Montana State University
© Copyright by Charles Wayland Conrad (1971)
Abstract:
Hydrocarbons are biosynthesized from acetate, propionate, and fatty acid substrates in P. americana
and are spread over the surface throughout adult life. The total amount of surface hydrocarbon is
approximately the same in teneral and fully-tanned adult roaches.
Since acetate-1-14C, propionate-l-l4c, and linoleate-l-l4C are not incorporated into hydrocarbons in
teneral adults, while other fatty acid substrates are incorporated, hydrocarbon biosynthesis via an
elongation-decarboxylation pathway does not occur at this time. An alternative mechanism for
hydrocarbon biosynthesis is head-to-head condensation of fatty acids. Propionate, and not isoleucine or
the S-methyl group of methionine,. is thought to be the source of the methyl side-chain in branched
hydrocarbons. Linoleate is believed to be the source of the unsaturated chain in diene hydrocarbons.
BIOSYNTHESIS OF HYDROCARBONS IN THE AMERICAN
COCKROACH, PERIPLANETA AMERICANA
by
CHARLES WAYLAND CONRAD
A t h e s i s s u b m itte d to th e G ra d u a te F a c u lty in p a r tia l
fu lfillm e n t o f th e re q u ire m e n ts fo r th e d e g re e
of
DOCTOR OF PHILOSOPHY
in
C h e m istry
A pproved:
H e a d , M a jo r D e p a rtm e n t
C h a irm a n , E xajm ning C o m m ittee
G ra d u a te D ean
MONTANA STATE UNIVERSITY
B ozem an , M o n ta n a
M a rc h , 19 7 Ii
ACKNOWLEDGMENT
The a u th o r w is h e s to th a n k h is w if e , w ho h e lp e d s u p p o rt him and
w ho ty p e d som e o f th e d ra fts o f th is w o rk . He a ls o w is h e s to th a n k h is
m a jo r a d v is o r , D r. L arry L. J a c k s o n , fo r h is g u id a n c e ,. te c h n ic a l a s s i s t ­
a n c e , w o rd s o f w is d o m , h e lp in .p rep arin g th is m a n u s c r ip t, and m oral
s u p p o rt d u rin g th e l a s t fe w y e a r s . T e c h n ic a l a s s i s t a n c e o f M elv in
A rm old, G a y le H ill, a n d .N e a l M a rx e r a re a c k n o w le d g e d . T his w ork w a s
in s p ir e d by th e r e s e a r c h o f D r. G raem e L . B aker an d w a s s u p p o rte d in
p a rt b y P e tro leu m R e s e a rc h Fund G ra n t N o . 2 3 5 3 -A 1 -5 , an d th e A g ric u l­
tu r a l E x p e rim en t S ta tio n an d C h e m istry D e p a rtm e n t o f M o n ta n a S ta te
U n iv e r s ity .
iv
TABLE OF CONTENTS
CHAPTER
PAGE
IN T R O D U C T IO N ............................................................................................... . . . .
P h y s io lo g y o f th e I n s e c t C u tic le
I
...........................................................
I
C u tic u la r L ip id s o f I n s e c ts a n d O th e r O r g a n i s m s ..........................
3
B io s y n th e s is o f F a tty A c i d s ........................................................................
3
N orm al F a tty A c i d s ........................................
3
B ran ch ed F a tty A c i d s ............................................................................
-5
B io s y n th e s is o f H y d r o c a r b o n s ..................................................................
8
H e a d - to - h e a d C o n d e n s a tio n P a t h w a y .......................................
8
E lo n g a tio n - d e c a rb o x y la tio n P a t h w a y ...........................................
11
O c c u rre n c e o f Both P a th w a y s in. th e Same
O rg a n ism
.........................................................................................
13
H y d ro c a rb o n s o f P e rip la n e ta a m e ric a n a ..............................................
13
B io s y n th e s is o f H y d ro c a rb o n s in P e rip la n e ta a m e r i c a n a .............
16
HYPOTHESIS
........................................................................
EXPERIMENTAL . . . ...................................................................................................
G row th C o n d itio n s
....................................
18
19
19
A ttem p ts to P re p a re a C e ll- f r e e B io s y n th e tic S y s t e m ....................
19
I n je c tio n o f S u b s tr a te s an d I n h i b i t o r s .................................................
21
In c u b a tio n o f I n je c te d C o c k ro a c h e s
.....................................................
21
.....................................................................
22
C h ro m a to g ra p h ic P u rific a tio n o f H y d ro c arb o n F ra c tio n s . . . . . .
22
E x tra c tio n o f S u rfa c e L ip id s
V
CHAPTER
PAGE
EXPERIMENTAL (c o n tin u e d )
A n a ly s is o f In te rn a l L ip id F ra c tio n s
....................................................
27
L iq u id S c in tilla tio n C o u n tin g o f L ip id F r a c t i o n s ..............................
29
O x id a tir e D e g ra d a tio n o f B ran ch ed S a tu ra te d H y d ro c a rb o n s . .
29
R E SU L T S............................................................................................................................. '3 1
In te g u m e n t H y d ro c a rb o n s o f T e n e ra l C o c k ro a c h e s
. . .................
32
H y d r o c a r b o n s ............................................................................................
33
In c o rp o ra tio n o f L a b e le d S u b s tr a te s in to S u rface
In c o rp o ra tio n o f L a b e le d S u b s tr a te s in to In te rn a l L ip id
F ra c tio n s
........................................................................................
D e g ra d a tio n o f B ran ch ed H y d ro c a rb o n s
36
....................................... ... .
36
. ..................................................
38
In h ib itio n w ith Sodium C i t r a t e ..................................................................
39
In h ib itio n w ith Sodium A c e t a t e ................................
40
In h ib itio n w ith T r ic h lo ro a c e tic A cid
D IS C U S S IO N ...........................................................
41
In c o rp o ra tio n o f A c e ta te an d F a tty A cids in to H y d ro c a rb o n s . .
42
In c o rp o ra tio n o f L in o le a te in to D ie n e H y d r o c a r b o n s ....................
42
P re c u rs o rs o f B ran ch ed H y d ro c arb o n s
.................................................
43
..............................................
45
P a th w a y s o f H y d ro c arb o n B io s y n th e s is
M e ta b o lic D iffe re n c e s D uring th e L ife C y c le
.................................
48
In h ib itio n , o f M e ta b o lic P a t h w a y s ...........................................................
49
P o te n tia l A reas fo r F u rth e r R e s e a r c h .....................................................
49
vi
CHAPTER •
PAGE
SUMMARY . . ......................................
52
LITERATURE CITED
SE
APPENDIX
..................................................................................................
60
v ii
LIST OF TABLES
TABLE
I.
PAGE
3'
In c o rp o ra tio n o f P a lm lta te -9 ,1 0 - B in to H y d ro c arb o n s by
C e ll- f r e e P r e p a ra tio n s o f M a le P. a m e ric a n a a n d Buffer
C o n t r o l s ....................................................
II.
W e ig h ts o f S u rfa c e H y d ro c arb o n s in N ew ly -m 'o lted -,an d
T w o -m o n th O ld e r A dult P. a m e r i c a n a ..............................................
H I.
34
In c o rp o ra tio n o f L a b e le d S u b s tr a te s in to In te rn a l L ip id s
o f A dult M ale. P. a m e ric a n a . ................................................................
V.
32
In c o rp o ra tio n o f L a b e le d S u b s tr a te s in to S u rfa ce H y d ro ­
c a rb o n F ra c tio n s in A dult P. a m e r i c a n a ...........................................
IV.
31
37
T r ic h lo ro a c e ta te In h ib itio n o f A c e ta te In c o rp o ra tio n in to
H y d ro c a rb o n s in. A dult M a le R a m e ric a n a 1-4 W e e k s
a f te r th e Im a g in a l E c d y s is ' ................................................................
VI.
38
Sodium C itra te In h ib itio n , o f L in o le a te In c o rp o ra tio n in to
H y d ro c a rb o n s in A dult M a le P. a m e ric a n a 1-4 W e e k s
a f te r th e Im a g in a l E c d y s is .......................................
V II.
39
In h ib itio n by 2 M Sodium A c e ta te o f In c o rp o ra tio n o f
L a b e le d S u b s tr a te s in to H y d ro c a rb o n s of A dult M a le
P. a m e ric a n a 1-4 W e e k s a f te r th e Im a g in a l E c d y s i s .................
40
v iii
LIST OF FIGURES
FIGURE
1.
PAGE
S te p s in th e de novo s y n th e s is a n d e lo n g a tio n , o f f a tty
a c id c h a i n s ..................................................................................................
2.
P ro p o se d p a th w a y fo r in c o rp o ra tio n o f is o le u c in e in to
b ra n c h e d f a tty a c id s in t o b a c c o ................................................. ... .
3.
.........................................................................................1 0
P ro p o se d e lo n g a tio n - d e c a r b o x y la tio n p a th w a y to form a
h y d ro c a rb o n m o le c u le from a f a tty
7.
a c id m o l e c u l e ...................
12
S tru c tu re s o f h y d ro c a rb o n s w h ic h h a v e b e e n re p o rte d for
P . a m e r i c a n a ...............................................................................................
8.
9
P ro p o se d m e c h a n ism fo r fo rm a tio n o f c o ry n o m y c o lic a c id
in C_. d ip h th e ria e
6.
7
P ro p o se d c o n d e n s a tio n o f tw o f a tty a c id m o le c u le s, to
form a h y d ro c a rb o n m o le c u le . ...........................................................
5.
6
P ro p o se d m e c h a n is m for s y n th e s is o f b ra n c h e d f a tty
a c id s from a c e t i c a c id an d p ro p io n ic a c i d .................................
4.
4
14
14
T im e -c o u rs e o f in c o rp o ra tio n o f a c e t a t e - I - C ,
14
14
d e c a n o a te - 1 - C , an d lin o le a te - 1 - C in to s a tu r a te d an d
u n s a tu r a te d h y d ro c a rb o n s o f m a le P. a m e ric a n a .........................
9.
23
A n a ly tic a l th i n - l a y e r p la te o f lip id s e x tra c te d w ith
h e x a n e from th e s u rfa c e o f fe m a le P. a m e r i c a n a .......................
24
Ix
FIGURE
10.
'
PAGE
A n a ly tic a l th i n - l a y e r p la te o f h y d ro c a rb o n s from th e
s u rfa c e o f P.,a m e ric a n a a f te r s e p a r a tio n from o th e r
s u rfa c e lip id s on a s i l i c a g e l c o l u m n ...........................................
11.
G as c h ro m a to g ra p h ic a n a ly s is o f h y d ro carb o n , fr a c tio n s
from a s u rfa c e e x tr a c t o f P. a m e ric a n a . . . .................................
1 2.
25
28
In c o rp o ra tio n o f p ro p io n a te a s th e p e n u ltim a te u n it in
e lo n g a tio n to form 3 -m e th y lp e n ta c o s a n e
....................................
44
13 . I P o s s ib le m e a n s o f b io s y n th e s is o f d ie n e h y d ro c a rb o n s
. in f u lly - ta n n e d a d u lt P. a m e r i c a n a .................................................
1 4.
46
P o s s ib le c o n d e n s a tio n in b io s y n th e s is o f d ie n e h y d ro ­
c a rb o n s in te n e r a l a d u lt P. a m e r i c a n a ...........................................
47
X
ABSTRACT
H y d ro c a rb o n s a re b io s y n th e s iz e d from a c e t a t e , p r o p io n a te , and
f a tty a c id s u b s tr a te s in R a m e ric a n a an d a re s p re a d o v e r th e s u rfa c e
th ro u g h o u t a d u lt l i f e . The to ta l am o u n t o f s u rfa c e h y d ro c a rb o n is
a p p ro x im a te ly th e sam e in te n e r a l an d f u lly - ta n n e d a d u lt r o a c h e s .
S in c e a c e t a t e - l - ^ C , p r o p i o n a t e - l - l ^ c , a n d li n o l e a t e -1 -1 4 C a re not
in c o rp o ra te d in to h y d ro c a rb o n s in te n e r a l a d u l t s , w h ile o th e r f a tty a c id
s u b s tr a te s a re in c o r p o r a te d , h y d ro c a rb o n b io s y n th e s is v ia a n
e lo n g a tio n - d e c a r b o x y la tio n p a th w a y d o e s n o t o c c u r a t th is tim e . An
a lte r n a tiv e m e c h a n ism for h y d ro c a rb o n b io s y n th e s is is h e a d - to - h e a d
c o n d e n s a tio n o f f a tty a c i d s . P r o p io n a te , an d n o t is o le u c in e o r th e
S-m e th y l group o f m e t h i o n i n e i s th o u g h t to be th e s o u rc e o f th e m eth y l
s id e - c h a i n in b ra n c h e d h y d ro c a rb o n s . L in o le a te is b e lie v e d to be th e
s o u rc e o f th e u n s a tu r a te d c h a in in d ie n e h y d ro c a rb o n s .
INTRODUCTION
It is o fte n th e c a s e in s c ie n tif ic r e s e a r c h th a t th e fu n c tio n o f a
b io c h e m ic a l i s fo u n d lo n g b e fo re th e id e n tity o r th e m e c h a n ism of s y n ­
t h e s i s o f th e c h e m ic a l is d is c o v e r e d . S u ch is th e c a s e o f th e "w axy"
s u b s ta n c e s w h ic h c o v e r th e c u tic le s o f m any p la n ts a n d a n im a ls .
Among th e a n im a ls w ith lip id c u tic u la r c o v e r in g s .a r e th e i n s e c t s .
D u sh am (I) re p o rte d th a t " o ily " s u b s ta n c e s w ere s e c r e te d b y w ax g la n d s
in c o c k r o a c h e s , b u t s u c h g la n d s h a v e n o t b e e n o b s e rv e d b y o th e r
w o r k e r s . D u sh am found th a t a t l e a s t p a rt o f th e s e c r e tio n o f th e w ax
g la n d s w a s tru e w a x ( e s te r s o f fa tty a c id s a n d f a tty a lc o h o ls ) w h ic h
c o a te d th e s u r f a c e s o f th e c o c k r o a c h e s . In 1935 R am say (2) d em o n ­
s tr a te d th a t w a te r l o s s from th e c u tic le o f th e A m erican c o c k r o a c h ,
P e r ip la n e ta a m e r ic a n a , w a s due to p a s s iv e p h y s ic a l f a c t o r s , ra th e r th a n
to a n y a c tiv e p h y s io lo g ic a l p h e n o m e n o n . S u b s e q u e n t to t h e s e tw o d i s ­
c o v e r ie s , i n t e r e s t in d e te rm in in g th e i d e n titie s a n d m e ta b o lic fu n c tio n s
o f th e s e c u tic u la r lip id m a te ria ls h a s g ro w n .
P h y s io lo g y o f th e I n s e c t C u tic le
L ite ra tu re c o n c e rn in g th e s tru c tu re an d fu n c tio n s o f i n s e c t c u tic u ­
la r lip id s h a s b e e n r e c e n tly re v ie w e d b y J a c k s o n an d B aker (3 ).
A lthough D u s h a m 's (I) d is c o v e ry o f w a x g la n d s a s th e s i t e o f s y n th e s is
for c u tic u la r lip id s in tw o s p e c ie s o f c o c k ro a c h e s w a s m ade m any y e a rs
a g o , no s ite o f c u tic u la r lip id s y n th e s is com m on to a ll i n s e c t s p e c ie s
2
h a s b e e n fo u n d . P iek (4) c o n c lu d e d th a t h y d ro c a rb o n b io s y n th e s is in
A pis m e llife ra o c c u rre d in o e n o c y t e s .
In th e p a s t d e c a d e r e s e a r c h h a s c e n te r e d aro u n d th e c o n tro v e rs y
o v e r th e m e c h a n ism b y w h ic h lip id s a re tra n s p o rte d to a n d la id dow n
u p o n th e s u rfa c e o f th e i n s e c t b o d y . B ase d o n e x p e rim e n ta l d a ta th a t
th e w a te r tr a n s p ir a tio n r a te th ro u g h th e i n s e c t c u tic le c a n b e a lte r e d by
te m p e ra tu re an d a d s o r p tiv e d u s t s , H u rst (5) and B eam en t (6,7) h ad p o s t ­
u la te d a p ro te in -b o u n d b a s e lip id la y e r u p o n w h ic h a re la y e r e d e ith e r a n
o rd e re d o r a n u n o rg a n iz e d c o v e rin g o f o th e r lip id m a te r ia ls w h ic h c o n ­
s titu te th e m ain b a rrie r to w a te r l o s s b y th e i n s e c t in a n e n v iro n m e n t o f
lo w r e la tiv e h u m id ity (8 -1 1 ). R e c e n t e le c tr o n m ic ro s c o p ic s tu d ie s by
L ocke (12,13) a n d o th e rs (14,15) h a v e sh o w n th e e x is te n c e o f w ax c a n a ls
e x te n d in g from n e a r th e m o n o c e llu la r h y p o d e rm is la y e r to th e e x te r n a l
c u tic u la r s u r f a c e . A c co rd in g to th is m o d e l, lip o p h ilic a n d h y d ro p h ilic
c h a n n e ls in th e s e w a x c a n a ls a re r e s p o n s ib le for tr a n s p o r t o f lip id
m a te r ia ls an d w a te r , r e s p e c tiv e ly .
H o ld g a te a n d S e a l (16) fou n d t h a t th e c u tic le o f te n e r a l (n e w ly m o lted ) T e n e b rio m o lito r w a s m ore h ig h ly p e rm e a b le to w a te r th a n th e
c u tic le o f a n i n s e c t a t a la te r s ta g e in i t s a d u lt l i f e . T h ey a ls o observed
t h a t th e s u rfa c e w a x o f a te n e r a l i n s e c t m e lte d a t a lo w e r te m p e ra tu re
th a n d id th a t o f a n o ld e r i n s e c t .
B ase d on th e s e o b s e r v a tio n s , th e y c o n ­
c lu d e d th a t a v e ry th in la y e r o f lip id c o v e re d th e c u tic le an d ra p id ly
3
s p re a d o v e r i t im m e d ia te ly a f te r e c d y s is ; a h e a v ie r , m ore p e rm a n e n t
lip id la y e r w a s s e c r e te d s u b s e q u e n tly .
C u tic u la r L ip id s o f I n s e c ts an d O th e r O rg a n ism s
C o m p o sitio n s o f c u tic u la r lip id s a n d th e id e n titie s o f s e v e r a l i n ­
d iv id u a l c o m p o u n d s h a v e b e e n d e te rm in e d fo r s e v e r a l i n s e c t s . Among
t h e s e a re b e e s (4), s c a l e i n s e c t s (17-20), silk w o rm s (21), c r ic k e ts (22,
23), s to n e f lie s (24), a n ts (25), an d c o c k r o a c h e s (3,2 6 -3 1 ). T h e se lip id s
c o n s i s t o f f a tty a c id s , a lc o h o ls , a ld e h y d e s , e s t e r s , s te r o ls , and
h y d ro c a rb o n s in v a ry in g a m o u n ts (3 ,4 ,1 7 -3 1 ), d e p e n d in g u p o n s p e c ie s
a n d , in som e c a s e s , s e x o f th e i n s e c t (2 8 ). S e v e ra l r e c e n t re v ie w s i n ­
d ic a te th a t th e ty p e s o f s u rfa c e lip id s o f a n im a ls (32), p la n ts (33),
b a c te r ia (34), a n d i n s e c t s (3,35) a re s im ila r .
' B io s y n th e s is o f F a tty A cids
S in c e th e p a th w a y s o f h y d ro c a rb o n s y n th e s is w h ic h h a v e b e e n
p ro p o s e d in th e lite r a tu r e in v o lv e f a tty a c id s a s p r e c u r s o r s , th e l i t e r a ­
tu re c o n c e rn in g f a tty a c id b io s y n th e s is h a s b e e n i n v e s t ig a te d .
N orm al F a tty A c id s .
F a tty a c id b io s y n th e s is h a s r e c e n tly b e e n
re v ie w e d (36), an d th e b a s ic s te p s a re p r e s e n te d in F ig u re I to p ro v id e a
b a s i s fo r d i s c u s s i o n . A lm ost a ll o rg a n is m s are c a p a b le o f s y n th e s is o f
norm al f a tty a c id s ; h o w e v e r, o n ly p la n ts an d som e m ic ro o rg a n is m s ,
4
COr
COOH
O
C H 3-C -S -C o A
b io tin
C H 2-C -S -C o A
H -S -P
R -C H 2-C -S -C o A
\
X
H -S -P
H -S -C o A
H -S -C o A
COOH
R -C H 2-C H - C H 2- C - S - P
F IG . I .
S te p s in th e de novo s y n th e s is and e lo n g a tio n o f fa tty
a c id c h a in s (3 6 ).
5
n o n e o f th e a n im a ls so fa r s tu d ie d , a re c a p a b le o f s y n th e s is o f p o ly ­
u n s a tu r a te d f a tty a c i d s .
B ran ch ed F a tty A c id s .
K aneda (37) h a s p ro p o s e d p a th w a y s fo r i n ­
c o rp o ra tio n o f som e b ra n c h e d am in o a c id s in to to b a c c o f a tty a c i d s ,
w h ic h w e re th e n c o n s id e re d to b e s u b s tr a te s in th e s y n th e s is o f
b ra n c h e d h y d ro c a rb o n s . H is p ro p o s e d p a th w a y for in c o rp o ra tio n o f
is o le u c in e in to b r a n c h e d .f a tty a c id s is sh o w n in F ig u re 2 . Albro and
D ittm e r (38) h a v e in v e s tig a te d in c o rp o ra tio n o f am ino a c id s in to fa tty
a c id s in th e m ic ro c o c c u s S a rc in a l u t e a . T hey d e te rm in e d th a t is o le u c in e
w a s in c o rp o ra te d in to a n te i s o (m eth y l b ra n c h on th ird c a rb o n from th e
m e th y l end) b ra n c h e d f a tty a c i d s , a n d v a lin e w a s in c o rp o ra te d in to is o
(m eth y l b ra n c h o n s e c o n d c a rb o n from th e m e th y l end) b ra n c h e d fa tty .
a c i d s , p ro b a b ly by a m e c h a n ism s im ila r to th a t p ro p o s e d b y K a n ed a .
A no th er p o s s ib le m e c h a n ism o f s y n th e s is o f b ra n c h e d fa tty a c id s
h a s b e e n o ffe re d by S a z an d W e il (39), w ho found th a t in A s c a r is , p ro ­
p io n ic a c id w a s in c o rp o ra te d in to a n te i s o b ra n c h e d f a tty a c id s by th e
p ro p o s e d m e c h a n ism sh o w n in F ig u re 3 . T h is m e c h a n ism w a s s u p p o rte d
by th e r e s u l t s o f K a n e d a 1s e x p e rim e n ts in v o lv in g s e v e r a l m ic ro o rg a n ­
is m s (4 0 ).
S till a n o th e r com m on s o u rc e o f m e th y l g ro u p s in b io lo g ic a l s y s ­
te m s is th e S-m e th y l group o f m e th io n in e . A lthough J a u re g u ib e rry e t a l .
(41) d e m o n s tra te d in c o rp o ra tio n o f th is group in to th e in te r n a l b ra n c h o f
1 0 -m e th y lo c ta d e c a n o ic a c i d , Albro an d D ittm e r (38) c o u ld n o t sh o w th a t
6
H NHo
I
I 3
CHg--CHg—C —C —C —O ©
% 9
R—C —C —OH
T ra n s a m in a tio n
NH„ O
I 3 Il
R—C ----- C —O ©
I
H
H O C
I
Il Il
C H q—C H 0—C —C —C —O ©
o
2 I
CH0
H—S—CoA
+
NADH0
CHg—CHg—C —C —S—CoA
E lo n g atio n
(F ig . I)
CH0
F IG . 2
P ro p o se d p a th w a y fo r in c o rp o ra tio n o f is o le u c in e in to
b ra n c h e d f a tty a c id s in to b a c c o (3 7 ).
7
Il
C H 3- C - O H
Il
+ C H 3- C H 2- C - O H
^
H2 O
O H O
II
I Il
C H q—C —C —C —OH
J
I
CH3
NADPH2
V 9.
nadp®
HO H O
I
I Il
C H 3- C - C - C - O H
H
CH3
^
H2O
O
Il
C H q- C = C - C - O H
3 I
I
H CH3
FM NH2
FMN
H O
I
li
C H 3- C H 2- C - C - O H
E lo n g atio n
(F ig . I)
CH3
F IG . 3 .
P ro p o se d m e c h a n ism fo r s y n th e s is o f b ra n c h e d fa tty
a c id s from a c e tic a c id a n d p ro p io n ic a c id (3 9 ).
8
th e 8 -m e th y l group o f m e th io n in e w a s in c o rp o ra te d in to th e b ra n c h e d
h y d ro c a rb o n s o f S ♦ l u t e a . J a c k s o n a n d B aker (3) h a v e d e m o n s tra te d th a t
th e S-m e th y l group o f m e th io n in e w a s n o t in c o rp o ra te d in to e ith e r th e
s a tu r a te d o r u n s a tu r a te d fr a c tio n o f R a m e ric a n a h y d ro c a rb o n s .
B io s y n th e s is o f H y d ro c arb o n s
O f th e p o s s ib le p a th w a y s fo r b io s y n th e s is o f h y d r o c a r b o n s , tw o
h a v e re c e iv e d s e r io u s c o n s id e r a tio n . T h e se a re (a) h e a d - to - h e a d c o n ­
d e n s a tio n o f tw o f a tty a c i d s , fo r w h ic h th e o u tlin e o f a p o s s ib le p a th w a y
i s sh o w n in F igure 4 , an d (b) e lo n g a tio n o f a fa tty a c id w ith s u b s e q u e n t
d e c a r b o x y la tio n .
H e a d - to - h e a d C o n d e n s a tio n P a th w a y .
C h an n o n an d C h ib n a ll (42)
p ro p o s e d h e a d - to - h e a d c o n d e n s a tio n o f tw o fa tty a c id s in h y d ro c a rb o n
b i o s y n t h e s i s , b a s e d on th e ir d is c o v e ry o f 1 5 -n o n a c o s a n o n e in b ro c c o li.
A cco rd in g to th e ir th e o ry , th is k e to n e w a s a n in te rm e d ia te d e c a rb o x y la ­
tio n p ro d u c t w h ic h w a s re d u c e d to n o n a c o s a n e , th e p re d o m in a n t h y d ro ­
c a rb o n fo u n d in b r o c c o li. In more r e c e n t w o rk , K aneda (3 7 ,4 3 ) w ith
to b a c c o , a n d A lbro an d D ittm e r (3 3 ,3 8 ,4 4 -4 6 ) w ith S_. lu te a h a v e in d i­
c a te d th a t s e v e r a l o rg a n is m s m ay form h y d ro c a rb o n s v ia s u c h a c o n d e n ­
s a tio n p a th w a y .
G a s ta m b id e -O d ie r an d L e d e re r (47) p ro p o s e d a c o n d e n s a tio n
m e c h a n ism fo r p ro d u c tio n o f c o ry n o m y c o lic a c id (F ig . 5) in
C o ry n e b a c te riu m d ip h th e r ia e . T his a c i d , th e y s u g g e s te d , m ig h t b e an
9
R -C H
O
Il
P
+
O
Il
H O -C -R '
\y
I —C
° —S
I—C
P
C —R1
A
\v
R—CHg—CHg—R
FIG. 4 .
P ro p o se d c o n d e n s a tio n o f tw o fa tty a c id m o le c u le s to
form a h y d ro c a rb o n m o le c u le (3 7 ).
10
O
R - C H 2- C - O H
+
O = C -C H g -R
I
/
O = C —C H 2—R
NADPHr
NADPv
O
Il
R—C H - C —OH
I
H O - C H - C H 2- R
F IG . 5 .
P ro p o se d m e c h a n ism for fo rm atio n o f c o ry n o m y c o lic a c id
in C . d ip h th e ria e (4 7 ).
11
in te rm e d ia te in. h y d ro c a rb o n b io s y n th e s is v ia th e c o n d e n s a tio n
p a th w a y .
A v a r ia tio n o f th e c o n d e n s a tio n p a th w a y h a s b e e n p ro p o s e d by ’
A lbro an d D ittm e r (34) in th e ir s tu d ie s o f £h l u t e a . T hey s u g g e s t r e d u c ­
tio n o f f a tty a c id s to a ld e h y d e s w h ic h a re th e n in c o r p o r a te d , a s th e ir
v in y l e t h e r s , in to n e u tra l p la s m a lo g e n s t r u c t u r e s . U pon r e le a s e from
th e s e p la s m a lo g e n s , e a c h m o le c u le o f v in y l e th e r w a s p ro p o s e d to c o n ­
d e n s e w ith a f a tty a c id m o le c u le to form a h y d ro c a rb o n u n s a tu r a te d a t
th e p o s itio n o f th e d o u b le bond in th e v in y l e th e r .
E lo n g a tio n - d e c a rb o x y la tio n P a th w a y . As w a s sh o w n in F igure I ,
f a tty a c i d s , w h e th e r norm al o r b r a n c h e d , s a tu r a te d o r u n s a t u r a t e d , m ay
be e lo n g a te d by s u c c e s s i v e a d d itio n o f tw o -c a rb o n u n its from m a lo n a te .
The th e o ry o f th e e lo n g a tio n p a th w a y i s th a t fo rm atio n o f a lo n g - c h a in
f a tty a c id i s fo llo w e d by i t s d e c a rb o x y la tio n to form a h y d ro c a rb o n
-
(F ig . 6). W ork' by K o la ttu k u d y an d c o w o rk e rs (4 8 -5 5 ), fo llo w in g th e
w ork o f C h an n o n a n d C h ib n a ll (42), d e m o n s tra te d th a t lo n g - c h a in k e t
to n e s w e re n o t p re c u rs o r s o f h y d ro c a rb o n s in b r o c c o li. K o lattu k u d y
(48,49) s u g g e s te d th a t th e s e h y d ro c a rb o n s w e r e , in f a c t , s y n th e s iz e d
v ia a n e lo n g a tio n - d e c a r b o x y la tio n p a th w a y , ra th e r th a n v ia c o n d e n s a tio n
o f f a tty a c id s a s h a d b e e n p ro p o s e d b y C h an n o n an d C h ib n a ll (4 2 ). T his
c o n c lu s io n w a s b a s e d , in p a r t, on th e f a c t th a t tr ic h lo r o a c e tic a c id
in h ib ite d b o th e lo n g a tio n <Ojf s h o r t- c h a in f a tty a c id s (16 c a rb o n s or
12
O
Il
R—CHg—C —S —P
NADH2 + FM N H 2
NAD
+ FMN + H2O
*?
R—(C H g )2^—C H 2—C —S ~ P
H2O
CO2 + H - S - P
R-(CHz)2 -C H g
FIG . 6.
P ro p o se d e lo n g a tio n - d e c a r b o x y la tio n p a th w a y to form a
h y d ro c a rb o n m o le c u le from a fa tty a c id m o le c u le .
13
sh o rte r) an d th e ir in c o rp o ra tio n in to h y d ro c a rb o n s , w h ile in c o rp o ra tio n
o f s t e a r i c a c id in to h y d ro c a rb o n s w a s n o t a f f e c te d .
O c c u rre n c e o f Both P a th w a y s in th e Sam e O rg a n is m . Albro and
D ittm e r (46) b e lie v e th a t h e a d - to - h e a d c o n d e n s a tio n m ay b e th e p r e ­
fe rre d m e c h a n ism o f h y d ro c a rb o n b io s y n th e s is in Sv lu te a ; h o w e v e r,
th e ir d a ta in d ic a te th a t e lo n g a tio n - d e c a r b o x y la tio n m ay b e r e s p o n s ib le
fo r som e o f th e h y d ro c a rb o n s y n t h e s i s . T hey b a s e d th is c o n c lu s io n on
th e f a c t t h a t d iff e r e n t p a tte r n s o f in c o rp o ra tio n o f la b e le d f a tty a c id s
w e re o b s e r v e d w h en c e ll s w e re grow n u n d e r d iff e r e n t c o n d itio n s of
m e d ia fo rm u la tio n , c e ll c o n c e n tr a tio n , a n d c o fa c to r p r e s e n c e .
H y d ro c a rb o n s o f P e rip la n e ta a m e ric a n a
P e rip la n e ta a m e r ic a n a , th e A m erican c o c k r o a c h , w a s c h o s e n for
s tu d y b e c a u s e i t s th re e p r in c ip a l h y d ro c a rb o n s ( n - p e n t a c o s a n e , 12%;
3 - m e th y lp e n ta c o s a n e ,, 2 0%; c i s , c i s -.6 ,9 - h e p ta c o s a d ie n e , 65%) (2 6,30,31)
r e p r e s e n t th re e d i s tin c tly d iff e r e n t s tr u c tu r a l ty p e s (F ig . 7 ). In a d d itio n ,
m in o r q u a n titie s o f o th e r h y d ro c a rb o n s h a v e b e e n f o u n d , in c lu d in g
m o n o u n s a tu ra te d h y d ro c a rb o n s o f 41- an d 4 3 -c a rb o n c h a in le n g th s (2 9 ).
H y d ro c a rb o n s o f b o th m ale an d fe m a le P. a m e ric a n a a re a lm p s t i d e n t i c a l ,
a lth o u g h m ore i s p r e s e n t in fe m a le s (2 6 ,2 9 ,5 6 ). S in c e o n ly th re e com ­
p o u n d s c o m p rise 97% o f th e h y d ro c a rb o n s o f th is i n s e c t , s tu d y of th e
p a th w a y s le a d in g to th e ir b io s y n th e s is w a s p a r tic u la r ly a ttr a c t iv e . P re ­
su m ab ly , b io s y n th e tic p a th w a y s fo r e a c h o f th e s e h y d ro c a rb o n s c a n be
14
C H 3-(C H 2 ) 23-C H 3
n - p e n ta c o s a n e
CH3-(C H g)^ -C H 3
n - h e p ta c o s a n e
C H 3-( C H 2)27-C H 3
n -n o n a c o sa n e
CH„
I 3
C H 3-C H 2-C H -(C H 2) 21- C H 3
3 -m e th y lp e n ta c o s a n e
CH3-(C H 2)^ -C H = C H -C H 2-C H = C H -(C H 2)13- C H 3
'C isfC is-6,9 - h e p ta c o s a d ie n e
C H 3-( C H 2)13-C H = C H -(C H 2) 24- C h 3
15 - h e n te tr a c o n te n e
C H 3- ( C h 2)13-C H = C H -(C H 2 )26- C H 3
15- t r i t e t r a c o n te ne
’ F IG . 7.
S tru c tu re s o f h y d ro c a rb o n s w h ic h h a v e b e e n re p o rte d fo r
R a m e ric a n a (2 6 ,2 9 ,3 0 ).
d e te rm in e d by in v e s tig a tio n o f th e m e ta b o lism o f p re c u rs o r m o le c u le s
w ith s im ila r p a r tia l s t r u c t u r e s .
I n v e s tig a tio n o f h y d ro c a rb o n b io s y n th e s is in i n s e c t s h a s b e e n
v e ry lim ite d . L am brem ont an d c o w o rk e rs (57) h a v e d e m o n s tra te d in c o r p o ra tio n o f a c e t a t e -
14
■
C in to b o th f a tty a c id s an d h y d ro c a rb o n s of th e .
b o ll w e e v il. S im ila r o b s e r v a tio n s h a v e b e e n m ade b y Lamb an d M onroe
(58) w ith th e c e r e a l l e a f b e e t l e s , B loch e t a l . (59) w ith h id e b e e t l e s ,
a n d R obbins e t a l . (60) w ith the h o u sefly . W ork by N e ls o n (61) h a s d e m 14
o n s tr a te d in v iv o in c o rp o ra tio n o f r a d io a c tiv ity from b o th a c e ta te - 1 - C
'14
an d p a lm ita te - 1 - C in to h y d ro c a rb o n s in th e fa t b o d y a n d in te g u m e n t o f
R a m e r ic a n a . B ase d on in v itro in c o rp o ra tio n o f r a d io a c tiv ity in to
15
h y d ro c a rb o n s o f th e in te g u m e n t, b u t n o t th e fa t body. N e ls o n c o n c lu d e d
th a t th e e p id e rm a l c e ll s w e re th e s ite o f h y d ro c a rb o n s y n t h e s i s .
L o u lo u d e s e t a l . (62) h a d p r e v io u s ly fo u n d in c o rp o ra tio n o f a c e ta te - 1 - C
in to b o th s a p o n if ic a b le a n d u n s a p o n if ia b le lip id s o f Pr a m e r ic a n a .
P re v io u s w ork in th is la b o ra to ry (3) h a s d e m o n s tra te d th e in c o r­
p o ra tio n o f s e v e r a l d iff e r e n t s u b s tr a te s in to s a tu r a te d a n d u n s a tu r a te d
f r a c tio n s o f th e h y d ro c a rb o n s o f R a m e r ic a n a . T h e se r e s u lts sh o w ed
th a t in liv e r o a c h e s a c e ta te an d m a lo n a te w e re in c o rp o ra te d in to b o th
f r a c tio n s e q u a lly , w h ile p a lm ita te an d p ro p io n a te w ere in c o rp o ra te d p r e ­
f e r e n tia lly in to th e s a tu r a te d f r a c tio n , a n d lin o le a te w a s in c o rp o ra te d
m ore in to .th e u n s a tu r a te d f r a c tio n . S tu d ie s o f s u b s tr a te s a p p lie d to
e x c is e d in te g u m e n ts o f th is i n s e c t sh o w e d th e sam e p a tte r n o f in c o r­
p o ra tio n o f s u b s t r a t e s , a lth o u g h n o t n e a r ly a s m uch in c o rp o ra tio n w a s
o b s e r v e d a s in v i v o , e x c e p t in th e c a s e o f p a lm ita te . S u b s e q u e n t to
th is p u b lic a tio n , i t w a s fo u n d th a t th e p a lm ita te s u b s tr a te s w h ic h h ad
b e e n u s e d in th e s e s tu d ie s w e re c o n ta m in a te d w ith la b e le d h y d ro c a rb o n s
from som e unknow n s o u r c e , an d d a ta from th e s e e x p e rim e n ts in v o lv in g
p a lm ita te m u st be d is r e g a r d e d . T h e se w o rk e rs a ls o sh o w e d th a t t r i c h lo r o a c e ta te in je c te d in to th e ro a c h e s h a d a n in h ib ito r y e f f e c t upon
in c o rp o ra tio n o f a c e t a t e , an d ch lo ro fo rm a n e s th e tiz a tio n re d u c e d in c o r­
p o ra tio n o f m a lo n a te in to h y d ro c a rb o n s .
In a d d itio n to h y d ro c a rb o n s , w h ic h m ake up a b o u t 75% o f th e s u r ­
fa c e lip id s o f P. a m e ric a n a (2 6,29), "other lip id s p e c ie s h a v e b e e n
16
id e n tif ie d . Both B aker e t a l . (26) a n d G ilb y an d C ox (30) h a v e o b s e rv e d
f a tty a c id s an d lo n g - c h a in a lc o h o ls an d som e o f th e ir e s t e r s . In a d d i­
t io n , G ilb y an d C ox (30) h a v e re p o rte d th e p r e s e n c e o f lo n g - c h a in
,
a ld e h y d e s in s u b s ta n tia l q u a n t i t i e s . T his o b s e r v a tio n h a s n o t b e e n
m ade in o u r la b o ra to ry , le a d in g to th e c o n c lu s io n th a t a ld e h y d e s m ay
v
p o s s ib ly be form ed a s p ro d u c ts o f a u to x id a tio n o f th e u n s a tu r a te d h y d ro ­
c a rb o n s o f th e c o c k ro a c h c u t i c l e . A tk in so n and G ilb y (63) h a v e d e te r ­
m in ed th a t th is p h e n o m en o n m ay b e r e la te d to th e h a rd e n in g o f lip id
m a te ria l on th e c u tic le a n d o o th e c a .
B io s y n th e s is o f H y d ro c a rb o n s in P e rip la n e ta a m e ric a n a
F a tty a c id s u b s tr a te s p a r tic ip a te n o t o n ly in h y d ro c a rb o n b io s y n ­
t h e s i s , b u t a ls o in c a ta b o lic an d s to ra g e p a th w a y s . F o rm atio n of
g ly c e r id e s an d o th e r e s t e r s d im in is h e s th e p o o l o f fre e f a tty a c i d s , m ak ­
in g them l e s s a v a ila b le fo r h y d ro c a rb o n b i o s y n th e s is . P ro d u c tio n of
a c e ty l- c o e n z y m e A u n its w h ic h c a n p a r tic ip a te in a n u m b er o f w e llknow n p a th w a y s (th e " a c e ta te e f f e c t" ) r e s u lts from |3 -o x id a tio n of fa tty
a c i d s , a n d th e r e la te d e lo n g a tio n o f f a tty a c i d s .
The p u rp o s e -’o f - th is : r e s e a r c h w a s to in v e s tig a te th e fa te of
la b e le d f a tty a c id s an d o th e r s u b s t r a t e s , p a r tic u la r ly in th e ir re la tio n to
th e p a th w a y o r p a th w a y s le a d in g to th e b io s y n th e s is o f th e m ajo r h y d ro ­
c a rb o n s o f P. a m e r ic a n a . The a p p ro a c h w a s to a llo w ly o p h iliz e d in te g u ­
m ent h o m o g e n a te s a n d liv in g c o c k r o a c h e s to m e ta b o liz e v a rio u s la b e le d .
17
s u b s tr a te s a n d to s tu d y th e in c o rp o ra tio n o f th e s e s u b s tr a te s in to p ro ­
d u c t h y d ro c a rb o n s . S tu d ie s w e re c a rrie d o u t u s in g te n e r a l (n e w ly m o lted ) a d u lt ro a c h e s an d a d u lts w h o se c u tic le s h a d ta n n e d c o m p le te ly .
F u rth e rm o re , a tte m p ts w e re m ade to b lo c k c o m p etin g p a th w a y s by a d m in ­
is te r in g s e v e r a l c o m p o u n d s w h ic h w e re e x p e c te d to d e m o n s tra te
in h ib ito r y e f f e c t s .
HYPOTHESIS
H y d ro c a rb o n s o f P e rip la n e ta a m e ric a n a a re b io s y n th e s iz e d from
f a tty a c id p re c u rs o r s v ia a p a th w a y o f e lo n g a tio n an d d e c a rb o x y la tio n o f
f a tty a c id s a n d /o r a p a th w a y in v o lv in g h e a d - to - h e a d c o n d e n s a tio n o f
tw o f a tty a c i d s . B io s y n th e s is o f h y d ro c a rb o n s c a n be s tu d ie d th ro u g h
th e p a tte r n o f in c o rp o ra tio n o f f a tty a c id s an d th e ir p re c u rs o r s in to
d iff e r e n t h y d ro c a rb o n , f r a c t i o n s .
EXPERIMENTAL
G row th C o n d itio n s
P e rip la n e ta a m e ric a n a w e re re a re d in c o v e re d g a rb a g e c a n s
e q u ip p e d w ith e le c tr i c f e n c e s to p re v e n t e s c a p e o f th e r o a c h e s . No
a tte m p t w a s m ade to k e e p d iff e r e n t d e v e lo p m e n ta l s ta g e s s e p a r a t e d , an d
a d u lts w e re a llo w e d to b re e d f r e e ly . Food an d w a te r w e re p la c e d in th e
c a g e s w ith th e c o c k r o a c h e s . D ie t w a s g ro u n d G rav y T rain dog fo o d .
W a te r w a s p ro v id e d in th e form o f a n a g a r -w a te r. (1:99) g e l. In o rd e r to
d e te rm in e m e ta b o lic d if f e r e n c e s d u rin g th e lif e c y c l e , a d u lt c o c k ro a c h e s
w e re c o lle c te d a t v a r io u s .tim e s fo llo w in g th e im a g in a l e c d y s i s . O ne
• s e r ie s o f e x p e rim e n ts w a s p erfo rm ed u s in g te n e r a l a d u lts w h ic h h a d n o t
y e t b e g u n to t a n . T h e se w e re c o lle c te d an d in je c te d w ith s u b s tr a te
s o lu tio n s w ith in tw o h o u rs fo llo w in g e c d y s i s . A n o th er group u s e d for
e x p e rim e n ta tio n w a s c o lle c te d from a fe w d a y s a fte r th e im a g in a l
e c d y s is th ro u g h s e v e r a l w e e k s fo llo w in g th is e c d y s i s . S till a th ird
group o f a d u lts w a s c o lle c te d a t l e a s t tw o m o n th s fo llo w in g th e im a g in a l
e c d y s is .
A ttem p ts to P rep a re a C e ll- f r e e B io s y n th e tic S y stem
It is re c o g n iz e d th a t in a w h o le a n im a l, in d e e d in a w h o le c e l l ,
s e v e r a l m e ta b o lic s y s te m s c o m p ete fo r s u b s tr a te m o le c u le s . In o rd e r to
s tu d y o n e p a r tic u la r m e ta b o lic s y s t e m , th e r e f o r e , i t is d e s ir a b le to i s o ­
la te th a t s y s te m from o th e r e n z y m e s . To t h a t e n d , a n a tte m p t w a s m ade
20
to p re p a re in v itro a c u ltu re c a p a b le o f h y d ro c a rb o n b i o s y n t h e s i s , w ith ­
o u t c o m p e tin g e n z y m e s .
■ S e v e ra l c o c k r o a c h e s w ere f r o z e n .a t -2 5 C fo r 20 m in; th e ir h e a d s
an d a p p e n d a g e s w e re rem o v ed w ith f o r c e p s , an d th e in te r n a l o rg a n s
w e re rem o v ed by d i s s e c t i o n . T h e se e x c is e d in te g u m e n ts (w h ich J a c k s o n
a n d B aker (2) h a d d e te rm in e d w e re c a p a b le o f s y n th e s iz in g h y d ro c a r­
b o n s) w e re th e n p la c e d in a s m a ll am o u n t o f c o ld p h o s p h a te b u ffe r,
pH 7.2 (p re v io u s ly d e te rm in e d to be th e pH o f c o c k ro a c h in te g u m e n t
h o m o g e n a te ). In a n e ffo rt to d e s tr o y a s fe w p ro te in m o le c u le s a s p o s ­
s i b l e , d is ru p tio n o f th e e x c is e d in te g u m e n t^ w a s d o n e a t h ig h s p e e d in
. •
'
■
a V irtis h o m o g e n iz e r for 10 s e c an d in a m o to riz e d m o rta r an d p e s tle fo r
2 m in . The slu rry , w h ic h w a s n o t c h e c k e d fo r h y d ro c a rb o n s y n th e tic
a c tiv ity , w a s th e n tr a n s f e r r e d to a c e n trifu g e tu b e w ith a s m a ll am o u n t
o f a d d itio n a l b u f f e r , a n d c e n trifu g e d a t 5000 x g fo r.15 m in to rem ove c e ll
fra g m e n ts . Up to th is p o in t, a ll w ork w a s done a t 4 C in, o rd e r to p r e ­
s e rv e en zy m e in te g r ity . The s u p e r n a ta n t, w h ic h w a s th o u g h t to c o n ta in
m o s t s o lu b le e n z y m e s , w a s th e n c o a te d on th e in s id e o f a f la s k and
q u ic k ly fro z e n in a d ry ic e - b u ta n o l b a th . The f la s k w a s a tta c h e d to a
v a cu u m a p p a r a tu s an d ly o p h lliz e d o v e r a p e rio d o f s e v e r a l h o u r s . T his
m eth o d p ro v id e d a c o n v e n ie n t m e a n s o f s to ra g e fo r th e p r e p a r a tio n .
A p p ro x im a te ly 35 to 50 mg o f p o w d e r w a s p re p a re d from, e a c h in te g u m e n t.
In e a c h e x p e rim e n t 25 mg o f l y o p h i l y s a t e , w h ic h h a d b e e n s to re d a t
-2 5 C , w a s s u s p e n d e d in 2 ml o f f r e s h b u ffe r, an d in c u b a tio n w as
21
a llo w e d to p ro c e e d a t room te m p e ra tu re fo r 6 h r. C o n tro ls c o n ta in in g
o n ly b u ffe r an d s u b s tr a te s w ere run c o n c u r re n tly .
I n je c tio n o f S u b s tr a te s an d In h ib ito rs
In e a c h e x p e rim e n t 1.0 pi o f a s o lu tio n o f a s u b s tr a te in w a te r or
to lu e n e s o lu tio n (d e p e n d in g upon s o lu b ility ) w a s in je c te d in to th e
th o r a c ic h a e m o c o e l o f e a c h c o c k r o a c h . I n s e c ts d id n o t see m to s u ffe r
a n y to x ic e f f e c ts from to lu e n e . R a d io b io c h e m ic a ls w e re p u rc h a s e d from
Volk an d A m e rs h a m -S e a rle . In a ll c a s e s th e in je c tio n c o n s is te d o f a p ­
p ro x im a te ly 0.25 pm ole o f a s u b s tr a te w ith s p e c if ic a c ti v ity ap p ro x i-m a te ly I p C /p m o le . A n a ly tic a l th i n - l a y e r c h ro m a to g ra p h y w a s u s e d to
d e te rm in e th e p u rity o f f a tty a c id s u b s t r a t e s , an d p r e p a r a tiv e th in - la y e r
c h ro m a to g ra p h y w a s u s e d to rem ove la b e le d h y d ro c a rb o n c o n ta m in a n ts
14
14
w h ic h w e re found in p a lm ita te -9 ,1 0 - C an d p a lm ita te - 1 6 - C . C are w a s
ta k e n to in j e c t a t a p o in t l a t e r a l to th e v e n tr a l n e rv e c o rd th ro u g h th e
v e n tr a l c o n ju n c tiv a l m em brane b e tw e e n th e m e ta th o ra x an d th e f ir s t
a b d o m in a l s e g m e n t. N o s ig n if ic a n t lo s s o f s u b s tr a te due to b le e d in g
w a s o b s e r v e d . In e x p e rim e n ts in w h ic h in h ib ito r s w e re u s e d , w a te r
s o lu tio n s o f th e s e com p o u n d s w e re in je c te d in th e sam e m a n n er 30 m in
p rio r to s u b s tr a te in je c tio n .
In c u b a tio n o f I n je c te d C o c k ro a c h e s
A fter i n j e c tio n , e a c h c o c k ro a c h w a s p la c e d in to a ro u n d -b o tto m
f l a s k , w h ic h w a s th e n c o v e re d w ith alum inum fo il in w h ic h s e v e ra l
22
s m a ll h o le s h a d b e e n m a d e . The i n s e c t w a s then, k e p t in th is fla s k for
18 hr a t room te m p e ra tu re in o rd e r to a llo w a d e q u a te c ir c u la tio n and
m e ta b o lism o f th e s u b s t r a t e s . In c u b a tio n tim e o f 18 h r w a s c h o s e n ,
s in c e th e to ta l am o u n t o f r a d io a c tiv ity from s e v e r a l s u b s tr a te s in c o r­
p o ra te d in to b o th th e s a tu r a te d an d u n s a tu r a te d h y d ro c a rb o n s a p p e a re d
to sh o w no in c r e a s e a f te r th is tim e (F ig . 8 ).
E x tra c tio n o f S u rfa c e L ip id s
i
A fter th e in c u b a tio n p e r io d , a b o u t 150 ml o f h e x a n e w a s p o u red
in to e a c h f la s k . The h e x a n e b o th k ille d th e ro a c h e s an d e x tr a c te d th e ir
s u rfa c e l i p i d s ., A tim e s tu d y (F ig . 9) sh o w e d th a t a f te r a p e rio d o f
3 Omin in h e x a n e a lm o s t a ll o f th e s u rfa c e h y d ro c a rb o n s h a d b e e n e x ­
tr a c te d from th e r o a c h e s . T h e re fo re a n e x tr a c tio n tim e o f 30 to 35 m in ,
d u rin g w h ic h tim e e s s e n t i a l l y a ll o f th e s u rfa c e h y d ro c a rb o n s an d v e ry
.l i t t l e in te r n a l (n o n -h y d ro c a rb o n ) lip id w a s r e c o v e r e d , w a s u s e d in a ll
e x p e r im e n ts .
■ C h ro m a to g ra p h ic P u rific a tio n o f H y d ro c arb o n F ra c tio n s
H e x a n e w a s re m o v e d from th e sa m p le by ro ta ry e v a p o ra tio n u n d e r
a s p ir a to r v a c u u m . The r e s id u e w a s tr a n s f e r r e d to a P a s te u r p ip e tte
co lu m n c o n ta in in g 50 mg o f colum n s i l i c a g e l (A d so rb o sil 100-140 m esh)
an d e lu te d in to a v ia l w ith 7 ml h e x a n e . The colum n r e ta in e d th e p o la r
lip id s an d a llo w e d a ll h y d ro c a rb o n s (1.0 to 1.7 mg p e r i n s e c t . C f.
J a c k s o n (29) ) to p a ss, th ro u g h to g e th e r in th e h e x a n e x> (F ig . 10A).
23
A c e ta te
161
90
OO
o
X
T3
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4-1
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L in o le a te
52
20
Time (hr)
FIG . 8 .
14
T im e -c o u rs e o f in c o rp o ra tio n o f a c e t a t e - 1 - C ,
d e c a n o a t e - 1 - , a nd l i n o l e a t e - 1 - i n t o s a tu r a te d (A) an d u n s a tu r a te d
(B) h y d ro c a rb o n s o f m a le P. a m e r ic a n a .
24
S a tu ra te d h y d ro c a rb o n s
U n s a tu r a te d h y d ro c a rb o n s
O rig in (n o n -h y d ro c a rb o n
m a te ria l)
c
E
o o
E
O
C
E
O
LO
CO
Cs]
P
E
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CO
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I
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55
c
nO
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I
C
O
C
F IG . 9 .
A n a ly tic a l th in - la y e r p la te o f lip id s e x tr a c te d w ith
h e x a n e from th e s u rfa c e o f fe m a le P. a m e r ic a n a . Five ro a c h e s w ere
s e r ia ll y e x tr a c te d for te n -m in u te p e r io d s .
P la te is s i l i c a (A d so rb o sil-3 )
d e v e lo p e d in h e x a n e a n d c h a rre d a f te r s p ra y in g w ith a s o lu tio n of
c h ro m ic an d s u lfu ric a c i d s .
25
I
s a tu r a te d
m o n o e n es
d ie n e s
B
A
F IG . 10.
A n a ly tic a l th in - la y e r p la te o f h y d ro c a rb o n s from th e
s u rfa c e o f P. a m e ric a n a a f te r s e p a r a tio n from o th e r s u rfa c e lip id s on a
s i l i c a g e l (A d so rb o sil 1 0 0 -1 4 0 m esh) c o lu m n . C h ro m a to g ra p h ic c o n d i­
tio n s w e re th e sam e a s in F ig . 9 .
S am p le B w as s p ra y e d w ith a s o lu ­
tio n o f s ilv e r n itr a te in e th a n o l b e fo re d e v e lo p in g th e p l a t e .
26
H e x a n e w a s e v a p o ra te d u n d e r a n itro g e n s w e e p . The to ta l h y d ro c a rb o n
sa m p le w a s tr a n s f e r r e d w ith a s m a ll am o u n t o f h e x a n e to a s i l i c a g e l
(A d so rb o sil 3 im p re g n a te d w ith s ilv e r n itr a te ) th i n - l a y e r p l a t e , on w h ic h
i t w a s p re p a r a tiv e ly c h ro m a to g ra p h e d w ith h e x a n e a s th e d e v e lo p in g
s o lv e n t (24, 6 4 ). The h y d ro c a rb o n s w e re th u s s e p a r a te d in to th re e f r a c ­
tio n s : s a tu r a te d , m o n o u n s a tu ra te d , a n d d iu n s a tu r a te d (from top o f p la te )
(F ig . 10B). S e p a ra tio n o f fr a c tio n s w a s v is ib le u n d e r u ltr a v io le t lig h t.
The s i l i c a h ad b e e n im p re g n a te d w ith rh o d am in e 6-G in o rd e r to f a c i l i ­
ta t e v i s i b i l i t y .
• E ach fra c tio n w a s th e n s c r a p e d in to a m edium p o ro s ity s in te re d
g l a s s B uchner fu n n e l an d e lu te d from th e s i l i c a g e l w ith 100 ml to lu e n e .
A fter ro ta ry e v a p o r a tio n o f th e t o l u e n e , e a c h sa m p le w a s tra n s fe rre d to
a liq u id s c i n t i l l a t i o n v ia l w ith h e x a n e , w h ic h w a s th e n e v a p o ra te d o ff
u n d e r a n itro g e n s w e e p . In o rd e r to s e p a r a te n orm al from b ra n c h e d
s a tu r a te d h y d r o c a r b o n s , th e s a tu r a te d fr a c tio n w a s th e n d is s o lv e d in a
h ig h ly - b ra n c h e d s o lv e n t , 2 ,2 ,4 -tr im e th y lp e n ta n e , an d s h a k e n w ith L inde
m o le c u la r s e iv e 5A for 3 h r. S tr a ig h t- c h a in co m p o u n d s p a s s e d , in to th e
s e i v e , w h ile b ra n c h e d c o m p o u n d s, in c lu d in g th e s o lv e n t , w e re e x ­
c lu d e d (6 5 ). T his s o lu tio n , c o n ta in in g b ra n c h e d h y d ro c a rb o n s o n ly w a s
tr a n s f e r r e d to s e p a r a te c o u n tin g v i a l s , le a v in g th e s e iv e c o n ta in in g
n o rm al h y d ro c a rb o n s in th e o rig in a l v i a l . The 2 ,2 ,4 -trim e th y lp e n ta n e w a s
e v a p o r a te d o ff u n d e r n itro g e n from th e. v ia l c o n ta in in g th e b ra n c h e d
27
h y d ro c a rb o n f r a c tio n . F igure 11 i s a re p ro d u c tio n o f g a s ch ro m ato g ram s
o b ta in e d in a n a ly s is o f th e c o n te n ts o f t h e s e h y d ro c a rb o n f r a c tio n s .
A n a ly s is o f In te rn a l L ip id F ra c tio n s
A fter h e x a n e e x tr a c tio n o f s u rfa c e l i p i d s , ro a c h c a r c a s s e s w ere
h o m o g e n iz e d in a m o to riz e d m o rtar a n d p e s tl e in c h lo ro fo rm . A few
d ro p s o f h y d ro c h lo ric a c id w e re a d d e d in o rd e r to e n s u re th a t a ll fa tty
a c id s w e re in th e s o lu b le fre e a c id fo rm , r a th e r th a n in th e s a l t fo rm ,
w h ic h is in s o lu b le in c h lo ro fo rm . A fter re m o v a l o f p a r tic u la te m a te ria l
by f iltr a tio n th ro u g h g la s s w o o l, th e ch lo ro fo rm w a s re m o v e d by ro ta ry
e v a p o r a tio n . The re s id u e w a s tr a n s f e r r e d to a v ia l w ith ch lo ro fo rm
w h ic h s u b s e q u e n tly w a s rem o v ed b y e v a p o ra tio n u n d e r n itr o g e n . A s m a ll
p o rtio n o f th e sa m p le w a s tr a n s f e r r e d w ith ch lo ro fo rm to a th in - la y e r
p l a t e . The p la te w a s e lu te d f ir s t w ith h e x a n e to s e p a r a te h y d ro c a rb o n s ,
w h ic h m oved to th e to p o f th e p la te in h e x a n e , from p o la r c o m p o u n d s ,
w hich, re m a in e d n e a r th e o rig in . N e x t, th e p la te w a s e lu te d w ith
h e x a n e : d ie th y l e t h e r : a c e tic a c id (8 5 :1 5 :1 , v / v / v ) in o rd e r to s e p a r a te
th e p o la r f r a c tio n s . T hree fr a c tio n s w e re s c r a p e d from th e p la te : h y d ro ­
c a r b o n s , g ly c e r id e s , an d o th e r e s t e r s , a n d o rig in , w h ic h c o n ta in e d fre e
f a tty a c id s an d o th e r h ig h ly p o la r l i p i d s . The c o m p o u n d s w e re e lu te d
from th e s i l i c a g e l w ith to lu e n e , w h ic h in tu rn w a s re m o v e d by ro ta ry
e v a p o r a tio n . S a m p le s w e re tr a n s f e r r e d to liq u id s c i n t i l l a t i o n v ia ls in
th e sa m e m a n n er a s s u rfa c e h y d ro c a rb o n f r a c t i o n s .
'
28
I S o lv e n t
2 7 d ie n e
T o tal h y d ro c a rb o n s
S a tu ra te d
B ran ch ed
M o n o en e
D ie n e
FIG . 11.
G as c h ro m a to g ra p h ic a n a ly s is o f h y d ro c a rb o n fra c tio n s
from a s u rfa c e e x tr a c t o f P. a m e r ic a n a . C o m p lete a s s ig n m e n ts o f s t r u c ­
tu re h a v e b e e n d o n e by o th e r w o rk e rs (2 6 ,2 9 ).
29
L iquid S c in tilla tio n C o u n tin g o f L ip id F ra c tio n s
A 15-m l a liq u o t o f flu o r s o lu tio n (0.4% PPO in to lu e n e ) w a s 'a d d e d
to e a c h o f th e c o u n tin g v ia ls c o n ta in in g lip id f r a c t i o n s . C o u n tin g for
10 m in w a s d o n e in th e
14
C c h a n n e l o f th e B eckm an LS-IOO liq u id s c i n t i l ­
la tio n c o u n tin g s y s te m . By e x te r n a l s ta n d a rd c a l i b r a t i o n , c o u n tin g
e f f ic ie n c y w a s d e te rm in e d to be a b o u t 90% . S in c e d ir e c t c o u n tin g o f
s a m p le s c o n ta in e d in m o le c u la r s e iv e d id n o t g iv e c o n s ta n t e ffic ie n c y ,
th e c o u n tin g r a te s fo r f r a c tio n s o f n o rm al h y d ro c a rb o n s w e re d e te rm in e d
b y s u b tr a c tin g th e c o u n ts in th e b ra n c h e d fr a c tio n from th e to ta l num ber
o f c o u n ts in th e s a tu r a te d h y d ro c a rb o n f r a c tio n s . D a ta h a v e b e e n c o n ­
v e r te d to th e fr a c tio n o f a d m in is te r e d s u b s tr a te r a d io a c tiv ity in c o rp o r­
a te d in to lip id f r a c t i o n s . S in c e th e r e s u ltin g nu m b ers w e re v e ry sm all,*
3
th e y h a v e b e e n m u ltip lie d by 10 to c o n v e rt them to w h o le n u m b ers for
p r e s e n ta tio n in th e R e s u lts s e c tio n .
O x id a tiv e D e g ra d a tio n of B ranch ed S a tu ra te d H y d ro c arb o n s
In o rd e r to d e te rm in e th e p o s itio n o f in c o rp o ra tio n o f th e r a d io ­
a c ti v ity from p ro p io n a te in to th e b ra n c h e d s a tu r a te d h y d r o c a r b o n s ,
o x id a tiv e d e g ra d a tio n of th e b ra n c h e d fr a c tio n w a s p e rfo rm ed (2 5 ,6 6 ).
The sa m p le (0.3 mg) w a s o x id iz e d w ith 7 mg ch ro m ic a n h y d rid e (CrOg) in
0.5 ml g la c i a l a c e t i c a c id in a t e s t tu b e fo r 4 hr a t 65 C . A c e tic a c id w a s
rem o v ed by e v a p o ra tio n u n d e r a s tre a m o f n itr o g e n . M e th y la tio n o f th e
f a tty a c id s p ro d u c e d w a s a c c o m p lis h e d b y re flu x in g th e r e s id u e in 3 ml
30
o f a 14% s o lu tio n o f bo ro n triflu o rid e in m e th a n o l fo r IO m in on a w a te r
b a th . A fter th e s o lu tio n h a d c o o le d , i t w a s d ilu te d w ith tw o v o lu m e s o f
w a te r an d w a s h e d w ith h e x a n e th re e tim e s , an d th e h e x a n e s o lu tio n w a s
c o n c e n tr a te d u n d e r n itro g e n an d tra n s f e r r e d to a th i n - l a y e r p l a t e , w h ic h
w a s th e n e lu te d w ith h e x a n e : d ie th y l e th e r : a c e tic a c id (8 5 :1 5 :1 ) . The
s i l i c a g e l w a s d iv id e d in to tw o p o rtio n s b y a lin e d raw n b e lo w th e
h y d ro c a rb o n s p o t; e a c h p o rtio n w a s th e n s c r a p e d in to a s in te r e d g la s s
fu n n e l a n d th e lip id s e x tr a c te d w ith to lu e n e .
H y d ro c arb o n s a n d fa tty
a c id m e th y l e s t e r s w e re id e n tif ie d b y c o m p arin g th e ir g a s c h ro m ato g ram s
w ith th o s e o f know n s t a n d a r d s .
RESULTS
T ab le I c o n ta in s r e s u lts o f e x p e rim e n ts in w h ic h p a lm ita te -9 ',IO-3H
w a s in c o rp o ra te d in to s a tu r a te d an d u n s a tu r a te d h y d r o c a r b o n s . No
a p p r e c ia b le d iffe re n c e w a s o b s e rv e d b e tw e e n c e ll - f r e e p re p a ra tio n s an d
c o n tr o ls . The lo w r a d io a c tiv ity c o u ld h a v e b e e n d u e e n tir e ly to th e
p r e s e n c e o f c o n ta m in a tin g h y d ro c a rb o n s in th is s u b s t r a t e . T h e se r e s u lts
in d ic a te th a t th e c e l l - f r e e p re p a ra tio n s w e re n o t c a p a b le o f h y d ro c a rb o n
b i o s y n t h e s i s . T h e re fo re , th e s e a tte m p ts to p re p a re a c e l l - f r e e h y d ro ­
c a rb o n b io s y n th e tic s y s te m w e re abandoned in fa v o r o f in v iv o e x p e r i­
m e n ts . S in c e J a c k s o n a n d B aker (3) h a d re p o rte d th a t liv e c o c k ro a c h e s
c o n s titu te d a b e tte r s y s te m fo r b io s y n th e s is o f h y d ro c a rb o n s th a n d id
e x c is e d in te g u m e n ts , liv in g a d u lt ro a c h e s w ere u s e d in th e re m a in in g
e x p e r im e n ts .
'
TABLE I
3
In c o rp o ra tio n o f P a lm ita te - 9 ,1 0 - H in to H y d ro c arb o n s by C e ll- f r e e
P re p a ra tio n s o f M a le P. a m e ric a n a an d B uffer C o n tro ls
P e rc e n t o f S u b s tra te In c o rp o ra te d x 10
P re p a ra tio n
C e ll- f r e e ly o p h ily s a te
C o n tro l
3
S a tu ra te d
U n s a tu ra te d
12
11
9
14
32
In te g u m e n t H y d ro c a rb o n s o f T e n e ra l C o c k ro a c h e s
It h a s b e e n fo u n d p re v io u s ly (29) th a t h y d ro c a rb o n s o f th e A m eri­
c a n c o c k ro a c h c o m p rise a b o u t 75 to 85% o f th e e x te r n a l lip id s (1.7 to
2.6 mg p e r i n s e c t ) . In T ab le II d a ta a re g iv e n d e m o n s tra tin g th a t th e
to ta l am o u n t o f h y d ro c a rb o n m a te ria l on th e c u tic le s o f te n e r a l an d o ld e r
a d u lt r o a c h e s w a s a p p ro x im a te ly th e s a m e . G as c h ro m a to g ra p h ic a n a ly , s i s sh o w e d th e h y d ro c a rb o n fr a c tio n s o f te n e r a l a d u lts to b e q u a li­
ta tiv e ly an d q u a n tita tiv e ly th e sam e a s th o s e o f o ld e r a d u lts (F ig . 11).
TABLE II
W e ig h ts o f S u rfa c e H y d ro c a rb o n s in N e w ly -m a lte d ^andrl T w o-m onth
O ld e r Adult. P. a m e ric a n a
M a le
F em ale
T e n e ra l
1.40 mg
1.39 mg
T w o-m onth
1.2 7 mg
1.65 mg
Age
33
In c o rp o ra tio n o f L a b e le d S u b s tr a te s in to S u rfa c e H y d ro c arb o n s
R e s u lts o f e x p e rim e n ts c o n c e rn in g th e in c o rp o ra tio n o f s u b s tr a te s
in to c u tic u la r h y d ro c a rb o n s o f P. a m e ric a n a a t v a rio u s a d u lt a g e s a re
sh o w n in T a b le II I. A w id e v a r ia tio n w a s o b s e rv e d in th e d a ta (s e e
A ppendix) w h ic h w e re a v e ra g e d to. th e v a lu e s sh o w n in th e t a b l e . It d id
n o t se e m r e a s o n a b l e , t h e r e f o r e , to a tte m p t to o b s e rv e s u b tle d iff e r e n c e s
in in c o rp o ra tio n o f d iff e r e n t s u b s t r a t e s . ' H o w e v e r, la rg e d iff e r e n c e s in
t h e s e v a lu e s w e re c o n s id e re d to r e p r e s e n t tru e d iff e r e n c e s in m e ta b ­
o lis m .
1
A c e ta te w a s in c o rp o ra te d , l e s s in to th e four ty p e s o f h y d ro c a rb o n
s tr u c tu r e s in te n e r a l c o c k r o a c h e s th a n in e ith e r group o f o ld e r c o c k ­
ro a c h e s . A lthough p ro p io n a te a ls o w a s n o t in c o rp o ra te d in to h y d ro c a r­
b o n s in te n e r a l r o a c h e s , i t w a s in c o rp o ra te d a p p re c ia b ly in to th e
b ra n c h e d h y d ro c a rb o n s in o ld e r a d u l t s . N e ith e r is o le u c in e n or th e
S-m e th y l group o f m e th io n in e s e e m e d to be in c o rp o ra te d in to b ra n c h e d
h y d ro c a rb o n s . N orm al s a tu r a te d f a tty a c id s w ere in c o rp o ra te d in to m o st
h y d ro c a rb o n fr a c tio n s in ro a c h e s o f a ll a g e s t e s t e d , in d ic a tin g th a t
h y d ro c a rb o n s w e re b e in g form ed c o n tin u o u s ly d u rin g a d u lt l i f e . In b o th
g ro u p s o f t a n n e d 'a d u l t s , few m ajo r q u a lita tiv e d if f e r e n c e s in th e in c o r • p o ra tio n o f s u b s tr a te s w e re o b s e r v e d . S te a ra te w a s w e ll-in c o rp o ra te d
in to h y d ro c a rb o n s in th e te n e r a l r o a c h e s . L in o le a te w a s n o t in c o rp o r­
a te d in to h y d ro c a rb o n s i n te n e r a l c o c k r o a c h e s , w h ile lin o le a te in c o rp o r­
a tio n in to d ie n e h y d ro c a rb o n s w a s h ig h in th e ta n n e d a d u l t s .
34
TABLE III
In c o rp o ra tio n o f L a b e le d S u b s tr a te s in to S u rfa ce H y d ro c arb o n F ra c tio n s
in A dult P. a m e ric a n a
R o a c h e s w e re a llo w e d to d ig e s t s u b s tr a te s fo r 18 hr a f te r a d m in is tra tio n .
3
N u m b ers r e p r e s e n t p e r c e n t o f a d m in is te r e d s u b s tr a te in c o rp o ra te d x 10 .
N = n o rm al s a tu r a te d h y d ro c a rb o n s . B = b ra n c h e d s a tu r a te d h y d ro c a rb o n s .
M = m onoene h y d ro c a rb o n s . D = d ie n e h y d ro c a rb o n s . t = I e s s th a n 10.
Males
S u b s tra te
tim e
14
2 :0 -1 - C
(a c e ta te )
0
14
3 :0 -1 - C
(p ro p io n a te ) '
14
10: 0- 1 - 0
(d e c a n o a te )
14
12: 0 - 1 - 0
(Ia u ra te )
B
M
D
B
t
22
19
t
N
M
D
12
30
t
t
1-4 wk
35
54
47 113
66
74
29 188
2+ mo
77
36
16 159
53
39
12 132
t
't
t
t
t
t
t
t
100
90
t
t
28
85
t
t
33 138
t
t
18
56
f
t
47
0
1-4 wk
2+ mo
14
8 :0 -1 - C
(o c ta n o a te )
N
Females
t
t
t
26
11
16
29
■ 1-4 wk
20
20
42
68
59
55
15 144
2+ mo
73
55
28
92
74
31
12
60
0
13
t
t
30
27
t
19
50
1-4 wk
42
22
13
46
■ 40
2+ mo
2 6 ,, 53
14
35
46
30
t
62
t
16
t
15
24
t
20
54
1-4 wk
78
25
20
38
81
65
43
98
2+ mo
43
27
22
39
52
27
15 105
0
Og
46 - 32 108
35
TABLE III (con tin u ed )
M a l e s _____
S u b s tra te
14:0 - I - 14C '
(m y rista te )
14
1 6 :0 -1 - C
(p a lm ita te )
, 14
1 6 :0 -1 6 - 0
(p a lm ita te )
tim e
0
14
1 8 :0 -1 8 - C
( s te a r a te )
14
1 8 :2 -1 - C
(Iin o le a te )
14
I s o le u c in e - U L - C
■M
D
N
•B
■M
D
t
t
.t
16
t
t
95
. 1-4 wk
82
16
36
47
60
43
45
53
2+ mo
29
13 . 15
19
51
20
15
64
.0
. 11
t
t
24
15
.t
t
39
l r 4 wk
23
19
36
37
19
19
14
53
2+ mo
24
t
17
21
16
t
t
24
0
t
17
22
19
• 24
19
24
1-4 wk
t
33 . 45
64
131
29
31
1:970
0
85 . 15
25
26
85
33
23
46
47
23
40
58
36
29
36
16 . 25
17
34
t
11
38
. t
t
20
23
t
t
32
13
66
28 ' 12 • 14.
44
48
1-4 wk
t
2+ mo
25
0
35
L
. 1-4 wk
t
17
47
22
19
12
38
42
2+ mo
41
t
t
29
68
t
t
56
0
10
t
t
12
t
t
t
t
t ' 10
31
t
12
12
75
1-4 wk
t
2+ mo
28
14
t
.71
30
21
11 136
t
t
t
55
40
. t
18. 142
16 . 29
•t
19
t
t
•U'
0
1-4 wk
14
M e th io n in e -C H g -. C
B
t
2+ mo
14
1 8 :0 -1 - C
( s te a r a te )
N
F e m .a I e s
.1-4 wk
t
t
36
In c o rp o ra tio n o f L a b e le d S u b s tra te s in to In te rn a l L ip id F ra c tio n s
T e n e ra l a d u lt P. a m e ric a n a d id n o t in c o rp o ra te a s ig n if ic a n t p ro p o r­
tio n o f th e a d m in is te r e d s u b s tr a te s in to in te r n a l h y d r o c a r b o n s , a s
sh o w n by th e d a ta in T a b le IV. On th e o th e r h a n d , a d u lt r o a c h e s o f tw o
m o n th s or lo n g e r in c o rp o ra te d a s ig n if ic a n t p o rtio n o f la b e le d a c e ta te
a n d p ro p io n a te in to in te r n a l h y d r o c a r b o n s . The p a tte r n o f in c o rp o ra tio n
o f f a tty a c id s in to th e o th e r tw o fr a c tio n s — e s t e r s , in c lu d in g g ly c e r ­
i d e s , an d o r ig in , w h ic h c o n ta in e d f a tty a c id s and p h o s p h o lip id s — w a s
s im ila r in som e c a s e s a n d d iff e r e n t in o t h e r s . In g e n e r a l , h o w e v e r,
a lm o s t a ll of th e la b e l from f a tty a c id s u b s tr a te s re m a in e d in th e fa tty
a c id f r a c t i o n s .
D e g ra d a tio n o f B ran ch ed H y d ro c arb o n s
O x id a tiv e d e g ra d a tio n o f b ra n c h e d h y d ro c a rb o n s w a s a tte m p te d
a f te r in je c tio n o f o ld e r ro a c h e s w ith p ro p io n a te (2 5 ,6 6 ). F o llo w in g th is
p r o c e d u r e , th e h e x a n e e x tr a c t w a s c h ro m a to g ra p h e d to s e p a r a te h y d ro ­
c a rb o n s from f a tty a c id m e th y l e s t e r s . O th e r b ra n c h e d h y d ro c a rb o n s ,
p r e v io u s ly tr e a te d th is w a y in o u r la b o ra to ry , w ere n o t fo u n d to be d e ­
g ra d e d (6 7 ). In. th is c a s e , no s p o t o th e r th a n th e h y d ro c a rb o n fra c tio n
w a s id e n tif ie d by g a s c h ro m a to g ra p h y a s h a y in g 2 6 c a rb o n s ? no fa tty
a c id m e th y l e s t e r w a s id e n tif ie d w h e n th e e x tra c t- o f th e lo w e r p o rtio n o f
th e p la te w a s s u b je c te d to g a s c h ro m a to g ra p h y . T h u s , th is a tte m p t to
d e g ra d e b ra n c h e d h y d ro c a rb o n s w a s u n s u c c e s s f u l.
J
37
TABLE IV
In c o rp o ra tio n o f L a b e le d S u b s tr a te s in to In te rn a l L ip id s of
A dult M a le P. a m e ric a n a
N u m b ers in d ic a te p e r c e n t o f in te r n a l la b e l in c o rp o ra te d in to e a c h
f r a c tio n .
A dult tim e
H y d ro c arb o n s
E s te rs
2: 0- I - 14C
0
2+ mo
3
25
68
34
30
41
0
2+ mo
6
401
60
46
' 35
14 ■
8: 0- I - 14C
0
41
.21
53
73
14
1 0 :0 -1 - C
2± m p
0
2+ mo
6
6
16
3
34
.51
50
46
12:0 - I - 14C
0 "
2+ mo
2
2
83
96
14
.2
14; O -I-14C
'0
2+ mo
I
I
77
46
22
53
■ 2
■ 2
42
.7 6
O rig in
0
2+ mo
16:0-^S -14C
0
.2+ mo
2
I ■
31
. 3
67
96
18; O -I-14C
0
2+ mo
0
0 ■
8
36
92
64
1 8 :0 - 1 8 - 4C
0
2+ mo
2
6
30
93
68
I
0
6
. 92
I
I
—1
O)
O
I
T-
- ffI
*—
I
I
O
CO
S u b s tra te
14
1 8 :2 -1 - C
.
56
22
38
In h ib itio n w ith T r ic h lo ro a c e tic A cid
K olattulcudy (48.) h a s s u g g e s te d th a t tr ic h lo r o a c e tic a c id (TCA) is
a n in h ib ito r 'o f f a tty a c id e lo n g a tio n . W h en s e v e r a l d iff e r e n t c o n c e n tra ­
tio n s o f TCA w ere a d m in is te r e d to. P. a m e ric a n a 30 m in p rio r to in je c tio n
w ith a c e t a t e - l - C
14
C , h y d ro c a rb o n s w e re la b e le d a s sh o w n in T ab le V. It
w a s o b s e r v e d th a t TCA c a u s e d re d u c tio n o f h y d ro c a rb o n b io s y n th e s is to
a b o u t h a lf th e norm al le v e l w h en c o n c e n tra tio n s o f 8% TCA o r l e s s w ere
u s e d . W hen th e le v e l o f 10% TCA w a s u s e d , fu rth e r in h ib itio n w as .
n o te d . U n fo rtu n a te ly , th is c o n c e n tra tio n o f in h ib ito r c a u s e d th e d e a th
o f a ll th e ro a c h e s b e fo re th e en d o f th e 1 8 -h o u r in c u b a tio n p e rio d ; som e
o f th e ro a c h e s w h ic h h a d b e e n in je c te d w ith o n ly 5 to 8% TCA d ie d
w ith in th is p e rio d .
TABLE V
T r ic h lo ro a c e ta te In h ib itio n o f A c e ta te In c o rp o ra tio n in to H y d ro c arb o n s in
A dult M a le P. a m e ric a n a 1-4 W e e k s a f te r th e Im a g in a l E c d y s is
TCA S o lu tio n
TCA S o lu tio n
S u b s tra te In c o rp o ra te d
In h ib itio n
(P er C ent)
(Ml)
(P er C e n tx lO ^ )
(P er C ent)
. 0
. 10
264
——
2
10
138
48
5
. 10
140
47
6
10
138
8
10
178
33
. 10
10
106
60
•
48
39
In h ib itio n w ith Sodium C itra te
)
To e lim in a te th e a c e ta te e f f e c t , so d iu m c i t r a t e , a w e ll-k n o w n
in h ib ito r o f (3- o x id a tio n ( 68), w a s in je c te d p rio r to in c u b a tio n w ith
l i n o l e a t e - I - . If th e e x p e c te d in h ib itio n a c tu a lly h a d o c c u r r e d , a d e c r e a s e d in c o rp o ra tio n o f
14
C from lin o le a te in to th e s a tu r a te d fr a c tio n s
r e la tiv e to th e d ie n e fr a c tio n w o u ld h a v e b e e n o b s e r v e d . T rea tm e n t w ith
c itr a te r e s u lte d in no re d u c tio n o f th is r e la tiv e in c o rp o ra tio n , a s sh o w n
by th e d a ta in T ab le VI.
TABLE VI
Sodium C itra te In h ib itio n o f L in o le a te In c o rp o ra tio n in to H y d ro c arb o n s in
A d u lt M a le P. a m e ric a n a 1-4 W e e k s a f te r th e Im a g in a l E c d y sis
Sodium C itra te
S o lu tio n
P er C e n t o f S u b s tra te In c o rp o ra te d in to H y d ro c arb o n s
^ in 3
S a tu ra te d
D ie n e s
M onoenes '
N one
20
14
38
10 m 10%
27
12
26
40
In h ib itio n w ith Sodium A c e ta te
A fu rth e r a tte m p t to e lim in a te th e a c e ta te e f f e c t w a s m ade by
u s in g p ro d u c t in h ib itio n o f |3 -o x id a tio n . S in c e a c e ta te is th e m ajo r en d
p ro d u c t o f f a tty a c id c a ta b o lis m , th is w a s in je c te d in to r o a c h e s p rio r to
in c u b a tio n w ith s u b s t r a t e s . In s e v e r a l e x p e r im e n ts , a c e ta te in h ib itio n
w a s n o t sh o w n to a lte r th e p a tte r n o f in c o rp o ra tio n o f s u b s tr a te s in to
h y d ro c a rb o n s a p p re c ia b ly (T able V II).
TABLE VII
■I n h ib itio n by 2 M Sodium A c e ta te o f In c o rp o ra tio n o f L a b e le d
S u b s tr a te s in to H y d ro c a rb o n s o f A dult M a le P. a m e ric a n a
1-4 W e e k s a f te r th e Im a g in a l E c d y s is
P er C e n t o f S u b s tra te In c o rp o ra te d in to
3
N aO A c1 _____________ H y d ro c arb o n s x 10____________
S u b s tra te
14
M a lo n a te - 1 ,3 - C
16:0 - I - 14C
14
1 8 :0 -1 - C
(ill)
N orm al
0
10
20
. 11
8
6
. 26
22
21
0
10
2.2
8
o'
6
6
10
B ran ch ed
D ien e
T o tal
9
17
18
49
66
95
113
44
89
19
21
35
•47
31
40
107
116
16
6
25
11
19
26
66
M o n o en e
49
D ISC U SSIO N
S e v e ra l a s s u m p tio n s m u st be m ad e b e fo re th e r e s u l t s p re s e n te d
h e re in a re d i s c u s s e d . F ir s t, i t i s a s s u m e d th a t m o st o f th e r a d io a c tiv ity
from, la b e le d s u b s tr a te s .-in c o rp o ra te d in to e a c h fr a c tio n w a s in c o rp o ra te d
in to th e m a jo r c o m p o n en t o f th a t f r a c tio n . T his is p ro b a b ly a v a lid
a s s u m p tio n s in c e a s in g le com pound c o m p ris e s th e b u lk o f e a c h o f th e
m o st a b u n d a n t f r a c t i o n s . N e x t, i t is a s s u m e d th a t th e b io s y n th e tic
p a th w a y s fo r e a c h h y d ro c a rb o n w e re th e s a m e in b o th m a le and fem ale
r o a c h e s . I t is u n lik e ly t h a t d iff e r e n t p a th w a y s h a v e e v o lv e d by w h ic h
m em bers o f e a c h s e x p ro d u c e th e sa m e co m p o u n d s in th e sam e r a t i o s .
F in a lly , i t is a s s u m e d t h a t , s in c e c o c k r o a c h e s s e e m in c a p a b le o f s y n ­
t h e s i s o f p o ly u n s a tu ra te d f a tty a c id s (3 6), I in o le a te is th e o n ly s o u rc e
o f u n s a tu r a te d h y d ro c a rb o n c h a in to b e in c o rp o ra te d in to th e d ie n e h y d ro c a rb q n f r a c tio n . It is n o te d th a t th e d is ta n c e s o f th e d o u b le b o n d s
from th e te rm in a l m e th y l en d o f lin o le a te a re th e sam e a s in
c i s , c i s - 6 ,9 - h e p ta c o s a d ie n e .
D u rin g th e p e rio d in th e lif e c y c le o f P* a m e ric a n a 1-4 w e e k s a f te r
•th e im a g in a l e c d y s i s , m e ta b o lic c h a n g e s o c c u r re la te d , to th e re p ro d u c t­
iv e fu n c tio n o f- th e s e i n s e c t s .(6 9 ). A h ig h e r ra te o f m e ta b o lism , is know n
d u rin g th is tim e th a n a t o th e r tim e s in th e lif e c y c le . As a r e s u l t , in c o r ­
p o ra tio n o f f a tty a c id s u b s tr a te s in to h y d ro c a rb o n s w a s e x p e c te d to be
m ore p ro n o u n c e d in fe m a le s th a n in m a le s .
42
In c o rp o ra tio n o f A c e ta te an d F a tty A cids in to H y d ro c arb o n s
A c e ta te w a s in c o rp o ra te d in to b o th in te g u m e n t a n d in te rn a l h y d ro ­
c a rb o n s o f f u lly - ta n n e d a d u lt P. a m e r ic a n a . T his in c o rp o ra tio n w as n o t
found in te n e r a l a d u l t s . T his s u g g e s ts tw o p o s s i b i l i t i e s : e ith e r a c e ta te
w a s n o t in c o rp o ra te d in to p re c u rs o r s o f h y d ro c a rb o n s d u rin g th e te n e r a l
p e r io d , o r th e p re c u rs o r s w e re n o t m e ta b o liz e d to form th e h y d ro c a r­
b o n s . O n th e o th e r h a n d , m any o f th e f a tty a c id s u b s tr a te s w e re in c o r ­
p o ra te d in to h y d ro c a rb o n s a lm o s t a s w e ll d u rin g th is te n e r a l p e rio d a s
th e y w e re a t l a t e r p e rio d s d u rin g th e a d u lt s ta g e . T h is in d ic a te s th a t
f a tty a c id s y n th e s is w a s in h ib ite d d u rin g th e te n e r a l p e rio d . I t.i n d i­
c a te s fu rth e r t h a t , a lth o u g h h y d ro c a rb o n s w ere form ed to a lm o s t th e
sa m e e x te n t d u rin g th e p e rio d im m e d ia te ly fo llo w in g e c d y s is a s la te r in
a d u lt lif e (a s in d ic a te d b o th by th e c o m p a ra b le a m o u n ts o f c u tic u la r
h y d ro c a rb o n s o f b o th g ro u p s an d b y th e in c o rp o ra tio n o f f a tty a c id s u b ­
s tr a te s ) , th is s y n th e s is in th e te n e r a l p e rio d w a s n o t due to in c o rp o ra ­
tio n o f a c e ta te u n its in a n e lo n g a tio n - d e c a r b o x y la tio n p a th w a y .
In c o rp o ra tio n o f L in o le a te in to D ie n e H y d ro c arb o n s
T e n e ra l c o c k r o a c h e s w e re c a p a b le o f s y n th e s is o f d ie n e h y d ro c a r­
b o n s from la b e le d n o rm al s a tu r a te d f a tty a c i d s , b u t d id n o t in c o rp o ra te
th e la b e le d c a rb o x y l group o f l i n o l e a t e . In o ld e r a d u l t s , th is c a rb o x y l
group o f lin o le a te w a s in c o rp o ra te d w e ll in to d ie n e s . I t w ill b e r e c a lle d
t h a t th e p o s itio n s o f u n s a tu r a tio n in th e m a jo r d ie n e h y d ro c a rb o n
43
(C ls7C is- 6,9- h e p ta c o s a d le n e ) o f P. a m e ric a n a a re th e sam e a s th o s e in
l i n o l e a t e , c o u n tin g from th e m eth y l e n d . In o ld e r a d u lt c o c k r o a c h e s , a s
w e ll a s in th o s e in th e te n e r a l p e r io d , s a tu r a te d f a tty a c id s w e re a ls o
in c o rp o ra te d in to d ie n e s to a la rg e e x te n t.
.I t is p o s s ib le to e x p la in in c o rp o ra tio n o f lin o le a te by e ith e r e lo n ­
g a tio n to form a 2 8-c a r b o n f a tty a c id w h ic h w a s th e n d e c a rb o x y la te d to
th e 2 7 -c a rb o n h y d ro c a rb o n ( e lo n g a tio n - d e c a r b o x y la tio n p a th w a y ) or
c o n d e n s a tio n o f lin o le a te o r o n e o f i t s e lo n g a tio n p ro d u c ts w ith a
s h o r te r , norm al f a tty a c id to form th e h y d ro c a rb o n . In te n e r a l a d u l t s ,
h o w e v e r, th e re w a s n e ith e r in c o rp o ra tio n o f a c e ta te (no e lo n g a tio n p a th w a y ) n o r in c o rp o ra tio n o f th e c a rb o x y l c a rb o n o f l i n o l e a t e ., T h u s,
no h y d ro c a rb o n fo rm a tio n from lin o le a te d u rin g th e te n e r a l p e rio d c o u ld
h a v e o c c u rre d e x c e p t by m e a n s o f a c o n d e n s a tio n in w h ic h th e c a rb o x y l
group o f lin o le a te w a s l o s t .
P re c u rs o rs o f B ran ch ed H y d ro c a rb o n s '
P ro p io n a te w a s in c o rp o ra te d , in to b ra n c h e d h y d ro c a rb o n s in ta n n e d
a d u l t s , b u t n o t in te n e r a l a d u lts . N e ith e r is o le u c in e n o r th e S'-m eth y l
group o f m e th io n in e w a s in c o rp o ra te d - in to b ra n c h e d h y d ro c a rb o n s in th e
g ro u p s o f o ld e r a d u l t s . From th e s e o b s e r v a tio n s , i t c a n b e c o n c lu d e d
t h a t p ro p io n a te p ro b a b ly w a s in c o rp o ra te d in to f a tty a c id s in ta n n e d
a d u lts by a m e c h a n ism s im ila r to th a t a lre a d y m e n tio n e d (F ig . 3 ). H ow ­
e v e r , s in c e fre e b ra n c h e d f a tty a c id s h a v e n o t b e e n ,re p o rte d :.in
sI
44
P. a m e ric a n a (3), b ra n c h e d fa tty a c id s , w ith o u t r e le a s e from h y d ro c a rb o n
b io s y n th e s is e n z y m e s , th e n m ig h t h a v e b e e n c o n v e rte d to h y d ro c a rb o n s
by e ith e r th e e lo n g a tio n - d e c a r b o x y la tio n p a th w a y o r th e c o n d e n s a tio n
p a th w a y . A lte rn a tiv e ly , p ro p io n a te c o u ld h a v e b e e n in c o rp o ra te d a s th e
p e n u ltim a te u n it d u rin g e lo n g a tio n o f a n orm al fa tty a c id (F ig . 12). To
in v e s t ig a te th is p o s s i b i l i t y d e g ra d a tio n o f b ra n c h e d h y d ro c a rb o n s w a s
a tte m p te d . R e s u lts from th e am in o a c id e x p e rim e n ts sh o w e d th a t in c o r ­
p o ra tio n o r p ro p io n a te w a s th e o n ly m e c h a n ism o f s y n th e s is o f b ra n c h e d
h y d ro c a rb o n s o b s e r v e d . T h e re fo re , i t a p p e a rs th a t b ra n c h e d h y d ro c a r­
b o n s w e re n o t form ed d u rin g th e te n e r a l p e rio d .
CHo O
I ^
O
Il
Il
C H 9-C -S -C o A
.
V
CHoO
I
3 II
C H 3-( C H 2) 20-C -S -C o A ----- -----------------> C H 3-(C H 2) 20- C H 2- C H - C - S - C oA
H -S -C o A
1) C 9 a d d itio n
------ ------------------------- >
2) d e c a rb o x y la tio n
F IG . 12.
C H o-(C H 2) -C H - C H 9- C H 9
In c o rp o ra tio n o f p ro p io n a te a s th e p e n u ltim a te u n it in
e lo n g a tio n to form 3 - m e th y lp e n ta c o s a n e .
45
P a th w a y s o f H y d ro c arb o n B io s y n th e s is
It h a s b e e n o b s e r v e d th a t a c e ta te w a s n o t in c o rp o ra te d in to h y d ro ­
c a rb o n s d u rin g th e te n e r a l p e rio d o f a d u lt P. a m e r ic a n a , w h ile f a tty a c id
s u b s tr a te s w e re in c o rp o ra te d . T h is ru le s o u t e lo n g a tio n a s a p a th w a y o f
h y d ro c a rb o n b i o s y n t h e s i s , a f l e a s t d u rin g th e te n e r a l p e rio d . T h u s , th e
m a jo r p a th w a y o f h y d ro c a rb o n b io s y n th e s is d u rin g th is p e rio d m u st h a v e
b e e n by w ay o f c o n d e n s a tio n o f f a tty a c i d s .( F i g .-13).. H o w e v e r, th e d a ta
a re in c o n c lu s iv e a s to w h ic h o f s e v e r a l p o s s ib le c o n d e n s a tio n s
o c c u r re d .
O f th e p o s s ib le c o n d e n s a tio n s , o n e in w h ic h lin o le a te c o n d e n s e d
w ith d e c a n o a te , w ith lo s s o f th e c a rb o x y l group from th e l i n o l e a t e , m ay
h a v e b e e n r e s p o n s ib le for fo rm atio n o f d ie n e h y d ro c a rb o n s . On th e
o th e r h a n d , th e c a rb o x y l c a rb o n o f lin o le a te m ay h a v e b e e n in c o rp o ra te d
in o ld e r ro a c h e s by e lo n g a tio n o f th e a c id , fo llo w e d by e ith e r c o n d e n ­
s a tio n w ith a n o th e r f a tty a c id o r a n e lo n g a tio n - d e c a r b o x y la tio n p a th w a y
(F ig . 13). The la c k o f in c o rp o ra tio n o f th e c a rb o x y l carb o n , in to th e
d ie n e h y d ro c a rb o n s o f th e te n e r a l a d u lt ro a c h e s s u g g e s ts t h a t a c o n d e n ­
s a tio n h a d o c c u rre d (F ig . 14).
O th e r c l a s s e s o f h y d ro c a rb o n s p ro b a b ly w ere fo rm ed a ls o by c o n ­
d e n s a tio n o f f a tty a c id s d u rin g th e te n e r a l p e rio d . F a tty a c id s u b s tr a te s
w e re in c o rp o ra te d in to th e s e o th e r h y d ro c a rb o n s in te n e r a l c o c k ro a c h e s ,
a lth o u g h a c e ta te w a s n o t. The la rg e in c o rp o ra tio n o f s te a r a te r e la tiv e
to o th e r f a tty a c id s s u g g e s ts th a t i t m ay h a v e b e e n th e m a in f a tty a c id
C H 3 (C H 2 )4 C H = C H C H 2 C H = C H (C H 2) 6COOH
C 2 a d d itio n
.C H 3(C H 2 ) 8COOH
c o n d e n s a tio n
C H 3(C H 2 )4 C H = C H C H 2 C H = C H (C H 2 ) gCOOH
4
C 2 a d d itio n s
f
\
C H 3 (C H 2) 6COOH
c o n d e n s a tio n
COr
v/
C H 3(C H 2)4 C H = C H C H 2 C H = C H (C H 2) ^ C H 3
FIG . 13.
P . a m e r ic a n a .
P o s s ib le m e a n s o f b io s y n th e s is o f d ie n e h y d ro c a rb o n s in fu lly -ta n n e d a d u lt
47
C H 3(C H 2) 4 C H = C H C H 2 C H = C H (C H 2 )*C O O H
+
C H 3(C H 2) 8 COOH
CO2
N/
C H 3(C H 2)4 C H = C H C H 3 C H = C H (C H 2) ^ C H 3
FIG . 14.
P o s s ib le c o n d e n s a tio n in b io s y n th e s is o f d ie n e h y d ro ­
c a rb o n s in te n e r a l a d u lt P. a m e r ic a n a .
48
in th e c o n d e n s a tio n w h ic h r e s u lts in b io s y n th e s is o f th e s e o th e r
c la s s e s of h y d ro c a rb o n s.
M e ta b o lic D iffe re n c e s D uring th e L ife C y c le
The s trik in g s im ila r ity in th e to ta l am o u n t o f c u tic u la r h y d ro c a r­
bon m a te ria l on te n e r a l a d u lts an d o n th o s e w h o se c u tic le h ad
th o ro u g h ly ta n n e d s u g g e s te d th a t h y d ro c a rb o n c o n te n t o f th e c u tic le w a s
c o n s ta n t th ro u g h o u t a d u lt l i f e . S in c e s y n th e s is o f h y d ro c a rb o n s w as
o b s e rv e d in a ll a g e s s t u d i e d , w ith th e e x c e p tio n o f b ra n c h e d h y d ro c a r­
b o n s in te n e r a l a d u l t s , it.a p p e a r s th a t a c o n s ta n t tu rn o v e r o f s u rfa c e
lip id s o c c u r s to r e p la c e m a te ria l l o s t b y a b r a s io n .
Im m e d ia te ly a f te r e c d y s i s , m e ta b o lic p r o c e s s e s in th e in te g u m e n t
n o rm a lly a re r e la te d to ta n n in g o f th e c u t i c l e . D uring th is p e r io d ,
c h a n g e s in th e m e ta b o lism o f am in o a c id s h a v e b e e n o b s e r v e d (70,71).
It s e e m s from th e r e s u lts p r e s e n te d h e re th a t c h a n g e s in lip id m e ta b ­
o lis m a ls o o c c u r re d . A c e ta te w a s n o t u tiliz e d in th e fo rm a tio n o f fa tty
a c id s , an d th e p a tte r n o f in c o rp o ra tio n o f lin o le a te in to h y d ro c a rb o n s
w a s m uch d iff e r e n t in th e te n e r a l p e rio d th a n in o ld e r a d u l t s . Few m ajo r
d iff e r e n c e s w e re o b s e r v e d in in c o rp o ra tio n o f fa tty a c id s u b s tr a te s in to
h y d ro c a rb o n s o f I - to 4 -w e e k a d u lts , w ho a re e n g a g e d in re p ro d u c tiv e
a c tiv ity , an d th o s e o f o ld e r a d u lt r o a c h e s , in w h ic h re p ro d u c tiv e fu n c ­
tio n s e s s e n t i a l l y h a v e b e e n c o m p le te d .
49
In h ib itio n o f M e ta b o lic P a th w a y s
A lthough s e v e r a l a tte m p ts w e re m ade to a lte r in c o rp o ra tio n o f
s u b s tr a te s by in h ib itio n o f m e ta b o lic p a th w a y s n o n e w a s s u c c e s s f u l .
A lthough i t w ou ld h a v e b e e n d e s ir a b le to s tu d y e a c h m e ta b o lic p a th w a y
in v o lv e d in th e lip id m e ta b o lism s e p a r a te ly , th is w a s im p o s s ib le in th e
liv in g s y s te m . If a c e ta te w ere n o t in c o rp o ra te d in to fa tty a c i d s , o r if
f a tty a c id s w e re n o t d e g ra d e d to form a c e t a t e , th e n a tu re o f th e in te r ­
lo c k in g p a th w a y s w o u ld n o t h a v e b e e n so c o m p le x . In h ib ito rs .e ith e r d id
n o t p ro d u c e th e e x p e c te d in h ib ito r y e f f e c ts o r c a u s e d d e a th to th e c o c k ­
ro a c h e s .
D uring th e c o u rs e o f th e s e e x p e r im e n ts , a s y s te m w a s d is c o v e re d
in w h ic h th e a c e ta te e f f e c t w a s n o t p ro m in e n t. In te n e r a l c o c k ro a c h e s
th e s h o rt s u b s tr a te s — a c e ta te an d p ro p io n a te — a p p e a re d n o t to be i n ­
c o rp o ra te d in to f a tty a c id s o f h y d r o c a r b o n s , th e re b y s im p lify in g th e
m e ta b o lis m . S in c e th is s y s te m s y n th e s iz e d h y d ro c a rb o n s n a tu ra lly ,
w ith no a r tif i c ia l co m p o u n d s a d d e d , i t w a s th o u g h t to b e p re fe ra b le to
th e in h ib ite d s y s t e m s .
P o te n tia l A reas for F u rth e r R e s e a rc h
O ne a re a fo r e x te n s io n o f th is p r o je c t c a n be th e id e n tif ic a tio n o f
th e p rim ary b lo c k o f f a tty a c id s y n th e s is w h ic h w a s fo u n d in te n e r a l
c o c k r o a c h e s . T his b lo c k m ay h a v e b e e n in th e fo rm atio n o f a c e t y l c o e n z y m e A, a lth o u g h th is is u n lik e ly b e c a u s e o f th e n e e d for th is
so
com pound in m any m e ta b o lic p a th w a y s .. It i s p o s s ib le th a t fix a tio n o f
c a rb o n d io x id e in th e fo rm atio n o f m a lo n y l-c o e n z y m e A w a s b lo c k e d , or
th a t o n e o f th e e n z y m e s o f f a tty a c id s y n th e s is w a s in h ib ite d . The
b lo c k w a s p o s s ib ly th e sam e one w h ic h a ls o b lo c k e d p ro p io n a te in c o r­
p o ra tio n in to h y d r o c a r b o n s . It a p p e a rs th a t no b ra n c h e d h y d ro c a rb o n
s y n th e s is from p ro p io n a te o c c u rre d d u rin g th e te n e ta l p e r io d , s u g g e s tin g
th a t c o n tin u o u s p ro d u c tio n o f th e s e c o m p o u n d s w a s n o t n e c e s s a r y d u r­
in g th is ti m e .
-
H o ld g a te an d S e a l (16) in d ic a te d th a t th e ra te o f s y n th e s is o f
c u tic u la r lip id s w a s h ig h e r in te n e r a l T. m o lito r th a n in o ld e r i n s e c t s .
In th e p r e s e n t s t u d i e s , no d iff e r e n c e w a s found in P. a m e r ic a n a , in d i­
c a tin g th a t h y d ro c a rb o n s y n th e s is h a d o c c u rre d p rio r to th e im a g in a l
m o lt. A s tu d y o f h y d ro c a rb o n b io s y n th e s is d u rin g th e p r e - e c d y s ia l
p e r io d , w h en s y n th e tic r a te s m ay be h ig h e r , th e re fo re c o u ld c o n trib u te
in fo rm a tio n n e c e s s a r y for a b e tte r u n d e rs ta n d in g o f th e m e ta b o lic p a th ­
w a y s in v o lv e d .
,
In sum m ary, th e p r e s e n t r e s e a r c h h a s sh o w n th a t som e h y d ro c a r­
bon b io s y n th e s is in a d u lt P e rip la n e ta a m e ric a n a o c c u rre d c o n s ta n tly ,
a lth o u g h d iff e r e n t m e c h a n is m s w e re p o s s ib le in te n e r a l a n d o ld e r c o c k ­
r o a c h e s . F a tty a c id s y n th e s is from a c e ta te d id n o t o c c u r a p p re c ia b ly in
th e te n e r a l a d u lt. I t a p p e a re d th a t h y d ro c a rb o n s y n th e s is o c c u rre d v ia
a p a th w a y in w h ic h f a tty a c id s w e re c o n d e n s e d , w ith th e p o s s ib il ity
th a t a n e lo n g a tio n - d e c a r b o x y la tio n m e c h a n ism m ay a ls o h a v e e x is te d in
I
51
f u lly - ta n n e d a d u l t s . In e x p e rim e n ts w ith i n h i b i t o r s , lip id m e ta b o lism
w a s n o t a lte r e d s u f f ic ie n tly to w a rra n t c o n tin u e d s tu d y ; h o w e v e r, o n e o f
th e d e s ir e d e f f e c ts — e lim in a tio n o f th e a c e ta te e f f e c t — w a s o b s e rv e d to
o c c u r d u rin g th e te n e r a l p e rio d .
/
C
SUMMARY
The i n s e c t e p ic u .tic le g e n e r a lly c o n ta in s lip id s w h ic h a c t a s a
b a r r ie r to w a te r lo s s an d in tr u s io n o f h arm fu l c h e m i c a ls . E lec tro n
m ic ro s c o p y s tu d ie s h a v e sh o w n th a t th e se , lip id m a te r ia ls a re p ro b a b ly
form ed in or n e a r th e h y p o d e rm is an d a re tra n s p o rte d to th e s u rfa c e
th ro u g h lip o p h ilic c h a n n e ls in "w a x c a n a ls " .
D uring th e lif e o f th e A m erican c o c k r o a c h , P e rip la n e ta a m e r ic a n a ,
s e v e r a l e v e n ts o c c u r w h ic h h a v e a b e a rin g on th e d e v e lo p m e n t o f th e
c u t i c l e . E ach i n s e c t grow s from h a tc h in g to a d u lt b y m e an s o f e c d y s i s ,
Or m o ltin g , w h ic h o c c u rs s e v e r a l tim e s d u rin g th e lif e c y c le . In th is
p r o c e s s , th e c u tic le , is s h e d , th e i n s e c t g r o w s , and. f in a lly th e new
c u tic le ta n s an d h a r d e n s . A dult P. a m e ric a n a h a v e th e a d d itio n a l fu n c ­
tio n o f r e p ro d u c tio n , a n d th e fe m a le is r e s p o n s ib le a ls o fo r p ro d u c tio n
o f th e c u tic le o f th e o o t h e c a , o r eg g p o d . A fter a b o u t tw o m o n th s , th e
e x te r n a l g e n ita lia o f a d u lt fe m a le s w e re o b s e rv e d to a tro p h y , an d th e
fe m a le w a s no lo n g e r c o n c e rn e d w ith fo rm a tio n o f a c u tic le o th e r th a n
her ow n.
In P. a m e r ic a n a , a lm o s t a ll o f th e s u rfa c e lip id s a re p a ra ffin
h y d ro c a rb o n s .. T here a re th re e m ajo r h y d ro c a rb o n s — n - p e n ta c o s a n e ,
3 - m e th y lp e n ta c o s a n e , c i s , c i s - 6 ,9 - h e p ta c o s a d ie n e — a n d s e v e r a l m inor
c o m p o n en ts'. T h e se c a n be s e p a r a te d c h ro m a to g ra p h ic a lly in to four
fr a c tio n s : norm al s a t u r a t e d , b ra n c h e d s a t u r a t e d , n o rm al m o n o e n e s , an d
n o rm al d i e n e s .
53
Two p a th w a y s o f h y d ro c a rb o n s y n th e s is h a v e b e e n s u g g e s te d in
th e lit e r a t u r e . H e a d - to - h e a d c o n d e n s a tio n o f f a tty a c id s i s sh o w n in
F igure 4 . The e lo n g a tio n - d e c a r b o x y la tio n p a th w a y i s o u tlin e d in F ig ­
u re 6 .
S e v e ra l la b e le d f a tty a c id an d o th e r p o s s ib le p re c u rs o r s u b s tr a te s
w e re in je c te d in to r o a c h e s a t v a rio u s a g e s a f te r th e im a g in a l e c d y s i s .
A fter 18 h r , e a c h ro a c h w a s p la c e d in h e x a n e for e x tr a c tio n o f s u rfa c e
h y d r o c a r b o n s , w h ic h w e re s e p a r a te d in to th e four f r a c tio n s m e n tio n e d
p re v io u s ly an d c o u n te d to d e te rm in e th e p a tte rn o f in c o rp o ra tio n o f
l a b e l . R e s u lts o f th e s e e x p e rim e n ts a re sh o w n in T a b le III.
A c e ta te w a s in c o rp o ra te d in to h y d ro c a rb o n s to a fa r g re a te r e x te n t
in f u lly - ta n n e d a d u lts th a n in te n e r a l a d u l t s . T his s u g g e s te d th a t an y
h y d ro c a rb o n s y n th e s is d u rin g th e te n e r a l p e rio d c o u ld n o t h a v e b e e n by
e lo n g a tio n o f f a tty a c i d s .
H o w e v e r, s e v e r a l o f th e f a tty a c id s u b ­
s t r a t e s w e re in c o rp o ra te d in to h y d ro c a rb o n s , in d ic a tin g th a t h y d ro c a rb o n ■
s y n th e s is h a d o c c u r r e d . T h e se r e s u lts s u g g e s t th a t a c o n d e n s a tio n
p a th w a y w a s a c tiv e d u rin g th e te n e r a l p e rio d .
In c o rp o ra tio n o f th e c a rb o x y l c a rb o n o f lin o le a te in to d ie n e s w as'
p ro n o u n c e d in f u lly - ta n n e d a d u l t s , b u t no in c o rp o ra tio n o f th is c a rb o x y l
c a rb o n w a s o b s e rv e d in te n e r a l a d u l t s . T his is c o n s i s t e n t w ith th e p ro ­
p o s e d c o n d e n s a tio n p a th w a y in te n e r a l r o a c h e s , s in c e o n e o f th e c a r ­
b o x y l g ro u p s o f c o n d e n s in g f a tty a c id s m u s t b e l o s t in s u c h a p a th w a y .
In o ld e r a d u l t s , in c o rp o ra tio n o f th is c a rb o x y l c a rb o n m ig h t be
54
a c c o m p lis h e d by a p a r tia l e lo n g a tio n - c o n d e n s a tio n p a th w a y , th e
e lo n g a tio n - d e c a r b o x y la tio n p a th w a y , o r p -o x id a tio n to form a c e ta te
u n its w h ic h a re s u b s e q u e n tly u s e d in f a tty a c id s y n t h e s i s .
F o rm atio n o f b ra n c h e d h y d ro c a rb o n s w a s in v e s tig a te d b y th e p a t­
te rn o f in c o rp o ra tio n o f th re e p o s s ib le p re c u rs o r s : p r o p io n a te , is o l e u ­
c in e , a n d th e S-m e th y I group o f m e th io n in e . In c o rp o ra tio n o f p ro p io n a te
in ta n n e d , b u t n o t t e n e r a l , a d u lts w a s o b s e r v e d . N e ith e r is o le u c in e n o r
m e th io n in e w a s in c o rp o ra te d in to b ra n c h e d h y d ro c a rb o n s . T his s u g g e s ts
th e m e c h a n ism sh o w n in F ig u re 3 fo r in c o rp o ra tio n o f a m e th y l b ra n c h .
It w a s d e te rm in e d a ls o th a t n e w ly -m o lte d , o r t e n e r a l , a d u lts h a d
a b o u t th e sam e am o u n t o f s u rfa c e h y d ro c a rb o n a s ta n n e d a d u l t s . T his
s e e m e d to in d ic a te th a t no h ig h e r r a te o f h y d ro c a rb o n s y n th e s is
o c c u rre d in te n e r a l ro a c h e s an d th a t m o st h y d ro c a rb o n s y n th e s is fo r th e
n e w c u tic le p ro b a b ly o c c u rre d b e fo re e c d y s i s .
An in v e s tig a tio n o f in c o rp o ra tio n o f s u b s tr a te s in to in te rn a l lip id
f r a c tio n s r e v e a le d th a t lo w e r p ro p o rtio n s o f a c e ta te a n d p ro p io n a te w e re
in c o rp o ra te d in to h y d ro c a rb o n s in te n e r a l a d u lts th a n in o ld e r a d u lt
c o c k r o a c h e s . T his fu rth e r in d ic a te d a la c k o f h y d ro c a rb o n s y n th e s is
from v e ry s m a ll s u b s tr a te s d u rin g th e p e rio d im m e d ia te ly fo llo w in g
e c d y s is .
In c o n c lu s io n , i t a p p e a rs th a t lip id b io s y n th e s is c h a n g e s d u rin g
th e a d u lt lif e o f P. a m e r ic a n a . The to ta l am p u n t of h y d ro c a rb o n is th e
sam e in te n e r a l a n d o ld e r a d u l t s , b u t th e p a tte rn o f in c o rp o ra tio n o f
55
s u b s tr a te s i s d if f e r e n t. M o st h y d ro c a rb o n s y n th e s is p ro b a b ly o c c u rs
p rio r to e c d y s i s . H e a d - to - h e a d c o n d e n s a tio n o f f a tty a c id s is p ro b a b ly
r e s p o n s ib le for h y d ro c a rb o n b io s y n th e s is d u rin g th e te n e r a l p e rio d ,
w h ile o th e r m e ta b o lic p a th w a y s m ay c o n trib u te to th e s y n th e s is in o ld e r
a d u lt c o c k r o a c h e s . P ro p io n a te s e e m s to b e th e s o u rc e o f th e m eth y l
s i d e - c h a i n in b ra n c h e d h y d ro c a rb o n s .
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APPENDIX
61
TABLE A -I
In co rp o ra tio n o f L a b eled . S u b s tr a te s in to N orm al S atu rated
H ydrocarbons- in A dult M a le P. a m erica n a
S u b s tr a te
14
2 :0 - 1 - C .
14
3 :0 - 1 - C
8: 0 - I - 14C
14
1 0 :0 -1 - C
A dult tim e
D a ta
0
t
15
16
t
t
1-4 wk
35
35
2+ mo
38
116
77
t
t
t
1-4 wk
100
100
. 2+ mo
15
57
27
33
t
t
t
1-4 wk
20
20
- 2+ mo
83
62
73
12
14
13
42
42
32
20 .
26
o.
0
0
1-4 wk
2+ mo
'
Average
62
TABLE A -I (c o n tin u e d
S u b stra te
A dult tim e
14
1 2 :0 -1 - C
0
D a ta
t
t
78
78
59
26
43
t
t
1-4 wk
82
82
2+ mo'
16
42
29
10
11
11
1-4 wk
23
23
2+ mo
16
32
24
t
t
t
1-4 wk
t
t
2+ mo
93
77
85
64
32
48
1-4 wk
t
t
2+ mo
35
14
25
35
35
1-4 wk
t
t
2+ mo
41
41
1-4 wk
2+ mo
14
1 4 :0 -1 - C
14
1 6 :0 -1 - C
16:0-IO-14C
18:0 - I - 14C
18:0 -I S -14C
A verage
0
0
0
0
0
.
63
TABLE A -I (c o n tin u e d )
S u b s tr a te
A dult tim e
D a ta
18:2- I - 14C
0
10
10
1-4 wk
t
t
2+ mo
28
27
28
Ile u - U L - 14C
0
- 1-4 wk
t
t
16
A verage
t
16
64
TABLE A -II
In co rp o ra tio n o f L a b ele d S u b s tr a te s in to N orm al S atu rated
H yd rocarb on s in A dult F em a le R a m erica n a
S u b s tr a te
"""1 I
•“H
I
O
C\]
14
3 :0 - 1 - C
14
8 :0 - 1 - C
Cf
I
ID
—
O*
O
14
1 0 :0 -1 - C
A dult tim e
D a ta
A verage
0
19
19
1-4 wk
66
66
2+ mo
53
53 '
0
t
. t
t
1-4 wk
28
28
2+ mo
18
18
0
11
11
1-4 wk
59
59
2+ mo
128
20
74
22
16
. 44
27
1-4 wk
40
40
2+ mo
24
68
46
24
28
19
24
1-4 wk
81
81
2+ mo
42
61
52
0
0
/
65
TABLE A -II (co n tin u ed )
S u b s tr a te
A dult tim e
D a ta
Average
14:0 - I - 14C
0
. 15
12
21
16
1-4 wk
60
60
2+ mo
60 '
42
51
16
14
15
15
1-4 wk
19
19
2+ mo
18
14
16
0
24
24
1-4 wk
131
131
2+ mo
85
85
0
58
58
1-4 wk
34
34
2+ mo
23
22
23
0
28
28
1-4 wk
19
19
2+ mo
68
68
0
' .t
t
t
1-4 wk
t
t
2+ mo
30
30
1-4 wk
t
t
14
1 6 :0 -1 -0 '
0
f
* I— '
I— I
CO
O
I
I— ‘
1 6 : 0 - 1 6 - 4C
14
18 :0 -1 8 - C
'
18:2-1-14C
M e t - C H 3-14C
66
TABLE A -III
In co rp o ra tio n o f L a b ele d S u b str a te s in to Branched S a tu ra ted H ydro­
ca rb o n s in A dult M ale. P. a m erlca n a
S u b s tr a te
A dult tim e
14
2 :0 - 1 - C
0
t
58
43
62
3
54
26
46
36
t
t
t
127
81
73
54
113
.9 0
62
234
119
• 138
t
t
t
1-4 wk
20
20
2+ mo
70
39
55
t
t
t
1-4 wk
22
22
2+ mo
21
85
53
2+ mo
0
1-4 wk
2+ mo
8: 0- I - 14C
10:0 - I - 14C
A verage
t
t
t
t
1-4 wk
3 : 0 - I - 14C
D a ta
v
0
0
67
TABLE AmIII (co n tin u ed )
S u b stra te_______________A dult tim e_________________D ata
14
1 2 :0 -1 - C
14:0 - I - 14C
14
1 6 :0 -1 - C ■
0
16
16
1-4 wk
25
25
2+ mo
36
. 18
27
t
t
1-4 wk
1,6
16
. 2+ mo
12
13,
13
t
t
t
18
10
30
19
t
16
t
13
20
17
35
31
33
33
33
29
t
15
36
58
47
14
18
16
t
t
t
0
0
1-4 wk
2+ mo
16:0-IG-14C
0
1-4 wk
2+ mo
18:0 - I - 14C
A verage
0
1-4 wk
2+ mo
68
TABLE A -III (c o n tin u e d )
S u b s tr a te
18: O-IS-14C
A dult tim e
0
t
15
19
17
t
14
t
0
t
t
1-4 wk
t
t
2+ mo
13
15
. 14
t
t
t
29
29
2+ mo
Ile u - U L - 14C
A verage
t
1-4 wk
14
1 8 :2 -1 - C
D a ta
0
1-4 wk
69
TABLE A-IV
In co rp o ra tio n o f L a b e le d S u b s tr a te s in to B ranched Satu rated
H yd rocarb on s in A dult F em a le P. a m erica n a
S u b s tr a te ______________ A dult tim e________________D a ta __________ A verage
14
2 :0 - 1 - C
0
1-4 wk
2+ mo
SrO -I-14C
0
72
' 71
79
74
39
39
t
t '
t
85
2+ mo
56
56
0
16
. 16
1-4 wk
"78
44
44
54
55
38
23
31
t
t
t
t
55
. 58
45
26
46
18
42
30
2+ mo
10:0 - I - 14C
t
120
50
1-4 wk
8: 0 - 1 - 4C
t
0
1-4 wk
2+ mo
70
TABLE A-IV (co n tin u ed )
S u b s tr a te
A dult tim e
12: O-I-14C
0
D a ta
1-4 wk
2+ mo
14
1 4 :0 -1 - C
; 0
1-4 wk
2+ mb
0
16:0 - I - 14C
,
•'
1-4 wk
'
*
2+ mo
16:0-IG-14C
A verage
t
t
t
t
56
57
78
67
65
24
29
27
t
t
t
t
43
43
42
43
15
25
20
t
t
• 13
. t
19
19
23
17
19
t
16'
t
0
19
19
1-4 wk
22
39
25
29
33
33
2+ mo
71
TABLE A-IV (c o n tin u e d )
S u b s tr a te
14
1 8 :0 -1 - u
A dult tim e
D a ta
A verage
0
36
36
1-4 wk
t
19
14
t
t
t
t
t
0
12
12
1-4 wk
t
18
19
12
t
15
t
t
t
t
16
15
15
15
t
12
2+ mo
21
21
1-4 wk
t
' t
2+ mo
14
1 8 :0 -1 8 -0
2+ mo
14
1 8 :2 -1 -0
0
1-4 wk
14
M e t - C H 3- C
72
TABLE A-V
In co rp o ra tio n o f L a b ele d S u b s tr a te s in to M o n o en e
H yd rocarb on s in A dult M a le P. a m erica n a
S u b s t r a t e ________ ;_____ A dult tim e________________ D a ta ___________ A verage
14
2 :0 - 1 - C
0
1-4 wk
2+ mo
3 : 0 - I - 14C
0
1-4 wk
2+ mo
8 :0 - I - 14C
0
1-4 wk
2+ mo
t
t
t
11
.
t
72
56
56
7124
28
25
47
12
19
16
C
t
t
t
t
t
t
t
t
t
t
t
11
17
74
64
54
27
18
36
19
t
42
28
73
TABLE A-V (co n tin u ed )
S u b stra te
A dult tim e
14
1 0 :0 -1 - C
0
1-4 wk
2+ mo
14
12: 0 -1 - C
0
1-4 wk
2+ mo
14:0 - I - 14C
0
1-4 wk
2+ mo
14
1 6 :0 -1 - C
0
1-4 wk
2+ mo
D a ta
A verage
t
-t
t
14
14
14
10
13
16
. 11
14
t
23
20
20
16 ■
t
~
20
27
17
22
t
t
35
28
46
48
24
36
19
10
15
t
t
t
68
15
28
40 '
27
36
12
22
17
74
TABLE A-V (co n tin u ed )
S u b s tr a te
14
1 6 :0 -1 6 - C
A dult tim e
D a ta
A verage
0
12
31
22
49
32
40
66
39
45
30
20
25
19
27
23
1-4 wk
2+ mo
14
1 8 :0 -1 - C
0
1-4 wk
2+ mo
18:0 -I S -14C
0
• • 1-4 wk
2+ mo
34
24
15
15
32
30
t
14
25
t
13
52
53
59
42
38
55
32
47
t
13
t
75
TABLE A-V (co n tin u ed )
S u b s tr a te
A dult tim e
14
1 8 :2 -1 - C
0
1-4 wk
2+ mo
I l e u - U L - 4C
0
1-4 wk
D a ta
A verage
t
t
t
11
12
18
10
t
t
t
t
t
t
t
t
76
TABLE A-VI
In corp oration , o f L a b ele d S u b s tr a te s in to M o n o en e H yd rocarb ons
in A dult F em a le P. a'm ericana
CO
O
i
L-
S u b s tr a te
A dult tim e
D a ta
A verage
0
12
12 .
1-4 wk
32
28
28
29
12
12
t
t
.t
t
t
t
t
t
t
t
0
29
29
1-4 wk
20
13
. 12
15
15
11
13
. 12
17
16
23
19
39
38
20
32
t
16
t
2+ mo
14
3 :0 - 1 - C
0
1-4 wk
2+ mo
SrO-I-14C
. 2+ mo
IOrO-I-14C
0
1-4 wk
2+ mo
77
TABLE A-VI (co n tin u ed )
S u b stra te
A dult tim e
D a ta
A verage
14
1 2 :0 -1 - C
0
26
21
12
20
■18'
39
50
63
.43
16
. 14
15
t
t
t
t
64
39
63
14
45
15
15
15
1-4 wk
2+ mo
14
1 4 :0 -1 - C
0
1-4 wk
2+ mo
14
1 6 :0 -1 - C
0
t
16
16
11
12
14
t
t
t
0
24
24
1-4 wk
33
25
34
31
1-4 wk
2+ mo
16: O-IG-14C
t
t '
10
2+ mo
23
.
23
78
TABLE A-VI (co n tin u ed )
S u b stra te
A dult tim e
D a ta
A verage
14
1 8 :0 -1 - C
0
29
29
1-4 wk
12
21
t
11
t
13
t
. t
11
t
37
39
38
t
12
t
t
11
t
15
. 16
16
t
12
2+ mo
11
11
1-4 wk
t
t
2+ mo
18:0 -I S -14C
0
1-4 wk
2+ mo
14
1 8 :2 -1 - C
0
1-4 wk
M e t - C H 3-
79
TABLE A-VII
In co rp o ra tio n o f L a b ele d S u b s tr a te s in to D ie n e H ydrocarb ons
in A dult M a le P. a m erica n a
S u b s tr a te
A dult tim e
2; O -I-14C
0
1-4 wk
2+
3: O -I-14C
mo
0
1-4 wk
2+ mo
D a ta
________ A verage
23
38.
t
27
22
121
no
. 76
150
68
120
149
113
82
236
159
t
t
t
t
t
t
t
t
t
t
t
t
t
80
TABLE A-VII (co n tin u ed )
S u b stra te
A dult tim e
D a ta
A verage
14
8 :0 - 1 - C
0
36
15
26
165
.61
58
49
35
37
68
96
87
92
1-4 wk
-
2+ mo
10; O-I-14C
41
19
1-4 wk
64
53
39
49
26
46
41
28
35
0
15
15
1-4 wk
37
26
51
42
32
38
48
29
39'
,
2+ mo..
14
1 2 ;0 -1 - C
2+ mo
O
CO
o
.
81
TABLE A-VII (co n tin u ed )
S u b s tr a te
A dult tim e
14:0 - I - 14C
0
1-4 wk
2+ mo
14
1 6 :0 -1 - C
0
1-4 wk
2+ mo
16; O-IG-14C
0
1-4 wk
2+ mo
D a ta
t
" A verage
t
27
46
65
49
47
10
19
15
20
28
24
39
52
46
24
22
37
17
24
21
17
21
19
31
92
59
74
64
33 ■
19
26
82
TABLE A-VII (co n tin u ed )
S u b stra te
14
1 8 :0 -1 - C
A dult tim e_________________D ata____________A verage
0
41
39
40
11
t
t
t
33
40
34
17
16
23
20
66
66
t
12
21
10
41 •
48 -
22
23
35
29
0
12
12
1-4 wk
18
29
47
31
1-4 wk
2+ mo
1 8 :0 -1 8 -
14
C
0
1-4 wk
2+ mo
18:2- I - 14C
2+ mo
14
Ile u -U L -C
0
1-4 wk
68
74
'
7-1
43
67
55
19
19
83
TABLE A-VIII
I n c o rp o ra tio n o f L a b e le d S u b s t r a t e s in to D ie n e H y d ro c a rb o n s in
A dult F e m a le P. a m e r ic a n a
S u b s tr a te
14
2 :0 - 1 - C
A dult tim e_______________ D a ta __________ A verage
0
30
30
169
192
203
188
132
132
t
t
t
t
t
t
t
t
t
t
0
47
47
1-4 wk
142
129
97
208
144
80
39
60
76
29
46
50
1-4 wk
2+ mo
SrO-I-l4C
0
1-4 wk
2+ mo
14
8 :0 - 1 - C
2+ mo
10:0 - I - 14C
0
1-4 wk
2+ mo
107
155
99
71
41
83
■
108
62
84
TABLE A-VIII (co n tin u ed )
S u b stra te
14
1 2 :0 -1 - C
A dult tim e_____ ___________ D ata____________A verage
75
49
37
54
189
60
64
79
98
80
130
105
35
66
185
95
61
60
39
53
. 68
59
64
74
18
25
39
49
70
35
60
53
15
33
24
0
. 19-
19
1-4 wk
79
68
62
70
46
46
0
1-4 wk
2+ mo
14
1 4 :0 -1 - C
o'
1-4 wk -
2+ mo
14
1 6 :0 -1 - C
0
1-4 wk
2+ mo
14
1 6 :0 -1 6 - C
2+ mo
85
TABLE A-VIH (co n tin u ed )
S u b stra te
14
1 8 :0 -1 - C
A dult tim e_________________D ata______
0
36
36
1-4 wk
26
47
30
48
38
30
34
32
0
44
44
„1-4 wk
33
35
57
42
2+ mo
56
56
0
t
19
t
96
93
65
85
. 37
75
2+ mo
136
136
1-4 wk
t
t
2+ mo
14
1 8 :0 - 1 8 - 1
14
1 8 :2 -1 - C
l-4 w k
M e t - C H 3-
A verage
86
TABLE A-IX .
W e ig h ts o f H y d ro c a rb o n s O b ta in e d from th e S u rfa c e o f T en eral
1
.
a n d 2 - M o n th A dult P. a m e r ic a n a
A v e ra g e s w e re r e p o r te d in Table II.
Males
Age o f R o a c h e s
T e n e ra l
D a ta
A v erage
Females
D a ta
A verage
1.56 mg
1.2 5 mg
. 1.3 5
1.66
1.61
1.15
1.18
1.39 mg
1.40 mg
2 -M o n th
1.40 mg
1.70 mg
1.38
1.59
1.04
1.65
1.2 7 mg
1.65 mg
3 1762
V