Studies on a growth factor in lettuce, Lactuca sativa var. romana Hort., required by the grasshoppers
Melanoplus bilituratus (Wlk.) and M. bivittatus (Say)
by Joseph Bernard Kreasky
A THESIS Submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree
of Doctor of Philosophy in Entomology at Montana State College
Montana State University
© Copyright by Joseph Bernard Kreasky (1960)
Abstract:
The grasshoppers Melanoplus bilituratus (Wlk.) and M. bivittatus (Say) require a water soluble factor
in lettuce, Lactuca sativa var. romana Hort0. for nymphal growth. Manipulation of water extracts of
dried lettuce showed that the active factor is heat stable, dialyzable, adsorbed on Norite A, and is
cationic. The acid soluble nucleotides of lettuce, synthetic nucleic acid derivatives, and the
ninhydrin-positive components of a fractionated, aqueous lettuce extract all failed to satisfy the need
for the lettuce factor. The active component in lettuce is probably different from a factor in corn leaves
which is required for growth of the European corn borer, Pyrausta nubilalis.
The nutritional needs of the two species of grasshoppers used in this study apparently differ from those
of their relative, the desert locust, Schistocerca gregaria.
STUDIES ON A GROWTH FACTOR IN LETTUCE, LACTUCA SATIVA
VAR. ROMANIA HORT. , REQUIRED BY THE GRASSHOPPERS
MELMOPLUS BILITURATUS ( WLK.) AND M. BIVITTATUS (SAY)
Ly
JOSEPH B. KREASKY
A THESIS
Submitted to th e Graduate F a c u l t y
in
p a r t i a l f u l f i l l m e n t of t h e requiremehth
f o r th e degree of
Doctor of Philosophy in Entomology
at
Montana S t a t e Co llege
Approved:
Head, ^ j o r ' D e p ^ ^ ^ e n t ^ "
Deah0 Graduate D i v l i ion
Bozeman, Montana
May, i960
2
TABLE OF CONTENTS
Page
A b s t r a c t -----------.------- --------------------------------------------------------------------------------
4
I n t r o d u c t i o n ---------:------------------- —------------------------ i.-----------------------------------
5
Acknowledgments— ----- -----------------------------:-------------------- ---------------------------
Il
Method------:------------ --------------- --------------------- ;-------------------:-------------------:-------
11
Rearing the t e s t i n s e c t s - ---------------- --------------------- -------- ------------ .—-
11
The b a s i c d i e t -------- :— -------------------------------------------------------------------
12
P r e p a r a t i o n of b o i l e d w a te r e x t r a c t s of d r i e d l e t t u c e -----------------
13
Treatment w it h d i a l y s i s ------------------------------------------------------------------
15
Treatment w it h N o r i t e A— -------------------------------- -----------------------------
15
Treatment w it h Dowex 50 and Dowex I ------- ------ ------------------ .---------—
16
The S e m i - p u r i f i e d e x t r a c t ——.--- ;------------------------ ------- -— ----------------
16
Chromatographic d e t e r m i n a t i o n of p u r i n e and pyri m id in e b a s e s ——
17
Chromatographic s e p a r a t i o n of n i n h y d r i n - p o s i t i v e compounds---------
18
S y n t h e t i c n u c l e i c a c i d d e r i v a t i v e s - - --------------.------- .------------------------
19
Acid s o l u b l e n u c l e o t i d e s of f r e s h l e t t u c e -------------------------------------
19
Treatment w it h p a r t i a l l y d e a c t i v a t e d c h a r c o a l s --------------,---------.—
19
Treatment of t h e b o i l e d w a te r e x t r a c t w it h a c i d ---------------------------
20
R e s u l t s ------------------------ :----------------------------------------------------------------------------
21
Growth re s p o n se s to unmodified and m odif ie d w a te r e x t r a c t s of
. ...
/
d r i e d l e t t u c e ------------------------------------------ ----------------- :----------------------
21
Unmodified b o i l e d w a t e r e x t r a c t s and ashed l e t t u c e --------------
21
D i a l y z a b l e and n o n d i a l y z a b l e f r a c t i o n s —--------------.----------------
23
N o r i t e A f i l t r a t e s and e l u a t e s --------------- 1----- ,—— — :-----------
23
3
Dowex 50 and Dowex I e f f l u e n t s ---------—:--------------------- ----------- -
25
N i n h y d r i h - p o s i t i y e components---------- ------------—=----------- ------------
28
E f f e c t of a c i d on th e b o i l e d w a te r e x t r a c t —.------------------------
30
P a r t i a l l y d e a c t i v a t e d c har co a l e l u a t e s ---------.----- ------------------
32
Growth re s p o n se s to n u c l e i c a c id d e r i v a t i v e s ----------------- ---------------
32
S y n t h e t i c n u c l e i c a c id d e r i v a t i v e s ----------------------------------------
32
Nu c le ic a c i d d e r i v a t i v e s of l e t t u c e --------------------------------------
34
D is cu ssi on and C o n c l u si o n s — .--------------------------- -------------------------------------
40
L i t e r a t u r e C i t e d ------------------------------ ------------------- .------------------------------------
49
Appendix
57
4
ABSTRACT
The g r a s s h o p p e rs Melanoplus b i l i t u r a t u s (Wlk.) and M„ b i v i t t a t u s
(Say) r e q u i r e a w a te r s o l u b l e f a c t o r in l e t t u c e , Lactuca s a t i v a v a r.
romana H o r t 0. f o r nymphal growth. Manipulation of w at er e x t r a c t s of
d r i e d l e t t u c e showed t h a t th e a c t i v e f a c t o r i s heat s t a b l e , d i a l y z a b l e ,
adsorbed on N o r i t e A, and i s c a t i o n i c . The a c i d s o l u b l e n u c l e o t i d e s of
l e t t u c e , s y n t h e t i c n u c l e i c a c i d d e r i v a t i v e s , and th e n i n h y d r i n - p o s i t i v e
components of a f r a c t i o n a t e d , aqueous l e t t u c e e x t r a c t a l l f a i l e d to
s a t i s f y the need f o r the l e t t u c e f a c t o r . The a c t i v e component in
l e t t u c e i s probably d i f f e r e n t from a f a c t o r in corn le av e s which i s
r e q u i r e d f o r growth of t h e European corn b o r e r , Py r au s ta n u b i l a l i s .
The n u t r i t i o n a l needs of th e two s p e c i e s of g ra s sh o p p e rs used in t h i s
stu dy a p p a r e n t l y d i f f e r from th o s e of t h e i r r e l a t i v e , the d e s e r t l o c u s t ,
S c h i s t o c e r c a q Jregaria .
5
INTRODUCTION
In i t s b r o a d e s t usage the term "growth f a c t o r " might be consi de red
as any exogenous substanceGs)-,- i d e n t i f i e d o r u n i d e n t i f i e d , which i s n e c e s ­
s a r y f o r , o r has a f a v o r a b l e e f fecit upon, growth of an organism under
s p e c i f i e d ex p er im en t al c o n d i t i o n s .
In a d d i t i o n , a growth f a c t o r i s
u s u a l l y thought of as an a c c e s s o r y s u b st a n c e to the s o - c a l l e d main com­
ponents of food, I . e . , fats-, p r o t e i n s , c a r b o h y d r a t e s , m in e r a l s a l t s ,
and w a t e r .
While i n v e s t i g a t i n g th e n u t r i t i o n a l re qu ire me nts of two
s p e c i e s of g r a s s h o p p e r s . - Melanoplus b i l i t u r a t u s (Walker) and M. b i v i t t a t u s (Say), a f a c t o r which i s p r e s e n t i n an aqueous l e t t u c e e x t r a c t
was found n e c e s s a r y f o r growth of nymphs fed an a r t i f i c i a l d i e t .
The
purpose of t h i s study i s to p r o v i d e - i n f o r m a t i o n on th e p r o p e r t i e s of
t h i s growth f a c t o r .
Woods (1953) d i v i d e s growth f a c t o r s i n t o two gro ups:
s ub st a nc es
of u l t i m a t e c a t a l y t i c function-; and" s u b s ta n c e s t h a t a r e i n c o r p o r a t e d
into cell constituents.
A c o n s i d e r a t i o n of th e c a t a l y t i c f u n c t i o n of a
growth f a c t o r would i n c l u d e - t h e p o s s i b i l i t y of i t s r o l e as a coenzyme or
as a component of a coenzyme.
Ne ilands and Stumpf (1958) have a r b i t r a r i l y s e p a r a t e d t h e coenzymes
i n t o the hy dro g e n -c ar ry in g and th e g r o u p - c a r r y i n g ty p e s.
Examples of the
h y d ro g e n -c a r ry in g coenzymes and t h e i r v it a m in m o i e t i e s a r e t h e p y r i d i n e
n u c l e o t i d e s , f l a v i n n u c l e o t i d e s , and l i p o i c a c i d .
Diphosphopyridine
n u c l e o t i d e and t r i p h o s p h o p y r i d i n e n u c l e o t i d e f u n c t i o n as hydrogen c a r ­
r i e r s by means of t h e i r n i c o t i n a m i d e m o i e t i e s .
Pullman (1953) has
6
p r e s e n t e d ev idence to show t h a t a r e v e r s i b l e re d u c t i o n occurs a t the para
p o s i t i o n of t h e pyridine^Ti^c^eTi? and^’Loewus e t - a l „
t h i s r e d u c t i o n i s s t e r e o s pec ifi e r.
(1956) have shown t h a t
The f l a v i n n u c l e o t i d e s , of which r i b o ­
f l a v i n ( v it a m in Bg) i s a component, a re b e l i e v e d to t r a n s p o r t hydrogen by
means of a r e v e r s i b l e - r e d u c t i o n of t h e i s o a l l o x a z i n e n u c le u s .
S in ge r and
Kearney (1950) demonstrrateti" t h a t s ever a I i s o a l l o x a z i n e d e r i v a t i v e s are
c ap ab le of c a t a l y z i n g t h e t r a n s f e r of hydrogen from th e reduced p y r i d i n e
n u c l e o t i d e s to- cytoehrome"e.
A more r e c e n t a d d i t i o n to t h e l i s t of
h y d r o g e n - c a r r y i n g 1 Coen-Zymes i s l i p o i c a c i d .
Reduction of t h e s u l f u r
atoms a f t e r openings o f t h e " e y e Tfc d f s u l f i d e - r i n g may account f o r the
a c t i v i t y of t h i s v it a m in .
I t s p a r t i c i p a t i o n in the o x i d a t i o n of pyruv at e
in S s c h e r l c h i a -coii- has been r e p o r t e d (Hager and Gunsalus, 1953).
. Among"the gro up- c a r r y r n g coenzymes t h e r o l e of th e adenos ine phos­
p h a t e s and t h e tram sp ho sp hory las es i s r e c ogniz e d in the s y n t h e s i s of
high energy p h o s p h a te bonds.
The a den os ine pyrophosphate compounds may
then -be*"Used^iw’pWsphoryl'atioTi^r e a e t i ows @S'weiT as in many o t h e r b i o ­
l o g i c a i v' p r o ce s s e s."' Besi d e s th e adenosine pyrophosphates, r e s e a r c h during
t h e p a s t decade"has i m p l i c a t e d o t h e r n u c l e i c a c i d d e r i v a t i v e s as p a r t i c i ­
p a n ts in enzymatic r e a c t i o n s .
U r id in e d ip hos ph at e g lu c o se has been shown
to p a r t i c i p a t e ' a s a coenzyme in t h e c onver sio n of g g l a c t o s e - l - p h o s p h a t e to
gIu c o s e - 1 - p hOs g h a t e - (Cauutto e t aI . . 1950); c y t i d i n e dip h o s p h at e c h ol in e
f u n c t i o n s in tire p ro c e ss of i n c o r p o r a t i n g " phos p h o ry ic h o li n e i n t o l e c i t h i n
(Kennedy and Weiss, 1956); and guanosine t r i p h o s p h a t e i s r e q u i r e d to phosp h o r y l a t e a deno si ne d ip ho s pha te ( Sanadi and Ayengar, 1954; Sanadi e t a l . .
7
1956).
A number of vit a m in s B a r e components of g r o u p - c a r r y i n g coenzymes.
Thiamine (v it a m in B^) i s known to a c t as a coenzyme in d e c a r b o x y la ti o n
r e a c t i o n s Such as th o s e mediated by py ru v ic a c i d oxida se, a - k e t o g l u t a r i c
o x id a s e , and t r a n s k e t o l a s e .
C o enzyme
Lohman and S c h u s te r (1937) showed t h a t t h i s
i s thiamin e pyrophos phate ( c o c a r b o x y l a s e ) .
The d is c o v e r y t h a t
p a n t o t h e n i c a c i d ( v it a m in Bg) is. a. component of coenzyme A led to the
e l u c i d a t i o n of th e f u n c t i o n of t h i s v i t a m i n .
Coenzyme A i s concerned
w it h th e t r a n s f e r of a c e t y l groups-by means of the a l t e r n a t e a c e t y l a t i o n
and d e a c e t y l a t i o n of t h e termi-naT s u l f h y d r y l groups ( Lynen, 1953) „
Pyri-
d o x a l- 5 - p h o s p h a te , a r e l a t i v e of p y r i d o x i n e ( v it am in B&), i s r e q u i r e d as
a coenzyme in dec a rb o x y la ti o n ,- t r a n s a m i n a t i o n , and r a c e m iz a ti o n of amino
a c i d s ( S n e l l , 1952).
F o l i c Oeidvknown a l s o as p t e r o y l g l u t a m i c ac id ,
p a r t i c i p a t e s in the t r a n s f e r of th e formimino group (S ager et. a L , , 1956;
M i l l e r and Waelsch, 1956)„
O th e r o r g a n ic compounds such as b i o t i n , g l u t a t h i o n e , vita mi n Bi2°
a s c o r b i c a c i d and the f a t s o l u b l e - v i t a m i n s have impor tan t p h y s i o l o g i c a l
m a n i f e s t a t i o n s , but thei-r s t a t u s as co enzymes i s ye t to be made c l e a r .
I t i s p r o b a b le t h a t many of t h e s e compounds w i l l be i d e n t i f i e d as cbenzymes or components of coenzymes in th e f u t u r e .
Besides th e r o l e of o rg a n ic compounds i n c a t a l y t i c p r o c e s s e s , t r a c e
elements a r e known to be concerned w it h many enzymatic r e a c t i o n s .
Ir on,
copper, z i n c , magnesium, manganese, molybdenum, c o b a l t , pota ssi um , and
calcium a r e c o n ta i n e d in v a r i o u s enzymes or f u n c t i o n as enzyme a c t i v a t o r s .
8
A rev ie w of t ha -metal enzymes- has- been -p r e s e n t e d by Williams (1953)»
The second group a f " g r o w t h f a c t o r s , th o s e which u l t i m a t e l y become
p a r t of t h e main c e l l c o n s t i t u e n t s , could concei va bly i n c l u d e a whole
ho s t of v a r i o u s compounds.
A few examples might in c l u d e amino a c i d s ,
p e p t i d e s , f a t t y a c i d s , and- ctrol-ines
Chattaway- e t a l . (1944) and Chat-
taway dt a_l. (1949) f o u n d - t h a t a s e r i n e - g l y c i n e - g l u t a m i c a c i d p e p t i d e
had growth promoting a c t i v i t y f o r Corynebacterium diptetheriae. S t r e p t o ­
coccus f a e c a l i s R0, and L a c t o b a c i l l u s e a s e l .
I t has been sug ges te d by
C h e l d e l i n (1954) t h a t th e need f o r p e p t i d e s may be the r e s u l t of poor
a s s i m i l a t i o n of th e component amino a c i d s or th e i n a b i l i t y on th e p a r t
of th e organism to c o n ju g a t e s p e c i f i c amino a c i d s .
The e s s e n t i a l f a t t y
a c i d s , l i n o l e i c a c i d , l i n o l e n i c a c id , and a r a c h i d o n i c a c i d , a r e a t l e a s t
in p a r t concerned w it h s t r u c t u r a l t i s s u e s .
A l k a l i n e h y d r o l y s i s of t i s s u e
g i v e s a g r e a t e r y i e l d of t h e s e a c i d s than does e x h a u s ti v e e x t r a c t i o n with
s o l v e n t s ( Rieckehof f et. aj.. „ 1949; R e i s e r , 1950) „
The n u t r i t i o n a l l y
im po rt an t compound c h o l i n e i s known to be i n c o r p o r a t e d i n t o S t r u c t u r a l
t i s s u e s and to p ro vid e a s ourc e of l a b i l e methyl groups f o r t r a n s m e t h y l ­
a t i o n r e a c t i o n s (Wendell and Nyc0 1954).
Because of the many d i f f e r e n t compounds t h a t could be a c t i v e b i o ­
l o g i c a l l y , i t i s e v i d e n t t h a t a growth s t i m u l u s imparted by an unknown
s u b s t a n c e could be due to any of a l a r g e a r r a y of bio c h e m ic a ls .
However,
from the o u t l i n e j u s t p r e s e n t e d , the v i t a m i n s , n u c l e i c a c i d d e r i v a t i v e s ,
t r a c e m e t a l s , amino a c i d s , p e p t i d e s , f a t t y a c i d s , and so f o r t h a r e l i k e l y
groups of which a growth f a c t o r could be a member.
9
I n v e s t i g a t i o n s of th e b a s i c n u t r i t i o n a l re qu ire me nts of i n s e c t s
have produced d a ta concerning the need f o r growth f a c t o r s by s e v e r a l
insects.
The b e e t l e Tene bri o m o l i t o r r e q u i r e s a s u b s ta n c e f o r l a r v a l
growth t h a t i s w a te r s o l u b l e ; i n s o l u b l e in f a t s o l v e n t s , he a t s t a b l e ,
a c i d r e s i s t a n t , and not adsorbed on c h a r c o a l ( F r a e n k e l , 1951a; 1951b),
This f a c t o r was shown to be c a r n i t i n e , a r e l a t i v e of c h o l i n e ( C a r t e r
et. elL , 1952) „
The European corn b o r e r , P y r a u s ta nubi l a l i s , grows
b e s t when a d i a l y z a b l e and heat s t a b l e f a c t o r in corn i s added to the
d i e t (Beck, 1953)„
An unknown f a c t o r o r f a c t o r s a f f e c t emergence of
the A s i a t i c r i c e b o r e r , Chilo s u p p r e s s a l i s . from i t s pupal case ( I s h i i ,
1956) and, r e c e n t l y , i t was. found t h a t i n o s i t o l would s a t i s f y the need
f o r a growth f a c t o r f o r t h e b o l l w e ev il , Anthohomous q r a n d i s ( Vanderz a n t , 1959).
Most of th e l i t e r a t u r e t h a t d e a l s w i t h g r a s s h o p p e r n u t r i t i o n i s
concerned w it h th e e f f e c t of ho s t p l a n t s on growth and egg pro d u c ti o n
(Hodge, 1933a; Chauvin, 1939; Sanderson, 1939; T a u b e r e t ^ a l . . , 1945;
B r e t t , 1947; P f a d t , 1949; Smith e t ' a l . .
P i c k f o r d , 1958; Smith, 1959).
1952; Barnes and Nerney, 1953;
Only a few workers have conducted s t u d i e s
de signed to p ro vid e i n f o r m a t i o n on th e chemical n a t u r e of th o s e sub­
s t a n c e s p r e s e n t i n ho s t p l a n t s which a r e r e l a t e d to t h e n u t r i t i o n a l needs
Of the g ra s s h o p p e r .
Hodge ( 1933b) r e p o r t e d t h a t when wheat le av e s a re
b o i l e d f o r 10 minutes some s u b s ta n c e n e c e s s a r y f o r growth of M. d i f f e r e n t i a l i s i s destroyed.
M. b i v i t t a t u s i s unab le to absorb c ar bohy drate
polymers from i t s food but r e a d i l y a s s i m i l a t e s monosaccharides (Brown,
10
1937)o
Smith and N o r t h c o t t (1951) m a i n t a i n t h a t t h e r e i s a p o s i t i v e
c o r r e l a t i o n between an i n c r e a s e i n n i t r o g e n c o nt en t of a wheat host and
g r e a t e r v i t a l i t y of M. mexicanus ( b i l i t u r a t u s ) as measured by s u r v i v a l ,
growth r a t e , and egg p r o d u c t i o n , w hil e wheat having a high phosphorous
c o n te n t l e s s e n s v i t a l i t y of t h i s s p e c i e s (Smith, 1960)„
One worker has
succeeded i n r e a r i n g S c h i s t o c e r c a q r e q a r i a on a s e m i - s y n t h e t i c d i e t and
has i n d i c a t e d t h a t v ita m in A ( o r a d e r i v a t i v e ) and a s c o r b i c a c i d are
r e q u i r e d f o r growth (Dadd, 1957)„
For g e n e r a l i n f o r m a t i o n c onc ernin g th e n u t r i t i o n a l needs of the
phytophagous, i n s e c t s th e r e a d e r i s r e f e r r e d to th e review by F r ie nd
(1958).
O th e r im po r ta nt reviews a re th o s e by Levinson (1955), Lipke and
F r a e n k e l (1956), Tr ag e r (1953), and Wigglesworth (1950).
P r e l i m i n a r y work in t h i s study c o n s i s t e d of t e s t i n g t h r e e a r t i ­
f i c i a l d i e t s as, p o s s i b l e s t a r t i n g p o i n t s in an i n v e s t i g a t i o n of the
b a s i c n u t r i t i o n a l needs of th e g r a s s h o p p e r s p e c i e s p r e v i o u s l y mentioned.
One of th e d i e t s used was ch em ic al ly d e f i n e d and has been used s u c c e s s ­
f u l l y f o r t e a r i n g th e onion maggot, Hy-Iemva a n t i qua ( F r i e n d and Pa tt o n ,
1956).
The second was a s e m i - s y n t h e t i c d i e t which i s a deq ua te f o r growth
of t h e pink bollwo-rm, Pe c ti n o o h o ra q o s s y p i e l l a (Vanderzant and R ei se r,
1956b).
The t h i r d , a l s o a s e m i - s y n t h e t i c d i e t , was desi gne d to support
growth of a wide v a r i e t y of organisms (Luckey, 1954).
d i e t s ( s e e Appendix
All of th e s e
f o r compositions) f a i l e d to su pport growth of
t h e g r a s s h o p p e r nymphs beyond the second m ol t.
I t was l e a r n e d , however,
t h a t growth to adulthood can be obta in e d i f th e pink boI !worm d i e t i s
11
supplemented w it h a w a t e r s o l u b l e f r a c t i o n of d r i e d l e t t u c e .
95 p e r c e n t e t h a n o l e x t r a c t s of l e t t u c e proved i n e f f e c t i v e .
E th er and
As a r e s u l t
of t h e s e p r e l i m i n a r y f i n d i n g s - , - t h e problem of e l u c i d a t i n g some p r o p e r ­
t i e s of the a c t i v e l e t t u c e component evolved.
ACKNOWLEDGMENTS
A p p re c i a ti o n i s extended to th e fo ll o w i n g pe rs on nel a t Montana
S t a t e C o ll e g e :
D rs . J-. E 0' Gander and D0 J 0 Reed, Department iof Chemistry,
f o r h e l p f u l s u g g e s t i o n s and t e c h n i c a l a s s i s t a n c e ; Drs0 J 0 H. Pepper, E0 B0
Harvey, Department-' of" Zoology and Entomology.,,, and K0 G oe ri n g , Department
of Chemistry, f o r a i d ’ i n 1the' p r e p a r a t i o n of t h e m a n u s c r i p t 0 Acknowledg­
ment i s a l s o made t o - Mrr F. T. Cowan, In Charge, Grasshopper I n v e s t i g a ­
t i o n s , U. S. Department of A g r i c u l t u r e , Bozeman, Montana, who suggested
t h a t a s tu d y be made of t h e n u t r i t i o n a l req u ir e m e n ts of g r a s s h o p p e r s .
In
c o o p e r a t i o n w i t h Montana S t a t e C o ll e g e ; the"work r e p o r t e d h e r e i n was done
w hi le I was a" member o f t h e s t a f f " o f th e above-mentioned l a b o r a t o r y .
METHOD
Rearing t h e t e s t i n s e c t s .
The’ egg pods o f two s p e c i e s of g r a s s h o p p e r s , Melanoplus b i l i t u r a t u s ..
(Walker) and M. b i v i t t a t u s ( S a y ) , were c o l l e c t e d a t s i t e s in Lake and Teton
c o u n ti e s , Mont a n a , and w e st e rn Oklahoma i n t h e autumn of 1955, 1956, 1957,
and 1958.
The pods were mixed w it h mois t s o i l , brought i n t o th e l a b o r a ­
t o r y , and s t o r e d in lo o s e l y - c a p p e d Mason j a r s a t 4°C0
When needed f o r
u s e t h e d e s i r e d number of pods was removed from the r e f r i g e r a t o r and
p la c e d on mo is t b l o t t i n g p a p er in a p e t r i d i s h .
The pods were in c ubat ed
12
a t a c o n s t a n t te m p e r a t u r e of SO0C0 u n t i l h a t c h i n g o c c u r r e d , which u s u a l l y
r e q u i r e d a week to 10 days.
Each newly ha tched nymph was removed from
t h e p e t r i d i s h as soon as p o s s i b l e a f t e r h a tc h i n g and p la c e d i n a clean
g l a s s tube ( 6 ” X I " ) ,
The r e a r i n g tu b e s were s to ppe r ed a t each end with
ga uz e -c oy er ed c o t t o n p l u g s .
These nymphs were r e a r e d a t a c o n s t a n t
te m p e r a t u r e of SOoC0 and i n co nti nuous l i g h t .
Ex perim ental d i e t s were pre p a re d as a g a r g e l s and c o n s i s t e d of a
b a s i c d i e t (Table I) to which was added aqueous l e t t u c e f r a c t i o n s or
S y n t h e t i c compounds.
P o r t i o n s of t h e a u t o c l a y e d d i e t to be fe d were
t r a n s f e r r e d from a 300 ml. Erlenmyer f l a s k to a p e t r i d i s h t y s t e r i l e
technique.
I n d i v i d u a l f e e d i n g s were made by t r a n s f e r r i n g s m al l p o r t i o n s
of t h e d i e t from th e - p e t r i d i s h to th e r e a r i n g tubes once each 24 h o u r s .
Care was taken to m a i n t a i n s t e r i l i t y in th e main r e s e r v o i r o f food.
The c r i t e r i o n u s e c h f o r t h e e f f e c t i v e n e s s of a d i e t i n promoting
growth was t h e number of mo lts ach ieved by t h e nymphs fe d e x c l u s i v e l y
on t h e t e s t d i e t .
E i t h e r f i v e o r s i x m ol ts occu r from egg to a d u l t in
t h e s e s p e c i e s ( S h o t w e l l , 1941);
The d u r a t i o n of the s t a d i a between
mo lts v a r i e d w i d e l y and depended On th e composition of th e medium being
fed.
Approximately seven days were r e q u i r e d f o r each stadium when th e
nymphs were fed a d i e t c ap a bl e of producing a d u l t s .
The l e n g t h of any
one t e s t ranged from t h r e e to s ix 'w e e k s and depended on th e number of
molts a chi ev ed by t h e nymphs Of a p a r t i c u l a r t e s t group.
The b a s i c d i e t .
The d i e t shown i n Table I w i l l be r e f e r r e d to th rou ghout t h i s stud y
13
as t h e b a s i c d i e t and was used as th e base to which v a r i o u s a d d i t i o n s were
made.
This d i e t was p a t t e r n e d a f t e r t h a t used by Vanderzaut and R e i s e r
(1956b) w it h c e r t a i n m o d i f i c a t i o n s .
A sc orb ic a c i d was added as an a n t i ­
ox id a n t and b r e w e r ' s y e a s t was s u b s t i t u t e d f o r the v it a m in S o l u t i o n used
in th e o r i g i n a l d i e t .
Fanderzanf^and R e i s e r used 2 M pota ssi um hydroxide
to d i s s o l v e th e c a s e i n ; bu t i n t h e s e experiments t h i s was p m i t t e d because
t h e d i s s o l v e d p r o t e i n tended to p r e c i p i t a t e a f t e r a u t o c l a v i n g .
Likewise,
sodium a l g i n a t e , a component o f the^ o r i g i n a l d i e t , was o m it te d because i t
caused th e a g a r medium to b o i l o u t . o f th e f l a s k s during a u t o c l a v i n g .
The
f i n a l w a te r c o n te n t was a d j u s t e d to 83 p e r c e n t .
The b a s i c d i e t was p r e p a r e d by d i s s o l v i n g the s u c r o s e in a p o r t i o n
of th e w a te r and adding t h i s s o l u t i o n to th e r e s t of th e i n g r e d i e n t s .
Agar was excluded a t t h i s s t a g e .
in a Waring Blendbr.
The m ix tu r e was then tho ro ughl y blended
A f t e r p l a n in g up to 190 grams of th e blended mix­
t u r e in 300 ml. Erlenmyer f l a s k s , a gar and e xperim ent al a d d i t i v e s were
in t r o d u c e d i n t o t h e i n d i v i d u a l f l a s k s .
The m ix tu r es were then a u to ­
cl ave d a t 20 pounds p r e s s u r e 1 f o r 20 m in ute s .
An even d i s t r i b u t i o n of
i n g r e d i e n t s was i n s u r e d 1by s w i r l i n g the f l a s k s during th e c a bli ng p e r io d .
S t e r i l e media were r e f r i g e r a t e d u n t i l needed.
'
P r e p a r a t i o n of b o i l e d w a t e r - e x t r a c t s of d r i e d l e t t u c e .
Boile d w a te r e x t r a c t s of d r i e d l e t t u c e were pre p a re d by adding one
l i t e r of d i s t i l l e d w a te r to 12, 24, or 36 grams of l e t t u c e powder and
b o i l i n g g e n t l y f o r one hour.
L e tt u c e powder was o b ta in e d by p u l v e r i z i n g
d r i e d romaine l e t t u c e , Lactuca s a t i v a v a r . romana H o r t . . in a Waring
14
Tabl e I 0
Composition o f t h e B asic D i e t
Gms
Inaredient
Gms i ■
Casein
5 .0
I -Cyst i n e
0.1
Choline c h l o r i d e
0 .1
Glycine
0 .1 5
C e l l u l o s e (powdered)
4.0
Sucrose
8.0
Bre w er' s yeaist
1.5
Corn o i l
1.0
A sco rbi c a c id
0.5
Chplesterol
0.3
Agar
4.0
Tntrredieh t
-
Wesson's s a l t s
Water
.
0.7
125.0
15
Blendor.
The m ix tu r e was b o i l e d and then f i l t e r e d through s e v e r a l l a y ­
e r s of cheese c l o t h .
The-pulp was- d i s c a r d e d .
A second f i l t r a t i o n
through a double l a y e r of muslin was used to remove the more f i n e l y d i v ­
ided p a r t i c l e s , and the tT i l t r a t e w a s - c o n c e n tr a te d by g e n t l e b o i l i n g in
th e open a i r .
P r e c i p i t a t e s t h a t formed during the p e r i o d of b o i l i n g
were removed by f r e q u e n t f i l t r a t i o n .
The f i n a l f i l t r a t e was ev aporated
to 25 ml. before- a d d i t i o n t o t h e b a s i c d i e t .
Treatment w it h d i a l y s i s .
The w a te r e x t r a c t was- c o n c e n t r a t e d to 75 ml. by h e a t i n g and
p la ce d in a tube-made of c e l l u l o s e casing (V-isking. C o ., Chicago, 1 11.) .
The d i a l y s i s t u b e and"a m a g n e t i c - s t i r r i n g . rod were p la ce d in two l i t e r s
of d i s t i l l e d w a t e r ' i n an Erlenmyer f l a s k .
A f t e r a u t o c l a v i n g a t 20 Ibis,
p r e s s u r e f o r - 20 mi nute s, th e flask- was p la c e d on a Cole-Parmer "Magne=
s t i r " and t h e w a te r was a g i t a t e d f o r 15 hoursi
Both d i a l y z a b l e and
n o n d i a l y z a b i e - f r a c t i o n s were reduced to 25 ml. by g e n t l e b o i l i n g in the
open a i r b e f o r e i n c l u s i o n in' t h e - b a s i c d i e t .
Treatment w it h lN o r i t e A.
The d i a l y z a b l e f r a c t i o n o f the b o i l e d w ater e x t r a c t was s t i r r e d
w i t h N o r i t e A a t pH 6 .5 u n t i l c om ple te ly d e c o l o r i z e d .
This u s u a l l y
r e q u i r e d from 8 t o 10 grams o f N o r i t e A to t r e a t an e x t r a c t d e ri v e d
from 24 grams of d r i e d l e t t u c e .
A f t e r f i l t r a t i o n , the c h a r c o a l was
s t i r r e d w it h an e l u a n t c o n s i s t i n g of 300 ml. o f e i t h e r 50 p e r c e n t e t h ­
anol o r 50 p e r c e n t e th a n o l p lu s 2 p e r c e n t c o n c e n t r a t e d ammonium
hydrox ide .
A filtrate,
an e th a n o l e-luate, and an ammoniacal e th an ol
16
e l u a t e of N o r i t e .'A were p r e p a r e d using two- s e p a r a t e b o i l e d w a te r e x t r a c t s ,
each d e r i v e d from 24 grams o-f d r i e d l e t t u c e .
The r e s u l t i n g f i l t r a t e and
e l u a t e s were c o n c e n t r a t e d - t o 2-5 m-T. over an open flame b e f o r e i n c l u s i o n
in t h e b a s i c d i e t s
Treatment w it h Dowex 50-a nd Dowex I .
The ammoniaca} e t h a n o l eluate- of N o r i t e A was t r e a t e d w it h Dowex I
(ap anion exchanger in the-^chlo-rider-form) and Dowex 50 ( a c a t i o n exchan­
g e r in th e hydrogen fo r m ).
G lass-tubing-(diameter:
9 / l 6 i n . ) was
packed w it h th e re s in s- to p r o v i d e - a bed s i x inc hes long.
The et han ol
and ammonia were removed-f rom three -amito nia cal e th an ol e l u a t e s b y h e a t ­
ing.
One e l u a t e was passe d through-a-column of Dowex I , t h e second e l u ­
a t e was pass ed through a column of" Dowex- 5 0 ,"and the t h i r d e l u a t e was
t r e a t e d wit h both exchange-"resins. ~ Because th e Dowex I became exhausted
duri ng pa ssa ge of the- e l u a t e , i t was r e p l a c e d as o f t e n as needed.
Exhaustion of t h i s r e s i n was"noted when t h e e n t i r e r e s i n bed became
discolored.
Replacement of th e Dowex-50 r e s i n was u nnece ssa ry as i n d i ­
c a te d by th e con tin ue d dis pla cem en t o f hydrogen ions by th e e l u a t e .
Flow r a t e s were maintained" as r a p i d as t h e p h y s i c a l n a t u r e of the r e s i n s
would p e r m i t .
The Dowex-I e f f l u e n t was c o n c e n t r a t e d over an open flame
b e f o r e a d d i t i o n to t h e b a s i c medium.
The Dowex 50 e f f l u e n t , which con­
t a i n e d d i s p l a c e d hydrogen i o n s , was c o n c e n t r a t e d under vacuum and then
a d j u s t e d to pH 6 . 5 w it h pota ssi um hydroxide.
The s e m i - p u r i f i e d e x t r a c t .
The Dowex I e f f l u e n t , j u s t d e s c r i b e d , was d e s ig n a te d as th e semi-
17
p u r i f i e d e x t r a c t and w i l l be r e f e r r e d to by t h a t term in subsequent
e xp er im en ts .
A r e c a p i t u l a t i o n of th e ev en ts le a d i n g to the s e m i - p u r i f i e d
e x t r a c t i s as f o ll o w s ;
1.
E x t r a c t i o n of 24 grams of d r i e d l e t t u c e with b o i l i n g wa ter.
2.
D i a l y s i s of t h e waiter f i l t r a t e .
3.
D e c o l o r i z a t i o n of th e d i a l y z a b l e f r a c t i o n of t h e f i l t r a t e
j
w it h N o r i t e A.
4.
E l u t i o n of t h e N o r i t e A with 50 p e r c e n t e th a n o l p l u s 2 p e rc en t
c o n c e n t r a t e d ammonium hydroxide.
5.
Treatment of th e ammoniacal e t h a n o l e l u a t e of N o r i t e A with
Dowex I .
Chromatographic d e t e r m i n a t i o n of pu ri n e and pyri m idi ne b a s e s .
Attempts were made to de te r m in e th e pre s en c e of f r e e p u r i n e and
pyr im id in e base s in th e s e m i - p u r i f i e d e x t r a c t by, usi ng t h e chromato­
g r a p h i c method of V is ch e r and Chargaff (1948).
A small amount of the
e x t r a c t was p la ce d on a pap er s t r i p w it h a m i c r o p i p e t t e and th e chrom­
atogram was run w it h b u t a n o l ; e t h a n o l :w a te r (50:15:35) as t h e so lv e n t
system.
For p u r i n e s ;
th e pap er s t r i p was dipped in 0 . 2 5 M me rcuric
n i t r a t e in 0 . 5 N n i t r i c a c i d , bathed in 0 . 5 N n i t r i c a c i d , and sprayed
w it h ammonium s u l f i d e .
For p y r i m i d i n e s :
t h e paper s t r i p was pla ced in
b u f f e r e d 0. 0 1 M m e rc u ri c a c e t a t e s o l u t i o n of pH 6 .2 f o r 30 seconds
( I p a r t 0 . 1 M m e rc uri c a c e t a t e , 3 p a r t s I M sodium a c e t a t e , and 6 p a r t s
w a t e r ) , bath ed f o r e x a c t l y 20 seconds in slowly renewed w a t e r , and
dipped i n ammonium s u l f i d e s o l u t i o n .
The p r e s e n c e of the m e rc u ri c s a l t s
18
of t h e ba se s i s i n d i c a t e d by a bla ck c o l o r fo ll o w i n g th e ammonium s u l f i d e
treatment.
Chromatographic s e p a r a t i o n of n i n h y d r i n - p o s i t i v e compounds.
Gross s e p a r a t i o n s of th e n i n h y d r i n - p o s i t i v e compounds were c a r r i e d
out by means of pa pe r chroma to gr ap hy; . Three b o i l e d w at er e x t r a c t s , each
d e r i v e d from 24 grams of d r i e d l e t t u c e were p re pa re d.
One of the
e x t r a c t s was m odif ied i n - t h e manner s p e c i f i e d f o r th e s e m i - p u r i f i e d
e x tra c t (Norite A step omitted).
The d i a l y z a b l e f r a c t i o n s of th e o t h e r
two e x t r a c t s were passed' through s e p a r a t e columns of Dowex 50.
The
r e s i n columns were eluted" with" 500 nri. of 4 N ammonium hydroxide and the
r e s u l t i n g e l u a t e s were c o n c e n t r a t e d under vacuum.
was added to t h e b a s i c d r eIr dir e c tl y ."
One of th e e l u a t e s
The -s e m i ^ p u r i f i e d e x t r a c t and the
o t h e r Dowex 50- e i u a t e were each a p p l i e d to f o u r s h e e t s of Whatman 3 MM
chromatography paper" (22%" X T8%") by s t r e a k i n g w it h a p i p e t t e .
The
s o l v e n t system .used was b u t a n o l : a c e t i c a c i d : w a te r which was prepa red
by s a t u r a t i n g b u ta n o l w i t h a s o l u t i o n of I p a r t g l a c i a l a c e t i c a c id
and 5 p a r t s w a te r .
The d i r e c t i o n of t h e s o l v e n t flow was descending.
Thin s t r i p s cut from"the c e n t e r and s i d e s of each paper s h e e t were
developed w it h a 0 . 2 p e r c e n t s o l u t i o n o f n in h y d r i n in a c e to n e .
Bands
of n in h y d r i n - p o s i ti v e - c o m p o u n d s were l o c a t e d on the undeveloped areas
by us in g t h e developed s t r i p s as r e f e r e n c e p o i n t s .
The s h e e t s c o n t a i n ­
ing th e s e m i - p u r i f i e d e x t r a c t were cut in h a l f which p r ov id e d low and
high Rf f r a c t i o n s .
The bands on t h e s h e e t s c o n ta i n in g th e Dowex 50
e l u a t e were s e p a r a t e d - b y - p l a c i n g a prominent band having an Rf of .50
19
i n t o one f r a c t i o n and combining the remaining bands i n t o a second f r a c ­
tion.
The p o r t i o n s of t h e - s h e e t s were e l u t e d w it h d i s t i l l e d w at er,
c o n c e n t r a t e d to 25 ml. by h e a t i n g ; and added to the b a s i c d i e t .
S y n t h e t i c n u c l e i c a c i d d e r i v a t i v e s ^,
S y n t h e t i c n u c l e i c a c i d d e r i v a t i v e s were in c lu d e d in th e b a s i c d i e t
e i t h e r i n w a t e r s o lu tio n- ( f a c i l i t a t e d by he ati ng), o r by d i r e c t a d d i t i o n .
These were o b t a i n e d f r o m - t h e - N u t r i t i o n a T Biochemicals C orp o ra ti o n ,
Cl ev ela nd, Ohio.
.Acid s o l u b l e n u c l e o t i d e s o f f r e s h l e t t u c e .
A maximum of the a c i d s u TubTe n u c l e o t i d e s was o b t a i n e d by the
I '
method of B e r g v i s t (1956). A homogenate of 350 girams oil: f r e s h l e t t u c e
;
and 1% l i t e r s of 10 p e r c e n t p e r c h l o r i c a c i d was prepared in a Waring
.
Blendor.
A fter f i l t r a t i o n ,
th e pulp was r e - e x t r a c t e d w it h % l i t e r of
5 p e r c e n t p e r c h l o r i c a c i d . - Both e x t r a c t s were combined ahd t r e a t e d with
10 grams of N o r i t e A.
T h e" ch arco al was then e l u t e d w it h a s o l u t i o n of
25 p e r c e n t e th a n o l and 0*5 p e r c e n t c o n c e n t r a t e d dmmonium hydrox ide .
The
e l u a t e was c o n c e n t r a t e d under vacuum, f i l t e r e d through a S e i t z f i l t e r ,
and added to th e s t e r i l e b a s i c d i e t .
Tr eat me nt with' p a r t i a l l y ' d e a c t i v a t e d c h a r c o a l .
F u r t h e r p u r i f i c a t i o n of th e s e m i - p u r i f i e d e x t r a c t was attem pted by
usi ng t h e method of A sa to or and B a l g l i e s h (1956).
This method i s based
on th e s e l e c t i v e a d s o r p t i o n of a rom at ic s u b s ta n c e s by p a r t i a l l y d e a c t i Vated c h a r c o a l s .
A c co rd in gly , c h ar co a l. w as d e a c t i v a t e d to varying
de gre es by s t i r r i n g , i t w i t h a 1.5 p e r c e n t s o l u t i o n (w/v) of s t e a r i c a cid
20
in e t h a n o l f o r one hour-,.. The m ix tu r e was then d i l u t e d w it h w a te r a t a
r a t e of 9 l i t e r s of w a te r p e r l i t e r of e t h a n o l .
The c h a r c o a l was c o l ­
l e c t e d i n a Buchner f u n n e l , washed w ith w a t e r , and a i r d r i e d .
A series
of 6 10-gram b a tc h e s of c h a r c o a l was t r e a t e d in the above manner.
An
amount of s t e a r i c a c id s o l u t i o n was added to each b a t c h of c h a r c o a l so
t h a t th e s t e a r i c a c i d c o n s t i t u t e d 15, 10, 7» 4, 2, and 0 p e r q e n t of the
weight of t h e c h a r c o a l .
A s e m i - p u r i f i e d e x t r a c t , d e ri v e d from 24 grams
of d r i e d l e t t u c e , was p r e p a r e d and t r e a t e d f i r s t w it h t h e 15 p e r c e n t
deactivated charcoal.
The r e s u l t i n g f i l t r a t e was then t r e a t e d w it h the
10 p e r c e n t d e a c t i v a t e d c h a r c o a l .
This p r o c e s s was r e p e a t e d by t r e a t i n g
each f i l t r a t e w ith th e nex t lower d e a c t i v a t e d c h a r c o a l .
Each of the
c h a r c o a l s was e l u t e d w it h 300 ml. of a 7 . 2 p e r c e n t aqueous phenol e l u a n t .
Phenol was removed by e v a p o r a t i n g th e e l u a t e S to dryness i n a drying oven.
The te m p e r a t u r e i n th e oven was not p e r m i t t e d to exceed 70° C.
The
d r i e d e l u a t e s were d i s s o l v e d i n 25 ml. of d i s t i l l e d w a te r b e f o r e i n c l u ­
s i o n i n th e b a s i c d i e t .
Treatment of th e b o i l e d w a t e r e x t r a c t w i t h a c i d .
To t e s t the e f f e c t of low pH on th e a c t i v i t y of the growth f a c t o r ,
h y d r o c h l o r i c a c i d was used to a d j u s t , an u n f r a c t i o n a t e d , w a t e r e x t r a c t
to pH 2.
The a c i d i f i e d e x t r a c t was p e r m i t t e d to stand' o v e r n i g h t in a
r e f r i g e r a t o r a f t e r which i t was reduced to 25 ml. under vacuum.
The
Co nc e nt ra te d e x t r a c t was then a d j u s t e d t o pH 6 w i t h sodium hydroxide
b e f o r e being added to t h e b a s i c d i e t .
21
RESULTS
GROWTH RESPONSES TO UNMODIFIED MD MODIFIED BOILED WATER EXTRACTS OF
DRIED LETTUCE.
Unmodified b o i l e d w a te r e x t r a c t s and ashed l e t t u c e .
The r e s u l t s of fe e d in g th e ash of d r i e d l e t t u c e and v a r i o u s concen­
t r a t i o n s of b o i l e d w a te r e x t r a c t s of d r i e d l e t t u c e a r e shown in Table I I .
Grasshopper nymphs fe d d i e t s la ck in g l e t t u c e e x t r a c t s f a i l e d to develop
beyond t h e second molt in most i n s t a n c e s .
There was no a p p a r e n t d i f f e r ­
ence between th e stirtiuli imparted by t h e e x t r a c t s d e ri v e d from 12 or 24
grams of l e t t u c e , but t h e e x t r a c t d e ri v e d from 36 gram's of l e t t u c e impart
t e d a weaker Stimulus than did th e s m a l l e r amounts.
One p o s s i b i l i t y f o r
t h i s i n h i b i t i o n of growth i s th e t o x i c e f f e c t of l a r g e c o n c e n t r a t i o n s of
th e r e q u i r e d s u b s ta n c e o r of e xtr a neo us m a t e r i a l s .
Trace elements a r e not t h e cause of th e growth s t i m u l u s , s i n c e
nymphs f e d ashed l e t t u c e f a i l e d to e l i c i t a growth r e s p o n s e .
Nymphs fed d i e t s l a c k i n g l e t t u c e e x t r a c t s appeared to be normal
u n t i l s e v e r a l days a f t e r t h e second m olt.
At t h i s time s i g n s of s t a r v a ­
t i o n appeared and th e nymphs wasted away slowly u n t i l they d i e d .
Diets
c o n t a i n i n g " t h e e x t r a c t s r e s u l t e d in a d u l t s t h a t were s l i g h t l y Smaller
than th o s e r e a r e d on n a t u r a l d i e t s .
Some had deformed wings but appeared
normal o t h e r w i s e .
The growth f a c t o r i s h e a t s t a b l e and r e s i s t s a i r o x i d a t i o n , s i n c e
c o n c e n t r a t i n g t h e e x t r a c t over an open flame and a u t o c l a v i n g does not
inactivate i t .
22
Table I I .
Growth Responses by M. b llitu r a tu s to Unmodified Boiled
Water E xtracts of D ried Lettuce and Ashed, Dried Lettuce.
Added to 150 gms. b a s i c d i e t 9
No--.■i n s e c t s
in t e s t
No. in s ec ts , completing
I - 6 molts
4
5
I -> r;-;
2
3
6b
D i s t i l l e d w a te r
15 .
15
12
I
0
0
0
Water e x t r a c t 12 gms. d r i e d
lettuce
15
15
15
15
15
14
13
do.
24 gms„
do.
15
14
14
14
14
13
13
do.
36 gms.
do.
15
15
14
14
7
4
0
15
15
14
0
0
0
0
Ash 24 gms. d r i e d l e t t u c e
a All a d d i t i o n s reduced o r d i l u t e d to 25 ml.
^The s i x t h molt marked the-appea-rance of th e a d u l t in t h i s s p e c i e s .
23
D i a lv z a b le and n o n d i a l y z a b l e f r a c t i o n s .
The d a ta i n Table I I I were in te n d e d to compare growth res pons e s to
th e low and high m o l e c u l a r weight f r a c t i o n s of the b o i l e d w a t e r e x t r a c t .
D i a l y s i s thr ough c e l l u l o s e c as in g was used to e f f e c t a g r o s s s e p a r a t i o n
of th e smal l and l a r g e m o le c ule s .
Heavy m o r t a l i t y and poor v i t a l i t y of th e newly, hatched nymphs o c c u r ­
r e d du rin g t h i s t e s t .
Such d i f f i c u l t i e s appeared from time to time
duri ng th e course of t h e e xpe ri me nt s.
I t was not uncommon f o r egg pods
c o l l e c t e d a t t h e same s i t e and the same time to giv e r i s e to vigorous
nymphs a t one h a tc h i n g and- to weak nymphs a t a n o th e r .
This could be the
r e s u l t . o f t h e i n n a t e c o n d i t i o n of t h e eggs o r of s t o r a g e c o n d i t i o n s in
th e l a b o r a t o r y .
The f a c t t h a t t h e growth resp onse by th o s e i n s e c t s f e d the u n f r a c ­
t i o n a t e d w a te r e x t r a c t was f a r weaker than t h a t p r e v i o u s l y o b t a i n e d
(T abl e I I ) p r o v id e s a d d i t i o n a l evidence t h a t the c o n d i t i o n of the nymphs
would have r e s u l t e d in poor growth r e g a r d l e s s of d i e t a r y i n c l u s i o n s .
T h e r e f o r e , t h e assumption Was madg t h a t t h e d i a l y z a b l e f r a c t i o n was the
a c t i v e one, s i n c e the r e s p o n s e - t o t h i s f r a c t i o n more n e a r l y p a r a l l e l e d
t h a t of the whole e x t r a c t .
That t h i s assumption was c o r r e c t i s shown
in th e next experiment.
N o r i t e A f i l t r a t e and e l u a t e s .
Table IV shows t h a t d a ta o b ta in e d from feeding th e f i l t r a t e of the
N o r i t e A t r e a t e d d i a l y z a b l e f r a c t i o n of th e w a te r e x t r a c t and two N o r it e
eluates.
Also , th e e f f e c t on growth by th e n o n d i a l y z a b l e f r a c t i o n was
24
Table. I I I .
Growth Responses by M. b ilitu r a tu s to Dialyzed, Boiled
Water E xtracts of Dried L ettuce.
Added to 150 gnrs. b a s i c d i e t 3
Nb. i n s e c t s
in t e s t
No. i n s e c t s completing
1 - 6 molts
I
2
3 . 4
5
6
Water e x t r a c t 12 gms. d r i e d l e t t u c e
10
3
3
2
0
0
0
D ia ly ga bl e f r a c t i o n w a t e r e x t r a c t
10
4
4
3
0
0
0
N on di a ly za bl e f r a c t i o n w a t e r e x t r a c t
10
2
I
0
0
0
0
3All a d d i t i o n s reduced to 25 ml.
25
repeated.
I t i s e v i d e n t from-Table IV t h a t t h e N o r i t e A tr e a t m e n t re n d e rs a
w a t e r e x t r a c t i n e f f e c t i v e i n s t i m u l a t i n g growth of the nymphs.
The 50
p e r c e n t e t h a n o l e l u a n t fa-Ms' to dislodge"-the a c t i v e f a c t o r from the
c h a r c o a l , bu t th e ammoniacaI e t h a n o l e l u a n t a p p a r e n t l y accomplishes
this.
That th e growth promoting f a c t o r i s d i a l y z a b l e i s a l s o confirmed
by t h e d a t a .
The -nondial-yzablBrrfra-c11on imparted a s l i g h t s t i m u l u s to
t h e nymphs which might have b e o n \t h e r e s u l t of incomplete removal of the
a c t i v e s u b s ta n c e during" d i a l y s i s .
Dowex I and Dowex 50 e f f l u e n t s . ■
The e f f e c t of Dowex I (Cl"") and Dowex 50 (H+) exchange r e s i n s on
th e a c t i v e f a c t o r in l e t t u c e i s shown in Table V.
Growth was s l i g h t l y
i n f e r i o r on t h e d i e t c o n t a i n i n g t h e ammpniacal e l u a t e of N o r i t e A
compared w it h t h e same- experiment i n Table IV.
Reduced growth a c t i v i t y
was observed i n t h e e i u a t e r t r e a t e d w it h Dpwex I , but growth was f a r
s u p e r i o r than w i t h t h o s e e l u a t e s t r e a t e d w it h Dowex 50.
The ammoniacal
e t h a n o l e l u a t e - of N o r i t e --A t r e a t e d w it h Dowex I w i l l be r e f e r r e d to ,
h e r e a f t e r , as- t h e s e m i - p u r i f i e d e x t r a c t .
In t h o s e e x t r a c t s which were pass ed through Dowex 5ti columns, the
high a c i d i t y of t h e e f f l u e n t s pre ve nte d t h e a gar media from s o l i d i f y i n g
a f t e r s t e r i l i z a t i o n in t h e a u t o c l a v e .
w i t h sodium hydroxide.
cation.
These d i e t s were a d j u s t e d to pH 6
Add ition of more a g a r then r e s u l t e d in s o l i d i f i ­
Because excess a c i d might have caused chemical changes in the
d i e t , t h i s p a r t of t h e experiment was r e p e a t e d w it h th e f o ll o w i n g
26
Table BF„
Growth Responses by & M litu r a tu s to the B ialyzable Fraction
of Water E x t r a c t s T r e a te d w it h N o r i t e JK, to N o r i t e A E l u a t e s ,
and to th e Non dia lyz ab le F r a c t i o n ,
No, i n S e c t s
in t e s t
Added to ISO gms, b a s i c d i e t
No,, i n s e c t s completing
1 - 6 molts
2
3
4
5
6
I
10
8
4
6
O
O
O
10
6
5
2
O
O
O
D + N + EAc"
10
8
7
.7
7
7
5
NDd
10
7
6
4
2
O
O
E x t r a c t 24 gms. d r i e d l e t t u c e
10
8
8
8
7
6
5
25 ml, d i s t i l l e d w a te r
10
7
3
0
O
O
O
D + Na
-
D + N + Eb
'
a D i a l y z a b l e f r a c t i o n of boTTed= -Water-Oxtr a c t de ri v e d from 24 gms, d ri e d
l e t t u c e t r e a t e d w it h 10-gms0: N o r i t e JV a t pH 6 , 5 , •
^3Sarae as (a) w i t h Norite? JV e l u t e d w it h 300 ml, 50 p e r c e n t e t h a n o l ,
■
c Same as ( a) w it h -Norite- JV- e l u t e d wit h 300 ml, 50 p e r c e n t e t h a n o l plu s
2 p e r c e n t c o n c e n t r a t e d ammonium hydroxide,
^No ndi al yz abl e f r a c t i o n of. b o i l e d w a te r e x t r a c t de ri v e d from 24 gms,
dried le ttu c e .
27
Table V0
Growth Responses by M„ b i l i t u r a t u s to Boiled Water E x t r a c t #
of D r ie d L e t t u c e 1T r e a te d w ith D i a l y s i s , N o r i t e A, Dowex I
and Dowex SO0
... ............. R......... .. ......................., , ,
Added to 150 gms„ b a s i c dr&b
No".' i n s e c t s
in t e s t
.. ......■
,, .
No. i n s e c t s completing
I - 6 mo lts
3
2
4
5
I
6
D + N + EAa
10
6
5
5.
5
4
2
(D + N + BA) + Dowex I
10
6
5
5
5
3
0
(D + N + BA) + Dowex.50"
10
5
3
0
0
0
0
(D + N + BA) + Dowex I & 50
10
5
I
0
0
0
0
a Dialyzed f r a c t i o n Of" w a t e r e x t r a c t d e r i v e d from 24 gms. d r i e d l e t t u c e
t r e a t e d w it h 10 gms, No rit e" A a t pH 6 . 5 ; N o r i t e A e l u t e d with. 300 ml.
50 p e r c e n t e t h a n o l p l u s 2 -pe Tcent-c o n c en tr at e d ammonium hydroxide.
28
Changes0
The e f f l u e n t from t h e Dowex 50 column was c o n c e n t r a t e d under
vaocuum u s i n g a minimum of h e a t and n e u t r a l i z e d with sodium hydroxide
b e f o r e a d d i t i o n to t h e b a s i c d i e t .
The r e s u l t s of t h i s r e t e s t were
e s s e n t i a l l y th e same as w i t h th e a c i d media.
The a c t i v e f a c t o r i s ,
t h e r e f o r e , e i t h e r r e t a i n e d on th e Dowex 50 r e s i n o r i n a c t i v a t e d by i t .
E f f o r t s to e l i c i t a growth re sp onse by i n c o r p o r a t i n g some of the
n i n h y d r i n - p o s i t i y e components of the s e m i - p u r i f i e d e x t r a c t i n t o the
basic d iet f a i l e d . .
Paper chromatography was used with two d i f f e r e n t
s o l v e n t syste ms , phfenol:w a te r (80:20) and b u t a n o l s a t u r a t e d w it h a s o l u ­
t i o n of I p a r t g l a c i a l a c e t i c a c i d and 5 p a r t s w at er.
Since th e d a ta in Table V showed t h a t Dowex 50 re nde re d a w a te r
e x t r a c t i n a c t i v e , i t was hoped t h a t e l u t i o n of t h i s r e s i n would r e s u l t
in re c o v e ry of the a c t i v i t y .
In o r d e r to t e s t the p o s s i b i l i t y t h a t a
n i n h y d r i n - p o s i t i v e compound*is r e s p o n s i b l e f o r growth s t i m u l a t i o n and
i s r e t a i n e d on th e Dowex 50 column, the d i a l y z a b l e f r a c t i o n s of two
b o i l e d w a te r e x t r a c t s , each d e ri v e d from 24 grams of d r i e d l e t t u c e , were
p a ss e d thr oug h s e p a r a t e columns of Dowex 50.
A f t e r each of th e Columns
was e l u t e d w it h 500 ml. of 4 N ammonium hydrox ide , th e e l u a t e s were con­
c e n t r a t e d un d e r vacuum.
One of th e e l u a t e s was chromatogrammed with
b u t a n o l : a c e t i c a c i d : w a te r as the s o l v e n t system; the o t h e r was added to
the basic d ie t in t a c t .
The r e s u l t s a r e shown i n Table VI.
The d a t a i n Table VI show t h a t th e a c t i v e m a t e r i a l was. abse nt from
th e Dowex 50 e l u a t e s , s i n c e th e i n t a c t e l u a t e f a i l e d to p r o v i d e a growth
29
Table VI q Growth Responses-by- # Q b i l i t u r a t u s to some Chromatdgrammed
N i n h y d r i n - P o s i ti v e - Components of l e t t u c e .
'.................. •
.
'
;
Added to 190 gms. b a s i c d i e t
‘
No. i n s e c t s
in t e s t
' ' T........... ' '
V """1
No i n s e c t s completing
I - 6 m ol ts
3
4
5
I
2
6
Low Rf bands Powex 50 e I u a t e a"
10
8
7
O
O
O
O
High Rf bands Dowex 50 e l u a t e
10
10
9
O
O
O
O
I n t a c t DOwex 50 e l u a t e
10
10
10
I
O
O
O
25 ml. d i s t i l l e d w at er
10
9
6
O
O
O
O
aThe a re a covered by t h e n i n h y d r i n - p o s i t i y e bands on th e p a p e r s h e e t s
was cut approximately, in h a l f , thus p ro v id i n g two f r a c t i o n s —low and
high Rf compounds.
30
response.
.An a dve r se e f f e c t on the growth f a c t o r due to t h e n a t u r e of
t h e e l u a n t does nq t seem l i k e l y because ammonium hydroxide in et han ol
was used w it h s u cc e ss to e l u t e N o r i t e A.
I t i s p o s s i b l e t h a t the e l u a n t
was i n e f f e c t i v e i n d i s p l a c i n g th e growth f a c t o r from t h e r e s i n , assuming
t h a t i n a c t i v a t i o n did not occu r.
Since t h e supply of M. b i l i t u r a t u s eggs became d e p l e t e d during the
w i n t e r of 1957-1958, i t was n e c e s s a r y to use th os e of M. b i v i t t a t u s .
It
was determined t h a t t h i s s p e c i e s had th e same require me nts f o r the w at er
s o l u b l e l e t t u c e f a c t o r as Mi b i l i t u r a t u s (T able V II ) .
At t h i s time the
e f f e c t o f th e n i n h y d r i n - r p o s i t i v e compounds i n th e s e m i - p u r i f i e d e x t r a c t
was r e p e a t e d .
The N o r i t e A s t e p in the- p r e p a r a t i o n of th e s e m i - p u r i f i e d
‘. Ie x t r a c t was o m itt e d be cause t h e = e l u t i o n of th e c har co a l might be a s i g ­
n i f i c a n t cause of l o s s of t h e a c t i v e f a c t o r .
The development of r e f e r ­
ence s t r i p s from th e chromatograms- r e v e a l e d an u n u s u a l l y heavy concen­
t r a t i o n of a s u b s t a n c e having an
of .5 0 .
This band was e l u t e d
s e p a r a t e l y ; th e remaining bands were combined.
The n i n h y d r i n - p o S i t i v e compounds agai n f a i l e d to im pa rt a st im ul us
to th e nymphs when M. ■b i v i t t a t u s was used as th e t e s t i n s e c t .
E f f e c t of a c i d on t h e b o l t e d ' w a t e r e x t r a c t .
In view of th e i n a b i l i t y to o b t a i n a s t i m u l u s w it h f r a c t i o n s from
t h e pap er chroma t og rants , t h e - e f f e c t of a c i d on the e x t r a c t was t e s t e d
because of t h e a c i d - n a t u r e o f - t h e s o l v e n t systems.
Also, t h e growth
f a c t o r was s u b j e c t e d t o a c i d c o n d i t i o n s duri ng tr e a tm e n t w i t h Dowex 50.
In a d d i t i o n to t e s t s w it h a c i d , t h e a c t i v i t y qf the s e m i - p u r i f i e d e x t r a c t
31
Table VII.
Growth Responses by M. b i v i t t a t u s to Chromatogrammed
N i o h y d r i n - P o s i t i v e Components of th e Se mi -P urif ed E x t r a c t .
Added to 190 gms. b a s i c d i e t
No. i n s e c t s
in t e s t
No. i n s e c t s completing
........... I 4 6 mb its
I
2
3
4
5
6
.50 R| band
IQ
9
Q
O
O
O
O
Remaining bands
10
9
I
O
O
O
O
Water e x t r a c t 24 gms. d r i e d
lettuce
lo
9
9
9
9
25 ml. d i s t i l l e d w a te r
10
10
8
O
*A e q u a ls a d u l t .
8
4
(4A*)
O O O
The number of molts r e q u i r e d to produce t h e a d u l t in
t h i s s p e c i e s v a r i e d between 5 and 6.
Those nymphs t h a t ach ieved
adu lthoo d a f t e r th e f i f t h molt a re so d e s i g n a t e d .
32
was re -c h e c k e d in o r d e r to be c e r t a i n t h a t p re v io u s r e s u l t s were r e p r o ­
ducible,
These d a t a a r e shown i n Table V I I I .
The d a t a su ppo rt t h e p re v io u s f i n d i n g t h a t a reduced but obvious
s t i m u l u s i s e l i c i t e d by t h e s e m i - p u r i f i e d e x t r a c t and t h e a c t i v e f a c t o r
must, t h e r e f o r e , be p r e s e n t p r i o r to chromatography.
e f f e c t on t h e a c t i v i t y of th e w a te r e x t r a c t .
Low pH has l i t t l e
The p o s s i b i l i t y t h a t the
growth f a c t o r i s l a b i l e to a d s o r p t i o n on pa per would be d i f f i c u l t to
r e c o n c i l e w it h i t s s t a b i l i t y on c h a r c o a l .
P a r t i a l l y deactivated charcoal e lu a t e s .
The p a r t i a l l y d e a c t i v a t e d c h a r c o a l method of Asatoor and D a lg li e sh
(1956) f o r p u r i f i c a t i o n of a ro ma tic s u b s ta n c e s was a l s o u n s u c c e s s f u l in
e f f e c t i n g a f u r t h e r p u r i f i c a t i o n of t h e a c t i v e su b st a n c e i n t h e semipurified extract.
D i f f i c u l t i e s i n e f f e c t i n g f u r t h e r p u r i f i c a t i o n s might be e xpla in ed
by th e p r o g r e s s i v e l o s s of t h e a c t i v e component a f t e r each tr e a tm e n t
u n t i l t h e lower t h r e s h o l d of th e growth s t i m u l u s i s r e a ch e d.
I t wquld
seem a p p r o p r i a t e to i n c r e a s e many time s t h e amount of s t a r t i n g m a t e r i a l ,
but t h i s would n e c e s s i t a t e t h e hand lin g of l a r g e amounts of ex traneous
m aterials.
O th e r methods would have to be devise d r a t h e r than the ones
used h e r e i n .
GROWTH RESPONSES TO NUCLEIC ACID DERIVATIVES.
Synthetic nucleic acid d e riv a tiv e s.
F i g u r e I shows th e r e s u l t s of scanning the s e m i - p u r i f i e d e x t r a c t a t
pH 7 and pH 9 . 5 i n th e u l t r a v i o l e t range.
Twp peaks a r e c l e a r l y ev id en t
33
T able V I I I 0
Growth R espbnses by M. b i v i t t a t u s to th e S e m i-P u rifie d
E x tra c t and an Unfr a c tib n a te d , A c id ifie d Water E x tra c t
o f L e ttu c e .
Added to 190 gms. b a s ic d ie t
No. in s e c ts
in t e s t
No. in s e c ts com pleting
1 - 6 m o lts
2
3
4
5
6
I
A c id ifie d e x t r a c t 8
10
9
9
8
8
6
(4A)
2
S e m i-p u rifie d e x tr a c t
10
9
9
9
7
I
(IA)
0
25 m l. d i s t i l l e d w ater
10
10
5
0
0
0
0
aW ater e x t r a c t o f '2 4 gms. d rie d l e t t u c e reduced to 500 m l ., a c i d i f i e d to
pH 2 w ith h y d ro c h lo ric a c id , allow ed to s ta n d o v e rn ig h t, reduced to
30 m l, under vacuum, a d ju s te d to pH 6<;5 w ith sodium h y d ro x id e.
34
a t pH 7; one occurs a t 258 mu, and th e o th e r a t 263 mu.
Both peaks a re
l o s t a t pH 9 ,5 ,
S ince th e n u c le ic a c id d e r iv a tiv e s a b so rb , g e n e r a lly , in th e re g io n
of 260 mu, and have been im p lic a te d in th e n u t r i t i o n o f ' D ro so p h ila (S c h u ltz
et, a l . , 1946) and th e onion maggot (F rie n d and P a tto n , 1956), th e p o s s i­
b i l i t y was c o n sid e re d th a t th e s e compounds a re p re s e n t in th e s e m i-p u ri­
f ie d e x t r a c t .
The chrom atographic method of V isch e r and C h arg aff (1948)
f o r th e d e te rm in a tio n of f r e e p u rin e and p y rim id in e b a se s re v e a le d th e
absence Of p u rin e s b u t was- in c o n c lu s iv e f o r p y rim id in e s .
The t e s t fo r
p y rim id in e s appeared to be p o s itiv e a t f i r s t b u t a tte m p ts to d u p lic a te
th e t e s t w ere in c o n s i s te n t.
N e v e rth e le s s , a t e s t was conducted in which
v a rio u s p y rim id in e b a se s , p u rin e and p y rim id in e n u c le o s id e s , and
d e o x y rib o n u c le ic a c id were fed in th e b a s ic d i e t .
A ll th e compounds te s t e d f a i l e d to im p art a grow th S tim u lu s as
shown in T able IX.
A nother p y rim id in e , 5-h y d rO x y m eth y lcy to sin e, was a ls o
found to be i n e f f e c t i v e .
N u cleic a c id d e r iv a tiv e s o f l e t t u c e .
When th e s e m i- p u r if ie d e x tr a c t was chromatogramnred Ori p a p er s t r i p s ,
two d i s t i n c t f lu o r e s c e n t a re a s appeared on th e s t r i p s when th ey were
scanned w ith a s h o rt wave u l t r a v i o l e t lamp.
In O rder to d eterm in e th e
e f f e c t of th e s e m a te r ia ls On grow th, th e s e m i-p u rifie d e x t r a c t d eriv e d
from 24 grams of d rie d l e t t u c e was reduced to ap p ro x im ately 10 ml. and
a p p lie d to la r g e s h e e ts of Whatman 3 MM chrom atography p a p e r.
The ascen d ­
in g s o lv e n t system, c o n s is te d o f b u ta rio ls e th a n ik w ater (5 0 :1 5 :3 5 ).
The
x
Absorbance
0.8-
pH 9 .5
2
& iio
Wave Length in m illim ic ro n s
U ltr a v i o l e t Spectrum of th e S e m i-P u rifie d L e ttu c e E x tra c t.
C hem istry, Montana S ta te C o lleg e)
(G. Baker, Dept, of
3'6
T able IX.
Growth Responses by M„ b i l i t u r a t u s * to P y rim id in e s,
N u c leo sid e s, and-"D eoxyribonucleic Acid.
Added to 190 gms. b a s ic d ie t
No. in s e c ts
in t e s t
No. in s e c t s com pleting
1 - 6 m o lts
I
2, .3
4
5
6
I cc. p y rim id in e s o lu tio n 3
10
8
I
0
0
0
0
2 cc.
do.
10
9
2
0
0
0
0
10 c c .
do.
10
10
3
0
0
0
0
DNA p lu s n u c le o s id e s ^
10
10
8
0
0
0
0
W ater e x tr a c t 24 gms. l e t t u c e
10
10
10
10
10
10
9
a Each c c, p y rim id in e s o lu tio n c o n ta in e d in w a te r:
4 mg. each of u r a c i l ,
thym ine, c y to s in e , 5-m e th y lc y to s in e HCl, and o r o ti c a c id .
^200 mg. DNA p lu s 10 mg. each of in o s in e , a d en o sin e , guanO sine, u r id in e ,
and c y tid in e .
^ C o lle c te d in Oklahoma.
37
f lu o r e s c e n t bands were c u t o u t of th e p a p e r, e lu te d w ith d i s t i l l e d
w a te r, and added to th e b a s ic d i e t ,
No grow th s tim u la tio n was observed.
The fo re g o in g d a ta c a s t doubt on th e r o l e of n u c le ic a c id d e riv a ­
ti v e s in s tim u la tin g th e grow th of th p nymphs.
I t would be h e lp fu l i f
t h i s e n t i r e c la s s o f compounds could be e lim in a te d from s u s p ic io n w ith
some degree of c e r t a i n t y .
S ince s y n th e tic n u c le o tid e s were o m itted
from th e t e s t d i e t s , th e method of B e rg v is t (1956) was used to i s o l a t e
th e a c id s o lu b le n u c le o tid e s of fr e s h l e t t u c e .
The r e s u l t s of in c o r ­
p o ra tin g t h i s f r a c tio n in to th e b a s ic d i e t a re shown in T ab le X.
A s l i g h t stim u lu s o c c u rre d in th e d i e t c o n ta in in g th e n u c le o ­
ti d e s compared w ith th e n e g a tiv e c o n tr o l.
While i t cannot be s a id
t h a t th e a c id s o lu b le n u c le o tid e s of l e t t u c e a re i n e f f e c t i v e in stim u ­
l a t i n g grow th, i t seems d o u b tfu l th a t th ey p la y a very s i g n i f i c a n t r o le .
F u rth erm o re, th e r e i s no ev id en ce th a t th e grow th f a c to r i s a n u c le ic
a c id d e r iv a tiv e .
A diagram m atic r e p r e s e n ta tio n of th e r e s u l t s of th e experim ents
i s giv en in F ig u re 2.
38
T able X.
Growth Responses by M0 b i l i t u r a t u s to A cid S o lu b le
N u c le o tid e s of L e ttu c e .
Added to 190 gms. b a s ic d ie t
No. in s e c ts
in t e s t
No. in s e c ts com pleting
1 - 6 m o lts
1
2
3
4
5 6
Acid s o lu b le n u c le o tid e s
10
9
8
4
1
0
0
25 m l. d i s t i l l e d w ater
10
10
5
0
0
0
0
39
I n a c tiv e o r A c tiv ity
Much Reduced
A c tiv e
95% ETHANOL EXTRACT*--------------— DRIED LETTUCE----------- >BOILED WATER EXTRACT
ETHER EXTRACT
HCl to pH2
d ia ly s is
NONDIALYZABLE4
FRACTION
N o rite A
FILTRATE 4
BOILED WATER EXTRACT
d ia ly s is
BOILED WATER EXTRACT
+DIALYZABLE FRACTION
DIALYZABLE FRACTION
50% e th a n o l
50% e th a n o l
ELUATE4
IRITE A
+ ELUAJE
2% cone. NH1OH
EFFLUENT 4
ELUATE f
Dowex 50 (H+)
4 N NHylOH
NINHYDRIN-POS 4
COMPOUNDS
ELUATE
lowex I (C I" )
DOWEX 50 (H+)
EFFLUENT
chrom atography
p a r t i a l l y d e a c tiv a te d
c h a rc o a ls , aq. phenol
ACID SOLUBLE<—p e r c h l o r i c --------FRESH LETTUCE
NUCLEOTIDES
a c id
SYNTHETIC DNA
AND NUCLEOSIDES
F ig u re 2.
Diagrammatic R e p re s e n ta tio n of A c tiv ity of V arious L e ttu c e
F r a c tio n s and Some S y n th e tic Compounds When Added to th e B asic D iet.
40
DISCUSSION .AND CONCLUSIONS
I t i s tem pting to conclude from th e r e s u l t s th a t M0 b i l i t u r a t u s
and M. b i v i t t a t u s nymphs have an a b s o lu te req u irem en t f g r th e growth
f a c t o r in q u e s tio n t in view of th e r a t h e r c o n s is te n t grow th re sp o n se s
dem onstrated by w ild p o p u la tio n s from b o th Montana and Oklahoma.
While
i t i s p o s s ib le th a t t h i s i s th e c a se , i t i s c o n c e iv a b le t h a t p o p u la tiq n s
of th e s e s p e c ie s m ight be encoutitered which vary w idely in t h e i r
re sp o n se s to aqueous l e t t u c e e x t r a c t s .
The e f f e c t s of th e g e n e tic c o n s ti tu tio n on th e m etabolism of
organism s i s t r e a t e d e x te n s iv e ly by Wagner and M itc h e ll (1 9 5 5 ), however,
th e s e a u th o rs a sk :
"But th e q u e stio n a r i s e s :
in th e absence of muta­
tio n and in a c o n s ta n t environm ent, i s i t to be expected th a t th e
m e ta b o lic p ro c e s s e s of a c e l l w i l l rem ain c o n sta n t? " ^
E vidence i s accu­
m u la tin g in su p p o rt of a n e g a tiv e answer to t h i s q u e s tio n .
The e f f e c t s
of crowding on th e p h y siology, of p o p u la tio n s of sm all mammals has been
of i n t e r e s t to s e v e ra l in v e s tig a to r s .,
C h itty (1957) re p o rte d th a t
h y p ertro p h y of th e sp le e n o ccu rs in denise b re e d in g p o p u la tio n s of v o le s
and C h r is tia n (1955) found a- d i r e c t r e l a tio n s h ip between a d re n a l hyper­
tro p h y and in c re a s in g p o p u la tio n d e n s ity .o f m ice.
C lark e (1953) has
su g g e ste d t h a t i h t r a s p e c i f i c s t r i f e i s re s p o n s ib le f o r a re d u c tio n in
f e r t i l i t y and a d e c re a se d b re e d in g season in high p o p u la tio n s of v o le s .
In O ther experim ents w ith m ice, C h r is tia n and Lemunyan <1958) were not
only a b le to show th a t crow ding.produced a d v erse e f f e c t s on T a c ta tin g
fe m a les, b u t a ls o t h a t th e s e e f f e c t s p e r s i s t e d in th e progeny f o r a t
l e a s t two g e n e r a tio n s .
Few experim ents such as th o se j u s t m entioned have been perform ed
u sin g i n s e c t s as t e s t a n im a ls, b u t W ellin g to n (1957) made o b s e rv a tio n s
^Wagner, R .P. and M itc h e ll, H.K.
1955.
John Wiley and Sons, New York, p. 390.
G e n e tic s and M etabolism .
41
on p o p u la tio n s of th e t e n t c a t e r p i l l a r ,- -Malacosoma p l u v i a l e „ and proposed
th a t changes in th e v ig o r o f p o p u la tio n s a re r e f l e c t e d in a v a r ia tio n of
th e ran g e of in d iv id u a l re q u ire m e n ts and re sp o n se s w ith r e s p e c t to e n v i­
ronm ental f a c t o r s .
S tu d ie s on th e m ig ra to ry l o c u s t , LocuSta m iq r a to r ia
m ia r a to r io id e s . have re v e a le d th a t Crowding a d u lt fem ales can reduce
th e ir f e r t i l i t y
(A lb re c h t -et a l . . 1958).
The h y p o th e sis th a t th e p h y s io lo g ic a l s t a t e o f anim al p o p u la tio n s
v a r ie s due to t h e i r own numbers and t h a t t h i s v a r i a b i l i t y , in tu r n , p ro ­
duces changes in re sp o n se s o f th e p o p u la tio n s to th e environm ent is
c u r r e n tly re c e iv in g th e a t t e n t i o n of c e r t a i n groups who a re in t e r e s t e d
in th e dynamics of g ra s sh o p p e r p o p u la tio n s in th e w estern U n ited S ta te s .
2
I f t h i s i s found to be t r u e ; changes in th e n u t r i t i o n a l ; re q u ire m e n ts of
g ra s sh o p p e r p o p u la tio n s could be m a n if e s ta tio n s of a dynamic p h y sio lo g y .
D uring th e c o u rse o f t h i s s tu d y , f i e l d c o lle c te d eggs gave r i s e to
nymphs o f d i f f e r e n t v i t a l i t y (s e e page 23 and T able I I I ) .
W hether th e s e
changes in v i t a l i t y of" th e nymphs can be a t t r i b u t e d to m ix tu re s Of
p h y s io lo g ic a lly d i f f e r e n t eggs i s open to s p e c u la tio n .
The v a r ia b le s to
which th e eggs were s u b je c te d d u rin g s to ra g e make an answ er d i f f i c u l t .
U n fo rtu n a te ly , l i t t l e i s known about th e e f f e c t s of te m p e ra tu re , 2
2
*
R egional G rasshopper R esearch..(.C ooperative R egional P r o je c t W-37, R evised,
Jan u ary I , 1960).
C o operating A g en cies:
Experim ent s t a t i o n s of C olorado,
Montana, Wyoming, C a l if o r n ia , Idaho, A rizo n a, and th e U. S. Department
of A g ric u ltu re .
42
m o is tu re , and gas te n s io n s of th e atm osphere on s to re d eggs.
The s i m i l a r i t y of th e l e t t u c e f a c t o r to a corn le a f f a c t o r which
Beck (1953) found to be n e c e s sa ry f o r optimum grow th of th e European
corn b o re r i s Of p a r t i c u l a r i n t e r e s t .
T able XI shows a com parison of
th e two f a c t o r s . .
Exam ination of T able XI w ill re v e a l th e s i m i l a r i t y Of c h a r a c te r ­
i s t i c s between th e two grow th f a c t o r s .
Only in th e case of th e c h arco al
f i l t r a t e f r a c t i o n do they d i f f e r b u t t h i s p ro v id e s ev id en ce t h a t th e two
f a c to r s do n o t have th e same s t r u c t u r e , assuming th a t b o th c h a rc o a l
tre a tm e n ts were c a r r ie d out under th e same c o n d itio n s .
In th e i s o la tio n
of c a r n i t i n e , an e s s e n t i a l n u tr i e n t f o r th e mealworm, T en eb rio m o lito r .
F ra e n k e l (1951) rem arked t h a t th e i n a b i l i t y of th e c h a rc o a l to adsorb
t h i s compound p ro v id ed a s i g n i f i c a n t s te p in i t s p u r i f i c a t i o n .
The su cc e ss of Dadd (1957) w ith h is s e m i-s y n th e tic d i e t f o r th e
d e s e r t lo c u s t, S c h is to c e rc a q r e q a r i a . i s s i g n i f i c a n t b ecau se of th e ta x ­
onomic r e l a tio n s h ip between th e g en era S c h is to c e rc a and U e la n o p lu s .
The
d i e t , w ith which t h i s a u th o r was a b le to r e a r from 20 to 40 p e rc e n t of
th e pymphs to a d u lth o o d , i s g iv en i n T able X II.
Dadd’ s d ie t c o n ta in s th r e e in g r e d ie n ts th a t a re n o t p re s e n t in th e
b a s ic d i e t used f o r M9- b iT ituratu-s and M„ b i v i t t a t u s . nam ely, g lu c o se ,
wheat germ o i I , and e u ro te n e- but i t i s d o u b tfu l th a t th e s e su b sta n c es
could supply th e m issin g f a c to r re q u ire d by th e g ra ssh o p p e rs used in
th e s e t e s t s .
G lucose- i-s a- component of th e onion maggot d i e t (se e
Appendix f o r compos-Ttiorr)1which was found to be i n e f f e c t i v e in prom oting
43
T able X I„
A com parison of Growth F a c to rs R equired in th e N u tr itio n of
th e European Corn B orer, P y ra u s ta n u b i l a l i s . and th e G ra ss­
hoppers M. b i l i t u r a t u s and M„ b i v i t t a t u s .
F r a c tio n t e s t e d 3
U nm odified w a ter c o n c e n tra te .
.
Growth a c t i v i t y
Growth a c t i v i t y
corn borer*3
crrasshODoers
-Active
A ctiv e
Ashed c o n c e n tra te
I n a c tiy e
I n a c tiv e
H eat c o a g u la b le f r a c tio n
I n a c tiv e
I n a c tiv e
H eat unc o a g u la b le f r a c tio n
-Active
A ctive
E th er s o lu b le
do.
I n a c tiv e
I n a c tiv e
E th e r in s o lu b le
do.
A c tiv e
A c tiv e
150% a ce to n e in s o lu b le
do.
I n a c tiv e
?
50% a c e to n e S o lu b le
do.
A c tiv e
?
C harcoal f i l t r a t e
A c tiv e
In a c tiv e
D ia iy z a b le f r a c tio n
A c tiv e
A ctive
N o n d ia ly za b le
I n a c tiv e
I n a c tiv e
a S q u rc e s :
do.
corn le a v e s f o r corn b o re r and l e t t u c e f o r g ra ssh o p p e rs
^3Data from Beck (1953)
44
T able X II.
C om position of a S em i-S y n th e tic D ie t f o r S c h is to c e rc a
a r e a a r ia F orm ulated by Dadd (1 9 5 7 ).
:
«
Gmsi, . In a r e d ie n t
I n a r e d ie n t
C e llu lo s e powder
G lucose
. . .
Gms.
2 ,0
C h o le s te ro l
0 .5
5 .0
C aro ten e (88 p e rc e n t
B -c aro ten e )
0.025
Sucrose
5 .0
A sc o rb ic a c id
0 .1
S teeh b o ck f s s a l t
m ix tu re ho. 40
0 .5
C h o lin e c h lo rid e
0 .1
C asein
3 .0
Wheat germ o i l
1.0 m l.
D ried, d e b itte r e d
yeast
3 .0
Water
2 .5 ml.
45
nymphal grow th.
C arotene and r e l a t e d compounds a re w a te r in s o lu b le and
would n o t be expected to be p re s e n t in w a te r e x tr a c ts of l e t t u c e .
They
would be p re s e n t in e th e r e x t r a c t s and, s in c e th e se e x t r a c t s produced no
s tim u li when added to th e b a s ic d i e t , i t must be concluded th a t th e c a ro ­
te n e s p la y no r o le in th e n u t r i t i o n of M. b i l i t u r a t u s and M. b i v i t t a t u s
when nymphal grow th i s used as th& c r i t e r i o n .
Again, on th e b a s is of
S o l u b i l i t y , i t i s u n lik e ly th a t wheat germ o i l could be a so u rc e of th e
w a te r s o lu b le l e t t u c e f a c t o r .
I t seems re a so n a b le to assume, th e r e f o r e ,
th a t th e r e a re im p o rtan t d if f e r e n c e s between th e n u t r i t i o n a l req u irem en ts
f o r th e g ra ssh o p p e rs used in t h i s stu d y and S. q r e q a r ia . d e s p ite t h e i r
taxonom ic r e l a t i o n s h i p .
The p re s e n t f in d in g s e a s t some doubt on th e v a l i d i t y of th e con­
c lu s io n by Hodge (1933b) t h a t b o ilin g wheat f o r 10 m inutes caused th e
d e s tr u c tio n of some n e c e s sa ry n u tr i e n t f o r th e g ra ssh o p p e r M. d if f e r e n tia lis .
I f th e f a c t o r re q u ire d f o r t h i s s p e c ie s i s th e same as th a t
en co u n tered in t h i s s tu d y , i t would be ex p ected th a t Hodge m erely e x tr a c ­
te d i t from th e p la n t t i s s u e r a th e r than d e stro y e d i t .
That th e Stadium between th e second and th i r d m o lts i s c r i t i c a l
w ith r e s p e c t to th e grow th f a c to r i s ev idenced by th e u s u a l f a i l u r e of
th e nymphs to ach ie v e th e t h i r d m olt w ith o u t th e in c lu s io n of a le ttu c e
p re p a ra tio n in th e d i e t .
I t m ight be in f e r r e d from t h i s t h a t a s p e c if ic
d ie ta r y need i s m a n ife st a t t h i s tim e.
However, i t i s th e w r i t e r ’ s
o p in io n th a t th e newly hatfched nymphs p o s se ss an endogenous.supply of
th e re q u ire d m e ta b o lite which i s s u f f i c i e n t to su p p o rt grow th u n t i l
46
a f t e r th e second m olt o c c u rs, s in c e s ta r v a ti o n symptoms a re n o t e x h ib ite d
im m ediately fo llo w in g th e second m o lt.
The p ro g re s s iv e w a stin g of th e
body, which f i n a l l y r e s u l t s in d e a th , b e g in s s e v e ra l days a f t e r m o ltin g .
I t rem ains to be shown w hether d i f f e r e n t p la n t s p e c ie s c o n ta in
v a ry in g amounts of th e grow th f a c to r en co u n tered in t h i s s tu d y .
A
re q u irem en t f o r an ad eq u ate amount o f th e n u t r i e n t f o r m aintenance and
egg p ro d u c tio n as w ell as grow th in g ra ssh o p p e rs m ight a ls o be worthy of
i n v e s t ig a ti o n .
The- re a so n s f o r d i f f e r e n t e f f e c t s of h o st p la n ts on egg
p ro d u c tio n ( s e e - I n tr o d u c tio n page 9) co u ld be a s s o c ia te d w ith v ary in g
amounts of th e in g e s te d f a c t o r .
Pepper and H astin g s (1943) were a b le to
show th a t th e fe c u n d ity of th e su g ar b e e t webwprm, L oxostege s t i c t i c a l i s .
was p o s i t i v e l y c o r r e la te d w ith th e l i n o l e i c a c id c o n te n t of th e h o st
p la n t t i s s u e and G rison (1952) re p o rte d th a t th e fe c u n d ity and lo n g e v ity
of th e C olorado p o ta to b e e tle y -Lep tin o ta r s a d e c e m lin e a ta . depended on
th e l e c i t h i n c o n te n t of i t s h o st p la n t.
The problem of how th e b a s ic n u t r i t i o n a l needs of in s e c t s f i t in to
th e mechanisms in v o lv e d in in s e c t- h o s t p la n t r e la tio n s h ip s has been th e
S u b je c t of c o n s id e ra b le d is c u s s io n by i n v e s t i g a t o r s .
F ra e n k e l (1953) i s
of th e o p in io n th a t h o s t p la n t s p e c i f i c i t y i s determ ined by th e p resen ce
o r absence o f chem ical s u b sta n c e s which a c t as s tim u li f o r th e sense
o rg an s of sm ell and t a s t e .
He s t a t e s :
" . . . l e a v e s from d i f f e r e n t p la n ts
d i f f e r r e l a t i v e l y l i t t l e in chem ical c o m p o sitio n .a s f a r Os th e n u t r i t i o n a l
needs of in s e c ts f o r food su b sta n c e s p ro p e r a re concerned, i . e . p r o te in ,
c a rb o h y d ra te s , f a t , m in e ra ls , s t e r o l s , and v ita m in s , and t h a t th e
47
s p e c i f i c i t y of food p la n ts f o r v a rio u s in s e c t s i s based on th e p resen ce
of chem ical compounds which s e rv e as chem ical s tim u li f o r th e sen so ry
o rgans of Smell and t a s t e , "
He goes on to sa y :
"The r e s u l t s of
numerous i n v e s t ig a ti o n s have shown c o n c lu s iv e ly th a t th e b a s ic food
re q u ire m e n ts of a l l in s e c ts which have so f a r been examined a re e ss e n ­
t i a l l y s i m i l a r . W h i l e t h i s may be tr u e f o r th e main n u t r i t i o n a l
re q u ire m e n ts of i n s e c t s , d if f e r e n c e s in need f o r a c c e sso ry f a c to r s can
h a rd ly be ig n o re d when t h e i r om ission from th e d ie t r e s u l t s in d eath
o r poor grow th Of th e t e s t organism s.
On th e o th e r hand, i t seems
re a so n a b le th a t r e p e l l e n t s , f o r in s ta n c e , could make a p la n t u n d e s ir ­
a b le as a h o s t d e s p ite i t s s u i t a b i l i t y o th e rw is e .
For a d is c u s s io n in
o p p o s itio n to F r a e n k e l1S view , see Beck (1 9 5 6 ).
As th e b a s ic n u t r i t i o n a l needs of more; phytophagous in s e c ts a re
in v e s t ig a te d , i t ap p ears th a t th e need f o r new growth f a c to r s w ill be
d is c o v e re d .
T his in fo rm a tio n w ill be v i t a l in h e lp in g to u n ra v e l th e
p u z z le of in s e c t- h o s t p la n t r e la tio n s h ip s as w e ll as in a s s e s s in g th e
d i r e c t n u t r i t i o n a l e f f e c t s of th e environm ent on in s e c t p o p u la tio n s .
The r e s u l t s Of th e experim en ts conducted in t h i s stu d y have p ro ­
v id e d in fo rm a tio n on some g e n e ra l c h a r a c t e r i s t i c s of " le ttu c e f a c to r " .
^ F rq en k e l, G. 1953.
I n s e c ts .
4
Ibidem .
The N u tr it io n a l Value o f Green P la n ts f o r
T ran s. IX th I n t e r n t l . Congr. E n t. 2:9 0 -1 0 0 , p. 90.
48
T his o rg a n ic su b sta n c e in w a te r s o lu tio n e x h ib its extrem e s t a b i l i t y to
p ro lo n g ed p e rio d s of h e a tin g and i s r e s i s t a n t to a i r o x id a tio n .
d ia ly z a b le th ro u g h a c e ll u lo s e membrane.
I t is
L e ttu c e f a c t o r i s s tro n g ly
adsorbed on c h a rc o a l from which i t can be e lu te d w ith ammoniacal e th a n o l.
An aqueous s o lu tio n c o n ta in in g l e t t u c e f a c t o r i s re n d e red in a c t iv e a f t e r
tre a tm e n t w ith a cat-ion-exchange r e s in (Dowex 50 H^), b u t i s la r g e ly
u n a ffe c te d a f t e r tre a tm e n t w ith an anion exchange, r e s in (Dowex I C l" ).
The a c tiv e m a te r ia l i s r e s i s t a n t to a c id s and to m ild a lk a l in e co n d i­
tio n s .
L e ttu c e f a c to r i s n o t a n u c le ic a c id d e r iv a tiv e .
49
LITERATURE CITED
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1958.
DETERMINATION DE
LA FERTILITE PAR L5EFFET DE GROUPE CHEZ LE CRIQUET MIGRATEUR
( LOCUSTA MIGRATOR!A MIGRATORIOIDES R. E T F . ) .
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B u ll, B io l. France
349-427.
A satoor, A ., and D a lg lie s h 0 C0 E0
1956.
THE USE OF DEACTIVATED CHAR­
COALS FOR THE ISOLATION OF AROMATIC SUBSTANCES,
P a rt I I
2291-2299.
B arnes, 0. L. and N erney0 N0 J .
Beck, S. D„
1953.
LARVAL GROWTH.
1956.
1953.
U npublished d a ta .
NUTRITION OF THE EUROPEAN CORN BORER,
NUBILALIS (HBN.) I I I .
Beck, S. D.
J o u r. Chem0 Soc0
PYRAHSTA
AN UNIDENTIFIED DIETARY FACTOR REQUIRED FOR
J o u r. Gen. P h y s io l. 36:
317-325.
THE EUROPEAN CORN-BORER, PYRAUSTA NDBILALIS (HBN.).
AND ITS PRINCIPAL HOST PLANT.
II.
THE INFLUENCE OF NUTRITIONAL
FACTORS ON LARVAL ESTABLISHMENT AND DEVELOPMENT OF THE CORN
PLANT.
Ann. E nt. Soc0 A m erica_49:
582-588.
Beck, S. D00 L i l l y , 3i. H ,, and S ta u f f e r , J 0 F.
1949.
EUROPEAN CORN BORERi PYRAUSTA NUBILALIS (HBN.).
NUTRITION OF THE
I,
OF A SATISFACTORY PURIFIED DIET FOR LARVAL GROWTH.
DEVELOPMENT
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1950,
THE PRINCIPLES OF INSECT PHYSIOLOGY,
D utton and C o ,, N0 Y0, Ch0 I l 0
W illiam s, R0 J 0 P0, 1953,
METAL IONS IN BIOLOGICAL SYSTEMS... B io l,
Rev. Cambridge P h il o s 0 Spc. 28: 381-453 (B io l, A b s,)
Woods, D0 D0
1953.
THE INTEGRATION OF RESEARCH ON THE NUTRITION AND
METABOLISM OF MICRO-ORGANISMS.
J o u r, Gen0 M ic ro b io l, 9 : 151-173.
57
APPENDIX
C om position of E xperim ental D iets,U sed in P re lim in a ry T e s ts .
I.
P in k boI !worm d i e t , V anderzant and R e is e r, ( 1956b)
I n g r e d ie n t
G m s./100 Gms„ Mediurn
C asein
5 .0
L -c y s tin e
0 .1
G lycine
0 .1 5
Sucrose
8 .0
Corn o i l
1.0
C h o le s te ro l
0 .3
W esson's s a l t s
0 .7
C holine c h lo rid e
0 .1
V
C e llu lo s e (powdered)
4 .0
Agar ■
3.0
Sodium a lg in a te
0 i5
V itam in s o lu tio n
1.0 ml.
2 M p o tassiu m hydroxide
to pH 6 .5
Water to 100 grams
The com position of th e v ita m in s o lu tio n was such th a t I ml.
c o n ta in e d th e fo llo w in g amounts of v ita m in s , V anderzaiit and
R e is e r, (1 9 5 6 a ):
Thiamin
0 .6 0 mg.
R ib o fla v in
0 .9 0
Calcium p a n to th e n a te
2.00
58
N iacin
6.25
P y rid o x in e
0 .8 0
F o lic a c id
0 .1 2 5
B io tin
0 .0 1 8
P--M inobenzoic a c id
1.50
V itam in
0.005
2
A s u b s t i t u t e v ita m in p re p a ra tio n ("H om icebrin", E li L i l l y & Co
used in p la c e of th e above v ita m in s o lu tio n , was s p e c if ie d as
having th e fo llo w in g c o n c e n tra tio n s o f v ita m in s in I cc:
Thiamin c h lo rid e
0 .2 0 mg.
R ib o fla v in
0 ,2 4 ,
P y rid b x in e HCl
0 .2 0
N ic o tin a m id e ........
2.00
V itam in Bjg -
0,0006
A scorbic a c id
12.00
V itam in A-
600 U .S .P . U n its
V itam in I)
200 U .S .P . U n its
U n iv e rs a l D ie t No. I " , Luckey (19154)
Gms. /K ilo , of D iet
p u r i f i e d c a s e in
Corn o i l
300
80
Cprn s ta r c h
300
C e llu lo s e (powdered)
120
Sucrose
120
59
V itam in Ji
10,000 IU
V itam in D
2,000 IU
a-T ocopherol
0 .1
V itam in K
0 .0 1
A sc o rb ic a c id
10.0
Thiamin Cl
0 .0 2
R ib o fla v in
0 ,0 2
N icotinam ide
S
O
Calcium p a n to th e n a te
0 .0 5
In o s ito l
2.0
C hpline c h lo rid e
2.0
P y rid d x in e Cl
0 .0 2
B io tin
0.001
F o lic a c id
0 .0 2
V itam in
0.0005
K a c e t a t e - .......
20.0
CaCO3
18.0
CaHPp4
13.5
Na2HPO4 .
12.0
NaCl
3.0
KI
0 .0 4 5
MgSO41TH3O
4 .5
MgO
4 .0
MnSO414H20
0 .7 5
60
3.
Fe(G6H5O j)2
4 .5
CuSO^^SHgO
0 .2 3
CoCl„6H2O
0 .0 3
ZnSO4 -TH2O
0 .0 6
Na2B4Oj.IOH2O
0 .0 3
AlK(SO4) 2. IOH2O
0 .0 4 5
Onion Maggot D ie t, F rie n d and P a tto n (19 5 6 ),
Gms.
Agar
25.0
R ib b n u c le ic a c id
1.0
In o s in e
0 .0 3
Thymine
0 .0 0 4
C h o le s te ro l
0 .1
D extrose
15.0
Wesson’ s s a l t s
2.0
C asein
24.21
L -c y s tin e
0 .5 0
G lycine
0 .7 0
D i s t i l l e d w a ter
1000.0
'
uGms.
B io tin
20.0
V itam in B12
40.0
Calcium p a n to th e n a te
C holine c h lo rid e
6 , 000.0
2 0, 000.0
61
Coenzyme A ...
1 ,0 0 0 .0
F o lic a c id
6 ,0 0 0 .0
N ic o tin ic " a c id
10,0 0 0 .0
P y rid o x in e HCl
3 0 ,0 0 0 .0
R ib o fla v in
2 ,4 0 0 .0
p -Anin oberrzo i c a c id
1 ,5 0 0 .0
Thiamin
1 ,5 0 0 .0
T h io c tic a c id
In o s ito l
500.0
2 0 ,0 0 0 .0
M ONTA NA S T A T F
__________
3 1762 10010956 8
/
D378
K87s
cop. 2
*
I
MHwwiW W ^ 140969
K reasky, J . B.
S tu d ie s on a growth f a c to r in
I a f- f - 11 f o
NAME An d
aoorm s
H57S
A'5 ?5
14096a