Serological studies of moth proteins with special reference to specific immune bodies and their
phylogenetic significance
by Saxon Martin
A THESIS Submitted to the Graduate Committee In partial fulfillment of the requirements for the
Degree of Master of Science in Botany and Bacteriology at Montana State College
Montana State University
© Copyright by Saxon Martin (1933)
Abstract:
no abstract found in this volume SEROLOGICAL STUDIES OF MOTH PROTEINS WITH SPECIAL
REFERENCE TO SPECIFIC IMMUNE BODIES AND
THEIR PHYLOGENETIC SIGNIFICANCE
by
SAXON MARTIN
A THESIS
S u b m itte d t o t h e G r a d u a te C om m ittee I n
p a r t i a l f u l f i l l m e n t o f t h e r e q u i r e m e n ts
f o r t h e D e g re e o f M a s te r o f S c ie n c e
i n B o ta n y a n d B a c t e r i o l o g y a t
M ontana S t a t e C o lle g e
'In C h a rg:ee o f jM
a pa j o r Work
C h airm an G r a d u a te C om m ittee
Bozem an, M ontana
J u n e , 1933
M-513
M "5^5"
2
TABLE OE CONTENTS
P ag e
I n t r o d u c t i o n .........................................................................................
3
M a t e r i a l s a n d M e th o d s * ............................
7
E x p e rim e n t w ith p h y s i o l o g i c a l s a l i n e
e x t r a c t . . ..........................................
9
E x p e r im e n ta l D a ta ..............................................................................
11
E x p e rim e n t w i t h a l c o h o l i c i n s o l u b l e p o r t i o n
o f e x t r a c t . . . . . . ................
22
G e n e r a l D i s c u s s i o n . . . . ................
24
Summary a n d C o n c l u s i o n s . . . ...............
25
L i t e r a t u r e C i t e d . . . . . .......... .................
27
44550
3
SEROLOGICAL STUDIES OF MOTH PROTEINS WITH SPECIAL
REFERENCE TO SPECIFIC IMMUNE BODIES AND
THEIR PHYLOGENETIC SIGNIFICANCE
INTRODUCTION
The s i g n i f i c a n c e a t t a c h e d t o im m u n o lo g ic a l r e a c t i o n s a n d e s p e c i a l l y
t o t h e s o - c a l l e d s p e c i f i c immune b o d y i n im m u n o lo g ic a l r e a c t i o n s o f v a r ­
i o u s b a c t e r i a h a s b e e n p r e s e n t e d by K rum ireide and N o b le ( 1 2 ) , H e i d e lb e r g e r
a n d A v e ry ( 8 ) , G u n n iso n a n d S h o e n h o lz ( 6 ) , H itc h c o c k ( 1 1 ) , Sugg a n d N e i l l
(2 0 ), L a n c e fle ld
(1 6 ), and o t h e r s .
The p r e s e n c e o f t h e s e b o d i e s i n t h e
h i g h e r fo rm s h a s b e e n d e m o n s tr a te d by L a n d s t e l n e r (1 5 ) i n h i s w ork on
h o rse s a liv a .
A s p e c i f i c Immune b o d y h a s b e e n i s o l a t e d fro m c o l l e c t i o n s o f F e l t l a
d u c e n s W lk. b e lo n g in g t o t h e f a m i ly P h a l a e n i d a e .
The w r i t e r h a s d e m o n s tra ­
t e d t h a t t h e p r e c i p i t i n r e a c t i o n c a n b e u s e d t o d e te r m in e a p h y l o g e n e t ic
r e l a t i o n s h i p b e tw e e n m em bers o f t h i s g r o u p .
T h e re h a s b e e n l i t t l e
w ork d o n e o n t h e s p e c i f i c immune b o d ie s
o c c u r r i n g i n m em bers o f v a r i o u s p h y l a o f t h e a n im a l kin g d o m a n d no a v a i l ­
a b l e l i t e r a t u r e a s r e g a r d s su c h w ork on i n s e c t s .
K rum w iede a n d N o b le (1 2 ) d e s c r i b e d a r a p i d m eth o d f o r t h e p r o d u c t i o n
o f p r e c i p i t i n a n t i g e n fro m b a c t e r i a .
H e i d e l b e r g e r a n d A v e ry (8 ) d e ­
s c r i b e d a m eth o d u s e d i n o b t a i n i n g t h e s o l u b l e s p e c i f i c s u b s ta n c e fro m
P n eum ococcus i n w h ic h t h e y i s o l a t e d t h e s o l u b l e s p e c i f i c s u b s ta n c e by
th e c o n c e n tr a tio n o f b r o th c u l t u r e s , p r e c i p i t a t i o n w ith a lc o h o l, r e p e a te d
4
r e - s o l u t i o n and p r e c i p i t a t i o n .
T h is was f o llo w e d b y a c a r e f u l s e r i e s
o f f r a c t i o n a l p r e c i p i t a t i o n s w i t h a l c o h o l o r a c e to n e a f t e r a c i d i f i c a t i o n
o f th e s o lu t i o n w ith a c e t i c a c id and r e p e a te d f r a c t i o n a l p r e c i p i t a t i o n
w i t h ammonium s u l p h a t e a n d d i a l y s i s o f t h e a q u e o u s s o l u t i o n o f t h e
a c tiv e f r a c tio n s .
T h e i r m a t e r i a l c o n t a i n e d 1 .2 p e r c e n t o f n i t r o g e n
a n d y i e l d e d 79 p e r c e n t o f r e d u c i n g s u g a r s on h y d r o l y s i s .
The m a t e r i a l
g a v e a s p e c i f i c r e a c t i o n i n a d i l u t i o n a s h i g h a s 1 : 5 ,0 0 0 ,0 0 0 .
L a n d s t e i n e r and L e v e n e (1 4 ) o b t a i n e d a n a c t i v e s u b s t a n c e fro m
d r i e d h o r s e k id n e y s w h ic h i n h i b i t e d h e m o ly s is i n h ig h d i l u t i o n s .
B a lls
(2 ) fo u n d a c o n s i d e r a b l e d e g r e e o f s p e c i f i c i t y i n i d e n t i f y i n g c e r t a i n
y e a s t s w ith t h e p r e c i p i t i n t e s t .
He s u g g e s t e d t h a t a g r o u p in g o f n e a r l y
r e l a t e d s p e c i e s c o u ld b e m ade by t h i s m e th o d .
The p r e c i p i t i n r e a c t i o n
was fo u n d t o b e h i g h l y s p e c i f i c f o r tw o t y p e s o f B a c i l l u s b o t u l l n u s v an
Ermengem a n d f o r t h e s u b - g r o u p s w i t h i n t h o s e t y p e s b y G u n n iso n and
S c h o e n h o lz ( 6 ) .
H e k to e n (1 0 ) w orked w i t h t h e p r e c i p i t i n r e a c t i o n o n b o d y c o n s t i ­
t u e n t s a n d f o u n d t h a t seru m p r o t e i n s , h e m o g lo b in , a n d s p e c i f i c p r e ­
c i p i t i n o g e n i n se ru m w e re fo u n d t o b e s p e c i f i c f o r t h e s p e c i e s .
L an d -
s t e i n e r a n d L e v e n e (1 4 ) w o rk in g o n a l c o h o l i c e x t r a c t s fro m h o r s e k id n e y s
o b t a i n e d a w a t e r s o l u b l e s u b s ta n c e w h ic h t h e y p u r i f i e d w i t h p y r i d i n e ,
c h lo r o f o r m , a n d a l c o h o l .
The f i n a l r e s i d u e was h y d r o ly z e d w i t h s u l p h u r i c
a c i d an d t h e a q u e o u s s o l u t i o n o f t h e p r o d u c t o f h y d r o l y s i s was t i t r a t e d
w i t h F e h l i n g 1S s o l u t i o n g i v i n g a r e d u c t i o n c o r r e s p o n d in g t o 28 m g. o f
g lu c o s e .
H itc h c o c k (1 1 ) i n h i s w ork on t h e s e p a r a t i o n o f a s e r o l o g i c a l
5
g ro u p d e s c r i b e d a m eth o d i n w h ic h h e u s e d so d iu m h y d r o x id e t o e x t r a c t
t h e a n t i g e n i c s u b s ta n c e fro m s t r e p t o c o c c i .
Ando ( I ) u s e d a m o d i f i c a t i o n
o f H e i d e l b e r g e r ’ s an d A v e r y 's m eth o d i n w o rk in g w ith s t r e p t o c o c c u s
v i r i d a n s S c h o t m u l le r , B a c i l l u s d y s e n t e r l a e S h ig a , a n d B a c i l l u s m a l l e i
H o e f l e r a n d S c h u tz and s u g g e s t e d i t s u s e i n t h e d i a g n o s i s o f g l a n d e r s *
E n d e rs (5 ) fo u n d a t y p e - s p e c i f i c s u b s ta n c e i n t h e a u t o l y t i c p r o d u c t s
o f Type I p n e u m o c o c c i d i s t i n c t fro m t h e s p e c i f i c c a r b o h y d r a te s *
He d i s ­
t i n g u i s h e d i t fro m t h e c a r b o h y d r a t e s b y t h e f a c t t h a t i t w as n o t s t a b l e
when b o i l e d i n w e a k ly a l k a l i n e s o l u t i o n *
T i l l e t , G o e b e l, a n d A very (2 1 )
w orked o n t h e c h e m ic a l a n d im m u n o lo g ic a l p r o p e r t i e s o f a s p e c i e s - s p e c i f i c
c a r b o h y d r a te o f p n e u m o c o c c i*
They s u s p e n d e d t h e o rg a n is m s i n 50 cc* o f
s a l i n e an d b r o k e them down by r e p e a t e d l y f r e e z i n g a n d th a w in g th e m .
The
f i n a l s o l u t i o n a f t e r t r e a t m e n t w ith a c e t i c a c i d and so d iu m h y d r o x id e
c o n t a i n e d f r a c t i o n C w h ic h was fo u n d to b e n o n - s p e c i f i c .
Sugg an d N e i l l (2 0 ) d e s c r i b e d a m eth o d f o r t h e e x t r a c t i o n o f a n t i ­
g e n s i n t h e i r s t u d i e s o n t h e r e l a t i o n s h i p s among t h e p n e u m o c o c c i i n w h ich
t h e a n t i g e n w as p r e p a r e d by b o i l i n g c e l l s i n *05 n o rm a l HCl a n d a d d in g
a c e t i c a c id to p r e c i p i t a t e th e p r o t e i n s .
The a c t i v e s u b s t a n c e was th e n
p r e c i p i t a t e d w ith a lc o h o l *
L a n d s t e i n e r ( 1 5 ) , w o rk in g on h o r s e s a l i v a , o b t a i n e d a n a c t i v e s u b ­
s t a n c e b y t r e a t m e n t w i t h a c i d an d a c e t o n e a n d f r a c t i o n a t i o n w i t h a l c o h o l ,
w h ic h g a v e a weak b i u r e t r e a c t i o n a n d on h y d r o l y s i s y i e l d e d 48,5% r e d u c i n g
su g a r.
6
Z o z a y a a n d Wood (2 4 ) fo u n d t h a t t h e p o l y s a c c h a r i d e a n d t h e " n u c l e o p r o t e l n " o b t a i n e d fro m d i f f e r e n t t y p e s o f m e n in g o c o c c i, g o n o c o c c i, a n d
M ic ro c o c c u s e a t a r r h a l i s P f e i f f e r , h a d s i m i l a r im m u n o lo g ic a l p r o p e r t i e s
a n d w e re n o t t y p e - s p e c i f i c .
They a l s o fo u n d t h a t t h e c a r b o h y d r a t e -
p r e c i p i t a b l e s u b s t a n c e i n immune s e r a a p p e a r e d i n t h e f o u r t h m onth o r
m o re i n t h e im m u n iz a tio n o f a n i m a l s .
Z o zay a (2 2 ) show ed t h a t p o l y s a c c h a r i d e s c a n b e r e n d e r e d a n t i g e n i c
by h a p to g e n i c a d s o r p t i o n upon a c o l l o i d c a r r i e r .
W ith t h e p o l y s a c c h a r i d e
o f T ype I I I p n e u m o c o c c i h e was u n a b le w i t h h i s m ethod o f im m u n iz a tio n o f
r a b b i t s i n s i x w eeks t o p r o d u c e a n y d e t e c t a b l e p r o t e c t i v e a n t i b o d i e s ,
b u t w as a b l e t o p r o d u c e a n t i c a r b o h y d r a t e a n t i b o d i e s .
c a r b o h y d r a t e s w ere n o n - a n t l g e n l c when u s e d a l o n e .
A ll o f th e b a c te r ia l
Z o z a y a (2 3 ) a l s o fo u n d
t h a t t h e s y n t h e t i c p o l y s a c c h a r i d e p r o d u c e d b y L e u c o n o s to c m e s e n t e r i o i d e s
(C ie n k o w s k i) Van Tiegheen fro m s a c c h a r o s e , r e a c t e d lm m u n o lo g ic a lly w i t h
a n t i s e r a fro m p n e u m o c o c c i, some o f t h e S a lm o n e lla a n d some o f t h e t y p e s
o f S t r e p t o c o c c u s v l r l d a n s B a rg e n .
He s a y s f u r t h e r , " t h i s im m u n o lo g ic a l
r e l a t i o n s h i p i s in d e p e n d e n t o f t h e s p e c i f i c a n t i p o l y s a c c h a r i d e a n t i ­
b o d i e s o f t h e s e s e r a , s u g g e s t i n g t h e e x i s t e n c e o f a d i s t i n c t a n tib o d y
p ro d u c e d by a n a c t i v e g ro u p o f t h e s p e c i f i c b a c t e r i a p o l y s a c c h a r i d e ,
w h ic h i s s i m i l a r o r i d e n t i c a l t o t h e a c t i v e g ro u p o f t h e d e x t r a n p o ly ­
s a c c h a r id e ."
"T h ese f in d in g s w a rra n t th e g e n e r a li z a ti o n t h a t a n tig e n s
n e e d n o t b e o f a co m p le x p r o t e i n n a t u r e , b u t t h a t some s u b s t a n c e o f a
c o llo id n a tu re i s re s p o n s ib le f o r a n ti g e n ic i ty .
The c o l l o i d may i n
c e r t a i n c a s e s b e a p r o t e i n , b u t a n y c o l l o i d w h ic h c a n a d s o r b t h e s p e c i f i c
7
p r in c i p le can s u f f i c e .
The p h y s i c a l a tt a c h m e n t o f t h e s p e c i f i c
p r i n c i p l e t o t h e c o l l o i d c a r r i e r m akes t h e c o m b in a tio n a n a n t i g e n .
I n t h e c a s e o f p u r e p r o t e i n s , we no d o u b t h a v e a s p e c i a l r a d i c a l i n
t h e w h o le m o le c u le w h ic h i s t h e a c t i v e g ro u p a n d g i v e s t h e a n t i b o d y
its
s p e c if ic ity ."
H e i l d e l b e r g e r a n d K e n d a ll (9 ) fo u n d f r a c t i o n s w h ic h y i e l d e d
s p e c i f i c p r e c i p i t a t e s w i t h Type I I I a n tip n e u m o c o c c u s h o r s e se ru m b u t
f a i l e d t o p r e c i p i t a t e ho m o lo g o u s r a b b i t a n t i s e r a , g i v i n g r i s e o n ly t o
s p e c ific in h ib itio n .
L a n c e f i e l d (1 6 ) show ed t h a t t h e h e m o l y ti c s t r e p t o c o c c i c a n b e
d i f f e r e n t i a t e d s e r o l o g i c a l l y by m eans o f t h e p r e c i p i t i n r e a c t i o n i n t o
d i s t i n c t a n d s h a r p l y d e f i n e d g ro u p s w h ic h a r e n o t d i s c l o s e d by t h e
a g g lu tin a tio n r e a c tio n .
S he u s e d h e a t a n d h y d r o c h l o r i c a c i d e x t r a c t s
o f t h e m ic r o o rg a n is m s a n d t h e s e r a o f r a b b i t s im m unized w i t h f o r m a l l n Iz e d c u ltu r e s .
MATERIALS AHD METHODS
The m o th s b e lo n g i n g t o t h e f a m i ly P h a l a e n i d a e ( N o e tu id a e ) * w e re
o b t a i n e d fro m t h e D e p a rtm e n t o f E n to m o lo g y , M ontana A g r i c u l t u r a l E x p e r i­
m en t S t a t i o n , a n d w e re c o l l e c t e d i n t h e s t a t i o n l i g h t t r a p s i n v a r i o u s
p a r t s o f M o n ta n a .
The s p e c im e n s u s e d w e re c h e c k e d t o g e n u s a n d s p e c i e s
b y D r. H. E . W all o f t h e D e p a rtm e n t o f E n to m o lo g y .
w e re :
E uxoa a l b l p e n n i s G r t .
♦ L e p i d o p te r a
The s p e c i e s u s e d
8
E uzoa g u a d r l d e n t a t a G.&R.
E uzoa t r l s t i c u l a M o rr•
F e ltla v e n e r a b llls a rld a C k lle
F e l t l a d u c e n s Wlk.
F e l t l a yaneou v e re n s ls G rt.
S ld e m la d e v a s t a t o r B ra c e
A g ro tla y p a llo n I t o t t e
A g r o t l s o r t h o g o n l a M o rre
S p a e lo tls h a v lla e G rte
P r o ta g r o tla n lv e lv e n o s a G rte
S ta e r ln th u s c e r i s y l K ir b y
Y anessa o a rd u l Le
Scotogram m a t r l f o l l l R o t t e
E u m lc h tls I o d a S t k r e
H ypocoena r u f a s t r l g a t a P a c k e
A u to g ra p h a b r a s s i e a e R i l e y
A u to g r a p h s c a l l f o m i c a S p e y e r e
H e lio th ls o b s o le te F a b re
C h o riz a g ro tls a u z l l l a r l s G r te
The s p e c im e n s w h ic h h a d n o t b e e n h e l d lo n g enough t o becom e d r y w ere
d r i e d i n a n o v e n w i t h t h e te m p e r a t u r e a t 57* C» o r I o w e r e
O nly t h e
t h o r a c e s o f t h e m o th s w ere u s e d i n t h e e x p e r im e n ts b e c a u s e o f t h e f a c t
t h a t t h e g r e a t e s t d e v e lo p m e n t o f m u s c le s o c c u r s i n t h i s b o d y d i v l s l o n e
The l e g s a n d w in g s w e re rem oved t o a v o id t h e c h i t i n o u s m a t e r i a l o f w h ic h
9
th e y c h ie f ly c o n s i s t .
The abdom en w as rem oved t o a v o id t h e p o s s i b i l i t y
o f i n t e r f e r e n c e o f f a t s , o i l s , an d i n t e s t i n a l c o n t e n t s w i t h t h e r e ­
a c tio n s .
The h e a d was rem oved i n o r d e r t o a v o id t h e p o s s i b i l i t y o f
t h e p r e s e n c e o f an o r g a n s p e c i f i c s u b s t a n c e w hich m ig h t i n t e r f e r e w ith
th e r e a c tio n o f th e s p e c ie s s p e c i f ic s u b s ta n c e .
I n o r d e r t o rem ove t h e a p p e n d a g e s fro m t h e t h o r a x , e a c h m oth was
h e l d w ith t h e t h o r a x b e tw e e n th e thum b a n d f o r e - f i n g e r a n d r o l l e d i n
s u c h a m an n e r t h a t t h e a p p e n d a g e s w e re b ro k e n o f f .
R o llin g in f r o n t o f
an e l e c t r i c fa n g r e a tly f a c i l i t a t e d th e m r k .
E x p e rim e n t w i t h P h y s i o l o g i c a l S a l i n e E x t r a c t
The t h o r a c e s w e re g ro u n d u p a s f i n e a s p o s s i b l e w ith a m o r t a r an d
p e s tle .
The g ro u n d m a t e r i a l w as e x t r a c t e d w ith p h y s i o l o g i c a l s a l i n e
f o r two d a y s i n t h e r e f r i g e r a t o r a t a t e m p e r a t u r e o f 3* t o 6* C. b e f o r e
each in n o c u la tio n .
The d e t a i l s o f t h e r a b b i t im m u n iz a tio n a r e g iv e n
i n T a b le I .
The r a b b i t s w e re b l e d a s e p t i c a l l y fro m t h e h e a r t s i x d a y s a f t e r
th e l a s t in n o c u la tio n .
The seru m was s e p a r a t e d i n t o s t e r i l e t u b e s an d
p la c e d in th e r e f r i g e r a t o r .
10
TABLE I
RABBIT IMMUNIZATION
F h a la e n id a e
Number o f i n n o c u l a t i o n a n d am ount o f in o c u lu m
Iuxoa
a lb ip e n n is
E uxoa
q u a d rid e n ta ta
Euxoa
tris tic u la
F e ltia
v e n e ra b ilis a r i
F e ltia
ducens
F e ltia
v a n c o u v e r e n s is
S id e m ia
•
Ol
o v
♦ R a b b it N o. 3 d i e d a f t e r t h e f i r s t i n n o c u l a t i o n
P h y s io lo g ic a l s a li n e
(* 8 5 # N a C l) e x t r a c t s o f t h e 11 s p e c im e n s
l i s t e d i n T a b le I I w e re m ade by p l a c i n g 100 mg. o f t h e g ro u n d t h o r a c e s
a n d 2 c c . o f s a l i n e i n w e ig h e d c e n t r i f u g e t u b e s .
T h e s e w e re k e p t i n t h e
r e f r i g e r a t o r f o r 24 h o u r s a f t e r w h ich t h e y w e re c e n t r i f u g e d a n d a s much
a s p o s s ib le o f th e s u p e rn a ta n t liq u id p ip e tte d in to la b e lle d tu b e s .
The
r e s i d u e s w e re t h o r o u g h l y d r i e d a n d w e ig h e d t o d e te r m in e t h e l o s s i n
w e ig h t I d iic h was c o n s id e r e d a s t h e w e ig h t o f t h e m a t e r i a l e x t r a c t e d .
The
d i l u t i o n s w e re m ade fro m t h e s e w e i g h ts w i t h c o r r e c t i o n s f o r t h e w e ig h t
o f t h e so d iu m c h l o r i d e .
T u b es i n w h ic h t h e r e a c t i o n s w e re r e a d w ere m ade fro m g l a s s t u b i n g
h a v in g a n i n s i d e d i a m e te r o f 4 mm. a n d a l e n g t h o f 6 cm.
A p p ro x im a te ly
0 .1 5 c c . o f s e r u n was r u n i n t o t h e b o tto m o f a tu b e a n d t h e sam e am ount
11
o f d i l u t e d e x t r a c t r u n i n a b o v e t h e s e ru m , c a r e b e in g t a k e n t o o b t a i n
a p r e c i s e l i n e o f c o n t a c t b e tw e e n t h e two f l u i d s .
B e c a u se o f t h e f a c t t h a t t h e s e r a a n d e x t r a c t d i l u t i o n s w ere k e p t
i n t h e r e f r i g e r a t o r , t h e r e a c t i o n s w e re r e a d a t t h e end o f t h e f i r s t
an d s e c o n d h o u r s a f t e r b e in g s e t up*
EXPERIMENTAL DATA
The r e s u l t s o f t h e p r e c i p i t i n r e a c t i o n s a r e g iv e n i n T a b le s I I ,
III,
17, and 7 .
I n e a c h t a b l e , t h e se ru m a g a i n s t w h ic h t h e e x t r a c t s
o f t h e s p e c i e s l i s t e d w ere r u n i s d e s i g n a t e d a s t h e a n tis e r u m *
I n r e c o r d i n g t h e r e a c t i o n s , a p r o f u s e p r e c i p i t a t e w as r e a d a s
f o u r p l u s , a d i s t i n c t r i n g w as r e a d a s t h r e e p l u s , a n d t h e l i g h t e r r i n g s
a s tw o a n d o n e p l u s .
A r e a c t i o n i n w h ic h a d e f i n i t e r i n g w as n o t fo rm ed
b u t i n w h ic h a s l i g h t p r e c i p i t a t e w as p e r c e p t a b l e was r e c o r d e d a s a
p lu s - m in u s r e a c t i o n .
TABLE II
PRECIPITIN REACTIONS
S u b - f a m ily
A g r o tin a e
(a n tise ru m )
H e lio th in a e
A g r o tin a e
A g r o tin a e
H a d e n in a e
C u c u l li n a e
A c r o n y c tin a e
A c r o n y c tin a e
P lu s iih a e
S p e a ie s
U ndi Iute<
T -IO
*
F e ltia
ducens
H e lio th is
o b s o le te
E uzoa
q u a d rid e n ta te
1 -2 0
-H-H
D ilu tio n o f E x tra c t
1 -4 0
1 -8 0
1 -1 0 0
HH
1 -1 6 0
1 -5 0 0
H H
-H-H
+
HH
-H-H
HH
H+
+
H H
HH
H H
H
H
H H
HH
±
-
w
HH
H H
H+
±
±
F e l t i a Y e n e ra b ilis
a rid a
*
-
H++
+
HH
Scotogram m a
trlfo lil
mm
HH
HH
HH
HH
E u m ie h tis
Io d a
HH
HH
-
—
H H
H H
+
-
-
S id e m ia
d e v a s ta to r
H+
H ypocoena r u fa s trig a ta
H H
HH
-
-
—
HH
HH
±
-
-
—
-
-H-H-
H+
t
-
-
A u to g ra p h a
b ra s s ic a e
1 -2 0 0
HH
++++
-
F a m ily
S p h in g id a e
S m e rin th u s
c e r i s y i K irb y
4-H-
+++
F a m ily
N y m p h a lid a e
V anessa
c a rd u i L.
•*
-
+
+
•»
H+
-
±
-
♦ S ig n s i n u p p e r p a r t o f s p a c e a r e r e a d i n g s f o r 1 s t h r . , lo w e r p a r t f o r 2 n d . h r .
Si*
TABLE III
PRECIPITIN REACTIONS
I
S u b - f a m ily
S p e c ie s
Z
7
A g r o t in a e
A n tis e r u m I
2
S
4
5
6
A c r o n y c tin a e
7
8
A g r o t in a e
9
10
A c r o n y c tin a e
11
-
D ilu tio n o f E x tra c t
1 ,0 0 0
I - 5 ,0 0 0
1 s t* h r*
Euxoa
a lb ip e n n is
E uxoa
q u a d rid e n ta ta
Euxoa
tris tic u la
F e ltia v e n e ra b i l i s a rid a
F e ltia
ducens
F e l t i a Van­
couver ens i s
S id e m ia
d e v a s ta to r
A g ro tis
y p s ilo n
A g ro tis
o rth o g o n ia
S p a e lo tis
h a v iIa e
P r o ta g ro tis
n iv e iv e n o s a
I - 1 0 ,0 0 0
2 n d .h r * l s t . h r . 2 n d . h r . l s t . h r . 2 n d .h r .
-H-H-
++++
+-
H
+*•
4—
H-H
HH
■
fw
HH
HH
H
+-
H
HH
HH
4-
HH
HH
_
"f1
-
5
/
HH
HH
HH
H++
HH
H-H-
HH
HH
H-H-
HH
HH
-HH
+—
—
H
+
H
4—
+—
TABLE 17
PRECIPITIN REACTIONS
S p e c ie s
I
A n ti­
se ru m 2
3
4
5
6
7
8
9
10
11
Euxoa
a lb lp e n n is
Euxoa
q u a d rid e n ta ta
E uxoa
tris tic u la
F e ltia v e n e ra b i l i s a rid a
F e ltia
ducens
F e ltia vanc o u v e re n s is
S id e m ia
d e ra s ta to r
A g ro tis
y p s ilo n
A g ro tis
o r th o g o n ia
S p a e lo tis
h a T ila e
P ro ta g ro tis
n iT e iT e n o s a
D i l u t i o n ojE* E x t r a c t
I - 1 0 ,0 0 0
I - 1 1 ,0 0 0
ls t i ir .2 n c L li n l s t . h r . 2 n d .h r .
++
-H-H-
++
++
D ilu tio n o f E x tra c t
I - 1 .0 0 0
I - 5 ,0 0 0
I - I C ,0 0 0
ls r t.h r. 2 n d .h r l s t . h r . 2nd.hr. I s U ir . BndJrr.
I
-H-H
H++
H
H
2
HH
HH
H
H
3
A n ti­
serum 4
HH
HH
H
H
HH-
HH
+"f
H
+-
4*4*
5
HH
HH
H
H
+-
+-
«*
+—
6
HH
HH
H
H
mm
mm
7
HH
HH
H
H
8
HH
HH
H
++
+H+
H++
++
H
mm
++
++
—
mm
mm
9
mm
mm
10
HH
HH
H
H
mm
mm
11
HH
+Hf
H
H
_
H
H
_
TABLE 17 (Continued)
PRECIPITIN REACTIONS
H
i
Ul
D ilu tio n o f E x tra c t
OOO
I - 1 0 .0 0 0
ist.hr* 2 n d . h r . l s t . h r . 2 n d . h r .
S p e c ie s
I
2
3
4
A n ti­
serum
5
6
7
8
9
10
U
E uzoa
a lb ip e n n is
Suzoa
q u a d rid e n ta ta
E uxoa
tris tic u la
F e ltia v e n e ra b i l i s a rid a
F e ltia
ducens
F e l t i a Van­
co u v er ens i s
S id e m ia
d e v a s ta to r
A g ro tis
y p s ilo n
A g ro tis
o rth o g o n ia
S p a e lo tis
h a v iIa e
P ro ta g ro tis
n iv e iv e n o s a
++++
-H-H
++++
-H-H
—
++++
-H-H
_
++++
HH
_
++++
H H
-
-H-H-
HH
++++
HH
++++
H++
-H-H-
H++
-H-H
HH
-H-H-
HH
-K-
H
_
H
_
TABLE V.
PRECIPITIN REACTIONS
D ilu tio n o f E x tra c t
I - 5.000
S p e c ie s
I
2
3
4
5
A n ti­
seru m 6
7
8
9
IO
11
E uxoa
a lb in e n n is
Euxoa
q u a d rid e n ta ta
Euxoa
tris tic u la
F e ltia v e n e ra b i l i s a rid a
F e ltia
ducens
F e l t i a Van­
couverens i s
S id e m ia
d e v a s ta to r
A g ro tis
y p s ilo n
A g ro tis
o rth o g o n ia
S p a e lo tis
h a v ila e
P ro ta g ro tis
n iv e iv e n o s a
D ilu tio n o f E x tra c t
I - 10.000
I ■
* 5.000
1 s t . h r . 2nd ^ ir. l s t J i r . 2 n d . h r .
I - 10.000
-H-
I
-H
-H
+-
+-
+—
2
H
H
+-
+-
3
-H
H
H
+++
■
»
•
m
•
+##
+-
H
+-
+-
-H
+
+-
4
H
H
H
H
+-
5
H
H
H
H
-H
6
A n ti­
se ru m 7
H
H
H
H
-H+
H
H
-H-
H
+—
H
+-
+W
*
+-
+-
H
-H-
H
H
H
+-
I - 11.000
L s t . h r .2 n d .h r , 1 s t Jir^ n c U h r , l s t . h r . 2 n d . h r .
-
-
-
-Hf
8
H
H
H
++
9
H
+-H
H
-H+
IO
H
H
+*•
-Hf
11
H
H
—
*
17
T he r e a c t i o n s r e c o r d e d i n T a b le I I a r e t h e r e s u l t s o f r e a c t i o n s
o f e x t r a c t s r u n a g a i n s t t h e se ru m fro m t h e r a b b i t im m unized a g a i n s t
t h e e x t r a c t o f F e l t l a d u c e n s o f t h e f a m i l y P h a la e n id a e #
The n e g a t i v e
r e a c t i o n s o f a s p h in x m o th , S m e rin th u s o e r i s y l o f t h e f a m i ly S p h i n g i d a e ,
a n d o f a b u t t e r f l y , V a n e s s a c a r d u i o f t h e f a m i l y N y m p h a lid a e , i n d i l u ­
t i o n s o f 1 -2 0 o r ab o v e, p o in t to a d e f i n i t e s e r o lo g ic a l d i f f e r e n c e
b e tw e e n t h e f a m i l i e s *
The p o s i t i v e r e a c t i o n s o f F e l t i a d u c e n s an d F e l t l a v e n e r a b l l i s
a r i d a i n t h e d i l u t i o n o f 1 -2 0 0 w h ile t h e r e p r e s e n t a t i v e s o f t h e o t h e r
f a m i l i e s show ed no p o s i t i v e r e a c t i o n s i n t h e d i l u t i o n , p o i n t t o a s e r o ­
l o g i c a l d i f f e r e n c e b e tw e e n t h e s u b - f a m i l i e s i n t h e f a m i ly P h a l a e n i d a e .
The e x t r a c t o f E uxoa q u a d r l d e n t a t a g a v e a p l u s - m in u s r e a c t i o n i n t h e d i l u ­
t i o n o f 1 -1 6 0 w h ic h show s i t s c l o s e r e l a t i o n s h i p t o th e o t h e r tw o g e n e r a
o f t h i s fa m ily .
The f a c t t h a t H e l i o t h i s o b s o l e t e , w h ich i s p l a c e d i n t h e
s u b - f a m i l y H e l i o t h i n a e by B a r n e s a n d McDunnough (3 ) g a v e a t w o - p lu s r e ­
a c t i o n i n 1-160^ p o i n t s t o t h e f a c t t h a t t h i s s p e c i e s and p o s s i b l y t h e
s u b -fa m ily H e lio th in a e a r e c lo s e ly r e l a t e d to th e s p e c ie s s tu d ie d o f th e
s u b - f a m i l y A g r o tin a e *
D ro u d t i n S e i t z
(1 9 ) s a y s t h a t a lt h o u g h i n h i s
w ork h e h a s s e p a r a t e d t h i s s m a l l , w e l l d e f i n e d g ro u p i n t o t h e s u b - f a m i l y
H e lio th in a e , i t s
r i g h t p l a c e i s i n t h e s u b - f a m i l y A g r o t in a e . H . o b s o l e t e
(7 )
i s p l a c e d i n t h e s u b - f a m i l y A g r o tin a e b y Hampson / a n d a l s o b y B a m e s and
McDunnough ( 3 ) , b u t McDunnough e x c lu d e s i t fro m A g r o tin a e i n h i s r e v i s i o n
o f t h e A g r o t id m o th s ( 1 7 ) .
18
The r e a c t i o n s I n T a b le I I I w e re r u n a g a i n s t t h e seru m o f t h e r a b b i t
im m unized a g a i n s t t h e e x t r a c t o f B . a l b i p e n n i s w h ic h i s p l a c e d i n t h e
s u b -fa m ily A g ro tin a e .
The e x t r a c t o f E . a l b i p e n n i s g a v e a t w o - p lu s r e ­
a c t i o n a g a i n s t i t s a n ti s e r u m i n t h e d i l u t i o n o f 1 -5000«
A ll o f th e o th e r
s p e c i e s s t u d i e d w h ic h h a v e b e e n p l a c e d i n t h i s s u b - f a m i l y b y B a rn e s a n d
McDunnough (3 ) a l s o g a v e r e a c t i o n s i n t h i s d i l u t i o n .
3 , d e v a s t a t o r an d
P r o t a g r o t l s n i v e i v e n o s a w h ic h a r e p l a c e d i n t h e s u b - f a m i l y A c r o n y c tin a e
d id n o t r e a c t in t h i s d i l u t i o n .
P . n i v e i v e n o s a , w h ic h was p l a c e d i n t h e
s u b - f a m i l y A g r o t in a e by b o t h B a rn e s an d McDunnough (3 ) a n d by D ro u d t i n
S e itz
(1 9 ), d id n o t r e a c t above 1 -1 0 0 0 .
T h i s l a c k o f r e a c t i o n w ould
i n d i c a t e t h a t t h e c h a n g e fro m t h e s u b - f a m i l y A g r o t in a e t o t h e s u b - f a m i l y
A c r o n y c tin a e i s a c o r r e c t o n e .
P r o t a g r o t l s n i v e i v e n o s a w h ich w as p l a c e d i n t h e s u b - f a m i l y A g r o tin a e
b y B a rn e s a n d McDunnough (3 ) an d i s now p l a c e d i n t h e s u b - f a m i l y A c ro n y c t i n a e b y McDunnough
(18)
d i d n o t r e a c t a b o v e 1 -1 0 0 0 a g a i n s t t h e serum
o f E . a l b i p e n n i s w h ic h i s p l a c e d i n t h e s u b - f a m i l y A g r o t in a e ( T a b le I I I ) .
McDunnough (1 8 ) s a y s t h a t t h e g en u s P r o t a g r o t l s f a l l s , on g e n e t a l i a ,
n e a r S id e m ia .
I t s r e a c t i o n was s i m i l a r t o t h a t o f S . d e v a s t a t o r a g a i n s t
t h e same se ru m , b u t a g a i n s t t h e seru m o f S_. d e v a s t a t o r ( T a b le V ),
l l v e l v e n o s a d id n o t r e a c t i o n a b o v e 1 - 5 0 0 0 .
T h is r a i s e s t h e q u e s t i o n a s
t o w h e th e r o r n o t i t b e lo n g s t o e i t h e r s u b - f a m i l y .
The e x t r a c t o f A g r o t l s o r t h o g o n la w as p o s i t i v e i n t h e h i g h e s t d i ­
l u t i o n s a g a i n s t t h e a n t i s e r a o f F . v e n e r a b i l i s a r i d a , E . d u c e n s , and
19
S . d e v a s ta to r*
I t g a v e a s l i g h t l y p o s i t i v e r e a c t i o n w i t h t h e a n ti s e r u m
o f E. a lh ip e n n is .
The e x t r a c t o f
E . t r i s t i e u l a gave p o s it i v e r e a c tio n s
in th e h ig h e s t d ilu tio n s a g a in s t th e a n ti s e r a o f E. a lh ip e n n is , F . v a n e ra b l l i s a r i d a , S . q u a d r i d e n t a t a , a n d s l i g h t l y p o s i t i v e r e a c t i o n s w ith t h e
a n t i s e r a o f F . V a n c o u v e re n s i s , a n d S . d e v a s t a t o r .
The i n c o n s i s t e n c y o f
t h e r e s u l t s fro m t h e s e two e x t r a c t s w as a t f i r s t t h o u g h t p o s s i b l y due t o
i n a c c u r a t e w e ig h in g o r d i l u t i n g o f t h e e x t r a c t s .
H ow ever, t h e s e p o i n t s
w e re c h e c k e d , a n d t h e sam e r e a c t i o n s r e - o c c u r r e d .
T a b le T I g i v e s t h e r e s u l t s o f r e a c t i o n s o f e x t r a c t s r u n a g a i n s t
s e r a o b t a i n e d fro m r a b b i t s im m unized a g a i n s t e x t r a c t s o f A . c a l i f o r n i c a ,
E . a l h i p e n n is , and C h o r lz a g r o tis a u x l l l a r l s .
E . a l h i p e n n i s . w h ic h i s p l a c e d i n t h e s u b - f a m i l y A g r o t I n a e , g a v e
a f o u r - p l u s r e a c t i o n i n t h e h i g h e s t d i l u t i o n a g a i n s t t h e a n ti s e r u m o f
A . C a l i f o r n i a w h ic h a c c o r d in g t o
th e s u b -fa m ily P lu s lin a e ,
Barnes a n d McDunnough (3 ) i s p l a c e d i n
T h is e x t r a c t a l s o w as t h e o n l y o n e r e a c t i n g
a g a i n s t i t s own a n t i s e r u m i n th e d i l u t i o n o f 1 - 2 0 ,0 0 0 ,
M o re - o v e r,
a g a i n s t t h e a n t i s e r u m o f C h o r l z a g r o t i s a u x l l l a r l s i t was t h e o n l y e x t r a c t
r e a c t i n g i n t h e d i l u t i o n o f 1 - 2 0 ,0 0 0 .
T h e se r e a c t i o n s do n o t te n d t o f o l l o w t h e d i f f e r e n t i a t i o n o b t a i n e d
in th e p re v io u s r e a c t i o n s .
H ydrogen i o n c o n c e n t r a t i o n d e t e r m i n a t i o n s
w e re r u n on a l l f i v e e x t r a c t s by m eans o f a p o t e n t i o m e t e r t o d e te r m in e
w h e th e r o r n o t t h i s f a c t o r was c a u s in g a d i s c r e p a n c y i n t h e r e a c t i o n s .
The d e t e r m i n a t i o n s a r e g i v e n i n T a b le 7 1 1 .
20
TART.Te VI
TITRATION OF SERA
1-1,000
Tat.hr.2nd.hr.
I u to g r a p h a
c a lifo m ic a I
Suzoa
a lb ip e n n is 2
3 h o riz a g ro tis
a u x ilia ris 3
1-5,000
I a t.h r .2nd.hr.
1-10,000
S a lin e
lat.h r.2n d -hr. la t .h r .Chaika
-H-H-
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e*
PRECIPITIN REACTIONS
1 - 5 ,0 0 0
1 - 1 0 ,0 0 0 1 - 2 0 ,0 0 0 1 - 4 0 ,0 0 0
2nd. 1 s t. 2nd. 1 s t. 2nd. 1 s t. 2nd.
S ub­
f a m i ly
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21
TABLE V I I
HYDROGEN ION DETERMINATIONS ON EXTRACTS
(POTMTI (METRIC)
E x tra c ts
M illiv o lts
PH
A u to g r a p h s c a l i f o m i c a
I
5 4 .5
4 .9 3
Boxoa a l b i p e n n i s
2
56
4 .9 3
C h o riz a g ro tis a u x llia r ls 3
57
4 .9 4
S id e m ia d e v a s t a t o r
4
57
4 .9 4
E uxoa t r i s t i c u l a
5
5 6 .5
4 .9 3
N o te : T he e x t r a c t s w ere a llo w e d t o r e a c h room t e m p e r a t u r e
b e f o r e t h e s e r e a d i n g s w e re m a d e .
E x tra c ts
M illiv o lts
A u to g r a p h s C a l i f o r n i a
I
53
4 .8 8
E uxoa a l b i p e n n i s
2
56
4 .9 3
C h o riz a g ro tis a u x ilia r is 3
57
4 .9 4
S id e m ia d e v a s t a t o r
4
57
4 .9 4
Euxoa t r i s t i c u l a
5
57
4 .9 4
N o te :
T h e s e r e a d i n g s wer e t a k e n a f t e r t h e e x t r a c t s h a d
r e m a in e d a t room t e m p e r a t u r e f o r two h o u r s .
22
S in c e t h e g r e a t e s t d i f f e r e n c e b e tw e e n t h e h y d ro g e n i o n c o n c en ­
t r a t i o n s i s o n l y 0 .0 6 i t i s u n l i k e l y t h a t t h i s f a c t o r c a u s e d t h e
v a r ie tio n in r e a c tio n s .
Due t o t h e l a c k o f m a t e r i a l i t w as n o t p o s s i b l e
t o r u n n i t r o g e n d e t e r m i n a t i o n s on t h e s e d i l u t e d
t h e m ethod o u t l i n e d by D a ja n l ( 4 ) .
e x t r a c t s a c c o r d in g t o
I t i s v e r y p r o b a b le t h a t su c h d e ­
d e t e r m i n a t i o n s w ould e x p l a i n t h e d i f f e r e n c e s i n r e a c t i o n .
E x p e rim e n t w ith a l c o h o l i c i n s o l u b l e p o r t i o n o f e x t r a c t
I n a n e x p e rim e n t t o o b t a i n t h e s p e c i f i c immune b o d y , 10 g r . o f
c r u s h e d t h o r a c e s o f F . d u c e n s was e x t r a c t e d f o r two d a y s w ith I l i t e r
o f d i s t i l l e d w a te r.
A t t h e end o f t h e f i r s t d a y t h e s o a k e d m a t e r i a l
was r e g r o u n d t o b r e a k up a s many o f t h e c e l l s a s p o s s i b l e .
A t t h e end
o f t h e s e c o n d d a y t h e s u s p e n s i o n w as c e n t r i f u g e d a n d 2& v o lu m e s o f 95 p e r
c e n t e th y l a lc o h o l added to th e s u p e rn a ta n t l i q u i d .
T h is m ix t u r e was
a llo w e d t o s t a n d i n t h e r e f r i g e r a t o r f o r two d a y s a f t e r w h ic h t h e p r e ­
c i p i t a t e w as r e c o v e r e d b y c e n t r i f u g a t i o n .
The p r e c i p i t a t e was d r i e d
i n a vacuum d e s s i c a t o r o v e r s u l p h u r i c a c i d a n d d i l u t i o n s m ade by w eig h ­
i n g o u t a known am ount o f t h e d r i e d m a t e r i a l .
T h is m a t e r i a l w as d i s s o l v e d i n p h y s i o l o g i c a l s q l i n e a n d r u n a g a i n s t
t h e a n ti s e r u m o f F . v e n e r a b i l i s a r i d a an d g a v e d e f i n i t e r e a c t i o n s i n d i ­
l u t i o n s a s h i g h a s 1 - 5 ,0 0 0 ,0 0 0 .
I n an a t t e m p t t o show t h e p r e s e n c e o f g l u c o s e i n t h i s a l c o h o l i c
p r e c i p i t a t e d m a t e r i a l , 15 g r . o f g ro u n d t h o r a c e s o f m is c e ll a n e o u s
P h a l a e n i d s was e x t r a c t e d w i t h I l i t e r o f p h y s i o l o g i c a l s a l i n e s o l u t i o n
f o r 36 h o u r s a t a t e m p e r a t u r e o f 3 " t o 6 6 C.
The s u s p e n s i o n was c e n t r l -
23
fu g ed ,
l i t e r s o f 9 5 p e r c e n t e t h y l a l c o h o l a d d e d , a n d t h e m ix tu r e
p l a c e d i n t h e r e f r i g e r a t o r ( 3 e t o 6» 0 . ) f o r 24 h o u r s a f t e r w h ich t h e
p r e c i p i t a t e was r e c o v e r e d by c e n t r i f u g i n g .
a f t e r d ry in g .
The t o t a l y i e l d was .4 5 0 g r .
T h re e h u n d r e d a n d f i f t y m i l l i g r a m s o f t h e d r i e d , g ro u n d
m a t e r i a l w as h y d r o ly z e d w ith 35 c c . o f 3 p e r c e n t HgSO4 f o r 2 4 h o u r s a t
100* C .
The p r o t e i n s w e re t h e n p r e c i p i t a t e d w ith so d iu m t u n g s t a t e .
The r e s u l t i n g c l e a r l i q u i d w as p l a c e d i n a p o l a r o s c o p e a n d w as fo u n d t o
c a u s e no r e f r a c t i o n o f l i g h t .
T w enty o n e a n d n i n e - t e n t h s c u b ic c e n t i ­
m e t e r s o f t h e m a t e r i a l c a u s e d no r e d u c t i o n when r u n a g a i n s t 3 c c . o f a
q u a n t i t a t i v e B e n e d i c t 's s o l u t i o n o f w h ic h 25 c c . was r e d u c e d b y 4 4 .1 5 mgm.
o f g l u c o s e when s t a n d a r d i z e d .
I n a s e c o n d e x p e rim e n t c r u s h e d t h o r a c e s o f A. c a l i f o m i c a w ere
t r e a t e d a s a b o v e an d r e a c t i o n s r u n w ith d i l u t i o n s o f t h e m a t e r i a l a g a i n s t
t h e s e r a o f E . a l b i p e n n i s , Gh. a u x l H a r i s , a n d S . d e v a s t a t o r .
The r e ­
a c t i o n s a r e g iv e n i n T a b le V I I I .
TABLE V I I I
PRECIPITIN REACTIONS
1 -^0 0 0
1 - 5 ,0 0 0
1-10,000
1-20^000
1-40V300
I-SO1QOC
H
-
-
A n tis e r u m
A. c a l i f o m i c a I
++++
HH
+H+
H
-HH
E. a lb ip e n n is
2
-H-H-
H
HH
H
HH
C h. a u x i l i a r i s 3
-H-H
+-H ++H
S . d e v a s ta to r
HH
+H -HH
4
H
-HH
++H H++
HH
+H
+
+H
-
H
*
-
H
*
I
24
The d e f i n i t e r e a c t i o n i n t h e a l c o h o l i c p r e c i p i t a t e d e x t r a c t o f
—* .S&A.lf o r n l c a I n t h e d i l u t i o n o f 1 —4 0 ,0 0 0 a n d i t s s l i g h t r e a c t i o n i n
t h e d i l u t i o n o f 1 - 8 0 ,0 0 0 a g a i n s t i t s o e n se ru m w h ile i t d i d n o t r e ­
a c t i n t h e s e d i l u t i o n s a g a i n s t t h e o t h e r s e r a shows t h a t a h i g h e r de­
g r e e o i d i f f e r e n t i a t i o n may b e o b t a i n e d w ith t h e a l c o h o l i c p r e c i p i t a t e d
e x t r a c t t h a n w ith t h e s a l i n e e x t r a c t .
s u b - f a m i l y P l u s i i n a e w h ic h i s t h e f i f t h
A. c a ll f o m ic a i s p la c e d in th e
s u b -fa m ily f o llo w in g th e sub­
f a m i l y A c r o n y c tin a e by B a rn e s and McDunnough ( 3 ) .
B . a l b i p e n n i s . G.
- u^ - . ^ alr A,3 * nnd J3. t r i s t l c u l a a r e p l a c e d i n t h e s u b - f a m i l y A g r o tin a e
a n d S . d e v a s t a t o r i n t h e s u b - f a m i l y A c r o n y c ti n a e .
GENERAL DISCUSSION
The r e m a r k a b le d e v e lo p m e n t o f t h e c h e m i s tr y o f s p e c i f i c immune
b o d i e s , a n d t h e a p p l i c a t i o n o f im m u n o lo g ic a l r e a c t i o n s i n t h e d e t e r m i n a t i o n
o f r e l a t i o n s h i p s b e tw e e n s p e c i e s o f v a r i o u s o rg a n is m s h a s b r o u g h t t o t h e
h a n d o f t h e s y s t e m a t l s t a new a n d e x a c t m ethod o f d e te r m i n in g p h y lo ­
g e n e t i c r e l a t i o n s h i p s w h ic h s h o u ld i n tim e o v e rsh a d o w t h e lo n g u s e d , a n d
o f t e n q u e s t i o n e d , m o r p h o lo g ic a l c h a r a c t e r s .
I t is o f c o u rse n e c e ssa ry
t o u s e t h e r e s u l t s o f p r e c i p i t i n r e a c t i o n s a n d t h e m o r p h o lo g ic a l c h a r a c t e r s
t o g e t h e r , b u t t h e r e a c t i o n s can b e u s e d i n d e c id i n g r e l a t i o n s h i p s w h ich
a r e q u e s t i o n a b l e when b a s e d s o l e l y upon some m o r p h o lo g ic a l c h a r a c t e r i s t i c .
The taxonom y o f t h e m o th s a n d t h e i r r e l a t i o n s h i p s a r e w orked o u t
by v a r i o u s a u t h o r s u s i n g d i f f e r e n t m o r p h o lo g ic a l c h a r a c t e r s
( s e e p a g e s 17
a n d 1 8 ) , w ith t h e r e s u l t t h a t nam es o f s p e c i e h a v e b een c h a n g e d .
S p e c ie s
h a v e b e e n t r a n s f e r e e fro m one g e n u s t o a n o t h e r , a n d g e n e r a h a v e been
25
c h a n g e d fro m o n e a u b - f a m i l y t o a n o t h e r .
The s e r o l o g i c a l r e a c t i o n s c a n ,
w ith o u t d o u b t , b e u s e d i n d e c i d i n g t h e c o r r e c t n e s s o f t h e s e c h a n g e s .
The e x t e n t o f v a r i a t i o n o f t h e p r e c i p i t i n r e a c t i o n s n e c e s s a r y t o
show d e f i n i t e d i f f e r e n c e s b e tw e en sp e c im e n s o r c l o s e r e l a t i o n s h i p s b e ­
tw e e n th em c o u ld b e s t b e d e te r m in e d a f t e r r u n n in g a l a r g e num ber o f r e ­
a c tio n s .
H ow ever, t h e t w o - p lu s r e a c t i o n o f H . o b s o l e t e
( T a b le I I ) i n
t h e d i l u t i o n o f 1 - 1 6 0 a g a i n s t t h e se ru m o f t h e r a b b i t im m unized a g a i n s t
t h e e x t r a c t o f JF. d u c e n s show s i t s
A g ro tin a e .
c lo s e r e l a t i o n s h i p t o t h e s u b -fa m ily
T h is r e l a t i o n s h i p h a s b e a n b o r n e o u t by t h e s y s t e m a t i s t s
(se e page 1 7 ).
The r e a c t i o n s o f a l l o f t h e s p e c i e s p l a c e d i n t h e s u b ­
f a m i l y A g r o t in a e i n t h e d i l u t i o n o f 1 - 5 ,0 0 0 (T a b le I I I ) t o t h e e x c l u s i o n
o f t h e r e a c t i o n s o f t h e tw o s p e c i e s p l a c e d i n t h e s u b - f a m i l y A c r o n y c tin a e
i n t h i s d i l u t i o n show t h a t t h e r e a d i n g o f a p lu s - m in u s r e a c t i o n i s p o s s i b l e
a n d i s d e f i n i t e l y o p p o s a b le t o a n e g a t i v e r e a c t i o n .
The f o u r - p l u s r e ­
a c t i o n o f B . q u a d r i d e n t a t a i n t h e d i l u t i o n o f 1 - 1 0 ,0 0 0 a g a i n s t t h e homo­
lo g o u s seru m i s a n e x c e l l e n t r e a d i n g a g a i n s t t h e t w o - p lu s an d p lu s - m in u s
r e a c t i o n s o f t h e o t h e r s p e c i e s w h ich r e a c t e d i n t h i s d i l u t i o n
(T a b le 1 7 ) .
SUMMARY AND CONCLUSIONS
1 . S e ro lo g ic a l s tu d ie s
( p r e c i p i t i n r e a c t i o n ) w e re m ade o f 14 g e n e r a
a n d 20 s p e c i e s o f m o th s p l a c e d i n 6 s u b - f a m i l i e s o f t h e f a m i l y P h a l a e n i d a e .
R e a c ti o n s w e re a l s o r u n u s i n g s p e c im e n s fro m t h e f a m i l i e s S p h in g id a e an d
N y m p h a lld a e .
2 . A s p e c i f i c immune b o d y was i s o l a t e d fro m c o l l e c t i o n s o f F . d u c e n s
w h ic h g a v e d e f i n i t e r e a c t i o n i n d i l u t i o n s a s h i g h a s 1 - 5 ,0 0 0 ,0 0 0 .
26
3» The s p e c i f i c immune s u b s ta n c e w h ic h w as i s o l a t e d w as n o t h y d ro ­
l y z a b l e t o r e d u c i n g s u g a r s a s shown by i t s
f a i l u r e to a f f e c t p o l a r i z e d
l i g h t o r t o r e d u c e q u a n t i t a t i v e B e n e d i c t 1S s o l u t i o n a f t e r t r e a t m e n t w i t h
H2 SO4 a t 1 00" Ce f o r 2 4 h o u r s e
4e E x p l a n a t i o n s o f t h e r e a c t i o n s a r e p r e s e n t e d a lo n g w ith t h e p l a c e ­
m en t o f t h e v a r i o u s s p e c i e s a c c o r d in g t o a num ber o f a u t h o r i t i e s e
Se I t i s e v i d e n t fro m t h e r e a c t i o n s o f t h e a b o v e m e n tio n e d s p e c i ­
m ens t h a t t h e p r e c i p i t i n r e a c t i o n may b e u s e d i n d e te r m i n in g a p h y lo ­
g e n e t i c r e l a t i o n s h i p b e tw e en g e n e r a an d s u b - f a m i l i e s o f t h e f a m i ly
Pbalaanidae.
27
LITERATURE CITED
! • A ndo, K#
A s im p le m eth o d o f o b t a i n i n g s o l u b l e s p e c i f i c s u b ­
s t a n c e s fro m v a r i o u s b a c t e r i a .
2 . B a l l s , A. K.
J o u r . Imm. 1 7 : 5 5 5 .
1929.
T he p r e c i p i t i n t e s t i n t h e i d e n t i f i c a t i o n o f th e
y e a s ts .
J o u r . Imm. 1 0 : 7 9 7 .
3 . B a r n e s , t t n ., a n d McDunnough, J .
o f B o r e a l A m e ric a .
4 . D a J a n i, S . W.
1925.
C heck l i s t o f t h e L e p id o p te r a
1917.
H e r a ld P r e s s . D e c a tu r , 111*
C h e m is try o f t h e h e a t c o a g u l a t i o n o f p r o t e i n s .
( G lo b u lin fro m hemp s e e d ) ♦
U n p u b lis h e d t h e s i s .
M a s te r
o f S c ie n c e D e g re e i n C h e m is tr y , M ontana S t a t e C o l l e g e .
1933.
5 . E n d e rs , J o h n F .
A ty p e s p e c i f i c s u b s ta n c e d i s t i n c t fro m t h e s p e c i ­
f i c c a r b o h y d r a t e s i n P n eum ococcus Type I*
5 2 :2 3 5 .
1930.
6 . G u n n is o n , J . P » , a n d S h o en h o I z , P .
S t u d i e s on t h e s e r o l o g i c a l
c l a s s i f i c a t i o n o f B. b o tu lin u s .
7 . Ham pson, S i r G eo rg e F .
J o u r . Imm. 1 3 :2 3 7 .
V o l. IV .
Pg. 657.
o f P n e u m o c o c c u s.
T a y lo r &
T he s o l u b l e s p e c i f i c s u b s ta n c e
J o u r . E x p . M ed. 3 8 :7 3 .
9 . H e i d e l b e r g e r , M ic h a e l a n d K e n d a l l, F o r r e s t E .
b o d ie s .
1903.
London.
8 . H e i d e l b e r g e r , M ., a n d A v e ry , 0 . T .
re a c tio n .
1927.
C a ta lo g u e o f t h e N o o tu id a e i n t h e c o l l e c t i o n
o f t h e B r i t i s h Museum.
F ra n c is .
J o u r . E z p . M ed.
1923.
S tu d ie s on p r e c i p i t i n
P r e c i p i ta ti n g h a p te n s ; s p e c ie s d if f e r e n c e s in a n t i ­
J o u r . j$xp. M ed. 5 7 : 3 , 3 7 3 .
1933.
28
IO e H e k to e n , Ludw ig*
O b s e rv a tio n s w ith th e p r e c i p i t i n r e a c tio n *
J o u r . Bnm. 1 4 : 1 .
1 1 . H i t c h c o c k , C. H .
1927.
S tu d ie s on i n d i f f e r e n t s tr e p to c o c c i*
t i o n o f a s e r o l o g i c a l g r o u p -T y p e I .
393.
I S e p a ra ­
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MONTANA STATE UNIVERSITY LIBRARIES
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