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Some studies in the nutrition and metabolism of the yellowfever mosquito, Aedes aegypti L. and the common house mosquito, Culex pipiens L. Part I. A study of mosquito larvae nutrition inthe relations to microorganisms, the vitamins or accessory growth factors,and the utilization of solutes by the larvae. Part II. The survival of thelarvae in media of different concentrations of the hydrogen ion

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UNIVERSITY OR MARYLAND
STATE
HO RTICU LTURA L
DEPARTMENT
COLLEGE
OFFICE
OF THE
STATE
PARK.
MARYLAND
ENTOM OLOGIST
A rthur
ft. B u d d in g to n , D o cto r o f P h ilo s o p h y , 1 9 4 0
T i t l e o f T h e s is :
Some S t u d ie s i n th e N u t r it io n and M eta b o lism o f
t h e Y ello w F ev er M o sq u ito , Aedes a e g y p t i L» and t h e Common House
M o sq u ito , P u le x p ip ie n s L*
Part I .
A s tu d y o f M osquito L arvae N u t r it io n i n th e R e la t io n s
t o M icro o rg a n ism s, th e V ita m in s or A c c e s s o r y Growth F a c to r s , and
t h e U t i l i z a t i o n o f S o lu t e s b y th e L a rv a e.
Part I I .
The S u r v iv a l o f th e Larvae i n Media o f D if f e r e n t
C o n c e n tr a tio n s o f th e Hydrogen I o n .
T h e s is and a b s t r a c t approved:
May 1 1 , 1940
SOME STUDIES IN THE NUTRITION AND METABOLISM OF THE YELLOW FEVER MOSQUITO,
AEDES AEGYFTI L . AND THE COMMON HOUSE MOSQUITO, CULEX PIPIENS L .
PART I .
A STUDY OF MOSQUITO LARVAE NUTRITION IN THE RELATIONS TO
MICROORGANISMS, THE VITAMINS OR ACCESSORY GROWTH FACTORS,
AND THE UTILIZATION OF SOLUTES BY THE LARVAE.
PART I I , THE SURVIVAL OF THE LARVAE IN MEDIA OF DIFFERENT CONCENTRATIONS
OF THE HYDROGEN ION.
by
A rth ur R . B ud dington
Pee*
T h e s is su b m itte d to th e F a c u lty o f th e G raduate S c h o o l o f t h e U n i v e r s it y
o f M aryland i n p a r t i a l f u l f i l l m e n t o f th e req u irem en ts f o r th e d e g r e e o f
D o cto r o f P h ilo so p h y *
Ju n e, 19AO.
UMI Number: DP70083
All rights reserved
IN FO R M ATIO N TO A LL U SER S
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a note will indicate the deletion.
UMI
Dissertation Publishing
UMI D P70083
Published by ProQ uest LLC (2015). C opyright in the Dissertation held by the Author.
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AC K N O W L E D G M E N T S
The a u th o r -w ishes t o e x p r e s s h i s p e r s o n a l thanks and s in c e r e
a p p r e c ia t io n t o D r. E r n e s t N . Cory f o r h i s d i r e c t s u p e r v is io n and
v a lu a b le s u g g e s t io n s i n t h i s p rob lem .
The w ork er f e e l s th a t h i s lo n g
a s s o c i a t i o n w it h P r o fe s s o r Cory h as u n q u e s tio n a b ly had a g r e a t b ea r­
in g upon th e p r o d u c tio n o f t h i s d i s s e r t a t i o n .
To D r. ¥ . C. S u p p lee o f th e C h em istry Departm ent o f t h e U n iv e r s it y
o f M aryland f o r h i s a id i n t h e b io c h e m ic a l p h a se s o f t h i s s tu d y , th e
a u th o r i s in d e b te d .
To Dr* L . P . Pitm an o f the E ntom ology Departm ent
f o r h i s a s s i s t a n c e , c r i t i c i s m s , and eq uipm ent, th e a u th o r w is h e s t o
e x p r e s s h i s s in c e r e th a n k s and a p p r e c a tio n *
To D r. N. E . P h i l l i p s
o f th e Z o o lo g y D epartm ent, f o r s u g g e s t io n s and eq u ip m en t, th e w r it e r
i s in d e b t e d .
For s u p p ly in g some o f t h e s a l t s , e x p e n s iv e v it a m in s , and con­
c e n t r a t e s , th e a u th o r i s in d e b te d to D r. Hugo N e ils o n o f th e U n ited
S t a t e s Bureau o f F i s h e r i e s h e r e a t C o lle g e Park*
The a u th o r a l s o w is h e s t o th ank Mr. Raymond Young o f th e De­
p artm en t o f B a c t e r io lo g y f o r h i s a id i n th e i s o l a t i o n o f m icro­
organ ism s o f v a r io u s pond w a te r s and su p p ly in g pure c u lt u r e s o f
v a r io u s b a c t e r ia and y e a s t s *
M osquito-
c u l t u r e s w ere o b ta in e d a t v a r io u s tim e s from t h e
Army M ed ica l C en ter, W a lte r Reed H o s p ita l*
TABLE OF CONTENTS
FART I#
INTRODUCTION ................................................................................
Page
1
REVIEW OF LITERATURE..........................................................
13
METHODS
32
..
.
R E S U L T S .......................................................................................
35
D I S C U S S I O N .......................................................................................
57
SU M M A R Y ..............................................................................................
62
LITERATURE C I T E D .................................................................
63
PART I I .
INTRODUCTION
................................................................................
REVIEW OF LITERATURE.......................................... .
*
.
69
70
M E T H O D S ..............................................................................................
R E S U L T S ......................................................................................
DISCUSSION
75
................................................................................
SUM M ARY....................................
LITERATURE CITED
74
.*
...................................
86
88
89
IV
TABLES
Page
P art I
S u r v iv a l o f S t e r i l e M osq uito L arvae i n
P r e se n c e o f E . c o l i .
36
S u r v iv a l o f S t e r i l e M osquito L arvae i n
P r e se n c e o f B . s u b t i l i s .
37
T able I I I . S u r v iv a l o f S t e r i l e M osquito Larvae i n
P r e se n c e o f S a c c h a r o n y c e ts .
38
Table I*
Table I I .
T ab le IV .
T a b le V .
T able V I.
T able VIC.
S u r v iv a l o f S t e r i l e M osquito Larvae i n
P r e se n c e o f A u to cla v ed Pond W ater.
42
S u r v iv a l o f S t e r i l e M osquito L arvae i n
P r e se n c e o f B e r k e fe ld W F i l t e r e d Pond W ater
43
S u r v iv a l o f S t e r i l e M osquito L arvae i n
P r e se n c e o f S t e r i l i z e d Y ea st
44
S u r v iv a l o f S t e r i l e M osquito L arvae i n
P r e se n c e o f A u to cla v ed Y e a st
46
T ab le V I I I . S u r v iv a l
P r e se n c e
o f S t e r i l e M osquito
o f A u to c la v e d Y ea st
L arvae i n
and Thiamin HC1
47
S u r v iv a l
P r e se n c e
o f S t e r i l e M osquito
o f A u to cla v ed Y e a st
L arvae i n
and R ib o f la v in
48
S u r v iv a l
o f S t e r i l e M osquito
P r e se n c e
o f A u to cla v ed Y ea st
and Thiam in HC1
Larvae i n
and R ib o f la v in
T ab le IX .
T able X .
T able X I.
S u r v iv a l o f S t e r i l e M osquito L arvae i n
P r e se n c e o f R ib o f la v in and Thiam in HC1
Table X I I . S u r v iv a l o f S t e r i l e M osquito Larvae i n
P r e se n c e o f R ib o f la v in , Thiamin HC1, N i c o t i n i c
A c id , and A sc o r b ic A cid
49
53
54
T able X I I I . S u r v iv a l o f S t e r i l e M osquito L arvae i n
P r e se n c e o f A u to cla v ed Y e a s t, R ib o f la v in
and Thiam in HC1.
55
T able XIV. S u r v iv a l o f Non—S t e r i l e M osquito Larvae
on a S o lu t e D ie t
56
V
P art I I *
^
Page
T a b le XV*
T able XVI*
S u r v iv a l o f A edes a e g y p t i a t D if f e r e n t pH*
Rang e s . ( g e n e r a l t a b l e )
77 - 80
S u r v iv a l o f C u lex p ip ie n s a t D i f f e r e n t
pH. R anges ( g e n e r a l t a b le )
81 - 83
T able X V II. S u r v iv a l o f A edes a e g y p t i a t D i f f e r e n t
pH* Ranges (c o m p o s ite t a b le )
84
Table X VIII S u r v iv a l o f C u lex p ip ie n s a t D if f e r e n t
pH* Ranges (c o m p o s ite t a b le )
84
v i.
LIST OF FIGURES
F ig . 1 .
Curve Showing th e E f f e c t o f pH* on th e D evelopm ent o f
A edes a l g y p t i and C u lex p i p i e n s .
P age
g^
PART I
A STUDY OF MOSQUITO LARVAE NUTRITION IN THE RELATION TO
MICROORGANISMS, THE. VITAMINS OR ACCESSARY GROWTH FACTORS,
AND THE UTILIZATION OF SOLUTES BY THE LARVAE.
INTRODUCTION
In t h e l i f e
o f any organism t h e r o l e o f grow th f a c t o r s or
grow th s u b s ta n c e s can by no means be o v e r lo o k e d .
I t has b een known
f o r a lo n g tim e t h a t th e la r v a e of m o sq u ito e s a r e a s s o c i a t e d in
t h e i r d evelop m en t w ith m ic r o o r g a n ism s5 n a m ely , b a c t e r i a , y e a s t s ,
m o ld s, a l g a e , d a sm id s, and p ro to zo a *
B i o l o g i s t s h ave known t h a t
t h e s e low form s o f l i f e p ro b a b ly su p p ly th e n e c e s s a r y fo o d f o r t h e
m osq u ito la r v a e and a l s o t h e grow th s u b s ta n c e s or f a c t o r s .
H ow ever, i n
r e c e n t y e a r s i t h a s been t h e ten d en cy o f r e s e a r c h w orkers t o remove
t h e m icro o rg a n ism s from t h e d i e t s o f t h e l a r v a l s t a g e s o f m o sq u ito e s
and o th e r f l i e s , su p p ly t h e v a r io u s f o o d s , s a l t s , p r o t e i n s , carb o­
h y d r a t e s , and p o s s i b l y f a t s , and a ttem p t t o add t h e a c c e s s o r y fo o d
f a c t o r s or v ita m in s t h a t seem t o be s u p p lie d by m icroorgan ism s in
n a tu r e .
A ccord in g t o R. A* G ortner i n h i s te x tb o o k on B io c h e m is tr y ,
’’V ita m in s may be d e f in e d a s o r g a n ic s u b s ta n c e s w hich must be
s u p p lie d i n t h e d i e t o f a n im a ls or may be s y n t h e s iz e d in an im als
from e s s e n t i a l d ie t a r y or m e ta b o lic p r e c u r s o r s , and w hich e x e r t a
h o r m o n e -lik e or enzyme a c t io n in th e anim al b o d y .*
G ortner f u r t h e r
p o in t s out t h a t th e v ita m in s a r e d e f i n i t e n u t r i t i v e s u b s ta n c e s
and t h e n u t r i t i o n a l q u a n t it a t iv e req u irem en ts f o r them a r e
r e l a t i v e l y v er y sm a ll j u s t as a r e t h e q u a n t it a t iv e n eed s f o r
in o r g a n ic s a l t s i n t h e an im al body.
2
In p a r t I o f t h i s r e p o r t t h e a u th o r h a s ap proached t h e problem
o f m o sq u ito la r v a e n u t r i t i o n a lo n g t h r e e l i n e s o f r e a s o n in g ;
F ir s t,
a c o n s id e r a t io n o f t h e grow th o f t h e la r v a e o f t h e y e llo w f e v e r m o sq u ito
A edes a e g y p t i and t h e common h o u se m osq u ito C u lex p ip ie n s i n p u re
c u lt u r e s o f l i v i n g b a c t e r ia and y e a s t j
S eco n d , a c o n s id e r a t io n o f
t h e e f f e c t s on l a r v a l developm ent o f pond w a ter w hich h as been
a u to c la v e d and B e r k e fe ld f i l t e r e d ;
T h ir d , t h e e f f e c t s o f v a r io u s
a c c e s s o r y grow th f a c t o r s or v ita m in s under s t e r i l e c o n d it io n s upon
l a r v a l d ev elo p m en t.
I n t h i s l a s t p h ase o f t h e n u t r i t i o n a l problem
one exp erim en t was perform ed t o a s c e r t a in t h e e f f e c t s o f s u b s ta n c e s
i n pure s o lu t i o n o f t h e grow th o f m osq u ito la r v a e *
Thus th e
g e n e r a l o u t l i n e o f t h e a u th o r * s i n t e n t i o n s has b een p r e s e n te d .
W ith a id ren d ered by t h e Departm ent of B a c t e r io lo g y a t th e
U n iv e r s it y o f M aryland some i s o l a t i o n a n a ly s e s w ere made on pond
w a ter i n w hich C u lex p i p i en s was found b r e e d in g .
Members o f tJae
g en era Aedes and A n op h eles w ere a l s o p r e s e n t b u t i n much s m a lle r
num bers.
The m ost common form s o f m icroorgan ism s p r e s e n t w ere:
b a c t e r ia :
E s c h e r ic h ia ( B a c i l l u s ) c o l i , A ero b a cter a e r o g e n e s ,
B a c i l l u s s u b t i l i s » B a c i l l u s c e r e u s , and o th e r sp o re form ing b a c i l l i ;
chrom ogens o f t h e genus F la v o b a c te r iu m ; unknown w h ite m old s;
A c tin o m y c e te s > common y e a s t .
I t was th o u g h t a d v is a b le t o o b ta in pure c u lt u r e s o f th e
common i n t e s t i n a l f e c a l b a c te r iu m , E s c h e r ic h ia ( B a c i l l u s ) c o l i ,
t h e sp o r e form in g rod -sh ap ed b a c te r iu m , B a c i l l u s s u b t i l i s » and th e
comnon b r e w e r 's y e a s t , Sac char omy c e t e s c ere v i s a e , and stu d y t h e
grow th o f m o sq u ito la r v a e in each o f t h e s e o rg a n ism s.
T h ese were
3
o b ta in e d from Mr* Raymond young o f t h e Departm ent o f B a c t e r io lo g y
and s u b -c u ltu r e d on an a u to c la v e d medium composed o f o y s t e r m eals
d r ie d y e a s t , g l u c o s e , and O sborne-M endel s a l t m ixtu re*
is
T h is medium
one d e s c r ib e d by t h e a u th o r in h i s t h e s i s , "Some S tu d ie s i n t h e
B io lo g y and M etab olism o f C u lex p ip ie n s L*", 1939* U n iv e r s it y o f
M aryland ( u n p u b lis h e d ) .
H ence th e r o l e o f t h r e e m icroorgan ism s
v e r y abundant i n th e n a tu r a l l i f e
o f m osq u ito la r v a e c o u ld be
s tu d ie d *
The e f f e c t s o f a u t o e la v in g and B e r k e fe ld W f i l t e r i n g o f
pond w a ter on C u lex p ip ie n s h ave been p r e v io u s ly r e p o r te d by t h e
au thor*
a eg y p ti *
T h ese same e x p e rim en ts have been r e p e a te d u sin g Aedes
The s u r v iv a l o f m osqu ito la r v a e i n a u to c la v e d o y s t e r m e a l,
d r ie d y e a s t , g lu c o s e and O sborne-M endel s a l t m ix tu re was l i k e w i s e
s tu d ie d and t h e r e s u l t s w i l l be p r e s e n te d l a t e r i n t h i s rep o rt*
In
t h i s p h ase o f t h e p ro b lem , a n o th er exp erim en t was c a r r ie d on w hich
seems u n iq u e i n i n s e c t n u t r i t i o n a l s t u d ie s *
An ap p aratu s was d e­
s ig n e d w h erein t h i s s y n t h e t ic medium was a u to c la v e d i n an E rlenm eyer
f l a s k co n n e cted t o a n o th e r f l a s k by means o f a s t e r i l i z e d S e i t z
filte r .
I n t h i s o th e r f l a s k t h e same s y n t h e t ic medium was n o t
s t e r i l i z e d and i t
c o n ta in e d many b a c te r ia *
Hence by r a i s i n g and
lo w e r in g one o f th e f l a s k s on a r in g s ta n d th e s o lu b le p r o d u c ts o f
b a c t e r i a l m eta b o lism co u ld f lo w th r o u g h t th e s t e r i l e f i l t e r i n t o th e
a u to c la v e d ( s t e r i l e ) medium*
I t was th o u g h t t h a t b a c t e r ia and
m a t e r ia ls n o t i n t r u e s o lu t io n co u ld be k ep t from t h e s t e r i l e
c u l t u r a l medium.
I f t h e grow th f a c t o r s th a t m icroorgan ism s produce
a r e s o l u t e s th e y sh o u ld p a ss th rou gh th e f i l t e r under p r e s s u r e
4.
(o n e f l a s k lo w er th a n o th e r ) t o t h e o th e r f la s k *
S t e r i l e eg g s or
s t e r i l e la r v a e w ere in o c u la t e d i n t o t h e s t e r i l e f la s k *
In t h e t h ir d p h ase o f P art I o f t h i s r e p o r t t h e a u th o r
s tu d ie d th e grow th o f t h e l a r v a l s t a g e o f Aedes a e g y p ti on d i e t s o f
known ch e m ic a l c o m p o sitio n f r e e from m ic r o o rg a n ism s.
A b a sa l d ie t
o f 0.1/£ o y s te r m e a l, 0 .1 $ d r ie d y e a s t , 0 .1 ^ g l u c o s e , and 0*1^
O sborne-M endel s a l t m ix tu r e t h a t had been a u to c la v e d was used i n
t h e s e e x p e r im e n ts .
S in c e a u to c la v e d y e a s t s u p p lie d a h e a t - s t a b l e
grow th f a c t o r i t was r e p la c e d i n some t e s t s w ith pure ch em ica ls to
d eterm in e i f any o f them was t h e grow th f a c t o r .
The v ita m in s
employed in t h i s problem a r e w a ter s o lu b le and were added t o th e
a u to c la v e d m edia by means o f t h e s t e r i l e B e r k e fe ld .
The c o m p o sitio n o f t h e O sborne-M endel s a l t m ix tu r e u sed
th ro u g h o u t t h e s e ex p erim en ts i s a s f o llo w s :
C alcium ca rb o n a te ...........
1 3 4 .8
grams
............................
2 8 .9
"
Magnesium c a rb o n a te
Sodium ca r b o n a te (Anhydrous)
.....................
3 4 .2
"
**
P o ta ssiu m c a rb o n a te ( d r ie d a t 180°C) ......................... 1 4 1 .3
P h o sp h o ric a c id
.1 1 9 .3
**
H y d r o c h lo r ic a c i d . ........................................
1 4 8 .3
"
S u lp h u r ic a c i d
9 .6
11
C itr ic a c i d
111*1
"
F e r r ic c i t r a t e ............................................................................. 7 .4 4
"
P o ta ssiu m io d i d e ........................................................................... 0 .0 2 0 w
Magnesium s u lp h a te
............................. ................................. 0 -1 1 7
"
Sodium f l u o r i d e ...................... ...................................................
0 .0 6 2
"
P o ta ssiu m alum ............................................................................. 0 .0 4 4
These c h e m ic a ls w ere mixed th o r o u g h ly in a " b a ll m ill" t o a very
f i n e powder w hich was uniform in c o m p o sitio n th r o u g h o u t.
The f a t s o lu b le v ita m in s o f h ig h e r a n im a ls , v ita m in s A» Dj E ,
have n o t been c o n s id e r e d by t h e w r it e r in h is e x p e r im e n ts .
w hich c h e m ic a lly i s
V itam in A
c l o s e l y r e la t e d t o th e c a r o te n e s o f p la n t s i s
a p p a r e n tly s y n t h e s iz e d by a n im a ls a lo n e .
T h is f a c t o r i s n o t found
in p la n t s a lth o u g h i t s fo r e r u n n e r s or p a ren t compounds a r e p r e s e n t
i n no s m a ll q u a n t i t i e s .
About 90 % o f t h i s v ita m in p r e s e n t in th e
body o f a n im a ls i s s to r e d i n th e l i v e r .
Cod l i v e r o i l and o th e r
f i s h o i l s a r e good s o u r c e s o f t h i s s u b s ta n c e .
Lack of v ita m in A
c a u se s i n r a t s and man an a trop h y o f e p ith e liu m c e l l s and a
k e r a t i n i z a t i o n o f th e e p it h e liu m , p a r t ic u l a r l y t h e eyes*
H eat in
t h e p r e s e n c e o f oxygen d e s tr o y s v ita m in A*
A ccord in g t o G ortn er (1 9 3 8 ) " e r g o s t e r o l and o th e r s t e r o l s ,
w h eth er i n p la n t s or a n im al t i s s u e , a re tra n sfo rm ed i n t o v ita m in D
under t h e i n f lu e n c e o f th d p rop er l i g h t ra y s and o th e r form s o f
en er g y ’*•
I t i s n o t c l e a r in t h e mind o f t h e p r e s e n t w r it e r J u st
e x a c t ly what v ita m in D i s c h e m ic a lly *
C o rtn er p r e s e n t s e v id e n c e
from t h e ch e m ic a l l i t e r a t u r e t h a t p o s s i b l e n in e form s o f t h e D v ita m in
e x is t.
E r g o s t e r o l and c a l c i f e r o l a r e s t e r o l s v ery c l o s e l y r e la t e d
t o c h o l e s t e r o l which can r e a d il y be d e t e c t e d i n a n im a l t i s s u e .
C a l c i f e r o l i s d e r iv e d from e r g o s t e r o l by v a r io u s form s o f i r r a d i a ­
t i o n , has b een i s o l a t e d in pure c r y s t a l l i n e form and i s now known
as v ita m in D2#
mammals t h e D v ita m in s a r e a s s o c ia t e d w ith th e
m eta b o lism o f t h e b o n e, and t h e u t i l i z a t i o n o f ca lciu m and p h osp h oru s,
and p r e v e n t r i c k e t s , h en ce th e name a n t ir a c h a t ic v ita m in .
H eat in
t h e p r ese n c e o f oxygen i s g e n e r a lly c o n sid e r e d d e s t r u c t iv e t o t h e s e
v ita m in s o f th e D group*
V itam in E i s t h e s t e r i l i t y v ita m in o f mammals and i s known as
A -to c o p h e r o l.
B o th :th e m ale and fem a le an im al a re u n ab le t o produce
f e r t i l e r e p r o d u c tiv e c e l l s i n t h e a b se n c e o f t h i s v ita m in .
V itam in E
i s p r e s e n t i n n a tu r e i n t h e germ o i l s o f b a r l e y , o a t s , r i c e , w h ea t,
c o r n , and many o th e r p l a n t s .
T h is f a c t o r i s v ery s e n s i t i v e t o
o x id a t io n and i s d e s tr o y e d by a u t o c la v in g in t h e p rese n c e o f oxygen .
The i n s e c t r eq u irem en ts o f v ita m in s A, D, and E a re n o t th o r o u g h ly
u n d ersto o d by t h e p r e s e n t w r i t e r .
As he w i l l show in th e r e v ie w o f
l i t e r a t u r e t h e m a jo r ity o f t h e e v id e n c e te n d s t o supp ort h is b e l i e f
t h a t i n s e c t s i n g e n e r a l do n o t r e q u ir e any of t h e s e t h r e e s u b sta n c e s
u n le s s th e y th e m s e lv e s s y n t h e s iz e them from v a r io u s fo r e r u n n e r s .
The e x p e r im e n ta l e v id e n c e p r e s e n te d by t h i s w orker seems t o show
any n eed by m o sq u ito la r v a e f o r A> D» and E i s so sm a ll t h a t m easu re­
ment o f i t can n ot be made from our p r e s e n t know ledge o f v ita m in s .
V itam in B , known t o t h e ch em ist a s th ia m in or i t s d e r iv a t iv e
th ia m in c h lo r id e h y d r o c h lo r id e , i s a w ater s o lu b le su b sta n c e p r e s e n t
i n y e a s t , wheat germ , r i c e p o lis h in g and o th e r p r o d u c ts .
In n a tu re
i t s prim ary so u r c e i s m icro organ ism s but i t i s now r e a d ily s y n t h e s iz e d
by t h e c h e m is t.
I n th e p r e s e n c e o f a l k a l i t h i s v ita m in i s r e a d ily
d e s tr o y e d by h e a t but i n v ery a c id medium (pH. 3 .5 ) i t r e s i s t s d e­
s t r u c t io n by h e a t .
F o r a lo n g tim e th e e f f e c t o f v ita m in B on th e
n erv o u s sy stem h as been known.
I t r a p id ly cu res p o l y n e u r it is i n
p ig e o n s and r a t s and i s th e a n t i - b e r i b e r i f a c t o r o f man.
Thiam in
i s v ery d e f i n i t e l y a s s o c ia t e d w ith th e m etab olism o f c a r b o h y d r a te s.
The s t r u c t u r e s o f th ia m in and th ia m in c h lo r id e h y d r o c h lo r id e a re
g iv e n below*
The a u th o r used pure c r y s t a l l i n e th ia m in c h lo r id e
h y d r o c h lo r id e in h i s e x p e r im e n ts.
7.
H
OH
H
H
Thiam in HcT
T
R -= = C
K
1
1 *
H— C--- C
1l l
H
I
Is
J
1l
/
----- c
1
H
•
H
1
G
I
H
Cl
I
N-
-G
-
f
- r
fT
^
>qh
H
H
H
Thiamin
V itam in
w a ter s o l u b l e .
o r G, known t o th e b io c h e m ist a s d. r i b o f l a v i n , i s
I t i s c h e m ic a lly r e la t e d t o th e y e llo w - g r e e n f lu o r e s c e n t
pigm ent known as t h e " y e llo w enzyme*.
T h is su b sta n c e has commanded
a t t e n t i o n o f c h e m ists and b i o l o g i s t s from 1879 t o th e p r e s e n t tim e*
I n th e p r e se n c e o f b lo o d th e y e llo w pigm ent tu r n s w h ite but r e tu r n s
to i t s
o r i g i n a l c o lo r on ex p o su re t o oxygen .
R ib o f la v in i s r e l a t i v e l y
h e a t - s t a b l e b u t a u to c la v in g f o r a lo n g tim e i n t h e p r e se n c e o f oxygen
w i l l d e s tr o y i t *
I t was f i r s t d is c o v e r e d i n y e a s t but i s now known
t o be w id e ly d i s t r i b u t e d i n b oth th e p la n t and a n im a l w orlds*
It
has been i s o l a t e d from egg w h it e , m ilk , l i v e r , k id n e y , u r in e , b a r le y
m a lt , d a n d e lio n b lo ss o m s , g r a s s e s , egg y o l k , and r e t in a s o f f i s h
ey es*
I t i s r e a d i l y s o lu b le i n w a t e r , in s o l u b l e i n t h e f a t s o lv e n t s ,
s t a b l e i n t h e p r e s e n c e o f s tr o n g a c id s and
by a l k a l i *
(s e lls *
heat
but r e a d ily d e str o y e d
V itam in Bg i s su p p osed t o b e r e q u ir e d by a l l l i v i n g
T h is i s a lm o st u n d ou b ted ly tr u e b e c a u se o f i t s
r e la tio n to
t h e o x id a t io n - r e d u c t io n enzyme and i t s w id esp read o ccu rren ce in
n a tu r e.
The la c k o f r i b o f l a v i n i n t h e d i e t o f r a t s r e s u l t s i n th e
appearance o f p e l l a g r a - l i k e s k in l e s i o n s , h en ce th e name ’h n t i—
p e lla g r a " v ita m in *
p e lla g r a " •
C hickens r e q u ir e th e v ita m in t o p r e v e n t " ch ick
I f i t i s removed from t h e d i e t o f dogs a c o n d it io n o f
b la c k to n g u e r e s u l t s .
The p r e s e n t worker u sed pu re c r y s t a l l i n e
r i b o f l a v i n i n h i s ex p erim en ts and th e in f lu e n c e o f i t
o f m o sq u ito la r v a e w i l l be shown.
on th e l i f e
The form ula f o r d* r ib o f l a v i n i s
g iv e n below s
H-C
-C
0—
C
H
H—
H-
d . r ib o f l a v i n
H
V itam in C o r 1* a s c o r b ic a c id (a ^ so c e v it a m ic a c id ) i s a w a te r s o l u b l e , h ig h ly u n s ta b le s u b s ta n c e , p r e s e n t i n a l l c i t r u s f r u i t
(le m o n s , o r a n g e s , g r a p e f r u it in p a r t i c u l a r ) , sp in a ch , t u r n ip g r e e n s ,
p ep p e r, c a b b a g e, and many o th e r g reen v e g e t a b le s *
M ilk i s a n o th e r
r ic h so u r ce o f a s c o r b ic a c id but i t i s o f t e n d e s tr o y e d i n p a s t e u r iz a ­
t io n *
I n mammals i t i s im p o rta n t i n calciu m m eta b o lism and i s
a s s o c ia t e d w ith th e d evelop m ent o f b o n e , t e e t h , and m u scles*
V itam in C
p r e v e n ts scu rv y and i s known a s t h e a n t is c o r b u t ic f a c t o r o f h ig h e r
v e r t e b r a te s *
The r o l e o f a s c o r b ic a c id in i n s e c t n u t r i t i o n w i l l be
d is c u s s e d la t e r *
T h is w orker u sed pure c r y s t a l l i n e 1 . a s c o r b ic a c id
i n h i s e x p e r im e n ts.
The fo rm u la f o r t h i s v ery h e a t " la b i le v ita m in
i s shown b elow :
H
HO-(jj
1
0
H— C
1____________________ ^
HC— OH
H
1* a s c o r b ic a c id
The v ita m in s
(th ia m in ) and B2 ( r i b o f l a v i n ) have been i s o l a t e d
from t h e B com plex and t h e i r s t r u c t u r e s and p r o p e r t ie s d e te r m in e d , a s
h a s been d e s c r ib e d in th e p r e c e d in g pages*
H owever, t h e r e a re s t i l l
o th e r s in t h e w a t e r - s o lu b le B com plex whose ch em istry and p h y s io lo g y
a r e n o t d e f i n i t e l y known.
th o se su b sta n c e s.
The a u th o r would l i k e t o m en tion t h r e e o f
One o f them was c a r e f u l l y s tu d ie d i n th e e x p e r i­
m ents on m o sq u ito la r v a e .
A nother was o b ta in e d i n a v ery sm a ll
q u a n tity t o o l a t e f o r t h e a u th o r to o b ta in s i g n i f i c a n t r e s u l t s from
i t s u se.
The t h ir d has been s y n t h e s iz e d o n ly i n th e l a s t th r e e
months and i s n o t y e t a v a i l a b l e .
In th e p a s t fe w y e a r s I t h as b een cla im ed by s e v e r a l w orkers
t h a t a p e lla g r a - p r e v e n t in g f a c t o r ( P .P .) e x i s t s w hich i s th o u g h t t o
be n i c o t i n i c a c id or a d e r i v a t i v e , n i c o t i n i c a c id am ide.
T h is
su b sta n c e i s th o u g h t t o be r e q u ir e d i n a d d it io n t o r i b o f l a v i n i n
t h e p r e v e n tio n o f p e l l a g r a .
C r y s t a llin e n i c o t i n i c a c id was o b ta in e d
and i t s e f f e c t s on th e grow th o f m osq u ito la r v a e d eterm in ed .
The
form u la f o r n i c o t i n i c a c id i s shown b elo w .
OH
N i c o t i n i c A cid
The seco n d o f t h e s e n o n e -to o -w e ll-u n d e r s to o d B v ita m in s i s
B6 o r aderm in.
I t i s th e r a t a n t ip e ll a g r a or a n t i- d e r m a t it is f a c t o r
o f Gyorgy ( 1 9 3 5 ) .
The work of C h ick , Copping and Edgar (1 9 3 0 -3 5 ) and
o f Copping (1 936) p r e s e n ts fu r th e r e v id e n c e t o c h e m ists and p h y s i o l o g i s t s
th a t t h is f a c to r does e x is t*
is
I t i s v e r y h e a t and a l k a l i s t a b le b u t
d e s tr o y e d by l i g h t on lo n g exp osure*
So l i t t l e
o f t h i s c h e m ic a l
was a v a i l a b l e th a t t h e a u th or was u n a b le t o com p lete h i s stu d y o f th e
e f f e c t o f Bg*
The form u la f o r aderm in i s g iv e n below *
H
1
Adermin
A t h ir d f a c t o r o f th e unknown B v ita m in s ap pears t o b e th e
f a c t o r o f W illia m s ( 1 9 3 4 0 3 8 ), who c a l l s i t " p a n to th en ic a cid " and
c la im s i t i s th e b io s f a c t o r r e q u ir e d i n th e developm ent o f y e a s t .
I n t h e p a s t few months W illia m s and h i s a s s o c i a t e s have s y n th e s iz e d
t h i s su b sta n ce*
I t i s p r e s e n t in la r g e q u a n t i t ie s i n y e a s t and l i v e r .
" P a n to th en ic a cid " a c c o r d in g to W illia m s w ith sto o d a u t o c la v in g f o r
n in e te e n h o u rs i n a c id m edia but was d e s tr o y e d by a u to c la v in g i n
a l k a l i n e media*
The au th or has b een u n ab le t o s e c u r e " p a n to th en ic
a cid " and h as n o t s e e n i t s c h e m ic a l form ula*
At some tim e i n th e n ear f u t u r e some ex p erim en ts w i l l be
perform ed on t h e e f f e c t s o f
o f m o sq u ito es*
b£
and th e Y f a c t o r on l a r v a l growth
As th e a u th o r ’ s r e s u l t s d e f i n i t e l y w i l l show , a
12.
t h i r d h e a t s t a b l e f a c t o r e x i s t s i n y e a s t and l i v e r v ery v i t a l in
th e grow th and developm ent o f m osq u ito larva© *
In th e l a s t fe w weeks o f t h i s stu d y in m osq u ito la r v a e n u t r i t i o n
th e a u th o r was a b le to s e c u r e i n pure c r y s t a l l i n e form t h e f o llo w in g
amino a c id s } l e u c i n e , g l y c i n e , h i s t i d i n e , tr y to p h a n e , a s p a r g in e , d .
c y s t i n e , and 1 . t r y o s i n e .
C on cen trated s o lu t i o n s o f t h e s e amino a c id s
w ere made and combined w ith Q .l^ l i v e r e x t r a c t , 0 * 1 % g lu c o s e , 0 .1 ^
O sborne-M endel s a l t m ix tu r e , p lu s V itam in s B, , B , B - , (e a c h 1 m g ./5 0 0 c c .)
X
and s t e r i l i z e d by f i l t e r i n g throu gh B e r k e fe ld W*
M
^
Time p e r m itte d
o n ly a stu d y o f t h i s d i e t i n t h e p r e s e n c e o f m ic ro o rg a n ism s.
However,
t h e a u th o r f e e l s t h a t h i s r e s u l t s are s i g n i f i c a n t enough t o r e p o r t and
w i l l be p r e s e n te d in t h e c h a p te r d ev o te d to r e s u l t s -
REVIEW OF LITERATURE
The e n to m o lo g ic a l l i t e r a t u r e on t h e s u b j e c t s o f i n s e c t d ig e s ­
t i o n , n u t r i t i o n , m e ta b o lism , and grow th s u b s ta n c e s i s v o lu m in o u s.
Dr* W ig g lesw o rth i n h i s new t e x t b o o k , The P r in c i p l e s o f I n s e c t
P h y s io lo g y , l i s t s and p a r t i a l l y r ev iew s two hundred and s i x t y - f i v e
s c i e n t i f i c p u b lic a t io n s i n h i s ch a p te r on d i g e s t i o n and n u t r i t i o n and
tw o hundred and t h r e e r e f e r e n c e s a r e found i n t h e c h a p te r d e v o te d t o
m etabolism *
I t i s beyond t h e a b i l i t y o f t h i s w orker t o r e v ie w by any
means a l l o f t h e s e p u b l i c a t i o n s .
At t h e same t im e , t h e f a c t o r s o f
tim e and sp a c e a r e a l s o p r o h i b i t i v e .
H ow ever, many o f t h e r e s e a r c h
p a p ers d i r e c t l y and i n d i r e c t l y r e la t e d t o t h i s problem w i l l be con­
s id e r e d i n t h i s review *
Boyce and L ew is (1 9 1 0 ) n o t ic e d t h a t m osq u ito la r v a e i n o rd in a ry
ta p w a ter ca u sed an i n c r e a s e i n t h e numbers o f b a c t e r ia p r e s e n t .
L oeb , J* ( 1 9 1 5 ) , i n some o f h i s e a r ly w ork, s tu d ie d t h e r o le o f
in o r g a n ic s a l t s and m icroorgan ism s i n t h e developm ent o f D r o s o p h ila *
A cco rd in g t o t h i s g r e a t s c i e n t i s t , t h e la r v a e o f t h e f r u i t f l y r e ­
q u ir e o n ly p o ta ssiu m a c id p h osp h ate and magnesium s u lp h a te a s t h e
in o r g a n ic s a l t com ponents o f t h e i r d ie t s *
In a d d itio n t o t h e s e , grape
s u g a r , cane s u g a r , ammonium t a r t r a t e , and b a c t e r ia w ere p r e se n t*
T hese s u b s ta n c e s m ixed w ith w a ter in chopped up b i t s o f a s h le s s f i l t e r
p a p er .w er e u sed as a d i e t and norm al growth r e s u lt e d *
In a n o th e r d i e t ,
Loeb o b ta in e d norm al growth w ith grap e sugar and t h e two in o r g a n ic
s a l t s w ith t h e a d d itio n o f t h e amino a c id s g lu ta m ic a c id and a la n in e *
T h ese tw o amino a c id s w ere t h e o n ly n itr o g e n compounds r e q u ir e d , and
e i t h e r one o f th em , p r o v id e d t h e o th e r was p r e s e n t , c o u ld be r e p la c e d
w ith ammonium t a r t r a t e o r s u c c in a t e .
p resen t in th e d ie ts *
Of c o u r s e , b a c t e r ia and y e a s t w ere
Sodium c h lo r id e and l i k e w i s e ca lciu m c h lo r id e
w ere u n a b le t o r e p la c e e i t h e r o f t h e two s a lt s *
A tk in and B a co t (1 9 1 7 ) s tu d ie d t h e e f f e c t s o f b a c t e r ia and y e a s t s
on t h e d evelop m ent o f t h e la r v a e o f Aedes a e g y p t i*
They co n clu d ed t h a t
t h e la r v a e o f t h i s m o sq u ito grow o n ly e x c e p t i o n a lly on dead f o o d s , and
t h a t l i v i n g b a c t e r i a and y e a s t s a re a much more s u i t a b l e fo o d th an
dead forms*
N orthrup (1 9 1 7 ,2 6 ) d id e x t e n s iv e r e s e a r c h on t h e r o l e o f y e a s t i n
t h e n u t r i t i o n o f D r o s o p h ila la r v a e *
He found t h a t t h e t o t a l numbers
o f em erging f l i e s w ere g r e a t e r on d e f i n i t e q u a n t i t i e s o f y e a s t when
b an an a, c a s e i n , or su g a r was added*
Growth o f t h e la r v a e was more
ra p id on a m ix tu r e o f banana and y e a s t th a n on y e a s t a lo n e , p ro v id ed
t h a t a t l e a s t t h i r t y - t h r e e p e r c e n t o f t h e d i e t was y e a s t*
Northrup
found t h a t k id n e y , l i v e r , and p a n crea s w ere a l s o ad eq u a te s o u r c e s o f
fo o d f o r t h e l a r v a e .
I n h i s l a t e r p ap er t h i s w orker re a r e d a s e p t i c
D r o s o p h ila la r v a e f o r two hundred and t h i r t y g e n e r a t io n s in b r e d i n t h e
dark on h e a t - k i l l e d y e a s t and banana*
H en ce, h e c o n c lu d e s t h a t t h e
a b se n c e o f m icro o rg a n ism s has no e f f e c t on t h e d u r a tio n o f l i f e and
grow th o f t h e f r u i t f l y .
Baumberger (1 9 1 9 ) s t u d ie d t h e form er problem o f Loeb on t h e r e ­
l a t i o n o f m icro o rg a n ism s and t h e i r s u b s tr a ta and t h e i r r o le in th e
n u t r i t i o n o f D r o so p h ila la r v a e .
He u sed t h e s u g a r s , in o r g a n ic s a l t s
(p o ta ssiu m a c id p h o sp h a te and magnesium s u lp h a t e ) , ammonium t a r t r a t e ,
and a g a r -a g a r as a r e a r in g medium*
He o b se rv ed t h a t t h i s medium when
s t e r i l i z e d by a u t o c la v in g su p p o rted t h e grow th o f la r v a e from s t e r i l e
e g g s , b u t th e y l i v e d f o r o n ly f i v e days and showed no in c r e a s e i n s iz e *
Baumberger th e n in o c u la t e d t h e medium w ith l i v i n g y e a s t s and th e la r v a e
grew a t a norm al r a t e ,
p a t in g n o r m a lly .
r e a c h in g t h e i r maximum s i z e
In th e p resen ce o f liv in g y e a s t ,
n it r o g e n was ammonium t a r t r a t e *
i n t e n days and pu­
t h e o n ly so u r ce o f
T h is w orker c o n c lu d e s t h a t s u g a r s ,
s a l t s and t h e n u c le o p r o t e in o f y e a s t a re an ad eq u a te fo o d f o r D r o so p h ila
la r v a e .
H ow ever, a s t h e p r e s e n t a u th o r would l i k e t o p o in t o u t , d u rin g
t h e e r a o f L oeb , N orthrup ( h i s e a r ly w o r k ), and Baum berger, m ost o f t h e
r e s e a r c h w orkers th o u g h t a lo n g t h e l i n e s o f p r o t e in req u irem en ts and
o v er lo o k ed i n many in s t a n c e s t h e im p ortan ce o f t h e a c c e s s o r y grow th
f a c t o r s or v it a m in s , many o f w hich a r e s y n t h e s iz e d by t h e m ic r o s c o p ic
form s o f l i f e *
B acot and Harden (1 9 2 2 ) perform ed some v ery sim p le ex p erim en ts on
t h e v ita m in r eq u ire m e n ts o f D r o s o p h ila *
They a r e p o s s ib l y t h e e a r l i e s t
w orkers t o c o n s id e r v ita m in s and i n s e c t n u t r it io n *
U sin g b u t t e r f a t a s
a so u r c e o f v ita m in A> y e a s t a s a so u r c e o f B , and
lemon j u ic e f o r C>
t h e y s tu d ie d t h e n ee d s o f t h e s e t h r e e f a c t o r s by D ro so p h ila la r v a e
s t e r i l e c o n d itio n s *
under
I t was r e p o r te d by them t h a t developm ent o f a s e p t i c
l a r v a e occu rred in t h e p r e s e n c e o f v ita m in s A and B*
The la r v a e d id n ot
r e q u ir e v ita m in C i n lemon j u i c e f o r t h e i r developm ent under s t e r i l e
c o n d itio n s *
The b a s a l d i e t u sed by t h e s e w o rk ers, w hich c o n s is t e d o f
c a s e in o g e n , s t a r c h , and s a l t s , was a u to c la v e d a lo n g w ith t h e v a r io u s
s o u r c e s o f t h e v ita m in s*
As w i l l be p o in te d out l a t e r by t h e p r e s e n t
w r i t e r , i t seem s odd t h a t grow th co u ld b e o b ta in e d w ith t h e a u to c la v e d
y e a s t s i n c e t h e im p o rta n t f a c t o r th ia m in (B^) i s c e r t a i n l y d e s tr o y e d by
t h e h e a t o f a u t o c la v in g .
G la se r (1 9 2 4 ) d eterm in ed t h e r e la t io n , o f m icroorgan ism s t o t h e
d evelop m ent and lo n g e v i t y o f f l i e s *
Working w ith D r o s o p h ila m e la n o g a s te r ,
Musca d o m e s tic a , Stom oxys c a l c i t r a n s %and L y p e r o sia i r r i t a n s h e found
t h a t t h e c o m p le tio n o f t h e l a r v a l s t a g e o f f l i e s was dependent upon
c e r t a i n a c c e s s o r y grow th f a c t o r s w hich must be in g e s t e d w ith t h e f o o d .
The n e c c e s s a r y grow th s u b s ta n c e s a r e o b ta in e d a c c o r d in g t o D r. G la se r
from b a c t e r ia and y e a s t s , a s w e l l a s from h ig h e r p la n t s and anim al
tis s u e *
He s t e r i l i z e d m ed ia a t h ig h te m p e r a tu r e s and o b serv ed t h a t
t h e s e v i t a l s u b s ta n c e s w ere d e s tr o y e d .
On t h e o th e r han d, c o n ta m in a tin g
t h e m edia w ith l i v i n g b a c t e r i a o r y e a s t s brou gh t ab out norm al grow th
when
in o c u la t e d w ith s t e r i l e e g g s .
T hese s u b s ta n c e s c o u ld a l s o be r e ­
tu rn ed t o c u l t u r a l m edia by ad d in g p la n t j u i c e s o r la r g e q u a n t it i e s o f
dead b a c t e r ia or y e a s t k i l l e d a t lo w h e a t .
G la s e r th e n co n c lu d e s t h a t
"m icroorganism s and t h e i r a c t i v i t i e s a r e n o t a b s o lu t e ly e s s e n t i a l t o
norm al g ro w th , d ev elo p m en t, and lo n g e v it y o f t h e f l i e s i n v e s t i g a t e d . 4*
However t h e m ic r o o r g a n ic f l o r a "may be one o f t h e p r in c ip a l so u r c e s
f o r t h e a c c e s s o r y grow th f a c t o r s o f some l a r v a l f l i e s found b r e e d in g i n
c e r t a in ty p e s o f m edia i n a s t a t e o f n a tu r e but cannot be regard ed as
a proved f a c t . 1* The p r e s e n t au th or w i l l l a t e r show t h a t a l l t h e
n e c e s s a r y grow th f a c t o r s ( a c c e s s o r y ) r e q u ir e d by m osq u ito la r v a e a r e
p resen t in y e a s t.
Boyd (1 9 2 6 ) r e p o r ts t h a t y e a s t s e r v e s as an ad eq u ate fo o d f o r t h e
la r v a e o f A n op h eles q u a d rim acu latu s and A n op heles c r u c ia n s .
Wolim an (19 26) r e a r ed t h e roach B l a t e l l a germ anica f o r tw e lv e
g e n e r a t io n s f r e e from m icro o rgan ism s and f r e e from v it a m in s .
17
He a l s o c la im s t h a t v ita m in s A and B f e d to o th e r in d iv id u a ls had
no e f f e c t i n in c r e a s in g th e grow th r a t e .
Seldom d o e s one re a d i n
th e l i t e r a t u r e such a rem a rk a b le a r t i c l e a s t h i s o n e .
None o f th e
Am erican r e s e a r c h w o r k er s u s in g B l a t e l l a a s a t e s t an im al have come
to t h e s e c o n c l u s i o n s .
R ich a rd so n (1 9 2 6 ) s tu d ie d th e growth o f th e M ed iterran ean f l o u r
moth (E p h e s tia k u e h n ie lla Z .) i n w h ea t f l o u r .
He found t h a t h igh ly-
m ille d w h eat f l o u r w as n o t a good r e a r in g medium f o r the la r v a e o f
t h i s m oth u n l e s s an a l c o h o l i c e x t r a c t o f y e a s t w as ad d ed .
He c la im s
t h a t v ita m in B p r e s e n t i n y e a s t , a l s o i n th e embryo o f th e w h eat
k e r n e l, i s r e q u ir e d f o r norm al d ev e lo p m e n t.
However, th e a d d it io n o f
egg y o lk and b u t t e r a l s o ca u sed an im proved grow th r a t e .
H ence,
Dr. R ich ard son b e l i e v e s t h a t v ita m in A i s l i k e w i s e n eed ed b y t h i s
in s e c t.
E th er and ch lo ro fo r m e x t r a c t e d t h i s s u b s ta n c e from egg y o lk
and b u t t e r .
B arb er (1 9 2 7 ) r e p o r t s on th e fo o d o f A n o p h elin e la r v a e and i n 1928
on th e fo o d o f C u lic in e la r v a e *
A u to c la v e d pond w a ter su p p o rted th e
grow th o f A n o p h elin e l a r v a e f o r one m onth, when th e y d ie d a t th e " h a lf grown" s t a g e .
E it h e r a u to c la v e d pond w a te r or b r o th f a i l e d to y i e l d
pupae under s t e r i l e c o n d i t i o n s .
C u lic in e la r v a e co u ld l i v e f o r tw e lv e
d a y s i n a u to c la v e d b r o th b u t cou ld n o t p u p a te .
However, when th e b r o th
was c o n ta m in a te d , norm al grow th was r e s t o r e d i n b o th groups o f m osquito
la r v a e .
C o n tin u in g h i s ex p er im en ts w it h pure c u lt u r e s o f l i v i n g y e a s t
p lu s th e p r o to z o a , C olp id iu m or a pure c u lt u r e o f th e a l g a , Scenedesm us,
B arber was a b le to r e a r to m a tu r ity A nop h eles c r u c ia n s . A. q u a d rim a cu la tu s»
A edes a r g e n te u s ( a e g y p t i ) , and C u lex f a t ig a n s ♦
He d e f i n i t e l y shows
t h a t m icroorgan ism s a r e im p o r ta n t i n th e norm al l i f e o f m osquito l a r v a e .
18.
Sweetman and Palm er (1 9 2 8 ) i n v e s t i g a t e d th e v ita m in req u irem en ts
o f t h e f l o u r b e e t l e , T rib o liu m confusum , D u v a l.
They u sed a s a b a s a l
d i e t o r r a t io n p u r i f i e d c a s e i n , d e x t r in , and s a lt s *
Removal o f t h e
of
e x t r a c t o f t h e s e m a t e r ia ls p ro lo n g ed growth but t h e o ccu rren ce p u p ation
show£di
t h a t T rib o liu m d id n o t a b s o lu t e ly n eed a f a t - s o l u b l e v ita m in .
However, p r o lo n g e d e x t r a c t io n w ith a lc o h o l p r e v e n te d p u p a tio n w hich
d em o n stra ted a n eed f o r one or more v ita m in s o f t h e B complex*
H ig h ly
p u r i f i e d v ita m in B from y e a s t when added t o t h e a lc o h o l e x t r a c te d
r a t io n r e s u lt e d i n th e p r o d u c tio n o f pupae i n o n e - h a lf t h e p e r io d
n o rm a lly req u ired *
shown*
Hence t h e need f o r one or more o f t h e B v ita m in i s
However f a t o f some n a tu r e a lth o u g h f r e e from v ita m in A d id
sh o r te n t h e l a r v a l p e r io d but t h i s may have been a fo o d f a c t o r .
C lev ela n d (1 9 2 8 ) d e s c r ib e s h i s a d d i t i o n a l o b s e r v a tio n s and e x p e r i­
m ents on t h e s y m b io s is betw een t e r m it e s and t h e i r i n t e s t i n a l p rotozoa*
When t e r m it e s o f t h e g en era R e t i c u l i t e r n e s > C a lc a r it e r n e s , N eo term es,
and K a lo te r n e s w ere t r e a t e d w ith oxygen a t s i x t y pounds p r e s s u r e f o r one
hour a l l o f t h e i r i n t e s t i n a l p r o to z o a w ere rem oved.
The t e r m it e s w hich
were u n in ju red by t h i s p r o c e s s w ere th e n f e d on wood, c e l l u l o s e , c o t t o n ,
and o th e r p r o d u c ts. In no c a s e co u ld th e y s u r v iv e and th e y d ie d o f
s ta r v a tio n .
S p ir o c h a e te s w ere removed by f e e d in g t e r m it e s 5% aqueous
a c id f u c h s in p lu s c e l l u l o s e l e a v in g t h e p ro to zo a i n s i d e th e d i g e s t i v e
tr a c t*
The t e r m it e s were s t i l l a b le t o d ig e s t wood and c e l l u l o s e ; h e n c e ,
s p ir o c h a e t e s p la y no r o le i n t h e d i g e s t io n of wood and c e l l u l o s e .
o th e r hand p r o to z o a a r e in d is p e n s a b le in t h e l i f e
On th e
o f th e t e r m it e s .
R u d o lfs and Lackey (1 9 2 9 ) r e p o r te d some u n u su a l o b s e r v a tio n s o f t h e
e f f e c t o f fo o d upon th e p h o to tr o p h ic b eh a v io r o f m osq u ito la r v a e .
Working
w ith C u lex p i p i e n s . Aed.es s y l v e s t r i s , and Aedes ca n a d e n sis th e y ob served
19*
t h a t so lo n g a s t h e la r v a e o f t h e s e t h r e e m o sq u ito e s f e d on a m ixed
d i e t t h e y were p o s i t i v e l y p h o to tr o p ic *
H ow ever, on a d i e t o f pure
c i l i a t e s t h e s ig n o f t h e i r p h o to tr o p ism was changed w it h in two days from
p o s i t i v e t o n e g a t iv e *
On a d i e t o f d i n o f l a g e l l a t e s , Chlamydomonas o r
E u glen a t h e i r p o s i t i v e s ig n was g r e a t l y i n t e n s i f i e d and rem ained so
u n t i l t h e p u p a tio n p erio d *
MacGregor (1 9 2 9 ) r e a r e d t h e la r v a e o f A edes
a r g e n te u s ( a e g y p t i)
t o m a tu r ity i n 1 0 -1 5 d ays a t 3 0 °G on a s t e r i l e d i e t on o n e -te n th p e r c e n t
" stan d ard bread'* t h a t had b een a u to c la v e d f o r 1 5 -2 0 m in u tes a t 120°C*
T h is exp erim en t h a s b een r e p e a te d b y s e v e r a l w orkers in c lu d in g th e
p r e s e n t a u th o r (u n p u b lish e d M a s te r 's T h e s is ) and no one h as been a b le t o
d u p lic a t e MacGregor*s r e s u l t s .
He c la im s t h a t " b a c te r ia th e m s e lv e s
f u r n is h t h e la r v a e w ith some im p o rta n t a c c e s s o r y fo o d f a c t o r s (v ita m in s ? )
from t h e i r own b o d ie s or produce t h i s f a c t o r by c o n v e r s io n o f th e m a t e r ia ls
o f th e b read " .
He f u r t h e r p o in t s out t h a t p o s s ib ly i n t h e b ak in g o f
ord in a ry b read t h e y e a s t v ita m in s a r e n o t d e str o y e d *
L ik e w ise t h e a u t o -
c la v in g from MacGregor*s v ie w p o in t may have no e f f e c t on th e a c c e s s o r y
foo d f a c t o r or f a c t o r s .
He a r g u es t h a t " it i s q u e s tio n a b le w h eth er v ita m in s
can e f f e c t t h e l i f e o f some i n s e c t s a t l e a s t , s in c e many e x h ib it a
s u c c e s s f u l e x i s t e n c e on such m a t e r ia ls a s p a p e r , s ta r c h p a s t e , spun c o t t o n ,
and s i l k , etc *
H ow ever, i n th e l i g h t o f D r. C le v e la n d 's work on th e r o le
o f p r o to z o a i n t h e d i g e s t i v e t r a c t s o f t e r m i t e s , i t does seem t h a t wood
and pure c e l l u l o s e
f a l l in t o t h e c a te g o r y o u t lin e d by MacGregor*
He f a i l s t o c o n s id e r t h a t b a c t e r i a , y e a s t s , m o ld s, and p r o to z o a a r e c e r ­
t a i n l y p r e s e n t "on such m a t e r ia ls as p a p e r , s ta r c h p a s t e , spun c o t t o n , and
s i l k , e t c ."
M atheson and Hinman (19 29) o b se rv ed t h a t Chara f r a g i l i s was t o x i c t o
*the la r v a e o f C u lex p i p i e n s , Aedes vex a n s , and A nopheles p u n c tip e n n is .
Ghara i s a s i l i c e o u s , u n i c e l l u l a r a l g a , r a r e ly e a te n a s fo o d by a n im a ls .
In a q u a r ia c o n t a in in g Chara t h e s e w ork ers found t h a t la r v a e o f t h e above
m o sq u ito s p e c ie s were k i l l e d in a lm o st 100 p e r c e n t o f t h e i r o b s e r v a t io n s .
W atery s o l u t i o n s c o n t a in in g one h a l f t o one ounce o f d r ie d Ghara t o ev ery
t w e lv e and one h a l l p i n t s o f w a ter w ere a l s o l e t h a l .
T hese n o t a t io n s a r e
v er y i n t e r e s t i n g i n t h a t an a l g a l form o f a d e tr im e n ta l n a tu r e i s r e p o r te d
s in c e m ost a lg a e a r e im p o rta n t i n m osq u ito la r v a e n u t r i t i o n .
Howland (1 9 3 0 ) s tu d ie d t h e r e l a t i o n of a l g a l fo o d t o t h e n u t r it i o n
of m o sq u ito la r v a e .
She d i s s e c t e d t h e la r v a e of Aedes a r g e n te u s ( a e g y p t i)
and fou nd many form s o f a lg a e p r e s e n t and con clu d ed t h a t t h e y form ed an
im p ortan t p a r t o f t h e l a r v a l d i e t .
On t h e o th e r hand she was a b le t o
re a r them on o th e r d i e t s c o n ta in in g no a lg a e *
Thus t h i s worker b e l i e v e s
th a t o th e r o r g a n ic form s assum e t h e r o l e p la y e d by a lg a e in n a tu r e .
T hese o th e r form s w ere u n d o u b ted ly b a c t e r ia and y e a s t s s in c e Howland made
no attem p t t o s t e r i l i z e
e i t h e r m edia or e g g s .
The R u ssia n worker B ek lem ish ev (1 9 3 0 ) by means o f m ic r o d is s e c t io n
te c h n iq u e i n v e s t i g a t e d t h e fo o d o f A n op h elin e l a r v a e .
He r e p o r ts t h a t th e
la r v a e o f A n op h eles m a c u lip e n n is u t i l i z e p a r t i c l e s s m a lle r th a n
o mu
and d e v e lo p a s f a r a s t h e fo u r th i n s t a r on c o l l o i d f r a c t i o n s o f o r g a n ic
su b sta n c e s p r e s e n t i n w a te r .
b r in g about m a tu rity *
T h ese sm a ll p a r t i c l e s were i n s u f f i c i e n t t o
L arvae o f" th e fo u r th i n s t a r u t i l i z e d c o a r s e r
p a r t i c l e s and album inous s u b s ta n c e s .
B ek lem ish ev c o n c lu d e s t h a t c o l l o i d a l
p a r t i c l e s a r e v e r y im p ortan t in t h e n u t r it i o n o f young A n o p h elin e la r v a e
w hich f e e d c h i e f l y by f i l t e r i n g .
B a c t e r ia were p r e s e n t i n th e pond w ater
* 1 mu s 1 m icro n , 0 .0 0 0 2 5 i n c h , 0 .0 0 1 m ill im e t e r .
1 m. mu • *001 m ic ro n , 0 .0 0 0 0 0 2 5 i n c h , 0 .0 0 0 1 m illim e t e r .
21.
and m o sq u ito la r v a e a n a ly z e d by t h i s worker*
S h i p i t z i n a (1930> 35) l i k e w i s e s t u d ie d t h e n u t r it i o n problem o f
m o sq u ito la r v a e a s a f f e c t e d by o r g a n ic c o l l o i d s and u sed fe e d in g e x p e r i­
m ents and m ie r o d is s e c t io n tec h n iq u e *
She f e d f i r s t i n s t a r la r v a e o f
A n op h eles m a c u lip e n n is s o lu t i o n s o f C h in ese in k ( p a r t i c l e s i z e , 1 mu t o
0 . 1 mu i n d ia m e t e r ) , ca rm in e, and m e d ic a l p r e p a r a tio n o f c o l l o i d a l s i l v e r
(d ia m e te r * 2 0 .m. m u).
In h e r l a t e r p ap er sh e r e p o r ts t h e u se o f q u artz
sand o f v a r y in g d e g r e e s o f f i n e n e s s and c o l l o i d a l g o ld and h aem oglob in .
S h i p i t z i n a ' s o b s e r v a tio n s r e v e a le d t h a t t h e f i l t e r i n g a p p a ra tu s o f th e
la r v a e was c a p a b le o f c a tc h in g c o l l o i d a l p a r t i c l e s , a s th e c o n c e n tr a tio n
of t h e s u b s ta n c e s t e s t e d was much g r e a t e r in t h e i r g u ts th an i n t h e w a ter.
P a r t i c l e s o f s o lu b le s ta r c h (5 m. mu.) were n o t f i l t e r e d from th e w a ter
by th e f i r s t i n s t a r l a r v a e .
liVhen w ater was p a sse d th rou gh f i l t e r paper
t h e grow th o f la r v a e i n i t was r e ta r d e d show ing t h a t c o a r s e r p a r t i c l e s
in s u s p e n s io n s a r e im p o rta n t as fo o d .
F i r s t i n s t a r la r v a e f i l t e r e d out
sand p a r t i c l e s from 2 2 .8 mu. t o 3 4 .2 mu. (d ia m e te r o f p a r t i c l e s - 20^
head w id t h ) .
L arvae o f t h e f o u r th i n s t a r were a b le t o f i l t e r p a r t i c l e s
of sand as la r g e a s 68 mu. t o 1 6 5 .3 mu. ( 3 1 .2 ^ d ia m eter o f head w id th ).
C o llo id a l g o ld p a r t i c l e s ( 2 6 - 3 1 .5 m. m u.) and haem oglobin p a r t i c l e s
( 2 - 4 m. mu.) w ere r e c o v e r e d from th e g u ts o f a l l i n s t a r s .
The work o f t h e s e two R u ssia n s w i l l be d is c u s s e d l a t e r as i t m ight a id
in t h e e x p la n a tio n f o r t h e f a c t s t h a t a u t o c la v in g and B e r k e fe ld f i l t e r i n g
o f pond w ater r e n d e r s i t in c a p a b le o f su p p o r tin g growth o f m osq u ito la r v a e
beyond t h e f o u r th i n s t a r .
Hinman (1 9 3 0 , 3 2 ( a ) > 3 2 (b ) » 33) dicL e x t e n s iv e r e s e a r c h on th e fo o d o f
m o sq u ito la r v a e .
u tiliz a tio n
He s tu d ie d t h e r o l e s of m ic ro o rg a n ism s, s o l u t e s , and th e
of w a ter c o l l o i d s .
From d i s s e c t i o n s o f t h e la r v a e o f e ig h t
s p e c i e s o f A e d e s , s i x s p e c ie s o f C u lex , A n op h eles p u n c t ip e n n is ..
T h e o b a ld ia d y a r i , Th. i n o m a t a , and Wycomyia s m i t h ii he found t h a t " d i f ­
f e r e n t s p e c ie s o f m o sq u ito la r v a e ( w it h in t h e same g en era ) from th e same
h a b it a t n o rm a lly d i g e s t about t h e same m a te r ia l" *
He f u r t h e r ob served
t h a t t h e la r v a e a r e n o t s e l e c t i v e i n t h e i r f e e d in g but sweep a c o n s ta n t
stream o f w a ter c o n t a in in g organ ism s and o th e r m a t e r ia ls in t o t h e i n t e s ­
t in a l tr a c t*
Prom inent among t h e b i o t a were V o r t i c e l l a * c i l i a t e s ,
T rach elm on as» E u g le n a » r o t i f e r s , many f ila m e n to u s g reen a l g a e , u n i c i l l u l a r
green a l g a e , d e sm id s, d ia to m s , b lu e g reen a l g a e , and c r u sta c e a *
Hence
Hinman i s o f t h e o p in io n t h a t m o sq u ito e s cannot be c o n t r o lle d by th e
e lim in a t io n o f l a r v a l food*
on s t e r i l e m edia*
He th e n t r i e d t o r e a r la r v a e o f Aedes a e g y p t i
A u to cla v ed pond w a ter i f uncon tam in ated f a i l e d t o
sup p ort t h e grow th o f t h e la r v a e o f t h i s s p e c ie s beyond th e t h ir d in s t a r *
On th e o th e r hand many o f th e co n tam in ated c u lt u r e s su p p orted growth
throu gh p u p a tio n and em ergence.
S im ila r r e s u l t s were o b ta in e d w ith
Culex p ip ie n s a s t h e t e s t anim al*
As Hinman s t a t e s " a u to c la v in g s o f w ater
from b r e e d in g p o o ls d e s tr o y e d c e r t a in elem en ts n e c e s s a r y f o r la r v a e t r a n s ­
form ation"*
L ik e w ise b a c t e r i a l c e l l s k i l l e d c h e m ic a lly f a i l e d t o support
grow th under s t e r i l e c o n d it io n s .
With B e r k e fe ld W or N f i l t e r e d pond
w a ter h e was a b le t o r e a r t o m a tu r ity sm a ll numbers o f Aedes a e g y p ti under
a s p p t ic c o n d itio n s *
R ep eated ex p erim en ts w ith s p e c ie s o f C ulex or
A n op h eles r e s u lt e d i n f a i l u r e t o o b ta in a d u lt s i n s t e r i l e B e r k e fe ld f i l t e r e d
pond w a te r .
In some l a t e r ex p erim en ts (1932) Hinman s tu d ie d d ia ly z e d w ater
t h a t was s t e r i l i z e d by f i l t r a t i o n .
D uring d i a l y s i s m ost o f th e c o l l o i d s
a r e removed from th e medium and t h e d ia ly z e d pond w ater f a i l e d t o support
th e grow th o f C u lic id la r v a e *
A n op h elin e la r v a e l i v e d f o r e le v e n days in
S eitz -W erk a f i l t e r e d pond w a te r w hereas i n pure s o lu t io n th e la r v a e cou ld
s u r v iv e f o r o n ly t h r e e days*
f o r s i x days*
C o llo id s o f sm a ll m o le c u le s sup p orted grow th
The co m p lete a b se n c e o f s o lu t e s cau sed d ea th a lth o u g h on a
s o l u t e d i e t a lo n e t h e la r v a e l i v e d f o r t h e s h o r t e s t p e r io d s in a l l o f th e
e x p e r im e n ts .
The p r e s e n t w r it e r a t t h i s tim e would l i k e t o b r in g out th e
f a c t t h a t a c c e s s o r y grow th f a c t o r s a r e o f te n w ater s o lu b le and hence
s o lu t e s *
At th e same tim e t h e r e i s t h e p o s s i b i l i t y t h a t Hinman may have
removed a l l o f h i s s a l t s i n th e d i a l y s i s o p e r a tio n s*
W hite (1 9 3 1 ) r e p o r t s t h a t s t e r i l e m aggots f o r s u r g ic a l u se can be
rea r ed on a u to c la v e d g l u c o s e , p e p to n iz e d a g a r , and meat*
Hobson (1 9 3 1 , 3 2 ( a ) > 3 2 ( b ) » 3 2 ( e ) ’ 3 3 * 3 5 ( a ) » 3 5 ( b ) » 3 3 ( c ) ) h as con~
t r ib u t e d much t o th e f i e l d o f i n s e c t n u t r i t i o n and m etab olism in a s e r i e s
o f p ap ers on th e p h y s io lo g y o f b lo w fly la r v a e *
In an e a r ly paper (32)
he r e p o r te d t h a t b lo w fly ( L u c i l ia ) la r v a e c o u ld be reared on s t e r i l e b ra in
(g u in e a p ig ) mush and th a t th e r a t e o f grow th was th e same a s on i n f e c t i v e
tis s u e .
T h e r e fo r e he co n clu d ed t h a t m icroorgan ism s p la y ed no p art in
i n t e s t i n a l d ig e s t io n *
Hobson a l s o n o t ic e d t h a t when s t e r i l e unheated
m u scle was u sed as a r e a r in g medium f o r a s e p t i c la r v a e t h e i r growth r a t e
was d ecreased *
When t h i s m u scle was in f e c t e d w ith B a c illu s c o l i norm al
grow th was r e s to r e d *
same th in g *
The a d d it io n o f y e a s t e x t r a c t t o th e m u scle d id th e
T h is w orker th e n con clu d ed t h a t some growth prom oting f a c t o r
was la c k in g in s t e r i l e m u s c le .
He th en s tu d ie d th e growth o f b lo w fly la r v a e
on b lo o d w hich a l s o was d e f i c i e n t in growth su b stan ce*
S t e r i l e h o r se
b lood was a b so rb ed on s t e r i l e c o tto n and a llo w e d t o c o a g u la te *
Hobson
found t h a t f o r norm al grow th t o o ccu r under a s e p s is w ith t h i s b lo o d d i e t ,
a t l e a s t t h r e e grow th f a c t o r s must be added!
aqueous or a l c o h o l i c e x t r a c t s o f y e a s t s ,
a su b sta n c e a b se n t from
b u t p r e s e n t in a s o lu b le form in
y e a s t a u t o l y s a t e , a h e a t - l a b i l e f a c t o r which co u ld be s u p p lie d by P e t e r s
" a n t i n e u r i t ic c o n c e n tr a te 1*, a h e a t s t a b le f a c t o r p r e s e n t i n a u to c la v e d
y e a s t e x tr a c t*
In t h e a n a l y s i s o f t h e r e s u l t s o f ex p erim en ts on m osq u ito
la r v a e t h a t a re t o be p r e s e n te d l a t e r , t h e p r e s e n t w r it e r w i l l d em o n strate
a t l e a s t t h r e e f a c t o r s p r e s e n t i n y e a s t s im ila r t o t h o s e d e s c r ib e d by
H obson.
A cco rd in g t o Hobson " t e s t s w ith a u to c la v e d y e a s t have shown t h a t
b lo w fly la r v a e r e q u ir e a h e a t - l a b i l e f a c t o r which i s jprobably a n a lo g o u s
t o members o f t h e v ita m in B com plex and th e y may a l s o be i d e n t i c a l w ith
v ita m in s r e q u ir e d by h ig h e r a n im a ls" .
One o f t h e f a c t o r s p r e s e n t i n y e a s t
i s v e r y r e s i s t a n t and s t a b l e in t h e p r e s e n c e o f b oth h ea t and a l k a l i s t h e
o th e r s e n s i t i v e t o a l k a l i and somewhat l a b i l e t o lo n g p e r io d s o f h e a tin g *
Hobson d is c o v e r e d a n o th e r f a c t o r r e q u ir e d by b lo w fly la r v a e , >a f a t s o lu b le
su b s ta n c e o f w hich i n an e a r l i e r pap er he s t a t e s t h a t " th e d i s t r i b u t i o n
o f th e b lo w fly f a c t o r shows t h a t i t i s n o t v ita m in A, D , or Ew.
However
he l a t e r shows t h a t t h i s f a t s o lu b le f a c t o r i s c h o l e s t e r o l w hich i s
c h e m ic a lly r e la t e d t o v ita m in D*
The e x t e n s iv e work o f t h i s E n g lis h i n s e c t
p h y s i o l o g i s t has been a g r e a t a id to t h e p r e s e n t w r it e r i n h i s ex p erim en ts
on t h e v ita m in req u irem en ts o f m o sq u ito la r v a e *
M ic h e lb a c h e r , H o s k in s , and Herms (1 9 3 2 ) i n v e s t i g a t e d t h e adequacy of
s t e r i l e s y n t h e t ic d i e t s i n t h e n u t r i t i o n o f th e f l e s h f l y l a r v a , L u c ili a
s e r ic a ta *
T hese w ork ers r e a re d b lo w fly la r v a e under a s e p t ic c o n d it io n s on
p u r i f i e d c a s e i n , brew ers y e a s t pow der, F le ish m a n n 's y e a s t (bakerssjl b u tt e r
f a t s or d o d - liv e r o i l ,
s a l t m ix tu r e , c y s t i n e , and agar*
The a d d it io n o f th e
amino a c id c y s t i n e had a rem arkable e f f e c t in in c r e a s in g t h e numbers o f
pupae*
te n t.
I t can be n o ted t h a t c a s e in i s a p r o t e in v ery low in c y s t i n e con ­
C y s tin e or r e la t e d s u lp h u r -c o n ta in in g amino a c id s i s a v ery im por­
t a n t su b s ta n c e in t h e n u t r i t i o n o f h ig h e r an im als*
Cook and S c o t t (1 9 3 3 ) r e p o r te d t h a t t h e t e r m it e Zo o te r m o p sis
a u g u s t i c o l l i a must h ave i n i t s d i e t v ita m in s A, B, D, and G. They fed
c o n s is tin g
t h i s t e r m i t e on a d i e t o f s u c r o s e , c a s e i n , C r is c o , s a l t s , and agar g e l .
V itam in s A and D w ere g iv e n as t h e n o n - s a p o n if ia b le p o r t io n s o f cod l i v e r
o i l and B and G i n r i c e p o l i s h i n g s .
The fo o d and c o n ta in e r s w ere changed
d a ily t o i n h i b i t t h e grow th o f b a c t e r ia and m o ld s.
I t must be remembered
t h a t t h e s e w orkers d id n o t c o m p le te ly c o n t r o l t h e e f f e c t s o f m icroorgan ism s
(cQmjblete s t e r i l i t y ) n o r d id t h e y u se c h e m ic a lly pure m a t e r ia ls (v it a m in s ) *
H oog, v a n 't (1 9 3 5 ,3 6 ) s tu d ie d t h e e f f e c t of v ita m in s on t h e a s e p t ic
c u ltu r e o f D r o so p h ila m e la n o g a s te r .
He c la im s t h a t t h e l i f e h i s t o r y of t h i s
s p e c ie s can be com p leted in t h e a b sen ce o f v ita m in s A, D, and E .
However
D r o so p h ila la r v a e need t h e n o n - s a p o n if ia b le f r a c t i o n o f f a t s in t h e i r food*
The a c t i v e p r i n c i p l e a c c o r d in g t o t h i s worker was c h o l e s t e r o l f o r w hich t h e
f o llo w in g c o u ld be s u b s t i t u t e d :
e r g o s t e r o l i but i t
lu m i,s t e r o l.
s i t o s t e r o l , p h y t o s t e r o l , s t i g m a s t e r o l, or
c o u ld n o t be r e p la c e d in th e food by c a l c i f e r o l or
A d i a l y s a t e o f y e a s t e x t r a c t c o n ta in s a l l o f t h e n e c e s s a r y
w ater s o lu b le f a c t o r s *
I r r a d ia t io n o f th e d i a l y s a t e by means o f v i s i b l e
l i g h t ren d ered i t i n a c t i v e and t h e a d d it io n o f l a c t o f l a v i n p a r t i a l l y r e s to r e d
th e a c t iv it y .
V an 't Hoog i n f e r s t h a t t h e v ita m in s B^, Bg» and o th e r w ater
s o lu b le f a c t o r s of y e a s t a r e r e q u ir e d by D r o so p h ila la r v a e *
N e lso n and Palm er (1 9 3 5 ) d eterm in ed t h e phosphorus c o n te n t o f th e e g g s ,
la r v a e , p u p ae, and a d u lt s o f T rib o liu m confusum and s tu d ie d th e need f o r
v ita m in D.
A n a ly s is showed t h a t phosphorus c o n te n t o f th e e g g s , la r v a e ,
p u p a e, and a d u lt s was Q.445/£, 0 * 4 5 6 ^ , Q.522J&, 0*598/£ o f t h e d r ie d w eig h t
r e s p e c tiv e ly .
They a l s o o b served t h a t v ita m in D was n o t needed f o r any
p h y s i o l o g i c a l fu n c tio n t h a t co u ld be d e t e c t e d by le n g th t>f tim e t o p u p a tio n ,
a l s o t h a t th e phosphorus c o n te n t o f t h e pupae was in no way e f f e c t e d by
26.
v ita m in D*
S t r e e t and Palm er (1 9 3 5 ) s t u d ie d t h e r eq u irem en ts o f T rib o liu m confusum
f o r v ita m in s o f B group*
They u se d a b a s a l d i e t c o n s is t in g o f 15 p a r t s o f
c a s e i n , 3 p a r t s o f C r is c o , 4 p a r t s O sborne-m endel s a l t m ix tu r e , and 10 p a r t s
of d e x tr in .
A 5 gram p o r t io n o f t h e b a s a l d i e t was m ixed w ith t h e v a r io u s
v ita m in req u irem en t su p p lem en ts and p la c e d i n a v i a l w ith tw en ty tw o -h o u r
o ld l a r v a e .
A l l v i a l s w ere in c u b a te d a t 32°G and 10% h u m id ity and a v er a g e
tim e o f p u p a tio n record ed *
p u p a tio n r e s u lt e d *
p u p a tio n r e s u lt e d *
In t h e p r e s e n c e o f a v ita m in
c o n c e n tr a te no
L ik e w is e i n t h e p r e s e n c e o f 5% a u to c la v e d y e a s t no
On t h e o th e r hand when t h e s e w orkers m ixed a v ita m in
c o n c e n tr a te (2% r i c e p o l i s h ) and 1% a u to c la v e d y e a s t t h e a v era g e p u p a tio n
tim e was 192 d a y s.
Thus i t
can be seen t h a t a h e a t - l a b i l e f a c t o r (B^) and
a h e a t s t a b l e f a c t o r a r e r e q u ir e d by t h e la r v a e o f t h e c o n fu sed f l o u r
b e e tle .
S t r e e t and Palm er a l s o o b se rv ed t h a t a t a pH* o f 13 t h e h e a t s t a b le
f a c t o r was d e s tr o y e d by fo u r h ou rs o f a u t o c la v in g .
Rozeboom (1 9 3 5 ) s t u d ie d t h e r e l a t i o n o f b a c t e r ia and b a c t e r i a l f i l t r a t e s
t o t h e developm ent o f m o sq u ito la r v a e *
He found t h a t t h e f i l t e r i n g o f pond
w a ter by means o f a B e r k e fe ld f i l t e r f a i l e d t o support t h e grow th of Aedes
a e g y p t i , C ulex p i p i e n s » C* t e r r i t a n s , and C* s a li n a r i u s under a s e p t i c con­
d it io n s *
When t h e same f i l t e r e d pond w a ter was in o c u la t e d w ith a m ixed c u l ­
t u r e o f E s c h e r ic h ia c o l i , B a c i l l u s s u b t i l i s , and B* m egatherium i t was n o tic e d
t h a t 144 la r v a e o f A* a e g y p t i y ie ld e d 60 fo u r th i n s t a r w ater la r v a e and 4
a d u lts .
'With t h i s same m ix tu r e 230 f i r s t i n s t a r la r v a e o f C ulex sp p . f a i l e d
t o e n te r t h e secon d in s t a r *
On t h e o th e r hand a con tam in ated bread m ixtu re
produced 90 a d u l t s , 102 f o u r th i n s t a r la r v a e , out o f 147 in o c u la t e d f i r s t
i n s t a r la r v a e o f Aedes a e g y p t i .
Both e x t r a - c e l l u l a r and i n t r a - c e l l u l a r
e x t r a c t s o f E* c o l i and B* s u b t i l i s were p rep ared and added by means o f th e
s t e r i l e B e r k e fe ld f i l t e r t o a medium composed o f a u to c la v e d dove f e c e s in
w a te r .
Growth c o u ld n o t p ro ceed on e i t h e r o f t h e s e d ie t s *
Hence Rozeboom
c o n c lu d e s t h a t t h e grow th f a c t o r s w ere n o t i n t r a or e x t r a - c e l l u l a r p ro d u cts
o f b a c t e r i a l m e ta b o lism .
Pond w a ter when a u to c la v e d gave n e g a t iv e r e s u l t s
but when co n ta m in a ted produced good grow th .
T ra g e r (1 9 3 5 a , 3 5^ , 3 6 , 37) and T r a g e r , M i l l e r , and Rhoads (1938) have
i n v e s t i g a t e d th e grow th s u b s ta n c e s r e q u ir e d by th e la r v a e o f Aedes a e g y p ti
under s t e r i l e c o n d it io n s .
T rager in h i s e a r l i e r work d em on strated t h a t th e
grow th o f t h i s m o sq u ito t o m a tu r ity c o u ld occu r on a s t e r i l e medium com­
p osed o f v it a m in - f r e e c a s e i n , s a l t s , and e x t r a c t s from E l i L i l l y l i v e r
e x t r a c t s No. 343 and H a r r is y e a s t e x tr a c t*
I t was con clu d ed by Trager th a t
tw o a c c e s s o r y grow th f a c t o r s , one i n l i v e r , and a n o th e r in y e a s t were r e ­
q u ire d by m o sq u ito la r v a e f o r normal d evelop m en t.
F a c to r A* in l i v e r ,
was found t o be s o lu b le i n w a ter and d i l u t e a l c o h o l , h ea t s t a b l e , but was
d e s tr o y e d by a l k a l i and ab sorbed by f u l l e r ' s
e a rth .
shown t o be p r e s e n t i n s m a ll q u a n t i t ie s i n y e a s t .
T h is f a c t o r was l a t e r
F a c to r B , found in y e a s t
and y e a s t e x t r a c t , a l s o in egg w h ite and w h ea t, was both h ea t and a l k a l i
s t a b l e and was n o t ab sorbed by f u l l e r ' s e a r th .
L a te r Trager rea red mos­
q u it o la r v a e on t h e y e a s t e x t r a c t m a t e r ia l w ith o u t t h e l i v e r e x tr a c t*
H owever, th e c o n c e n tr a tio n o f t h e y e a s t had t o be g r e a t ly in c r e a s e d in
o rd e r t o sup p ort grow th w ith o u t t h e l i v e r e x tr a c t*
v ita m in
When l^ercks c r y s t a l l i n e
was a u to c la v e d and u sed t o r e p la c e th e y e a s t e x t r a c t th e la r v a e
d ie d i n t h e f i r s t or secon d in s t a r *
H ow ever, when s t e r i l i z e d by f i l t r a t i o n
th ro u g h a S e i t z f i l t e r t h e la r v a e co u ld rea ch th e t h ir d i n s t a r , but none
rea ch ed th e fo u r th .
r e q u ir e d .
At t h e same tim e r e l a t i v e l y la r g e amounts o f
were
T rager d e f i n i t e l y s t a t e s t h a t v ita m in B^ was n o t t h e l i v e r growth
f a c t o r r e q u ir e d by m osq u ito la r v a e *
The a b i l i t y o f la r v a e t o u t i l i z e s o lu t e s
was a l s o i n v e s t i g a t e d by ’t h i s w ork er.
He found t h a t la r v a e o f A edes a e g y p t i
c o u ld e x i s t on s o l u t e s a lo n e a s f a r a s t h e f o u r t h i n s t a r .
Calcium io n s had
t o b e p r e s e n t i n t h e s o l u t e d i e t , i n o rd er f o r t h e la r v a e t o rea ch th e
f o u r t h in s t a r *
T r a g e r , M i l l e r , R hoads, (3 8 ) o b se rv ed t h a t e x t r a c t s p rep ared
from t h e u r in e o f norm al i n d i v i d u a ls o r p a t ie n t s w ith a p l a s t i c anem ia
c o n ta in e d a s u b s ta n c e w h ich “en hances t h e grow th o f t h e la r v a e o f th e mos­
q u i t o , A edes a eg y p ti* *.
T h is s u b sta n c e a c c o r d in g t o them , i s p o s s i b l y a
f l a v i n e or a f l a v i n e compound.
I t i s “la c k in g or i s p r e s e n t i n s m a lle r
q u a n t i t i e s or e x t r a c t s from th e u r in e o f p e r n ic io u s anem ia p a t ie n t s show ing
sym ptons o f t h e d i s e a s e " .
H ow ever, when t h e s e same p a t ie n t s w ere g iv e n
a d eq u a te t r e a tm e n t , t h e u r in e th e n c o n ta in e d a s much o f t h e su b s ta n c e a s
norm al u r in e e x t r a c t s .
F r o s t , Harms*, and H osk in s (1 9 3 6 ) h ave s t u d ie d th e n u t r i t i o n a l r e q u ir e ­
m ents o f t h e la r v a e o f th e m o sq u ito , T h eo b a ld ia in c id e n s ThoijcU
T h ese
w ork ers fo u n d t h a t y e a s t (F leisehm ann* s ) was an a d e q u a te fo o d f o r t h e l a r v a e .
The m ost f a v o r a b le c o n c e n t r a tio n was 2—2 .5 m illig r a m s p e r cc*
B rew ers
y e a s t i n a c o n c e n t r a tio n o f 1 m illig r a m p er c c . was a l s o s a t i s f a c t o r y .
T h eo b a ld ia in c id e n s was r ea r ed t o m a tu r ity un der a s e p t i c c o n d i t i o n s , b u t
m ost o f t h e ex p erim en ts w ere made under non s t e r i l e c o n d it io n s by a d a i l y
t r a n s f e r o f th e la r v a e t o f r e s h f o o d s .
T e c h n ic a l and p u r i f i e d c a s e i n a lo n e
proved t o be in a d e q u a te a s r e a r in g m edia but were im proved by t h e a d d i t i o n
o f c e r t a i n in o r g a n ic s a l t B .
The a d d it io n of d r ie d y e a s t t o v ita m in f r e e
c a s e i n sup p orted t h e grow th o f la r v a e t o m a tu r it y .
The e s s e n t i a l grow th
f a c t o r c o n tr ib u te d by y e a s t a c c o r d in g t o t h e s e w orkers was th e r m o s ta b le
and n o t f a t s o lu b le and was th o u g h t t o be v ita m in G or a member o f t h e G
com plex*
“P r o te in s a r e by f a r t h e m ost im p o rta n t c l a s s o f fo o d s t u f f s , and
c a r b o h y d r a te s and f a t s a r e r e q u i r e d ,i f a t a l l , i n s m a ll am ounts", a c c o r d in g
t o t h e s e w o r k e r s.
They a l s o c la im t h a t v ita m in s A, B , C, D, and E d id n ot
ap p ear t o be n eed ed f o r th e d evelop m en t o f t h i s i n s e c t .
The te c h n iq u e o f F r o s t , H e m s , and H osk in s was ad op ted fo r two e x p e r i­
m ents s in c e i t
o c c a s io n s much l e s s l o s s o f tim e b eca u se o f co n ta m in a tio n
and c o n se q u e n t n e c e s s i t y o f r e p e a tin g e x p e r im e n ts.
th a t i t s
H owever, i t i s l i k e l y
r e s u l t s a r e n o t so a c c u r a te as t h o s e o b ta in e d by th e u se o f s t e r i l e
m ed ia , s in c e m icro o rg a n ism s may i n 24 h ou rs produce s u f f i c i e n t v ita m in s to
v i t i a t e t h e r e s u lt s *
J a co b s and R aichoudhury (1 9 3 7 ) rea red t o m a tu r ity E p h e s tia
(M ed iterra n ea n f l o u r moth) under a s e p t i c c o n d ito n s .
k u e h n ie lla Z.
They u sed w holem eal
f l o u r t h a t had been k ep t a t 1 8 0 °C f o r two hou rs and no b a c t e r i a l growth was
o b se r v e d .
Eggs o f t h i s moth were immersed in 0*1^ m ercu ric c h lo r id e s o lu t io n
f o r t e n m in u tes and in o c u la t e d i n t o t h e medium.
T hese workers n o ted t h a t
grow th was slo w e r and t h a t em ergence o f th e f i r s t moths occu rred fo u r days
l a t e r on t h e s t e r i l i z e d m ed ia , th a n on th e same unheated media*
I t was
cla im ed by them t h a t t h e s lo w in g o f th e growth r a t e was p robab ly due t o th e
d e s t r u c t io n o f t h e A and B v it a m in s .
Rakhmanova (1 9 3 7 ) i n v e s t i g a t e d th e r o l e o f b a c t e r ia in t h e growth o f
t h e la r v a e o f A n op h eles m a c u lip e n n is .
In s u s p e n s io n s of B a c illu s f lu o r e s c e n s
t h e la r v a e g r a d u a lly d ie d o f f i n t h e co u rse o f t h e i r d ev elo p m en t.
H owever,
i n pure c u lt u r e s o f B* c o l i i n s t e r i l i z e d ta p w a te r , norm al growth o f la r v a e
o c c u r r e d , and a d u lt s w ere o b ta in e d i n h ig h c o n c e n tr a tio n s o f t h e b a c t e r ia .
He th o u g h t t h a t t h e medium was n o t s u f f i c i e n t l y n o u r is h in g to a llo w th e
b a c t e r i a t o in c r e a s e i n a p p r e c ia b le numbers and t o become " t o x ic ” .
In to o
low a b a c t e r i a l c o n c e n t r a t io n , t h e larva© d ie d o f h u n ger.
Melampy and Maynard (1 937) s tu d ie d th e n u t r it i o n o f th e c o c k r o a c h ,
B l a t e l l a g erm an ica.
They found d i e t s o f m ilk and m ilk p ro d u cts in a d e q u a te
f o r th e grow ing ro a ch .
Ground wheat and d r ie d skim m ilk or ground wheat and
m eat w ere v e r y s u i t a b l e d i e t s .
An e t h e r e x t r a c t o f m ilk w hich w ith sto o d
s a p o n i f i c a t i o n produced a grow th s t im u la t in g s u b s ta n c e .
c o u ld n o t be s u b s t i t u t e d f o r t h e o i l ( f a t ) from w h eat.
Lard or b u tte r o i l
A v ita m in o f th e
B com p lex w hich was h e a t and a l k a l i s t a b l e was a l s o r e q u ir e d .
C ro w ell and McCay (1 9 3 7 ) r e p o r t t h a t t h e webbing c lo t h e s m oth,
T in e o la b i s s e l l i e 11a r e q u ir e s p a r t o r a l l o f th e v ita m in B complex*
t h e f a t s o lu b l e v ita m in s were n o t n eed ed by t h i s in s e c t *
F a t and
They a l s o r e p o rt
t h a t p u r i f ie d c a s e in was n o t an ad eq u ate p r o t e in f o r optimum d evelop m en t,
b u t when p u r i f i e d la c ia lb u m in was ad d ed , norm al grow th o f T in e o la r e s u lte d *
C y s tin e co u ld n o t r e p la c e la c ta lb u m in in t h e d ie t *
B u sn e l (1 9 3 7 ) i n an a n a to m ic a l and p h y s i o l o g i c a l stu d y o f th e la r v a e
o f E p h e s tia k u e h n ie lla r e p o r t s t h a t t h e la r v a e co u ld n o t m ature on f l o u r
w hich had i t s
v ita m in s removed by trea tm en t w ith X -ra y s or h e a t .
M.cCay (1 9 3 8 ) s tu d ie d t h e n u t r i t i o n a l req u irem en ts o f B la t e 11a
g e r a a n ic a *
He s t a t e s t h a t " th e combined e v id e n c e from a l l o f t h e s e e x p e r i­
m ents in d ic a t e d t h a t t h i s s p e c ie s has l i t t l e i f any n eed f o r v ita m in s A
and D, but t h a t t h e r e i s a requ irem ent f o r some o f t h e f a c t o r s o f y e a s t " •
The y e a s t f a c t o r s were i n p a rt s o lu b le i n w ater and i n p a r t i n t h e f a t
s o lv e n ts .
B u s h n e ll (1 9 3 8 ) r e p o r ts t h a t th e la r v a e o f T rib o liu m confusum r e q u ir e
t h e a d d it io n o f y e a s t t o t h e i r cornm eal r e a r in g m edia f o r a h ig h p e r c e n ta g e
o f em ergen ce.
At th e same tim e th e d ev elo p m e n ta l p e r io d was red uced by
t h i r t e e n days when y e a s t was added t o t h e cornm eal d i e t .
Ludwig and Fox (1 9 3 8 ) s tu d ie d t h e grow th and s u r v iv a l o f Jap an ese
b e e t l e la r v a e in d i f f e r e n t media*
They ob served t h a t w heat or y e a s t , when
added t o s o i l s c o n t a in in g b e e t l e la r v a e and p la n t m a t e r i a l s , in c r e a s e d th e
numbers o f s u r v iv in g b e e t l e s , shortened th e m etam orphic p e r io d , and produced
i n d i v i d u a l s o f la r g e r s i z e .
The f a c t s were a t t r ib u t e d by Ludwig and Fox
t o th e p r e se n c e o f a c c e s s o r y food f a c t o r s b e lo n g in g to t h e v ita m in B com plex
in wheat and y e a s t •
G la s e r (1 9 3 8 ) r e v e a le d th a t th e common h o u s e f l y , Musea d o m e s tic a ,
co u ld be rea red under s t e r i l e c o n d ito n s on a medium composed o f a u to c la v e d
y e a s t , l i v e r , w a te r , and sa w d u st.
C h iu , S h in Foon and McCay (1 9 3 9 ) have c o n tin u e d th e stu d y o f th e
n u t r i t i o n a l req u irem en ts o f th e co n fu sed f lo u r b e e t l e , T rib oliu m confusum
Duv*
T hese w orkers a l s o s tu d ie d th e food req u irem en ts o f th e bean b e e t l e ,
A c a n t h o s c e lid e s o b te c tu s Say*
developm ent o f T r ib o liu m .
A h ig h p r o t e in d i e t was needed f o r th e
A p u r if ie d d i e t composed o f c a s e i n , corn s t a r c h ,
y e a s t , c o t to n seed o i l , and G sborne-M endel S a lt m ix tu re was s u f f i c i e n t f o r
norm al d ev elo p m e n t.
V itam in s
add G ( l a c t o f l a v i n ) a lo n e did not f u l f i l l
t h e r eq u ir em en ts f o r th e grow th o f T rib o liu m .
They con firm ed th e p r e v io u s
work o f McCay, t h a t th e f a t s o lu b le v ita m in s A and D were not needed by th e
c o n fu se d f lo u r b e e t l e .
One or more a d d it io n a l f a c t o r s p r e s e n t i n y e a s t
were e s s e n t i a l f o r th e growth o f t h i s b e e t l e .
T rib o liu m la r v a e grew
n o rm a lly when f e d d e x tr o s e as t h e i r o n ly so u r ce o f ca rb o h y d ra te.
METHODS
The r o l e o f m icro o rg an ism s i n th e n u t r i t i o n and m eta b o lism o f
i n s e c t s h a s b e e n c o n s id e r e d i n th e p o r t io n o f t h i s t h e s i s d ev o ted
t o t h e r e v ie w o f l i t e r a t u r e *
I t can b e r e a d i l y s e e n t h a t f o r
s i g n i f i c a n t r e s u l t s e x p e r im e n ts m ust b e con d u cted on s t e r i l e m edia
in o c u la t e d w it h e i t h e r s t e r i l e e g g s o r s t e r i l e la r v a e *
P r e lim in a r y e x p er im en ts w ere co n d u cted b y the p r e s e n t w r it e r
t o d ete rm in e a s u c c e s s f u l te c h n iq u e f o r s t e r i l i z i n g th e eg g s o f
m o sq u ito es*
"White (1 9 3 1 ) and T rager (1 9 3 5 ) recommended a s o lu t io n
composed o f m e rcu ric c h lo r id e 0*25 gram, sodium c h lo r id e 6*5 gram s,
h y d r o c h lo r ic a c i d 1 .2 5 c c * , e t h y l a lc o h o l (9 5 /0 250 c c * , and d i s ­
t i l l e d w a te r 7 5 0 cc*
They exp osed the e g g s f o r p e r io d s o f f i v e to
t e n m in u te s and t r a n s f e r r e d them b y means o f a s t e r i l e lo o p or s t e r i l e
p i p e t t e t o th e v a r i o u s m edia*
O ther w o rk ers have u se d B0% a lc o h o l ,
0 .1 $ aqueous m ercu ric c h l o r i d e , and 5$ fo rm a lin *
The a u th o r ad op ted
th e te c h n iq u e d e s c r ib e d b y Hinman (1 9 3 2 ) and l a t e r u se d b y Rozeboom
( 1 9 3 5 )•
Eggs w ere submerged i n a d i l u t e so a p s o lu t io n f o r f i v e
m in u te s , r in s e d i n s t e r i l e w a te r , and th e n immersed i n a 1 /1 0 0 0
d i l u t i o n o f h e x y l r e s o r c i n o l (com m ercial S .T .3 7 ) f o r a p e r io d o f
two m in u tes*
A ll m ethods have two w e a k n e s s e s , t h e d i f f i c u l t y o f o b ta in in g
s t e r i l e c u l t u r e s and o f o b ta in in g a r e a s o n a b le p e r c e n ta g e o f hatch*
Many tu b e s had t o b e d is c a r d e d b e c a u s e o f co n ta m in a tio n *
The
m o sq u ito egg p r e s e n t s a r e l a t i v e l y l a r g e s u r fa c e and p e n e t r a t io n
o f t h e d i s i n f e c t a n t th rou gh th e c h o r io n k i H s t h e em bryo.
The
a u th o r c o n s id e r e d 6 0 -8 0 $ h a tc h o f t h e t r e a t e d e g g s f r e e from m icro­
o rg a n ism s v e r y fo r tu n a t e *
S t e r i l i t y was ch eck ed e i t h e r b y p la t in g
a p o r t io n o f th e r e a r in g m edia on P e t r i —d is h e s on sta n d a rd b a c t e r io ­
l o g i c a l n u t r i e n t a g a r or b y making s t r e a k s on a g a r p l a n t s .
A n o th er problem i n te c h n iq u e was th e m a tte r o f egg m anipula­
tio n .
The m ethod o f MacGregor (1 9 2 9 ) o f u sin g g l a s s "boats" proved
t o b e t h e m ost f e a s i b l e .
G la ss c o v e r s l i p s w ere c u r le d i n th e flam e
o f a Bunsen b u r n e r and e g g s w ere p i p e t t e d i n t o t h e s e “b o a ts" w here
t h e y ad h ered t o th e in n e r s u r f a c e s .
m a n ip u la ted w it h s t e r i l e f o r c e p s .
The "boats" w ere e a s i l y
The r a f t - l i k e egg m asses o f
C u lex p i p i e n s had t o b e t e a s e d a p a r t and th e e g g s sep a ra ted # .
o f A edes a r e l a i d s i n g l y and a re more r e a d i l y h a n d le d .
Eggs
I n many ex­
p e r im e n ts s t e r i l e e g g s w e re in o c u la t e d d i r e c t l y i n t o t e s t tu b e s o r
E rlen m eyer f l a s k s c o n t a in in g th e d e s ir e d d i e t s .
However, i f e x c e s s
o f t h e m a t e r ia ls to make up one o f th e d i e t s was on hand th e eg g s
w ere t r a n s f e r r e d to s t e r i l e w a te r and a f t e r t h e la r v a e had h a tch ed
t h e y w ere t r a n s f e r r e d t o t h e d i e t .
This l a t t e r method enhances th e
p o s s i b i l i t i e s o f c o n ta m in a tio n b u t has an ad van tage when a low p er­
c e n ta g e o f h a tc h in g o c c u r s .
A nother problem en c o u n te r e d i n n u t r i t i o n a l s t u d i e s i n i n s e c t
p h y s io lo g y i s i n t h e p r e p a r a tio n o f d ie t s *
M icroorgan ism s can be
e a s i l y d e s tr o y e d i n fo o d s t u f f s b y means o f h e a t ( u s u a l ly b o i l i n g
or a u t o c la v ip g ) , c h e m ic a ls , o r u l t r a v i o l e t l i g h t .
S in c e b io c h e m is ts
h ave p r e s e n te d d a ta show in g t h a t th e u s e o f h e a t , u l t r a v i o l e t or
ev en v i s i b l e l i g h t d e s t r o y s s e v e r a l o f th e v it a m in s , i t was f o r ­
tu n a te t h a t th e v ita m in s u se d b y t h i s a u th o r a r e s o lu b le i n w a te r
and c o u ld b e s t e r i l i z e d b y b e in g p a s s e d through a s t e r i l e B e r k fe ld ¥
filte r .
The o y s t e r m eal p r o t e in u sed i n t h e s e ex p erim en ts was
v i t a m i n - f r e e m a t e r ia l and was a u t o c la v e d .
G lu co se and th e O sborne-
Mendel s a l t m ix tu re and d r ie d y e a s t w ere a u t o c la v e d .
Two r e a r in g c a g e s w ere m a in ta in ed i n a la r g e in c u b a to r
JL
(2B 0°C . — 1 ° ) .
I n one ca g e A edes a e g y p t i c u lt u r e s w ere m a in ta in ed
th r o u g h o u t th e y e a r .
C u lex p ip ie n s was b red i n th e o th e r c a g e .
M ales o f b o th s p e c i e s w ere f e d on m o is t r a i s i n s and th e fe m a le s on
l i v i n g p lu c k e d p ig e o n s .
E it h e r s p e c ie s o v i p o s i t s r e a d i l y on f i l t e r
p a p er f l o a t i n g on l a r g e f in g e r b ow ls o f w a te r .
o c c u r s on th e to p o f w a t e r .
O v ip o s it io n a l s o
Eggs o f A edes a e g y p t i a r e l a i d s i n g l y
w h erea s e g g s o f C u lex p ip ie n s a r e l a i d i n r a f t s o f 200—300 or m ore.
35
RESULTS
Growth o f M osq uito L arvae i n Pure C u ltu r e o f M icro o rg a n ism s.
I n t h e s e e x p e r im e n ts i t w as th o u g h t a d v is a b le to a l t e r th e b a s a l
d i e t o f 0*1$ o y s t e r m ea l, d r ie d y e a s t , g lu c o s e , and Osborn e-M endel
s a l t m ixtu re*
A t t h i s c o n c e n t r a tio n o f o y s t e r m ea l, d r ie d y e a s t , and
g lu c o s e th e b a c t e r i a l grow th i s to o g r e a t f o r t h e w e ll- b e in g o f m osquito
la r v a e *
T h e re fo r e a d i e t o f 0 .0 5 $ o y s t e r m eal, 0*05$ d r ie d y e a s t ,
0*05$ g l u c o s e , and 0 .1 $ O sborne-M endel s a l t m ix tu re was made i n d i s ­
t i l l e d w a te r and a u to c la v e d *
P o r tio n s 2 0 -5 0 c c . i n volum e w ere tr a n s ­
f e r r e d t o s t e r i l e t e s t tu b e s or f l a s k s and in o c u la t e d w ith th e d e s ir e d
o r g a n ism s.
T w en ty-fou r h ou rs l a t e r , s t e r i l e d a y -o ld la r v a e o f th e
m o sq u ito e s w ere in o c u la t e d i n t o th e v a r io u s tu b e s and f la s k s *
T able I shows th e r e l a t i o n o f E . c o l i t o t h e growth o f th e
l a r v a e , T ab le I I , th e r e l a t i o n o f B . s u b t i l i s * and Table I I I , th e
r e l a t i o n o f £>* c e r e v i s a e *
Thus i t can b e se e n t h a t o f th e th r e e m icro o rg a n ism s, o n ly
two o f them , B a c i l l u s s u b t i l i s * and S acch arom ycetes c e r e v is a e ( y e a s t )
c o u ld su p p o r t th e grow th o f m osq u ito la r v a e i n pure c u l t u r e .
The
r e s u l t s o f t h e t e s t s w it h E s c h e r ic h ia c o l i r e v e a l t h a t some o f th e
fo u r th i n s t a r la r v a e w ere a b le t o p u p a te b u t none o f them em erged.
T his may b e c o n s id e r e d u n u su a l i n t h a t when an i n s e c t s p e c ie s p u p a tes
i t i s g e n e r a lly a b le t o em erge.
Many d i e t s w i l l su p p o rt th e growth
o f m o sq u ito la r v a e t o th e fo u r th i n s t a r b e f o r e d ea th o c c u r s .
On i n ­
a d eq u a te o r in c o m p le te d i e t s the p u p a l s t a g e i s a lm o st n e v e r r e a c h e d .
T a b le I#
S u r v iv a l o f S t e r i l e M osquito L arvae on A u to cla v ed 0*05%
O y ste r M eal, 0 .0 5 $ D ried Y e a s t, 0*05$ G lu c o se , and 0 .1 $
O'sb.-M endel S a l t M ix tu r e . I n o c u la t e d -with E s c h e r ic h ia
c o l i . p .H . 6 * 8 -7 * 0 - NaOH - KH2P0^
A edes a e g y p t i
N o . Day
Old L arvae
No* 4-th
I n s t a r L arvae
N o.
Pupae
No*
A d u lts
1
2
3
4
5
6
7
8
9
10
20
20
20
20
20
20
20
20
20
20
14
11
16
12
14
10
11
8
12
15
2
0
3
1
2
0
0
2
4
1
0
0
0
0
0
0
0
0
0
0
TOTAL
200
123
15
0
T e s t N o.
C u lex p ip ie n s
T e s t N o.
N o. Day
O ld Larvae
No* 4 t h
I n s t a r Larvae
No.
Pupae
No.
A d u lts
1
2
3
4
5
6
7
8
9
10
10
10
10
10
10
10
10
10
10
10
2
4
2
3
5
2
3
1
3
2
0
1
0
0
2
1
0
0
1
1
0
0
0
0
0
0
0
0
0
0
TOTAL
100
27
6
0
T a b le XX.
S u r v iv a l o f S t e r i l e M osq u ito L arvae on A u to cla v ed 0 .0 5 ^
O y ster M e a l, 0*05% D ried Y e a s t , 0.0 5 % G lu c o se , and 0*1%
C sb .-M en d el S a lt M ix tu r e . I n o c u la t e d w ith B a c i l l u s
s u b t i l i s . p .H . 6 . 8 - 7 . 0
A edes a e g y p t i
T e s t N o.
N o. Day
Old L arvae
No. 4 th
I n s t a r Larvae
No.
Pupae
No.
A d u lts
1
2
3
4
5
6
7
8
9
10
20
20
20
20
20
20
20
20
20
20
15
12
16
14
12
13
16
15
12
15
14
12
15
14
12
10
13
12
12
14
14
12
14
12
12
9
13
11
12
14
TOTAL
200
140
128
123
C u lex p ip ie n s
T e s t N o.
No. Day
Old L arvae
N o. 4 th
I n s t a r L arvae
No.
Pupae
No.
A dult s
1
2
3
4
5
6
7
8
9
10
10
10
10
10
10
10
10
10
10
10
7
8
7
5
7
6
5
8
7
8
4
4
3
2
5
2
2
4
3
3
4
3
3
2
4
2
2
3
3
3
TOTAL
100
68
32
29
T a b le I I I .
S u r v iv a l o f S t e r i l e M osquito Larva© on A u to cla v ed 0 .0 5 ^
O y ster M e a l, 0*05^ D ried Y e a s t , 0 .0 5 ^ G lu c o se , and 0*1%
Osb*-M endel S a lt M ixture* I n o c u la te d w ith S acch arom ycetes
c e r e v i s a e . p.H* 6 . 8 - 7 . 0
Aedes a e g y p ti
T e s t No.
No. Day
Old Larvae
No. 4 th
I n s t a r Larvae
No.
Pupae
No.
A d u lts
1
2
3
4
5
6
7
8
9
10
20
20
20
20
20
20
20
20
20
20
18
16
16
17
15
18
17
16
14
15
18
15
16
15
14
16
17
15
12
12
16
15
16
14
14
14
16
15
12
12
TOTAL
200
162
150
144
No.
Pupae
No.
A d u lts
C u lex p ip ie n s
T e s t No.
No. Day
Old Larvae
No. 4 th
I n s t a r Larvae
1
2
3
4
5
6
7
8
9
10
10
10
10
10
10
10
10
10
10
10
9
8
7
6
8
8
9
6
8
9
9
8
6
6
7
8
8
6
8
8
9
8
6
6
6
8
8
6
7
8
TOTAL
100
78
74
72
Of A edes a g e y p t i, 62*5$ o f th e la r v a e rea ch ed th e fo u r th
i n s t a r and 7*5$ p u p a ted when r e a r e d on a pure c u lt u r e o f E . c o l i ;
27$ o f t h e la r v a e o f C u lex p ip ie n s rea ch ed th e fo u r th i n s t a r and
6$ p u p a ted on t h i s same c u l t u r e .
When r e a r e d on a p u re c u lt u r e o f B . s u b t i l i s 70$ o f th e la r v a e
o f A edes a e g y p t i r e a c h e d th e fo u r th i n s t a r , 6^$ p u p a ted , and 62*5$
a p p eared a s a d u l t s ; 68$ o f th e la r v a e o f C u lex p ip ie n s rea ch ed th e
fo u r th i n s t a r , 32$ w ere a b le t o p u p a te , and 29$ appeared a s a d u lt s
on a c u lt u r e o f t h i s same b a c t e r i a l organism *
On & pure c u lt u r e o f y e a s t 81$ o f t h e la r v a e o f Aedes a e g y p t i
rea c h e d th e f o u r th i n s t a r , 75$ s u r v iv e d t o th e p u p al s ta g e and 72$
a p p ea red a s a d u l t s ; 78$ o f th e la r v a e o f C u lex p ip ie n s w en t i n t o th e
f o u r t h i n s t a r , 7 5 $ p u p a te d , and 72$ o f them emerged a s a d u lts *
Growing on p u re c u lt u r e s o f th e f i r s t two m icro o rg a n ism s,
A* a e g y p t i seem ed t o b e a more v ig o r o u s ty p e than C * p ip ie n s *
Growth o f M osq u ito L arvae i n A u to cla v ed and B e r k e fe ld F i l t e r e d Pond
W ater
Some o f th e n a tu r a l pond w a ter i n the v i c i n i t y o f th e campus
a t C o lle g e P ark , i n w h ic h numerous m osq u ito la r v a e w ere fo u n d , w as
b r o u g h t i n t o t h e la b o r a t o r y and f i l t e r e d through c o t t o n to remove
a l l o f th e d e b r i s .
P a r t o f th e pond w a ter was a u to c la v e d i n l a r g e
t e s t tu b e s a t 15 pounds p r e s s u r e ( 1 2 0 ° C .) , f o r t h i r i y m in u te s .
Eggs
s t e r i l i z e d b y t h e d e s c r ib e d h e x y lr e s o r c in o l method w ere in o c u la t e d
in to th e tu b e s.
R e s u lt s a r e p r e s e n te d i n Table IV .
Some o f th e same pond w a te r d e s c r ib e d above was f i l t e r e d
th rou gh c o t t o n to remove th e d e b r is and f i l t e r e d th rough th e B e r k e fe ld ¥
to remove th e m icroorgan ism s*
A f t e r f i l t r a t i o n th e s t e r i l e w a te r was
t r a n s f e r r e d t o t e s t tu b e s o r s m a ll E rlenm eyer f l a s k s and s t e r i l e e g g s
w ere i n o c u la t e d i n t o them*
The r e s u l t s a r e shown i n Table V*
Thus i t ca n b e r e a d i l y s e e n t h a t th e a u t o c la v in g o f pond w a ter
d e s t r o y s i t s c h e m ic a l, p h y s i c a l , o r b i o t i c f a c t o r or f a c t o r s n e c e s s a r y
f o r d ev elo p m en t o f m osq u ito l a r v a e s in c e o n ly 12*9$ o f th e la r v a e o f
A ed es a e g y p t i and 7*6$ o f th e la r v a e o f C u lex p ip ie n s rea ch ed th e
f o u r th i n s t a r b e f o r e d e a th o ccu rred *
Those la r v a e r e a ch in g th e
f o u r th i n s t a r w ere s m a ll and v e r y i n a c t iv e *
The B e r k e fe ld f i l t r a t i o n o f pond w a te r l i k e w i s e r e n d e r s i t
u s e l e s s f o r th e d evelop m en t o f m osq u ito l a r v a e .
O nly £* 3% o f th e
la r v a e o f A* a e g y p t i and 4*7% o f th e la r v a e o f C u lex s u r v iv e d t o th e
f o u r th in s t a r *
Growth o f M osq uito L arvae on A lc o h o l and H eated S t e r i l i z e d Y ea st
From th e r e s u l t s p r e s e n te d i n th e au th or f s M aster t h e s i s
( i b i d ) and t h e a n a l y s i s o f th e r e s u l t s o f o th e r w o r k e r s, i t can b e
s e e n th a t y e a s t p la y s an im p o rta n t r o l e i n th e d evelop m ent o f
m o sq u ito l a r v a e .
I t w as d e c id e d t o s tu d y th e r e l a t i o n o f d r ie d
y e a s t s t e r i l i z e d b y means o f 70% e t h y l a lc o h o l t o th e n u t r i t i o n
o f m o sq u ito la r v a e *
The a lc o h o l was p la c e d over a r e l a t i v e l y la r g e
amount o f y e a s t ( d r ie d ) and a llo w e d to rem ain f o r p e r io d s o f 4 -6
w eek s*
From tim e t o tim e t h e m ix tu re was a g i t a t e d i n o rd er t h a t th e
a l c o h o l c o u ld p e n e t r a te a l l p a r t s o f th e y e a s t and f la s k *
a lc o h o l
w as removed b y vacuum d i s t i l l a t i o n *
The
B a c te r io lo g ic a l
t e c h n iq u e was em ployed i n ord er t o p rev en t c o n ta m in a tio n .
A heavy s u s ­
p e n s io n o f t h i s c h e m ic a lly s t e r i l i z e d y e a s t was m ixed w ith 0.1% g lu c o s e
and 0*1% O sborne-M endel s a l t m ix tu re and p o r t io n s o f i t were t r a n s f e r r e d
t o s t e r i l e t e s t tu b e s or f l a s k s .
tu b e s and f l a s k s .
S t e r i l e d a y -o ld la r v a e were added t o th e
The r e s u l t s a r e shown i n T a b le V I.
The e f f e c t s o f a u to c la v e d y e a s t (0.1%) in a d d it io n t o a u to c la v e d 0.1%
o y s t e r m e a l, G.1% O sbom e-M endel s a l t m ix tu r e , and 0*1% g lu c o s e were th en
s tu d ie d .
T h ese s u b s ta n c e s a f t e r a u to c la v in g w ere t r a n s f e r r e d t o s t e r i l e
t e s t tu b e s and sm a ll s t e r i l e f l a s k s and s t e r i l e la r v a e o f Aedes a e g y p t i.
T h ese r e s u l t s a r e p r e s e n te d i n T ab le V I I •
S in c e i t was th o u g h t t h a t s o lu b le p ro d u cts o f b a c t e r i a l m etab olism
m igh t c o n t a in grow th f a c t o r s r e q u ir e d by m osq uito l a r v a e , an attem p t was
made t o r e a r la r v a e i n a s t e r i l e s o lu t io n c o n s is t in g of a u to c la v e d 0.1/a
d r ie d y e a s t , 0.1% o y s t e r m e a l, 0.1% g lu c o s e , and 0.1% Osborne-W endel s a l t
m ix tu r e t h a t was co n n ected t o a con tam inated s o lu t io n o f t h e s e same
s u b s ta n c e s by means o f a s t e r i l e S e it z f i l t e r .
H owever, in every in s t a n c e
b a c t e r i a were a b le t o grow th rou gh t h e a s b e s t o e s f i l t e r and co n ta m in a tio n
occurred.
I t can be e a s i l y s e e n by a n a l y s i s o f T a b le VI t h a t a lc o h o l s t e r i l i z e d
y e a s t b e s id e s b e in g a s u i t a b l e fo o d f o r t h e la r v a e o f m o s q u ito e s , p r o v id e s
a l l o f th e a c c e s s o r y fo o d (grow th) f a c t o r s .
As th e r e s u l t s i n T ab le VI
show , 75% o f la r v a e o f Aedes a e g y p ti reach ed th e fo u r th i n s t a r , 71% p u p a ted ,
and 69.5% were a b le t o emerge as a d u l t s .
In th e c a s e o f C ulex p ip ie n s
74% o f t h e la r v a e r ea ch ed th e fo u r th i n s t a r , 71% p u p a ted , and 65% em erged.
On t h e a u to c la v e d y e a s t d i e t none o f th e la r v a e o f e it h e r s p e c ie s was
a b le t o le a v e t h e f o u r t h i n s t a r .
Of th e day o ld la r v a e o f Aedes a e g y p ti
17.9% rea ch ed t h e fo u r th i n s t a r b e fo r e d ea th o c c u r r e d , w hereas o n ly 10.6%
o f t h e larva© o f C u lex p ip ie n s reach ed th e fo u r th i n s t a r s t a g e .
T a b le IV .
S u r v iv a l o f M osquito Larvae from S t e r i l i z e d E ggs
I n o c u la t e d i n t o A u to cla v ed Pond Water* p*H* 6 * 8 - 7 .0
Aedes a e g y p ti
T e s t No*
No.
Eggs
N o.
L arvae H atch .
No. 4 th
I n s t a r L arvae
No.
Pupae
No.
A d u lts
1
2
3
4
5
6
7
8
9
10
15
15
15
15
15
15
15
15
15
15
12
8
11
7
7
12
8
9
10
11
1
0
0
2
1
3
0
2
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
TOTAL
150
95
12
0
0
No.
Pupae
No.
A d u lts
*C u lex p ip ie n s
T e s t No#
1
2
3
4
5
6
7
8
9
10
TOTAL
N o.
Eggs
5
10
30
15
5
5
12
22
14
5
, 133..-
N o.
L arvae H atch .
No. 4th
I n s t a r Larvae
2
6
18
9
3
2
10
16
9
3
0
0
2
2
0
0
1
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
78
6
0
0
^ R e s u lts on C u lex p ip ie n s i n t h i s t a b l e ta k en from
a u t h o r 's M a s te r 's T h e s is (N o n -B u ffered M edia)*
T a b le V*
S u r v iv a l o f M osquito Larva© from S t e r i l i z e d Eggs
I n o c u la t e d i n t o Pond W ater F i l t e r e d Through B e r k e fe ld W.
A edes a e g y p t i
T e s t N o.
1
2
3
4
5
6
7
8
9
10
total
.
No.
E ggs
No.
L arvae H a tch .
15
15
15
15
15
15
15
15
15
15
7
8
8
9
7
10
6
11
11
7
0
0
1
150
84
No. 4 th
I n s t a r L arvae
No.
Pupae
No.
A d u lts
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
7
0
0
a
0
l
l
0
a
* C u lex p ip ie n s
T e s t N o.
No.
E ggs
No.
L arvae H atch .
1
6
12
8
13
9
15
12
2
8
3
10
6
8
5
0
0
0
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
75
42
2
0
0
a
3
4
5
6
7
TOTAL
No. 4 th
I n s t a r L arvae
No.
Pupae
No.
A dult s
• R e s u lt s on C ulex p ip ie n s in t h i s t a b le ta k en
from a u t h o r 's M a s te r 's T h e s is (Non—Buff© red M edia*)
44.
S u r v iv a l o f S t e r i l e M osquito L arvae on Heavy S u sp en sio n
o f A lc o h o l K i l l e d Y ea st p lu s A u to cla v ed 0*1^ G lu co se
and 0 .1 $ O sb-M endel S a lt M ixture* p*H* 6*8-7*0
Aedes a e g y p t i
No* Day
Old Larvae
1
z
3
4
5
6
7
8
9
10
TOTAJ
No* 4 th
I n s t a r L arvae
20
20
20
20
20
20
20
20
20
20
17
10
16
13
17
18
16
12
15
16
200________________
No.
Pupae
17
10
15
12
15
16
16
12
15
14
No.
A d ult
16
10
15
11
15
16
16
11
15
14
ISO______________ 142___________ 139
C u lex p ip ie n s
T est
1
2
3
4
5
6
7
8
9
10
T T l 'V A '
No. Day
No*
4 th
No*
No.
Old L arvae______ I n s t a r Larvae_________ Pupae_________A d u lts
10
10
10
10
10
10
10
10
10
10
8
10
7
8
9
7
6
7
6
6
8
9
7
8
9
7
5
6
6
6
6
9
7
8
9
7
5
4
4
6
100
74
71
65,
45.
The r e s u l t s o f T a b le VI and T a b le V II show t h a t a t l e a s t one or more f a c t o r s
p r e s e n t i n t h e y e a s t a r e d e s tr o y e d by h e a t ( a u t o c la v in g ) .
Growth o f M o sq u ito L arvae on S t e r i l e M edia w ith th e A d d itio n o f
V itam in s
S in c e h e a t in g d e s tr o y e d t h e grow th p rom otin g q u a l i t i e s o f y e a s t , th e
a u th o r th e n s tu d ie d t h e e f f e c t s o f ad d in g pure c r y s t a l l i n e v it a m in s .
S in c e
t h e v ita m in s o f y e a s t (B com plex) a r e w ater s o l u b l e , t h i s co u ld be done
under a s e p t i c c o n d ito n s by means o f th e s t e r i l e B e r k e fe ld f i l t e r #
A u to-
c la v e d v i t a m in - f r e e o y s t e r m e a l, g l u c o s e , and O sbom e-M en del s a l t m ix tu r e
was u sed a s a b a s a l d i e t .
Y ea st was added t o t h i s b a s a l d i e t p r e v io u s t o
a u t o c la v in g i n some o f t h e t e s t s and i t was o m itted i n o th e r s*
T hiam in( B^} ,
a v ita m in found i n y e a s t , was t e s t e d a lo n e i n th e presen ce o f y e a s t .
f l a v i n (B 2 )
9
a l s o fou nd i n y e a s t , was l i k e w i s e t e s t e d *
R ibo­
A co m b in a tio n o f
a u to c la v e d y e a s t , t h ia m in , and r ib o f l a v i n was n e x t t r i e d t o d eterm in e th e
e f f e c t s o f th e t h r e e s u b s ta n c e s on l a r v a l grow th and d ev elo p m en t.
T ab le V III
g iv e s t h e r e s u l t s o f grow th under a s e p t ic c o n d ito n s on t h e b a s a l d i e t p lu s
th ia m in h y d r o c h lo r id e and a u to c la v e d y e a s t ,
a u t o c la v e d y e a s t 5
c la v e d y e a s t }
T a b le IX p lu s r i b o f l a v i n and
T a b le X p lu s th ia m in h y d r o c h lo r id e , r i b o f l a v i n , and a u to ­
T a b le XI p lu s th ia m in h y d r o c h lo r id e and r i b o f l a v i n (no
y e a st).
A n a ly s is o f T a b le s V I I I , IX , X , and XI r e v e a ls t h a t t h r e e su b sta n c e s
a r e r e q u ir e d f o r t h e grow th and d evelop m ent o f th e la r v a e o f Aedes a e g y p ti
and C u lex p i p i e n s .
The v ita m in s th ia m in h y d r o c h lo r id e (B^) and r ib o f la v i n
(B 2 ) a r e r e q u ir e d i n a d d it io n t o a t l e a s t one h ea t s t a b l e f a c t o r p r e s e n t
i n a u to c la v e d y e a s t .
I f any one o f th e t h r e e f a c t o r s , B^, B2» ° r a u to ­
c la v e d y e a s t w ere rem oved, grow th co u ld n o t p roceed beyond th e fo u r th i n s t a r .
S u r v iv a l o f M osquito Larvae from S t e r i l i z e d Eggs
I n o c u la t e d i n t o A u to cla v ed 0*l/£ O y ster M eal, 0 .1 %
Y e a s t, O.lf* G lu c o s e , and 0*1^ Osb -Mendel S a lt
M ix tu r e , p .H . 6 .8 -7 * 0
A edes a e g y p t i
1
2
3
4
5
6
7
8
9
10
taj
No.
E ggs
No.
L arvae H atch.
15
15
15
15
15
15
15
15
15
15
12
11
9
9
13
11
15
10
12
15
No. 4 th
I n s t a r L arvae
2
0
0
3
6
1
2
1
2
4
150_________ 117_____________ 21____________ '
No.
Pupa
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
jE
No.
Pupae
*Ni
lu;
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
o
o
o
0
0
0
0
0
C ulex p ip ie n s
>st
1
2
3
4
&
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
No*
E ggs
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
No.
L arvae Hatch*
6
7
4
8
5
4
4
6
10
6
8
9
4
9
4
5
10
8
7
6
200_______ 1 3 2
No* 4 th
I n s t a r JLarvae
0
2
0
1
0
0
0
2
2
0
0
3
0
0
0
1
1
0
1
1
14________________
0
0
0
0
0
0
0
0
0
0
0
Q
47.
T a b le V I I I .
S u r v iv a l o f M osquito Larvae from S t e r i l i z e d Eggs
I n o c u la t e d i n t o A u to cla v ed 0 . 1 % O y ste r M ea l, 0*1% Dr.
Y e a s t , 0 . 1 / G lu c o s e , and 0 . 1 / Qsb—
M endel S a lt M ixtu re
p lu s Thiam in HGL ( l m g ./ 5 0 0 c c .) p .H . 6 . 8 - 7 . 0 Thiam in
added by B e r k e fe ld W
Aedes aegypti
No.
E ggs
No.
L arvae H a tch .
1
2
3
4
5
6
7
8
9
10
n
12
13
14
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
12
9
8
8
10
11
11
7
9
10
13
12
12
10
13
TOTAL
225
155
T e s t N o.
No. 4 th
I n s t a r L arvae
No.
Pupae
No.
A d u lts
0
2
0
0
2
1
1
0
3
0
0
1
1
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
13
0
0
No.
Pupae
No.
A d u lts
C u lex p ip ie n s
No.
Eggs
No.
L arvae H atch .
1
2
3
4
5
6
7
8
9
10
15
15
15
15
15
15
15
15
15
15
9
a
12
10
6
9
9
10
7
9
0
0
2
1
0
3
2
2
2
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
TOTAL
150
89
15
0
0
T e s t No.
N o. 4 th
I n s t a r L arvae
43.
T a b le XX*
S u r v iv a l o f M osq uito Larvae from S t e r i l i z e d E ggs I n o c u la te d
i n t o A u to c la v e d Q.L% O y ste r Meal* 0 .1 ^ D ried Y east* 0 .1 ^
G lu co se* and 0*1/£ Osb—M endel S a lt M ixtu re p lu s R ib o f la v in
( l m g*/ 5 0 0 c c .) p*H* S*8~7.Q R ib o f la v in added B e r k e fe ld W
Aedea a e g y p ti
T e s t No*
No*
__ S g g s
N o.
L arvae H atch .
No. 4 th
I n s t a r Larvae
No.
Pupae
No.
A dult s
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
12
9
12
11
8
8
14
8
15
12
12
8
9
14
10
2
0
3
1
1
0
0
2
4
3
0
1
2
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
TOTAL
225
162
20
0
0
C u lex p ip ie n s
T e s t No*
No.
Eggs
No.
Larvae H a tch .
No. 4 th
I n s t a r Larvae
No.
Pupae
No.
A d u lts
1
2
3
4
5
6
7
8
9
10
15
15
15
15
15
15
15
15
15
15
12
9
10
8
8
14
12
9
5
8
1
2
4
1
0
2
4
2
1
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
TOTAL
150
95
19
0
0
T a b le X*
S u r v iv a l o f M osq uito Larva© from S t e r i l i z e d Eggs
I n o c u la t e d i n t o A u to cla v ed 0 . 1 / O y ster M eal, 0 . 1 / D ried
Y e a s t , 0 * 1 / G lu c o s e , and 0 . 1 / OdD-Mendel S a lt M ixtu re
p lu s Thiam in HCL and R ib o f la v in (B oth v ita m in s 1 m g*/ 500cc*
and added th r u B e r k e fe ld W.) p*H. 6 * 8 - 7 .0
Aedes a e g y p ti
N o.
_Eggs
No.
L arvae H atch .
No. 4 th
I n s t a r Larvae
1
2
3
4
5
6
7
8
9
10
15
15
15
15
15
15
15
15
15
15
12
12
11
9
13
14
14
12
9
13
12
12
10
9
12
12
14
10
6
11
12
11
10
9
10
12
13
10
7
11
11
11
9
9
10
11
13
10
7
10
TOTAL
150
119
110
105
101
No.
Pupa©
No.
A dult s
T e s t No*
No.
Pupa©
No.
A d u lts
C u lex p ip ie n s
T e s t No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
TOTAL
No.
E ggs
No.
Larvae H a tch .
No. 4 th
I n s t a r Larvae
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
12
8
11
9
9
14
6
10
13
8
8
10
12
8
5
10
8
11
8
7
12
6
8
11
6
8
8
12
7
5
11
8
11
7
7
12
6
8
10
6
8
8
11
7
5
11
8
11
7
7
12
6
8
10
6
8
8
11
7
5
225
143
127
125
125
50.
In "the p r e s e n c e o f v ita m in B-^ and a u to c la v e d y e a s t 8 .3 ^ o f th e la r v a e
o f A edes a e g y p t i rea c h e d th e fo u r th i n e t a r and 1 6 .8 ^ o f th e la r v a e o f G ulex
p ip i e n s rea ch ed t h i s same s t a g e .
I n t h e p r e s e n c e o f v ita m in B 2 and a u to c la v e d y e a s t , s i m i l a r r e s u l t s
w ere o b t a in e d .
Of th e Aedes a e g y p t i la r v a e , o n ly 1 2 .3 ^ o f them reach ed th e
f o u r t h i n s t a r b e f o r e d ea th occurred*
In th e c a se o f C u lex p ip ie n s
1 9 .8 ^
o f t h e la r v a e s u r v iv e d t o t h e f o u r th i n s t a r .
I t can be s e e n t h a t when a co m b in a tio n of th ia m in h y d r o c h lo r id e (B^
) 9
r i b o f l a v i n (Bg) » and a u to c la v e d y e a s t i s u sed as a r e a r in g medium, b oth
s p e c ie s o f m o sq u ito e s rea ch ed m a tu r it y .
Aedes a e g y p ti 91.6/C o f t h e la r v a e
rea ch ed th e fo u r th i n s t a r , 8 8 .2 ^ p u p a te d , and 84/C ap p eared as a d u lt s on
t h i s medium i 88*8^ o f t h e la r v a e o f C u lex p ip ie n s reach ed t h e f o u r th in s t a r ,
8 7 .4 ^ pupat e d , and 8 7 .4 ^ em erged.
I n t h e p r e s e n c e o f th ia m in h y d r o c h lo r id e and r i b o f l a v i n but w ith o u t
th e a d d it io n o f a u to c la v e d y e a s t grow th beyond t h e f o u r th i n s t a r was im­
p o s s ib le *
The r e s u l t s show t h a t 6*3^ o f th e A* a e g y p t i la r v a e and 1 6 .3 ^
o f t h e C. p ip ie n s la r v a e rea ch ed t h e f o u r th i n s t a r .
I t was th e n d e c id e d t o remove t h e a u to c la v e d y e a s t and add o th e r
v ita m in s t o th e B i and B 2 d i e t .
V itam in C ( a s c o r b ic a c id ) and n i c o t i n i c
a c id r e p la c e d t h e y e a s t .
I t can be s e e n t h a t n i c o t i n i c a c id and a s c o r b ic a c id c o u ld n o t r e p la c e
t h e a u to c la v e d y e a s t i n t h e d i e t of m osq u ito la r v a e as grow th beyond th e
fo u r th i n s t a r was im p o s s ib le .
The r e s u l t s show t h a t 14.7^. o f th e la r v a e
o f A edes a e g y p t i and 2 3 .4 $ o f C ulex p ip ie n s reach ed th e fo u r th i n s t a r .
At a v er y l a t e d a te a sm a ll q u a n tity o f a d en n in (Bg) was o b ta in ed
th ro u g h th e c o u r te s y o f D r. Hugo N e ils o n o f th e U n ited S t a te s Bureau o f
F i s h e r i e s a t C o lle g e P a rk .
T h is v ita m in was s u b s t it u t e d f o r th e a u to c la v e d
51.
y e a s t and com bined w ith th ia m in h y d r o c h lo r id e and r ib o f l a v i n in a p rep ared
d ie t.
H ow ever, b e ca u se o f c o n ta m in a tio n , cau sed p o s s ib ly by a m in u te h o le
in t h e B e r k e fe ld f i l t e r , t h e a u th o r was u n a b le t o se c u r e s i g n i f i c a n t r e s u l t s .
An a ttem p t was made t o employ th e d a i l y t r a n s f e r o f la r v a e * te c h n iq u e
d e s c r ib e d by F r o s t , H e r n s, and H oskins (1 9 3 6 ) but by t h a t tim e t h e su p p ly
o f v ita m in Bg was ex h a u sted and t h e exp erim en t m ust be r e p e a te d a t a l a t e r
d a te.
A n oth er s o u r c e o f w a ter s o lu b le v it a m in s , t h e e x a c t n a tu r e o f which
i s unknown, i s l i v e r .
The a u th o r s u b s t it u t e d t h e 0.1j£ a u to c la v e d y e a s t
w ith a Q«l/£ a u to c la v e d c o n c e n tr a te o f l i v e r .
Thiam in h y d r o c h lo r id e and
r i b o f l a v i n w ere added by means o f t h e B e r k e fe ld f i l t e r t o th e l i v e r con ­
c e n t r a t e (0*1^ ) w hich had b een a u to c la v e d w ith th e o y s t e r m e a l, g lu c o s e ,
and G sborne-M endel s a l t m ix tu r e .
Thus i t
The r e s u l t s a re p r e s e n te d i n T a b le X XII.
can b e s e e n t h a t a u to c la v e d l i v e r , a s d oes a u to c la v e d y e a s t ,
c o n t a in s one o r more h e a t s t a b l e s u b s ta n c e s r e q u ir e d by m osq u ito la r v a e .
The r e s u l t s show t h a t 9 1 .
o f t h e la r v a e o f Aedes a e g y p ti reach ed th e
f o u r t h i n s t a r , 81*8^ pupated and 19*3% emerged as a d u l t s ;
w hereas 90*2%
o f t h e la r v a e o f C u lex p ip ie n s reach ed t h e fo u r th i n s t a r , 85*8% p u p a ted ,
and 81.6/& ap p eared a s a d u lts *
I n o rd er t o stu d y t h e e f f e c t s o f m a t e r ia l i n t r u e s o lu t io n upon th e
growth and d evelop m ent o f m o sq u ito l a r v a e , t h e amino a c i d s , l e u c i n e ,
g l y c i n e , h i s t i d i n e , t r y p to p h a n e , a s p a r g in e , c y s t i n e , and t y r o s in e w ere
o b ta in e d and a s tr o n g s o lu t i o n o f them was made.
Thiamin h y d r o c h lo r id e ,
r i b o f l a v i n , ad erm in , Q .l^ l i v e r c o n c e n t r a t e , g lu c o s e , and O sbom e-M endel
s a l t m ix tu r e w ere added t o th e amino a c id s o lu t io n and f i l t e r e d th rou gh
t h e s t e r i l e B e r k e fe ld .
A s h o r ta g e o f tim e fo r c e d th e a u th o r t o in o c u la t e
n o n - s t e r i l e day o ld la r v a e i n t o t h i s d i e t .
T a b le XIV*
The r e s u l t s can be s e e n in
The r e s u l t s show t h a t no la r v a e w ere a b le t o d ev elo p beyond t h e fo u r th
i n s t a r on t h i s d ie t *
In t h e c a s e o f Aedes a e g y p t i 71/C o f t h e la r v a e
r e a c h e d t h e fo u r th i n s t a r 9 w hereas 63.5/C o f t h e la r v a e o f C ulex p ip ie n s
rea ch ed t h i s s t a g e .
T a b le
X I.
S u r v iv a l o f M osq uito Larva© from S t e r i l i z e d Eggs
I n o c u la te d i n t o A u to cla v ed 0 .1 ^ O y ster M ea l, 0 .1 ^ G lu c o se ,
and Q *lfa Osb-Mendel S a lt M ixtu re p lu s Thiam in HCL and
R ib o f la v in (B oth v ita m in s 1 m g ./ 500cc* and added th r u
B e r k e fe ld W-) p.H . 6 . 8 - 7 . 0
Aedes a e g y p t i
No.
Eggs
No.
L arvae H a tch .
1
2
3
4
5
6
7
a
9
10
15
15
15
15
15
15
15
15
15
15
6
8
10
5
7
8
8
11
6
10
TOTAL
150
79
T e s t N o.
No. 4 th
I n s t a r Larvae
No.
Pupae
No.
A dult s
0
0
1
0
2
0
0
1
0
1
0
0
0
0
0
0
0
Q
0
0
0
0
0
0
0
0
0
0
0
0
5
0
0
No.
Pupae
No •
A d u lts
0
0
0
0
0
0
0
0
0
0
C u lex p ip ie n s
T e s t No.
1
2
3
4
5
6
7
8
9
10
TOTAL
No.
E ggs
No.
L arvae H atch .
N o. 4 th
I n s t a r Larvae
15
15
15
15
15
15
15
15
15
15
12
9
9
8
10
15
8
8
11
8
2
0
0
0
1
4
2
3
2
3
0
0
0
0
0
0
0
0
0
0
150
98
17
0
0
54.
S u r v iv a l o f M osq uito L arvae from S t e r i l i z e d Eggs
I n o c u la t e d in t o A u to cla v ed 0 .1 ^ O y ste r M ea l, 0.1/C G lu c o s e ,
0*1/4 Osb—M endel S a lt M ix tu re p lu s Thiam in HCL, R i b o f la v in ,
A s c o r b ic A c id , N i c o t i n i c A cid ( A ll v ita m in 1 m g ./ 5Q 0cc.
added th r u B e r k e fe ld W). pH 6 . 8 - 7 . 0 - NaOH - KH2 PO4
Aedes a e g y p t i
T est
1
2
3
4
5
6
7
8
9
10
TOTA3
No.
E ggs
No.
L arvae H atch.
15
15
15
15
15
15
15
15
15
15
10
12
8
11
10
10
150
No. 4 th
I n s t a r Larvae
2
2
0
1
No.
Pupae
No.
A d u lts
12
8
12
2
1
1
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
102
15
0
0
4
9
C ulex p ip ie n s
T est
1
2
3
4
5
6
7
8
9
10
No.
Eggs
15
15
15
15
15
15
15
15
15
15
150
No.
Larvae Ha~tch.
No. 4 th
N o.
No.
I n s t a r Larvae______Pupae_____ A d u lts
8
10
12
4
IP
9
0
0
8
6
8
7
7
2
6
1
2
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
20
0
0
3
T a b le X III*
S u r v iv a l o f M o sq u ito Larva© from S t e r i l i z e d E ggs
I n o c u la t e d i n t o A txtoclaved 0*1% O y ster M ealj 0*1% L iv e r
Cone* , and 0 * 1 / Osb—M endel S a lt M ixtu re p lu s Thiam in
HCL and R ib o f la v in (B oth v ita m in s 1 m g*/ 5© 0cc. and
added th r u B e r k e fe ld ¥ • ) p.H* 6 .8 -7 * 0
A edes a e g y p t i
N o.
E ggs
N o.
L arvae H a tc h .
N o. 4 th
I n s t a r Larvae
1
2
3
4
5
6
7
8
9
10
15
15
15
15
15
15
15
15
15
15
10
12
14
11
11
12
13
10
12
11
8
12
12
11
10
12
12
8
11
10
8
10
12
9
9
10
12
8
9
8
8
10
11
9
. 8
10
12
8
8
8
TOTAL
150
116
106
95
92
T e s t N o.
_
No.
Pupae
No.
A d u lts
C u lex p ip ie n s
NO.
E ggs
No.
L arvae H a tch .
N o• 4 th
I n s t a r Larvae
1
2
3
4
5
6
7
8
9
10
15
15
15
15
15
15
15
15
15
15
12
8
8
9
7
7
12
10
12
7
10
7
8
7
6
7
11
9
11
7
10
7
8
6
6
7
10
8
11
6
9
7
8
6
5
7
9
8
10
6
TOTAL
150
92
83
79
75
T e s t No.
N o.
Pupae
NoA d u lts
56*
Tabli
S u r v iv a l o f M osquito L arvae ( N o n - S t e r ile ) I n o c u la t e d i n t o a
S t e r i l e B e r k e fe ld F i l t e r e d S o lu t e D i e t , C o n s is t in g o f: 0 * 1 /
G lu c o s e , 0*1% Obs-M endel S a lt M ix tu r e , 0*1% L iv e r Cone*,
T h iam in , R i b o f la v in , Adermin (Each v ita m in 1 rag*/ 5 0 0 c c .)
and S even Amino A c id s . pH 6 . 8 - 7 . 0
A edes a e g y p t i
T est
1
2
3
4
5
6
7
8
9
10
TOTAJ
N o. Day
No.
4 th
N ol
No.
Old L arvae______ I n s t a r L arvae______ Pupae___________ A d u lts
20
20
20
20
20
20
20
20
20
20
14
15
11
9
10
16
16
18
15
18
0
0
0
0
0
0
0
0
0
0
200______________ 142________________ 0___________
0
0
0
0
0
0
0
0
0
0
0
C u lex p ip ie n s
T e st
1
2
3
4
5
6
7
8
9
10
No* Day
No*
4 th
No*
No.
Old L arvae______I n s t a r _Larvae_______ Pupae__________ A d u lts
20
20
20
20
20
20
20
20
20
20
14
16
9
11
13
8
12
14
15
15
200
___________ 127
0
0
0
0
0
0
0
0
0
0
______
0
0
0
0
0
0
0
0
0
0
0________________ 0
*yConcesnfcrated S o lu tio n o f : L e u c in e , G ly c in e , H i s t i d i n e ,
T ryp top h d n e, A sp a r g in e , C y s t in e , and T y r o s in e .
DISCUSSION*
I t lia s b e en shown b y th e w r it e r t h a t th e la r v a e o f Aedes a e g y p t i
and C u lex p i p i e n s i n t h e p r e s e n c e o f c e r t a i n ty p e s o f l i v i n g b a c t e r ia
and y e a s t s grew and d e v e lo p e d i n th e norm al manner*
B a c illu s s u b lit is
and S a c c a r o m y c e te s c e r e v i s a e w ere v e r y u s e f u l organ ism s i n th e d i e t s
o f m o sq u ito la r v a e *
However* i f E s c h e r ic h is c o l i produced s u it a b le
grow th s u b s ta n c e s* t h e y w ere i n q u a n t i t i e s t o o s m a ll t o b e e f f e c t i v e *
On t h e o t h e r hand* E* c o l i may have p rod u ced s u b s ta n c e s u n fa v o r a b le
o r t o x i c i n t h e medium*
O ddly enough, Hobson (1 9 3 2 ) was a b le to r e s t o r e
t h e grow th o f b l o w f ly la r v a e on s t e r i l e m u scle b y th e a d d it io n o f a
c u l t u r e o f t h i s b a cteriu m *
Rafchmanova (1 9 3 7 ) c o n clu d ed t h a t pure
c u l t u r e s o f th e c o lo n b a c i l l u s i n a u to c la v e d t a p w a te r was n o t " t o x ic ”
t o th e l a r v a e o f A n o p h eles m a c u lip e n n is *
However* he o b se rv ed t h a t
B a c i l l u s f lu o r e s c e n s would n o t su p p o rt t h e grow th o f Anopheles la r v a e *
The a u t o c la v in g and B e r k e fe ld f i l t e r i n g o f pond w a te r d e s tr o y e d
i t s p r o p e r t i e s a s a r e a r in g medium f o r t h e la r v a e o f b o th s p e c ie s *
S h i p i t z i n a (1 9 3 0 , 1 9 3 5 ) h a s shown th e v a lu e o f c o l l o i d s i n m o sq u ito
la r v a e n u t r i t i o n a s h a s B ek lim sh en (1 9 3 0 )*
Many o f th e c o l l o i d s a r e
rem oved by b o th th e a u t o c la v in g and B e r k e fe ld f i l t e r i n g te c h n iq u e *
S h i p i t z i n a o b se r v e d t h a t t h e s m a ll la r v a e ( 1 s t th rou gh 3rd i n s t a r s )
u t i l i z e d v e r y s m a ll c o l l o i d s * b u t t h a t t h e fo u r th i n s t a r la r v a e
r e q u ir e d t h e c o a r s e r p a r t i c l e s i n s u s p e n s io n s f o r food*
Her o b s e r v a tio n s
a r e a n a la g o u s t o t h o s e o f B e k lem ish ev who n o te d t h a t t h e la r v a e o f
A n o p h eles m a c u lip e n n is c o u ld d e v e lo p o n ly a s f a r a s t h e fo u r th i n s t a r s
on p a r t i c l e s 5 mm* o r l e s s i n d ia m eter*
Hinman (1 9 3 2 ) found t h a t upon
d i a l y s i s o f pond w a te r A n o p h elin e la r v a e c o u ld l i v e f o r o n ly t h r e e
d a y s , w h erea s i n S e i t z —Werke f i l t e r e d pond w a te r la r v a e o f t h i s same
58,
group s u r v iv e d f o r e le v e n d a y s*
The d ia ly z e d w a te r c o n s is t e d o f
s u b s ta n c e s i n t r u e s o l u t i o n , w h erea s t h e S eitz -W e r k e f i l t r a t e
c o n t a in e d s m a ll c o l l o i d s i n a d d i t i o n t o s o l u t e s .
The p r e s e n t a u th o r w ou ld l i k e t o p o in t o u t t h a t a u t o c la v in g
had o t h e r im p o rta n t e f f e c t s on pond w a t e r , and on th e o y s t e r m e a l,
d r ie d y e a s t s u b s t r a t e .
The p rim ary e f f e c t was t h e d e s t r u c t io n o f
t h e m icro o rg a n ism s p r e s e n t i n t h e s e m e d ia .
Should t h e grow th f a c t o r s
b e p rod u ced b y l i v i n g o r g a n ism s, and t h e a u t h o r 's r e s u l t s j u s t
p r e s e n te d h ave shown t h i s t o b e t r u e , t h e rem oval o f t h e m ic r o sc o p ic
f l o r a rem oved t h e p r i n c i p a l s o u r c e o f t h e a c c e s s o r y fo o d f a c t o r s .
H eat a l s o b r e a k s down many c h e m ic a ls and t h e d e s t r u c t io n o f t h e
h e a t - l a b i l e grow th f a c t o r s by a u t o c la v in g i s in e v i t a b l e *
However, th e
e x p la n a tio n o f t h e i n a c t i v a t i o n o f pond w a te r b y t h e B e r k e fe ld
f i l t e r i n g tr e a tm e n t i s somewhat hard to e x p la in .
I f gorwth s u b s ta n c e s
a r e w a te r s o l u b l e , h en ce s o l u t e s , t h e y sh ou ld r e a d i l y p a s s th rou gh t h e
f i l t e r and a p p ear i n th e f i l t r a t e .
T h is may b e t r u e , b u t i n t h e
a b se n c e o f l a r g e r c o l l o i d ^ th e ap p aren t n eed f o r w h ich t h e a u th o r h as
d i s c u s s e d , grow th was l i k e w i s e r e t a r d e d ,
(T rager (1 9 3 5 ) c la im e d th a t
t h e grow th f a c t o r s r e q u ir e d b y t h e la r v a e o f A edes a e g y p t i w ere s o lu t e s
and t h a t t h e l a r v a e on s o l u t e s a lo n e s u r v iv e d t i l l th e fo u r th i n s t a r ,
Rozeboom ( 1 9 3 5 ) , a s m en tio n ed b e f o r e , w as a b le to r e a r th e la r v a e o f
v a r io u s m o sq u ito e s on a co n ta m in a ted medium o f b read m ix tu re o r one o f
d ove f e c e s .
H owever, when t h e d ove f e c e s w ere a u to c la v e d and in t r a ­
c e l l u l a r and e x t r a c e l l u l a r e x t r a c t s o f E , c o l i and B , s u b t i l i s added,
grow th t o m a tu r ity was im p o s s ib l e .
The work o f t h i s l a t t e r a u th o r
c o n f u s e s t h e n u t r i t i o n a l r e q iir e m e n t o f m osq u ito la r v a e problem a lo n g
th e e x p e r im e n ta l r e a s o n in g s o f B ek lim sh ev , S h ip it z a n a , Hinman, T r a g e r ,
and t h e p r e s e n t a u th o r i n t h a t he had c o l l o i d a l m a t e r ia ls and th e
f a c t o r s o f b o th t y p e s o f b a c t e r i a l m eta b o lism p r e s e n t i n h is d i e t .
H owever, i t i s th e o p in io n o f t h e p r e s e n t w orker t h a t amounts o f growth
s u b s ta n c e s e x t r a c t e d from b a c t e r i a l c e l l s b y no means compare w ith th e
amount and n a tu r e o f t h e grow th s u b s ta n c e s produced b y l i v i n g b a c t e r i a
on a n u t r i e n t m edium .
The u s e o f b a c t e r i a l f i l t r a t e s i n i n s e c t
n u t r i t i o n a l s t u d i e s h as p rod u ced no p o s i t i v e r e s u l t s , w h ereas a c a r e f u l
e x a m in a tio n o f t h e p h y s i o l o g i c a l l i t e r a t u r e r e v e a ls numerous c a s e s o f
n e g a t iv e d a t a .
F or i n s t a n c e , t h e work o f Hobson n o te d above on t h e
n u t r i t i o n a l r e q u ir e m e n ts o f b lo w f ly la r v a e shows t h a t b lo w f ly la r v a e
r e q u ir e d i n a d d i t i o n t o two aqueous o r a l c o h o l i c e x t r a c t a b le f a c t o r s
p r e s e n t i n y e a s t , a t h i r d s u b s ta n c e “a b s e n t from aqueous o r a l c o h o l i c
e x t r a c t s o f y e a s t s . 1* I t i s u n fo r tu n a te t h a t a p p a ra tu s h as n o t a s y e t
b e e n p e r f e c t e d w h e r e in m o sq u ito la r v a e c o u ld grow i n a s t e r i l e medium
c o n n e c te d b y means o f a s t e r i l e sem i—p erm eab le membrane f o some n a tu r e
w it h a c o n ta m in a ted medium o f th e same c o m p o sitio n *
I f t h i s c o u ld b e
d o n e, th e n an y grow th s u b s ta n c e s produced b y th e c e l l u l a r a c t i v i t i e s
o f m ic ro o rg a n ism s c o u ld b e b e t t e r in v e s t ig a t e d *
The a u th o r *s e x p e r i­
m ents w it h th e S e i t z f i l t e r f a i l e d b e c a u se b a c t e r i a w ere a b le t o grow
th ro u g h t h e f i l t e r *
A c a r e f u l c o n s id e r a t io n m ust b e g iv e n t h e v ita m in s a s t h e y
i n f l u e n c e t h e norm al grow th , m e ta b o lism , and d evelop m ent o f th e la r v a e
o f A edes a e g y p t i and C u lex p i p i e n s .
The r e s u l t s d e f i n i t e l y show t h a t
th ia m in ( th ia m in h y d r o c h lo r id e ) , r i b o f l a v i n , and a h e a t s t a b le su b sta n c e
p r e s e n t i n y e a s t o r a w a te r c o n c e n t r a te o f l i v e r w ere r e q u ir e d b y
m o sq u ito l a r v a e .
Ni&dt&nic a c id o r a s c o r b ic a c id f a i l e d t o supplem ent
th e h e a t —s t a b l e f a c t o r o f y e a s t o r l i v e r *
The p r e s e n t au th o r w ould
l i k e t o compare t h e s e s u b s ta n c e s w it h t h e f a c t o r s t h a t Hobson (1932)
h a s shown t o be r e q u ir e d b y L u e i l i a la r v a e *
Hobson* s r e s u l t s i l l u s t r a t e
th e p r e s e n c e o f t h r e e f a c t o r s i n y e a s t .
One was h e a t - l a b i l e f a c t o r
w h ich c o u ld b e s u p p lie d b y P e te r * s ’’a n t in e W r itic c o n c e n t r a t e ,11
a h e a t - s t a b l e f a c t o r i n a u to c la v e d y e a s t , and a s o lu b le f a c t o r i n
y e a s t a u to ly s a te .
I t i s f e l t b y th e p r e s e n t au thor t h a t s in c e
P e te r * s fla n t i n e u r i t i c c o n c e n t r a t e 1* i s a r i c h so u r c e o f th e h e a t l a b i l e v itim in
d e r r iv ia t iv e •
t h i s f a c t o r p r o b a b ly was th ia m in or i t s
The same s t a b l e y e a s t f a c t o r o f b lo w f ly n u t r it i o n may
h a v e b e e n th e same h e a t - s t a b l e y e a s t f a c t o r o f m osq u ito l a r v a l n u t r i­
tio n .
R ib o f la v in may p o s s i b l y have b een th e s u b s ta n c e i n Hobson* s
y e a s t a u t o ly s a t e .
G yorgy (1 9 3 5 ) i n v e s t i g a t e d th e G com plex r e q u irem en t o f r a t s #
I n a d d i t i o n to n l a c t o f l a v i n e ( r i b o f l a v i n ) a seco n d a n t id e r m a t it is
f a c t o r w as n e e d e d .
T h is l a t t e r f a c t o r was h e a t - s t a b l e even i n th e
p r e s e n c e o f s tr o n g a l k a l i .
C h ick , C opping, and Edgar (1 9 3 5 ) s tu d ie d
t h e 3 com plex and co n firm ed t h e r e s u l t s o f Gyorgy t h a t v e r y h e a t — and
a l k a l i - s t a b l e f a c t o r w as p r e s e n t i n th e com plex#
c a l l e d v ita m in B^#
T h is f a c t o r h a s b een
U n f o r tu n a t e ly , t h e p r e s e n t w r i t t e r can n ot i n t h i s
t h e s i s r e p o r t t h e e f f e c t o f t h i s v ita m in on m osq u ito l a r v a e .
W illia m s
( 1 9 3 4 , 193S) and co -w o rk ers h ave s tu d ie d th e B v ita m in s and t h e i r
e f f e c t on th e grow th o f y e a s t #
These w o rk ers c4®im t h a t a f i l t r a t e
f a c t o r or **p an toth en ic?! a c id w h ich i s h e a t - s t a b l e i n th e p r e s e n c e o f
a c i d i s q u ir e d f o r t h e normal grow th o f y e a s t c e l l s #
c a l l e d t h i s s u b s ta n c e t h e Y f a c t o r #
W illia m s has
Dann and SubbanfcSfl* (1 9 3 8 ) i n a
s tu d y o f r a t d e r m a t it is f a c t o r s and c h ic k d e r m a t it is f a c t o r s c o n clu d e
t h a t " th e v ita m in
com plex c o n ta in s t h e f o llo w in g f o u r d i f f e r e n t
s h a r p ly c h a r a c t e r iz e d e n t i t i e s ; r i b o f l a v i n , n i c o t i n i c a c i d , v ita m in
61
B^, and th e f i l t r a t e f a c t o r * 11
I n t e r e s t i n g r e s u l t s can b e e x p e c te d
when t h e s e s u b s ta n c e s a r e s tu d ie d under c o n t r o l le d c o n d it io n s i n i n s e c t
n u t r i t i o n e s p e c i a l l y i n th e d i e t s o f m osq u ito la r v a e *
T ra g er (1 9 3 5 ) h a s d eterm in ed two f a c t o r s r e q u ir e d b y m osq u ito
l a r v a e , b o th o f w h ic h a r e s o lu t e s *
t h e la r v a e beyond th e f o u r t h i n s t a r .
However, T rager was u n ab le to r e a r
I t i s u n u su a l t h a t T rager g o t
m o sq u ito la r v a e t o grow under s t e r i l e c o n d it io n s i n t h e p r e s e n c e o f
a u t o c la v e d l i v e r and y e a s t e x t r a c t s i n t h a t h e a t d e s t r o y s v ita m in
v e r y r e a d i l y and h a s a marked e f f e c t on r i b o f l a v i n .
S in c e a l l th r e e
grow th f a c t o r s r e q u ir e d b y m o sq u ito la r v a e a re p r e s e n t i n y e a s t , i t i s
o n ly n a t u r a l t h a t F r o s t , H e m s , and H osk in s (1 9 3 3 ) co u ld r e a r th e la r v a e
o f T h e o b a ld ia in c id e n s t o m a tu r ity on y e a s t a lo n e *
^he work o f I^dwing
and Fox (1 9 3 3 ) on Jap an ese b e e t l e l a r v a e , and th e work o f C hiu and
McCay (1 9 3 9 ) w it h t h e c o n fu se d H o u r b e e t l e te n d s t o f u r t h e r show t h e
v a lu e o f y e a s t i n p r o v id in g grow th s u b s ta n c e s t h a t a r e e s s e n t i a l f o r
l a r v a l grow th*
SUMMARY.
1«
M osq uito la r v a e can d ev elo p on p u re c u lt u r e o f some b a c t e r i a
and ca n n o t d d v elo p on o t h e r s ,
E s c h e r ic h ia c o l i f a i l s t o su p p o rt th e
grow th o f t h e la r v a e o f A edes a e g y p t i qnd C u lex p ip ie n s *
B a c illu s
s u b t i l i s and S a cch a ro m y cetes c e r e v is a e su p p o rt th e gorw th o f la r v a e
t o m a tu r ity *
2*
grow th o f
A u to c la v e d and B e r k e fe ld Wf i l t e r e d pond
w a ter su p p o r ts th e
m o sq u ito la r v a e o n ly a s f a r a s t h e fo u r th i n s t a r under
s t e r i l e c o n d itio n s *
3*
M osquito la r v a e d e v e lo p to m a tu r ity on a lc o h o l s t e r i l i z e d
y e a s t , b u t ca n n o t d e v e lo p beyond th e f o u r th i n s t a r on a u to c la v e d
y e a s t a lo n e under s t e r i l e c o n d itio n s *
4*
M osquito la r v a e r e q u ir e f o r d evelop m en t to m a tu r ity a t
l e a s t th r e e f a c t o r s ; th ia m in h y d r o c h lo r id e (B ^ ), r i b o f l a v i n (B 2 )> and
a h e a t —s t a b l e f a c t o r i n y e a s t under a s e p t i c c o n d it io n s *
5*
N i c o t i n i c a c id and a s c o r b ic a c id do n o t r e p la c e th e h e a t -
s ta b le fa c to r in y e a st*
6*
The h e a t - s t a b l e f a c t o r o f y e a s t i s a l s o p r e s e n t i n a u to ­
c la v e d l i v e r c o n c e n tr a te *
7*
v ita m in
The h e a t - s t a b l e f a c t o r h as p r o p e r t ie s s im i l a r t o t h o s e o f
and pantothe'lm iic a c id *
8*
M osquito la r v a e f a i l e d t o d e v e lo p beyond t h e f o u r th i n s t a r
on a s o l u t e d i e t c o n t a in in g amino a c i d s , v it a m in s , g lu c o s e , and
Osborne-MendftL s a l t s *
LITERATURE CITED
A t k in , E . E .
& B a c o t , A*
1917
The r e l a t i o n betw een t h e h a tc h in g o f th e
eggs and t h e developm ent of t h e la r v a e o f
S tegom yia f a s c i a t a . and t h e p r e s e n c e o f
b a c t e r ia and y e a s t* P a r a s it o lo g y 9:
4 8 2 -5 3 6 .
B a c o t , A*
& H arden, A*
19 22
V itam in req u irem en ts o f D r o so p h ila I
V itam in B and C Biochera* J o u r. 1 6 : 1 4 8 152.
B a r b er , M. A*
1927
The fo o d o f A n o p h elin e la r v a e * Food o r­
gan ism s in pure c u ltu r e * U n ite d S t a t e s
P u b lic H e a lth S e r v ic e No. 22: 1 4 9 4 -1 5 1 4 .
1928
The fo o d o f C ull c in e la r v a e . Food o r­
ganism s i n p u re c u lt u r e . U n ite d S t a t e s
P u b lic H e a lth S e r v ic e No. 43: 1 1 -1 7 .
Baum berger, J . P .
1919
A n u t r i t i o n a l stu d y o f i n s e c t s w ith
s p e c i a l r e f e r e n c e t o m icroorgan ism s and
t h e i r su b s tr a ta *
Jour* Exp* Z o o l. 28:
1 -8 2 .
B e k le m ish e v , V. Iff.
1930
The im p ortan ce o f c o l l o i d o - d i s p e r s i v e
s u b s ta n c e s i n t h e n u t r i t i o n o f t h e la r v a e
o f A n oph eles (In R u s s ia n ). Mag* P a r a s.
Z o o l. A cad. S ci* U* R* S. S. 1: 2 7 -3 6 .
(R eview ed i n R eview o f App. E n to .)
B od an sk y, M*
1934
I n t r o d u c t io n t o P h y s io lo g i c a l C h em istry.
John 7 /ile y & S o n s, I n c . , New York &
London.
B o y c e , R*
& L e w is , F* G*
1910
The e f f e c t o f m osq u ito L arvae upon
d r in k in g w a te r . Ann. Trop. Med. and
P a r a s it o l.
3: 5 9 1 -5 9 4 .
(R eview ed by
Hinman)
B oyd, M. F*
1926
A n o t e on t h e r e a r in g o f A n op h elin e
la r v a e . B u ll. E n t. Res* 1 6 . p . 1 0 8 .
B u s n e l, R* G.
1937
C o n tr ib u tio n a r 1 * etu d e anatom ique e t
p h y s io lo g iq u e de l a c h e n i l l e d 1E p h e s tia
k e u h n ie lla 2 - R ev. V eg. P a r is 24:
1 3 7 -1 6 2 .
(A b str a c te d in R ev. Appl* E n to .)
B u s h n e ll, R. J*
1938
The r e l a t i o n s o f n u t r i t i o n a l l e v e l s to
t h e growth o f p o p u la tio n s o f T rib o iiu m
confusum , Duv. Ann. E n t. 6oc* Amer. 31:
3 4 5 -3 5 1 .
C h ic k , H«
& C op p in g, A.
1930
The co m p o site n a tu r e o f t h e w a t e r - s o lu b le
v ita m in B^, and th e a n t i- d e r m a t i t i s
v ita m in Bg. Biochem . J o u r. 24: 1 7 6 4 1779.
C h ic k , H . , C opping,
A* , E d g a r , C» E.
1935
The w a t e r - s o lu b le B - v ita m in s . XV. The
com ponents o f v ita m in Bg* Biochem* J o u r .
29: 7 2 2 -7 3 4 .
C h iu , S h in Foo
& McCay, C. M<
1939
N u t r i t i o n a l s t u d ie s o f t h e c o n fu sed
f l o u r b e e t l e , T rib o liu m confusurn Duv.
and t h e bean b e e t l e , A c a n t h e s c e lid e s
o b t e c t u s , Say- Ann. Ent* S o c. Amer. 32:
1 6 4 -1 7 0 .
C le v e la n d , L* R.
19 28
F u r th e r o b s e r v a tio n s and ex p erim en ts
on t h e s y m b io s is betw een t e r m it e s and
t h e i r i n t e s t i n a l p r o to z o a . B i o l . B u l l.
54 : 2 3 1 -2 3 9 .
Cook, S . F .
& S c o t t , K* G•
1933
The n u t r i t i o n a l req u irem en ts o f
Z o o term o p sis a n g u s t i c o l l i s . Jour.
& Comp. P h y s io l. 4: 9 5 -1 0 9 .
C e ll.
C r o w e ll, M* F .
& McCay, C« M.
1937
N u t r i t io n a l s t u d ie s o f t h e w ebbing
c lo t h e s m oth. P h y s io l. Zool* 1 0 :
372.
368-
Dann, W. J .
& Subbarow, Y*
1938
D i f f e r e n t i a t i o n o f t h e r a t d e r m a t it is
f a c t o r and t h e c h ic k d e r m a t it is f a c t o r
from n i c o t i n i c a cid . J o u r . N u t r it io n . 16:
1 8 3 -1 9 5 .
1936
The n u t r i t i o n a l req u irem en ts o f t h e la r v a
o f t h e m o sq u ito , T h e o b a ld ia in c id e n s
Thom. J o u r. E xp. Zool* 73: 461—479*
19 24
The r e l a t i o n o f m icroorgan ism s t o t h e
d evelop m ent and lo n g e v it y o f f l i e s .
Amer
J o u r. T rop. Med* 4: 8 5 -1 0 7 .
1938
A m ethod f o r th e s t e r i l e c u ltu r e o f
h o u s e f l i e s . J o u r . P a r a s it o lo g y 25:
179.
F r o s t , F* M .,
H erm s, W. B * ,
& H osk im s, W.
G la s e r , R. W*
177-
G o r tn e r , R. A*
1938
O u tlin e s o f B io c h e m istr y . John W iley
& S o n s, I n . New York & London.
G yorgy, P a u l
1935
I n v e s t i g a t i o n s on t h e v ita m in Bg com plex.
I I I . The i n a c t i v a t i o n o f l a c t o f l a v i n e
and v ita m in Bfi by v i s i b l e l i g h t . Biochem
J o u r . 29: 7 6 ? -7 7 5 .
65.
Hi nma n , E. H#
1930
A stu d y o f t h e fo o d o f m osq u ito la r v a e
( C u l ic i d a e ) . Amer# J o u r . Hyg# 12: 2 3 8 270.
1932
(a )
The u t i l i z a t i o n o f w ater c o l l o i d s and
m a t e r ia ls i n s o lu t io n by a q u a tic a n im a ls
w ith e s p e c i a l r e f e r e n c e t o m osq u ito
l a r v a e . Q uart. R ev. B i o l . 7: 2 1 0 -2 1 7 .
1932
(b )
The r o le o f s o lu t e s and c o l l o i d s in t h e
n u t r i t i o n o f A n o p h elin e l a r v a e . P relim #
R ep o rt. Amer# Jour# Trop# Med* 12:
263271.
1933
H obson, R. P#
Enzymes in t h e d i g e s t i v e t r a c t o f mos­
q u ito la r v a e # Ann. Ent# S o c. Amer#
26: 4 5 - 5 2 .
1931
S tu d ie s on t h e n u t r it i o n o f B lo w fly
l a r v a e . I S tr u c tu r e and f u n c t io n o f t h e
d ig e s tiv e t r a c t .
Jour* Exp. B i o l . 9:
1 2 8 -1 3 8 .
1932
(a )
S tu d ie s on t h e n u t r i t i o n o f B lo w fly
la r v a e X I. The r o l e o f t h e i n t e s t i n a l
f l o r a in d ig e s t io n #
Jour* E xp. B io l#
9 : 128-138#
1932
(b )
S tu d ie s on t h e n u t r it i o n o f B lo w fly
la r v a e I I I . The l i q u e f a c t i o n o f m uscle#
Jour# Exp* B i o l . 9 : 3 5 9 -3 6 5 .
1932
(c)
S t u d ie s on t h e n u t r i t i o n o f B lo w fly
la r v a e IV# The norm al r o l e o f m ic r o ­
organism s in l a r v a l growth* Jour# Exp.
B i o l . 9: 3 6 6 -3 7 7 .
1933
Growth o f B lo w fly la r v a e on b lo o d and
serum X# R esp on ses o f a s e p t i c la r v a e
t o v ita m in B* Beochem# J o u r . 27: 1 8 9 9 1909.
1935 (a )
Growth o f B lo w fly la r v a e on b lo o d and
serum I I # Growth in a s s o c i a t io n w ith
b a c te r ia * Biochem . J o u r . 29: 1286-1291#
1935 (b )
On a f a t s o lu b le growth f a c t o r r e q u ir e d
by B lo w fly la rv a e# I D is t r ib u t io n and
P r o p e r t ie s . Biochem* J o u r . 29: 129 2 1296.
1935 ( c )
On a f a t s o lu b le growth f a c t o r r e q u ir e d
by B lo w fly la r v a e . I I I d e n t i t y o f
grow th f a c t o r w ith c h o l e s t e r o l . Biochem .
J o u r. 29: 2 0 2 3 -2 0 2 6 .
66
H oog,
e.
G. van* t
Howland, L* J .
1935
A s e p tic c u lt u r e o f i n s e c t s in v ita m in
resea rch * Z e it s c h r V ita m in fo r sc h 4s 3 0 0 344.
(A b str a c te d in B i o l . A b s tr a c ts )
1936
A s e p tic c u lt u r e o f i n s e c t in v ita m in r e ­
search * Z e it s c h r . V ita m in fo r sc h . 5; 1 1 8 1 2 6 . (A b s tr a c te d i n B i o l A b s tr a c ts )
1930
The n u t r i t i o n o f m osq u ito laratae w ith
s p e c i a l r e f e r e n c e t o t h e i r a l g a l fo o d .
B u l l . E n t. Res* 2 1 J 4 3 1 -4 4 0 .
J a c o b s , 3* E*
1937
& RalchoucLhury, D* P*
Some o b s e r v a tio n s o f E p h e s tia k u e h n ie lla 2 .
rea red under a s e p t i c c o n d it io n s . Ann.
A p p l. B i o l . 24; 6 3 2 -6 5 0 .
L oeb , J*
1915
The s a l t s r e q u ir e d f o r th e developm ent
o f in se c ts *
J o u r . B i o l . Chem* 23; 4 3 1 434.
1915
The s im p le s t c o n s t it u e n t s r e q u ir e d f o r
grow th and c o m p le tio n o f t h e l i f e c y c le
i n an i n s e c t (D r o s o p h ila ) ♦ S c ie n c e 41 ;
1 6 9 -1 7 0 .
Ludw ig, D*
& F o x , H.
1938
Growth and s u r v iv a l o f Ja p a n ese b e e t l e
larva© r ea red in d i f f e r e n t m edia* Ann.
E n t. S o c . Amer. 31; 4 4 4 -4 5 6 .
Math©aon, R.
& Hinman, E* H*
1928
Chara f r a g i l i s and m o sq u ito d ev elo p m en t.
Amer. J o u r. Hyg* 7; 2 2 9 -2 9 2 .
M acGregor, M. E .
19 29
The s i g n i f i c a n c e o f t h e pH. i n th e
d evelop m ent o f m osq u ito la r v a e *
P a r a s it o lo g y 21; 1 3 2 -1 5 7 .
1938
The n u t r i t i o n a l req u irem en ts o f B l a t e l l a
g erm a n ica * P h y s io l. Z o o l. 1 1 ; 8 9 —1 0 1 .
1937
N u t r it i o n a l s t u d ie s w ith t h e co ck ro a ch .
P h y s i o l. Z o o l. 10: 3 6 -4 5 .
McCay, C* M*
Melampy, R. M.
& M aynard. L. A
M ic h e lb a c h e r , A* E , , 1932
H o s k in s , W. M»,
& H e m s , W. B.
N e ls o n , J . W.
& P filo isr, L« S*
1935
The n u t r i t i o n o f f l e s h f l y la r v a e ,
L u c ilia s e r i c a t a I* The adequacy o f
s t e r i l e s y n t h e t ic d i e t s . J o u r. Exp. Zool*
64: 1 0 9 -1 3 2 .
The phosphorus c o n te n t and req u irem en ts
o f t h e f lo u r b e e t l e , T rib o liu m confusum
Duv. and a stu d y o f i t s need f o r v ita m in
D* J o u r . Agr* R e s. 50: 8 4 9 -8 5 2 .
67.
N o r th ru p , J* H.
1917
The r o le o f y e a s t in t h e n u t r it i o n of
an i n s e c t ( D r o s o p h ila ) • Jour* B io l*
Ghem* 30: 181-187*
1926
D u ra tio n o f l i f e o f an a s e p t ic D r o s o p h ila
c u lt u r e in b red i n th e dark f o r 230
g e n e r a tio n s * J o u r . G en. P h y s i o l . 9: 7 6 3 770*
Rakhmanova, P . I .
1937
R o le d es b a c t e r ia dans l a v i e d es la r v e s
d es c u l i c i d a e
(I n R u s s ia n ). Med. P a r a s i t .
6 s 2 0 -3 6 .
(A b s tr a c te d i n R ev. App* E n t .)
R ic h a r d so n , C* H.
1926
A p h y s i o l o g i c a l stu d y o f t h e grow th o f th e
M ed iterra n ea n f lo u r m oth, E p h esta
k u e h n ie lla Z. i n wheat f l o u r . J o u r . A gr.
Res* 32s 8 9 5 -9 2 9 .
Rozeboom
1935
The r e l a t i o n o f b a c t e r ia and b a c t e r i a l
f i l t r a t e s t o th e develop m ent o f m osq u ito
l a r v a e . Amer. J o u r . Hyg. 21s 1 6 7 -1 7 9 .
R u d o lfs , W.
& L a ck ey , J . B.
1929
E f f e c t o f fo o d upon t h e p h o to tr o p ism o f
m o sq u ito la r v a e . Amer. J o u r . H yg. 10s
2 4 5 -2 5 2 .
S h i p i t z i n a , N. K-
1930
On t h e r o l e o f o r g a n ic c o l l o i d s o f w a ter
in t h e f e e d in g o f t h e la r v a e o f A n op h eles
m a c u lip e n n is * ( i n R u ssia n ) B u l l . I n s t .
R ech. B i o l . U n iv . Perm. 7? 1 7 1 - 1 9 4 .( R .A .E .)
1935
Grandeur maximum e t minimim d es p a r t i c l e s
pouvant e t r e a v a t e e s p ar l e s la r v e s
d f A n op h eles m a c u lip e n n is m e ss a e . ( i n
R u ssia n ) Med* P a r a s i t . 4s 3 8 1 -3 8 9 .
(A b str a c te d in Rev* App. E n t .)
S t r e e t , H. R.
& P a lm er, L. S.
1935
R eq uirem en ts o f th e f lo u r b e e t l e ,
T rib o liu m confusum , f o r v ita m in s o f th e
B grou p . P r o c . S o c. Exp. B i o l . & Med.
32: 1 5 0 0 -1 5 0 1 .
Sweetm an, M. D*
& P a lm er, L . S*
19 28
I n s e c t s a s t e s t a n im a ls in v ita m in r e ­
s e a r c h . I V itam in req u irem en ts f o r th e
f lo u r b e e t l e , T rib o liu m confusum Duv.
Jour* B i o l . Chem. 77: 3 3 -3 8 .
T rag© r, W illia m
1935
(a )
1935 (b)
The c u lt u r e o f m osq u ito la r v a e f r e e from
l i v i n g m icro o rg a n ism s. Amer. J o u r . Hyg.
2 2 : 1 8 -2 5 .
On th e n u t r i t i o n a l req u irem en ts o f m os­
q u ito la r v a e ( Aedes a e g y p t i) . Amer. Jou r.
Hyg. 22: 4 7 5 -4 9 3 .
68*
T r a g e r , Y 7 illiam
1936
The u t i l i z a t i o n o f s o lu t e s by m osq u ito
la r v a e * B u ll* No* 2s 3 4 3 -3 5 1 .
1937
A grow th f a c t o r r e q u ir e d by m o sq u ito
la r v a e * J o u r , E xp. B i o l . 14s 240—251.
T r a g e r , W illia m ,
M i l l e r , D. K. ,
& R h oad s, C_. P.
1938
The ab sen ce from th e u r in e o f p e r n ic io u s
anem ia p a t ie n t s o f a m o sq u ito la r v a e
grow th f a c t o r p r e s e n t i n norm al u r in e .
J o u r . Exp* Med. 64: 469-480*
W h ite , G. P .
1931
P r o d u c tio n o f s t e r i l e m aggots f o r
s u r g ic a l u s e . A b s tr a c ts P r o c . Amer.
Soc* P a r a s i t o l o g i s t s . J o u r. P a r a s i t o l .
D ec. 1 9 3 1 .
1939
The P r in c i p l e s o f I n s e c t P h y s io lo g y .
E* P . D u tton Co. , I n c . New Y ork.
W ig g le s w o r th ,
B*
W illia m s , R. J*
1934
& S a u n d e rs, D* H*
The e f f e c t s o f i n o s i t o l , c r y s t a l l i n e
v ita m in B^> an d^ panoth en ic a c i d ” on t h e
grow th o f d i f f e r e n t s t r a i n s o f y e a s t .
B iochem . J o u r. 28: 1887-1900*
W illia m s , R. J . ,
1938
T r u e s d a ll, J* H. ,
e t o th ers
P a n o th e n ic a c i d . I I .
I t s c o n c e n tr a tio n
and p u r i f i c a t i o n from l i v e r . Jour* Amer*
Chem* S o c . 60: 2 7 1 9 -2 7 2 3 .
W ollman, E*
O b se r v a tio n s su r une l i g n e e a s e p tiq u e
de B l a t l e s ( B l a t e l l a germ an ica) d a n ta n t
de c in q ans* C. R . S o c . B i o l . 9 5 : 1 6 4 165.
(A b s tr a c te d i n R e s . App. E n t .)
1926
THE SURVIVAL OF MOSQUITO LARVAE IN DIFFERENT CONCENRATXONS
OF THE HYDROGEN ION.
INTRODUCTION.
P a r t I o f t h i s d i s s e r a t i o n t lie e f f e c t s o f m icroorgan ism s *
v it a m in s , and s o l u t e s on th e norm al grow th and d evelopm ent o f th e
la r v a e o f A edes a e g y p t i and C u lex p ip ie n s h as b een c o n sid e r e d *
H ow ever, b e f o r e s u c c e s s f u l e x p e r im e n ts on t h e d ie t a r y n eed s o f m o sq u ito
la r v a e c o u ld b e p erfo rm ed , a know ledge o f t h e optimum pH ra n g es was
g r e a tly need ed .
As th e r e v ie w o f th e l i t e r a t u r e w i l l show , la b o r a t o r y
e x p e r im e n ta tio n on th e i n f l u e n c e o f t h e hydrogen io n on t h e la r v a e
o f m o s q u ito e s , h a s n e v e r b e en c a r e f u l l y c o n s id e r e d .
H ence, th e
p r e s e n t w orker deemed i t h i g h l y d e s ir a b l e t o s tu d y t h i s problem, b e f o r e
t h e grow th e x p e r im e n ts w ere p erfo rm ed .
S in c e P a r t I I o f t h i s t h e s i s i s o f a su p p lem en ta ry n a tu r e and
th e a u th o r d o es n o t t h in k i t e i t h e r n e c e s s a r y o r a d v is a b le t o d is c u s s
o r e x p la in th e p h y s ic o - c h e m ic a l phenomena a s s o c i a t e d w it h t h e hyd rogen
io n and i t s a c t i v i t y #
B r i e f l y s t a t e d , e l e c t r o l y t e s i n aqueous
s o l u t i o n a r e c a p a b le o f d i s s o c i a t i o n in t o t h e i r com ponents o r io n s #
T h is d i s s o c i a t i o n r e s u l t s i n th e p r o d u c tio n o f an a c i d , n e u t r a l, o r
b a s i c ( a l k a l i n e ) c o n d it io n i n w a te r s o l u t i o n s .
The m easurem ent o f t h e
a c i d i t y o r a l k a l i n i t y o f a sy stem c o n s t i t u t e s w h at i s known a s a
m easurem ent o f th e pH (h yd rogen io n c o n c e n t r a t io n ) .
The a u th o r h a s s t u d ie d t h e in f lu e n c e o f d i f f e r e n t c o n c e n t r a tio n s
o f t h e hydrogen i o n on t h e d evelop m ent o f th e la r v a e o f th e y e llo w
f e v e r m osq u ito and t h e common h o u se m osq u ito*
The f a c t o r s o f s i z e o f"
th e larVste sat a d e f i n i t e a g e and p e r c e n ta g e em ergence o f th e a d u lt s
h ave b een c o n s id e r e d #
REVIEW OF LITERATURE
MacGregor (1 9 2 1 * 2 9 ) en d eavored to s tu d y th e in f lu e n c e o f th e
h y d ro g en i o n c o n c e n t r a t io n upon th e d evelopm ent o f m osquito la r v a e *
I n h i s e a r l y work he n o t ic e d t h a t an a c id c o n d it io n (pH. 4 V 0 was
l e t h a l to A n o p h eles m a c u lip e n n is , a . b if u r c a t u s , and O c h le r o ta tu s
n em o ro so u s.
On t h e o th e r hand a pH- range o f 8 .2 - 8 * 4 t o 9 . 6 was
f a v o r a b le .
H is e a r l y paper c o n s is t e d o f a few f i e l d o b s e r v a tio n s ,
b u t l a t e r MacGregor perform ed some e x p erim en ts on t h e e f f e c t o f
pH. under la b o r a t o r y c o n d i t io n s .
He o b serv ed t h a t under s t e r i l e
c o n d it io n s on h i s a u to c la v e d ” stan d ard b rea d ” d i e t , w h ich o th e r
w o rk ers have b ee n -unable to d u p l i c a t e , th e la r v a e o f Aedes a r g e n te u s ( a e g y p t i ) d e v e lo p e d e q u a lly w e l l a t hydrogen io n c o n c e n tr a tio n s o f
4 * 0 , 6 . 0 , and 8 . 0 .
I n a 0.5% r a i s i n d i e t th e pH. a t f i r s t was
4 —6 .5 b u t on s ta n d in g f o r tw en ty —fo u r h ou rs i t
dropped to a 2—3
ra n g e; " N e v e r t h e le s s ,” a c c o r d in g to MacGregor, " th e la r v a e a t th e
end o f 2 4 h o u rs a r e u n a f f e c t e d and i n t h i s medium d e v e lo p a lm o s t a s
w e l l a s t h e y d o i n th e sta n d a rd b rea d medium .*1
A l l o f th e la r v a e
d ie d on d i e t s -whose c o n c e n t r a tio n s o f th e hydrogen io n w ere b etw een
8 .5
and 9 . 0 , made up w it h d i l u t e ammonia to in c r e a s e th e a l k a l i n i t y .
But when t h e s e same d i e t s w h e r e in la r v a e had d ie d b e c a u s e o f e x c e s s
a l k a l i w ere made a c id w it h a c e t i c a c id t o pH .4 * 0 , in o c u la t e d la r v a e
w ere a b le t o d e v e lo p and rea ch m a t u r it y .
MacGregor u n f o r t u n a t e ly
made th e m is ta k e o f u sin g u n b u ffe r e d m ed ia .
Throughout h i s p a p er
he c o n s t a n t ly e x p la in s th e sudden ch a n g es i n hydrogen io n con cen ­
tr a tio n .
B arber and Komp (1 9 2 2 ) i n t h e i r w a ter su rv ey s o b serv ed t h a t
A n o p h eles p u n c tip e n n is «
A .
g u a d r im a c u la tu s. and A ,c r u c ia n s w ere found
b r e e d in g i n w a t e r s w hose pH. ranged from 5 .0 t o 8 . 0 .
M o r is h ita
(1 9 2 5 ) w orking w it h C u lex f a t i g a u s i n Japan n o t ic e d
t h a t a l l b r e e d in g w a te r s v a r ie d i n pH. from 6 .5 t o 7 . 6 .
However,
b etw ee n 5*3 and 9*6 u nder la b o r a t o r y c o n d itio n s t h i s m osqu ito co u ld
s u r v iv e .
He c o n c lu d e d th e hydrogen io n c o n c e n t r a tio n w as o f l i t t l e
im p o rta n ce i n m osq u ito d e v e lo p m e n t.
P r u th i (1 9 2 6 ) i n v e s t i g a t e d th e r o le o f pH
o f th e May—f l y la r v a C loen d ip teru m L .
i n th e d evelop m en t
He o b se rv ed tha t i n a pH
ra q g e o f 6 . 0 — 9*5 t h e la r v a d id n o t s u f f e r a n y g r e a t harm .
mum r a n g e s w ere 7 . 0 — 9*5*
O p ti­
D eath w as e a r l i e r below 6 . 0 th an above
9 . 5 and a t a pH o f 3 . 0 d e a th o ccu rred i n 8 h o u r s .
P r u th i c o n c lu d e s
t h a t " i t d o e s n o t r e q u ir e a lo n g argum ent to show t h a t hydrogen
io n c o n c e n t r a t io n , as s u c h , i s a l s o a f a c t o r o f g r e a t im p o rta n ce."
H arold (1 9 2 6 ) o b se r v ed th a t A edes m a c u la tu s , A . a n n u lip e s ,
A.
g e n i c u l a t u s , and A. r u s t ic a n s w ere found o n ly i n a c id w a t e r s ,
b u t t h a t o t h e r members o f th e same g e n u s, Aedes p u n c to r , A . c in e r e u s
a lo n g w it h T h eo b a ld ia m o r sita n s w ere found i n w a te r s a s a lk a li n e a s
p H .7 .5 .
A n o p h eles s p e c ie s w ere found i n w a te r s from 6 . 0 t o 6 . 8 .
C u lex p ip ie n s was n ev e r found i n a c i d w a t e r s .
S in c e t h i s w orker*s
o b s e r v a tio n s w ere made i n t h e f i e l d o v er a sm a ll a rea th e y c o n t r ib u t e
but l i t t l e
to th e p ro b lem .
Many o t h e r f a c t o r s , e s p e c i a l l y h o s t s f o r
th e a d u lt fe m a le , g o v ern th e b r e e d in g p la c e s f o r m o s q u ito e s .
B ek lem ish ev and M itro fa n o v a (1926) s t u d ie d the problem o f
l a r v a l d i s t r i b u t i o n o f A n op h eles m a c u lip e n n is .
They n e v e r reco rd ed
72.
th e la r v a e o f t h i s m o sq u ito i n w a te r w hose pH w as b elo w 7 * 0 .
A pH
ra n g e from 7*1 - 7 .9 5 a lw a y s p r e s e n te d th e g r e a t e s t number o f l a r v a e .
S e n io r -W h ite (1 9 2 6 ) made an e x t e n s iv e su rv ey o f th e m o sq u ito e s
o f C ey lo n and s t u d ie d th e p h y s i c a l f a c t o r s i n m osquito p o p u la t io n s .
This w o rk er o b se r v e d t h a t
n a t u r a l w a te r s i n C eylon had a pH ran ge
from 5 * 4 t o 9*2 b u t t h a t m osq u ito la r v a e
i n a pH ra n g e o f 5*8 t o 8 . 6 .
i n g e n e r a l w ere found w ith ­
He a l s o n o t ic e d t h a t m o sq u ito e s l i v i n g
i n moving w a te r s had w id e r t o le r a n c e s th an th o s e i n sta n d in g w a t e r s .
A n o p h e lin e s had w id e r t o le r a n c e s th an C u l i c i n e s .
R u s s e l (1 9 2 7 ) s t u d ie d th e hydrogen io n c o n c e n t r a tio n o f b r e e d ­
in g w a t e r s i n M aced on ia.
She found t h a t th e A n o p h elin es o f t h i s a rea
p r e f e r r e d a ran ge o f pH 8 .0 t o 9»5*
However th e C u lic in e s w ere n o t
s o s e l e c t i v e a s th e y w ere fou nd i n a pH ran ge o f 6 .5 t o 1 0 . 0 .
R u d o lfs and L ackey (1 9 2 9 ) r e p o r te d t h a t th e c o n c e n t r a tio n o f
th e hydrogen io n had l i t t l e
e f f e c t on t h e d evelop m en t o f A edes
c a n a d e n s is a s f a r a s th e la r v a e th e m se lv e s w ere co n cern ed , b u t t h a t
i t p r o b a b ly d eter m in e d t o
a c o n s id e r a b le e x t e n t th e abundance o f
d ia to m s , p r o to z o a , f u n g i ,
and o th e r food form s.
S in o r o d in z e n and Adowa (1 9 3 0 ) r e p o r te d t h a t th e y found
A n o p h eles m a c u lip e n n is b r ee d in g i n w a te r s whose pH ran ged b etw een
5 .0 and 1 0 . 0 .
However a pH o f 7 .5 was c o n s id e r e d to b e optimum.
Buchmann ( 1931 ) s t u d ie d th e r o l e p la y e d b y hydrogen io n con­
c e n t r a t i o n i n th e l i f e o f C u lex p ip ie n s and s p e c ie s o f A ed es.
A ccord in g t o t h i s w orker th e la r v a e o f C# p ip ie n s w ere i n d i f f e r e n t
to pH v a lu e s from 4*4 t o 8 . 5 - 9 * 0 .
The la r v a e o f A edes s t i c t i c i u s
A . y e x a n s and A . c a s p iu s d e v e lo p e d o n ly i n w a te r betw een 6 .5 andS.O .
73.
V a r a sh i (1 9 3 1 ) n o t ic e d t h a t th e la r v a e o f A n op h eles m a c u lip e n n is
w ere found i n w a te r s w hose pH v a r ie d b etw een 5 * 8 and 7 . 7 .
B r a d le y (1 9 3 2 ) h a s s t u d ie d th e b r e e d in g o f A n op h elin e m o sq u ito e s
under f i e l d c o n d it io n s *
I n arters w hose pH v a r ie d b etw een 6 .8 and 9 . 2
A n o p h elin e la r v a e w ere fo u n d .
However a t 6 . 8 and above 8 .3 o n ly v e r y
s m a ll numbers o f la r v a e o c c u r r e d .
B etw een a pH o f 7 . 0 and 7 . 8 th e
g r e a t e s t nunber o f la r v a e w ere o b se r v e d .
B e a t t i e (1 9 3 2 ) w ork in g i n T rin id a d s tu d ie d th e b r e e d in g o f
A n o p h e les t a r s im a c u la tu s i n th e f i e l d .
Her o b s e r v a tio n s r e v e a le d
t h a t t h e l a r v a e c o u ld b e found b etw een a pH o f 5 .8 and one o f 7*5*
She s t a t e s th a t no d e f i n i t e c o r r e l a t i o n e x i s t e d b etw een th e hydrogen
io n c o n c e n t r a t io n and numbers o f m osq u ito l a r v a e .
W o o d h ill (1 9 3 8 ) r e p o r te d t h a t v a r i a t io n s i n t h e pH from A .2
to 6 . 8 up t o 9 . 0 s l i g h t l y r e ta r d e d th e d ev elo p m en t o f th e la r v a e
to th e a d u l t s t a g e b u t had no e f f e c t on th e t o t a l numbers o f th e
a d u l t s p r o d u ce d .
METHODS
I n P a r t I I o f t h i s r e p o r t no a tte m p t was made t o r e a r th e la r v a e
u n d er s t e r i l e c o n d it io n s s i n c e optimum grow th o c c u r s i n th e p r e s e n c e
o f m icro o rg a n ism s*
A H m edia w as b u f f e r e d and ch eck ed a t th e end o f
24- h o u rs i n c a s e an y ch a n g es i n pH had occu rred *
O c c a s io n a lly th e
pH o f a b u f f e r e d m edia w ould su d d e n ly change and t h e Jar had t o be
d is c a r d e d *
A t e v e r y pH c o n s id e r e d 8 t e s t s w ere p e r fo r e d w ith 25—d ay
o ld la r v a e p e r t e s t *
A t * 1 1 c o n c e n t r a tio n s o f t h e hydrogen io n from
5 - 8 ( in c lu d in g t h is o n e ) up t o 9 *0 , th e m edia w ere b u ff e r e d w it h NaOH
and KH2P0^*
B elow 5*8 th e a u th o r u se d NaOH and KH p h th a la t e t o
b u f f e r t h e m edia*
Hydrogen i o n d e te r m in a tio n s w ere made w it h a s l i d e com parator
and s e r i e s o f d yes m an ufactu red b y
W. A. T a y lo r and Co*
T h is p i e c e
o f eq uipm ent was lo a n e d b y D r. W* C. S u p p lee o f t h e D epartm ent o f
C h em istry h e r e a t t h e U n iv e r s it y *
The a c c u r a c y o f t h i s in str u m e n t
w as ch eck ed a g a i n s t a Beckman e le c t r o m e t r ic pH m e te r , u s in g b u f f e r s
o f a known hydrogen i o n c o n c e n tr a tio n *
A ll la r v a e w ere r e a r e d on a 6 * 3 # o y s t e r m e a l, 0 . 1 # d r ie d y e a s t ,
0*1# g l u c o s e , and o . l # O sborne-M endel s a l t m ix tu re d i e t i n t h e p r e s e n c e
o f m icro o rg a n ism s*
H ow ever, th e a u th o r w ould l i k e t o p o in t o u t t h a t
a t t h e h yd rogen io n c o n c e n t r a t io n s o f 4 *0 , 5 *0 , 5 *2 , 5 *4-* and 5*6
t h e p h t h a la t e io n was p r e s e n t ( i n b u f f e r ) *
A lth o u g h i t was g iv e n no
e x p e r im e n ta l c o n s id e r a t i o n , th e a u th o r fee&& t h a t t h e p h t h a la t e io n
w as n o t a t o x i c f a c t o r *
A n a ly se s o f r e s u l t s w ere made on t h e p e r c e n ta g e o f a d u lt em ergence
and s i z e o f t h e la r v a e a t t h e end o f 5 d a y s .
RESULTS.
The r e l a t i o n o f pH t o t h e d evelop m en t o f A edes a e g y p t i a r e
p r e s e n te d i n th e g e n e r a l t a b l e , T ab le XV, and t h e c o m p o site t a b l e ,
T a b le XVII 3 w h er ea s th e r e s u l t s f o r C u lex p ip ie n s a r e shown i n th e
g e n e r a l t a b l e , T a b le XVI, and t h e c o m p o site t a b l e , T able X V III.
A t a pH o f 4 * 0 i t can b e s e e n t h a t a l l o f t h e la r v a e o f b o th
s p e c i e s c o u ld n o t s u r v iv e f o r a 24 hour p e r io d ; a t 5 .0 a l l la r v a e
w ere d ead a t th e end o f 4-8 h o u r s; and a t 5*2 t h e la r v a e c o u ld s u r v iv e
f o r 7 2 h o u rs b e f o r e d ea th o c c u r r e d .
The la r v a e o f A edea a e g y p t i w ere a b le t o d e v e lo p a t a pH o f
5 . 4 and 24$ o f them em erged a s a d u l t s .
On t h e o th e r hand, t h e la r v a e
o f C u lex p i p i e n s l i v e d f o r 120 h ou rs (5 d a y s) and d e a th o c c u r r e d .
B etw een t h e pH ra n g e o f 5 . 6 - 6 . 0 la r v a e o f b o th s p e c ie s resp on ded
s i m i l a r l y a s shown b y th e t a b l e s .
I n f a c t , a t a pH o f 6 . 0 b o th Aedes
and C u lex y i e l d e d 60$ em ergence*
C o n c e n tr a tio n s o f th e hydrogen io n b etw een a pH ra n g e o f 6 .2 ~
7 . 4 w ere v e r y f a v o r a b le f o r t h e grow th and d ev elo p m en t o f t h e la r v a e
o f b o th A . a e g y p t i and £ . p i p i e n s .
H owever, a t a pH o f 7 . 4 th e la r v a e o f C u lex p ip ie n s d id n o t
s u r v iv e i n a s g r e a t a number a s d id t h e la r v a e o f A edes a e g y p t i .
At
t h i s h yd rogen i o n c o n c e n t r a t io n o n ly 64 $ em ergence was o b se rv ed f o r
C u le x p i p i e n s « w h erea s 80$ em ergence was n o te d f o r A edes a e g y p t i .
A t a pH ra n g e o f 7 . 6 t h e d i f f e r e n c e i n th e em ergence p e r c e n ta g e
b etw een t h e two s p e c i e s i s l i k e w i s e v e r y w id e .
o f Cule x p i p i e n s em erged .
A edes a e g y p t i em erged .
O nly 4$ o f t h e l a r v a e
On th e o th e r hand, 24$ o f t h e la r v a e o f
I n b o th c a s e s t h e f a l l i n t h e em ergence p e r c e n ta g e
compared t o a pH o f 7 * 4 i s trem en d o u s.
The la r v a e o f b o th s p e c i e s c o u ld s u r v iv e f o r 7 2 h ou rs a t a
pH o f 7 * 8 j a t 8*0 d e a th o ccu rred i n 48 h o u r sj a t 9*0 i n 24 hours*
The la r v a e o f b o th A edes a e g y p t i and C u lex p ip ie n s a t t h e
end o f 5 d a y s w ere v e r y s m a ll a t pH r a n g e s 5*4—5*8*
However, on th e
h ig h e r h yd rogen i o n c o n c e n t r a t io n s i d e , t h a t i s a pH range o f 7 * 4 7 *6 , no d i f f e r e n c e c o u ld b e s e e n when compared w it h a 5 day o ld
l a r v a e grown a t a pH ra n g e o f 6 *4—7*2*
T a b le XV*
E f f e c t o f pH* on t h e D evelopm ent o f Aedes a e g y p t i*
pH* 4 .0 - NaOH - K H phthalate
8 t e s t s w ith 25 la r v a e i n t e s t
A l l la r v a e dead a t end o f 24 h o u rs.
pH* 5 .0 - NaOH - K H phthalate
8 t e s t s w ith 25 la r v a e in t e s t
A l l la r v a e dead a t end o f 48 hours*
pH* 5 . 2 - NaOH - K H phthalate
8 t e s t s w ith 25 la r v a e i n t e s t
A l l la r v a e dead a t t h e end o f 72 hours*
pH* 5*4 - NaOH - K H phthalate
T e st No*
No*
L arvae
10
25
25
25
25
25
25
25
25
25
25
TOTAL
AVER.
250
25
1
2
3
4
5
6
7
8
9
No*
A d u lts
2
5
7
6
8
7
8
5
4
8
60
6
T a b le XV* - C on tin u ed .
pH. 5 .6 NaOH - KH2 PO4
pH* 5 .8 NaOH - KH^PO^
No.
Larvae
No.
A d u lts
T e s t Ho.
8
12
1
2
5
7
3
9
9
7
3
4
5
11
9
10
25
25
25
25
25
25
25
25
25
25
9
10
TOTAL
AVER*
250
25
T e s t No.
1
2
3
4
5
6
7
8
9
80
8
1
2
3
4
5
6
7
8
9
10
8
TOTAL
AVER*
No.
A d u lts
25
25
25
25
25
25
25
25
25
25
15
9
250
25
120
12
12
12
8
17
12
10
14
11
pH. 6 .2 NaOH - KHg PO^
pH. 6 .0 NaCH - KH2 PQ4,
T e s t No .
6
7
No.
Larvae
No.
Larvae
No*
A d u lt s
25
25
25
25
25
25
25
25
25
25
17
13
15
12
10
No*
T e s t No. Larvae
1
2
3
4
5
18
6
20
7
17
15
13
8
TOTAL________ 250______ 150
AVER*__________ 25________15
9
10
TOTAL
AVER*
25
25
25
25
25
25
25
25
25
25
______ 250
25
No.
A d u lts
22
19
24
20
17
20
24
22
16
17
201
20
T a b le XV* — C ontinued*
pH • 6*4 NaOH - KH^PO^
pH. 6 .6 NaOH - KBgPO^
No.
L arvae
No.
A d u lts
24
10
25
25
25
25
25
25
25
25
25
25
25
25
18
19
24
23
18
23
TOTAL
AVER*
250
25
220
22
T e s t No*
1
2
3
4
5
6
7
8
9
21
No.
Larvae
No.
^ d u lt s
10
25
25
25
25
25
25
25
25
25
25
25
25
25
24
23
25
24
25
23
TOTAL
AVER.
250
25
240
24
1
2
3
4
5
6
7
8
9
No.
Larvae
No*
A d u lts
25
10
25
25
25
25
25
25
25
25
25
25
TOTAL
AVER.
250
25
240
24
1
2
3
4
5
6
7
8
9
22
23
25
25
24
22
24
25
25
pH* 7 .0 NaOH - KHgPO^
pH» 6 .8 NaOH - KHgBO^
T e s t No •
T e s t No •
21
T e s t No .
1
2
3
4
5
6
7
8
9
10
TOTAL
AVER.
No.
Larvae
No.
A dult s
25
25
25
25
25
25
25
25
25
25
25
24
24
24
250
25
240
24
22
24
23
25
24
25
T a b le XV* - C ontinued*
pH. 7 .2 NaOH - KHgEQ^
pH* 7 .4 NaOH - KH^PO^.. .
No.
L arvae
No.
A d u lt s
25
25
25
25
25
25
25
25
25
25
18
23
19
24
250
TOTAL
AVER.__________25
220
T e s t No.
1
2
3
4
5
6
7
8
9
10
20
8
24
25
10
4
7
7
10
25
25
25
25
25
25
25
25
25
25
TOTAL
AVER.
250
25
3
4
5
6
7
8
9
200
20
7
No.
A dult s
1
2
250
TOTAL
AVER*_________ 25
20
N o.
L arvae
8
3
5
No *
A dult s
17
18
24
23
22
No.
Larvae
25
25
25
25
25
25
25
25
25
25
1
2
3
4
5
pH. 7 .6 NaOH - KH^O^. ......
T e s t No.
T e st No.
6
9
22
20
21
24
22
18
20
18
pH. 7*8 NaOH - KHgP-O^
8 t e s t s w ith 25 la r v a e i n t e s t
A l l la r v a e dead a t end of 72 h ours
pH* 8 .0 NaOH - KRgPOx
8 t e s t s w ith 25 la r v a e in t e s t
A l l la r v a e dead a t end o f 48 hours
8
6
5
7
60
6
pH* 9 .0 NaOH - KHgEQ^
8 t e s t s w ith 25 la r v a e in t e s t
A l l la r v a e dead a t end of 24 hours
81
T ab le XVI.
E f f e c t o f pH* on t h e Developm ent o f C ulex p i p i ens *
pH* 4*0 - NaOH - K H phthalate
8 t e s t s w ith 25 la r v a e i n t e s t
A l l la r v a e dead a t end o f 24 hours*
pH* 5*0 - NaOH - K H phthalate
25 la r v a e in t e s t
A l l la r v a e dead a t end o f 48 hours*
8 t e s t s w ith
pH* 5 .2 - NaOH - K H phthalate
8 t e s t s w ith 25 la r v a e in t e s t
A l l la r v a e dead a t end o f 72 h o u r s.
pH* 5 . 4 - NaOH - K H phthalate
8 t e s t s w ith 25 larsrae in t e s t
A l l la r v a e dead a t end o f 120 hours*
pH* 5*6 NaOH - K H phthalate
T e s t No-
No*
Larvae
10
25
25
25
25
25
25
25
25
25
25
TOTAL
AVER.
250
25
1
2
3
4
5
6
7
8
9
No.
A d u lts
pH. 5 .8 NaOH - KHgRG^.
T est No.
9
7
8
6
10
25
25
25
25
25
25
25
25
25
25
63
7
TOTAL
AVER.
250
25
6
3
5
1
2
7
3
4
5
10
8
7
2
pH* 6 .0 NaOH - KHgRO-4 -
6
9
No.
A d u lt s
17
10
25
25
25
25
25
25
25
25
25
25
18
16
13
16
17
10
TOTAL
aver .
250
25
160
15
TOTAL
AVER.
1
2
3
4
5
6
7
8
9
11
12
18
22
No.
A dult s
15
110
12
11
13
9
8
8
14
11
12
9
pH. 6 .2 NaOH - KHgPO^
N o.
L arvae
T e s t No .
No.
L arvae
T e st No-•
1
2
3
4
5
6
7
8
9
No.
Larvae
No.
A dult s
25
25
25
25
25
25
25
25
25
25
14
17
250
25
151
15
21
13
11
16
16
18
10
15
T a b le XVI* - C o n tin u ed .
pH. 6 .4 NaOH - KHgEO^ '
T e s t No.
1
2
3
4
5
6
7
8
9
10
TOTAL
AVER.
No.
L arvae
No. .
A d u lt s
25
25
25
25
25
25
25
25
25
25
17
250
25
180
18
22
TOTAL
AVER.
250
25
220
22
9
6
7
21
17
23
25
20
24
23
pH. 7 .0 NaOH - KHgBO^
10
TOTAL
AVER.
250
25
240
24
8
9
10
8
25
7
No.
A dult s
24
18
25
21
25
25
25
25
25
25
25
25
25
25
6
No.
Larvae
25
25
25
25
25
25
25
25
25
25
17
15
No.
A d u lts
3
4
5
1
2
3
4
5
No.
Larvae
1
2
T e st No
23
17
18
15
15
pH* 6 .8 NaCH - KH2p o4
T e st No.
pH. 6 .6 NaOH - KH^PO^
22
25
22
25
24
25
T e s t No.
1
2
3
4
5
6
7
22
8
25
25
9
10
TOTAL
AVER.
No.
Larvae
No.
A d u lts
25
25
25
25
25
25
25
25
25
25
18
23
250
25
200
20
21
17
15
22
22
23
18
21
T a b le XVT* - C ontinued*
pH* 7 . 2 NaOH - KH2 P0^
pH* 7 .4 NaOH - KH2 PO4 .
No.
Larvae
No*
A d u lts
16
10
25
25
25
25
25
25
25
25
25
25
TOTAL
AVER.
250
25
180
18
T e s t No*
1
2
3
4
5
6
7
3
9
21
14
TOTAL
AVER.
250
25
10
1
9
8
9
17
19
11
9
20
19
21
16
16
pH* 7 .8 NaOH - KH^FO^
8 t e s t s w ith 25 la r v a e in t e s t
10
7
160
16
7
0
0
1
2
0
1
1
2
8
250
25
19
23
16
19
25
25
25
25
25
25
25
25
25
25
6
TOTAL
AVER.
6
No.
A d u lts
3
4
5
10
22
No.
L arvae
1
2
No.
A dult s
12
18
12
No.
Larvae
25
25
25
25
25
25
25
25
25
25
1
2
3
4
5
pH. 7 .6 NaOH - KH
T e s t No •
T est No.
3
0
A l l la r v a e dead a t end o f 72 hours
pH* 8 .0 NaOH - KH^PO^,
8 t e s t s w ith 25 la r v a e in t e s t
A l l la r v a e dead a t end o f 48 hours
pH* 9 .0 NaOH - KH2 PO4
8 t e s t s w ith 25 la r v a e i n t e s t
A l l larva© dead a t end o f 24 hou rs
T a b le
pH.
4 .0
5*0
5 .2
5 .4
5 .6
5 .8
6 .0
6 .2
6 .4
6 .6
6 *8
7 .0
7 .2
7 .4
7 .6
7.8 '
8 .0
9 .0
T a b le
pH.
4 .0
5 .0
5 .2 ;
5 .4
5 .6
5 .8
6 .0
6 .2
6 .4
6 .6
6 .8
7 .0
7 .2
7 .4
7 .6
7 .8
8 .0
9 .0
C om posite T a b le Showing E f f e c t o f pH. on Developm ent o f th e
L arvae o f A edes a e g y p t i. A verage o f 8 t e s t s .
X V II*
No.
L arvae
25
25
25
25
25
25
25
25
- 25
25
25
25
25
25
25
25
25
25
X V III.
End
24 Hrs*
A l l dead
L iv in g
H
tt
End
48 H rs.
A l l dead
L iv in g
m
tt
tt
tt
tt
tl
•I
tl
II
It
It
tt
tt
tt
ft
tt
tt
tt
«%
tt
M
tt
tt
tt
tt
tt
End
72 H rs.
A l l dead
L iv in g
tt
»
tt
tt
tt
tt
tt
t>
tt
tt
tt
No.
A d u lts
%
A d u lts
24
6
8
12
32
48
60
80
15
20
22
88
24
24
24
96
96
96
22
88
20
6
80
24
A l l dead
A l l dead
A l l dead
C om posite T a b le Showing E f f e c t o f pH* on Developm ent of th e
L arvae o f C u lex p i p i e n s . A verage of 8 t e s t s .
N o. .
L arvae
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
End
24 H rs.
A l l dead
L iv in g
«
End
48 H rs.
A ll dead
L iv in g
tt
tt
«
«
m
it
tt
tt
tt
tt
tt
tt
it
tt
tt
tt
n
tt
t$
tt
it
«
«.
m
tt
n
A l l dead
A l l dead
End
7 2 .Hrsi.
No.
A d u lts
%
A d u lts
A H dead
L iv in g L arvae dead end 5 days
»t
28
7
tt
48
12
tt
15
60
t»
15
60
»
76
18
t*
tt
tt
tt
n
w
A ll dead
22
88
24
96
80
76
64
4
20
18
16
1
85
10 0
C.
30
PIPIENS
—
A . AEGYPTI
60 —
40
20
40
4.8
4.4
pH
F ig * X*
OF
5.2
5.6
6.0
6.4
6.8
7.2
7.6
MEDIA
E f f e c t o f pH* on th e Developm ent o f Aedes
a S g y p ti and C u lex p ip ie n s
SO
DISCUSSION
t
The a u th o r h a s shown th a t th e la r v a e o f A edes a e g y p t i s u r v iv e i n
m edia w hose h yd rogen io n c o n c e n t r a tio n v a r ie d b etw een a pH* ran ge o f
5*4 — 7 * 6 w h ereas th e la r v a e o f C u lex p ip ie n s s u r v iv e d a ran ge o f pH*
b etw een 5*6 - 7 * 6 .
MacGregor (1 9 2 9 ) w orking w it h Aedes a e g y p t i cla im e d
t h a t th e la r v a e d e v e lo p e d e q u a lly a s w e l l a t 4 * 0 , 6 * 0 , and 8 * 0 .
However
when t h e e x p e r im e n ta l te c h n iq u e em ployed b y MacGregor i s exam ined i t can
b e s e e n t h a t he f a i l e d t o u s e b u f f e r e d m edia o f an y t y p e .
Under t h e s e
c o n d it io n s th e p r e s e n t w ork er c a n n o t f e e l th a t t h i s w ork er (MacGregor)
h as drawn s c i e n t i f i c c o n c lu s io n s .
I n th e p r e s e n t w o r k e r ^ own work he
h a s o b se r v e d t h a t t h e o y s t e r m eal and d r ie d y e a s t medium (a lo n g w ith
g lu c o s e and Osborne M endel s a l t s ) when l e f t u n b u ffe r e d changed from d ay
to day.
T h is was p r o b a b ly due t o a c t io n o f b a c t e r i a l m eta b o lism and
m e ta b o lic a c t i v i t y o f t h e la r v a e t h e m s e lv e s .
The o b s e r v a tio n s o f M o r is h it ia (1 9 2 5 ) r e v e a le d t h a t Cu l e x f a t i g a n s
la r v a e was found o n ly i n b r e e d in g w a te r s w hose pH. v a r ie d , from 6*5 t o 7 . 6
By com paring t h e s e o b s e r v a tio n s w it h th e r e s u l t s p r e s e n t i n t h i s r e p o r t
c l o s e agreem ent can b e seen *
H arold (1 9 2 6 ) c la im e d t h a t C u lex p ip ie n s la r v a e w ere n e v e r fou nd i n
a c id w a t e r s .
However, th e p r e s e n t w orker w ould l i k e t o p o in t out t h a t
H a r o ld ts o b s e r v a tio n s w ere made o v e r a v e r y s m a ll a rea u n d er f i e l d con­
d itio n s .
O th er f a c t o r s o f t e n govern th e b r e e d in g p la c e s f o r m o sq u ito e s
and i t i s in c o n c e iv a b le th a t a d u lt fe m a le s can s e l e c t b r e e d in g w a te r s
w hose h yd rogen io n c o n c e n t r a tio n i s f a v o r a b le t o l a r v a l d e v e lo p m e n t.
B arb er and Komp (1 9 2 2 ) on t h e o t h e r hand d id co n d u ct e x t e n s iv e
s u r v e y s on th e b r e e d in g p la c e s o f A n o p h elin e la r v a e and r e p o r te d t h a t
b etw een a pH. range o f 5*0 — 8 . 0 la r v a e d id o c c u r .
These r e s u l t s do c l o s e l y
87.
ap p ro x im a te t h e r e s u l t s o b ta in e d b y th e p r e s e n t w orker b r e e d in g Aedes
a e g y p t i and C u lex p ip ie n s i n t h e la b o r a t o r y .
The o b s e r v a tio n s o f S e n io r-W h ite (1 9 2 6 ) w ere i n agreem ent t o a
c e r t a i n e x t e n t m t h t h o s e p r e s e n te d b y th e p r e s e n t w o rk er.
T his w orker
o b se r v e d t h a t m o sq u ito la r v a e i n C eylon w ere found w it h in a pH. ran ge o f
5 .8 t o 6 . 6 .
I t i s u n fo r tu n a te t h a t o n ly a few la b o r a t o r y ex p erim en ts have b een
c o n d u c ted a lo n g t h e s e l i n e s o f r e s e a r c h .
SUMMARY
X.
The la r v a e o f A edes a e g y p t i s u r v iv e i n m edia w hose hydrogen
io n c o n c e n t r a t io n v a r i e s b etw een a pH. ran ge o f 5*4 ~ 7 * 6 ; w h erea s th e
la r v a e o f C u lex p ip ie n s s u r v iv e i n a pH. ran ge o f 5*6 — 7 #6
2.
The optimum ra n g es f o r b o th s p e c ie s l i e b etw een a pH# ran ge o f
6*2 - 7 *4 .
3#
The la r v a e o f A edes a e g y p t i are a b le t o d e v e lo p i n w id e r ra n g es
o f h yd rogen i o n c o n c e n t r a tio n s than th e la r v a e o f C u lex p i p i e n s #
4#
M osq uito la r v a e a r e s m a lle r when r e a r e d i n m edia w hose pH#
ra n g e i s b e tw ee n 5 * 4 - 5*8 th a n i n th o s e c l o s e r t o n e u t r a lit y *
LITERATURE CITED
B a rb e r , M. A*
& Komp, w.
1922
The hydrogen io n c o n c e n tr a tio n o f w a te r s
i n r e l a t i o n t o A n op h elin e p rod u ction *
U* S* P u b lic H e a lth Emergency A b str a c t
J 712.
B e a t t i e , M. F . V .
1932
The p h y s ic o -c h e m ic a l f a c t o r s o f w a ter
i n r e l a t i o n s t o m osq u ito b r e e d in g i n
T r in id a d . B u l l . E n t. R es. 23: 4 7 7 -5 0 0 .
B e k le m ish e v , V*
& M itr o fa n o v a , F .
1926
On t h e e c o lo g y o f t h e la r v a e o f A n op h eles
m a c u lip e n n is * The problem o f d is t r ib u t io n *
( i n R u s s ia n ) . B u ll* I n s t . R esch erch es
b i o l . & Sta* B io l* U n iv . Perm. (A b str a c te d
i n R ev. Appl* E n t .)
BodLansky, M-
1934
I n t r o d u c t io n t o P h y s i o l o g i c a l C h em istry.
John W iley & S o n s, I n c . New York & London.
B r a d le y , G. H.
1932
Some f a c t o r s a s s o c ia t e d w ith t h e b r e e d in g
o f A n op h eles m o s q u ito e s . J o u r . A gr. R e s.
4 4 : 3 8 1 -3 9 9 .
Buchmann, T A T .
1931
U ntersuchungen ttber d ie bed eu tun g d er
was s e r s t o f fr o n e n k ^ n z e n tr a tio n f u r d ie
e n tw ic k lu n g d e r m u ck en larven . Z Angew.
E n t . 17: 4 0 4 -4 1 7 .
(A b s tr a c te d i n R ev.
Appl* E n t .)
C la r k , ?/• M*
1928
The D e te r m in a tio n o f H ydrogen I o n s . 3rd
E d it io n . The W illia m s & W ilk in s Co.
B a ltim o r e .
1938
O u tlin e s o f B io c h e m is tr y . John W iley &
S o n s, I n c . New York & London.
H a r o ld , C. H* H.
1926
S t u d ie s on m osqu ito b io n o m ic s .J l* R. A*
M« C* 4 7 : 8 1 - 9 4 .
(A b str a c te d in R ev.
A p p l• E n t .)
M acGregor, M. E*
1921
The in f l u e n c e o f t h e hydrogen io n c o n c e n tr a ­
t i o n i n th e developm ent o f m osq u ito la r v a e .
P a r a s it o lo g y * 13: 3 4 8 -3 5 1 .
19 29
The s ig n i f i c a n c e o f t h e pH. i n th e d e v e lo p ­
ment o f m osq u ito la r v a e * P a r a s it o lo g y .
21: 1 3 2 -1 5 7 .
G o r tn e r , R.
h.
A*
W.
90
M o r is h it a , H.
19 28
E x p erim en ta l o b s e r v a tio n s o f t h e h a b it s o f
C ulex f a t i g a n s -with s p e c ia l r e f e r e n c e t o
th e i n f lu e n c e o f hydrogen io n c o n c e n tr a ­
t i o n upon i t s developm ent*
( i n Jap an ese*)
J l* Med* Soc* Formosa* No* 2 4 7 , 37pp.
(A b str a c te d in Rev* Appl* E n t .)
P r u t h i , Hem Sin gh
1926
The in f lu e n c e o f some p h y s ic a l and
ch em ica l c o n d it io n s o f w a ter on M a y -fly
la r v a e * ( C loeon d ipteru m L*) B u l l . Ent*
R e s. 16: 2 7 9 -2 8 4 .
R u d o lp h s, a .
& L a c k e y , J* B*
19 29
The c o m p o sitio n o f u n te r and m osq uito
b r e e d in g . Amer. J o u r. Hyg* 9: 1 6 0 -1 8 0 -
R u s s e l , H*
1927
N o tes on th e r e a c t io n s o f th e b r e e d in g
p la c e s o f A n o p h elin es i n M acedon ia.
B u l l . E n t. R e s . 18: 155-163*
SenLor-Tfifriite, R*
1926
P h y s ic a l f a c t o r s in m osq u ito eco lo g y *
B u ll* E n t. R es. 1 6 : 1 8 7 -2 4 8 .
Sm orodinzew , I* A*
& Adowa, A. W*
1930
Sur l e r o l e de l a r e a c t io n du m ilie u dans
l ' e c o l o g i e d es la r v e s d*A n op heles
m a c u lip e n n is * B u ll- Soc* P a th . e x o t .
23: 3 8 - 4 3 .
(A b s tr a c te d i n R ev. Appl*
E n t .)
Y a r a s h i, V*
1931
Zur b i o l o g i e d e r A n o p h e le sla r v e s (A n op h eles
m a c u lip e n n is ) i n verb in d u n g m it den
w a s s e r fa k to r e n i n K a lc h is . A rch. S c h i f f s u-Tropenhyg* 35: 336-345*
(A b str a c t i n
R ev. Appl* E n t .)
W o o d h ill, A. R.
1938
S a l i n i t y t o le r a n c e and pH. range o f C ulex
f a t ig a n s W ie d ., w ith n o t e s on th e a n a l
p a p i l l a e o f s a l t w ater m o s q u ito e s . P r o c.
L in n . S o c. N* S* W* 63: 2 7 3 -2 8 1 .
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