Title: Dietary amino acid requirements of the almond moth, Cadra cautella (Walker)
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Title: Dietary amino acid requirements of the almond moth, Cadra cautella (Walker) based on radiometric and carcass analysis techniques
Physical Description: xi, 110 leaves : ill. ; 28 cm.
Language: English
Creator: Heller, Jack Myron, 1943-
Publication Date: 1975
Copyright Date: 1975
 Subjects
Subject: Moths   ( lcsh )
Entomology and Nematology thesis Ph. D
Dissertations, Academic -- Entomology and Nematology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis--University of Florida.
Bibliography: Bibliography: leaves 101-109.
Statement of Responsibility: by Jack Myron Heller.
General Note: Typescript.
General Note: Vita.
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Bibliographic ID: UF00099399
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000408111
oclc - 02164077
notis - ACF4524

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DIETARY I;:iNc, AOCID FESQUIiREMENTS OF
THE ALMOND MOTNI, CADrLO CAUTELLA (WALKER),
BASED O1: RADIOMEiTRIC AND CARCASS ANALYSIS TUCHNIQUiES











By

JACK MYRON HELLER


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFIL IENT OF THE PEQUIREIHENTS FOR THE
DEGIE OF DOCTOR OF PHILOSOPHY





UNIVERSITY OF FLORIDA


1975














ACKNOWLEDGEMENTS


I would like to thank the many individuals who assisted me

throughout the study and preparation of this dissertation.

My profound thanks' go to Dr. R. E. Waites, who served as

Chairman of the supervisory committee and whose help was

invaluable throughout this study.

Appreciation is also expressed to Dr. W. G. Eden, Chairman of

the Department of Entomology, and Drs. D. L. Silhacek, B. J.

Smittle, and D. S. Anthony, members of my supervising committee.

Special appreciation goes to my wife, Barbara, for her

patience and help during this period of graduate study, and for

assisting in the preparation of this manuscript.












TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS .................................... ii

LIST OF TABLES ...................................... V

LIST OF FIGURES ..................................... viii

ABSTRACT .............................. ................ x

INTRODUCTION ........................................ 1

LITERATURE REVIEW ................................... 4

Radioisotopes and the Determination of
Amino Acid Requirements ..................... 4

Carcass Analysis for the Estimation of
Quantitative Amino Acid Requirements ........ 11

General Insect Nutrition ....................... 12

Distribution and Metabolism of Amino
Acids and Proteins ........................... 13

Methodology .................................... 14

MATERIALS AND METHODS ............................... 22

Rearing .............. ........................... 22

Radiochromatography and Autoradiography
of U-14C-glucose ........................... 22

Radioactive Medium Preparation ................. 24

Protein Extraction ............................. 25

Protein Hydrolysis ............................. 26

Thin-Layer Chromatography of Amino Acids ....... 29

Radioactivity Measurements ..................... 30

Carcass Analysis ............................... 33






iii













TABLE OF CONTENTS Continued


Page


Gas Chromatography of Amino Acids for
Carcass Analysis ............................

Microbiological Study ..........................

RESULTS AND DISCUSSION .............................

Qualitative Amino Acid Requirements by
the Indirect Radioactive Method and
Thin-Layer Chromatography ...................

Carcass Analysis for the Determination of
Quantitative Amino Acid Requirements
Using Gas Chromatography ....................

SUMMARY .............................................

APPENDIX ............................................

REFERENCES CITED ....................................

BIOGRAPHICAL SKETCH .................................
































iv


35

40

42




42




74

87

89

101

110











LIST OF TABLES


Table Page

1 Amino Acid Requirements of Some Insects Deter-
mined by the Radioactivity Method ............. 6

2 Experimental Parameters Used to Determine
Amino Acid Requirements by the Indirect
Radioactivity Method .......................... 8

3 Rf and RLeucine Values of 22 Amino Acid
Standards Separated by Two-Dimensional
Thin-Layer Chromatography on Cellulose ........ 45

4 Cpm/Carbon Atom of Amino Acids from Acid
and Base Hydrolysates of Protein Extracted
from Almond Moth Larvae Reared on Medium
Containing 14C-Glucose ........................ 47

5 Radioactivity in Amino Acids from Acid
and Base Hydrolysates of Proteins Extracted
from Larval Rearing Media Containing
14C-Glucose ................................... 53

6 Cpm/Carbon Atom of Free Amino Acids Extracted
from Sterile Larval Rearing Medium Containing
14C-Glucose and That Had Almond Moth
Larvae Reared on It ........................... 58

7 Cpm/Carbon Atom of Free Amino Acids Extracted
from Non-Sterile Larval Rearing Medium that was
Incubated with 14C-Glucose for 12 Weeks and Had
'To Larvae Reared on It ........................ 59

8 Cpm/Carbon Atom of Free Amino Acids Extracted
from Sterile Larval Rearing Medium Containing
14C-Glucose and on Which No Larvae
were Reared ................................... 60

9 Result of Microbiological Study on Almond
Moth Eggs and Larvae and 14C Media to Deter-
mine the Source of Radioactive Free Amino
Acid Synthesis ................................ 64

10 Specific Activity of Dried Supernatant Fractions
from the Extraction of Fifth-Instar Almond
Moth Larvae Reared on Medium Containing
14C-Glucose ................................... 66












LIST OF TABLES Continued


Table Page

11 Specific Activity of Dried Supernatant
Fractions from the Extraction of Larval
Rearing Medium Containing 14C-Glucose and
That Had Almond Moth Larvae Reared on It ...... 67

12 Specific Activity of Dried Supernatant
Fractions from the Extraction of Larval Rearing
Medium Containing 14C-Glucose and That Had
Mo Larvae Reared on It ........................ 68

13 Weights of Dried Supernatant Fractions from
the Extraction of Fifth-Instar Almond Moth
Larvae Reared on Medium Containing
14C-Glucose ................................... 69

14 Weights of Dried Supernatant Fractions from the
Extraction of Larval Rearing Medium Containing
14C-Glucose and That Had Almond Moth Larvae
Reared on It .................................. 70

15 Weights of Dried Supernatant Fractions from the
Extraction of Larval Rearing Medium Containing
14C-Glucose and That Had To Larvae
Reared on It .................................. 71

16 Pattern of Amino Acids in Fifth-Instar Almond
Moth Larvae ................................... 75

17 Percent Composition of Amino Acids in Fifth-
Instar Almond Moth Larvae ..................... 76

18 Retention Time, Relative Retention Time, and
Sensitivity of 19 Trimethylsilyl Amino Acid
Standards ..................................... 79

19 Amino Acid Mixture Patterned After Carcass
Analysis of Fifth-Instar Almond Moth Larvae ... 82












LIST OF TABLES Continued


Table Page

A-i Effect of Thin-Layer Adsorbent and Thin-Layer
Adsorbent and Ninhydrin in Combination on
Counting System Background .................... 92

A-2 Quenching Properties of 20 Non-Visualized
Amino Acid Standards Adsorbed on Cellulose
Thin-Layers Using the Internal
Standardization Method ........................ 94

A-3 Quenching Properties of 20 Amino Acid
Standards Adsorbed on Cellulose Thin-Layers
and visualized with 1/2 percent Ninhydrin
in Acetone Spray Using the Internal
Standardization Method ....................... 95

A-4 Quenching Properties of Visualized Amino Acids
from Larval Protein Hydrolysates Separated
by TLC and Using Automatic External
Standardization ............................... 96

A-5 Quenching Properties of Five Supernatant
Fractions from the Extraction of Larval Proteins
Using the Internal Standardization Method ..... 98

A-6 Quenching Properties of Supernatant Fractions
from the Extraction of Larval Protein Using
Automatic External Standardization ............ 99












LIST OF FIGURES


Figure Page

1 Principle of the radioactivity method for
determining amino acid requirements ........... 5

2 Flow diagram for the extraction and clean-up
of proteins used in this study ................ 27

3 Separation of amino acids present in an
acid hydrolysate of protein extracted
from fifth-instar almond moth larvae reared
on medium containing 14C-glucose .............. 43

4 Separation of amino acids present in a
base hydrolysate of protein extracted
from fifth-instar almond moth larvae reared
on medium containing 14C-glucose .............. 44

5 Spot map of 22 amino acid standards separated
on cellulose thin-layer plates ................ 46

6 Separation of amino acids present in an
acid hydrolysate of protein extracted
from larval rearing medium which contained
14C-glucose and on which larvae
were maintained ............................... 51

7 Separation of amino acids present in a
base hydrolysate of protein extracted
from larval rearing medium which contained
14C-glucose and on which larvae
were maintained ............................... 52

8 Separation of free amino acids extracted from
sterile larval rearing medium containing
14C-glucose and on which larvae
were maintained ............................... 55

9 Separation of free amino acids extracted
from non-sterile larval rearing medium
containing 14C-glucose and that had
no larvae reared on it ........................ 56











LIST OF FIGURES--Continued


Figure Page

10 Separation of free amino acids extracted from
sterile larval rearing medium containing
14C-glucose and that had no larvae
reared on it .................................. 57

11 Gas-liquid chromatogram of TMS amino acid
derivatives from protein hydrolysate
of fifth-instar almond moth larvae ............ 77

12 Gas-liquid chromatogram of TMS-free
amino acids extracted from fifth-instar
almond moth larvae ............................ 78

13 Gas-liquid chromatogram of TMS protein
amino acid standards .......................... 80

14 Gas-liquid chromatogram of a TMS
derivatization of an alkaline acetone
fraction from the extraction of protein
from almond moth larvae ....................... 83

A-1 One-dimensional co-chromatography of
14C-Glucose with 1/2% glucose standard
(in 3% aqueous ethanol) followed by auto-
radiography of the thin-layer plate
on Kodak No-Screen X-ray Film ................. 91











Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy



DIETARY AMINO ACID REQUIREMENTS OF
THE ALMOND MOTH, CADRA CAUTELLA (WALKER),
BASED ON RADIOMETRIC AND CARCASS ANALYSIS TECHNIQUES

by

Jack Myron Heller

August, 1975

Chairman: Robert E. Waites
Major Department: Entomology and Nematology

The almond moth, Cadra cautella (Walker), synthesizes

alanine, aspartic acid, glutamic acid, glycine, proline, and

serine from U-14C-glucose. These amino acids are considered

nutritionally non-essential. Amino acids that contained no

radioactivity and are considered nutritionally essential include

arginine, histidine, isoleucine, leucine, lysine, methionine,

phenylalanine, threonine, tryptophan, tyrosine and valine.

Cystine and cysteine contained an intermediate amount of activity

and are still unclassified with respect to dietary need.

Radioactive essential and non-essential free amino acids were

isolated from larval rearing media. The specific activities were

substantially higher in amino acids extracted from medium that

had larvae reared on it than medium that had no larvae reared on

it. There appears to be an insect-microorganism relationship at

work here. However, the exact nature of this relationship and to











what extent it contributes to the insects' nutrition are at

present undetermined. Since there was no detectable

incorporation of radioactive essential free amino acids from the

medium into the larval proteins (with the possible exception of

cysteine), the importance of this relationship seems questionable

when the techniques I employed for this study are used.

The study to isolate the source of the insect associated

microorganisms is at present inconclusive. However, from the

data available presently, it appears that the microorganisms come

from within the almond moth eggs.

Carcass analysis of fifth-instar almond moth larvae showed

that proline, tyrosine, and glutamic acid made up almost 70

percent of the total free amino acids present in the larvae. At

this stage in larval development prior to pupation, cuticular

tanning and thickening are beginning and proline and tyrosine are

major participants in these events.

The protein amino acids, which contain a high percentage of

glutamic acid and aspartic acid, in combination with the free

amino acids are the basis for a dietary amino acid mixture at the

2 percent and 3 percent levels. By substituting this mixture for

casein as the protein source in the almond moth diet, the

requirements for vitamins and minerals can be determined more

effectively.
















INTRODUCTION


The study of insect nutrition, in particular the formulation

of synthetic diets utilizing specific amino acids, vitamins, and

minerals, has in recent years greatly expanded. When the

specific nutrient requirements of an insect are known, many other

facts about it become much clearer (i.e., physiological and

biochemical). Once nutrition has been eliminated as a variable,

many other aspects of the insect can be studied such as genetics,

control (i.e., toxicant evaluation), biochemical relationships to

higher or related organisms, etc.

Indirect nutritional procedures have come into widespread use

to study organisms that cannot be reared axenically or on defined

media. They also support and enhance information gained through

classic nutritional techniques in addition to adding information

on the metabolic pathways of many nutrients.

The indirect radioactivity method was first demonstrated in

the work of Black, Kleiber, and Smith (1952) on the cow with

radioactive carbonate and fatty acids being incorporated into

non-essential amino acids. Shortly thereafter, Steele (1952)

found that when U-14C-sucrose was ingested by the adult mouse,

radioactivity appeared in the non-essential but not in the

essential amino acids extracted from the carcass proteins.

Kasting and McGinnis (1958) confirmed this relationship for the











blowfly, Phormia regina (Meig), using U-14C-glucose. Their data

were supported by the results from the classical deletion

procedure which had been done earlier on this insect.

The similarity in amino acid composition of the whole animal

carcass and the pattern of amino acid requirements as determined

by nutrition studies has been noted by Wu and Hogg (1952) for

protozoa and Williams et al. (1954) for larger animals. The

pattern of amino acids from carcass analysis studies has been

used in several instances to formulate chemically defined diets

with maximum larval growth being achieved (Auclair and Cartier

1963, Rock and King 1967b, and Rock and King 1967c).

The almond moth, Cadra cautella (Walker) (Lepidoptera:

Pyralidae) is a common pest of tobacco, cocoa beans, dried

fruits, and nuts throughout much of the world (Bach 1930,

Wadsworth 1933, Fraenkel and Blewett 1946a). Therefore, it was

chosen as the subject of this study. The objective of the study

was to determine the qualitative (i.e., indispensable) amino acid

requirements of the almond moth. This was to be followed by

determination of the amount of each protein amino acid present in

the carcass, with the aim of determining the quantitative amino

acid requirements (both dispensable and indispensable) of this

insect.

Once the qualitative and quantitative requirements had been

established, a synthetic amino acid mixture could be developed.







3




By substituting this mixture for casein as the protein source in

the almond moth diet, the requirements for vitamins and minerals

can be determined more effectively. This is because dietary

compounds such as casein often contain contaminants or substances

which confuse nutritional research (i.e., vitamins, minerals,

etc.). Once a completely defined diet has been established,

nutrition could be eliminated as a variable.factor in future

studies on physiology, biochemistry, ecology, and ultimate

control.

These two nutritional techniques were applied to my study of

the almond moth.
















LITERATURE REVIEW


Radioisotopes and
the Determination of Amino Acid Requirements

Since the classic work of Steele (1952), many investigators

have used carbon-14 labeled substrates to determine the

nutritionally essential amino acids of a variety of different

insects.

This procedure requires a compound that is normally present

in the diet and readily metabolized as the carbon-14 substrate.

Following administration by incorporation in the diet, injection,

or some other suitable means, the organism is allowed time to

metabolize the substrates.

Metabolism of the substrate results in incorporation of label

in the synthesized compounds (i.e., nutritionally non-essential)

and no incorporation in the essential compounds that must be

supplied in the diet. (See Figure 1.)

The indirect radioactivity method has been applied to a

number of phytophagus or other insects which cannot be reared on

chemically defined media (Kasting and McGinnis 1958, 1960, 1962,

1964, Kasting et al. 1962, Strong and Sakamoto 1963, Rodriguez

and Hampton 1966, Rock and King 1968, Rock and Hodgson 1971). A

summary of the amino acid requirements of the above insects

determined by this method-is shown in Table 1.












RADIOACTIVE GLUCOSE SUBSTRATE


RADIOACTIVE -


NUTRITIONALLY
NON-ESSENTIAL

SALANINE <-


ASPARTIC
ACID

GLYCINE

GLUTAMIC
ACID

SERINE

ETC.


CACCICACCCC*

C*02 x
INTERMEDIATES
CACAC*
PYRUVATE

C'C'CACCC*
o -KETO
GLUTARATE
CCACAC*
OXALOACETATE
\__


NUTRITIONALLY
ESSENTIAL


VALINE

LEUCINE

ISOLEUCINE

THREONINE

ARGININE

LYSINE

HISTIDINE

ETC.


\ NATURAL
FOOD SUPPLY


Figure 1. Principle of the radioactivity method for
determining amino acid requirements.*



* Kasting, R., and A. J. McGinnis. 1966. Radioisotopes and the
determination of nutrient requirements. Ann. N.Y. Acad. Sci.
139: 99.


NO
RADIO-
ACTIVITY


I












Table 1. Amino Acid Requirements of Some Insects Determined by
the Radioactivity Method



Amino Acid Blow Pale Wire- Green Wheat Two- Red- Boll
Fly Western worm Peach Stem Spotted Banded Worm
Cutworm Aphid Sawfly Spider Leaf
Mite Roller


- +


Glutamic acid -
Aspartic acid -
Alanine +'
Proline +'
Serine
Glycine
Histidine +


Threonine + +? +


Leucine


+ + + + + + +


+ + + +


+ + + + + + + +


Isoleucine + +
Valine + +
Tyrosine + +
Phenylalanine + +
Lysine + +
Arginine + +
Methionine + +


Cystine
Cysteine


+ +


+ + + + + +
+ + + + + +
+ + + + +
+ + + + +
+ + + + +
+ + + + + +


+ +


+ + +


+ = nutritionally essential
- = nutritionally non-essential
+?= some synthesis, possibly essential











There are several important factors to consider when using

the indirect radioactivity method to determine nutrient

requirements. The first of these is the method by which the

radioactive substrate is administered. The labeled compound may

be administered as a single dose or the organism may be

continuously exposed to the radioactive substrate. Organisms are

often continuously exposed to a radioactive substrate by having

it incorporated in their diet (Strong and Sakamoto 1963,

Rodriguez and Hampton 1966, Rock and King 1968, Rock and Hodgson

1971). Most studies involve the administration of only a single

dose of substrate injection. Kasting and McGinnis (1964) have

devised a method of vacuum infiltration for organisms that are

not easily injected.

The radioactive substrate administered to the organism can

affect both what compounds are labeled and the specific activity

of these compounds. This was clearly shown in the study of Black

et al. (1952) using bovine tissues. In this study, the labeling

of amino acids varied depending on whether labeled acetate,

propionate, butyrate, or bicarbonate was administered. Table 2

presents a summary of some of the substrates used, methods of

administration, and metabolism periods for the determination of

amino acid requirements using the indirect radioactivity method.











Table 2. Experimental Parameters Used to Determine Amino Acid
Requirements by the Indirect Radioactivity Method


Organism Radioactive Method of Metabolism Source of Reference
Substrate Adminis- Period Isolated
tration (Hours) Amino Acid


Acetate-
I-14C
Acetate-
II-14C


Single
injection


3, 10, 22,
34


Casein


Black
et al.
1957


Frog & Glucose- Single
Tadpole U-14C injection




Mouse Sucrose- Single
U-14C feeding




Rat Glucose- Single
U-14C injection

Blow fly Glutamic Single
acid U-14C injection


Wheat Glucose- Vacuum
Stem U-14C infiltra-
Sawfly tion

Green Glucose- Continuous
Peach U-14C feeding
Aphid 24 hr.

Red- Glucose- Continuous
Banded U-14C feeding
Leaf 48 hr.
Roller

Boll Glucose- Continuous
Worm U-14C feeding
48-72 hr.


4, 8, 12 Liver,
tail,
muscle,
carcass


Whole
mouse
(minus
intestines)


Nakagawa
et al.
1964


Steele
1952


2.5 min.- Brain and Gaitonde
2 hr. liver et al. 1965


48, 63


60-84


Whole Kasting &
carcass McGinnis
1960

Whole Kasting &
carcass McGinnis
1964

Whole Strong &
carcass Sakamoto
1963

Whole Rock &
carcass King
1968


Whole Rock &
carcass Hodgson
1970











The length of the metabolism period can affect the order of

specific radioactivities among the protein and free amino acids.

The level of specific radioactivity in free amino acids is not

necessarily related to that of the protein amino acids. Nakagawa

et al. (1964) showed that with a metabolism period of 4 hr. in

the frog, the free amino acids that were labeled were not

necessarily labeled in the protein amino acids from the same

tissues. With a longer metabolism period following single

administration of a radioactive substrate, there is generally

greater incorporation of radioactivity into the protein amino

acids, while radioactivity in the free amino acids may reach a

peak and start to decline. The metabolism period often appears

to be chosen by trial and error. Schaefer (1964) and Kasting et

al. (1962) used the rate of production of 14C02 and the total

amount produced following administration of radioactive

substrates as a guide to selecting the optimum metabolic period.

To obtain reliable results using the radioactivity method,

another factor that must be considered is the purity of isolated

compounds (i.e., amino acids) before radioactivity measurement.

This can be accomplished by simple two-dimensional thin-layer

chromatography (TLC) with some organisms. However, with free

amino acids from insect preparations, an ion exchange column

followed by band paper chromatography in at least three solvent

systems may be required to remove interfering materials (Kasting

and McGinnis, 1960).











The tissues from which amino acids are isolated also have a

significant effect on the results obtained with the indirect

radioactivity method. Specific radioactivities will depend on

whether amino acids are isolated from the free or protein

fractions (Nakagawa et al., 1964), or the whole carcass or

specific organs (Nakagawa et al. 1964, Black et al. 1952,

Gaitonde 1965). However, with most organisms, the different

synthetic abilities of different organs are not a problem because

the amino acids are isolated from the whole animal. A problem

arises when amino acids with low or intermediate specific

activities are isolated from whole insect larvae. The low level

of radioactivity in certain amino acids may be due to dilution of

these compounds synthesized in one organ by unlabeled amino acids

from other tissues or organs. Studies such as those carried out

by Lipke et al. (1965) on the biosynthetic capabilities of the

different tissues and organs of the cockroach will aid in

interpretation of results by demonstrating which organs and

tissues are capable of synthesizing specific amino acids.

The specific radioactivities of isolated amino acids'may also

vary depending on the concentration in which they are present.

However, this has not been found with a number of insects

(Kasting et al. 1962, Kasting and McGinnis 1962, 1964).

In many instances, the classical deletion technique has shown

tyrosine to be a non-essential amino acid while the indirect











radiometric technique has indicated that it is essential (i.e.,

lacks radioactivity). Fukuda (1956) and Kasting and McGinnis

(1962) have shown that tyrosine is synthesized from the essential

amino acid phenylalanine and thus, even though it lacks

radioactivity, it can still be classed as non-essential in many

insects.

Kasting and McGinnis (1966) have thoroughly reviewed the

subject of radioisotopes and the determination of nutrient

requirements.

Carcass Analysis for the Estimation of
Quantitative Amino Acid Requirements

To determine quantitative amino acid requirements of an

insect, feeding tests on graded levels of an amino acid mixture,

such as one resembling a casein hydrolysate or some other

protein, are evaluated on the basis of growth and development of

the insect.

A problem with this approach is that the initial balance of

amino acids, both dispensable and indispensable, is probably far

from optimum. Often, little consideration is given to the

dispensable amino acid balance. Breuer et al. (1964) showed the

importance of dispensable amino acid balance on total amino acid

balance in the rat.

Several workers have used new methods to gain a better

starting point from which to develop an optimum amino acid

mixture for various organisms.











Wu and Hogg (1952) using protozoa and Williams et al. (1954)

using the rat, chick, and pig noted the similarity in amino acid

composition of the whole animal carcass and the pattern of amino

acid requirements determined by nutritional studies. Auclair and

Cartier (1963) successfully reared the pea aphid Acyrthosiphon

pisum (Harris) on an amino acid diet based on the average

concentration of these compounds in the blood and excreted

honeydew. Rock and King (1967c) estimated the amino acid

requirements for growth in the codling moth, Carpocapsa pomonella

(Linneus) by carcass analysis. Rock and King (1967b) found that

the quantitative pattern of amino acids in 1-day-old pupae of

Argyrotaenia velutinana (Walker) supported maximum larval growth

when this insect was reared axenically on a chemically defined

diet. Rock and King (1966) studied the amino acid composition in

hydrolysates of the red-banded leaf roller during development.

Their work indicated a shift in amino acid requirements during

growth and development. This shift in requirements is probably

mediated by the requirements of the particular tissue that is

being formed in the rapidly developing and growing larvae.

General Insect Nutrition


Several excellent reviews covering various phases of insect

nutrition are currently available in the literature. Reviews

concerning the general subject of insect nutrition include House

(1961, 1962), Lipke and Fraenkel (1956), and Fraenkel (1959).











Friend (1962) discusses the nutritional requirements of

phytophagus insects while House (1959) deals with the parasitoid

Pseudosarco phaga affinis (Fall) and other insects. The current

status of and future possibilities for research on the axenic

culture of arthropods are discussed by Rodriguez (1966).

Richards and Brooks (1958) and Henry (1962) reviewed the

significance of internal symbiosis and microorganisms in insect

nutrition. Fraenkel and Blewett (1946b, 1946c) and Waites and

Gothilf (1969) have studied the dietary requirements of the

almond moth and several other closely related lepidopterous

insects.


Distribution and Metabolism of
Amino Acids and Proteins


Numerous articles and reviews on the distribution and

metabolisms of proteins and amino acids in insect tissues and

fluids are available in the literature.

Florkin (1958) reviewed the subject of free amino acids in

insect hemolymph and Chen (1962) the broader subject of free

amino acids in insects. The free amino acids in Prodenia

eridania, Culex pipiens, Glossina palpalis, and Drosophile

melanogaster have been studied by Levenbook (1962), Chen (1963),

Balogun (1969) and Mitchell and Simmons (1961), respectively.

In the area of amino acid metabolism, Brunet (1965) reviews

the subject of aromatic compounds while Bheemeswar (1958)











discusses general amino acid metabolism covering the subject of

major biochemical reactions (i.e., deamination, transammation,

decarboxylation, and peptide and protein synthesis). Chen (1966)

has written a very extensive review covering the subject of amino

acid and protein metabolism in insect development. He follows

the changing patterns of amino acid composition throughout the

life stages of various insects. Henry and Block (1961) report on

the metabolism of sulfur-containing amino acids in the German

cockroach, Blattella germanica (L.). Bursell (1963) follows the

changing pattern of free amino acids in the thorax of the male

tsetse flies during the hunger cycle and flight activity.


Methodology


Thin-Layer Chromatography


Several two-dimensional solvent systems were investigated for

the separation of complex amino acid mixtures before the one

giving the best resolution was found.

Heathcote and Jones (1965) devised a pair of solvent systems

for ascending two-dimensional chromatography on cellulose thin-

layers. This system produced unambiguous separation of 23

naturally occurring amino acids, including leucine and isoleucine,

in 6 hr. This method required no tank saturation and using a

ninhydrin staining solution could detect less than 1 pg of amino

acid.











The solvent systems were 2-propanol-formic acid-water

(40:2:10 by volume) for development in the first dimension and

tertiary butyl alcohol-methyl ethyl ketone-NH40H-distilled water

(50:30:10:10 by volume) for development in the second dimension.

Jones and Heathcote (1966) used the same solvent system to

separate the amino acids in protein hydrolysates. However, this

time ninhydrin-collidine reagent was used to visualize the amino

acids. This system made for easier identification of certain

amino acids due to their characteristic color on staining.

Haworth and Heathcote (1969) modified their previous method

and accomplished the separation of up to 63 compounds. This new

solvent system consisted of 2-propanol-methyl ethyl ketone-lN HCI

(60:15:25 v/v) for development in the first dimension and

tertiary butyl alcohol-methyl ethyl ketone-acetone-methanol-

NH40H-distilled water (40:20:20:1:14:5 v/v) for development in

the second dimension. With this new solvent system, a large

spread was produced between the amino acid spots. In addition,

highly reproducible results were obtained making it possible to

use the Rf's of the various spots to identify the amino acids

present in a protein hydrolysate. Using a ninhydrin-cadmium

acetate dye system 5 X 10-4 moles of amino acid can be detected

following two-dimensional TLC. Heathcote and Haworth (1969a)

again modified their solvent system to the following composition:

tertiary pentyl alcohol-methyl ethyl ketone-acetone-methanol-











NH4OH-distilled water (50:20:10:5:15:5 v/v). Heathcote et al.

(1970) used selective staining to identify complex mixtures of

amino acids and nitrogen containing metabolites separated by TLC.

De Zeeuw (1968a) compared the use of saturated and

unsaturated TLC chambers. He obtained better separation using an

unsaturated chamber in addition to obtaining good reproducibility

of R 's and good spot shape. Other factors that affect

separation and spot shape and should be kept constant are

temperature, relative humidity, solvents, adsorbent, and geometry

of the chamber. De Zeeuw (1968b) also studied the influence of

humidity variations on the TLC of hypnotics. He showed that Rf's

changed considerably with variations in relative humidity of the

TLC room. R 's increased with increasing humidity and then fell

sharply at higher humidities.

Heathcote and Washington (1967) and Heathcote and Haworth

(1969b) discussed the quantitation of small amounts of amino

acids separated by thin-layer or paper chromatography using

colorimetric or densitometric techniques respectively.

Stahl (1968) stressed the need for standardization of terms

in the literature so techniques could be repeated in other

laboratories and results could be compared between laboratories.

Several excellent texts on TLC include Smith (1960), Stahl

(1969), and Pataki (1966).











Gas Chromatography


A fairly recent advance in the quantitative analysis of amino

acids was the adaptation of gas chromatography to these

compounds. It is a very sensitive technique that requires only

small amounts of the compound of interest to be injected into the

instrument.

Gehrke et al. (1969) did an extensive study of the

trimethylsilyl (TMS) derivatives of protein amino acids examining

such factors as chromatographic separation, precision and

accuracy of the method, silylation as a function of reaction

temperature and time, molar excess of reactants, stability of the

TMS derivatives, quantitative analysis of a synthetic amino acid

mixture, and application to biological samples. Gehrke and

Leimer (1970b) studied the effect of solvents on derivatization

of amino acids using bis (trimethylsilyl) trifluoroacetamide

(i.e., BSTFA). They found using polar solvents for the

derivatization reaction produced two chromatographic peaks for

glycine and one for arginine. In non-polar solvents, only the

first chromatographic peak for glycine and no peaks for arginine

were obtained. Gehrke and Leimer (1971) improved upon the

previous work on trimethylsilylation of the 20 protein amino

acids. Their major aim, which they accomplished, was to achieve

a single derivatization, single injection method for the analysis

of the 20 protein amino acids as the TMS derivatives in complex










biological substances. Amino acids were reproducibly converted

in a single step, closed tube reaction to the TMS derivatives in

2.5 hr. at 150 C. Excellent separation of the 20 TMS protein

amino acid derivatives was achieved on a single 6 m X 2 mm I.D.

column packed with 10 percent OV-11 on 100/200 mesh Supelcoport

in 60-80 min. Data from amino acid analysis of ribonuclease,

B-casein, K-casein, soybean meal, and blood are in good agreement

with values obtained by classical ion-exchange methods, and

establishes the use of the TMS-/gas-liquid chromatographic (GLC)

method for quantitative analysis of amino acids in biological

materials.

Several other derivatives are available for amino acid

analysis by GLC. Vance and Feingold (1970) and Pisano and

Bronzert (1972) studied the methylthiohydantoin derivatives of

amino acids and Fu and Mak (1971) the N-acyl amino acid alkyl

esters.

Along with the TMS amino acids, the other major derivatives

for GLC are the N-trifluoroacetyl (N-TFA) n-butyl ester and, in

some cases, the methyl ester (Islam and Darbre 1969, Roach and

Gehrke 1969a, 1969b, Casagrande 1970, Pellizzari et al. 1971,

Gehrke et al. 1971).

Zumwalt et al. (1970) used the N-TFA n-butyl esters for

quantitative analysis of amino acids in complex biological

substances such as urine and blood plasma. He used ion-exchange











resins for sample clean-up and obtained quantitative recovery of

amino acids from the exchange columns.

Gehrke and Leimer (1970a) studied the effect of salts on the

derivatization and chromatography of N-TFA n-butyl esters of

amino acids. They found that inorganic salt at a ratio of 1:1,

salt to total amino acids, was not a serious problem for

qualitative work. However, for quantitative work, the following

salts should be removed by ion-exchange chromatography: oxalate,

manganese (III, cobalt (II), nickel, zinc, tin (II), lead (II),

chromium (III), and iron (III).

Gehrko et al. (1971) used the N-TFA n-butyl ester derivatives

to search for amino acids in hydrolysates of lunar fines from

Apollo 11 and 12 missions.

Zumwalt et al. (1971a, 1971b) refined the N-TFA n-butyl ester

systems to the point where nanogram and picogram amounts of amino

acids could be analyzed.

Gehrke et al. (1971) have an excellent review article on the

TMS and N-TFA n-butyl ester systems for the analysis of the 20

protein amino acids in biological samples.

Burchfield and Storrs (1962) have a general text on the

biochemical applications of gas chromatography.











Radioisotope Techniques


The use of radioisotopes in metabolic studies has greatly

increased the number of scientific questions that can be answered

and the limits of detection that can be reached.

Snyder (1965) reports on quantitative radioassay methods for

TLC. lie compares zonal versus autoradiographic scans and prefers

zonal scans due to their greater resolving power and speed.

Snyder (1966) also compares zonal versus strip scans of thin

layer chromatograms and again prefers the zonal scans due to

their greater sensitivity and resolving power. This is

especially true with weak beta emitters such as 3H-labeled

compounds in biological specimens. Three excellent reviews

covering the subjects of TLC radioassay, instrumentation and

procedures for 14C and 3H radioassay by TLC and liquid

scintillation radioassay of thin layer chromatograms are

presented by Snyder (1968, 1969a, 1969b).

Bell and Hayes (1958) review many aspects of liquid

scintillation counting.


Protein and Amino Acid Extraction and Treatment


Many texts are available covering the vast subject of protein

and amino acid extraction, hydrolysis, clean-up, etc. Some of

the more complete works include Block and Weiss (1956), Alexander

and Block (1960a, 1960b), and Blackburn (1968).







21




Roach and Gehrke (1970) developed a new rapid acid hydrolysis

technique for proteins. Using aqueous 6N HC1 at a ratio of 1 mg.

of protein to 1 ml. of acid, they heated the mixture in a sealed

test tube containing a N2 atmosphere for 4 hr. at 145 C. +2.

Essentially, equivalent hydrolysis and yield were obtained when

this method was compared with the standard 11000. +10 for 26 hr.

using ribonuclease as a protein source.
















MATERIALS AND METHODS


Rearing


A stock culture of the almond moth was maintained on a diet

consisting of the following ingredients: 4 parts cornmeal, 4

parts whole wheat flour, 2 parts finely ground dog food, 1 part

brewer's yeast, 1 part oatmeal, 1/2 part wheat germ, 1 part

honey, and 1 part glycerine. These ingredients were mixed

thoroughly and placed in 1/2 gal. wide-mouth mason jars. The

medium was then innoculated with several hundred eggs and the

jars covered with 12 cm. filter paper discs and sealed with metal

jar rings. Following emergence of adult moths, the filter paper

discs were replaced with screen wire discs. The jars were then

inverted in a rack and eggs were collected in petri dishes as

they dropped through the screen. These eggs were used to

innoculate the next generation of the culture.

Radiochromatography and Autoradiography
of U-7C-Glucose


Uniformly labeled 14C-glucose was used throughout this study.

The purity of the 14C-glucose was checked by TLC and

autoradiography of the chromatograms.











Twenty micrograms of unlabeled carrier glucose in a volume of

5 pl. was applied to an Eastman Chromagram Sheet of Silica Gel G.

The spot was then dried in a cool air current. An aliquot of

radio labeled glucose containing 0.01 pCi. of activity was then

applied on top of the unlabeled carrier spot. In a second lane

next to the 14C-glucose, 10 1l. of an unlabeled 1/2 percent

aqueous glucose solution was spotted.

The solvent system used for development was n-butanol-

isopropanol-water (5:3:1). The chromatogram was allowed to

develop until the solvent front reached 1/2 in. from the top edge

of the plate. Following development, the chromatogram was

thoroughly dried to remove all traces of solvent. The plate was

then sprayed with a solution consisting of 0.9 g. oxalic acid and

1.8 ml. aniline in 200 ml. of H20 and heated to 105C. for 15

min. to visualize the glucose spot. The dried chromatogram was

then wrapped in a single thickness of plastic film in order to

protect the x-ray film from any substances on the thin-layer

plate which might cause fogging. Then, in a dark room with a

safety light on, a sheet of unexposed x-ray film was placed in

direct contact on top of the plastic wrapped chromatogram. A

small notch was cut in the film and chromatogram. The separated

film and plate could then be realigned after development by

aligning the notches. The film and chromatogram were then placed












between two pieces of wood cut approximately the same size as the

x-ray film. The two pieces of wood were held together securely

by placing several elastic bands around them. This procedure

kept the film and chromatogram aligned correctly. The wood

blocks were then wrapped in several layers of aluminum foil and

placed in a drawer for 20 hr. Following this, the x-ray film was

developed, washed, and air dried.

Rf's for the radioactive and non-radioactive glucose spots

were then determined and compared and the x-ray film examined for

the 14C-glucose spot and any impurities or streaking.


Radioactive Medium Preparation


Radioactive medium was prepared by pipetting 160 vCi. of

14C-glucose* onto 30 g. of standard rearing medium. The medium

was thoroughly mixed and placed in an 8 oz. baby food jar. It

was then innoculated with approximately 200 eggs and the jar

sealed with a filter paper disc and metal jar ring. Following

development, mature larvae were removed from the radioactive

medium for extraction of protein.






* Supplied by Amersham/Searle. Specific Activity 309 mCi./mM.










Protein Extraction


A weighed amount of mature larvae that had been reared on

medium containing 14C-glucose was placed in a tissue grinder and

homogenized with 20 ml. of cold 10 percent trichloroacetic acid

(TCA). The grinder was rinsed with 10 ml. of cold 5 percent TCA

and the liquid combined with the larval homogenate. The

homogenate was then centrifuged for 5 min. and the supernatant

decanted off for further analysis. The pellet containing the

protein fraction plus other components (i.e., lipids, nucleic

acids, etc.) was dried under a stream of N2.

Twenty milliliters of acetone, made alkaline with NH14011, was

added to the centrifuge tube. The mixture was then placed in a

70 C. water bath and stirred gently for 5 min. Following this,

it was again centrifuged for 5 min. and the supernatant poured

off and saved. The pellet was then dried under a stream of N2.

The acetone extraction, centrifugation, and drying steps were

then repeated. The pellet was subjected to this treatment again,

first using 95 percent ethanol and then using ether. It was

repeated twice with each solvent and all supernatant fractions

were saved for weighing and scintillation counting.

The pellet plus 10 ml. of 5 percent TCA were then placed in a

900C. water bath for 15 min. with continuous stirring. The tube

and its contents were then cooled under running water,

centrifuged, the supernatant poured off and the pellet dried











under N2. The protein pellet was then washed with 3-5 ml.

portions of 5 percent TCA centrifuged, dried, and weighed.

The flow diagram in Figure 2 will help illustrate what

components were extracted with the different solvents.

As a control, protein was extracted from the radioactive

media. Two types of samples were analyzed; medium that had

larvae reared on it and medium that had no larvae reared on it.

This protein was extracted and analyzed in the same way as the

insect protein.

Free amino acids from the media, which were contained in the

first TCA supernatant fraction, were also analyzed. Before TLC

could be used to separate the amino acids in this fraction, it

first had to be cleaned up using the column method for carcass

analysis of free amino acids.

Protein and free amino acids were also extracted from mature

larvae that had been reared on non-radioactive medium. These

were used in the carcass analysis study.

Protein Hydrolysis


Proteins extracted from the larvae and media were subjected

to acid and base hydrolysis so analysis of the amino acids could

be accomplished by TLC and GLC.











Cold 10% TCA and larvae

Homogenize


Centrifuge


Supernatant T
(glycogen, sugars, free (
amino acids, vitamins,
nucleotides, etc.)



Neutral lipids









Phospholipids


CA precipitate
Lipids, nucleic acids, proteins)

Alkaline acetone extraction



Residue

Ethanol extraction
Ethanol extraction


Ether extraction
1


Residue


Hot TCA extraction


Nucleic acid


Residue
(protein)


Figure 2. Flow diagram for the extraction and clean-up of
proteins used in this study.











Acid Hydrolysis:

Ten milligrams of protein were placed in a 125 mm. screw top

test tube with a Teflon-lined cap. The tube was flushed with a

stream of filtered N2 and 10 ml. of 6N HC1 were added. The tube

was again flushed with N2, sealed, and heated for 4 hr. at 1450C.

The protein hydrolysate was then evaporated to dryness under

vacuum in a 60 C. water bath. The residue was taken up in 2 ml.

of 10 percent aqueous 2-propanol (v/v) and again evaporated to

dryness. This step was repeated once again after which the

residue was dissolved in 1/4 ml. of 10 percent aqueous 2-propanol

for TLC. For GLC analysis, the amino acid residues were

dissolved in 1 ml. of 0.05 N aqueous IHC1.


Base Hydrolysis:

Base hydrolysis was used for the study of amino acids that

were partially or completely destroyed by acid hydrolysis, such

as tryptophan. Ten milligrams of protein, 65 mg. of Ba(OH)2*8H20

and 1 ml. of 1120 were placed in a screw top test tube. The top

of this tube had a small hole drilled in it and a silicone rubber

septum from a gas chromatograph injection port was placed in the

top. The top was screwed on and a hypodermic needle that had

been attached by means of rubber tubing to a vacuum line was

inserted into the tube through the hole in the top. The tube was

then evacuated with the rubber septum keeping it air tight.











The tube was heated for 24 hr. at 1250-1300C. after which it

was cooled. The protein hydrolysate was then adjusted to pH 6

with 2N 12SO4 and then heated to boiling. The tube and its

contents were then centrifuged to separate the BaS04. The BaS04

pellet was washed with a little water and the combined

supernatant and washing were evaporated to dryness. The residue

was then dissolved in 1/4 ml. of 10 percent aqueous 2-propanol

for separation of the amino acids by TLC.


Thin-Layer Chromatography of Amino Acids


Amino acids from the protein hydrolysates were separated and

identified by TLC. Five microliters of hydrolysate were spotted

on a 20 X 20 cm. Eastman Chromagram Sheet of cellulose without

fluorescent indicator. The starting point was 1/2 in. from the

edges of the plate at the bottom left hand corner. The spot was

positioned by marking the edges of the plate with a soft lead

pencil. The solvent front was also marked in this manner, care

being taken so as not to disturb the thin-layer and cause

distortion of the spots during chromatography. After application

to the thin-layer plate, the spot was dried in a stream of warm

air.

Separation of 20 amino acids required the use of

two-dimensional chromatography. The solvent systems used were

2-propanol-methyl ethyl ketone-lN HC1 (60:15:25 v/v) for











development in the first dimension and 2-methyl-2-butanol-methyl

ethyl ketone-acetone-methanol-water-concentrated NH4OH

(50:20:10:5:15:5 v/v) for development in the second dimension.

Development in each phase was allowed to continue until the

solvent front reached 1/2 in. from the top edge of the plate.

Between development in the first and second dimensions, the plate

was dried for 2 hr. in a fume hood. Following development in the

second dimension, the plate was allowed to dry overnight.

Visualization of the amino acids was accomplished by spraying

the dried plates with 1/2 percent ninhydrin in acetone and then

heating for 20 min. at 600C.

A standard plate (i.e., spot map) was prepared to facilitate

identification of the amino acids. Standard solutions (0.025 M)

of the 22 amino acids of interest were made up in 10 percent

aqueous 2-propanol. These standards were chromatographed and

their positions on the plate noted along with their R 's in both

dimensions.


Radioactivity Measurements


The supernatant fractions from the larval and media protein

extractions were placed in numbered scintillation vials that had

been previously weighed. The liquid was then evaporated to

dryness under a N2 stream and heat lamp and the vials again

weighed. After the weight of each supernatant fraction was











known, 1 or 2 ml. of Soluene,* a sample solubilizer, was added to

each vial. The samples were set aside for 48 hr. to allow the

solubilizer to work. Fifteen milliliters of scintillation fluid

consisting of 5 g. 2,5-diphenyloxazole (PPO), 0.250 g. 1,4

bis-2-(4-methyl-5 phenylox-axolyl)-benzene (dimethyl POPOP), and

1 L. of toluene were added to each vial. The vials were then

placed in a Packard Tri-Carb Liquid Scintillation Spectrometer

and allowed to temperature equilibrate (3-4 C.) for 1 hr. These

samples were counted for only 10 min. due to the high activity

present. The counts per minute (cpm) were corrected for any

quenching with automatic external standardization (AES).

Following separation and visualization, the individual amino

acids were scraped off the thin-layer plates into scintillation

vials. The spots from 10 plates were pooled for each amino acid.

Fbr scintillation counting, a cocktail similar to the previous

one was used except that Cab-0-Sil** (4 percent w/w) was added.

Cab-O-Sil is a gelling agent which aided in suspension of the

amino acids on the thin-layer adsorbent. The vials were then

placed in the scintillation counter and allowed to temperature

equilibrate for 1 hr. They were counted for 100 min.



* Packard Instrument Company, Inc.

** Rohm and Haas.












AES was used to correct the counts for any quenching in the

samples. Several vials containing different amounts of thin-

layer adsorbent were also counted to determine if the adsorbent

caused any increase in activity due to fluorescence.

The quenching properties of 20 amino acids and the

supernatant fractions from the larval protein extraction were

studied using internal standardization. This study was

undertaken to test the validity of the external standards method.

Twenty microliters of a 1 mg./ml. standard of each of 20

amino acids were pipetted onto a cellulose thin-layer plate that

had been divided into 20 sections. Each section of the plate

contained a single amino acid standard. Twenty scintillation

vials each containing 15 ml. of the same scintillation cocktail

used in the counting of amino acids from the protein hydrolysate,

10 Pl. of 14C-glucose, and 2-3 drops of Bio-solv TM* solubilizer

were counted in a Beckman LS-200 Liquid Scintillation

Spectrometer prior to the addition of a single amino acid; each

vial was recounted to see how much quenching resulted.

This same study was repeated again with the exceptions that

this time only 5 p1. of 14C-glucose were used and the amino acids

were visualized by the method noted previously before they were

added to the scintillation vials.



* Beckman Instrument Company.











The quenching properties of supernatants from the protein

extractions were studied in a similar manner. The supernatant

fractions, from the extraction of protein from larvae reared on

non-radioactive medium, were dried, weighed, and solubilized as

previously discussed. Each one was then added to a separate vial

which contained 15 ml. of the same scintillation fluid used

previously for counting supernatant fractions. These vials also

contained 10 pi. of 14C-glucose and 1-2 drops of Bio-solvT

solubilizer. They had been counted before the addition of the

supernatant fractions and were now counted a second time to

determine the extent of quenching caused by the various

fractions.

Carcass Analysis


Fifth-instar larvae were analyzed for the total amount of

each amino acid they contained. The protein and free amino acids

were analyzed separately and then combined later to arrive at a

total for each individual amino acid. The carcass analysis was

replicated twice using two separate groups of larvae.

Protein extraction and subsequent hydrolysis for the

liberation of amino acids were accomplished using the same method

as described previously for the thin-layer work. The free amino

acids were obtained from the supernatant of the larval-TCA

homogenate following centrifugation. Before the free amino acids

could be derivatized for GLC, they had to-be separated from any











interfering biological substances with an ion-exchange column.

The resin, Amerlite CG-120 (100/200 mesh), was prepared as

follows: resin was placed in a 500 ml. beaker and covered with

3N NH40H. It was then placed on a magnetic stirrer and swirled

for 60 min. The resin was allowed to settle and the NHiOH

decanted off. This process was repeated twice more and the resin

was then washed with double distilled water until it was

approximately neutral. The resin was then regenerated by

swirling for 30 min. three times with 3N HC1. It was then washed

with double distilled water until it was approximately neutral.

The columns, which consisted of 125 mm. test tubes with a 2 mm.

hole in the bottom, were then filled to the 3/4 mark with wet

resin. The level of liquid was never allowed to fall below the

surface of the resin. The liquid in the column was then allowed

to fall to approximately 3 mm. above the resin surface and the

sample was added with a pasteur pipette. The entire 30 ml. of

TCA supernatant fraction were passed through the column.

Following this, 5-10 ml. portions of distilled water were used to

wash the resin. The washes were discarded. The amino acids were

then eluted from the column using five separate 2 ml. portions of

3N NH4Mt. This was followed by five, 5 ml. portions of distilled

water. The flow rate through the column was approximately 1-2

ml./min.











The effluent from the column was collected in a 125 ml. round

bottom flask. It was evaporated to dryness on a rotary

evaporator with the flask immersed in a 600C. constant

temperature water bath. The residue was dissolved in 1 ml. of

aqueous 0.05N HC1 for derivatization. Free amino acids extracted

from the rearing media were dissolved in 1/4 ml. of 10 percent

aqueous 2-propanol for TLC.


Gas Chromatography of Amino Acids
for Carcass Analysis


Column Packing and Preparation:

Twenty grams of Supelcoport* 100/200 mesh were placed in a

round bottom flask and just covered with methylene chloride. The

methylene chloride had been dried by running it through a silicic

acid column and was then distilled into an all glass bottle to

protect it from atmospheric moisture.

OV-11 (2.22 g.) dissolved in a minimal amount of methylene

chloride, was then added to the round bottom flask containing the

Supelcoport. This gives a 10 percent loading of OV-ll on the

solid phase. The flask was then placed on a rotary evaporator



* Supelco Inc.










and the methylene chloride slowly evaporated at room temperature

until the column packing was just damp. The flask was then

immersed in a 600C. water bath while under full vacuum on the

rotary evaporator until no odor of methylene chloride remained.

A 12 ft. by 2 mm. I.D. glass column was then silylated to

prepare it for the column packing. The column was first filled

with a 10 percent v/v solution of dimethyldichlorosilane in

toluene and allowed to stand for 15 min. with the solution in it.

The column was then flushed and filled with absolute methanol.

After 5 min., the methanol was removed and the column was washed

twice with acetone. It was then placed in an oven to dry. The

packing of 10 percent OV-ll on 100/200 mesh Supelcoport was then

added to the column.

The column was then placed in the gas chrcmatograph oven and

flushed with N2 carrier gas for 30 min. Following this, it was

no-flow conditioned at 325 -330 C. for 12-15 hr. The oven was

then cooled to room temperature. A flow of 10-15 ml./min. of N2

carrier gas was used for the rest of the conditioning. The oven

was then temperature-programmed to 300C. at a rate of lC./min.

and allowed to remain undisturbed at this temperature for at

least 24 hr.


Derivatization of Amino Acids:

An aqueous aliquot of protein hydrolysate or free amino acid

extract, containing from 0.5-6 mg. of total amino acids, was











added to a 65 mm. screw top culture tube with a Teflon-lined cap.

The amino acid solution was just evaporated to dryness in a 700C.

sand bath while passing a stream of regulated, filtered N2 into

the tube. Methylene chloride (0.5 ml.) was then added to the

tube and evaporated just to dryness. This last step was repeated

two more times. A known amount of internal standard, in this

case decanoic acid in acetonitrile, was then added to the tube.

The amount of internal standard should correspond to about the

amount of each individual amino acid in the test tube and there

should be 0.25 ml. of acetonitrile for each mg. of total amino

acid. Therefore, in the standard tube which contained 0.1 mg. of

each of 20 amino acids for a total of 2 mg. of amino acid, an

internal standard of 0.2 mg./ml. acetonitrile would be used. By

adding 0.5 ml. of this solution, the required 0.1 mg. of internal

standard and 0.5 ml. of acetonitrile for the 2 mg. of total amino

acid would be added.

In derivatizing the protein hydrolysates, an aliquot

corresponding to 4 mg. of total amino acids was used. Since the

amount of each amino acid in the hydrolysate varied, 0.1 mg. of

internal standard was chosen as the arbitrary amount to use.

An additional problem with the free amino acid extracts was

that the amount of total amino acids was not known. Therefore,

these several equal aliquots were derivatized using different

total amounts of acetonitrile.











Following addition of the internal standard, 0.25 ml. of bis

(trimethylsilyl) trifluoroacetamide (BSFTA) was added for each 1

mg. of total amino acids in the tube. Different amounts of BSFTA

were also tried in the free amino acid derivatizations. The

tubes were then securely closed and placed in an ultrasonic bath

for 1 min. to insure complete mixing.

The trimethylsilyl (TMS) derivatives of the amino acids were

made by heating the tubes for 2.5 hr. at 150 C. in an oil bath.

The tubes should be only 1/4 full and not immersed in the oil

above the level of liquid. A reagent blank containing everything

but amino acids was also run to check for extraneous peaks.

In addition to the protein hydrolysates and free amino acid

extracts which were used for carcass analysis, several other

samples were studied. Supernatant fractions from the extraction

of protein from non-radioactive larvae were studied to see if

there was any loss of amino acids during the extraction

procedure. The first of each of the following fractions were

studied: alkaline acetone, ethanol, ether, and hot TCA. These

fractions were cleaned up using the same procedure as that used

for the larval free amino acids.

A second larval protein extraction was then performed and the

same fractions were analyzed. However, this time the fractions

were evaporated to dryness and then hydrolyzed with 6N HC1. The

hydrolysates were then cleaned up using the same column procedure











as that for the larval free amino acids. Both sets of fractions

were then derivatized in the same manner as the protein

hydrolysate samples.


Gas Chromatography of Trimethylsilyl Amino Acids:

The carcass analysis samples were chromatographed on a model

2100 Varian Aerograph using a flame ionization detector. Five

microliters of sample were injected directly onto the column.

The following parameters were used for the separation and

detection of amino acids: injector temperature, 2750C.; detector

temperature, 300 C.; N2 carrier gas flow rate, 17 ml./min.; oven

temperature, initial 1000C.; 3-min. hold after start of solvent

peak, 40C./min. increase to 3000C.; attenuator settings, 32 X

0-11.

The samples prepared from the various protein extraction

supernatants were analyzed on a Tracor MT 220 gas chromatograph

using a flame ionization detector. The following chromatographic

conditions were used with this machine: injector temperature,

270C.; detector temperature, 2250C.; N2 carrier gas flow rate,

17 ml./min.; oven temperature, initial 1000C.; 3-min. hold after

start of solvent peak, 5C./min. increased to 2100C.; attenuator

settings 8 X 10.











Microbiological Study


When analysis of several free amino acid fractions from

radioactive media, both with and without larvae on them, showed

radioactive amino acids to be present, a study was undertaken to

find the source of their synthesis.

The third replicate of the essential amino acid study was run

under aseptic conditions. The larval rearing medium, containing

14C-glucose, was heat sterilized at 15 lb. pressure for 15 min.

The medium was sterilized in the rearing jar, which was covered

with aluminum foil. Almond moth eggs were surface sterilized by

placing them in a 3 percent zephiran chloride solution, in

sterile distilled water, for 15 min. The eggs were rinsed well

with sterile distilled water and put on sterile filter paper in a

sterile petri dish. All work was carried out in a sterile hood.

The eggs were then placed on the sterile, radioactive medium and

the rearing jar was placed in a closed TLC tank to minimize air

movement and maintain a sterile environment. The free amino

acids in this rearing medium were analyzed after the larvae were

taken off for protein extraction. The free amino acids from both

sterile and non-sterile medium, containing 14C-glucose but no

larvae, were also extracted and analyzed as a control.

The sterility of the almond moth eggs and radioactive medium

was checked by incubating them separately in nutrient broth at

37 C. for 4 days and then streaking on nutrient agar plates. The







41



plates were then incubated at 370C. for 4 days and read as +

(i.e., growth) or (i.e., no growth). Other materials studied

in this way or simply by streaking on nutrient agar plates were:

non-sterile eggs, non-sterile medium, sterile larvae, and sterile

medium that had sterile larvae reared on it. Three replicates

were run on each material studied.















RESULTS AND DISCUSSION


Qualitative Amino Acid Requirements by
The Indirect Radioactive Method and
Thin-Layer Chromatography


Two-dimensional TLC permitted the identification of 19 amino

acids (Figures 3 and 4) from acid and base hydrolysates of

protein from fifth-instar almond moth larvae. The larvae had fed

ad libitum on medium made radioactive with 14C-glucose for

approximately 3 weeks. Identification of amino acid spots was

made with the aid of Rf and Rleucine values (Table 3) calculated

from a spot map (Figure 5) of 22 amino acid standards. The

cpm/carbon atom (Table 4) of amino acids isolated from larval

protein shows that alanine, aspartic acid, glutamic acid,

glycine, proline, serine, and an unknown ninhydrin positive

compound from the acid hydrolysis fraction were highly labeled.

Because these amino acids were synthesized from glucose by the

almond moth, they are considered nutritionally non-essential.

The cpm/carbon atom of arginine, histidine, isoleucine, leucine,

lysine, methionine, phenylalanine, threonine, tryptophan, valine,

tyrosine, and two ninhydrin positive unknowns were not

significantly above background. These amino acids were therefore

not synthesized to any appreciable extent from glucose and must

be considered nutritionally essential. Cystine and cysteine

contained an intermediate amount of radioactivity. This



























Ala Tyr









ioQ
A p





Ser
Gly(





His
CySH0


X Origin



2nd Dimension



Figure 3. Separation of amino acids present in an acid
hydrolysate of protein extracted from fifth-instar
almond moth larvae reared on medium containing
14C-glucose.


* Unknown ninhydrin positive compounds.


I*O














Ile 0 M




Ar. ( Trp
SGluO co- Pro





SLyGly



iHis
O-



S(CyS)2
X Origin

2nd Dimension


Figure 4. Separation of amino acids present in a base
hydrolysate of protein extracted from fifth-instar
almond moth larvae reared on medium containing
14C-glucose.


* Unknown ninhydrin positive compound.











Table 3. Rf and RLeucine Values of 22 Amino Acid Standards
Separated by Two-Dimensional Thin-Layer Chromatography
on Cellulose


Amino acid First dimension* Second dimension**
Rf Rleucine Rf Rleucine
X 100 X 100 X 100 X 100

Alanine 55 63 12 25
Arginine 16 18 4 8
Asparagine 20 23 7 15
Aspartic acid 42 48 3 6
Cysteine 8 9 2 4
Cystine 4 5 2 4
Glutamic acid 52 60 3 6
Glutamine 26 30 8 17
Glycine 34 39 9 19
Histidine 9 10 13 27
Hydroxyproline 42 48 10 21
Isoleucine 86 99 44 92
Leucine 87 100 48 100
Lysine 15 17 8 17
Methionine 74 85 37 77
Phenylalanine 78 90 50 104
Proline 55 63 16 33
Serine 37 43 15 31
Threonine 46 53 40 83
Tryptophan 64 74 45 94
Tyrosine 69 79 31 65
Valine 76 87 29 60


* First dimension
(60:15:25, v/v).


= 2-propanol-methyl ethyl ketone-iN HCI


** Second dimension = 2-methyl-2-butanol-methyl ethyl
ketone-acetone-methanol-water-ammonium hydroxide
(50:20:10:5:15:5).






46









Il Lou










01 LyP

Hyp OrigTh





Glu(NH2)

O Asp (NH2)
Mg Lys


2 YS 2 His

X Origin

nd
2nd Diension >



Figure 5. Spot map of 22 amino acid standards separated on
cellulose thin-layer plates. Solvent system: first
dimension: 2-propanol-methyl ethyl ketone 1N HC1
(60:15:25, v/v); second dimension: 2-methyl-2-
butanol-methyl ethyl ketone-acetone-methanol-water-
concentrated ammonium hydroxide (50:20:10:5:15:5, v/v).











Table 4. Cpm/Carbon Atom of Amino Acids from Acid and Base
Hydrolysates of Protein Extracted from Almond Moth
Larvae Reared on Medium Containing 14C-Glucose


Amino acid Carbon Mg. of Cpm+/carbon atom
atoms/ amino Rep. Rep. Rep.
amino acid/ I II III
acid 10 TLC
plates

Alanine 3 .125 112.5 532.5 276.0
Arginine 6 .075 1.0 0.5 0.5
Aspartic acid 4 .211 62.0 270.7 86.0
Cysteine 3 11.0 16.0 13.5
Cystine 6 5.0 10.8 5.8
Glutamic acid 5 .271 74.0 328.5 116.5
Glycine 2 .070 81.0 347.0 182.0
Histidine 6 0.0 0.0 0.0
Isoleucine 6 .102 0.0 0.0 0.0
Leucine 6 .154 0.0 0.0 0.0
Lysine 6 .128 0.0 0.3 0.3
Methionine 5 .030 1.2 2.6 2.6
Phenylalanine 9 .081 0.1 0.2 0.0
Proline 5 .078 15.5 63.8 30.8
Serine 3 .083 55.0 256.5 25.5
Threonine 4 .059 1.3 0.0 0.0
Tryptophan 11 0.7 0.9 0.9
Tyrosine 9 .103 0.2 0.0 0.0
Valine 5 .109 0.4 0.0 0.0
Unknown I acid
hydrolysate* 4 33.7 41.0 41.0
Unknown II acid
hydrolysate* 4 0.8 0.0 0.0
Unknown III base
hydrolysate* 4 2.2 3.1 2.6


+ Cpm corrected for background and quenching.
* Calculated on the basis of four carbon atoms per molecule.










situation could indicate one of several possibilities. These two

amino acids may be synthesized to only a limited extent and still

need to be supplied in the diet. Another possibility is that

dietary cysteine or methionine may spare the need for

biosynthesis. There may actually be no radioactivity in these

compounds and the activity observed may be coming from labeled

contaminants which have been detected at the origin of the

thin-layer plate by liquid scintillation counting. -Only cysteine

was found in the acid hydrolysis fraction. The cystine, if

present, may have been substantially destroyed by the hydrolysis

procedure and/or converted to cysteine (Blackburn 1968). Only

cystine, or what appeared to be cystine, was found in the base

hydrolysis fraction which is unusual since this procedure readily

destroys both cysteine and cystine (Blackburn 1968). These two

compounds are often found to be among the lowest in concentration

of any amino acids present in the insect which complicates their

detection and quantitation (Strong and Sakamoto 1963, Rodriguez

and Hampton 1966, Rock and King 1968, and Rock and Hodgson

1971). Even though many insects do not require cysteine or

cystine (Strong and Sakamoto 1963, Rodriguez and Hampton 1966,

Rock and King 1968, and Rock and Iodgson 1971), I would supply

these amino acids in the diet until further study could be done

for the following reasons. The three carbon amino acids

cysteine, serine, and alanine which are derived from pyruvate,

should have a very high specific activity if they are indeed

synthesized (Black et al. 1957, Rock and King 1968). Dilution


__











with unlabeled carbon, and thus low specific activity, would be

expected at the level or stage of metabolism where proline is

synthesized. The synthetic route to the carbon chain of proline

is indirect and multiple intermediates exist where dilution of

labeled carbon would be expected from unlabeled dietary

components. Also, these amino acids being present in such low

concentrations, as was noted previously, precludes extensive

dilution of labeled cysteine and cystine with unlabeled cysteine

and cystine.

Tyrosine, which contained no radioactivity, was not

synthesized from the carbon chain of glucose. It must,

therefore, be classified as nutritionally essential.

Phenylalanine is known to be the principal precursor of tyrosine

in the rat (Steele 1952). In insects, the synthesis of tyrosine

from phenylalanine has been demonstrated in the silkworm larvae

(Fukuda 1956), the pale western cutworm (Kasting and McGinnis

1962), and the prairie grain wireworm (Kasting et al. 1962). In

the above cases when phenylalanine is supplied in sufficient

amounts, tyrosine can be classified as nutritionally

non-essential.











As a control, protein was extracted from two types of

radioactive media; one medium had larvae reared on it and one

medium had no larvae reared on it. Figures 6 and 7 are spot maps

of an acid and base hydrolysate of proteins extracted from larval

rearing media. Table 5 presents the data from radiometric

analysis of these proteins. There was no synthesis of any

radioactive protein amino acids in the medium by any organism

which might have contributed to the activity in the proteins of

the insect.

Several extractions of the larval rearing media for free

amino acids were attempted. However, upon TLC, no satisfactory

separation of amino acids was achieved. The amino acids tended

to clump in three undistinguishable groups. These groups were

pooled, counted by liquid scintillation, and found to contain

activity. The ion exchange clean-up procedure which was used in

the carcass analysis section of this paper was then tried prior

to TLC of these free amino acids. This procedure resulted in a

clean preparation, which upon TLC produced a high resolution,

unambiguous separation of the amino acids present. Readable

thin-layer plates were only obtained from the third replicate of

this study which was carried out under aseptic conditions. The

free amino acids from a sample of each of three radioactive media

were studied. These were sterile medium that had larvae reared

















* Val


Ala



Asp/"^\
ApQ Ser

Gly




CySi/ His


X Origin


1 o00"

let he
met


STyr


(E Thr


2 Dimension ---


Figure 6. Separation of amino acids present in an acid
hydrolysate of protein extracted from larval rearing
medium which contained 14C-glucose and on which larvae
were maintained. The same pattern and amino acids
were present in an acid hydrolysate of protein from
radioactive medium on which no larvae were reared.















1e Leu
Val 0i 0 u

OGQr 2Phe

rMet r p he












X Origin
Ala








Lys Hi



X Origin


2nd Dimension


Figure 7. Separation of amino acids present in a base
hydrolysate of protein extracted from larval rearing
medium which contained 14C-glucose and on which larvae
were maintained. The same pattern and amino acids
were present in a base hydrolysate of protein from
radioactive medium on which no larvae were reared.











Table 5. Radioactivity in Amino Acids from Acid and Base
Hydrolysates of Proteins Extracted from Larval Rearing
Media Containing 14C-Glucose


Amino acid Medium containing Medium containing
larvae no larvae
cpm/carbon atom* cpm/carbon atom*

Alanine 0 0.2
Aspartic acid 0 0
Cysteine 0 0
Glutamic acid 0 0
Glycine 0 0
Histidine 0 0
Isoleucine 0.1 0.1
Leucine 0.3 0.2
Lysine 0 0
Methionine 0 0
Phenylalanine 0.3 0.4
Proline 0 0
Serine 0 0
Threonine 0.3 0
Tryptophan 0 0.2
Tyrosine 0.3 0
Valine 0 0.3


* Corrected for
replicates.


background and quenching. Average of three











on it, non-sterile medium that had no larvae reared on it, and

sterile medium that had no larvae reared on it. Figures 8, 9,

and 10, respectively, show the amino acids present in these

samples of media. Tables 6, 7, and 8 present the results of

radiometric analysis of these amino acids. The amino acids

extracted from sterile medium which had no larvae reared on it

contained no radioactivity. This finding is self-explanatory and

requires no further comment. The amino acids from non-sterile

medium, which also had no larvae reared on it, contained

substantial radioactivity. All 15 amino acids and the four

ninhydrin positive unknown compounds contained activity to one

degree or another.

Most microorganisms are known to be capable of synthesizing

all the protein amino acids. Even though the larval rearing

medium is quite dry and this should preclude the need for aseptic

conditions (Fraenkel 1959), the synthesis of amino acids that is

occurring is probably microbial in origin.

The free amino acid extract from the sterile medium that had

larvae reared on it contained 22 free amino acids and five

ninhydrin positive unknowns, all but one substantially labeled.

The activity of the amino acids in this extract ranged from 3 to

28 times the activity in the corresponding amino acids from the

non-sterile extract. The presence of almond moth larvae from

surface sterilized eggs in the sterile rearing medium somehow



















IO
GluO


IlI*


Hyp
Ser


Va 1 T

Tyr


le
O"h

0 Pe

QfP.


c Thr


(CHS) is
(CyS) 2


X Origin


nd
2nd Dimension


Figure .8. Separation of free amino acids extracted from sterile
larval rearing medium containing 14C-glucose and on
which larvae were maintained.


* Unknown ninhydrin positive compounds.


























Gly Ser


OPhe


aI

Asp (H2)
Arg
A His
CVO"-
ArgCySH

X Origin

2nd Dimension



Figure 9. Separation of free amino acids extracted from
non-sterile larval rearing medium containing
14C-glucose and that had no larvae reared on it.
(Incubated with 14C-glucose for 12 weeks.)


* Unknown ninhydrin positive compounds.



















Uene


OGlu Ala r


W -o DA -
s 2Ser


b GIlu(NH2)
A Asp (NH2)

161 DHis
CYSHO Q
X Origin

2n Dimension



Figure 10. Separation of free anino acids extracted from sterile
larval rearing medium containing 14C-glucose and that
had no larvae reared on it.


* Unknown ninhydrin positive compounds.











Table 6. Cpm/Carbon Atom of Free Amino Acids Extracted from
Sterile Larval Rearing Medium Containing 14C-Glucose
and That Had Almond Moth Larvae Reared on It


Amino acid Carbon atoms/ Cpm+/carbon
amino acid atom

Alanine 3 189.5
Arginine 6 263.6
Asparagine 4 152.7
Aspartic acid 4 223.7
Cysteine 3 1398.0
Cystine 6 162.0
Glutamic acid 5 191.5
Glutamine 5 156.3
Glycine 2 1248.0
Histidine 6 71.8
Hydroxyproline 5 169.0
Isoleucine 6 41.8
Leucine 6 46.6
Lysine 6 414.8
Methionine 5 56.5
Phenylalanine 9 18.0
Proline 5 30.0
Serine 3 113.0
Threonine 4 85.3
Tryptophan 11 5.3
Tyrosine 9 20.6
Valine 5 132.8
Unknown I* 4 213.0
Unknown II* 4 55.7
Unknown III* 4 69.0
Unknown IV* 4 54.3
Unknown V* 4 155.0


+ Cpm corrected for background and quenching.
Cpm/carbon atom calculated on the basis of four carbon atoms
per molecule.












Table 7. Cpm/Carbon Atom of Free Amnino Acids Extracted from
Mon-Sterile Larval Rearing Medium That was Incubated
with 14C-Glucose for 12 Weeks and Had No Larvae Reared
on It


Amino acid Carbon atoms/ Cpm+/carbon
amino acid atom

Alanine 3 50.0
Arginine 6 41.0
Asparagine 4 24.0
Aspartic acid 4 53.0
Cysteine 3 50.0
Glutamic acid 5 32.3
Glycine 2 250.0
Histidine 6 7.0
Hydroxyproline 5 29.3
Isoleucine 6 14.2
Leucine 6 13.8
Phenylalanine 9 3.9
Serine 3 38.5
Tyrosine 9 5.9
Valine 5 33.3
Unknown I* 4 12.7
Unknown II* 4 15.3
Unknown III* 4 9.0
Unknown IV* 4 11.0


+ Cpm corrected for background and quenching.
* Cpm/carbon atom calculated on the basis of four carbon atoms
per molecule.











Table 8. Cpm/Carbon Atom of Free Amino Acids Extracted from
Sterile Larval Rearing Medium Containing 14C-Glucose
and on Which No Larvae were Reared


Amino acid Carbon atoms/ Cpm+/carbon
amino acid atom

Alanine 3 0.0
Arginine 6 0.0
Asparagine 4 0.0
Aspartic acid 4 0.0
Cysteine 3 0.0
Glutamic acid 5 0.3
Glutamine 5 0.0
Glycine 2 0.0
Histidine 6 0.0
Hydroxyproline 5 0.0
Isoleucine 6 0.0
Leucine 6 0.0
Lysine 6 0.0
Methionine 5 0.0
Phenylalanine 9 0.0
Proline 5 0.0
Serine 3 0.0
Threonine 4 0.0
Tryptophan 11 0.0
Tyrosine 9 0.0
Valine 5 0.0
Unknown I* 4 0.0
Unknown II* 4 0.0
Unknown III* 4 0.0


+ Cpm corrected for background and quenching.
Cpm/carbon atom calculated on the basis of four carbon atoms
per molecule.










initiated the production of radioactive amino acids in the

medium. This could have been accomplished either through the

introduction of microorganisms into the medium by the larvae or

through the excretion of radioactive amino acids into the medium

by the larvae. Another possibility is that the sterile medium

became contaminated from an external source. Even if this was

the case, the amino acids from the sterile medium containing the

larvae had much more activity than the amino acids in the

non-sterile medium. This was in spite of the fact that the

period of metabolism was only 4 weeks for the sterile medium

containing the larvae as opposed to 12 weeks for the non-sterile

medium that had no larvae on it. It is possible that the insect

larvae from the surface sterilized eggs innoculated the medium

with gut microorganisms which were received transovarially from

the previous generation. An alternative is that the

microorganisms were contained within the egg and released upon

hatching of the larvae. Whatever the source of the radioactive

free amino acids, and I suspect it to be microorganisms in the

medium, the presence of the larvae seems to enrich the medium and

enhance microbial synthesis. The other possibility is that the

microbes from the insects are more efficient or adapted to the

synthesis of amino acids than simple contaminating organisms.

These free amino acids in the medium do not seem to contribute to

the synthesis of proteins in the insect, since none of the










insect's essential amino acids that were labeled in the medium

were labeled in the insect proteins. A calculation was made to

determine the ratio of activity in the medium free amino acid as

compared to the 14C-glucose activity added to the same amount of

medium (see Appendix). The activity contributed by the free

amino acids to the medium at the end of the incubation period was

equal to 1.4 percent of the activity contributed by the

14C-glucose (see Appendix). In this low a proportion, it is

difficult to see how this would help the insect meet its needs

for the nutritionally essential amino acids in nature. However,

with the extremely high specific activity in the cysteine, from

medium free amino acids, it is possible that this could be the

source of the radioactivity in the cysteine isolated from insect

proteins. The free amino acids were not extracted from insect

tissues to ascertain whether there was any internal synthesis of

nutritionally essential free amino acids or incorporation of

radioactive nutritionally essential medium free amino acids into

the free amino acid pools of the insect.

In a great many studies where the continuous exposure

radioactivity method was used to determine nutrient requirements

and sterile technique was not used, there was synthesis of

so-called essential amino acids (Kasting and McGinnis 1966).

This was not the case with the present study where there was no










evidence of synthesis of essential amino acids. The almond moth

larvae were reared in the presence of 14C-glucose (in non-sterile

medium for the first two replicates) for approximately 3 weeks

and allowed to feed ad libitum. This metabolism period is

approximately seven times longer than any other continuous

feeding period noted in the literature. Even with this extended

metabolism period, there was no incorporation of radioactive

essential amino acids into insect protein. This would seem to

minimize the importance of any symbiotic type of relationship, if

indeed one is present, between the almond moth and any

microorganism. The synthesis of nutritionally essential amino

acids in the medium may be due to simple contamination of the

medium with the larval presence merely enhancing the growth

potential of the microbe, or true innoculation with insect

associated microorganisms. This insect innoculum, if present,

may be simple gut microorganisms that are insect associated but

have little nutritional significance. By either route, this

situation seems of little benefit to the insect with regard to

essential amino acid synthesis.

A small microbiological study was undertaken to try and

determine the source of the essential amino acid synthesis in the

medium. Table 9 presents the results of this study which was

carried out on the third replicate (i.e., sterile) of the

radiometric study that was just discussed above. As can be seen











Table 9. Result of Microbiological Study on Almond Moth Eggs and
Larvae and '1C Media to Determine the Source of
Radioactive Free Amino Acid Synthesis


Material Studied Replicate Replicate Replicate
I II III

Sterile medium before -
innoculation

Sterile eggs before -
innoculation

Non-sterile medium + + +

Non-sterile eggs + + +

Larvae from sterile + + +
eggs that had been
reared on sterile
medium for 1 week

Sterile medium that + + +
had larvae from sterile
eggs reared on it for
1 week

2-week old sterile -
medium that had no
larvae reared on it










from the table, the medium and eggs started out in an initially

sterile state. Upon hatching and spending 1 week in the sterile

medium, both the larvae and medium became contaminated with some

type of microorganism. The control sterile medium that had no

larvae reared on it was still sterile after 2 weeks. Sterile

eggs placed on a number of plates hatched and the larvae burrowed

into the agar. Within several days, there was microbial growth

on these plates. From such a limited study, it is difficult to

determine whether the microbial innoculum came from within the

sterile eggs or from some form of external contamination which

was enhanced by the presence of larvae on the medium. However,

since there was microbial growth when larvae from sterile eggs

hatched on the agar plates, it appears that the microbial

innoculum came from within the almond moth eggs.

During the protein extraction procedure, the various

fractions from the larval and media extractions were dried,

weighed, and counted using liquid scintillation. Tables 10

through 15 present the results of these analyses. The extracts

from the larval protein extraction all contain substantial

activity as was expected. The cold TCA fraction contains

glycogen, sugars, free amino acids, vitamins, and nucleotides.

The activity in this fraction is probably contained in the

glycogen which is synthesized from dietary carbohydrate (i.e.,

14C-glucose), the nutritionally non-essential free amino acids











Table 10. Specific Activity of Dried Supernatant Fractions from
the Extraction of Fifth-Instar Almond Moth Larvae
Reared on Medium Containing 14C-Glucose


Supernatant fractions* Specific Cpm /g. % of total
activity of insect counts
(cpm**/mg.
of extract)

Replicate 1

TCA (cold) 585 173,372 9.04
Alkaline acetone 27,672 1,102,132 57.44
95% ethanol 7,456 111,699 5.82
Ether 810 2,443 0.13
TCA (hot) 379 59,391 3.10
Residue (protein) 1,852 469,852 24.49

Replicate II

TCA (cold) 735 196,107 10.26
Alkaline acetone 10,479 1,028,242 53.80
95% ethanol 3,852 20,775 1.09
Ether 2,015 3,073 0.16
TCA (hot) 612 42,595 2.23
Residue (protein) 3,542 620,558 32.47

Replicate III

TCA (cold) 504 149,667 9.37
Alkaline acetone 17,066 1,016,207 63.59
95% ethanol 3,790 78,692 4.92
Ether 996 2,512 0.16
TCA (hot) 360 40,539 2.54
Residue (protein) 1,534 310,482 19.43


* See Figure 2 for explanation of fractions.
** Cpm corrected for background and quenching.











Table 11. Specific Activity of Dried Supernatant Fractions from
the Extraction of Larval Rearing Medium Containing
14C-Glucose and That IIad Almond Moth Larvae Reared on
It


Supernatant fractions* Specific Cpm**/g. % of total
activity of medium counts
(cpm**/mg.
*of extract)

Replicate I

TCA (cold) 5,244 1,385,239 87.63
Alkaline acetone 10,393 235,479 9.85
95% ethanol 21,781 45,851 1.97
Ether 545 310 0.02
TCA (hot) 130 11,508 0.54

Replicate II

TCA (cold) 7,323 1,599,430 81.72
Alkaline acetone 4,160 297,146 15.19
95% ethanol 7,196 53,416 2.73
Ether 119 237 0.02
TCA (hot) 75 6,870 0.35

Replicate III

TCA (cold) 6,512 1,722,520 82.53
Alkaline acetone 6,094 193,509 14.03
95% ethanol 14,095 38,751 2.73
Ether 267 289 0.02
TCA (hot) 124 10,635 0.69


* See Figure 2 for explanation of fractions.
** Cpm corrected for background and quenching.












Table 12. Specific Activity of Dried Supernatant Fractions from
the Extraction of Larval Rearing Medium Containing
14C-Glucose and That Had No Larvae Reared on It


Supernatant fractions* Specific Cpm**/g. % of total
activity of medium counts
(cpm**/mg.
of extract)

Replicate I

TCA (cold) 98 25,285 33.11
Alkaline acetone 1,319 41,496 54.35
95% ethanol 835 7,287 9.55
Ether 67 47 0.06
TCA (hot) 25 2,244 2.94

Replicate II

TCA (cold) 89 23,388 30.50
Alkaline acetone 795 42,172 55.01
95% ethanol 953 8,466 11.04
Ether 102 66 0.08
TCA (hot) 25 2,583 3.37

Replicate III

TCA (cold) 132 31,882 41.83
Alkaline acetone 768 33,221 43.59
95% ethanol 1,708 8,774 11.51
Ether 40 38 0.05
TCA (hot) 18 2,302 3.02


* See Figure 2 for explanation of fractions.
** Cpm corrected for background and quenching.











Table 13. Weights df Dried Supernatant Fractions from the
Extraction of Fifth-Instar Almond Moth Larvae Reared
on Medium Containing 14C-Glucose


Supernatant fractions* Weight Mg./g. % of total
(g.) of insect weight
extracted extracted

Replicate I

Weight of insect extracted 4.5215
TCA (cold) 1.3483 296.6 29.81
Alkaline acetone 0.3443 75.7 7.61
95% ethanol 0.1164 25.8 2.59
Ether 0.0265 5.9 0.60
TCA (hot) 0.7128 156.9 15.77
Residue weight (protein) 1.1534 253.7 25.50

Replicate II

Weight of insect extracted 0.8627 -
TCA (cold) 0.2295 266.8 26.68
Alkaline acetone 0.1436 165.9 16.59
95% ethanol 0.0076 8.8 0.88
Ether 0.0027 3.2 0.32
TCA (hot) 0.0599 69.6 6.96
Residue weight (protein) 0.1513 175.2 17.52

Replicate III

Weight of insect extracted 3.6124 -
TCA (cold) 1.0718 296.9 29.68
Alkaline acetone 0.3845 106.4 10.64
95% ethanol 0.1413 39.1 3.91
Ether 0.0177 5.0 0.50
TCA (hot) 0.4074 112.7 11.27
Residue weight (protein) 0.7225 200.3 20.02


* See Figure 2 for explanation of fractions.











Table 14. Weights of Dried Supernatant Fractions from the
Extraction of Larval Rearing Medium Containing
14C-Glucose and That Had Almond Moth Larvae
Reared on It


Supernatant fractions* Weight Mg./g. % of total
(g.) of medium weight
extracted extracted

Replicate I

Weight of medium extracted 1.0640 -
TCA (cold) 0.2814 264.0 26.41
Alkaline acetone 0.0517 48.6 4.86
95% ethanol 0.0083 7.7 0.77
Ether 0.0013 1.2 0.12
TCA (hot) 0.0943 88.7 8.87
Residue weight (protein) 0.3672 345.0 34.49

Replicate II

Weight of medium extracted 0.8241 -
TCA (cold) 0.1819 220.9 22.09
Alkaline acetone 0.1011 122.2 12.22
95% ethanol 0.0144 17.0 1.70
Ether 0.0041 5.1 0.51
TCA (hot) 0.0763 92.2 9.22
Residue weight (protein) 0.1670 202.4 20.27

Replicate III

Weight of medium extracted 1.2132 -
TCA (cold) 0.3211 264.5 26.47
Alkaline acetone 0.0737 60.4 6.08
95% ethanol 0.0074 6.1 0.61
Ether 0.0024 2.0 0.20
TCA (hot) 0.1043 86.0 8.60
Residue weight (protein) 0.4116 339.5 33.97


* See Figure 2 for explanation of fractions.












Table 15. Weights of Dried Supernatant Fractions from the
Extraction of Larval Rearing Medium Containing
14C-Glucose and That Had No Larvae Reared on It


Supernatant fractions* Weight Mg./g. % of total
(g.) medium weight
extracted extracted

Replicate I

Weight of medium extracted 1.4412 -
TCA (cold) 0.3727 258.9 25.89
Alkaline acetone 0.0964 67.0 6.70
95% ethanol 0.0265 18.3 1.83
Ether 0.0021 1.5 0.15
TCA (hot) 0.1305 90.6 9.06
Residue weight (protein) 0.5896 409.5 40.94

Replicate II

Weight of medium extracted 1.5365
TCA (cold). 0.4031 262.4 26.24
Alkaline acetone 0.1529 99.6 9.96
95% ethanol 0.0287 18.7 1.87
Ether 0.0021 1.3 0.13
TCA (hot) 0.1590 103.5 10.35
Residue weight (protein) 0.4206 273.7 27.41

Replicate III

Weight of medium extracted 1.4820
TCA (cold) 0.3584 241.8 24.18
Alkaline acetone 0.1202 81.0 8.10
95% ethanol 0.0155 10.4 1.04
Ether 0.0028 1.9 0.19
TCA (hot) 0.1863 125.7 12.57
Residue weight (protein) 0.3173 214.0 21.39


* See Figure 2 for explanation of fractions.










which are also synthesized from 14C-glucose and the purine and

pyrimidine bases of the nucleotides. The pyrimidine ring

contains carbon atoms that originate from CO2 and aspartic acid.

Both of these compounds are labeled via synthesis from

14C-glucose. The purine ring structure contains carbon atoms

that come from CO2 and'glycine. Both of these compounds are also

labeled via synthesis from 14C-glucose. The alkaline acetone, 95

percent ethanol, and ether fractions contain the extracted

neutral and phospholipids. Dietary carbohydrate is a major

precursor of lipids (Friend 1962, House 1965). A large quantity

of activity would therefore be expected in the lipid extracting

fractions (Table 10). This was indeed the case with

approximately 83 percent of the activity from the three

replicates concentrated in the three lipid extracts. The

specific activity in these fractions was also high. The TCA,

which was the final extracting solvent, contained nucleic acids.

The activity in the nucleic acids comes from the labeling of

purine and pyrimidine bases which was discussed above with

respect to labeling in nucleotides.

In the extraction of protein from media that had larvae

reared.on them, most of the radioactivity was concentrated in the

cold TCA fraction as opposed to the lipid fractions for the











larval protein extraction. Most of this activity is not simply

11C-glucose being extracted off the media since the cold TCA

fraction from medium that had no larvae reared on it contained

only about 1/60 of the amount of activity that this medium had on

it. The lipid fractions from this medium extraction did not

contain a large percentage of the total counts present but they

did have a very high specific activity.

The fractions from the medium that had no larvae reared on it

contained small amounts of activity compared to the other medium

extracted. The specific activity of these fractions was also

many times lower than comparable ones from the medium that had

larvae reared on it. The third replicate was carried out in

duplicate for the medium that had no larvae reared on it. The

sterile replicate was used only for extraction of free amino

acids while the non-sterile replicate was used for all other

extractions.

Several small studies related to the radiometric analysis

were carried out. These are presented in the appendix.











Carcass Analysis for the Determination of
Quantitative Amino Acid Requirements
Using Gas Chromatography


Results of the carcass analysis of fifth-instar almond moth

larvae are presented in Tables 16 and 17 and Figures 11 and 12.

The gas-liquid chromatogram of 19 TMS amino acid standards along

with retention time, relative retention time, and sensitivity,

used to identify and quantitate the amino acids in the larvae,

are presented in Table 18 and Figure 13.

Of the free amino acids in the almond moth larvae, proline,

glutamic acid, and tyrosine were present in the highest

concentrations making up almost 70 percent of the free amino

acids present. Proline and tyrosine which participate in

cuticular tanning prior to and during pupation would be expected

at a high concentration in larvae this close to pupation (Chen

1962, 1966). Glutamic acid has also been reported in high

concentrations among the free amino acids of insect larvae (Rock

and King 1966, 1967a).

Among the protein amino acids, there was a very uniform

distribution with respect to amount present. Glutamic acid and

aspartic acid were the only amino acids present in substantially

greater quantity than the others. This has been noted for other

insects also (Rock and King 1966, 1967b, 1967c).












Table 16. Pattern
Larvae


of Amino Acids in Fifth-Instar Almond Moth


Amino acid Mg. of amino acid/ Mg. of amino
q. of larvae acid/g. of
Free Protein Total protein
amino amino amino
acids acids acids

Alanine .290 10.433 10.723 62.3
Valine .150 9.134 9.284 54.5
Leucine .224 12.864 13.088 76.8
Isoleucine .096 8.548 8.644 51.0
Glycine .119 5.866 5.985 35.0
Proline 2.218 6.495 8.713 38.8
Serine .275 6.914 7.189 41.3
Threonine .103 4.944 5.047 29.5
Hydroxyproline 1.215 1.215 7.3
Aspartic acid .040 17.640 17.680 105.3
Methionine .057 2.472 2.529 14.8
Glutamic acid 1.098 22.668 23.766 135.3
Phenylalanine .050 6.788 6.838 40.5
Arginine .134 6.285 6.419 37.5
Lysine .172 10.685 10.857 63.8
Tyrosine .531 8.590 9.121 51.3

Total 5.557 141.541 147.098 845.0












Table 17. Percent Composition of Amino Acids in Fifth-Instar
Almond Moth Larvae


Amino acid % of total % of total % of total amino acids
free protein
amino amino free protein total
acids acids amino amino amino
acids acids acids

Alanine 5.22 7.37 .20 7.09 7.29
Valine 2.70 6.45 .10 6.21 6.31
Leucine 4.03 9.09 .15 8.75 8.90
Isoleucine 1.73 6.04 .07 5.81 5.88
Glycine 2.14 4.14 .08 3.99 4.07
Proline 39.91 4.59 1.51 4.42 5.93
Serine 4.95 4.89 .19 4.70 4.89
Threonine 1.85 3.49 .07 3.36 3.43
Hydroxyproline .86 .83 .83
Aspartic acid .72 12.46 .03 11.99 12.02
Methionine 1.03 1.75 .04 1.68 1.72
Glutamic acid 19.76 16.02 .75 15.41 16.16
Phenylalanine .90 4.80 .03 4.62 4.65
Arginine 2.41 4.44 .09 4.27 4.36
Lysine 3.10 7.55 .12 7.26 7.38
Tyrosine 9.56 6.07 .36 5.84 6.20

Total 100.01 100.01 3.79 96.23 100.02






















































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Table 18. Retention Time, Relative Retention Time, and
Sensitivity of 19 Trimethylsilyl Amino Acid Standards


Amino Acid Retention Relative Sensitivity
Time Retention (ng./mm.
(sec.) Time of recorder
deflection)

Alanine .525 .432 11.6
Valine 725 .597 10.4
Leucine 825 .679 10.9
Isoleucine 870 .716 21.7
Glycine 895 .737 9.3
Proline 930 .765 16.1
Serine 1000 .823 8.5
Threonine 1035 .852 9.6
Internal Standard 1215 1.000 -
(Decanoic acid)
Hydroxyproline 1300 1.070 16.1
Aspartic acid 1320 1.086 9.4
Methionine 1370 1.128 17.2
Cysteine 1400 1.152 35.7
Glutamic acid 1475 1.214 18.5
Phenylalanine 1565 1.288 12.2
Arginine 1735 1.428 22.7
Lysine 1890 1.556 11.4
Tyrosine 2020 1.663 11.9
Tryptophan 2500 2.058 26.3
Cystine 2520 2.074 71.4


NOTE: Standard column and conditions Column: 10 percent OV-11
on Supelcoport 100/120 mesh. 12 ft. x 2 mm. I.D. Conditions:
injector temperature 2750C.; detector temperature, 3000C.; oven
temperature, initial 1000C.; 3-min. hold after start of solvent
peak 40C./min. increased to 3000C.; attenuator settings 32 x
10-1i; N2 carrier gas flow, 17 ml./min.; flame ionization
detector; internal standard, Decanoic acid.





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A small amount of hydroxyproline was detected among the

protein amino acids. This is very unusual since hydroxyproline

is not commonly found in proteins. It has, however, been found

in fibrous proteins, such as collagen, and in plant proteins.

Table 19 is a proposed mixture, at the 2 percent and 3

percent dietary level, of amino acids based on results from

carcass analysis of fifth-instar almond moth larvae. Tryptophan

is not included in this table since it is destroyed by acid

hydrolysis and no base hydrolysates were analyzed. Histidine,

cysteine, and cystine were not detected with the GLC method as

they were with TLC. Cysteine and cystine are often in very low

concentrations in late instar lepidoptera larvae (Rock and King

1966, 1967c).

A study was done to determine if there was any loss of amino

acids during the protein extraction procedures. Figure 14

presents the results of this study. The derivatized extraction

fractions contained no significant chromatographic peaks, aside

from the internal standard, both before and after acid

hydrolysis. This is not surprising since amino acids and

peptides are markedly hydrophilic and thus very insoluble in

non-aqueous solvents. The organic solvents used for the

extraction procedure (i.e., alkaline acetone, ether, and 95

percent ethanol) would have very little affinity for amino acids

and not contribute to the loss of these compounds. The aqueous
I











Table 19. Amino Acid Mixture Patterned After Carcass Analysis of
Fifth-Instar Almond Moth Larvae


Amino Acid 2% Dietary 3% Dietary Dietary
level level requirement
(mg./100 g. (mg./100 g. from 14C
of diet) of diet) study

Alanine 146 219
Valine 126 189 +
Leucine 178 267 +
Isoleucine 118 177 +
Glycine 82 123
Proline 118 177
Serine 98 147
Threonine 68 102 +
Aspartic acid 240 360
Methionine 34 51 +
Glutamic acid 324 486
Phenylalanine 94 141 +
Arginine 88 132 +
Lysine 148 222 +
Tyrosine 124 186 +
Hydroxyproline 16 24


+ = Not synthesized nutritionally essential
- = Synthesized nutritionally non-essential.





















































































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cold TCA fraction takes out the free amino acids from the insect

tissue in the first extraction step and it was this fraction that

was analyzed for free amino acids of the insect tissues.

The almond moth required the same 10 amino acids as did the

eight insects in Table 1, in addition to tryptophan. It did not

synthesize or require any amino acid that had not been reported

previously for another insect.

The percentage composition of amino acids in the almond moth

was quite similar to that found in other insects (Rock and King

1966, 1967b, 1967c). Free amino acids consisted mainly of

proline, glutamic acid, and tyrosine (Chen 1962, 1966, Rock and

King 1966, 1967a) while, among the protein amino acids, glutamic

acid and aspartic acid predominated with the others being present

in fairly equal concentration (Rock and King 1966, 1967b, 1967c).

The presence of some type of insect-microorganism

relationship in the almond moth has not been noted previously.

House (1962) notes that symbiotes usually occur in species that

feed exclusively on substances such as plant sap, vertebrate

blood, and certain stored-products that are deficient in specific

nutrients. Fraenkel (1959), on the other hand, states that the

need by many stored product insects for eight or nine B-complex











vitamins, even when reared in the absence of axenic conditions,

minimizes the nutritional importance of microorganisms that are,

no doubt, present in the intestine. He also states that due to

the dry, powdery nature of the diet of stored product insects,

microbial actions in the medium would be almost non-existent. In

the almond moth medium, there is substantial microbial action, as

evidenced by the large number of substantially labeled free amino

acids isolated here. However, the nutritional importance of this

action seems at present of only slight value. With the possible

exception of cysteine, there was no incorporation of labeled

essential medium free amino acids into the protein of the larval

almond moth. With the continuous exposure method of isotope

administration, there is often microbial synthesis of essential

free amino acids, with subsequent incorporation into the insect

larvae (Kasting and McGinnis 1966). This was not the case with

the almond moth, even though it was exposed to the 14C-glucose

for a much longer time period than in most continuous exposure

nutritional studies.

With the incorporation of the data from the radiometric study

with those obtained from the carcass analysis study, the dietary

amino acid requirements of the almond moth have been identified.

These data have also been used to formulate an amino acid ration

which should be an excellent starting point for formulating a







86



completely defined diet for the almond moth. Once the almond

moth can be reared on a completely defined diet, nutritional

state can be eliminated as a variable in any future studies with

this insect pest.
















SUMMARY


The almond moth synthesizes alanine, aspartic acid, glutamic

acid, glycine, proline, and serine from 14C-glucose. These amino

acids are considered nutritionally non-essential. Arginine,

histidine, isoleucine, leucine, lysine, methionine,

phenylalanine, threonine, tryptophan, tyrosine, and valine were

not synthesized and are considered nutritionally essential.

Cysteine and cystine were synthesized to a limited degree and

cannot yet be classified.

Radioactive essential and non-essential free amino acids were

isolated from larval rearing media. The specific activities were

substantially higher in amino acids extracted from medium that

had larvae reared on it than from medium that had no larvae

reared on it. There appears to be an insect-microorganism

relationship at work here. However, the exact nature of this

relationship and to what extent it contributes to the insects'

nutrition are at present undetermined. This situation seems to

contradict what some authors have stated that, due to the dry

nature of the stored product insects' diet, microbial

associations or contamination should not be a factor (Fraenkel

1959). However, since there was no detectable incorporation of

radioactive essential free amino acids from the medium into the

larval proteins (with the possible exception of cysteine), the










nutritional importance of this relationship seems questionable

when the techniques I employed in this study are used.

In other radiometric nutrition studies with insects, where

the continuous exposure method of isotope incorporation was used,

there appeared to be synthesis of essential amino acids by the

insect (Kasting and Mctinnis 1966). This synthesis was

presumably microbial in nature. Even though the period of

exposure to the 14C-glucose was approximately seven times longer

in my study than with other continuous exposure studies, there

was still no appreciable synthesis of essential amino acids by

the larvae or any related microorganisms. This factor in

addition to the one stated previously tends to minimize the

nutritional importance of the insect-microorganism relationship

with the techniques employed in this study.

The study to isolate the source of the insect associated

microorganisms is at present inconclusive. However, from the

data available presently, it appears that the microorganisms come

from within the almond moth eggs.

From carcass analysis of fifth-instar almond moth larvae, it

was determined that proline, tyrosine, and glutamic acid make up

almost 70 percent of the total free amino acids present. Proline

and tyrosine participate in cuticular tanning at this time just

prior to pupation. The protein amino acids, which contain a high

percentage of glutamic acid and aspartic acid, in combination

with the free amino acids are the basis for a dietary mixture at

the 2 percent and 3 percent levels (Table 19).


































APPENDIX




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