S-ALANINE METABOLISM IN THE HOUSEFLY,
MUSCA DOMESTlCA 7L.

Thesis for the Degree of Ph“ D.
MICHlGAN STATE UNWERSITY
RICHARD H. ROSS, JR.

1972

LIBRARY

Michigan State
TH 55!. University

 

This is to certify that the

thesis entitled .- i _‘ I' e

B—ALANINE METABOLISM IN THE HOUSEFLY, 4

v

MUSCA DOMESTICA L .

presented by

Richard H. Ross, Jr.

has been accepted towards fulfillment
of the requirements for

Eh I] degreein Entomology

 

ABSTRACT

B-ALANINE METABOLISM IN THE
HOUSEFLY, MUSCA DOMESTICA L.

 

By

Richard H. Ross, Jr.

Housefly larvae, Musca domestica L., were reared on a B—alanine—

 

free diet. The adults were fed a synthetic diet also lacking B-alanine.
At various developmental stages the B-alanine was extracted, purified,
derivatized, and quantitatively assayed by gas-liquid chromatography.
Prior to pupation, wandering larvae synthesized B-alanine so that a
peak concentration of 362.2 i 27.2 ug/g wet weight was found in the
white puparium stage. Another peak of 290.1 ug/g was found in the
fourth day of puparial development prior to adult emergence. The
adults contained more than 600 ug/g which they maintained for at least
the first week after eclosion.

B-Alanine synthesis was studied at the time of pupation in the
housefly. The larvae were reared aseptically and upon pupation were
injected with uracil—6-3H, aspartate-U-IAC, pantothenate-l—14C,
propionate-2-14C, or malonate-2-14C. It was found by radioassay
that relative to the other compounds tested uracil contributed 56.2%
and aspartate 24.2% while pantothenate, propionate, and malonate con-

tributed 9.4%, 7.1%, and 3.1%, respectively, to B-alanine synthesis.

Richard H. Ross, Jr.

Aseptically reared houseflies in the early white puparium stage
were injected with B-alanine-l—lac, B-alanine-2-14C, or B—alanine—
3-14C to study the utilization of B-alanine during pupal sclerotization.
On analysis, less than 5% of the radioactivity was expired as 14C02 in
24 hr and less than 1% was incorporated into the total lipids from
each of the radiolabelled compounds. B—Alanine labelled at carbons
l, 2, or 3 gave 1 to 2%, 2 to 4%, and l to 2% in the other aqueous
fraction, respectively. It was found that the radioactivity in the
amino acids decreased from about 60% at 0.5 hr after injection to
about 10 to 20% by 4 hr for each of the labelled compounds. The
decline in amino acid radioactivity had a corresponding elevation in
activity incorporated into the residue; it increased from about 5% to
over 40% in the same time period. From 4 hr to 24 hr after injection

the amino acid radioactivity remained relatively constant at about 10%

while the radioactivity in the residue remained at about 40 to 50%.

B-ALANINE METABOLISM IN THE HOUSEFLY,

MUSCA DOMESTICA L.

 

By

Richard H. Ross, Jr.

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Entomology

1972

LIST OF TABLES .

LIST OF FIGURES

PART I:

Introduction .

Materials and Methods

Results .
Discussion
References

Introduction .

Materials and Method

Results .
Discussion
References

Introduction .

Materials and Methods

Results .
Discussion
References

Literature
References

Review

PART II:

PART III:

STUDIES

STUDIES

TABLE OF CONTENTS

B-ALANINE CONCENTRATIONS
AND ADULTS

ON B-ALANINE SYNTHESIS IN THE

EARLY PUPARIUM

ON B-ALANINE UTILIZATION IN THE

EARLY PUPARIUM

ii

Page
iii

iv

NOU'IUJN

16
18
20
24
26

29
31
33
41
43

44
47

Table

10.

11.

LIST OF TABLES

B—Alanine concentrations during the development of
larvae, puparia, and adults of Musca domestica
reared on an aseptic synthetic diet . . . . . . . .

 

Treatment data of radiolabelled compounds injected
into early puparia of the housefly . . . . . .

Recovery of injected radioactivity in the residue,
lipids, amino acids, and other aqueous from early
puparia of the housefly . . . . . . . . .

Incorporation of
injection into

radioactivity into B-alanine after
early puparia of the housefly . . .

Treatment data of radiolabelled B-alanine injected
into early puparia of the housefly . . . . . . . . .

Average per test

of the live weight and per cent

recovery for the various times after injection of
B-alanine-14C into early puparia of the housefly . .

Average per test

of the accumulative 14CO production

after injection of B-alanine into early puparia of

the housefly .

Average per test
into the total
140 into early

Average per test
into the other
14C into early

Average per test
into the amino
14C into early

Average per test

of the incorporation of the radiolabel
lipids after injection of B-alanine—
puparia of the housefly . . . . .

of the incorporation of the radiolabel
aqueous after injection of B—alanine—
puparia of the housefly . . . . . .

of the incorporation of the radiolabel
acids after injection of B-alanine-
puparia of the housefly . . . . . .

of the weight and incorporation of the

radiolabel into the residue after injection of B-
alanine-IAC into early puparia of the housefly . .

iii

Page

20

22

23

33

34

36

37

38

39

40

Figure
l.

2.

LIST OF FIGURES

The mass spectrum of N-TFAjnfbutyl—B-alanine . . .

B-Alanine concentrations in different life stages of
houseflies reared on an aseptic synthetic diet .

iv

PART I: B-ALANINE CONCENTRATIONS IN THE LARVAE,

PUPARIA, AND ADULTS

INTRODUCTION

B-Alanine was incorporated into the cuticle of insects during
the hardening process (SEKI, 1962; JACOBS, 1966; BODNARYK and LEVENBOOK,
1969; BODNARYK, 1970; DUFFY, 1970; GILBY and MCKELLAR, 1970; and
SRIVASTAVA, 1971). LEVENBOOK E£.§l' (1969) found that B-alanine
occurred as a dipeptide, sarcophagine, in the fleshfly, Sarcophaga
bullata; this dipeptide was not found in the housefly, Musca domestica,

or the blowfly, Phormia regina. BODNARYK and LEVENBOOK (1968) reported

 

that B-alanine occurred in carnosine (B—alanyl-L—histidine) in the
larvae of P, regina.

PANT and LAL (1970) found that the B-alanine titre in Sarcophaga

 

ruficornis varied during metamorphosis. This was also shown for

 

§, bullata (LEVENBOOK £5 31., 1969; BODNARYK and LEVENBOOK, 1969) and
for P, regina (LEVENBOOK and DINAMARCA, 1966). In order to study the
metabolism of B-alanine in the housefly, this study was undertaken to
quantitatively determine the B-alanine titre during various develop-

mental stages from larvae to adults.

MATERIALS AND METHODS

The houseflies used were a maximum longevity strain obtained
from the Insect Physiology Laboratory, USDA, Beltsville, Maryland. The
flies were routinely reared on CSMA medium (ANON., 1959) and the adults
fed a 1:1 mixture of sucrose and nonfat dry milk. The eggs were col-
lected and sterilized in 0.1% hypochlorite solution for 20 minutes.

The larvae were reared aseptically according to MONROE (1962) and were
periodically washed out of the culture flasks to obtain samples. The
puparia were collected, weighed and held for various lengths of time,
and the adults maintained on a synthetic diet (MONROE and LAMB, 1968).
The casein used in both larval and adult diets was obtained from
Fisons Pharmaceuticals Limited, Loughborough, England.

The casein was analyzed for B-alanine as well as the dried milk
and sugar used in the routine rearing of the flies. Samples were ex-
tracted with methylene chloride in a Soxhlet extractor for 24 hrs. The
samples were then hydrolyzed in 5 ml 6 N H01 at 110°C for 26 hrs, dried
on a rotating evaporator and added to a 1.1 X 7 cm Dowex 50-X12 (H+ form)
cation exchange column. The column was washed with 100 ml water and the
amino acids were taken off with 100 ml 10% ammonium hydroxide. The re-
sulting aqueous was evaporated to dryness and the N-TFAjnfbutyl esters
of the amino acids made according to ROACH and GEHRKE (1969). The

samples were then analyzed by gas—liquid chromatography (GLC).

4

The houseflies were analyzed in triplicate; they were homogenized
in water, refluxed for 90 min. in acetone-ethanol (1:1) and vacuum
filtered (KAPLANIS g£_§l,, 1960). The lipids were then extracted 3
times with ethyl ether, the aqueous added to a 1.1 X 7 cm cation ex-
change column, and the amino acids analyzed by GLC as above. In a
preliminary experiment the B-alanine extracted from a sample of house—
fly puparia was first partially purified by ascending paper chromato-
graphy. The amino acids were streaked on a sheet of Whatman No. 1
filter paper. The mobile phase was the upper layer of an Efbutanol-
acetic acid-water (250:60:250) mixture. The chromatogram was developed
for 12 hrs., dried and redeveloped in the same system. The B-alanine
was eluted from the paper with water and the N-TFAjnfbutyl derivative
made as above. Identity was proven by GLC and mass spectrometry.

The GLC analysis in all experiments was accomplished using a
Research Specialties Corp., Series 600 GLC equipped with a dual hydro-
gen flame detector. A 2 m X 4 mm ID glass column packed with Tabsorb
(Regis Chemical Co., Chicago, Illinois) was used to separate the amino
acid derivatives. The temperature was programmed from 94°C to 210°C

at 3.5°C/min; N was at 94 ml/min. The peak areas were computed by

2
disc integration and compared to a standard curve of B-alanine. Be-
cause B-alanine and leucine could not be separated completely (in the
samples where the B-alanine peak was smaller than leucine) the areas

were compared to those obtained by adding B—alanine to a leucine.

standard that gave a comparable area to that of the unknown.

RESULTS

Figure 1 shows the mass spectrum of extracted and derivatized
B-alanine from housefly puparia which was identical to that obtained
for authentic derivatized B—alanine (N-TFAjnfbutyl-B-alanine). The
peak at m/e 241 represents the molecular ion. The base peak at m/e
55 represents [CH2=CH-CH-CH3]+ while its complementary peak is m/e 186.
The loss of water from m/e 186 yields m/e 168 (CF3C0NHCHZCH2CEO+).

Table l and Figure 2 show the changes in the titre of B-alanine
in various stages of houseflies reared on a B-alanine-free diet. There
is a sharp increase in B-alanine concentration beginning in the larval
wandering stage (51.4 i 2.0 ug/g wet wt.) and reaching a peak of
362.2 i 27.2 ug/g shortly after pupation. There is a second peak of
390.1 ug/g prior to eclosion during the fourth day after puparium forma—
tion. The adults also accumulate a very high level of B-alanine which
they maintain through at least the first week after eclosion. In a
preliminary sample where the adults were fed the dry milk-sugar diet
which was found to contain B-alanine, the adults acquired a comparable
concentration but at a slightly faster rate. All points on the graph
except the 4-day-old puparial peak represent the average of triplicate
injections from each of 3 samples containing between 50 and 250 in-
dividuals. The peak concentration for the 4-day-old puparia represents
the value obtained for 1 sample and indicates that the B-alanine con-

centration reaches a maximum in pharate adults prior to eclosion.

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TABLE l.--B-A1anine concentrations during the develOpment of larvae,
puparia, and adults of Musca domestica reared on an aseptic
synthetic diet.

 

 

B-Alanine concentration

 

Stage of development

 

(age in hrs.) ug/individual ug/g wet weight
Larvae
72 (3)* 0.7 i 0.2 38.8 i 3.
96 (4) 0 i 0.0 51.4 i 2.0
Puparia
l (0.04) 4.1 i 0.1 362.2 : 27.2
(0.25) 2.0 i 0.3 148.0 i 11.6
12 (0.5) 1.2 i 0.1 125.1 i 19.1
24 (1) 1.0 i 0.4 96.1 i 4.2
48 (2) 0.5 i 0.0 64.9 i 2.4
72 (3) 0.5 i 0.1 52.0 i 3.4
84 (3.5) 0.5 i 0.0 55.4 i 0.7
96 (4) 0.6 i 0.1 74.8 i 1.8
>96 (>4) 3.9** 290.1**
Adults after emergence
1 (0.04) 1.3 i 0.1 178.1 i 13.1
12 (0.5) 0.8 i 0.2 104.9 i 9.6
24 (l) 1.2 i 0.0 139.2 i 7.2
48 (2) 4.0 i 0.3 471.4 1 25.5
72 (3) 5.2 i 0.6 566.5 i 40.2
96 (4) 5.5 i 0.3 602.7 i 52.0
120 (5) 6.4 i 0.1 648.2 f 25.0
144 (6) 6.4 i 0.2 631.8 i 24.6
168 (7) 6.6 i 1.2 633.3 i 27.3

 

*For rapid comparison age in days in parentheses.

**Based on one sample.

 

 

 

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1 n l n n P n l 1 1 n 1 1 1 1

3 4 0 I 2 3 44 0 l 2 3 4 5 6 7

larvae puparia l OdUHS after emergence
age (days)

Figure 2.--B-Alanine concentrations in different life stages of
houseflies reared on an aseptic synthetic diet.

DISCUSSION

PANT and LAL (1970) found that B-alanine in S, ruficornis

 

reached a peak concentration in the white puparium stage and that it
could be related to cuticular protein formation which also occurred
at this stage; however, they did not find a peak prior to eclosion.
LEVENBOOK gt a1. (1969) found that B-alanyl—L-tyrosine in S, bullata
reached a peak concentration in wandering larvae while BODNARYK and
LEVENBOOK (1969) showed that free B—alanine reached a peak sometime
after pupation and prior to the initiation of cuticular darkening.
LEVENBOOK and DINAMARCA (1966) reported that B-alanine reached a peak
concentration in the puparia of P, regina, but they did not find a
definite peak before adult emergence. In working with houseflies,
LORD and SOLLY (1964) found that B-alanine was a major amino acid in
the adults.

This study showed that houseflies maintained a rather low level
of B-alanine during larval development and that there was a very rapid
synthesis of B-alanine in the late wandering or white puparium stage.
Upon the initiation of puparium darkening the B-alanine concentration
decreased, which could be due at least partially to the incorporation
of B-alanine into the cuticle. The occurrence of B—alanine in the
pupal sheath has been well documented for houseflies (SEKI, 1962;

FUKUSHI and SEKI, 1965; FUKUSHI, 1967), Drosophila sp. (SEKI, 1962;

 

10
JACOBS and BRUBAKER, 1963; JACOBS, 1966; FUKUSHI, 1967; and JACOBS,
1968a), Lucilia cuprina (GILBY and MCKELLAR, 1970), the fleshfly,
S, bullata (BODNARYK and LEVENBOOK, 1969) and the wax moth, Galleria

mellonella (SRIVASTAVA, 1971).

 

In one trial, there was also a peak B—alanine concentration in
4-day-old puparia prior to adult emergence. Because the other 3 trials
did not show a peak until the newly emerged adult stage, the synthesis
of B-alanine occurred rapidly sometime during the fourth day and prior
to eclosion; biological variation may have contributed to a variation
of several hours even under identical rearing conditions. As the newly
emerged adults' cuticle hardened the B-alanine concentration decreased.
After the flies had fed for several hours the B-alanine concentration
increased to about twice the concentration of either of the peaks
present during pupal formation and development. The study showed that
this buildup occurred when B-alanine was present or absent in the diet.
LORD and SOLLY (1964) also found that B-alanine was a major component
of the adult housefly amino acids. The function of B-alanine has not
been determined in adult flies although JACOBS (1968a) has shown that

injected B-alanine in Drosgphila melanogaster adults inhibited CO

 

2

excretion from glucose and several other sugars, and JACOBS (1968b)
indicated that B—alanine may inhibit glucose oxidation by inhibiting
phosphorylation. JACOBS (1970) also showed that B—alanine inhibited

the catabolism of phenylalanine in 2, melanogaster, and VERESHTCHAGIN

 

.gg'gl. (1961) showed that B-alanine depressed the electrical activity

in the nerve chain of the pine moth caterpillar, Dendrolimus pini.

 

JACOBS and BRUBAKER (1963) and JACOBS (1966) showed the presence of a

11

B-alanine oxidase in Q, melanogaster which indicated that B—alanine

 

may be very active in adults.
Complete understanding of the function of B—alanine in insects,

however, awaits further investigations.

REFERENCES

ANONYMOUS (1959) The Feet-Grady method. Official method of the
Chemical Specialties Manufacturers Association for evaluating
liquid household insecticides, latest revision. Soap Sanitary
Chem. Spec. Blue Book and Catalog, 32nd year, 219-220.

BODNARYK R.P. (1970) Effect of dopa-decarboxylase inhibition on the
metabolism of B-alanyl—L-tyrosine during puparium formation in
the fleshfly Sarcophagg bullata Parker. Comp. Biochem. Physiol.
35, 221-227.

 

BODNARYK R.P. and LEVENBOOK L. (1968) Naturally occurring low-
molecular-weight peptides from the blowfly Phormia regina.
Biochem. J. 110, 771-773.

 

BODNARYK R.P. and LEVENBOOK L. (1969) The role of B-alanyl-L-tyrosine
(sarc0phagine) in puparium formation in the fleshfly Sarcophaga
bullata. Comp. Biochem. Physiol. 30, 909-921.

 

DUFFY J.P. (1970) Beta—alanine—l-IAC metabolism in Tenebrio molitor.
Science Studies 26, 35—41.

 

FUKUSHI Y. (1967) Genetic and biochemical studies on amino acid
compositions and color manifestation in puapl sheaths in insects.
Jap. J. Genet. 42’ 11-210

FUKUSHI Y. and SEKI T. (1965) Differences in amino acid compositions
of pupal sheaths between wild and black pupa strains in some
species of insects. Jap. J. Genet. 40, 203-208.

GILBY A.R. and MCKELLAR J.W. (1970) The composition of the empty
puparia of a blowfly. J. Insect Physiol. 16, 1517-1529.

JACOBS M.E. (1966) Deposition of labeled beta-alanine in ebony and
non-ebony Drosophila melanogaster with notes on other amino
acids. Genetics 53, 777-784.

 

JACOBS M.E. (1968a) B-Alanine use by ebony and normal Drosophila
melanogaster with notes on glucose, uracil, d0pa, and dopamine.
Biochem. Genetics 1, 267-275.

 

 

12

13

JACOBS M.E. (1968b) Effect of beta-alanine on glucose and fructose
catabolism in Drosophila melanogaster with notes on beta-

aminoisobutyric and gamma—aminobutyric acids. J. Insect Physiol.
14, 1259-1265.

 

JACOBS M.E. (1970) Effect of noradrenaline, dopamine, and beta-
alanine on phenylalanine and glucose catabolism in Drosophila
melanogaster. J. Insect Physiol. 16, 55—60.

 

 

JACOBS M.E. and BRUBAKER K.K. (1963) Beta—alanine utilization of
ebony and non-ebony Drosgphila melanggaster. Science 129,
1282-1283.

 

KAPLANIS J.N., ROBBINS W.E. d TABOR L.A. (1960) The utilization
and metabolism of 4-C -cholesterol by the adult house fly.
Ann. Ent. Soc. Am. 53, 260-264.

LEVENBOOK L., BODNARYK R.P. and SPANDE T.F. (1969) B-Alanyl-L-tyrosine.
Chemical synthesis, prOperties and occurrence in larvae of the
fleshfly Sarcophaga bullata Parker. Biochem. J. 113,837-841.

 

LEVENBOOK L. and DINAMARCA M.L. (1966) Free amino acids and related
compounds during metamorphosis of the blowfly Phormia regina.
J. Insect Physiol. 12, 1343-1362.

 

LORD K.A. and SOLLY S.R.B. (1964) Effect of insecticides, especially
diazinon, on the amino acids of adult houseflies Musca domestica.
Biochem. Pharmacol. 13, 1341-1349.

 

MONROE R.E. (1962) A method for rearing house fly larvae aseptically
on a synthetic medium. Ann. Ent. Soc. Am. 55, 140.

MONROE R.E. and LAMB N.J. (1968) Effect of commercial proteins on
house fly reproduction. Ann. Ent. Soc. Am. 61, 456-459.

PANT R. and LAL D.M. (1970) Variation of free amino acids in
Sarcophaga ruficornis during metamorphosis. Indian J. Biochem.
7, 57-59.

 

ROACH D. and GEHRKE C.W. (1969) Direct esterification of the protein
amino acids. Gas-liquid chromatography of N-TFA beUtYl esters.
J. Chromatog. 44, 269-278.

SEKI T. (1962) Absence of beta alanine in hydrolyzate of the pupal
sheaths of ebony mutants of Drosophila virilis. DrOSOphila
Information Service 36, 115.

 

SRIVASTAVA R.P. (1971) The amino acid composition of cuticular pro-
teins of different developmental stages of Galleria mellonella.
J. Insect Physiol. 17, 189-195.

 

l4

VERESHTCHAGIN S.M., SYTINSKY I.A. and TYSHCHENKO V.P. (1961) The
effect of y-aminobutyric acid and B—alanine on bioelectrical
activity of nerve ganglia of the pine moth caterpillar
(Dendrolimus pini). J. Insect Physiol. 6, 21-25.

 

PART II: STUDIES ON B-ALANINE SYNTHESIS IN THE

EARLY PUPARIUM

15

INTRODUCTION

In housefly puparia HIJIKURO (1968) found that B—alanine was
synthesized from 14C—aspartic acid and 3H—uracil. A wild type fly
strain utilized both precursors, while a black puparium strain was
unable to incorporate 14C-aspartic acid into B-alanine. NAKAI (1971)
also found that aspartic acid and uracil were precursors of B-alanine
in wild type housefly pupae. He concluded that in the wild type strain
the puparial B-alanine was probably derived primarily from aspartic
acid.

WHITE 25 El“ (1964) stated that B-alanine catabolism could go
through malonic semialdehyde, malonic acid, and acetic acid and that
an alternate pathway of synthesis and utilization would be through
malonic semialdehyde and B-hydroxypropionyl CoA to propionyl CoA.

HAYAISHI g£_§l. (1961) showed that in Pseudomonas fluorescens B-

 

alanine transaminated with pyruvic acid to give malonic semialdehyde
and L-a—alanine. KUPIECKI and COON (1957) showed that B—alanine also
transaminated reversibly with a-ketoglutarate to give malonic semi-
aldehyde, and they also suggested that in plants propionate was a
precursor of B-alanine. It has also been demonstrated that propionate
may serve as a B—alanine precursor in animal tissues (RENDINA and

COON, 1957).

16

17
This study reports the relative contributions of uracil—6-3H,
D-pantothenate-l-lac, malonate-Z-IAC, propionate-Z-IAC, and L-
aspartate-U—l4C in B-alanine synthesis during the housefly white

puparial stage.

MATERIALS AND METHODS

The houseflies used were a maximum longevity strain obtained
from the Insect Physiology Laboratory, USDA, Beltsville, Maryland.
The flies were routinely reared on CSMA medium (ANON., 1959) and the
adults fed a 1:1 mixture of sucrose and nonfat dry milk. For these
studies eggs were collected and sterilized in 0.1% hypochlorite solu-
tion for 20 min and the larvae were reared aseptically according to
MONROE (1962). The wandering larvae were washed out of the diets and
allowed to pupate. Upon pupation 1 pl of a given radiolabelled com—
pound was injected into the posterior portion of the newly formed white
puparium. The uracil—6-3H and D—pantothenic acid-l—14C (sodium salt)
were obtained from New England Nuclear Corp., Boston, Mass. The
sodium malonate-2-14C, L-aspartic acid-U-lAC, and sodium propionate-
2-14C were obtained from Amersham/Searle, Des Plaines, Illinois. The
injected puparia were then aged for 1 hr., weighed, and frozen at -32°C
until further analysis. From 20 to 30 puparia were used in each test
and each compound was analyzed in triplicate. In order to determine
the time needed for maximum incorporation, larvae were also injected
at intervals of several hours prior to pupation.

The puparia were homogenized in water, refluxed for 90 min in

acetone—ethanol (1:1) at 4 times the aqueous volume and vacuum filtered

(KAPLANIS §£_§l,, 1960). The residue was dried, weighed, and combusted

18

19

to 14CO in a combustion flask previously reported by HOPKINS and

2
LOFGREN (1968). The 14CO2 was trapped in 10 ml monoethanolamine-
methyl cellosolve (1:2) and radioassayed with a Nuclear Chicago Unilux 1
(model 6850) liquid scintillation spectrometer.

The lipids were extracted 3 times with ethyl ether, dried over
sodium sulfate, and radioassayed. The aqueous after lipid extraction
was added to a 1.1 x 7 cm Dowex 50-X12 cation exchange column and the
wash from 100 ml water (subsequently referred to as other aqueous) was
evaporated to 5 ml and radioassayed. The amino acids were eluted with
100 ml 10% ammonium hydroxide (v/v); they were evaporated to 5 ml and
radioassayed.

For all samples except those injected with pantothenate-l-IAC
the amino acids were dried and the N‘TFAfEbetYl derivatives made
according to ROACH and GEHRKE (1969). The derivatives were then
analyzed by a gas—liquid chromatograph equipped with a dual hydrogen
flame detector (Research Specialties Corp., Series 600). The separa-
tions were accomplished using a 2m x 4mm ID glass column packed with
Tabsorb (Regis Chemical Co., Chicago, Illinois). The temperature was
programmed from 94 to 210°C at 3.5°C/min. A 10:1 splitter was put on
the column outlet and the amino acid derivatives collected with hexane

in a dry ice-acetone bath. The relative per cents of Bealanine were

then calculated by radioassay.

RESULTS

In all samples where the compounds were injected prior to pupa—
tion, if injection was done more than 1 to 2 hrs before the larvae
pupated, there was a decrease in total activity recovered as well as
a decrease in activity incorporated into B-alanine with increasing
time before pupation. The results presented here, therefore, were
obtained by injecting the compounds as pupation occurred and holding
the puparia for 1 hr prior to analysis. Table 2 summarizes the number
of puparia, their weight, and the injection data for each of the
radiolabelled compounds analyzed.

TABLE 2.--Treatment data of radiolabelled compounds injected into
early puparia of the housefly.

 

 

Radio
chemical Total Total Total
Radiolabelled purity No. weight dpm ug
compound % puparia mg injected injected
Uracil-6-3H 99 67 990.9 3,507,182 0.015
Aspartate-U-14C 98 90 1,491.6 2,913,030 0.800
Pantothenate-l-14C 99 75 1,349.4 1,152,524 27.825
Malonate-2—14C 98 90 1,021.0 1,162,080 4.185
Propionate-2-14C >99 79 1,329.6 1,092,254 3.934

 

20

21

Table 3 presents the data on the recovery of the injected
radioactivity in the residue, lipids, amino acids, and other aqueous
for each of the compounds. These data show that very little radio—
activity was recovered in the lipids of all the samples. Uracil-6-
3H incorporated most of its label, 32.1% and 25.6%, into the other
aqueous and amino acid fractions, respectively, while 6.1% was re-
covered in the residue. With aspartate-U-IAC, 20.4% was found as
amino acids, 13.9% as other aqueous, and 6.1% as residue. After
injection of pantothenate-l-14C the major portion (70.5%) was re-
covered in the other aqueous fraction, while only 4.0% and 0.5%
were recovered in the amino acids and residue, respectively. Malonate-
2-14C had 48.0%, 13.9%, and 3.2% in the other aqueous, amino acids,
and residue, respectively. After injection with propionate-Z-IAC
the amino acids had the most radioactivity (24.6%), followed by the
other aqueous (14.7%) and the residue (5.0%).

Table 4 presents the amount of radioactivity found as B-
alanine after gas-liquid chromatographic separation, subsequent trap-
ping, and radioassay. It was found that relative to the other radio-
labelled compounds examined, uracil accounted for 56.2% of the radio-

labelled B-alanine and asparate for 24.2%. Pantothenate, propionate,

and malonate accounted for 9.4%, 7.1%, and 3.1%, respectively.

22

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muo>ououun.n mqn<a

23

TABLE 4.--Incorporation of radioactivity into B—alanine after injection
into early puparia of the housefly.

 

Radioactivity in B-alanine

 

 

Radiolabelled % of Relative
compound ug-equivalents injected %**
Uracil-6-3H 0.003 t 0.000 23.9 1 2.3 56.2
Aspartate-U-lAC 0.083 1 0.010 10.3 i 1.3 24.2
Pantothenate-l-14C* 1.124 t 0.309 4.0 i 1.1 9.4
Malonate-2-14C 0.054 t 0.021 1.3 i 0.5 3.1
Propionate-2-14C 0.118 1 0.023 3.0 i 0.6 7.1

 

*Based on all of amino acid

radioactivity as B-alanine.

**Based on the total radioactivity contributed by all compounds

tested.

DISCUSSION

ROSS and MONROE (1972) reported that B-alanine concentration
reached its peak in the l-hr-old white puparium stage. They proposed
that B-alanine was synthesized primarily after the start of pupation.
These radiolabelled experiments would support the conclusion that
B-alanine synthesis occurred primarily upon pupation on at most 1 to
2 hrs prior to its onset.

This study indicated that for thisstrain of houseflies most of
the B-alanine was synthesized from uracil and aspartate with these two
compounds accounting for over 80% of the radioactivity recovered in
the B-alanine fraction. This per cent probably represented a minimal
figure as it was expected that uracil and aspartate were diluted more
than the remaining three compounds tested upon their admixing with the
biological pool. These results are similar to those found by NAKAI
(1971) with his wild housefly strain. He found that uracil incorporated
about 50% of the injected radioactivity into B-alanine while aspartate
accounted for 10% to 20%. HIJIKURO (1968) also indicated that uracil
and aspartate were used in the synthesis of B-alanine in housefly
pupae. Because both uracil and aspartate incorporated some radio-
activity into the residue, it was possible that a small amount of the
synthesized B-alanine had been utilized in cuticle formation in the

early pupal stage within 1 hr after the onset of pupation.

24

25

These experiments showed that pantothenate contributed nearly
10% to B-alanine synthesis; however, in reality its contribution was
probably negligible. Because of the specific activity a relatively
large amount was injected thereby increasing the available panto—
thenic acid in the biological pool by several fold. Simple hydrolysis
of pantothenic acid would yield B-alanine and pantoic acid.

Together, malonate and pr0pionate contributed about 10% of the
B—alanine found. Because the biological pools of these compounds were
theoretically low and higher weights of them were also injected, it
was thought that these two compounds also contributed negligibly to
B-alanine synthesis.

These experiments indicated conclusively that of the five most
probable precursors in B-alanine synthesis by various pathways, that
this strain of the housefly, M, domestica, used only uracil and
aspartate. The experiments were unable to show, however, whether
B-alanine synthesis from aspartate occurred through uracil synthesis

and catabolism or via a direct aspartate decarboxylase system.

REFERENCES

ANONYMOUS (1959) The Peat-Grady method. Official method of the
Chemical Specialties Manufacturers Association for evaluating

liquid household insecticides, latest revision. Soap Sanitary
Chem. Spec. Blue Book and Catalog, 32nd year, 219-220.

HAYAISHI O., NISHIZUKA Y., TATIBANA M., TAKESHITA M., and KUNO S.
(1961) Enzymatic studies on the metabolism of B-alanine. J.
Biol. Chem. 236, 781-790.

HIJIKURO S. (1968) Studies on the biosynthetic pathways of B—
alanine in wild and black puparium strains of the housefly.
Jap. J. Genet. 43, 419.

HOPKINS T. L. and LOFGREN P.A. (1968) Adenine metabolism and urate
storage in the cockroach Leucophaea maderae. J. Insect Physiol.
14, 1803-1814.

 

KAPLANIS J.N., ROBBINS W.E., and TABOR L.A. (1960) The utilization
and metabolism of 4-C14—cholesterol by the adult house fly.
Ann. Ent. Soc. Am. 53, 260-264.

KUPIECKI F.P. and COON M.J. (1957) The enzymatic synthesis of
B—aminoisobutyrate, a produce of valine metabolism, and of

B-alanine, a product of B-hydroxypropionate metabolism. J.
Biol. Chem. 229, 743-754.

MONROE R.E. (1962) A method for rearing house fly larvae aseptically
on a synthetic medium. Ann. Ent. Soc. Am. 55, 140.

NAKAI S. (1971) Genetic and biochemical studies on the B-alanine
metabolism in the housefly, Musca domestica L. Jap. J. Genet.
46, 53-60.

 

RENDINA G. and COON M.J. (1957) Enzymatic hydrolysis of the coenzyme
A thiol esters of B-hydroxypropionic and B-hydroxyisobutyric
acids. J. Biol. Chem. 225, 523-534.

ROACH D. and GEHRKE C.W. (1969) Direct esterification of the protein

amino acids. Gas-liquid chromatography of N—TFA Efbutyl esters.
J. Chromatog. 44, 269-278.

26

27

ROSS R.H. JR. and MONROE R.E. (1972) B-Alanine metabolism in the
housefly, Musca domestica: Concentrations in the larvae,
pupae, and adults. J. Insect Physiol. 18, 791-796.

 

WHITE A., HANDLER P., and SMITH E.L. (1964) Principles of Biochemistry
3rd Ed. McGraw-Hill Book Co., New York, 534—535.

PART III: STUDIES ON B-ALANINE UTILIZATION

IN THE EARLY PUPARIUM

28

I‘llllllll’l'l'li
I lilllil‘li

INTRODUCTION

Injection of labelled B-alanine into rats by PIHL and FRITZSON

(1955) showed that B-alanine-l—IAC was very rapidly oxidized, B-alanine-

2-140 was much more slowly oxidized, and B-alanine-3—14C was inter-

mediate. They suggested that B-alanine was deaminated to formylacetic

acid, then decarboxylated to CO and acetaldehyde which was further

2
oxidized to acetic acid. JACOBS and BRUBAKER (1963) and JACOBS (1966)

also showed that B-alanine labelled at carbons 1 and 2, after injection

into Drosgphila melanogaster pupae, was respired as 14CO They further

2.
showed that B-alanine was incorporated into the pupal sheaths. DUFFY

 

(1968) indicated that Q. melanogaster adults possessed a B-alanine

 

decarboxylase and a B-alanine oxidase.
BODNARYK (1971a) showed by kinetic studies that B-alanine was
probably incorporated as an N-terminal amino acid in the puparia of

Sarcophaga bullata. BODNARYK (1971b) showed that over 50% of the

 

bound B-alanine was incorporated in_S. bullata puparia within 3 hr

after the onset of sclerotization. He also indicated that the amount

of incorporation was directly related to the extent of sclerotization.
R088 and MONROE (1972a, 1972b) showed that B-alanine concentration

increased in the white puparium stage of Musca domestica and after the

 

onset of sclerotization decreased over a 24 hr period. They indicated

that the B-alanine was primarily synthesized from uracil and aspartic

29

30
acid at the time of pupation. The purpose of this study was to
determine where the B-alanine was being utilized during the first 24

hr after pupation.

MATERIALS AND METHODS

The houseflies used were a maximum longevity strain obtained
from the Insect Physiology Laboratory, USDA, Beltsville, Maryland.
Eggs were collected from a stock culture, surface sterilized in 0.1%
hypochlorite solution for 20 min and the larvae reared aseptically
according to MONROE (1962). The wandering larvae were washed out of
the diets and allowed to pupate. Within 15 min after the onset of
pupation 1 ul of the radiolabelled B-alanine was injected into the
posterior end of the white puparium. The B-alanine-l-IAC was obtained
from New England Nuclear Corp., Boston, Mass. The B-alanine—Z-IAC and
B-alanine-3—14C were obtained from International Chemical and Nuclear
Corp., Irvine, California. After injection, samples were analyzed for

14CO2 production in a respiration train modified from that reported by

HOPKINS and LOFGREN (1968). The 14CO2 was collected at various time
intervals in monoethanolamine-methyl cellosolve (1:2) and radioassayed
with a Nuclear Chicago Unilux I (model 6850) liquid scintillation
spectrometer. Other samples were aged for various time intervals,
weighed, and frozen at -32°C until further analysis. Values were
obtained from triplicate samples of 20 puparia each.

The puparia were homogenized in water, refluxed for 90 min in

acetone-ethanol (1:1) at 4 times the aqueous volume and vacuum filtered

(KAPLANIS g; 31., 1960). The residue was dried, weighed, and combusted

31

32

14

to CO and water in a combustion flask previously reported by

2
HOPKINS and LOFGREN (1968). The 14CO2 was trapped in 10 ml mono-
ethanolamine-methyl cellosolve (1:2) and radioassayed.

The lipids were extracted 3 times with ethyl ether, dried over
sodium sulfate, and radioassayed. The aqueous after lipid extraction
was added to a 1.1 x 7 cm Dowex 50—X12 cation exchange column and the
wash from 100 ml water (subsequently referred to as other aqueous)
was evaporated to 5 ml and radioassayed. The amino acids were eluted

with 100 ml 10% ammonium hydroxide (v/v); they were evaporated to

5 ml and radioassayed.

RESULTS

Table 5 shows the injection data for the B-alanine—14C labelled
at each of the carbon atoms. In all tests the same weight was injected
into each puparium. Table 6 presents the average live weights and
total per cent recovery for each test of 20 puparia. The average
weights per puparium ran from 11.8 to 17.9 mg while the average per

cent recoveries ranged from 31.3% to 92.8%.

TABLE 5.--Treatment data of radiolabelled B-alanine injected into
early puparia of the housefly.

 

 

Radio-
chemical No. Total Total
Radiolabelled purity puparia dpm ug
compound % per test injected injected
B-Alanine-l-IAC 99 20 546,680 27.80
B-Alanine-2-14C 99 20 74,180 27.80
B-Alanine—3-14C 98 20 777,880 27.80

 

33

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34

 

 

 

 

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35
14

In Table 7, which shows the average accumulative CO2 production,
the data clearly indicate that during the first 24 hr after pupation
that little 14CO2 was produced from any of the carbon positions of
B-alanine. Carbons labelled in the l and 3 position produced 4 to 5%
while the 2 position produced about 3% of the initial radioactivity
into l4C02.

Table 8 shows that virtually no radioactivity was incorporated
into the total lipid fraction during the first 24 hr after pupation.
In Table 9 the data show that little radioactivity was found in the
other aqueous portion of the puparial extracts. B-Alanine-l—IAC in-
corporated about 1% into this fraction while the 2 and 3 positions
incorporated 2 to 3% and l to 2%, respectively.

Table 10 shows a marked decrease in the radioactivity found in
the amino acids following injection of each of the compounds. In all
cases the levels decreased over the first 3 hr after pupation and
reached fairly constant levels by about the 4th hr. The majority was
lost in the first 2 hr after puparial formation. In Table 11 the data
indicate a corresponding increase in the radioactivity incorporated
into the residue during the first 2 to 3 hr following the onset of
pupation. At 0.5 hr there was less than 8% in the residue and by
3 to 4 hr between 40% and 50% of the injected radioactivity had been

incorporated into the residue. After 4 hr the radiolabelled residue

remained fairly constant for the duration of the experiment.

36

 

 

 

 

 

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0.N H 0.00 00.0 H 00.NH 5.H H 0.00 0.N H 0.00 00.0 H 00.0H 0.N H 0.N0 0.5H H 0.00 00.0 H H0.0H 0.H H 0.05 0
0.0 H 0.N0 50.H H 00.0H 5.N H H.00 0.0 H 5.00 0H.H H 05.0H 0.N H 0.50 0.0 H 0.HN 00.N H 00.0 0.H H N.00 N
H.0 H N.0N 00.0 H 05.0 0.H H 0.00 N.N H 0.NH 00.0 H N0.0 0.0 H N.00 5.0 H 5.0 N0.H H 00.N ~.N H 0.50 H
0.H H 0.5 5N.0 H 0H.N 0.H H 0.00 0.0 H 0.0 0H.0 H 0N.H 0.H H 0.00 0.0 H 0.0 N0.0 H 50.H 5.0 H 5.~5 0.0
0000 0:0H000 05 0000 0:0H000 06 0000 0:0«000 0! u:
0000000H 00:0H0>H000 «00:0H03 0000000H 00:0H0>H=00 «00:0H03 0000000H 0000H0>H000 «00:0H03 aOHHUOnaH
00 N :0: 00 N 10: 00 N -0: 00000
OIHH
00HI0100H00H<10 00HIN100H00H<10 0H|H100H00H<10

 

.AH00000: 0:0 00 nHuoaan hHuoo ounH 00H|00H00H010
mo 00H00000H 00000 «0:0H000 0:0 000H H000H0H000 0:0 00 aoHuuuoahounH 0:0 0:0«93 0:0 00 0000 Hon 00000><II.HH mAn<H

DISCUSSION

These experiments indicated that during puparial formation and
subsequent sclerotization B-alanine was almost exclusively incorporated
into the residue. The data showed that very little radioactivity was
recovered as 14C02, total lipids, and other aqueous. Because the
original radiolabelled compounds had 1 to 2% impurities, the data show
that less than 6% was recovered in the total of these three fractions
in the first 24 hr after pupation. This indicated that the pathways
suggested by PIHL and FRITZSON (1955) in rats and DUFFY (1968) in

g, melanogaster were not being used during puparial formation and

 

sclerotization.

These experiments showed that B-alanine was incorporated directly
into the residue and that this process occurred rapidly during the
initial period of sclerotization. These data showed that fully 50% of
the B-alanine was incorporated within 3 hr after pupation Which agrees
with that found by BODNARYK (1971b) for §, bullata.

ROSS and MONROE (1972a) indicated that B-alanine reached a
maximum concentration in 1 hr old white puparia and that the level had
dr0pped off very rapidly by 6 hr but that it took 12 - 24 hr for it to
level off. These experiments indicated that the maximum rate of incor-
poration occurred from 0.5 - 3 hr after injection of early puparia

within 15 min after the onset of pupation. The data for y} domestica

41

42
tend to support the findings of BODNARYK (1971a, 1971b) that S,
bullata rapidly incorporated B-alanine into the puparial case during
sclerotization and that B-alanine was found as an N—terminal amino
acid in cuticular proteins.
The metabolic fates of B-alanine in the housefly at times other
than during puparial formation and sclerotization await further in-

vestigations.

REFERENCES

BODNARYK R.P. (1971a) N-Terminal B-alanine in the puparium of the
fly Sarcophaga bullata: Evidence from kinetic studies of its
release by partial acid hydrolysis. Insect Biochem. 1, 228-236.

 

BODNARYK R.P. (1971b) Studies on the incorporation of B-alanine into
the puparium of the fly, Sarcophaga bullata. J. Insect Physiol.
17, 1201-1210.

 

DUFFY J.P. (1968) B-Alanine metabolism in Drosophila melanogaster
adults. J. Insect Physiol. 14, 199-204.

 

HOPKINS T.L. and LOFGREN P.A. (1968) Adenine metabolism and urate
storage in the cockroach Leucophaea maderae. J. Insect Physiol.
14, 1803-1814.

 

JACOBS M.E. (1966) Deposition of labeled beta-alanine in ebony and
non-ebony Drosgphila melanogaster with notes on other amino
acids. Genetics 53, 777-784.

 

JACOBS M.E. and BRUBAKER K.K. (1963) Beta-alanine utilization of
ebony and non-ebony Drosophila melanogaster. Science 139,
1282-1283.

 

KAPLANIS J.N., ROBBINS W.E., and TABOR L.A. (1960) The utilization
and metabolism of 4-014—cholesterol by the adult house fly.
Ann. Ent. Soc. Am. 53, 260-264.

MONROE R.E. (1962) A method for rearing house fly larvae aseptically
on a synthetic medium. Ann. Ent. Soc. Am. 55, 140.

PIHL A. and FRITZSON P. (1955) The catabolism of CIA-labelled 8-
alanine in the intact rat. J. Biol. Chem. 215, 345-351.

ROSS R.H. JR. and MONROE R.E. (1972a) B—Alanine metabolism in the
housefly, Musca domestica: Concentrations in the larvae, pupae,
and adults. J. Insect Physiol. 18, 791—796.

 

ROSS R.H. JR. and MONROE R.E. (1972b) B—Alanine metabolism in the
housefly, Musca domestica: Studies on anabolism in the early
puparium. J. Insect Physiol. In press.

 

43

APPENDIX

LITERATURE REVIEW

The metabolism of B-alanine has been studied in a variety of
microorganisms and animals. CAMPBELL (1957) showed that B-alanine

was a product of uracil degradation in Clostridium uracilicum, and

 

Pseudomonas fluorescens was shown to have a B—alanine transaminase
(HAYAISHI £5 31., 1961). Deamination was also shown to be a route
for catabolism in rats (GRAFF and HOBERMAN, 1950). PIHL and FRITZSON
(1955) showed that B-alanine was rapidly decarboxylated in rats and
postulated that the degradation occurred by deamination to formyl—
acetic acid, subsequent decarboxylation to acetaldehyde, which was
oxidized to acetic acid.

Insects also contain B-alanine. SEKI (1962) showed that the
pupal sheaths of certain flies contain B-alanine and this was supported
by the studies of KUME and SEKI (1962), FUKUSHI and SEKI (1965), and

HIJIKURO (1968b). In certain strains of Drosophila melanogaster,

 

Bombyx mori, and Musca domestica B-alanine might have been related to

 

 

puparium color (SEKI gt 21., 1966; HIJIKURO, 1967; FUKUSHI, 1967).

JACOBS (1966) showed that wild type 2, melanogaster pupae incorporated

 

more B—alanine than did the ebony strain. GILBY and MCKELLAR (1970)
found B-alanine in the blowfly puparial sheath and SRIVASTAVA (1971)

also found it in the cuticular proteins of Galleria mellonella pupae.

 

JACOBS and BRUBAKER (1963) demonstrated that ebony Q. melanogaster

 

44

4S
deposited less 14C from labelled B-alanine in pupal sheaths and
decarboxylated and oxidized it much more rapidly than did non-ebony
flies.
The concentrations of B-alanine vary during metamorphosis in
Sarcophaga ruficornis (PANT and LAL, 1970). They found that the B-
alanine concentration decreased upon pupation, and this was also found

to be true in Phormia regina (LEVENBOOK and DINAMARCA, 1966). BODNARYK

 

and LEVENBOOK (1968) found that B-alanyl-L-histidine (carnosine) was
a constituent of g, regina, and LEVENBOOK g£_al, isolated B-alanyl-

L-tyrosine (sarcophagine) from the larvae of Sarcophaga bullata.

 

BODNARYK and LEVENBOOK (1969) demonstrated that B-alanine from the
dipeptide was incorporated into the cuticle after pupation, and

BODNARYK (1970) hypothesized that the sarcophagine was cleaved prior

to the incorporation of B-alanine into the cuticle. BODNARYK (1971a)
demonstrated that the utilization was under hormonal control via a
dipeptidase and DOPA—decarboxylase which were ecdysterone-induced
enzymes. BODNARYK (1971c) showed that fully 50% of the total amount

of bound B-alanine in the empty pupal case had been incorporated

within 3 hr after the onset of pupation. HIJIKURO (1968a) had suggested

that the B-alanine was an N—terminal amino acid in 2. melanogaster

 

pupae, and this was further shown by kinetic studies in_§. bullata
(BODNARYK, 1971b).

Few studies have been done on B-alanine biosynthesis in insects.
JACOBS (1968a) has indicated that uracil was a precursor to B-alanine

in Q, melanogaster, and uracil and aspartic acid were found to be

 

precursors in M, domestica puparia (NAKAI, 1971).

46

B-Alanine is metabolically active in adults also. CURTIS gt
_31. (1959) showed that B-alanine depressed spinal neurones in the
cat. The inhibition of motor activity was also shown in worms and
gypsy moth caterpillars (VERESHTCHAGIN gt al., 1963) and also in the
pine moth caterpillar (VERESHTCHAGIN gt 31., 1961). LORD and SOLLY
(1964) showed that B-alanine was an abundant amino acid in M. domestica
adults. DUFFY (1968) demonstrated the presence of a B—alanine oxidase

and a B-alanine decarboxylase in D, melanogaster adults. B-Alanine

 

inhibited glucose but not glucose-6-phosphate metabolism (JACOBS,
1968a, 1968b) and JACOBS (1970) showed that B-alanine also inhibited
the metabolism of other compounds also. The overall importance of
B—alanine in the metabolic scheme of insect adults remains to be

studied and understood.

REFERENCES

BODNARYK R.P. (1970) Effect of DOPA—decarboxylase inhibition on the
metabolism of B-alanyl-L-tyrosine during puparium formation in
the fleshfly Sarcophaga bullata Parker. Comp. Biochem. Physiol.
35, 221-227.

 

BODNARYK R.P. (1971a) Effect of exogenous molting hormone (ecdysterone)
on B-alanyl-L-tyrosine metabolism in the larva of the fly
Sarcophaga bullata Parker. Gen. Comp. Endocrinol. 16, 363-368.

 

BODNARYK R.P. (1971b) N-Terminal B-alanine in the puparium of the fly
Sarcophaga bullata: Evidence from kinetic studies of its release
by partial acid hydrolysis. Insect Biochem. 1, 228-236.

 

BODNARYK R.P. (1971c) Studies on the incorporation of B-alanine into
the puparium of the fly, Sarcophaga bullata. J. Insect Physiol.
17, 1201—1210.

 

BODNARYK R.P. and LEVENBOOK L. (1968) Naturally occurring low-
molecular-weight peptides from the blowfly Phormia regina.
Biochem. J. 110, 771-773.

 

BODNARYK R.P. and LEVENBOOK L. (1969) The role of B-alanyl-L—
tyrosine (sarcophagine) in puparium formation in the fleshfly
Sarcophaga_bullata. Comp. Biochem. Physiol. 30, 909-921.

 

CAMPBELL L.L., JR. (1957) Reductive degradation of pyrimidines.
II. Mechanism of uracil degradation by Clostridium uracilicum.
J. Bacteriol. 73, 225-229.

 

CURTIS D.R., PHILLIS J.W., and WATKINS J.C. (1959) The depression
of spinal neurones by y-aminofnfbutyric acid and B-alanine.
J. Physiol. 146, 185-203.

DUFFY J.P. (1968) B-Alanine metabolism in Drosophila melanogaster
adults. J. Insect Physiol. 14, 199-204.

 

FUKUSHI Y. (1967) Genetic and biochemical studies on amino acid
compositions and color manifestation in pupal sheaths of insects.
Jap. J. Genet. 42, 11-21.

47

48

FUKUSHI Y. and SEKI T. (1965) Differences in amino acid compositions
of pupal sheaths between wild and black pupa strains in some
species of insects. Jap. J. Genet. 40, 203-208.

GILBY A.R. and MCKELLAR J.W. (1970) The composition of the empty
puparia of a blowfly. J. Insect Physiol. 16, 1517-1529.

GRAFF J. and HOBERMAN H.D. (1950) On the metabolism of B-alanine.
J. Biol. Chem. 186, 369-372.

HAYAISHI 0., NISHIZUKA Y., TATIBANA M., TAKESHITA M., and KUNO S.
(1961) Enzymatic studies on the metabolism of B-alanine. J.
Biol. Chem. 236,781-790.

HIJIKURO S. (1967) Studies on the content of beta-alanine and the
body color of Drosophila. Drosophila Information Service 42,
87.

 

HIJIKURO S. (1968a) 0n the binding state of beta-alanine in the
pupal sheaths of D, melanogaster. DrOSOphila Information
Service 43, 175.

 

HIJIKURO S. (1968b) Studies on the biosynthetic pathways of B-
alanine in wild and black puparium strains of the housefly.
Jap. J. Genet. 43, 419.

JACOBS M.E. (1966) Deposition of labeled beta-alanine in ebony and
non-ebony Drosophila melanogaster with notes on other amino
acids. Genetics 53, 777-784.

 

JACOBS M.E. (1968a) B-Alanine use by ebony and normal Drosophila
melanogaster with notes on glucose, uracil, dopa, and dopamine.
Biochem. Genet. 1, 267-275.

 

 

JACOBS M.E. (1968b) Effect of beta-alanine on glucose and fructose
catabolism in Drosophila melanogaster with notes on beta—amino-

isobutyric and gamma-aminobutyric acids. J. Insect Physiol.
14, 1259-1264.

 

JACOBS M.E. (1970) Effect of noradrenaline, dopamine, and beta-
alanine on phenylalanine and glucose catabolism in Drosophila
melanogaster. J. Insect Physiol. 16, 55-60.

 

 

JACOBS M.E. and BRUBAKER K.K. (1963) Beta-alanine utilization of
ebony and non-ebony Drosophila melanogaster. Science 139,
1282-1283.

 

KUME Y. and SEKI T. (1962) Difference in B-alanine content of pupa
sheath between wild and black pupa strains of B, mori and M.
domestica. Jap. J. Genet. 37, 396.

49

LEVENBOOK L., BODNARYK R.P., and SPANDE T.F. (1969) B-Alanyl-L-
tyrosine. Chemical synthesis, pr0perties and occurrence in

larvae of the fleshfly Sarcophaga bullata Parker. Biochem. J.
113, 837-841.

 

LEVENBOOK L. and DINAMARCA M.L. (1966) Free amino acids and related
compounds during metamorphosis of the blowfly Phormia regina.
J. Insect Physiol. 12, 1343-1362.

 

LORD K.A. and SOLLY S.R.B. (1964) Effect of insecticides, especially
diazinon, on the amino acids of adult houseflies Musca domestica.
Biochem. Pharmacol. 13, 1341-1349.

 

NAKAI S. (1971) Genetic and biochemical studies on the B—alanine
metabolism in the housefly, Musca domestica L. Jap. J. Genet.
46, 53-600

 

PANT R. and LAL D.M. (1970) Variation of free amino acids in
Sarcophaga ruficornis during metamorphosis. Indian J. Biochem.
7, 57-59.

PIHL A. and FRITZSON P. (1955) The catabolism of CIA-labeled B-
alanine in the intact rat. J. Biol. Chem. 215, 345-351.

SEKI T. (1962) Absence of beta-alanine in hydrolyzate of the pupal
sheaths of ebony mutant of D, virilis. Drosophila Information
Service 36, 115.

SEKI T., FUKUSHI Y., and KIKKAWA H. (1966) A close relationship
between the color of puparium and B-alanine in some species of
insects. Drosophila Information Service 41, 144.

SRIVASTAVA R.P. (1971) The amino acid composition of cuticular
proteins of different deve10pmenta1 stages of Galleria mellonella.

 

J. Insect Physiol. 17, 189-196.

VERESHTCHAGIN S.M., SYTINSKY I.A., and TYSHCHENKO V.P. (1961) The
effect of y-aminobutyric acid and B-alanine on bioelectrical
activity of nerve ganglia of the pine moth caterpillar
(Dendrolimus pini). J. Insect Physiol. 6, 21-25.

 

VERESHTCHAGIN S.M., SYTINSKY I.A., and TYSHCHENKO V.P. (1963) The
effect of y—aminobutyric acid and B-alanine on the motor effects

and bioelectrical activity of annelids and arthropods. Biol.
Abstracts 41, 16734.

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