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thesis entitled
STUDIES ON THE BIOSYNTHESIS 0P (H-ll-TETRADECENYL
ACETATE AND (y-ll-TETRADECEN-YL ACETATE IN
PHEROMONE GLANDS 0F PLATYNOTA STULTANA
(LEP I DOPTERA:TORTRI C I DAE

presented by
JONATHAN JEWELL NEAL

has been accepted towards fulfillment
of the requirements for

M.S. degree in ENTOMOLOGY

 

 

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STUDIES ON THE BIOSYNTHESIS 0F (§)-ll-TETRADECENYL
ACETATE AND (§)-ll-TETRADECENYL ACETATE IN

PHEROMONE GLANDS OF PLATYNOTA STULTANA

 

(LEPIDOPTERA:TORTRICIDAE)

BY

Jonathan Jewell Neal

A THESIS

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

MASTER OF SCIENCE

Department of Entomology

1980

ABSTRACT

STUDIES ON THE BIOSYNTHESIS OF (§)-11-TETRADECENYL
ACETATE AND (Z)-ll-TETRADECENYL ACETATE IN
PHEROMONE GLANDS OF PLATYNOTA STULTANA
(LEPIDOPTERA:TORTRICIDAE)

 

BY

Jonathan Jewell Neal

Potential precursors of (§)-ll-tetradecenyl acetate
were injected into hemocaels of fifth instar, pupae, and adult
Platynota stultana, or tOpically applied to the pheromone
glands of adult moths. Glands were analyzed for (§)-ll-

tetradecenyl acetate content and specific activity of 14

C
or for percentage (E) and (E) composition of the 11-
tetradecenyl acetates.

14

l- C-myristic acid applied tOpically to adult moths

was incorporated into (§)-ll-tetradecenyl acetate. Evidence

concerning 1-14C-acetate or 1-14

C-myristyl acetate was incon-
clusive. Application of (§)-ll-tetradecenol or (§)-ll-
tetradecenol to moths did not alter the naturally occurring
ratio (88:12) of (§)-ll-tetradecenyl acetate to (§)-ll-
tetradecenyl acetate.

Myristic acid was discovered to be retained by a 3%

OV—l column and liberated upon subsequent injections.

Jonathan Jewell Neal

Some samples of (§)-11—tetradecenyl acetate from

moths treated with l4C-myristic acid were ozonized and

chromatographed. l4C appeared in the ll-ethoxyundecanal

collected but not in the ll-tetradecenyl acetate fraction,
strong evidence that 14C from myristic acid is incorporated

into (§)-ll-tetradeceny1 acetate.

ACKNOWLEDGMENTS

I would like to thank my major professor, Dr. George
S. Ayers for his support, his contributions to the design of
these experiments, and for sharing with me his philOSOphy of
education.

I am most grateful to Dr. Ring T. Carde for pro-
viding many resources to make this project possible, for his
interest and support for my professional development and his
promotion of good communication between research groups.

I would like to thank Dr. James R. Miller for pro-
viding the gas chromatoqraph used in this research, for his
many ideas, suggestions and willingness to discuss problems,
and for his insistence on replication and search for the
truth.

I thank Dr. Loran L. Bieber for his suggestions and
helpfulness.

I would like to thank Mr. Jeff Scott for assistance
in some of the experimental procedures and Mr. Reginald
Webster for his willingness to negotiate insect trades, his
suggestions, and his friendship.

Ms. Diana Luedeman deserves a special note of thanks

for her work in preparation of this manuscript.

ii

To many others who have aided myself and this pro-

ject I would like to express my appreciation.

iii

TABLE OF CONTENTS

Page
LIST OF TABLES O O O O O O O O O O O O O 0 v
LIST OF FIGURES. . . . . . . . . . . . . . vi
INTRODUCTION. . . . . . . . . . . . . . . 1
MATERIALS AND METHODS. . . . . . . . . . . . 6
Rearing. . . . . . . . . . . . . . 6
Gas Chromatography . . . . . . . . . . . . 6
Radioassays . . . . . . . . . . . . . . 7
Chemicals . . . . . . . . 8
Introduction of Test Chemicals to Moths . . . . 8
Pheromone Extraction, Purification and Quantitation . 9
Ozonolysis of (§)-ll- tetradecenyl Acetate . . . . 10
RESULTS 0 O O O O O O O O O O O O O O 0 ll
Topical Application of (E) and (g) ll-tetradecenol
to Pheromone Glands . . . . . . . . . . . 11
Injection of 4 14C Compounds as a Function of Life
Stage. . . . . . . 13
Injection vs. TOpical Application of 4 14C Compounds
to Moths. . . . l3
Topical Application of 1-14C- -Myristic Acid to l and
2 Day Old Moths . . . . . . . . . . . . 18
DISCUSSION . . . . . . . . . . . . . . . 19
Variability in the Data Collected . . . . . . 19
Gas Chromatographic Analysis of Materials Used in
Labeling Experiments. . . . 20
Ozonolysis of Moth Produced (§)-ll-tetradecenyl
Acetate . . . . . . . . . . . . . . . 23
Summary. . . . . . . . . . . . . . . . 25
REFERENCES . . . . . . . . . . . . . . . 29

iv

Table

LIST OF TABLES

The effect of tOpical application of (E) or
(Z) 11— tetradecen-l- OL on the ratio of (E)
to (Z) isomers of 11- -tetradecenyl acetate . .
The effect of injection timing on the 14C
activity in the (E)-ll-tetradecenyl acetate
fraction from Platynota stultana treated with
four different compounds. . . . . . . .

 

The effect of tOpical application and injection
of four 4C labeled compounds to moths on the
14C activity in the (E)-ll-tetradecenyl acetate
fraction . . . . . . . . . . . . .

The effect of timing of tOpical application of
1-14C myristic acid on the 14C activity
appearing in the (E)-ll-tetradecenyl acetate
fraction . . . . . . . . . . . . .

Contaminating 14C activity in the (E)-ll-
tetradecenyl acetate fraction contributed by
var1ousC1abeling materials. . . . . .

The effects of GC column history on 14C activity
in myristic acid samples collected from a 3%
OV-l GLC column. . . . . . . . . .

The effect of ozonolysis of (E)-ll-tetradecenyl
acetate samples from three groups of l-
myristic acid treated moths on the 14C
activity distribution in those samples

Page

12

14

15

18

22

24

26

LIST OF FIGURES
Figure Page

1. Several hypothetical pathways for the bio-
synthesis of (E)-ll-tetradecenyl acetate . . 5

vi

INTRODUCTION

The importance of pheromones as an integral part of
insect communication systems has been established for many
insect Species. Pheromones are in some cases essential to
species recognition and reproduction and have been impli-
cated as important agents in speciation, caste determination,
tracking and defensive behavior (Birch, 1974). Undoubtedly
as more is learned about these complex systems, novel stra-
tegies will evolve to make use of this knowledge.

Although much attention has been focused on identi-
fication of the chemical components of pheromone systems,
there are still many unanswered questions regarding other
aspects of the chemical communication system. Pheromone bio-
synthesis is one such aspect. An extensive literature search
yields only a few papers concerning this tOpic.

Three basic techniques have been used to obtain
information in previous studies of pheromone biosynthesis.
These include labeling with radioactive isotopes, use of
isomeric precursors to alter the ratio of isomeric pheromone
components produced, and analysis of the contents of the
pheromone-producing tissue. Jones and Berger (1978) showed

14

that C acetate was incorporated into cis-7-dodecenyl

acetate, the pheromone of Trichoplusia 21- Schmidt and

 

l4C oleic acid

Monroe (1976) demonstrated incorporation of
into nonanal and undecanal, pheromone components of Galleria

mellonella. They also obtained small amounts of incorporation
l4 l4

 

with C acetate and C prOpionate. Thompson and Mitlin
(1978) showed incorporation of tritium labeled nerol and
geraniol into 4 monoterpene compounds comprising male boll
weevil pheromone. Kasang, g2 21° (1974) found large amounts
of (E)-2-methyl-7-octadecene in Porthetria dispar pheromone
glands and used tritium labeled material to show its incor—
poration into the pheromone cis-7,8-epoxy-2-methyloctadecane.

14C and 35

Crewe and Ross (1975) used 8 labeling to show
incorporation of the CH3-S-moity from methionine into
dimethyl disulphide and dimethyl trisulphide (the status of
these compounds as pheromones is unproven). Renwick g; 3;.
(1976) showed that the relative amounts of cis and trans

verbenol produced by Ips paraconfusis could be controlled

 

by the relative amounts of (-) and (+) a-pinene in the diet.

Platynota stultana, the omnivorous leaf roller (OLR)

 

has several traits which makes it a good test insect for
studying pheromone biosynthesis. It can be reared on an
artificial diet, has a relatively short period of develop-
ment, is large enough to be easily injected and has a phero-
mone that can be obtained in relatively pure form in quan-
tities large enough for analysis. Pheromone production
appears to be confined to an eversible gland on the dorsal
side of the abdomen. Its pheromone, identified by Hill and

Roelofs (1975), consists of a mixture of (E)- and (E)-ll-

tetradecenyl acetate in a ratio of 88 to 12. No papers con—
cerning pheromone biosynthesis of this species have yet been
published.

As the framework for a working hypothesis, several
closely related possible biosynthetic pathways are prOposed
(Figure 1). This hypothesis assumes that pheromone bio-
synthesis starts with acetate and proceeds through normal
fatty acid biosynthesis to either myristic acid (tetra-
decanoic acid) or ll-tetradecenoic acid. The acid would
undergo subsequent reduction to the corresponding alcohol
and esterification to an acetate. If myristic acid were
involved, the introduction of the double bond could occur at
any of the steps shown.

The double bond is of prime interest because of its
constant cis/trans ratio and because the same acetates ((E)-
and (E)-ll~tetradecenyl acetate) in different ratios are used
as the pheromone in several closely related Species of leaf
rollers (Klun g; gl., 1974; Roelofs 23 gl., 1974; Hill g3 gl.,
1974). It is important that female moths maintain precise
control of this ratio in order to be attractive to male moths
of only the same species (Baker g3 gl., 1975). Insights into
regulation of this aspect of pheromone biosynthesis may shed
light on aspects of species evolution as well as provide
insight into the possibility of developing resistence to
control strategies utilizing pheromones. In addition to
these insights such knowledge might lead to novel pheromone

based control strategies.

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MATERIALS AND METHODS

Rearing

Adult Platynota stultana were allowed to mate in and

 

deposit egg masses on the sides of 8 liter polyethylene bags.
Egg masses were surface-sterilized with .05% sodium hypoch-
lorite solution, rinsed with distilled water, dried, and
allowed to hatch in glass shell vials with cotton plugs.
Approximately 50 first instar larvae were transferred with a
camel hair brush to waxed 16 02. paper cups containing
modified pinto bean diet (Shorey & Hale, 1965) and kept in

a constant environment chamber (photOperiod = 16L:8D, tem-
perature = 24°C, humidity = 65%-85%). After 21 days, pupae
were removed from the diet, sexed, and allowed to emerge in

1 liter polyethylene bags containing paper toweling to absorb
molting fluid. Adults were transferred every 24 hours to 1

liter polyethylene bags containing a wetted cotton wick.

Gas Chromatography
Gas liquid chromatographic (GLC) analysis was done
on a Packard Series 7300 Gas Chromatograph. GLC columns
(glass, 2 m x 4 mm) were packed with either 3% OV-l on

100-120 mesh Gas Chrom Q or 10% GEXF-1150 on 100-120 mesh

AW—DMCS treated Gas Chrom W. Flame ionization detection
was used for quantification. When used for purification,
the entire effluvia from the column was diverted through
glass capillary tubes sandwiched between slabs of dry ice.
Samples were recovered by rinsing the capillary tubes with
93. 0.5 ml C82. Fraction cuts were made in relation to the
retention time of standards run immediately prior to sample

injection.

Radioassays

Quantitation of 14C was done on a Searle Isocap/300
Liquid Scintillation System with high background reject, or
a (refrigerated) Packard Tricarb. Samples were placed in
glass scintillation vials containing 15 m1 of a fluor mixture
(0.67 l toluene, 0.33 1 triton X100, 4 g PPO, 0.5 g dimethyl
POPOP) . I

Background counts were determined by counting blank
vials prepared identically to those containing samples (blank
vials contained 15 ml of cocktail plus amount of solvent used
for dissolving sample). Channels ratio in conjunction with
14C quench series was used for converting counts to dis-
integrations and was checked against an external standard.
Both methods gave good agreement. Samples and blanks were

counted for consecutive 10 min intervals until the level of

significance reached 90% (Seelye, 1975).

Chemicals

 

(E)-11-tetradeceny1 acetate, (E)-1l-tetradecenyl

acetate, (E)-ll-tetradecen-l-ol and (E)-11-tetradecen-1-ol

14

were purchased from Farchan Chemicals Inc., 1- C acetic acid

and 1-14C- stearic acid from ICN Pharmaceuticals Inc. and

14 14

1- C tetradecanoic acid and 1- C tetradecanol from DHOM

14

Products Inc. 1- C tetradecyl acetate was synthesized by

addition of excess AR grade acetyl chloride (Mallinckrodt

14C tetradecanol. The reaction was

Chemical Inc.) to 1-
monitored at 10 minute intervals by GLC (3% OV-l at 160°)
until complete and purified by collection from the same
system. Purity was confirmed by TLC (Silica gel G/Benzene:
ether-9:1) and by GLC (3% OV-l at 160° and 10% GEXF-llSO at
150°C). All solvents used were distilled prior to use.

Introduction of Test Chemicals
to Moths

 

All chemicals were administered by means of injection
or topical application using an ISCO model M microapplicator.
Needles were made by pulling 2 mm glass tubing and inserting
the blunt end into a half hole rubber septum. The septum
assembly was fitted onto a calibrated 25 ul fixed needle
syringe with the needle shortened to 1 cm. Injections were
made intersegmentally into the abdominal cavity. Topical
applications were made by pressing the abdomen to evert the
pheromone gland and placing the tip of the needle directly

on the gland.

Pheromone Extraction, Purification
and Quantitation

 

Pheromone glands were dissected by pressing the
abdomen to evert the gland, and excising the everted tissue
with micro-dissecting scissors. Glands were immediately
placed in 1 dram vials containing 0.5 m1 methylene chloride
and extracted for 24 hr at -18°C. Extracts were then filtered
through glass wool and reduced to gg. 5 ul for GLC injection.
Samples were collected from the 3% OV-l column which was used
to separate 14 carbon alcohols from their corresponding
acetates. The fraction from :1 minute of the center of the
(E)-11-tetradecenyl acetate peak was reduced to approximately
5 ul and injected onto the 10% GEXF-llSO column. Collections
from -1.5 to -0.5 min (tetradecyl acetate peak) and -0.5 to
+0.5 min of the center of the (E)-11-tetradeceny1 acetate
peak were made.

The fraction containing (E)-ll-tetradeceny1 acetate
was diluted to 5m1 with C82. Palmitoleic acid methyl ester
was added as an internal standard. Two 0.5 m1 aliquots were
placed in scintillation vials for 14C analysis and two 0.5
m1 aliquots were reduced to gg. 5 ul and analyzed for (E)-
ll-tetradecenyl acetate on a 10% GEXF-llSO column. The
fraction from -l.5 min to -0.5 from the center of the (E)-
11-tetradeceny1 acetate peak was submitted to liquid scin-

l4

tillation for C analysis.

10

Ozonolysis of (E)—11-tetradecenyl acetate

Samples containing (E)-ll—tetradecenyl acetate in
CS2 were placed in a dry ice-acetone bath. Ozone was bubbled
through the mixture until a distinct blue color appeared,
indicating saturation. Nitrogen was then bubbled through
the mixture to drive off excess oxygen and 93. 2 mg tri-
phenylphosphine was added and allowed to react for 30 min.
The solvent was then reduced to gg. 5 ul for GLC. The 11-
ethoxy-undecanal was purified by collection from a 3% OV-l
column and analyzed for 14C content. The propanal produced
during ozonolysis was lost during the solvent reduction

step.

RESULTS
Topical Application of (E) and (Z) 11-
tetradecenol to Pheromone Glands

Table 1 shows the effects of tepical application of
0.05 ul of both (E)- or (E)-11-tetradecenol on ll-tetradecenyl
acetate production in adult female OLR's. Listed in the
table are the date the replicate was treated, the (E)- and
(E)-percentages of the moth produced 11-tetradeceny1 acetate
and the amount of (E)-ll-tetradeceny1 acetate per tip.

The data show that with the exception of the 3/28/78
experiment, (E)-11-tetradecenol treated moths had ll-tetra-
decenyl acetate compositions very close to the 11% (E) isomer
- 89% (E) isomer blend found in the control moths. The
replicate from 3/28/78 is thought to have been accidently
contaminated during analysis, but was included for complete-
ness. The other three samples are all within the limit of
experimental variation expected.

Moths treated with (E)-1l-tetradecenol, with the
exception of the 2/23/78 replicate, have ll-tetradecenyl
acetate composition close to that of the control moths. The
2/23/78 experiment is again thought to have been accidently
contaminated during analysis but was included for complete—

ness.

ll

12

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Injection of 4 14C Compounds as a
Function of Life Stage

 

l4c labeled

Female OLR's were injected with four
compounds at different life stages and at different times
within those life stages as shown in Table 2. Listed are
the date the replicate was injected, the amount of moth pro-
duced (E)-1l-tetradecenyl acetate per aliquot tested, the
disintegrations per minute in that aliquot and the ratio of

the moles of 14

C to the moles of (E)-1l-tetradeceny1 acetate.
Groups of 50 females were used; each moth received at least
50,000 dpm.

The data show that injection of the long chain com-
pounds (myristic acid, myristanol, and myristyl acetate)

into female OLR's did not result in 14

C incorporation into
(§)-11-tetradecenyl acetate. Injected 14C acetate, however,
resulted in activity in the (E)-11-tetradecenyl acetate
fraction. These activities showed sizable variation but in
general were about the same for all study periods. Experi-
ments done in 1977 show activity, while those done after 1977

do not. There were no differences between procedures used

before and after the end of 1977.

Injectiopyvs. T0pical Application of
4 I"C Compounds to Moths

14

Table 3 compares the C activity in the (E)-ll-

tetradecenyl acetate peak resulting from both injection and

14

tepical application of four C labeled compounds. The table

shows the date the label was applied, the amount of (E)-11-

214

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tetradecenyl acetate per aliquot, the disintgegration rate

14C to the moles

per aliquot, and the ratio of the moles of
of (E)-ll-tetradecenyl acetate for each of 4 compounds
applied. Groups of 50 females were used and each moth

received at least 50,000 dpm.

The data show that the method of application for

14 14

C acetate had little effect on the amount of C in the
(E)-11-tetradecenyl acetate fraction. Both methods produced
only low levels of activity with much variation.

Moths treated topically with 14

C myristic acid

showed much higher activity in the (E)-ll-tetradecenyl acetate
fraction than moths treated by injection. This treatment
produced the greatest activity and highest incorporation
ratios of any combination of possible precursor and appli-
cation method tested. The results show much variation and
there is no linear relationship between quantity of (E)-1l-

14

tetradecenyl acetate and C activity.

There is little evidence to suggest that myristanol

is incorporated into (E)—ll-tetradecenyl acetate. l4C

activity in the pheromone fraction occurred only once where
moths were treated by topical application, and not at all
where treatment was by injection.

Treatment of female moths with 14

14

C-myristyl acetate
by injection produced no C activity in the tetradecenyl
acetate fraction and gave variable activity when applied

tepically. On the 10% GEXF-llSO column tetradecyl acetate

elutes l min prior to the (E)-ll-tetradecenyl acetate peak

17

under the conditions of these experiments. Because of the

14

possibility of C myristyl acetate contamination, the

fraction 1 min prior to the (E)-ll-tetradecenyl acetate

14C content. In no

fraction was trapped and analyzed for
case did this fraction produce activity above background,
indicating that tetradecyl acetate was not interfering with
analysis.

Topical Application of 1-14C-Myristic Acid

to l and 2 Day Old Moths

Table 4 compares the activity appearing in the
(§)-ll-tetradecenyl acetate fractions from moths treated
as 0-24 hr old adults vs. those treated as 24-48 hr old
adults. Fifty thousand dpm myristic acid was used as the

14C label and glands were dissected 24 hr after treatment.

The data show no difference between the 2 treatments.

18

Table 4.--Effect of timing of togical application of 1-14C-

myristic acid on the 1 C activity appearing in the
(E)-1l-tetradeceny1 acetate fraction.a

 

 

 

 

0-24 Hour Adults 24-48 Hour Adults

Date b

ng/sample dpm ratioC ng/sample dpm ratio
1/24/77 12 109 .0112
1/20/78 21 32 .0018
2/25/78 69 17 .0005
5/7/78 70 39 .0012
9/22/78 86 250 .0055 135 97 .0014
9/22/78 148 37 .0005 333 61 .0003
1/1/79 84 50 .0012
6/12/79 7 Bd -
8/24/79 54 Ed -

 

a(E)-ll-tetradecenyl acetate was collected first
from a 3% OV-l GLC column, then from 10% GEXF-1150.

bng/sample of (E)-11-tetradeceny1 acetate.

CRatio of the moles of 14

tetradecenyl acetate.

C to the moles of (E)-1l-

dB = Background.

DISCUSSION

VariabilityEof the Data Collected
14

 

The results from C tracer studies are quite vari-

able. Isolated results taken one at a time both support and

refute 14

C incorporation and the results from the experiments
described previously must be viewed as a whole if reasonable
conclusions are to be derived.

The large variations in the data, particularly those

14

of the ratio of C activity to the quantity of (E)-11-

tetradecenyl acetate argue against incorporation. A large
amount of 14C in one replicate and no measurable 14C in
another without diminished (E)-11-tetradecenyl acetate con-
tent is suggestive of some underlying difference in tech-
nique or unidentified methodological artifact. Great care,
however, was taken to insure that experimental conditions
were consistent. Method and amount of injection, timing of
the injections, timing of the dissections, and methods of
analysis were unchanged throughout. Rearing of insects went
unchanged and age of the insects in each group was care-
fully determined. Compounds tested were kept cold and in

the dark when not in use. Standards were always gas chroma-

tographed prior to submission of moth extracts. GC columns

19

20

were evaluated for performance, and when below standard,
were repacked with packing from the same batch to enhance
consistency. GC traces were free of significant visable
contamination. The main scintillation counter used was
equipped with high background reject such that any sample
containing high energy counts greater than 1% of the total

counts in the 14

C channel would be rejected. When a second
instrument was used, a portion counted was often counted

as well in the primary instrument. In all cases the two
instruments agreed well.

Gas Chromatoqraphic Analysis of Materials
Used in Labeling Experiments

The 4 compounds used in the 14

C labeling experiments
were chromatographed to check for contaminants and the
possibility of starting material ending up in the final
sample.

Using column conditions identical to those used for
isolating (E)-11-tetradecenyl acetate, acetic acid, myristic
acid, myristanol and myristyl acetate were injected onto a
3% OV-l column. No peaks which could be construed as con-
taminants were observed. Myristyl acetate appears to be the
only compound capable of interfering with future analysis.
On a 10% GEXF-llSO column, myristyl acetate and (E)-ll-
tetradecenyl acetate have retention times about 1 min apart
(not quite baseline separation). Because myristyl acetate

is also produced by the pheromone gland the fraction 1 min

prior to the (E)-11-tetradecenyl acetate peak was collected

21

14 14

and analyzed for C in all studies. In no case was C
activity observed in this fraction. This points against the
possibility of contaminants from a tailing myristyl acetate
peak.

l4C myristyl acetate

It is interesting to note that
is not present in moths treated with 14C myristyl acetate
only 24 hr prior to extraction. This suggests that myristyl
acetate is either degraded very rapidly, transported else-
where within the moth or released from the pheromone gland
during that interval.

Other experiments in this series give a different

picture. In these studies the 14

C labeled myristanol,
myristic acid, myristyl acetate and acetate used in labeling
experiments were injected onto GC columns and trapped to

see if 14

C contaminants in them could interfere with analy-
sis. These were treated identically to moth samples; they
were first trapped from 3% OV-l and subsequently trapped

from 10% GEXF-llSO. Approximately luCi, the amount that
would have been applied to samples of 50 moths, was used.
Results are shown in Table 5. The data indicate that clearly
it is possible for contaminants to appear in the fraction
which would contain (E)-ll-tetradecenyl acetate. The results
are quite variable and further experimentation indicated

that this resulted from the past history of the column rather
than from compounds with long retention times. For example,

the myristic acid from 12/16/79 was trapped from a differ-

ent GEXF-llSO column than 12/18/79 and 12/20/79 samples

22

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23

with different results. Another demonstration of the effects

of column history is seen in Table 6. Injection of 14C

myristic acid onto 3% OV-l gave 1029 counts in the (E)-ll-
tetradecenyl acetate region. The following shot of un-
labeled myristic acid had 10,826 counts in the (E)-ll-
tetradecenyl acetate region. Apparently, introduction of

14

cold material can release C labeled material that has hung

up on the column. From this information alone, one could

conclude that contamination is responsible for all the

14

measured C activity. This would also account for the large

amount of variation as well as the nonlinearity between the
amount of tetradecenyl acetate recovered and the measured

14C content.

Ozonolysis of Moth Produced (E)-ll-
tetradecenyl Acetate

 

In order to gain more information, samples from

14C myristic acid treated moths showing relatively high

14G activity and still having an adequate amount of sample
left were subjected to ozonolysis. These samples were from
fractions that originally were collected from 3% OV-l and
10% GEXF-1150 columns and correspond to the (E)-1l-tetra-
decenyl acetate areas. The GC traces of the samples on the
GEXF-llSO column were free of visual contaminants with a
detection limit of somewhat less than 1 ng. In no case did
the fraction 1 min prior to the (E)-ll-tetradecenyl acetate

14

fraction show any potentially contaminating C activity.

24

Table 6.--The effects of GC column history on 14C activity in myristic acid
samples collected from a 3% OV-l GLC column.

 

 

 

 

12/18/79 12/20/79
Sample Markers
AIdpm)‘ B(dpm)b AIdme° B(dpm) a C(dpmle
Retention
Time (Min)
0-4 20 565 4 49 38
4-7 (E)-ll-tetradecenol 33 125 Bf 30 1
7-9 39 370 5 27 64
9-12 (E)-ll-tetradeceny1 1253 23546 1004 10801 16
Acetate

283 12
12-15 1173 6717 116

41 29
15-30 2855 9136 359
30-45 1352 3884 166
45-60 999 1194 156
60-75 105
75-90 129

 

a12/18/79 A. luCi of 1-1‘c myristic acid was injected into the column
at time - 0, after column was unused for several days.

b12/18/79 B. luCi of 1-14C myristic acid was injected onto the column
at time = O, 60 min after the 12/18/79 A injection.

c12/20/79 A. luCi of 14C myristic acid was injected onto the column
at time = 0, after column went unused for 2 da.

d12/20/79 8. long of unlabeled myristic acid were injected onto the
column at time = 0, 90 min after the 12/20/79 A
injection.

e12/20/79 C. luCi of 14C myristic acid was injected onto the column
at time = 0, 30 min after 12/20.79 B was injected.

fB = Background.

25

Half of each sample was counted directly, the other half

that underwent ozonolysis, was collected from 3% OV-l and

analyzed for 14C content. Table 7 gives the experiment from

which the sample came, the amount of 14

14

C in the half sample

counted directly, the C found in each fraction collected,

the markers used and their corresponding retention times.

The data are not complete due to high energy background

present in some samples. In the 1/3/79 experiment, the 14C

activity appears to be Spread throughout all the fractions.

14

In the 9/22/78 ozonized samples, the C activity

has been shifted from the fraction containing (E)-11-
tetradecenyl acetate to the fraction containing the 13-

carbon ozonolysis fragment. This is reasonable proof that

14

that C activity in those samples is in fact incorporated

into (E)-ll-tetradecenyl acetate. Unfortunately, it was

not possible to analyze all samples in this manner.

Summary

In view of the evidence presented, it can be con-

14

cluded that C labeled myristic acid, at least in some

instances, is incorporated into (E)-1l-tetradecenyl acetate.
It is not known whether the myristic acid is incorporated
directly, or if first is broken down--perhaps to acetate--
and if so whether the label appears throughout the chain or
in the acetate portion of the molecule. The fact that

samples from 1-14C-acetate labeled moths had lower ratios

14

of C to (E)-1l-tetradecenyl acetate weights against

26

Table 7.--The effect of ozonolysis of (E)-1l-tetradecenyl acetate samples
from three groups of 1-14C myristic acid treated moths on the
14C activity distribution in those samples.a

 

1/1/79 Studya

 

Retention
Time (Min)b Markers dpm
0-5.2 Propanal 31
5.2-8.0 13 Carbon Ozonolysis Fragment 22
8.0-12.0 (E)-11-tetradeceny1 acetate 36
Directly Counted Half 90

 

 

0

9/22/78 Study

 

 

dpm
Retention
Time (Min)b Markers 24-481 24-4811
d

0-2 Propanal B 4
2-4.5 3 3
4.5-6.7 13 Carbon Ozonolysis fragment 36 15
6.7-7.2 5 l
7.2-9.0 (E)-ll-tetradecenyl acetate 4 HBe
9.0-12.o use 0
12.0-15.0 7 HBe

Directly Counted Half 53 28

 

aThis is the same sample listed in Table 3 under myristic acid
for 1/1/79.

bRetention time on a 3% OV-l column (150°) from which ozonized
samples were collected.

cThese are the same samples listed in Table 4 under 24-48 hour
adults.

dB = Background.

eHB = Rejected because of high background.

27

incorporation through breakdown to acetate. Also, l-l4C-

stearic acid which would be expected to behave similarly to
myristic acid in any breakdown to acetate, failed to produce
any l4C labeled (E)-1l-tetradecenyl acetate. (These ex-
periments were done on 1/20/78 and 2/25/78 but not listed

in Table 3.)

Evidence that 14

C acetate or myristyl acetate is
incorporated into (E)-ll-tetradecenyl acetate is inconclu-
sive. It is doubtful that myristanol is incorporated into

(E)-ll-tetradeceny1 acetate because of the low 14C content

14

measured in samples from C myristanol treated moths. For

the long chain compounds, tOpical application gives much

higher 14

C counts in the final sample than injection and may
therefore be a better technique than injection in certain
instances.

Relative to tetradecenyl acetate biosynthesis in
Platynota stultana, myristic acid incorporation shows that
the lipid pool is involved, but no particular pathway can
be pinpointed. The large experimental variation may mean
that myristic acid is not a direct precusor of (E)-ll-
tetradecenyl acetate. However, if myristic acid is incor-
porated directly, the lack of incorporation of myristanol
would indicate that the next step in the pathway is not
reduction to myristanol. This of course assumes myristanol
penetrated the pheromone gland and was available for subse-

quent biosynthesis. The lack of effect of (E) or (E) 11-

tetradecenol on the percentage composition of (E) and (E)-

28

ll-tetradecenyl acetates would imply that regulation of the
pheromone blend would occur after alcohol formation or that
the alcohol itself is not directly in the line of synthesis.
This again assumes alcohol penetration into the gland. Large
amounts of ll-tetradecenol can still be found in extracts
of the gland 24 hours after treatment with (E) or (E) 11-
tetradecenol. Hill and Roelofs have found that 14 carbon
alcohols found in the pheromone gland have the same isomeric
ratio as that of acetates. Since the alcohol is found in
higher quantities in the glands of older moths, it is
possible that the alcohol results from the breakdown of the
corresponding acetate. If this is the case, then the bio-
synthetic pathway for (E)-ll-tetradecenyl acetate is more
complicated than the scheme originally hypothesized.

It is evident from this study that pheromone bio-
synthesis occurs in adult moths. Production occurs at least
on both the first and second days after eclosion and possibly

throughout the life of the moth.

REFERENCES

REFERENCES

AliNiazee & Stafford. "Evidence of a Sex Pheromone in the
Omnivorous Leafroller." Ann. Ent. Soc. AM. Vol. 64,
p. 1330, 1971.

E. L. Atkins Jr., 23 21° "The 'Omnivorous Leafroller,‘
Platynota stultana Weshm., on Cotton in California:
Nomenclature, Life History and Bionomics (LepidOp-
tera:Tortricidae)." Ann. Ent. Soc. Am. Vol. 50,
p. 251, 1957.

 

Baker, John E; El. "Sex Pheromone Field Trapping of the
Omnivorous Leafroller, Platynota stultana."
Environmental Ent. Vol. 4, p. 10, 1975.

 

Birch, Martin. Pheromones. North Holland Publishing Co.,
1974.

 

Crewe, R. M. & F. P. Ross. "Pheromone Biosynthesis: The
Formation of Sulphides by the Ant Paltothyreus
Tarsatus." Insect Biochem. Vol. 5, p. 839, 1975.

 

Hill, A.; Carde, R.; Comeau, A.; Bode, W.; & Roelofs, W.
"Sex Pheromones of the Tufted Apple Bud moth.
Platnota ideausalis." Environ. Entomol 3:249-252,
1974.

 

Hill, A. & W. Roelofs. "Sex Pheromone Compounds of the
Omnivorous Leafroller Moth, Platynota stultana."
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Hughes, Patrick R. "Myrcene: A Precursor of Pheromones in
Ips Bettles," J. Insect Physiol. Vol. 20, p. 1271,
1974.

Jones, I. F. & R. Berger. "Incorporation of (1-14C) Acetate
into cis-7-Dodecen-l-ol Acetate, a Sex Pheromone in
the Cabbage Looper (Trichoplusia Ei)-" Environ.
Entomol. 7:666-669, 1978.

 

29

30

Kasang, G.; D. Schneider; & M. Beroza. "Biosynthesis of the
Sex Pheromone Disparlure by Olefin-Epoxide Conner-
sion." Naturwissenschaften. Vol. 61, p. 130, 1974.

Klun, J. A.; O. L. Chapman; K. C. Mattes; R. W. Wojtkowski;
M. Beroza; & P. E. Sonnet. "Insect Sex Pheromones:
Minor Amount of Opposite Geometrical Isomer Critical
to Attraction." Science 181:661-663, 1973.

Miller, J. R. & W. Roelofs. "Sex Pheromone Development
Correlated with Pheromone Gland Development and Age
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velgtinana." Ann. Ent. Soc. AM. Vol. 70, p. 136,
1977.

 

Mitlin, Norman & P. A. Hedin. "Biosynthesis of Glandlure,
the Pheromone of the Boll Weevil, Antggnomus Grandis,
from Acetate, Mevalenate, and Glucose." J. Insect
Physiol. Vol. 20, p. 1825, 1974.

 

Renwick, J. A. A.; P. R. Hughes; & I. S. Krull. "Selective
Production of cis- and trans- Verbenol from (-) and
(+)-a-Pinene by a Bark Beetle." Science. Vol. 191,
p. 199, 1967.

Roelofs, W.; A. Hill; R. Carde; J. Tette; H. Madsen; & J.
Vakenti. "Sex Pheromones of the Fruitree Leafroller

Moth, Archips argyrospilus." Environ. Entomol. 3:
747-751, 1974.

Schmidt, Steven P. & R. E. Monroe. "Biosynthesis of the
Waxmoth Sex Attractants." Insect Biochem. Vol. 6,
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Seelye, James G. "Counting Times for Low Level Radioactive
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