(Z)-11-Octadecenyl acetate in Drosophila funebris : formation, transfer, catabolism and aggregation
activity
by Russell Dean Leu
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in
Biochemistry
Montana State University
© Copyright by Russell Dean Leu (1988)
Abstract:
(Z)-11-Octadecenyl acetate (Z11 - 18 : Ac) was identified as the most abundant (2300 ng/fly) hexane
extractable component of the ejaculatory bulb of sexually mature virgin male D. funebris. Virgin
female flies did not have Z11 - 18 : Ac at any age. There was a rapid increase in Z11 - 18 : Ac in virgin
male flies during the first three days after eclosion. In mature male flies, about, 1500 ng of Z11 - 18 :
Ac was in the ejaculatory bulb and about 800 ng was on the surface of the fly. During mating about
1200 ng of Z11 -18 : Ac was transferred to the female fly, but was not transferred into the reproductive
tract. The female fly then looses approximately 60 ng of the Z1 1-18:Ac to the media. This amount is
independent of the time spent in the holding vial. The amount of Z11 - 18:Ac on mated females
decreased to undetectable levels at 12 hours post-mating. Concurrent with the Z11-18:Ac decrease
there is an increase in the concentration of 14, 16 and 18 carbon fatty acids. The female is able to
metabolize external Iy applied Z11 -18:Ac, vaccenol , vaccenic acid, oleyl acetate and stearyl acetate.
Sexually immature (6-8 hours old) virgin females can metabolize applied Z11-18:Ac. A combination of
Z11 -18:Ac and volatile, polar components from the male hexane extract comprise the aggregation
pheromone in Eu. funebris. (Z)-II-OCTADECENYL ACETATE IN DROSOPHILA EUNEBRIS:
FORMATION, TRANSFER , CATABOLISM AND
AGGREGATION ACTIVITY
by
Russell Dean Leu
A thesis submitted in partial fulfillment
of the requirements for the degree
of
Master of Science
in
Biochemistry
MONTANA STATE UNIVERSITY
Bozeman, Montana
March 1988
■
;
•
/V3-72
LC7
ii
APPROVAL
of a thesis submitted by-
Russell Dean Leu
This
thesis has been read by each member of the thesis
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and has been found to be
satisfactory
regarding
c o n t e n t , English u s a g e , f o r m a t , citations,
bibliographic
s t y l e , and consistency,
and is ready for submission to the
College of Graduate studies.
Date
^
lraduate
Committee
Approved for the Chemistry Department
Approved for the College of Graduate Studies
7 7-Ff
"
Date
Graduate Dean
iii
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from this
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thesis
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Brief quotations
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Signature
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iv
TABLE OF CONTENTS Page
LIST OF TABLES ..........'..................................
LIST OF FIGURES
...........................................
v
vi
ABSTRACT ................................................... .
INTRODUCTION
Objectives
.................................
. ................................................
%
MATERIALS AND METHODS ............................
Ejaculatory Bulb Removal and Analysis .................
Extraction of Whole Flies ................................
Formation of the Ejaculatory Bulb Compound with Age ...
Transfer During Mating .......................
Female Reproductive Tract Removal ..................
Ejaculatory Bulb Compound Loss by the Female ..........
Application of Compounds to Virgin Females ............
Collection of Volatiles from Hexane Extracts ..........
Bioassays ......................................
RESULTS AND DISCUSSION
....................................
Identification ...........................................
Formation .................................................
Transfer During Mating ..................................
Catabolism .............................................
Application of Zl1-18:Ac ...................
Application of Vaccenic Acid ...............
Application of Vaccenol ................
Application of Similar Acetates ...........
Application of Zl1-18:Ac to
D . melanogaster . . . ...............................
Bioassay Response .............
CONCLUSIONS
................................................
REFERENCES CITED
........................
5
6
7
7
8
8
9
11
^
^
13
17
1
22
24
25
25
28
34
V
LIST OF TABLES
. .
Table
1.
2.
3.
4.
5.
Page
Drosophila species with identified
aggregation pheromones ......
2
A comparison of the amounts of Zl I-18:Ac
sequentially extracted from D^ funebris and
D . melanogaster mated females ........................
15
Recovery of Zl1-18:Ac from mated and virgin
D . funebris ....... *...... ..... ......................
17
Aggregation response of several extracts relative
to the mature male JX funebris hexane extract ......
30*
5
Response of JX_ funebris to the male hexane extract
and two components of the extract ........... .
32
vi
LIST OF FIGURES
Figure
'I.
2.
3.
Page
(Z)-II-Octadecenyl acetate (Z 11- 18 :Ac) in male
D . funebr i s with age ..................................
12
Seven day old
funebris matings with the
subsequent removal of the ejaculatory bulb,
reproductive tract and hexane soak of the fly ......
14
Loss of (Z)-I I-Octadecenyl acetate (Z11-18:Ac) from
mated D . funebris females with time .................
18
4.
Application of I I00 ng of (Z )-11-OctadecenyI acetate
(Z 11- 18 :Ac) to 7 day old virgin female
funebris .. 20
5.
Application of 2500 ng of vaccenic acid and 1000
ng of vaccenol to mature D^_ funebris females ...... . .23
6.
Application of I 100 ng of (Z)-II-Octadecenyl acetate
(Z11-18:Ac), 1000 ng of oleyl and stearyl acetate to
7 day old virgin female JX funebris .................
267
Application of 500 ng of (Z)-II-Octadecenyl acetate
(Z11- 18:Acj to 4-5 day old virgin female
D. melanogaster ....... ...............................
27
7.
v ii
ABSTRACT
(Z)-II-Octadecenyl acetate (ZI I- I8:Ac)
was identified
as the most abundant (2300 ng/fly) hexane ext ractable
component of the ejaculatory bulb of sexually mature virgin
male
f u n.ebj”i ^ . Virgin female flies did not have Zll18:Ac at any age.
There was a rapid increase in Z I I- I8 :A c
in virgin male flies during the first three days after
eclosion.
In mature male flies,, about, 1500 ng of Z11 - 18 :A c
was in the e ja cul at ory bulb and about 800 ng was on the
surface of the fly.
During mating about 1200 ng of Zll18 :A c was t r a n s f e r r e d to the f e m a l e fly, but was not
transferred into the reprodu ctive tract.
The female fly
then looses app rox im at el y 60 ng of the Zl I- 18:A c to the
media.
This amount is independent of the time spent in the
holding vial.
The amount of Z I I-18: Ac on mated females
decreased to un det ect abl e l e v e l s at 12 hours post-mating.
Concurrent with the Z I I-18:Ac decrease there is an increase
in the concentration o f . 14, 16 and 18 carbon fatty acids.
The female is able to me tab oli ze external Iy applied Zll1 8 :Ac, v a c c e n o I , vaccenic acid, oleyl acetate and stearyl
acetate.
Sexually immature (6-8 hours old) virgin females
can me ta b o l i z e applied Z11-18:Ac. A combination of
Zll18 :Ac and
volatile, polar components from the male hexane
extract
comprise the aggregation pheromone in Eu. funebris.
I
INTRODUCTION
fu n
is
cosmopolitan
in
species
temperate
southern
of
the
cold
adapted
of
Drosophil a found
primarily
(T),(2)
woodlands
and
considered
one
northern
a facultative
(I)
most
a n,d is
latitudes
fungal
common
(3).
at
the
Although
species
(4),
mos t
it
is
funebris
does well on a synthetic Drosophila diet (Instant Drosophila
Medium 4-24, Car oli na Biological).
Many c l o s e l y related
species are obligate fungal feeders (4) but D. funebris will
not hybridize with them (2).
Previous studies of jX funebris have included dispersal
rates
(5),(6),
accessory
(7);
fitness
gland, secretory
parameters
proteins
(10),
(8),
phenomenon
aggregation
species
(15),
pheromones
in JX me I ano gaster
have
D £ £ £ 0£h Il1L£.
been
(16),
in
is probably
demonstrated
group
(12),
in
(13),
s i m u l a ns (17),
D^ yakuba and jX
and in D^ mu I I e M
and
a
Mal e- p r o d u c e d
in D^ an a n a s sae and
in 2^ maur it i a na,
in D^ h^dei, (21),
I.
the
of the jX v i.r:(:Ms species
m a l e r k o t l i an a (18),
(19),
in
(I)
(11).
Aggregation using aggregation pheromones
general
(9),
IX
seven
(14),
in
EU
b i^ec t ^ n a t e
jia M
(20),
(22), as shown in Table
2
Table I.
Drosophila species with identified aggregation
pheromones .
GENUS DROSOPHILA
SUBGENUS SOP HOP HO R A
GROUP MELANOG ASTER
SUBGROUP
MELANOGASTER
-melanogaster
-mauritiana
-simuIans
-yakuba
SUBGROUP
ANANASSAE
-ananassae
-bipectinata
-malerkotliana
SUBGROUP
SUZUKII
-r ajasekai
SUBGENUS DROSOPHILA
GROUP VIRILIS
GROUP REPLETA
SUBGROUP
-americana
SUBGROUP
HYDEI
-borealis
MULLERI
-hydei
-Iittoralis
-mulleri
-Iummei
-novamexicana
-texana
-virilis
Djl m e l a n o g a s t e r ,
yakuba,
simulans,
JX
m a ur iLt iLan a, JX
rajasekari, D. ananassae, D. bipectinata, and
m a I e r ko 11 JlB n a
subgenus
JX
GROUP FUNEBRIS
-funebris
are all
Sophophora
octadeceny I acetate
and
in the mel anoga ster
all
of
these
(Z 11 — lSsAc)
as
pheromone, except JDj^ rna^JLe^r_ko^t_JLi_a^iia^ and
use Z I I- 20 :A c.
group of the
species
the ir
use Z - I 1-
aggregation
^
—— — that
The rest of the species studied to date are
in the Dr oso phi la subgenus and the aggregation pheromones
are hydrocarbons,
esters,
and ketones,
of two classes of these compounds
pheromone.
and usually
comprises
a blend
the aggregation
In all cases the pheromone attracts nearly equal
3
numbers
of
both sexes
in the wind-tunnel
olfactometer,
and
the pheromone is synergistic with food related odors.
In
most species, the aggregation
mature male e ja cul at ory
rep rod uct ive
tract
bulb,
pheromone is present in the
transferred
during mating,
and transferred by the
females to the food media within hours
(17),
(18),
(19),
studies
me lanogaster where it was
(23),
after mating (16),
(20).
N on-pheromonal
mating
to the female
of ZI I- I8 :A c include work on D,
reported to inhibit
courtship
but this was proven to be incorrect
Zl I - 18:Ac has
been quantitated
female JX me Iano gas ter
ap pr ox ima te ly
300
ng
( 16),
(20%
of
in
virgin
(24),
their
(25).
ma le
Males
Z I I - 18:Ac)
(24),
and
and
(25).
mated
transfer
into
the
female’s reproductive tract with approximately 20 ng located
on the cuticle.
The female.then
loses a majority of the
transferred Z I I- 18 :Ac to the vial within 6 hours.
No p r ev iou s pheromonal work with _Dj_ fHll®.^IL-LiL has been
reported.
distant
thus
D. fu n e b r i s
relative
far.
in
the
Sophophora
of the m e l anogaster
Experiments
with JX
group
fune bris
after previous pheromone studies (16),
(17).
subgenus
species
are
is
a
studied
patterned
4
Objectives
The
most
ejacu la to ry
abundant
bulb
identification,
female,
hexane
will
be
extractable
characterized
2) rate of synthesis,
4) loss by the female, and
aggregation pheromone
system.
compound
of
the
including:
I)
3) transfer to the
5) i n v o l v e m e n t in the
5
METHODS AND MATERIALS
D.
fu ne b r i s,
wild
type,
Biology-University of Milan,
from
Milan,
the
(Department
of
Italy) were raised on a
diet of yeasted Instant Droso phi la medium 4-24 (Carolina
Bi olo g i c a l
Supply
Co.,
Burlington,
North
ambient lab temperatures using a 16 hour
dark cycle.
Carolina)
at
light and 8 hour
At less than 24 hours after
ec Ios ion, flies
(anesthesized with carbon dioxide) were separated by sex.
A p p r ox ima te ly 10 flies were put into a rearing vial (10 cm x
3 cm ID) until
a specified age.
D_j_ m e l a n o g a s t e r Canton S
were reared and handled as reported previously (16).
Ejaculatory
Male s
were
Bulb
killed
approximately 30 minutes.
Removal
by
placing
of
the
abdomen
them
at
-IO0
C.
for
A thin dissecting pin was used to
fasten the fly to a cork board.
the tip
and Analysis
Under
(near, the
20 X magnification,
genitalia)
was
grasped
between two pins and this section of tissue which included
the
ejaculatory bulb was removed. The ejaculatory bulb was
carefully
separated
in a 0.5 ml conical
from the
surrounding tissue
vial containing
and placed
10 u I of hexane and I
ug of nonadecane as a quantitative internal
standard.
A pin
6
point was used to smash the bulb, and release the contents
for
extraction.
The hexane extract
(2-3 u I ) was analyzed in a Varian
3700 gas chromatograph fitted with a 15 meter Megabore DB-1
capillary column
(J and W Scientific,
ionization detector.
temperature
I 30° C , i n c r e a s e d
standards
CA) and flame
The temperature program was initial
temperature of 300°C.
synthetic
Folsom,
at
I O 0 C Z m i n to
Chromatographic retention times of
of the hydrocarbons, esters
previously observed
a fin al
and ketones
as aggregation pheromones were compared
to the retention time of the major chr omatographic peak in
the D. funeb r is eja cu la to ry bulb extract.
Compounds with
similar retention times to the peak from D. f u n e b ris were
chr omatographed on a 30 meter D B - 225 c api ll ary column
(J
and W Scientific, Folsom, CA) programmed from 130 to 200° C
at 5°/min.
Electron impact mass spectra were obtained on a VG MMI6
mass spectrometer using
a 30 meter DB-5 capillary GCoolumn
for introduction of the sample.
The double bond was located
by ozonolysis and GC of the products (26).
ExtracJ^i_o_n
The ejaculatory
o_f^ Who 1_£ _Fl_i_e„s
bulb compound
could
be extracted
from
whole mature male flies by soaking them for 45 minutes in 10
uI of hexane containing I ug of nonadecane as a quantitative
internal standard.
Likewise,
the remainder of the fly after
7
removal
of the ejaculatory bulb was soaked in hexane for 45
minutes and the extract analyzed by GC.
Formation
of
the. Ejaculatory
Bulb Compound with Age
Within two hours of eclosion and each day thereafter up
to 6 days,
the
ejaculatory
extracted and analyzed.
bulbs
of males
were
removed,
The remainder of the flies was also
extracted and analyze.
■ Transfer During Mating
Seven
day-old
experiments.
X 6 mm
flies
were
used
in
the
mating
Flies to be mated were placed in (9mm diameter
height)
chambers
without
Immediately
upon
completion
predetermined
times
after
flies were k i l l e d by
the
of
copulation
aid
of
anesthesia.
mating
or
at
began,
the
heavy ether anesthesia,
into separate 0.5-1 .0 ml conical
various
individual
then placed
vials and stored at - IO0C
until subsequent extraction and GC analysis.
Femal e Reproductive Tract Remo v a I
■The
repro duc tiv e
tract
of
mated
female
flies
was
removed by first fastening a fly to a cork board with a pin.
The ovipositor was clasped with a pair of forceps and pulled
out.
Two
dissecting
pins
were
used
to
separate
the
reproductive tract excluding the ovaries from the intestine.
The reproductive tract excluding the ovaries was placed into
8
a I ml conical
vial with 10 Ul of hexane containing I ug of
nonadecane as a quantitative internal standard.. Analysis by
GC was performed as previously described.
^ J.££y.I.£^.°£.Z Bu l^b _Com££U rid^ Los^ _by t^h£ Fe m a le
Immediately
specified time,
after
completion
a 4 ml conical
vial
specified time,
the
vial
at - I O 0 C.
removed
placed
washed
concentrating
nonadecane,
at
a
Ten females were placed in
for
30 minutes,
into
3 times
the
After a
containing the females was placed
a separate
extraction and GC analysis
was
or
fitted with a wire mesh cap.
in a freezer
vial
m a tin g,
females were removed from the mating chamber
using carbon dioxide anesthesia.
and
of
the
conical
as p r e v i o u s l y
with
extract
a qu anti tat ive
100
females
uI
were
via l
described.
of
hexane.
under
nitrogen,
internal
standard
I
was
for
The
After
ug
of
added.
GC-analysis was performed as previously described.
Ap p l ication of Compounds to V irgin Females
Seven
day old
ap prox imate ly
virgin
10 minutes
females
to make
were
refrigerated
them easier
for
to handle.
They were then placed in a petri dish which sat on crushed
ice.
Using
acetone
a 1.0 uI
containing
syringe
a
(Hamilton Co.) 0.2-0.3 uI of
specified
amount
applied to the posterior end of the fly.
into a 4 ml conical
of
material
was
Ten flies were put
vial fitted with a wire mesh cap for
a
9
predetermined period of time.
as previously described,
chromatograph
c ap il la ry
fitted
column
and analyzed
with
a
30
150°
in' a Varian
meter
(J&W Scientific,
ionization detector.
temperature,
Flies and vial were extracted
3700 gas
Megabore
Folsom,
D B- 225
CA) and flame
The temperature program was initial
increased
at
2°
C/min
to
a
final
temperature of 200°. An equal volume of Meth-Prep I (Applied
Science) to met hy la te the fatty acids was coinjected with
the sample.
Collection
of
Volatiles
from Hexane Extracts
A 2 cm long column of Tenax porous polymer 35/60 Mesh
(Applied Science) was formed in a cap i l l a r y tube (2mm ID X
95mm L) with glass wool placed on either side.
An apparatus
was
nitrogen
ass emb lied
ambient
vapors
so
that
a gentle
stream
of
at
temperature evaporated a 0.5 ml sample, causing the
to
pass
into
the
Tenax
column.
After
evaporation which took approximately 10 minutes,
complete
the Tenax
was eluted with 250 uI of pentane and the resulting extract
was quantitated by GC and Used in further tests.
Bioassays
Flies for the bioassays were removed from the rearing
jars when 0-2 days old,
starved overnight in the wind-tunnel
olfactometer and tested the following morning.
The bioassay
procedure and apparatus were fully described by Bartelt and
Jackson
large
(12).
Briefly,
enough
to
allow
the win d-tunnel
free
approximately 1000 flies.
flight
olfac tomet er was
and was
stocked
with
A sample to be tested was applied
to a filter paper strip inserted around the lip of a glass
vial.
Two vials to be compared were placed on the floor of
the ol fa ct om et er in the upwind end.
drop of water,
Each vial contained a
which was not itself
caused the flies that entered the vial
the test.
attractive but which
to remain throughout
Tests lasted for 3 minutes after which the vials
were capped and flies counted. Each bioassay experiment used
the
balanced
treatments
combinations.
incomplete
were
The
tested
bioassay
block
in
design,
pairs
data
was
in
in
all
whi ch
the
possible
transformed
to
the
I o g (x + I) scale before analysis to stabilize variance
and
ana lysis was done by the method of Yates (27).
Normally,
12 tests could be run before the number of flies became too
low to give good results.
RESULTS AND DISCUSSION
Identi f ication
The
hexane
extract
of mature
male
ejacul ato ry
contained only one GC peak of appreciable size.
of
retention
tim es
on
the
non
polar
bulbs
Comparison
DB-I
column
of
previously used synthetic compounds showed a match with (Z)1 I-octadecenyI acetate (Z I 1- I8 :Ac).
The ejacul ato ry bulb
compound and ZlI-ISsAc also had matching
the polar DB-225 column.
retention times on
Mas s-spectra and GC analysis of
the ozonolysis products of the ejaculatory bulb compound and
Z11-18:Ac
were
structure
of
oct adecenyI
identical.
the
All
eja cul ato ry
evidence
bulb
supported
compound
as
the
(Z)-1 I-
acetate.
Z I I- 1 8:Ac has been identified from the ejaculatory bulb
of a number of species in the m
was
the
first
report
of Zl1-18:Ac
I L B J i g^oup, but this
outside
the m e l anogaster
group of the Sophophora subgenus.
Formation
There was no det ec ta ble
females
of
any
ag e;
ZlI-IBsAc
however,
there
present
was
a
in virgin
dramatic
increase in Z I I- 18: Ac in male flies during the first three
days,
as shown in Figure I. After three days, the level of
12
2400
Nanograms of Zll-ISrAc / fly
2200
2000
1800
0
1
2
3
Age
Figure I.
4
5
6
(Days)
(Z)-Il-Octadecenyl acetate (Zll-ISrAc) in
male D. funebris with a g e . Shaded areas
represent Zll-lSrAc in the ejaculatory bulb
while clear areas represent Zll-ISrAc on the
rest of the fly (N= 2 sets of 3 f l i e s ) .
13
Z 11-I 8:Ac
ng/fly.
reached
a
plateau
level of approximately 2300
In comparison, ejaculatory bulbs of JX me lanogaster
had ap pro xi ma te ly 1 600 ng/ fl y of Z I I- 18 :Ac (24),
extracts
of whole
at age 5 days
flies
(16)
of JX_ m e l anogaster had
and JX. s i m u l an s had
hexane
1400 ng/fly
1000 ng/fly
(17).
In male JX_ f u n e_b£ Jl^ of all ages, most of the Z I I- I8 :A c was
located in the ejaculatory bulb,
there
was
ap pr ox ima te ly
remainder
of
the
fly,
800
as
however after 2 days of age
ng/fl y
shown
of
Zl I- 18:A c on
in F i g u r e
the
I. This
was
confirmed by making two I second hexane dips of mature male
flies which removed 820 ng of ZI I- I8:Ac.
Transfer During M ating
Z l 1-18:Ac
(1000-1400
ng) was
transferred
male JX funebris to the female during mating.
from
virgin
Over half of
the Zl I- 18 :Ac was transferred within the first five minutes
of mating, as illustrated in Figure 2,
with a maximum of it
being
13
transferred
in
th e
a p p r ox im ate ly 16 minute mating.
unexpected
results.
The
first
minutes
transferred
an
JX funebrJ^s produced some
female
reproductive
contained 240 + 45 ng Z I I-18:Ac/fIy which was
20% of the
of
Z I I- I8 :A c. After
three
tracts
approximately
minutes, the
amount present in the reproductive tract was independent of
time.
Eighty
fu n e b r is
percent
females
was
of the Z I I- I8 :Ac transferred
easily
ext ra ctab le
with
to JX
hexane,
suggesting that it was on or near the surface of the fly, as
14
(■--- B) Zll -18 :Ac in
the male ejaculatory
bulb
( V — '■) Z l l -18:Ac on
the remainder of the
male fly
O ---A) Zll - 1 8 :Ac in
the female reproductive
tract
(A--- A) Z ll -18:Ac on
the remainder of the
female fly
Nanograms of Zll-18:Ac / fly
2400
2000
1600
1200
800
400
0
0
3
8
Mating Time
Figure 2.
13
(Minutes)
Seven day old ETl funeb ris matings with the
subsequent removal of the ejaculatory b u l b ,
reproductive tract and hexane soak of the
f l y . (N=3 sets of 3 flies)
15
shown in Table 2 and Figure 2,
unlike what was observed
with D . me I a n o g a s t e r . Transfer of Z I 1-18:Ac in D . fune b r is
must
be
occurring
di ffe re nt ly
than JX
me JL a n o g a s t e r . It
appeared that the female reproductive tract became "filled"
within 3 minutes and the rest of the transferred Z I I- 18 :Ac
was deposited on or near the surface of the cuticle.
the mated
pair
was
still
coupled
when
they
were
Since
frozen,
there should be no way for the female to lose the Z 11- 18:Ac
from her reproductive tract.
Table 2.
A c o m p a r i s o n of the a m o u n t s of Z l 1 - 1 8 : Ac
sequentially extracted from D. funebris and
D . melanogaster mated females.
Zl1-18:Ac (ng/fly)
2nd one
sec dip
(N=7 sets of 3 flies)
Total
4 5 min
soak
Mated female
flies
1st one
sec dip
JX funebris
560+210
260+80
170+90
980+320
42 + 9
15 + 5
360+80
410+80
JX melanogaster
In D . me I a n o g a s te r , ap prox imate ly 300 ng of Z I I- I 8 :Ac
was transferred (24) and the transfer was complet e within
the first six minutes
(25).
of an
appro ximat ely
20 min mating
In IX me I ano g a s t er the Z I I- 18:Ac was
deposited
into the female’s reproductive tract during mating with only
a small
amount (60 ng) found on the female’s cuticle
(Table
2; 25). In comparison, two I second hexane dips of mated JX
16
funebris
but
females
only
removed
approximately
approximately
60
ng
800 ng of Z I I- I8:Ac
of
Z11-18:Ac
from
D♦
me Ianogaster mated females, as presented in Table 2.
The po s s i b i l i t y was considered that mated Djl fune b r i s
females
were
releasi ng
the
Z I I- 18:Ac
transfer to their food source.
legs
from mated
females
to
their
legs
for
Removal and extraction of
revealed the legs had 81+68 ng/fly
while the remainder of the fly had 990 + 230 ng/fly of Zll18:Ac. This small
amount could result from rubbing her legs
against her cuticle.
If JX
portion
f u n ebrjijs males
of the
Z I I- 18:Ac
t r a c t , how was
such
female?
method
One
Interrupting
showed
that
into
a large
a mating
th e re
were
was
no
the
depositing
female's
quantity
could
just
only
be
additional
repro ducti ve
transferred
cuticle
as the pair
a small
to
started
to the
cuticle.
(0-1 minute)
Zl I- 18:A c
surface of mating male flies than on virgin flies,
on
the
as shown
in Table 3. The males had transferred over 200 ng of Zll1 8 :Ac to the females with no corresponding increase in the
level of Zl1-18:Ac extracted from their cuticle.
The males
were not transferring a large quantity of Zl I- 18: Ac to their
cuticle
and
then
"rubbing
it"
on
the
female's
cuticle.
Neither was the female using her legs to "rub" the Z11- 18:Ac
from the males's cuticle onto her cuticle. The small
amount
(80 ng) of Z I I- I8 :Ac located on her legs could result from a
17
preening action with the Z I I- I8 :Ac being removed from her
own cuticle.
Table 3.
Recovery of Zl1-18:Ac from mated and virgin
D . funebris.
ng Z11-18:Ac/fly (+ SB)
(N=5 sets of 3 flies)
Complete
mating
Virgin
fly
120+50
150+40
I20+120
2nd one sec dip of male
220+120
140+50
I80+1 10
45 min soak of male
740+430
450+240
750+260
45 min soak of female
240+90
1260+320
0-1 min
mating
Treatment
I St
one sec dip of male
Catabolism
24
Within
hours,
the
female
had
lost
transferred Zl1-18:Ac with approximately 75%
all
of
the
lost within 3
hours, as illustrated in Figure 3. Extraction of the
female
holding vial
showed that <100 ng of Zl1-18:Ac was deposited
in the vial.
The transfer to the vial was independent of
time and
showed a
slight
increase
for the first
9 hours
and then
decreased. This amount could be rubbed from the
cuticle as the female came into contact with the surface of
the vial.
Females were
their surroundings.
"losing" Zl I- I 8 :A c , but not to
In comparison,
when
the male deposits
18
1200
Nanograms ZlI -18:Ac / fly
1000
Time after Mating
Figure 3.
(Hours)
Loss of (Z)-Il-Octadecenyl acetate (Zll-18:Ac)
from mated
fu nebris females with time.
(■— — ■) represents Zll-18:Ac on the female.
( ► — •■) represents Zll -18 :Ac emitted by the
female into the vial. (N=l set of 10 flies)
19
the Zl1-18:Ac into the female's reproductive tract,
within 6
hours after mating both D. simulans (17) and D_^ m e lanogaster
(16) lose a majority of "the transferred Z I I- 1 8 :Ac to their
holding vials.
.App l ic ation of ZII-ISiAc
Females may be
or
m e t a b oli zi ng
sequestering
it.
A dose
acetone was applied
surface
1100
ng
of
Zl I-ISiAc
to 7 day old virgin females and
extracts, of
Compared with mated
loss by virgin
of
the Z11 - 18:Ac internal Iy
the
flies
females,
were
a similar
females occurred,
analyzed
in
the
hourly.
rate of Zl I- 1 8 1Ac
as shown in Figure 4, with
a rapid disappearance for the first four hours and then a
gradual
loss..
The
quantity of Z l l - 1 8 1Ac
in
the holding
vials was similar to the quantity emitted by mated females.
Ap pr o x i m a t e l y 5% of the applied Z I I- 1 8 :Ac was lost to the
vial
and
the
amount
deposited
was
independent
of
time.
Virgin females treated the applied Z I I- I8:Ac sim i l a r l y to
the transferred Z I I— I 8 ;A c of mated females, but it is not
clear whether the Z I I- I 8 :Ac was sequestered int er nal ly or
metabolized.
Flies
to which
increased level of 14,
Z l 1-18:Ac had been applied
16 and 18 carbon fatty acids
a peak of app rox im at el y
600 ng
at 3 hours
levels within 12 hours,
an
reaching
followe d
rapid decrease from 3 to 4 hours and then a slow
to normal
showed
by a
decrease
as shown in Figure 4.
20
1200
Nanograms / fly
1000
2
3
4
5
6
7
8
Post Application Time
Figure 4.
9 10 11 12
(Hours)
Application of 1100 ng of (Z)-Il-Octadecenyl
acetate (Zll-18:Ac) to 7 day old virgin female
D. fun eb ri s . (*-— * ) represents Zll -18 :Ac
remaining on the f l y . (■— — ■) represents
the total amount of cuticular fatty acids.
(N= 5 sets of 10 flies)
21
Female
flies
to which
only
acetone
was applied
showed
<100 ng of 14, 16, 18 carbon fatty acids whether the female
was
extracted
and
analyzed
immediately
(0 hour) or allowed
to remain.in the holding vial for 3 hours.
critical
in determining
acids.
A 45
minute
compounds without
compounds.
It
compounds
since
the
Soaking time was
concentrations
hexane
soak
of these
removed
the
fatty
external
leaching out very much of the internal
was
important
the se
to
fatty
remove
acids
were
only
surface
also
present
internal Iy in both the male and female flies and significant
amounts could be extracted with a 3 hour or longer hexane
soak.
Since
the
and then
concentration
decreased
further metabolic
rather
of these
than
fatty
remain
processes were
acids
increased
relatively
constant,
indicated.
lower starting temperature for the GC
Using a much
analysis,
no new
peaks appeared as the concentration of the three fatty acids
decreased.
either
This implied that the three fatty acids
degraded
transferring
was
not
ve r y
rapidly
them internally
removing
them.
or
else
the
and the 45 minute
A 24
hour
hexane
were
female
was
hexane
soak
soak
removed
si gn if ica nt ly more of the three fatty acid products but it
was
not
whether
known whether
the products were natural Iy there or
some of the applied
material
was
taken
internally.
If I I 0 0 ng of Z 1 I- 18 :Ac was applied to very young (6-8
hour, old)
virgin
females,
(
they possessed
the
same
amount
22
of Z 11 - I8 :A c (approximately 3 5 0 n g ) after 3 hours and lost
the same amount to the vial
mature
flies did.
(approximately 20 ng) as the
Thus, the transfer system was functional
at a very young age.
Further
the female
tests to show that Zl I- 18:Ac was metabolized by
involved
the application
acid and vaccenol to the female.
Zl I-18:Ac would
ester,
presumably
of synthetic
A degradation process of
involve hydrolyzing
oxidizing the alcohol
vaccenic
to an acid,
the acid to shorter chain compounds.
the acetate
and then oxidizing
If vaccenic acid
and vaccenol were on the degradation pathway then the enzyme
system(s) involved would treat them similar to Zl1-18:Ac and
metabolize
them.
Appl ication of V accenic Acid
When a large dosage ( 1000 ng was utilized too rapidly to
accurately
measure)
of
vaccenic
acid
(2500 ng) was
applied
to a mature virgin female,
it was utilized very rapidly,
illustrated in Figure 5.
Within 2 hours approximately 80%
of the applied
A 24
hour
vaccenic
as
vaccenic acid was "lost" from the cuticle
hexane
soak
showed
app ro xim at ely
acid was present within the fly.
1100
ng
of
The ratio of
internal vaccenic acid to oleic acid was relatively constant
for
the
rapidly
Hexane
first
with
rinses
2 hours
no
after
vaccenic
of the holding
application
acid
detected
vials
then
after
decreased
5
hours.
showed that no vaccenic
23
2500
Nanograms / fly
2000
1500
1000
Post Application Time
Figure 5.
(Hours)
Application of 2500 ng of vaccenic acid
(■— — ■) or 1000 ng of vaccenol ( ► — b )
to 7 day old
funebris females.
(N= I set of 10 flies)
24
acid was
lost to the surroundings.
In addition,
no Zll-
18:Ac or vaccenol was seen in any of the chromatograms.
Comparing
the free fatty acid
extracted
by the
24 hour hexane soak after vaccenic acid was applied,
showed
that the 16:0 and 16:1 fatty acid
levels
l e v e l s were
relatively
constant with time (approximately 1.3 times the 18:1 levels)
while the 14:0 and 14:1 levels increased slightly with time.
Thus, the decrease in
vaccenic acid levels in the fly does
not change the internal fatty acid profile very much.
Application
When
female,
o f V accenol
10 0 0
ng
of
vaccenol
was
applied
a mature
the disappearance of vaccenol was similar to the
disappearance of vaccenic acid with a rapid
first 3-4 hours and then a gradual
5.
to
loss,
loss within the
as shown in Figure
In addition, the 45 minute hexane soak con sis tentl y
removed a small amount of Zl I - 18:Ac (approxim ately 30 ng)
and
vaccenic
system(s)
to
acid
involved
(approximately
10 ng).
in this transformation
The
seems
enz yme
to be able
both oxidize the alcohol to the acid and acetyl ate the
alcohol to the acetate ester.
soak removed a small
24 ho ur
vaccenic
hexane
acid.
if any vaccenol
vaccenic acid
nor
soak
While the 45 minute hexane
amount of Z11-18:Ac from the surface,
removed
Rinses
of the
was released
no
vials
to the
Zl1-18:Ac was
internal
showed
a
Zl I - I 8 : A c or
that
surroundings.
detected in vial
little
Neither
rinses.
25
Appl ication of simil ar acetates
Since the female was able to m eta bo liz e Z I I- I8:Ac and
its degradation products,
the degradation
of two cl o s e l y
related acetates (stearyl acetate, an 18 carbon saturated
acetate and oleyl
acetate,
an 18 carbon unsaturated acetate
with the double bond located in the 9 position) were tested.
Utilization
similar
of the two ^acetates
to the p r e v i o u s l y
applied
at 1000 ng/fly was
applied compounds with a rapid
initial loss that tapers off, as shown in Figure 6.
A small
amount of both compounds (approximately 50 ng) was deposited
into the holding
and
did
not
double
slightly
Thus,
involved,
This
increase
me ta bo liz ed
ace ta te .
vial.
a
amount was relatively
with
time.
s i ow er
Oleyl
a c e t a t e was
than Z l 1— ISsAc
non-specific
enz y m e
constant
or
stearyl
syst em(s)
was
because lack of a double bond or movement of the
bond
d o e s n ’t
affect
the e n z y m e ’s
ability
to
metabolize the applied compounds.
Appl ication of ZlI-ISsAc to
~~
D_j_ melanogaster
The ability of JX funebris to met ab o l i z e Z I I- I8:Ac
and
closely
related
m e l anogaster.
in
this
(16).
cuticle
When
virgin females,
in D. funebris occurred,
the
lead us to investigate JX
ZtI-ISsAc was also the aggregation
species
applied to
compounds
within
500
ng
of
pheromone
ZlI-ISsAc
was
the same utilization pattern as
with 9 0% of the Z 11- 18:Ac lost from
5 hours,
as
shown
in
Figure
7.
In
26
■Zll-18:Ac remaining on
the fly
800
I
I
IOleyl acetate
Ion the fly
remaining
I—
I
I Stearyl acetate remaining
I on the fly
Nanograms / fly
600
400
200
0
Post Application Time
Figure 6.
(Hours)
Application of 1100 ng (Z)-Il-Octadecenyl
acetate (Zll-18:A c ) , 1000 ng of oleyl and
stearyl acetate to 7 day old virgin female
D. funebris (N=l set of 10 flies).
27
Nanograms
Z11 -18:Ac / fly
600
400
200
0
0
1
2
3
4
Post Application Time
Figure 7.
5
(Hours)
Application of 500 ng of (Z)-11-Octadecenyl
acetate (Zll-18:Ac) to 4-5 day old virgin
female D. m e lariogaster (N=l set of 10 flies).
28
addition,
Soaking
holding
the
vial
flies
for
internal Z11-18:Ac.
rinses
24
contained
hours
no
removed
Z I I- 18 :Ac.
no
additional
Thus, it appeared that the ability to
metabolize Z11-18:Ac was common to both species.
Bioassay Response
Identification of the aggregation pheromone began by
preparing a crude hexane extract of 7 day old flies.
the
flies
extracted
to
7 days
ensured
that
they
were
Aging
mature
when
and a re prod uci ble a m o unt of material could be
removed by extraction.
When
the
flies
were
first
put
into
the wind-tunnel
olfactometer, they tended to form tight aggregation units in
the corners.and on the upwind screen.
temperature
of
20o - C. was
used
to
An overnight holding
reduce
stress.
temperature kept the flies tightly aggregated.
This
One half
hour before the tests began, the temperature was increased
to 24°
C.
and
lights
were
turned
on,.
This
aided
in the
relatively uniform dispersal of the flies and also increased
the frequency of flies that were in flight.
the
bioassay
tests,
the
cage
While running
temperature
increased to a final temperature of 26°C.
was
slowly
This resulted in
a better bioassay response than if a constant temperature
was maintained.
number
of
responding
In response to an active
airborne
to
an
flies
active
increased.
preparation,
preparation
However,
1/2-2/3
the
when
of
the
29
responding flies crawled up the sides and into the vials.
It became very evident within 30 seconds if a vial contained
an active
toward
vial,
preparation because the number
the
vial
flies
increased
usually
of flies
dramatically.
remained
Once
for the duration
crawling
inside
the
of the test.
The same response behaviors were exhibited toward both flyderived
and synthetic
catches
for
any
materials.
treatment
varied
The abs olute
from
day
to
bioassay
day,
due
p ri ma ri ly to the overnight hol di ng temperature and in the
number
and
condition
of
the
flies
in
the
win d-tunnel
olfactometer.
Initial attempts to demonstrate an aggregation response
in Dl_ funebhis were patterned
me lanogaster
after pre vio us work with Dj.
(16) and Di simulans (17). The mature male and
female fly (7 day old) hexane extracts
male
and
female
bioassayed
to
in Table 4.
attractive,
fly
(0-24
determine
hours)
along
hexane
the aggregation
Hexane extract of mature
with
immature
extracts
activity,
males
were
as shown
was c l ear ly
catching an average of 23.1 flies per test with
55% of the flies being male.
Both the immature male
and
immature female extracts showed little activity and were not
s i g n if ic an tl y different from the hexane control.
Mature
female extract was significantly different from the control
but showed
little
activity.
males
and
It was decided to work with
extracts
from mature
determine
produced
the aggregation response.
what
component(s)
30
Table 4.
Aggregation response of several extracts relative
to the mature male D . funebris hexane extract.
Relative Response15
(N= 8 )
Hexane Extract of :a
100*
Mature Males
Immature (0-24hr) Males
3
Mature Females
4*
-5
Immature (0-24 hr) Females
Hexane Control
flies/test)
(1.3
flies/test);
mature
males
(23.1
a)
All extracts used at one fly equivalent
b)
(Extract - Control)
Relative Response = _____________________ ___________ X 10(
(Mature Male Extract - Control)
#
Denotes significant difference vs control at the
0.05 level
The hexane extract from 7 day old virgin male flies was
fractionated
using
eluting with hexane;
10%
an
5 %,
methanol/methylene
open
column
acid
and
10%, 25% and 50% ether-hexane;
and
chloride.
of
The
silicic
three
most
polar
fractions showed some activity, but various combinations,
when compared to the male hexane extract,
an equivalent aggregation response.
'
would not produce
31
Examining pre vi ou s
pheromone
of
work
mu I I e ri
showed that the aggregation
(22 ) contained
volatile, polar component.
an
unidentified,
To test this in JX f un ejDi-i^, a
sample of the male hexane extract was taken to dryness under
nitrogen
and
an equivalent
amount of hexane was added back
to the remaining residue.
"evaporated"
male
hexane
The aggregation response of the
extract
was
reduced
over 50% when
compared to the untreated male hexane extract. Clearly,
volatile
component of the male hexane extract was
also essential for aggregation activity.
why
a
combination, of
account for all
The
combined
silicic
acid
a
lost and
This would explain
fractions
could
not
the act ivity of the male hexane extract.
fractions
had
been partial Iy evaporated under
nitrogen to achieve the proper concentration for bioassay
tests.
A fraction was prepared
the
volatile
Tenax.
component
the male
In the wind-tunnel olfactometer,
produced a good response,
equivalent
to
combination
of the Tenax
hexane
from
for bioassay tests by trapping
the
male
hexane
extract
the Tenax volatiles
as shown in Table 5, although not
hexane
e xtr ac t's
volatiles
fraction in bioassay
with
resp onse.
hexane
A
the 5% ether-
produced a response equivalent
to the response of the hexane extract of males.
showed
in
Since GC
Zl1-18:Ac to be the major component in the 5% etherfraction,
volatiles.
Zl I-ISzAc
Tenax volatiles
was
tested
wi t h
the
Tenax
accounted for approximately 40%
32
of the. male hexane e x t r a c t ’s activity,
but when added to
Z 11 - 18:Ac the response of the combination was
statistically
e qu iva le nt to the male hexane e x t r a c t , as shown in Table 5 .
Table 5.
Response of JX funebris to the male hexane
extract and two components of the extract.
Mean Bioassay Catch
CN=12)
Treatment3
Male Hexane Extract
20.7a
Tenax Volatiles from
Male Hexane Extract &
Zl1-18:Ac (2200 ng )
2 1 .8a
Tenax Volatiles from
Male Hexane Extract
LU
CTi
O
7 .9b
Zl1-18:Ac (2200 ng)
a)
All fl y - d e r i v e d fractions, extracts and synthetic
compounds were used at I fly equivalent per test.
b)
Means followed by different letters were significantly
different at the 5 % level (LSD).
Tenax volatiles collected from the mature female hexane
extract and analyzed
on the GC showed
the male Tenax extract.
little
similarity
to
This could account for the minimal
aggregation response of the mature female hexane extract.
Thus
far,
attempts
to
isolate
and
identify
component(s) in the Tenax fraction were unsuccessful.
appeared
to
results with
be
more
the
than
volatile
one
component
but
the
There
reproducing
c o m p o u n d (s) was difficult.
A
33
volatile
component
identified
viriI is
(22),
in JX
mu I Ie r i 1s pheromone
and an attract ive
(12) remained.unidentified.
has been developed and tested,
polar
Until
was
never
component
in IX
a new procedure
the volatile component(s) of
D. funebris* pheromone system will
remain unidentified.
34
CONCLUSIONS
1.
(Z)-II-Octadecenyl acetate
was
identified as the
most
abundant component of the male ejaculatory bulb.
2.
Z11— 18;Ac was produced by males at a rapid rate during
the
first
three
days
after
eclosion
to
a
level
of
approximately 2200 ng/fly of which approximately 1500 ng
was stored in the ejaculatory bulb and approximately 800
ng was on the surface of the fly.
3.
During mating the male transferred approximately 1200.ng
of Zl I- 18:Ac to the female with over half transferred in
the first five minutes of an average 16
■ Most
and
reproductive
The
mating.
of the Z l 1-18:Ac was on the cuticle of the mated
female
4.
minute
approximately
2 0 % was
in
the
female
tract.
mated female fly released approximately 5% of the
1200 ng of transferred Zl1-18:Ac to the food media.
c a t a b o I i zed
the
remaining
hours after mating.
Z I I- I 8 :Ac
within
They
about
12
Mature virgin females c a t a b o Iize
Z11- I8 :Ac, v accenoI, vaccenic acid , stearyl acetate,
and
oleyl acetate.
5.
Volatile,
along with
system
in
polar components from the male hexane extract
Zl1-18:Ac comprise the aggregation pheromone
D.
f._uH
HA— .
REFERENCES CITED
Mer re 11 , D.J.195 1. Interspecific competition between
Drosophilafunebris
and Drosophila me I anogaster.
Am.
NatT~85:159-l69.
E w i n g , A.W. 1979.
Complex courtship songs in the
Dr o sop h ila fu ne b ris Species Group: Escape
from
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