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THESIS

This is to certify that the

thesis entitled
ISOLATION AND PRELIMINARY CHARACTERIZATION
OF THE PHEROMONE OF A GREENHOUSE FUNGUS GNAT,
BRADYSIA IMPATIENS (JOHANNSEN) (DIPTERA:SCIARIDAE)
presented by

Richard Andrew Kurnot

has been accepted towards fulfillment
of the requirements for

MS - degree inihfimm

 

 

 

Major professor

Date .Desember 10 . 1981

0-7639

 

 

 

 

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LIBRARIES remove this ChECkOUt from
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ISOLATION AND PRELIMINARY CHARACTERIZATION
OF THE PHEROMONE OF A GREENHOUSE FUNGUS GNAT,

BRADYSIA IMPATIENS (JOHANNSEN) (DIPTERA:SCIARIDAE)

 

By

Richard Andrew Kurnot

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE

Department of Chemistry

1981

ABSTRACT

ISOLATION AND PRELIMINARY CHARACTERIZATION
OF THE PHEROMONE OF A GREENHOUSE FUNGUS GNAT,

BRADYSIA IMPATIENS (JOHANNSEN) (DIPTERA:SCIARIDAE)

 

By

Richard Andrew Kurnot

L57//'/fng/’

The pheromone of a greenhouse Fungus gnat, Bradvsia

_—__.\__—

impatiens, has been isolated and partially characterized.

 

Behavioral studies indicate that the pheromone appears to
be comprised of one component, a 0-1“ hydrocarbon with
three degrees of unsaturation including at least one double
bond. Further investigations are needed to determine the

exact structure of the active component.

ACKNOWLEDGMENTS

The author would like to express sincere gratitude to
Drs. Donald Farnum and Ring Cardé, and to committee member
Dr. William Reusch.

A special thanks is also extended to Liga Dindonis, for
her help with the culture maintenance.

The author would also like to thank Dr. Matthew Zabik

for the use of his GC-MS facility.

11

TABLE OF CONTENTS

Chapter Page

LIST OF TABLES O O O O O O O I O O O O O O O O O O 0 iv
LIST OF FIGURES . . . . . . . . . . . . . . . . . . v

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

METHODS AND MATERIALS . . . . . . . . . . . . . . . 3
Insect Rearing and Culture
Maintenance . . . . . . . . . . . . . . . . 3
Extract Preparation . . . . . . . . . . . . . . A
Biological Assays . . . . . . . . . . . . . . . A
Isolation of Active Component . . . 5
Quantitative Analysis of Active
Component . . . . . . . . . . . . . . . . . . . 7
Characterization of Active
Component . . . . . . . . . . . . . . . . . . . 7

RESULTS 0 O O O 0 O O O O I O O O O O 0 O O O O 0 O 1 0
Insect Rearing and Culture
Maintenance . . . . . . . . . . . . . . . . . . 10
Isolation of Active Component . . . . . . . . . 10
Quantitative Analysis of GC

Isolation . . . . . . . . . . . . . . . . . . . 1“
Characterization of Active

Component . . . . . . . . . . . . . . . . . . 1A
GC— Mass Spectrometry of Active

Component . . . . . . . . . . . . . . . . . 18

DISCUSSION. . . . . . . . . . . . . . . . . . . . . 21
BIBLIOGRAPHY. . . . . . . . . . . . . . . . . . . . 2“

iii

Table

LIST OF TABLES

Page

Biological Activity of XF-llSO

Fractions . . . . . . . . . . . . . . . . . ll
Biological Activity of OV-l

Fractions . . . . . . . . . . . . . . . . . 15
Biological Activity of Eluent

From Reaction Mixtures of

Characterization Tests. . . . . . . . . . . 17

iv

Figure

anuom

LIST OF FIGURES

Page
OV-l Chromatogram of Active
Fraction. . . . . . . . . . . . . . . . . . 12
Regions of OV-l Chromatogram. . . . . . . . 13
Quantification of Active Component. . . . . 16
Mass Spectrum of Active Component . . . . . l9
GC-MS Chromatogram of Active
Fraction. . . . . . . . . . . . . . . . . . 20

INTRODUCTION

Relatively few Dipteran pheromones have been identified
(Fletcher 1977). Of the ones which have, most appear to
be comprised of one or more long-chain hydrocarbons, usually
with some unsaturation present. Carlson et El- (1971)
reported the isolation of (Z)-9-tricosene as the compound
which attracts the male house fly, Musca domestica, to the
female. Uebel g§_§1. (197“) have identified the components
a pheromone which elicited mating strikes by male face
flies (Musca autumnalis) as (Z)-1A-nonacosene, (Z)-l3-non-
acosene, and (Z)-9-heptacosene. Other dipteran pheromones
identified by Uebel g§_gl. include (Z)-9-hentriacontene
and (Z)-9-tritriacontene as components of a system which
induces mating strikes by male stable flies (Stomoxys
Icalcitrans) (1975), and (Z)-11-hentriacontene as the male

 

copulatory stimulant for Fannia pusio, a common poultry

pest (1978).
One species of sciarid fly in which the pheromone has

 

been identified is Lycoriella mali. Kostelc 33 a1. (1980)
reported n-heptadecane as a male sex-attractant.

Alberts 33 al. (1981) have established the existence of
a sex pheromone in the sciarid fly Bradysia impatiens.

This study will involve the isolation and preliminary

characterization of the chemical components of this

pheromone system.

MATERIALS AND METHODS

Insect Rearing and Culture Maintenance

B. impatiens were reared using a modified version of

 

Kennedy's procedure (1973). Glass shell vials, 25 x 95 mm,
were filled with 15 m1 of 2% non—nutritive agar solution.
Vials were slanted while cooling to maximize surface area
of solidified agar. Vials were immediately plugged with
foam stoppers to prevent contamination from molds. The
vials were allowed to desiccate for one day, at which time
granular agar was sprinkled over the agar slant to absorb
any remaining moisture which might entrap insects.

Active insects could be placed in each vial for mating
and oviposition with an aspirator. This transfer method
was more convenient than the "cooling" method used by
Alberts gt_§l. (1981), and precluded any adverse behavioral
effects which can accompany a transfer which involves
anesthetization (Fletcher) (1977). Two males and one fe-
male were placed in each vial. Hatched larvae were fed a
l:A mixture of Brewer's yeast and finely chopped, auto-
claved alfalfa. The alfalfa was used as a support for the
nutrient Brewer's yeast. The culture was maintained at

25il°C with a 16:8 L:D cycle in a controlled environment

chamber.

Extract Preparation

Pheromone extracts were prepared by placing whole
anesthetized femals in 10-15 m1 of hexane for ca. 1 hr. at
20-25°C. Extracts were filtered, dried over anhydrous sodium

sulfate, and stored at -10°C.
Biological Assays

Biological assays were conducted in glass orientation
tubes, 1 m x 3 cm diam. Each glass tube was connected to
a 3-way, 105° connecting tube. Prior to reaching the ob-
servation tubes, air was forced through a water trap (to
prevent insect desiccation) at 1.9 m/sec.

The biological assay procedure involved placing 5-10
active male insects in each observation tube via aspiration.
Insects were allowed to acclimate to air flow for 15 min.
prior to testing. Test samples were pipetted on to 1.5 cm
diam. filter papers, which were suspended in the 105° con—
necting tubes from cork stoppers by metal wire. Male in-
sect wing-fanning (excitation response) and orientation
were monitored at 15, 30, and 60 sec. After sample intro-
duction. A positive orientation occurred when an insect
moved into the upwind 10 cm of the tube. Percent response

was calculated using the formula

 

percent = positive _ background
response responses responses
n - background

responses

where n equals the number of insects tested. Background
responses were determined by monitoring insect activity for
60 sec. prior to introduction of test samples.

The wing-fanning response, which occurred only in the
presence of pheromone, was used to determine biological
activity.

All biological assays were conducted under optimal
conditions of response with regard to time, temperature,
insect age, and extract concentration (Alberts 93 a1.)
(1981). Experiments were conducted at 15-1600 hr. after
the onset of photophase in a 16:8 L:D cycle. Only 1-day-
old males were tested, and no females younger than l-day-
old were extracted. Extract concentrations were greater
than 50 female equivalents (FE) in all cases, and the con-
trolled environment chamber in which bio-assays were con—
ducted was kept at 25t1°C.

Bio-assay experiments were conducted using a randomized

complete block design.

Isolation of Active Component

The active component was isolated using gas chromato-

graphy (GC). Extracts were initially separated into

fractions using a polar column (10% XF-1150 on 100-120 mesh
Gas Chrom Q, 1.8 m x 2 mm ID at 92°C). Fractions were
tested for biological activity through the assay method
described earlier. The active fraction was then re-in-
Jected on to a nonpolar column (3% OV-l on 100-120 mesh
Gas Chrom Q, 1.8 x 2,mm ID at 100°C) and its chromatogram
recorded. The chromatogram was then divided into regions
before peaks, regions of peaks, and regions after peaks,
and biological activity determined for each region.
Extracts containing 100 FE were prepared, dried over
anhydrous sodium sulfate, and concentrated under dry N2
to 10 ul prior to injection. The eluent from the XF-1150
column was trapped at 5 min. intervals in 20 cm x 1 mm ID
capillaries cooled with dry ice. Fractions were rinsed
out with 20 ul of hexane and tested for biological activity.
. Additional extracts containing 500 FE were prepared and
the active fraction collected from the XF-1150 column.
The fraction was rinsed out with 10 ul of C82, which was
concentrated to 2 ul. This concentrate was then injected
on to the OV-l column, its chromatogram recorded, and divided
as described earlier. Fractions corresponding to these
divisions were collected from additional 100 FE extracts,

and tested for biological activity.

Quantitative Analysis of Active Component

The amount of active component in an extract can be
determined using gas chromatography by comparing active
peak area with that of an apprOpriate external standard.
Tridecane was chosen as this standard based on its similar
retention time (TR = 15 min on OV-l at 100°C) with that of

the active peak (TR = 1A.6 min. on OV-l at 100°C).
Characterization of Active Component

A partial characterization can be performed on the
. active component by conducting reactions on the extracts
mwhich alter specific functional groups. If the specific
functional group were present, a successful reaction which
altered that functionality would modify the GC retention
time of the active component and probably eliminate its
biological activity. Therefore, elimination of the bio-
logical activity of the eluent at the retention time of
the active component is evidence that a specific functional
group is present, and has been altered in the reaction.

A A00 FE extract was prepared and divided into A
equivalent portions. The following reactions were per-

formed on each of three portions:

Acetylation

The extract was stirred with 100 pl of acetyl chloride
for 3 hr, after which 1 m1 of H20 was added. The mixture
was extracted with 10 m1 of ether, which was subsequently

dried and concentrated to 10 ul for injection.

LAH Reduction

The extract was added with stirring to 10 m1 ether and
0.1 g LiAlHu. The mixture was refluxed for A hr, following
which Nastu-loHZO was added to deactivate any remaining
LiAlHu. The reaction mixture was filtered through Celite

and concentrated to 10 ul for injection.

Microhydrogenation

The extract was concentrated and dissolved in 2 m1
ethanol. Addition of 0.1 g Pd/C was followed by bubbling
H2 through the solution for the duration of the reaction.
The mixture was stirred and refluxed for A hr., filtered

through Celite and concentrated to 10 ul for injection.

The fourth portion of the extract was used as the con-
trol. This was similarly concentrated for injection.

The four concentrates were then injected on to OV-l
and the eluent from each trapped at the retention time of

the active component. Each eluent was then tested for

biological activity, a lack of which would suggest func-

tional group alterations.

GC-Mass Spectroscopy of Active Components

A mass spectrum of the active component was made by
performing GC-MS on the active fraction trapped from the
XF-llSO column. An OV-l column was coupled with a DuPont
321 GC-MS, EI source, 60 eV, so that the active component
could be separated as in the isolation procedure. The
OV-l column was programmed from 125 to 150°C, at A°C/min.
Injection size was 5 ul. The solvent used was C82. Sample
size, as determined by comparison with external standard

tridecane, was 20 ng.

RESULTS

Insect Rearing and Culture Maintenance

Bradysia impatiens is a monogenic species, i.e., flies
emerging from each vial are of the same sex. At 25t1°C,
male insects emerged approximately 1“ days after mating,
while females emerged in about 17 days. Twenty-two percent
of mated vials emerged as males, while twenty-one percent
emerged as females for an overall success rate of forty-
three percent. Considerably more males emerged per suc-

cessful vial than females.

Isolation of Active Compgnent

The biological activity of each fraction of the XF-
1150 eluent is recorded in Table 1. The active fraction
appears to be no. A.

Injection of the active fraction from a 500 FE extract
on to OV—l gave the chromatogram in Figure l. The chro-
matogram was divided into Regions I, II, and III as des-
cribed earlier (Figure 2), and eluent collected from
additional 100 FE extracts according to the retention time

of each region. Biological activity for each region is

10

11

Table 1. Biological Activity of XF-1150 Fractions.

 

 

 

Retention Percent Percent
Fraction Time Wing-Fanning Orientation N
1 0-10 min 0 12.5 21
2 10-15 0 “.5 20
3 15-20 0 7.7 60
A 20-25 65 12.7 60
5 25-30 0 0 61
6 30-60 0 9.1 22

 

 

N number tested.

Column Temperature = 92°C.

12

 

 

 

 

 

 

 

 

 

Wl

 

W

 

 

 

I 1 L I __I
20 15 10 5 0
TRin min

 

Figure 1. OV-l chromatogram of active fraction trapped
from XF-1150. Column temperature - 100°C; '
Injection - 500 FE in 2 ul CS2; Attenuation
- 8 x 10’12.

13

 

 

 

 

 

 

 

 

 

MW)

(g 4~5>

Region III Region I

(15-30 min) k (0-1ll min)
Region II (Active Region)

(1A-15 min)

 

 

 

 

 

 

Figure 2. Regions of 3% OV—l chromatogram; region of
activity.

1A

recorded in Table 2. From these results, Region II con-

taining a single peak appears to be active.
Quantitative Analysis of GC Isolation

The amount of active component in the 500 FE extract
was determined to be ca. 2 ng by comparison of active
peak area with that of the external standard, tridecane.
Comparison of the 500 FE extract chromatogram with that of
a 2 ng injection of tridecane under similar conditions
(Figure 3) shows that peak areas are similar. This infor-
mation indicates that the isolation procedure yielded

about A pg of active component per insect extracted.
Characterization of Active Component

Concentrates from the three reaction mixtures and the
control were injected on to OV-l, and the eluent from each
trapped at the retention time of the active component
(TR = 1A-15 min at 100°C). These four samples were then
tested for biological activity (Table 3). These tests in-
dicate the presence of unsaturation and no evidence of

carbonyl or alcohol functional groups.

15

Table 2. Biological Activity of OV-l Fractions.

 

 

 

Retention Percent Percent
Fraction Time Wing-Fanning. Orientation N
Region I 0-1A min 0 17.A 29
Region II lA—15 78.1 16.7 32
Region III 15-30 0 0 32

 

 

N number tested.

Column Temperature = 100°C.

l6

 

 

 

Active
Component

(a. wwf/ 7
i w J
TWW

TR

Figure 3. Quantification of active component. Curve (a)
500 FE active fraction from XF-1150 (peak area=

139.5); (b) 2 ng tridecane external standard
(peak area=138.7).

 

 

 

 

 

 

 

 

 

 

 

 

 

17

Table 3. Biological Activity of Eluent from Reaction Mix-
tures of Characterization Tests.

 

 

 

Percent . Percent
Test Wing-Fanning Orientation N
Blank 71.A 16.7 21
LAH Reduction 5A.5 10.5 22
Acetylation 65.0 7.1 20
Microhydrogenation 0 0 23

 

 

N Number tested.

All eluents were trapped at TR = 1A-15 min from OV-l at 100°C.

l8

GC-Mass Spectrometry of Active Component

GC-MS of a fraction which contained 20 ng of the active

component yielded a weak signal at m/e = 192, and a moderate

signal at m/e 177 (Figure A) when the active component
was ionized. No signals were observed at higher m/e ratios
while signals at m/e ratios lower than this range were
rendered useless due to background interference. However,
the two signals are potentially useful, as signals which
differ by 15 mass units are usually due to the molecular
ion (M+) and the ion resulting from the loss of a methyl
fragment (M-l5).

Figure 5 shows the chromatogram from the GC-MS. Points

of ionization for background and active component mass

spectra are labelled.

19

 

(a)

 

 

WWW

 

 

(b) 160 180 200
m/e
Figure A. (a) Mass spectrum of active component, 20 ng

(b)

sample.
Background (mass spectrum of solvent).

20

 

 

Background Ionization

Active Component
Ionization

 

 

 

 

 

 

\/

TR

 

 

Figure 5.

Chromatogram from GC-MS. 5 ul injection of active
fraction from 10% XF—1150, 5000 FE extract, in CS
GC—MS column: 3% OV—l on 100-120 mesh Gas Chrom Q.
Column Temperature: 125°C programmed at A°C/min

to 150°C.

DISCUSSION

The results from Table II and Figure 1 indicate that

the Bradysia impatiens sex pheromone has been isolated as

 

a single peak in the OV—l chromatogram. No evidence was
present, such as multiple areas of activity on either column,
which would suggest that this peak contains more than one
component.

The strategy of injecting whole extracts onto the OV-l
column, and analyzing the resulting active fraction was in
effect a "clean-up" procedure. This initial fractionation
allows active material to be separated from biologically
inactive nonpolar components which elute with the solvent.
The reinjection of the remaining active fraction onto the
OV-l column similarly allows separation from inactive
polar components.

This gas chromatography clean-up method is preferential
to a standard column chromatography clean-up procedure in
that minimal amounts of solvent are used. While solvent
quantities on the order of milliliters are normally needed
to elute the various fractions from a standard packed
column, only 10-20 ul of solvent were required to rinse
the trapped eluent from the capillary tubes. The latter

method decreases interference from solvent impurities

21

22

which become increasingly important when dealing with
extremely small amounts of sample.

The use of GC-MS precludes the necessity of actually
trapping the isolated active peak in the OV-l chromatogram
of the XF-ll50 active fraction. However, if quantities of
essentially pure active component were desired, they could
be secured by either trapping the eluent at the proper
retention time or by employing a GC splitter.

The limited mass spectral information obtained with 20
ng of active component necessitates a larger sample to be
used in further GC-MS investigations. However, the signals
at m/e = 192 and m/e = 177 are probably due to M+ and M-15
ions. A molecular weight of 192 is indicative of a C-lA
hydrocarbon with three degrees of unsaturation. This
information coupled with the evidence of alkene functionality
encountered in the characterization tests indicates that the
active component iSElC-lA hydrocarbon with at least one
double bond and two other degrees of unsaturation, such as
additional double bonds or rings.

Care must be taken when interpreting the results of
the characterization tests, however. While a lack of
biological response is evidence for a functionality, a
continued response does not necessarily indicate the lack
of that functionality. If the latter assumption is falsely
made, cases are ignored where reactions are proceeding, but
not to completion. One way around this problem would be

to dilute the reaction mixture to'a threshhold concentration

23

and compare with the threshhold concentration of the active
starting material. Any significant deviations would in-
dicate a partial reaction.

The quantification of active material was based on the
assumption that the detector response would be equivalent
for both theclét3external standard and the active component.
This is a valid assumption when using a flame ionization
detector if weight rather than mole quantities are com-
pared.

In conclusion, evidence has been presented which in-
dicates that the female produced sex pheromone of Bradysia

impatiens has been isolated as a single component. Mass

 

spectrosc0py coupled with functional group characterization
tests indicate that this component may be a C-lA hydrocarbon
with three degrees of unsaturation including at least one
double bond. Further investigations using larger quantities
of material will be required to identify the exact struc-

ture of this compound.

BIBLIOGRAPHY

BIBLIOGRAPHY

Alberts, S. A., Kennedy, M. K., and Carde, R. T., 1981.
Pheromone-Mediated Anemotactic Flight and Mating Behavior
of the Sciarid Fly Bradysia impatiens, Environ. Entomol.

 

Carlson, D. A., Mayer, M. S., Silhacek, D. L., James, J.
D., Beroza, M., and Bierl, B. A., 1971. Sex Attractant
Pheromone of the Housefly: Isolation, Identification,
and Synthesis. Science 17A:76.

Fletcher, B. S. 1977. Behavioral Responses of Diptera to
Pheromones, Allomones, and Kairomones. In: Chemical
Control Q; Insect Behavior, Eds. H. S. Shorey and J.
J. McKelvey, Jr. Wiley Interscience.

 

Kennedy, M. K. 1973.- A Culture Method for Bradysia 13¢
patiens (Diptera: Sciaridae). Ann. Entomol. Soc.
Am. 66:1163.

Kostelc, J. G., Girard, J. E., Hendry, L. B. 1980. Iso-
lation and Identification of a Sex Attractant of a
Mushroom - Infesting Sciarid Fly. J. Chem. Ecol. 6:1.

Uebel, C., Sonnet, P. E., Miller, R. W., and Beroza, M.,
197A. Sex Pheromone of the Face Fly, Musca automnalis
(DeGeer). J. Chem. Ecol., 1:195.

Uebel, E. C., Sonnet, P. E., Bierl, B. A., Miller, R. W.,
1975. Sex Pheromone of the Stable Fly: Isolation and
Preliminary Identification of Compounds That Induce
Mating Strike Behavior. J. Chem. Ecol., 1:377.

Uebel, E. C., Meyer, Schwarz, Menzer, R. E., and Miller,
R. W., 1978. Mating Stimulatnt Pheromone and Cuticu-
lar Lipid Constituents of Fannia pusio (Wiedemann),
J. Chem. Ecol. A:73.

 

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