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Jurenka and Subchev
108
Archives of Insect Biochemistry and Physiology 43:108–115 (2000)
Identification of Cuticular Hydrocarbons and the Alkene
Precursor to the Pheromone in Hemolymph of the Female
Gypsy Moth, Lymantria dispar
Russell A. Jurenka1* and Mitko Subchev2
1
2
Department of Entomology, Iowa State University, Ames
Institute of Zoology, Bulgarian Academy of Sciences, Sofia, Bulgaria
Hydrocarbons were extracted from the surface of the cuticle
and from the hemolymph of adult female gypsy moths. GC and
GC/MS analysis indicated that the cuticular hydrocarbons with
chain lengths >21 carbons were the same as those found in the
hemolymph. These consisted of mostly saturated straight chain
hydrocarbons with heptacosane the major component. Methyl
branched hydrocarbons were also identified including a series
of tetramethylalkanes with chain lengths of 30, 32, and 34 carbons. In addition to those found on the cuticle surface, the
hemolymph contained the alkene pheromone precursor, 2methyl-Z7-octadecene and two saturated analogues, 2-methyloctadecane and 2-methyl-hexadecane. No evidence was
obtained for the presence of the pheromone 2-methyl-7,8-epoxy-octadecane in the hemolymph. Pheromone gland extracts
indicated that small amounts (<1 ng) of the alkene precursor were also present in the gland. Relatively larger amounts
of the alkene precursor were found in the hemolymph at the
time when pheromone titers were higher on the gland. The
presence of the hydrocarbon pheromone precursor in the
hemolymph is discussed in relation to possible biosynthetic
pathways for producing the gypsy moth pheromone. Arch.
Insect Biochem. Physiol. 43:108–115, 2000. © 2000 Wiley-Liss, Inc.
Key words: disparlure; hemolymph transport; tetramethylalkane; methylbranched alkane
INTRODUCTION
Female moths usually utilize a blend of
chemical components for attracting a conspecific
mate. These chemical components are based on a
10-21 hydrocarbon chain with an oxygenated functional group that includes esters, aldehydes, and
alcohols. Several families of moths utilize hydrocarbons or epoxides of hydrocarbons as the sex
pheromone. This group includes the families
Geometridae, Arctiidae, Lymantriidae, and some
Noctuidae (Arn et al., 1992).
© 2000 Wiley-Liss, Inc.
Abbreviations used: Shorthand notation for hydrocarbons is
as follows: XX is the number of carbons in the linear chain,
n- = straight chain alkane, me = methyl group. Thus n-C27
= n-heptacosane, 2-me-C18 = 2-methyl-octadecane, 2-me-Z7C18 = 2-methyl-Z7-octadecene.
Contract grant sponsor: NSF; Contract grant number:
INT9813419.
*Correspondence to: Russell A. Jurenka, Department of Entomology, Iowa State University, 411 Science II, Ames, IA
50011-3222. E-mail: [email protected]
Received 16 September 1999; accepted 22 November 1999
Pheromone Precursor in Hemolymph
Hydrocarbons are also found on the cuticle
where they aid in maintaining internal water balance by preventing desiccation (Blomquist et al.,
1987). Almost all insects have at least some hydrocarbon on their surface. For those moths that utilize hydrocarbons or derivatives of hydrocarbons as
a sex pheromone it is of interest to identify the hydrocarbons found on the cuticular surface. These
insects may utilize related longer chain hydrocarbons as protectants against desiccation.
The gypsy moth, Lymantria dispar (Lymantriidae), is thought to utilize a single component
for male attraction (Bierl et al., 1970). Disparlure,
(2-methyl-7,8-epoxy-octadecane), is utilized by several different Lymantria species as a sex attractant
(Arn et al., 1992). Several related compounds were
recently identified from the nun moth, Lymantria
monacha, that are thought to play a role in species
separation (Gries et al., 1996). In fact, the alkene
precursor to disparlure is thought to be attractive
to male nun moths (Grant et al., 1996) but inhibitory to male gypsy moths (Cardé et al., 1973).
The biosynthetic pathways for most of the
hydrocarbon and epoxide pheromone components
utilized by moths is unknown. The di- and triunsaturated components with n-6 or n-3 double
bond configurations are probably derived from linoleic and linolenic acids, respectively. This was
demonstrated for the Arctiid moths, Estigmene
acrea and Phragmatobia fuliginosa, where linolenic acid was shown to be incorporated into the
21-carbon pheromone component Z3, Z6-9,10-epoxy-heneicosadiene (Rule and Roelofs, 1989). In
addition, it was shown that 2-methyl-heptadecane, found in the arctiid moths belonging to
the genus Holomelina, was biosynthesized de
novo with the amino acid leucine contributing
the methyl carbon (Charlton and Roelofs, 1991).
These studies were conducted by injecting labeled precursor into pupae or adults in order
to test for incorporation into pheromone. Recently, it was demonstrated that the hydrocarbon pheromone, at least in Holomelina
aurantiaca, was biosynthesized in cells found
associated with the epidermis (oenocytes) and
transported to the pheromone gland by the lipid
carrier lipophorin (Schal et al., 1998). Thus, the
pheromone glands in H. aurantiaca do not
biosynthesize the pheromone but store and release it during the calling period.
109
It is unknown how the gypsy moth makes
disparlure, which has both a methyl group and
an epoxide. Only one study has demonstrated that
the precursor 2-methyl-Z7-octadecene (2-me-Z7C18) was converted to the epoxide pheromone if
injected into pupae (Kasang and Schneider, 1974).
Here we report that this precursor is found in
the hemolymph of female gypsy moths and is correlated with the titer of disparlure found in the
pheromone gland. We also report the identification of other hydrocarbons found in the hemolymph and on the cuticle.
MATERIALS AND METHODS
Insects
Female pupae were obtained from the Gypsy
Moth Rearing Unit at the Otis Plant Protection
Center (Otis ANGB, MA) and were kept at a 16:8
L:D cycle and 22°C. They emerged in approximately 7–8 days.
Hydrocarbon Extraction
Pheromone glands, which are located in the
intersegmental membrane between the 8th and
9th segments (Hollander et al., 1982), were collected by excising the 8th segment with scissors
and immediately placing the terminal portion of
the abdomen in solution for extraction. If the
glands were placed in hexane, they were removed
after 5 min. If placed in chloroform:methanol (1:2
v:v) they were removed after 24 h. Hemolymph
was collected by puncturing the cuticle on the dorsal side of the abdomen and gently squeezing the
insect. The exuded hemolymph was collected with
a microcapillary pipet. The amount of hemolymph
collected was recorded and mixed with an equal
volume of methanol. Lipids were extracted with
hexane and then purified by a small silica gel column (40 mg in a 2 × 90 mm glass pipet). Hydrocarbons were eluted with hexane and epoxides
with 90:10 hexane:ether. Whole adult females
were extracted by immersing in hexane (approximately 1 ml per female) for 5 min followed by
another 1 ml hexane rinse for 1 min. This extract was then purified by a silica gel column (300
mg in a 6 × 145 mm glass pipet) and hydrocarbons eluted with hexane.
110
Jurenka and Subchev
Chromatography
A Hewlett-Packard 5890 GC equipped with
a flame ionization detector and an EC-1 (Alltech,
Deerfield, IL) capillary column (30 m × 0.25 mm)
was used for analysis. The oven was temperature
programmed at 80°C for 1 min, then 20°/min to
150°, and then 7°/min to 320°C and held for 20
min. A Hewlett-Packard 5972 series mass selective detector was used to obtain mass spectra. It
was connected to a HP-5890 GC with a DB-5
(J&W Scientific, Folsom, CA) capillary column (30
m × 0.25 mm) that was temperature programmed
at 80°C for 1 min, then 10°C/min to 320°C and
held for 20 min. For quantification Z9-heneicosene
was used as an internal standard. Dimethyl disulfide derivatives were made as described (Francis and Veland, 1981) and purified by a silica gel
column eluted with hexane:ether (9/1 v/v).
RESULTS
Hexane extracts of the entire female were purified and the hydrocarbon fraction analyzed by GC.
Identifications were confirmed by GC/MS (Fig. 1 and
Table 1). n-Alkanes (C21-C32) made up the majority of hydrocarbons found on the cuticle of 2-dayold female moths. Heptacosane (n-C27) was the
major hydrocarbon found, comprising about 40% of
the total. Methyl-branched hydrocarbons collectively
made up about 30% of the total. Of these, only two
were found to have a methyl group in the 2 position and had chain lengths of 30 and 32 carbons.
These were identified by relative elution position
and mass spectra showing a strong M-43 ion and
ion 69 >70. The other methyl-branched hydrocarbons were identified based on equivalent chain
lengths and mass spectrometry diagnostic ions
(Table 1). The major methyl-branched hydrocarbons
were identified as a 11,15-dimethyl series with chain
lengths of C31, C33, and C35 and a series of
tetramethylalkanes that coeluted with 7-methylC31, -C33, and -C35. Shown in Figure 2A is a partial mass spectrum of the peak that contains
7-methylhentriacontane (7-me-C31) and 4,8,12,16tetremethyltriacontane (4,8,12,16-tetrame-C30). We
interpret this mass spectrum to indicate the presence of the 7-methyl branch based on the abundance of ion fragment at 112 and 31 carbons based
on a 0.6 reduction in elution time relative to C32.
The tetramethylalkane was indicated by the pres-
ence of the odd-mass fragments at 141, 211, and
281, which suggest methyl branches at carbon 8,
12, and 16, respectively. Odd-numbered mass
fragments usually indicate the presence of at least
a second methyl branch in the fragment, thus the
141 indicates another methyl group in the fragment (Blomquist et al., 1987). The even-mass
fragment 224 indicates an internal cleavage on
methyl-branched carbon 16. The odd-mass fragments at 295, 365, and 435 suggest methyl
branches at carbon 12, 8, and 4, respectively. In
addition, ion 463 (M-15) indicates a M = 478,
which is consistent with a hydrocarbon having a
total of 34 carbons. The other tetramethyl hydrocarbons with chain lengths of C32 and C34 were
identified in a similar manner. These mass spectra are consistent with those published by Nelson
et al. (1988) on the identification of tetramethyl
alkanes in the teste fly, Glossina brevipalpis.
Hemolymph, collected from 2-day-old adult
females, was extracted with hexane and the hydrocarbons purified. GC/MS analysis indicated
that these hydrocarbons were the same hydrocarbons that were found on the cuticular surface
(Fig. 1 and Table 1). In addition, the alkene 2me-Z7-C18 was found as well as its saturated
analogue. These were identified based on retention times and mass spectra (Fig. 2B,C). The alkene 2-me-Z7-C18 has a base peak of 56 and a M
= 266. The spectra was identical to an authentic
standard (Sigma Chem. Co., St. Louis, MO). The
position of the double bond was determined by
dimethyl disulfide derivatives with the results indicating a double bond at carbon 7. Mass spectrometry of the derivative indicated prominent
ions at 159 and 201, with a molecular ion at 360
(Fig. 2D). The double bond configuration was not
determined but is presumed to be Z as indicated
in a previous identification from compounds isolated from the pheromone gland (Bierl et al.,
1972). The saturated analogue, 2-me-C18, had an
equivalent chain length 0.3 longer and had a base
peak of 57, which is typical for saturated hydrocarbons. It was identified as 2-methyl due to the
prominent M-43 ion at 225 and ion 69 >70 (Fig.
2B). Also identified was 2-me-C16, which had an
M-43 at 197 and ion 69 >70 (mass spectra not
shown). The 16 and 18 carbon compounds were
not found on cuticular washes of females but were
found in extracts of pheromone glands.
Pheromone Precursor in Hemolymph
111
Fig. 1. Representative partial chromatograms of the hydrocarbons obtained from the cuticle surface (A) and ex-
tracted from the hemolymph (B) of 2-day-old adult female
L. dispar.
The percent composition of the hydrocarbons
identified indicated similar distribution between
those found on the cuticle and in the hemolymph
(Table 1). However, there was a larger total percentage of methyl-branched hydrocarbons found
in the hemolymph.
The time of appearance of the alkene in the
hemolymph with pupal and adult development
was determined. Small amounts of 2-me-7-C18
were first observed in pharate adults about 3 h
before eclosion (Table 2). The amount of the alkene increased after eclosion and was found to
reach about 1 ng/µl hemolymph in 24-h-old females. The hemolymph of older females (up to 4
days old) also contained about the same amount
of the alkene and its saturated analogue as did
24-h-old females (data not shown). Disparlure was
first detected on the pheromone gland in 3-h-old
112
Jurenka and Subchev
TABLE 1. Hydrocarbons Found on the Cuticle and in the Hemolymph of 2-Day-Old Adult Female
Lymantria dispar
% Compositionb
Hydrocarbon
ECLa
2-me-C16
2-me-Z7-C18
2-me-C18
n-C21
n-C22
n-C23
n-C24
n-C25
n-C26
n-C27
n-C28
n-C29
n-C30
2-me-C30
n-C31
7-me-C31 +
4,8,12,16-tetrame-C30
16.6
18.3
18.6
21.0
22.0
23.0
24.0
25.0
26.0
27.0
28.0
29.0
30.0
30.6
31.0
31.4
11, 15-dime-C31
n-C32
2-me-C32
7-me-C33 +
4, 8, 12, 16tetrame-C32
11, 15-dime-C33
7-me-C35 +
4,8,12,16-tetrame-C34
31.6
32.0
32.6
33.4
11, 15-dime-C35
a
b
33.6
35.4
35.6
Diagnostic ions
197 (M-43)
266 (M+) 56 base
225 (M-43)
296
310
324
338
352
366
380
394
408
422
393 (M-43) 69>70
436
112
141, 211, 281, 224, 295,
365, 435, 463, (M-15)
168, 323; 239, 252
450
421 (M-43) 69>70
112
141, 211, 281, 252, 323,
393, 463, 491 (M-15)
168, 351, 239, 280
112
141, 211, 281, 280, 351,
421, 491
168, 379, 239, 308
% n-alkanes
% methylalkanes
Cuticle
Hemolymph
—
—
—
0.1 ± 0.1
0.2 ± 0.3
1.5 ± 0.3
0.2 ± 0.1
5.0 ± 2.0
1.5 ± 0.3
38.8 ± 3.6
1.8 ± 0.7
18.4 ± 3.3
0.7 ± 1.0
1.1 ± 0.4
1.3 ± 0.2
0.3 ± 0.3
1.8 ± 0.9
1.1 ± 0.9
0.6 ± 0.7
0.3 ± 0.2
9.0 ± 3.0
0.6 ± 0.2
11.1 ± 2.7
1.9 ± 0.4
26.1 ± 4.8
0.8 ± 0.4
5.7 ± 2.4
0.4 ± 0.3
0.2 ± 0.1
0.2 ± 1.6
5.1 ± 2.2
11.8 ± 7.8
2.8 ± 1.0
0.8 ± 0.9
2.5 ± .5
3.7 ± 0.8
3.8 ± 1.2
0.2 ± 0.1
1.8 ± 1.9
6.8 ± 1.8
7.1 ± 1.6
2.9 ± 0.6
5.6 ± 2.0
4.6 ± 0.4
4.7 ± 1.4
70.2
29.8
2.1 ± 1.4
58.4
41.6
ECL = equivalent chain length.
Values are the means ± S.D., n = 4.
females. In contrast, the longer chain hydrocarbons (> C21) were present in the hemolymph of
pharate adults at least 24 h before eclosion but
the alkene was not found (data not shown).
DISCUSSION
The hydrocarbons found on the cuticle surface of female L. dispar contained primarily
straight chain alkanes with heptacosane as the
major alkane. The major methyl-branched components were a series of tetramethyl even-numbered alkanes and a series of 11,15-dimethyl
odd-numbered alkanes. The tetramethylalkanes
coeluted with a 7-methyl odd-numbered alkane
and based on the mass spectra the tetramethyl
alkanes made up most of the peak area. Tetra-
methylalkanes have been identified in a number
of different insects including Glossina brevipalpis
where 4,8,12,16-tetramethyl alkanes have been
described (Nelson et al., 1988). The methylbranched hydrocarbons found in the gypsy moth
started with a chain length of 30 carbons and no
shorter chain length methyl-branched alkanes
were found on the cuticle.
A relatively small amount of the 2-methyl
branched hydrocarbons were present. Of those
identified, only 2-me-C30 and 2-me-C32 were
found on the cuticle and these comprised only
about 3.5% of the total hydrocarbons. The pheromone disparlure has a 2-methyl branch with 18
carbons in the linear chain and we found that
this branching pattern is not utilized to a great
extent in longer chain alkanes. Instead, the
Pheromone Precursor in Hemolymph
Fig. 2. Mass spectra of the GC peaks containing (A)
4,8,12,16-tetrame-C30 + 7-me-C31, (B) 2-me-C18, and (C)
2-me-7-C18 obtained from extracts of the hemolymph of 2day-old adult female L. dispar. D: Mass spectrum of the dimethyl disulfide derivative of 2-me-7-C18.
branching pattern for the longer chain alkanes is
more internal starting at carbon four. Also no alkenes were found on the cuticle surface. Thus, of
the hydrocarbons found on the surface of the cuticle, none are of the same homologous series as
the hydrocarbon pheromone precursor. This indicates that the 2-methyl branching pattern and
unsaturation is reserved for the biosynthesis of
113
pheromone. It will be of interest to find out if
other moths that utilize hydrocarbons or derivatives of hydrocarbons as sex pheromones also do
not have similar homologous hydrocarbon structures on the cuticle surface. This is in contrast to
other insects where hydrocarbon pheromone biosynthesis has been studied in detail. In the house
fly, Musca domestica, the sex pheromone component Z9-C23 is produced by vitellogenic females,
whereas previtellogenic females do not have this
hydrocarbon but instead have longer chain length
homologous C27, C29, and C31 alkenes (Dillwith
et al., 1983). During female adult maturation, the
chain length of the alkenes is altered to produce
the shorter chain length sex pheromone component by a change in the elongation enzymes
(Tillman et al., 1992). In the German cockroach,
Blattella germanica, the oxygenated (ketone)
pheromone components have the same methylbranching pattern and chain length as hydrocarbons found on the cuticular surface (Jurenka et
al., 1989). These examples indicate that hydrocarbons normally found on the cuticle can be altered and used as sex pheromones.
Hydrocarbons are thought to be produced by
specialized cells called oenocytes that are associated with abdominal epidermal cells or fat body
(Diel, 1975; Wigglesworth, 1970). The hydrocarbons are transported to epidermal cells throughout the body by the lipid carrier protein lipophorin
(Chino, 1985). Lipophorin is a multifunctional
transport protein in that it will carry a variety of
lipophilic compounds including sex pheromones.
The hydrocarbon sex pheromones of Drosophila
melanogaster were shown to be transported by
lipophorin (Pho et al., 1996). The methyl-ketone
sex pheromone of female German cockroaches was
shown to be carried by lipophorin and deposited
on the cuticle surface (Gu et al., 1995). Recently,
it was proposed that the hydrocarbon pheromone
2-me-C17, utilized by the arctiid moth, Holomelina aurantiaca, was biosynthesized by oenocytes and transported to the pheromone gland by
lipophorin (Schal et al., 1998). The sex pheromone
was specifically unloaded at the pheromone gland
because very little of the longer chain-length hydrocarbons were found in the gland. Thus, the
pheromone glands in Holomelina do not biosynthesize the pheromone but store and release
it during the calling period (Schal et al., 1998).
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Jurenka and Subchev
TABLE 2. Pheromone (Disparlure), Pheromone Precursor (2-me-Z7-C18), and 2-me-C18 Amounts Found in the
Pheromone Gland and Hemolymph of L. dispar Females of Different Ages*
Hemolymph (ng/µl)b
Pheromone gland (ng/gland)
Age
a
Pharate adult
Newly eclosed
3h
24 h
Disparlure
2-me-Z7-C18
2-me-Z7-C18
2-me-C18
< 0.1 (4)
< 0.1 (4)
0.7 ± 0.3 (4)
6.9 ± 1.9 (8)
< 0.1 (4)
< 0.1 (4)
< 0.1 (4)
0.7 ± 0.3 (8)
0.09 ± 0.02 (7)
0.04 ± 0.01 (4)
0.30 ± 0.08 (4)
1.28 ± 0.27 (8)
0.05 ± 0.01 (7)
0.02 ± 0.01 (4)
0.15 ± 0.03 (4)
0.50 ± 0.10 (8)
*Values are the means ± S.D., n = number in ( ).
a
Pharate adults were sampled about 3 hrs before eclosion. Newly eclosed were sampled at 15 to 30 min after eclosion;
3 and 24 hr indicate sampling times after eclosion.
b
Mean amounts of hemolymph that were collected for each age: pharate, 100 µl; newly eclosed, 200 µl; 3 hr, 125 µl; 24 hr,
60 µl.
We are proposing that a similar process occurs in the gypsy moth where the alkene precursor to the pheromone is produced in oenocytes
and transported by lipophorin to the pheromone
gland. The alkene precursor would be picked up
by pheromone gland cells and then converted to
the epoxide disparlure. In support of this hypothesis, we found the alkene in the hemolymph along
with two saturated homologues, 2-me-C18 and
2-me-C16. These compounds were not found on
the cuticle, but were found associated with the
pheromone gland. In addition we did not find
any evidence for the presence of disparlure in
the hemolymph. Also, we found the alkene precursor levels increased at about the same time
as the levels of disparlure increased on the
gland in adult females.
Relatively large amounts of the alkene precursor were found in the hemolymph of 24-h-old
females (about 77 ng total) compared to the
amount of disparlure in the gland (about 7 ng/
gland). This may indicate that the conversion rate
of the alkene to the epoxide is relatively slow or
that the enzymes involved in making the epoxide
are low in abundance. However, a previous study
has shown that the female will continually release pheromone throughout the photoperiod with
an average of about 7 ng/hr from a laboratory
reared population (Charlton and Cardé, 1982).
Therefore, the female is apparently continuously
synthesizing disparlure for release. Future work
is needed to understand the rate of emission compared to the biosynthesis of disparlure.
Hydrocarbons are biosynthesized through
fatty acid biosynthesis and when an appropriate
chain-length is reached the carboxyl group is removed resulting in a hydrocarbon one carbon
shorter (Blomquist et al., 1987). Apparently only
the oenocytes have a decarboxylation type of enzyme system for removing the carboxyl group
from fatty acids to produce a hydrocarbon. The
evidence available thus far indicates that this enzyme system has not evolved in any other cells.
Thus, those insects that utilize hydrocarbons or
derivatives of hydrocarbons, for specialized
functions, such as in chemical communication,
the hydrocarbon must be biosynthesized in the
oenocytes and then transported to a target tissue such as a pheromone gland (Schal et al.,
1998). This is in contrast to other moth pheromone glands that are capable of biosynthesizing
the pheromone de novo from acetate (Jurenka
and Roelofs, 1993).
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