Identification of (Z)-4-tridecene from defensive secretion of green lacewing, Chrysoperla carnea

Article abstract of DOI:10.1023/A:1005587113469

We report the identification of a defensive secretion from the green lacewing, Chrysoperla carnea. By using combined gas chromatography-electroantennographic detection (GC-EAD), we found one major compound in the solvent extract of this secretion that elicited a significant EAD response from the antenna. Based upon its characteristic fragments from gas chromatography-mass spectrometry (GC-MS) analysis, the compound was identified as a tridecene. Dimethyldisulfide derivatization suggested that a double bond was located between positions 4 and 5 in the carbon chain. Thus, the compound was tentatively identified as a 4-tridecene. Coinjection of the extract with a mixture of the Z or E form of the synthetic 4-tridecene revealed that the unknown was (Z)-4-tridecene. EAG dose-responses showed a direct correlation to dose. Single sensillum recordings from sensilla trichodea situated on the antennae suggested the presence of receptor neurons specifically responding to this compound. An arrestment behavior was observed when tested in the Y-tube olfactometer. Preliminary field trapping results indicate that the compound is an antagonist to attraction. The avoidance behavior of predatory ants, observed when tested with the synthetic compound of this secretion further suggested a defensive function.

Full text of DOI:10.1023/A:1005587113469

Journal of Chemical Ecology, Vol. 26, No. 10, 2000
IDENTIFICATION OF (Z)-4-TRIDECENE FROM
DEFENSIVE SECRETION OF GREEN LACEWING,
Chrysoperla carnea
JUNWEI ZHU,1,* RIKARD C. UNELIUS,2 KYE-CHUNG PARK,1
SAMUEL A. OCHIENG,1 JOHN J. OBRYCKI,1 and
THOMAS C. BAKER1
1Department of Entomology, Iowa State University
Ames, Iowa 50010
2Department of Chemistry, Royal Institute of Technology
SE-100 44 Stockholm, Sweden
(Received December 30, 1999; accepted June 5, 2000)
Abstract—We report the identification of a defensive secretion from the green
lacewing, Chrysoperla carnea. By using combined gas chromatography–elec-
troantennographic detection (GC-EAD), we found one major compound
in the solvent extract of this secretion that elicited a significant EAD
response from the antenna. Based upon its characteristic fragments from gas
chromatography–mass spectrometry (GC-MS) analysis, the compound was
identified as a tridecene. Dimethyldisulfide derivatization suggested that a
double bond was located between positions 4 and 5 in the carbon chain.
Thus, the compound was tentatively identified as a 4-tridecene. Coinjection
of the extract with a mixture of the Z or E form of the synthetic 4-tridecene
revealed that the unknown was (Z)-4-tridecene. EAG dose–responses showed
a direct correlation to dose. Single sensillum recordings from sensilla
trichodea situated on the antennae suggested the presence of receptor neurons
specifically responding to this compound. An arrestment behavior was
observed when tested in the Y-tube olfactometer. Preliminary field trapping
results indicate that the compound is an antagonist to attraction. The avoidance
behavior of predatory ants, observed when tested with the synthetic compound
of this secretion further suggested a defensive function.
Key Words—Defensive secretion, green lacewing, Chrysoperla carnea,
(Z)-4-tridecene, electroantennography, single sensillum recording, alarm
pheromone, Chrysopidae, Neuroptera.
*To whom correspondence should be addressed.
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0098-0331 00 1000-2421$18.00 0  2000 Plenum Publishing Corporation
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ZHU ET AL.
INTRODUCTION
The Chrysopidae, the so-called green lacewings have been extensively stud-
ied because many species are predators of agricultural insect pests (New, 1975;
Tauber et al., 2000). Many are resistant to a wide range of insecticides commonly
used in crop pest control, which has led to interest in their use in integrated con-
trol systems (Ridgway and Kinzer, 1974). Larvae or adults of several chrysopids
are predators that feed on soft-bodied insects or their eggs. Lacewings are sub-
ject to predation by other invertebrates, such as ants, or parasitized by some
hymenopteran species (Evans, 1978; Blum, 1981; Ruberson et al., 1995). Many
arthropods have evolved to optimize the effectiveness of their defensive system
as means of deterring predators. We observed that adults of the green lacewing,
Chrysoperla carnea, discharge a secretion with a distinctive odor from a pair of
glands situated in the anterior part of the prothorax. Sometimes, the entire head
is covered with this offensive-smelling, pale yellowish fluid. The constituents
of this secretion could function as defensive compounds against insectivorous
invertebrates or perhaps vertebrates.
Although there has been a steady and significant increase in our knowl-
edge of the chemical communication in arthropods over the past 30 years,
much is yet to be learned, particularly in the defensive chemistry of inver-
tebrates. In the present paper, we report on recordings of coupled gas
chromatography–electroantennographic responses from antennae of adult C.
carnea to their own exuduates. We also identified the main constituent that may
function as an alarm pheromone for defensive purposes. In addition, we made
single sensillum recordings from sensilla trichodea of antennal neurons respon-
sive to the identified chemical and one selected plant volatile, (Z)-3-hexenyl
acetate. Behavioral assays with the synthetic compound conducted in a Y-tube
olfactometer indicated that lacewing adults exhibit arrestment of upwind move-
ment to this compound, and field trapping tests demonstrated antagonistic effects
on attraction of adults in the field.
METHODS AND MATERIALS
Insects and Defensive Secretion Extraction. Larvae of Chrysoperla carnea
were purchased from Rincon-Vitova (Ventura, California) and reared on a regime
of 20 C and 16L : 8D period. Females were mated and produced eggs. Larvae
were fed with pea aphids, Acyrthosiphon pisum, and frozen Ephestia kuekniella
eggs until pupation. Emerged adults were sexed and kept in separate cages with
a supply of aphid honeydew coated leaves and water until they were used in
experiments.
Two methods were used to collect defensive secretions. Three 4-day-old
adults were either dissected into three parts, head with thorax, abdomen, and

(Z)-4-TRIDECENE IN C. carnea
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wings, or adult lacewings were forced to secrete defensive fluids by clasping
their body and wings with a pair of soft foreceps. The fluids were then collected
with a 10-ml micropipet. Both body parts and collected fluids were extracted
with distilled hexane containing an internal standard, (Z)-8-tridecenyl acetate,
for qualitative and quantitative analyses. The extracts were stored at 20 C.
Electrophysiological and Chemical Analysis. A Hewlett Packard 5890 Series
II gas chromatography was equipped with a DB-5 column (30 m × 0.25 mm ID;
J&W Scientific, Folsom, California). An effluent split at 1 : 1 ratio allowed
simultaneous flame ionization (FID) and electroantennographic detection (EAD)
responses of extracts. Helium was used as the carrier gas with a flow rate of approx-
imately 30 ml min for both FID and EAD. Extracts were injected in the splitless
/
mode. The injector temperature was 250 C, and the split valve was opened 1 min
after injection. The column temperature started at 80 C for 1 min following the
injection and then linearly increased to 250 C at a rate of 20 C min. The outlet
/
for the EAD was continuously supplied with a purified airstream flowing over the
antennal preparation at a speed of 0.5 m sec. A dissected lacewing head with its
/
antennae was used for EAD recordings. A capillary recording Ag–AgCl electrode
filled with the electrode gel (Spectra 360, Parker Laboratories, New Jersey) was
placed in contact with the cut tip of the antenna. The other electrode, filled with the
same gel, was connected with the head and served as the ground. The EAD setup
with a high-impedance DC amplifier with automatic baseline drift compensation
was purchased from Syntech (Hilversum, The Netherlands). A GC-EAD program
(version 2.3) developed by Syntech was used to record and analyze the amplified
EAD and FID signals on a Pentium II MICRON computer.
GC-MS analyses of the GC-EAD active defensive compound were per-
formed by using a Hewlett Parkard 5890 Series II gas chromatograph linked
to a Hewlett Parkard 5972 mass selective detector (MSD), which was equipped
with a second DB-5 column used for the GC-EAD analysis described above.
The GC operating conditions were the same as those described for the GC-EAD
analysis. Spectra were recorded from 30 to 550 amu after electronic impact ion-
ization at 70 eV. The chemical identification of the defensive compound was
performed by comparing the retention time and mass spectrum with that of the
synthetic standard.
Electroantennogram Responses and Single Sensillum Recording. EAG
recordings were made by connecting an electrogel-filled (Spectro 360, Parker
Laboratory, New Jersey) glass electrode to the cut distal end of a dissected lace-
wing antenna; the other electrode filled with the same gel was placed in connec-
tion with the base of the antenna. Antennal responses to different doses of the
identified synthetic defensive compound from both sexes were recorded. Serial
dilutions of the tested compound were made in redistilled HPLC-grade hexane
at dosages of 1, 10, 100, and 1000 mg. The tested synthetic compounds were
applied to filter-paper strips (0.5 × 2.5 cm, Whatman No. 1) in 10 ml of solvent.

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ZHU ET AL.
The filter-paper strips were inserted into Pasteur pipet (15 cm long). A control
puff of hexane was applied after each puff of a tested stimulus. The sequence of
exposure to the stimulus compound on each antenna proceeded from the lowest
to the highest concentration.
Single sensillum recordings were performed by using the penetration tech-
nique (Hubel, 1957) with tungsten electrodes. Tungsten wire electrodes (0.2 mm
diam.) were sharpened electrolytically to a tip diameter <0.3 mm. A C. carnea
adult was restrained with wire loops on a dental wax stub, and the antennae were
fixed in the desired position. The tip of the recording electrode was inserted at the
sensillum base by using a micromanipulator; the other electrode was inserted into
the lacewing abdomen with a second micromanipulator. The recording electrode
was connected to a high impedance amplifier (Syntech) that was also connected
to a loudspeaker to provide an indication of the quality of the contact. The signal
was recorded on a Vetter model 420F four-channel FM recorder. The recorded
responses were transferred from tapes to a Micron Millennia Pentium computer
and analyzed by using a Syntech analog-to-digital interface with Autospike for
Windows 95 software (Syntech).
Alkylthiolation for Determining Double Bond Position. Dimethyldisulfide
(DMDS) derivatization of the extract was conducted as described in Buser et al.
(1983). First 100 ml of DMDS (Sigma) and 10 ml of iodine solution (120 mg I2
in 4 ml diethyl ether) were added to the head and thorax extracts of 10 lacewing
equivalents. The reaction was performed at approximately 40 C for 12 hr. After
adding 100 ml Na2S2O3 solution to reduce the excessive amount of iodine to
iodide, 1 ml hexane was added to extract the product. The hexane layer was
then transferred and concentrated to 200 ml, and 2 ml were injected for GC-MS
analysis.
Chemicals. (Z)-4-Tridecene was prepared by a Wittig reaction as described
below. Potassium t-butoxide (13.4 g, 120 mmol) was dissolved in 100 ml tetra-
hydrofuran (THF), and n-butyltriphenylphosphonium bromide (16 g, 40 mmol)
was added to the stirred solution. After reflux for 30 min, the t-butanol formed
was distilled off (10 ml). The reaction was cooled to room temperature, and a
solution of octanal (5.68 g, 40 mmol) in THF (6 ml) was added. The result-
ing mixture was stirred for 2 hr at 25 C before it was poured into cold satu-
rated NH4Cl (100 ml). The organic phase was separated, and the aqueous phase
extracted with pentane (2 × 50 ml). The combined organic phases were then
dried with MgSO4. After filtration, the product was subjected to fractional dis-
tillation. The yield was 4.4 g in an isomeric purity of 94% of Z isomer by GC-
MS. 1H NMR (CDCl3) d: 5.31–5.40 (m, 2H), 1.98–2.04 (m, 4H), 1.27–1.41 (m,
14H), 0.90 (t, 3H), 0.88 (t, 3H). 13C NMR (CDCl3 d: 130.1, 129.6, 31.9, 29.8,
29.5, 29.3, 29.3, 27.2, 27.2, 22.9, 22.7, 14.1, 13.8. MS m z (relative intensity):
/
182 (M, 18), 125 (3), 111 (10), 97 (20), 83 (33), 69 (39), 56 (39), 55 (80), 43
(43), 41 (100).

(Z)-4-TRIDECENE IN C. carnea
2425
Behavioral Response Y-Tube Olfactometer, Defensive Evaluation, and Field
Trapping. The behavioral response of adult lacewings to the identified defensive
secretions and or alarm pheromone was first conducted under laboratory condi-
/
tions (23 C and daylight intensity) in the Y-tube olfactometer. In the olfactome-
ter, freshly field-collected lacewings were offered a choice between the synthetic
(Z)-4-tridecene (500 mg) and the control (hexane). The numbers of C. carnea
adults entering either chamber were recorded. The duration of each choice test
was limited to no more than 5 min. Control runs were also performed in the
absence of any test stimuli to ensure that there was no bias for a particular cham-
ber of the olfactometer.
The defensive value of this compound was evaluated by placing one honey
bar inside each of two Petri dishes with Crematogester and Tapinoma ant species
(N 20). The honey bar in one of the dishes was encircled by a 3-mm-wide ring
of the synthetic (Z)-4-tridecene (5 mg). The circle, outlined in pencil, was about
10 mm from the edge of the honey bar. The other Petri dish served as the control,
with only the solvent application. The ants’ forging behavior was observed, and
the number of times ants avoided or retracted from the treated source after an
attempted approach was recorded.
A field trapping test to investigate possible antagonistic effects of the com-
pound on lacewing attraction was conducted in an alfalfa field in Ames, Iowa
(May–July, 1999). Synthetic compounds were prepared in hexane or methy-
lene chloride, and medical peerless cotton rolls (5 cm long, 100% cotton) were
used for dispensers. The loading dosage for baits containing 2-phenylethanol (a
known attractant for C. carnea) (Zhu et al., 1999) or (Z)-4-tridecene was 10
mg. Traps similar to Rebell-type traps, made of four Pherocon AM sticky traps
back to back, were used for trapping in the field test (Trece Inc.). Traps were
´ ´
hung from the bent end of metal posts, 10–15 cm above the canopy of alfalfa
plants. Within a replicate (N 3), traps were set at least 10 m apart. Traps were
checked every other day for six days, and the trap position within a series was
randomzied to minimize the effects of habitat heterogeneity.
Scanning Electron Microscopy. Antennae of adult C. carnea were first
fixed in 70% ethanol, then dehydrated in absolute ethanol, critical point-dried,
and coated with gold–palladium alloy (60 : 40) before examination under a Jeol
5800LV scanning electron microscope operated at 10–15 kV.
RESULTS AND DISCUSSION
GC-EAD analyses of the secretion exuded by both sexes of C. carnea
demonstrated the presence of one minor and one major peak, with only the latter
eliciting a significant EAG response from antennae (Figure 1). GC-MS analy-
ses of extracts showed that the main compound contained characteristic ions of

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ZHU ET AL.

(Z)-4-TRIDECENE IN C. carnea
2427
FIG. 2. Mass spectrum of the EAD active compound from extracts of the defensive secre-
tion of adult green lacewings, Chrysoperla carnea.
an alkene, with a molecular ion at m z 182 (Figure 2). The fragmentation pat-
/
terns and mass spectrum suggested a tridecene. Thus, an alkylthiolation was car-
ried out to determine the double-bond position. After dimethyldisulfide (DMDS)
derivatization of the extracts, the characteristic mass fragments with ions at m z
/
103 and 173 and the molecular ion at m z 276 indicated that the double bond of
/
this tridecene was between carbons 4 and 5 (Figure 3). Geometric isomerism of
the 4-tridecene was determined by coinjection of synthetic 4-tridecene contain-
ing both the Z and E isomers together with the lacewing extracts. The E isomer
eluted earlier than the Z isomer, and the Z isomer coeluted with the EAG active
compound, which, therefore, was assigned as the (Z)-4-tridecene. Quantitative
analyses of extracts showed about 5.5 mg of (Z)-4-tridecene from the secretion
of a single female and 3.9 mg from one male.
EAG responses of both sexes of C. carnea to (Z)-4-tridecene increased with
increasing doses, and the highest response was observed at a dose of 1000 mg
(Figure 4). By using the tungsten recording technique, we successfully recorded
the response to (Z)-4-tridecene from single sensilla trichodea situated on the
antennae (Figure 5), but not to one of the most dominant components from
alfalfa, (Z)-3-hexenyl acetate (Figure 6). To our knowledge, this is the first sin-
gle sensillum recording conducted on a lacewing species. The response to (Z)-
4-tridecene consisted of a large variety of action potential amplitudes, which

2428
ZHU ET AL.
FIG. 3. Mass spectrum of DMDS-derivates of the EAD active compound from extracts
of the defensive secretion of adult Chrysoperla carnea showing diagnostic mass spectral
fragments of 4-tridecene.
suggests the presence of at least two receptor neurons in the trichoid sensillum.
Of special interest is the discovery that both sexes of C. carnea can detect
(Z)-4-tridecene, based on the significant EAG responses elicited from the anten-
nae during stimulation. Furthermore, both sexes responded behaviorally to (Z)-4-
tridecene, with 87% of both deterred from entering the arm of the Y-tube olfac-
tometer with (Z)-4-tridecene (Figure 7). This suggests that the compound may
also function as an alarm signal for nearby conspecific individuals, in addition
to being a possible defensive compound against predators. In addition, 93% of
tested ants avoided the sugar bar surrounded with the synthetic (Z)-4-tridecene.

(Z)-4-TRIDECENE IN C. carnea
2429
FIG. 4. EAG dose–response of male and female green lacewings, Chrysoperla carnea,
to the identified defensive component, (Z)-4-tridecene. Means ( standard error) with
different letters are significantly different (ANOVA followed by FPLSD test, P < 0.05).
In contrast, more than 95% of tested ants fed on the sugar bar without the appli-
cation (Figure 8). Our preliminary field trapping results demonstrated antagonis-
tic effects, with only two lacewings caught in traps baited with the compound
together with 2-phenylethanol, a newly identified attractant (Zhu et al., 1999). In
contrast, an average of more than 15 lacewings were captured in traps containing
only 2-phenylethanol.
Alkenes, such as 4-tridecene, have been reported as constituents of the
Dufour’s gland secretion of several ant species (Lanne et al., 1988). These chemi-
cals combined with other straight chain or branched hydrocarbons or terpenes
function as alarm, territory, or trail signals or act as defense signals in several
subfamilies of Formicidae (Attygalle and Morgan, 1984). This study is the first
report of (Z)-4-tridecene identified from the putative defensive secretion of a
lacewing species. Adults of C. carnea are active mostly at night. During the
day, when they are resting on vegetation, they can become prey to invertebrate
predators, such as ants, wasps, and beetles (Blum and Wallace, 1973; Jones et
al., 1977; Evans, 1978; Clancy, 1946). Because of the possible alarm and defen-
sive nature of (Z)-4-tridecene in ant species, C. carnea might be able to use this
compound for defensive purposes. It might also be effective for defense against
vertebrate predators, for example, birds and bats.

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ZHU ET AL.

(Z)-4-TRIDECENE IN C. carnea
2431
FIG. 6. Single-sensillum recordings from sensillum trichodeum of a female green lace-
wing, Chrysoperla carnea, stimulated for 0.2 sec (bar) with hexane (A), 100 mg of (Z)-
4-tridecene (B), and 100 mg of (Z)-3-hexenyl acetate (C).
FIG. 7. Behavioral response of Chrysoperla carnea to 500 mg of (Z)-4-tridecene in the
Y-tube olfactometer. Bars represent the mean SE. Different letters at the top of bars
indicate means are significantly different at P < 0.05 (Student’s t test).

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ZHU ET AL.
FIG. 8. Percentages of ants touching the sugar sources with or without treatment with (Z)-
4-tridecene. Bars represent the mean SE. Different letters at the top of bars indicate
means are significantly different at P < 0.05 (Student’s t test).
Blum and Wallace (1973) found skatole and 1-tridecene in the secretions
of another lacewing species, Chrysopa oculata. It is interesting that the two dif-
ferent lacewing species produce quite specific defensive secretion constituents.
One possible explanation could be that these two species have different predatory
ant species, which produce different alarm pheromones, since both 1-tridecene
and (Z)-4-tridecene have been identified from the Dufour glands of several ant
species (Lanne et al., 1988). In contrast to many other species of chrysopids not
producing defensive secretions, the currently investigated C. carnea and previ-
ously reported C. oculata do produce secretions and have been cited as being the
only two odorous lacewing species (McLachlan, 1874). The advantage of pro-
ducing defensive secretions could be one of the selective forces for maintaining
their populations, and partly explain why they are two of the more common
species in Europe and North America.

(Z)-4-TRIDECENE IN C. carnea
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Sound communication between the two sexes of conspecific lacewing
species has been widely studied (Henry, 1979). However, chemical communica-
tion in lacewings has not been as well studied as in other invertebrate species,
such as moths, ants, and wasps. Two recently published papers reported that
adult C. carnea and C. cognata respond to volatiles released from either their
aphid prey or plants (Boo et al., 1998; Zhu et al., 1999). Here we demonstrate
that C. carnea adults not only produce defensive secretions against predation,
but probably also use this compound as an alarm pheromone.
Acknowledgments—The authors would like to express their thanks to Bess Lewis, Anna Keyte,
and Brad Tucker for rearing the predatory insects and to Marcus Ruda for helping with the chemi-
cal synthesis. We also thank Tracey Pepper for assisting on scanning electron microscope at the
ISU Bessey Microscopy Facility. This project was supported by a grant (98-72) from the Leopold
Center for Sustainable Agriculture. Journal Paper No. J-18702 of the Iowa Agricultural and Home
Economics Experiment Station, Ames, Iowa, Project No. 3437, and supported by Hatch Act and
State of Iowa Funds.
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