Journal of Chemical Ecology, Vol. 31, No.

12, December 2005 ( #2005)

DOI: 10.1007/s10886-005-8406-z

ELECTROPHYSIOLOGICAL AND BEHAVIORAL

RESPONSES TO CHOCOLATE VOLATILES
IN BOTH SEXES OF THE PYRALID MOTHS
Ephestia cautella AND Plodia interpunctella

P.-O. CHRISTIAN OLSSON,1,* OLLE ANDERBRANT,1

CHRISTER LÖFSTEDT,13 ANNA-KARIN BORG-KARLSON,2
and ILME LIBLIKAS3
1
Department of Ecology, Lund University, Ecology Building, SE-223 62 Lund, Sweden
2
Organic Chemistry, Royal Institute of Technology, Teknikringen 30,
SE-100 44 Stockholm, Sweden
3
Laboratory of Ecochemistry, Estonian Agricultural University,
EE-510 05 Tartu, Estonia

(Received November 15, 2004; revised August 11, 2005; accepted August 28, 2005)

Abstract—Volatiles from chocolate mediate upwind flight behavior in

Ephestia cautella and Plodia interpunctella. We used gas chromatography
with electroantennographic detection and found 12 active compounds derived
from three different chocolate types, i.e., plain, nut-containing, and rum-
flavored. Eight of the compounds were identified with mass spectrometry, and
the activity of three compounds, ethyl vanillin, nonanal, and phenylacetalde-
hyde (PAA), was subsequently confirmed in both electrophysiological and
behavioral assays. In the electroantennogram experiment, PAA and nonanal
were consistently eliciting responses in both species and sexes. Ethyl vanillin
was active in males of both species, and also in P. interpunctella females.
E. cautella females showed no antennal activity in response to ethyl vanillin.
All three volatiles were attractive to E. cautella males and P. interpunctella
females in a flight tunnel. E. cautella females were significantly attracted only
to ethyl vanillin. P. interpunctella males were attracted to PAA. Ethyl vanillin
is a novel insect attractant, whereas both nonanal and phenylacetaldehyde
mediate behavior in many insect species. A final experiment revealed that a
blend of the three volatiles was required to induce landing in the flight tunnel
bioassay, and that the landing rate was dependent on dose. The three-compo-

* To whom correspondence should be addressed. E-mail: christian.olsson@ekol.lu.se

2947
0098-0331/05/1200-2947/0 # 2005 Springer Science + Business Media, Inc.
2948 OLSSON ET AL.

nent blend attracted both sexes of P. interpunctella and females of E. cautella,

whereas E. cautella males were not attracted.

Key Words—Pyralidae, Lepidoptera, GC-EAD, GC-MS, EAG, flight tunnel,

food volatiles, ethyl vanillin, phenylacetaldehyde, nonanal.

INTRODUCTION

Adult moths (Lepidoptera) of the Pyralidae family rarely feed, but volatiles
from the larval host may still be used by females to locate oviposition sites and
possibly by males to locate habitats with females (Ramachandran et al., 1990).
A number of studies have identified chemicals mediating both oviposition and
flight in pyralid females, e.g., both phenylacetaldehyde (PAA) and terpenes in
Ostrinia nubilalis (Hübner) (Cantelo and Jacobson, 1979; Maini and Burgio,
1990; Binder et al., 1995; Binder and Robbins, 1997). Female Amyelois
transitella (Walker) flew toward crude almond oil (Phelan and Baker, 1987),
and the mediating chemicals were later identified as oleic acid and linoleic acid
(Phelan et al., 1991).
Two pyralid moth species found in indoor habitats, e.g., pet food stores and
chocolate factories, are the almond moth, Ephestia cautella (Walker), and the
Indian meal moth, Plodia interpunctella (Hübner). A number of studies have
shown that wheat odors induce flight and oviposition in gravid E. cautella
females (Barrer, 1977; Barrer and Jay, 1980; Gothilf et al., 1993). Similarly,
corn (Phillips and Strand, 1994), nuts and almonds (Hoppe, 1981), and walnut
oil (Nansen and Phillips, 2003) induce upwind flight and/or oviposition of
gravid females of P. interpunctella. A 1:1 mixture of acetic acid and isoamyl
alcohol (3-methyl-1-butanol), initially developed from host plants of noctuid
moths, has been used to trap female P. interpunctella in Hungary (Toth et al.,
2002). Both males and gravid females of E. cautella and P. interpunctella fly
toward a variety of chocolate products and extracts (Olsson et al., 2005).
Several studies have identified the volatile constituents of chocolate and
hundreds of organic compounds were found (Ziegleder and Stojacic, 1988;
Schnermann and Schieberle, 1997; Counet et al., 2002). However, their
behavioral effect on insects has not been investigated. Our objectives were to
identify electrophysiologically active volatiles in extracts of the chocolate
varieties that eventually could be used in monitoring traps of the pyralid moths.
We tested synthetic references of the identified substances with electroantenno-
grams (EAG) and in a flight tunnel to observe their activity, both electrophys-
iologically and behaviorally. Finally, we tested a blend of the active volatiles to
investigate if it was more behaviorally active than the individual volatiles when
presented separately.
MOTH RESPONSES TO CHOCOLATE VOLATILES 2949

METHODS AND MATERIALS

Insects. Laboratory cultures of E. cautella, originating from a Swedish

chocolate factory, and P. interpunctella, provided by the Danish Pest Infestation
Laboratory at Lyngby, were used. Larvae were reared on (100 g) of a diet of a
10:2:1 mixture of wheat germ, Brewer’s yeast, and glycerol. Insects were
separated by sex at the pupal stage, and the adults were kept in separate climate
chambers with L17:D7 photoperiod, at 24-C and 60% relative humidity. All
insects used in the experiments were 2–5 d old.
Headspace Extracts. Volatiles from 15 g of either plain, nut-containing, or
rum-flavored chocolate were collected on a charcoal filter at room temperature
for 24 hr, using a closed loop stripping apparatus (Bestmann et al., 1988). The
filter was extracted with 80 ml of dichloromethane, and the extract was diluted
with 720 ml cyclohexane. The filter was cleaned with a 2:1 mixture of methanol
and acetone between collections.
Steam Distillation Extracts. The steam distillation extraction technique
was used to obtain more volatiles in higher concentrations from the chocolate
samples. Water was boiled at elevated pressure, and the steam transferred to a
vessel containing 200 g of chocolate maintained at 100-C. During 2 hr, the
steam from the stirred water–chocolate mixture was collected and cooled to
room temperature. The collected water layer was extracted three times with
diethyl ether. The organic layer (300 ml), with a strong smell of chocolate, was
dried with MgSO4 and concentrated under reduced pressure to 2 ml.
Gas Chromatography with Electroantennographic Detection. A Hewlett
Packard 5890 series II gas chromatograph with a nonpolar HP-1 column
(30 m  0.25 mm i.d.) was used. Samples were injected splitless, and the
injector temperature was 225-C. The carrier gas was hydrogen. The column
temperature was kept at 40-C for 2 min then increased by 10-C/min up to
230-C. The final temperature was kept at 230-C for 10 min. The injected
sample was split between two outlets allowing simultaneous recording of
response of the flame ionization detector (FID) and the electroantenno-
graphic detector (EAD). Nitrogen was used as a make-up gas for the split.
The antenna was cut off at the first basal segment and at the tip segment,
then placed between two glass capillaries filled with saline solution and
containing a silver wire (Baker et al., 1991). The antenna was placed
approximately 10 mm from the GC outlet tube. Recordings from antennae of
virgin females and males were made to determine electrophysiologically active
components in the chocolate extracts. Three antennae of each sex and species
were tested for each of the extracts. All gas chromatography with electro-
antennographic detection (GC-EAD) recordings were analyzed with Autospike
32 software (Syntech, The Netherlands).
2950 OLSSON ET AL.

Gas Chromatography-Mass Spectrometry. Chocolate extracts were ana-

lyzed with a Hewlett Packard 5890 series II gas chromatograph equipped
with an HP-1 nonpolar column. Helium was used as carrier gas. The initial
temperature was maintained at 50-C for 2 min and then increased by 10-C/
min to the final temperature 250-C. The effluent was analyzed with an
HP5972 mass spectrometer in scan mode. Substances that repeatedly elicited
antennal responses were identified by comparing retention times (maximum
deviation T 0.02 min) with synthetic references, and with mass spectra from
MS libraries (NBS75k and Wiley275, match quality >90%) and synthetic
references.
Chemicals. All synthetic references were >95% pure when checked by the
gas chromatography-mass spectrometry (GC-MS) method described above. The
chemicals were purchased from Sigma-Aldrich (cyclohexanone, ethyl vanillin,
and vanillin), Acros Organics (nonanal), ICN Biochemicals (phenylacetalde-
hyde), and Fisher Scientific (two isomers of a-pinene and cyclohexanol).
Electroantennograms. Four doses, ranging from 10j2 to 10 mg, of the
identified substances were tested for their electrophysiological activity. For
a-pinene, both isomers were tested to determine which one was electrophys-
iologically most active. In the case of ethyl vanillin, 100 mg were also tested,
to compensate for the lower vapor pressure of this chemical (vapor pressures:
ethyl vanillin 8.84  10j4 Torr, nonanal 0.532 Torr, and PAA 0.368 Torr). The
antenna was mounted and humidified as above. A 10-ml aliquot of a com-
pound to be tested diluted in cyclohexane was applied on a 7  13 mm
filter paper, which was inserted into a Pasteur pipette. The tip of the Pasteur
pipette was inserted in a hole 200 mm from the opening of an 8-mm tube
leading to the antenna, and air was puffed for 0.5 s at 5 ml/s flow rate with
a stimulus controller (CS-02, Syntech). As positive standard, 5 ng of the
four-component pheromone blend of P. interpunctella (Zhu et al., 1999) were
used for males and 10 ml of headspace extract of rum-flavored chocolate,
corresponding to 18 min collection in the closed loop apparatus, were used
for females. Ten ml of cyclohexane were used as a negative control to dis-
tinguish olfactory from mechanical responses. The stimulation procedure
was as follows: first the standard was used, followed by the negative con-
trol, and then the standard again. The antenna was then stimulated with the
test substances with a standard stimulation every third time. This procedure
was repeated for each dose, starting with the lowest to avoid adaptation of
the receptors. At least 15-sec intervals were maintained between stimula-
tions. The standardized EAG response of a substance was calculated by di-
viding the EAG response with the mean of the standard puffed after and
before the substance (Hansson et al., 1991). Ten antennae from virgin moths
were tested for each species and sex. The EAG recordings were analyzed with
Autospike 32 software (Syntech, The Netherlands).
MOTH RESPONSES TO CHOCOLATE VOLATILES 2951

Flight Tunnel Experiments. A 0.7  0.7  2.0 m flight tunnel was used
for all behavioral experiments (Zhu et al., 1999). Light intensity was 10 lx,
temperature was maintained at 23 T 2-C, and relative humidity at 30–60%. All
flight tunnel experiments were conducted during the first 2 hr into the
scotophase, and each moth was observed for 3 min. Mated females and
unmated males were used for all experiments, as mating has a positive effect on
female behavior and none on male behavior (Olsson et al., 2005). The
chemicals were tested in the flight tunnel by using an ultrasonic sprayer
(El-Sayed et al., 1999) at a frequency range of 290–320 kHz, to avoid the
disturbing effects on moths hearing the ultrasound (Skals et al., 2003). The
chemicals were released at 50 ng/min (10 ng/ml at 5 ml/min) rate after
quantification (standard 10 ng decyl acetate) of the active components in the
behaviorally active chocolate extracts (Olsson et al., 2005). The released dose
was never more than 10 times greater than those found in the chocolate ex-
tracts, thereby minimizing the risk of arrestment behavior in response to too
high concentrations. Cyclohexane was used as a negative control.
In a subsequent flight tunnel experiment, a blend of ethyl vanillin, nonanal,
and phenylacetaldehyde was tested. Gas chromatography was used to analyze
the three chocolate types extracted with the two different techniques and to
determine the ratio between the volatiles in the blend used. Because nonanal
was present in all extracts, it was set as a reference for the comparison, and
given the value of 1 in the blend. A 2:1:2 blend of ethyl vanillin, nonanal, and
phenylacetaldehyde was chosen as representative for authentic chocolate odors.
The blend was released at three doses ranging from 2.5 (e.g., 0.5 ng/ml at 5 ml/
min, in which 0.5 ng/ml equals 0.2 ng ethyl vanillin, 0.1 ng nonanal, and 0.2 ng
PAA/ml) to 250 ng/min, and tested for both species and sexes. For all tests
orientated upwind, flight at least halfway to the odor source and landing on the
source were observed.
Statistics. EAG data were analyzed with ANOVA followed by Tukey’s
post-hoc test at P < 0.05 level in SPSS 10.0 for Macintosh. The flight tunnel
data were analyzed at the P < 0.05 level with Ryan’s multiple comparison test
for proportions (Ryan, 1960).

RESULTS

Twelve electrophysiologically active compounds were found when

analyzing the different chocolate types with GC-EAD (Figure 1). Some
compounds were found in all samples, whereas others varied with both the
type of chocolate and the extraction technique used (Table 1). Compounds 1, 3,
7, 8, and 10–12 were identified by comparing their mass spectra with synthetic
samples. Compound 9 (Figure 1) was tentatively identified as 3-ethyl-2,5-
2952 OLSSON ET AL.

FIG. 1. Examples of GC-EAD runs showing (a) E. cautella female response to headspace
extract of nut-containing chocolate, (b) E. cautella male response to steam distillation
extract of rum-flavored chocolate, (c) P. interpunctella female response to headspace extract
of rum-flavored chocolate, and (d) P. interpunctella male response to steam distillation
extract of nut-containing chocolate. Chemical names corresponding to the peak numbers are
found in Table 1.
MOTH RESPONSES TO CHOCOLATE VOLATILES 2953

TABLE 1. LIST OF ELECTROPHYSIOLOGICALLY ACTIVE SUBSTANCES FOUND IN THREE

CHOCOLATE PRODUCTS, DEPENDING ON EXTRACTION TECHNIQUE
AND CHOCOLATE TYPE

Headspace Steam distillation

Peak no. Chemical Plain Nut Rum Plain Nut Rum

1 Cyclohexanone j + j j j j
2 Unidentified j j + j j j
3 a-Pinene j + j j j j
4 Unidentified j j + j j j
5 Unidentified j j j j + j
6 Unidentified j j j j j +
7 Phenylacetaldehyde j j j + + +
8 Cyclohexanol j + + j j j
9 3-Ethyl-2,5-dimethyl-pyrazine + j j j j +
10 Nonanal + + + + + +
11 Vanillin + + + + j j
12 Ethyl vanillin + + + + j +

+: The chemical is present in the extract; j: the chemical is not detected.

Peak numbers correspond to numbers in Figure 1.

dimethylpyrazine, by comparing its mass spectrum with a library mass

spectrum. Remaining compounds (peaks 2 and 4–6 in Figure 1) were not
identified. E. cautella females responded to 11 of the active substances and the
males to 10 (Table 2). Both sexes of P. interpunctella responded to eight
substances each, of which six were in common (Table 2).
In both sexes of both species, the two highest doses (1 and 10 mg) of
phenylacetaldehyde and the highest dose (10 mg) of nonanal elicited EAG
responses that were significantly different from the control (Figures 2 and 3). E.
cautella males also responded to the second highest dose (1 mg) of nonanal
(Figure 2b). All adults, except E. cautella females, responded to 100 mg of ethyl
vanillin, and the males of both species also responded to 10 mg of ethyl vanillin
(Figures 2 and 3). All other substances and doses were not electrophysiolog-
ically active when compared to the control. The difference in EAG response
between active and nonactive substances was larger in males than in females for
both species.
In the first flight tunnel experiment, males of E. cautella responded
significantly to nonanal, PAA, and ethyl vanillin, whereas females only flew
upwind to ethyl vanillin (Figure 4a). Males were generally more responsive than
females, especially to nonanal and PAA where the intersexual differences were
significant. In the case of P. interpunctella, females responded to nonanal, PAA,
and ethyl vanillin, whereas males only responded significantly only to PAA
2954 OLSSON ET AL.

TABLE 2. SUMMARY OF THE ELECTROPHYSIOLOGICAL ACTIVITY OF THE DIFFERENT

SUBSTANCES FOUND IN THE CHOCOLATE PRODUCTS, DEPENDING ON SPECIES AND SEX

Ephestia cautella Plodia interpunctella

Peak no. Chemical Females Males Females Males

1 Cyclohexanone j j + +
2 Unidentified + + j +
3 a-Pinene + j j j
4 Unidentified + + j +
5 Unidentified + + j j
6 Unidentified + + + j
7 Phenylacetaldehyde + + + +
8 Cyclohexanol + + + +
9 3-Ethyl-2,5-dimethyl-pyrazine + + + j
10 Nonanal + + + +
11 Vanillin + + + +
12 Ethyl vanillin + + + +

N = 3 for each combination of species and sex. Peak numbers correspond to numbers in Figure 1.
+: Electrophysiological activity in at least two out of the three runs; j: no electrophysiological
activity.

(Figure 4b). As with E. cautella males, P. interpunctella males were more

responsive than females, with a significantly higher male response to PAA.
However, when tested separately, PAA did not induce any significant landing
on the source of either sex in either species.
In the second flight tunnel experiment, significantly more E. cautella
females flew upwind to the intermediate dose (25 ng/min) of the three-
component blend, and all doses induced significantly more landings at the
source than in the control (Figure 5a). In contrast, E. cautella males were not
attracted to the blend at any concentration tested. The frequency of upwind
flight was positively correlated with the dose for P. interpunctella females, as
the two highest doses (25 and 250 ng/min) were significantly different from the
control (Figure 5b). Male upwind flight behavior was significantly higher at all
doses compared with the control. The highest proportions of both P.
interpunctella males and females landing on source were observed at the
intermediate dose (25 ng/min), which was significantly different from the
controls (Figure 5b).

DISCUSSION

The attraction of females to chocolate volatiles could be considered as

adaptive, because they would guide females to suitable oviposition sites (Olsson
MOTH RESPONSES TO CHOCOLATE VOLATILES 2955

FIG. 2. EAG responses of E. cautella males and females to different doses (0.01–10 mg,
except ethyl vanillin for which 100 mg were also tested) of chocolate-derived chemicals.
*Data points are significantly different from control, when tested at the P < 0.05 level
with Tukey’s post-hoc test (ANOVA, males: F = 73.40, df = 33, P < 0.001; females: F =
7.11, df = 33, P < 0.001). Control was cyclohexane.
2956 OLSSON ET AL.

FIG. 3. EAG responses of P. interpunctella males and females to different doses (0.01–
10 mg, except ethyl vanillin for which 100 mg was also tested) of chocolate-derived
chemicals. *Data points are significantly different from the control, when tested at the
P < 0.05 level with Tukey’s post-hoc test (ANOVA, males: F = 38.60, df = 33, P <
0.001; females: F = 4.70, df = 33, P < 0.001). Control was cyclohexane.
MOTH RESPONSES TO CHOCOLATE VOLATILES 2957

FIG. 4. Proportion of E. cautella and P. interpunctella males and females flying upwind
in response to individual chemicals released at a rate of 50 ng/min. Bars with the same
letters do not differ significantly at P < 0.05 level (Ryan’s test). *Significant intersexual
difference for that chemical. Control was cyclohexane.

et al., 2005). For males, a positive response could lead them to sites containing
receptive females, and thus increase their reproductive fitness. The findings
show that different chocolate products contain substances that elicit both
electrophysiological and behavioral responses in both E. cautella and P.
interpunctella. However, there are a number of interspecific and intersex
differences in both the EAG and behavioral responses that are somewhat
unexpected. A good example of differences between the EAG and behavioral
responses is seen with E. cautella females and ethyl vanillin. While ethyl
vanillin stimulation resulted in no significant EAG response, it did induce
upwind flight, possibly due to the different techniques used to deliver the test
material in the two experiments. Ethyl vanillin has a low volatility, so fewer
molecules may be released in the EAG setup, resulting in low responses as
fewer receptors are activated (Schiestl and Marion-Poll, 2002). In contrast, we
used a sprayer in the wind tunnel so the volatility of ethyl vanillin would not
influence release rates.
2958 OLSSON ET AL.

FIG. 5. Proportion of E. cautella and P. interpunctella males and females flying upwind
and landing in response to a three-component blend of ethyl vanillin, nonanal, and
phenylacetaldehyde, released at three different doses. Bars within the same category with
the same letters do not differ significantly at P < 0.05 level (Ryan’s test). *Significant
intersexual difference within each behavioral step and dose. Control was cyclohexane.

Both sexes of P. interpunctella and E. cautella females exhibited better

upwind flight to the blend than to individual components; E. cautella males
responded to the individual components but not to the blend. Similarly, more
males than females landed in response to the intermediate dose of the blend.
This is surprising given that chocolate is more attractive to females than males
(Olsson et al., 2005). Therefore, additional experiments will be required to
elucidate the biological importance of these differences, including the
possibility that there are still some unidentified compounds that elicit behavior.
MOTH RESPONSES TO CHOCOLATE VOLATILES 2959

Ethyl vanillin, identified from samples of milk chocolate, is a synthetic

form of vanilla flavor not naturally occurring outside stored product environ-
ments (Hurst and Martin, 1982). The observed responses suggest that this is an
adaptation to a man-made compound, which would allow moths to locate food
sources suitable for larval development.
PAA is an attractant for several other lepidopteran species, e.g., the above-
mentioned O. nubilalis, for both sexes of Trichoplusia ni (Hübner) (Creighton
et al., 1973), Autographa californica (Speyer) (Landolt et al., 2001), and
Pseudoplusia includens (Walker) (Meagher, 2002). It was also used in the
impressive multispecies survey of 72 different moths (Cantelo and Jacobson,
1979). Our results suggest that nonanal and PAA, both chocolate volatiles
(Schnermann and Schieberle, 1997; Counet et al., 2002), might be useful in
traps for monitoring females of the two indoor moth species and may work as a
pheromone synergist enhancing male attraction to pheromone traps.

Acknowledgments—We thank Mats Ekeberg for providing chocolate products; Karin Johnson,
Annika Söderman, and Martin Ohsé for assistance with the flight tunnel experiments; and two
anonymous reviewers as well as members of the Pheromone Group for their comments on earlier
versions of the manuscript. The study was a part of the research program BPheromones and
Kairomones for Control of Pest Insects^ (Biosignal) sponsored by the Swedish Foundation for
Strategic Environmental Research (MISTRA) and Cerealia R&D.

REFERENCES

BAKER, T. C., FRANCKE, W., MILLAR, J. G., LÖFSTEDT, C., HANSSON, B. S., DU, J. W., PHELAN,
P. L., VETTER, R. S., YOUNGMAN, R., and TODD, J. L. 1991. Identification and bioassay of sex
pheromone components of carob moth, Ectomyelois ceratoniae (Zeller). J. Chem. Ecol.
17:1973–1988.
BARRER, P. M. 1977. The influence of airborne stimuli from conspecific adults on the site of
oviposition of Ephestia cautella (Lepidoptera: Phycitidae). Entomol. Exp. Appl. 22:13–22.
BARRER, P. M. and JAY, E. G. 1980. Laboratory observations on the ability of Ephestia cautella
(Walker) (Lepidoptera: Phycitidae) to locate, and to oviposit in response to a source of grain
odor. J. Stored Prod. Res. 16:1–7.
BESTMANN, H. J., ERLER, J., and VOSTROWSKY, O. 1988. Determination of diel periodicity of sex
pheromone release in three species of Lepidoptera by Fclosed loop-stripping_. Experientia 44:
797–799.
BINDER, B. F. and ROBBINS, J. C. 1997. Effect of terpenoids and related compounds on the
oviposition behavior of the European corn borer, Ostrinia nubilalis (Lepidoptera: Pyralidae).
J. Agric. Food Chem. 45:980–984.
BINDER, B. F., ROBBINS, J. C., and WILSON, R. L. 1995. Chemically mediated ovipositional
behaviours of the European corn borer, Ostrinia nubilalis (Lepidoptera: Pyralidae). J. Chem.
Ecol. 21:1315–1327.
CANTELO, W. W. and JACOBSON, M. 1979. Phenylacetaldehyde attracts moth to bladder flower and
to blacklight trap. Environ. Entomol. 8:444–447.
COUNET, C., CALLEMIEN, D., OUWERX, C., and COLLIN, S. 2002. Use of gas chromatography–
2960 OLSSON ET AL.

olfactometry to identify key odorant compounds in dark chocolate. Comparison of samples

before and after conching. J. Agric. Food Chem. 50:2385–2391.
CREIGHTON, C. S., MCFADDEN, T. L., and CUTHBERT, E. R. 1973. Supplementary data on
phenylacetaldehyde: an attractant for Lepidoptera. J. Econ. Entomol. 66:114–115.
EL-SAYED, A., GÖDDE, J., and ARN, H. 1999. Sprayer for quantitative application of odor stimuli.
Environ. Entomol. 28:947–953.
GOTHILF, S., SHAAYA, E., and LEVSKI, S. 1993. Effect of sex, age and mating on attraction of Cadra
cautella (Walker) (Lep., Phycitidae) to stored food. J. Appl. Entomol. 116:139–144.
HANSSON, B. S., VAN DER PERS, J. N. C., HÖGBERG, H.-E., HEDENSTRÖM, E., ANDERBRANT, O., and
LÖFQVIST, J. 1991. Pheromone perception in male pine sawflies, Neodiprion sertifer
(Hymenoptera: Diprionidae). J. Comp. Physiol. A 168:533–538.
HOPPE, V. T. 1981. Food preference, oviposition and development of the Indian meal moth Plodia
interpunctella Hübner on different products of the chocolate industry. Z. Angew. Entomol.
91:170–179.
HURST, W. J. and MARTIN, R. A. JR: 1982. The HPLC analysis of ethyl vanillin in chocolate
products. Am. Lab. 14:74–75.
LANDOLT, P. J., ADAMS, T., REED, H. C., and ZACK, R. S. 2001. Trapping alfalfa looper moths
(Lepidoptera: Noctuidae) with single and double component floral chemical lures. Environ.
Entomol. 30:667–672.
MAINI, S. and BURGIO, G. 1990. Influence of trap design and phenylacetaldehyde upon field capture
of male and female Ostrinia nubilalis (Hb.) (Lepioptera: Pyralidae) and other moths. Boll. Ist.
Entomol. Guido Grandi Univ. Studi Bologna 45:157–165.
MEAGHER, R. L. JR: 2002. Trapping noctuid moths with synthetic floral volatile lures. Entomol. Exp.
Appl. 103:219–226.
NANSEN, C. and PHILLIPS, T. W. 2003. Ovipositional responses of the Indian meal moth, Plodia
interpunctella (Hübner) (Lepidoptera: Pyralidae) to oils. Ann. Entomol. Soc. Am. 96:524–531.
OLSSON, P.-O., ANDERBRANT, C., and LÖFSTEDT, O. 2005. Flight and oviposition behavior of
Ephestia cautella and Plodia interpunctella in response to odours of different chocolate
products. J. Insect Behav. 18:363–381.
PHELAN, P. L. and BAKER, T. C. 1987. An attracticide for control of Amyelois transitella
(Lepidoptera: Pyralidae) in almonds. J. Econ. Entomol. 80:779–783.
PHELAN, P. L., ROELOFS, C. J., YOUNGMAN, R. R., and BAKER, T. C. 1991. Characterization of
chemicals mediating ovipositional host-plant finding by Amyelois transitella females. J. Chem.
Ecol. 17:599–613.
PHILLIPS, T. W. and STRAND, M. R. 1994. Larval secretions and food odors affect orientation in
female Plodia interpunctella. Entomol. Exp. Appl. 71:185–192.
RAMACHANDRAN, R., KHAN, Z. R., CABALLERO, P., and JULIANO, B. O. 1990. Olfactory sensitivity
of two sympatric species of rice leaf folders (Lepidoptera: Pyralidae) to plant volatiles.
J. Chem. Ecol. 16:2647–2666.
RYAN, T. A. 1960. Significance tests for multiple comparisons of proportions, variances, and other
statistics. Psychol. Bull. 57:318–328.
SCHIESTL, F. P. and MARION-POLL, F. 2002. Detection of physiologically active flower volatiles
using gas chromatography coupled with electroantennography, pp. 173–198, in J. F. Jackson
and Linskens H. F. (eds.). Analysis of Taste and Aroma. Springer, Berlin, Germany.
SCHNERMANN, P. and SCHIEBERLE, P. 1997. Evaluation of key odorants in milk chocolate and cocoa
mass by aroma extract dilution analysis. J. Agric. Food Chem. 45:867–872.
SKALS, N., PLEPYS, D., EL-SAYED, A. M., LÖFSTEDT, C., and SURLYKKE, A. 2003. Quantitative
analysis of the effects of ultrasound from an odor sprayer on moth flight behavior. J. Chem.
Ecol. 29:71–82.
MOTH RESPONSES TO CHOCOLATE VOLATILES 2961

TOTH, M., REPASI, V., and SZÖCS, G. 2002. Chemical attractants for females of pest pyralids and
phycitids (Lepidoptera: Pyralidae, Phycitidae). Acta Phytopathol. Entomol. Hung. 37:375–384.
ZHU, J., RYNE, C., UNELIUS, C. R., VALEUR, P. G., and LÖFSTEDT, C. 1999. Reidentification of the
female sex pheromone of the Indian meal moth, Plodia interpunctella: evidence for a four-
component blend. Entomol. Exp. Appl. 92:137–146.
ZIEGLEDER, G. and STOJACIC, E. 1988. Changes of flavour of milk chocolate during storage. Z.
Lebensm. Unters. Forsch. 186:134–138.