Electrophysiological And Behavioral

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 ¨ FSTEDT,13 ANNA-KARIN BORG-KARLSON,2 CHRISTER LO 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 rumflavored. Eight of the compounds were identified with mass spectrometry, and the activity of three compounds, ethyl vanillin, nonanal, and phenylacetaldehyde (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: [email protected]

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 (Hu¨bner) (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 (Hu¨bner). 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 electroantennograms (EAG) and in a flight tunnel to observe their activity, both electrophysiologically 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 electroantennographic 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 electroantennographic detection (GC-EAD) recordings were analyzed with Autospike 32 software (Syntech, The Netherlands).

2950

OLSSON

ET AL.

Gas Chromatography-Mass Spectrometry. Chocolate extracts were analyzed 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 (phenylacetaldehyde), 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 electrophysiologically 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 compound 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 distinguish olfactory from mechanical responses. The stimulation procedure was as follows: first the standard was used, followed by the negative control, 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 stimulations. The standardized EAG response of a substance was calculated by dividing 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 extracts, 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.

2953

MOTH RESPONSES TO CHOCOLATE VOLATILES

TABLE 1. LIST OF ELECTROPHYSIOLOGICALLY ACTIVE SUBSTANCES FOUND CHOCOLATE PRODUCTS, DEPENDING ON EXTRACTION TECHNIQUE AND CHOCOLATE TYPE Headspace

IN

THREE

Steam distillation

Peak no.

Chemical

Plain

Nut

Rum

Plain

Nut

Rum

1 2 3 4 5 6 7 8 9 10 11 12

Cyclohexanone Unidentified a-Pinene Unidentified Unidentified Unidentified Phenylacetaldehyde Cyclohexanol 3-Ethyl-2,5-dimethyl-pyrazine Nonanal Vanillin Ethyl vanillin

j j j j j j j j + + + +

+ j + j j j j + j + + +

j + j + j j j + j + + +

j j j j j j + j j + + +

j j j j + j + j j + j j

j j j j j + + j + + 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 electrophysiologically 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 Peak no.

Chemical

Females

Males

1 2 3 4 5 6 7 8 9 10 11 12

Cyclohexanone Unidentified a-Pinene Unidentified Unidentified Unidentified Phenylacetaldehyde Cyclohexanol 3-Ethyl-2,5-dimethyl-pyrazine Nonanal Vanillin Ethyl vanillin

j + + + + + + + + + + +

j + j + + + + + + + + +

Plodia interpunctella Females + j j j j + + + + + + +

Males + + j + j j + + j + + +

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 threecomponent 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 environments (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 abovementioned O. nubilalis, for both sexes of Trichoplusia ni (Hu¨bner) (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 So¨derman, and Martin Ohse´ 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., LO¨FSTEDT, 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., GO¨DDE, 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., HO¨GBERG, H.-E., HEDENSTRO¨M, E., ANDERBRANT, O., and LO¨FQVIST, 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 Hu¨bner 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 (Hu¨bner) (Lepidoptera: Pyralidae) to oils. Ann. Entomol. Soc. Am. 96:524–531. OLSSON, P.-O., ANDERBRANT, C., and LO¨FSTEDT, 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., LO¨FSTEDT, 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 SZO¨CS, 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 LO¨FSTEDT, C. 1999. Reidentification of the female sex pheromone of the Indian meal moth, Plodia interpunctella: evidence for a fourcomponent 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.