ANTENNAL RESPONSES TO SERIAL DILUTIONS OF APPLE VOLATILE CHEMICALS BONNIE JEAN OHLER

Transcription

1 FEMALE CODLING MOTH, CYDIA pomonella (LEPIDOPTERA: TORTRICIDAE), ANTENNAL RESPONSES TO SERIAL DILUTIONS OF APPLE VOLATILE CHEMICALS By BONNIE JEAN OHLER A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE IN ENTOMOLOGY WASHINGTON STATE UNIVERSITY Department of Entomology AUGUST 2010

2 To the faculty of Washington State University: The members of the Committee appointed to examine the thesis of BONNIE JEAN OHLER find it satisfactory and recommend that it be accepted. Richard S. Zack, Ph.D.,Chair Peter J. Landolt, Ph.D. Vincent R. Hebert, Ph.D ii

3 ACKNOWLEDGEMENTS I would like to express my gratitude to everyone who helped me throughout the completion of my degree. Firstly I thank my family, my husband, Heath Ohler, and my daughter, Molly Paynter, for their support and patience as I have worked towards finishing this thesis project. I thank my advisors, Vince Hebert, Rich Zack and Pete Landolt, for their insights into my research, their guidance and for providing me with a research assistantship. I also thank Christelle Guédot for her help, scientific advice, friendship and mentoring. I thank Dave Horton who advised me on the statistics I used to complete these studies. I thank Daryl Green for the laboratory support he provided as a member of the chemical ecology research team at the USDA-ARS Yakima Agricultural Research Laboratory in Wapato. This research was supported by USDA, ARS Specific Cooperative Agreement S Management of Insect Pests of Potato and Tree Fruits. This research was performed at the USDA-ARS Yakima Agricultural Research Laboratory in Wapato. iii

4 FEMALE CODLING MOTH, CYDIA pomonella (LEPIDOPTERA: TORTRICIDAE), ANTENNAL RESPONSES TO SERIAL DILUTIONS OF APPLE VOLATILE CHEMICALS Abstract by Bonnie Jean Ohler Washington State University August 2010 Chair: Richard S. Zack Volatile chemicals from apples collected during each of the two codling moth, Cydia pomonella, flights in central Washington were analyzed using gas chromatography coupled with electroantennal detection (GC-EAD) using serially diluted samples. More concentrated immature apple volatile chemical collections elicited significantly more antennal responses than less concentrated volatile chemical collections. Antennal responses of wild codling moths were compared to laboratory-reared moths at one concentration with no significant difference found. Four immature apple volatile chemicals elicited an antennal response at the lowest concentration and three of these were identified. These chemicals are: (Z)-3-hexenol, (d)-linalool, and (E,E)-αfarnesene. The fourth chemical eluted with the solvent peak during analysis preventing structural determination and indicating low boiling point and low molecular weight. These chemicals are of interest for understanding how codling moths use kairomones to find and select host for oviposition and, ultimately, developing attractants for female codling moths. iv

5 TABLE OF CONTENTS Page ACKNOWLEDGEMENTS... ABSTRACT... LIST OF TABLES... LIST OF FIGURES... iii iv vi vii SECTION 1. BACKGROUND AND LITERATURE REVIEW INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES v

6 LIST OF TABLES 1. Mean percent (±SEM) antennal responses of laboratory reared versus wild codling moths to volatile chemical collections at 1.0 apple hour equivalent for each peak. (N=5) Mean (±SEM) number of antennal responses to different amounts of immature apple volatile chemicals collected during two codling moth flights, June 18 through June 22 and July 28 through August 1, (N = 5) Percent antennal response of codling moth females to peaks for each concentration analyzed by GC-EAD/FID and mean (± SEM) amount (µg) per apple hour equivalent (AHE 1 ) for apple volatile chemicals collected during the first and second flights (N = 5)...21 vi

7 LIST OF FIGURES 1. GC-EAD (top) with GC-FID (bottom) of 1 apple hour equivalent (AHE) collected during the first codling moth flight to a mated female codling moth antenna separated on the DB-1 column. Peak numbers correspond to peak numbers in Tables 1 and 3. Peaks which elicited at least one response at 0.1 AHE: peak 2 (Z)-3-hexenol, peak 7 d-linalool and peak 11 (E,E)- α-farnesene...18 vii

8 Dedication This thesis is dedicated to my parents who taught me the value of hard work and the satisfaction of finishing things I begin and to my husband and daughter, without whose support I would not have been able to finish. viii

9 Background and Literature Review Chemical ecology is the study of the way living things use chemicals to communicate. These chemicals are collectively called semiochemicals. Semiochemicals can be produced and perceived within a species as pheromones or between species as allelochemicals. Pheromones can serve different functions. Sex pheromones attract mates. Alarm pheromones alert other individuals of danger. Aggregation pheromones can attract others of the same species for protection or for aid in overcoming a resource s defenses. Pheromones can serve other functions as well, especially with social insects. Allelochemicals can be classified into three major groups. If the chemical message benefits the sender, but not the receiver, it is called an allomone. An allelochemical that benefits the receiver, but not the sender, it is a kairomone. A chemical signal that benefits both the sender and the receiver is a synomone. The research reported here deals exclusively with kairomones or more specifically, chemicals produced by host plants that codling moths use to locate and exploit them. Host plants produce and release these chemicals as part of their own metabolic processes, evolved to benefit themselves, but codling moths have evolved the capacity to detect and utilized them as many herbivorous insects have evolved the capacity to locate their hosts (Finch, 1986). Volatile organic chemicals are released from the plant and dispersed by the wind forming a complex and dynamic plume of odor chemicals. There has been much work aimed at describing the use of odor plumes by insects in their search for both mates and hosts (Murlis et al. 1992). There are different plume structures to consider. In the case of phytophagous insects locating hosts, the odor plumes released by hosts can be composed of a variety of volatile compounds, as is the case for apples. Codling moths must locate apples within a complex matrix of background odors. 1

10 Phytophagous insects can use visual, tactile, or odor stimuli to locate host plants. Flying insects, especially Diptera and Lepidoptera, most often use a combination of visual and odor chemical stimuli as they search for, perceive, approach and arrive at a host. The insects then use chemical, tactile and other senses to evaluate and accept or reject the host. Ultimately, the insect will oviposit on or near an accepted host and leave in search of the next suitablehost (Finch, 1986). Codling moth flight takes place, mostly after the beginning of scotophase (Howell,1991). As a consequence, it is less likely that visual cues are responsible for host location. Visual cues may play a part, but it is my hypothesis that female codling moths rely on kairomones emitted by the host plant to locate fruit for their offspring. Bioassays aimed at testing for attraction to host plant odor can be problematic (Finch 1986). Since host location kairomones may function as arrestants at short range (Finch 1986). The behavior observed in a host location bioassay might be very different from the behavior observed in the field where the moth approaches the host from a greater distance than can be observed in the laboratory. The host odor may function as an attractant, but at close range cause the insect to slow down and arrest short of the target, leaving the insect to evaluate other stimuli in order to reach their ultimate goal (Finch 1986). Moreover, host location behavior can be selected against in laboratory populations of insects. It has been shown that codling moths display different oviposition behavior after being reared in the laboratory (Witzgall et al. 2005). In a laboratory colony with an absence of host plant odor the moths that need host plant odors to oviposit would quickly be eliminated from the population selecting for those individuals that do not display this behavior. Several studies have evaluated the odor profile of codling moth hosts, focusing mainly on the major host, apple (Hern and Dorn, 2001, Coracini et al. 2003, Bäckman et al. 2001, 2

11 Bengtsson et al. 2001, Casado et al. 2006). These studies have employed several different sampling techniques including extraction, headspace sampling by solid phase microextraction (SPME) and dynamic sampling using gas pushed and/or pulled through an adsorbent or trap. These samples have been analyzed by different research groups using gas chromatography with flame ionization detection (GC-FID), mass spectral detection (GC-MSD), and electroantennal detection (GC-EAD). Another study compared volatile chemical emissions of different host plant species with non-host plant species (Witzgall, 2005). This combined work has contributed to a very long list of volatile chemicals from apple, pear and walnut that might be important in codling moth behavior. Despite this information, we do not yet understand how codling moths use host kairomones to locate and select oviposition sites. This may be a result of the sheer magnitude of the list of chemicals. These compounds have very different volatilities and would also require different methods to achieve controlled delivery rates. Reading the many studies on this subject a list of 41 chemicals can be compiled (see below) which have been identified in codling moth host plants and elicit antennal response in GC-EAD analysis (Hern and Dorn, 2001, Coracini et al. 2003, Bäckman et al. 2001, Bengtsson et al. 2001, Casado et al. 2006). This list illustrates the magnitude of the problem. 1. benzyl aldehyde 22. (Z)-3-hexenyl acetate 2. hexanol 23. (Z)-3-hexenyl butanoate 3. benzyl alcohol 24. (Z)-3-hexenyl benzoate 4. octanal 25. (Z)-3-hexenyl hexanoate 5. nonanal 26. (E,Z)-2,4-decadienoate 6. decanal 27. β-pinene 7. (E)-2-hexenal 28. β-myrcene 8. 6-methyl-5-hepten-2-one 29. (E)-β-ocimene 9. methyl salicylate 30. (Z)-jazmone 10. propyl hexanoate 31. limonene 11. butyl acetate 32. 4,8-dimethyl-1,3,(E)7-nonatrien 12. butyl butanoate 33. linalool 3

12 13. butyl 2-methyl butanoate 34. α-copaene 14. butyl hexanoate 35. β-caryophyllene methyl butyl acetate 36. α-caryophyllene 16. hexyl acetate 37. (E)-β-farnesene 17. hexyl propionate 38. germacrene D 18. hexyl butanoate 39. (Z,E,)-α-farnesnene 19. hexyl 2-methyl butanoate 40. (E,E)-α-farnesnene 20. hexyl hexanoate 41. farnesol 21. (Z)-3-hexen-1-ol Electroantennal detection has been, in some cases, the only criteria by which the chemicals have been evaluated as kairomones. Electroantennal detection cannot be relied on exclusively for the identification of insect attractants (Finch, 1986). Antennal response does not necessarily predict attraction. Antennal response only indicates that the antenna is capable of detecting the compound. For example, the compound in question may be a repellant or an arrestant. There is no way to tell from the antennal response. Potential kairomones can be screened for antennal response by electroantennal detection, but behavioral activity can only be assessed using behavioral assays, either in the lab (e.g. flight tunnel) or in the field (e.g. trapping). An ultimate goal of research on host plant kairomones is to understand how pest insects locate their hosts in the field and perhaps to find a way to interfere with or enhance this behavior. Only field tests and field observation can truly show success in this aim. GC-EAD is a useful means toward this end and not an end by itself. The first published study on the subject of codling moth host kairomones (Sutherland and Hutchins 1972) reported the sesquiterpene α-farnesene to be an attractant to codling moth larvae. α-farnesene was later shown to stimulate adult females to increase oviposition in olfactometer and arena assays (Sutherland and Hutchins, 1972). Codling moth eggs are most often deposited by their mothers on leaves near an apple which requires the neonate larvae to be able to locate and penetrate the apple (Jackson, 1978). While this sequiterpene was shown in several bioassays 4

13 to be attractive to larvae (Sutherland and Hutchins 1972, Landolt et al. 2000), it has not been shown to be an attractant in adult moths, except in a Y-tube olfactometer (Hern and Dorn 1999). The Y-tube olfactometer assay is an ambulatory assay, which is a different response than flight towards an odor source as would be necessary for successful attraction from a distance and for trapping. Trapping and wind tunnel experiments using this chemical have not shown attraction. It could be argued that in commercial orchards female codling moths may not search for and approach hosts from a distance, because they emerge as adults very near to a host plant. Female moths could randomly approach nearby trees, only evaluating potential hosts after contact or at close range. In this case the arrestant properties of the host kairomones would be more important. However, observational evidence (Witzgall et al. 1999) as well as the successful trapping of female codling moths using fruit inside traps (Landolt and Guédot, 2008) support the hypothesis that female codling moths are capable of long range search and host fruit perception. Plant odor chemistry is complex with many of the components having enantiomers as well as cis/trans isomers. Codling moth host plants produce many volatile chemicals including several terpene and terpenoid compounds, which can be very difficult to definitively identify. Many of these chemicals are not available for purchase and must therefore be synthesized and/or purified before confirmation of identity and behavioral activity can take place. Terpene chemistry is a large and exciting discipline, with several economically important compounds discovered so far. These chemicals are used in a range of applications, from medicinal to flavor and fragrance. Terpenes are chemicals constructed from isoprene units. Each isoprene unit has five carbons and is a major building block of natural chemicals. Terpenoids are derivatives of terpenes that have added functional groups like alcohol in the case of linalool. Terpenes and terpenoids can be classified by the number of isoprene units which they contain. Hemiterpenes 5

14 and hemiterpenoids are made of one isoprene unit, while monoterpenes are constructed from two isoprenes. Several sesquiterpenes, consisting of three isoprene units, all with the same molecular mass of 204, along with a few monoterpenes have been identified in apple and pear extracts and volatile collections. There are many well established techniques for isolating and identifying terpenes within natural sources. The host plants produce very small amounts of each of the several components, making it difficult to collect and separate enough material to allow positive identification. Some of the identification techniques used for terpenes historically have required the purification of milligram amounts. Progress has been made recently in developing techniques specialized for utilizing sub-microgram amounts of unknown compounds, including sub-microgram NMR techniques (Nojima et al., 2004), sub-microgram scale collection techniques from gas chromatography Nojima et al., 2008) and micro scale reactions (Attygale, 1998). 6

15 Introduction The codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), is the key insect pest of apples (Malus spp.) throughout the world. Codling moth larvae also damage pears (Pyrus spp.), walnuts (Juglans spp.) and quinces (Cydonia spp.). Codling moth management can result in the use of large amounts of pesticides resulting in large monetary investments by apple orchardists (Williamson et al. 1996). A search for more selective alternatives to agrochemically intensive broad spectrum insecticide treatments has lead to advances in using the natural chemistry of insects (Kahn et al. 2008). Female codling moths produce a sex pheromone which is a blend of short chained aliphatic alcohols with the corresponding aldehyde and acetate (Arn et al. 1985). The major component is codlemone (E, E-8,10-dodecadienol) (Roelofs et al. 1971). This female-produced sex pheromone is used in monitoring traps to allow the timing of pesticide sprays to impact vulnerable egg and neonate stages of the codling moths, making pesticide applications more efficient and reducing the total number needed (Batiste et al. 1973). Codling moth female-produced sex pheromone formulations are also used for mating disruption with demonstrated success (Brunner et al. 2002). It has been shown that much of the success of mating disruption can be attributed to the delay of male moths finding mates (Knight, 2007). Mating disruption has limitations, however, including decreased efficacy in orchards with large codling moth populations and in locations where immigration of mated females occurs (Witzgall et al. 2008). In situations where mating disruption does not provide the desired level of control, an effective management strategy that targets female codling moths would be ideal (Witzgall et al., 2008). Female codling moths oviposit single eggs near or on individual fruit (Jackson, 1978). Thus, female host location behavior could be targeted with a codling moth management strategy 7

16 utilizing attractants based on kairomones. Because codling moths fly mostly during the scotophase (Howell, 1991) and are attracted to host fruit in traps (Landolt and Guédot, 2008), we can deduce that they probably use olfactory cues to locate host fruit, at least from a distance. Other cues, such as texture and taste could be involved in host evaluation and acceptance. Female codling moths have been shown to be attracted to odors from host fruit in the laboratory (Reed and Landolt 2002, Vallat and Dorn 2005) and in the field with traps (Coracini et al. 2004, Landolt and Guédot 2008). Numerous investigations have also identified hostderived volatile chemicals that elicit antennal response in codling moths (Bäckman et al. 2001, Bengtsson et al. 2001, Casado et al. 2006, and references therein). While these studies have resulted in a long list of host odorants, behavioral evaluations have not yet led to an understanding of specific volatile chemicals responsible for host location and host selection by codling moth females. This may be due in part to the inherent difficulty of using behavioral assays to evaluate host finding, as discussed by Finch (1986). This difficulty may be due to several factors including the structural, temporal, and spatial complexity of host plant odor in an orchard setting, and the low concentration of these chemicals being released by the host. The volatile chemicals released by apples have been shown to vary by season and by time of day (Vallet and Dorn, 2005; Casado et al., 2006). A majority of behavioral studies were conducted with laboratory-reared moths, and it is possible that this has affected results of these studies. Laboratory-reared codling moths may no longer possess the necessary traits for normal host location and oviposition behavior due to selection pressures involved in establishing a laboratory colony. Indeed, one generation after starting a laboratory-reared colony of codling moths, the females no longer exhibit the same oviposition behavior in the absence of host odor as wild moths (Witzgall et al., 2005). The first objective of the present study was to compare the 8

17 antennal responses to volatile chemicals collected form immature apples of wild moths to laboratory reared moths. Given the extensive characterization of the odor and flavor chemistry of apple providing a comprehensive list of volatile chemicals (Mehinagic et al. 2006, and references therein), the codling moth host location kairomones have, most likely, been previously reported as apple odorants. However, the problem lies in determining which of these chemicals are important for codling moth host location and host selection behavior, and how they should be presented to elicit host location behavior. The ultimate goal would be the development of a long range attractant for use in codling moth traps or as part of an attract-and-kill device. Prior to this time, determinations of compounds that elicit antennal responsse by electroantennal detection (EAD) from apples have been performed using concentrated apple extractions or concentrated volatile chemical collections. Insect antennal response is a concentration dependant phenomenon, with concentration thresholds determining behavioral response. The concentration threshold for a given chemical can be indicative of the behavioral importance of a chemical (Kendra et al. 2009). Small amounts of volatile chemicals are emitted by apples which are then diluted through diffusion and manipulated by air currents to make dilute and dynamic odor plumes (Murlis et al. 1992). It is possible that many antennally active chemicals identified in prior studies may not elicit antennal responses at concentrations likely to be present in real host location situations. This study examines concentration dependant response to construct a list of apple volatile chemicals that are active at low concentrations. Serial dilutions of volatile chemical collections from whole immature apples that yet elicit antennal responses are more likely to approximate the concentration encountered in the apple orchard as a female codling moth approaches a host from a distance. In the present study serially 9

18 diluted volatile chemical collections were analyzed by gas chromatography with flame ionization detection coupled with electroantennal detection (GC-FID/EAD) to 1) determine whether the number of antennal responses decreases with decreasing concentration of volatile chemical collections and 2) identify the apple odor chemicals eliciting antennal responses in female codling moths at dilute concentrations. 10

19 Materials and Methods Insects Laboratory-reared codling moths were obtained as pupae from the insectary at USDA- ARS Yakima Agricultural Research Laboratory, Wapato, Washington, U.S.A. Larvae were reared on artificial diet (Toba and Howell, 1991), under 16:8 (Light:Dark) light cycle at 25 C and ambient relative humidity (40 50% RH) and allowed to pupate inside 3 cm wide roles of corrugated cardboard. The pupae were then removed from the corrugated cardboard. In order to compare laboratory-reared to wild codling moth antennal responses to volatile chemical collections from immature apples wild codling moth larvae were obtained by picking infested fruit from unmanaged volunteer or feral apple trees of unknown variety near the Wapato laboratory on July , July 18, 2008 and July 20, These apples were placed inside plastic containers with corrugated cardboard strips in a greenhouse with natural light. The 3 cm wide cardboard strips were placed along all four sides of the plastic containers 6 to 10 cm above the bottom. The containers were covered with organdy cloth attached with 19 mm wide twosided tape (3M, St. Paul, Minnesota, U.S.A.). The cardboard strips were replaced each week and the codling moth pupae were removed. Both laboratory-reared and wild pupae were sorted by sex and set in 237 ml waxed paper cups in cages at a ratio of two males to one female to increase likelihood of mating. The maximum number of pupae per cup was 200. Cages were kept in reversed light cycle conditions (16:8, L:D) with lights turning off at 10:00 PST at 22 C and ~60% RH. After emergence, moths were given sugar water and water in cotton balls in 6 cm diameter Petri dishes. For EAD antennal preparations 4 to 6 day old female moths were taken from cages throughout the scotophase. The right antenna was removed by pulling the scape of the antenna free from the head. The distal 2-3 antennal segments were excised and the antenna was then connected 11

20 between two pulled glass capillary pipettes filled with Ringer s solution with gold wire electrodes inserted. The female codling moths were then dissected to determine mating status. The presence of (a) spermatophore(s) in the bursa copulatrix indicated that the female was mated. Only antennae from mated females were used. Apple Volatile Chemical Collections Apples Apples were collected from an orchard located north of Moxee, Yakima County, Washington State, U.S.A. Apples were picked at 6:00 PST each morning. Collection of volatile chemicals began within one hour from the time of picking. All apples were Gala variety apples and were picked from the same orchard. This orchard was being managed as a certified organic production orchard with codling moth managed primarily by pheromonal mating disruption. Volatile chemical collections from apples were performed for four hours each morning June and 28 July 1 August 2008, each period falling within codling moth flights in the orchard used in this study. The codling moth flights in this orchard were monitored using traps baited with acetic acid and pear ester (Landolt and Guédot 2008). Volatile Chemical Collection Apparatus Apple diameters and number of apples were recorded before the apples were placed into a 3.8 liter glass jar. Commercial air (Oxarc, Inc., Spokane, WA, U.S.A.) was purified by charcoal filter (R & D Hydrocarbon Trap, R & D Separations, Rancho Cordova, California, U.S.A.) The charcoal purified air was delivered into the glass jar at a rate of 300 ml/min, determined by flow meter (Aarlborg Instruments, Monsey, New York, U.S.A), through 0.2 cm ID Teflon tubing. Teflon tubing of the same diameter was used to connect the jar to a glass connector with an o-ring to allow insertion of the chemical trap. A vacuum flow of 250 ml/min was applied after the trap. The chemical trap used was a 30 mg Super-Q polymeric adsorbent trap (Agricultural Research Science, Inc., Gainesville, Florida, 12

21 U.S.A.). Collections were performed for 4 hours preceded by a one hour system blank collection. Volatile chemicals were rinsed from the chemical trap with 250 µl methylene chloride. Five micrograms dodecyl acetate (5 µl of 1 mg/ml in methylene chloride) was added to make 20 µg/ml eluate as an internal standard. These samples were then stored for analysis in 1 ml autosampler vials in a freezer at -20 C. The Super-Q traps were extracted five times with 500 µl methylene chloride prior to use and prior to storage. Chemical Analysis Gas Chromatography with Flame Ionization Detection coupled with Electoantennal Detection (GC-FID/EAD) Antennae were prepared and mounted according to methods described above. The samples were analyzed using a Hewlett Packard 5890 Series II gas chromatograph (Hewlett Packard, Palo Alto, California, U.S.A) and an IDAC-232 data acquisition interface with a micromanipulator assembly type IRN-5 (Syntech, The Netherlands). The gas chromatograph was equipped with a DB-1 fused silica capillary column (J & W Scientific, Folsum, California, U.S.A.) 60 m (length) x 0.25 mm (ID) x 0.25 µm film thickness. Samples were injected manually using a 10 µl syringe (Hamilton Company, Reno, Nevada, U.S.A.) in splitless mode. The injector temperature was 250 C and the carrier gas was helium. The column temperature program started at 40 C for two min, increased to 200 C at a rate of 10 C/min, and was held at 200 C for 12 min for a total run time of 30 min. The column effluent was split at a ratio of 2:1 using an OSS-2 splitter (SGE Analytical Science, Austin, Texas, U.S.A.) between the EAD and the FID. Nitrogen was used as a makeup gas at the splitter at a flow rate of 40 ml/min. Apple volatile chemical collections were analyzed by GC-FID/EAD at three different concentrations. For the purpose of this study concentrations of each apple volatile chemical collection sample are described using apple hour equivalents (AHE) following the equation: 13

22 (number of apples x hours of collection)/(volume of sample). The three different concentrations analyzed by GC-FID/EAD were: 0.1 AHE, 1 AHE, and 10 AHE per injection. Samples collected during the second flight were also analyzed five times each with both wild and laboratory-reared codling moth antennae at one AHE to compare antennal responses for wild and laboratory-reared moths. Gas Chromatography with Mass Spectrometry (GC-MS) The samples were analyzed using an Agilent 6890 chromatograph with a 5973 electron impact mass selective detector (Agilent Technologies, Palo Alto, California). This GC was also equipped with a DB-1 fused silica capillary column (J & W Scientific, Folsum, California) 60 m (length) x 0.25 mm (ID) x 0.25 µm film thickness. Each sample was injected to allow for preliminary identification of chemicals. These analyses were conducted using the same temperature and pressure program as the GC- FID/EAD described above to allow for comparison of chromatograms. Chemical Identification Chemicals were chosen for identification if antennal responses were elicited at 0.1 AHE during at least one trial and if responses were elicited during both first and second flight analysis. Three compounds were identified using the methods described here and confirmed by dual column retention time comparison and mass spectral comparison against purchased or purified standards as described. Dual column comparison was accomplished by analyzing the volatile chemical collection samples using an Agilent 6890 chromatograph with flame ionization detection (Agilent Technologies, Palo Alto, California, U.S.A.) equipped with a DB-1MS column (see above) followed by a DB-WAXETR fused silica capillary column (J & W Scientific, Folsom, California, U.S.A.) 60 m (length) x 0.25 mm (ID) x 0.25 µm film thickness. (Z)-3-hexenol was preliminarily identified using comparison with NIST spectral library followed by confirmation by retention time and spectral comparison with purchased reference standards 14

23 (Sigma-Aldrich, St. Louis, Missouri, U.S.A.). Linalool was identified and confirmed using the same methods as for (Z)-3-hexenol. The reference standard was racemic linalool (Sigma- Aldrich, St. Louis, Missouri, U.S.A.). The enantiomeric determination was carried out by retention time comparison to racemic linalool on an Agilent 6890 chromatograph equipped with a RT-βDEXse chiral column (Restek, Bellefonte, Pennsylvania, U.S.A.) 30 m (length) x 0.25 mm (ID) x 0.25 µm (film thickness). The linalool in immature apple volatile chemical collections matched the retention time of d-linalool, (S)-(+)-linalool. The analysis was conducted in splitless injection mode with an inlet temperature of 250 C. The oven temperature started at 40 C for one min, ramped at 5 C/min to 200 C and held for seven min. (E,E)-αfarnesene was extracted and purified from peals of stored ripe apples using high performance liquid chromatography with ultraviolet detection (HPLC-UV) by procedures adapted from Whitaker et al. (1997). The purified extract was analyzed for purity by GC-MS and HPLC-UV. The dry weight was obtained and a reference standard solution made. This purified standard was used for identity confirmation. Quantification We estimated the amounts of eleven substances by comparison with internal standard peak area and calculated the mean and standard error of the amount for each peak. These chemicals were chosen based on an antennal response of over 60% at one concentration during at least one trial Data Analysis GC-FID/EAD Chromatograms with electroantennograms were imported from the GC-EAD software (Syntech, The Netherlands) as an ASCII file to an automated EAD analysis program for analysis (Slone and Sullivan 2007). This program transformed the data and identified antennal responses from background noise using three separate algorithms based on amplitude, peak 15

24 shape, and the combination of the both amplitude and peak shape. Retention times of responses recognized by all three algorithms, were tabulated and percent response was calculated for each peak. The total number of responses for each GC-FID/EAD analysis was also tabulated and compared between concentration treatments using SAS Version 9.1 for Windows (SAS Institute 2002) with an analysis of variance using PROC MIXED. Comparisons of percent response were made between wild and laboratory-reared moths using the Student s t-test. 16

25 Results Figure 1. GC-EAD (top) with GC-FID (bottom) of 1 apple hour equivalent (AHE) collected during the first codling moth flight to a mated female codling moth antenna separated on the DB- 1 column. Peak numbers correspond to peak numbers in Tables 1 and 3. Peaks which elicited at least one response at 0.1 AHE: peak 2 (Z)-3-hexenol, peak 7 d-linalool and peak 11 (E,E)- α-farnesene. Eleven chemicals, described as peaks 1 11, elicited antennal responses in 60 percent of trials to at least one concentration of immature apple volatile chemical collections (Figure 1). The antennal responses of wild and laboratory-reared codling moths to volatile chemicals from immature apples showed no significant differences for any chemicals at the concentration of 1 AHE (Table 1). Peaks 1, 3, 5 and 9 elicited one or zero total antennal responses during this trial. 17

26 Table 1 Mean percent (±SEM) antennal responses of laboratory reared versus wild codling moths to volatile chemicals from immature apples at 1.0 apple hour equivalent for each peak. (N=5) Peak RtI 2 Codling Mean Response Statistics for t-test Number 1 moths Ratio Lab 68 ± 5 t = -1.18, df = 4 Wild 80 ± 9 P = Lab 32 ± 10 t = -1.20, df = 4 Wild 56 ± 17 P = Lab 88 ± 12 t = 1.43, df = 4 Wild 64 ± 12 P = Lab 100 ± 0 t = n/a, df = 4 Wild 100 ± 0 P = n/a Lab 28 ± 19 t = 0.60, df = 4 Wild 16 ± 7 P = Lab 44 ± 19 t = -0.92, df = 4 Wild 64 ± 10 P = Lab 64 ± 15 t = -0.68, df = 4 Wild 76 ± 10 P = Internal 1589 Lab 96 ± 4 t = -1.00, df = 4 Standard Wild 100 ± 0 P = These peaks were chosen because they showed two or more responses during this test, peak numbers correspond to Figure 1. 2 RtI retention index DB-1 60m x 0.25mm ID x 0.25µm Film Thickness Both first and second codling moth flight samples showed a significant interaction between AHE concentration and number of antennal responses (F = 38.65; df = 2, 8; P < and F = 24.01; df = 2, 8; P = respectively). Electroantennograms of lower concentrations showed significantly fewer responses than higher concentrations (Table 2). Immature apple volatile chemical collections from the first flight at a concentration of 0.1 AHE produced significantly fewer responses than 1 AHE (Tukey adjusted P = ) and 10 AHE (Tukey 18

27 adjusted P < ), while 1 AHE elicited significantly fewer responses than 10 AHE (Tukey adjusted P = ). Analysis of apple odorants collected during second flight showed that at the concentration of 0.1 AHE significantly fewer responses where produced than at 1 (Tukey adjusted P = 0.04) and 10 AHE (Tukey adjusted P = ), while the 1 AHE concentration volatile chemical collections elicited significantly fewer responses than 10 AHE (Tukey adjusted P = 0.01). Table 2 Mean (±SEM) number of antennal responses to different amounts of immature apple volatile chemicals collected during two codling moth flights, June 18 through June 22 and July 28 through August 1, (N = 5) Number of AHE 1 Responses First Flight ± 0.8a ± 0.7b ± 0.6c Second Flight ± 1.2x ± 0.6y ± 1.9z 1 AHE apple hour equivalent, (number of apples x number of hours collected)/volume of sample 2 Means within the same flight followed by different letters were significantly different at P <

28 Table 3 Percent antennal response of codling moth females for each concentration analyzed by GC-EAD/FID to apple volatile chemicals collected during the first and second codling moth flights and mean (± SEM) amount of each chemical per apple hour equivalent (AHE 1 ) (N = 5). 1st Flight 2nd Flight Peak Mean(± SEM) Mean(± SEM) Number 2 RtI 3 AHE AHE AHE (µg/ahe) AHE AHE AHE (µg/ahe ± ± ± ± < < ± ± < ± ± < ± ± ± < ± ± < < < ± (number of apples x number of hours collected) of sample 2 Peaks shown elicited at least 60 percent responses at one or more concentration, peak numbers correspond to Figure 1. 3 RtI - retention index DB-1 60m x 0.25mm ID x 0.25µm Film Thickness Only four apple odorants elicited any responses at the lowest concentration tested (Table 3). Three of these chemicals were identified: peak 2 (Z)-3-hexenol (DB-1 retention index (RtI), DB-WAXETR RtI: 826, 1316), peak 7 d-linalool (1086, 1472), and peak 11 (E,E)-αfarnesene (1499, 1673). Peak 1 (DB 1 RtI: 744) elicited one antennal response to first flight apple volatile chemical collections at 0.1 AHE concentration, however, peak 1 did not elicit any responses at any concentration of the second flight samples. 20

29 Discussion Antennal responses to volatile chemical collections from immature apples of laboratory-reared moths were comparable to wild moths. Witzgall et al. (2005) showed that oviposition behavior in response to host plant odors differed between laboratory-reared moths and wild codling moths. The current study demonstrated that this difference in behavior is likely not caused by the inability of the laboratory-reared moths to perceive host odors. While laboratory-reared moths do not require host odor to stimulate oviposition (Witzgall et al. 2005), this does not necessarily preclude them from detecting the odor chemicals as indicated by antennal response. A relationship between the concentration of immature apple volatile chemical collections and the number of responses elicited from antennae of mated female codling moths was shown. These results are consistant with a prior study which used prepared standards at two concentrations (Ansebo et al., 2004). In the present study serial dilutions of apple volatile chemical collections were analyzed to approximate host odors at a distance from a source in the field. These results may indicate which chemicals are most likely to be perceived by a moth from a distance and be used for host location. The chemicals reported in this study, (Z)-3-hexenol, d-linalool and (E,E)-α-farnesene, have previously been identified from immature apple volatile chemical collections and were found to be antennally active in codling moths (Bäckman et al., 2001; Bengtsson et al., 2001; Vallet and Dorn, 2005; Casado et al., 2006). However, the enantiomeric determination of linalool was previously unreported. This determination is important because insect olfactory systems have been shown to be highly sensitive to enantiomeric purity (Ulland et al., 2006). d- linalool consistently elicited antennal responses at all concentrations even though it was not the most abundant chemical present in immature apple volatile chemical collections. Previous 21

30 studies assessed responses to racemic linalool. These studies included antennal responses to prepared standards (Ansebo et al., 2004), flight tunnel assays in combination with sex pheromone (Yang et al., 2004) and field trapping assays (Coracini et al., 2003). Both male and female codling moth antennae responded to racemic linalool (Ansebo et al., 2004) while the behavioral assays did not show any attraction (Yang et al., 2004; Coracini et al., 2003). Both (Z)-3-hexenol and (E,E)-α-farnesene also elicited antennal responses at the lowest concentration. (Z)-3-hexenol synergizes male codling moth attraction to sex pheromone (Yang et al., 2004), but has otherwise not been reported as being behaviorally active to codling moths. Casado et al. (2006) reported that male antennal responses to (Z)-3-hexenol were of higher magnitude than responses of mated females. Coracini et al. (2003) included (Z)-3-hexenol in host odor blends they tested as attractants in traps. None of the blends that included (Z)-3- hexenol caught significantly more codling moths than the control. (E,E)-α-farnesene is an attractant for codling moth larvae (Sutherland and Hutchins, 1972) and stimulates increased oviposition by female moths inside olfactometer and Petri dish assays (Wearing and Hutchins, 1973). Hern and Dorn (1999) demonstrated a sexually dimorphic response of codling moths to (E,E)-α-farnesene in Y-tube olfactometer assays. Males were more attracted to (E,E)-α-farnesene versus a control at very high doses while females were attracted by low doses only. (E,E)-α-farnesene alone was not attractive in flight tunnel assays (Yang et al., 2004) or in traps in the field (Coracini et al., 2003). Host location behavior is likely to be mediated by a blend of chemicals, perhaps even necessitating specific ratio and/or concentration of constituents. The three chemicals identified in this study as being antennally active at the lowest concentration, (Z)-3-hexenol, d-linalool, and (E,E)-α-farnesene, are of interest in future testing to determine the specific blend and ratio of 22

31 chemicals that codling moths use as host location kairomones. Future work should also include similar analysis of volatile chemicals produced by other codling moth host species and varieties. Perhaps the analysis of other host species will yield additional chemicals or reinforce the selection of the above three chemicals. 23

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