IDENTIFYING NOVEL ATTRACTANTS FOR INSECT PESTS FROM PLANT-PRODUCED CHEMICALS IN CRANBERRY

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1010345
• Project Start Date: Sep 1, 2017
• Project End Date: Aug 31, 2020
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218

Performing Department
Entomology

Non Technical Summary
The proposed research will identify plant-produced chemicals that insect pests utilize to identify and locate their host for egg laying in the cranberry system. The main insect pests of cranberry are two Lepidoptera: cranberry fruitworm Acrobasis vaccinii and sparganothis fruitworm, Sparganothis sulphureana. To date, no research has addressed the identification of cranberry-produced chemicals that these moths are using to identify and locate egg laying sites on their cranberry host. In the first objective of this study, we will isolate host plant volatile chemicals from cranberry plants using an active air collection system. In the second objective, the antennal responses of S. sulphureana and A. vaccinii adult females will be assessed to the volatile chemical blends from air collections of cranberry plants, using a gas chromatograph coupled with an electroantenno detector. The third objective will identify and quantify the emission of chemicals that trigger antennal responses in S. sulphureana and A. vaccinii adult females using a gas chromatograph coupled with a mass spectrometer. In the last objective, we will evaluate the behavioral response of S. sulphureana and A. vaccinii to the antennally-active compounds to identify the blend and ratio of compounds for optimal attractiveness using flight tunnel bioassays and field tests. The identification of cranberry-produced chemicals will provide the basis for improving monitoring methods for these major commercial pests and for implementation in attract-and-kill strategies.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
211	1121	1130	100%

Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
1121 - Cranberry;

Field Of Science
1130 - Entomology and acarology;

Keywords

kairomone

cranberry

insects

pests

management

monitoring

Goals / Objectives
The overall goal is to identify cranberry-produced chemicals that may trigger behavioral responses in the two most damaging insect pest species in the Wisconsin cranberry industry.The proposed research will be divided into four objectives:Objective 1. Isolate host plant volatile chemicals from cranberry plants at relevant phenological stages that coincide with moth species activity.Objective 2. Determine the antennal responses of S. sulphureana and A. vaccinii adult females to the volatile chemical blends from headspace collections of cranberry plants at the selected phenological and temporal stages. Objective 3. Identify and quantify the emission of chemicals that trigger antennal responses in S. sulphureana and A. vaccinii adult females.Objective 4. Evaluate the behavioral response of S. sulphureana and A. vaccinii adult females to the antennally active compounds to identify the optimal blend and ratio of compounds for optimal attractiveness.

Project Methods
Objective 1. We will isolate host plant volatile chemicals from cranberry plants at relevant phenological stages that coincide with moth species activity. Specifically, we will compare the chemical profiles of plants setting fruit and plants at fruit maturity to coincide with the oviposition period of the two flights of S. sulphureana (fruit set and fruit maturity) and the one flight of A. vaccinii (fruit set). For each time period, paired samples will be collected of plants with fruits and vegetative parts of the plants bearing leaves only to separate green leaf chemicals from fruits chemicals. Because S. sulphureana females lay their egg masses on the upper side of the cranberry leaves while A. vaccinii females lay eggs on the calyx of the fruit (Averill and Sylvia, 1998), attractive compounds for ovipositing females may originate from the leaves, fruit, or a combination of both. Because female moths are active at dusk, we will also compare the profiles of plants at fruit set during the photophase and the scotophase. For the photophase, collections will be conducted between 10AM and 4PM; for the scotophase, collections will be conducted between 8-11PM. For all collections, we will go to a commercial cranberry marsh in Central Wisconsin and use plants of the cultivar Stevens, the most commonly grown variety of cranberry in Wisconsin.We will collect the chemicals produced by plants at the different phenological stages (fruit set and fruit maturity), light phases (photophase and scotophase), and plant parts (leaves and fruit) using an active portable volatile collection system consisting of an electric portable air section pump, flow meters, gas sampling bags, and chemical adsorbent traps (e.g. poropak). Air will be pulled from three different sampling bags simultaneously at 300ml/min in each bag, one containing five cranberry uprights with fruit, five cranberry uprights with no fruit or flower, and an empty bag that will serve as a control. Bags will be placed over the uprights and closed up with clamps around the stems of the plant. Collections will be performed for a period of 1hr following a 15 min purge period of the system. Each collection will be performed five times. Following the collections, adsorbent traps will be sealed and brought back to the laboratory for extraction of the chemical compounds collected. Adsorbent traps will be extracted with 200 ml of dichloromethane and an internal standard, such as n-octane, will be added.Objective 2. We will assess the antennal responses of S. sulphureana and A. vaccinii adult females to the cranberry-produced chemicals from different plant parts and at the relevant phenological and temporal stages identified in objective 1.Adults of both species will be sexed daily and placed in mesh-lidded 355 mL clear plastic cups with a ratio of one female to two males for 48 hrs. to increase the likelihood of mating. Cages will be kept at 22 °C and ~60% RH in reversed light cycle conditions with a L16:D8 photoperiod. Females used in the electro antennodetection tests will be sugar-fed and 2-6 days post-emergence to insure optimal response to host plant chemicals. The right antenna of a female will be removed from the head and placed between 2 electrodes and 1 ml aliquots of headspace collections will be injected in the gas chromatograph (GC) equipped with a flame ionization detector (FID) and coupled with an electro antennodetection (EAD) system. One antenna per air collection will be used. The GC-FID/EAD system consists of an Agilent 7890 B Series GC-FID and a Syntech EAD IDAC-232 data acquisition interface and universal single ended probes (Syntech, The Netherlands). The GC will be equipped with a DB-1MS fused silica capillary column (Agilent Technologies) 30 m (length) x 0.25 mm (ID) x 0.25 µm film thickness. The column temperature program will start at 40 °C for two min, increased to 200°C at a rate of 10°C/min, and held at 200°C for 12 min for a total run time of 30 min. The column effluent will be split at a ratio of 2:1 between the EAD and the FID.Objective 3. We will identify and quantify the chemicals that triggered antennal responses in S. sulphureana and A. vaccinii adult females.In sub-objective A, a 1µl aliquot of each headspace collection sample will be analyzed using a Thermo Trace 1310 GC equipped with a Thermo ISQ mass spectrometer (MS) with electron ionization (EI) and a flame ionization detector (FID). Dual column comparison will be accomplished by analyzing the volatile chemical collection samples using the Agilent 7890B Series GC-FID equipped with a DB-1MS column followed by a DB-WAX fused silica capillary column of the same dimensions and using the same temperature and pressure program as described above.In sub-objective B, a 1ml aliquot of each headspace collection sample will be analyzed using the 7890 GC-FID equipped with the same DB-1MS capillary column and same column temperature program. For each behaviorally active compound, the integrated peak area data will be quantified using the GC-FID response to increasing quantities (5-50 ng) of an authentic standard.Objective 4. We will evaluate the behavioral response of S. sulphureana and A. vaccinii adult females to the antennally active compounds identified in objective 3 to characterize the attractiveness of selected compounds at biologically relevant concentrations and naturally occurring ratios of chemicals produced by cranberry plants. This objective will be divided into two sub-objectives: A) evaluate the behavioral response of adult females of both species in flight tunnel bioassays and B) evaluate the attractiveness of selected compounds to S. sulphureana and A. vaccinii in a field setting at a commercial cranberry marsh.In sub-objective A, the same methods described above in objective 2 for obtaining, rearing, and maintaining both insect species will be used for the adult moths used in behavioral bioassays. Bioassays will consist of a wind tunnel (ARS Analytical Research Systems) 41x41x152 cm placed in a dark room equipped with red lights and kept at ~60% RH. Each day, assays will be conducted with treatments randomized and a total of 10 females tested per treatments. While the total number of treatments will not be revealed until the EAD-active chemicals are identified, in the first experiment, treatments will consist of 1) solvent control, 2) complete blend of EAD-active chemicals; 3, 4, 5...) each individual compounds individually. Chemical compounds will be applied to filter paper as a substrate with 200 µl of each treatment applied to a filter paper. The flight tunnel wind speed will be set at 0.2 m/sec. Mated and fed S. sulphureana and A. vaccinia females will be assayed individually to attractants. Each female moth will be given 2 min to locate the source of the chemicals. For each moth, we will record whether the female displayed upwind flight (anemotaxis), plume tracking, and whether or not the moth contacted the filter paper.In sub-objective B, we will conduct field testing of the chemicals to confirm the attractiveness in commercial cranberry. Focusing on the compounds that triggered upwind flight in sub-objective A, we will conduct a second experiment called "drop-out" consisting of 1) solvent control, 2) blend of attractive chemicals; 3) attractant blend minus one chemical; 4) attractant blend minus another chemical; etc. This drop out study will allow us to determine whether all of the compounds identified in the attractant blend are necessary to account for the attraction. We will use P2 traps baited with the different treatments dispensed from rubber septa. Traps will be set out for four weeks and checked weekly for the presence of S. sulphureana and A. vaccinii and the numbers of each males and females for each species will be counted.

Progress 09/01/17 to 08/31/20

Outputs
Target Audience:The main audiences are the Wisconsincranberry growers,cranberry consultants, and University of Wisconsin - Madison Division of Extension outreach specialists. Theresults of this research areapplicable acrossthe entire cranberry industryat the state, national andinternational levels. This research will also be shared outside the cranberry growing community, with researchers who coulduse methods and technologies developed here to further research on similar topics in different systems. Changes/Problems:This study failed to find chemical attractants for either species of moth. None of the chemicals that were identified proved to be attractive in our behavioral assays and the field study in 2020. There are several factors that could explain this. One possibility is that one or all of the three unidentified compounds are important to attraction. There are a few additional techniques that could be utilized in effort to identify these chemicals. All of them would require collecting more samples from cranberry vines and learning new analytical chemistry techniques, possibly necessitating purchasing additional equipment. Other factors that can be investigated are the release rates and ratios of chemicals as well as the type of traps used in the field tests may not have been ideal. Further tests could try different release rates, ratios and blends of chemicals and different traps. The most limiting factor with these additional tests is time. These insect species only fly for a few weeks once per year for cranberry fruitworm and twice for sparganothis fruitworm which limits the number of combinations that can be investigated each year. The objectives of this study state that laboratory behavioral assays would be used to determine attractiveness. Several techniques were tried including y-tube olfactometry, flight tunnel, and arena assays. None of these techniques allowed us to show any attraction of cranberry volatile chemicals when compared to a blank. There are many possible reasons for this. We may not have discovered the correct abiotic conditions preferred by the moths whensearching for a host. Cranberry fruitworm require a very significant investment of timeto collectand rearing methods are not well established with high mortality and only one generation per year,leading to limited supply for testing. The conditions the moths experience in cranberry marshes at night when they are searching for host plants are hard to reproduce in the laboratory, including high humidity and moderate to cold temperatures. Additional attempts at reproducing these conditions may prove successful. Laboratory behavioral assays could be crucial to developing an attractant as they make it possible to conduct tests throughout the year while field testing doesnot. We plan to continue to address these challenges to identify plant compounds that could help the cranberry industry. What opportunities for training and professional development has the project provided?A PhD student learned how to develop methods and equipment toconduct all of the above statedaccomplishments, including: field volatile chemical collections from plants, rearing both species of moths in the laboratory, installing,operating, and maintainingthe chromatography equipment (GC-EAD and GC-MS), how to set up and run a flight tunnel and y-tube olfactometer forbehavioral testing of these chemicals. The student alsolearntto use a completelynew software for chemical analysis, to communicatewith cranberry growers and state agencies, and learnt about the cranberry system as a whole. The student also deepened her knowledge ofinsect taxonomy and analytical chemistry, and entomology as a whole. She also participated actively throughout her project with the professional societies of entomology. She gained leadership experience with the Entomological Society of Americaas a member ofthe student affairs committees at the branch and national levels and as a treasurerand organizer of the branch meeting held in Madison in 2018. She also was an active participant of the Entomogical Graduate Student Association throughout her tenure at UW-Madison. How have the results been disseminated to communities of interest?Research has been disseminated to the cranberry growers at the 2020 Wisconsin Cranberry School, as well as at professional societies (Entomological Society of America national and branch meetings)as described in the product section. As the graduate student finishestowriteher dissertation, we anticipate three manuscripts to be submitted to peer-reviewed scientific journals. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1: Volatile chemicals were collected from cranberry vines in the field during four time periods: 1. Before bloom, 2. During Bloom, 3. After fruit set with small unripe fruit, and 4. Before ripening with large unripe fruit. A portable volatile chemical collection apparatus was assembled to allow collecting these air collectionsin the field for 4 hours after sunset to coincide with the major pest flight times. Five replicates were collected in 2017 and 2018. Objective 2: The antennal responses ofS. sulfureana (sparganothis fruitworm)andA. vaccinii (cranberry fruitworm)adult femalesto the fivevolatile chemical blends collected in objective 1, were assessed using a gas chromatograph coupled with an electro antenno detector.Antennal responses were tabulated and led to a total of 12 chemicals triggering antennal responses between the two insect species. Three of these chemicals elicited antennal responses from sparganothis fruitworm only, while 5 were only antennally active for cranberry fruitworm. The remaining four elicited antennal responses from both species. Objective 3: Nine chemicals were identified using gas chromatography and mass spectrometry. Two chemicals were too volatile and were impossible to separate from the solvent peak and another chemical was present is such small amounts that none of these three chemicals wereidentifiable with our methods. The chemicals which elicited antennal responses insparganothis fruitworm alone were prenyl acetate, phenol, and Z-3-hexenyl acetate. The chemicals which elicited antennal responses incranberry fruitworm alone were: 2 ethyl hexanol, acetophenone, and bicyclopentanone. The chemicals that both species responded towere identified as: nonanal, 2 ethyl hexanyl acetate, and methyl salicylate. The method used to collect the volatile chemicals only allowed the determination of the relative ratio of the chemicals present in the samples with approximation of amount. Chemicals were confirmed using authentic standards and assesed for antennal responses from both species to confirm responses. Objective 4: The chemicals identified in objective 3 were formulated into lures using commercialstandards and loaded in the ratioof chemicals determined in objective 1. The chemicalstested in the field using traps to assess the attractiveness of the chemicals to both insect species during their flight period in cranberry. Inexperiment 1, each chemical was tested individually, with 10 replicates for two weeks in June 2020.In experiment 2, the entire blend of chemicals was tested along with every combination that included the full blend with onechemical removed. This experiment was also conducted with 10 replicates for two weeks in June 2020.During both experiments, zero cranberry fruitworm were caught in any oftreatmenttraps (data not shown) andlow numbers of sparganothis fruitworm moths were caught in some traps, but no treatments were significantly different from the controls (blanktraps with no chemical lures) in either experiment.

Publications

Progress 10/01/18 to 09/30/19

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A PhD student has been hired and has been trained or has researched and learned how to conduct all of the above stated accomplishments, including: field volatile chemical collections from plants, rearing both species of moths in the laboratory, operation of GC-EAD and GC-MS for use in this study including learning to use new software for analysis, communication with cranberry growers and state agencies, and currently, how to set up and run a flight tunnel and y-tube olfactometer for behavioral testing of these chemicals. How have the results been disseminated to communities of interest?Research results to date were presented at the Annual Conference of the Entomological Society of America in St Louis, MO in November 2019-. Results will also be presented in January 2020 to the Wisconsin Cranberry School where 400 cranberry growers attended. What do you plan to do during the next reporting period to accomplish the goals?We will complete objectives 3 and 4. Behavioral testing will happen in the laboratory in early parts of 2020 and field assessments in summer 2020.

Impacts
What was accomplished under these goals? Objective 2 was completed in 2019 and data has been analyzed. Objective 3 is being conducted. The chemicals have been quantified and identification of the chemicals still requires comparison with authentic standards to be confirmed. Twelve chemicals were identified to trigger antennal responses between the two species of moths. Seven by sparganothis fruitworm, nine by cranberry fruitworm with four that overlapped between species. The chemicals have been tentatively identified and are being checked against standards. Objective 4 is yet to be conducted once chemical identification has been confirmed and standards available for behavioral assays.

Publications

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The main audiences are the cranberry growers, cranberry consultants, and University of Wisconsin - Extension agents. The results of this research could, potentially, be applicable across the entire cranberry growing region at the state, national and international level. This research will also be shared outside the cranberry growing community, with researchers who could use methods and technologies developed here to further research on similar topics in different systems. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A PhD student has been hired and has been trained or has researched and learned how to conduct all of the above stated accomplishments, including: field volatile chemical collections from plants, rearing both species of moths in the laboratory, operation of GC-EAD and GC-MS for use in this study including learning to use new software for analysis, communication with cranberry growers and state agencies, and currently, how to set up and run a flight tunnel and y-tube olfactometer for behavioral testing of these chemicals. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period we plan to continue, and finish objectives 2 and 3, which are the analyses using GC-EAD and GC-MS of the cranberry volatile chemicals to determine which chemicals are likely to be involved in the location of cranberry plants for oviposition. We plan to start the behavioral testing of these chemicals in the laboratory, as well (Obj 4).

Impacts
What was accomplished under these goals? In the first year of this project, volatile chemicals have been collected from cranberry plants using a portable volatile collection apparatus in a cranberry marsh at 4 different phenological plant stages that correspond to adult moth activity: pre-bloom, during bloom, after bloom with very small fruit, and later with large unripe fruit (Obj 1). These volatile chemicals are, currently, being analyzed using GC-EAD with mated cranberry fruitworm and sparganothis fruitworm adult female moths (Obj 2). These samples are also being evaluated using GC-MS (Obj 3). This work is still in progress, with a few chemicals of interest having been already identified. Insects of both species have been collected from the field and are being reared in the lab for use in future testing using the GC-EAD as well as for the behavioral testing we plan to do in the laboratory (Obj 4).

Publications

Progress 09/01/17 to 09/30/17

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We plan to continue objective 1 and start on objective 2. Moths are being reared in the laboratory and chemicals are being analyzed and compared with standards to run on the GC-EAD.

Impacts
What was accomplished under these goals? We started collecting host plant chemicals from cranberry plants at relevant phenological stages (Objective 1)

Publications