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2019 Evaluation of candidate pheromone blends for mating disruption of the invasive swede midge (Contarinia nasturtii) Elisabeth Ann Hodgdon University of Vermont
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Recommended Citation Hodgdon, Elisabeth Ann, "Evaluation of candidate pheromone blends for mating disruption of the invasive swede midge (Contarinia nasturtii)" (2019). Graduate College Dissertations and Theses. 1063. https://scholarworks.uvm.edu/graddis/1063
This Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. EVALUATION OF CANDIDATE PHEROMONE BLENDS FOR MATING DISRUPTION OF THE INVASIVE SWEDE MIDGE (CONTARINIA NASTURTII )
A Dissertation Presented
by
Elisabeth Ann Hodgdon
to
The Faculty of the Graduate College
of
The University of Vermont
In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Specializing in Plant and Soil Science
May, 2019
Defense Date: March 22, 2019 Dissertation Examination Committee:
Yolanda H. Chen, Ph.D., Advisor Kimberly F. Wallin, Ph.D., Chairperson David S. Conner, Ph.D. Scott C. Merrill, Ph.D. Cynthia J. Forehand, Ph.D., Dean of the Graduate College
ABSTRACT
Swede midge ( Contarinia nasturtii , Diptera: Cecidomyiidae) is a small invasive fly that is currently threatening Brassica vegetable and oilseed production in the Northeastern U.S. and Canada. Larvae feed on plant meristems, resulting in deformed leaves, stems, and heads. Extremely low damage thresholds for heading Brassica vegetables, multiple overlapping generations, and lack of effective organic insecticide options present serious challenges for managing this pest. Pheromone mating disruption (PMD), which involves confusing male insects with unnaturally large doses of sex pheromones, is particularly promising for swede midge management because it prevents mating and subsequent oviposition. One major challenge to PMD for swede midge management is that the chiral female pheromone blend, a 1:2:0.02 blend of (2 S, 9 S)- diacetoxyundecane, (2 S, 10 S)-diacetoxyundecane and ( S)-2-acetoxyundecane, is expensive to synthesize due to the structural complexity of the compounds. Here, we explored three ways to reduce the cost of swede midge PMD: the use of lower-cost racemic pheromones containing all possible stereoisomers, single-component blends, and the possibility of using timed pheromone dispensers by testing for diel patterns of midge reproductive behavior. Although we found that males were not attracted to blends containing the racemic stereoisomers of the main pheromone component, (2 S, 10 S)-diacetoxyundecane, racemic blends functioned equally as well as chiral blends in confusing males and altering female behavior in PMD systems. We observed 95% and 87% reductions in males caught in monitoring traps in three-component chiral and racemic PMD plots of broccoli, respectively. In addition to confusing males, we also found that females altered their reproductive behavior in response to both chiral and racemic pheromones. Females released pheromones more frequently when exposed to three-component chiral and racemic blends, and were less likely to mate afterward. Single-pheromone treatments containing either chiral or racemic 2,10-diacetoxyundecane neither confused males nor influenced female behavior. We identified a total of eight hours during the day and night when midges do not exhibit mate-seeking behavior, during which programmable PMD dispensers could be turned off to save pheromone inputs. We found that up to 81% of females released pheromones to attract males for mating in the early morning shortly after dawn. Most females emerged in the morning as well, releasing pheromones soon after eclosing. Because midges are receptive to mates shortly after emergence, they may mate at their emergence site. Overall, we found relatively high levels of crop damage in our pheromone-treated plots, likely due to the migration of mated females into our plots. If midges mate at emergence sites, rotation of Brassica vegetable crops may result in overwintered midges emerging in fields where host plants are not currently grown. Further research is needed to determine where midges mate in order to determine where to install PMD dispensers. CITATIONS
Material from this dissertation has been accepted for publication in The Canadian Entomologist on January 17, 2019 in the following form:
Hodgdon E.A., Hallett R.H., Stratton C.A., Chen Y.H.. (2018). Diel Patterns of Emergence and Reproductive Behaviour in the Invasive Swede Midge (Diptera: Cecidomyiidae). The Canadian Entomologist.
AND
Material from this dissertation has been submitted for publication to the Journal of Chemical Ecology on February 5, 2019 in the following form:
Hodgdon, E.A., Hallett R.H., Stratton C.A., Chen Y.H.. (2019). Racemic Pheromone Blends Disrupt Mate Location in the Invasive Swede Midge ( Contarinia nasturtii ). Journal of Chemical Ecology.
ii ACKNOWLEDGEMENTS
I express a great deal of gratitude to my advisor, Dr. Yolanda Chen, for her mentorship over the past four years. I can attribute a great deal of my growth as a scientist to Yolanda, who taught me the value of critical and original thinking in research.
I also thank my committee members, Dr. David Conner, Dr. Rebecca Hallett, Dr. Scott
Merrill, and Dr. Kimberly Wallin for helping shape my research projects and providing insightful advice during my Ph.D. program.
I am grateful for the companionship of my labmates in the Insect Agroecology and Evolution Lab. Chase, Kristian, Jorge, and Andrea, I appreciate your friendship and support over the years. To the Hallett Lab at the University of Guelph, thank you for welcoming me into your lab and helping me through 24-hour experiments and tedious field work every summer. I also thank Jamie Heal, the UVM Greenhouse staff,
Pfenning’s and Elora Research Station farm staff, and countless student research assistants for all their help with my experiments. I thank Amy Saenger for her patience and last-minute assistance formatting this dissertation.
Last but not least, I thank my soon-to-be husband, Ben, and my family for their continuous support for my studies. One of my fondest memories during my Ph.D. is when Ben helped me observe midge mating at 5 am before he went to work. To my dad,
Andy, the hardest worker I know, I owe my work ethic and love of insects. To my mom,
Nadine, I owe my attention to detail and love of learning. Thank you.
iii
TABLE OF CONTENTS
CITATIONS…………………………………………………………………………… ii
ACKNOWLEDGEMENTS…………………………………………………………...iii
LIST OF TABLES…………………………………………………………………... viii
LIST OF FIGURES……………………………………………………………………ix
CHAPTER 1: INTRODUCTION…………………………………………………….. 1
1.1 Introduction ...... 1 1.1.2 Pheromone Mating Disruption ...... 1 1.1.3 Mating Disruption in Annual Systems ...... 1 1.2 The Invasive Swede Midge ...... 3 1.2.1 Life Cycle and Biology ...... 3 1.2.2 Management ...... 5 1.3 Opportunities for Economical Mating Disruption of Swede Midge ...... 6 1.3.1 Racemic Pheromone Blends...... 7 1.3.2 Efficient Pheromone Application ...... 8 1.4 Research Objectives ...... 9 1.5 References cited ...... 11
CHAPTER 2: RACEMIC PHEROMONE BLENDS DISRUPT MATE LOCATION IN THE INVASIVE SWEDE MIDGE ( CONTARINIA NASTURTII )………………………………………………………………………….. 18
2.1 Acknowledgements ...... 19 2.2 Abstract ...... 20 2.3 Introduction ...... 21 2.4 Methods and Materials ...... 25 2.4.1 Swede Midge Colony Rearing ...... 25 2.4.2 Test Insects ...... 26 2.4.3 Swede Midge Pheromone...... 26 iv 2.4.4 Male Dose-Response to Chiral Pheromone ...... 28 2.4.5 Pheromone Choice ...... 30 2.4.6 Male Flight and Courtship Behavior in Response to Pheromone Blends ...... 31 2.4.7 Simulated Pheromone-Mating Disruption...... 33 2.5 Results ...... 35 2.5.1 Male Dose-Response to Chiral Pheromone ...... 35 2.5.1 Pheromone Choice ...... 35 2.5.2 Male Flight and Courtship Behavior in Response to Pheromone Blends ...... 36 2.5.3 Simulated Pheromone-Mating Disruption...... 37 2.6 Discussion ...... 37 2.7 References ...... 40 2.8 Tables ...... 44 2.9 Figures ...... 47
CHAPTER 3: SYNTHETIC PHEROMONE EXPOSURE INCREASES CALLING AND REDUCES SUBSEQUENT MATING IN FEMALE SWEDE MIDGE (DIPTERA: CECIDOMYIIDAE)…………………………………………. 52
3.1 Acknowledgements ...... 53 3.2 Abstract ...... 54 3.3 Introduction ...... 55 3.4 Methods and Materials ...... 59 3.4.1 Swede Midge Colony Rearing ...... 59 3.4.2 Laboratory Calling Experiments ...... 62 3.4.3 Field Calling Experiment ...... 65 3.4.4 Propensity to Mate Experiment ...... 67 3.5 Results ...... 69 3.5.1 Laboratory Calling Experiments ...... 69 3.5.2 Field Calling Experiment ...... 70 3.5.3 Propensity to Mate Experiment ...... 71 3.6 Discussion ...... 71 3.7 References ...... 77 3.8 Tables ...... 82 3.9 Figures ...... 83 v CHAPTER 4: FIELD TESTS OF CANDIDATE PHEROMONE BLENDS FOR MATING DISRUPTION OF THE INVASIVE SWEDE MIDGE (DIPTERA: CECIDOMYIIDAE)…………………………………………………………………..88
4.1 Acknowledgements ...... 89 4.2 Abstract ...... 90 4.3 Introduction ...... 91 4.4 Methods and Materials ...... 95 4.4.1 Experimental Sites ...... 95 4.4.2 Mating Disruption Treatments ...... 97 4.4.3 Evaluation of Mating Disruption ...... 98 4.4.4 Statistical Analyses ...... 100 4.5 Results ...... 100 4.5.1 Trap Shutdown ...... 100 4.5.2 Plant Damage and Yield Assessment ...... 102 4.6 Discussion ...... 103 4.7 References ...... 110 4.8 Tables ...... 115 4.9 Figures ...... 118
CHAPTER 5: DIEL PATTERNS OF EMERGENCE AND REPRODUCTIVE BEHAVIOUR IN THE INVASIVE SWEDE MIDGE (DIPTERA: CECIDOMYIIDAE)…………………………………………………………………122
5.1 Abstract ...... 123 5.2.1 Résumé ...... 124 5.2 Introduction ...... 125 5.3 Materials and Methods ...... 128 5.3.1 Swede Midge Colony Rearing ...... 128 5.3.2 Sex-Based Differences in Diel Emergence ...... 130 5.3.3 Diel Patterns of Female Calling ...... 131 5.3.4 Diel Periodicity of Male Response to Pheromone Traps ...... 133 5.4 Results ...... 134 5.4.1 Sex-Based Differences in Diel Emergence ...... 134
vi 5.4.2 Diel Patterns of Female Calling ...... 135 5.4.3 Diel Periodicity of Male Response to Pheromone Traps ...... 136 5.5 Discussion ...... 137 5.6 Acknowledgements ...... 142 5.7 References ...... 143 5.8 Figures ...... 147
COMPREHENSIVE BIBLIOGRAPHY…………………………………………... 150
APPENDIX A………………………………………………………………………...161
vii LIST OF TABLES
Table 2.1 Stereoisomers of pheromone components in blends used for behavioral assays ...... 44 Table 2.2 Pheromone treatments used for pheromone attraction and preference experiments in olfactometer and wind tunnel ...... 45 Table 2.3 Pheromone treatments in each arm used in simulated pheromone mating disruption experiment ...... 46 Table 3.1 Pheromone blend treatments used in laboratory experiments ...... 82 Table 4.1 Plot and broccoli production characteristics at experimental sites ...... 115 Table 4.2 Amounts of pheromone components per reservoir dispenser in three- and single-component experimental experiments ...... 116 Table 4.3 Mean damage ratings (± SEM) at three and six weeks after transplanting and at harvest ...... 117 Table A.1 Swede midge natural pheromone compounds and compounds produced in the synthetic racemic blend (Hillbur et al. 2005) ...... 161
viii LIST OF FIGURES
Fig. 2.1 Percentage of males flying toward the pheromone source in the y-tube olfactometer at increasing doses ...... 47 Fig. 2.2 Percentage of males choosing pheromone blends when given a choice between two blends in a y-tube olfactometer ...... 48 Fig. 2.3 Percentage of males landing within 60 cm, 5 cm, and making contact with the pheromone source in the wind tunnel ...... 49 Fig. 2.4 Percentage of males fanning wings after landing in response to pheromone blends in the wind tunnel ...... 50 Fig. 2.5 Observed incidences of mating in the y-tube olfactometer simulated pheromone mating disruption experiment ...... 51 Fig. 3.1 Females calling in dose response experiment using three-component chiral pheromone blend ...... 83 Fig. 3.2 Females calling in pheromone blend experiment ...... 84 Fig. 3.3 Midges calling for at least half of the time points (continuous callers) within each pheromone treatment in the laboratory calling experiment ...... 85 Fig. 3.4 Incidences of calling by individual females at 15-minute intervals following emergence ...... 86 Fig. 3.5 Females calling in untreated (control) or three-component chiral pheromone mating disruption plots ...... 87 Fig. 4.1 Mean numbers of males caught in monitoring traps in three-component mating disruption plots each week ...... 118 Fig. 4.2 Mean numbers of males caught in monitoring traps in single-component mating disruption experiment plots each week ...... 119 Fig. 4.3 Broccoli marketable yield in three-component experiment mating disruption plots in (A.) 2016 and (B.) 2017 ...... 120 Fig. 4.4 Broccoli marketable yield in single-component experiment mating disruption plots in (A.) 2017 and (B.) 2018 ...... 121 Fig. 5.1 Mean percent emergence of total male and female adult swede midge from soil from Swiss (A.) and Ontarian (B.) populations over three 24 hour periods 147 Fig. 5.2 Percentage of females calling each half-hour in (A.) Swiss and (B.) Ontarian colonies ...... 148 Fig. 5.3 Mean percent of total males caught in traps at each hour per day in Ontario field trial ...... 149
ix CHAPTER 1: INTRODUCTION
1.1 Introduction
1.1.2 Pheromone Mating Disruption
Pheromone mating disruption is an insect pest management tactic that interferes with mate location by confusing male insects with synthetic sex pheromones (Cardé and
Minks 1995; Howse et al. 1998). Mating disruption has been highly successful for managing several important lepidopteran pests of perennial fruit crops, with nearly one million treated hectares annually (Welter et al. 2008; Witzgall et al. 2010; Miller and Gut
2015). Despite the widespread use of mating disruption for Lepidoptera in perennial cropping systems (Miller et al. 2015), it is seldom used to manage insects in other orders and in annual systems. Applying mating disruption protocols for Lepidoptera to insects in other orders with varying life histories and ecologies can be challenging, particularly in more complex annual cropping systems (Milli et al. 1997; Fadamiro et al. 1999; Smart et al. 2013).
1.1.3 Mating Disruption in Annual Systems
The development of pheromone mating disruption in annual systems is complicated by crop rotation. Major challenges in mating disruption systems for vegetable and other annual crop pests include migration of mated females, lack of proper pheromone formulations, prohibitive pheromone costs, and overall lack of efficacy
(Fadamiro et al. 1999, Michereff Filho et al. 2000, Schroeder et al. 2000, Megido et al.
2013). Crop rotation in annual systems can result in a mismatch between the location of overwintering pests and the next year’s crop. Alternate mating sites outside the crop field
1 render mating disruption dispensers less effective in preventing mating, resulting in considerable “edge effects” when the treated area is not large enough (Milli et al. 1997;
Fadamiro et al. 1999; Smart et al. 2013). Mating disruption has been tested for several lepidopteran pests of vegetable crops, including diamondback moth ( Plutella xylostella ;
Plutellidae), European corn borer ( Ostrinia nubilalis ; Crambidae), beet armyworm
(Spodoptera exidua ; Noctuidae), and others, with varying success without resulting in commercial adoption (Fadamiro et al. 1999; Kerns 2000; Schroeder et al. 2000; Wu et al.
2012).
Despite the aforementioned challenges, the most widely used mating disruption system within annual crops has been for tomato pests, including tomato pinworm
(Keiferia lycopersicella ; Lepidoptera: Gelechiidae) and the tomato leafminer ( Tuta absoluta ; Lepidoptera: Gelechiidae). Pheromone mating disruption is particularly promising for these two pests because larvae feed within the leaf mesophyll, within the fruit or underneath the calyx, and contact with foliar insecticides can be difficult if their application is not timed carefully (Jiménez et al. 1988; Cocco et al. 2013). Mating disruption for these two pests has been particularly useful in areas where excessive insecticide use resulted in resistance (Jiménez et al. 1988; Trumble and Alvarado-
Rodriguez 1993; Schuster et al. 2000; Vacas et al. 2011). Tomato mating disruption systems have been the most successful in highly contained greenhouses with mesh- covered ventilation systems versus in open fields (Vacas et al. 2011; Cocco et al. 2013).
Mated females from untreated areas are less likely to invade treated enclosed greenhouses.
2 Pheromone mating disruption may also be particularly useful for galling midges in the family Cecidomyiidae, which contains several economically-important pests of both annual and perennial crops (Gagne 1989; Hall et al. 2012). Adult midges are small, and males are highly responsive to minute amounts of female sex pheromone (Hall et al.
2012). Because cecidomyiid females produce pheromone amounts measured in picograms compared with micrograms in Lepidoptera, significantly less pheromone would be needed to confuse midges versus moths (Hall et al. 2012). Pheromone mating disruption was successfully demonstrated for swede midge ( Contarinia nasturtii Kieffer), but additional research and development is necessary for commercial adoption (Samietz et al. 2012).
1.2 The Invasive Swede Midge
1.2.1 Life Cycle and Biology Since its introduction to North America from Europe in the 1990’s, swede midge has caused serious losses of Brassica vegetable and oilseed crops in the Northeastern
U.S. and Canada (pers. obs. , Hallett and Heal 2001). Adult midges are small (~2 mm) and are not easily seen without magnification. Adults live for approximately 1-3 days, mating and ovipositing into the meristems of Brassica spp. vegetables and oilseed crops, including broccoli, cauliflower, cabbage, and Brussels sprouts ( B. oleraceae ), as well as kale, mustard, and canola (B. napus) (Readshaw 1961; Gagne 1989; Hallett 2007) . Larval feeding on meristematic tissue disrupts growth and renders crops unmarketable.
Cecidomyiid pests are particularly difficult to manage because of their unique ability to manipulate plant growth. Larval feeding on plant tissues alters nutrient allocation and plant hormone dynamics (Tooker and De Moraes 2007; Tooker and 3 Moraes 2010), often resulting in irreversible damage to crops (Stratton et al. 2018). While swede midge do not induce true gall formation in host plants similarly to other cecidomyiids (Gagne 1989), larval feeding within the meristem damage causes distorted stem and leaves. In heading Brassica vegetables, severe damage results in multiple smaller heads or complete lack of head formation. Damage symptoms typically appear after midge larvae have dropped off the plant to pupate in the soil (Chen et al. 2011). As a result, some growers unfamiliar with the pest in North America initially mistake swede midge damage symptoms for nutrient deficiencies because no insects may then be found on damaged plants (Hallett and Heal 2001).
Due to challenging aspects of larval feeding, alternative management tactics to insecticides are urgently needed. Similarly to tomato leafroller and pinworm, feeding larvae are protected by plant tissue from contact with sprays, and foliar insecticides are largely ineffective (Wu et al. 2006a; Hallett et al. 2009a; Seaman et al. 2014; Evans and
Hallett 2016). Once damage is visible, it is typically too late for growers to take action to prevent losses. Additionally, heading Brassica crops have extremely low damage thresholds, where only one feeding larva can render a cauliflower plant unmarketable, and host plants are vulnerable to damage from seedling stage until head formation
(Stratton et al. 2018).
Because swede midge populations are present throughout the growing season
(Hallett et al. 2009b), crops must be protected all season long. Midges pupate in the soil under their host plants and later emerge as adults, with timing dependent upon soil moisture and temperature (Readshaw 1966). As the growing season progresses, an increasing number of midges enter diapause, with cocoon-like pupae visibly different
4 from typical pupae (Readshaw 1961; Des Marteaux et al. 2012). While most overwintering pupae emerge the following spring, a small proportion of midges remain in the soil for a second or third winter (Readshaw 1961; Des Marteaux et al. 2015). As a result, growers rotating host crops can have multiple emergence sites after several years of swede midge infestation. At least two emergence phenotypes and four generations of swede midge in Ontario result in midge pressure from May until October (Hallett et al.
2009b).
1.2.2 Management
Currently, the two most effective options for certified-organic growers are to use spatially and temporally wide crop rotations and insect exclusion netting (Hodgdon et al.
2017). However, crop rotation and netting are not always feasible for growers. It is recommended that growers rotate susceptible crops at least 1 km away from infested fields, or wait to plant crops until after spring emergence ceases (Chen et al. 2011;
Hodgdon et al. 2017). However, small-scale growers with a limited land base cannot achieve adequate distance away from former Brassica fields. Additionally, insect exclusion netting can be cost prohibitive due to the small mesh size required to exclude swede midge (C. Hoepting, pers. comm.; unpubl. data).
Currently, there are no insecticides approved for certified-organic production that are effective for swede midge (Seaman et al. 2014; Evans and Hallett 2016). Repellent plant essential oils and plant defense elicitors are appropriate chemical controls for organic production systems and have shown some potential in laboratory and field trials, but require additional research (C.A. Stratton, pers. comm.). Systemic insecticides followed by calendar sprays of foliar insecticides are currently recommended to manage 5 swede midge in conventional systems; however, they are not always effective when swede midge populations are large (Hallett et al. 2009a; Chen and Shelton 2010; Chen et al. 2011).
Because of the limitations to chemical and physical control, researchers have sought biological control options. Exploration in Europe for specialist natural enemies as candidates for classical biological control were unsuccessful (Abram et al. 2012). While several generalist insect predators, parasitoids, and nematodes have been observed feeding on swede midge, none provide sufficient control in the field (Corlay et al. 2007;
Evans and Hallett 2016). In Ontario and Québec, parasitoids have been observed using swede midge as hosts at rates insufficient to provide crop protection (C.-E. Ferland, pers. comm.).
1.3 Opportunities for Economical Mating Disruption of Swede Midge
In Europe, Samietz et al. (2012) successfully demonstrated reduction of swede midge damage to Brussels sprouts using pheromone mating disruption. However, they speculate that mating disruption may not be economically feasible for swede midge due to the high cost of pheromone synthesis. The swede midge pheromone consists of
1:2:0.02 mixture of (2 S,9 S)-diacetoxyundecane, (2 S,10 S)-diacetoxyundecane and ( S)-2- acetoxyundecane (Appendix 1; Hillbur et al. 2005). Due to the complexity and chirality of the compounds, synthesis is costly and requires a highly skilled chemist (C.
Oelschlager, pers. comm.). Because few other alternatives to insecticides are available for swede midge management, strategies to reduce the cost of mating disruption will be worthwhile in order to allow pheromone mating disruption to be commercially-feasible for swede midge mating disruption on vegetable farms. 6 1.3.1 Racemic Pheromone Blends
To reduce the cost of pheromone inputs in mating disruption systems, unattractive racemic or simplified pheromone blends are viable options for some insect pests.
Racemic blends, or unpurified mixtures of compounds containing all possible stereoisomers (three-dimensional structures of the molecules), are often cheaper to produce because they allow the chemist to skip costly purification processes to isolate specific stereoisomers. Simplified blends, or blends containing only one or two compounds that comprise an insect’s pheromone blend, are also more economical because they require the synthesis of fewer compounds. Although the natural and most attractive pheromone blends have long been considered the most effective for mating disruption, (Roelofs 1978; Minks and Carde 1988), there is increasing evidence that alternative blends can be promising. For example, unnatural racemic blends substituted for stereo-specific chiral compounds are effective for mating disruption in pests across diverse cropping systems, such as gypsy moth ( Lymantria dispar ; Lepidoptera: Erebidae) in forests, the white grub beetle ( Dasylepida ishigakiensis ; Coleoptera: Scarabaeidae) in sugarcane, and the obliquebanded leafroller ( Choristoneura rosaceana ; Lepidoptera:
Tortricidae) in apple orchards (Evenden et al. 1999; Onufrieva et al. 2008; Arakaki et al.
2013).
Racemic blends may be cheaper to use for swede midge mating disruption.
However, the racemic blend of the main pheromone component, (2 S,10 S)- diacetoxyundecane, is unattractive to males (Boddum et al. 2009). Unexpectedly,
(Boddum et al. 2010) found that males possess antennal receptors for at least one of the non-natural stereoisomers in 2,10-diacetoxyundecane. Midges exposed to racemic 2,10-
7 diacetoxyundecane exhibited behavior consistent with repellency. The ecological relevance for males possessing receptors for pheromones not produced by female swede midge is unknown. Despite their unattractiveness to males, racemic compounds may function to disrupt mating.
1.3.2 Efficient Pheromone Application
Reducing the amount of pheromones released by dispensers can further reduce costs of mating disruption. Reducing pheromone inputs can be achieved by lowering the loading rate in individual dispensers, using partial or incomplete blends omitting one or more compounds in an insect’s pheromone blend, installing fewer dispensers in a field, or by using programmable dispensers that turn off during particular times of the day. For example, timed-release aerosol canister dispensers can release pheromone solely during the times of the day when insects are naturally active. For nocturnal moth species, dispensers can be programmed to release pheromones only at nighttime while still successfully managing the pests (Rama et al. 2002; Stelinski et al. 2007; Higbee and
Burks 2008; Casado et al. 2014). In order to use programmable timed pheromone release for swede midge, one must first determine whether the midge exhibits diel periodicity of mating behavior. Several other cecidomyiid pests have predictable patterns of mating at specific times of the day (Modini 1987; Bergh et al. 1990; Pivnick 1992; Heath et al.
2005). If midges mate at particular times of the day, programmable dispensers could be turned off when midges are not searching for mates.
8 1.4 Research Objectives
Here, I examine the reproductive behavior and responses of swede midge to chiral and racemic pheromone blends to evaluate candidate blends for mating disruption and explore opportunities for reducing mating disruption cost. Because racemic pheromone blends are promising for mating disruption due to their potential for cost savings, I study both male and female behavioral responses, including attraction and mating disruption, to partial and complete racemic blends compared with chiral blends. Using my observations of midge behavior and results from our field tests, I identify the most promising blend for swede midge mating disruption.
In Chapter 2, I examine male sensitivity, preferences, and mating disruption potential of chiral and racemic pheromone blends in laboratory settings. First, I observe whether male midges fly toward and display courtship behavior in response to chiral, racemic, chiral/racemic, and solvent-only treatments in a series of y-tube olfactometer and wind tunnel experiments. Second, I test whether the same pheromone blends confuse males and prevent mate location in a simulated mating disruption setup in a y-tube. Using the results of my simulated mating disruption experiment, I identify promising pheromone blends to test in the field in Chapter 4.
While Chapter 2 considers the effects of pheromones on male behavior, Chapter 3 tests for effects of pheromones on female reproductive behavior. Female “autodetection” and response to their own sex pheromones are seldom considered in mating disruption studies, which typically focus on males (Holdcraft et al. 2016). Additionally, there are currently no published studies on autodetection in Cecidomyiidae (pers. obs.). In Chapter
3, I test whether females alter their pheromone-releasing behavior while exposed to
9 synthetic pheromones and whether intense pheromone pre-exposure alters subsequent propensity to mate. Although increased calling and decreased propensity to mate has been shown in Lepidoptera (Stelinski et al. 2006, 2014; Kuhns et al. 2012; Rehermann et al. 2016; Holdcraft et al. 2016), it is unknown how swede midge females respond to pheromones. Here, I consider the implications for female autodetection on swede midge pheromone mating disruption efficacy.
In Chapter 4, I test the efficacy of three-component and single-component chiral and racemic pheromone blends to confuse males and provide crop protection in small- plot broccoli test systems in Ontario and Québec, Canada. To determine whether pheromones confuse males, I test for trap shutdown, or a lack of males caught in monitoring traps set up within the mating disruption plots. Because crop protection is the ultimate goal for mating disruption, I measure swede midge damage to the broccoli crop at three points during the season. Using my data from both trap shutdown and crop protection in conjunction with results from Chapter 2, I determine the most effective swede midge candidate pheromone blend for mating disruption.
Lastly, in Chapter 5 I examine diel periodicity of swede midge emergence and reproductive behavior in order to determine whether programmable pheromone dispensers are a possibility for cost-effective pheromone mating disruption. By observing the behavior of male and female mating behavior over 24-hour periods, I identify the times of day when swede midge typically mates, thus determining the times of day when dispensers could be turned off to save pheromone inputs.
As swede midge distribution increases annually and growers are faced with few management options, additional research and development of swede midge control tactics
10 is urgently needed. A better understanding of swede midge responses to pheromones and possible mechanisms of mating disruption is necessary to advance field implementation of this pest management technique. Additionally, swede midge mating disruption research may help advance the understanding of how to implement this pest management tactic for non-Lepidopteran pests and in annual cropping systems. Pheromone mating disruption, while typically utilized for moth pests in perennial fruit crops, is far less frequently used for other pests and in annual crops. Perhaps in the future with additional research, mating disruption technologies will be possible for other challenging pests, such as swede midge.
1.5 References Cited
Abram PK, Haye T, Mason PG, et al (2012) Identity, distribution, and seasonal phenology of parasitoids of the swede midge, Contarinia nasturtii (Kieffer) (Diptera: Cecidomyiidae) in Europe. Biol Control 62:197–205. doi:10.1016/j.biocontrol.2012.04.003
Arakaki N, Hokama Y, Nagayama A, et al (2013) Mating disruption for control of the white grub beetle Dasylepida ishigakiensis (Coleoptera: Scarabaeidae) with synthetic sex pheromone in sugarcane fields. Appl Entomol Zool 48:441–446. doi:10.1007/s13355-013-0202-6
Bergh JC, Harris MO, Rose S (1990) Temporal patterns of emergence and reproductive behavior of the Hessian fly (Diptera: Cecidomyiidae). Ann Entomol Soc Am 83:998–1004
Boddum T, Skals N, Hill SR, et al (2010) Gall midge olfaction: Pheromone sensitive olfactory neurons in Contarinia nasturtii and Mayetiola destructor . J Insect Physiol 56:1306–1314. doi:10.1016/j.jinsphys.2010.04.007
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17 CHAPTER 2: RACEMIC PHEROMONE BLENDS DISRUPT MATE LOCATION IN THE INVASIVE SWEDE MIDGE (CONTARINIA NASTURTII )
ELISABETH A. HODGDON *1 (0000-0002-2432-9201), REBECCA H. HALLETT 2 (0000-0002-4316-7877), KIMBERLY F. WALLIN 3,4 (0000-0002-6849-6351), CHASE STRATTON 1, and YOLANDA H. CHEN 1 (0000-0001-9439-5899)
1Department of Plant and Soil Science, University of Vermont, 63 Carrigan Dr., Burlington, Vermont, USA 05405
2School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada N1G 2W1
3Rubenstein School of Environment and Natural Resources, 81 Carrigan Drive, Burlington, Vermont, USA 05405
4United States Department of Agriculture Forest Service, Northern Research Station, 707 Spear St., South Burlington, Vermont, USA 05403
*Corresponding author contact information: Elisabeth A. Hodgdon Email: [email protected] Telephone: +1 (802) 522-6914
18
2.1 Acknowledgements
We thank Ylva Hillbur from the Swedish University of Agricultural Sciences for providing advice regarding pheromone doses and wind tunnel designs, Guiseppe Petrucci from the University of Vermont (UVM) Department of Chemistry for assisting with pheromone solution preparation, and William Louisos from the UVM College of
Engineering and Mathematical Sciences for measuring the wind speed in our wind tunnel. We also thank Oliver Bevan, Paolo Filho, Christian DeRoy, Kathryn Jacobs,
Brennan Kensey, Laurel Martinez, Ross Pillischer, Julia Ramseyer, Leila Rezvani,
Rebecca Roman, Dylan Samson-McKenna, Justine Samuel, Chase Stratton, and Samuel
Zuckerman for assistance with swede midge rearing. A USDA NIFA Crop Protection and
Pest Management grant (2014-70006-22525) provided funding for these projects.
19
2.2 Abstract
Swede midge, ( Contarinia nasturtii Kieffer), is an invasive cecidomyiid pest that is causing serious losses of Brassica oilseed and vegetable crops in the Northeastern U.S. and Canada. Currently, few alternatives to systemic insecticides exist for its management.
Because only one feeding larva can render heading Brassica crops unmarketable, management strategies that prevent oviposition are urgently needed. Pheromone mating disruption is a promising management approach for swede midge because it prevents mating and subsequent crop damage. While the swede midge pheromone has been identified, one of the major barriers to adoption is the high cost of chemical synthesis.
Racemic blends consisting of natural and non-natural stereoisomers could be promising candidates for mating disruption because they are more cost-effective to produce.
However, it is unclear whether males are attracted to racemic pheromone mixtures and whether they can prevent males from mating with females. Here, we studied whether males behaved differently in response to chiral and racemic pheromone blends by examining: 1) Male attraction to varying pheromone doses; 2) Whether males discriminate between chiral and racemic pheromone blends in a y-tube olfactometer and wind tunnel; and 3) Whether males can locate and successfully mate with females within pheromone-permeated y-tubes. We found that picogram amounts of pheromone attracted males and prevented them from locating females in y-tubes. While males were more attracted to the chiral blends compared to the racemic blends, all blends prevented nearly all males from mating with females. Therefore, low dose racemic blends may be promising for pheromone mating disruption.
20 2.3 Introduction
Contarinia nasturtii Kieffer (swede midge; Diptera: Cecidomyiidae) is a small galling fly that is a serious pest of Brassica vegetable and oilseed crops in Europe,
Northeastern Canada and the U.S. (Hallett and Heal 2001; Chen et al. 2011). Larvae feed within the plant meristem, causing deformed and scarred leaves and stems, and in severe cases cause complete loss of heads in broccoli, cauliflower, cabbage, and other related
Brassica crops. Recently, vegetable growers in the U.S. states of New York and Vermont have reported up to 100% yield loss of organic kale and broccoli. No insecticides that are approved for certified-organic production are effective in controlling the midge (Seaman et al. 2014; Evans and Hallett 2016). Due to the severe economic losses inflicted by this pest, some small diversified organic growers in the region now avoid Brassica production entirely (Y. Chen, pers. obs .).
Several aspects of swede midge biology create difficulty managing this pest. The presence of multiple overlapping generations and prolonged crop susceptibility to damage necessitates protection throughout the growing season (Hallett et al. 2009;
Stratton et al. 2018). Larvae are protected from foliar insecticides within the meristem
(Wu et al. 2006). Compounding these challenges is an extremely low damage threshold for vegetables, for example, Stratton et al. (2018) found that a single larva can render a cauliflower plant unmarketable. While some growers use calendar sprays of conventional insecticides to manage swede midge, reliance on chemical controls represents a loss of years of progress toward integrated pest management of vegetable pests (Andaloro et al.
1983; Chen et al. 2011). Given that a single larva can lead to unmarketable Brassica
21 crops, management approaches that prevent oviposition, such as pheromone mating disruption, are urgently needed.
Although cecidomyiids can be highly difficult to manage, certain aspects of their biology and ecology present opportunities for economical pheromone mating disruption systems. Pheromone mating disruption involves deploying large doses of synthetic female sex pheromone to interfere with the males’ ability to find mates. Adults are very short-lived, with discrete diel periodicity of mating (Gagne 1989; Bergh et al. 1990;
Harris et al. 1999; Hodgdon et al. Accepted ). Timed pheromone devices, releasing pheromones only at the particular times of day when adults are sexually active, could eliminate the use of pheromone when the insects are not naturally looking for mates. In our previous studies, we found that swede midge males are responsive to pheromones in the morning (Hodgdon et al., Accepted ). Because male cecidomyiid antennae are acutely sensitive to minute amounts of pheromone, pheromone mating disruption dispensers may be effective in releasing much smaller, and thus cheaper, amounts of material compared with systems for pests in other insect orders. Females of some economically-important cecidomyiids release quantities of pheromone measurable in picograms hourly (Hall et al.
2012), compared with exponentially larger amounts measured in micrograms in
Lepidoptera.
Due to its structural complexity and chirality, the swede midge pheromone is very costly to synthesize (Hillbur et al. 2005; Samietz et al. 2012), which may limit its commercial feasibility. However, Samietz et al. (2012) demonstrated successful swede midge mating disruption using the chiral pheromone blend. Currently, the chiral swede
22 midge sex pheromone is primarily used for monitoring and to inform insecticide spray programs for some crops (Hallett et al. 2007; Hallett and Sears 2013). The swede midge pheromone components, consisting of a 1:2:0.02 ratio of (2 S,9 S)-diacetoxyundecane,
(2 S,10 S)-diacetoxyundecane and ( S)-2-acetoxyundecane (Hillbur et al. 2005), each contain at least one chiral center, and therefore multiple possible stereoisomers. Failure to remove other non-natural stereoisomers during synthesis of the main compound,
(2 S,10 S)-diacetoxyundecane, reduces the cost of the pheromone blend, but causes a total loss of male attraction (Boddum et al. 2009).
Unexpectedly, Boddum et al. (2010) found that males possess receptors for at least one of the non-natural stereoisomers in the racemic blend of 2,10- diacetoxyundecane. The purpose for male swede midge possessing receptors for pheromones not produced by conspecific females is unknown. It is possible that swede midge congeners produce other stereoisomers in the 2,10-diacetoxyundecane blend. For some insects, the ability to detect and avoid pheromone plumes from closely related species aids in discrimination for members of their own species (Symonds and Elgar
2008). Although male swede midge are not attracted to pheromone blends containing non-natural stereoisomers of 2,10-diacetoxyundecane, they may present a lower-cost alternative for pheromone-based pest management technologies such as pheromone mating disruption, for which attraction may not be necessary (Evenden et al. 1999;
Stelinski et al. 2008; Miller and Gut 2015).
More economical racemic pheromone blends have the potential to be useful for pheromone mating disruption, although their efficacy for swede midge is yet unknown.
23 Racemic or non-natural stereoisomers of pheromones have been successfully used for pheromone mating disruption of other important pest species, including gypsy moth
(Lymantria dispar ; Lepidoptera: Lymantriidae) and the white grub beetle ( Dasylepida ishigakiensis ; Coleoptera: Scarabaeidae), even though the blends are not attractive to males (Onufrieva et al. 2008; Arakaki et al. 2013). Although Boddum et al (2009; 2010) investigated male swede midge attraction and physiological responses to racemic blends, additional research is necessary to determine whether they can disrupt mating.
We hypothesized that although the complete racemic blend is not attractive to male swede midge (Boddum et al. 2009), it may be effective in confusing males and preventing mate location in a simulated mating disruption system. We tested male behavioral responses when exposed to three pheromone blends to determine candidate blends for mating disruption: the complete chiral (natural) blend, the complete racemic blend containing all possible stereoisomers of each compound, and a chiral/racemic blend. The chiral/racemic blend contained chiral (2S,10 S)-diacetoxyundecane, necessary to attract males, and the racemic blends of the other components (2,9- and 2,10- diacetoxyundecane). Specifically, our research questions were: 1) How does pheromone dose influence male attraction; 2) Which pheromone blends do male midges prefer; 3)
Which pheromone blends elicit male upwind flight and courtship behavior; and 4) Which pheromone blends prevent males from locating and mating with females in a controlled laboratory setting? Here, we use our findings to identify candidate pheromone blends for future mating disruption trials in the field.
24 2.4 Methods and Materials
2.4.1 Swede Midge Colony Rearing
We reared swede midge in a laboratory colony for our behavioral assays. The midge colony originated from the Swiss Federal Research Station for Horticulture in
Wädenswil, Switzerland, and was previously reared at the University of Guelph in
Ontario, Canada prior to importing the colony to our lab (USDA APHIS permit number
P526P-13-03136). To avoid genetic bottlenecking in the colony, we periodically added field-collected midges from Vermont, U.S. The colony was kept at 22.4 ± 1.2ºC °C and
40.7 ± 11.4% relative humidity with a 16:8 light:dark photoperiod. We used Brassica oleracea group Botrytis ‘Snow Crown’ (cauliflower; Harris Seeds, Rochester, NY) for rearing due to its suitability as a swede midge host (Hallett 2007). Plants received fertilizer at a rate of 150 ppm with two parts 21-5-20 and one part 15-0-14 with supplemental magnesium and were grown in Fafard 3B soilless potting medium (Sun Gro
Horticulture, Agawam, MA, U.S.A). We introduced 6-8 week old plants into rearing cages for oviposition when the cauliflower buds were approximately 3 cm in diameter.
After plants were exposed to adult midges for 24 - 72 hours, we moved plants to separate cages to allow larvae to develop. Once larvae reached the third instar stage after 14 days, we cut the stems of the cauliflower plants and inserted the buds into the potting media to facilitate movement of larvae into the media for pupation. When ready to pupate, larvae jump from or crawl down the stems of their host plants into the soil below for pupation
(Readshaw 1961). We then returned the infested pots to the oviposition cages.
25 2.4.2 Test Insects
We used unmated midges that were less than 24h old. Adult swede midge typically live for one to three days. Females mate only once, usually within the first day after eclosion (Readshaw 1961). We used a combination of individuals emerging from the laboratory colony and from isolated single female progenies. In our laboratory setting, the majority of midges emerge shortly after dawn (Hodgdon et al., Accepted ). We captured individuals as they emerged from the soil from the main colony and transferred males and females to separate containers to prevent mating prior to the behavioral assays.
To prevent midge mating prior to behavioral assays, we reared offspring cohorts from individual females in deli containers (Webstaurant Store, Lititz, PA, USA) to separate the emerging males and females. A majority of cecidomyiid females produce either only male or only female progeny (Stuart 1991; Benatti et al. 2010), which may be a strategy to prevent inbreeding (Tabadkani et al. 2011). To produce unmated adults, we caged one female and two or three males from the main colony in a modified plastic deli container (two 946 ml containers fastened together), each with an eight to ten week-old cauliflower plant. We cut the cauliflower meristems and inserted them partially into the soil after 14 days, similarly to our colony rearing protocol. We aspirated adult offspring emerging in the containers approximately 18-21 days later singly into vials and held them in the experiment room for at least 30 minutes prior to our trials.
2.4.3 Swede Midge Pheromone
For all of our behavioral trials, we formulated the pheromone blends so that the amounts of the naturally-produced chiral stereoisomers for each component were equal
26 across chiral and racemic blend treatments, similar to those used by Boddum et al. (2009) in their wind tunnel studies with swede midge. We obtained >98% optically-pure swede midge pheromone components from ChemTica Internacional (Santo Domingo, Heredia,
Costa Rica) and formulated the following blends for each behavioral assay: solvent-only
(hexane) control, chiral blend containing each of the three naturally-produced stereoisomers, racemic blend containing all possible stereoisomers for each compound, and a chiral/racemic blend (Table 1). The chiral/racemic blend contained (2 S,10 S)- diacetoxyundecane, required for male attraction, and the racemic blends of the other components (2,9- and 2,10-diacetoxyundecane), which do not inhibit attraction (Boddum et al. 2009 ). Because the racemic blend of 2,9- and 2,10-diacetoxyundecane includes RR,
RS, SS and SR (or meso-) stereoisomers due to the presence of two chiral centers (Table
1; Hillbur et al. 2005), one quarter of these mixtures contained the SS stereoisomers, therefore four times as much of the racemic blend was required to deliver equal amounts of the SS stereoisomers. We needed only twice as much 2-acetoxyundecane because this compound has only one chiral center, and the racemic blend contains only two stereoisomers (Hillbur et al. 2005). For each experiment, we delivered the pheromones in
10 µl solutions with HPLC-grade hexane (Fisher Scientific, NH, USA) onto VWR qualitative #413 white filter paper (VWR International, Radnor, PA, USA), similarly to
Hillbur et al. (2005). Our doses ranged greatly, measured in picograms for the olfactometer trials and in nanograms for the wind tunnel experiment (Table 2) due to the differing sizes and air volumes of the treated areas in these devices.
27 2.4.4 Male Dose-Response to Chiral Pheromone To determine which doses to use in our subsequent olfactometer pheromone choice experiments, we conducted a sensitivity experiment to test male attraction to varying doses of the chiral pheromone blend in a y-tube olfactometer. We recorded whether midges were attracted to and moved toward the pheromone source, the solvent- only control, or exhibited no upwind movement. Lack of movement is an indicator of both excessively small and large pheromone doses (Shorey 1973; Farkas et al. 1974).
Although it is unknown exactly how much pheromone a single swede midge female produces (“female equivalent” doses), gland extracts from the congener C. pisi (pea midge) yielded a few picograms (Hall et al. 2012). Our highest dose (4 ng of (2 S,10 S)- diacetoxyundecane with the other two components following in ratio) was based upon estimates of female equivalents from Hessian fly (Y. Hillbur, pers. comm. ). Using 4 ng of
(2 S,10 S)-diacetoxyundecane as a starting point, we tested whether males would respond to decreasing serial dilutions (0.4, 0.04, 0.004, and 0.0004 ng of (2 S,10 S)- diacetoxyundecane, with (2 S,9 S)-diacetoxyundecane and ( S)-2-acetoxyundecane following in a 1:0.02 ratio) for a total of five pheromone doses to create our dose- response curve. We placed the pheromones into one arm of the y-tube. Our control treatment, in the other arm, consisted of 10 µl hexane only.
The olfactometer (Sigma Scientific, Micanopy, FL) consisted of an air compressor delivering air through activated carbon filters and into Teflon tubing. The tubing attached to two 10 cm long glass odor adapters that were fitted onto both stems of a y-tube. The inner diameter of the y-tube was 1.8 cm, the distance from the end of the stem to the junction was 14.5 cm, and the arms were 8 cm long. The olfactometer was set 28 up in separate room with a temperature of 22.8. ± 0.54ºC. Because swede midge are small
(~2 mm in length) and relatively weak fliers in a y-tube ( pers. obs. ), we set the airflow setting through each arm of the y-tube at a rate of 0.3 L/min.
We tested male responses to pheromones within three hours after the onset of photophase, when female midges typically release pheromone (Hodgdon et al.,
Accepted ). Within the y-tube, each male had five minutes to respond to the stimulus before being removed. If a midge traveled >2.5 cm past the y-tube junction and remained there for at least 15 seconds, we recorded a positive response to the pheromone treatment.
When midges did not make a choice within the time limit, they were removed from the y- tube and were not tested again. To remove directional bias, we switched the orientation of the y-tube after each replicate, and randomly selected a different concentration to test after every five midges. We tested the treatments in a random order, with five males comprising one block, and a total of eight blocks, for a total of n = 40 replicate midges for each treatment. Between each block, we cleaned the glassware with hexane and allowed the pieces to air dry. We recorded male responses with a binary scoring system: flight toward the pheromone (1), or either no flight or flight toward the solvent-only control (0).
To test whether the number of midges flying toward the pheromone differed significantly from 50%, we used a series of binary exact tests for each pheromone dose.
Because we used the same pheromone source (filter paper) for five midges within treatment groups, we first conducted chi-square tests to determine if the pheromone source (individual filter paper) significantly influenced the distribution of midge responses within each pheromone treatment. Because we found that pheromone source 29 (filter paper) did not significantly differ within all of our pairwise comparisons
(P > 0.05), we did not include pheromone source as a variable in our final analyses. For all statistical analyses, we used SPSS statistical software version 22 (International
Business Machines, Armonk, NY, USA). We interpreted statistical significance of our results using α = 0.05 and Bonferroni corrected P values where necessary for multiple comparisons.
2.4.5 Pheromone Choice
We tested if midges prefer one pheromone blend to another (Table 1) using a series of six pairwise comparisons in a y-tube olfactometer. Three of the comparisons consisted of the control treatment (hexane only) to either the chiral, chiral/racemic, or racemic pheromone blend, and the remaining comparisons consisted of one pheromone blend versus another (chiral versus chiral/racemic, chiral versus racemic, and chiral/racemic versus racemic). For comparisons of a pheromone blend versus the control, we loaded the total most attractive dose of the pheromone blend (4 pg; Fig. 1) into one arm and the other with solvent only (Table 2). For the remaining comparisons comparing different pheromone blends, the total amount of pheromone delivered to males in each arm was equal to half the dosage used in pheromone-control setups so that the total amount delivered to males was equal to the most attractive amount.
We used unmated males for the experiments using the same olfactometer protocol as described for the sensitivity assays, for a total of n = 70 replicate midges for each comparison experiment. Unlike in the dose-response experiments, we excluded males that did not make a choice from further analysis, based on the protocol used by
30 Andersson et al. (2009) and because we previously determined that our dosages were appropriate based on our sensitivity experiments. Unresponsive midges may have had differing pheromone sensitivity and/or differing circadian patterns of sexual activity, or may have been harmed during handling. We used binomial exact tests to examine differences between the proportions of midges choosing one pheromone treatment versus another in each of our y-tube setups, similar to our dose-response analyses.
2.4.6 Male Flight and Courtship Behavior in Response to Pheromone Blends
We tested whether male midges exhibited upwind movement and courtship behavior in response to single pheromone blends in a wind tunnel similar to the one used by Hillbur et al. (2005) and Boddum et al. (2009) for swede midge. Swede midge courtship behavior is similar to other plant-feeding midges and lepidopterans. After detecting an attractive pheromone, male midges fly upwind in a zigzag pattern, using the edges of the pheromone plume to guide them toward areas of higher concentration, and ultimately, the female or pheromone source (Gagne 1989; Hillbur et al. 2005; Boddum et al. 2009). When males get closer to the pheromone source or female, they fan (vibrate) their wings. When a pheromone signal is lost, adulterated, or unattractive, males may cease to travel farther upwind (Shorey 1973). Therefore, we assumed that the farther a midge traveled upwind within the tunnel, the more attracted it was to the pheromone treatment.
The tunnel (50 x 50 x 170 cm) consisted of an acrylic structure with activated carbon filters and mesh screens on both ends to remove contaminates and smooth air flowing through. We used a household box fan (51 x 51 cm) to push air through the
31 tunnel. We confirmed that the filter slowed the air to a speed of 0.5 cm/s using a hot wire anemometer (model 55P16, Dantek Dynamic, Skovlunde, Denmark). Using smoke from a smoke pen placed at the upwind end, we confirmed that the airflow through the wind tunnel was laminar. We placed filter papers with pheromone into a bent wire paper clip holder on an overturned glass beaker serving as a platform at the upwind end of the wind tunnel. To minimize external stimuli such as light and odors, we set up the wind tunnel in a room free of plants and insects. The building’s ventilation system exchanged air in the room approximately every eight minutes and was on average 26.7 ± 1.1ºC. The tunnel received sunlight through windows as well as 40W fluorescent lights hung directly above and parallel to the tunnel. We used a handheld light meter (Enviro-Meter, Control
Company, Webster, TX, USA) to adjust the setup so that the light levels were approximately equal at both ends of the tunnel.
Using a randomized complete block design, we observed the responses of n = 50 males to the four pheromone treatments using the same doses as Boddum et al. (2009;
Table 2). To avoid contamination between pheromone treatments and airborne pheromone buildup in the experiment room, we tested 10 male responses to only one pheromone treatment per day. We conducted the experiments between two to four hours after the onset of photophase, the peak hours of male mate-searching activity (Hodgdon et al., Accepted ). After releasing single males from glass vials into the tunnel 120 cm away from the platform, we gave each male three minutes to respond. We chose a three- minute timeframe because the pheromones dissipated quickly from the paper, and because midges typically responded within the first one or two minutes ( pers. obs.) . We categorized male attraction by recording whether they exhibited the following behaviors: 32 wing fanning, flight and/or landing at least halfway to the pheromone source (60 cm), flight and/or landing within 5 cm of the pheromone source, and flight and/or landing on the filter paper. We replaced the filter paper after every three minutes or a maximum of two replicates. We cleaned the tunnel with 70% ethanol and allowed the fan to push clean air through the tunnel after each block for at least one hour to remove residual pheromone. We used chi-square analyses of wing fanning, flying halfway, within 5cm, and making contact with the pheromone source to determine whether males responded to the pheromone treatments differently.
2.4.7 Simulated Pheromone-Mating Disruption
Using the y-tube olfactometer, we created a simulated pheromone-mating disruption system to test whether male midges could successfully locate and mate with intermittently calling females against a background of synthetic pheromone. Both arms contained a filter paper loaded with equal doses of one pheromone blend (Table 3). In the other arm, beyond the wire screen separating the filter paper in the odor adapter and the y-tube, we placed five unmated females with access to the male. We gave males ten minutes to mate with the females after being released into the y-tube.
We used pheromone doses tenfold higher than the most attractive dose to the midges in the olfactometer dose-response experiment (Table 2, Fig. 1), and loaded both arms with the equal doses of the same pheromone blend on filter papers. This was the lowest dose at which midges in our sensitivity experiment demonstrated behaviors consistent with arrestment (Fig. 1). Arrestment, when males exhibit reduced mate- searching behavior in the presence of high ambient levels of pheromone due to sensory
33 impairment or other factors, is one mechanism in which mating disruption prevents mate location (Miller and Gut 2015). Our picogram pheromone doses were exponentially (1e -6) lower than Samietz et al.’s (2012) microgram loading rates for swede midge mating disruption dispensers for agricultural field use (50 µg (2 S,9 S)-diacetoxyundecane, 100 µg
(2 S,10 S)-diacetoxyundecane and 1 µg ( S)-2-acetoxyundecane) due to the exponentially smaller volume of air in our y-tube compared with open-air field plots.
We conducted the mating disruption simulation experiments in the morning
(within three hours following the onset of photophase) and in the evening (four hours prior to scotophase), which is when males and females display mate-seeking behavior
(Chapter 5; Hodgdon et al., Accepted ). We used a binary scoring system to record whether or not males for each pheromone treatment (n = 32) mated with at least one female. As described by Readshaw (1961), we recorded successful mating when copulation behavior occurred with the abdomens joined for at least five seconds. In between each replicate, we cleaned the glassware with hexane and replaced the pheromone sources to avoid contamination between pheromone treatments. We tested the pheromone treatments using a randomized complete block design, with one replicate per treatment per block.
We tested how pheromone blend, the time of observation (morning or evening), and their interaction influenced the probability of mating using a binary logistic regression model. Because both time and the interaction term were not significantly associated with mating success, we pooled data from our morning and evening mating
34 observations together for the final model. We used a series of chi square tests to evaluate pairwise comparisons between pheromone treatments.
2.5 Results
2.5.1 Male Dose-Response to Chiral Pheromone
We found that pheromone concentrations varied in their attractiveness to male midges. The 0.004 ng dose of (2 S,10 S)-diacetoxyundecane (with remaining compounds in ratio) was the only dose that attracted significantly more than 50% of midges
(P = 0.017). Males were less attracted to lower and higher doses (Fig. 1). The highest dose, 4 ng, was the least attractive, attracting only 12.5% of midges. Midges that did not fly toward the pheromone treatments either avoided the pheromone by entering the control arm of the y-tube, or exhibited arrestment by remaining stationary within the release point of the stem.
2.5.1 Pheromone Choice
When given a choice between two pheromone blends in a y-tube, males were approximately three times more likely to prefer the chiral and chiral/racemic pheromone blends compared to the control and racemic pheromone blend treatments ( P < 0.05 for each comparison; Fig. 2). Males did not exhibit a significant preference between the chiral and chiral/racemic blends, and the difference between the number of males choosing the chiral pheromone blend over the chiral/racemic mixture was not significant
(P = NS ). When given a choice between the racemic and control treatments, four times more males ( P < 0.001; 79%) chose the control treatment over the racemic treatment.
35 Males behaved as though repelled by the racemic blend, flying into the only location within the y-tube not permeated with the racemic compounds—the arm containing the solvent-only control.
2.5.2 Male Flight and Courtship Behavior in Response to Pheromone Blends
Males were clearly more attracted to the chiral and chiral/racemic pheromone treatments, as few to no males flew toward the control or racemic treatments. The chiral pheromone blend attracted the most males landing within 5 cm from the pheromone source (66%, Fig. 3) compared with all other pheromone treatments ( P < 0.05) except for the chiral/racemic blend ( P > 0.05). Males were 16 times more likely to fly within 5 cm of the chiral blend versus the racemic blend. Percentages of midges flying within 5 cm of the racemic (4%) and control (0%) treatments did not significantly differ ( P > 0.05). Few males flew halfway (60 cm) when exposed to these treatments (4% and 0%, respectively), and most exhibited no forward flight at all. When exposed to the racemic blend, more than half of the males (55%) appeared to be repelled, reversing their flight direction in the tunnel and landing on the back wall at the farthest point away from the pheromone source. Across all pheromone treatments, on average one in ten midges did not make a choice in the y-tube and was excluded from analysis.
The pheromone blends also differed in terms of eliciting wing fanning, a male courtship behavior following landing near females or attractive pheromones. The likelihood of wing fanning varied significantly across the different pheromone treatments
2 (χ 3 = 57.43, P < 0.001). We observed the highest number of males (54%) fanning their wings in response to the chiral treatment ( P < 0.05; Fig. 4). Males were almost three
36 times as likely to fan their wings when exposed to the chiral blend versus the chiral/racemic blend. Few (2% or 0%) males fanned their wings in response to the racemic or control treatments, respectively, indicating that they were not stimulated by these blends.
2.5.3 Simulated Pheromone-Mating Disruption
In our simulated pheromone mating disruption system, all pheromone blend treatments significantly reduced the ability of males to successfully locate and mate with
2 calling females ( χ 3 = 38.017, P < 0.001). Out of 32 replicate midges for each treatment, only one male was able to copulate with a female in each of the chiral and racemic treatment groups. No males mated under the chiral/racemic treatment, whereas 14 males mated in the control treatment (Fig. 5). On average, 18% of males across all of the pheromone treatments exhibited arrestment (failure to leave the stem of the y-tube) versus 0% of control males, and the remaining entered the y-tube arms but did not copulate with females.
2.6 Discussion
Despite the widespread belief that the most effective pheromone blends for mating disruption are the most attractive blends, less attractive synthetic blends can also confuse males (Evenden et al. 1999; Thorpe et al. 1999; Arakaki et al. 2013; Miller and
Gut 2015). We argue that less attractive blends should be considered for mating disruption systems if they function equally as well as the natural blends and are more economical. In our simulated laboratory pheromone mating disruption, we found that
37 unattractive racemic swede midge pheromone blends functioned similarly to attractive chiral blends and prevented males from mating. Given that the racemic blend is potentially less expensive than the chiral blend, it may be useful for mating disruption systems in field settings.
Although two of our pheromone blend treatments contained racemic compounds, males responded differently to them. One of our blends, chiral/racemic, contained non- natural stereoisomers of 2,9-diacetoxyundecane and 2-acetoxyundecane, but only the naturally-produced stereoisomer of the main pheromone component, (2 S,10 S)- diacetoxyundecane. This blend functioned similarly to the complete chiral blend in eliciting male flight, as males do not possess receptors for any of the non-natural stereoisomers in 2,9-diacetoxyundecane and 2-acetoxyundecane (Boddum et al. 2010).
However, we did observe significantly more wing fanning in response to the chiral versus chiral/racemic blend in the wind tunnel, indicating that males were ultimately more stimulated by the natural blend at close range. When we used racemic blends for all three compounds, midges were repelled. Because we did not observe a significant difference in overall male attraction for the chiral or chiral/racemic blends, the chiral/racemic blend may be a lower cost alternative to the chiral blend, enabling more affordable monitoring lures and pheromone mating disruption systems.
Both racemic blends appeared to be equally effective in preventing mating in our y-tube setup. Only one male was able to locate and mate with females in each of the chiral and racemic treatments. These individuals may have had heightened sensitivity to pheromones, enabling them to cue in on calling female pheromone plumes amidst the
38 background of synthetic pheromone. Additionally, many males exposed to pheromone treatments exhibited arrestment, remaining in the stem of the y-tube and not searching for mates in our simulated mating disruption setup. Males that did not search for mates may have been over-stimulated or desensitized by attractive pheromones, not attracted by the particular pheromone blend treatment, or could have been unresponsive to pheromones due to differing circadian rhythms compared to the majority of the population. While our tests help determine which pheromone blends prevent mating in the laboratory, further exploration into the mechanisms of disruption of midge behavior could further refine pheromone blend choices for a commercial mating disruption system.
Non-natural pheromone blends can elicit a range of behaviors that can contribute to mating disruption. For example, some pheromone blends target multiple receptors in the antennae and could potentially elicit more debilitating behavioral effects than others, such as 2, 10-diacetoxyundecane in swede midge. Miller et al. (2015) argue that pheromone blends eliciting multiple behavioral impairments are necessary for successful mating disruption. For example, some synthetic pheromones desensitize male sensory systems and prevent normal response to calling females, which can include arrestment, sensory impairment and/or habituation (Cardé and Minks 1995; Daly and Figueredo
2000; Judd et al. 2005; Stelinski et al. 2008). Synthetic blends can also mix with female pheromones, adulterating the chemical composition of the female plume and decreasing male attraction to the signal (Miller and Gut 2015). Ultimately, all of these behaviors can reduce mating success, especially in combination via additive effects. Additional behavioral testing will be necessary to further elucidate the mechanisms of disruption by chiral and racemic pheromone blends for swede midge. 39 Our results can be used to inform future mating disruption trials in the field. Our results indicate two promising attributes of swede midge chemical ecology for more economical pheromone mating disruption: 1) Midges are responsive to minute pheromone amounts, indicating that little pheromone material is needed to confuse males; and 2) Two lower-cost racemic blends compared with the chiral blend are equally suitable for mating disruption. Because there are currently no insecticides approved for organic management of swede midge that are effective (Seaman et al. 2014; Evans and
Hallett 2016), pheromone mating disruption may be a viable alternative for managing this pest in organic cropping systems. Future research testing the utility of racemic pheromones in field tests of mating disruption is a practical next step in the development of this pest management tactic for swede midge.
2.7 References
Andaloro JT, Hoy CW, Rose KB, Shelton AM (1983) Evaluation of insecticide usage in the New York processing-cabbage pest management program. J Econ Entomol 76:1121– 1124. Andersson MN, Haftmann J, Stuart JJ, et al (2009) Identification of sex pheromone components of the hessian fly, Mayetiola destructor . J Chem Ecol 35:81–95. doi:10.1007/s10886-008-9569-1 Arakaki N, Hokama Y, Nagayama A, et al (2013) Mating disruption for control of the white grub beetle Dasylepida ishigakiensis (Coleoptera: Scarabaeidae) with synthetic sex pheromone in sugarcane fields. Appl Entomol Zool 48:441–446. doi:10.1007/s13355- 013-0202-6 Benatti TR, Valicente FH, Aggarwal R, et al (2010) A neo-sex chromosome that drives postzygotic sex determination in the Hessian fly ( Mayetiola destructor ). Genetics 184:769–777. doi:10.1534/genetics.109.108589 Bergh JC, Harris MO, Rose S (1990) Temporal patterns of emergence and reproductive behavior of the Hessian fly (Diptera: Cecidomyiidae). Ann Entomol Soc Am 83:998– 1004. Boddum T, Skals N, Hill SR, et al (2010) Gall midge olfaction: Pheromone sensitive olfactory neurons in Contarinia nasturtii and Mayetiola destructor . J Insect Physiol 56:1306–1314. doi:10.1016/j.jinsphys.2010.04.007 40 Boddum T, Skals N, Wirén M, et al (2009) Optimisation of the pheromone blend of the swede midge, Contarinia nasturtii , for monitoring. Pest Manag Sci 65:851–856. doi:10.1002/ps.1762 Cardé RT, Minks AK (1995) Control of moth pests by mating disruption: successes and constraints. Annu Rev Entomol 40:559–85. doi:10.1146/annurev.ento.40.1.559 Chen M, Shelton AM, Hallett RH, et al (2011) Swede midge (Diptera: Cecidomyiidae), ten years of invasion of crucifer crops in North America. J Econ Entomol 104:709–716. doi:10.1603/EC10397 Daly KC, Figueredo AJ (2000) Habituation of sexual response in male Heliothis moths. Physiol Entomol 25:180–190. doi:10.1046/j.1365-3032.2000.00184.x Evans BG, Hallett RH (2016) Efficacy of biopesticides for management of the swede midge (Diptera: Cecidomyiidae). J Econ Entomol 109:2159–2167. doi:10.1093/jee/tow192 Evenden ML, Judd GJR, Borden JH (1999) Pheromone-mediated mating disruption of Choristoneura rosaceana : Is the most attractive blend really the most effective? Entomol Exp Appl 90:37–47. doi:10.1023/A:1003598114512 Farkas SR, Shorey HH, Gaston LK (1974) Sex pheromones of Lepidoptera: Influence of pheromone-concentration and visual cues on aerial odor-trail following by males of Pectinophora gossypiella . Ann Entomol Soc Am 67:633–638. Gagne RJ (1989) The plant-feeding gall midges of North America. Cornell University Press, Ithaca, NY Hall DR, Amarawardana L, Cross J V., et al (2012) The chemical ecology of cecidomyiid midges (Diptera: Cecidomyiidae). J Chem Ecol 38:2–22. doi:10.1007/s10886-011-0053-y Hallett RH (2007) Host plant susceptibility to the swede midge (Diptera: Cecidomyiidae). J Econ Entomol 100:1335–1343. doi:10.1603/0022- 0493(2007)100[1335:HPSTTS]2.0.CO;2 Hallett RH, Goodfellow SA, Weiss RM, Olfert O (2009) MidgEmerge, a new predictive tool, indicates the presence of multiple emergence phenotypes of the overwintered generation of swede midge. Entomol Exp Appl 130:81–97. doi:10.1111/j.1570- 7458.2008.00793.x Hallett RH, Goodfellow SA, Heal JD (2007) Monitoring and detection of the swede midge (Diptera: Cecidomyiidae). Can Entomol 139:700–712. doi:10.4039/n05-071 Hallett RH, Heal JD (2001) First Nearctic record of the swede midge (Diptera: Cecidomyiidae). Can Entomol 133:713–715. doi:10.4039/Ent133713-5 Hallett RH, Sears MK (2013) Pheromone-based action thresholds for control of the swede midge, Contarinia nasturtii (Diptera: Cecidomyiidae), and residual insecticide efficacy in cole crops. J Econ Entomol 106:267–76. doi:10.1603/ec12243 Harris MO, Galanihe LD, Sandanayake M (1999) Adult emergence and reproductive behavior of the leafcurling midge Dasineura mali (Diptera: Cecidomyiidae). Ann Entomol Soc Am 92:748–757. Hillbur Y, Celander M, Baur R, et al (2005) Identification of the sex pheromone of the swede midge, Contarinia nasturtii . J Chem Ecol 31:1807–1828. doi:10.1007/s10886- 005-5928-3
41 Hodgdon EA, Hallett RH, Stratton CA, Chen YH (2018) Diel patterns of emergence and reproductive behaviour in the invasive swede midge (Diptera: Cecidomyiidae). Manuscript accepted for publication . Judd GJR, Gardiner MGT, DeLury NC, Karg G (2005) Reduced antennal sensitivity, behavioural response, and attraction of male codling moths, Cydia pomonella , to their pheromone (E,E)-8,10-dodecadien-1-ol following various pre-exposure regimes. Entomol Exp Appl 114:65–78. doi:10.1111/j.0013-8703.2005.00231.x Koppenhofer AM, Behle RW, Dunlap CA, et al (2008) Pellet formulations of sex pheromone components for mating disruption of oriental beetle (Coleoptera: Scarabaeidae) in turfgrass. Environ Entomol 37:1126–1135. doi:10.1603/0046- 225x(2008)37[1126:pfospc]2.0.co;2 Miller JR, Gut LJ (2015) Mating disruption for the 21st century: Matching technology with mechanism. Environ Entomol 1–27. doi:10.1093/ee/nvv052 Miller JR, Gut LJ, de Lame FM, Stelinski LL (2006) Differentiation of competitive vs. non-competitive mechanisms mediating disruption of moth sexual communication by point sources of sex pheromone (Part I): Theory. J Chem Ecol 32:2089–2114. doi:10.1007/s10886-006-9134-8 Onufrieva KS, Thorpe KW, Hickman AD, et al (2008) Gypsy moth mating disruption in open landscapes. Agric For Entomol 10:175–179. doi:10.1111/j.1461-9563.2008.00375.x Readshaw JL (1961) The biology and ecology of the swede midge, Contarinia nasturtii (Kieffer), (Diptera; Cecidomyiidae). 132. Samietz J, Baur R, Hillbur Y (2012) Potential of synthetic sex pheromone blend for mating disruption of the swede midge, Contarinia nasturtii . J Chem Ecol 38:1171–1177. doi:10.1007/s10886-012-0180-0 Seaman A, Lange H, Shelton AM (2014) Swede midge control with insecticides allowed for organic production, 2013. Arthropod Manag Tests 39:E64–E64. doi:10.4182/amt.2014.E64 Shorey HH (1973) Behavioral responses to insect pheromones. Annu Rev Entomol 18:349–380. doi:10.1146/annurev.en.18.010173.002025 Stelinski LL, Miller JR, Rogers ME (2008) Mating disruption of citrus leafminer mediated by a noncompetitive mechanism at a remarkably low pheromone release rate. J Chem Ecol 34:1107–1113. doi:10.1007/s10886-008-9501-8 Stratton CA, Hodgdon EA, Zuckerman SG, et al (2018) A single swede midge (Diptera: Cecidomyiidae) larva can render cauliflower unmarketable. J Insect Sci 18:1–6. doi:10.1093/jisesa/iey062 Stuart JJ and JHH (1991) Genetics of sex determination in the Hessian fly, Mayetiola destructor . doi:10.1093/jhered/82.1.43 Symonds MRE, Elgar MA (2008) The evolution of pheromone diversity. Trends Ecol Evol 23:220–228. doi:10.1016/j.tree.2007.11.009 Tabadkani SM, Khansefid M, Ashouri A (2011) Monogeny, a neglected mechanism of inbreeding avoidance in small populations of gall midges. Entomol Exp Appl 140:77–84. doi:10.1111/j.1570-7458.2011.01130.x Thorpe KW, Mastro VC, Leonard DS, et al (1999) Comparative efficacy of two controlled-release gypsy moth mating disruption formulations. Entomol Exp Appl 90:267–277. doi:10.1046/j.1570-7458.1999.00447.x
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43 2.8 Tables
TABLE 2.1 STEREOISOMERS OF PHEROMONE COMPONENTS IN BLENDS USED FOR BEHAVIORAL ASSAYS
Pheromone blend Diacetoxyundecane Acetoxyundecane 2S,9 S 2R,9 R- 2S,10 S 2R,10 R 2, S 2, R 2R,9 S- meso-10 2S,9 R- Chiral X - X - X - Chiral/Racemic X X X - X X Racemic X X X X X X Control ------
44 TABLE 2.2 PHEROMONE TREATMENTS USED FOR PHEROMONE ATTRACTION AND PREFERENCE EXPERIMENTS IN OLFACTOMETER AND WIND TUNNEL
Pheromone blend Diacetoxyundecane Acetoxyundecane (2 S,9 S) 2,9 (2 S,10 S) 2,10 2S 2 Olfactometer (control vs. pheromone) Chiral 2 pg - 4 pg - 0.04 pg - Chiral/Racemic - 8 pg 4 pg - - 0.08 pg Racemic - 8 pg - 16 pg - 0.08 pg Control ------Olfactometer (pheromone vs. pheromone) Chiral 1 pg - 2 pg - 0.02 pg - Chiral/Racemic - 4 pg 2 pg - - 0.04 pg Racemic - 4 pg - 8 pg - 0.04 pg Control ------Wind tunnel Chiral 10 ng - 20 ng - 0.2 ng - Chiral/Racemic - 40 ng 20 ng - - 0.4 ng Racemic - 40 ng - 80 ng - 0.4 ng Control ------
45 TABLE 2.3 PHEROMONE TREATMENTS IN EACH ARM USED IN SIMULATED PHEROMONE MATING DISRUPTION EXPERIMENT
Pheromone blend Diacetoxyundecane Acetoxyundecane 2S,9 S 2,9 2S,10 S 2,10 2S 2 Chiral 20 pg - 40 pg - 0.4 pg - Chiral/Racemic - 80 pg 40 pg - - 0.8 pg Racemic - 80 pg - 160 pg - 0.8 pg Control ------
46 2.9 Figures
80
60
40
20
Males Responding (%) Responding Males 0 4 0 00 400 0 .00 .0040 .0 .4000 0 0 0 0 0 4. Pheromone Concentration (ng (2 S,10 S) -diacetoxyundecane)
FIG. 2.1 PERCENTAGE OF MALES FLYING TOWARD THE PHEROMONE SOURCE IN THE Y-TUBE OLFACTOMETER AT INCREASING DOSES. PHEROMONE CONCENTRATIONS ARE DISPLAYED IN AMOUNTS OF (2S, 10 S)-DIACETOXYUNDECANE, THE MAIN COMPONENT IN THE SWEDE MIDGE PHEROMONE BLEND, WITH (2 S,9 S)- DIACETOXYUNDECANE AND ( S)-2-ACETOXYUNDECANE FOLLOWING IN THE RATIO PRODUCED BY FEMALES (2:1:0.02, RESPECTIVELY) (HILLBUR ET AL 2005). 0.0040 NG OF (2S, 10 S)- DIACETOXYUNDECANE WAS THE ONLY DOSE THAT ATTRACTED NUMBERS OF MIDGES SIGNIFICANTLY GREATER THAN 50% (P = 0.017)
47 Chiral Control ***
Chiral/Racemic
Control ***
d n
e Racemic
l B
Control ***
e n o Chiral
m NS o
r Chiral/Racemic
e h
P Chiral Racemic **
Chiral/Racemic Racemic ***
0 20 40 60 80 100 Males Choosing Pheromone Blend (%)
FIG. 2.2 PERCENTAGE OF MALES CHOOSING PHEROMONE BLENDS WHEN GIVEN A CHOICE BETWEEN TWO BLENDS IN A Y-TUBE OLFACTOMETER. NS , **, AND *** INDICATE NON-SIGNIFICANCE (P > 0.05), AND STATISTICAL SIGNIFICANCE ( P < 0.01 AND P < 0.001, RESPECTIVELY) FOR INDIVIDUAL PAIRWISE PHEROMONE COMPARISON TESTS
48 100 *** *** *** a Control a Racemic a a a Chiral/Racemic 50 a Chiral
b b b Males Landing (%) Landing Males 0 c b b <60 <5 Contact Landing Distance from Pheromone (cm)
FIG. 2.3 PERCENTAGE OF MALES LANDING WITHIN 60 CM, 5 CM, AND MAKING CONTACT WITH THE PHEROMONE SOURCE IN THE WIND TUNNEL. DATA POINTS WITH THE SAME LETTER WITHIN THE SAME LANDING DISTANCE ARE NOT STATISTICALLY SIGNIFICANTLY DIFFERENT BASED ON CHI SQUARE POST HOC TESTS ( P > 0.05). *** INDICATES STATISTICAL SIGNIFICANCE OF THE OVERALL MODEL FOR EACH DISTANCE AT P < 0.001.
49 60 a ***
40
b 20
c c
Males Fanning Wings (%) Wings Fanning Males 0 c c iral ntrol Ch emi emi o ac ac C R l/R a Chir Pheromone Blend
FIG. 2.4 PERCENTAGE OF MALES FANNING WINGS AFTER LANDING IN RESPONSE TO PHEROMONE BLENDS IN THE WIND TUNNEL. TREATMENTS INDICATED WITH THE SAME LETTER ARE NOT STATISTICALLY SIGNIFICANTLY DIFFERENT (P > 0.05) BASED ON POST HOC CHI SQUARE TESTS AND *** INDICATES STATISTICAL SIGNIFICANCE OF THE OVERALL MODEL AT P < 0.001
50 20 *** a 15
10 Matings Matings 5
Number of Successful Successful of Number b b b 0 l o emic ontr Chiral cemic c C a Ra
Chiral/R Pheromone Treatment
FIG. 2.5 OBSERVED INCIDENCES OF MATING IN THE Y-TUBE OLFACTOMETER SIMULATED PHEROMONE MATING DISRUPTION EXPERIMENT. TREATMENTS WITH THE SAME LETTER ARE NOT STATISTICALLY SIGNIFICANTLY DIFFERENT ( P > 0.05) BASED ON POST HOC CHI SQUARE TESTS AND *** INDICATES STATISTICAL SIGNIFICANCE OF THE OVERALL MODEL AT P < 0.001
51 CHAPTER 3: SYNTHETIC PHEROMONE EXPOSURE INCREASES CALLING AND REDUCES SUBSEQUENT MATING IN FEMALE SWEDE MIDGE (DIPTERA: CECIDOMYIIDAE)
ELISABETH A. HODGDON *1 (0000-0002-2432-9201), REBECCA H. HALLETT 2 (0000-0002-4316-7877), JAMES D. HEAL 2, ANDREA E. M. SWAN 1, AND YOLANDA H. CHEN *1 (0000-0001-9439-5899)
1Department of Plant and Soil Science, University of Vermont, 63 Carrigan Dr., Burlington, Vermont, USA 05405
2School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada, N1G 2W1
*Corresponding author contact information: Elisabeth A. Hodgdon Email: [email protected] Telephone: +1 (802) 522-6914
52
3.1 Acknowledgements
Funding for our research was provided by the following sources: USDA NIFA
Crop Protection and Pest Management grant (2014 - 70006 - 22525) to YHC, USDA
Northeast SARE Graduate Student grant (GNE16 - 121) to EAH, and the Ontario
Ministry of Agriculture, Food and Rural Affairs University of Guelph Partnership to
RHH. We thank Jamie Bauman, Kylie Barwise, Jarrett Blair, Charles-Étienne Ferland,
Paolo Filho, Kate Lindsay, Charlotte MacKay, Laurel Martinez, Matthew Muzzatti, Ross
Pillischer, Elizabeth Porter, Dylan Samson-McKenna, Justine Samuel, Linley Sherin,
Emily Sousa, and Chase Stratton from the University of Guelph and University of
Vermont for their assistance with our experiments. We also acknowledge David Conner,
Ylva Hillbur, Alan Howard, Scott Merrill, and Kimberly Wallin for their advice regarding experimental design and statistical analysis.
53
3.2 Abstract
Pheromone mating disruption confuses male insects with large amounts of synthetic pheromone and has been tremendously successful for managing important agricultural pests. However, “male confusion” may be an overly simplified explanation for how pheromone treatments actually disrupt mating. It is unclear exactly how unnaturally large doses of synthetic sex pheromone impact the behavior of female insects, particularly those in non-lepidopteran insect species. For some insects,
“autodetecting” females possess receptors for their own pheromone and respond to ambient pheromones by altering their mating behavior, including increasing pheromone release and preferring to call rather than mate with receptive males. We examined whether autodetection occurs in the galling fly family Cecidomyiidae, which contains several important agricultural pests such as the invasive swede midge ( Contarinia nasturtii Kieffer). Our objective was to test whether exposure to various synthetic candidate pheromone blends for mating disruption influence calling and subsequent propensity to mate in female swede midge, a pest of Brassica vegetable and oilseed crops. Here we show that females exposed to both chiral and racemic three-component pheromone blends called significantly more frequently and for longer durations versus midges in control treatments in both laboratory and field settings. However, midges exposed to incomplete blends did not increase calling. Additionally, midges pre-exposed to three-component pheromone blends were less likely to mate following exposure, indicating that these blends may be promising for effective mating disruption.
54 3.3 Introduction
Despite the tremendous success of pheromone mating disruption for pest management, exactly how high levels of synthetic pheromones influence insect behavior is not entirely known (Miller and Gut 2015). Pheromone mating disruption is thought to prevent mating in insect pests by confusing males and attracting them to pheromone dispensers rather than to females, impairing the male sensory system, and otherwise preventing males from finding females (Cardé and Minks 1995; Miller and Gut 2015).
However, these explanations may offer only a partial scenario for how mating disruption actually prevents mating, and does not consider how half of the insect population— female insects—may be responding to unnaturally large doses of synthetic pheromones.
Despite increasing evidence that females are able to “autodetect” and alter their reproductive behavior in response to their own pheromones (Stelinski et al. 2006, 2014;
Gocke et al. 2007; Kuhns et al. 2012; Bakthavatsalam et al. 2016; Rehermann et al.
2016), there are relatively few studies exploring the mechanisms of pheromone mating disruption mechanisms that have considered females.
Autodetection may confer fitness benefits to female insects by enabling them to infer the number of nearby females. For some lepidopteran females, high ambient pheromone levels could cue them to disperse and avoid competition for mates and to ensure sufficient resources for future offspring (Saad and Scott 1981). In other species, however, autodetection triggers the opposite response. Sex pheromones for some species also serve as aggregation pheromones for other females, strengthening their pheromone plumes to attract more males and provide more choices for mates (Arakaki et al. 2003;
Lim and Greenfield 2008). In addition to serving as cues for movement, sex pheromones 55 can influence calling behavior. In many lepidopteran species, females increase calling behavior when exposed to conspecific females or synthetic sex pheromone (Stelinski et al. 2006, 2014; Gocke et al. 2007; Lim and Greenfield 2007; Kuhns et al. 2012;
Rehermann et al. 2016). If synthetic pheromones cause females to aggregate and emit more pheromone resulting in a stronger signal, males may be more likely to locate them amidst mating disruption treatments. Alternatively, synthetic pheromones could cause females to disperse from pheromone-treated fields, where they may be more successful in mating. Therefore, high levels of synthetic pheromones could impact female behavior in a variety of ways that may enhance or hinder pheromone mating disruption efforts.
In addition to influencing movement and calling behavior, pre-exposure to synthetic pheromones can cause female insects to be less likely to mate afterwards. For example, pre-exposed females rejected males in favor of continuing calling (Kuhns et al.
2012). They suggested that the females either perceived high doses of synthetic pheromone as competition and therefore an unsuitable environment for their offspring, or that synthetic female sex pheromone may have masked male aphrodisiacs. If females do not detect male aphrodisiac compounds due to sensory blockage or inhibition by sex pheromones, they may not be receptive for mating in a mating disruption system.
Ultimately, the exact causes for reduced or delayed mating following female pheromone exposure are unknown, but could present important implications for overall mating disruption success (Mori and Evenden 2013; Holdcraft et al. 2016). An understanding of how different pheromone blends disrupt female mating behavior could be highly useful in choosing blends for a mating disruption system.
56 There is limited knowledge on how non-lepidopteran female insects respond to female sex pheromones in the field. In a recent review of autodetection, 79% of 42 published papers used lepidopteran study subjects, mostly observed in laboratory settings
(Holdcraft et al. 2016). Despite potential logistical difficulties, observing females in field settings may offer a more complete understanding of how female populations respond to pheromone mating disruption treatments. More research is needed to understand how non-lepidopteran species autodetect and alter their behavior in response to pheromones.
Differing life histories, biology, and ecology of non-lepidopteran species may impact the ecological relevance of autodetection in other insect orders.
Pheromone mating disruption is a promising new tactic for managing swede midge ( Contarinia nasturtii Kieffer; Diptera: Cecidomyiidae) in North America. The invasive midge is particularly difficult to manage for multiple reasons. First, female midges oviposit into the meristem of cruciferous ( Brassica spp.) vegetables and oilseed crops, where feeding larvae distort and scar heads, stems, and leaves, reducing marketable yield (Hallett 2007). Due to extremely low thresholds for damage for cruciferous vegetables, management strategies that prevent mating and subsequent oviposition are promising. Stratton et al. (2018) found that feeding damage from a single swede midge larva can render a cauliflower head unmarketable. Second, foliar insecticides, biological control, resistant crop varieties, and cultural controls are ineffective or non-existent for this pest (Chen et al. 2011; Seaman et al. 2014; Evans and
Hallett 2016). Third, due to the emergence of multiple overlapping generations and prolonged sensitivity of host crops to feeding, heading cruciferous vegetables appear to be susceptible throughout the growing season (Hallett et al. 2009; Stratton et al. 2018). 57 As a result, few practical alternatives to conventional insecticides exist for control of this species (Chen et al. 2011).
Samietz et al. (2012) found that the pheromone blend naturally produced by females, a 1:2:0.02 mixture of (2 S,9 S)-diacetoxyundecane, (2 S,10 S)-diacetoxyundecane and ( S)-2-acetoxyundecane (Hillbur et al. 2005), confused males and led to higher yields of cruciferous vegetable crops. However, they speculate that mating disruption may not be economically feasible at a commercial scale due to the high cost of pheromone synthesis, due to the three-dimensional structural complexity and stereoisomeric specificity (chirality) of the pheromone blend (Hillbur et al. 2005). To reduce the costs of pheromone inputs in a mating disruption system, racemic blends containing synthetic stereoisomers may present an opportunity for cost savings by omitting purification steps during chemical synthesis (C. Oelschlager, pers. comm.).
Although complete racemic blends are unattractive to male swede midge, they may function to confuse males in a mating disruption system (Boddum et al. 2009).
Despite being less attractive to males, non-natural racemic pheromones have been successful other insect pests, (Onufrieva et al. 2008; Arakaki et al. 2013). Since racemic pheromone blends are low-cost, they may be promising candidates for swede midge mating disruption from an economic perspective. Therefore, understanding how male and female midges respond to these compounds is important for informing whether the racemic blend is as effective as the chiral blend in preventing mating.
While mating disruption is a new promising management tactic for swede midge, it is currently unknown how females respond to candidate pheromone blends for this
58 technique; exactly how pheromone mating disruption treatments influence females of non-lepidopteran species in general is largely unknown. While one can assume that the primary mechanisms of mating disruption will apply to insects in other orders, autodetection has never been studied in Cecidomyiidae (Holdcraft et al. 2016). Here, we evaluate the effects of synthetic pheromones on female reproductive behavior from individuals from both European and North American populations in laboratory and field settings to determine whether females are impacted by mating disruption treatments. If particular pheromone blends negatively impact female mating behavior in addition to confusing males, they may be ideal candidates for mating disruption. Specifically, we asked: 1) How does exposure to single- and three-component chiral and racemic pheromone treatments influence calling frequency and duration in laboratory and field settings? and; 2) How does pre-exposure to three-component chiral and racemic pheromone blends influence females’ propensity to mate?
3.4 Methods and Materials
3.4.1 Swede Midge Colony Rearing
We reared two colonies using similar protocols for our experiments: one
(“Ontarian colony”) at the University of Guelph in Ontario, Canada, and another (“Swiss colony”) at the University of Vermont in Burlington, Vermont, USA. Using two colonies allowed us to test for autodetection phenomena across populations of differing geographical origins. The Ontarian colony was initiated from wild individuals collected in Ontario in 2016 and was supplemented with over 1,000 wild individuals annually that were collected from infested canola ( Brassica napus L.) plants. The Swiss colony was
59 initiated in 2014 and consisted of predominately Swiss midges imported from the Swiss
Federal Research Station for Horticulture in Wädenswil, Switzerland, with the exception of 122 wild individuals from Vermont added to the colony in 2015. Midges from both locations shared similar diel patterns of emergence and calling (Chapter 5; Hodgdon et al.
Accepted ), and males responded similarly to pheromones (Chapter 4; (Hillbur et al. 2005;
Hallett and Sears 2013)). We used the Ontarian colony for our experiments testing for increased calling when females were exposed to synthetic pheromone treatments in the laboratory and field. To test if pre-exposure to pheromones reduced mating, we used midges from the Swiss colony. Due to limitations to moving individuals from the Swiss colony, as outlined in our permit (USDA APHIS permit number P526P-13-03136), we were unable to use Swiss midges in our field experiments.
We used cauliflower ( Brassica oleracea var. Botrytis ‘Snow Crown’) to rear the midges because of its large bud size and high susceptibility to swede midge (Hallett
2007). We transplanted cauliflower into either 13 (Ontarian) or 10 cm (Swiss) diameter pots filled with soilless potting media. Plants received all-purpose synthetic 20-20-20 fertilizer weekly (Ontario) or two parts 21-5-20 and one part 15-0-14 thrice weekly
(Swiss). When plants reached the 8-10 leaf stage and produced large buds, we exposed the plants to emerging adults in oviposition cages. After 24-72h, we moved plants into separate larval rearing cages for ten days. We maintained both adults and larvae in
Plexiglas cages at approximately 25 °C with relative humidity >30% under at 16L:8D photoperiod. When swede midge larvae are ready to pupate, they vacate the plant stems by dropping off or crawling down the stem into the top few centimeters of soil underneath their host plants (Readshaw 1961). To facilitate movement of third instars
60 into the soil for pupation, we cut cauliflower stems at 5 cm below the base of the bud and pushed the stems into the potting media 10-12 days following oviposition. We then returned the infested pots to the oviposition cages for adult emergence.
We used unmated newly-emerged females for our calling and mating experiments. Since females only release pheromones prior to mating and mate only once
(Readshaw 1961), we reasoned that unmated females would be most likely to autodetect and subsequently alter their behavior. For the calling experiments, we collected females from the Ontarian colony as they emerged from the soil during the peak emergence period for females, within the first three hours after dawn (Chapter 5; Hodgdon et al.
2018). For the mating experiments, we used a combination of newly-emerging midges from the Swiss colony, as well as <24h-old unmated males and females from separate single-female progenies.
Due to the genetics of sex determination in cecidomyiids, the majority of female swede midge (~ 80%, pers. obs.) generate single-sex offspring (Readshaw 1961; Benatti et al. 2010). To isolate the progeny of individual females, we caged one female with two or three males with an eight to ten week old cauliflower plant in double-stacked one-quart
(946 ml) deli containers (WebstaurantStore, Lititz, PA, USA). Rearing offspring separately from individual females allowed us to generate separate containers of emerging males and females, preventing mating prior to our experiments. We allowed the midges to mate and oviposit within the containers, and after 14 days, we cut the plant stems and inserted them into the soil. Any adults emerging from cohorts with both sexes were not used for experiments, because they may have already mated prior to our
61 experiments. By using a combination of newly-emerged individuals from the main colony and over 100 single-female progenies over 17 weeks, we were able to sample a wide selection of individuals from the Swiss population for the mating experiment.
3.4.2 Laboratory Calling Experiments
To test for influences of pheromones on calling, we observed the behavior of
Ontarian females exposed to large doses of synthetic pheromones (ChemTica
Internacional S.A., Heredia, Costa Rica; Table 1) in flasks. We used 1-cm cotton roll dispensers and applied pheromones in HPLC-grade hexane. Hillbur et al. (2005) found cotton roll dispensers to be more effective than rubber septa for attracting males because they release the pheromone components in biologically-active ratios. We adjusted the pheromone doses to hold the biologically-active portion (chiral compounds) equivalent across treatments, similarly to other pheromone-response studies using swede midge
(Hillbur et al. 2000; Boddum et al. 2009). Because each biologically-active stereoisomer represents either one quarter ((2 S,9 S)- and (2 S,10S)-diacetoxyundecane) or one half (2 S- acetoxyundecane) of the racemic mixture of each swede midge pheromone component
(Boddum et al. 2009), we either quadrupled or doubled the amount of each racemic mixture. For our single-component experiments, we applied the same amounts of either
(2 S,10 S)- or 2,10-diacetoxyundecane (which comprises approximately one third of the complete swede midge pheromone blend) as were applied within the three-component treatments (Table 1).
To determine the biologically-active pheromone dose to use in the experiment, we first conducted a dose-response test to determine the sensitivity of the females to their
62 natural (three-component chiral) pheromone. Because single-female equivalent doses of pheromones are currently unknown for swede midge (Y. Hillbur, pers. comm.), we chose our doses based on those that elicit responses in males. We observed the calling behavior of n = 42 females exposed to two doses of pheromone (Table 1): a dose inflicting inhibition or arrestment behavior in males in an olfactometer (“low dose”; Chapter 2), and then to a dose 10 times that amount (“high dose”). We chose doses causing inhibition in males because they may be more representative of mating disruption scenarios.
Because calling did not significantly differ between the high and low pheromone doses
(Fig. 1), we used the lower dose in the subsequent laboratory calling and mating experiments testing effects from chiral and racemic pheromone blends.
We observed midges over 10 mornings using a randomized complete block design, observing up to five females per pheromone treatment per morning (block) depending upon midge emergence rates and mortality. We observed female calling behavior from 0930-1330, which included the morning hours when females are most likely to call, as well as one hour into the inactive period (1230-1330; Chapter 5;
Hodgdon et al., 2018). Females were housed singly with dental wicks in 125-ml
Erlenmeyer flasks with silicone stoppers. An air pump circulated air through the flasks via Teflon tubing through holes in the stoppers at a rate of approximately 200 cm 3/min.
Air exited the flask through a second hole in the stopper and was removed from the room by an overhead exhaust duct. The experimental setup was housed in a room kept at
25.2 ± 0.5ºC and 49.2 ± 4.2% RH with overhead lighting at 196 ± 47 lux.
Following our dose-response experiment, we tested for differences in calling
63 between Ontarian females (22 blocks (days), n = 50) exposed to chiral, racemic, three- component, or single-component blend blends (Table 1). The control treatment consisted of hexane only. We used the same protocol for both the dose- and blend-response experiments (described previously).
We recorded a female midge as “calling” if the terminal segments of the abdomen containing the pheromone glands and ovipositor were protruding and extended at least one third of their combined total length, which is characteristic calling behavior of cecidomyiids (Gagne 1989; Harris et al. 1999). Every fifteen minutes, we scored whether each midge was calling using a binary scoring system (yes or no). Each female was used only once in the trials. Midges that did not call or died during the study were omitted from the data set.
We tested if pheromone treatment, block, and dose influenced the probability of calling over time, using a generalized estimating equations (GEE) extension of the generalized linear model (GLM) menu with a binomial distribution within SPSS statistical software (version 24, IBM, Armonk, NY). We used GEEs because they account for repeated-measures and non-normally distributed data, are robust to unbalanced designs, and generate population-level estimates of model parameters (Zeger et al. 1988). For all statistical analyses, we evaluated statistical significance using α =
0.05, excluding non-significant variables from our final models. Where descriptive statistics are presented, numbers following the ± symbol indicate standard errors of the mean.
64 To determine whether the pheromone treatments caused differences in the duration of calling, we gave each midge that called for at least eight consecutive time points (80 minutes or 50% of our observational period) a score of 1, and the remaining midges a score of 0. We used a binary logistic regression model to examine differences in the distribution of “continuous callers” using pheromone and block as predictor variables, and chi square tests to make post hoc pairwise comparisons between pheromone treatments with a Bonferroni correction.
3.4.3 Field Calling Experiment
We tested whether Ontarian females increased calling when exposed to pheromone mating disruption treatments in treated and untreated plots of broccoli at the
University of Guelph Elora Research Station in Ariss, Ontario, Canada. As part of a separate study (Chapter 4), we had established 16 x 16m plots to test the efficacy of the three-component chiral pheromone blend to confuse males (cause trap shut-down) and reduce swede midge crop damage versus an untreated control treatment. The treated and untreated plots were situated at least 500m apart to avoid drift of pheromone into the untreated plots. Each plot contained 20 rows of ‘Everest’ broccoli with pheromone dispensers situated within the rows.
Each plot contained 80 mating disruption dispensers. The bags hung from wire stakes 25 cm from the ground. We used reservoir-type semi-permeable polyethylene bag dispensers (ChemTica Internacional S.A., Heredia, Costa Rica). Each bag contained a microcentrifuge tube with a small hole drilled in the top, containing 50 µg (2 S,9 S)- diacetoxyundecane, 100 µg (2 S,10 S)-diacetoxyundecane, and 1 µg (2 S)-
65 acetoxyundecane. We have found that bags release pheromones in quantities sufficient to cause trap shutdown and confuse males for 12-15 weeks (Chapter 4). Trap shutdown, a metric for mating disruption efficacy, occurs when few to no males are found in traps baited with commercial pheromone lures installed within an area treated with mating disruption (Lance et al. 2016). Although we did not measure ambient pheromone levels in the plots, we found that traps in treated plots caught >80% fewer males compared to the untreated plots, indicating that the pheromones effectively disrupted male mate- seeking behavior (Chapter 4).
Although we were only able to observe female calling behavior in both one treated and one untreated plot at a time, we repeated the experiment across six experimental periods (for a total of n = 33 midges) in late July and August in 2017 and
2018, during 6 – 12 weeks after installing dispensers. During each period, we observed the calling behavior of 4-10 midges. Performing the study in more than one mating disruption plot across two years allowed us to minimize plot-specific factors that could have influenced our midge responses in our plots. Across the six observational periods, environmental conditions were quite similar. Weather during these periods ranged from overcast to sunny and clear, with an average temperature of 21.3 ± 0.67 ºC and
57.5 ± 3.6% RH (Government of Canada 2018).
Using multiple observers trained to perform the same behavioral observation protocol, we recorded midge calling in both control and treated plots concurrently between 3.5 and 6.5 hours after dawn. To control for bias associated with individuals observing midges, we rotated observers between chiral and control treatments between
66 each block. To minimize the stress associated with transporting adult midges, we first brought infested potting media from the Ontario colony to the field site so that the midges could emerge under field conditions. We aspirated the emerging females between one to three hours after the onset of photophase and transferred them singly into 20 ml glass scintillation vials with tops covered in fine mesh. We then randomly selected half of the midges for the control plot, and the other half for the pheromone-treated plot. The vials rested on their side on top of overturned 2-gallon (7.6 L) pots at approximately 25 cm above the ground so that they would be at the same height as the dispensers and receive adequate air circulation. Midges that died or did not call during our experiments were excluded from statistical analysis.
We tested whether the distributions of binomial calling data significantly differed between midges in treated versus untreated plots used a GLM model with GEE extension.
We included experimental day and day*pheromone treatment in our preliminary models to determine whether environmental conditions that varied across individual days influenced calling. Because we only observed midges in two plots per treatment (one per year), we also included year and year*pheromone in our initial models to explore whether plot conditions influenced calling. We also used a binary logistic regression model to test whether the pheromone-treated versus untreated plots differed in their influence on continuous calling behavior (calling for 50% of consecutive time points).
3.4.4 Propensity to Mate Experiment
We tested whether pre-exposure to pheromone subsequently influenced the propensity for Swiss females to mate. We exposed unmated females to either the three-
67 component chiral, three-component racemic, or solvent-only pheromone treatments in flasks using the same dosages that we used in our pheromone blend calling experiment
(Table 1), using the previously described setup conditions. Afterward, we transferred females from flasks to one-quart (946 ml) deli containers containing moistened potting media and four unmated males. In order to determine whether pheromone exposure altered female courtship behavior, we observed female behavior for 20 minutes following the pre-exposure treatments and recorded the following using binary (yes or no) scales: 1) whether females were calling every two minutes; 2) if females rejected courting males at least once; and 3) if the midges successfully mated. Based on the observations of mating behavior by Readshaw (1961), we recorded successful mating when males mounted females and remaining joined for at least five seconds. We also recorded female rejection, which consisted of observing a female move away or otherwise rebuff a male that approached with fanning wings trying to mount her. Measuring calling and rejection across pheromone blends allowed us to better understand why mating did or did not occur. We used a randomized complete block design and conducted our experiment across 30 mornings for a total of n = 32 replicates during one to three hours after dawn.
Due to the time constraints of the relatively short peak midge calling period, we observed
1-2 replicates of each treatment per day. Neither males nor females were reused for additional testing following each observational period.
We used a binary logistic regression model to test whether pheromone pre- exposure using different pheromone blends influenced the probability of mating. We also included hour (0700-0800, 0800-0900, or 0900-10000) and experimental day within the initial models to determine whether timing influenced mating. In a separate binary 68 logistic regression model, we tested whether pheromone blends influenced the probability that females would reject advancing males at least once during their period of peak receptivity. Lastly, we tested whether the pheromone blend influenced the probability of midge calling (yes or no) during our observational period if they did not mate using a
GLM with GEE extension.
3.5 Results
3.5.1 Laboratory Calling Experiments
In the dose-response experiment, we found that both the low and high dose
2 increased calling compared to the solvent-only control treatment (Fig. 1; ( χ 2 = 8.066,
P = 0.018). Up to 31% more midges exposed to pheromone called versus midges in control treatments during our observational time periods. Calling did not significantly differ between the low and high dose based on post hoc pairwise comparison tests
(P > 0.05).
When we observed female calling response to different pheromone blends, we found that some blends increased calling compared with the control treatment, but others did not. Females were twice as likely to call when exposed to the three-component chiral and racemic blends compared with the control ( P < 0.001). However, midges exposed to the one-component treatments were not more likely to call (Fig. 2; P > 0.05). Midges exposed to the three-component chiral blend were the most likely to call, followed by the three-component racemic treatment. Experimental day and day*pheromone were significant variables within our model ( P < 0.001). We observed some daily differences
69 in calling between days, possibly due to our low numbers of replicates per treatment within each block.
The treatment blends altered calling duration; some blends induced females to continuously call. On average, we observed individual midges in the three-component chiral treatment calling for twice as much time as the control midges (Figs. 3, 4). We found that the pheromone blend significantly influenced the number females that
2 continuously called ( χ 4 = 17.425, P = 0.002). Out of all of the pairwise comparisons, the probability of continuous calling was only significantly different between the control and three-component chiral treatments (Fig. 4).
3.5.2 Field Calling Experiment
In the field, a higher percentage of female midges called when exposed to chiral pheromone mating disruption treatments versus the untreated control (Fig.
2 5; χ 1 = 15.169, P < 0.001). On average, twice as many midges called in the mating disruption plots at any given time (44.1 ± 1.4% vs. 19.8. ± 2.2% of control midges).
Additionally, pheromone exposure significantly influenced the number of continuously-
2 calling midges ( χ 1 = 4.524, P = 0.033). Midges were eight times more likely to call continuously in the pheromone-treated plot versus the control plots (24% vs. 3% of total midges). None of the other variables significantly influenced calling in the model (day, year, day*pheromone treatment, or year*pheromone treatment), indicating that environmental and plot variation did not significantly influence midge response to pheromone between years.
70 While female calling activity typically decreases in the afternoon (Chapter 5), we found that a greater number of midges exposed to mating disruption treatments called after 1200 hours versus control midges (Fig. 5). While less than 20% of control females continued to call from 1200-1230 hours, approximately twice as many midges called during this time in the mating disruption plots. However, we did not observe prolonged calling in our laboratory experiments (Figs. 1, 2).
3.5.3 Propensity to Mate Experiment
Midges were significantly less likely to mate following pre-exposure to the three- component chiral and racemic pheromone blends versus the solvent-only control
2 treatment ( χ 2 = 7.354, P = 0.025). While 68% of the midges mated in the control treatment, only 42% and 35% mated in the chiral and racemic treatment groups, respectively. We did not observe significant differences between likelihoods of mating at different times during our observational periods. Hour and day were not significant in the models. For the midges that did not mate during the observational periods, neither calling nor rejection varied significantly across pheromone treatments within our models
(P > 0.05).
3.6 Discussion
Female swede midge adults clearly autodetect in both laboratory and field settings. We believe our findings are the first documented case of autodetection in
Cecidomyiidae, a family including several challenging agricultural pests. When exposed to synthetic pheromone, female midges behaved as though they perceived competition in their environment by calling more frequently and for longer periods of time. Thus,
71 exposure to high ambient pheromone levels appears to increase female pheromone release to improve the probability that males will be attracted to their pheromone plume.
We observed the greatest increase in calling when midges were exposed to the three-component chiral pheromone blend, the blend naturally produced by females. This finding is not surprising, given that this is the same blend released by females, and is the most attractive to males (Chapter 2; Boddum et al. 2009). Females were less likely to exhibit these behaviors when exposed to single pheromone components or non-natural stereoisomers. Decreased response to racemic and single-component blends is also seen in males (Boddum et al. 2009). Males are not attracted to racemic blends of the “main”
(most prevalent) pheromone compound, 2,10-diacetoxyundecane, and possess antennal receptors for at least one of the unnatural stereoisomers of this compound (Boddum et al.
2009, 2010). Perhaps females respond less strongly to racemic blends due to mixing of the natural and unnatural compounds in solution, resulting in adulteration of the signal.
Electroantennagram tests would be necessary to determine whether females have receptors for non-natural compounds of 2,10-diacetoxyundecane, similarly to males.
Because no other insect is currently known to produce the other stereoisomers in the racemic 2,10-diacetoxyundecane blend, it is unclear how possessing receptors for these compounds is ecological relevant for males and potentially females (Boddum et al.,
2010).
Although females increased calling in response to the three-component chiral and racemic blends, it is unclear whether increased calling alone would impact the probability of midge mating in a pheromone mating disruption system. If females increase
72 pheromone release within mating disruption systems, males may be more likely to detect their plume within a background of synthetic pheromone. However, releasing more pheromone may have adverse fitness effects for females. Swede midge do not feed as adults and have short 1-3 day lifespans (Readshaw 1961), so higher pheromone production may require additional energy where internal resources are limited. Some moth species, such as Lobesia betrana and Cydia pomonella (Tortricidae), have shorter lifespans when continuously exposed to synthetic pheromones (Harari et al. 2015). Harari et al. (2015) also found that L. betrana laid fewer eggs following exposure to conspecific calling females. Alternatively, midges may not experience decreased fitness if they habituate and eventually ignore pheromone mating disruption treatments. Habituated females may resume normal pheromone-releasing behavior later, experiencing little extra energy expenditure. In future experiments, one could observe calling females for longer periods of time to determine if habituation occurs and how long-term pheromone exposure influences female behavior.
Although female midges appear to be more receptive to males and call more while exposed to pheromones, females pre-exposed to pheromones and then placed in clean air were less likely to mate. Pheromone pre-exposure can disrupt normal courtship and mating behavior in other insects. Kuhns et al. (2012) found that moths pre-exposed to pheromones preferred to call rather than mate. However, we observed neither increased calling nor rejection when swede midge were pre-exposed to pheromone. Similar to our other experiments, it is possible that the females perceived synthetic pheromone as competition, and would have dispersed to other areas, preferring to mate in a location with fewer females. However, we did not observe females flying within the flasks or 73 plastic containers in an attempt to migrate. On the contrary, we typically observed females remaining stationary while calling on the sides or mesh tops of their holding containers with the males. Occasionally, we observed females pre-exposed to racemic pheromones calling within a couple of centimeters away from males, but males did not approach the females. It is possible that inhibitory stereoisomers from the racemic blend remained on the cuticle or other body parts of the female midges and counteracted the attractive pheromone plumes released by their pheromone glands.
Females may be less likely to mate following pheromone pre-exposure if pheromones masked male aphrodisiacs. If males release aphrodisiacs to court females, females may be less likely to detect these compounds if their antennal receptors are impaired by sex pheromones. Kuhns et al. (2012) hypothesized that male aphrodisiacs may not be detected by females rebuffing males following pheromone pre-exposure.
However, male swede midge are not known to produce pheromones. Due to the minute amounts of pheromones produced by cecidomyiids and difficulties extracting them (Hall et al. 2012), it is possible that males do produce pheromones, but they have not yet been identified. Female swede midge criteria for accepting mates are unknown.
Another possible explanation for reduced propensity to mate is that the two populations differ in autodetection behavior, where pheromones may promote mating in the Ontarian population while they may not in the Swiss population. However, we have evidence that aspects of reproductive behavior is consistent across populations. We found that males from both populations are attracted to the same monitoring lures in both
Ontario and Switzerland (Chapter 4; (Hillbur et al. 2005; Hallett and Sears 2013). The
74 diel periodicities of mating behaviors in males and females are also consistent across populations (Chapter 5; Hodgdon et al 2018). It is possible that pheromone pre-exposure and transfer to clean air disrupted female behavior in a manner that we were unable to observe and identify, despite calling more frequently during exposure. Testing for increased calling during pheromone exposure in the Swiss population would have allowed us to better understand how Swiss midges responded to pheromones. Further studies are needed to elucidate how females from varying geographical locations may differ in their responses to pheromones. Because female autodetection in general is a lesser-studied topic, we are unaware of any studies testing how female autodetection differs across populations of the same species.
Lastly, it is possible that our method for measuring calling did not fully capture the insect behavior in such a way that allows a reconciliatory explanation with our propensity to mate data. By recording whether or not a female was displaying pheromone-releasing behavior, we found that females increased the frequency of pheromone-releasing behavior. While one can assume that females displaying pheromone-releasing postures more frequently equates to more pheromone released from the females’ pheromone glands, this may not be the case. Females may have released pheromone more frequently, but in smaller amounts when exposed to synthetic pheromone. Additionally, insects may not always release pheromone when exhibiting the pheromone-releasing posture. The posture may serve as a visual cue for males that they are receptive for mating. Females that are less attractive to males may release less pheromone when in the presence of conspecific females to take advance of their pheromone plumes as a strategy to conserve their own resources while still attracting 75 mates. For example, when some female insects aggregate, a portion of the less attractive female population, “satellite females,” position themselves next to other calling females
(Yasui et al. 2007; van Wijk et al. 2017). By positioning themselves near attractive calling females, they are better able to secure mates than if they are alone. Satellite females within an attractive pheromone plume may release less pheromone themselves in order to conserve internal resources while still gaining access to males (Holdcraft et al.
2016). If so, this strategy may help explain why some females immediately taken from pheromone-laden environments were less attractive to males.
Understanding longer-term effects of mating disruption treatments on midge fitness would provide further insight into the impacts of autodetection on mating disruption efficacy. In the future, observing midges for longer periods of time would allow testing for persistence of behavioral effects on female midges as a result of pheromone exposure. Exposure to elevated levels of ambient pheromone could result in multiple longer-term effects, such as shorter lifespans, older insect age at mating, and decreased fecundity of mated females. Next steps in swede midge autodetection research could include allowing females a range of “recovery” times after pre-exposure to test for potential delays in mating. It is possible that females could have recovered and mated with males following our 20-minute testing period, experiencing a delay in mating.
Overall female fitness and numbers of offspring can be reduced when mating delays occur and females are older at the time of mating (Mori and Evenden 2013; Harari et al.
2015). Mori and Evenden (2013) argue that it is common for delayed mating to occur and mated females to be present in agricultural landscapes treated with mating disruption, despite observing trap shutdown and overall crop protection. If midges have long 76 recovery times to receptivity to males following pheromone pre-exposure, this may contribute to mating disruption success. Since midges typically live for only 1-5 days
(Readshaw 1961), a delay may have serious fitness consequences. However, if recovery time is minimal, reduced mating in the short term after exposure to synthetic pheromone may be trivial.
Pheromone mating disruption is a promising management tactic for many difficult pests. To inform decisions on the most effective candidate pheromone blend for mating disruption, both male and female behavior should be considered. Increased calling indicates increased willingness for females to mate when exposed to pheromone.
However, we observed a decrease in female propensity to mate after exposure to pheromones. Insect courtship behavior, while sometimes seemingly simple to the human eye in observational studies, can be complex. The entire sequence of events involved in mate searching and receptivity for swede midge may not yet be fully understood.
Specifically, many questions remain to be answered to further elucidate the role of female sex pheromone in mediating female reproductive behavior. Our experiments offer novel insight into how cecidomyiid females autodetect and alter their reproductive behavior in response to pheromones. We hope that our results inspire future research into this intriguing potential mating disruption mechanism, particularly in Cecidomyiidae.
3.7 References
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81 3.8 Tables
TABLE 3.1 PHEROMONE BLEND TREATMENTS USED IN LABORATORY EXPERIMENTS
Diacetoxyundecane Acetoxyundecane Pheromone blend 2S,9S 2,9 2S,10S 2,10 2,S 2 Dose-response experiment Three-component chiral 10 ng - 20 ng - 0.2 ng - (High dose) Three-component chiral 1 ng - 2 ng - 0.02 ng - (Low dose) Control (solvent only) ------Blend-response experiment
Three-component chiral 1 ng - 2 ng - 0.02 ng -
Three-component racemic - 4 ng - 8 ng - 0.04 ng
Single-component chiral - - 2 ng - - - ((2 S, 10 S)-) Single-component racemic - - - 8 ng - - (2, 10-) Control (solvent only) ------
82 3.9 Figures
100 * b Control 80 a 2 ng 60 a 20 ng
40
20 Females Calling (%) Calling Females 0 Time After 0 50 100 150 200 250 Emergence (Min) 0915 1005 1055 1145 1235 1325 Hour of Day
FIG. 3.1 FEMALES CALLING IN DOSE RESPONSE EXPERIMENT USING THREE-COMPONENT CHIRAL PHEROMONE BLEND. TREATMENTS WITH THE SAME LETTER TO LEFT OF LEGEND ARE NOT SIGNIFICANTLY DIFFERENT BASED ON POST HOC PAIRWISE COMPARISON TESTS ( P > 0.05) AND ASTERISK INDICATES OVERALL MODEL SIGNIFICANCE ACROSS ALL TIME POINTS AT ( P < 0.05)
83 100 *** c Control 80 c (2 S, 10 S)- 60 a 3-Component Chiral c 2, 10- 40 b 3-Component Racemic 20 Females Calling (%) Calling Females 0 0 50 100 150 200 250 Time After Emergence (Min) 0915 1005 1055 1145 1235 1325 Hour of Day
FIG. 3.2 FEMALES CALLING IN PHEROMONE BLEND EXPERIMENT. TREATMENTS WITH THE DIFFERENT LETTERS TO THE LEFT OF LEGEND ARE SIGNIFICANTLY DIFFERENT BASED ON POST HOC PAIRWISE COMPARISON TESTS ( P > 0.05), AND ASTERISKS INDICATE OVERALL MODEL SIGNIFICANCE ACROSS ALL TIME POINTS AT ( P < 0.001)
84 40 ** b b 30
20 a a 10
Continuous Callers (%) Callers Continuous a 0 al ic )- ir S , 10- 2 Control , 10 Ch p. Racem S . (2 m p -Co m 3 -Co 3 Pheromone Blend
FIG. 3.3 MIDGES CALLING FOR AT LEAST HALF OF THE TIME POINTS (CONTINUOUS CALLERS) WITHIN EACH PHEROMONE TREATMENT IN THE LABORATORY CALLING EXPERIMENT. TREATMENTS WITH DIFFERENT LETTERS ARE SIGNIFICANTLY DIFFERENT BASED ON POST HOC PAIRWISE COMPARISON TESTS (P > 0.05), AND ASTERISKS INDICATE OVERALL MODEL SIGNIFICANCE AT ( P < 0.01)
85 5 A. 5 B. B. 10 10 15 15 20 20 25 25 30 30 35 35 Midge Individual Midge 40 40 45 45 50 50 75 150 225 75 150 225 Time After Emergence (Min) 1030 1145 1300 1030 1145 1300 Hour of Day
FIG. 3.4 INCIDENCES OF CALLING BY INDIVIDUAL FEMALES AT 15- MINUTE INTERVALS FOLLOWING EMERGENCE. DARKENED CELLS INDICATE OBSERVED CALLING UNDER THE (A.) CONTROL AND (B.) THREE-COMPONENT CHIRAL PHEROMONE TREATMENTS.
86 100 *** Control 80 3-Component Chiral 60
40
20 Females Calling (%) Calling Females 0 0 50 100 150 200 Time After Emergence (Min) 0940 1020 1200 1020 1250 Hour of Day
FIG. 3.5 FEMALES CALLING IN UNTREATED (CONTROL) OR THREE- COMPONENT CHIRAL (3C-N) PHEROMONE MATING DISRUPTION PLOTS. ASTERISKS INDICATE OVERALL MODEL SIGNIFICANCE ACROSS ALL TIME POINTS AT ( P < 0.001)
87 CHAPTER 4: FIELD TESTS OF CANDIDATE PHEROMONE BLENDS FOR MATING DISRUPTION OF THE INVASIVE SWEDE MIDGE (DIPTERA: CECIDOMYIIDAE)
ELISABETH A. HODGDON* 1 (0000-0002-2432-9201), REBECCA H. HALLETT 2 (0000-0002-4316-7877), JAMES D. HEAL 2, CHASE A. STRATTON 1, CHRISTINE A. HOEPTING 3, and YOLANDA H. CHEN 1 (0000-0001-9439-5899)
1Department of Plant and Soil Science, University of Vermont, 63 Carrigan Dr.,
Burlington, Vermont, USA 05405
2School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada, N1G 2W1
3Cornell Cooperative Extension – Orleans County, Cornell University, 12690 Route 31, Albion, NY, 14411
*Corresponding author contact information: Elisabeth A. Hodgdon Email: [email protected] Telephone: +1 (802) 522-6914
88
4.1 Acknowledgements Funding for our research was provided by the following sources: USDA NIFA
Crop Protection and Pest Management grant (2014 - 70006 - 22525) to YHC, Northeast
SARE Graduate Student grant (GNE16 - 121) to EAH, and the Ontario Ministry of
Agriculture, Food and Rural Affairs University of Guelph Partnership to RHH. We are grateful for assistance from Kylie Barwise, Jamie Bauman, Jarrett Blair, Josée Boisclair,
Thierry Boislard, Charles-Étienne Ferland, Paolo Filho, Laurence Jochems-Tanguay,
David Kells, David Laurie, Kate Lindsay, Charlotte MacKay, Ted Marois, Matthew
Muzzatti, Cameron Ogilvie, Elizabeth Porter, Jorge Ruiz-Arrocho, Linley Sherin, Leah
Slater, Emily Sousa, Andrea Swan, and Jonathan Williams from the Institut de recherche et développement en agroenvironnement, University of Guelph, and University of
Vermont for our field trials. We also thank Pfenning’s Organic for providing land, technical assistance, and broccoli seedlings for our field trials at their farm.
89
4.2 Abstract Swede midge, Contarinia nasturtii (Diptera: Cecidomyiidae), is a challenging invasive pest of Brassica vegetable and oilseed crops in Canada and the Northeastern
U.S. Midge larvae feed within the meristem of their host plants, causing deformed heads, stems, and leaves. Due to extremely low damage thresholds, pheromone mating disruption is particularly promising for swede midge management in high value vegetable crops because it prevents mating and ultimately oviposition, and is allowable for organic production. However, a major challenge is that the naturally-produced swede midge pheromone, a 1:2:0.02 blend of (2 S,9 S)-diacetoxyundecane, (2 S,10 S)-diacetoxyundecane and ( S)-2-acetoxyundecane, is costly to synthesize due to the chirality of the compounds.
In field plots of broccoli, we tested whether chiral, racemic, and single-component pheromone blends confused males causing trap shutdown, and whether they reduced crop damage compared to untreated controls. We found a significant reduction in numbers of males caught in three-component chiral and racemic pheromone plots, but not in the single-component pheromone treatments. While marketable broccoli yields were not higher overall in the pheromone-treated plots compared with the untreated controls, yields were statistically significantly higher in the three-component chiral treatment in year two. Therefore, the three-component chiral blend appears to be the most promising pheromone blend for swede midge mating disruption. However, due to relatively high levels of midge damage across all treatments, additional research is necessary to optimize pheromone mating disruption in complex annual cropping systems.
90 4.3 Introduction Swede midge, (Contarinia nasturtii Kieffer), a galling fly in the family
Cecidomyiidae, is a challenging invasive pest of Brassica crops in North America. Since its introduction to Ontario, Canada from Europe in the 1990’s (Hallett and Heal 2001), swede midge has spread to several Canadian provinces and U.S. states. Severe economic losses of canola (Brassica napus L.), broccoli and cabbage ( Brassica oleracea L.), and other related crops have been reported in Ontario, Québec, New York, and Vermont
(unpubl. data, Hallett and Heal 2001; Chen et al. 2011). Climactic models predict that swede midge has the potential to establish in important vegetable production areas in the
Eastern U.S. and canola-producing provinces within the Canadian Prairies (Mika et al.
2008), threatening the economic viability of Brassica vegetable and oilseed production in
North America. Due to challenges associated with identifying and managing swede midge, populations have grown to devastating levels on individual farms, leading to reports of up to 100% crop loss (Hallett and Heal 2001; Chen et al. 2011).
Although swede midge do not induce gall formation similarly to other cecidomyiids, feeding larvae have the unique ability to manipulate plant growth, often resulting in irreversible crop damage. Adult midges oviposit into the meristems of host plants. Midge larvae feed within the meristematic tissue, causing scarred and deformed growth, rendering leaves, stems, and heads unmarketable (Readshaw 1966; Hallett 2007;
Chen et al. 2011; Stratton et al. 2018). Digestive secretions by cecidomyiid larvae break down plant cells and alter plant nutrient allocation and hormone dynamics within the plant (Tooker and De Moraes 2007; Tooker and Moraes 2010). Heading Brassica vegetables are particularly sensitive to larval feeding, where a single larva renders a
91 cauliflower plant unmarketable, and plants are susceptible to damage from seedling stage until heading (Stratton et al. 2018). Because damage symptoms are often visible only after larvae vacate the plant to pupate within the soil, growers often mistake midge feeding damage for nutrient deficiencies (Hallett and Heal 2001).
The cryptic feeding behavior of larvae poses challenges for the use of insecticides for swede midge management. Because larvae are protected within new leaves in the meristem, foliar insecticides are seldom reliably effective due to poor contact with the feeding insects (Hallett et al. 2009a; Seaman et al. 2014; Evans and Hallett 2016).
Current recommendations for control of swede midge include applications of systemic insecticides, followed by calendar sprays of foliar insecticides (Hallett et al. 2009a; Chen and Shelton 2010). The recommendation to use calendar sprays of insecticides negates decades of integrated pest management recommendations for reduced reliance on insecticides (Andaloro et al. 1983; Chen et al. 2011) There are presently no known certified pesticides allowable for certified-organic production that are effective for swede midge management (Seaman et al. 2014; Evans and Hallett 2016). As a result, growers managing their crops organically must rely on alternatives to insecticides and ecologically-based management strategies.
Additionally, aspects of swede midge ecology present management difficulties.
Due to the presence of more than one emergence phenotype and multiple overlapping generations (Hallett et al. 2007; Hallett et al. 2009b), swede midge is present throughout the growing season in North America. Therefore, management strategies that provide continuous protection are urgently needed. Several commonly-used biological, cultural,
92 and physical control tactics are not feasible for swede midge management. Exploration in
Europe for natural enemies rendered no suitable candidates for biological control programs (Corlay et al. 2007; Abram et al. 2012). While some alternative management strategies, such as insect exclusion netting and wide crop rotations away from infested fields, can be effective as alternatives to insecticides for swede midge management (Chen et al. 2011; Hodgdon et al. 2017), these options are often neither economically nor logistically feasible for many growers (Hodgdon et al., unpubl. data ).
Because only one larva feeding on the apical meristem can render a heading
Brassica vegetable unmarketable (Stratton et al. 2018), and because larvae are difficult to control when protected within the leaves of the meristem, management strategies that prevent oviposition are urgently needed. While “scout and spray” pest management algorithms are effective for other pests of Brassica crops, for example, lepidopteran pests with highly visible larval stages and higher damage thresholds (Andaloro et al. 1983), swede midge damage prevention may be more complex. An emphasis on preventing oviposition and larval feeding within the meristem rather than curative measures will be necessary to prevent crop damage from this devastating pest.
Pheromone mating disruption is a pest management strategy that involves the application of large quantities of synthetic sex pheromone to crops to confuse males and prevent mating. Pheromone mating disruption is promising for swede midge management because it prevents mating and ultimately oviposition. This tactic has been very successful for lepidopteran pest management in orchard and vineyard systems, decreasing insecticide use and limiting impacts on non-target organisms (Welter et al. 2008;
93 Witzgall et al. 2008). However, it is seldom used for non-lepidopteran pests and pests of annual crops (Miller and Gut 2015). Cost and difficulties demonstrating crop protection due to migrating gravid females are typically cited as challenges associated with using mating disruption in annual crops (Fadamiro et al. 1999; Welter et al. 2008; Vacas et al.
2011).
Samietz et al. (2012) demonstrated that pheromone mating disruption can be effective for swede midge in Europe; however, considerable economic challenges exist for commercial adoption. The female swede midge sex pheromone, a 1:2:0.02 mixture of
(2 S,9 S)-diacetoxyundecane, (2 S,10 S)-diacetoxyundecane and ( S)-2-acetoxyundecane
(Hillbur et al. 2005), is costly to synthesize due to the presence of either one or two chiral centers in each component. Therefore, multiple stereoisomers (three-dimensional configurations) are possible for each compound. After synthesis, chemists need to isolate the biologically-active stereoisomers from the racemic blend of components, increasing the cost of synthesis (C. Oelschlager, pers. comm.). Racemic pheromone compounds, or unpurified mixtures of all possible stereoisomers of the pheromone compounds, may present a more economical mating disruption system for swede midge. Although non- natural stereoisomers of the “main” (most abundant) swede midge pheromone component in the blend, (2 S,10 S)-diacetoxyundecane, inhibit male attraction (Boddum et al. 2009), they may still be useful for mating disruption.
Mating disruption deploying unattractive and racemic non-natural pheromone stereoisomers (Onufrieva et al. 2008; Arakaki et al. 2013) as well as single-component treatments (Mafi et al. 2005; Higbee and Burks 2008) have been successful in confusing
94 males of other species and has led to considerable cost savings. In addition to racemic blends containing all three compounds within the swede midge blend, deploying the most attractive compound alone in its natural (2 S, 10 S)-diacetoxyundecane or racemic form may present another opportunity for lower-cost mating disruption systems for this pest.
Unexpectedly, male midges possess antennal receptors for at least one of the non-natural stereoisomers within the racemic blend of 2,10-diacetoxyundecane (Boddum et al. 2010).
Racemic and single-component pheromone blends have yet to be tested for swede midge mating disruption.
Here, we assessed the efficacy of chiral, racemic and single-component pheromone mating disruption treatments in small field plots of broccoli. Using reservoir- type mating disruption dispensers, we tested whether male trap counts (trap shutdown) and crop damage differed between plots treated with either three-component, single- component, chiral, and racemic pheromone blends. We use our results to identify the most promising candidate pheromone blends for mating disruption for swede midge.
Lastly, we discuss future research directions to address ecological challenges associated with implementing this management tactic in complex annual cropping systems.
4.4 Methods and Materials 4.4.1 Experimental Sites We tested our mating disruption treatments at a total of three field sites in Ontario and Québec, Canada for two field seasons per experiment. For the three-component pheromone experiment, our test plots were located in Ontario, Canada at the University of Guelph Elora Research Station (Elora) and at a large commercial vegetable farm in
95 New Hamburg, Ontario (New Hamburg) in 2016 and 2017. Swede midge was first documented near this region in North America in 2000 (Hallett and Heal 2001), thus local populations are well-established there. For the single-component experiment conducted in 2017 and 2018, we used three field sites: Elora, New Hamburg, and a third site at the Institut de recherche et de développement en agroenvironnement (IRDA) in St-
Bruno-de-Montarville, Québec, Canada (St-Bruno). Two of our three sites (New
Hamburg and St-Buno) were certified organic.
To ensure swede midge pressure, we situated the experimental plots at each location in close proximity to, but not within, fields where Brassica oilseed or vegetable crops were grown in previous years. At the Elora site, wheat ( Triticum aestivum L.), corn
(Zea mays L.), canola ( Brassica napus L.), and soybean ( Glycine max (L.) Merr.) comprised the major components of the surrounding cropping systems. Mixed vegetable crops (including Brassica and Raphinus spp.) were grown at the New Hamburg site.
Mixed vegetable crops and grassland comprised a majority of the landscape at the St-
Bruno site.
We used randomized complete block designs to test three pheromone treatments-- chiral, racemic, and an untreated control--in 16x16m plots of broccoli, based on experimental designs used by Samietz et al. (2012). We tested three- and single- component treatments in separate experiments. Each block contained one plot of each treatment (three plots per block), with a total of n = 6 (three-component) and n = 4
(single-component; Table 1) replicate plots. To avoid the spread of pheromone plumes from one plot to another, we separated plots by a minimum of 425 m. We situated each of
96 the three plots per block as equidistant as possible from known infested fields to ensure equal swede midge pressure.
Each plot consisted of 20 rows of broccoli using 30 cm within-row and 76 cm between-row spacing (~1,040 plants per plot). We used ‘Everest’ broccoli, with the exception of ‘Windsor’ being planted at one site in one year (Stokes Seed Ltd., Thorold,
ON). Both varieties are marketed for late-season crops and medium crown size. Hallett
(2007) found no significant differences in swede midge susceptibility between the two varieties. We seeded broccoli a minimum of five weeks prior to transplanting, which occurred in May or June (Table 1), corresponding with the time of year when overwintering midges begin to emerge from the soil in Ontario (Hallett et al. 2009b).
Seedlings were produced with conventional (Elora) or certified organic (New Hamburg and St-Bruno) peat-based potting media and fertilizers in heated greenhouses and were transplanted when they had 2-5 true leaves. We irrigated plots immediately following transplanting using overhead irrigation, but the plots only received natural rainfall throughout the remainder of the experiment with the exception of one site (St-Bruno), which received drip irrigation throughout the season. Broccoli production practices, including fertilization and weed management, followed typical regimes for the region.
Plots were hand weeded as needed until head formation and received no pesticide treatments.
4.4.2 Mating Disruption Treatments We used reservoir-type pheromone dispenser bags to test our pheromone treatments (ChemTica Internacional, S.A., Heredia, Costa Rica) using spacing and release rates similar to those used by Samietz et al. (2012). Each dispenser consisted of a 97 brown polyethylene bag that contained the pure pheromone held within a microcentrifuge tube. Each tube had a small hole to release the pheromone. The bag was designed for ultraviolet light protection and slow release of the pheromone through the semi- permeable material (C.A. Oelschlager, pers. comm .). Each dispenser contained 100 times the pheromone amount used for monitoring (Samietz et al 2012). Amounts of each component were quadrupled or doubled for the racemic treatment so that the biologically- active stereoisomers were equal across pheromone treatments (Table 2). We installed the dispensers within the same day that the broccoli was transplanted, hanging the dispenser bags approximately 25 cm above the ground on hooked wire flag posts arranged in the field in a staggered 2 x 2m grid pattern. No bags or posts were set up within the control plots. Bags were installed once and were not replaced during the season.
4.4.3 Evaluation of Mating Disruption We used trap shutdown to assess whether the pheromone treatments disrupted the males’ ability to locate attractive pheromone lures in monitoring traps. When mating disruption treatments are effective, there are few to no males caught in sticky traps baited with commercial pheromone lures (Howse et al., 1998). We installed four traps within the plots in random locations, at least 5 m from the edges of the plots and apart from each other, one week after transplanting and dispenser installation. Each trap consisted of a delta trap body with a sticky card liner (Solida Distributions, St-Ferréol-les-Neiges, QC) and polyethylene cap commercial lure containing 500 ng (2 S,9 S)-diacetoxyundecane, 1
µg (2 S,10 S)-diacetoxyundecane, and 10 ng ( S)-2-acetoxyundecane (PheroNet Swede
Midge Lures, Andermatt Biocontrol, Grossdietwil, Switzerland ). Traps hung from wooden stakes 25 cm above the soil surface. Lures were replaced twice during the 98 experiment, at 30 and 60 days after transplanting. We counted males caught on the sticky cards in the traps weekly for 12 weeks. For trap counts, plant damage and yield assessments, we used a plot-level unit of measurement, calculating mean measurements from our subsamples for each plot.
We evaluated plants for swede midge damage to vegetative plant parts at 3 and 6 weeks after transplanting using a four-point damage scale of increasing damage severity by Hallett (2007), where: 0 = no swede midge damage, 1 = minor swelling, scarring, or deformation of meristem, petioles, and leaves, 2 = moderate to severe swelling, scarring, or deformation of meristem, petioles, and leaves, and 3 = complete death of apical meristem. Using a random number generator, we selected three locations per row of broccoli and scored three plants per row for a total of 60 plants per plot.
At the end of the season, we obtained a yield estimate of the broccoli in each plot.
We evaluated swede midge damage to broccoli crowns using a six-point damage scale by
Hallett and Heal (2011) when broccoli heads were ready for harvest, between 9-12 weeks after transplanting. Plants with no damage received a score of zero. Plants with petiole scarring, head unevenness, and/or accompanying deformity due to larval feeding received scores of 1-4 with increasing severity. Complete death of the apical meristem resulted in a score of 5. We then used a separate binary scoring system to further categorize heads as either marketable or unmarketable, counting heads receiving a damage score of 0 as marketable, and 1-5 as unmarketable based on USDA quality standards for insect damage
(United States Department of Agriculture 2006).
99 4.4.4 Statistical Analyses To test for differences in numbers of males trapped in plots over time, we used the generalized estimating equations (GEE) extension for generalized linear models (GLM) using SPSS version 24 (IBM, Armonk, NY). We used GEE extensions because they are robust to non-normally distributed count data and allow for repeated measures (Zeger et al. 1988; Muff et al. 2016). We analyzed pooled data across years as well as each year separately, which allowed us to test both overall (pooled) and individual yearly results.
We specified “plot” as subjects in the GEE menu. Trap counts followed Poisson distributions, and pheromone and block were included as variables in all models. When we pooled data across years, we included year and year*pheromone variables.
For our plant damage assessments at each time point, we used ordinal logistic regression with similar variables. Because our yield data consisted of non-normally distributed ranks, we used non-parametric Kruskal-Wallis tests with pairwise post hoc
Mann-Whitney U comparison tests to determine whether our counts of marketable broccoli crowns differed across treatments. For all models, we evaluated significance using α = 0.05.
4.5 Results 4.5.1 Trap Shutdown We captured significantly fewer males in monitoring traps in the three-component chiral and racemic pheromone-treated plots compared with the control (pooled:
2 χ2 = 79.30, P < 0.001). In 2017, the pairwise comparisons indicated that male trap counts significantly differed between the chiral and racemic plots; however, they were
100 not significantly different in 2016 (Fig. 1). The mean numbers of males caught in traps were 4.8 ± 1.4 and 11.3 ± 4.0 per week for the chiral and racemic treatments respectively, compared with 86.8 ± 15.7 males in the control plots across both years. These trap counts represent 95% (chiral) and 87% (racemic) reductions in trap counts compared with the untreated control.
Unlike in the three-component study, we did not observe trap shutdown in the single-component experiment (Fig. 2). The pheromone treatments did not influence
2 weekly male trap counts in 2017 or in the pooled data set (pooled: χ2 = 1.373, P > 0.05).
Numbers of males fluctuated weekly within control plots, presumably according to patterns of emergence associated with the multiple generations of midges and differing
Ontario emergence phenotypes (Hallett et al. 2009b; Fig. 2). Overall, the trap counts were lower in our single-component experiment compared to the three-component experiment.
At one site (St-Bruno), swede midge populations were very low (<1 male per trap per day) for several weeks at the start of the experiment. However, we believe that increases in midge population density in the final weeks of the experiment allowed us to test for trap shutdown.
In addition to differences in trap counts due to pheromone treatments, trap counts fluctuated widely, ranging from zero to hundreds of males per week in the control plots.
These fluctuations, which we mainly observed at three-week intervals, were presumably due to multiple generations of midges at our experimental sites, which is typical for
Ontario (Hallett et al. 2007, 2009b). Midge numbers varied by week within the pheromone-treated plots as well, but were dampened by trap shutdown effects. In 2016,
101 we observed few midges before the ninth week of the experimental period, which we attribute to local drought conditions that may have hindered emergence of midges. A critical level of soil moisture is required for swede midge to complete pupation
(Readshaw 1966; Chen and Shelton 2007).
4.5.2 Plant Damage and Yield Assessment Overall, we found little swede midge damage in plots at the third week assessment, but found increasing damage during the sixth week ratings (Table 3).
Pheromone treatment was not a significant predictor of plant damage at three weeks in either experiment ( P > 0.05 for all models), likely due to lower midge populations at the beginning of the season and/or delay in onset of damage symptoms. However, we found that the three-component treatments influenced the incidence of broccoli damage at six
2 weeks and at the final damage assessment in 2017 ( χ2 = 6.309, P = 0.043 at six weeks,
2 χ2 = 19.775, P < 0.001 at harvest), but not in 2016. In 2017, mean harvest damage ratings were over three times as high in control versus chiral plots (Fig. 3). Damage ratings were statistically significantly lower in the chiral versus control plots, but damage did not vary significantly between the control and racemic, or chiral and racemic plots
(P > 0.05).
Because damage ratings were lower in the chiral plots, we observed a
2 corresponding significant increase in marketable yields in 2017 ( Η2 = 7.200, P = 0.027).
While half (55.56 ± 4.59%) of the broccoli heads were marketable in the chiral-treated plots, only 6.67 ± 1.22% of broccoli heads (a ninefold decrease) were marketable in the control plots (Fig. 3). Although not statistically significant, we also observed marketable yield increases in the racemic plots compared with the control, where 25.0 ± 5.0% of 102 broccoli was undamaged. The numbers of marketable broccoli heads did not significantly differ across treatments in our single-component experiment (pooled model: H2 = 0.787,
P > 0.05; Fig. 4).
4.6 Discussion Developing effective commercial pheromone mating disruption systems involve many years of experimentation in order to identify the most effective pheromone blends, dispenser types and densities, and other setup logistics for a particular pest. Our research represents the first swede midge mating disruption study in North America. Here, we aimed to identify the most effective pheromone blend for mating disruption—a critical first step in mating disruption research and development. Despite the potential for cost savings by racemic and single-component pheromones, we believe that the three- component chiral blend is most promising for swede midge mating disruption. We observed both statistically significant trap shutdown and decreases in swede midge damage to broccoli in this treatment only. Our results appear to confirm the long held belief that the most attractive pheromone blend is the most effective for mating disruption
(Minks and Carde 1988; Cardé and Minks 1995).
While the three-component racemic blend did not result in a significant increase in marketable broccoli yield compared with the untreated control, we did observe trap shutdown in plots treated with this blend. Reductions in weekly trap counts within both chiral and racemic plots compared with control trap counts fell within the typical range for trap shutdown in commercial mating disruption systems, between 80-90% (Miller and
Gut 2015). Both blends confused males. It is possible that males, despite being unable to 103 locate lures in monitoring traps, we able to find and mate with females amidst racemic pheromone treatments.
However, although not statistically significant, we did observe a 23% and 20% increase in marketable broccoli heads in three-component racemic plots compared with both untreated and three-component chiral plots, respectively, in the first year of our study. We also observed a 19% increase in marketable yield in racemic plots compared with the untreated plots in the second year (Fig. 3). Overall, swede midge pressure was higher in 2017 than in 2016, with mean trap counts in untreated plots equaling 101 (2017) versus 79 (2016) midges per trap per week during the experimental period. Perhaps racemic blends are more effective in preventing crop damage when midge populations are lower, although additional testing would be necessary to demonstrate differential effects of midge population sizes on pheromone blend efficacy.
Despite the potential for cost savings, we found that the single-component treatments were not promising for mating disruption. The pheromone blends did not cause trap shutdown or protect the broccoli crop. Males are not attracted to (2 S,10 S)- diacetoxyundecane without the other two compounds in the natural swede midge pheromone blend ((2 S,9 S)-diacetoxyundecane and ( S)-2-acetoxyundecane) and are repelled by the racemic 2,10-diacetoxyundecane (Boddum et al. 2009). The partial and unattractive pheromone blends released from single-component dispensers did not effectively mask pheromone plumes from the attractive lures in our monitoring traps.
Thus, males were likely able to find and mate with females amidst our single-component pheromone treatments.
104 Despite deploying the pheromone treatments, we observed high levels of damage in all of the treated plots. In our most successful mating disruption treatment, the three- component chiral blend in 2017, only 56% of heads were marketable. Migration of mated females into pheromone-treated plots is a common challenge for pheromone mating disruption in both annual and perennial cropping systems, resulting in high levels of crop damage despite trap shutdown (Jiménez et al. 1988; Cardé and Minks 1995; Fadamiro et al. 1999; Vacas et al. 2011). We have increasing evidence that swede midge mates shortly after emergence (Hodgdon et al, accepted ) and that they do not preferentially mate in the presence of their host plants (Hodgdon et al, unpubl. data ). If swede midge mate at their emergence sites and then migrate to host plants to oviposit, mating disruption treatments in the current year’s Brassica crops will be largely ineffective in providing crop protection, regardless of male confusion and trap shutdown. Because midges overwinter in the soil and exhibit multiple emergence phenotypes (Hallett et al.
2009b), they often emerge over prolonged periods of time from multiple fields on vegetable farms where growers rotate their Brassica crops. Emerging in last year’s
Brassica fields may result in a mismatch between emergence, mating, and oviposition sites. Rather, pheromone dispensers would be more useful if they were installed at emergence sites.
Further exploration into the location of swede midge mating and migration patterns will be necessary to inform the installation of dispensers within a farm landscape. Annual cropping systems can be challenging environments for the deployment of pheromone mating disruption systems. Mating disruption has typically been most successful for perennial crops, where the area to be treated is straightforward, and for 105 pests with fewer generations per year. Mating at emergence sites and post-mating migration also occur in other cecidomyiid pests in annual cropping systems, such as the brassica pod midge ( Dasineura brassicae Winn.) and orange wheat blossom midge
(Sitodiplosis mossellana Géhin) (Sylven 1970; Williams et al. 1987; Smith et al. 2007).
Having to treat multiple emergence sites as well as the current crop field would greatly increase the cost of a mating disruption system. In addition to needing to treat more than one field, pheromone dispensers would need to be in place all season long due to the presence of multiple overlapping generations in North America (Hallett et al. 2009b).
We may have also experienced high levels of crop damage because of our small plot size. Milli et al. (1997) found significant variability in ambient pheromone levels within 10 m of treated field borders. Thus, pheromone mating disruption may be more effective when larger areas are treated. We may have experienced significant edge effects due to our small plot size, with inadequate coverage of pheromone to suppress mating.
Due to the resource-intensive nature of pheromone mating disruption experiments, achieving adequate replication using large-scale plots is challenging and often cost prohibitive. However, for swede midge, larger tracts of land may need to be treated based on crop rotation history, which varies by farm. Perhaps larger, whole-farm proof-of- concept mating disruption experimental designs will be necessary to determine whether this tactic can be successful for swede midge management. Small-scale, organic vegetable farms would be ideal candidates for demonstration.
Although swede midge is a problematic pest in Europe, invasive populations in
North America appear to be much more challenging, potentially due to lack of
106 specialized natural enemies or a natural enemy complex (Corlay et al. 2007). Despite using a similar experimental design, Samietz et al. (2012) did not observe high rates of crop damage in their swede midge mating disruption study in Europe. Swede midge damage in their Brussels sprouts test crops did not exceed 2% in treated plots. However, significantly lower swede midge populations at their test sites may have allowed for a greater reduction in crop damage. Their weekly mean trap counts never exceed 50 males and in control plots, damage never exceeded 20%. In our study, we commonly observed more than 10 times their numbers of males per trap during weekly checks at peak emergence periods during July and August (Figs. 1 and 2). We observed almost complete yield loss in several of our untreated plots. For areas with higher swede midge pressure, a combination of management tactics may be necessary to sufficiently reduce damage. If mating disruption dispensers are needed for extended periods of time in multiple fields in addition to other management tactics, then further research is needed to reduce the cost of swede midge mating disruption for the system to be commercially feasible.
Although the cheaper single-component and racemic blends were not successful, other methods can be used to decrease the cost of pheromone mating disruption, such as reducing dispenser densities and strategically turning off dispensers at certain times of the day. For example, deploying fewer yet more efficient dispensers could allow for labor savings associated with installation. Our dispenser density (80 dispensers per 16x16m plot) was quite high and somewhat unrealistic for vegetable growers to implement (A.
Jones, pers. comm .). Aerosol “mega-dispensers” requiring fewer devices per unit area are economical and effective for some lepidopteran pests. To further reduce costs, aerosol dispensers can be programmed to release pheromone only during the times of day when 107 insects are active based on known diel patterns of activity (Rama et al. 2002; Stelinski et al. 2007; Higbee and Burks 2008; Casado et al. 2014; Mori and Evenden 2015). Such devices are turned off when insects are naturally inactive, thus saving pheromone inputs.
Swede midge exhibits diel periodicity of mating, (Chapter 5; Hodgdon et al., accepted ), which introduces the potential to turn off programmable aerosol dispensers during the afternoon and night when the insects are inactive.
Our field trials demonstrate the considerable potential for pheromone mating disruption to contribute to managing swede midge. Future research and development efforts, particularly related to midge mating and migration patterns to inform optimal dispenser locations, will be necessary as next steps toward commercial adoption. Without a knowledge of where midges mate, pheromone mating disruption for swede midge may not become commercially viable. Because crop rotation is not feasible for growers with small land bases, and no organic insecticides are currently effective for swede midge
(Seaman et al. 2014; Evans and Hallett 2016), small-scale organic growers will benefit the most from a new ecologically-based swede midge management strategy. Logical next steps include testing mating disruption for swede midge by installing dispensers at emergence sites based on cropping histories and pest damage records on small-scale farms.
As swede midge populations continue to build in North America with the potential to spread to important vegetable producing regions (Mika et al. 2008; Chen et al. 2011), effective management strategies are desperately needed to prevent economic losses on farms. Both our research results and Samietz et al’s (2012) study in Europe
108 indicate potential for effective swede midge pheromone mating disruption. Mating disruption may be most effective for swede midge in conjunction with other management strategies, such as insecticides, crop rotation, or netting. While mating disruption for lepidopteran pests, such as codling moth, have benefited from decades of research and development (Welter et al. 2008; Witzgall et al. 2008), swede midge mating disruption is still in very early stages. With additional research, pheromone mating disruption has the potential to offer another important tool for swede midge management.
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114 4.8 Tables
TABLE 4.1 PLOT AND BROCCOLI PRODUCTION CHARACTERISTICS AT EXPERIMENTAL SITES
Site Number Broccoli Seeding Transplanting Growth stage at of variety date date transplanting replicates Three-component experiment 2016 Elora 1 Windsor 3-May 24-Jun 3-5 leaf stage New Hamburg 2 Everest Unknown 21-Jun 2-3 leaf stage 2017 Elora 1 Everest 24-Apr 29-May 2-3 leaf stage New Hamburg 2 Everest Unknown 22-May 2-4 leaf stage Single-component experiment 2017 Elora 1 Everest 24-Apr 29-May 2-3 leaf stage New Hamburg 1 Everest Unknown 22-May 2-3 leaf stage St-Bruno-de- 1 Everest Unknown 13-Jun 2-3 leaf stage Montarville 2018 Elora 1 Everest 23-Apr 30-May 3-4 leaf stage
115 TABLE 4.2 AMOUNTS OF PHEROMONE COMPONENTS PER RESERVOIR DISPENSER IN THREE- AND SINGLE-COMPONENT EXPERIMENTAL EXPERIMENTS. NO DISPENSERS WERE INSTALLED IN CONTROL (UNTREATED) PLOTS.
Pheromone blend Diacetoxyundecane Acetoxyundecane treatment (2 S,9 S)- 2,9- (2 S,10 S)- 2,10- (S)-2- 2- Three-component experiment Chiral 50 µg - 100 µg - 1 µg - Racemic - 200 µg - 400 µg - 2 µg Control ------Single-component experiment Chiral - - 100 µg - - - Racemic - - - 400 µg - - Control ------
116 TABLE 4.3 MEAN DAMAGE RATINGS (± SEM) AT THREE AND SIX WEEKS AFTER TRANSPLANTING AND AT HARVEST
Pheromone Mean damage rating (± SEM) treatment Year 1 Year 2 3 weeks z 6 weeks Harvest y 3 weeks 6 weeks Harvest Three-component experiment Chiral 0.0 ± 0.0 0.1 ± 0.0 2.0 ± 0.6 0.1 ± 0.1 0.4 ± 0.2 b w 1.2 ± 0.2 b Racemic 0.1 ± 0.1 0.1 ± 0.0 1.7 ± 1.1 0.1 ± 0.1 0.4 ±0.1 b 2.2 ± 0.5 b Control 0.1 ± 0.0 0.1 ± 0.1 1.9 ± 0.6 0.4 ± 0.1 1.2 ± 0.3 a 3.6 ± 0.2 a 2 2 2 2 2 2 Test statistic χ2 = 0.106 χ2 = 0.199 χ2 = 0.127 χ2 = 2.501 χ2 = 6.309 χ2 = 19.775 Significance x NS NS NS NS * * Single-component experiment Chiral 0.1 ± 0.0 0.5 ± 0.3 2.2 ± 1.0 0.0 v 0.6 0.8 Racemic 0.1 ± 0.0 0.4 ± 0.1 2.1 ± 1.1 0.0 1.2 3.0 Control 0.1 ± 0.0 0.4 ± 0.2 2.0 ± 1.0 0.1 0.3 3.5 Test statistic χ 2 = 0.354 χ 2 = 0.072 χ 2 = 0.192 2 2 2 - - - Significance NS NS NS
zThree and six week damage ratings were conducted using a four-point scale of vegetative damage, where 0 = no swede midge damage, 1 = mild twisting or scarring of petioles, leaves, and/or meristem swelling, 2 = moderate/severe twisting or scarring of petioles, leaves, and/or meristem swelling, and 3 = complete death of apical meristem yHarvest damage ratings were conducted using a six-point scale of increasing scarring of pedicels within the broccoli crown and accompanying deformity of head due to larval feeding, where 0 = no damage, 1 = mild scarring and deformity, 2 = moderate scarring and deformity, 3 = moderate/severe scarring and deformity, 4 = severe scarring and deformity, and 5 = complete death of apical meristem (no main broccoli crown) xNS and * refer to non significance ( P < 0.05) and statistical significance at P < 0.05, respectively, of overall Kruskal-Wallis tests wMeans indicated by different letters are statistically different ( P < 0.05) according to Mann-Whitney U post hoc pairwise comparisons vSingle-component damage rating means not followed by SEM due to one replicate in year 2
117 4.9 Figures
400 2016 Control *** a 3-Component 300 Chiral
)
M 3-Component
E 200 Racemic
S
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k 100
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e b
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T
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s
e
l ***
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M 300
f
o
. o 200 a
N
100 b c 0 0 2 4 6 8 10 12 Weeks After Transplanting
FIG. 4.1 MEAN NUMBERS OF MALES CAUGHT IN MONITORING TRAPS IN THREE-COMPONENT MATING DISRUPTION PLOTS EACH WEEK. *** INDICATES STATISTICAL SIGNIFICANCE OF OVERALL MODEL WITH PHEROMONE AS A PREDICTOR OF TRAP COUNTS (2016: 2 2 χχχ2 = 33.079, P < 0.001, 2017: χχχ2 = 103.384, P < 0.001). TREATMENT LINES MARKED WITH DIFFERENT LETTERS ARE SIGNIFICANTLY DIFFERENT BASED ON POST HOC PAIRWISE COMPARISON TESTS (P < 0.05)
118 400 2017 Control NS (2 S,10 S)- 300 2,10-
) 200
M
E
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a
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400 r 2018
e
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s
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. 200
o
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FIG. 4.2 MEAN NUMBERS OF MALES CAUGHT IN MONITORING TRAPS IN SINGLE-COMPONENT MATING DISRUPTION EXPERIMENT PLOTS EACH WEEK. LACK OF ERROR BARS FOR 2018 DUE TO THE PRESENCE OF ONLY ONE REPLICATE IN THIS YEAR. NS INDICATES NONSIGNIFICANT DIFFERENCES IN TRAP COUNTS 2 ACROSS PHEROMONE TREATMENTS IN 2017 ( χχχ2 = 2.565, P > 0.05)
119 80 80 A. NS B. * b 60 60
40 40 a
20 20 a
Marketable Broccoli (%±SEM) Broccoli Marketable 0 0 l l l ic ol c tro tr n hira m mi C ce on Chira ce Co a C R Ra Pheromone Treatment
FIG. 4.3 BROCCOLI MARKETABLE YIELD IN THREE-COMPONENT EXPERIMENT MATING DISRUPTION PLOTS IN (A.) 2016 AND (B.) 2017. NS INDICATES NONSIGNIFICANT YIELD DIFFERENCES IN 2016 ( H2 = 4.908, P > 0.05) AND * INDICATES STATISTICALLY SIGNIFICANT YIELD DIFFERENCES IN 2017 ( H2 = 7.200, P = 0.027). TREATMENTS INDICATED BY DIFFERENT LETTERS ARE STATISTICALLY DIFFERENT ( P < 0.05)
120 60 60 A. NS B.
40 40
20 20
Marketable Broccoli (%±SEM) Broccoli Marketable 0 0 l - - ) )- - S 10 S 0 0 ,1 ontro 10 2, 1 2 C , , S Control S (2 (2 Pheromone Treatment FIG. 4.4 BROCCOLI MARKETABLE YIELD IN SINGLE-COMPONENT EXPERIMENT MATING DISRUPTION PLOTS IN (A.) 2017 AND (B.) 2018. NS INDICATES NONSIGNIFICANCE ( H2 = 0.828, P > 0.05). LACK OF ERROR BARS FOR 2018 DUE TO THE PRESENCE OF ONLY ONE REPLICATE IN THIS YEAR
121 CHAPTER 5: DIEL PATTERNS OF EMERGENCE AND REPRODUCTIVE BEHAVIOUR IN THE INVASIVE SWEDE MIDGE (DIPTERA: CECIDOMYIIDAE)
Elisabeth A. Hodgdon *1 , Rebecca H. Hallett 2, Chase A. Stratton 1, and Yolanda H. Chen *1
1Department of Plant & Soil Science, University of Vermont, 63 Carrigan Drive, Burlington, Vermont, United States of America 05405
2School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada N1G 2W1
*Corresponding author contact information: Elisabeth A. Hodgdon Email: [email protected]
Yolanda H. Chen Email: [email protected] Phone: +1 (802) 656-2627
122
5.1 Abstract
Swede midge ( Contarinia nasturtii Kieffer) is a serious invasive pest of Brassica oilseed and vegetable crops in Canada and the United States of America. Pheromone mating disruption is a promising new tactic for managing this difficult pest, but research is needed to determine how pheromone delivery can be optimized. With an understanding of swede midge diel mating patterns, pest managers could limit pheromone release to periods when midges are sexually active. We conducted a series of 24 hour trials to test whether swede midge exhibit diel periodicity of emergence, female calling, and male capture in pheromone traps. We found that females began releasing pheromones almost immediately following emergence within the first five hours after dawn. In the field, we found that males were most active from dawn until late morning, indicating that midges mate primarily during the first five hours of photophase. Low levels of reproductive activity during midday and night time hours present opportunities to turn off dispensers and reduce the cost of pheromone inputs in a mating disruption system.
123
5.2.1 Résumé
La cécidomyie du chou-fleur ( Contarinia nasturtii Kieffer) est un ravageur envahissant des oléagineux et des légumes du genre Brassica au Canada et aux États-Unis d’Amérique. La confusion sexuelle par phéromones est une nouvelle tactique prometteuse pour lutter contre ce ravageur difficile à gérer, mais plus de recherche est encore nécessaire pour optimiser la méthode de diffusion des phéromones. Avec une meilleure connaissance des schémas d'accouplement diurnes de la cécidomyie du chou- fleur, les personnes responsables de la lutte contre ce ravageur pourraient restreindre la distribution de phéromones aux périodes où la cécidomyie du chou-fleur est sexuellement active. Nous avons donc fait une série d’expériences de 24 heures pour déterminer si la cécidomyie du chou-fleur démontre des schémas diurnes d’émergence, de l’appel des femelles, et de la capture des mâles dans les pièges de phéromones. Nous avons constaté que les femelles commencent à émettre des phéromones presque immédiatement après leur émergence, durant les premières heures suivant l’aube. Dans les champs, nous avons constaté que les mâles sont le plus actif de l’aube jusqu’à la fin du matin, en indiquant ainsi que la cécidomyie du chou-fleur s’accouple pendent les cinq premières heures de la photophase. Les faibles niveaux d'activité sexuelle durant le milieu de la journée et pendant la nuit offrent des occasions d'éteindre les diffuseurs programmables et pour
économiser les intrants de phéromones dans un système de la confusion sexuelle de la cécidomyie du chou-fleur.
124 5.2 Introduction
Many important agricultural insect pests across diverse orders show predictable diel periodicity of sexual communication (Bergh et al. 1990; Rodriguez et al. 1992;
Knight et al. 1994; Groot 2014; Mori and Evenden 2015). Circadian clocks govern the schedules of pheromone release by females, male response to pheromone, and copulation in insects (Saunders 1997; Groot 2014; Gadenne et al. 2016). Pest managers can take advantage of species-specific reproductive diel patterns when using pheromone mating disruption, which involves the release of high doses of female pheromones, preventing males from finding receptive females by masking their pheromone plumes, attracting males away from females, causing male habituation, and other mechanisms (Welter et al.
2008). By understanding when pest species are receptive to mating, pest managers can limit the release of pheromones to the times of day when insects are most actively mating. Modern pheromone mating disruption dispenser types commonly used for lepidopteran pests, such as aerosol dispensers, can be programmed to release pheromones exclusively at particular times of the day. For example, identifying the nocturnal mating schedules of moth pests has led to synchronized night time pheromone release, which has been successful in disrupting mating, providing crop protection, and/or limiting unnecessary pheromone inputs when the insects are not active (Rama et al. 2002;
Stelinski et al. 2007; Higbee and Burks 2008; Casado et al. 2014; Mori and Evenden
2015).
Swede midge ( Contarinia nasturtii Kieffer) is a challenging invasive pest of
Brassica ( Brassicaceae ) crops in North America, including B. oleracea (broccoli, cabbage, cauliflower, and kale) and B. napus (canola). A member of the family 125 Cecidomyiidae, which contains several challenging agricultural pests, swede midge is uniquely difficult to manage because of its ability to distort plant growth. Since its introduction to North America from Europe in the 1990’s, swede midge has been responsible for serious economic losses of canola and broccoli crops in Ontario and
Québec, Canada and the northeastern United States of America (Hallett and Heal 2001;
Chen et al. 2011). Larvae cause plant damage by feeding within the meristem of their host plants and distorting growth, causing leaves, heads, and other plant parts to become unmarketable. Because they are enclosed within the newly forming meristematic leaves of their host plants, feeding larvae are protected from contact with foliar insecticides. As a result, foliar insecticides are largely ineffective for this pest (Chen et al. 2011).
Alternative management practices are needed to prevent adult midges from ovipositing on host plants. Only a single swede midge larva per plant can render a cauliflower head unmarketable (Stratton et al. 2018). Because of this extremely low damage threshold, pheromone mating disruption is a promising tactic for this pest because it prevents mating and subsequently provides crop protection from feeding larvae. Although pheromone mating disruption has been tremendously successful for lepidopteran pests (Welter et al. 2008; Witzgall et al. 2010; Miller and Gut 2015), it has not yet been developed for dipteran pests. The use of cecidomyiid and other dipteran pheromones in pest management is problematic, as they are structurally complex and particularly costly to synthesize (Hillbur et al. 2005; Hall et al., 2012; Samietz et al.
2012). However, for difficult to manage pests such as swede midge, pheromone mating disruption may present an alternative to calendar sprays of conventional insecticides, which is currently the most widely used tactic for this pest (Chen et al 2011). Although 126 Samietz et al. (2012) demonstrated that pheromone mating disruption was successful in providing crop protection from swede midge in Europe, they reported that their system was likely not economically feasible at the commercial level due to prohibitive costs of swede midge pheromone synthesis. Timed pheromone-releasing devices could allow for mating disruption to be economically viable by reducing pheromone inputs. However, far fewer studies have described the chemical ecology and diel patterns of cecidomyiid behaviours compared with lepidopterans due to their small size, difficulty to rear in laboratory conditions, and sometimes immeasurably small amounts of pheromone that they produce compared with other insect pests (Hall et al. 2012).
A better understanding of the diel reproductive behaviour of swede midge and other cecidomyiids can aid in the development of more economical pheromone mating disruption systems for these pests. Many economically-important cecidomyiids display sexually dimorphic emergence and pheromone-releasing (“calling”) behaviours that are governed by circadian clocks (Modini et al. 1987; Bergh et al. 1990; Pivnick and Labbé
1992; Heath et al. 2005). Because most midges are very short lived, synchronization of reproductive behaviour between males and females must be precise. Female midges emerge from the soil, mate shortly after eclosion, locate host plants, oviposit, and die shortly thereafter (Gagné 1989; Hall et al. 2012). Male swede midge and other male cecidomyiid sibling cohorts typically emerge on average one to two days before females
(Gagné 1989; Bergh et al. 1990; Hillbur et al. 2005). While females begin releasing pheromones and are receptive to males almost immediately after eclosion, male cecidomyiids require additional time to be able to fly in search of mates, presumably to allow for sufficient cuticle sclerotization ( ibid ). Newly emerged cecidomyiid females are 127 highly attractive to males for mating (Gagné 1989; Bergh et al. 1990). While some species call during an extended period from morning until evening (apple leaf-curling midge, Dasineura mali Kieffer), others call during a shorter window of time, primarily during the end of scotophase through early morning hours (sorghum midge, Contarinia sorghicola Coquillett, and Hessian fly, Mayetiola destructor Say) or during the evening only (orange wheat blossom midge, Sitodiplosis mosellana Géhin) (Modini et al. 1987;
Bergh et al. 1990; Pivnick and Labbé 1992; Heath et al. 2005). While Hillbur et al.
(2005) and Boddum et al. (2009) observed calling and mating for swede midge shortly after the onset of photophase, they did not formally test their observations.
Here, we studied the diel periodicity and sex-based differences of emergence and reproductive behaviour of swede midge in laboratory and field settings to determine when adults are sexually active. Specifically, we tested the following research questions:
1) Do diel emergence patterns differ by sex? 2) Do newly-emerged females display diel patterns of calling? and; 3) Do males show diel patterning in their response to pheromones?
5.3 Materials and Methods
5.3.1 Swede Midge Colony Rearing
We used two laboratory colonies for the study, one at the University of Vermont in Burlington, Vermont, United States of America, and the second at the University of
Guelph in Guelph, Ontario, Canada. The “Swiss colony,” maintained at the University of
Vermont since 2014, originated predominantly from the Swiss Federal Research Station for Horticulture in Wädenswil, Switzerland, with an input of 122 field-collected midges
128 from northwestern Vermont to increase genetic diversity of the colony in 2015. The
“Ontarian colony” consisted solely of midges collected from fields Ontario in 2016 and
2017. We reared the Swiss colony at 22.4 ± 1.2ºC temperature and 40.7 ± 11.4% relative humidity (RH) with a 16:8 (L:D) photoperiod. With the following exceptions, we reared midges in the Ontarian colony under similar conditions: the RH in the rearing room was on average 50-70%, and the photoperiod was shifted one hour later. Because our experiments with each colony were temporally and spatially separate due to their rearing locations, we did not include population as a variable in the statistical analyses of experiment results described in later sections and analysed our data for both populations separately.
We used cauliflower, B. oleracea group Botrytis (Brassicaceae) ‘Snow Crown’
(Harris Seeds, Rochester, New York, United States of America), for rearing because of its large bud size and high susceptibility to swede midge (Hallett 2007). We seeded cauliflower in Fafard 3B soilless potting medium (Sun Gro Horticulture, Agawam,
Massachusetts, United States of America), and transplanted the seedlings into 10 or 15 cm pots at approximately four weeks after seeding. Plants received synthetic fertilizer application thrice weekly following transplanting at a total rate of 150 ppm consisting of two parts 21-5-20 and one part 15-0-14 with supplemental magnesium. We introduced plants to the swede midge ovipositional rearing cages after approximately 8-12 weeks of age, when buds began to form but were less than 1 cm in diameter. The plants were exposed to adult midges for 24-72 hours in the main oviposition cages. We then transferred the exposed plants to separate cages for larval development for 10-14 days.
When ready to pupate, midge larvae vacate the meristem by crawling or jumping onto the 129 soil, pupating within the top few centimeters of the soil surface (Readshaw 1961). To facilitate larval movement into the potting medium, we cut the top 5 cm of the cauliflower apical meristems and inserted them into the potting medium. We placed the pots with pupae into the ovipositional rearing cages, where they would then emerge as adults.
5.3.2 Sex-Based Differences in Diel Emergence
We tested whether male and female adults differ in diel patterns of emergence by conducting 24 hour observational trials. To set up the emergence trials, we grew single 8-
12 week old cauliflower plants and exposed them to adult midges in the ovipositional cages 20-22 days before the trials. We counted the numbers of midges emerging from pupae-infested potting media in plant pots from the Swiss and Ontarian colonies in separate experiments in June-September 2015 and June-August 2017, respectively. Each experiment consisted of three 24 hour observational periods. To capture midges emerging from the soil during each hour, we removed the potting media containing cauliflower roots and swede midge pupae from the plant pots and placed them into one-quart (946 ml) deli containers fitted with insect netting on the lid. At the end of each hour for 24 hours, we counted the number of male and female midges that had emerged in each container. To observe midge emergence during the scotophase without disturbing midge behaviour with bright artificial lighting, we used 25W red incandescent light bulbs because flies are less sensitive to light in this wavelength range (Fingerman and Brown
1952).
130 To test if the numbers of emerging male and female midges differed at each hour, we used generalized linear mixed models (GLMMs) because they can account for repeated measures and multiple predictors and are robust to non-normally distributed data
(Bolker et al. 2009). The number of midges emerging each hour followed Poisson distributions. Using separate GLMMs with repeated measures frameworks for each colony, we tested whether 1) the total number of midges; and 2) the numbers of males and females emerging were equal at each hour of the day. Within each model, individual deli containers or pots containing potting media from individual host plants were considered the subjects or units of replication ( n = 8 pots from the Swiss colony and n =
34 pots from the Ontarian colony) and hour within day was specified in the repeated measures field. We specified hour, day of the experiment, sex, and the interaction term hour*sex as predictors. In this model and GLMMs for subsequent datasets, we used
Satterthwaite’s Approximation to generate degrees of freedom due to small sample sizes and/or the unbalanced structures within our data. We used SPSS statistical software version 24 (International Business Machines Corporation, Armonk, New York, United
States of America) and confidence intervals of 95% for these and all subsequent statistical analyses. Where descriptive statistics are displayed, standard error follows the mean.
5.3.3 Diel Patterns of Female Calling
We tested whether midges display diel patterns of pheromone-releasing behaviour by observing female calling over three 24 hour experiments for each colony separately in laboratory settings in June-September 2015 (Swiss) and June-August 2017 (Ontarian).
We aspirated unmated female midges as they emerged from potting media during 131 morning peak periods of female emergence for each colony (Fig. 5.1), and began our experiment approximately one hour following the peak emergence period to ensure that the midges were the same age. We placed females singly in 20 ml glass scintillation vials covered with insect netting containing 1 cm2 pieces of white filter paper moistened with distilled water from which the midges could drink.
We categorized calling behaviour using a binary scoring system for whether or not midges exhibited the characteristic cecidomyiid pheromone-releasing posture described by Harris et al. (1999): terminal segments of the abdomen protracted to expose the pheromone gland and ovipositor extended. We observed midges for calling twice per hour, on the hour and half hour, using a 20x hand lens. A score of 1 was given when midges were observed calling either once or twice per hour, and a score of 0 was given when no calling was observed. We excluded midges that died or did not call (non-callers) during each 24 hour period from further analysis. Non-callers may have mated prior to capture, or called while unobserved. Our final sample size consisted of n = 107 (Swiss) and n = 91 (Ontarian) calling midges.
For each colony, we used separate GLMM models to analyze the relationship between hour of day and the probability of female calling to test the null hypothesis that the probability of midge calling was equal throughout the 24 hour period. For each model, we used hour and day of experiment as predictors, individual midges coded as subjects, and hour within day specified as the unit of repeated measures. Calling behaviour followed a binomial distribution.
132 5.3.4 Diel Periodicity of Male Response to Pheromone Traps
Because female calling patterns represent the reproductive behaviour of only half of the population, we also tested for diel patterns of male mate-seeking. We tested whether the number of males caught in pheromone traps each hour, our proxy for response to pheromone and mate-searching, followed diel patterns at a commercial vegetable farm in New Hamburg, Ontario, Canada, at which we previously measured very high levels of swede midge infestation (> 30 males per pheromone trap per day;
Hallett et al 2009). Using cardboard Jackson (delta) traps with commercial pheromone lures (Solida Distributions, Saint-Ferréol-les-Neiges, Québec, Canada) we counted numbers of males trapped on sticky cards once each hour for 24 hours. For each 24 hour observation period, we used four traps set up in each of four different vegetable fields with emerging swede midge populations. Fields contained either living or mowed crop residues from Brassica vegetable crops, including broccoli, cauliflower, kale, collard greens, and daikon radishes ( Raphinus sativus var. longipinnatus ). We placed traps at least 100 m apart within the fields, which is twice the recommended spacing for avoiding trap interference (Allen 2009). We placed new lures in each trap at the start of each set of trials, which was repeated three times, on the 17-18 and 27-28 July, and 17-18 August
2016, for a total of 16 replicates per 24-hour observation period (48 total). Due to the lack of males caught in 11 traps over the 24-hour periods, our final dataset consisted of counts from n = 37 traps. During the second and third 24 hour periods, we measured temperature, RH, and wind speed in each field at every hour. Using individual traps as experimental units, we used a GLMM with repeated measures and Poisson distribution to
133 examine whether trap counts differed significantly by hour of day and by day of the experiment.
5.4 Results
5.4.1 Sex-Based Differences in Diel Emergence
We found that both male and female adults from both populations emerged according to a diel pattern (Swiss: F 47,1547 = 4.488, p < 0.001 and Ontarian: F 49,334 =
4.712, p < 0.001). Over the three 24 hour periods, we observed a total of 383 Swiss and
287 Ontarian midges emerging from the colony potting media. Females from both colonies emerged predominantly in the morning, with a smaller peak in emergence in the late afternoon in the Swiss colony (Fig. 5.1). Emergence of males was split primarily between the morning and late afternoon (Fig. 5.1).
In the Swiss colony, hour was a significant predictor of emergence ( p < 0.001) and the interaction term hour*sex was significant (p = 0.013), indicating that males and females emerged at different times of the day. The majority of Swiss females (63% of the total) emerged within the first three hours after dawn (0500-0800; Fig. 5.1A). During the same time period only 36% of Swiss males emerged. More males emerged in the late afternoon and evening compared with females, with 36% of total emerging males eclosing between 1500-2100, whereas only 10% of total females emerged during this time period.
Similarly to the Swiss colony, Ontarian midges exhibited diel sex-based differences in emergence, as hour and the interaction term hour*sex were both statistically significant predictors of emergence in the final model ( p < 0.001). The
134 female emergence peak occurred within the first six hours of the photophase (0600-
1200), when 75% of females emerged (Fig. 5.1B). Females from the Ontarian colony, unlike those from the Swiss colony, did not emerge in the afternoon. The majority of males (63%) emerged between four peaks throughout the photophase, with the highest peak in the afternoon at 1400.
5.4.2 Diel Patterns of Female Calling
The majority of both Swiss and Ontarian females (>50%) called in the morning
(Fig. 5.2). In both colonies, the calling patterns were very similar to the emergence patterns, indicating that females are most receptive to males shortly after they emerge
(Fig. 5.1). We observed many females with protruding ovipositors as they were emerging from the soil. Hour of day was a significant predictor of female calling ( p < 0.001) for both the Swiss and Ontarian colonies within the overall models (F 25, 2541 = 22.626, p <
0.001 and F 25,2152 = 13.714, p < 0.001, respectively). Consistent with the characteristic calling behaviours of other cecidomyiids (Gagne 1989), the females were stationary while calling in the vials, extruding and sometimes waving their ovipositors.
Occasionally, we observed small droplets of pheromone at the end of the ovipositors, and some females wiped their ovipositors onto the sides of the vials leaving a visible trail of pheromone. A small portion of midges (2 Swiss and 13 Ontarian individuals) did not call during the experimental period and were excluded from analyses.
The Swiss colony showed a bimodal pattern of calling at dawn and dusk (Fig.
5.2A). Over 80% of all Swiss midges called at 0500, the peak of calling. On average,
70% of all midges called during the first two hours of the photophase (0500-0700). A
135 smaller peak in calling activity occurred in the late afternoon, with 60% of midges calling between 1500-2100. Groups of midges calling during the morning and afternoon were not exclusive; the same midges called during both periods. The number of calling midges dropped by 49% after the onset of the scotophase at 2100. Fewer than 20% of midges called between 1000-1400 and 2200-0000.
In the Ontarian colony, the peak calling time occurred four hours after the onset of photophase (1030), when 66% of midges called (Fig. 5.2B). During the first five hours of the photophase (0600-1100), an average of 54% of midges called at each time point, and most midges stopped calling at 1200. Similarly to the Swiss colony, calling in the
Ontarian population was bimodal with a small peak in activity in the evening. During the evening peak in calling, 31% of females called between 2000-0000.
5.4.3 Diel Periodicity of Male Response to Pheromone Traps
We found evidence that males show diel periodicity in their attraction to the female sex pheromone. The number of males caught in the pheromone traps varied significantly throughout the day (F 19,939 = 3.656, p < 0.001). We captured 73% of males in pheromone traps within the first five hours after dawn (0600-1100; Fig. 5.3).
Despite variation in weather, phases of the moon, sunrise, and sunset, the day of the experiment was not a significant predictor within the model. The weather during our three 24 hour experimental periods was variable, with periods of sun and clouds throughout each day and two brief periods of rain in the evening and night during two of the three 24 hour periods. Temperature averaged 27.8 ± 4.4ºC with 56.7 ± 14.4% RH during the photophase, and 22.0 ± 2.2ºC with 76.8 ± 8.3% RH during the scotophase.
136 Wind was highly variable, with the strongest gusts observed from 1000-1300, ranging from 0 to 69 m/s. We observed virtually no wind (<1 m/s) from 1900-1000. Sunrise and sunset occurred at approximately 0600 and 2100, respectively.
5.5 Discussion
Swede midge show clear diel and sex-based patterns of adult emergence and mating behaviour. Despite testing our research questions across different populations in varying geographical locations, the patterns we observed were remarkably consistent.
Our study is the first comprehensive test for diel periodicity of swede midge behaviour, offering information that may be used in future management programs for this challenging pest.
We observed our largest peak in emergence of females within the first five hours after dawn. However, Readshaw (1961) observed peaks in emergence of females later in the day, from approximately 1000-1200 until 1800 in the field and from 1400-2200 in laboratory settings with varying temperature regimes. In both settings, he observed more males emerging in the morning prior to females. Although these findings appear to contradict our results, in both field and laboratory experiments Readshaw did not observe emergence from 2200 until 0800, and may have missed emergence of midges around dawn. Hillbur et al. (2005) observed emergence of swede midge primarily during the night and first hour of photophase, outside of Readshaw’s observation window. In our
Swiss colony, the secondary peak in emergence of males and females, from 1500-2000, more closely matches with Readshaw’s findings. However, in our Ontarian colony, we observed many males but few females emerging during this time period. These
137 differences may be due to the origin of the colonies, where the Swiss colony primarily represents a European population, while the Ontarian colony was initiated from North
American insects.
We found that the times of female emergence and calling were synchronized, and that females began calling almost immediately after eclosing in the morning, suggesting that swede midge mate at their emergence sites. One of the major challenges surrounding implementing a pheromone mating disruption system within annual systems is complex crop rotation practices. Unlike in perennial cropping systems, pheromone mating disruption dispensers may be more effective in annual cropping systems for midges at the sites of midge emergence, rather than within the current crop. Many annual crops are rotated between fields from year to year, which could separate emerging midges from where their host plants are grown in the current year. In other midge species that infest annual crops, such as brassica pod midge ( Dasineura brassicae Winnertz) and the orange wheat blossom midge, overwintering individuals emerge in fields where their host crops were grown in the previous year (Williams et al. 1987; Smith et al. 2007). Males emerging first linger at the emergence site and mate with females after they eclose and begin calling. The mated females then migrate to other fields to lay their eggs on their host plants if hosts are not already present at the emergence site ( ibid ). In the future, experiments to directly test where swede midge mate will aid in determining where to install pheromone mating disruption dispensers.
We found that female calling behaviour and male responsiveness to pheromone followed a similar timeline. Our data suggest that midges mate primarily within the first
138 five hours after dawn. Although the majority of adults were synchronized in their reproductive behaviour, we found a noticeable difference in the reproductive activity between males and females during the late afternoon and early evening. While a large number of females called during the late afternoon hours in the Swiss colony, Ontarian females in the laboratory and males in the field were less active in the field during these times. Calling periods from our Ontarian colony more closely match our results from the males in the field, as these populations are of similar origin. However, the calling periods of the Ontarian females are slightly longer than the periods of time when we captured males. Perhaps males are more likely to search for females under differing seasonal environmental conditions not represented during our warm summer experiments.
Otherwise, releasing pheromones while males are not responsive may incur a seemingly unnecessary fitness cost to females.
Overall, we observed more variation in periodicity of activity in the Ontarian midges versus the Swiss midges. The Swiss population had been exposed to the selective pressures of colony rearing for many more years than the Ontarian colony, which was initiated more recently from field populations. Laboratory rearing conditions can impose bottlenecking and selection pressures unlike those in the wild within very few generations
(Hoffman and Ross 2018). However, bottlenecking during the introduction of swede midge to North America may have occurred as well, limiting the genetic diversity of the wild population in Ontario. Studies on the population genetics of swede midge would be necessary to determine differences between European and North American populations.
Despite differing origins and exposure to potentially different bottlenecking pressures,
139 our results indicate that the Swiss colony has retained diel behavioural patterns generally similar to Ontarian midges.
In addition to differences in colony age, our two swede midge colonies were established from wild individuals from different geographical locations. Our Swiss colony originated from Europe, closer to the origin of the species, while our Ontarian colony represents the invasive North American population. Geography is known to play a role in influencing differences in diel patterns across insect populations of the same species found in different locations. Geographical differences in calling and mate-seeking patterns can be due to the presence of multiple strains of the insect and/or due to climactic differences in their environments. Differences in local photoperiods, temperature, humidity, and wind introduce variation in timing of behaviours (Delisle and
McNeil 1986; Blackwell 1997; Rund et al. 2012; Pellegrino et al. 2013; Groot 2014). Had we tested midges from both populations during the same experiments and included population origin in our models, we would have more confidence making comparisons between the two colonies. Because we found similar patterns across the colonies, the use of both populations strengthen our conclusion that swede midge primarily mate in the morning.
Our data provide a timeline of daily reproductive events that may be useful for designing more efficient pheromone mating disruption systems and potentially other management practices. The diel reproductive behaviour of swede midge presents an opportunity to turn off mating disruption dispensers at the times of the day when the insects are not active, primarily between a five hour period midday, and during a three
140 hour night time period. During these time periods, less than 20% of males were captured and less than 20% of females called. Turning off dispensers during these eight hours would reduce the cost of pheromone inputs by 33%. With these cost savings, pheromone mating disruption could be a more viable commercial pest management strategy for swede midge in the future.
A predicted increase in the threat of invasive agricultural pests (Paini et al. 2016), paired with a growing market for organic produce in Canada and the United States of
America (Agriculture and Agri-Food Canada 2017, United States Department of
Agriculture Economic Research Service 2017), creates an imminent need for continuing research and development of more economical ecologically-based pest management strategies. While pheromone mating disruption is not a new pest management technique, its use for dipteran pests is novel and may require additional design considerations compared with those used for lepidopteran pests. Species-specific knowledge of life cycles, ecology, and biology for introduced species in their new environments will be necessary for exploiting their behaviour for future pest management strategies.
141 5.6 Acknowledgements
We acknowledge funding from a United States Department of Agriculture
National Institute of Food and Agriculture Crop Protection and Pest Management grant
(2014 - 70006 - 22525), the Vermont Agency of Agriculture Specialty Crop Block Grant
Program (02200 – SCBGP - 9 - 1), and the Ontario Ministry of Agriculture, Food, and
Rural Affairs – University of Guelph Partnership. We thank the following people for technical support at the University of Guelph and University of Vermont: Jamie Bauman,
Jarrett Blair, Raina Brot-Goldberg, Charles-Étienne Ferland, Paolo Filho, James Heal,
Kathryn Jacobs, David Laurie, Kate Lindsay, Charlotte MacKay, Ted Marois, Cameron
Ogilvie, Ross Pillischer, Elizabeth Porter, Rebecca Roman, Justine Samuel, Leah Slater,
Jonathan Williams, and Samuel Zuckerman. We also thank Pfenning’s Organic
Vegetables, Inc. for allowing us to conduct our field experiments at their farm, the
University of Vermont campus greenhouses for swede midge colony plant production,
Alan Howard from the University of Vermont Statistical Consulting Services for assistance with statistical analysis, and Scott Merrill, Kimberly Wallin, David Conner, and Marion Harris for advice on experimental designs and data interpretation. Lastly, we thank Charles-Étienne Ferland and Vincent Martineau for their assistance with French translation of our abstract.
142
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146 5.8 Figures
35 Male A. 30 Female 25 20 15 10
SEM) 5 ± 0 0 5 10 15 20 Hours after onset of photophase 0500 1000 1500 2000 0100 Hour of day
35 B. 30 25 20 15 Mean percent emergence ( emergence percent Mean 10 5 0 0 5 10 15 20 Hours after onset of photophase 06001100 1600 2100 0200 Hour of day
Fig. 5.1. Mean percent emergence of total male and female adult swede midge from soil from Swiss (A.) and Ontarian (B.) populations over three 24 hour periods. The values of “0” on the upper x-axes indicate artificial dawn (lights on). Both GLMM models of calling using hour, sex, and hour*sex as predictors were statistically significant at p < 0.001.
147
100 A. 80
60
40
20
0 0 5 10 15 20 Hours after onset of photophase 0500 1000 1500 2000 0100 Hour of day
100
Percent of total females calling females total of Percent B. 80
60
40 20 *** 0 0 5 10 15 20 Hours after onset of photophase 0600 1100 1600 2100 0200 Hour of day
Fig. 5.2. Percentage of females calling each half-hour in (A.) Swiss and (B.) Ontarian colonies. The GLMMs for both populations using hours as predictors were statistically significant (p < 0.001).
148 SEM)
± 40
30
20
10
0 0 5 10 15 20 Hours after onset of photophase
Mean percent trap capture ( capture trap percent Mean 0600 1100 1600 2100 0200 Hour of day
Fig. 5.3. Mean percent of total males caught in traps at each hour per day in Ontario field trial. The GLMM with hour of day as a predictor of trap captures was significant at p < 0.001.
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160 APPENDIX A ntarinia IS OFIS THE SWEDE ND COMPOUNDSND AL. 2005). COMPOUNDS IN 10.1002/ps.1762 BORATORY SYNTHES on the pheromone of blend the of swede midge, 10886-005-5928-3 ication the pheromone of sex the of swede midge, Co E PRODUCED DURING LA
, for monitoring. , for Pest Sci Manag 65:851–856. doi: TABLE A.1 SWEDE NATURAL MIDGE PHEROMONE COMPOUNDS A THE RACEMIC BLEND AR PRODUCED IN THE SYNTHETIC RACEMIC BLEND (HILLBURET Hillbur M, Y, Celander Baur R, Identifet al (2005) nasturtii. J Chem Ecol 31:1807–1828. doi: 10.1007/s Contarinia nasturtii References: References: Boddum T, Skals M,N, Wirén et al (2009) Optimisati
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