3 Improved Attraction of Anthonomus Eugenii to Traps Using Semiochemicals

Improved detection and monitoring of pepper weevil (Anthonomus eugenii) in Ontario peppers

Cassandra J. Russell

A Thesis presented to The University of Guelph

In partial fulfilment of requirements for the degree of Master of Science in Environmental Sciences

Guelph, Ontario, Canada

ABSTRACT

IMPROVED DETECTION AND MONITORING OF PEPPER WEEVIL (ANTHONOMUS

EUGENII) IN ONTARIO PEPPERS

Cassandra J. Russell Advisor: University of Guelph, 2021 Dr. Rebecca H. Hallett

The pepper weevil (Anthonomus eugenii Cano) is a serious invasive pest of peppers in Ontario. The goal of this research was to improve detection and monitoring tools for A. eugenii to minimize losses in cultivated peppers. In lab trials, fewer A. eugenii escaped from sticky cards compared to other trap types, and sticky cards coated with new adhesives formulated for A. eugenii retained the most adults. Trécé pheromone lures attracted the highest number of A. eugenii in field trials compared to other lures. The addition of kairomones did not synergize A. eugenii response to pheromone lures in lab or greenhouse trials, possibly due to experimental conditions that may have affected adult response and behaviour. Therefore, the use of kairomones in A. eugenii monitoring requires further investigation. Until then, the currently available pheromone lures and sticky cards should continue to be used for monitoring for A. eugenii in Ontario pepper crops.

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ACKNOWLEDGEMENTS

First and foremost, I would like to thank my advisor, Dr. Rebecca Hallett. Your encouragement and positive outlook through unsuccessful experiments, colony crashes, and countless other roadblocks was instrumental. Thank you for not only supporting my development as a researcher, but also encouraging my career development and building my self-confidence throughout this entire journey.

A very special thank you to Dr. Angela Gradish for the many hours spent correcting my countless spelling and grammar mistakes and helping me through all the writing stages. Thank you to my committee members, Dr. Roselyne Labbé and Dr. Cynthia Scott-Dupree for all your feedback and guidance, and thanks to Dr. Michelle Edwards for answering all my repetitive stats and SAS coding questions.

To my wonderful lab mates and friends, Charles-Étienne Ferland, Carol McLennan, Graham Ansell, Jenny Liu, Marlee-Ann Lyle, Matthew Muzzatti, and the other biocontrol and SES colleagues I met along the way – thank you all for getting me through this and I can’t wait to see all your faces again (without masks). A very special thanks out to my relentless Room 2112 motivational team: Jenny Liu and Sahba Shahmohamadloo. I think I owe you each a steak dinner from our weekly goal setting activities.

I am incredibly thankful to all the cooperating greenhouse and field pepper growers and funding support from the Ontario Greenhouse Vegetable Growers, the Entomological Society of Canada travel scholarship, and the OMAFRA-UofG HQP Program and Production Systems (Plants) Program.

To my OMAFRA pepper weevil team; Amanda Tracey, Cara McCreary, Denise Beaton, and Hannah Fraser - thank you for sharing all your pepper weevil expertise, helping me get connected to the right growers, and being amazing role models.

To the pepper weevil crew at the AAFC-Harrow, thank you for making me feel welcome and helping me with my experiments. To all the summer students over the years in the Hallett lab including my PW colony superstars Kylie, Averyl, Emily and Serena: a million thanks for sticking it out with moldy peppers 3 times a week and keeping the colony going.

Thank you to my past roommates for being so supportive and allowing bugs in freezer, and to Lily for helping me to turn a vision into the greatest pepper weevil diagram that ever was.

To my parents, Karen and Steve, thank you for all your support to follow my passions and pursue graduate studies. To my entire family and the Di Vincenzo’s, thank you for understanding that weevils don’t observe holidays and for working family get- togethers around my crazy schedule so that I could keep my weevils alive. To my grandmother, Bev, I would have never applied for graduate school without your support and encouragement. You have always been my inspiration to grow and learn and I thank you for every ounce of wisdom and love you’ve provided.

Lastly, to my partner, Nick. Your unconditional love and support over the last 3+ years of my master’s has been instrumental to my success. From helping me stay fueled with caffeine for those long nights and early mornings, to offering a hug and a shoulder to cry on, to reminding me that a failed experiment wasn’t the end of the world – thank you and I love you. You’ve been a part of this journey since day one and I wouldn’t have it any other way.

TABLE OF CONTENTS

Abstract ...... ii

Acknowledgements ...... iii

Table of Contents ...... v

List of Tables ...... viii

List of Figures ...... xi

1 Potential for improved detection and monitoring of Anthonomus eugenii in Ontario 1

1.1 Introduction ...... 1

1.2 Anthonomus eugenii pest status ...... 2

1.2.1 Geographic distribution ...... 2

1.2.2 Pest status in Canada...... 3

1.2.3 Fruit damage and economic impact ...... 4

1.3 Anthonomus eugenii biology ...... 6

1.3.1 Life cycle ...... 6

1.3.2 Host plants ...... 7

1.4 Anthonomus eugenii monitoring and management ...... 9

1.4.1 Use of semiochemicals in pest monitoring and management ...... 9

1.4.2 Current Anthonomus eugenii monitoring strategies ...... 13

1.4.3 Improved monitoring based on knowledge of host plant interactions ...... 17

1.4.4 Potential management strategies ...... 18

1.5 Research objectives ...... 21

2 Improved trapping of Anthonomus eugenii in field and greenhouse peppers ...... 23

2.1 Introduction ...... 23

2.2 Materials and Methods ...... 28

2.2.1 Study insect ...... 28

2.2.2 Assessment of Anthonomus eugenii movement on and escape from commercial sticky cards under laboratory conditions ...... 29

2.2.3 Evaluation of alternative trap designs for the capture of Anthonomus eugenii adults under laboratory conditions ...... 32

2.2.4 Evaluation of novel adhesive formulations on yellow sticky cards for Anthonomus eugenii retention under laboratory conditions ...... 34

2.3 Results ...... 36

2.3.1 Assessment of movement of Anthonomus eugenii on commercial sticky cards under laboratory conditions ...... 36

2.3.2 Assessment of escapes of Anthonomus eugenii from commercial sticky cards under laboratory conditions ...... 38

2.3.3 Evaluation of alternative trap designs for capture of Anthonomus eugenii adults under laboratory conditions ...... 41

2.3.4 Laboratory evaluation of novel adhesive formulations for Anthonomus eugenii retention...... 44

2.4 Discussion ...... 45

3 Improved attraction of Anthonomus eugenii to traps using semiochemicals ...... 52

3.1 Introduction ...... 52

3.2 Materials and Methods ...... 57

3.2.1 Field evaluation of commercial lures for Anthonomus eugenii ...... 57

3.2.2 Addition of kairomones to pheromone lures for increased attraction of Anthonomus eugenii ...... 59

3.2.3 Evaluation of kairomone and pheromone lures for Anthonomus eugenii in an experimental greenhouse ...... 62

3.3 Results ...... 66

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3.3.1 Field evaluation of commercial lures for Anthonomus eugenii ...... 66

3.3.2 Addition of kairomones to pheromone lures for increased attraction of Anthonomus eugenii ...... 66

3.3.3 Evaluation of kairomone and pheromone lures for Anthonomus eugenii in an experimental greenhouse ...... 70

3.4 Discussion ...... 72

4 General conclusions and discussion ...... 79

5 References ...... 88

6 Appendix ...... 98

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LIST OF TABLES

Table 2.3: Odds ratios of Anthonomus eugenii adult escapes on 5 types of sticky cards. Male and female adults were manually placed on sticky cards lateral or ventral side down, and any weevil that had left the surface during the 48 h experimental period was counted as escaped. The experiment was conducted at 23 and 32 °C with young (< 7 d old) and mature (> 28 d old) adults. Confidence limits that contain the number 1.0 are not statistically significant. Significant ratios are indicated with an asterisk...... 42

Table 2.4: Results of an ANOVA comparing the number of adult Anthonomus eugenii captured for each of the 6 types of insect traps. Male and female adults, 8–12 d old, were introduced into a mesh cage containing each trap type, and the number of individuals that were captured on or in each trap was counted after 48 h. The experiment was conducted at 23 °C. Significant model effects are bolded (P ≤ 0.05). . 43

Table 2.5: Results of an ANOVA comparing the mean number of Anthonomus eugenii adult escapes from 5 different sticky card trap types. Male and female adults were dropped on the sticky cards ventral side down from 1 cm above the surface of each card in a grid layout. Any weevil that had left the surface of the sticky card over the 48 h experimental period was counted as escaped. The experiment was conducted at 23 °C with adult weevils (8–12 d old). Significant model effects are bolded (P ≤ 0.05)...... 44

Table 3.1: Stimuli used for the Y-tube olfactometer experiments and their components. Concentrations of kairomone blend components were based on ratios collected from headspace volatile collections (Addesso et al. 2011) and used in previous studies (Muñiz-Merino et al. 2014)...... 60

Table 3.2: List of treatments used for the infested greenhouse experiment to determine the response of Anthonomus eugenii to different semiochemical lures in the presence of

fruiting pepper plants. All lures were supplied by Trécé Inc. Some treatment details, such as concentration and release rate, have been omitted upon request of the proprietor...... 65

Table 3.3: Results of an ANOVA on the total number of captured Anthonomus eugenii adults on sticky cards baited with different lure treatments over a 2 week period in an infested jalapeno pepper field in Dresden, Ontario. Significant model effects are bolded (P ≤ 0.05)...... 67

Table 3.4: Results of chi-square analyses on percent responding Anthonomus eugenii from each cohort and each treatment within the cohorts in a Y-tube bioassay. Adults of the appropriate age and mating status were selected randomly from the colony and were given 15 min to respond to 1 of 2 stimuli in each bioassay. Adults were run individually through the Y-tube until 30 respondents were completed for each treatment. Significant differences between treatments are bolded (P ≤ 0.05)...... 70

Table 3.5: Results of an ANOVA comparing the total number of adult Anthonomus eugenii captured on sticky cards baited with 1 of 6 lure treatments in a greenhouse with fruiting pepper plants (Capsicum annuum var. Fascinato). Significant model effects are bolded (P ≤ 0.05)...... 71

Table 6.1: Pairwise comparisons (Tukey’s HSD) of mean minimum distance traveled by adult Anthonomus eugenii on 5 types of sticky cards. Male and female adults were manually placed on the sticky cards on their side or ventral side down, and the distance each weevil travelled across the card was recorded after 48 h. The experiment was repeated at 23 and 32 °C with young (< 7 d old) and old (> 28 d old) adults and results from all 4 experiments were analyzed together. Significant pairwise comparisons are bolded (P ≤ 0.05)...... 98

Table 6.2: Pairwise comparisons (Tukey’s HSD) of proportion of Anthonomus eugenii escapes ± SE from 5 types of sticky cards. Male and female adults were manually placed on the sticky cards on their side or ventral side down, and the distance each weevil travelled across the card was assessed for 48 h. The experiment was repeated at 23 and 32 °C with young (< 7 d old) and mature (> 28 d old) adults and results from all four experiments were analyzed together. Significant pairwise comparisons are bolded (P ≤ 0.05)...... 99

Table 6.3: Pairwise comparisons (Tukey’s HSD) of the mean number of Anthonomus eugenii adults captured by 6 types of insect traps. Male and female adults, 8–12 d old, were introduced into a mesh cage containing each trap type at 23 °C, and the number of individuals that were captured inside or on each trap was counted after 48 h. Significant pairwise comparisons are bolded (P ≤ 0.05)...... 100

Table 6.4: Pairwise comparisons (Tukey’s HSD) of mean number of Anthonomus eugenii adult escapes from 5 different sticky card traps types. Male and female adults

were dropped on the sticky cards, ventral side down from 1 cm above the surface of each card in a grid layout. Any weevil that had left the surface of the sticky card over the 48 h experimental period was counted as escaped. The experiment was conducted at 23 °C with adult weevils (8-12 d old). Significant pairwise comparisons are bolded (P ≤ 0.05)...... 101

Table 6.5: Pairwise comparisons (Tukey’s HSD) of the total number of adult Anthonomus eugenii captured on sticky cards baited with 1 of 6 lure treatments in a greenhouse with fruiting pepper plants (Capsicum annuum var. Fascinato). Significant pairwise comparisons are bolded (P ≤ 0.05)...... 102

LIST OF FIGURES

Figure 1.1: Life cycle of Anthonomus eugenii, including the duration and location (inside vs. outside the fruit) of each life stage. Life stages that occur inside of the fruit can be found in any part of the fruit interior, such as the seed core or fruiting wall. Larvae are typically localized in the seed cluster but are depicted here in the pericarp for clarity. .... 7

Figure 1.2: Pherocon® Pepper Weevil (PEW) Kits (Trécé Inc. Adair, OK, USA) packaging (A), sticky cards with plastic cover (B), and 2 pairs of lures: PEW I (C) and PEW II (D)...... 15

Figure 2.1: Anthonomus eugenii trap deployed at the edge of a pepper field. The trap has substantial amounts of insect by-catch collected over the course of a week...... 26

Figure 2.2: Candidate traps evaluated for their ability to capture A. eugenii. (A) boll weevil trap, (B) Unitrap, (C) homemade jar trap, (D) modified pyramid trap, (E) Tanglefoot-coated yellow card, (F) Pherocon sticky card...... 27

Figure 2.3: Grid layout for arrangement of A. eugenii on novel adhesive cards (A) before and (B) after all 18 adults were dropped on the surface of the card...... 35

Figure 2.4: Mean minimum distance traveled (±SE) by adult Anthonomus eugenii on 5 types of sticky cards. Male and female adults were manually placed on the sticky cards on their side or ventral side down, and the distance each weevil travelled across the card was recorded after 48 h. The experiment was conducted at 23 and 32 °C with young (< 7 d old) and old (> 28 d old) adults and results from all 4 experiments were analyzed and presented together. Bars with the same letter are not significantly different (Tukey’s HSD, P ≤ 0.05)...... 38

Figure 2.5: Proportion of Anthonomus eugenii escapes (± SE) from 5 types of sticky cards. Male and female adults were manually placed on the sticky cards on their side or ventral side down, and the distance each weevil travelled across the card was assessed for 48 h. The experiment was repeated at 23 and 32 °C with young (< 7 d old) and mature (> 28 d old) adults and results from all 4 experiments were analyzed together. Bars with the same letter are not significantly different (Tukey’s HSD, P ≤ 0.05)...... 41

Figure 2.6: Mean number (±SE) of Anthonomus eugenii adults captured by 6 types of insect traps. Male and female adults (8–12 d old) were placed in a mesh cage containing each trap baited with a Trécé pheromone lure. The number of adults captured inside or on each trap after 48 h was counted. The experiment was conducted at 23 °C. Bars with the same letter are not significantly different (Tukey’s HSD; P ≤ 0.05)...... 43

Figure 2.7: Mean number (±SE) of Anthonomus eugenii adult escapes from 5 types of adhesive formulations on yellow cards. Male and female adults were dropped on the

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sticky cards ventral side down from 1 cm above the surface of each card in a grid pattern. Any weevil that had left the surface of the sticky card over the 48 h experimental period was counted as escaped. The experiment was conducted at 23 °C with adult weevils (8–12 d old). Bars with the same letter are not significantly different (Tukey’s HSD, P ≤ 0.05)...... 45

Figure 3.1: Experimental set-up for the evaluation of commercial lures in a jalapeno pepper field infested with Anthonomus eugenii in Dresden, Ontario. Sticky traps were baited with 1 of 3 treatments (C: control [n = 10], T: Trécé lure [n = 10], R: Russell IPM lure [n = 10]), secured to pigtail stakes, and placed in the pepper field on September 20, 2018. Traps were checked twice weekly for 2 weeks. Captured adults found at each check were counted and removed...... 58

Figure 3.2: Experimental design for a greenhouse experiment to evaluate the attractiveness of different semiochemical lures to Anthonomus eugenii in the presence of fruiting pepper plants. Plants were spaced equally apart on metal plant benches, and the 5 lure treatments were attached to yellow sticky cards that were hung between pepper plants. Control sticky cards were unbaited. Forty (20 male, 20 female) A. eugenii were released 24 h prior to the start of the experiment at each release point...... 64

Figure 3.3: Mean ± SE number of adult Anthonomus eugenii captured over 14 d on sticky cards baited with a Russell IPM lure (n = 10) or a Trécé lure (n = 10). Control traps were unbaited (n = 10). Sticky traps and lures were attached to metal ground stakes and placed in a jalapeno pepper field in Dresden, Ontario on September 20, 2018. Bars with the same letter are not significantly different (Tukey’s HSD, P ≤ 0.05).67

Figure 3.4: Percent of adult Anthonomus eugenii that responded to 1 of 2 stimuli over 4 treatments (air vs. kairomone, air vs. pheromone, kairomone vs. pheromone and kairomone + pheromone vs. pheromone) during two-way choice bioassays in a Y-tube olfactometer for (A) young mated females, (B) young males, (C) young unmated females, and (D) mature unmated females (n = 30 for each cohort). Significant treatments (P < 0.05) are indicated with an asterisk...... 69

Figure 3.5: Mean (± SE) total number of captures of Anthonomus eugenii on sticky cards baited with 1 of 6 lure treatments in an infested greenhouse with fruiting pepper plants (Capsicum annuum var. Fascinato). Bars with the same letter are not significantly different (Tukey’s HSD, P ≤ 0.05)...... 71

1 Potential for improved detection and monitoring of Anthonomus eugenii in Ontario

1.1 Introduction

In 2017, Canada imported approximately CAD $3.7 billion in vegetables (Statistics

Canada 2018). The United States and Mexico were the first and second largest sources of imports, with vegetable imports valued at approximately $2.2 billion and $1 billion, respectively (Statistics Canada 2018). Large amounts of fresh peppers are included in these imports, and come from places where Anthonomus eugenii Cano (Coleoptera:

Curculionidae), commonly known as pepper weevil, is a widespread insect pest.

Anthonomus eugenii has been an established pest of peppers in Mexico, Central

America, and the United States since the early 1900s. Since the arrival of A. eugenii in

Ontario in 2009, monitoring efforts have been unable to detect infestations before economic damage occurs (Canadian Food Inspection Agency 2011, Ingerson-Mahar et al. 2015). The biology and chemical ecology of A. eugenii make it a difficult pest to detect using commercially available pheromone traps. By identifying optimal trapping devices and assessing A. eugenii responses to aggregation pheromones and plant volatiles, an improved monitoring strategy could be developed for A. eugenii.

Development of a reliable trapping device and an attractive, pheromone-based lure could also offer solutions for A. eugenii management through mass trapping techniques.

1.2 Anthonomus eugenii pest status

1.2.1 Geographic distribution

Anthonomus eugenii is a primary pest of peppers in the southern United States,

Mexico, Central America, and the Caribbean (Elmore et al. 1934, Ingerson-Mahar et al.

2015). It was first detected in Hawaii in 1933 and Puerto Rico in 1982, but has caused only sporadic infestations in those locations since its initial detection (Elmore et al.

1934, Abreu and Cruz 1985). Anthonomus eugenii was detected in the Netherlands in

2012 (NPPO-NL 2012) and in Italy in 2013 (Speranza et al. 2014), and it was immediately added to the European and Mediterranean Plant Protection Organization quarantine list (NPPO-NL 2012). Because timely detection and quarantine measures were implemented, A. eugenii is now considered absent in the EU (van der Gaag and

Loomans 2013).

Anthonomus eugenii hosts on plants in the Solanaceae family, in particular cultivars in the genus Capsicum, including C. annuum, C. frutescens, and C. chinense

(Elmore et al. 1934, Burke and Woodruff 1980, Andrews et al. 1986, Patrock and

Schuster 1992). Populations of A. eugenii persist only in areas where its host plants are available throughout the year, which largely limits its distribution to the southern United

States and Central America (Capinera 2010). However, because pepper transplants are shipped north each spring, A. eugenii is sometimes detected in more northern locations;

New Jersey has seen sporadic outbreaks since 1957 (Ingerson-Mahar et al. 2015), and

British Columbia documented its first infestation of A. eugenii in 1992 (Costello and

Gillespie 1993). In 2009, A. eugenii was first detected in pepper greenhouses in Essex

and Chatham-Kent Counties of southwestern Ontario (Canadian Food Inspection

Agency 2011), and numerous A. eugenii infestation events since then have caused significant yield losses to Ontario greenhouse and field pepper producers.

1.2.2 Pest status in Canada

Canada is involved in two-way trade of peppers with the United States and receives imports of peppers from Mexico during winter months or when local production does not meet demand. Both trade routes likely contribute to the northward spread of A. eugenii (Fernández et al. 2020). In 2010, the Canadian Food Inspection Agency (CFIA) investigated the pest status of A. eugenii in Canada, and although it possesses many characteristics of, and met the minimum criteria to be considered a quarantinable pest, it was not regulated (CFIA 2011). This decision partially was based on the low likelihood of A. eugenii surviving Canadian winters. It was determined that spread of A. eugenii within Canada would be caused by the transport of pepper plants and harvested fruit, and thus the pest could be managed by the pepper industry itself (CFIA 2011).

Furthermore, the CFIA (2011) determined that various control measures were available to eradicate A. eugenii from greenhouses in Ontario. Practices such as screening greenhouse vents and having a long clear-out period during the winter where no peppers would be available to the pest were proposed to mitigate A. eugenii dispersal and establishment. In addition, greenhouses that did not process imported peppers in close proximity to their greenhouse pepper production areas were considered to be unlikely to introduce this pest to their crop (CFIA 2011, Ingerson-Mahar et al. 2015, van de Vossenberg et al. 2019).

A study conducted in New Jersey confirmed that infested shipments of peppers from Mexico and the southern United States were the main source of A. eugenii introductions to more northern locations in North America (Ingerson-Mahar et al. 2015, van de Vossenberg et al. 2019), and A. eugenii-infested imports is the most accepted hypothesis for its occurrence in greenhouse and field pepper crops in southern Ontario

(Labbé et al. 2018, Fernández et al. 2020). When A. eugenii emerge from imported fruit during shipping or packaging, its dispersal into neighbouring greenhouses and fields is highly probable due to a lack of physical barriers, including exclusion screening, on greenhouse vents. Additionally, adults are able to fly or crawl out of greenhouses and orient toward field peppers or other wild solanaceous hosts in warmer months when outdoor host plants are growing (Ingerson-Mahar et al. 2015). Therefore, effective detection and monitoring tools are required to provide warning to growers immediately when A. eugenii is present to prevent dispersal from greenhouses to fields.

1.2.3 Fruit damage and economic impact

The 2019 farm gate value of greenhouse pepper crops in Canada was estimated at $441 million and was the highest value exported fresh vegetable crop at $420 million

(Statistics Canada 2020). Throughout Canada there was a total of 560 ha of greenhouse pepper production, 70% of which occurred in Ontario (Statistics Canada

2020). In 2019, Ontario produced approximately 40 million kg of field peppers (Statistics

Canada 2021) and 99 million kg of greenhouse peppers (Statistics Canada 2020), most of which were grown in Essex and Chatham-Kent Counties in southwestern Ontario.

Peppers are a valuable trade commodity, and therefore any damage that affects crop

yield can have a detrimental economic impact to this sector of the Canadian agricultural industry. Anthonomus eugenii can cause severe pepper crop damage and economic loss, with yield losses reaching up to 100% if it is left unmanaged (Campbell 1924;

Elmore et al. 1934; Goff and Wilson 1937; Genung and Ozaki 1972; Riley and Sparks

1995). In 2016, A. eugenii-related crop losses in Ontario field and greenhouse peppers exceeded $67 million (Niki Bennett, Ontario Greenhouse Vegetable Growers, personal communication).

Anthonomus eugenii primarily damages blossoms, buds, and immature fruit of pepper plants, which results in substantial yield reductions (Genung and Ozaki 1972).

Larvae feed on seeds and tissue inside the fruit, contaminating it with frass and leading to tissue decay (Elmore et al. 1934, Burke and Woodruff 1980, Capinera 2010, Seal and

Martin 2016). Fruit drop caused by internal larval feeding is the most obvious sign of infestation, but yellowing of the peduncle and premature colour change of fruit may also occur (Seal and Martin 2016). At high pest densities, adult feeding on the fruit exterior causes scarring and predisposes fruit to plant pathogens, rendering the fruit unmarketable (Burke and Woodruff 1980). In the absence of pepper blossoms and fruit, adults feed on the leaves and stems of peppers but cause no significant damage

(Campbell 1924).

1.3 Anthonomus eugenii biology

1.3.1 Life cycle

The life cycle of A. eugenii described below is from Elmore et al. (1934) unless otherwise stated (Figure 1.1). Anthonomus eugenii is multivoltine and completes its life cycle in 20–30 days. There may be 5–8 overlapping generations of A. eugenii per year, depending on environmental conditions. Adult females preferentially oviposit in young fruit but will also lay their eggs in flowers, buds, stems, and mature fruit (Campbell

1924). To oviposit, females pierce a hole in the skin of the fruit or stem with their mouthparts. They then lay a single egg in the hole and seal it with an anal secretion that hardens into a plug to protect the egg. A female can lay up to 600 eggs in her lifetime

(but averages closer to 341) at a rate of 5–7 eggs per day (Capinera 2010). Females prefer to lay one egg per fruit as larval competition can occur inside the fruit, and oviposition plugs are found to deter oviposition by other females (Addesso et al. 2007).

Development from egg to first instar larva takes 3–4 days. Larval instars are whitish-grey with a yellowish-brown head capsule, and 1–3.3 mm long. Larval development takes place through 3 instars over the course of 8–10 days. Larval feeding creates tunnels, which can become surrounded with brownish frass. Pupation, which lasts 3–6 days, takes place in a brittle cell formed from this material (Campbell 1924).

Newly eclosed adults spend 2–3 d sclerotizing and darkening within the fruit, until they chew a round exit hole to leave the fruit. Adults are typically dark black or dark maroon and measure between 2 and 3.5 mm long. White, grey or yellowish hairs cover the elytra and thorax. Toapanta et al. (2005) identified the optimal temperature for A.

eugenii population increase in jalapeno fruit to be 30 °C, with a lower developmental threshold of 9.6 °C. There is no evidence that A. eugenii diapauses (Elmore et al. 1934); however, they are suspected to overwinter in Ontario in structures and spaces that remain warm throughout the winter, such as greenhouses.

1.3.2 Host plants

Anthonomus eugenii is primarily a pest of all species of cultivated peppers

(Capsicum species), but it can also feed and reproduce on a variety of other solanaceous plants, such as wild American black nightshade (Solanum americanum

Mill.) and silverleaf nightshade (Solanum elaeagnifolium Cav.) (Tejada and Reyes 1986,

Patrock and Schuster 1992). These wild nightshades often serve as alternate host plants for A. eugenii when peppers are absent (Ingerson-Mahar et al. 2015) and commonly grow along the perimeters of greenhouses and fields where cultivated peppers are present. However, nightshade species not in the genus Capsicum are not as attractive to A. eugenii, likely because their fruit are relatively small and a single fruit may not be sizeable enough to support a single weevil (Fernández et al. 2017). Other solanaceous field crops are also susceptible to attack: tomatillo (Physalis philadelphica

Lam.) is a moderately susceptible host, and eggplant (Solanum melongena L.) grown in proximity to peppers will sometimes be injured (Patrock and Schuster 1992). Although tomatoes also belong to Solanaceae, A. eugenii was not found to oviposit on tomatoes in the field (Addesso and McAuslane 2009). However, Addesso et al. (2009) observed oviposition on tomato in the lab while testing additional substrates for A. eugenii rearing.

Because of their close association with Capsicum plant species, A. eugenii likely evolved alongside solanaceous plants in the American tropics (Patrock and Schuster

1992, Addesso and McAuslane 2009, Ingerson-Mahar et al. 2015).

To locate food or host plants, insects use host-specific visual, olfactory, mechanical, and/or gustatory cues to verify the suitability of a plant for feeding or oviposition (Gilbert 1979). Seal and Martin (2016) conducted a study to determine A. eugenii preference for different colours and sizes of pepper fruit. In preliminary field tests with 4 pepper cultivars, they found significantly more larvae in bell pepper fruit compared to smaller peppers, such as Hungarian wax and habanero. Bell peppers had

a larger cross-sectional fruit area, which A. eugenii likely preferred to ensure sufficient food material to guarantee larval survival (Seal and Martin 2016). Adult weevils also preferred medium-sized bell pepper fruit (> 1.5 cm long) over smaller fruit for feeding and oviposition (Seal and Martin 2016). Anthonomus eugenii displayed no preference between green and yellow fruit. Addesso et al. (2011) observed that pepper plants undergoing or that had undergone feeding damage were preferred by A. eugenii over undamaged fruit. Addesso and McAuslane (2009) determined that A. eugenii adults use volatiles released from host plants to orient to and evaluate host plant suitability.

1.4 Anthonomus eugenii monitoring and management

1.4.1 Use of semiochemicals in pest monitoring and management

Many insects use olfactory or chemical cues to locate food, host plants, or potential mates; or to mediate other ecological interactions. Chemicals that mediate interactions between organisms are referred to as semiochemicals. Semiochemicals are categorized as either allelochemicals or pheromones. Allelochemicals are semiochemicals that mediate interspecific communication, whereas pheromones mediate intraspecific communication (Tewari et al. 2014).

Pheromones are chemicals commonly used by insects as intraspecific cues for feeding, aggregation, mating, and resource marking. Pheromones used for mating are emitted to elicit a sexual response in a member of the opposite sex (Landolt and Phillips

1997, Reddy and Guerrero 2004). Pheromones that attract both sexes, commonly referred to as aggregation pheromones, can also be considered sex pheromones in

cases where they are used to attract potential mates (Landolt and Phillips 1997). Male- released aggregation pheromones attract both females and other males to gain access to resources such as host plants and mates. Males may employ and augment aggregation pheromones released by other males to locate and court females, either by using the same signaling site or by intercepting females attracted to signalling males

(Landolt and Phillips 1997).

Eller et al. (1994) identified the male-produced aggregation pheromone of

Anthonomus eugenii and its 6 major components: (Z)-2-(3,3- dimethylcyclohexylidene)ethanol, (E)-2-(3,3-dimethylcyclohexylidene)ethanol, (Z)-(3,3- dimethylcyclohexylidene)acetaldehyde, (E)-(3,3-dimethylcyclohexylidene)acetaldehyde,

(E)-3,7-dimethyl-2,6-octadienoic acid (geranic acid), and (E)-3,7-dimethyl-2,6-octadien-

1-ol (geraniol). Traps baited with a lure containing a synthetic pheromone blend consisting of these compounds captured adult A. eugenii in field tests, 50–67% of which were females, demonstrating that the pheromone was an aggregation pheromone (i.e., it attracts both sexes of A. eugenii) (Eller et al. 1994).

Eller and Palmquist (2014) assessed factors affecting pheromone production in

A. eugenii adult males. Peak pheromone production of 800 ng/h was observed around noon, and low production rates of 12 ng/h occurred during scotophase. Pheromone production increased with age up to 15 d and then tapered off. As male density increased, pheromone production decreased, but the proportion of geranic acid in the pheromone blend increased with male density. Interestingly, extremely low or

undetectable amounts of pheromone were produced when male A. eugenii were fed an artificial diet. Conversely, when males were fed a diet of fresh peppers, pheromone levels were high, indicating a strong relationship between the chemical ecology of A. eugenii and host plant dependency.

Host plants play a significant role in the chemical ecology of insects, not only by influencing production of pheromones as stated above, but also by affecting the behaviour of herbivores and natural enemies (Tewari et al. 2014). Plants emit a variety of volatile organic compounds (VOCs) in response to environmental changes (Baldwin

2010), and these VOCs have important roles in regulating plant-plant and plant-insect communication (Dicke and Baldwin 2010, Das et al. 2013). Volatile organic compounds released by host plants that are used in host finding by herbivorous insects are considered kairomones, a type of allelochemical that benefits the organism receiving the chemical signal, while putting the emitter at a disadvantage (Tewari et al. 2014).

An undamaged plant maintains a baseline level of volatiles that are released from the plant and/or from accumulated storage sites in the leaf (Pare and Tumlinson

1999). These stored VOCs, also called constitutive VOCs, can be volatized into the air by healthy and unhealthy plants. The primary functions of constitutive VOCs are aroma and pollinator attraction (Degenhardt and Lincoln 2006, Das et al. 2013). Tissue breakage from wounding, cutting, or herbivore feeding can cause higher concentrations of constitutive VOCs to be released into the atmosphere, which may serve as warning signals to nearby plants (Heil and Ton 2008). When plants are damaged by insect

feeding, the profile of the volatiles emitted is often distinctive from those of undamaged or mechanically damaged plants (Dicke and van Loon 2000). Studies have established that the compounds released specifically in response to insect damage are synthesized de novo and are not stored in the plant (Pare and Tumlinson 1997, Dicke and van Loon

2000), and they are thus referred to as induced VOCs (Dicke and van Loon 2000,

Kessler and Baldwin 2001, Dicke and Baldwin 2010).

The importance of plant-produced volatiles for host location by pest insects, including many Anthonomus species, has been widely documented (Szendrei et al.

2009). For example, the cranberry weevil (Anthonomus musculus Say) elicited strong antennal responses to 4 volatiles from blueberry buds and flowers (Szendrei et al.

2009). Experiments with apple blossom weevil (Anthonomus pomorum L.) have identified a blend of 6 volatile components from apple plants that are essential to maintain an attractive blend to adult weevils (Collatz and Dorn 2013). Addesso and

McAuslane (2009) determined that A. eugenii was attracted to constitutive volatiles, and specifically preferred damaged fruiting and flowering plants, and fruiting plants with actively feeding weevils (Addesso et al. 2011). These results suggest that A. eugenii are more strongly attracted to upregulated constitutive volatiles and induced plant volatiles

(Addesso et al. 2011). Muñiz-Merino et al. (2014) later showed that a blend of (E)-β- ocimene and 2-isobutyl-3-methoxypyrazine was attractive to both male and female A. eugenii in laboratory assays.

For many coleopteran species that aggregate on host plants, there are relationships between host plants and sex pheromone release (Landolt and Phillips

1997). For instance, palm weevil (Rhynchophorus palmarum L.) males are stimulated to release aggregation pheromone following exposure to ethyl acetate produced by their palm host plants (Jaffee et al. 1993). Aggregation pheromone production by male A. eugenii is also host dependent, and a lack of access to natural host plants for feeding results in males producing little to no aggregation pheromone (Eller and Palmquist

2014).

Kairomone-pheromone interactions have been evaluated and used in the monitoring and management of various Anthonomus pest species. In the case of the strawberry blossom weevil, Anthonomus rubi Herbst, the addition of floral volatiles to pheromone monitoring traps increased weevil response to the lures (Wibe et al. 2014).

The addition of kairomones from the host plant of cotton boll weevil, Anthonomus grandis Boheman, to aggregation pheromone lures resulted in an enhanced weevil response to monitoring traps (Dickens 1989), and demonstrated a synergized response to monitoring traps compared to pheromones alone (Magalhães et al. 2012).

1.4.2 Current Anthonomus eugenii monitoring strategies

Effective control of A. eugenii is hindered by problems associated with detecting adults before economic injury occurs (Genung and Ozaki 1972). Prior to the identification and commercial development of the male-produced aggregation pheromone, A. eugenii population estimates were best obtained by visual examination

and the use of un-baited yellow sticky traps (Segarra-Carmona and Pantoja 1988).

Although damage-based action thresholds have been described (Cartwright et al.

1990), pheromone trap captures (Eller et al. 1994) and visual counts of adults on terminal buds are the most widely used monitoring methods for adult A. eugenii

(Andrews et al. 1986).

In Ontario, commercially available Pherocon® Pepper Weevil (PEW) Kits (Trécé

Inc. Adair, OK, USA) (Figure 1.2) are recommended for monitoring for A. eugenii in greenhouses and fields (OMAFRA 2016). Each kit contains double-sided yellow sticky cards (Figure 1.2 B) and a two-part synthetic pheromone lure (Eller et al. 1994) (Figure

1.2 C and D). As per package instructions, lures should be replaced every 2–4 weeks and sticky cards every 2 weeks or as needed (Trécé Inc. Adair, OK, USA). The lures are placed on the top of the card and the card is then commonly secured to a ground stake or hung from a fixture. Traps should be placed a few rows into field pepper crops at 0.2 ha intervals (i.e., 2 traps per acre) to intercept adult A. eugenii. Traps also should be placed in or around cull piles, patches of solanaceous weeds, and the exterior of greenhouses; and at transplanting or packaging facilities to detect incoming adults

(CFIA 2011). In field peppers, traps should be placed 10–60 cm above the soil, or as close to the fruit canopy level as possible (Riley and Schuster 1994). Trap placement recommendations for greenhouse peppers are not published; however, many greenhouse growers hang traps from overhead wires in the top canopy of the pepper crop (personal observation). The lure used in the Pherocon traps is a two-part lure.

Figure 1.2: Pherocon® Pepper Weevil (PEW) Kits (Trécé Inc. Adair, OK, USA) packaging (A), sticky cards with plastic cover (B), and 2 pairs of lures: PEW I (C) and PEW II (D).

Lure I consists of a blend of 4 proprietary compounds known as: grandlure II, grandlure

III, grandlure IV, and geraniol; and lure II consists of geranic acid alone (Eller et al.

1994).

Eller et al. (1994) determined that geranic acid was required for the greatest attraction of adult weevils. The lure substrate/carrier is the commercially available

Hercon® laminate (Hercon Environmental, Emigsville, PA, USA) for a consistent release of geranic acid and protection from oxidation, light and hydrolysis (Kydonieus and

Beroza 1981, Eller et al. 1994). However, Eller et al. (1994) found that the Hercon laminate substrate released much less geranic acid than a single male weevil. In field

experiments, these pheromone traps were more attractive than unbaited traps, both early in the season before fruiting, and later in the season when fields were either tilled or in the absence of fruit (Eller et al. 1994). As the number of mature pepper plants increased, the effect of the pheromone lure decreased and fewer A. eugenii were detected on adjacent traps. This decline in trapping efficacy was attributed to the presence of a comparatively higher proportion of males (and therefore high amounts of naturally occurring male aggregation pheromone) and fruiting pepper plants (Eller et al.

1994).

Because of the low attraction of A. eugenii to pheromone monitoring traps when surrounded by fruiting pepper crops, pheromone trap-based action thresholds are set low at one A. eugenii/trap per field or greenhouse (Ingerson-Mahar et al. 2015). When

A. eugenii are caught on monitoring traps, the severity of an infestation cannot be determined; all that can be learned is that the pest is present. In many cases, growers have reported outbreaks of A. eugenii in field or greenhouses, yet no adults have been captured on traps (Ingerson-Mahar et al. 2015; Amanda Tracey, OMAFRA, personal communication). This observation calls into question not only the attractiveness of the pheromone lure, but also the efficacy of the current trap design. Sticky traps used outdoors for long durations are particularly vulnerable to deterioration as the surface often becomes saturated with debris and non-target insects, which limits their trapping potential (Sanders, 1986). It is also possible that A. eugenii may be able to escape from the currently used sticky cards, as has been reported anecdotally by growers and crop scouts (Amanda Tracey, OMAFRA, personal communication), so the effectiveness of

trap adhesives should also be investigated. In addition to efficacy, another factor that limits the use of A. eugenii monitoring traps is cost. Ontario greenhouses can reach up to 40 hectares in size. Costs for deployment of traps at the recommended interval (i.e.,

0.2 traps per ha) in these large facilities would exceed $80,000 over an 8-month growing period, excluding any labour costs associated with trap maintenance, which is a high cost for the monitoring of a single insect pest (Cara McCreary, OMAFRA, personal communication).

1.4.3 Improved monitoring based on knowledge of host plant interactions

Developing an improved A. eugenii lure remains a realistic possibility by exploiting knowledge gained from laboratory assays on A. eugenii host plant volatile preferences (Addesso et al. 2011), and on their responses to a combination of kairomones and aggregation pheromone (Muñiz-Merino et al. 2014). Because of similarities in biology and chemical ecology between A. eugenii and A. grandis, the use of kairomones in combination with pheromones shows promise for increasing the response of A. eugenii to pheromone monitoring traps. An improved lure would facilitate early detection of A. eugenii infestations and emergence, which would help with the timing of pest management strategies and the establishment of improved action thresholds. Creating a more attractive and effective A. eugenii trap should also allow crop scouts and growers to gain a better understanding of the dynamics of A. eugenii infestations and the relationship between trap captures and adult densities.

1.4.4 Potential management strategies

Mass trapping management strategies have been used to control a wide range of insect pests, most often lepidopteran, coleopteran, and dipteran species (El-Sayed et al.

2006). The primary distinction between mass trapping and monitoring approaches is that traps intended for mass trapping are deployed in greater densities than monitoring traps to help with population suppression or eradication (Steiner 1952). In some regions, mass trapping strategies have contributed to the eradication of invasive species such as the gypsy moth, Lymantria dispar L., and the cotton boll weevil,

Anthonomus grandis (El-Sayed et al. 2006). Mass trapping approaches use species- specific synthetic chemical lures, such as sex or aggregation pheromones, and host attractants, to attract insects to collection traps (El-Sayed et al. 2006, Gregg et al.

2018). Mass trapping strategies can use trapping mechanisms that either kill the target insect on collection by means of downing solutions, insecticidal vapour strips or from physical contact of insecticides or retain live adults in a collection area of the trap (El-

Sayed et al. 2006). These techniques have been used successfully for the control of other weevil species. For example, a combination of aggregation pheromones and pieces of host plants in traps successfully decreased the presence of Metamasius hemipterus L. and Rhynchophorus ferrugineus L. in palm plantations (Giblin-Davis et al.

1996, Hallett et al. 1999). It is important to note, however, that mass trapping management strategies often have the greatest probability of success when a pest is present at very low densities, which is often the case for invasive insect species like A. eugenii (Tewari et al. 2014).

Another management option that harnesses insect chemical ecology is a push- pull management approach. Similar to mass trapping, pheromones or other semiochemicals are used to attract or “pull” pests into predetermined areas or traps

(Tewari et al. 2014). Simultaneously, semiochemicals, such as host marking pheromones, are used to deter or “push” pests away from valuable resources to reduce the occurrence of infestations (Tewari et al. 2014). Push-pull management systems have successfully been used in apple orchards for the control of plum curculio

(Conotrachelus nenuphar Herbst) (Leskey et al. 2008) and with European cherry fruit fly

(Rhagoletis cerasi L.) (Aluja and Boller, 1992). With any of these potential management strategies, the density and efficiency of traps, as well as the strength and attraction of lures, need to be sufficient to maximize trap captures in an effort to reduce economic damage from the target pest (Tewari et al. 2014).

The A. eugenii aggregation pheromone has the potential to be used as part of a management strategy, just as the cotton boll weevil pheromone, grandlure, has proven to be effective for its management in cotton crops (Hardee et al. 1974). A combination of A. eugenii aggregation pheromone and pepper kairomones could increase weevil attraction to traps and be used in a mass trapping strategy targeting A. eugenii adults.

Research is needed to determine whether mass trapping of A. eugenii is best achieved through traps that kill collected adults or trap and collect live adult weevils.

Natural pheromone production by living weevils caught in traps could affect trap attractiveness. Since the proportion of geranic acid in the A. eugenii aggregation

pheromone increases proportionally to male density (Eller et al. 1994), a live collection trap design may increase trap attractiveness to both males and females who orient to the aggregation pheromone, but this has not yet been investigated. More research is needed to determine if a threshold of geranic acid begins to have the opposite effect and acts as an anti-aggregation pheromone that repels A. eugenii, serving as a signal of resource depletion. Anti-aggregation pheromones have been documented in other coleopteran species, specifically the mountain pine beetle (Dendroctonus ponderosae

Hopkins), where verbenone was found to deter beetles from lodgepole pines (Hunt et al.

1989, Lindgren et al. 1989). The release of verbenone from D. ponderosae signals tissue degradation and alters adult behaviour to minimize overcrowding within a host

(Lindgren and Borden 1993, Lindgren et al. 1996).

A highly attractive kairomone lure coupled with the existing aggregation pheromone lure could serve as the “pull” factor in a push-pull management strategy for

A. eugenii. Potential “push” factors have yet to be determined; however, a host fruit- marking pheromone in female oviposition plugs has been identified that deters other gravid females from ovipositing in that same fruit (Addesso et al. 2007). This pheromone could be useful in a push-pull strategy for A. eugenii, but the specific compounds that make up this pheromone have yet to be determined (Addesso et al.

2007).

Volatiles derived from non-host plants could also be used to mask host odors and render pepper fruit less attractive as oviposition substrates for A. eugenii (Addesso

et al. 2009). The use of non-host plant volatiles as repellent compounds for phytophagous insects has been studied in aphids (Myzus persicae Sulzer and

Acyrthosiphon pisum Harris) (Bruce et al. 2005), pollen beetles (Meligethes aeneus

Fabricius (Mauchline et al. 2005), and the multicoloured Asian lady beetle (Harmonia axyridis Pallas) (Riddick et al. 2000). However, the duration of their effectiveness is often limited to days or even hours as synthetic volatile formulations tend to volatilize quickly and require multiple applications (Isman 2006). It is anticipated that the combined application of male aggregation pheromones, female oviposition deterrent pheromones, and host plant volatiles (both constitutive and induced) will play a large role in development of an effective strategy aimed at the integrated management of A. eugenii.

1.5 Research objectives

The biology of A. eugenii makes it a difficult pest to monitor and control because most of its life stages occur within the pepper fruit (Elmore et al. 1934). An effective, reliable trapping system is needed to maximize detection of free-living A. eugenii adults.

As trap captures have been unreliable and adults may be escaping sticky cards, trap performance for A. eugenii retention should be evaluated. Adult preference for fruit and/or associated host plant volatiles, which are thought to play a role in the low capture rates observed on traps during fruit production (Eller et al. 1994, Addesso and

McAuslane 2009, Addesso et al. 2011), indicates that it should be possible to develop a more attractive trap. The potential of these kairomones to synergize response to the

aggregation pheromone in a field setting should also be investigated (Muñiz-Merino et al. 2014).

The goal of this research therefore was to improve monitoring and detection tools for A. eugenii in the presence of fruiting plants by:

1. Assessing alternative trapping mechanisms for their ability to trap and

retain A. eugenii, and

2. Determining whether the addition of plant volatiles and kairomones to

synthetic aggregation pheromone lures would increase A. eugenii

attraction to monitoring traps during pepper production.

By developing effective monitoring and management tools for A. eugenii, pepper growers could save millions of dollars’ worth of lost fruit yield.

2 Improved trapping of Anthonomus eugenii in field and greenhouse peppers

2.1 Introduction

Anthonomus eugenii has been an established pest of cultivated peppers

(Capsicum species) in Mexico and the southern United States since the early 1900s

(Elmore et al. 1934, Ingerson-Mahar et al. 2015). In 2009 it was identified in

Leamington, Ontario (Canadian Food Inspection Agency 2011) and was considered the number one threat to pepper crops from 2015 onward (Cara McCreary, OMAFRA, personal communication). Anthonomus eugenii likely arrived in Ontario through infested shipments of peppers from Mexico and the southern United States. Packaging and processing facilities are often attached to large commercial greenhouses where adults can survive winters while feeding on the immature plants (Fernández et al. 2020).

Females of A. eugenii lay eggs in blossoms, buds, and immature fruit of pepper plants (Campbell 1924, Genung and Ozaki 1972). Eggs hatch into larvae, which feed on the internal tissues of developing fruit, often resulting in premature fruit abortion and yield losses of up to 100% if left untreated (Campbell 1924, Elmore et al. 1934, Genung and Ozaki 1972, Ingerson-Mahar et al. 2015). When A. eugenii adults emerge from infested fruit in pepper greenhouses, they are likely to disperse into adjacent fields and other greenhouses because of a lack of physical barriers or exclusion screens on greenhouse vents (Ingerson-Mahar et al. 2015). Since all immature life stages of A. eugenii occur inside the fruit, where they are hidden and protected from insecticide treatments, early detection of adults is key for implementing an effective management

strategy. Therefore, effective detection and monitoring tools for A. eugenii are required to provide growers with timely information to rapidly detect A. eugenii and implement appropriate management strategies to minimize economic losses attributed to the presence of this crop pest.

Visual scouting for A. eugenii is difficult as adults are small and most damage to the fruit is internal. Pheromone-based monitoring traps are a viable addition to visual crop scouting for detection of A. eugenii adults in field and greenhouse pepper crops.

To monitor for A. eugenii, growers and scouts currently use Trécé Pherocon® Pepper

Weevil Kits (Trécé Inc. Adair, OK, USA), which contains a two-sided yellow sticky card and a two-part aggregation pheromone lure that attracts both sexes of A. eugenii (Eller et al. 1994). However, these monitoring traps have been unable to detect A. eugenii in cultivated peppers before economic damage occurs (Ingerson-Mahar et al. 2015), possibly because adults are able to escape from the traps. Over the past few years, many growers and scouts that have used Trécé monitoring kits have reported escapes of A. eugenii from sticky cards (Amanda Tracey, OMAFRA, personal communication).

Additionally, 2 A. eugenii trapping studies reported that weevils frequently escaped the lower rim of the trap within 2 h (Eller et al. 1994, Riley and Schuster 1994). Because of the overall low capture rates typically recorded for these pheromone-baited sticky cards, current action thresholds are correspondingly low, at one adult per trap per field or greenhouse.

Unreliable monitoring tools can directly affect A. eugenii detection and management. When such tools do not work effectively, significant crop damage, yield loss, and dispersal of invading adults towards new areas, including to packing lines, greenhouses or neighbouring field peppers, can occur. Improved monitoring tools are needed to ensure that a high proportion of A. eugenii adults that are invading a crop are being captured and retained for monitoring/counting purposes. Yellow sticky traps are easy to use and effective for capturing a variety of arthropod pests (Yaobin et al. 2012).

Enhancements to the adhesive formulation used on such traps, along with the pairing of traps with pheromone lures may result in improved retention of and a more reliable monitoring trap for A. eugenii. Since yellow sticky cards attract and trap many other insects, sticky cards used for A. eugenii monitoring will often be covered with non-target insects, known as bycatch (Figure 2.1). Bycatch on sticky cards reduces the adhesive surface area available to capture and retain target insect pests, which can reduce the efficacy of sticky cards as a monitoring tool. Therefore, alternative trapping methods that specifically target A. eugenii and reduce the amount of insect bycatch and debris in or on traps could improve the efficacy of A. eugenii monitoring.

In previous field experiments, traps that captured and retained live adults in a collection jar were most successful for trapping A. grandis (Leggett 1979). Since A. eugenii produces an aggregation pheromone similar in chemical composition to the aggregation pheromone produced by A. grandis (Eller et al. 1994), live trapping of A. eugenii could be a viable option and should be evaluated. Adult A. eugenii commonly fly short distances between plants and then walk to find suitable mates or fruit (personal

Figure 2.1: Anthonomus eugenii trap deployed at the edge of a pepper field. The trap has substantial amounts of insect by-catch collected over the course of a week.

observation), and based on this behavioural characteristic, several collection trap types that hold live adults may be suitable for trapping A. eugenii. Boll weevil traps and pyramid traps, commonly used for live captures of pest insects, require adults to land on the trap and then climb upward, where they are captured in collection jar (Figure 2.2

A,D). Alternatively, jar traps and unitraps have larger or easily accessed entry holes that target flying insects and often include a drowning solution to retain captured insects

(Figure 2.2 B,C).

The purpose of this study was to determine an enhanced trapping protocol to use with semiochemical lures for improving the monitoring of A. eugenii in greenhouses.

First, the ability of A. eugenii to escape from currently available commercial sticky traps was evaluated, and the distance A. eugenii were able to move on sticky cards under greenhouse conditions was quantified. Next, the capture rates of A. eugenii on sticky cards and alternative trap types were compared. Finally, laboratory trials were conducted to assess the efficacy of novel adhesive formulations to retain A. eugenii on sticky card surfaces. Together, findings from these studies will enable growers and scouts to use the best possible trapping tools available for monitoring A. eugenii in greenhouse pepper crops, ultimately minimizing the time devoted to visual crop detection and reducing monitoring costs for growers.

2.2 Materials and Methods

2.2.1 Study insect

A laboratory colony of A. eugenii was established at the University of Guelph in

October 2017 from infested bell pepper, Capsicum annuum L., fruit collected from commercial greenhouses, and laboratory-reared adults from the Agriculture and Agri-

Food Canada (AAFC)–Harrow Research and Development Centre. To maintain genetic diversity, infested fruit were collected from A. eugenii-infested commercial greenhouses in Essex and Chatham-Kent Counties in Ontario between July and November in 2017,

2018, and 2019, and were combined with laboratory-reared adults collected from the

University of Guelph and AAFC–Harrow Research and Development Centre colonies.

Adults and infested fruit were kept in ventilated containers (12-L plastic containers for adults and various sized containers for infested fruit) and maintained in a controlled environment cabinet set at 28 °C, 45–60% RH, and a 14:10 h light:dark photoperiod. Adult weevils were provided with water, peppers (C. annuum varieties

Felicitas, Fascinato, Karisma, or Jalapeno), 10% sucrose solution, and 25% honey solution. Water, sucrose, and honey solutions were provided separately in 30-mL plastic cups with a cotton dental wick and replaced every week. To maintain humidity inside the adult containers, a sponge lightly soaked in water was added to each adult container, and a piece of paper towel was placed on the inside bottom of the container to absorb excess moisture.

Peppers were sown every 2 months in LA4 potting mix (Plant Products,

Ancaster, ON, Canada) from seed (Stokes Seeds Ltd., Thorold, ON, Canada) at the

University in Guelph in greenhouses maintained between 18–24 °C under natural light and watered as needed. Peppers were fertilized weekly with starter fertilizer (10-52-10,

Plant Products, Ancaster, ON, Canada) from the true leaf stage to flowering, and all- purpose fertilizer (20-20-20, Plant Products, Ancaster, ON, Canada) post flowering.

Every 2–3 days, green pepper fruit with a diameter between 5 and 20 cm were harvested and sanitized with a 0.3 % hydrogen peroxide spray solution to reduce pathogen incidence. Sanitized peppers were hung from the tops of the adult weevil cages by a paper clip attached to the stem of the fruit. A minimum ratio of 1 pepper: 20 adults was maintained in each box, and a mix of pepper fruit varieties was provided.

When an insufficient number of peppers were available, pepper plant leaves were added to adult boxes to supplement feeding. After 2–3 d of hanging in adult cages, infested pepper fruit were removed and sanitized by wiping with a 0.3 % hydrogen peroxide solution. Infested peppers were placed into ventilated plastic emergence containers and sanitized regularly by wiping them with 0.3 % hydrogen peroxide solution every 2–3 d until A. eugenii adults emerged.

2.2.2 Assessment of Anthonomus eugenii movement on and escape from commercial sticky cards under laboratory conditions

The ability of A. eugenii adults to move on and escape from various sticky card types was assessed using a three-way factorial experimental design (5 adhesive trap treatments × 2 starting positions × 2 sexes). Experiments were conducted at both 23 °C

and 32 °C to account for possible changes to adhesives at different temperatures.

Young (< 7 d old) and mature (> 28 d old) adult weevils were tested separately at each temperature (4 experiments total). In total, 4 commercially available yellow sticky card trap types (Pherocon® [Trécé Inc. Adair, Oklahoma, USA], Horiver [Koppert Canada Ltd.

Scarborough, Ontario, CA], Bug-Scan® and Bug-Scan® Dry [Biobest Canada Ltd.

Leamington, Ontario, CA]), and yellow cards manually coated with 2 mm of Tanglefoot®

Tangle-Trap® (The Ortho Group, Marysville, OH, USA) were evaluated.

Twenty-four h prior to the start of each experiment, A. eugenii adults of each tested age category were randomly selected from the colony. Adults were sexed according to the methods of Eller (1995). Once sexed, individuals were marked on their elytra with 1 of 4 colours of Sharpie® water-based paint pen to distinguish sexes and starting position. Marked weevils were kept in a ventilated container with a water source and starved overnight for 16 h.

Each experimental sticky card was cut to 14 cm2 of adhesive area, and a paperclip was attached to the center of the non-adhesive top border of the card. Using a pin, 4 depressions were made in the centre of each card 3 cm away from each other in a square pattern. These marks served as the starting locations for the weevils. Cards were immediately hung from the paper clip in a mesh cage (H: 48.3 cm × W: 48.3 cm ×

L: 91.4 cm) (BugDorm, MegaView Science Co., Taiwan) in the experimental room to allow the adhesive to adjust to the assessment temperature for 24 h prior to the experiment.

For each card, 4 weevils, 1 of each of the 4 paint colours, were randomly assigned to a treatment and to 1 of the 4 starting locations on that corresponding card.

One weevil of each sex was placed lateral side down on the card, and one weevil of each sex was placed on the card ventral side down with legs in contact with the adhesive. The cards were then hung from the top of the mesh cage in a randomized order.

Traps were assessed 0.5, 1, 2, 6, 12, 24, 36, and 48 h after initial weevil placement. At each assessment period, the location of each weevil was marked and the straight-line (or minimum) distance between the previous and current location of each weevil was measured. Weevils that were no longer on the surface of the card were counted as escaped. After 48 h, the distances of each straight line were summed to calculate the total minimum distance each weevil traveled on a card.

An analysis of variance (ANOVA) was completed on pooled data from all 4 experiments to compare the effects of trap type, starting position, sex, temperature, age, and their interactions on the mean minimum distance traveled (MMDT) by adult A. eugenii after 48 h, and if the adults escaped after 48 h using PROC GLIMMIX (SAS

Studio, University Edition 2.6 9.4 M5). A lognormal distribution with identity link was used for the MMDT data, and a binomial distribution with a logit link was used for the escape data. For both MMDT and escapes, date of experiment and replicate were random effects. Date of experiment nested within replicate was included as a random effect in the model to account for the 4 experiments being conducted at different times.

Trap type, starting position, sex, age, and temperature were fixed effects. Three-way interactions were not included in the models. Means separations were performed for models with significant fixed effects using a Tukey-Cramer adjustment (α = 0.05). For

MMDT, LS Means and standard error were back-transformed for presentation in figures and tables. Odds ratios with a 95% confidence interval were used to show differences in escape between the different variables (trap type, sex, starting position, temperature, and age). The odds ratio for an escape (1) was used as a reference, and a ratio > 1 indicated a significant difference in potential to escape.

2.2.3 Evaluation of alternative trap designs for the capture of Anthonomus eugenii adults under laboratory conditions

Twenty-four h prior to the start of the experiment, adult A. eugenii (8–12 d old) were randomly selected from the colony and sexed as previously described. Once sexed, adults were placed in groups of 20 (10 males and 10 females) in ventilated containers and starved overnight for 14 h.

In this study, 6 insect trap types were evaluated and compared: a boll weevil trap

(Great Lakes IPM, Vestaburg, MI, USA), green Unitrap (Great Lakes IPM, Vestaburg,

MI, USA), homemade plastic jar trap (1-L clear plastic jar with red tape, with eight 3.5 cm diameter openings in the upper half of the jar covered in drywall mesh), modified hanging yellow pyramid trap (1-L clear plastic deli cup with metal mesh funnel fitted with a yellow vein trap, approximately 41 cm in height), yellow card covered with a 2-mm thick layer of Tanglefoot sticky coating on both sides (23 cm × 14 cm adhesive surface),

and Pherocon double sided yellow sticky card (Figure 2.2). Two replicates were run simultaneously and the study was repeated 3 times over time for a total 6 replicates.

Traps were placed individually in mesh cages (H: 91.4 cm × W: 48.3 cm × L: 48.3 cm, BugDorm, MegaView Science Co.) in a controlled environment room at 23 °C, 50–

60% RH, and a 14:10 h light to dark photoperiod. Cages were spaced a minimum distance of 20 cm apart in a randomized complete block design. Using twist ties, traps were hung on the nylon cord on the ceiling of each cage so that the top of each trap was 10 cm from the ceiling. To retain captured adults, a 4-cm-deep layer of 0.1% soap solution was added to Unitraps and jar traps. All traps were baited with both Trécé pepper weevil pheromone lures (PEW I and PEW II). Lure packages were opened and kept in a fume hood for 24 h prior to each experiment to ensure a consistent release of pheromones from the lures during the 48 h experimental period.

Groups of 20 adult A. eugenii (10 females and 10 males) were randomly assigned to each trap. Adults were released at the bottom center of the cage and the cage was closed. After 48 h, the number of adults captured by each trap was recorded.

An ANOVA was completed using PROC GLIMMIX to compare weevil captures for each of the 6 trap types (SAS Studio, University Edition 2.6 9.4 M5). Variance was partitioned into the fixed effect trap type and random effect replicate. Sex was not included in the model due to low captures. A Shapiro-Wilk test was used to confirm the data displayed a normal distribution, and residuals were analyzed to confirm all other assumptions were met. To satisfy assumptions of normality, one outlier observation was

removed (replicate 6, jar trap) after an analysis of residuals. Means were compared using a Tukey’s multiple comparison test (α = 0.05).

2.2.4 Evaluation of novel adhesive formulations on yellow sticky cards for Anthonomus eugenii retention under laboratory conditions

Twenty-four h prior to the start of the experiment, adult A. eugenii (8–12 d old) were randomly selected from the colony, sexed, and starved as previously described.

Four novel types of proprietary adhesive-coated yellow cards provided by Trécé were compared to the current yellow sticky card supplied in the Trécé Pherocon Pepper

Weevil (PEW) Kits. All cards were 15 cm wide and 30 cm long, with adhesive on both sides. One card of each type was randomly assigned to a single mesh cage (H: 48.3 cm

× W: 48.3 cm × L: 48.3 cm, BugDorm, MegaView Science Co.) and cages were prepared in a randomized complete block design. Experiments were conducted in a controlled environment room maintained at 24 °C, 50–60% RH, with a 14:10 h light:dark photoperiod. Each card was hung individually in a cage 24 h prior to the start of the experiment to allow the adhesive to adjust to the temperature of the room.

Groups of 18 (9 male, 9 female) adult A. eugenii were randomly assigned to each card. The adhesive surface of each card was divided into a 3 × 6 grid layout with each grid square measuring 5.0 × 4.2 cm. Alternating between males and females, adults were randomly and individually dropped onto the centre of each grid square ventral-side down, from a height of 1 cm (Figure 2.3). Based on observations of adult A. eugenii landing on traps during the trap design experiment, as well as other qualitative

Figure 2.3: Grid layout for arrangement of A. eugenii on novel adhesive cards (A) before and (B) after all 18 adults were dropped on the surface of the card.

observations made in a colony setting, it was determined that adults most often land on adhesive cards ventral side down. Therefore, starting position was removed as a factor in this experiment and all adults were placed ventral side down. Once all 18 adults were dropped onto the grid squares of each card, each card was hung in the mesh cage using a paper clip (Figure 2.3).

Cards were observed 0.25, 1, 4, 24, and 48 h after initial weevil introductions.

Any adult weevil that had escaped from a card was removed from the cage and sexed.

After 48 h, the total number of escaped adult weevils was recorded for each adhesive card treatment tested. The experiment was replicated 6 times between September and

October 2019.

A Shapiro-Wilk test was used to confirm the data displayed a normal distribution and residuals were analyzed to confirm all other assumptions were met. An analysis of variance was used to compare A. eugenii escapes from the 5 adhesive cards using

PROC GLIMMIX (SAS Studio, University Edition 2.6 9.4 M5). Sex and adhesive card type were fixed effects, and replicate was a random effect. An ANOVA was performed to determine the effect of sex, adhesive card type, and the interaction between sex and adhesive card type on the total number of escapes after 48 h. There were no significant effects in this model, and therefore, the data were reanalyzed with only adhesive card type included as a fixed effect in the model. Means comparisons using a Tukey’s test were performed for significant ANOVAs (α = 0.05).

2.3 Results

2.3.1 Assessment of movement of Anthonomus eugenii on commercial sticky cards under laboratory conditions

Trap type had a significant effect on the distance adult A. eugenii were able to travel on sticky cards (Table 2.1). The MMDT by adult weevils was significantly lower on

Tanglefoot-coated and Pherocon cards compared to the other 3 commercially available sticky cards (Figure 2.4; Appendix Table 6.1).

Sex, age, temperature, and starting position did not affect MMDT (Table 2.1).

However, the interaction of trap type and age was significant (Table 2.1). This interaction between trap type and age occurred because young weevils did not move as far as mature weevils on Tanglefoot or Bug-Scan sticky card treatments. The reverse was true for the other 3 sticky card treatments.

Table 2.1: Results of an ANOVA on the mean minimum distance traveled by Anthonomus eugenii adults on 5 types of sticky cards. Male and female adults were manually placed on the sticky cards on their side or ventral side down, and the distance each weevil travelled across the card was assessed for 48 h. The experiment was conducted at 23 and 32 °C with young (< 7 d old) and mature (> 28 d old) adults. A log distribution was used and back transformed data are reported. Significant model effects are bolded (P ≤ 0.05). Fixed effects Num df Den df F P Trap type 4 358 39.6 <0.0001 Sex 1 358 1.59 0.208 Temperature 1 358 2.51 0.114 Starting position 1 358 1.63 0.203 Age 1 358 0.38 0.539 Trap type*sex 4 358 0.23 0.922 Trap type*temperature 4 358 1.83 0.123 Trap type*starting position 4 358 1.76 0.137 Trap type*age 4 358 2.81 0.0253 Temperature*sex 1 358 0.27 0.607 Sex*starting position 1 358 2.75 0.0983 Age*sex 1 358 1.38 0.240 Temperature*starting position 1 358 4.69 0.0310 Age*temperature 1 358 0.21 0.650 Age*starting position 1 358 0.54 0.461

Random effects Estimate Standard Error Chi Sq Pr > Chi Sq Date 0.01820 0.0281 0.74 0.388 Replicate 0.00969 0.0216 0.00 1.000 Replicate (date) <0.001000 <0.00100 0.00 1.000 Residual 0.877 0 0.0655

The interaction between temperature and starting position was also significant

(Table 2.1). At 23 °C, adults that started on their feet traveled further than adults that started on their sides, but the same relationship was not observed at 32 °C.

2.3.2 Assessment of escapes of Anthonomus eugenii from commercial sticky cards under laboratory conditions

Trap type had a significant effect on the number of adult weevils that escaped from sticky cards (Table 2.2). Fewer weevils escaped from Tanglefoot and Pherocon

cards compared to the other sticky trap types (Figure 2.5; Appendix Table 6.2). There was no difference in the number of weevil escapes between Tanglefoot and Pherocon traps (Figure 2.5; Appendix Table 6.2). The highest proportion of adult escapes was observed from Bug-Scan Dry Touch sticky cards (Figure 2.5; Appendix Table 6.2). Adult weevils were on average 118 times more likely to escape from a Bug-Scan Dry Touch card as they were to escape a Tanglefoot-coated card (Table 2.3). Sex also significantly affected weevil escape (Table 2.2), with females being twice as likely to escape from sticky cards than males (Table 2.3).

The interactions between trap type and age, and between temperature and starting position, were also significant (Table 2.2). Young weevils moved further on the

Horiver cards (52.1 ± 8.00 mm) relative to mature weevils (37.2 ± 5.81 mm), and the reverse was true for the Bug-Scan cards (young: 46.4 ± 7.61 mm, mature: 60.7 ± 9.41 mm). All other treatments showed very little difference between the 2 age groups. With respect to the interaction between temperature and starting position, more adults in the

23 °C treatment that started on their feet escaped relative to adults that started on their sides. The opposite trend was observed at 32 °C: Adults that started on their sides had escaped more frequently than those that started on their feet.

Table 2.2: Results of an ANOVA comparing the number of adult Anthonomus eugenii escapes from 5 types of sticky cards. Male and female adults were manually placed on the sticky cards on their side or ventral side down, and any weevil that had left the surface of the sticky card over the 48 h experimental period was counted as escaped. The experiment was conducted at 23 and 32 °C with young (< 7 d old) and mature (> 28 d old) adults. A binary distribution was used and significant model effects are bolded (P ≤ 0.05). Fixed effects Num df Den df F P Trap type 4 429 19.7 <0.0001 Sex 1 429 4.84 0.0283 Temperature 1 429 1.16 0.282 Starting position 1 429 0.64 0.424 Age 1 429 0.16 0.691 Trap type*sex 4 429 0.22 0.925 Trap type*temperature 4 429 1.48 0.208 Trap type*starting position 4 429 0.96 0.429 Trap type*age 4 429 2.42 0.0481 Sex*temperature 1 429 0.01 0.918 Sex*starting position 1 429 2.95 0.0867 Age*sex 1 429 0.36 0.551 Temperature*starting position 1 429 6.76 0.0096 Age*temperature 1 429 0.39 0.535 Age*starting position 1 429 0.05 0.817

Random effects Estimate Standard Error Chi Sq Pr > Chi Sq Date 0.2110 0.285 1.11 0.146 Replicate 0.0980 0.194 0.00 1.000 Replicate (date) <0.00100 <0.0010 0.00 1.000 Residual <0.00100 <0.0010

2.3.3 Evaluation of alternative trap designs for capture of Anthonomus eugenii adults under laboratory conditions

Trap type was a significant source of variation for mean A. eugenii capture numbers

(Table 2.4). Tanglefoot-coated cards, Pherocon sticky cards, and jar traps captured significantly more adults than the other trap types (Figure 2.6; Appendix Table 6.3). The

Unitrap captured significantly more adults than the modified pyramid trap and boll weevil trap (Figure 2.6; Appendix Table 6.3).

Fixed effects Numerator Denominator Odds DF Lower Upper Significance Ratio Estimate Trap type Bug-Scan Dry Bug-Scan 1.89 429 0.983 3.65 Bug-Scan Dry Horiver 2.20 429 1.15 4.22 * Bug-Scan Dry Pherocon 27.9 429 11.0 71.0 * Bug-Scan Dry Tanglefoot 118 429 26.9 525 * Bug-Scan Horiver 1.16 429 0.621 2.18 Bug-Scan Pherocon 14.7 429 5.91 36.7 * Bug-Scan Tanglefoot 62.8 429 14.3 274 * Horiver Pherocon 12.7 429 5.09 31.4 * Horiver Tanglefoot 53.9 429 12.4 235 * Pherocon Tanglefoot 4.26 429 0.856 21.2 Sex Female Male 2.01 429 1.08 3.74 * Starting position Feet Side 1.27 429 0.702 2.32 Age Mature Young 1.21 429 0.466 3.16 Temperature 23 °C 32 °C 0.584 429 0.218 1.56

Random Effects Estimate Standard Error ChiSq Pr > ChiSq

Replicate 0.210 0.136 1.36 0.243 Residual 2.120 0.606

2.3.4 Laboratory evaluation of novel adhesive formulations for Anthonomus eugenii retention

Adhesive type had a significant effect on the number of weevils that escaped within

48 h (Table 2.5). Over all 6 replicates, no weevils escaped from adhesive treatment TR

270 during the experiment. In contrast, the highest number of weevils escaped from treatment TR 274 (Figure 2.7; Appendix Table 6.4). Significantly fewer weevils escaped from treatments TR 270 and TR 275 compared to treatment TR 274 (Figure 2.7;

Appendix Table 6.4). The number of weevils that escaped from TR 270 and TR 275 traps was not significantly different from the currently used Pherocon sticky card trap

(Figure 2.7; Appendix Table 6.4).

Fixed effects Num df Den df F P Trap type 4 20 5.74 0.003

Random Effects Estimate Standard Error ChiSq Pr > ChiSq

Replicate 0.230 1.46 0.02 0.880 Residual 11.2000 3.54

Figure 2.7: Mean number (±SE) of Anthonomus eugenii adult escapes from 5 types of adhesive formulations on yellow cards. Male and female adults were dropped on the sticky cards ventral side down from 1 cm above the surface of each card in a grid pattern. Any weevil that had left the surface of the sticky card over the 48 h experimental period was counted as escaped. The experiment was conducted at 23 °C with adult weevils (8–12 d old). Bars with the same letter are not significantly different (Tukey’s HSD, P ≤ 0.05).

2.4 Discussion

These results demonstrate that A. eugenii adults are able to move on and escape from commercially available sticky cards, confirming concerns expressed by growers and scouts that use these traps for detecting and monitoring A. eugenii. Adults moved less on, and fewer adults escaped from, Pherocon and Tanglefoot-coated traps, the traps currently used for A. eugenii monitoring. Although the MMDT for weevils was lowest on these 2 card types, adults were still able to move up to 99 mm and 136 mm on Tanglefoot and Pherocon cards, respectively. Adults were unable to directly fly off

sticky cards, needing first to walk to the card edges or to the non-adhesive top surface of cards before being able to escape (personal observation). These results suggest that adults that land closer to the edge of a card are more likely to escape relative to those that land closer to the middle. Similarly, Eller et al. (1992) reported that during field trials adult A. eugenii would walk to the bottom of Pherocon sticky cards used for monitoring and onto the wooden stakes that the cards were suspended on to escape. Since both measures of distance traveled and percent escape gave similar results, adult escapes from sticky cards are most likely occurring by walking to trap borders or non-adhesive areas of the cards rather than by flight alone. This finding suggests that distance traveled alone could be a good indicator for determining the rate of insect retention on sticky cards in future studies.

When Pherocon traps are set up in pepper fields or greenhouses, it is common for other insects and debris to be caught on sticky cards, which reduces the adhesive surface area. Experiments conducted in pepper fields or commercial greenhouses should examine the ability of A. eugenii to move on and escape from traps with reduced adhesive surfaces due to bycatch to determine if this increases their movement on and escape from sticky cards.

Weevil age, starting position, temperature, and sex had no consistent impact on A. eugenii movement on or escape from sticky cards. Although the interaction with weevil age and trap type was statistically significant, because weevil age on its own was not a significant source of variation and no patterns were observed in the treatment

responses of different weevil ages, the interaction of age and trap type was not considered further. Fewer males escaped from sticky cards, possibly because of the pronounced tibial spur on the hind legs of males (Eller 1995), or a greater motivation among females to move and seek out oviposition sites. Starting position also was a significant source of variation in several experiments, but through observations of weevil captures on traps in a greenhouse setting, it was determined that adults were unlikely to land on sticky traps on their side, and thus conducting experiments with adults starting on their feet was more representative of trap captures in field settings.

The high levels of escape and MMDT observed for adult weevils on 3 of the 5 sticky card types assessed (Bug-Scan, Bug-Scan Dry, and Horiver) suggests that these cards do not represent an advantage over the use of Pherocon cards, which are currently supplied in commercial monitoring kits. Furthermore, grower adoption of Tanglefoot as a monitoring tool for A. eugenii will likely be low as its use requires manual application of the adhesive to cards, presenting a challenge for quick field deployment and regular trap replacement. This factor, in addition to weevil escapes and distance traveled not being significantly reduced compared to Pherocon cards, suggests that Tanglefoot- treated cards would not be a good replacement for monitoring of A. eugenii.

In assessments of alternative trap types, boll weevil traps caught the fewest adults. Similarly, Patrock et al. (1992) observed that A. eugenii easily maneuvered in and out of boll weevil-style traps in a field setting. Both Pherocon and Tanglefoot sticky card traps and jar traps caught more insects than the traps that required a directed

walking movement by the weevils (i.e., walking up towards a collection jar), such as the boll weevil trap and the modified pyramid design. Although sticky cards are easy to use and inspect for A. eugenii adults, many insects are attracted to yellow, which increases bycatch and reduces the effective adhesive surface area, especially in field conditions

(Palumbo et al. 1995, Naranjo et al. 1995, Pinto‐Zevallos and Vänninen 2013). Thus, it is possible that such a general-purpose trap type may become less effective over time relative to other trap types. A jar trap may be effective in a monitoring program for A. eugenii, and bycatch of non-target insects or other debris may be reduced if the entry holes are sized specifically for A. eugenii. It is clear, however, that more research is needed to determine the optimal entry hole size and best trap placement within greenhouses and fields to optimize the use of jar traps as part of an integrated pest management strategy for A. eugenii. Jar traps may also be advantageous for use as part of a mass capture strategy for A. eugenii, particularly where the presence of A. eugenii has already been confirmed. However, sticky cards still represent effective tools that allow for quick and accurate visual checks for initial weevil detection. In contrast, jar traps involve more handling and inspection of the drowning liquid to find captured weevils, and thus, they may be better suited for a mass trapping management system, where less frequent trap handling is needed.

Adhesive formulation assessments revealed that treatment TR 270 from Trécé retained the highest number (100%) of A. eugenii over all replicates at 23 °C. These results indicate that this new formulation would be an excellent candidate for further field testing and eventual use in A. eugenii monitoring programs in greenhouses. To be

effective for insect monitoring, sticky traps must maximize captures of the target insect, and the likelihood of capture depends on if the trap can be effectively located by the target responder, the efficiency of the trapping mechanism (if the responder is able to engage with the trap and be captured), and if the responder is retained in or on the trapping mechanism for the desired amount of time (Miller et al. 2015). Of these factors, trap retention is the easiest to test and improve on, since retention is easy to measure in controlled experiments. Study organisms can be placed directly on or into the trap and subsequently observed to determine the rate of escape. Assessment of other factors, such as trapping efficiency, also sometimes referred to as trap adoption, rely more heavily on field trials, which allow for observation of how target organisms engage with the trapping mechanism in the relevant environment (Miller et al. 2015). This factor should be assessed for any new adhesive to quantify its performance in a field setting.

The experiments described in this study were conducted under relatively stable environmental conditions and in the absence of other insect pests. Therefore, further experiments should be conducted in a commercial greenhouse setting to determine adhesive performance under fluctuating environmental conditions and in the presence of non-target insects and plant debris.

Monitoring and trapping of A. eugenii in greenhouses is still a relatively new practice because populations of this pest historically have only been observed in field peppers (Ingerson-Mahar et al. 2015). Therefore, there is a lack of information on optimal trapping protocols for greenhouse monitoring of A. eugenii, and how the use of

traps may affect the abundance of other pests or beneficial insects present in commercial greenhouses. Most commercial greenhouse facilities have monitoring strategies in place for detection and quantification of other insect pests, such as whiteflies or thrips (Steiner et al. 1999), and rely largely on biological control agents for their control (Costello and Gillespie 1993, OMAFRA 2010). Furthermore, bumble bees

(Bombus species) are regularly placed throughout greenhouses to assist with crop pollination (OMAFRA 2010). Deploying sticky cards or jars with a solution that drowns arthropods indiscriminately may negatively impact biological control agents and bumble bees. Therefore, commercial pepper greenhouse trials are needed to determine whether A. eugenii monitoring programs can be effectively integrated with existing commercial crop production and pest management practices.

In conclusion, results from this chapter indicate the need for a more reliable trapping method for monitoring A. eugenii, especially in the greenhouse growing conditions of southwestern Ontario. Current action thresholds for A. eugenii are 1 adult/trap per field

(Ingerson-Mahar et al. 2015). Thus, any escape of A. eugenii from monitoring traps can significantly delay the implementation of a management program, leading to an increased risk of crop damage and economic losses in cultivated peppers (Natwick and

Trumble 2007). Results from these experiments indicate that sticky cards continue to be the most effective trap for monitoring A. eugenii. Two new adhesives show promise to be more effective than the current industry standard sticky trap adhesive but require additional experiments under field conditions. Additionally, since completion of these experiments, Trécé Pherocon Pepper Weevil Kits are now supplied with a new sticky

card with similar retentive properties to TR 270 but with a more photo-stable adhesive optimized for outdoor use (Brent Short, Trécé Inc., personal communication). As such, it is recommended that producers continue to use current Pherocon sticky traps with

Trécé pheromone lures, and to monitor the traps as frequently as economically possible throughout the pepper growing season, to reduce both the risk of adult A. eugenii escape and any delay to the application of management tools for the control A. eugenii.

3 Improved attraction of Anthonomus eugenii to traps using semiochemicals

3.1 Introduction

Anthonomus eugenii is the primary pest of peppers in much of North America, especially in Mexico, the Caribbean, and the southern United States where peppers are grown year-round (Elmore et al. 1934, Seal and Lamberts 2012, Ingerson-Mahar et al.

2015). Its high fecundity and multiple overlapping generations per season make A. eugenii a difficult pest to control (Capinera 2010, Ingerson-Mahar et al. 2015).

Therefore, timely detection of A. eugenii in cultivated peppers is important to ensure control measures are implemented before economic damage and losses occur (Elmore et al. 1934, Andrews et al. 1986, Servín et al. 2002).

Unreliable or delayed detection of A. eugenii can allow populations to build up rapidly and cause significant economic losses of cultivated peppers. Because visual scouting of adults and damage is ineffective and laborious, aggregation pheromone traps are widely used for monitoring and detecting A. eugenii in cultivated peppers (Eller et al. 1994, Bottenberg and Lingren 1998, Szendrei et al. 2011, Muñiz-Merino et al.

2014, Fernández et al. 2020). A male-released aggregation pheromone was identified by Eller et al. (1994) and was later produced for commercial use as part of a monitoring and detection tool for A. eugenii (Bottenberg and Lingren 1998). The aggregation pheromone consists of 6 components: (Z)-2-(3,3-dimethylcyclohexylidene) ethanol, (E)-

2-(3,3-dimethylcyclohexylidene) ethanol, (Z)-(3,3-dimethylcyclohexylidene) acetaldehyde, (E)-(3,3-dimethylcyclohexylidene) acetaldehyde, (E)-3,7-dimethyl-2,6-

octadienoic acid (geranic acid) and (E)-3,7-dimethyl-2,6-octadien-1-ol (geraniol) (Eller et al. 1994). Adult male and female A. eugenii orient to sticky traps baited with the aggregation pheromone lures, although the attraction of A. eugenii to these lures decreases in the presence of flowering and fruiting pepper plants (Patrock et al. 1992,

Eller et al. 1994).

Behaviours of phytophagous insects are often closely integrated with their host plants as insects not only feed, but often also meet, court, mate and/or oviposit on host resources (Landolt and Phillips 1997). Phytophagous insects will commonly use plant- derived semiochemicals, including plant volatiles, to locate their hosts (Landolt and

Phillips 1997, Reddy and Guerrero 2004, Addesso and McAuslane 2009, Dicke and

Baldwin 2010, Das et al. 2013). When phytophagous insects locate host plants and begin to feed, plants respond with changes to their volatile profile, which can be attractive or repellant to the insects (Dicke and van Loon 2000). Feeding-induced plant volatiles can affect the behaviour of insects by providing information to insects on the location of conspecific aggregations (Loughrin et al. 1996), or they can interact with insect-produced pheromones (Borden 1984). When plant volatiles affect the behaviour of a different species, in this case a phytophagous insect, they are referred to as kairomones (Kost 2008).

Kairomones can induce the production or release of sex pheromones (Borden

1984, Kalberer et al. 2001, Reddy and Guerrero 2004), or increase the effectiveness of other insect-produced pheromones (Reddy and Guerrero 2004). Kairomones have been

used for the monitoring of Anthonomus species, specifically the cranberry weevil (A. musculus) (Mechaber 1992; Szendrei et al. 2009), apple blossom weevil (A. pomorum)

(Collatz and Dorn 2013), and boll weevil (A. grandis Boheman) (Dickens 1989). In some species, when kairomones are coupled with pheromones, the response to the combination of semiochemicals can be greater than the sum of the response to the 2 semiochemicals, and this is referred to as synergism (Landolt and Phillips 1997).

Synergistic responses by phytophagous insects to 2 or more semiochemicals are commonly observed, especially in coleopteran species (Said et al. 2011). Behavioural synergy has been thoroughly examined in Rhynchophorus weevils where responses to aggregation pheromones are dramatically increased by the perception of host plant volatiles in field traps (Hallett et al. 1993). Similarly, the boll weevil, a congener of A. eugenii, has demonstrated a synergistic response to field traps when its aggregation pheromone (grandlure) is paired with green leaf volatiles (trans-2-hexen-I-ol, cis-3- hexen-l-ol, or l-hexanol) (Dickens 1989, Dickens et al. 1990). The discovery of this synergist effect led to the pheromone + cotton volatile traps becoming an integral part of the eventual eradication of the boll weevil in the United States (Hardee and Harris

2003).

Host plant kairomones have yet to be used for A. eugenii monitoring but could provide increased attraction or a synergized response to current adult pheromone monitoring tools. To determine host plant cues, Addesso et al. (2011) compared male and female A. eugenii attraction to different phenological stages of pepper plants that had been damaged by conspecific feeding with attraction to undamaged plants. They

determined that both males and females responded more to upregulated constitutive volatiles that were released by flowering and fruiting plants that had feeding damage over undamaged plants. Additionally, males and unmated females preferred plants with males actively feeding on them over plants with feeding females. Mated females, however, exhibited no preference between plants with males or plants with females, indicating that mating status influences attraction to semiochemicals in A. eugenii adults

(Addesso et al. 2011). Headspace volatiles of feeding-damaged plants were analyzed using gas chromatography and mass spectrometry, which revealed that plants upregulated the constitutive volatile E-β-ocimene when they were fed on, along with 13 other volatile compounds during fruiting and flowering (Addesso et al. 2011). Muñiz-

Merino et al. (2014) then tested the response of males and females to a series of volatile compounds alone and in combination, and determined that E-β-ocimene and 2- isobutyl-3-methoxypyrazine, a potent odorant of Capsicum species fruit (Zachariah and

Gobinath 2008), attracted an equal number of mated female A. eugenii and a higher number of male A. eugenii compared to the male-produced aggregation pheromone.

The combination of host plant volatile compounds that is attractive to male A. eugenii

(E-β-ocimene and 2-isobutyl-3-methoxypyrazine, hereafter referred to as kairomones)

(Muñiz-Merino et al. 2014) has yet to be tested on unmated female A. eugenii.

These studies suggest that unmated A. eugenii females are more likely to orient to aggregation pheromones coupled with kairomones than to kairomones alone, whereas the reverse is true for mated females (Addesso et al. 2011, Muñiz-Merino et al.

2014). Therefore, the attraction of unmated females to monitoring traps before mating

occurs may be improved by the use of the aggregation pheromone coupled with kairomones (Addesso et al. 2011, Muñiz-Merino et al. 2014). In addition, the attraction of A. eugenii to kairomones in the presence of fruiting pepper plants needs to be assessed to determine if the combination of kairomones and pheromones will outcompete natural semiochemicals in field or greenhouse settings. If a synergistic response by A. eugenii occurs to a combination of host plant volatiles and aggregation pheromone in the presence of fruiting plants, then integrating kairomones into synthetic lures may help to improve monitoring and management of A. eugenii in cultivated peppers.

The goal of this study was to determine if combining pepper kairomones with aggregation pheromone lures improves the attraction of A. eugenii to monitoring traps.

First, the attractiveness of current pheromone lures to A. eugenii in the presence of fruiting plants was determined. Second, previously identified kairomones were added to the most attractive lure to determine if response by A. eugenii to the pheromone could be increased or synergized in lab bioassays. Third, the attractiveness of commercial lures with pheromone and kairomone components to A. eugenii was evaluated in an experimental greenhouse. A semiochemical-based trap that is highly attractive to A. eugenii in the presence of fruiting pepper plants would improve detection and management of A. eugenii in cultivated peppers.

3.2 Materials and Methods

3.2.1 Field evaluation of commercial lures for Anthonomus eugenii

A field trial was conducted from September 20 to October 4, 2018 in an infested jalapeno pepper field in Dresden, Ontario to evaluate the relative attractiveness of commercial lures to A. eugenii. The field had not been sprayed with insecticides for 2 weeks prior to the experiment, and all plants were at fruiting stage, with a mix of mature and young fruit present on each plant.

The experiment was arranged in a randomized complete block design with 10 blocks spaced at least 20 m apart (Figure 3.1). Blocks were either on the field edges or in centre rows of the field. Treatments within a block were placed 3 m apart. For each treatment, a double-sided Pherocon yellow sticky card (Trécé Inc. Adair, Oklahoma,

USA) was secured to a metal pigtail stake (Hillman Group, Cincinnati, Ohio, USA) and stuck in the ground so the top of the card was approximately 70 cm above the soil. Each sticky card was baited with a Trécé pepper weevil lure (Trécé Inc. Adair, OK, USA) or a

Russell IPM pepper weevil lure (Russell IPM Ltd, Deeside, UK). Control sticky cards were unbaited (no lure). Lures were secured to the top of the sticky cards with a twist tie. Traps were checked twice a week, and sticky cards were replaced halfway through the experimental period on September 27. Any A. eugenii found on traps during bi- weekly checks were counted and removed.

A Shapiro-Wilk test was used to confirm the data displayed a normal distribution, and residuals were analyzed to confirm all other model assumptions were met. An analysis of variance (ANOVA) was used to compare total A. eugenii captures over the 2 weeks among the 3 lure treatments using PROC GLIMMIX (SAS Studio, University

Edition 2.6 9.4 M5). Lure treatment was a fixed effect and replicate was a random effect. Means were separated using a Tukey’s test (α = 0.05).

3.2.2 Addition of kairomones to pheromone lures for increased attraction of Anthonomus eugenii

Assays were conducted with laboratory-raised A. eugenii adults (see Chapter 2), and 4 cohorts of adults were tested: young unmated females, young females assumed to be mated, young males, and mature unmated females. Young adults were 3–6 d old and mature adults were 10–14 d old. Adults were sexed using the methods described by Eller (1995). Males and mated females of the appropriate age were selected at random from rearing containers and used for each of the 4 treatments. To obtain unmated females, infested peppers were kept individually in 475-mL plastic containers with ventilated lids and checked hourly for emergence. Emerged adults were sexed, and unmated females were placed individually in ventilated plastic containers (60 mL) with access to peppers, leaves, and water until they reached the appropriate age for bioassays.

Four treatments were assessed using 4 different stimuli (Table 3.1), which were chosen based on previous olfactometry studies (Addesso et al. 2011, Muñiz-Merino et al. 2014): clean air compared to the pheromone, clean air compared to the kairomone, the pheromone compared to the kairomone, and the pheromone compared to the kairomone + pheromone. Over 3 days, adults were tested until 30 responders for each treatment were completed (approximately 10 adults per day for each treatment). The order of treatments was randomized each day, and stimuli were randomly assigned to an arm of the Y-tube and switched after 6 respondents to eliminate side bias. An adult

Treatment Components, concentration, and purity Manufacturer

Air Clean air n/a

Pheromone PEW I and PEW II lures [grandlure II Trécé Inc. (48 %), (E)-grandlure II (32 %), (Adair, OK, USA) grandlure III (3 %), grandlure IV (2 %), geranic acid (13 %) and geraniol (2 %)]

Kairomone (E)-β-ocimene [10 μg/μL] (95%) Bedoukian Research Inc. (Danbury, CT, USA)

2-isobutyl-3-methoxypyrazine [0.5 Sigma Aldrich Canada μg/μL] (≥99%) (Oakville, ON, Canada)

Kairomone + Compounds and concentrations listed As above Pheromone above in combination

was considered to have made a choice once it passed a line halfway up an arm of the olfactometer (4.5 cm from the junction of the arm). If an adult did not respond after 15 min, it was excluded from the experiment.

Prior to bioassays, controls were run to ensure that physiological response by the weevils (walking, flying) was possible in the system, and that the system did not inhibit their natural movement. To ensure airflow was not eddying, smoke tests were conducted, and the same airflow rate was used as previous experiments that examined

A. eugenii responses in the same sized Y-tube (Addesso and McAuslane 2009,

Addesso et al. 2011). Finally, all other specifications (light source, room temperature,

elimination of external visual cues, Y-tube angle) were followed according to previous studies (Addesso and McAuslane 2009, Addesso et al. 2011, Muñiz-Merino et al. 2014).

A glass Y-tube olfactometer (ca. 14 cm common tube, 9 cm arms, 2 cm internal diameter; Analytical Research Systems, Gainesville, FL, USA) was used to evaluate the behavioural response of adult A. eugenii to pepper volatiles. Using methods published by Addesso and McAuslane (2009), medical-grade air was passed through a charcoal filter (Sigma Scientific LLC, Micanopy, FL, USA) and then through a Dudley bubbling tube containing deionized water. Airflow was split into 2 airstreams, and the flow rate of each airstream was adjusted to 250 mL/min with a flow meter (Key Instruments,

Trevose, PA, USA). Stimulus sources were held in 25 mL Pyrex™ filtering flasks with tubulation fitted with a plastic cork. Connections of the olfactometer used chemically resistant Tygon tubing (6.4 mm diameter, Hach Company, London, Ontario, CA), and all connections were sealed with Teflon tape. The Y-tube was ventilated passively through the base where adults were introduced into the olfactometer.

The Y-tube was placed at a 30-degree angle inside a cardboard box lined with white fabric to eliminate external visual cues. The bioassay room was maintained at 24–

26 °C and 30% RH, and the Y-tube was illuminated by an overhead suspended fluorescent light fixture (60W). Before each treatment, the Y-tube, connections, and stimulus jars were washed with phosphate-free soap and rinsed with deionized water 3 times. Clean glassware was then dried in an oven for 30 min, rinsed with acetone, and dried for 15 min in a fume hood before use. For the pheromone components, Trécé

pepper weevil pheromone lures (Trécé Inc. Adair, OK, USA) were opened 24 h prior to bioassays to allow for flash-off and kept in a fume hood at 21 °C until used in experiments. Intact commercial pheromone lures were placed directly into the stimulus jars. For treatments with the kairomone stimulus, 10 μL of solution was pipetted onto a piece of 2.1 cm filter paper. To ensure there was no interference from the solvent

(mineral oil) used for the kairomone mixture, tests were completed prior to the experiment to compare the solvent against clean air, and no significant differences were observed between treatments (data not shown).

Response data were analyzed as percent response using a chi-squared analysis

(Proc FREC) with an expected probability for Y-tube assays of 0.5 for each treatment

(SAS Studio, University Edition 2.6 9.4 M5).

3.2.3 Evaluation of kairomone and pheromone lures for Anthonomus eugenii in an experimental greenhouse

An experiment was conducted in an A. eugenii-infested greenhouse to determine the response of male and female A. eugenii to different semiochemical lures in the presence of fruiting plants. Prior to the start of the experiment, a large mesh cage

(approximately 5 m × 7.5 m) was constructed in a controlled-environment greenhouse at the University of Guelph. The greenhouse was held at a maximum of 23 °C during the day and a minimum of 18 °C at night, and 50–60% RH. The greenhouse was not artificially lit.

Twenty-four bell pepper plants (C. annuum var. Fascinato) were randomly distributed throughout the mesh cage on 4 benches (Figure 3.2). Plants were seeded on

June 6, 2019 and had a mixture of fruit, flowers, and buds at the time of the experiment.

Throughout the experiment, pepper plants were watered as needed and fertilized once per week with 20-20-20 solution (Master Plant-Prod Inc., Leamington, Ontario, CA).

Six treatments were tested for attraction of male and female adult A. eugenii in the presence of fruiting plants and were selected based on results from Muñiz-Merino et al. (2014): a sticky card without any lure (unbaited control), the pheromone lure currently used for A. eugenii monitoring (PEW), the PEW lure with a new PVC release device formulated specifically for the 2 lure components (PEW PVC), kairomone lures without the addition of any pheromones (TRE 2150 and TRE 2151), and a kairomone lure coupled with the PEW PVC pheromone lure (TRE 2152) (Table 3.2). All lures were formulated and provided by Trécé Inc.

Twenty-four h prior to the start of the experiment, 160 adult weevils (80 males and 80 females) that were 3–6 d old were randomly selected from the colony. Adults were placed into 4 vials (20 males and 20 females in each vial), and the vials were opened and placed on 1 of 4 cardboard platforms attached to the benches inside of the cage to allow the weevils to disperse throughout the greenhouse cage (Figure 3.2). The lures were opened and removed from their foil packets in a fume hood 24 h prior to placing them in the greenhouse to ensure a consistent release of compounds during the experiment.

At the start of the experiment, the lures were attached to Pherocon sticky cards with twist ties. Each sticky card trap was hung from a string between 2 plants in the experimental greenhouse. Each treatment was at least 1.6 m apart and treatment location was randomized within each replicate. Cards were hung so that the top of the card was 105 cm above the bench, approximately 35 cm above the plant canopy.

Table 3.2: List of treatments used for the infested greenhouse experiment to determine the response of Anthonomus eugenii to different semiochemical lures in the presence of fruiting pepper plants. All lures were supplied by Trécé Inc. Some treatment details, such as concentration and release rate, have been omitted upon request of the proprietor. Treatment name Components Release device Unbaited Control No lure None PEW PEW I and PEW II Centrifuge tube and rubber cap PEW PVC PEW I and PEW II PVC strips TRE 2150 E-β-ocimene Bubble cap TRE 2151 2-isobutyl-3-methoxypyrazine Bubble cap TRE 2152 E-β-ocimene, 2-isobutyl-3- PVC strips (PEW I and II) and methoxypyrazine, PEW I and bubble cap (other components) PEW II

The experiment consisted of 2 replicates and was repeated 3 times (6 replicates total) (Figure 3.2) between October 8 and 26, 2019. Traps were checked every 24 h, and any adults found on the adhesive surface were removed, sexed, and counted. After

5 days, all traps were removed from the greenhouse and additional weevils were added to the enclosed greenhouse area to account for any that had been captured or found dead. Sticky cards were replaced for each replication, but the same lures were used for all 3 of the experiments. The next pair of replicates was started 48 h later to allow any residual lure components to dissipate from the greenhouse.

A Shapiro-Wilk test was used to confirm the data displayed a normal distribution, and residuals were analyzed to confirm all other assumptions were met. An ANOVA was used to compare total A. eugenii captures among the 6 lure treatments over a 5-

day period using PROC GLIMMIX (SAS Studio, University Edition 2.6 9.4 M5). Lure treatment was a fixed effect, and replicate and date were random effects. Means comparisons using a Tukey’s test were performed for significant ANOVAs (α = 0.05).

This analysis was also performed for female captures and male captures separately.

3.3 Results

3.3.1 Field evaluation of commercial lures for Anthonomus eugenii

Lure treatment had a significant effect on the number of A. eugenii captures

(Table 3.3). Significantly more adults were captured on traps baited with the Trécé lure than the Russell IPM lure (P = 0.0220) and the unbaited sticky card (P = 0.0142) (Figure

3.3). Cards baited with the Russell IPM lure caught a single adult weevil over the course of the experiment. The majority of captures were recorded 4 d after the sticky cards were replaced with new cards. Adult weevils that were found on the sticky cards were all alive at the time of collection and most were observed near the edges of the sticky cards.

3.3.2 Addition of kairomones to pheromone lures for increased attraction of Anthonomus eugenii

No significant differences in the response of young mated female weevils were observed between treatment stimuli (Figure 3.4 A, Table 3.4); however, responses in the pheromone vs. the pheromone + kairomone treatment demonstrated a slightly higher, but non-significant, response to the pheromone stimulus without the added kairomones (Figure 3.4 A, Table 3.4).

Fixed effects Num df Den df F P Lure treatment 2 8 8.580 0.0102

Random effects Estimate Standard Error Chi Sq Pr > Chi Sq Replicate 0.133 0.379 0.15 0.351 Residual 1.030 0.517

Responses of young males did not significantly deviate from 50% for any treatment combination (Figure 3.4 B, Table 3.4); however, the male cohort was the only group out of the 4 cohorts tested that demonstrated a numerically higher, but non- significant, response to the pheromone + kairomone combination vs. the pheromone alone (Figure 3.4 B, Table 3.4).

Significantly more young unmated females responded to clean air over the kairomone treatment (Figure 3.4 C, Table 3.4), but this cohort did not show a significant difference in responses in other treatments (Figure 3.4 C, Table 3.4).

Finally, mature, unmated females did not show a significant response to any stimuli in any treatment combination (Figure 3.4 D, Table 3.4). Similar to results from the young unmated female cohort, the pheromone alone elicited a higher response compared to the combination of pheromone + kairomone but differences were not significant (Figure 3.4 D, Table 3.4). Mature unmated females responded less to the pheromone compared to clean air, but these differences were not significant (Figure 3.4

D, Table 3.4). Across all Y-tube bioassays, an average (±SE) of 24 ± 2.0% of weevils were considered non-responders.

Treatment Cohort Stimulus 1 Stimulus 2 χ² DF P Young Air Kairomone 2.13 1 0.144 females Air Pheromone 0.00 1 1.00 Kairomone Pheromone 1.20 1 0.273 Kairomone + Pheromone Pheromone 1.20 1 0.273 Young Air Kairomone 0.133 1 0.715 males Air Pheromone 1.20 1 0.273 Kairomone Pheromone 0.133 1 0.715 Kairomone + Pheromone Pheromone 0.533 1 0.465 Young Air Kairomone 10.8 1 0.001 unmated Air Pheromone 0.133 1 0.715 females Kairomone Pheromone 0.00 1 1.00 Kairomone + Pheromone Pheromone 2.13 1 0.144 Mature Air Kairomone 0.533 1 0.465 unmated Air Pheromone 2.13 1 0.144 females Kairomone Pheromone 0.533 1 0.465 Kairomone + Pheromone Pheromone 3.33 1 0.068

3.3.3 Evaluation of kairomone and pheromone lures for Anthonomus eugenii in an experimental greenhouse

Lure treatment had a significant effect on the total number of A. eugenii captures

(Table 3.5). Traps baited with TRE 2152 captured significantly more weevils than the current PEW lure and the unbaited control (Figure 3.5; Appendix Table 6.5). Traps baited with the standard pheromone lure with the PVC release device (PEW PVC)

Fixed effects Num df Den df F P Lure treatment 5 20 5.09 0.0036

Random effects Estimate Standard Error Chi Sq Chi Sq > P Date 2.320 2.810 2.51 0.113 Replicate 0.480 0.787 1.61 0.204 Trap location 0.281 0.380 0.39 0.534 Residual 2.760 1.040

caught the second highest number of weevils, but they did not catch significantly more than any other treatment (Figure 3.5; Appendix Table 6.5).

Captures of females were higher than males, and lure treatment was a significant source of variation (F = 3.92; df = 5, 20; P = 0.0122). Differences between lure treatments of female captures were consistent with the results of total captures and followed the same trend as shown in Figure 3.5. There were no differences in male captures among treatments (F = 2.24; df = 5, 20; P = 0.0899), likely due to low overall captures.

3.4 Discussion

During the field experiment, Trécé lures captured significantly more A. eugenii adults, and therefore, Trécé lures were used in subsequent experiments. Captures on

Trécé lure-baited traps in the field experiment were highest after the sticky cards were replaced with fresh cards. The adhesive surface area on sticky cards can be reduced over time from debris or bycatch (Riley and Schuster 1994, Bottenberg and Lingren

1998), which might explain why captures were highest after sticky cards were replaced.

Most of the adults captured on sticky cards were found alive, dorsal-side up, and near the borders of the cards, which indicates that those individuals were caught recently, could walk and find the edge of a card, and possibly escape the card. This observation reinforces the need to combine attractive semiochemical lures with traps that effectively retain captured A. eugenii individuals.

There were no significant differences in A. eugenii response to the pheromone and kairomone treatments in Y-tube olfactometry bioassays. In contrast, Muñiz-Merino et al. (2014) tested the same compounds and reported that males responded significantly more to the pheromone + kairomone stimulus over pheromone alone. In the present study, unmated females were tested in Y-tube bioassays to determine if responses would yield similar results to mated females or if mating status impacts semiochemical preference. Because no significant differences were found in either female cohort when comparing the pheromone and kairomone compounds, the hypothesis proposed by Addesso and McAuslane (2009) that mating status impacts semiochemical preferences was not supported by these results. In addition, female and male response to the aggregation pheromone was not significant in this experiment when compared to clean air, which is not consistent with results from other studies with

A. eugenii (Eller et al. 1994, Addesso and McAuslane 2009, Addesso et al. 2011,

Muñiz-Merino et al. 2014). Care was taken to ensure that standard Y-tube bioassay parameters and protocols used in previous studies were strictly followed, and therefore, it is very unlikely that the Y-tube design was a possible factor contributing to the lack of responses and significant results. However, a number of other factors alone or in combination, such as the low number of replicates, an issue with stimuli sources, or a lack of genetic diversity in the laboratory population used for the experiments might explain the lack of responses by A. eugenii observed in this study.

In this study, only 30 responders for each treatment were completed, whereas most other two-choice olfactory bioassays have included 50 or more responders

(Addesso et al. 2011, Collatz and Dorn 2013, Muñiz-Merino et al. 2014). The number of adults available for experiments was limited because of the absence of wild A. eugenii populations in Ontario and issues with achieving consistently high reproduction in the laboratory colony. With a greater number of responders, treatment effects may have been detected.

Contamination of one or more stimuli could explain the lack of response by A. eugenii; however, this is unlikely since standard cleaning and handling protocols for bioassay equipment were followed. Throughout the bioassays, multiple packaged pheromone lures from different shipments were used, reducing the likelihood that the lack of response to the pheromone lure, a known attractant to male and female A. eugenii, was simply due to a manufacturing error. However, as the lures were obtained through a commercial supplier, handling and storage procedures are unknown and could have affected lure performance. A headspace volatile analysis might have determined if the pheromone lures or other stimuli, such as the volatile blend, were not releasing appropriate ratios of semiochemicals to be detected by the weevils, but such an analysis was not performed.

The small size and genetic characteristics of the colony of A. eugenii used for experiments also could have impacted the response behaviour of adults used in experiments. The colony experienced multiple population bottlenecks: The colony was started from small samples of individuals from local greenhouse infestations and the

AAFC Harrow colony, and after establishment, it experienced multiple population

crashes. Because there were no sources of wild A. eugenii to supplement the colony with from December 2018 onward, the population from which adults were taken for experiments likely had reduced genetic diversity and may have been experiencing inbreeding depression. In turn, this may have impacted genetically based behavioural traits, like olfactory reception and/or response (Hansson and Stensmyr 2011, Leary et al. 2012), in the colony population, resulting in an unexpectedly high number of adults that did not respond to olfactory cues in the laboratory experiments.

The goal of the infested greenhouse experiment was to determine if the response by male and female A. eugenii to different semiochemicals reported by Muñiz-Merino et al. (2014) also would be observed in the presence of fruiting plants. Results from the infested greenhouse lure experiment did support some of the findings of Muñiz-Merino et al. (2014), namely that pheromone and kairomone stimuli together (TRE 2152) caught more weevils than the standard pheromone alone (PEW) or no lure. Although not significant, captures were numerically higher with kairomone lures (TRE 2151, TRE

2150) than the unbaited control and the current pheromone lure (PEW) in the present study. With more replicates, significant treatment differences may have been observed, further supporting previous findings that kairomones do elicit a higher response by females compared to no lure (Muñiz-Merino et al. 2014). Based on previous studies

(Addesso and McAuslane 2009, Addesso et al. 2011, Muñiz-Merino et al. 2014), it is likely that the TRE 2152 lure, or another lure that combines the male aggregation pheromone and the principal volatiles E-β-ocimene and 2-isobutyl-3-methoxypyrazine,

could be used to attract and trap male and unmated female A. eugenii and still maintain some attraction to mated females looking for oviposition sites.

Due to space limitations, the lures in the greenhouse experiment were less than

2 m apart, and therefore, overlap of lure plumes may have been a factor in the high captures observed on control cards and the lack of significant differences in captures between treatments. Further replicates could not be completed due to restrictions related to the COVID-19 pandemic. Trials with these lures in commercial greenhouses with wild populations were planned; however, there were no known commercial greenhouses infested with A. eugenii in 2019, and the pandemic prevented access to any commercial greenhouses in 2020. Experiments with these combinations of semiochemicals in infested fields or greenhouses should be conducted to determine male and female response to pheromone lures with added kairomones in the presence of fruiting plants, and if kairomones alone could be used to attract mated female A. eugenii, the most destructive cohort of A. eugenii, for monitoring or management strategies.

Although results from the Y-tube olfactometer bioassays and greenhouse experiments were inconclusive, other studies on Anthonomus species have found that host plant volatiles enhance adult response to pheromones in laboratory trials (Dickens

1989, Mechaber 1992, Szendrei et al. 2009). In a meta-analysis of behavioural manipulation of insect pests using plant volatiles, Szendrei and Rodriguez-Saona

(2010) emphasized that there is an abundance of published research on laboratory

interactions between plant volatiles and insects, but a lack of field evaluations of these relationships, especially when using them towards improving insect response for monitoring and management purposes. In field trials, a combination of pheromone and plant volatiles does not always attract more adult weevils than pheromone traps alone but can skew the sex ratio of trapped insects towards more female captures (Szendrei et al. 2011), which further emphasizes the importance of field trials to understand how semiochemical tools perform in a field or greenhouse setting with wild populations.

The presence of male-produced aggregation pheromones coupled with volatiles from host plants has the potential to affect the behavioural responses of male and female A. eugenii differently (Addesso and McAuslane 2009, Addesso et al. 2011).

Previous studies indicated that mating status might determine female response to semiochemicals, such as kairomones and aggregation pheromones (Addesso and

McAuslane 2009). Assuming that the primary goal of unmated females is to locate a mate, this would likely translate to an increased response to stimuli that include male- produced aggregation pheromones (Addesso et al. 2011). From these previous studies, it was hypothesized that unmated females would respond to the pheromone stimuli with or without added kairomones, whereas mated females would respond to the kairomone stimuli in the Y-tube bioassays without the need to combine with pheromones, but these patterns were not observed in the present study. More research is needed in both lab and field settings to test this hypothesis and identify optimal semiochemical blends that maximize attraction of both mated and unmated females to monitor and manage this pest successfully. Determining the optimal release rates of semiochemical components

is another important aspect of lure optimization to increase attraction to traps as insect olfactory receptors are sensitive to dose and purity when it comes to developing lures with synthetic compounds (Andersson et al. 2015). Dose response studies have not yet been conducted or reported in the literature for the plant volatiles tested in these experiments and should be considered.

The addition of the host plant volatiles E-β-ocimene and 2-isobutyl-3- methoxypyrazine shows promise for increasing A. eugenii captures on pheromone- baited sticky traps, and their use should be investigated further in greenhouse or field studies in the presence of fruiting pepper plants and wild A. eugenii populations. More specifically, attraction of A. eugenii of different mating statuses to specific volatiles at different doses warrants further investigation. As females pose the greatest threat to host plants through oviposition, semiochemical lures should be formulated to target females both before and after mating occurs to reduce the incidence of economic damage in cultivated peppers. In addition, developing a more attractive trap and lure combination in the presence of fruiting plants that optimizes captures is important to achieve more reliable population estimates for A. eugenii, which in turn would play a critical role in understanding population patterns and weevil distribution in cultivated peppers to allow for more targeted management protocol. Overall, a more attractive semiochemical lure would not only increase the efficacy of current monitoring tools but could also be integrated into management strategies for controlling A. eugenii populations, like mass trapping, to reduce economic damage to pepper crops.

4 General conclusions and discussion

Anthonomus eugenii is a relatively new pest to Ontario that caused approximately $67 million in damage, or up to 38% production loss, to greenhouse peppers in 2016 (Niki Bennett, Ontario Greenhouse Vegetable Growers Association, personal communication). Because most of its life stages occur within host fruit, A. eugenii is a challenging pest to manage, and therefore, effective monitoring to detect the arrival of adults to fields or greenhouses is critical. The goal of this research was to improve A. eugenii monitoring by quantifying trap captures of adults, identifying ways to maximize the retention of captures, and evaluating A. eugenii response to semiochemical lures to improve attraction to traps in the presence of fruiting plants.

The results from experiments that assessed A. eugenii adult movement on and escape from sticky cards, and the results from the alternative trap design experiments, suggest that the currently used Pherocon® sticky cards continue to be the most reliable type of monitoring trap for A. eugenii. However, given that A. eugenii were still able to move and escape from Pherocon cards, changes to the adhesive coating that improve the retention of A. eugenii are needed. Experiments with novel adhesives provided by

Trécé demonstrated that alternative adhesive formulations, such as TRE 270, could retain more A. eugenii than others under lab conditions (Chapter 2, Figure 2.7). Since the completion of these assays, a new adhesive formulation of Pherocon cards is now provided in Trécé Pherocon Pepper Weevil Kits that is similar in structure and retentive properties to TRE 270 (Brent Short, Trécé Inc., personal communication). Therefore,

the adhesive cards now supplied in Trécé Pherocon Pepper Weevil Kits should reduce the number of A. eugenii escapes from monitoring traps. Although these traps have yet to be tested specifically in field or greenhouse growing systems in Ontario, they currently are the best option available to growers for monitoring A. eugenii in cultivated peppers.

The finding that females were twice as likely to escape sticky cards than males was unexpected given that males possess an additional tibial spur (Eller 1992) that might provide more grip to walk over sticky surfaces. As this spur is absent in females, it is possible that their need to find suitable mates and oviposition sites may cause females to be more likely to walk to the edges of cards than males. Since all trapping experiments were conducted under laboratory conditions, a number of other independent variables that could impact A. eugenii escape were not assessed.

Abiotic conditions that could impact retentive properties of sticky cards were not examined through these experiments but could offer additional insights for A. eugenii monitoring programs, such as how often sticky traps should be checked under variable environmental conditions. Field peppers, in particular, experience significant changes in weather conditions such as exposure to sunlight, varied humidity levels, or rainfall events. Further experiments could be conducted to determine how retentive properties of sticky cards, and thus escape rates of A. eugenii, are impacted by abiotic factors in the field. With action thresholds for A. eugenii set at one adult per field, even a single escape can be costly and delay detection and management. Therefore, understanding

additional factors that affect adhesive performance and influence A. eugenii escapes in field traps would allow pest management specialists and extension agents to give improved recommendations on how often sticky cards should be checked and if certain abiotic conditions prompt more frequent checks of monitoring traps.

Although responses of A. eugenii to semiochemicals in Y-tube bioassays were inconclusive, further research should be conducted to determine if pheromone lures combined with kairomones can elicit a higher response from A. eugenii than aggregation pheromone lures alone. An increased response to pheromone lures with additions of host plant volatiles or kairomones has been demonstrated in other weevil pest species, such as Rhynchophorus palmarum L. (Oehlschlager et al. 1993),

Rhynchophorus ferrugineus Olivier (Hallett et al. 1999), Anthonomus grandis Boheman

(Dickens 1989), Anthonomus rubi Herbst (Wibe et al. 2014), and most recently in A. eugenii, where male response was increased by added kairomones in Y-tube assays

(Muñiz-Merino et al. 2014). A knowledge gap still exists in understanding how adding kairomones to pheromone lures affects the behaviour of unmated A. eugenii, as well as how male and female responses to lures are impacted by natural semiochemicals in the environment. Y-tube olfactory assays only provide a limited understanding of responder preference and do not take into consideration additional factors, such as environmental conditions, that drive insect response in a realistic setting (Ballhorn and Kautz 2013).

While results from the infested greenhouse experiment aimed to address these gaps, more data is needed to understand response to pheromone + kairomone lure treatments on a larger spatial scale and in the presence of fruiting plants.

The new Trécé pheromone lure coupled with a kairomone blend of (E)-β- ocimene and 2-isobutyl-3-methoxypyrazine captured more A. eugenii in the greenhouse experiment than the currently used pheromone lure and control, which suggests that kairomones may increase the attractiveness of the pheromone lure. However, this difference was not significant, and therefore, further testing of lures coupled with new adhesive sticky cards in the presence of fruiting plants is needed to conclusively determine if kairomone additions will improve the efficacy of A. eugenii monitoring in field and greenhouse conditions.

As most experiments were completed under laboratory conditions, an evaluation of new adhesives coupled with current or experimental lures in a field or greenhouse is still needed. Bycatch can be a significant factor limiting the success of monitoring A. eugenii with pheromone-baited sticky cards in cultivated peppers as the adhesive can become full of debris and non-target insect captures, often while the pheromone release rate is still effective (Riley and Schuster 1994). Increased bycatch reduces the adhesive surface area and retentive capacity of sticky traps, rendering them less effective overall.

In addition to other non-target captures that reduce adhesive surface area, several other weevil species that are morphologically similar to A. eugenii have been commonly found on A. eugenii monitoring traps throughout Ontario, including

Ceutorhynchus erysimi, Ceutorhynchus rapae, and Anthonomus signatus Say

(McCreary et al. 2019). Anthonomus eugenii shares components of its aggregation pheromone with other Anthonomus species, such as the strawberry blossom weevil (A.

rubi) (Innocenzi et al. 2001) and boll weevil (A. grandis) (Tumlinson et al. 1969), and therefore, C. erysimi, C. rapae, and A. signatus may be attracted to one or more components of the A. eugenii pheromone lure. It is possible that the addition of host plant compounds, such as (E)-β-ocimene, may also increase attraction of other non- target weevil species, such as A. signatus, a morphologically similar weevil found in

Ontario. Strawberry, the host plant of A. signatus, also releases (E)-β-ocimene (Kovach et al. 1999, Mozūraitis et al. 2020). Similar observations have been noted in experiments aimed to trap target weevil pests, such as the pea leaf weevil, Sitona lineatus, where use of specialized pheromone and kairomone baits captured high numbers of non-target weevils (St. Onge et al. 2017).

Some weevil species are morphologically similar to A. eugenii, and growers have misidentified non-target weevil trap captures as A. eugenii (McCreary et al. 2019).

Because control strategies should be implemented as soon as a single A. eugenii adult is detected on pheromone traps (Eller et al. 1994, Ingerson-Mahar et al. 2015), these false positive A. eugenii detections have led to unnecessary management actions in both field and greenhouse peppers (Cara McCreary, OMAFRA, personal communication). Management actions may include increased labour dedicated to thorough plant inspections and/or additional insecticide applications, both of which have costly impacts on pepper production (Fernández et al. 2020). Therefore, to improve A. eugenii monitoring it would be advantageous to examine solutions to reduce false positive identifications of A. eugenii, such as weevil identification training and resources or the development of rapid molecular detection techniques, that could be used to

positively identify A. eugenii within hours either in a nearby lab or by growers and scouts via an easy to use, portable lab test.

Interestingly, during the experiment to assess the efficacy of alternative trap types, jar traps caught an equivalent number of A. eugenii as sticky cards (Chapter 2,

Figure 2.6). Although jar traps might not be a suitable replacement for yellow sticky cards, which allow for quick visual inspection of A. eugenii adults, the use of jar traps as part of a mass trapping management strategy should be examined. Mass trapping is one of the older approaches to control insect populations (Steiner 1952) and has been successful for controlling weevil pests such as palm weevils R. ferrugineus (Hallett et al.

1999) and R. palmarum (Oehlschlager et al. 1993) where traps were baited with both pheromones and host plant materials. Mass trapping strategies have also been used successfully to eradicate the boll weevil, A. grandis, whereby host plant volatiles were used to synergize attraction to pheromone-baited collection traps (Hardee 1982,

Ridgway et al. 1990). The success of mass-trapping depends on a highly attractive semiochemical lure that can compete with natural sources of attraction, such as fruiting plants and aggregations of the target species (Jones 1998, El-Sayed et al. 2006).

Therefore, improved lures should be examined first before this management strategy is tested.

An important consideration for a mass trapping technique for A. eugenii is the need for a trap that kills the insect upon collection (Gregg et al. 2018). If jar traps paired with a highly attractive semiochemical lure were used in a field or greenhouse setting,

captured adults, specifically males, would need to be killed to prevent them from producing high levels of geranic acid (Eller and Palmquist 2014). High geranic acid production occurs when there is a high density of males in close proximity and is thought to signal resource depletion to other weevils (Eller and Palmquist 2014).

Therefore, a live trap that allows males to continuously produce pheromones in a localized spot would make the traps less attractive to incoming adults. Additionally, a semiochemical lure aimed at attracting mated and unmated female A. eugenii would be a very useful tool as females pose the greatest economic threat to cultivated peppers through oviposition (Campbell 1924). To date, kairomones have not been found to synergize female response to pheromone lures (Muñiz-Merino et al. 2014), but kairomone lures resulted in higher captures of females in the infested greenhouse experiments in the present study. If a highly attractive semiochemical lure can be developed that targets females, and jar traps can effectively capture and kill a large number of A. eugenii adults under field and greenhouse conditions, an mass trapping strategy could be another promising management tactic for this challenging pest. As initial infestations of A. eugenii in Ontario are usually localized and at low densities, these management strategies in combination with other currently used physical controls, such as mesh screening, and effective pest monitoring (McCreary et al. 2017), have a high probability of success (El-Sayed et al. 2006). Additionally, there is a lack of insecticides currently registered in Canada for A. eugenii, and the use of insecticides in greenhouses is not desirable because they can have negative impacts on biological

control agents (Labbé et al. 2020). Therefore, semiochemical-based mass trapping strategies show promise as part of an IPM program for A. eugenii.

This research determined that A. eugenii is currently most effectively monitored using the yellow sticky cards and aggregation pheromone lures provided in Trécé

Pherocon Pepper Weevil Kits (Trécé Inc. Adair, OK, USA). Although a small number of

A. eugenii escaped from the Pherocon cards during laboratory experiments, advancements to the retention properties of the adhesive on Pherocon cards have been made, and kits produced in 2020 and later have improved adhesive that should result in greater retention of A. eugenii. More investigation into the addition of kairomones to pheromone lures is needed both in lab and field trials. In Y-tube bioassays, neither unmated females nor mated females nor males responded more to pheromone lures coupled with a kairomone blend of (E)-β-ocimene and 2-isobutyl-3-methoxypyrazine compared to the pheromone alone. Dose responses to these individual compounds have not been reported in the literature and could be a next step in determining optimal compound ratios to increase response from A. eugenii adults. Sticky cards baited with the kairomone blend and the aggregation pheromone did have a significantly higher number of A. eugenii captures compared to unbaited cards and the pheromone control; however, additional experiments in a more realistic field or greenhouse setting are needed to determine if the kairomone blend is synergizing the response of A. eugenii to the pheromone lure. Future research should focus on optimizing semiochemical lures so they attract the maximum number of female A. eugenii, which would not only to

improve monitoring of the pest, but also contribute to development of better management tactics to be included in future A. eugenii IPM programs.

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6 Appendix

Standard Adjusted Treatment Treatment Estimate Error DF t value P Bugscan Dry Bugscan 0.0259 0.139 358 0.19 0.999 Bugscan Dry Horiver 0.214 0.137 358 1.56 0.522 Bugscan Dry Pherocon 1.20 0.147 358 8.14 <.0001 Bugscan Dry Tanglefoot 1.57 0.167 358 9.40 <.0001 Bugscan Horiver 0.188 0.139 358 1.35 0.660 Bugscan Pherocon 1.17 0.149 358 7.85 <.0001 Bugscan Tanglefoot 1.54 0.169 358 9.16 <.0001 Horiver Pherocon 0.986 0.147 358 6.69 <.0001 Horiver Tanglefoot 1.36 0.167 358 8.10 <.0001 Pherocon Tanglefoot 0.370 0.175 358 2.11 0.218

Standard Adjusted Treatment Treatment Estimate Error DF t value P Bugscan Dry Bugscan 0.638 0.333 429 1.91 0.312 Bugscan Dry Horiver 0.791 0.330 429 2.40 0.118 Bugscan Dry Pherocon 3.33 0.475 429 7.00 <.0001 Bugscan Dry Tanglefoot 4.78 0.756 429 6.32 <.0001 Bugscan Horiver 0.153 0.320 429 0.480 0.989 Bugscan Pherocon 2.69 0.465 429 5.79 <.0001 Bugscan Tanglefoot 4.14 0.751 429 5.51 <.0001 Horiver Pherocon 2.54 0.463 429 5.48 <.0001 Horiver Tanglefoot 3.99 0.749 429 5.32 <.0001 Pherocon Tanglefoot 1.45 0.816 429 1.78 0.389

Standard Treatment Treatment Estimate Error DF t value Adjusted P Modified Boll weevil trap pyramid trap -1.00 0.840 24 -1.19 0.837 Boll weevil trap Jar trap -12.7 0.873 24 -14.6 <.0001 Pherocon Boll weevil trap sticky card -14.3 0.840 24 -17.1 <.0001 Tanglefoot- Boll weevil trap coated card -14.0 0.840 24 -16.7 <.0001 Boll weevil trap Unitrap -8.17 0.840 24 -9.72 <.0001 Modified pyramid trap Jar trap -11.7 0.873 24 -13.4 <.0001 Modified Pherocon pyramid trap sticky card -13.3 0.840 24 -15.9 <.0001 Modified Tanglefoot- pyramid trap coated card -13.0 0.840 24 -15.5 <.0001 Modified pyramid trap Unitrap -7.17 0.840 24 -8.53 <.0001 Pherocon Jar trap sticky card -1.59 0.873 24 -1.83 0.469 Tanglefoot- Jar trap coated card -1.26 0.873 24 -1.44 0.701 Jar trap Unitrap 4.57 0.873 24 5.24 0.0003 Pherocon Tanglefoot- sticky card coated card 0.333 0.840 24 0.400 0.999 Pherocon sticky card Unitrap 6.17 0.840 24 7.34 <.0001 Tanglefoot-coated card Unitrap 5.83 0.840 24 6.94 <.0001

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Table 6.4: Pairwise comparisons (Tukey’s HSD) of mean number of Anthonomus eugenii adult escapes from 5 different sticky card traps types. Male and female adults were dropped on the sticky cards, ventral side down from 1 cm above the surface of each card in a grid layout. Any weevil that had left the surface of the sticky card over the 48 h experimental period was counted as escaped. The experiment was conducted at 23 °C with adult weevils (8-12 d old). Significant pairwise comparisons are bolded (P ≤ 0.05).

Treatment Treatment Estimate Standard Error DF t value Adjusted P 270 273 -3.83 1.93 20 -1.98 0.309 270 274 -8.33 1.93 20 -4.31 0.00280 270 275 -0.83 1.93 20 -0.430 0.992 270 STD -2.67 1.93 20 -1.38 0.647 273 274 -4.50 1.93 20 -2.33 0.177 273 275 3.00 1.93 20 1.55 0.543 273 STD 1.17 1.93 20 0.600 0.973 274 275 7.50 1.93 20 3.88 0.00740 274 STD 5.67 1.93 20 2.93 0.0565 275 STD -1.83 1.93 20 -0.950 0.874

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Standard Adjusted Treatment Treatment Estimate Error DF t value P 2150 2151 0.00139 0.891 20 0.00 1.00 2150 2152 -2.18 0.926 20 -2.35 0.221 2150 PEW 1.29 0.863 20 1.49 0.672 2150 PVC -1.20 0.812 20 -1.48 0.681 2150 UNB 1.59 0.839 20 1.90 0.433 2151 2152 -2.18 0.889 20 -2.45 0.186 2151 PEW 1.29 0.926 20 1.39 0.732 2151 PVC -1.20 0.835 20 -1.44 0.704 2151 UNB 1.59 0.863 20 1.84 0.464 2152 PEW 3.47 0.908 20 3.82 0.0119 2152 PVC 0.976 0.883 20 1.11 0.873 2152 UNB 3.77 0.894 20 4.21 0.00490 PEW PVC -2.49 0.893 20 -2.79 0.101 PEW UNB 0.301 0.906 20 0.33 0.999 PVC UNB 2.79 0.902 20 3.09 0.0554

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