Anthony Stephen Dimeglio Thesis Submitted to The
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Title: Murgantia histrionica (Hahn): new trapping tactics and insights on overwintering survival Name: Anthony Stephen DiMeglio Thesis submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Master of Science in Life Sciences In Entomology Thomas P. Kuhar, Chair Donald C. Weber, Co-Chair Dorothea Tholl September 12, 2018 Keywords: (Harlequin bug, Stink bug, Brassica, Trap, Aggregation pheromone, Murgantiol, Low temperature biology, pest management) Title Murgantia histrionica (Hahn): new trapping tactics and insights on overwintering survival ABSTRACT The harlequin bug, Murgantia histrionica (Hahn), is a serious pest of brassicaceous vegetables in southern North America, with limited establishment north of the 40°N latitude presumably due to low overwintering survival. Integrated Pest Management (IPM) requires knowledge of pest populations and tools to monitor them. For harlequin bug, knowledge of the number of successfully overwintered bugs, and development of an effective trap to monitor populations, are essential to its management. To gain insight into overwintering survival, I determined the supercooling points (SCPs) for Maryland and Virginia adult populations and found no significant difference between these populations. SCPs were similar for adults (푋̅ = -10.4oC; nd rd th th o 휎푋 = 2.5) and early (2 – 3 ) and late (4 – 5 ) instar nymphs (푋̅ = -11.0 C; 휎푋 = 4.9) and between st o adult males and females. However, SCPs for 1 instars (푋̅ = -21.6 C; 휎푋 = 1.5) and eggs (푋̅ = o -23.2 C; 휎푋 = 1.0) were significantly lower. Field survival of overwintering harlequin bug adults was significantly impacted (with 80-96% mortality) during widespread air temperatures lower than -15oC and sub-freezing soil temperatures in the mid-Atlantic region. Our results provide new information on M. histrionica overwintering biology, and thermal limitations to its distribution, which leads to improved predictive capabilities to forecast pest severity. To monitor harlequin bug activity for IPM recommendations an effective trap is necessary; at this time no such trap exists. The research presented herein contributes new knowledge of harlequin bug visual ecology, which will aid in the development of an effective trap. In both lab and field color choice experiments, harlequin bug adults and large nymphs responded positively to green and black colors, and statistically less frequently to yellow, white, purple or red with the exception of adult females, which were most attracted to red and green in the lab, but green and black in the field. To develop an effective trapping device for this pest, experiments were conducted in Virginia to assess factors to increase harlequin bug attraction to and arrestment at fixed artificial stimuli (“traps”) positioned within an agricultural landscape. In a laboratory experiment, harlequin bugs were effectively killed or severely impaired after a few minutes exposure to a synthetic pyrethroid-incorporated netting (D-Terrence®, Vestergaard-frandsen), and, thereafter, the netting was used as a toxicant on the trapping devices in the field. In one experiment, square corrugated plastic panels were wrapped with the insecticide netting and baited with harlequin bug aggregation pheromone, murgantiol. Bugs were effectively drawn to the panels, with green panels having significantly more dead harlequin bugs and fewer dead lady beetles (Coleoptera: Coccinellidae) at their base than yellow panels. Thus, green was chosen as the ideal trap color to use for another field experiment that evaluated three trap types – a corrugated plastic square panel, a pyramidal trap, and a ramp trap – each with three lure treatments, murgantiol alone or murgantiol plus a low or high rate of benzyl isothiocynate. More bugs were killed with the pyramidal trap than with the panel trap or the ramp trap, and more bugs were killed at traps containing murgantiol combined with benzyl isothiocyanate than at those with murgantiol alone. This research demonstrated that, with use of proper visual and semiochemical stimuli, harlequin bugs can be drawn to trapping devices and effectively killed after contact with deltamethrin-incorporated netting. GENERAL AUDIENCE ABSTRACT Harlequin bugs are orange and black aggregation pheromone emitting stink bug pests, specifically of cole crops such as kale, broccoli and collards. This nearly loyal crop preference makes an interesting challenge for trapping them and helping farmers predict pest severity. Harlequin bugs can be found in much of North America, and are a serious problem in the southeastern United States. Presumably their persistence into northern regions is limited by extreme winters. In 2014 and 2015 the arctic polar vortex extended into mid-latitudes bringing a blanket of sustained sub- freezing temperatures to much of the United States. We used these events to determine effects of extreme winter weather on harlequin bug survival. In both years we observed nearly identical low temperatures of -15oC and this linked to high (80-96%) harlequin bug mortality. In the lab we measured exact lethal freezing temperatures in harlequin bugs (i.e. supercooling points) to see if a physiological metric could be used to predict overwinter survival. Harlequin bug adults froze and died at -10.4oC, and similarly, their larger juvenile stages freeze at -11.0oC. Freshly hatched harlequin bugs and unhatched eggs froze at considerably lower temperatures with eggs forming ice crystals at -23.2oC and recent hatches at -21.6oC. Now with an understanding of how harlequin bugs likely survive winter extreme, we can then work on developing a trap to tally their populations in the spring and predict summer and fall pest severity. In the lab and field, harlequin bug adults and large nymphs were more likely found on green and black colors, and statistically less frequently on yellow, white, purple or red colors with the exception of adult females, which were most attracted to red and green in the lab, but green and black in the field. To increase harlequin bug attraction to and termination at traps square corrugated plastic panels were wrapped with an insecticide netting and baited with harlequin bug aggregation pheromone, murgantiol. Bugs were effectively drawn to the panels, with green panels having significantly more dead harlequin bugs and fewer dead beneficial lady beetles (Coleoptera: Coccinellidae) at their base than yellow panels. Thus, green was chosen as the ideal trap color to use for another field experiment that evaluated three trap types – a corrugated plastic square panel, pyramidal trap, and ramp trap – each with three lure treatments, murgantiol alone or murgantiol plus a low or high rate of mustard oil. More bugs were killed with the pyramidal trap than with the panel trap or the ramp trap, and more bugs were killed at traps containing murgantiol combined with benzyl isothiocyanate than at those with murgantiol alone. This research demonstrated that with the proper visual elements and odors, harlequin bugs can be drawn to traps and effectively killed after contact with insecticide- incorporated netting. Acknowledgements It is worth a listen: RadioLab host, Jab Abumrad, uses a theoretical biologist’s idea of the “adjacent possible” as a back-drop to describe how thoughtful stories culminate. Like an organism’s evolutionary history, this thesis is a fragment of a larger story of those most adjacent that provided the necessary environment to succeed. The ideas, experiments and narrative in this thesis were stimulated by many patient and understanding individuals. Dr. Donald C. Weber and many others at the USDA-ARS provided the mentorship and invaluable time in developing my critical eye on biological sciences. Dr. Thomas P. Kuhar, and his outstanding leadership, was vital in honing in many of my obscure ideas into formable research questions that emphasized on “do- good” outcomes. Dr. Dorothea Tholl shared a world novel to many, molecular biochemistry, and inspired me to push my limits of thought, inquiry and measurements. Dr. Tim Kring was an understanding force who kept the process going. A loud “thank you” is more than well deserved to my fellow Vegetable Insect Pest Research lab mates, and Department of Entomology and Biological Science colleagues for their support. As someone with bipolar, I understand how science can create both stimulating and degrading environments—and this thesis carries a legacy of extreme ups and downs. The people adjacent to me were affected greatly while I was ignorant to my condition. Learned from this thesis are lessons of openness and vulnerability. Through the patience of others, and surviving the struggles first hand, I was able to build the tools necessary to cope and grow past the condition. For those that are learning about their struggle with bipolar/depression as a daily survivor I encourage you to push forward and open up to those adjacent. The feelings you feel are OK and people will understand, and share their warmth. These financial donors made the successful defense of this thesis possible: Carolyn Elmore, Barbra Hiscock, Deborah Troehler, Meredith McQuoid-Greason, Gail Enright, Corinne Noirot, and Liane Ripley. Without their financial gifts, this thesis would be years delayed. Many grants and agencies supported this project and my coursework. Words cannot articulate well enough how the beautiful Lauris McQuoid-Greason was instrumentally vital in bringing this all together. She was there