Improved Trapping Strategies for Managing Harlequin Bug: Applying recent research and discovery of its aggregation pheromone as a tool for vegetable growers

Objective 1. Preference in harlequin bug for certain visual cues such as color and trap-architecture. (manuscript in press in Environmental Entomology 2017)

Obj. 1A. HB preference for colors

Materials and Methods

Insect cultures. Field-collected M. histrionica adults from Painter, VA (37^o 35’04.61” N, 75^o 49’15.64” W; elevation = 9 m) in mid-April 2015 initiated a colony for this study. Bugs were reared on potted collards (Brassica oleracea: VATES or Champion) in a greenhouse under summer-like conditions (16:8 L:D and 20 to 30^oC). Adults and large nymph (4^th through 5^th instars) were sourced in our laboratory color choice experiments.

Laboratory Color Choice Assay. Corrugated polypropylene (Coroplast, Inteplast Group, Vanceburg, KY) panels (dimensions at 0.6m × 0.6m × 4mm thickness) were assembled with clear packing tape into a hexagonal choice-test arena with in an interior diameter = 1.42 m interior diameter.   Reflectance spectra data was collected using a DH-2000-BAL light source (Ocean Optics Inc., Dunedin, FL), Ocean Optics SpectraSuite software (Ocean Optics Inc., Dunedin, FL) and a USB4000 Spectrometer (Ocean Optics Inc., Dunedin, FL) calibrated with a Labsphere Reflectance Standard AS-01158-060 USRS-99-010 BZ37A (Labsphere, Inc, North Sutton, NH). The arena was designed for a 6-way color choice test including these specific colors: green (510nm), red (624nm), black (no spectral peaks), white (420 - 880nm), yellow (568nm), and purple (440nm, 677nm). The hexagonal arena was illuminated under “cool-white” (4100K) fluorescent lighting   (Philips Lighting Corporation, Somerset, NJ) with ambient temperatures ranging from 20 – 23^oC, and rotated in a six times in order to eliminate panel location bias.

Randomly-selected harlequin bugs were grouped by sex and life stage and starved 12 to 16 h before conducting experiments.  Grouped bugs were organized into units of five individuals in a 200ml centrifuge tube and immobilized on ice for three minutes before gently introducing them into the center of the arena. Five minutes were allotted for all bugs to disperse and select a color panel once one of the five bugs regained motion. Color selection “choice” was determined if an individual bug made contact anteriorly or oriented within 2 cm of the colored panel.

With three replicates of the experiment, a total of 89 females, 86 males and 91 late-instar nymphs were assayed. Bugs tested had no prior experience to the arena and were not reused in subsequent assays.

Field experiment. A six-way color choice experiment using corrugated polypropylene, with the same dimension and colors listed in our lab color choice assay, was conducted at Kentland Farm in Whitethorne, VA (37^o 12’06.73”N, 80^o 33’52.27”W; elevation = 517 m) in early autumn 2015.   Color panels were deployed as traps in a bare tilled field without host plants to intercept local populations of harlequin bugs on September 25, 2015. Trap surfaces were treated with a pyrethroid insecticide bifenthrin (Bifenture 2E, United Phosphorous Corporation, King of Prussia, PA), at 0.2 g per liter applied with a manual pressurized backpack sprayer (Solo Sprayers, Newport News, VA) to knock down intercepted bugs. Naturally dispersing populations were assumed to be highest near a 2015 spring-and-summer mustard and collard field, and therefore we situated the trap transect   6 m distant from the edge of, and parallel to, the east-west axis of the field  with the faces of the square traps exposed north and south.

Six weeks before the field color choice experiment, the collard and mustard field was mowed (leaving ca. 0.5 m tall residue) and seeded with rye (Secale cereale L.) cover crop to encourage a slow dispersal of harlequin bugs out of the field. The color choice test transect was a randomized block design of five replicate series with traps spaced 3.66 m on center. One week before deploying the traps, the infested field was mowed low (leaving ca. 0.15 m host residue, at this point fairly desiccated) to encourage dispersal of harlequin bugs. Dead harlequin bugs were collected within a 0.2 m radius of each panel trap centroid 48 h after setting out traps, and every 72 h for 9 days. 

Statistical Analysis. All data were analyzed in JMP 11.0.0 (SAS Institute, Cary, NC). Residuals conformed to normality, therefore one-way and two-way ANOVA were used to test for statistical difference in color response from lab and field experiments, and Fisher’s exact test for determining group differences in lab assay.  Data reported were non-transformed with α set at =0.05. Mean numbers of bugs responding were separated with a post-hoc Tukey’s HSD test.

Obj. 1B: Lethal time on commercial deltamethrin-treated screens (long-lasting insecticidal mosquito nets)

In order to determine the feasibility of using deltamethrin-treated screens to kill harlequin bugs in a future trap device, we needed to first determine if the treated screens were effective at immobilizing and/or killing harlequin bugs.  

Dates: June 12 – 14, 2015

Treatments:

Black DeTerrance™ mesh from Vestergaard frandsen - cut 120mm circles + excised collard leaf - Exposure times: 90, 45, 30, 15, 5, 1 minutes    

Control no-mesh (collard leaf)

Design:

Four replicates of deltamethrin-treated mesh (DeTerrance™) were cut into 120mm circles to fit snug into respective Petri dishes. Field collected harlequin bug late instars (3^rd to 4^th) from Beltsville, MD were separated into random groups of 10 bugs. Each group of nymphs were gently placed ventral side down on the mesh/Petri dish combo, with the lid fit snug to limit upward movement onto untreated surfaces of the Petri dish. Exposure times followed at 90, 45, 30, 15, 5, & 1 minutes by removing the mesh from respective Petri dishes. Immediately after timed exposure bugs were asses for mortality, moribundity, and no visible effects on motor response. Bugs were retained in respective petri dishes for post exposure effects (moribund, mortality & no-effects) at 24, 32, & 56 hrs.  Data were analyzed using Simple Probit Analysis (JMP Software, SAS Institute: SimpleProbit.jmpaddin). Time in minutes was transformed logarithmically (alpha set at 0.05) to predict LT50, LT75, LT90 and LT95.

Obj. 1C: To evaluate the field response of HB to dark green versus yellow color in the presence of murgantiol pheromone

Dates: Sept.8.2015 – Sept.11.2015

Treatments:

Design:

Green versus Yellow Corrugated plastic panels (each baited with a single murgantiol lure) were wrapped in a single layer of deltamethrin treated mesh. For green panels we used black mesh since we did not have a commercially available green insecticide treated mesh. As for the yellow panels, we used yellow insecticide treated mesh.

Green and yellow panels were grouped in a two-way choice test blocked in four replicates around overwintered collard plot. Treatments were spaced 10m apart and replicates spaced 33m apart. The overgrown, weedy, collard plot was inspected for harlequin bugs prior to setting up the experiment. Panels were centered in 1.0m2 of bare soil, to limit inference from plant visual and volatile cues. Additionally, the trap was situated flush to the soil surface to ensure bugs were able to make an unobstructed contact with the trap.  With panels arranged the plot was high mowed (image #) to disperse resident insects. At 48hrs, dead harlequin bugs and coccinellid species (determined with field ID markers) were counted within 30cm of the trap centroid.

Obj. 2: To evaluate the efficacy of the harlequin bug aggregation pheromone, murgantiol with and without the addition of mustard oil volatiles.

Dates: Sept.8.2015 – Sept.11.2015

Field experiment was conducted at Kentland Farm in Whitethorne, VA to determine effective shape of dark-green trap devices, and determine significance of benzyl isothiocynate at high and low emissions as a synergist to murgantiol.

Treatments:

Square vs. Pyramid vs. Ramp trap combined with either murgantiol, murgantiol l+ low benzyl isothiocynate, or murgantiol + high benzyl isothiocynate

Design: Randomized complete block design.  A pyramid trap, used by Leskey et al. (2015) in brown marmorated stinkbug trapping traps, ramp trap used in trapping banana weevil and square panel used earlier in lab color choice test, were evenly coated with dark green spray paint (Rust-Oleum, Gloss Hunter Green, UltraCover Paint+Primer, Vermon Hills, IL). Traps are spaced nine feet apart from their centroid along the east-west transect of a field margin south of two fields with summer cole crops; the most adjacent were collards (var. champion) neighbored by a mustard trap crop experiment.

One week prior to establishing treatments host residue in neighboring plots was mowed low to encourage harlequin bug dispersal. Five days later cabbage seedlings were planted in-between trap locations, at three to a row by three rows.

Nine treatments in total for each of the four blocks, which were each baited with a single murgantiol septa (loaded at ##) with and without either low or high doses of benzyl isothiocynate (technical grade, 94%, Sigma Aldrich). Low and high volatile emissions rates were dispensed with one and two, respectively, 75mm capillary tubes with _#_ interior diameter (Fischer Scientific Co., Pittsburgh, Pa.; cat. no. 02-668-68). Once in the field capillary tube(s) were pierced through Teflon seal in a 1ml gc-vial () loaded with 200ul of BITC.  Individual vials and septum were affixed 15 – 25 cm from the soil surface to traps with a small green zip-tie, and stapled as necessary.

Trap surfaces were treated with a pyrethroid (Bifenthrin, United Phosphorous Corporation, at 3.2ml/ga + surfactant mixed in a backpack sprayer) to knockdown intercepting bugs for more accurate counts. Dead harlequin bugs were collected within 20cm radius of trap centroid 72hrs after setting out the traps. Since the bugs were so numerous (several thousand individuals), counts were tallied later in the laboratory, accounting for sex and life stage (adult versus nymph).