1. Bioassay Components
2. Arena Preparation
3. Starting Bioassays
4. Ending and Quantifying Bioassays
NOTE: The duration of each experimental replicate will depend on insect behavior and response timing, in general use a longer exposure, more responses tend to be more informative.
The terrestrial arena has been used to improve pest monitoring traps for red flour beetles14 and the aerial arenas for hessian flies15 and biting midges7. Although the cloverleaf arenas were similar, the conditions for each insect species were different and accommodated the evaluation of nocturnal or diurnal insects that can crawl or fly. More importantly these lab studies translated into field applications for monitoring insect pest population changes, introduction of invasive species, population suppression, and/or disease vector risk assessments.
The red flour beetles, a stored product pest, were evaluated in the terrestrial arena and filmed using an infrared camera14. Responses were considered positive for a color, if a beetle moved towards and contacted the LED. The arena setup was a competitive style with four lights or three lights and a dark blank for control. The trial data indicates the beetles were most attracted to near UV LED (390 nm) (Figure 4). This information was used to make a better red flour beetle trap using an octagonal UV LED array, which resulted in a 20% increase in collection compared to a 1% capture rate with the original pheromone attractant alone.
Hessian flies, wheat field crop pests were evaluated for photo attraction using the aerial arena with a diurnal setting15. Hessian flies were most attracted to green wavelengths with high intensities (Figure 5). Females preferred the green spectra of 502 and 525 nm. However, both sexes preferred high intensity light (16 W/m2). This is the first report of Hessian fly attraction to select emitted wavelengths and intensities from LEDs under controlled conditions. These results are being used to develop a better Hessian fly detection trap for uninfested wheat fields.
The disease vector biting midge, Culicoides sonorensis can transmit viruses, which in cervids, ovids, and bovids may result in epizootic hemorrhagic disease or blue tongue disease. C. sonorensis were tested using the aerial arena under nocturnal conditions to determine the optimal colors that attracted sugar seeking biting midges7. The highest proportions of biting midges were attracted to ultraviolet (UV) light and light intensity was important with the brightest lights being most attractive (Figure 6). Sugar-seeking and escape behaviors were triggered by 355 nm and 365 nm in wavelength respectively and the biting midges distinguished between the two-colored lights. Using these wavelengths, the attraction of C. sonorensis to light traps can be improved and the lights have been incorporated into insecticidal sugar traps16.
Figure 1: This drawing reflects the dimension of the Terrestrial arena. The release point at the middle of the arena as well as points of LED attachments at the apex of each half circle are labeled. Also presented is an example of a conical light projection from an LED. The optimal viewing angle of the LEDs is 45° although the arena design allows for more narrow or broad viewing angles as the half circles will limit light crossover except at the middle of the arena. The terrestrial arena has a lower profile compared to the aerial arena because the insects do not need space to fly, which helps video recordings stay focused on the insects. Please click here to view a larger version of this figure.
Figure 2: The aerial light assay arena constructed from clear acrylic although it has all the same design benefits of the terrestrial arena but allows for more vertical space for flying insect evaluation. Four collection containers have LEDs of various wavelengths illuminating their respective apex of the cloverleaf. This figure shows the arena set up competition style with red, green, blue, and UV lights. Please click here to view a larger version of this figure.
Figure 3: The electrical schematic of a 6 V DC power source attached to variable resistors (potentiometers) that control the power to each LED (light emitting diode) so the intensity of each LED can be adjusted independently. Neutral density paper can also be used to reduce the intensity without altering the emitted wavelengths. Wavelength and wavelength range are adjusted by selecting different LED chemistries. Please click here to view a larger version of this figure.
Figure 4: (Top) The movement of ten red flour beetles for 5 min was assessed in the cloverleaf arena. A visit was defined movement towards a color resulting in touching the LED. Tested colors were blue (410 nm) and UV (390, 380, and 360 nm). Standard error bars are indicated and significant differences are denoted by letters (p<0.0001), different letters indicate significantly different means. (Bottom) Further evaluation of movement with lower intensity colors was similar to above but with the colors UV (390 nm), green (555 nm), red (655 nm), and yellow (587 nm). (Figure 4 was reprinted from Duehl et al. 2011 with permission.) Please click here to view a larger version of this figure.
Figure 5: Males and female Hessian flies were evaluated for photo attraction separately to prevent cofounding factors. (A-C) are female fly responses and (D-F) are male. Significant differences are indicated by different letters (P <0.05), different letters indicate significantly different means. (A and D) Both males and females were significantly attracted to green (527 nm) compared to red (624 nm), amber (590 nm), and blue (472 nm). (B and E) Within the green spectra 502-525 nm was most attractive and (C and F) intensity of light was important. (Figure was reprinted from Schmid et al. 2017 with permission.) Please click here to view a larger version of this figure.
Figure 6: (Top) Culicoides sonorensis were attracted significantly more to UV light than blue, green, or red. Different letters indicate significantly different means (P <0.05), different letters indicate significantly different means. Sugar meals were provided prior to each replicate. (Bottom) Attraction to light intensity was assessed using Culicoides sonorensis movement towards the same UV light, but at different intensities (4, 8, and 12 watts) and a blue light (24 watts). (Figure was reprinted from Snyder et al. 2016 with permission.) Please click here to view a larger version of this figure.
Supplemental Table 1: General LED table for wavelengths. More narrow LED wavelengths do exist; this list just shows broad ranges of LEDs that exist in the insect's vision spectra. Please click here to download this file.
metal flashing material | |||
#10 stainless steel machine screw | Stock | ||
#10 stainless steel locking nut | Stock | ||
5-mm LED holder | Radio Shack Corp | 276-080 | |
matte black spray paint | Stock | ||
Fluon | Stock | ||
molded polyacrylic | |||
screw top Nalgene | Thermo Fisher Scientific | Nunc polymethylpentene | 125 mL, 64 mm outer diameter, 74 mm height |
Threaded Teflon pipes | Stock | 15 mm diameter, 60 mm length | |
StellarNet light spectrometer | Stellar Net, Inc | BLACK Comet C-SR-25 | |
LED infrared light source | Tracksys LTD | ||
infrared video camera | Panasonic Corp | WV-BP330 Panasonic CCTV camera | |
MEDIACRUISE software | Canopus Corp |
Optimized visual attractants will increase insect trapping efficiency by using the target insect's innate behaviors (positive photo-taxis) as a means to lure the insect into a population control or monitoring trap. Light emitting diodes (LEDs) have created customizable lighting options with specific wavelengths (colors), intensities, and bandwidths, all of which can be customized to the target insects. Photo-attraction behavioral bioassays can use LEDs to optimize the attractive color(s) for an insect species down to specific life history stages or behaviors (mating, feeding, or seeking shelter). Researchers must then confirm the bioassay results in the field and understand the limited attractive distance of the visual attractants.
The cloverleaf bioassay arena is a flexible method to assess photo attraction while also assessing a range of natural insect behaviors such as escape and feeding responses. The arena can be used for terrestrial or aerial insect experiments, as well as diurnal, and nocturnal insects. Data collection techniques with the arena are videotaping, counting contact with the lights, or physically collecting the insects as they are attracted towards the lights. The assay accounts for insects that make no-choice and the arenas can be single (noncompetitive) color or multiple (competitive) colors. The cloverleaf design causes insects with strong thigmotaxis to return to the center of the arena where they can view all the options in a competitive LED tests. The cloverleaf arena presented here has been used with mosquitoes, bed bugs, Hessian fly, house flies, biting midges, red flour beetles, and psocids. Bioassays are used to develop accurate and effective insect traps to guide the development and optimization of insect traps used to monitor pest population fluctuations for disease vector risk assessments, the introduction of invasive species, and/or be used for population suppression.
Optimized visual attractants will increase insect trapping efficiency by using the target insect's innate behaviors (positive photo-taxis) as a means to lure the insect into a population control or monitoring trap. Light emitting diodes (LEDs) have created customizable lighting options with specific wavelengths (colors), intensities, and bandwidths, all of which can be customized to the target insects. Photo-attraction behavioral bioassays can use LEDs to optimize the attractive color(s) for an insect species down to specific life history stages or behaviors (mating, feeding, or seeking shelter). Researchers must then confirm the bioassay results in the field and understand the limited attractive distance of the visual attractants.
The cloverleaf bioassay arena is a flexible method to assess photo attraction while also assessing a range of natural insect behaviors such as escape and feeding responses. The arena can be used for terrestrial or aerial insect experiments, as well as diurnal, and nocturnal insects. Data collection techniques with the arena are videotaping, counting contact with the lights, or physically collecting the insects as they are attracted towards the lights. The assay accounts for insects that make no-choice and the arenas can be single (noncompetitive) color or multiple (competitive) colors. The cloverleaf design causes insects with strong thigmotaxis to return to the center of the arena where they can view all the options in a competitive LED tests. The cloverleaf arena presented here has been used with mosquitoes, bed bugs, Hessian fly, house flies, biting midges, red flour beetles, and psocids. Bioassays are used to develop accurate and effective insect traps to guide the development and optimization of insect traps used to monitor pest population fluctuations for disease vector risk assessments, the introduction of invasive species, and/or be used for population suppression.
Optimized visual attractants will increase insect trapping efficiency by using the target insect's innate behaviors (positive photo-taxis) as a means to lure the insect into a population control or monitoring trap. Light emitting diodes (LEDs) have created customizable lighting options with specific wavelengths (colors), intensities, and bandwidths, all of which can be customized to the target insects. Photo-attraction behavioral bioassays can use LEDs to optimize the attractive color(s) for an insect species down to specific life history stages or behaviors (mating, feeding, or seeking shelter). Researchers must then confirm the bioassay results in the field and understand the limited attractive distance of the visual attractants.
The cloverleaf bioassay arena is a flexible method to assess photo attraction while also assessing a range of natural insect behaviors such as escape and feeding responses. The arena can be used for terrestrial or aerial insect experiments, as well as diurnal, and nocturnal insects. Data collection techniques with the arena are videotaping, counting contact with the lights, or physically collecting the insects as they are attracted towards the lights. The assay accounts for insects that make no-choice and the arenas can be single (noncompetitive) color or multiple (competitive) colors. The cloverleaf design causes insects with strong thigmotaxis to return to the center of the arena where they can view all the options in a competitive LED tests. The cloverleaf arena presented here has been used with mosquitoes, bed bugs, Hessian fly, house flies, biting midges, red flour beetles, and psocids. Bioassays are used to develop accurate and effective insect traps to guide the development and optimization of insect traps used to monitor pest population fluctuations for disease vector risk assessments, the introduction of invasive species, and/or be used for population suppression.