1. Bioassay Components
<ol>
	<li>Terrestrial arena construction
	<ol>
		<li>Use metal flashing material strips of 2.54 cm length. Bend each strip into a half circle having a diameter of 15.24 cm (Figure 1).</li>
		<li>Connect the ends of each half circle and form a cloverleaf shape from the four pieces. Connect a locking nut and a #10 machine screw to the ends of each half circle to hold them together.</li>
		<li>Drill a hole at the midpoint of each semicircle 0.79 cm from...

<ol>
	<li>Briscoe, A. D., Chittka, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+color+vision+in+insects.">The evolution of color vision in insects.</a> Annu. Rev. Entomol. 46, 471-510 (2001).</li><li>Srinivasan, M. V., Chahl, J. S., Nagle, M. G., Zhang, S. W., Srinivasan, M. V., Venkatesh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embodying+natural+vision+into+machines.">Embodying natural vision into machines.</a> From living eyes to seeing machines. , 249-265 (1997).</li><li>Srinivasan, M., Moore, R. J. D., Thurrowgood, S., Soccol, D., Bland, D., Barth, F. G., Humphrey, J. A. C., Srinivasan, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Biology+to+engineering:+insect+vision+and+applications+to+robotics.">From Biology to engineering: insect vision and applications to robotics.</a> Frontiers in sensing. , 19-39 (2012).</li><li>Allan, S. A., Day, J. F., Edman, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+ecology+of+biting+flies.">Visual ecology of biting flies.</a> Annu.Rev. Entomol. 32, 297-316 (1987).</li><li>Brown, A. W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+the+responses+of+the+female+Aedes+mosquito+Part+V.+The+role+of+visual+factors.">Studies of the responses of the female Aedes mosquito Part V. The role of visual factors.</a> Bull. Entomol. Res. 44, 567-574 (1953).</li><li>Brown, A. W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+responses+of+the+female+Aedes+mosquito+Part+VI.+The+attractiveness+of+coloured+cloths+and+Canadian+species.">Studies on the responses of the female Aedes mosquito Part VI. The attractiveness of coloured cloths and Canadian species.</a> Bull. Entomol. Res. 45, 67-78 (1954).</li><li>Snyder, D., Cernicchiaro, N., Cohnstaedt, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sugar-feeding+status+alters+biting+midge+photoattraction.">Sugar-feeding status alters biting midge photoattraction.</a> Med. Vet. Entomol. 30, 31-38 (2016).</li><li>Menzel, R., Shmida, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ecology+of+flower+colours+and+the+natural+colour+vision+of+insect+pollinators:+The+Israeli+flora+as+a+study+case.">The ecology of flower colours and the natural colour vision of insect pollinators: The Israeli flora as a study case.</a> Biological Reviews. 68, 81-120 (1993).</li><li>Walker, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+selected+thysanoptera+to+colored+surfaces.">Responses of selected thysanoptera to colored surfaces.</a> Environ. Entomol. 3, 295-304 (1974).</li><li>Lelito, J. P., Fraser, I., Mastro, V. C., Tumlinson, J. H., Baker, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+visual-cue-based+stickytraps+for+monitoring+of+emerald+ash+borers,+Agrilus+planipennis+(Col.,+Buprestidae).">Novel visual-cue-based stickytraps for monitoring of emerald ash borers, Agrilus planipennis (Col., Buprestidae).</a> J. Appl. Entomol. 132, 668-674 (2008).</li><li>Diclaro, J. W., Cohnstaedt, L. W., Pereira, R. M., Allan, S. A., Koehler, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+and+Physiological+Response+of+Musca+domestica+to+Colored+Visual+Targets.">Behavioral and Physiological Response of Musca domestica to Colored Visual Targets.</a> J. Med. Entomol. 49 (1), 94D100 (2012).</li><li>Steverding, D., Troscianko, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+role+of+blue+shadows+in+the+visual+behaviour+of+tsetse+flies.">On the role of blue shadows in the visual behaviour of tsetse flies.</a> Proc. R. Soc. Lond. B. 271, 16-17 (2004).</li><li>Cohnstaedt, L. W., Gillen, J. I., Munstermann, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-emitting+diode+technology+improves+insect+trapping.">Light-emitting diode technology improves insect trapping.</a> J. Am. Mosq. Control Assoc. 24, 331-334 (2008).</li><li>Duehl, A. J., Cohnstaedt, L. W., Arbogast, R. T., Teal, P. E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+light+attraction+to+increase+trap+efficiency+for+Tribolium+castaneum+(Coleoptera:+Tenebrionidae).">Evaluating light attraction to increase trap efficiency for Tribolium castaneum (Coleoptera: Tenebrionidae).</a> J. Economic Entomol. 104, 1430-1435 (2011).</li><li>Schmid, R. B., Snyder, D., Cohnstaedt, L. W., McCornack, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hessian+Fly+(Diptera:+Cecidomyiidae)+Attraction+to+Different+Wavelengths+and+Intensities+of+Light-EmittingDiodes+in+the+Laboratory.">Hessian Fly (Diptera: Cecidomyiidae) Attraction to Different Wavelengths and Intensities of Light-EmittingDiodes in the Laboratory.</a> Environ. Entomol. 46 (4), 895-900 (2017).</li><li>Cohnstaedt, L. W., Snyder, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+features+of+a+proposed+insecticidal+sugar+trap+for+biting+midges.">Design features of a proposed insecticidal sugar trap for biting midges.</a> Vet. Ital. 52 (3-4), 265-269 (2016).</li></ol>

Photo-attraction bioassays are an important tool to determine the optimal attractive color(s) and minimize the options for field trials of these colors. However, several factors must be considered when optimizing the bioassay for a specific insect including: Single Light vs. competitive light experiments, brightness, optimal spectral range, ambient light interference, state of the insects, and natural behaviors that may limit the possible responses.
Most insects have some phototaxis, ...

Almost all entomological surveillance depends on olfaction or visual attractants and often both. Volatile olfactory attractants may disperse throughout the environment resulting in a large attractive area. However, visual attractants may have a more limited range because of the invertebrate compound eye resolving images1,2,3. Therefore, visual attractants must be optimized to the insect of interest to maximize attraction and the trap designed to take advantage of the target insect&#39;s natural behaviors.
Visual attraction is based on wavelengths from the sun or other sources of light that are absorbed or reflected by an object&#39;s surface; organisms view this absorption/refraction of wavelengths as color. Insect vision has been found to include blue, green, and ultraviolet (UV) wavelengths1. Insects use their vision to aid in finding mates, food, and shelter4. Insects can visually define object sizes, colors, shapes, movements and contrasts5,6. Nocturnally active insects are generally attracted to light of differing contrast and intensity4, whereas diurnal insects can resolve colors and images, in addition to contrast because of greater photon availability during the day. Monitoring traps use the insect&#39;s visual cues to their advantage to optimize attraction and capture7.
The most common method of evaluating photo-attraction was observation of insect movement towards various colored shapes such as flowers8&#160;or objects (such as sticky cards9,10). Visual bioassays using colonized insects can help identify the optimal range of wavelengths and/or intensities, which reduces the number of field trials. Visual bioassays such as the &#34;Two-Sided Light Tunnel&#34; were designed for testing flies11. The problem with two sided light tunnels are that they do not account for insects that are not collected. Most insects will get stuck on internal corners and along edges in arenas. Also only two colors can be tested at one time. Other assays include the methods of Steverding &#38; Troscianko (2004)12, which narrowed tsetse fly attraction to broad bands (&#177;50 nm) of light colors. Light emitting diodes (LEDs) have been incorporated into traps to improve insect attraction by optimizing the wavelengths of emitted light1,13,14. Optimizing the visual attraction of these traps or monitoring devices will improve insect collection efficiency by using the insect&#39;s innate behaviors to lure insects. In this way, bioassay results are used to optimize existing trapping technology. The &#34;Terrestrial Arthropod Trap&#34; that improved the industry standard dome-type trap for red flour beetle surveillance (US patent# US8276314B2)) and the &#34;Method and Compositions for Improved Light Traps&#34; that incorporated of light emitting diodes into aerial insect traps (US patent# US2009/0025275A1). The two patents use LED technology that was optimized using the bioassay results to significantly improve insect traps.
This study describes a photo attraction bioassay arena and methods that allow investigators to evaluate the insect response to narrow wavelengths as a competitive or single attractive color. Equipment and experimental modifications are presented for nocturnal, diurnal, terrestrial, and aerial insects.

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key elements of photo attraction bioassay for insect studies or monitoring programs

Optimized visual attractants will increase insect trapping efficiency by using the target insect&#39;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.

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 monito...

Watch this Scientific Journal Video about Key Elements of Photo Attraction Bioassay for Insect Studies or Monitoring Programs at JoVE.com

Key Elements of Photo Attraction Bioassay for Insect Studies or Monitoring Programs

Photo-attraction bioassay arenas are used to determine the optimal light color(s) to maximize insect attraction; however bioassays and methods are specific to target insect behaviors and habitats. Customizable equipment and modifications are explained for nocturnal or diurnal and terrestrial or aerial insects.

Optimized visual attractants will increase insect trapping efficiency by using the target insect&#39;s innate behaviors (positive photo-taxis) as a means ...

This method can help answer key questions in insect surveillance and control, by improving the attraction of existing traps and incorporating colors or lights into the design of future monitoring and control traps. The main advantage of this technique is the initial evaluation of attractive colors and insect behaviors in the lab, prior to the costly and time consuming tests in the field. Insects have innate behaviors or instincts that should be used to make monitoring and trapping more effective.The bioassay arena is designed to be a flexible method to evaluate behaviors in any insect species, prior to field trials. This protocol uses an aerial arena, but a terrestrial arena design is also available. The critical difference between the aerial and terrestrial arena's is the use of vertical space, which changes the size of the arena.When selecting an arena type for an experiment consider the insect species being studied. The aerial arena has the ability to capture insects in a collection container, whereas the terrestrial arena is a two dimensional object and really is designed for recording visits to the lights. To begin, disassemble and clean the aerial arena with an odorless, non abrasive soap and warm water.Then dry the arena and allow it to finish air drying. Before each experiment record the pertinent environmental conditions, and monitor any trends that occur from experiment to experiment. First, use four supports and black linen cloth to set up a pedestal.Then place the arena baseplate on top of the pedestal and assemble each piece of the arena on the baseplate. Next, place the cloverleaf arena around the central release point in the baseplate. Then install the LEDs into the LED holders of the four collection containers, and set up the electrical equipment to control the lights.To prepare the arena for a competitive experiment, position LED lights with distinct characteristics on each cloverleaf. The arena set up is the most important and critical factor, because simple changes in the arena can alter the insect's behaviors. Therefore, many factors can be changed or altered to address insect behaviors, such as photo attraction.Behaviors with feeding, so blood fed, or sugar fed or starved. Different life history stages or different physiological responses. Next, to control for potential effect of light interference or environmental preferences, rotate the light positions after every replicate.Refer to the text protocol for further information on filming devices. First place the clear lid over the arena, parallel to the baseplate. Then introduce immobilized midges to the arena.After the experimental period record the pertinent environmental conditions. Finally, after euthanizing the midges count the responders in each collection container. In this study the movement of red flower beetles was evaluated in a competitive style terrestrial arena for five minutes, and filmed with an infrared camera.Test colors included blue, and three wavelengths of UV light. The trial data indicated that the beetles were most attracted to near UV light with a wavelength of 390 nanometers. The movement of Hessian flies was also evaluated in a competitive style aerial arena with a diurnal setting.The lights were most attracted to lights in green wavelengths with high intensities. Female flies preferred the green spectra of 502 and 525 nanometers. While both sexes preferred the high intensity 16 watts per square meter light.Biting midges were also tested in the aerial arena under nocturnal conditions, to determine optimal attractive colors. The highest proportions of midges were attracted to UV light, with the brightest lights being the most attractive. Once mastered, the photo attraction bioassay can be used to rapidly assess insect behavior, prior to costly and labor intensive field trials.

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Behavior

7.6K visualizações.  Agricultural Research Service, United States Department of Agriculture. This method can help answer key questions in insect surveillance and control, by improving the attraction of existing traps and incorporating colors or lights into the design of future monitoring and control traps. The main advantage of this technique is the initial evaluation of attractive colors and insect behaviors in the lab, prior to the costly and time consuming tests in the field. Insects have innate behaviors or instincts that should be used to make monitoring and trapping more effect...