Name: using flight mills to measure flight propensity and performance of western corn rootworm, diabrotica virgifera virgifera (leconte)
Uploaded: 2019-10-29T12:30:00
Description: Watch this Scientific Journal Video about Using Flight Mills to Measure Flight Propensity and Performance of Western Corn Rootworm, Diabrotica virgifera virgifera LeConte at JoVE.com

E.Y.Y.&#8217;s graduate assistantship was supported by the National Science Foundation I/UCRC, the Center for Arthropod Management Technologies, under Grant No. IIP-1338775, and industry partners.

1. Rear western corn rootworm for flight tests
NOTE: If the adult&#8217;s age must be controlled or known, adults must first be collected in the field followed by rearing their offspring to adulthood for testing. If the age of the beetle or a standardized rearing environment is not of concern, then directly testing field-collected adults may be possible, and the protocol can begin with step 2.
<ol>
	<li>Collect at least 500 western corn rootworm adults from a cornfield of interest to ensure ...

<ol>
	<li>Gillette, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabrotica+virgifera+Lec.+as+a+corn+root-worm.">Diabrotica virgifera Lec. as a corn root-worm.</a> Journal of Economic Entomology. 5 (4), 364-366 (1912).</li><li>Rice, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+rootworm+corn:+assessing+potential+agronomic,+economic,+and+environmental+benefits.">Transgenic rootworm corn: assessing potential agronomic, economic, and environmental benefits.</a> Plant Management Network. , (2004).</li><li>Gray, M. E., Sappington, T. W., Miller, N. J., Moeser, J., Bohn, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+and+invasiveness+of+western+corn+rootworm:+Intensifying+research+on+a+worsening+pest.">Adaptation and invasiveness of western corn rootworm: Intensifying research on a worsening pest.</a> Annual Review of Entomology. 54 (1), 303-321 (2009).</li><li>Martinez, J. C., Caprio, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IPM+use+with+the+deployment+of+a+nonhigh+dose+Bt+pyramid+and+mitigation+of+resistance+for+western+corn+rootworm+(Diabrotica+virgifera+virgifera).">IPM use with the deployment of a nonhigh dose Bt pyramid and mitigation of resistance for western corn rootworm (Diabrotica virgifera virgifera).</a> Environmental Entomology. 45 (3), 747-761 (2016).</li><li>Miller, N. J., Sappington, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+dispersal+in+resistance+evolution+and+spread.">Role of dispersal in resistance evolution and spread.</a> Current Opinion in Insect Science. 21, 68-74 (2017).</li><li>Spencer, J. L., Hibbard, B. E., Moeser, J., Onstad, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behaviour+and+ecology+of+the+western+corn+rootworm+(Diabrotica+virgifera+virgifera+LeConte).">Behaviour and ecology of the western corn rootworm (Diabrotica virgifera virgifera LeConte).</a> Agricultural and Forest Entomology. 11, 9-27 (2009).</li><li>VanWoerkom, G. J., Turpin, F. T., Barret, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+constant+and+changing+temperatures+on+locomotor+activity+of+adult+western+corn+rootworms+(Diabrotica+virgifera)+in+the+laboratory.">Influence of constant and changing temperatures on locomotor activity of adult western corn rootworms (Diabrotica virgifera) in the laboratory.</a> Environmental Entomology. 9 (1), 32-34 (1980).</li><li>Naranjo, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+flight+behavior+of+Diabrotica+virgifera+virgifera+and+Diabrotica+barberi+in+the+laboratory.">Comparative flight behavior of Diabrotica virgifera virgifera and Diabrotica barberi in the laboratory.</a> Entomologia Experimentalis et Applicata. 55 (1), 79-90 (1990).</li><li>Stebbing, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flight+behavior+of+methyl-parathion-resistant+and+-susceptible+western+corn+rootworm+(Coleoptera:+Chrysomelidae)+populations+from+Nebraska.">Flight behavior of methyl-parathion-resistant and -susceptible western corn rootworm (Coleoptera: Chrysomelidae) populations from Nebraska.</a> Journal of Economic Entomology. 98 (4), 1294-1304 (2005).</li><li>Dobson, I. D., 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=Analysis+of+long-range+reproductive+behavior+of+male+Diabrotica+virgifera+virgifera+LeConte+and+D.+barberi+Smith+and+Lawrence+to+stereoisomers+of+8-methyl-2decyl+propanoate+under+laboratory+conditions.">Analysis of long-range reproductive behavior of male Diabrotica virgifera virgifera LeConte and D. barberi Smith and Lawrence to stereoisomers of 8-methyl-2decyl propanoate under laboratory conditions.</a> Journal of Chemical Ecology. 13 (6), 1331-1341 (1987).</li><li>Spencer, J. L., Isard, S. A., Levine, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+flight+of+western+corn+rootworm+(Coleoptera:+Chrysomelidae)+to+corn+and+soybean+plants+in+a+walk-in+wind+tunnel.">Free flight of western corn rootworm (Coleoptera: Chrysomelidae) to corn and soybean plants in a walk-in wind tunnel.</a> Journal of Economic Entomology. 92 (1), 146-155 (1999).</li><li>Coats, S. A., Tollefson, J. J., Mutchmor, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+migratory+flight+in+the+western+corn+rootworm+(Coleoptera:+Chrysomelidae).">Study of migratory flight in the western corn rootworm (Coleoptera: Chrysomelidae).</a> Environmental Entomology. 15 (3), 620-625 (1986).</li><li>Coats, S. A., Mutchmor, J. A., Tollefson, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+migratory+flight+by+juvenile+hormone+mimic+and+inhibitor+in+the+western+corn+rootworm+(Coleoptera:+Chrysomelidae).">Regulation of migratory flight by juvenile hormone mimic and inhibitor in the western corn rootworm (Coleoptera: Chrysomelidae).</a> Annals of the Entomological Society of America. 80 (5), 697-708 (1987).</li><li>Kennedy, J. S., Ainsworth, M., Toms, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+studies+on+the+spraying+of+locusts+at+rest+and+in+flight.">Laboratory studies on the spraying of locusts at rest and in flight.</a> Anti-Locust Bull. L. 2, 64 (1948).</li><li>Krogh, A., Weis-Fogh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Roundabout+for+studying+sustained+flight+of+Locusts.">A Roundabout for studying sustained flight of Locusts.</a> Journal of Experimental Biology. 29, 211-219 (1952).</li><li>Attisano, A., Murphy, J. T., Vickers, A., Moore, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+flight+mill+for+the+study+of+tethered+flight+in+insects.">A simple flight mill for the study of tethered flight in insects.</a> Journal of Visualized Experiments. (106), e53377 (2015).</li><li>Okada, R., Pham, D. L., Ito, Y., Yamasaki, M., Ikeno, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+flight+ability+of+the+ambrosia+beetle,+Platypus+quercivorus+(Murayama),+using+a+low-cost,+small,+and+easily+constructed+flight+mill.">Measuring the flight ability of the ambrosia beetle, Platypus quercivorus (Murayama), using a low-cost, small, and easily constructed flight mill.</a> Journal of Visualized Experiments. (138), e57468 (2018).</li><li>Jones, V. P., Naranjo, S. E., Smith, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Insect ecology and behavior: laboratory flight mill studies. , (2010).</li><li>Abendroth, L. J., Elmore, R. W., Boyer, M. J., Marlay, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Corn Growth and Development. , (2011).</li><li>Meinke, L. J., Sappington, T. W., Onstad, D. W., Guillemaud, T., Miller, N. J., Kom&#225;romi, J., Levay, N., Furlan, L., Kiss, J., Toth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+corn+rootworm+(Diabrotica+virgifera+virgifera+LeConte)+population+dynamics.">Western corn rootworm (Diabrotica virgifera virgifera LeConte) population dynamics.</a> Agricultural and Forest Entomology. 11, 29-46 (2009).</li><li>Hammack, L., French, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+dimorphism+of+basitarsi+in+pest+species+of+Diabrotica+and+Cerotoma+(Coleoptera:+Chrysomelidae).">Sexual dimorphism of basitarsi in pest species of Diabrotica and Cerotoma (Coleoptera: Chrysomelidae).</a> Annals of the Entomological Society of America. 100 (1), 59-63 (2007).</li><li>Guss, P. 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+sex+pheromone+of+the+western+corn+rootworm+(Diabrotica+virgifera).">The sex pheromone of the western corn rootworm (Diabrotica virgifera).</a> Environmental Entomology. 5 (2), 219-223 (1976).</li><li>Hammack, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calling+behavior+in+female+western+corn+rootworm+beetles+(Coleoptera:+Chrysomelidae).">Calling behavior in female western corn rootworm beetles (Coleoptera: Chrysomelidae).</a> Annals of the Entomological Society of America. 88 (4), 562-569 (1995).</li><li>Minter, M., Pearson, A., Lim, K. S., Wilson, K., Chapman, J. W., Jones, C. 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Characterizing western corn rootworm flight behavior is important for devising effective resistance management plans. Flight behavior of this pest has been studied in the laboratory using various methods including actographs, flight tunnels, and flight mills. Flight mills, as described and illustrated in this paper, allow insects to make uninterrupted flights so that researchers can quantify flight parameters such as distance, duration, periodicity, and speed of individual flights, over an entire test period.
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The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), was identified as a pest of cultivated corn in 19091. Today, it is the most important pest of corn (Zea mays L.) in the U.S. Corn Belt, with larval feeding on corn roots causing most of the yield loss associated with this pest. The annual costs for management and corn production losses due to corn rootworm are estimated to exceed $1 billion2. The western corn rootworm is highly adaptable, and populations have evolved resistance to multiple management strategies including insecticides, crop rotation, and transgenic Bt corn3. Determining spatial dimensions over which tactics must be applied to mitigate local development of resistance, or a resistance hotspot, depends on a better understanding of dispersal4. Mitigation measures will not be successful if they are restricted to too small of a spatial scale around a resistance hotspot, because resistant adults will disperse beyond the mitigation area5. Understanding flight behavior of western corn rootworm is important to create effective resistance management plans for this pest.
Dispersal by flight plays an important role in adult western corn rootworm life history and ecology6, and the flight behavior of this pest can be studied in the laboratory. Several methods may be used to measure flight behavior in the laboratory. An actograph, which restricts flight in a vertical plane, can measure the amount of time an insect is engaged in flight. Actographs have been used to compare flight duration and periodicity patterns of western corn rootworm males and females at different ages, body sizes, temperatures, insecticide susceptibility, and insecticide exposure7,8,9. Flight tunnels, which consist of a tracking chamber and directed air flow, are especially useful for examining insect flight behavior when following an odor plume, such as candidate pheromone components10 or plant volatiles11. Flight mills are perhaps the most common method for laboratory studies of insect flight behavior and can characterize several aspects of flight propensity and performance. Laboratory flight mills have been employed in studies of western corn rootworm to characterize propensity to make short and sustained flights as well as hormonal control of sustained flight12,13.&#160;
Flight mills provide a relatively simple way to study insect flight behavior under laboratory conditions by allowing researchers to measure various flight parameters including periodicity, speed, distance, and duration. Many of the flight mills used today are derived from the roundabouts of Kennedy et al.14 and Krogh and Weis-Fogh15. Flight mills can be different in shape and size, but the basic principle remains the same. An insect is tethered and mounted on a radial horizontal arm that is free to rotate, with minimal friction, about a vertical shaft. As the insect flies forward, its path is restricted to circling in a horizontal plane, with the distance traveled per rotation dictated by the length of the arm. A sensor is typically used to detect each rotation of the arm caused by the flight activity of the insect. Raw data include rotations per unit time, and time of day flight occurred. The data are fed into a computer for recording. Data from multiple flight mills are often recorded in parallel, essentially simultaneously, with banks of 16 and 32 flight mills being common. The raw data are further processed by custom software to provide values for such variables as flight speed, total number of separate flights, distance and duration flown, and so forth.
Every insect species is different when it comes to the best method for tethering because of morphological variables such as overall size, size and shape of the target area for attaching the tether, softness, and flexibility of the insect, need and method for anesthetization, potential for fouling the wings and/or head with misplaced or overflow adhesive, and many, many more details. In the cases of visualized tethering of a plataspid bug16 and an ambrosia beetle17, the respective target areas for tether attachment are relatively large and forgiving of imprecise adhesive placement because the head and wings are somewhat well-separated from the attachment site. This is not to downplay the difficulty of tethering these insects, which is demanding for any species. But the western corn rootworm is a particularly challenging insect to tether: the pronotum is narrow and short, making very precise attachment with a minimal amount of adhesive (dental wax in this case) necessary to prevent interference with the opening of the elytra for flight and with the head, where contact with eyes or antennae can affect behavior. At the same time, the tether must be firmly attached to avoid dislodgement by this strong flyer. The demonstration of tethering of rootworm adults is the most important offering in this paper. It should be of help to others who work with this or similar insects where the method visualized here could be a useful option.
This paper describes methods used to effectively tether and characterize the flight activity of western corn rootworm adults that were reared at different larval densities. The flight mills and software used in this study (Figure 1) were derived from designs posted on the internet by Jones et al.18 Tethering techniques were modified from the description in Stebbing et al.9 An array of 16 flight mills was housed in an environmental chamber, designed to control lighting, humidity, and temperature (Figure 2). Using this or similar setup along with the following techniques allows for testing factors that may influence the flight propensity and performance of western corn rootworm, including age, sex, temperature, photoperiod, and many others.&#160;

<table border="0" cellpadding="0" cellspacing="0" style="width:800px;" width="799">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Butane multi-purpose lighter</td>
			<td style="width:191px;">BIC</td>
			<td style="width:117px;">UXMPFD2DC</td>
			<td style="width:233px;">To soften wax when tethering</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Clear polystyrene plastic vial (45-ml)</td>
			<td>Freund Container and Supply</td>
			<td>AS112</td>
			<td style="width:233px;">To hold beetle while anesthetizing</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dehydrated culture media, agar powder</td>
			<td>Fisher Scientific</td>
			<td>S14153</td>
			<td style="width:233px;">To make agar for holding moisture for adults</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:259px;">Delrin rod (1&#34; diameter, 3.75&#34; long)</td>
			<td style="width:191px;">Many suppliers: can use cheapest on the internet.</td>
			<td style="width:117px;"></td>
			<td style="width:233px;">For post of flight mill</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dental wax</td>
			<td>DenTek</td>
			<td>47701000335</td>
			<td style="width:233px;">Adheres wire tether to prothorax</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:259px;">Ferrite ring magnets (OD: 0.69&#8221;, ID: 0.29&#8221;, Thickness: 0.118&#8221;; 7oz pull)</td>
			<td style="width:191px;">Magnet Shop</td>
			<td style="width:117px;">63B06929118</td>
			<td style="width:233px;">Opposing - to generate the float.</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:259px;">Hall effect sensor</td>
			<td style="width:191px;">Optikinc</td>
			<td style="width:117px;">OHN3120U</td>
			<td style="width:233px;">Look under magnetic sensors on the left side of the Optekinc website then look for the part number. A link is given for current suppliers.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:259px;">Hypodermic tubing (22 gauge; 0.0358&#8221; OD x 0.01975&#8221; ID x 0.004&#8221; wall)</td>
			<td style="width:191px;">Small Parts, Inc.</td>
			<td style="width:117px;">HTX-22T-12</td>
			<td style="width:233px;">Used for flight mill arms and main axis rod.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Incubator (104.1 x 85.4 x 196.1 cm)</td>
			<td>Percival Scientific</td>
			<td>I-41VL</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="163">
			<td height="163" style="height:163px;width:259px;">LabVIEW Full Development System software, system-design platform</td>
			<td style="width:191px;">National Instruments (See http://www.ni.com/en-us/shop/labview/select-edition.html)</td>
			<td style="width:117px;">LabVIEW 2018 (Full Edition)&#160;</td>
			<td style="width:233px;">Provides environment needed to run flight mill files (.vi extensions) available for download from Jones et al.18 at http://entomology.tfrec.wsu.edu/VPJ_Lab/Flight-Mill.&#160; LabVIEW 2018 Full is compatible with Win/Mac/Linux operating systems.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Mesh cage (18 x 18 x 18 cm)</td>
			<td>MegaView Science Co. Ltd.</td>
			<td>BugDorm-4M1515</td>
			<td style="width:233px;">mesh size = 44 x 32, 650 &#181;m aperture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Needle tool</td>
			<td>BLICK</td>
			<td>34920-1063</td>
			<td style="width:233px;">For scoring soil surface for egg laying in laboratory</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:259px;">Nickel ring magnets (3/16&#8221; OD x 1/16&#8221; ID x: 1/16&#8221; thick)</td>
			<td style="width:191px;">K&#38;J Magnetics</td>
			<td style="width:117px;">R311</td>
			<td style="width:233px;">Used to trigger the digital hall effect sensor.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dish (100 mm x 15 mm)</td>
			<td>Fisher Scientific</td>
			<td>S33580A</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Plastic container (44-ml)</td>
			<td>Dart</td>
			<td>150PC</td>
			<td style="width:233px;">For initial rearing of young larvae</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic container (473-ml)</td>
			<td>Placon</td>
			<td>22885</td>
			<td style="width:233px;">For rearing of older larvae</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Round brush (size 2)</td>
			<td>Simply Simmons</td>
			<td>10472906</td>
			<td style="width:233px;">For transferring freshly hatched neonates to surface of roots</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sieve (250-&#181;m)</td>
			<td>Fisher Scientific</td>
			<td>08-418-05</td>
			<td style="width:233px;">To separate eggs from soil</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Steel wire (28-gauge)</td>
			<td>The Hillman Group</td>
			<td>38902350282</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:259px;">Teflon rod (3/8&#34; diameter, 3/4&#34; length)</td>
			<td style="width:191px;">United States Plastic Corporation</td>
			<td style="width:117px;">47503</td>
			<td style="width:233px;">To accept the rotating arm.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Vacuum&#160;</td>
			<td>Gast Manufacturing, Inc.</td>
			<td>1531-107B-G288X</td>
			<td style="width:233px;">For aspirating adults in laboratory</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">White poly chiffon fabric</td>
			<td>Hobby Lobby</td>
			<td>194811</td>
			<td style="width:233px;">To prevent escape of larvae from rearing container</td>
		</tr>
	</tbody>
</table>

using flight mills to measure flight propensity and performance of western corn rootworm, diabrotica virgifera virgifera (leconte)

The western corn rootworm, Diabrotica virgifera virgifera (LeConte) (Coleoptera: Chrysomelidae), is an economically important pest of corn in the northern United States. Some populations have developed resistance to management strategies including transgenic corn that produces insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt). Knowledge of western corn rootworm dispersal is of critical importance for models of resistance evolution, spread, and mitigation. Flight behavior of an insect, especially over a long distance, is inherently difficult to observe and characterize. Flight mills provide a means to directly test developmental and physiological impacts and consequences of flight in the laboratory that cannot be obtained in field studies. In this study, flight mills were used to measure the timing of flight activity, total number of flights, and the distance, duration, and speed of flights taken by female rootworms during a 22-h test period. Sixteen flight mills were housed in an environmental chamber with programmable lighting, temperature, and humidity control. The flight mill described is of a typical design, where a flight arm is free to rotate about a central pivot. Rotation is caused by flight of an insect tethered to one end of the flight arm, and each rotation is recorded by a sensor with a time-stamp. Raw data are compiled by software, which are subsequently processed to provide summary statistics for flight parameters of interest. The most difficult task for any flight mill study is attachment of the tether to the insect with an adhesive, and the method used must be tailored to each species. The attachment must be strong enough to hold the insect in a rigid orientation and to prevent detachment during movement, while not interfering with natural wing motion during flight. The attachment process requires dexterity, finesse, and speed, making video footage of the process for rootworms of value.

Figure 4 shows representative examples of outputs expected after flight testing. Flight data were obtained from experimental work conducted in the Department of Entomology at Iowa State University. Six-day-old, mated female western corn rootworm adults were tethered to flight mills and placed in a controlled environmental chamber set at 14:10 L:D, 60 % RH, and 25&#176; C. The beetles were left on the flight mills for 22 consecutive hours beginning 30 min before initiation of simulated dawn, ...

Watch this Scientific Journal Video about Using Flight Mills to Measure Flight Propensity and Performance of Western Corn Rootworm, Diabrotica virgifera virgifera LeConte at JoVE.com

Using Flight Mills to Measure Flight Propensity and Performance of Western Corn Rootworm, Diabrotica virgifera virgifera LeConte

Flight mills are important tools for comparing how age, sex, mating status, temperature, or various other factors may influence an insect&#8217;s flight behavior. Here we describe protocols to tether and measure the flight propensity and performance of western corn rootworm under different treatments.

Using Flight Mills to Measure Flight Propensity and Performance of Western Corn Rootworm, Diabrotica virgifera virgifera (LeConte)

The western corn rootworm, Diabrotica virgifera virgifera (LeConte) (Coleoptera: Chrysomelidae), is an economically important pest of corn in the northern ...

Insects of different ages, mating status, rearing conditions, et cetera, often differ in flight propensity or performance. Flight mill experiments can reveal the relative effects of such factors on flight behavior. Though difficult to study in the field, flight mills allow extensive probing of flight under controlled conditions.Managing a pest resistance to insecticides including transgenic crops requires understanding its flight behavior. Western corn rootworm disbursal is complex and flight mill studies are helping clarify it. With adaptation, this method can be applied to almost any insect species, whether small or large, pest or non-pest.Tethering any insect is the most difficult technique to master. Beetles with a small pronotum like rootworms are especially challenging. Practice tethering individuals unimportant to your actual experiments because many will be discarded.A long learning curve is expected so be patient with yourself. There are many nuances to tethering. Watching is worth a thousand words.To begin, bend a 40 millimeter length 28 gauge steel wire 90 degrees at the center. Wash hands to remove any debris, dirt, or oil. Take a small amount of dental wax slightly larger than a pinhead and roll it between the fingertips until a ball is formed.Place an adult beetle on a flat surface and position its dorsal side up. Reposition the legs if necessary so that the beetle lies completely flat on the surface. Use a butane lighter to briefly heat the dental wax on the wire for less than one second.Do not reuse the wax if it has been heated too long and fallen off the wire as it will not effectively adhere to the insect cuticle. After anesthetization of the beetle, carefully place the end of the steel wire with the melted dental wax on the dorsal surface of the pronotum while pointing the other end of the wire without dental wax along the midline of the abdomen. To avoid hindering flight, make sure that the melted wax does not get on the elytra or its sutures.There is only a short time window available between heating the wax on the tether and affixing it to the anesthetized beetle. But with enough practice, tethering will become second nature. Place the free end of the wire into the opening of the hollow metal tube of the flight mill arm.Ensure that the wire fits tightly enough to be held in place by friction. Immediately after mounting a beetle, tear a small piece of tissue paper from a larger tissue. Offer the tissue piece to the tethered beetle hanging from the flight mill for tarsal contact.This will greatly reduce initial escape or landing flight behavior. Tether additional beetles for flight testing. Eliminate human presence in the flight room at least 30 minutes before the test begins.Prior to flight testing, start the flight mill software program. Enter the information under the initialization tab. Set the start time and end time for the desired duration of the flight test.Set the minimum threshold to zero minutes. This ensures that any detection of the flight arm passing the sensor will be recorded. Set the maximum threshold to one minute which ensures that one minute must elapse between sensor detections of the flight arm before declaring an end to a flight.Enter a name for the file. Set raw data log interval to one minute to control the output of revolutions logged every minute. Under the subject information tab, fill in the columns labeled ID, species, age, sex, diet, and comment as desired.After that, return to the initialization tab and slide the button in the upper left of the screen display to auto. Then click on the start button. Once the current time matches the start time, the program begins collecting raw data for any flying beetles as seen in the status and debug tab.If the program is set to auto, the program stops collecting raw data once the current time matches the end time. Alternatively, click the stop button to stop the program before the scheduled end time. Click exit after the flight testing period has ended.Ensure that a TDMS file is saved under the filename entered during program initialization. Click on the TDMS file and save the document as a spreadsheet. After completion of a flight mill test, remove all flight tested beetles.Remove the wax bead connecting the tether to the pronotum and gently peel the wire away from the pronotum. The insect is available for further experimentation if desired. The six-day-old mated female Western corn rootworm adults were left on the flight mills for 22 consecutive hours beginning 30 minutes before initiation of simulated dawn and their flight activity was recorded.The first tab in the resulting spreadsheet summarizes the individual adults that were tested. For the female beetle tethered to flight mill number two, the spreadsheet displays the number of flights, total revolutions per flight, start and end time of each flight, and the duration of each flight. This female engaged in several independent flights most of which were very short.However, in flight number five, the female traveled 1, 258 meters over a 38-minute period of uninterrupted flight. The female beetle tethered to flight mill number one did not engage in flight during the test period so a blank spreadsheet is displayed. A summary of the flight parameters from the raw data was retrieved from the flight mill software.Total flight parameters refer to the sum of all flights of an individual during the 22-hour test period whereas the longest flight parameters refer to the longest uninterrupted flight during the test. It is very important to handle the beetle with care while tethering. During this process, the beetle is easily damaged or tethered improperly which will result in erroneous flight data.Easy removal of the tether wax from the cuticle allows testing the effects of flight and treatment groups of subsequent traits of interest such as fecundity and longevity.

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Research

JoVE Journal

Behavior

Use of the Operant Orofacial Pain Assessment Device (OPAD) to Measure Changes in Nociceptive Behavior

We present an operant system for the detection of pain in awake, conscious rodents. The Orofacial Pain Assessment Device (OPAD) assesses pain behaviors in a more clinically relevant way by not relying on reflex-based measures of nociception. Food fasted, hairless (or shaved) rodents are placed into a Plexiglas chamber which has two Peltier-based thermodes that can be programmed to any temperature between 7 &deg;C and 60 &deg;C. The rodent is trained to make contact with these in order to access a reward bottle. During a session, a number of behavioral pain outcomes are automatically recorded and saved. These measures include the number of reward bottle activations (licks) and facial contact stimuli (face contacts), but custom measures like the lick/face ratio (total number of licks per session/total number of contacts) can also be created. The stimulus temperature can be set to a single temperature or multiple temperatures within a session. The OPAD is a high-throughput, easy to use operant assay which will lead to better translation of pain research in the future as it includes cortical input instead of relying on spinal reflex-based nociceptive assays.

Use of the Operant Orofacial Pain Assessment Device OPAD to Measure Changes in Nociceptive Behavior

We present a user-friendly, high-throughput operant system for the evaluation of pain behaviors in awake, conscious rodents. The Orofacial Pain Assessment Device (OPAD) can assess pain through a reward/conflict paradigm thus providing a more humane way of testing. This protocol will yield more clinically relevant and translational data from rodents.

We present an operant  system for the detection of pain in awake, conscious rodents. The Orofacial  Pain Assessment Device (OPAD) assesses pain behaviors ...

Early Metamorphic Insertion Technology for Insect Flight Behavior Monitoring

Early Metamorphosis Insertion Technology (EMIT) is a novel methodology for integrating microfabricated neuromuscular recording and actuation platforms on insects during their metamorphic development. Here, the implants are fused within the structure and function of the neuromuscular system as a result of metamorphic tissue remaking. The implants emerge with the insect where the development of tissue around the electronics during pupal development results in a bioelectrically and biomechanically enhanced tissue interface. This relatively more reliable and stable interface would be beneficial for many researchers exploring the neural basis of the insect locomotion with alleviated traumatic effects caused during adult stage insertions. In this article, we implant our electrodes into the indirect flight muscles of Manduca sexta. Located in the dorsal-thorax, these main flight powering dorsoventral and dorsolongitudinal muscles actuate the wings and supply the mechanical power for up and down strokes. Relative contraction of these two muscle groups has been under investigation to explore how the yaw maneuver is neurophysiologically coordinated. To characterize the flight dynamics, insects are often tethered with wires and their flight is recorded with digital cameras. We also developed a novel way to tether Manduca sexta on a magnetically levitating frame where the insect is connected to a commercially available wireless neural amplifier. This set up can be used to limit the degree of freedom to yawing &#8220;only&#8221; while transmitting the related electromyography signals from dorsoventral and dorsolongitudinal muscle groups.

We present a novel surgical procedure to implant electrodes in Manduca sexta during its early metamorphic stages. This technique allows mechanically stable and electrically reliable coupling with the neuromuscular tissue to study flight neurophysiology dynamics. We also present a novel magnetic levitation platform for tethered studies of insect yaw.

Early Metamorphosis Insertion Technology (EMIT) is a novel methodology for integrating microfabricated neuromuscular recording and actuation platforms on ...

A Method for Investigating Age-related Differences in the Functional Connectivity of Cognitive Control Networks Associated with Dimensional Change Card Sort Performance

The ability to adjust behavior to sudden changes in the environment develops gradually in childhood and adolescence. For example, in the Dimensional Change Card Sort task, participants switch from sorting cards one way, such as shape, to sorting them a different way, such as color. Adjusting behavior in this way exacts a small performance cost, or switch cost, such that responses are typically slower and more error-prone on switch trials in which the sorting rule changes as compared to repeat trials in which the sorting rule remains the same. The ability to flexibly adjust behavior is often said to develop gradually, in part because behavioral costs such as switch costs typically decrease with increasing age. Why aspects of higher-order cognition, such as behavioral flexibility, develop so gradually remains an open question. One hypothesis is that these changes occur in association with functional changes in broad-scale cognitive control networks. On this view, complex mental operations, such as switching, involve rapid interactions between several distributed brain regions, including those that update and maintain task rules, re-orient attention, and select behaviors. With development, functional connections between these regions strengthen, leading to faster and more efficient switching operations. The current video describes a method of testing this hypothesis through the collection and multivariate analysis of fMRI data from participants of different ages.

This video presents a method of examining age-related changes in functional connectivity of cognitive control networks engaged by targeted tasks/processes. The technique is based on multi-variate analysis of fMRI data.

The ability to adjust behavior to sudden changes in the environment develops gradually in childhood and adolescence. For example, in the Dimensional ...

Multifunctional Setup for Studying Human Motor Control Using Transcranial Magnetic Stimulation, Electromyography, Motion Capture, and Virtual Reality

The study of neuromuscular control of movement in humans is accomplished with numerous technologies. Non-invasive methods for investigating neuromuscular function include transcranial magnetic stimulation, electromyography, and three-dimensional motion capture. The advent of readily available and cost-effective virtual reality solutions has expanded the capabilities of researchers in recreating &#8220;real-world&#8221; environments and movements in a laboratory setting. Naturalistic movement analysis will not only garner a greater understanding of motor control in healthy individuals, but also permit the design of experiments and rehabilitation strategies that target specific motor impairments (e.g. stroke). The combined use of these tools will lead to increasingly deeper understanding of neural mechanisms of motor control. A key requirement when combining these data acquisition systems is fine temporal correspondence between the various data streams. This protocol describes a multifunctional system&#8217;s overall connectivity, intersystem signaling, and the temporal synchronization of recorded data. Synchronization of the component systems is primarily accomplished through the use of a customizable circuit, readily made with off the shelf components and minimal electronics assembly skills.

Transcranial magnetic stimulation, electromyography, and 3D motion capture are commonly used non-invasive techniques for investigating neuromuscular function in humans. In this paper, we describe a protocol that synchronously samples data generated by all three of these tools along with the unique addition of virtual reality stimulus presentation and feedback.

The study of neuromuscular control of movement in humans is accomplished with numerous technologies. Non-invasive methods for investigating neuromuscular ...

Reversible Cooling-induced Deactivations to Study Cortical Contributions to Obstacle Memory in the Walking Cat

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For example, in the cat, visual information about an impending obstacle can modulate stepping for avoidance. Locomotor adaptation can also occur independent of vision, as sudden tactile inputs to the leg by an expected obstacle can modify the stepping of all four legs for avoidance. Such complex locomotor coordination involves supraspinal structures, such as the parietal cortex. This protocol describes the use of reversible, cooling-induced cortical deactivation to assess parietal cortex contributions to memory-guided obstacle locomotion in the cat. Small cooling loops, known as cryoloops, are specially shaped to deactivate discrete regions of interest to assess their contributions to an overt behavior. Such methods have been used to elucidate the role of parietal area 5 in memory-guided obstacle avoidance in the cat.

Complex locomotion in naturalistic environments requiring careful coordination of the limbs involves regions of the parietal cortex. The following protocol describes the use of reversible cooling-induced deactivation to demonstrate the role of parietal area 5 in memory-guided obstacle avoidance in the walking cat.

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For ...

Using Rapid Serial Visual Presentation to Measure Set-Specific Capture, a Consequence of Distraction While Multitasking

This method uses a rapid serial visual presentation (RSVP) paradigm to measure the cost of distraction when participants maintain multiple search goals. The protocol identifies two types of distraction within a single task - contingent attentional capture and set-specific capture - that represent different types of limitations of cognitive processing. Participants search for letters in two or more &#34;target&#34; ink colors (e.g., green and orange) within a continuous RSVP stream of heterogeneously colored letters, while ignoring two peripheral RSVPs of letters. Upon detecting a target, participants are to identify the letter. On some trials, target-colored distractors appear in the periphery just prior to the presentation of a target, causing a drop in target identification performance. Contingent attentional capture is observed by examining performance on trials in which the peripheral distractor is the same color as the target on that trial (e.g., both orange). Set-specific capture is represented by performance on trials in which the peripheral distractor is target-colored (e.g., orange), but not the same color as the target on that trial (e.g., green.) By varying the amount of time (i.e., the number of stimuli appearing) between the presentation of the distractor and the target, researchers can observe how participants recover from these distraction costs over time. As compared to static displays that are often used to measure contingent attentional capture, the dynamic display produces much larger effects, allowing the researcher to identify subtle effects of smaller manipulations. An unusual aspect of our design is that it employs a continuous display; &#34;filler&#34; stimuli connect one trial to the next seamlessly, and participants respond during this interval whenever they detect a target. The continuous display reduces chance performance to near-zero levels (rather than 50%) and provides researchers with a more sensitive measure of performance differences across trial types.

This method uses a dynamic visual display to index costs of distraction during visual search, including both &#34;contingent attentional capture&#34; and &#34;set-specific capture,&#34; which is a cost of distraction that occurs when the participants maintain multiple search goals simultaneously. This method has revealed basic mechanisms and limitations of visual attention.

This method uses a rapid serial visual presentation (RSVP) paradigm to measure the cost of distraction when participants maintain multiple search goals. ...

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific responses to changing temperatures according to their physiological tolerance and adaptability. Drosophila flies also possess a temperature sensing system that has become fundamental to understanding the neural basis of temperature processing in ectotherms. We present here a temperature-controlled arena that permits fast and precise temperature changes with temporal and spatial control to explore the response of individual flies to changing temperatures. Individual flies are placed in the arena and exposed to pre-programmed temperature challenges, such as uniform gradual increases in temperature to determine reaction norms or spatially distributed temperatures at the same time to determine preferences. Individuals are automatically tracked, allowing the quantification of speed or location preference. This method can be used to rapidly quantify the response over a large range of temperatures to determine temperature performance curves in Drosophila or other insects of similar size. In addition, it can be used for genetic studies to quantify temperature preferences and reactions of mutants or wild-type flies. This method can help uncover the basis of thermal speciation and adaptation, as well as the neural mechanisms behind temperature processing.

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Here we present a protocol to automatically determine the locomotor performance of Drosophila at changing temperatures using a programmable temperature-controlled arena that produces fast and accurate temperature changes in time and space.

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific ...

Using a Real-Time Locating System to Measure Walking Activity Associated with Wandering Behaviors Among Institutionalized Older Adults

A real-time locating system (RTLS) can be used to track the walking activity of institutionalized older adults in long-term care who are at risk for wandering behaviors. The benefits of a RTLS are objective and continuous measurements of activity. Self-report methods of activity, especially wandering, by health care staff are vulnerable to floor effects and recall bias, and continuous clinical or research observation over the long-term can be time-consuming and expensive. Health care staff also fail to recognize the onset and/or duration of wandering behaviors, which are associated with a variety of adverse health outcomes in this population but amenable to intervention. RTLS technologies can measure the walking activity of institutionalized residents with cognitive impairment over time with a high degree of accuracy. This is particularly useful for the study of wandering, defined as walking for at least 60 seconds with few (if any) breaks in activity. Wandering is associated with disease progression, hospitalizations, falls and death. Previous work suggests older adults with poor balance ability and high sustained walking activity may be particularly susceptible to poor health outcomes. RTLS&#39;s are used to assess cognitive impairment and factors associated with gait and balance; however, supplemental paper and pencil gait/balance tools may be used to further refine risk profiles. This project discusses the use of a RTLS to measure walking activity and also gait quality and balance ability measures on this population.

This paper discusses the use of a continuous and objective real-time locating system to measure walking activity associated with wandering behaviors, focusing on older adults with cognitive impairment. Walking activity is measured by walking distance, sustained walking distance, and sustained gait speed. Also assessed are gait quality and balance ability.

A real-time locating system (RTLS) can be used to track the walking activity of institutionalized older adults in long-term care who are at risk for ...

Biomechanical Analysis Methods to Assess Professional Badminton Players' Lunge Performance

Under the condition of simulating a badminton court in the laboratory, this study used the injury mechanism model to analyze the maximal right lunge movements of eight professional badminton players and eight amateur players. The purpose of this protocol is to study the differences in kinematics and joint moment of the right knee and ankle. A motion capture system and force plate were used to capture data of the joint movements of the lower extremity and the vertical ground reaction force (vGRF). Sixteen young men who did not have any sports injuries in the past 6 months took part in the study. The subjects performed a maximal right lunge from the start position with their right foot, stepping on and fully contacting with the force plate, hit the shuttlecock with an underhand stroke to the designated position in the backcourt, and then returned to the start/end position. All subjects wore the same badminton shoes to avoid a difference in impact from different badminton shoes. The amateur players showed a greater range of ankle movement and reverse joint moment on the frontal plane, and a larger internal joint rotation moment on the horizontal plane. The professional badminton players exhibited greater knee moment on the sagittal and frontal planes. Therefore, these factors should be considered in the development of the training program to reduce the risk of sports injuries in knee and ankle joints. This study simulates the real badminton court and calibrates the range of activities of each movement of the subjects so that the subjects complete the experimental action in a natural state with high quality. A limitation of this study is that it does not combine joint load and muscle activity. Another limitation is that the sample size is small and should be expanded in future studies. This research method can be applied to the lower limb biomechanical research of other footwork in the badminton project.

Here, we present a protocol to evaluate the differences in injury mechanisms between professional and amateur players when performing a badminton maximal right lunge movement by analyzing lower limb kinematics.

Under the condition of simulating a badminton court in the laboratory, this study used the injury mechanism model to analyze the maximal right lunge ...

A Novel Pavlovian Fear Conditioning Paradigm to Study Freezing and Flight Behavior

Fear- and anxiety-related behaviors significantly contribute to an organism&#8217;s survival. However, exaggerated defensive responses to perceived threat are characteristic of various anxiety disorders, which are the most prevalent form of mental illness in the United States. Discovering the neurobiological mechanisms responsible for defensive behaviors will aid in the development of novel therapeutic interventions. Pavlovian fear conditioning is a widely used laboratory paradigm to study fear-related learning and memory. A major limitation of traditional Pavlovian fear conditioning paradigms is that freezing is the only defensive behavior monitored. We recently developed a modified Pavlovian fear conditioning paradigm that allows us to study both conditioned freezing and flight (also known as escape) behavior within individual subjects. This model employs higher intensity footshocks and a greater number of pairings between the conditioned stimulus and unconditioned stimulus. Additionally, this conditioned flight paradigm utilizes serial presentation of pure tone and white noise auditory stimuli as the conditioned stimulus. Following conditioning in this paradigm, mice exhibit freezing behavior in response to the tone stimulus, and flight responses during the white noise. This conditioning model can be applied to the study of rapid and flexible transitions between behavioral responses necessary for survival.

Defensive behavioral responses are contingent upon threat intensity, proximity, and context of exposure. Based on these factors, we developed a classical conditioning paradigm that elicits clear transitions between conditioned freezing and flight behavior within individual subjects. This model is crucial for the understanding the pathologies involved in anxiety, panic, and post-traumatic stress disorders.

Fear- and anxiety-related behaviors significantly contribute to an organism&#8217;s survival. However, exaggerated defensive responses to perceived threat ...