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>
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		<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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