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 enough eggs are obtained for rearing adequate numbers of adults. Use a manual aspirator to collect adults from the field. 
	NOTE: It is recommended to collect adults during peak abundance, around late July in the U.S. Corn Belt, to ensure the collection of both sexes. Most adults will be males if collected earlier, whereas most will be females if collected later.</li>
	<li>Place the collected male and female adults into a mesh cage containing chopped corn ear, corn leaf tissue, 1.5% agar solid, and an oviposition substrate. An 18 x 18 x 18-cm cage (mesh size 44 x 32, 650 &#181;m aperture) can hold up to 500 adults at one time.
	<ol>
		<li>Use the corn grown in the field as a source of corn ear, which will be picked at the R3, or milk stage of kernel development19. The R3 kernel is yellow outside, while the inner fluid is milky white due to accumulating starch. Corn ears can be frozen and stored for up to a year until they are needed. To feed the rootworm, remove the husk and chop the corn into horizontal cross-sections about 3 cm thick. Chopped corn is the primary diet for the adults and should be changed out twice a week.</li>
		<li>Obtain leaves from greenhouse-grown corn plants of any age. Amount of leaf tissue will vary with the number of adults in the cage. Avoid using field plants, as they may introduce disease.</li>
		<li>To make the solid agar, mix 15 g of agar powder with 1 L of DI water. Heat the mixture until boiling. Pour the liquid into Petri dishes (100 mm x 15 mm) while it is hot. Place a lid on the Petri dish once cool and place them into cold storage (6&#176; C). Agar provides adults with a source of moisture and should be changed out twice a week.</li>
		<li>To prepare an oviposition substrate, place 40 g of sieved field soil (&#60;180 &#181;m) into a Petri dish. Moisten the soil with deionized water. Ensure that the soil at the bottom of the Petri dish appears wet. Score the top of the moistened soil with a needle tool. Remove the oviposition substrate weekly and place in an incubator at 25&#176; C and 60% RH for at least one month.</li>
	</ol>
	</li>
	<li>After incubating eggs for one month, wash the contents of the oviposition substrate through a 250-&#181;m sieve until all soil has been removed. Quantify the eggs by placing washed eggs in a 10-mL graduated cylinder. There are approximately 10,000 eggs per 1 mL.</li>
	<li>Place the quantified eggs into a 44-mL container and cover with sieved field soil (&#60;180 &#181;m). Western corn rootworm eggs undergo obligate diapause through the winter20. To break diapause, place eggs into cold storage (6&#176; C) for at least 6 months. 
	&#8203;NOTE: Eggs may be kept in cold storage for longer than 5 months, but egg viability decreases with time. After 12 months, there may be little to no hatch.</li>
	<li>After a minimum of 5 months, remove eggs from the cold storage and place in an incubator at 25&#176; C and 60% RH. Neonates hatch as early as 16 days after removal from cold storage.</li>
	<li>Once the eggs hatch, place three germinated kernels at the bottom of a 44-mL plastic container with roots exposed (i.e., not covered with soil). Use a soft bristle brush to transfer 12 neonates to the surface of the roots.</li>
	<li>Add 4.5 mL of DI water to 40 mL of sieved soil (&#60;600 &#181;m). Place the moistened soil over the germinated kernels that have been infested with neonates and cover the container with mesh fabric to prevent larvae from escaping.</li>
	<li>On the same day that the 44-mL plastic container is set up with neonates, prepare a 473-mL container with corn kernels that have not germinated. The rootworm larvae will be transferred to this larger container later. The number of kernels determines the desired larval density per plant. Add 120 g of soil mixture consisting of 50% sieved field soil (&#60;600 &#181;m) and 50% potting soil moistened with 20 ml of deionized water.</li>
	<li>After 7 days, transfer all contents of the 44-mL container to the 473-mL container. The larvae will be second instars at the time of transfer. 
	NOTE: This transfer to a larger container is necessary to supply larvae with enough root mass for feeding through pupation.</li>
	<li>Observe the emergence of adults typically around 26 days after egg hatch. Adults are active fliers upon emergence and may escape the 473-mL container when attempting to collect them by hand. Instead, use a vacuum with an aspirator to collect adults.</li>
	<li>Segregate adults by sex and/or date if needed for comparative testing. Sex of western corn rootworm can be determined by observing the morphology of the prothoracic basitarsi21. Males have broad, square-shaped prothoracic basitarsi, whereas those of females are narrow and conical-shaped.
	<ol>
		<li>Place beetle into a 45-mL clear polystyrene plastic vial and cover with a lid with 6 small (~1 mm diameter) holes.</li>
		<li>Anesthetize the beetle. Place the end of a tube attached to a CO2 tank regulator over the holes in the lid and allow a gentle flow of CO2 to enter the tube for approximately 10 to 15 s until the adult loses its grip on the wall of the vial. 
		NOTE: The anesthetized insect will remain immobilized for about 1 min.</li>
		<li>Place the anesthetized beetle, ventral side up, on an inverted plastic petri dish bottom. Carefully place the non-inverted lid of the petri dish over the beetle. Ensure that the tarsi of the beetle press against the lid, allowing easy observation of the prothoracic basitarsi under a dissecting microscope.</li>
	</ol>
	</li>
	<li>If the experiment requires that beetles be mated prior to flight, then use males at least 5 days old to mate with the newly emerged females. 
	NOTE: Use of older males ensures that they are sexually mature upon their introduction to virgin females. Females are sexually mature upon adult emergence, whereas males require 5 to 7 days of post-emergence development to reach sexual maturity22,23.</li>
</ol>
2. Start the flight mill software program prior to flight testing
NOTE: The flight mill program files (.vi file extensions which run in a commercial software platform, see Table of Materials) and details for their use are provided for download via links (&#34;data analysis routine&#34; and &#34;Circular Flight Mill Instructions&#34;, respectively) in the &#34;Flight mill wiring and software&#34; section on the Jones et al.18 website. If the programs no longer function in newer or future versions of the software platform, or if the user wants to add new capabilities, the routines provided by Jones et al. 18 can be modified by the user as needed.
<ol>
	<li>Open the flight mill software program (Figure 3).</li>
	<li>Enter the information under the Initialization tab.
	<ol>
		<li>Set the Start Time and End Time for the desired duration of the flight test. 
		NOTE: All adults should be tethered and mounted on flight mills by 30 min prior to the Start Time. It may take an experienced person 30 min to 45 min to tether and prepare 16 beetles for flight testing (see Section 3).</li>
		<li>Set the Min Threshold (min) to 0. This ensures that any detection of the flight arm passing will be recorded, and is the default recommended by Jones et al. 18.</li>
		<li>Set the Max Threshold (min) to 1. Here, 1 min was used. This value means that 1 min must elapse between sensor detection of the flight arm to &#34;call&#34; the end of a flight.</li>
		<li>Enter a name for the file.</li>
		<li>Set Raw Data Log Interval (min) to 1. This value controls the interval over which the raw data will be compiled for output reporting. Here, it is set to 1 min. Thus, the output of revolutions, for example, will be logged per minute. 
		NOTE: The actual time interval between electronic scanning of sensor activity is very short, but a 1-min interval allows logging at a fine enough scale for most research purposes, while restricting the number of lines in the spreadsheet output to a reasonable number for examining by eye.</li>
	</ol>
	</li>
	<li>Under the Subject Information tab, fill in the columns labeled ID, diet, sex, species, and comments as desired.</li>
	<li>Click on the START button located on the left side of the screen display. The program will begin collecting raw data once the Current Time matches the Start Time.</li>
</ol>
3. Tether western corn rootworm to flight mill
<ol>
	<li>Bend a 40-mm length 28-gauge steel wire 90&#176; at the center. 
	NOTE: The wire may also be of another metal such as copper or brass.</li>
	<li>Take a small amount of dental wax, slightly larger than a pinhead, and roll it between the fingertips until a ball is formed. Ensure that fingers are clean to prevent debris, dirt, and oil from incorporating into the wax, because it may prevent the wax from adhering to the insect.</li>
	<li>Push one end of the 40 mm the bent wire into the center of the ball of wax.</li>
	<li>Anesthetize the test adult with CO2 as described above (see 1.11.1 and 1.11.2).</li>
	<li>Place the anesthetized adult on a flat surface and position its dorsal side up. If the beetle does not lie completely flat on the surface, reposition the legs so that it does. It is important that the beetle lie as flat as possible on the surface to ensure the correct positioning of the wire.</li>
	<li>Briefly (&#60; 1 s) heat the dental wax on the wire with a butane lighter. If the wax is heated for too long, the melted wax will drop off of the wire. Do not reuse the wax if it has fallen off from the wire, as it will not effectively adhere to the insect cuticle.</li>
	<li>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, (i.e., the end without dental wax), along the midline of the abdomen. Alternatively, point the end of the wire without the dental wax toward the head if desired. In that case, a flying beetle will push the flight arm instead of pulling it. Be sure that the melted wax does not get on the elytra or its sutures, as it may prevent or hinder flight.</li>
	<li>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 tight enough to hold in place by friction. The tethered beetle may be positioned to fly either clockwise or counter-clockwise.</li>
	<li>Immediately after mounting a beetle, tear a small piece (~1-cm dia) of tissue paper from a larger tissue. Offer the tissue piece to the tethered beetle hanging from the flight mill for tarsal contact; most beetles will grasp the tissue and hold it against gravity until they release it at the beginning of their first flight activity. This will greatly reduce initial escape or landing flight behavior. 
	NOTE: Human presence in the flight-testing room should be limited to attaching and removing adults from the flight mills. The test period usually does not begin until at least 30 min have elapsed since attachment (see Note under 2.2.1), and humans should not be present in the flight room during this time or during the test period itself.</li>
	<li>Remove all flight-tested adults after completion of a flight mill test. Remove the wax bead connecting the tether to the pronotum by gently peeling the wire away from the pronotum. The wax will separate easily without damaging the cuticle, making the insect available for further experimentation if desired.</li>
</ol>
4. Save the data collected from the flight mill program.
<ol>
	<li>The program may be set to either MANUAL or AUTO. If the program is set to manual, then the user must end the program by clicking the STOP button. If the program is set to AUTO, then the program will stop collecting raw data once the Current Time matches the End Time.</li>
	<li>Click EXIT after the flight-testing period has ended.</li>
	<li>Ensure that a TDMS file is saved under the file name entered during program initialization (step 2.2).</li>
	<li>Click on the TDMS file and save the document as a spreadsheet (.xlsx).</li>
</ol>
5. Retrieve flight parameters from the saved spreadsheet (.xlsx)
NOTE: A spreadsheet can be custom designed to manipulate the raw data output from the flight mill software. Here, the software program was the same as described by Jones et al. 18, but an additional routine was added to recognize and summarize the longest uninterrupted flight by an individual insect during the test period.
<ol>
	<li>For each individual that engaged in flight activity, the spreadsheet will include the following information: flight number, total revolutions, start time, end time, and flight duration in minutes.
	<ol>
		<li>To calculate the total distance flown during the test period, sum the column labeled &#8216;Total Revs&#8217; and multiply it by the distance flown per revolution. Distance per revolution depends on the length of the flight arm from the central pivot to the attached insect. For example, if this distance is 15.9 cm, each revolution is equivalent to one meter flown. The total number of revolutions may also be found in the &#8216;Test Stats&#8217; tab.</li>
		<li>To calculate the total duration flown during the test period, sum the column labeled &#8216;Flight Duration (min)&#8217;.</li>
		<li>To determine the distance and duration of the longest uninterrupted flight, go to the &#8216;Test Stats&#8217; tab and look under the column labeled &#8216;Longest Flight #&#8217;.</li>
		<li>Flight speed can be calculated by dividing distance flown by flight duration. For insects, speed is commonly expressed in m/s or km/h.</li>
	</ol>
	</li>
</ol>

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<li>Riley, J. R., Downham, M. C. A., Cooter, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+the+performance+of+leafhoppers+on+flight+mills+with+that+to+be+expected+in+free+flight%5BTitle%5D)+AND+%22Entomologia+Experimentalis+et+Applicata%22%5BJournal%5D)">Comparison of the performance of leafhoppers on flight mills with that to be expected in free flight.</a> Entomologia Experimentalis et Applicata. 83, 317-322 (1997).</li>
<li>Isard, S. A., Spencer, J. L., Mabry, T. R., Levine, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+atmospheric+conditions+on+high-elevation+flight+of+western+corn+rootworm+%28Coleoptera%3A+Chrysomelidae%29%5BTitle%5D)+AND+%22Environmental+Entomology%22%5BJournal%5D)">Influence of atmospheric conditions on high-elevation flight of western corn rootworm (Coleoptera: Chrysomelidae).</a> Environmental Entomology. 33 (3), 650-656 (2004).</li>
<li>Chapman, J. W., Reynolds, D. R., Wilson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-range+seasonal+migration+in+insects%3A+mechanisms%2C+evolutionary+drivers+and+ecological+consequences%5BTitle%5D)+AND+%22Ecology+Letters%22%5BJournal%5D)">Long-range seasonal migration in insects: mechanisms, evolutionary drivers and ecological consequences.</a> Ecology Letters. 18, 287-302 (2015).</li>
<li>Spencer, J. L., Mabry, T. R., Vaughn, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+transgenic+plants+to+measure+insect+herbivore+movement%5BTitle%5D)+AND+%22Journal+of+Economic+Entomology%22%5BJournal%5D)">Use of transgenic plants to measure insect herbivore movement.</a> Journal of Economic Entomology. 96 (6), 1738-1749 (2003).</li>
<li>Isard, S. A., Spencer, J. L., Nasser, M. A., Levine, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aerial+movement+of+western+corn+rootworm%2C+Diabrotica+virgifera+virgifera+%28Coleoptera%3A+Chrysomelidae%29%3A+diel+periodicity+of+flight+activity+in+soybean+fields%5BTitle%5D)+AND+%22Environmental+Entomology%22%5BJournal%5D)">Aerial movement of western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae): diel periodicity of flight activity in soybean fields.</a> Environmental Entomology. 29 (2), 226-234 (2000).</li>
<li>Kim, K. S., Sappington, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+structuring+of+western+corn+rootworm+%28Coleoptera%3A+Chrysomelidae%29+populations+in+the+U.S.+based+on+microsatellite+loci+analysis%5BTitle%5D)+AND+%22Environmental+Entomology%22%5BJournal%5D)">Genetic structuring of western corn rootworm (Coleoptera: Chrysomelidae) populations in the U.S. based on microsatellite loci analysis.</a> Environmental Entomology. 34 (2), 494-503 (2005).</li>
</ol>

The authors have nothing to disclose.

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

using-flight-mills-to-measure-flight-propensity-performance-western

Research

JoVE Journal

Behavior

6.1K Views.  Iowa State University. Flight mills are important tools for comparing how age, sex, mating status, temperature, or various other factors may influence an insect’s flight behavior. Here we describe protocols to tether and measure the flight propensity and performance of western corn rootworm under different treatments.

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

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 ...

Measuring the Flight Ability of the Ambrosia Beetle, Platypus Quercivorus (Murayama), Using a Low-Cost, Small, and Easily Constructed Flight Mill

The ambrosia beetle, Platypus quercivorus (Murayama), is the vector of a fungal pathogen that causes mass mortality of Fagaceae trees (Japanese oak wilt). Therefore, knowing the dispersal capacity may help inform trapping/tree removal efforts to prevent this disease more effectively. In this study, we measured the flight velocity and duration and estimated the flight distance of the beetle using a newly developed flight mill. The flight mill is low cost, small, and constructed using commonly available items. Both the flight mill arm and its vertical axis comprise a thin needle. A beetle specimen is glued to one tip of the arm using instant glue. The other tip is thick due to being covered with plastic, thus it facilitates the detection of rotations of the arm. The revolution of the arm is detected by a photo sensor mounted on an infrared LED, and is indicated by a change in the output voltage when the arm passed above the LED. The photo sensor is connected to a personal computer and the output voltage data are stored at a sampling rate of 1 kHz. By conducting experiments using this flight mill, we found that P. quercivorus can fly at least 27 km. Because our flight mill comprises cheap and small ordinary items, many flight mills can be prepared and used simultaneously in a small laboratory space. This enables experimenters to obtain a sufficient amount of data within a short period.

Measuring the Flight Ability of the Ambrosia Beetle, Platypus Quercivorus Murayama, Using a Low-Cost, Small, and Easily Constructed Flight Mill

We developed a low cost and small flight mill, constructed with commonly available items and easily used in experimentation. Using this apparatus, we measured the flight ability of an ambrosia beetle, Platypus quercivorus.

The ambrosia beetle, Platypus quercivorus (Murayama), is the vector of a fungal pathogen that causes mass mortality of Fagaceae trees (Japanese oak wilt). ...

Environment

Building an Enhanced Flight Mill for the Study of Tethered Insect Flight

Makerspaces have a high potential of enabling researchers to develop new techniques and to work with novel species in ecological research. This protocol demonstrates how to take advantage of the technology found in makerspaces in order to build a more versatile flight mill for a relatively low cost. Given that this study extracted its&#160;prototype from flight mills built in the last decade, this protocol focuses more on outlining divergences made from the simple, modern flight mill. Previous studies have already shown how advantageous flight mills are to measuring flight parameters such as speed, distance, or periodicity. Such mills have allowed researchers to associate these parameters with morphological, physiological, or genetic factors. In addition to these advantages, this study discusses the benefits of using the technology in makerspaces, like 3D printers and laser cutters, in order to build a more flexible, sturdy, and collapsible flight mill design. Most notably, the 3D printed components of this design allow the user to test insects of various sizes by making the heights of the mill arm and infrared (IR) sensors adjustable. The 3D prints also enable the user to easily disassemble the machine for quick storage or transportation to the field. Moreover, this study makes greater use of magnets and magnetic paint to tether insects with minimal stress. Lastly, this protocol details a versatile analysis of flight data through computer scripts that efficiently separate and analyze differentiable flight trials within a single recording. Although more labor-intensive, applying the tools available in makerspaces and on online 3D modeling programs facilitates multidisciplinary and process-orientated practices and helps researchers avoid costly, premade products with narrowly adjustable dimensions. By taking advantage of the flexibility and reproducibility of technology in makerspaces, this protocol promotes creative flight mill design and inspires open science.

This protocol uses three-dimensional (3D) printers and laser cutters found in makerspaces in order to create a more flexible flight mill design. By using this technology, researchers can reduce costs, enhance design flexibility, and generate reproducible work when constructing their flight mills for tethered insect flight studies.

Makerspaces have a high potential of enabling researchers to develop new techniques and to work with novel species in ecological research. This protocol ...

Engineering

Dissection for Identifying Migratory Insects via Ovarian Reproductive Analysis

Dissection and Grading of Ovarian Development in Wild-Type Female Insects

Migratory insect pests pose serious challenges to food production and security all over the world. The migratory pests can be monitored and captured using searchlight traps. One of the most important techniques for migratory pest forecasting is to identify the migratory species. However, in most cases, it is difficult to get the information just by appearance. Therefore, using knowledge acquired by systematic analysis of the female reproductive system can help to understand the combined anatomic morphology of the ovarian mating sac and ovary developmental grading of wild-type migratory insects captured with searchlight traps. To demonstrate the applicability of this method, ovarian development status and egg grain development stages were directly assessed in Helicoverpa armigera, Mythimna separata, Spodoptera litura, and Spodoptera exigua for the ovarian anatomy, and the ovarian mating sacs were studied in Agrotis ipsilon, Spaelotis valida, Helicoverpa armigera, Athetis lepigone, Mythimna separata, Spodoptera litura, Mamestra brassicae, and Spodoptera exigua, to explore their relationships. This work shows the specific dissection method to predict wild-type migratory insects, comparing the unique reproductive system of different migratory insects. Then, both tissues, namely, the ovary and mating sacs, were further investigated. This method helps to predict the dynamics and the structural development of reproductive systems in wild-type female migratory insects.

Author Spotlight: Analysis of Ovarian Anatomy in Migratory Insects to Overcome Experimental Challenges 

The protocol demonstrates a simple and easy dissection method, suitable for wild-type migratory female insects captured with searchlight traps. This technique can significantly clarify the same species by comparing both reproductive tissues, namely the mating sac and ovarian development of wild-type female insects.

Migratory insect pests pose serious challenges to food production and security all over the world. The migratory pests can be monitored and captured using ...

A Simple Flight Mill for the Study of Tethered Flight in Insects

Flight in insects can be long-range migratory flights, intermediate-range dispersal flights, or short-range host-seeking flights. Previous studies have shown that flight mills are valuable tools for the experimental study of insect flight behavior, allowing researchers to examine how factors such as age, host plants, or population source can influence an insects' propensity to disperse. Flight mills allow researchers to measure components of flight such as speed and distance flown. Lack of detailed information about how to build such a device can make their construction appear to be prohibitively complex. We present a simple and relatively inexpensive flight mill for the study of tethered flight in insects. Experimental insects can be tethered with non-toxic adhesives and revolve around an axis by means of a very low friction magnetic bearing. The mill is designed for the study of flight in controlled conditions as it can be used inside an incubator or environmental chamber. The strongest points are the very simple electronic circuitry, the design that allows sixteen insects to fly simultaneously allowing the collection and analysis of a large number of samples in a short time and the potential to use the device in a very limited workspace. This design is extremely flexible, and we have adjusted the mill to accommodate different species of insects of various sizes.

Flight in insects is influenced by a number of factors and the propensity to disperse is an important variable in understanding insect ecology and biological control strategies. We describe the construction and use of a simple, relatively inexpensive, and flexible flight mill for measuring parameters of tethered flight in insects.

Flight in insects can be long-range migratory flights, intermediate-range dispersal flights, or short-range host-seeking flights. Previous studies have ...

Neuroscience

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although RNA interference (RNAi) has proved to be a powerful tool for studying WCR biology, it has its limitations. Specifically, RNAi itself is transient (i.e. does not result in long-term Mendelian inheritance of the associated phenotype), and it requires knowing the DNA sequence of the target gene. The latter can be limiting if the phenotype of interest is controlled by poorly conserved, or even novel genes, because identifying useful targets would be challenging, if not impossible. Therefore, the number of tools in WCR&#39;s genomic toolbox should be expanded by the development of methods that could be used to create stable mutant strains and enable sequence-independent surveys of the WCR genome. Herein, we detail the methods used to collect and microinject precellular WCR embryos with nucleic acids. While the protocols described herein are aimed at the creation of transgenic WCR, CRISPR/Cas9-genome editing could also be performed using the same protocols, with the only difference being the composition of the solution injected into the embryos.

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

Here we present protocols for collecting and microinjecting precellular western corn rootworm embryos for the purpose of performing functional-genomic assays such as germline transformation and CRISPR/Cas9-genome editing.

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although ...

Bioengineering

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

Biology

Measuring Crop Motility and Food Passaging in Drosophila

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. Disordered GI motility and gastric emptying cause multiple diseases and symptoms. As a powerful genetic model organism, Drosophila can be used in GI motility research. The Drosophila crop is an organ that contracts and moves food into the midgut for further digestion, functionally similar to a mammalian stomach. Presented is a protocol to study Drosophila crop motility using simple measurement tools. A method for counting crop contractions to evaluate crop motility and a method for detecting the distribution of food dyed blue between the crop and gut using a spectrophotometer to investigate the effect of the crop on food passaging is described. The method was used to detect the difference in crop motility between control and nprl2 mutant flies. This protocol is both cost-efficient and highly sensitive to crop motility.

Measuring Crop Motility and Food Passaging in Drosophila

The goal of this protocol is to measure crop contraction and quantify food distribution in the Drosophila gut.

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. ...

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

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Measuring Crop Motility and Food Passaging in Drosophila