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

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.