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

Summary

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.

Abstract

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.

Introduction

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 transg....

Protocol

1. Rear western corn rootworm for flight tests

NOTE: If the adult’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.

1. 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 recommend....

Results

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° C. The beetles were left on the flight mills for 22 consecutive hours beginning 30 min before initiation of simulated dawn, and t.......

Discussion

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.

Materials

List of materials used in this article

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