A FffiLD TEST KIT FOR THE DETERMINATION OF INSECTICIDE RESISTANCE IN HORN FLY POPULATIONS'"

J. E. Cilek and F. W. Knapp Department of Entomology

University of Kentucky Lexington, KY 40546

Abslract: In 1984 horn fly resistance \.0 permethrin was detected on a farm in the southwestern portion of Kentucky (Christian Co.). Due to the limited applicability of existing techniques to evaluate horn fly resistance in field situations, a field test kit was developed. This kit and its use in horn fly susceptibility studies in Kentucky are described.

Additional monitoring of horn ny susceptibility on this same farm during 1985. using the test kit, confirmed multiple resistance to permethrin and fenvalerate with cross resistance to nucythrinate. The level of perlllethrin resistance detected in October 1984 (over 30-fold) did not change appreciably in overwintering horn flies collected in April 1985. Therefore, pyrethroid ear tags were no\. used during 1985. In May and July the horn fly population approached susceptible levels.

The field t.est kit, used in this study, proved to be a rapid economical and reliable method for evaluating horn fly resistance. Results can be obtained in as little as 2 h for pyrethroid insecticides using this kit.

Key Words: Horn fly, pyrethroid, resistance, permcthrin, fenvalerate, flucythrinate.

J. Agric. Entomol. 3(3): 201-206 (July 1986)

The appearance of horn fly pyrethroid insecticide resistance has recently become one of increasing concern across the country (Kunz 1985; Schmidt et a1. 1985). Horn fly pyrethroid resistance has been attributed to the use of these compounds in slow release insecticide~impregnated ear tags for fly control on cattle. Resistance has usually been detected after ca. 3 years continuous use of the tags. Horn fly resistance to permethrin was first discovered in Kentucky using the Kerrville test kit developed by Kunz and Schmidt (1985) and Schmidt et a1. (1985) in 1984. However, after using this test kit, we found several drawbacks in its applicability for use in field evaluations. For example, a source of CO 2 or refrigeration was needed to allow transfer of flies into the test containers. Mortality was assessed at 24 h and the flies had to be furnished with a source of blood in the interim. This situation stimulated the development of a field test kit which could provide a technique that could more rapidly detect insecticide resistance levels in horn fly populations. This paper describes the field test kit, discusses its use and presents data on horn fly resistance to various synthetic insecticides in Kentucky as determined by this test kit.

MATERIALS AND METHODS

Test Kit Preparation The test kit utilizes insecticide treated 18 X 150 mm glass test tubes. The kit

is prepared by dispensing 0.25 ml of a mixture of insecticide and acetone into

J DrPTERA; Muscidae. 2 The investigation reported in Ihis puper (No. 86·7.98) is in connection with Ii project. of the Kentucky

Agricultural Experiment SWlion (lnd is published with approval of the director. A<:ceptcd for publication " September 1986. Fee paid for early publication.

201

202 J. Agric. Entomol. Vol. 3, No.3 (1986)

each tube. The tube is tilted until the liquid almost touches the inner lip and then rotated until the acetone evaporates. Untreated control tubes are coated with acetone only. One end of a 6.5 em 2 fine mesh cloth is then fastened with tape ca. 1 em from the mouth of the tube (Fig. 19).

Fly Collection and Tran.sfer Procedures Horn flies were collected from corralled cattle with a sweep net. A flashlight

battery powered mosquito aspirator (Hausherr's Machine Works, Tom's River, NJ) (Fig. 1b) was then used to collect the flies from the net. Transfer of flies from the aspil·ator into 24 X 82 mm glass holding vials (Fig. Ie) is accomplished by removing the aspirator suction tube (Fig. Id) and immediately placing the open end of the glass holding vial into the mouth of the aspirator (Fig. Ie). Turning the aspirator on and off regulates the number of flies being released into the holding vial. In our testing we used ca. 10 - 15 nonsexed flies/vial. To prevent escape of flies from the holding vial before transfer to the treated test tubes, place a thumb over the opening of the vial and then quickly insert the t.reated tube into this vial. Flies are then shaken from the holding vial into the treated test tube. A holding vial is needed in these series of transfer steps to allow one person to transfer flies into extra vials while another person is transferring flies from these vials into the treated tubes. This technique allows for rapid transfer of flies to the treated tubes. Also, a holding vial prevents contamination of the aspirator when transferring the horn flies into the treated test tubes. The inner dimensions of the aspirator tube, collection vial and treated test tubes are all of sufficient size that each fits very closely within the next in the transferring process.

Fig. 1. a) Fine mesh cloth secured with tape to treated test tube, b) mosquito aspirator, c) mouth of aspirator, d) aspirator suction tube, c) glass holding vial, 1) treated test tube covered with I'cmaining cloth and secured with tape.

CILEK and KNAPP: Field Kit for Determining Resistance in Horn Flies 203

Once mes have been transferred into the treated tube, the opening is covered with the remaining cloth and secured with tape (Fig. If). The treated test tubes are held horizontally within a standard test tube rack during the exposure period. Treated tubes should be held in a shaded ai' cool area to avoid moisture condensation internally. If this is a problem, the tubes can be placed into a styrofoam cooler containing an appropriate desiccant such as anhydrous calcium sulfate.

Determination of Hom Fly Insecticide Susceptibility Al least three replicates of each insecticide concentration are required for

adequate testing. Horn fly mortality is recorded at 2 h and immobile flies are counted as dead. If needed, the 2 h time interval used to collect dose mortality data in this test kit may be extended. However, control mortality in untreated tubes often exceeds 20% after 4 h and increases at successive hourly intervals to the point where data collected beyond 4 h may be invalid. This may be especially true during extremely wann weather.

Resistance Testing in Kentuchy In our investigation on horn fly resistance in Kentucky, glass test tubes were

used within 24 h of treatment with varying dosages of technical permethrin (95.2%) from Burroughs Welcome Co., Triangle Park, NC; fenvalerate (96%) from 80S Biotech Corp., Painesville, OH and flucythrinate (71.3%) from American Cyanamid, Princeton, NJ.

Serial dilutions were made from each of the above insecticides starting with a 1% wt/v solution dissolved in acetone. Five to ten residue levels, expressed in p.g/ cm 2, were used in these studies.

In October 1984 and during the spring and summer of 1985 suspected resistant horn flies (Pembroke strain) were collected from cattle on a farm located in the southwestern part of the State of Tennessee-Kentucky border (Christian Co.). For comparison, a susceptible horn fly strain (Spindletop) was collected from our University of Kentucky research farm located in the middle of the state. All testing was conducted while on the fann, Analysis of the collected data was accomplished by using a SAS (1982) probit computer package. Resistant ratios were calculated as the LC 50 of the resistant (Pembroke) strain divided by the LC 50 of the susceptible (Spindletop) strain.

Effect of Storage Methods on Test Kit The residual efficacy of the permethrin treated tubes was investigated when

stored either at 25°C or O°C. Three replications of each concentration ranging from 0.0015 to 0.118 p.g/cm 2 were storage tested. Control tubes were treated with acetone only. Each concentration was separately packaged in plastic bags to avoid possible contamination. Two replicates of the concentration series were treated and each placed into a sealed cardboard container to exclude light. The exclusion of light eliminated the confounding effects of photodecomposition on the surface residue of the glass tube. One container was held at room temperature (25°C) and the other container was held at O°C. The USDA-ARS, Kerrville, TX, pyrethroid susceptible horn fly strain was used in this test. Three day old nonsexed flies were exposed to the treated lubes on the day of treatment, 3 days and 1 wk posttreatment and at weekly intervals thereafter for 5 wk. Sufficient tubes were treated so that no tubes were used more than once.

204 J. Agric. Entomol. Vol. 3, No.3 (I 986)

Horn fly mortality due to the effects of fumigation for permethrin, fenvalerate, and ilucythrinate was determined by holding horn flies in surface treated 3.8 liter glass containers. About 20 flies were confined to a 16 X 67 mm glass vial which had both ends covered with a fine mesh cloth and securred with tape. The vial containing the flies was suspended in the middle of the treated 3.8 liter glass container ca. 8 em from the bottom surface. The residue concentrations of the three insecticides tested were ca. 10 X that of their respective LC 50 values (which had been determined previously from the pyrethroid susceptible Spindletop hom fly strain). Treated containers were used ca. 2 h after treatment, after which time the vials containing the horn flies were lowered into their respective containers and the mouth of the containers covered with plastic film to prevent the escape of any insecticide vapors. Control containers were treated with acetone only. Each fumigation test was replicated three times for each insecticide. Mortality was recorded at 2, 4 and 6 h.

RESULTS AND DISCUSSION

The resistant Pembroke hom fly population when compared with the susceptible Spindletop population was observed to be over 32 fold more resistant to permethrin in October 1984 (Table 1). Because the level of resistance remained constant in the April 1985 overwintering horn fly population, a decision was made not to use pyrethroid insecticide-impregnated ear tags on this farm in 1985. Previous to 1985, the history of insfcticide ear tag use was as follows: 1981 stirofos, 1982 ~ 83 fenvalerate and 1984 permethrin.

Table 1. Susceptibility of two hom fly strains to permethrin.

J1.g/cm 2

Strain LC 50 Confidence Interval Slope ± SE RR-

Spindletop 0.0009 0.0005·0.0012 2.32 ± 0.48

Pembroke (Oct 1984) 0.0293 0.0283·0.0304 0.84 ± 0.25 32.6

Pembroke (Apr 1985) 0.0246 0.0180·0.0350 1.08 ± 0.14 37.3

Pembroke (May 1985) 0.0015 0.0014·0.0016 1.00 ± 0.55 1.7

Pembroke (July 1985) 0.0012 0.0003·0.0028 1.26 ± 0.23 1.3

- Resistance Ratio = LC 50 Pembroke Strain LC 50 Spindlctop Strain

CILEJ< and KNAPP: Field Kit for Determining Resistance in Horn Flies 205

By May and July resistance decreased dramatically and approached susceptible levels. However, the slope of the dose mortality lines on these two collection dates was still lower than the susceptible (Spindletop) population, indicating that that resistant (Pembroke) population was still heterozygous with respect to resistant and susceptible genes. Therefore, if the resistant Pembroke population were again exposed to pel'methrin the appearance of resistance would be probable.

Multiple resistance to fenvalerate was observed to be about 4 fold on this farm in July (Table 2) while 7 fold resistance to Oucythrinate was detected during May. The existence of cross resistance to flucythrinate indicates that resistance to this pyrethroid occurs in multiresistant permethrin and fenvalerate populations.

Table 2. Susceptibility of two horn fly strains to flucythrinate and fenvalerate in p..g/cm 2.

Chemical Hom fly

Strain Coil Date LC 50

Confidence Intervals Slope± SE RR'

Fenvalerate Spindletop

Pembroke July 85

0.00028

000097

0.00025 - 0.00031

0.00045 - 0.00300

2.84 ± 0.20

1.17 + 0.28 3.5

Flucythrinate Spindletop

Pembroke May 85

0.00022

0.00143

0.00012 - 0.00029

0.00063 - 0.00234

3.26 ± 0.80

1.30 + 0.24 6.5

• Rcsi5t!mce Rlllio _ LC 50 Pembroke Strain LC 50 Spindlctop Strain

Comparision of the residual activity of pennethrin treated tubes at two storage regimes is presented in Table 3. Prepared tubes stored at 25°C and O°C lost some of their residual activity after 4 wk and were considered to be inadequate for dose mortality determinations. However, if the determination of the presence or absence of insecticide resistance is only needed then the residual efficacy of higher concentrations may be maintained for longer periods of time using these two storage methods.

Table 3. Comparison of two methods of storage on the residual effectiveness of permethrin on horn fly mortality.

% Mortelity

Cone. Day 0 3 days 1 wk 2 wk 4 wk 5 wk (p.g/cm 2) 25° O°C 25° O°C 25° O°C 25° O°C 25° O°C

0.0015 32.4 28.3 20.0 32.5 54.9 61.2 29.1 45.8 2.5 4.0 4.8 0.0029 63.2 83.6 75.6 59.5 83.8 64.1 54.3 60.0 45.8 15.8 14.6 0.0044 86.8 88.5 93.8 65.0 94.9 100.0 96.6 92.3 73.3 51.4 40.4 0.0059 93.8 92.4 98.6 93.3 97.7 100.0 97.4 97.3 89.4 96.2 72.1 0.0089 100.0 100.0 100.0 100.0 100.0 100.0 100.0 95.1 94.5 100.0 96.3 0.0118 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 93.2

206 J. Agric. Entomol. Vol. 3. No. 3 (1986)

Furthermore. there was no apparent horn fly mortality due to the effects of fumigation for permethrin, fenvalerate or flucythrinate at 2. 4 or 6 h post­treatment.

We feel that our test kit is a simple. reliable and economical method for the determination of the level of horn fly insecticide resistance in field situations. One or two concentrations can be used for the rapid detection of the presence or absence of resistance or several concentrations can be utilized to establish the ex.tent of resistance. OUf field test kit method eliminates the need to anesthetize horn flies for manipulation, reduces the need of holding the flies for long periods of time (e.g., 24 h) and also eliminates the need to provide them with a food source in the interim. Results from the test kit can be obtained in as little as 2 h, or the time interval may be expanded as needed to determine the susceptibility of horn fly popoulations to varying doses of one or several insecticides.

REFERENCES CITED

Kunz, S. E. 1985. Hom fly resistance. Animal NUlrition and Health. 40: 40-41. Kunz, S. E., and C. D. Schmidt. 1985. The pyrethroid resistance problem in the horn ny. J.

Agric. Entomol. 2: 358-363. SAS. 1982. SAS User's Guide. Cary, NC. Schmidt, C. D., S. E. Kunz, H. D. Petersen, and J. L. Robertson. 1985. Resistance of hom

flies (Diptera: Muscidae) to pennethrin and fenvalerate. J. Eeon. Enlomol. 78: 402­406.