An Extension Perspective on Monitoring Pesticide Resistance

Introduction

Pesticides play an important role in public health as part of sustainable integrated mosquito management (Rose, 2001). However, insecticide resistance is a threat to any long-term control program (Brogdon & McAllister, 1998a). Recently, McAbee et al. (2004) reported pyrethroid resistance in Culex pipiens,the northern house mosquito from California, being the first report of genetic resistance to this class of pesticides in North American mosquitoes. Therefore, periodic evaluation of insecticide susceptibility is a necessary part of good pest management practice. We describe here outreach activities with mosquito control programs focusing on measuring insecticide susceptibility of mosquito populations.

Background

Since 1964, the Public Health Entomology Research & Education Center (PHEREC) has been providing Extension services to Florida mosquito control programs. In 1992, PHEREC became a research center of the College of Engineering Sciences Technology and Agriculture at Florida Agricultural & Mechanical University, an 1890 land-grant institution.

In Florida, mosquito control has evolved from over-reliance on insecticide application for control of adult mosquitoes (adulticides) to integrated pest management programs (Florida Coordinating Council on Mosquito Control, 1998). Our goal is to empower mosquito control programs to monitor the efficacy of adulticides. The main defense against resistance is close surveillance of mosquito susceptibility to pesticides. Program directors must make timely management decisions based on current susceptibility data. Such information allows rational consideration of alternatives to adulticides, such as larvicides, biological control, and source reduction by mechanical means.

In 1998, the United States Environmental Protection Agency (USEPA) registered d-phenothrin, a synthetic pyrethroid, for use as a mosquito control adulticide. McAllister and Brogdon (1999) introduced the bottle bioassay, simplifying the procedure for monitoring insecticide susceptibility of adult mosquitoes. Although they provided the concentration of active ingredient of pesticide per bottle for most organophosphate and pyrethroid pesticides, no information was provided for d-phenothrin. September 2001, three Florida mosquito control programs requested assistance in evaluating the efficacy of d-phenothrin.

Methods

There are two different methods commonly used for the detection of insecticide resistance (Cochran, 1997; Brogdon & McAllister, 1998b):

• Concentration-mortality method (Figure 1). The pesticide d-phenothrin was evaluated at 4 concentrations, .025, 0.1, 0.25, 1 µg per ml of acetone. The test mosquitoes were Culex quinquefasciatus, the southern house mosquito. The response curve is bracketed by 95% confidence limits. These data allow estimation of LC₅₀, LC₉₀ and LC₉₅.

• Time-mortality method (Figure 2). The pesticide d-phenothrin was evaluated at 4 concentrations, 10.75, 21.5, 43, 86 µg per ml of acetone. The test mosquitoes were Culex quinquefasciatus. The response curves are not conducive to formal statistical testing, however, these data allow rapid comparison of field-collected mosquitoes with baseline (susceptible) populations.

Figure 1.
Concentration-Mortality Curve

Figure 2.
Time-Mortality Curve

In the former, a range of concentrations of the active ingredient is tested and percent mortality, at a pre-determined time, is recorded. In the latter, a diagnostic dose of active ingredient is tested and time to mortality is recorded. The diagnostic dose is the lowest concentration of insecticide that gives 100% mortality over the shortest achievable time (WHO 1998; Petersen, Floore, & Brogdon, 2004).

Each method has its advantages and disadvantages. An advantage of the concentration-mortality method is that statistical testing by probit analysis can be used. This procedure is widely accepted (Robertson & Preisler, 1991). Concentration-mortality is the preferred method for studies in which statistical confidence limits are desired. No standard statistical test is available for analyzing time-mortality data. Based on observations made during client/service visits throughout the state, Florida mosquito control programs use time-mortality data, rather than dose-mortality data to evaluate susceptibility of target mosquito species.

The advantages of the time-mortality method are that it is simple, rapid, and uses a single discriminating concentration of active ingredient that is compared to the response of known susceptible mosquitoes (Brogdon & McAllister, 1998b; McAllister & Brogdon, 1999; Petersen, 2002; Petersen et al., 2004; WHO 1963, 1970, 1981).

Program Description

The objectives of the PHEREC extension program are to:

• Establish the diagnostic dose for each insecticide used to control adult mosquitoes in Florida.
• Establish baseline susceptibility data for mosquito species of public health importance.
• Standardize methods of insecticide susceptibility testing throughout the state.
• Improve sustainable pest management strategies that maintain efficacy for mosquito control.

The procedure we teach is the bottle bioassay in which insects are exposed to the insecticide by tarsal contact as they walk on an insecticide-treated surface (Cochran, 1997; Brogdon & McAllister, 1998b). An advantage of our program is that the pesticide to be tested is off-the-shelf product actually used by the mosquito control program in contrast to reagent grade chemicals that would be purchased from a chemical supply company.

Extension Documents--PHEREC Technical Memoranda

As part of our Extension program we published a series of protocols detailing the preparation and execution of insecticide susceptibility bioassays. The memoranda are "how to" guides providing step-by-step instructions for preparing off-the-shelf pesticides for the resistance tests and are available online at http://www.pherec.org/bottleassay/technicalmemoranda.html.

These technical memoranda have been included in the most recent edition of the Florida Mosquito Control Handbook that is produced and distributed by the Florida Mosquito Control Association. Online version available at http://edis.ifas.ufl.edu/BODY_IN050

Recent Workshops

The concepts presented in this article were the core of two recent workshops:

• Florida Mosquito Control Association, Dodd Plenary Short Courses. January 26-30, 2004. Gainesville, Florida. The complete program is available at
  http://www.floridamosquito.org/Dodd2004/Doddprogram04.pdf

• Florida Agricultural & Mechanical University, College of Engineering Sciences Technology and Agriculture, John A. Mulrennan, Sr., Public Health Entomology Research and Education Center, Southeast Regional Conference, February 9-11, 2004. Panama City Beach, Florida. The complete program is available at
  http://www.pherec.org/pherecnews/Vol5No3/page9.html

To evaluate the impact of the workshops and the technical memoranda a survey, http://www.pherec.org/bottleassay/survey2004.html, was conducted during August 2004. Each of the 57 mosquito control programs recognized by the Bureau of Entomology and Pest Control of the Florida Department of Agriculture and Consumer Services was faxed a survey form. Twenty-six replies were received. We then followed up by telephone, contacting every one of the 57 programs, thus achieving 100% response. Forty-one programs had received training, and 37 programs were actually performing the resistance testing. Insecticide resistance to organophosphates was reported by 7, but none reported resistance to pyrethroids. We have identified mosquito control programs that acknowledge that they need training. We will program future training to meet these recognized needs.

Conclusions and Future Direction

This article presents a description of an extension program that provides simple, reliable, and inexpensive methods of evaluating insecticide susceptibility in a manner deemed appropriate for mosquito control programs. The program uses off-the-shelf insecticides that are readily at hand and familiar to mosquito control workers as opposed to reagent grade chemicals. Future work will focus on developing additional technical memoranda for pesticides used in Florida that are not included in the Florida Mosquito Control Handbook. Expected impacts include early warning of reduced efficacy of pesticides and reduced cost to mosquito control districts when ineffective products are discontinued.

Acknowledgment

This work was funded in part by the Florida Department of Agriculture and Consumer Services grant number 5491.

References

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Brogdon, W. G., & J.C. McAllister, J. C. (1998b). Simplification of adult mosquito bioassays through the use of time-mortality determination in glass bottles. J. Am. Mosq. Control Assoc. 14:159-165.

Cochran, D. G. (1997). Misuse of the tarsal-contact method for detecting insecticide resistance in the German cockroach (Dictyoptera: Blattellidae) J. Econ. Entomol. 90(6):1441-1444.

Florida Coordinating Council on Mosquito Control. 1998. Florida Mosquito Control: The state of the mission as defined by mosquito controllers, regulators, and environmental managers. University of Florida. Available at: http://fmel.ifas.ufl.edu/whitep/whitep.htm

McAbee, R. D., Kang, K-D., Stanich, M. A., Christiansen, J. A., Wheelock, C. E., Inman, A. D. Hammock, B. D. & Cornel, A. J. (2004). Pyrethroid tolerance in Culex pipiens pipiens var molestus from Marin County, California. Pest Management Science 60(4):359-368.

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[WHO] World Health Organization. (1981). Instructions for determining the susceptibility or resistance of adult mosquitoes to organochlorine, organophosphate and carbamate insecticides. Establishment of the base-line. Geneva, Switzerland: WHO/VBC/81.805.

[WHO] World Health Organization. (1998). Test procedures for insecticide resistance monitoring in malaria vectors, bio-efficacy and persistence of insecticides on treated surfaces. Geneva, Switzerland: WHO/CDS/CPC/MAL/98.12.