Recipient Organization
LOUISIANA STATE UNIVERSITY
202 HIMES HALL
BATON ROUGE,LA 70803-0100
Performing Department
Entomology
Non Technical Summary
Insecticides are effective toxins: they kill insects that are susceptible to them. Unfortunately, there is a small percentage of insects that are not susceptible and are not killed. These resistant insects remain after the susceptible insects are killed, and may pose serious problems as agricultural pests, or as vectors of human or animal diseases. The major goal of the research proposed here is to develop methods to identify how resistant insects survive exposure to insecticides that are normally lethal, then to use this knowledge to develop strategies for overcoming resistance. The specific objectives of this research are to measure the susceptibility to insecticides in agricultural pests and mosquitoes, and use biochemical tests to determine how resistant insects differ from susceptible insects such that they can survive insecticide treatments. In addition, the effects of insecticides used in mosquito abatement efforts on domestic honey bee production will be determined. Once this is known, insecticide use strategies can be modified such that only the most effective insecticides are used, and contamination of the urban and agricultural environments by overuse of potentially harmful chemicals is minimized.
Animal Health Component
65%
Research Effort Categories
Basic
35%
Applied
65%
Developmental
0%
Goals / Objectives
The major goal of this project is to optimize management strategies used against agricultural and urban pests in Louisiana by developing countermeasures for insecticide resistance where it develops, and by minimizing non-target effects of insecticide sprays. Specific objectives are to:1.) Use biochemical technologies to study mechanisms of resistance in Lepidopteran pests of agricultural crops.2.) Develop methods for detection and characterization of resistance mechanisms in populations of the Southern house mosquito, Culex quinquefasciatus, an important vector of human and animal diseases in Louisiana.3.) Test the hypothesis that organophosphorus insecticides, when used at sublethal doses, can synergize toxicity of ester- containing insecticides against the Southern house mosquito, Cx. quinquefasciatus; and4.) Determine effects of insecticides used in urban mosquito control programs (mainly, pyrethroids and organophosphates) on non-target organisms, including the honey bee, Apis mellifera.
Project Methods
Insects (i.e., Lepidopteran pests and mosquitoes) will be collected from the field and reared in the laboratory. My approach for detecting metabolic resistance in both groups of insect pest will be similar to that taken in studying pyrethroid resistance inHeliothisvirescens. These previous studies suggest that two major groups of detoxifying enzymes (esterases and P450 monooxygenases) are associated with resistance to pyrethroids in this insect. In the current study, pharmacokinetic and spectrophotometric assays will be used to examine the role of insecticide- metabolizing enzymes (predominantly P450 monooxygenases, esterases and glutathione S-transferases) in resistance to insecticides in laboratory- selected and field-collected strains of pest insects.For biological assays with lepidopteran pests (Objective 1), susceptibility to insecticides will be examined using diet incorporation or topical bioassays. For diet incorporation assays, larvae (third stadium, day 1) will be placed on artificial diet containing various concentrations of insecticide (technical grade). Methods for biological assays with mosquitoes (Objective 2) will differ. For immature mosquitoes, larvae will be reared in pans containing distilled water and, at the first day of the thrid instar, trasfered to dishes containing measured concetratons of insecticide. Mortality will be determined at 2 days post-treatment, with "death" defined as he inability to move within 30 seconds after being prodded with a fine hair paintbrush. At least five concentrations (or doses) will be tested using 10 larvae per assay. In addition, susceptibility of adult Cx. quinquefasciatus to insecticides will be assayed using the WHO bottle assay, as described by Brogdon et al. (1999). Following evaporation of solvent, 10 adult mosquitoes will be placed into 15 ml scintillation vials that have been pretreated with insecticide solutions in acetone. Mortality will be scored at 1, 12, 24, and 48 hours, and insects will be considered dead if they are incapable of coordinated movement. At least five concentrations of each insecticide will be tested, and three determinations will be made for each concentration. Data will be analyzed as described above.Larval tissues (fat bodies and midguts) from fifth instars Lepidoteran pests or whole bodies of third instar mosquitoes will be dissected and homogenized in ice cold, 1.15% KCl using a glass on glass tissue homogenizer. The resulting homogenate will be centrifuged (1,000g, 10 min, 4oC) to remove debris, and then diluted into buffer of appropriate pH for measurement of enzyme activities. Protein concentrations will be determined according to the method of Bradford (1976), using bovine serum albumin (fraction V, concentrations corrected for impurities) as the standard.Spectrophotometry with model (non-insecticide) substrates (for example, 2, 4- dichloronitrobenzene, p-nitroanisole, and S-methyl thiobutyrate) will be used to determine involvement of detoxifying enzymes. The major advantage of spectrophotometric assays (relative to the thin- layer chromatography assay) is that they are rapid, less labor- intensive, and high throughput. In addition, in most cases, the products of these reactions are colored; thus, these assays are amenable to modification for use in-field without a spectrophotometer.For synergists tests with mosquitoes (Objective 3), non-toxic doses of a number of organophosphate compounds will be tested as inhibitors of esterase activity (in biochemical assays) and as synergists of resmethrin toxicity (in biological assays). For purposes of comparison, activities of each compound in the two assays will be compared to that of S- S- S- tributylphosphorotrithioate (DEF). Compounds to be tested include: dibrom, acephate, etrimfos, and chlorpyrifos. Each of these compounds has a relatively low mammalian toxicity (< 400 mg/kg, rat oral), and is considered an appropriate candidate for potential field use as an insecticide synergist.In preliminary tests, the maximal sublethal concentrations of each compound will be determined in topical bioassays. Ten, adult Cx. quinquefasciatus will be treated topically on the thoracic dorsum with serial doses of each compound (1 µl, in acetone), then introduced into 15 ml scintillation vials capped with moistened cotton plugs. Mortality will be scored at 1, 12, 24, and 48 hours, and insects will be considered dead if they are incapable of coordinated movement. In subsequent assays, adult mosquitoes (10 per dose) from field-collected or laboratory-susceptible strains will be treated topically with sublethal concentrations of synergists then, 30 minutes later, introduced into scintillation vials containing residues of resmethrin at a concentration equivalent to the LC30. Once again, mortality will be scored at 1, 12, 24, and 48 hours, and insects will be considered dead if they are incapable of coordinated movement. Control vials will contain either acetone only, resmethrin only, or insects pretreated with synergists but not exposed to resmethrin. Each assay will be done in triplicate, and three determinations will be made for each compound. Compounds will be considered synergists if mortality in the presence of the compound is significantly greater than in the presence of resmethrin alone. Mortalities will be compared using a generalized linear model (SAS, PROC GLIMMIX) with a binomial distribution.For Objective 4, topical bioassays with honey bees will be used to determine relative toxicities of insecticides used in local mosquito abatement efforts. These data will provide a measure of relative toxicity of these insecticides that is internally consistent and will serve as a foundation for studies comparing insecticide toxicities among honey bee strains. In addition, we will use these bioassays to evaluate effects of pre-exposure to commonly used miticides on insecticide toxicity. We hypothesize that Russian, Carniolan, and Italian bees will differ in their susceptibility due to different grooming behaviors and overall size, and that mite management strategies will impact insecticide toxicity.Toxicity will be measured in topical assays with five commonly used mosquito adulticides from different insecticide classes. Insecticides tested will include: malathion, naled, resmethrin, etofenprox, and a dual-action insecticide containing phenothrin and prallethrin. For all bioassays, insecticides will be dissolved in acetone, and applied as a 1 μl droplet onto the prothoracic dorsum of 3 day old bees that were pre-weighed and anesthetized with carbon dioxide. Five to eight concentrations will be used to define log dose-probit relationships for each insecticide. Each assay will be performed in triplicate (10 bees per replicate) and determined 3-5 times on different days and with different groups of bees. Mortality will be defined as absence of coordinated movement within 15 seconds of being prodded with a sharpened pencil and will be assessed 48 hours after treatment. Results will be analyzed as described above.Similar biological assays will be used to measure possible synergists effects between miticides and insecticides. Adult bees will be pretreated topically with a maximum, sublethal concentration of miticide (Amitraz or Thymnol) then, 30 min later, a dose of insecticide corresponding to the LD50 will be topically applied. Mortality (defined and measured as above) will be compared in the absence and presence of the miticide, and synergism ratios (SRs) will be calculated as: (mortality measured in treatments with insecticide+ miticide)/(mortality with insecticide alone). Ratios significantly greater than 1 will used as an indication of synergism.