Name: Bioassays for Monitoring Insecticide Resistance (Video) | JoVE Journal
Uploaded: 2010-12-30T16:30:00.000+00:00
Duration: 6 min 30 s
Description: Learn bioassay techniques to monitor insecticide resistance in arthropod pests using adult vial tests. This method compares LC50 values to assess susceptibility and inform pest management strategies.

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5 minutes

We would like to thank Cotton Incorporated State Support Program #08-317MO, USDA/CSREES SR-IPM Grant 2009-34103-20018 and Landis International for funding research pertaining to this publication.

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<li>Lopez, J. D. Jr, Hoffman, W. C., Latheef, M. A., Fritz, B. K., Martin, D. E., Lan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adult+vial+bioassay+of+insecticidal+toxicity+against+cotton+fleahopper%2C+Pseudatomoscelis+seriatus+%28Hemiptera%3A+Miridae%29%5BTitle%5D)+AND+%22J.+Pest.+Sci.%22%5BJournal%5D)">Adult vial bioassay of insecticidal toxicity against cotton fleahopper, Pseudatomoscelis seriatus (Hemiptera: Miridae).</a> J. Pest. Sci.. 33, 261-265 (2008).</li>
<li>Lopez, J. D. Jr, Hoffman, W. C., Latheef, M. A., Fritz, B. K., Martin, D. E., Lan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+toxicity+of+selected+insecticides+against+thrips+on+cotton+in+laboratory+bioassays%5BTitle%5D)+AND+%22J.+Cotton+Sci%22%5BJournal%5D)">Evaluation of toxicity of selected insecticides against thrips on cotton in laboratory bioassays.</a> J. Cotton Sci. 12, 188-194 (2008).</li>
<li>Smith, T. P., Hammond, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+susceptibility+of+sweetpotato+weevil+%28Coleoptera%3A+Brentidae%29+to+selected+insecticides%5BTitle%5D)+AND+%22J.+Econ.+Entomol%22%5BJournal%5D)">Comparative susceptibility of sweetpotato weevil (Coleoptera: Brentidae) to selected insecticides.</a> J. Econ. Entomol. 99, 2024-2029 (2006).</li>
<li>Kanga, L. H. B., Wall, M. L., Plapp, F. W. Jr, Gardiner, E. M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pyrethroid+resistance+in+field-collected+boll+weevils+from+southeast+Arkansas+in+1994%5BTitle%5D)+AND+%22Southwest.+Entomol%22%5BJournal%5D)">Pyrethroid resistance in field-collected boll weevils from southeast Arkansas in 1994.</a> Southwest. Entomol. 20, 247-253 (1995).</li>
<li>Bayoun, I. M., Plapp, F. W. J. r, Gilstrap, F. E., Michels, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Toxicity+of+selected+insecticides+to+Diuraphis+noxia+%28Homoptera%3A+Aphidiae%29+and+its+natural+enemies%5BTitle%5D)+AND+%22J.+Econ.+Entomol%22%5BJournal%5D)">Toxicity of selected insecticides to Diuraphis noxia (Homoptera: Aphidiae) and its natural enemies.</a> J. Econ. Entomol. 88, 1177-1185 (1995).</li>
<li>Smith, T. R., Cave, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pesticide+susceptibility+of+Cybocephalus+nipponoicus+and+Rhyzobius+Iophanthae+%28Coleoptera+Cybocephalidae%2C+Coccinellidae%29%5BTitle%5D)+AND+%22Fla.+Entomol%22%5BJournal%5D)">Pesticide susceptibility of Cybocephalus nipponoicus and Rhyzobius Iophanthae (Coleoptera Cybocephalidae, Coccinellidae).</a> Fla. Entomol. 89, 502-507 (2006).</li>
<li>Sukontason, K., Olson, J. K., Hartberg, W. K., Duhrkopf, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Organophosphate+and+pyrethroid+susceptibilities+of+Culex+salinarius+adults+from+Texas+and+New+Jersey%5BTitle%5D)+AND+%22J+Am.+Mosquito+Contr%22%5BJournal%5D)">Organophosphate and pyrethroid susceptibilities of Culex salinarius adults from Texas and New Jersey.</a> J Am. Mosquito Contr. 14, 477-480 (1998).</li>
<li>Kanga, L. H. B., Plapp, F. W. J. r, Wall, M. L., Elzen, G. W., Lopez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monitoring+for+resistance+to+organophosphorus%2C+carbamate%2C+and+pyrethroid+insecticides+in+the+oriental+fruit+moth+%28Lepidoptera%3A+Torticidae%29%5BTitle%5D)+AND+%22Canadian+Entomol%22%5BJournal%5D)">Monitoring for resistance to organophosphorus, carbamate, and pyrethroid insecticides in the oriental fruit moth (Lepidoptera: Torticidae).</a> Canadian Entomol. , 131-441 (1999).</li>
<li>Plapp, F. W., Jackman, J. A., Campanhola, C., Frisbie, R. E., Graves, J. B., Luttrell, R. G., Kitten, W. F., Wall, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monitoring+and+management+of+pyrethroid+resistance+in+the+tobacco+budworm+%28Lepidoptera%3A+Noctuidae%29+in+Texas%2C+Mississippi%2C+Louisiana%2C+Arkansas%2C+and+Oklahoma%5BTitle%5D)+AND+%22J.+Econ.+Entomol%22%5BJournal%5D)">Monitoring and management of pyrethroid resistance in the tobacco budworm (Lepidoptera: Noctuidae) in Texas, Mississippi, Louisiana, Arkansas, and Oklahoma.</a> J. Econ. Entomol. 83, 335-341 (1990).</li>
<li>Mink, J. S., Boethel, D. J., Leonard, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monitoring+permethrin+resistance+in+soybean+looper+%28Lepidoptera%3A+Noctuidae%29+adults%5BTitle%5D)+AND+%22J.+Entomol.+Sci%22%5BJournal%5D)">Monitoring permethrin resistance in soybean looper (Lepidoptera: Noctuidae) adults.</a> J. Entomol. Sci. 28, 43-50 (1993).</li>
<li>Cook, D. R., Leonard, B. R., Gore, J., Temple, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Baseline+responses+of+bollworm%2C+Heliocoverpa+zea+%28Boddie%29%2C+and+tobacco+budworm%2C+Heliothis+virescens+%28F.%29%2C+to+indoxcarb+and+pyridalyl%5BTitle%5D)+AND+%22J.+Agricul.+Urban+Entomol%22%5BJournal%5D)">Baseline responses of bollworm, Heliocoverpa zea (Boddie), and tobacco budworm, Heliothis virescens (F.), to indoxcarb and pyridalyl.</a> J. Agricul. Urban Entomol. 22, 99-109 (2005).</li>
<li>Temple, J. H., Pommireddy, P. L., Cook, D. R., Maçon, P., Leonard, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Arthopod+management%3A+Susceptibility+of+selected+lepidopteran+to+pests+Rynaxypyr%2C+a+novel+insecticide%5BTitle%5D)+AND+%22J.+Cotton+Sci%22%5BJournal%5D)">Arthopod management: Susceptibility of selected lepidopteran to pests Rynaxypyr, a novel insecticide.</a> J. Cotton Sci. 13, 23-31 (2009).</li>
<li>Plapp, F. W., Vinson, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+toxicities+of+some+insecticides+to+the+tobacco+budworm+and+its+ichneumonid+parasite%2C+Campoletis+sonorensis%5BTitle%5D)+AND+%22Environ.+Entomol%22%5BJournal%5D)">Comparative toxicities of some insecticides to the tobacco budworm and its ichneumonid parasite, Campoletis sonorensis.</a> Environ. Entomol. 6, 381-384 (1977).</li>
<li>Xu, J., Shelton, A. M., Cheng, X. iaN. ian <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Variation+in+susceptibility+of+Diadegma+insulare+%28Hymenoptera%3A+Ichneumonidae%29+to+permethrin%5BTitle%5D)+AND+%22J.+Econ.+Entomol%22%5BJournal%5D)">Variation in susceptibility of Diadegma insulare (Hymenoptera: Ichneumonidae) to permethrin.</a> J. Econ. Entomol. 94, 541-546 (2001).</li>
<li>Snodgrass, G. L., Scott, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Seasonal+changes+in+pyrethroid+resistance+in+tarnished+plant+bug+%28Heteroptera%3A+Miridae%29+populations+during+a+three+year+period+in+the+delta+area+of+Arkansas%2C+Louisiana+and+Mississippi%5BTitle%5D)+AND+%22J.+Econ.+Entomol%22%5BJournal%5D)">Seasonal changes in pyrethroid resistance in tarnished plant bug (Heteroptera: Miridae) populations during a three year period in the delta area of Arkansas, Louisiana and Mississippi.</a> J. Econ. Entomol. 93, 441-446 (2000).</li>
<li>Miller, A. L. E., Way, M. O., Bernhardt, J., Stout, M. J., Tindall, K. V. Multi-state resistance monitoring of rice stink bug with a new and old insecticide. Proceedings Rice Technical Working Group, February 22-25, 2010, Biloxi, MS, , (2010).</li>
<li>Abbott, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+method+for+computing+the+effectiveness+of+an+insecticide%5BTitle%5D)+AND+%22J.+Econ.+Entomol%22%5BJournal%5D)">A method for computing the effectiveness of an insecticide.</a> J. Econ. Entomol. 18, 265-267 (1925).</li>
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<li>Preisler, H. K., Robertson, J. L. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPreisler%2C+HK+%22Pesticide+bioassays+with+arthropods%22">Pesticide bioassays with arthropods</a>. , CRC Press. Boca Raton, FL. (1992).</li>
<li>Hollingsworth, R. G., Steinkraus, D. C., Tugwell, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Responses+of+Arkansas+population+of+tarnished+plant+bugs+%28Heteroptera%3A+Miridae%29+to+insecticides%2C+and+tolerance+differences+between+nymphs+and+adults%5BTitle%5D)+AND+%22J.+Econ.+Entomol%22%5BJournal%5D)">Responses of Arkansas population of tarnished plant bugs (Heteroptera: Miridae) to insecticides, and tolerance differences between nymphs and adults.</a> J. Econ. Entomol. 90, 21-26 (1997).</li>
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No conflicts of interest declared.

The LC50 value can be used to establish as baseline susceptibility of a target population(s). The value of this data can be in future monitoring surveys or for the immediate purpose of comparing the current results to that of a previously determined LC50 to determine the susceptibility of the target population has shifted. Actual LC50 values can be compared among populations by examining the 95% confidence intervals; if the upper and lower limits do not overlap, then it is likely that the population has experienced a significant change in susceptibility and in some situations is an indication of resistance33. The LC50s can also be used to examine seasonal changes in insecticide esusceptibilitye28, or compare responses among species or insecticide AI s. Considerable work has also use these data to compare responses between males and females10 or between adults and immature stages,10,34. Sometimes confidence intervals are wide or not able to be calculated. To gain tighter confidence intervals conduct the bioassay with more insects and/or more concentrations.

<table><tbody><tr><td>50-100 ml glass beaker </td><td></td><td></td><td></td></tr><tr><td>Volumetric flask with stoppers (can use the amber colored flasks for light sensitive pesticides such as pyrethroids)</td><td></td><td></td><td></td></tr><tr><td>20ml glass scintillation vials with un-lined lids</td><td></td><td></td><td></td></tr><tr><td>A method for color coating vials (paint or markers)</td><td></td><td></td><td></td></tr><tr><td>Acetone</td><td></td><td></td><td></td></tr><tr><td>Commercial hotdog roller (heat element disconnected)</td><td></td><td></td><td></td></tr><tr><td>2 wash bottles (one modified such that the opening is large for fast delivery of the liquid and one so that the spout opening is small for slow delivery of the liquids)</td><td></td><td></td><td></td></tr><tr><td>Small weigh boat (make sure all plastic materials acetone safe)</td><td></td><td></td><td></td></tr><tr><td>Access to a balance with 0.001g readability or a higher precision.</td><td></td><td></td><td></td></tr><tr><td>Parafilm M</td><td></td><td></td><td></td></tr><tr><td>Repeater pipettor</td><td></td><td></td><td></td></tr><tr><td>Appropriate tips</td><td></td><td></td><td></td></tr><tr><td>Glass pipettes that fit in the volumetric flask.</td><td></td><td></td><td></td></tr><tr><td>Hood (used to remove the acetone smell)</td><td></td><td></td><td></td></tr><tr><td>A place to store insecticide solutions and vials [refrigerator (solutions), dark room or freezer depending on the chemical (vials)]</td><td></td><td></td><td></td></tr></tbody></table>

bioassays for monitoring insecticide resistance

Bioassays for Monitoring Insecticide Resistance

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products are labeled for an individual pest within a particular crop system, chemical control options are limited. Therefore, the same product(s) are used repeatedly and continual selection pressure is placed on the target pest. There are both financial and environmental costs associated with the development of resistant populations. The cost of pesticide resistance has been estimated at approximately $ 1.5 billion annually in the United States. This paper will describe protocols, currently used to monitor arthropod (specifically insects) populations for the development of resistance. The adult vial test is used to measure the toxicity to contact insecticides and a modification of this test is used for plant-systemic insecticides. In these bioassays, insects are exposed to technical grade insecticide and responses (mortality) recorded at a specific post-exposure interval. The mortality data are subjected to Log Dose probit analysis to generate estimates of a lethal concentration that provides mortality to 50% (LC50) of the target populations and a series of confidence limits (CL's) as estimates of data variability. When these data are collected for a range of insecticide-susceptible populations, the LC50 can be used as baseline data for future monitoring purposes. After populations have been exposed to products, the results can be compared to a previously determined LC50 using the same methodology.

This manuscript demonstrates and discusses techniques used to survey pesticide susceptibility and detect resistance to contact and systemic pesticides in arthropod pests.

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products ...

The overall goal of the following experiment is to determine if the insect has developed resistance to the insecticide. This is achieved by making concentrations to be tested by dissolving technical grade insecticide into acetone for contact insecticides as described by plaid AL in 1987. Or honey water solution for systemic insecticides as described by snores Etol in 2008.As a second step, Eloqua 0.5 milliliters of the acetone insecticide solution into a 20 milliliter glass insulation vial, and rotate vials on a commercial hotdog roller with the heating element turned off or Eloqua 0.5 milliliters of the honey water insecticide solution onto a piece of floral foam placed inside a vial. Next, collect the desired insect for the adult vial test in order to conduct the bioassay, which monitors for insecticide resistance results are obtained that show whether or not the insect has developed resistance to the insecticide based on comparing the lc 50 achieved via probate analysis to pre-existing lc fifties. Hello, my name is Audra Miller.I'm from the University of Missouri College of Agriculture, food and Natural Resources Delta Research Center in Portageville, Missouri. I'll be conducting an experiment out of Dr.Kelly Kindle's entomology lab. I'll be showing the procedure to detect whether or not insecticide resistance is occurring.Now let's get started. It is imperative to use proper safety precautions like safety goggles, masks and gloves, et cetera. When handling insecticides, consult the material safety data sheet for the recommended personal protective equipment of the specific pesticide being tested.In order to make a stock solution, adjust for purity and measure technical grade insecticide in a way. Boat in the hood partially fill a volumetric flask with acetone. Add the insecticide, rinse the whey boat with acetone into the flask.Fill the volumetric flask to the graduation line with acetone mixed by inverting several times. Make serial dilution as required color code the vials for the concentrations using paint or markers. Pour approximately half the volume as the number of vials to be made in a small beaker when the solution is not in use, cover it to minimize evaporation.Use a repeater pipetter to deliver 0.5 milliliters to the vials. Work from low to high concentration, starting with the control of acetone only in order to coat the inside of the vial with the technical grade insecticide. Place vials on a commercial hotdog roller with the heat turned off.Allow the vials to rotate until all the acetone has evaporated. Examine each vial individually to ensure dryness. Gather the insects to be tested using pheromone traps, sweep nets, or any other means of mass capture.Provide insects with food and moisture for eight to 24 hours. Select healthy active specimens for the bioassay with 10 to 25 insects per test.Concentration. Place insects in the vials such that they're exposed to the full range of the concentrations, not just one concentration at a time.After appropriate incubation time, assess mortality based on criteria of coordinated movement, such as the ability to write itself if placed on its s dorsal surface or coordinated movement when gently prodded. If the insect can write itself but falls over, there is no coordinated movement, so the insect should be assessed as dead. Record the number of live and dead individuals and calculate the percent survival for all concentrations.Prepare vials with the technical grade insecticide dissolved in 10%Honey water solution weigh honey to make a 10%by volume honey water solution transfer to a volumetric flask and add technical grade insecticide to the honey water solution and rinse the way boat. Repeat the process of making cereal dilution, but use honey water instead of acetone. Cut a piece of floral foam and place inside the vial Eloqua.Point five milliliters of solution onto the floral foam enclos insects with a cotton ball instead of a lid. Conduct the bioassay in the same manner as you would with the contact insecticides correct for mortality in the control. According to Abbott, determine the lethal concentration required to kill 50%of a population and 95%confidence intervals.This can be performed using a software program like SAS Proc probate or Polo Plus, for example, in this sample program for SAS dose represents the concentration used in the bioassay. N is the number of individuals tested at a given concentration and response is the number of dead individuals find the probability of 0.5 and this is the lc 50 with confidence limits of 0.1891 to 0.3273 micrograms per vile. If baseline data exists for the insect tested or an lc 50 obtained from a known susceptible population, the new lc 50 can be compared to the lc 50 of baseline data or susceptible populations.If the baseline lc 50 was 0.1487 micrograms per vile with confidence limits of 0.1167 to 0.1793 micrograms per vile, one could conclude there was a significant change in resistance because the confidence limits do not overlap. However, if the baseline lc 50 was 0.1487 micrograms per vile, but the confidence limits were 0.0958 to 0.1983 micrograms per vile, there would not be a significant increase in resistance because the confidence limits overlapped. This experiment can be used to help tell if insecticide resistance is a cause for the insecticide failure or not, but remember when working with insecticide, you should always use the proper protective equipment.Now.Thanks for watching and good luck with your experiments.

bioassays-for-monitoring-insecticide-resistance

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Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

In this video, we demonstrate the topical testing assay to determine the toxicity of a potential insecticide on adult female mosquitoes. Using a dopamine receptor antagonist that causes lethal inhibition of neurotransmission in mosquitoes, this method describes a way to evaluate the toxic effects of insecticides upon topical application.

Topical Testing Assay: A Technique to Evaluate the Insecticidal Potential of Dopamine Receptor Antagonists on Adult Female Mosquitoes by Topical Application

A robust and flexible approach to confirm herbicide resistance in weed populations is presented. This protocol allows the herbicide resistance levels to be inferred and applied to a wide range of weed species and herbicides with minor adaptations.

Protocols for Robust Herbicide Resistance Testing in Different Weed Species

Insecticide evaluations are often targeted against adult insects, rather than immature stages. Here, we present a protocol for evaluating insecticides against bed bug eggs with a comparison to the first nymphal bed bug stage. These protocols could be adjusted for other insects to evaluate insecticide efficacy in non-adult life stages.

A Method for Evaluating Insecticide Efficacy against Bed Bug, Cimex lectularius, Eggs and First Instars

Photo-attraction bioassay arenas are used to determine the optimal light color(s) to maximize insect attraction; however bioassays and methods are specific to target insect behaviors and habitats. Customizable equipment and modifications are explained for nocturnal or diurnal and terrestrial or aerial insects.

Key Elements of Photo Attraction Bioassay for Insect Studies or Monitoring Programs

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

Here, we present a protocol to produce house fly carboxylesterase proteins in vitro with a baculovirus mediated insect cell expression system and later functionally characterize their roles in metabolizing permethrin, thereby, conferring pyrethroid resistance by conducting cell-based MTT assay and in vitro metabolic studies.

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

The efficacy of public health pesticides targeting nuisance and disease-vector insects is not uniform across different ecological zones. Here we present a system of techniques using captive vector insects as sentinels for pesticide efficacy to derive electronic maps supporting the standard evaluation of pesticides across multiple environments.

Visualizing Efficacy of Pesticides Against Disease Vector Mosquitoes in the Field

We describe the methodology and importance of the topical application bioassay to measure insecticide susceptibility in mosquitoes and fruit flies. The presented assay is high-throughput, utilizes insect mass-thus allowing for calculating a mass-relativized lethal dose instead of concentration-and likely has lower variability than other similar methods.

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies

This protocol describes the obligate feeding assay to evaluate the potentially toxic effect of a phytochemical on the lepidopteran insect larvae. This is a highly scalable insect bioassay, easy to optimize the sublethal and lethal dose, deterrent activity, and physiological effect. This could be used for screening eco-friendly insecticides.

Developing a Feeding Assay System for Evaluating the Insecticidal Effect of Phytochemicals on Helicoverpa armigera

Learn bioassay techniques to monitor insecticide resistance in arthropod pests using adult vial tests. This method compares LC50 values to assess susceptibility and inform pest management strategies.

Bioassays for Monitoring Insecticide Resistance

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