INSECTICIDE RESISTANCE MANAGEMENT OF HOUSE FLIES AT ANIMAL PRODUCTION FACILITIES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0231048
• Project Start Date: Oct 1, 2012
• Project End Date: Sep 30, 2016
• Program Code: [(N/A)]- (N/A)

Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853

Performing Department
Entomology

Non Technical Summary
House flies, Musca domestica L. (Diptera: Muscidae), are cosmopolitan, ubiquitous, and transmit more than 100 human and animal diseases. House flies are also one of the most serious pests at dairy, horse, hog, sheep and poultry facilities worldwide. House fly activities result in exposure to debilitating disease causing agents, public health and nuisance concerns, lowered levels of milk and egg production, and reduced feed conversion. Economic losses and the cost associated with fly suppression are difficult to quantify, but costs of pesticides for fly control at poultry facilities alone are estimated at over $200 million annually in the USA. The primary means for control of house flies is with the use of insecticides. House flies have shown a remarkable ability to rapidly evolve resistance to each of the insecticides used against it. Resistance results in increased application frequencies and amounts. This results in decreased yields, increased environmental damage, increased costs, increased worker safety concerns, and outbreaks of human and animal diseases when vectors cannot be controlled. Thus, it is essential to understand the mechanisms by which insects become resistant so that we are able to intelligently design strategies to delay the onset of this potentially devastating problem. Pyrethroids are one of the largest and most widely used classes of insecticides, with several compounds registered for house fly control. The primary target site of pyrethroid insecticides is the voltage sensitive sodium channel (Vssc). Mutations in Vssc are an extremely important mechanism of resistance to pyrethroid insecticides. In house flies the first such mutation described was kdr (L1014F) and this mutation has been found worldwide. A second mutation, super-kdr (M918T + L1014F) conferring higher levels of resistance (for most pyrethroids, but not all) was subsequently detected. A third mutation, kdr-his (L1014H) was reported in 2001, and as now been found globally. Overall, the level of protection conferred by house fly Vssc mutations to pyrethroid insecticides is super-kdr > kdr > kdr-his, although there is very limited information about kdr-his. Despite this, at some the frequency of kdr-his is very high and the frequencies of kdr and super-kdr are low. This has led to speculation about what factors could be driving the high kdr-his allele frequencies in some populations. The goal of this study is to better understand the selective advantage of kdr-his flies, both when exposed to different pyrethroid insecticides, and in the absence of insecticides. Our long term goal is to understand the forces that select for kdr-his, and to determine the relative fitness cost of this allele in the absence of insecticides. We will isolate a strain of house fly having only the kdr-his resistance mechanism and determine its fitness through a series of experiments. To provide measures to delay the evolution of resistance we need to understand the forces which select for the trait and the relative rate at which the resistance alleles are lost from a population in the absence of insecticides. Results from this proposal will address this need.

Animal Health Component

30%

Research Effort Categories

Basic

70%

Applied

30%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
307	3110	1130	25%
311	3110	1130	25%
315	3110	1130	25%
723	5220	1130	25%

Knowledge Area
311 - Animal Diseases; 723 - Hazards to Human Health and Safety; 307 - Animal Management Systems; 315 - Animal Welfare/Well-Being and Protection;

Subject Of Investigation
3110 - Insects; 5220 - Pesticides;

Field Of Science
1130 - Entomology and acarology;

Keywords

resistance management

pyrethroid insecticides

kdr

super-kdr

kdr-his

Goals / Objectives
OBJECTIVES: House flies are serious pests at animal facilities and pyrethroid insecticides are one of the major tools used to suppress fly populations. However, resistance to pyrethroids has evolved in some populations. Our long term goal is to understand the forces that select for kdr-his, and to determine the relative fitness cost of this allele in the absence of insecticides. Toward this end we will pursue three specific objectives. Objective 1. Quantify the relative level of resistance conferred by the kdr-his mutation to commonly used pyrethroid insecticides. Objective 2. Determine the inheritance of kdr-his to pyrethroid insecticides. Objective 3. Examine the fitness of kdr-his relative to a susceptible allele in the absence of insecticide.

Project Methods
Objective 1. Determine the relative level of protection conferred by the kdr-his mutation to commonly used pyrethroid insecticides. The kdr-his allele will be introgressed into the background of the susceptible aabys strain using a series of crosses and backcrosses. The NC19 strain was collected from North Carolina and is homozygous for kdr-his, and aabys is a standard susceptible strain with morphological mutations on each of the five autosomes . We will cross unmated aabys females with NC19 males. The F1 males will be backcrossed to aabys females. From the resulting backcross offspring, unmated flies having the aa+ys phenotype will be isolated and crossed. Unmated males and females from this cross will be placed into a cup (one male and one female per cup) and eggs will be collected. After sufficient eggs have been collected, the adults will be genotyped. At least three lines in which both parents were homozygous for kdr-his will be maintained. We will isolate a minimum of three separate lines being homozygous for kdr-his. Adult female flies will be bioassayed by topical application using standard methods. Pyrethroids are available from commercial vendors such as ChemService. Resistance ratios for the kdr-his strain will be obtained by conducing side-by-side assays with the aabys strain. We will compare the resistance ratios for kdr-his (LD50 kdr-his/LD50 aabys) we obtain to those of kdr and super-kdr reported in the literature. In this way we will know what the relative selective advantage is for each resistance allele (when homozygous) for different pyrethroid insecticides. Pyrethroid insecticides (technical grade materials) will be selected based on their relative use in the USA and/or by the availability of published resistance ratios for kdr and super-kdr (for comparison purposes). We will test permethrin, cyfluthrin, deltamethrin, cypermethrin, pyrethrins + PBO and fenvalerate. Additional pyrethroids will be tested if time allows. Objective 2. Determine the inheritance of kdr-his to pyrethroid insecticides. We will cross unmated aabys females to kdr-his males and bioassay the F1 offspring with pyrethroid insecticides (see Objective 1 for list). The inheritance of the resistance for each pyrethroid will be calculated according to the method of Stone. The inheritance of kdr and super-kdr is usually incompletely recessive to most pyrethroids. In this way we will know what the relative selective advantages is for each resistance allele in heterozygous condition for different pyrethroid insecticides. Objective 3. Examine the fitness of kdr-his relative to a susceptible allele in the absence of insecticide. We will establish four replicate cages each containing unmated flies of identical age: Two cages (i.e. duplicates) of 100 male aabys + 300 female kdr-his and two cages (duplicates) of 100 male kdr-his + 300 female aabys. At each generation 100 flies will be genotyped from each replicate cage to determine which Vssc alleles they contain. It is estimated that we will allow each cage to run for 30 generations. Analyses of allele frequencies and genotypes will be conducted as described previously.

Progress 10/01/12 to 09/30/16

Outputs
Target Audience:Our work will directly serve the needs of those involved in the dairy and poultry industries. This is by nature a diverse group of stakeholders that includes women and minorities. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three technicians received training over the course of the project. They were all able to learn new skills and improve on existing skill sets. Several undergraduates (15) were involved in this project. Some had simple jobs and others were doing experiments (under supervision). All gained new skills and an appreciation for research. The PI continued his professional develppment through publication of research findings and presentations a scientific meetings. How have the results been disseminated to communities of interest?All results have (or will soon be) published in peer-reviewed journals, which are globally available. In addition, specific results from each state were sent to collaborators who work directly with animal production owners and staff. The results generated from the research have been used to educate stakeholders about the problem of insecticide resistance, what it costs them and what they can do about it. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Target site insensitivity because of mutations in the voltage-sensitive sodium channel gene (Vssc) is a major mechanism of resistance to pyrethroid insecticides in the house fly, Musca domestica. There are three known Vssc alleles that confer resistance to pyrethroids in the house fly: knock down resistance (kdr; L1014F), super-kdr (M918T1L1014F) and kdr-his (L1014H), but there has been no side-by-side comparison of the resistance levels that they confer. We compared the levels of resistance conferred by the three Vssc alleles in congenic strains to 19 structurally diverse pyrethroids, and compared the full-length Vssc cDNA sequences from each strain. Generally, the levels of resistance conferred were kdr-his
Publications

• Type: Journal Articles Status: Published Year Published: 2012 Citation: Wang, Q., Li, M., Pan, J., Di, M., Liu, Q., Meng, F., Scott, J. G. and Qiu, X. 2012. Diversity and frequencies of genetic mutations involved in insecticide resistance in field populations of the house fly (Musca domestica L.) from China. Pestic. Biochem. Physiol. 102: 153-159.
• Type: Journal Articles Status: Published Year Published: 2012 Citation: Rinkevich, F. D., Schweitzer, P. A. and Scott, J. G. 2012. Antisense sequencing improves the accuracy and precision of A-to-I editing measurements using the peak height ratio method. BMC Res. Notes. 5:63.
• Type: Journal Articles Status: Published Year Published: 2012 Citation: Gao, Q., Li, M., Sheng, C., Scott, J. G. and Qiu, X. 2012. Multiple cytochrome P450s overexpressed in pyrethroid resistant house flies (Musca domestica). Pestic. Biochem. Physiol. 104: 252-260.
• Type: Journal Articles Status: Published Year Published: 2012 Citation: Rinkevich, F. D., Hedtke, S. M., Leichter, C. A., Harris, S. A., Su, C., Brady, S. G., Taskin, V., Qiu, X. and Scott, J. G. 2012. Multiple origins of kdr-type resistance in the house fly, Musca domestica. PLOS One 7:e52761.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Rinkevich, F. D., Leichter, C. A., Lazo, T. A., Hardstone, M. C. and Scott, J. G. 2013. Variable fitness costs for pyrethroid resistance alleles in the house fly, Musca domestica, in the absence of insecticide pressure. Pestic. Biochem. Physiol. 105: 161-168.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Scott, J. G., Leichter, C. A., Rinkevich, F. D., Harris, S. A., Su, C., Aberegg, L. C. Roger Moon, R., Geden, C. J., Gerry, A. C., Taylor, D., Byford, R. L., Watson, W., Johnson, G., Boxler, D. and Zurek, L. 2013. Insecticide resistance in house flies from the United States: Resistance levels and frequency of pyrethroid resistance alleles. Pestic. Biochem. Physiol. 107: 377-384.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Li, M., Reid, W. R., Zhang, L., Scott, J. G., Gao, X., Kristensen, M., and Liu, N. 2013. A whole transcriptomal linkage analysis of gene co-regulation in insecticide resistant house flies, Musca domestica. BMC Genomics 14: 803.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Kavi, L. A. K., Kaufman, P. E. and Scott, J. G. 2014. Genetics and mechanisms of imidacloprid resistance in house flies. Pestic. Biochem. Physiol. 109:64-69.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: H�jland, D. H., Scott, J. G., Jensen, K.-M. V. and Kristensen, M. 2014. Autosomal male determination in a spinosad-resistant house fly strain from Denmark. Pest Manag. Sci. 70: 114-117.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Scott, J. G., Warren, W. C., Beukeboom, L. W., Bopp, D., Clark, A. G., Giers, S. D., Hediger, M., Jones, A. K., Kasai, S., Leichter, C. A., Li, M., Meisel, R. P., Minx, P., Murphy, T. D., Nelson, D. R., Reid, W. R., Rinkevich, F. D., Robertson, H. M., Sackton, T. B., Sattelle, D. B., Thibaud-Nissen, F., Tomlinson, C., van de Zande, L., Walden, K. K. O., Wilson, R. K. and Liu, N. 2014. Genome of the house fly (Musca domestica L), a global vector of diseases with adaptations to a septic environment. Genome Biol. 15: 466.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Hamm, R. L., Meisel, R. P. and Scott, J. G. 2015. The evolving puzzle of autosomal versus Y-linked male determination in Musca domestica. G3 5: 371-384.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Meisel, R. M., Scott, J. G. and Clark, A. G. 2015. Transcriptome differences between alternative sex determining genotypes in the house fly, Musca domestica. Genome Biol. Evol. 7: 2051-2061.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Sun, H., Tong, K.P., Kasai, S. and Scott, J. G. 2016. Overcoming super-kdr mediated resistance: Multi-halogenated benzyl pyrethroids are more toxic to super-kdr than kdr house flies. Insect Molec. Biol. 25: 126-137.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Meisel, R. P., Davey, T., Son, J. H., Gerry, A. C., Shono T. and Scott, J. G. 2016. Is multifactorial sex determination in the house fly, Musca domestica (L.), stable over time? J. Heredity 107: 615-625 (doi: 10.1093/jhered/esw051).
• Type: Journal Articles Status: Awaiting Publication Year Published: 2017 Citation: Scott, J. G. Evolution of pyrethroid resistance in Musca domestica. Pest Manag. Sci. DOI 10.1002/ps.4328).
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Kasai, S., Sun, H. and Scott, J. G. 2016. Diversity of knockdown resistance alleles in a single house fly population facilitates adaptation to pyrethroid insecticides. Insect Molec. Biol. DOI: 10.1111/imb.12267

Progress 10/01/14 to 09/30/15

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Undergraduate students havereceived training in molecular biology, basic entomology, insect rearing, toxicology and data analyses. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Several new Vssc mutations were recently found from a dairy in Kansas. These appear too linked to pyrethroid resistance, although the levels of resistance they confer is unknown. We will isolate isogenic strains for each of these mutations and text the toxicity of pyrethroid insecticides against the resistant and susceptible strains to characterize the levels of resistance they provide. We will also determine the frequency of these alleles in flies collected from the dairy in 2015. This information will provide us with information we need to effectively control house flies at animal production facilities.

Impacts
What was accomplished under these goals? Target site insensitivity due to mutations in the voltage-sensitive sodium channel gene (Vssc) is a major mechanism of resistance to pyrethroid insecticides in the house fly, Musca domestica. There are three known Vssc alleles that confer resistance to pyrethroids in house fly: kdr (L1014F), super-kdr (M918T + L1014F), and kdr-his (L1014H), but there has been no side-by-side comparison of the resistance levels they confer. We compared the levels of resistance conferred by the three Vssc alleles in congenic strains to 19 structurally diverse pyrethroids, and compared the full length Vssc cDNA sequences from each strain. Generally, the levels of resistance conferred were kdr-his < kdr < super-kdr. However, there was significant variation in this pattern, especially for super-kdr where both high and low resistance ratios were observed for several pyrethroids. We also examined the levels of resistance in heterozygotes. Resistance in each of the hybrids was generally inherited as an incompletely recessive trait, except for the kdr-his/kdr hybrids which showed incompletely to completely dominant resistance (i.e. had resistance levels comparable to kdr homozygotes). It was generally believed that the super-kdr allele conferred higher levels of resistance to pyrethroids than kdr or kdr-his. Our results indicate this is not the case for three pyrethroids with multi-halogenated benzyl groups (1R-trans fenfluthrin, tefluthrin and transfluthrin), as these show lower protection with the super-kdr mutation than with kdr. Conversely, for some pyrethroids the level of protection conferred by super-kdr is about 1000-fold greater than kdr. Thus, in house fly populations where super-kdr is common, our results suggest there are four pyrethroids (etofenprox, flumethrin, fenpropathrin and acrinathrin) which would be largely ineffective. Our results are consistent with heterologous recombination studies which have shown that some mutations may confer reduced insensitivity to one pyrethroid, but not to another (e.g. I1011M reducing sensitivity to permethrin, but not deltamethrin), and that the M918T mutation conferred protection of VSSC against cismethrin, cypermethrin, deltamethrin and permethrin, but not to fenfluthrin. The relative resistance levels we observed for kdr and kdr-his are similar to those proposed, based on in vitro studies for permethrin, but not for deltamethrin. Our finding of a slightly lower level of resistance to 1R-trans fenfluthrin, tefluthrin and transfluthrin in the JPskdr compared to the ALkdr strain suggests a slight antagonism of the protection afforded by the 1014F mutation in the super-kdr strain. A paper was submitted based in these results and was recently accepted for publication.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Meisel, R. P., J. G. Scott, and A. G. Clark. 2015. Transcriptome differences between alternative sex determining genotypes in the house fly, Musca domestica. Genome Biol. Evol. 7:2051-2061.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Hamm, R. L., R. P. Meisel, and J. G. Scott. 2015. The evolving puzzle of autosomal versus Y-linked male determination in Musca domestica. G3 5:371-384.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Undergraduate students have been trained with several molecular biology skills. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? The first set of allele competition studies will be completed. Bioassays will be completed on the strains and hybrids.

Impacts
What was accomplished under these goals? Isogenic strains, susceptible or containing one of the resistance mutations were isolated. Bioassays and allele competition studies have been started.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Scott, J. G., Warren, W. C., Beukeboom, L. W., Bopp, D., Clark, A. G., Giers, S. D., Hediger, M., Jones, A. K., Kasai, S., Leichter, C. A., Li, M., Meisel, R. P., Minx, P., Murphy, T. D., Nelson, D. R., Reid, W. R., Rinkevich, F. D., Robertson, H. M., Sackton, T. B., Sattelle, D. B., Thibaud-Nissen, F., Tomlinson, C., van de Zande, L., Walden, K. K. O., Wilson, R. K. and Liu, N. 2014. Genome of the house fly (Musca domestica L), a global vector of diseases with adaptations to a septic environment. Genome Biol. 15: 466.

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: The audience and intended beneficiaries are primarily those involved in dairy and poultry operations, as well as the consumers of products produced at these facilities. Our results are also likely to benefit other animal production facilities. Effective resistance management reduces pesticide use and extends the number of seasons that an effective control agent can be used. This resulted in less pesticide application, increased worker safety, reduced levels of pesticide residues and lower amounts of inputs being necessary for production. In addition, effective fly control is necessary to prevent litigation from those living in proximity to animal production facilities. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? There was training provided forfour undergraduates, one of whom completed an honors thesis. There was also training provided for two technicians and one graduate student. How have the results been disseminated to communities of interest? Results were presented at the National Entomological Society of America meetings in 2012. Some results have been published. What do you plan to do during the next reporting period to accomplish the goals? We will investigate the genetics and mechanisms of imidacloprid resistance in the house fly.

Impacts
What was accomplished under these goals? Although insecticide resistance is a widespread problem for most insect pests, frequently the assessment of resistance occurs over a limited geographic range. Herein, we report the first widespread survey of insecticide resistance in the USA ever undertaken for the house fly, Musca domestica, a major pest in animal production facilities. The levels of resistance to six different insecticides were determined (using discriminating concentration bioassays) in 10 collections of house flies from dairies in nine different states. In addition, the frequencies of Vssc and CYP6D1 alleles that confer resistance to pyrethroid insecticides were determined for each fly population. Levels of resistance to the six insecticides varied among states and insecticides. Resistance to permethrin was highest overall and most consistent across the states. Resistance to methomyl was relatively consistent, with 65-91% survival in nine of the ten collections. In contrast, resistance to cyfluthrin and pyrethrins + piperonyl butoxide varied considerably (2.9-76% survival). Resistance to imidacloprid was overall modest and showed no signs of increasing relative to collections made in 2004, despite increasing use of this insecticide. The frequency of Vssc alleles that confer pyrethroid resistance was variable between locations. The highest frequencies of kdr, kdr-his and super-kdr were found in Minnesota, North Carolina and Kansas, respectively. In contrast, the New Mexico population had the highest frequency (0.67) of the susceptible allele.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Scott, J. G., Leichter, C. A., Rinkevich, F. D., Harris, S. A., Su, C., Aberegg, L. C. Roger Moon, R., Geden, C. J., Gerry, A. C., Taylor, D., Byford, R. L., Watson, W., Johnson, G., Boxler, D. and Zurek, L. 2013. Insecticide resistance in house flies from the United States: Resistance levels and frequency of pyrethroid resistance alleles. Pestic. Biochem. Physiol. 107: 377-384.