Source: CORNELL UNIVERSITY submitted to NRP
MOLECULAR BIOLOGY, BIOCHEMISTRY AND GENETICS AND OF INSECTICIDE RESISTANCE
Sponsoring Institution
State Agricultural Experiment Station
Project Status
COMPLETE
Funding Source
STATE
Reporting Frequency
Annual
Accession No.
0198489
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2003
Project End Date
Sep 30, 2008
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
ENTOMOLOGY
Non Technical Summary
Insecticide resistance reduces crop yields and hinders control of disease vectors. The purpose of this project is to learn more about how insects evolve resistance.
Animal Health Component
40%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2113110115010%
3113110104010%
7113110115040%
7213110104040%
Knowledge Area
311 - Animal Diseases; 211 - Insects, Mites, and Other Arthropods Affecting Plants; 711 - Ensure Food Products Free of Harmful Chemicals, Including Residues from Agricultural and Other Sources; 721 - Insects and Other Pests Affecting Humans;

Subject Of Investigation
3110 - Insects;

Field Of Science
1040 - Molecular biology; 1150 - Toxicology;
Goals / Objectives
The objectives of the proposed research are to identify the CYP6D1 regulatory elements involved in insecticide resistance and to examine the role of different CYP6D1 alleles in resistance. To accomplish this objective, the following specific aims will be undertaken: 1. More fully understand the basis for enhanced transcription of CYP6D1 in resistant house flies. A. Identify regions in the CYP6D1 5' flanking sequence that bind nuclear proteins. B. Identify the proteins binding to the CYP6D1 5' flanking sequences. C. Determine if (and where) the transcriptional repressor Gfi-1 binds to the 5' flanking sequence of CYP6D1 from resistant and susceptible strains. D. Define the promoter region of CYP6D1. E. Compare the relative rates of transcription mediated by CYP6D1 5' flanking regions from susceptible and resistant strains. 2. Evaluate the role of the different CYP6D1 alleles in resistance.
Project Methods
We will identify regions in the CYP6D1 5' flanking region that bind nuclear proteins using gel shift assays and DNase I footprinting assays.

Progress 10/01/03 to 09/30/08

Outputs
OUTPUTS: Spinosad resistance has been selected for and characterized in several insect species, although the molecular basis of the resistance is still not known. In Heliothis virescens a strain was selected that was 317-fold resistant when exposed to treated diet and 1070-fold resistant by topical application. Resistance was incompletely recessive. A strain of Plutella xylostella was collected in Hawaii and selected with spinosad which produced a strain that was 18,600-fold resistant (leaf dip bioassay). Spinosad resistance could not be overcome by insecticide synergists, leading to the conclusion that metabolism mediated detoxification was probably not responsible for the resistance. Spinosad resistance was controlled by one locus and was recessive in P. xylostella from Hawaii and partially recessive in P. xylostella from Malaysia and China. Spinosad resistance has also been studied in Spodoptera exigua (85-fold), B. dorsalis (400-fold) and Musca domestica (below). Given the recessive nature of spinosad resistance, heterozygotes are very difficult to detect using insecticide bioassays.51) If the mutation responsible for resistance could be identified, a powerful resistance monitoring technique could be developed that would differentiate homozygous susceptible, homozygous resistant and heterozygous individuals. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Spinosad is a new and highly promising insecticide, derived from the bacteria Saccharopolyspora spinosa, with efficacy against a wide range of insects. The mechanism of action of spinosad appears to be unique, with a primary site of attack being the nicotinic acetylcholine receptor and a secondary site of attack being GABA receptors. Nicotinic acetylcholine receptors (nAChR) belong to the Cys-loop superfamily of ligand-gated ion channels that include -aminobutyric acid (GABA)-gated channels, glycine receptors, glutamate-gated Cl- channels and 5-hydroxytryptamine type 3 receptors. Neural nAChRs are composed of five subunits, with a minimum of 2 αs. Receptors consisting of only α subunits are known in vertebrates, but not in invertebrates. Each subunit possesses a large N-terminal extracellular domain that includes the acetylcholine (ACh) binding site and four transmembrane domains (M1-4) with M2 contributing most of the amino acids that line the ion channel. The relatively small number of nAChR subunits in insects is compensated for by diversification due to alternative exon use and RNA editing. Spinosad resistance has been selected for and characterized in several insect species. Generally, resistance is monofactorial, recessive and cannot be overcome by insecticide synergists. Spinosad resistance in the house fly maps to chromosome 1 and three nAChR subunit genes (α5, α6, and β3) are predicted to exist on chromosome 1 based on Drosophila/Musca homology maps. However, cloning and sequencing of Mdα5, Mdα6, and Mdβ3 from susceptible and spinosad resistant strains of house fly found no differences that could be associated with resistance.

Publications

  • Rinkevich, F. D., Hamm, R. L., Geden, C. J. and Scott, J. G. 2007. Dynamics of insecticide resistance alleles in two different climates over an entire field season. Insect Biochem. Molec. Biol. 37: 550-558.
  • Gao, J.-R., Deacutis, J. M. and Scott, J. G. 2007. The nicotinic acetylcholine receptor subunit gene Md6 from Musca domestica is diversified via post transcriptional modification. Insect Molec. Biol. 16: 325-334.
  • Gao, J.-R., Kozaki, T., Leichter, C. A., Rinkevich, F. D., Shono, T., and Scott, J. G. 2007. The A302S mutation in Rdl that confers resistance to cyclodienes and limited cross-resistance to fipronil is undetectable in field populations of house flies from the USA. Pestic. Biochem. Physiol. 88: 66-70.
  • Gao, J.-R., Deacutis, J. M. and Scott, J. G. 2007. The nicotinic acetylcholine receptor subunit gene Mdalpha2 from the housefly, Musca domestica. Arch. Insect Biochem. Molec. Biol. 64: 30-42.
  • Ozoe, Y., Ishikawa, S., Tomiyama, S., Ozoe, F. and Scott, J. G. 2007. Antagonism of the GABA receptor of dieldrin-resistant houseflies by fipronil and its analogues. In Synthesis and Chemistry of Agrochemicals Series VII, J. W. Lyga and G. Theodoritis, ed. Amer. Chem Soc., Washington, DC, pp 39-50.
  • Gao, J.-R., Deacutis, J. M. and Scott, J. G. 2007. Characterization of the nicotinic acetylcholine receptor subunits Mdalpha5 and Mdbeta3 on autosome 1 of Musca domestica indicate they are not involved in spinosad resistance. Insect Molec. Biol. 16: 691-701.
  • Hardstone, M.C., Leichter, C.A., Harrington, L.C., Kasai, S., Tomita, T. and Scott, J. G. 2007. Cytochrome P450 monooxygenase-mediated permethrin resistance confers limited cross-resistance in larvae of the southern house mosquito, Culex pipiens quinquefasciatus. (89: 175-184).
  • Hamm, R. L. Kaufman, P. E., Reasor, C. A., Rutz, D. A. and Scott, J. G. 2006. Resistance to cyfluthrin and tetrachlorvinphos in the lesser mealworm, Alphitobius diaperinus, collected from the eastern United States. Pest Manag. Sci. 62: 673-677.
  • Gao, J. and Scott, J. G. 2006. Role of the transcriptional repressor mdGfi-1 in CYP6D1v1-mediated insecticide resistance in the house fly, Musca domestica. Insect Biochem. Molec. Biol. 36: 387-395.
  • Rinkevich, F. D., Zhang, L., Hamm. R. L., Brady, S. G., Brian P. Lazzaro, B. P. and Scott, J. G., 2006. Frequencies of the pyrethroid resistance alleles of Vssc1 and CYP6D1 in house flies from the eastern United States. Insect Molec. Biol. 15: 157-167.
  • Paul, A., Harrington, L. C., Zhang, L. and Scott, J. G. 2005. Insecticide resistance in Culex pipiens from New York. J. Am. Mosq. Cont. Assoc. 21: 305-309.
  • Ponlawat, A., Scott, J. G. and Harrington, L. C. 2005. Insecticide susceptibility of Aedes aegypti and Aedes albopictus across Thailand. J. Med. Entomol. 42: 821-825.
  • Hamm, R., Shono, T. and Scott, J. G. 2005. A cline in frequency of autosomal males is not associated with insecticide resistance in house fly. J. Econ. Entomol. 98: 171-176.
  • Shono, T., Zhang, L. and Scott, J. G. 2004. Indoxacarb resistance in the house fly, Musca domestica. Pestic. Biochem. Physiol. 80: 106-112.
  • Scott, J. G., Leichter, C. A. and Rinkevich, F. D. 2004. Insecticide resistant strains of house flies, Musca domestica show limited cross-resistance to chlorfenapyr. J. Pestic. Sci. 29: 124-126.
  • Scott, J. G. and Kasai, S. 2004. Evolutionary plasticity of monooxygenase-mediated resistance. Pestic. Biochem. Physiol. 78:171-178.
  • Scott, J. G. 2008. Unraveling the mystery of spinosad resistance in insects. J. Pestic. Sci. 33: 221-227.
  • Scott, J. G. 2008 a. Insect Cytochrome P450s: Thinking Beyond Detoxification. In Recent Advances in Insect Physiology, Toxicology and Molecular Biology, N. Liu, ed. Research Signpost, Kerala, India. pp 117-124.
  • Kozaki, T., Kimmelblatt, B. A., Hamm, R. L. and Scott, J. G. 2008. Comparison of two acetylcholinesterase gene cDNAs of the lesser mealworm, Alphitobius diaperinus, in insecticide susceptible and resistant strains. Arch. Insect Biochem. Physiol. 67: 130-138. 142.
  • Deacutis, J. M., Leichter, C. A., Gerry, A. C., Rutz, D. A., Watson, W. D., Geden, C. J. and Scott, J. G. 2007. Susceptibility of field collected house flies to spinosad before and after a season of use. J. Agric. Urban Entomol. 23: 105-110.


Progress 10/01/06 to 09/30/07

Outputs
House flies were collected from four dairies in Maine, New York, North Carolina, and Florida where high levels of resistance to permethrin have been documented. Regions of two genes, CYP6D1 and Vssc1, having alleles that confer resistance to permethrin (and other pyrethroids) were analyzed from individuals at each collection site. The combinations of resistance alleles for Vssc1 and CYP6D1 were highly variable between each state. The resistance allele CYP6D1v1 was found at a high frequency (0.63-0.91) at all sites. Individuals homozygous susceptible for CYP6D1 were very rare and detected only at the dairy in Maine. In addition to the typical Vssc1 mutation responsible for resistance, kdr (L1014F), we also identified individuals with a L1014H mutation. Although house flies homozygous for the L1014H mutation had a lower level of resistance to permethrin, compared to L1014F, the H1014 resistance allele was frequently detected. No individuals with the super-kdr allele (M918T + L1014F) were detected from the field collections. The intron 3 bp downstream of the kdr mutation was found to be extremely variable, providing an opportunity to reconstruct a phylogeny of Vssc1 alleles. Based on this analysis it appears the kdr-his mutation had multiple evolutionary origins, but that the kdr mutation may have had a single origin.

Impacts
Our results show that the environment has an impact on the relative fitness imparted by insecticide resistance alleles. These results suggest that reistance management strategies will vary between locations with different climates.

Publications

  • Rinkevich, F. D., Zhang, L., Hamm. R. L., Brady, S. G., Brian P. Lazzaro, B. P. and Scott, J. G., 2006. Frequencies of the pyrethroid resistance alleles of Vssc1 and CYP6D1 in house flies from the eastern United States. Insect Molec. Biol. 15: 157-167.
  • Ozoe, Y., Ishikawa, S., Tomiyama, S., Ozoe, F. and Scott, J. G. 2007. Antagonism of the GABA receptor of dieldrin-resistant houseflies by fipronil and its analogues. In Synthesis and Chemistry of Agrochemicals Series VII, J. W. Lyga and G. Theodoritis, ed. Amer. Chem Soc., Washington, DC, pp 39-50.
  • Gao, J.-R., Deacutis, J. M. and Scott, J. G. 2007. The nicotinic acetylcholine receptor subunit gene Mdalpha2 from the housefly, Musca domestica. Arch. Insect Biochem. Molec. Biol. (64: 30-42).
  • Gao, J.-R., Kozaki, T., Leichter, C. A., Rinkevich, F. D., Shono, T., and Scott, J. G. 2007. The A302S mutation in Rdl that confers resistance to cyclodienes and limited cross-resistance to fipronil is undetectable in field populations of house flies from the USA. Pestic. Biochem. Physiol. 88: 66-70.


Progress 01/01/06 to 12/31/06

Outputs
Insecticides are one of the major tools for controlling vector populations and reducing the transmission of human pathogens. However, there are few new insecticides being developed and marketed for vector control. Herein we report on the toxicity of six novel insecticides to both adult and larval Aedes aegypti, and the toxicity of three novel insect growth regulators (IGRs) to larvae. Four insecticides were highly or moderately toxic to larvae with LC50 values of 16 (chlorfenapyr), 70 (hydramethylnon), 79 (indoxacarb) and 84 ng/ml (imidacloprid). Diafenthiuron and chlorfenapyr were moderately toxic to adult mosquitoes with LC50 values of 13 and 92 ng/cm2, respectively. Imidacloprid was strongly synergised by piperonyl butoxide (PBO) in Ae. aegypti adults, suggesting that neonicotinoids are intrinsically very toxic to adult mosquitoes (in the absence of detoxification). The effect of PBO on the toxicity in adults and larvae was considerably different, both in terms of the insecticides that were synergised (or antagonized in the case of chlorfenapyr vs. adults) and in terms of the degree of synergism. This result implies that the P450s involved in metabolism of these insecticides are different between adults and larvae. Pyriproxyfen was confirmed as a potent IGR (EC50 of 0.0017 ng/ml) for mosquitoes, although tebufenozide lacked activity.

Impacts
We have identified new insecticides with potential for control of mosquitoes.

Publications

  • Paul, A., Harrington, L.C. and Scott, J.G. 2006. Evaluation of novel insecticides for control of the dengue vector, Aedes aegypti. J. Med. Entomol. 43: 55-60.
  • Hardstone, M.C., Baker, S.A., Ewer, J. and Scott, J.G. 2006. Deletion of Cyp6d4 does not alter toxicity of insecticides to Drosophila melanogaster Pestic. Biochem. Physiol. 84: 236-242.
  • Gao, J. and Scott, J.G. 2006. Role of the transcriptional repressor mdGfi-1 in CYP6D1v1-mediated insecticide resistance in the house fly, Musca domestica. Insect Biochem. Molec. Biol. 36: 387-395.
  • Hamm, R.L. Kaufman, P.E., Reasor, C.A., Rutz, D.A. and Scott, J.G. 2006. Resistance to cyfluthrin and tetrachlorvinphos in the lesser mealworm, Alphitobius diaperinus, collected from the eastern United States. Pest Manag. Sci. 62: 673-677.
  • Gao, J. and Scott, J.G. 2006. Use of quantitative real-time PCR to estimate the size of the house fly (Musca domestica) genome. Insect Molec. Biol. 15: 835-837.


Progress 01/01/05 to 12/31/05

Outputs
Insecticides are the primary means to control Culex pipiens, an enzootic vector of West Nile virus, in the United States. To better understand how the evolution of resistance might be impacting control of this insect, we investigated the levels of resistance in populations collected from two metropolitan areas (Albany and Syracuse) to four larvicides (methoprene, phenothrin, Bacillus sphaericus (Bs) and Bacillus thuringiensis israelensis (Bti)) and one adulticide (phenothrin) registered for mosquito control in New York State. High levels of resistance were found only to Bti, and only at one site (Syracuse). Resistance levels to the other insecticides were less than 10-fold. Given the large difference in Bti resistance between Syracuse and Albany, it appears these Cx. pipiens populations do not rapidly mix, leading to localization of resistant populations.

Impacts
We demonstrated that insecticide resistance is not having a substantial negative impact on our ability to control Culex mosquitoes in New York.

Publications

  • Hamm, R., Shono, T. and Scott, J. G. 2005. A cline in frequency of autosomal males is not associated with insecticide resistance in house fly. J. Econ. Entomol. 98: 171-176.
  • Paul, A., Harrington, L. C., Zhang, L. and Scott, J. G. 2005. Insecticide resistance in Culex pipiens from New York. J. Am. Mosq. Cont. Assoc. 21: 305-309.


Progress 01/01/04 to 12/31/04

Outputs
The cytochrome P450 monooxygenases are an important metabolic system involved in the detoxification of xenobiotics, and are thus one of the major mechanisms by which insects evolve insecticide resistance. However, comparatively little is known about the evolutionary constraints of this insecticide resistance mechanism. We investigated the genetic basis of resistance in a strain of house fly (NG98) from Georgia, USA that had evolved 4,000-fold resistance to the pyrethroid insecticide permethrin, and compared this to other permethrin resistant strains of house flies from the United States and Japan. Resistance in NG98 was due to kdr on autosome 3 and monooxygenase-mediated resistance on autosomes 1, 2 and 5. These results indicate that the genes which evolve to produce monooxygenase-mediated resistance to permethrin are different between different populations, and that the P450 monooxygenases have some degree of plasticity in response to selection. Monooxygenase-mediated resistance appears to evolve using different P450s, and possibly different regulatory signals controlling P450 expression, even in strains selected with the same insecticide.

Impacts
These results portend great difficulty for the development of diagnostic tools that could be used reliably over large areas to monitor monooxygenase-mediated pyrethroid resistance.

Publications

  • Scott, J. G. and Kasai, S. 2004. Evolutionary plasticity of monooxygenase-mediated resistance. Pestic. Biochem. Physiol. 78:171-178.