Lygus Doug Lygus hesperus Tetranychus lauraticae Franklinella zalomcidentalis Adaptive Trait Evolution: Pesticide Resistance DOUGLAS WALSH, LAURA LAVINE, FRANK ZALOM USDA PMAP, WA ALFALFA SEED COMMISSION, WA HOPS COMMISSION, US AID, WSCPR, WSU EMERGING ISSUES
Program Objectives Identify insecticide/ miticide resistance genes from target pests Population genetic analyses of resistance for each gene of interest for each target pest Develop a rapid (and ultimately inexpensive) diagnostic tool for identification of resistance for target pests Use this information to make IPM decisions and recommendations to growers (sustainable & economical)
Pyrethroid Resistance in Onion Thrips
Wu, M., H. Gotoh, T. Waters, D. B Walsh, L. C. Lavine. 2013 Identification of an alternative Knockdown Resistance kdr-like mutation, M918L, and a novel mutation, V1010A, in the Thrips tabaci voltage-gated sodium channel gene. Pest Management Sci. DOI: 10.1002/ps.3638 Dose responses of Thrips tabaci populations from Washington State onion fields to lambdacyhalothrin. Conc. g/liter lambda-cyhalothrin
Partial nucleotide sequence chromatograms of the two mutation sites (M918L and V1010A) of the voltage-sensitive sodium channel subunit gdna from Thrips tabaci strain.
We identified two single nucleotide substitutions the T. tabaci voltage-gated sodium channel gene. One mutation resulted in the nonsynonymous substitution of methionine with leucine (M918L), which is well known to be responsible for super knockdown resistance in some pest species. Another non-synonymous substitution, a valine (GTT) to alanine (GCT) replacement at amino acid 1010 (V1010A) was identified in our study and was associated with lambdacyhalothrin resistance. All populations of T. tabaci Collected Washington State onion fields had these mutations. 100% resistance
Lygus bug objectives 2013 1. To investigate the toxicity of bifenthrin to L. hesperus in the field 2. To understand the mechanisms of bifenthrin resistance at the genetic level in these same L. hesperus field populations by characterizing: a) genetic mutations in the voltage-gated sodium channel (a known target for pyrethroid insecticides) b) The role of metabolic detoxification genes (such as cytochrome P450s or ABC transporters) in L. hesperus resistance to pyrethroids.
MATERIALS AND METHODS Insect Sampling and Collection 2013 L. hesperus were collected from: a)alfalfa forage field b)alfalfa seed field c)mustards in orchards and along roadsides
MATERIALS AND METHODS Sampling location A. Prosser B. Touchet C. Othello D. Yakima E. Lowden F. Prosser G.Grandview H. Gardena I. Wapato
MATERIALS AND METHODS Intensive Sampling- 2014 Lygus bugs were collected with a sweep net from the upper part of alfalfa plants Lygus bugs were aspirated from the sweep net collection and placed in a container with green bean pods (Phaseolus vulgaris L) for transport back to the lab in Prosser. Lygus bugs were then bioassayed with bifenthrin and then preserved for molecular work in Pullman in Fall 2013 & winter 2014
MATERIALS AND METHODS Toxicological Bioassays Adults of L. hesperus from each location were tested for resistance to insecticides bifenthrin (pyrethroid) by using a Potter Spraying Tower Five concentrations plus control was tested and each test was replicated three times. Each replicate consisted of 10 adults of L. hesperus. Mortality was quantified after 3, 24, and 48 hours after treatment (HAT)
MATERIALS AND METHODS Probit Analysis
Location Crops Slope±SE LC 50 (ppm)(95%fl) a LC 90 (ppm)(95%fl ) b RR 50 c RR 90 d Prosser Alfalfa forage 1.508 ± 0.257 3.579 (2.056 5.569) 25.323 (14.652 64.014) 1 1 Touchet Alfalfa forage 1.855 ± 0.253 18.393 (11.931 26.347) 90.263 (60.210 160.561) 5 4 Othello Alfalfa forage 2.155 ± 0.480 9.700 (5.368 14.883) 38.155 (25.076 78.705) 3 2 Yakima Alfalfa seed 1.891±0.400 9.659 (4.134 15.536) 46.002 (29.314 95.159) 3 2 Lowden Alfalfa seed 1.460±0.231 9.602 (1.904 22.292) 72.487 (30.939 425.063) 3 3 Gardena Rd, Touchet Alfalfa seed 2.056±0.386 21.597 (11.906 31.586) 90.730 (61.643 168.804) 6 4 Grandvie w Mustards 1.979±0.330 10.729 (5.957 16.434) 47.642 (30.805 89.447) 3 2 Prosser Mustards in orchard 1.390±0.238 3.651 (1.794 6.249) 30.503 (16.686-80.149) 1 1 Wapato, Yakima Mustards in orchard 2.295±0.518 12.112 (5.959 3.776) 43.819 (29.605 88.434) 3 2
These objectives are now being worked on-- 1. To understand the mechanisms of bifenthrin resistance at the genetic level in L. hesperus field populations by characterizing: a) genetic mutations in the voltage-gated sodium channel- We have not found any mutations like we did with thrips b) The role of metabolic detoxification genes (such as cytochrome P450s or ABC transporters) in L. hesperus resistance to pyrethroids. We re going that way now.
Metabolic Resistance Role of ABC transporters in resistance Tolerant L. hesperus Bifenthrin plasma membrane intracellular ABC ATP ADP Susceptible L. hesperus Bifenthrin plasma membrane ABC intracellular
Metabolic Resistance Target genes of ABC transporters in our studies are MDR genes 5, 6, 7, and 8 and ABCT genes 4 and 5
Normalized Fold Expression 14.00 12.00 10.00 8.00 6.00 4.00 2.00 0.00 The result of q-pcr indicates that some ABC transporters in resistant populations may show significantly different expression compared to the control (susceptible) population, most notably in MDR6 and MDR8 * * * * * * Metabolic Resistance * * * ABCB4 ABCB5 MDR5 MDR6 MDR7 MDR8 Target * * * O S T L OB Y Gv W The gene expression levels (relative to susceptible population) of six ABC transporters in L. hesperus populations: susceptible (S), RR50 =3 Othello (O), RR50 =5 Touchet (T), RR50 =3 Yakima (Y), RR50 =3 Lowden (L), RR50 = 1 Olsen Bros (OB), RR50 =3 Grandview (GV), and RR50 =3 Wapato (W).
Conclusions Almost all of the field population of L. hesperus collected from fields of alfalfa grown for seed in central Washington show increased levels of bifenthrin resistance ABC transporters in resistant populations show significantly different expression compared to the control (susceptible) population. Stay tuned for our results involving the voltagegated sodium channel genes.
Acaricide Resistance Spider Mites The two-spotted spider mite is the arthropod pest with the greatest documented number of resistance events worldwide. Genomics/transcriptomic resources are available
Leaf Disc Bioassay Transferred ten adult female mites to leaf discs placed on top of soaked cotton Exposed to 2 ml of varying concentration of candidate acaricides. Mites are held at 24C for 24 hrs, and evaluated for mortality
Results: Dose response curves of susceptible colony Abamectin Bifenazate
We have tested selected field populations of spider mites from a representative sample of hop yards and compare their dose response curves to susceptible. Twelve populations from hops, two from wine grapes, and one from potato in 2013.
In 2013, 13 T. urticae populations from hopyards were tested for abamectin resistance, and 12 of these 13 populations were tested for bifenazate resistance within 72 hr of collection. abamectin doses ranged from 0.225-39.3 mg a.i./l, bifenazate doses ranged from 8.99-899 mg a.i./l. Resistance ratios (RR) were estimated at the LC50 level as RR= LC50 of field strains/ LC50 of susceptible strains calculated in Objective f. Resistance ratios among hopyard-collected mites to abamectin were calculated to be between 1.29 and 107. The resistance ratio among hopyard collected mites to bifenazate was calculated to be between 2.29-96.3.
We are developing discriminating doses of candidate miticides that can be used to rapidly identify the prevalence of tolerance or resistance in a spider mite population to the candidate miticides. The discriminating dose we developed for bifenazate was 224 mg a.i./l (this is 1/4 the concentration of the field rate), And the discriminating dose we developed for abamectin was 22.5 mg a.i./l (this is the full field rate).
Developing Molecular Diagnostics for Managing Spider Mite Resistance in Hops Yards I. Increased detoxification Cytochrome P450s 11/82 ABC transports 9/103 pesticides
Developing Molecular Diagnostics for Managing Spider Mite Resistance in Hops Yard I. Increased detoxification Cytochrome P450s 11/82 ABC transports 9/103 So far 4 P450s have been identified and could serve as targets for pesticide resistance in field samples pesticides
Developing Molecular Diagnostics for Managing Spider Mite Resistance in Hops Yards II. Target mutations pesticides
Developing Molecular Diagnostics for Managing Spider Mite Resistance in Hops Field II. Target mutations Pesticides SNPs checked 1. Pyrethroids 7 SNPs in sodium channel 2. Abamectin 2 SNPs in Glutamate gate channel 1&3 3. Bifenazate 4 SNPs in Cytochrome b pesticides
Developing Molecular Diagnostics for Managing Spider Mite Resistance in Hops Field II. Target mutations Pesticides SNPs checked 1. Pyrethroids 7 SNPs in sodium channel 2. Abamectin 2 SNPs in Glutamate gate channel 1&3 3. Bifenazate 4 SNPs in Cytochrome b pesticides Mutations in cytochrome b were identified from 15 samples belonging to 4 field collections
Developing Molecular Diagnostics for Managing Spider Mite Resistance in Hops Yards II. Target mutations Pesticides SNPs checked 1. Pyrethroids 7 SNPs in sodium channel 2. Abamectin 2 SNPs in Glutamate gate channel 1&3 3. Bifenazate 4 SNPs in Cytochrome b Field samples pesticides This single mutation is linked to the populations of mites from Washington hopyards with the greatest levels of resistance to bifenazate
Abamectin Resistance ABC transporters are transmembrane proteins that utilize the energy of adenosine triphosphate (ATP) hydrolysis to translocate toxins across membranes pesticide extracellular plasma membrane intercellular ATP ADP
Increases in the titer of ABC transporters in the mite population over the course of the season is making them more tolerant to many toxins including abamectin.