Adaptive Trait Evolution: Pesticide Resistance

DOUGLAS WALSH, LAURA LAVINE, FRANK ZALOM

USDA PMAP, WA ALFALFA SEED COMMISSION, WA HOPSCOMMISSION, US AID, WSCPR, WSU EMERGING ISSUES

Lygus Doug Lygus hesperus

Franklinella zalomcidentalis

Tetranychus lauraticae

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

Conc. g/liter lambda-cyhalothrin

Dose responses of Thrips tabaci populations from Washington State onion fields to 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 non-synonymous 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 lambda-cyhalothrin 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.

L. hesperus were collected from:a)Alfalfa forage fieldb)Alfalfa seed fieldc)Mustards in orchards and along roadsides

Insect Sampling and Collection 2013

MATERIALS AND METHODS

Sampling locationA. Prosser

B. Touchet

C. Othello

D. Yakima

E. Lowden

F. Prosser

G.Grandview

H. Gardena

I. Wapato

MATERIALS AND METHODS

• 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

Intensive Sampling- 2014MATERIALS 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

•

MATERIALS AND METHODS

Location Crops Slope±SE LC50(ppm)(95%FL)a LC90(ppm)(95%FL)b RR50

c RR90d

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

Grandview 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.

Role of ABC transporters in resistanceRole of ABC transporters in resistance

ADPATP

plasma membrane

intracellular

Bifenthrin

ABC

Tolerant L. hesperus

plasma membrane

intracellular

Bifenthrin

ABC

Susceptible L. hesperus

Metabolic Resistance

Metabolic Resistance

Target genes of ABC transporters in our studies are MDR genes 5, 6, 7, and 8 and ABCT genes 4 and 5

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

ABCB4 ABCB5 MDR5 MDR6 MDR7 MDR8

Nor

mal

ized

Fol

d Ex

pres

sion

Target

OSTLOBYGvW

*

*

** *

*

* *

*

*

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Metabolic Resistance

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).

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

Conclusions

• Almost all of the field population of L. hesperuscollected 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 voltage-gated 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 cottonExposed 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

ABC transports

9/10311/82

pesticides

Developing Molecular Diagnostics for Managing Spider Mite Resistance in Hops Yard

I. Increased  detoxification

Cytochrome P450s

ABC transports

9/10311/82

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

Pesticides SNPs checked

1. Pyrethroids    7 SNPs in sodium channel2. Abamectin      2 SNPs in Glutamate gate

channel 1&33. Bifenazate      4 SNPs in Cytochrome b

II. Target mutations

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 Field

Pesticides SNPs checked

1. Pyrethroids    7 SNPs in sodium channel2. Abamectin      2 SNPs in Glutamate gate

channel 1&33. Bifenazate      4 SNPs in Cytochrome b

II. Target mutations

pesticides

Field samples

Developing Molecular Diagnostics for Managing Spider Mite Resistance in Hops Yards

Pesticides SNPs checked

1. Pyrethroids    7 SNPs in sodium channel2. Abamectin      2 SNPs in Glutamate gate

channel 1&33. Bifenazate      4 SNPs in Cytochrome b

pesticides

II. Target mutations

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

intercellular

extracellular

plasma membrane

ADPATP

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.