Update on the search for new toxicants and methods of control

James J. Becnel

1. Novel insecticide chemistries or formulations

2. Personal protection

3. Application technology

Toxicological evaluation of compounds

1) Cooperative Project with Chemical Companies

2) Cooperative Projects with ARS Natural Products Utilization Research Unit (NPURU)

Screening Protocols• Phase 1 – chemical screening using high throughput

Ae. aegypti assay with 1st instar larvae and categorize activity based on 24 hour mortality

• Phase 2 - Compounds >80% mortality evaluated with topical assays (hit rate of ~ 10%)

• Phase 3 - Determine LD50 values for the most active adulticides and LC50 values for the most active larvicides

• Day 1: Hatch Aedes aegypti eggs

• Day 2: Sort 1st instar larvae to 24-well plates, 5/well

Add 1ml of water and food

Add 10 ul of insecticide solution

• Day 3: Score bioassay data (dead/total)

High throughput screen

Phase 2 - Topical application

Accurate

Small Amount Fast

Easy

Maximum capacity: < 200 chemicals per week

Chemical

Diversity Samples from Chemical Library

Phase 1 Results for 9344 compounds

Chemicals tested against first instar Ae. aegypti

87%

2.5% 1.3% 1.5% 2.1% 5.2%

0100020003000400050006000700080009000

0 1 2 3 4 5

Number dead (out of 5)

Num

ber o

f che

mic

als

(3pp

m)

8.8% hit rate

Chemtura Summary

Four samples advanced to test against permethrin-resistant strain Puerto Rico: C2395, B6572, C3506, and F9842

C2395 active at 100 ppm, B6572 active at 25 ppm

C3506 and F9842 active at 12.5 ppm against adults

Chemical OrlandoLD50

Puerto RicoLD50

Relative PR-Orl resistance

C3506 8.0 ppm 81.0 ppm ~10.00×

F9842 16.0 20.0 1.25

Permethrin 2.2 25.6 ~11.00

ARS Natural Products Utilization Research Unit, Oxford, MS

Charles Cantrell

David Wedge

Kumudini Meepagala

Results: Chemicals from plants NPURU

Scientist #

Active (ppm)

Larval Adult

2 4 8 16 .02 .30 .60 1.2

Meepagala 55 2 2 13 - 0 0 0 7

Cantrell 53 0 2 3 - 0 2 2 2

Wedge/ Tabanca 509 1 5 6 14 1 6 7 8

Total 617 3 9 22 14 1 8 9 17

Chemical approach: Summary

1) Employed high throughput screening larvae assay and screened ~10,000 compounds; additional ~2,000 by end of year.

2) Two compounds with good adult activity, follow-up in progress

3) Screened hundred of natural plant chemicals; several showed high adulticidal activity

Molecular Pesticide DevelopmentThrough RNAi Technology

Molecular pesticide

Pesticide whose active ingredient is RNA.

Approach for the Developmentof Molecular Pesticides

(Aedes aegypti model)

Target Essential Pathways Programmed Cell Death,

Mitochondria Pathway, etc

Molecular Cloning(DNA, RNA, PCR)

Vector, Carrier, Formulation

(Aedes aegypti model)

Bioassays

Molecular Pesticide Construction

“Pesticidal Double Stranded RNA Composition and Method of Use

Thereof,” Pridgeon, Becnel and Strickman

Patent Application

Current Status of Application as of Nov. 9, 2009

Received a restriction requirement from the Patent Office

Estimate that within 6 months we'll receive a First Office Action on the merits

Status of IAP RNAi constructs as pesticides• In selection process of a single IAP construct

for mass-production. Contract in place (1-25-2010) to make 5 grams of IAP RNAi constructs.

• Investigating additional IAP targets (IAP2, IAP3, IAP4)

• Probed 44 regions of four IAP genes for activity

• 2640 of 7986 bp total (~32% of sequence info)

• Activity of these constructs being evaluated

IAP 2

IAP 3

IAP 1

IAP 4

PCR-select subtractive cDNA library

A revolutionary method for finding differentially expressed genes

Very well-suited for the identification of rare critical transcripts, which are typically the most difficult to obtain

Control Pesticide-treated

mRNA

cDNA synthesis

Rsa I digestion &adaptor ligation

cDNA hybridization/subtraction

TA cloning Sequencing

Annotation & QPCR

mRNA

cDNA synthesis

Rsa I digestion &adaptor ligation

Subtractive library clone designations

P450

Hypothetical/Unknown

Energy

Housekeeping

Other

Insecticide resistance

Orlando permethrin Puerto Rico permethrin Puerto Rico minus induced induced Orlando

Three P450 pulled out of Puerto Rico minus Orlando subtractive library.

Greater number of metabolism (energy) genes in Puerto Rico than Orlando.

P450 screening- ongoing and future goals

• Designed and generated dsRNA constructs to knock-down P450 genes.

• Currently determining ability to knock-down P450 gene expression (time to knock-down and duration).

• Functional studies with permethrin treatments to determine if specific knock-down of P450s increases susceptibility in Puerto Rico.

• Assess specificity using other mosquito spp.

1) Refining IAP constructs to select those with high efficacy for large scale production.

2) Molecular targets were identified by subtractive cDNA library after challenging the mosquitoes with pesticides.

3) Functional role of several P450s implicated in permethrin resistance to be evaluated.

Molecular approach: Summary

DWFP Toxicology Team Members

• Dr. Julia Pridgeon (Toxicologist & Molecular Biologist)• Dr. Liming Zhao (Molecular Biologist)• Dr. Monique Coy (Toxicologist & Molecular Biologist)• Bill Reid (Technician, Toxicology & Molecular Biology)• Neil Sanscrainte (Technician, Toxicology & Molecular Biology)• LT Anne Thornton (visiting scientist, Toxicology & Molecular

Biology)

All members in theMosquito and Fly Research Unit

Center for Medical, Agricultural, and Veterinary Entomology ARS, USDA and the Deployed War Fighter Protection Research Program

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