Progress in DWFP and Related Research

Ulrich R. BernierUSDA-ARS-CMAVE

Mosquito & Fly Research Unit352-374-5917

ubernier@gainesville.usda.ufl.edu2007 DoD Pest Management Workshop

12-16 February 2007

DWFP Projects to be Discussed

Chemical structure activity studies Development of portable devices Biting protection assessment of uniforms Development of inhibitor sustained

release delivery systems Laboratory and field studies with sand

flies in Egypt

Structure-Activity Studies of Known Arthropod Repellents and Toxicants Using

CODESSA and QSAR• Collaboration: Agreement with University of Florida Department of

Chemistry

• Research problem: Characterize the molecular structural and electronic properties that:

– correlate with level of repellent activity against multiple insect species– lead to insecticidal activity

• Key point: Since 1942, USDA has compiled records for thousands of compounds tested for repellent and insecticidal properties.

• Military relevance: Development of novel repellents and insecticides will enhance the arsenal of products to control insects that transmit disease to personnel.

Candidate repellent

in vivo in vitro

Topicalcage test

Cloth patch cage test

GC/MSPurity

Spatialinhibition

HITSS Olfactometer

MFRU Repellent Testing Paradigm

Toxicologytests

ProductCommercial

partner Registration

Candidatetoxicant

Toxicant track

Field evaluation

Also tested with infected mosquitoes

Experimental Classes of Repellents Used in Models

Duration of protection

On cloth patch On skin

Class 1 ineffective 0-60 min

Class 2 1-5 days 61-120 minClass 3 6-10 days 121-180 min

Class 4 11-20 days 181-299 min

Class 5 => 21 days => 300 min

Class 1 11(50%)

4

Class 2 4 2(14%)

1

Class 3 7 7 3(50%)

1 2

Class 4 1 2 1(50%)

Class 5 1(33%)

Class 1 Class 2 Class 3 Class 4 Class 5

Experimental Classes

Pred

icte

d C

lass

esModel-Predicted Classification vs.

Experimental Classification of Repellents

Carboxamides and Piperidines

N

O

CH3

CH3

CH3

N

O

CH3

NH

DEET

Piperidine

1-(3-methylbenzoyl)piperidine

Bioassay of Synthetic Piperidines as Candidate Attraction-Inhibitors(Candidates are coded P1-33)

0%

20%

40%

60%

80%

100%

US

DA

-I1 P6

P24

P13

P23

P19

P26 P2

P28 P3

P4

deet

P18

P31 P7

P21

P16

P12

P20

P25

P27 P1

P29

P17

P10 P5

P32

P11

P33

P14

P15

ATT

RP

30 P9

P22 P8

Treatment

Attr

actio

n

USDA Inhibitor Deet Attractant Blend

N

O

CH3

CH3

N

O

P13AI3-38739

P63-Methyl AI3-33510

• Current database contains over 30,000 compounds tested by USDA

• Spatial repellency has been assayed with the olfactometer for the 33 synthetic piperidines. Analysis of the carboxamides will take place in 2007.

• Work is in progress on 5 database sets:– House fly residual insecticides– Anopheles spp. larvicides– Ae. aegypti repellents– Anopheles spp. residual insecticides– Oc. taeniorhyncus insecticides

• Repellents assays (cloth patch test) completed for 6 piperidines- both CPT and MED have been determined—the remaining 27 piperidines and carboxamides should be completed by 30 Jun 2007.

Structure-Activity Studies (cont.)

• Collaboration: Agreement with University of Florida Dept. of Chemistry

• Research problem : An economical field device for airborne detection and quantification ULV spray fate and dispersal, repellents, and inhibitors is not currently available.

• Key point: FAIMS devices:

– are small, low-cost units– exhibit high chemical sensitivity and selectivity with low noise and can

separate isomers– can be used for continuous monitoring and work at atmospheric pressure

• Military relevance: Addition of a low-cost, field deployable device to mask attraction of personnel from biting insects can result in reduced disease risk to deployed personnel.

Development of Portable Devices for Detection and Quantification of Insecticides, Repellents, and Inhibitors

Field Asymmetric Ion Mobility Spectrometry (FAIMS)

Field-Deployable Devices

• Preliminary results with naled and malathion have demonstrated that this technique can separate isomers and be used in the field.

• Currently exploring FAIMS optimization of naled, malathion, permethrin, d-phenothrin, resmethrin, bifenthrin, cyfluthrin, and deltamethrin

• Next step is the evaluation of FAIMS as a stand-alone detector and determination of detection limits for target compounds

Development of Portable Devices (cont.)

Assessment of Biting Protection of U.S. Military Uniforms and Development of Criteria

to Protect Deployed Troops in the Future• Collaboration: Agreement with Natick Soldier Center—MIPR

initiated by USMC

• Research problems:– Determine if application of permanent press finish affected

performance of permethrin-treated Marine Corps Combat Utility Uniforms (MCCUUs)

– Develop a protocol to determine biological efficacy of factory-treated MCCUUs against mosquitoes

• Key point: DWFP component of this work is to establish baseline efficacy data of sand fly bite protection from factory-treated MCCUUs.

• Military relevance: Factory-treated MCCUUs that retain biologically efficacious levels of permethrin over their lifetime preclude the need for field-treatment of uniforms and may ultimately reduce disease risk.

Untreated sleeve - Female mosquitoes land andprobe in search of a blood meal

Treated sleeve after 2 min - Many females are still on the sleeve but few obtain blood

Treated sleeve after 8 min - Few mosquitoes remain on the sleeve and many have left the garment

0.030

0.040

0.050

0.060

0.070

0.080

0.090

0.100

0.110

0.120

0.130

0.140

0.150

0.160

0x Wash 20x Wash 50x Wash

Laundered Level of Permethrin-Treated First Article Specimens

Blouse 10 Blouse 11 Blouse 12 Trouser 10 Trouser 11 Trouser 12

American Apparel First ArticleQuantitation of Permethrin in Fabric

by Gas Chromatography (Natick Soldier Center)

Treated MCCUUs in 2004 (phase 2) exhibited rapid initial losses of permethrin when laundered.

0 washes 20 washes 50 washes

Assessing Biting Protection in Uniforms (cont.)

• Summary– Initial assessment of fabrics started on March 17, 2006– Three companies have qualified, one is undergoing first article

validation– There have been three failures, one company has failed twice

• Future– Bite protection for first article validation will be completed by 10

Mar 2007– Continue MCCUU evaluation as needed– ACU study involving field treatment– Evaluation of factory-treated MCCUU field performance (with

JAX NECE, CDR Rankin and LT Doud at Lejeune)– Development of new treatments – spatial effect

Development and Testing of Sustained Release Delivery Mechanisms for Repelling Mosquitoes and

Biting Insects• Collaboration: Agreement with Bioguard R&D to develop delivery devices for

field release of attraction-inhibitors

• Research problem: USDA-developed attraction inhibitors require encapsulation or binding of active ingredients in suitable non-reactive media to:

– produce a sustained release at efficacious delivery rates– to facilitate transport to the field where they will produce repellency in the field for 30

days.

• Key point: Inhibitors that prevent host detection by biting insects are a novel method of personal protection technology.

• Military relevance: Improvement of insecticide application technology, personal protection and vector inhibitors used in the field can yield more effective protection of deployed personnel and result in reduced disease risk.

Volatile samples are contained in pressurized canisters and delivered into an olfactometer port to test mosquito attraction or inhibition

Kairomones alert mosquitoes that a host is near. Mosquitoes follow the plumes upwind (anemotaxis) toward the source of the

odor.

Adapted from: Bernier, U.R., Laboratory Research and Development of Attractants, Inhibitors and Repellents, Tech. Bull. Florida Mosq. Control Assoc., 2006, 9:9-16.

Many of the less volatile repellents, like DEET, are a feeding deterrent or contact irritant that prevent biting. The chemical on the skin surface is unappealing for the mosquito to land, remain

on, or feed through.

Adapted from: Bernier, U.R., Laboratory Research and Development of Attractants, Inhibitors and Repellents, Tech. Bull. Florida Mosq. Control Assoc., 2006, 9:9-16.

An attraction-inhibitor decreases host-finding ability by masking or cloaking the kairomones, producing anosmia or hyposmia in the

mosquitoes.

?

Adapted from: Bernier, U.R., Laboratory Research and Development of Attractants, Inhibitors and Repellents, Tech. Bull. Florida Mosq. Control Assoc., 2006, 9:9-16.

Addition of a small amount of inhibitor to an attractant blend or to human emanations decreased

attraction of mosquitoes*

92.7

12.8

88.6

23.5

0

10

20

30

40

50

60

70

80

90

100

Attr

actio

n (%

)

Blend Blend + Inhibitor Hand (L) Hand (L) + Inhibitor

Treatment

Bioassay of Candidate Spatial Repellents for Use in Field-Deployed Devices to Cloak

Humans from Mosquitoes

0%10%20%30%40%50%60%70%80%90%

100%

Treatment

Attr

actio

n

Attractant Blend

Efficient Inhibition

Examples of carriers

Nanoclay containing attraction-inhibitors

Air-activated warmer to elevate and maintain a constant temperature

Outer packaging of durable material to contain the system

Development of Field Kits to Deliver Sustained Release of Attraction-Inhibitors

Second version of prototype release devicesPackage size approximately 3” x 3”

Current device to be tested in Egypt. It delivers 40-80 mg/day for 3-5 days at wind speed of 3-5 mph and

provides coverage up to 10-15 m from the device. Capsule is 1.5” diameter, 0.5” height.

• Advantages of prototype devices:– Predicted release of active ingredients at efficacious

levels for 30+ days at 1 m from source and 3-5 days at 10-15 m from source

– Next generation device is easily transportable to field in glass ampule

– Packaging meets DOT and IATA requirements for unregulated transport

• Final designs for laboratory and field testing are related to individual personnel protection (IPP) requirements.

• Future:– Preliminary design and evaluation of the broad area pack (BAP)– Design and evaluation of nano-carrier for permethrin-based

fiber systems for extended release-based longevity

Development of Sustained Release Mechanisms (cont.)

Laboratory and Field Experiments with Attractants, Inhibitors and Repellents against Sand Flies in Egypt

• Collaboration: LCDR David Hoel, NAMRU-3, Cairo, Egypt

• Research problems:– Explore chemical and physical means to enhance collections of

sand flies in commercial field traps– Explore inhibition of sand fly host-seeking in the laboratory and

field.– Develop baseline efficacy of MCCUUs against sand flies.

• Key point: NAMRU-3 conducts field experiments in Aswan—>90% Phlebotomus papatasi

• Military relevance: Permethrin-treated uniforms are less protective against sand fly bites than against mosquitoes. Development of novel personal protectants for use against sand flies will improve protection of troops.

Spacious laboratories for visiting scientists

Office space for the taking

Field Site in Bahrif, Aswan

LCDR Hoel working on a different trap study

Jerry Hogsette conducting researchwith fly traps

Trap site 1 - Typically the most productive site

Sample acquisitionHuman hair as one of the treatments

1-Octen-3-ol lure and “human” lure

Baited trap- Traps are operated overnight(~1700 to 0700 hrs)

Trap site 4Measuring overnight loss of lure

Aswan Bahrif MMX/Lure Study

0.0

20.0

40.0

60.0

80.0

100.0

120.0

140.0

P. pap

atasi

An. pharo

ansis

Cx . an

tennatu

s

Cx.

pipien

s

Cx. pe

rexiguu

s

Ae. ca

spiou

s

Cx. po

icilip

s

M. domes

tica

Species Trapped

Human hair Octenol + CO2 CO2 only Unbaited

Inse

cts/

Nig

ht

3832 32

Laboratory and Field Experiments in Egypt (cont.)• Three sets of field data have been collected August-September, 2006).

• No significant difference observed between trap lures.

• In future laboratory studies inhibitors will be tested against sand fly species in colonies at NAMRU-3

• Future field studies will involve:– exploration of trap location and physical arrangement – examination of best LED-lure-placement combination– incorporation of inhibitors in experiments– determination of daytime resting sites of sand flies