Experimental Use Permit 

Field Trial Information Requirements 

Complete one form for each cooperator/participant field trial(s). The information boxes 

will expand when completed in Microsoft Word. 

Participants and cooperators, i.e., persons who grant permission for an experimental use 

pesticide to be used on application sites they own or control: 

If participants change, the permit needs to be modified. 

Name of program participant or cooperator: 

James Mains, Ph. D. 

Address: 

MosquitoMate, Inc. 

1122 Oak Hill Drive 

Lexinaton, KY 40505-3322 

Telephone No., e-mail address: 

mosquitomate@gmail.com 

Office: (859) 317-9213 

Fax: (859) 317-9452 

Qualifications, Title, Position: 

Dr. James Mains, Medical Entomologist & 

Chief Operating Officer, MosquitoMate, Inc. 

Names of US state(s) or territory(ies) where the product is intended to be used: 

Kentuck , USA 

Names of state(s) or territory(ies) in which the product may be shipped for further 

distribution elsewhere: 

None 

Test material: 

What amount or quantity of active ingredient or test material to be used for testing? 

30,000 Aedes albopictus males infected with the wPip Wolbachia pipientis microbial pesticide per 

week for six months. 

What is the justification for the amount or quantity of active ingredient or test material 

proposed to be used, including how it was determined? 

Based on laboratorry research, a 15: 1 ratio of Aedes albopictus males infected with the V'.'olbachia 

pipientis microbial pesticide to each wild type Ae. albopictus male is needed to resu1t i11 c.. 

population suppression effect (Fig. 1 ). The number of target mosquitoes varies seasC'rc1lly 

throughout the year (Fig. 2). Based upon ongoing monitoring, we estimate a popt:l3~i'J'1 size of 

approximately 1,500 mosquitoes at each field site. To allow for mortality and potenti31 

underestimation of o ulation size, we lan to release u to 30,000 males er week. 

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3 )$00 

A 1600 

& 1400 

s 1200 

1000 4 

800 

- Un ated Population 600 

~ 

+ - llntr~ated Population 400 

.8 

E 

::> 

1 i - Wolbaohla Biopesti<:•de Treated Population 200 

c 0 !!! 

e 

~ 700 

600 

-;; B 1il 

5 

500 

400 

4 

300 

200 

- Wolbachia Biopestidde Treated Populanon 

100 

~J 0 

- Untreated Population 

0 

10 20 30 40 50 60 70 80 90 100 110 120 

Time (Week>) 

Figure 1. Replicate populations of Aedes albopictus are monitored in a closed laboratory 

model system. (A) represents two control populations that did not receive W. pipientis male 

releases and one treatment population that received repeated releases of W. pipientis 

infected males. (B) represents one control population that did not receive W. pipientis 

male releases and one treatment population that received repeated releases of W. pipientis 

infected males. Each incomaptible male release is represented by a shaded vertical bar in 

(A) and (B), with the height indicating the release size. All treatment populations were 

eliminated with repeated W. pipientis infected male releases. 

.. 

.0 

~ .. 

~ 

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a. 

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35 . 

-e- West Wood Orivt Neighborhood 

30 

10 • 

5 .; 

Error Ban • Standard Error 

Tlme(Weeko) 

Figure 2. Variation in size of the Ae. afbopictus population at two neighborhoods within 

Lexington, KY. Data displayed as mean adult Ae. albopictus females caught per BG trap 

per 24 hour sampling period. 

What is the test area acres, hectares, square feet, or square meters? 

The test area is located in the Westwood Court neighborhood of Lexington, KY. Total area of the 

property to be sampled is 0.17 acres (per Property Valuation Administrator Fayette County, KY). 

0 Ovitraps 

e ZAP Male Release 

Figure 3. Release area in Westwood Court neighborhood of Lexington, KY. White box indicates 

area to be sampled with black dots, white triangles and white diamonds indicating point of male 

releases and sam lin areas res ectivel . 

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What are the crop(s), animal(s), surface(s), or site(s) to be treated? 

Male A. albopictus mosquitoes infected with the wPip W. pipientis microbial pesticide will be 

released into a neighborhood. The male mosquitoes will mate with indigenous females, causing 

conditional sterility and resulting in population decline and potential elimination. 

The purpose of study is to determine pesticidal value, toxicity, or other properties. What 

are the objectives and values of this study, including what type(s) of data is/are to be 

collected? 

The objectives and values are to determine the pesticidal value of a W. pipientis microbial 

pesticide for the suppression and elimination of an A. albopictus mosquito population. While the 

results of contained trials are encouraging (Fig. 1 ), the competitiveness and suppressive effect of 

the released males should be assessed in the field. The experiment will test for a hypothesized 

increased frequency of A. albopictus eggs failing to hatch (i.e., due to W. pipientis induced 

conditional sterility) and a significant reduction in the A. albopictus population size compared to a 

control site, which is not being treated. 

What Is/are the site location(s) where the test material is to be used, i.e. GPS coordinates 

and/or other details to be able to find the site(s)? 

The potential site is in Lexington, Kentucky. 

Westwood Court neighborhood of Lexington, KY (38°01'32" N 84°33'00" W) 

What are the intended plot sizes, number of plots, number of replicates, and other relevant 

test parameters? 

The potential test site is 0.17 acre. Releases and mosquito sampling will be performed weekly at 

this site. Pending on results and permission, this experimental strategy is to be replicated at up 

to three additional sites, all of which are in the Lexington, KY area and totaling not more than 0. 7 

acre. 

How will the test material be used? What is/are the mode(s) of application and rate(s) of 

use or application? 

30,000 W. pipientis-bearing male Aedes albopictus mosquitoes will be released weekly at the 

selected site. The W. pipientis clade is from indigenous Culex pipiens mosquitoes. Mosquito 

populations will be sampled using BG trap sampling of the adult population and ovicups to 

sample eggs. BG trap adult collection and ovicup egg collection data from the experimental site 

will be compared to an untreated control site to examine for an effect of the release of the prociuct 

on the Ae. albo ictus mos uito o ulation. 

How will the test be supervised? 

Dr. Mains will oversee the distribution and monitorin 1 ~--~----~~--~~----~~~------~----~-----L----~~----------

What are the proposed time dates and duration for the experiment? 

Male releases should start in March 2013. The male releases will continue for ap~rc•xirr.ately six 

months from the start date. If delayed, these dates may be delayed accordingly. However, a :ater 

start will be an experimental complication, due to a potential increase in the targeted populati.>n J 

size, which means more released males are needed to im act the local o ulation. 

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How will the test material be stored and how will unused test materials and their 

containers be disposed of at the end of the experiment: 

Male mosquitoes are to be reared in Lexington, KY within a contained facility and released 

immediately upon maturation. At the conclusion of the experiment all equipment used in the 

rearing process (e.g., cages and larval pans) will be frozen to kill any remaining mosquitoes. 

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