## 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 , I nc. 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 Mai ns , Med ical Entomologist 

### & Chief Operating Officer, Mosqu itoMate , I nc. 

### 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 mi c rob ial 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 an d potenti31 underestimation of o ulation s ize , we lan to re lease u to 30, 000 males er week
.



### Page I ofS3 )$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

- Wolbachi a Biopestidde Treated P opu l anon 1 00

# ~J 0 - Untreated P opu lati on 0 10 20 30 40 50 60 70 80 90 100 110 120 T im e ( Week >) Figure 1. Replicate populations of Aedes albopictus are monitored in a closed laboratory model system. (A) represents two control populations that d id not rece ive 

### W. pipientis male releases and one treatment population that received r epeated releases of 

### W. pipient i s infected males
. (B) represents one contro l population that d id not rece ive 

### W. pi pi

entis male releases and one treatment population that received repeated re leases of 

### W. pipientis infected males
. Each i ncomaptible male re lease 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

### ~ 

# ..**~** 

### " :0**·~** a. E

### ~



### Page 2 of535 . -e- West W ood Or ivt Ne ighborhood 30

10 •
5 .; Error Ban • Standard Erro r
Tlm e(Weeko) Figure 2. Variation in size of the Ae. afbopictus populatio n at two neighborhoods with i n Lexington , KY
. Data displayed as mean adult Ae
. albopictu s 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 Lexingto n, KY
. Total area of the property to be sampled is 0 . 17 acres (per Property Valuation Adm inistrator Fayette County, KY)
.

0 Ovitraps 

### e ZAP Male Release

Figure 3. Release area i n Westwood Court ne igh borhood of Lexington , KY
. White box in d icates area to be sampled with black dots , white triangles and wh ite diamonds i ndicati ng po int of male re leases and sam lin areas res ective l
. 

### Page 3 of 5

### 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 steril ity 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 a nd 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 mosqu ito 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 c lade 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 wi ll 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
.



### Page 4 ofSHow 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 i n the rearing process (e
.g. , cages and larval pans) will be frozen to kill any remaining mosquitoes.



### Page 5 of 5