Summer Envisionship Initial Report
Matthew Allen and Tony DeRosa
Project Title: Determining the prevalence of Wolbachia infection and to identify the Wolbachia
strains present in common pest insects of organic and GMO-free crops in western Wisconsin.
Project Introduction: We are looking at insect pests of organic and no-GMO crops in western
Wisconsin. Organic and non-GMO farming is increasing in its popularity in western Wisconsin
and throughout the country. For these types of farmers, insect pest management is very
different from that of other commercial farmers. Non-organic farmers generally use chemical
pesticides and/or GMO crops to manage insect pests. Organic and non-GMO farmers must rely
on manual and natural control of insect pests. We were interested in determining the
Wolbachia infection rate and if possible the strains involved in the infection for these insect
pests. Potentially, an understanding of the Wolbachia infection rates and knowledge of the
strains involved in the Wolbachia infection could lead to novel non-chemical pest control. That
portion is outside the scope of this project but it was a motivator for our interest in finding out
about the presence of Wolbachia in these insect groups.
Sample Sites: We have collected samples from 5 different organic farms in northwestern
Wisconsin. We sampled farms from Eau Claire, Durand, Glenwood, Augusta, and Stanley. The
farms were in Eau Claire, Chippewa, Dunn, and Pepin counties.
Methods and Procedures:
For collections, we collected insects in the field using nets and tweezers. All insects
were placed live into 95% ethanol on ice and then stored in the freezer. When possible,
extractions and amplifications were done on the same day as the collection. Amplified samples
were kept refrigerated until run on the gels and then were frozen.
For extraction and amplification, Sigma Extract-N-AmpTM Tissue PCR Kits (product
number XNAT2-1KT) were used.
Extraction Protocol:
1. In a 1.5 ml microtube, combine 100µl of Extraction Solution and 25µl of Tissue Prep
Solution and mix by pipetting up and down
2. Add 2-10mg piece of insect abdomen or if small, whole insect to the solution and
macerate using a microtube pestle
3. Incubate at room temperature for 10 minutes
4. Incubate at 95°C for 3 minutes
5. All 100µl of Neutralization Solution B and mix by vortexing
6. Pipet off the liquid into a new tube and store at 4°C or continue to PCR immediately
Amplification Protocol:
1. In a PCR tube, combine 7µl PCR quality water, 10µl Extract-N-Amp PCR Ready Mix, 2µ
wspec F primer, 2µl wspec R primer, and 4µl of DNA for a total volume of 25µl
2. Run in thermalcycler with the following settings
a. 95°C 2 min
1 cycle
b. 94°C 30 sec
38 cycles
55°C 45 sec
72°C 90 sec
c. 72°C 10 min
1 cycle
d. 4°C
indefinitely
Electrophoresis Protocol:
1.
2.
3.
4.
Prepare a gel at 1% agarose using TBE
Mix 10µl of sample with 2µl of FOTO/Vision stain on Parafilm
Put 10µl of sample and stain mixture into the gel
Run at 100V for 30 minutes
Preliminary Results: We collected and tested samples from 5 different farms. Farm 1 was in
Eau Claire County. Farm 2 was in Dunn County. Farm 3 was in Pepin County. Farm 4 was in
Eau Claire County. Farm 5 was in Chippewa County. The following insects were collected at
each site and in parentheses are the number of positive results and the number of individuals
tested.
Farm 1:
Colorado Potato Beetle – Coleoptera
Flea Beetle – Coleoptera
Squash Bug – Hemiptera s.o. Heteroptera
Cabbage Butterfly – Lepidoptera
(0/4)
(13/13)
(8/15)
(0/2)
Farm 2:
Colorado Potato Beetle – Coleoptera
Cabbage Butterfly (larvae) – Lepidoptera
(0/5)
(0/5)
Farm 3:
Cucumber Beetle – Coleoptera
Fruit Fly – Diptera
(0/5)
(3/15)
Farm 4:
Flea Beetle – Coleoptera
(12/15)
Farm 5:
Colorado Potato Beetle (immature) – Coleoptera
Cabbage Butterfly – Lepidoptera
Cucumber Beetle – Coleoptera
(0/5)
(0/5)
(0/4)
See attached spread sheet for details of each sample run and gel photos.
Collections tab gives details on the collection dates and times.
Data tab gives all of the data and explanations of codes.
Individual gel tabs shows data for each gel and the gel photo