Wolbachia Population for the San José State University Metropolitan Area

WOLBACHIA POPULATION - Brandie J Schmidgall 1

Wolbachia Population for the San José State University Metropolitan Area Brandie J Schmidgall. James C. Enochs High School. Paper Submitted for Independent Research Project. Tutor Mr. Menshew. May 2012

Abstract Approximately sixty six percent of the entire insect population, which is about eighty percent of the animal population, is infected with Wolbachia.

Wolbachia is a parasitic bacterium that feminizes or kills off the males in the infected species. Nineteen various insects from the San José State University metropolitan area were collected, identified, and screened for Wolbachia using the Polymerase Chain Reaction. Even with a high PCR success rate, there was a low infection rate.

Keywords: Wolbachia, PCR, Polymerase chain reaction, San Jose, insects

Introduction Over the course of time, the few original species of insects have gone through speciation to create the many species we have today. They affect our everyday lives in ways that we may not realize. “They have a direct impact on agricultural food production by chewing the leaves of crop plants, sucking out plant juices, boring within the roots, stems or leaves, and spreading plant pathogens” said John Meyer [1]. Insects make up the vast majority, approximately eighty percent, of the animal kingdom. Of that eighty percent,

approximately sixty five out of every hundred, or sixty five percent, are infected with a “male-killing” bacterium called Wolbachia [2]. That means approximately fifty five percent of the animal kingdom, or 106 different species of insects [4], is infected with Wolbachia. Consequently, Wolbachia is the recently most diverse bacteria known to infect insects.

John Warren of Rochester University surveyed various species throughout the United States to determine that approximately

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twenty percent of the U.S. insect species are infected with Wolbachia [3]. How does this relate to a local area? There is only one way to find out, test the area for the infection. The insects used in this lab were collected in the San José State University Metropolitan Area

by Kristen Wolslegel. A local research area compared to the entire United States, or the

entire insect population, though large enough to work with. In this experiment, various insects were collected, identified and finally screened for Wolbachia using primers designed specifically for the Babec protocol [5]. Though there was a high PCR success rate, there was a low infection percentage.

Methods Insects were trapped into killing jars for DNA Isolation collection. For preservation, the specimens were placed in a holding jar with 95% Part 1: Preparation & Lysis of Sample ethanol.

A sterile 1.5ml microfuge tube was labeled with insect voucher number [5]. An Identifying the Order of the approximately 2 mm by 2 mm sample was Specimen taken of each specimen’s abdomen and A lab tissue (Kimwipe) was placed on placed in separate microfuge tubes with the the lab station while the specimen was corresponding voucher number. 200µl of Lysis carefully taken out of the holding jar, and buffer (2 ml 0.5M EDTA, 0.2 ml 5M NaCl, placed on the tissue. Voucher photographs of 2.5 ml 10% SDS, 1 ml 1M TRIS ph8, 94.3 ml the specimen were taken once the specimen dH2O) was then added and the insect was was dry for future identification. The specimen then macerated by hand. Next 800µl Lysis was placed in the Petri dishes after photos buffer was added to the labeled microfuge tube, for a total of 1000µl Lysis buffer. The were taken for future use. tube was cap locked and placed in a 99°C An online insect ID key, like the one at heat block for 5 minutes. The pressure inside discoverlife.org was found for identification of the tube was briefly released and the solution the specimen. The needed information about was mixed thoroughly in the tube. Then the the specimen was added and searched. The sample was centrifuged at ~10,000 rpm for 5 description that fit the specimen the most was minutes. found and recorded that as the order of the insect, as directed in the Babec protocol [5].

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Part 2: Removing Impurities A second sterile 1.5 ml microfuge tube was labeled with the corresponding voucher number. The initial tube was taken out of the centrifuge [5]. 400 µl of the supernatant was withdrawn from the first tube without disturbing the pellet. The supernatant was then transferred to the new tube, and old tube containing the pellet was discarded properly.

To the supernatant, 40 µl of 5M NaCl were added, and then shook to mix. Held on ice for 5-10 minutes, or until cloudy. The tube with NaCl was placed in the centrifuge and spun again [5].

Part 3: Isolate the DNA

TE/RNase buffer (100µl RNase A (5mg/ml), 4.9 ml TE buffer) was added to the pellet in the micorfuge tube, solution became cloudy and resuspension occurred. Solution was centrifuged for 1 minute as directed [5].

Polymerase Chain Reaction The voucher number was written onto a small PCR tube. 20µl of Master Mix (250µl 10x PCR Buffer II, 400µl 25mM MgCl2, 200µl 10mM dNTP mix, 40 µl BSA (10 mg/ml), 20 µl 5 U/µl standard AmpliTaq) was added into the labeled PCR tube [5]. To the Mater Mix, 20 µl of Primer Mix (1µM of each Wolbachia primer: Wspec Forward, Wspec Reverse, and 1 µl of each control cytochrome oxidase c Gene: COI Forward, COI Reverse, dH2O) was added. To the 40 µl of liquid, 10 µl of extracted DNA was added, according to the Babec protocol [5]. The tube was capped and placed in the thermal cycler with the voucher code in the corresponding box on the grid.

Cycling was run according to the Babec protocol [5].

A third sterile 1.5 ml microfuge tube was obtained and labeled with “DNA” and the corresponding identification voucher number.

The previous tube was retrieved from the centrifuge [5] and 300µl of the supernatant was withdrawn from the tube. The supernatant was transferred to the new tube, while the old tube and pellet were disposed of properly.

400µl of isopropanol was added to the tube Electrophoresis with transferred liquid. The 700µl of liquid in the tube was inverted several times to mix the The PCR tube was removed from the contents. thermal cycler and centrifuged briefly. A After the inversion of the microfuge fourth, and final, 1.5 ml microfuge tube was tube, it should be placed in a balanced labeled with the voucher number. 20 µl of the centrifuge, and centrifuged [5]. The 1.5ml PCR product and 2 µl of the loading dye were microfuge tube was retrieved from the dispensed into the tube and mixed. The PCR centrifuge and all the supernatant was product was placed on a rack for storage. 15removed, without the pellet being disturbed. 20 µl of the mixture was loaded into a well in The pellet was air-dried for about 5-10 the gel. 15-20 µl of the 100bp ladder was minutes with the cap open. 200µl of added into one well on each gel. The samples

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were electrophoresed at 150 V for 25-40 gel. The bands of DNA and/or infections were minutes [5]. Each gel was set in a well lighted made visible on the photographs. position and then a picture was taken of each

Results There were 2 earwigs, 1 spider, 6 flies, 2 beetles, 1 wasp, 1 isopod, 1 moth, 1 dragonfly, and 4 unidentified insects (which include 2 positive control insects) tested during the course of this experiment, for a total of 19 various insects collected from the San JosĂŠ State University Metropolitan Area.

Of these 19 specimens, 16 had a successful PCR run. This calculated out to be an approximately 94.7% success PCR run rate.

Of the 16 that turned out results, 14 did not show signs of Wolbachia infection, calculating out to an approximate 10.5% Wolbachia infection of the population.

GEL #1 Lane

Insect 1 Ladder 2 Positive 3 Earwig 4 Wasp 5 Negative 6 Positive 7 Fly 8 Fly 9 Fruitfly 10 Isopod 11 Diptera 12 Diptera

Infected?

Y N N N Y N N N N N

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GEL #2 Lane

Gel #3 Lane

The low infection rate could be due to a number of things. One possible reason is that the insects that showed no signs of infection were infected, but with levels that were too small to be detected. A second reason could be that the insects were collected from a very

Insect 1 Ladder 2 Positive 3 Earwig 4 Negative 5 Arachnid 6 Moth 7 8 9 10 11 12

Infected?

Y N N N N

Insect Infected?

1 ladder 2 postive control y 3 beetle n 4 negative control n 5 dragonfly n 6 n/a n 7 crtl insect y 8 lady bug y 9 y 10 ctrl insect y 11 fly n 12

narrow urbanized area, therefore not much chance for outside infections. There are endless possibilities of why this occurred, and no one could be absolutely sure of the “correct reason�.

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Discussion In this experiment I was trying to disprove that the environment was not a factor in insects becoming infected with Wolbachia.

collected. Not knowing this does not help in determining the weather around the insects.

The weather and the environment go hand-inhand with affecting insect’s lives.

In the protocol, the steps were generally easy to understand and follow. They compensated for many sources of error in the protocol itself. Though in the protocol [5], steps 18 is where it gets confusing. This step tells you to drain all of the liquid without disturbing the pellet. Though if the previous steps were done with no error, then it would be a perfect DNA sample, which would have no pellet. If there is no pellet, then how does one know much liquid should be drained from the tube? One doesn’t, for that step it is all a guessing game, unless you have the pellet. Instead of saying “remove all of the supernatant”, it should say “remove most of the supernatant” to compensate for the guess work.

The results appear to show that in the San José State University metropolitan area, for the samples collected, Wolbachia is not as prevalent as the national average. They showed that a small fraction of the insects were infected. Though with the other research that has been done on a much larger scale, this is minuscule in infection in the population. This is a great result because it may suggest that Wolbachia is not evenly distributed throughout the US. This helps show the importance of doing more studies in this and surrounding areas for better understanding of the Wolbachia distribution on a local, regional, and national level.

I knew the general area in which the insects were collected, San José State University metropolitan area; though this was not specific enough to determine the exact environment the insects were in. Another factor that would affect my theory would be the time of year in which the insects were

The PCR method chosen in this lab has proven to be very effective. Kristen Wolslegel said the success rate was “the highest I’ve ever seen in a workshop” when she e-mailed the results to Dave Menshew. Therefore the three specimen that did not give any results was not by fault of the PCR protocol written up by Babec.

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Conclusion Through collection, identification, and screening, as directed by Babec [5], I was able to determine the percent infection with Wolbachia. There was a small percent of infected insects in the sample collected in the San JosĂŠ State University metropolitan area. The results gained from this lab do differ from the national study, the results are

lower than the national average. To get a better understanding of the Wolbachia distribution in the United States, future studies with more samples at each location are needed.

These studies would help determine whether or not Wolbachia is evenly distributed in the United States, or if there are regional/local variations to the distribution.

Acknowledgements The author would like to extend gratitude to: Chris Thomas, Dave Menshew, KimMarie Hansen, Kristen Wolslegal, Drew Davis, The Bay Area Biotechnology Education Consortium (BABEC), Michael Coats and Chris Fiscus. Without their advice, encouragement, and incredible support this project would not have been possible.

This project was funded by BABEC through the BABEC: Discover the Microbes Within Teacher Workshop, which was held at CaĂąada College.

The instructors, KimMarie and Kristen, were very encouraging, helpful, and understanding. They were very patient with Drew and I through the entire process. They were quick to encourage me in my career path and make me feel comfortable around the teachers in the training.

References 1. Meyer,

John: A Class of Distinction.

NC http://www.cals.ncsu.edu/course/ent425/text01/index.html. Jan 21 2007.

2. Timmer, John: Meet Wolbachia: Arstechnica.com, 2011.

the

male-killing,

gender-bending,

State

University.

gonad-eating

bacteria.

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3. Knight, Jonathan: Meet the Herod bug. Macmillan Magazines Ltd. Nature Magazine. Volume 412. July 2001. 12-14 4. Werren, JH, Baldo, L, and Clark, ME: Wolbachia: master manipulators of invertebrate biology.

Macmillan Publishers Limited. Nature Reviews: Microbiology. Volume 6. October 2008. 741-751.

5. Wolbachia PCR Teacher Guide. BABEC. Babec.com. 2011 6. Discover Life. Discoverlife.org 7. Charlat, S, Hurst, GDD, MerŇŤot, H: Evolutionary consequences of Wolbachia infections. Elsevier.

TRENDS in Genetics. Vol 19. No 4. April 2003. 217-223.

Appendix BABEC: Discover the

microbes within workshop

On November 19, 2011 BABEC hosted a teacher workshop called Wolbachia PCR Lab: Discover the Microbes Within Workshop. It was held at CaĂąada College, room 16-212.

It started at 8:30 am, and ended at 4:00 pm. We ran through the entire lab in one day, whereas in the class room it would take about nine days. It was a little fast-paced and vigorous, but mainly a move at your own pace lab. Drew Davis and I were the first students to be able to attend this workshop. It was created to help teachers understand the lab so that they could teach it in their class. . The instructors were very helpful and encouraging and made the protocol easy to understand.