Researching Pelecinids – Part 6

Last year I chronicled my experience researching Pelecinus polyturator in Dr. Norman Johnson’s lab. This research journey included learning the biology and behavior of P. polyturator, photographing specimens, collecting in town using Malaise traps, extracting DNA and amplifying it using polymerase chain reaction (PCR), learning the importance of literature reviews, and trying to collect live adults at Zaleski State Forest. Take a look at my previous blog posts to know more about these topics and catch up on my research so far.

author using laptop

The author analyzes DNA sequences using the Sequencher software

Over the last six months I continued working on my research, and now I have several exciting updates to share. While I took a hiatus from the lab in autumn semester to focus on my academic coursework, the research process did not stop. The DNA we extracted was sent out to be  commercially sequenced. Once I returned in spring semester, it was a matter of assembling those sequences and performing phylogenetic analysis before we could start drawing conclusions.

The focus of our study was the sex ratio variation between northern and southern populations of P. polyturator. Using molecular techniques we amplified sequences of the cox1 gene, which can be used as a genetic barcode to differentiate species. Additionally, we amplified the ftsZ gene in Wolbachia, an intracellular symbiotic bacterium that can effect sex ratio in insects. We suspected that if different strains of Wolbachia were present in the different P. polyturator populations, this could be a contributor of the observed sex ratio variation.

The Wolbachia turned out to be nearly identical from every locality sampled. On the other hand, the cox1 gene varied greatly between the northern and southern populations, and even between different localities of the southern population. From this information we concluded that the Wolbachia strain was not a direct cause of the sex ratio variation. The differences in the cox1 gene, however, suggested that there may be speciation occurring between the northern and southern populations.

Doing the research and analyzing the results was only a part of the process. Science is a collaborative endeavor, and it is important for a scientist to be able to communicate the results and ideas to others in a clear and concise way.

On March 1, I had the opportunity to present my research at the CFAES Undergraduate Research Forum. This is an event where students within the CFAES (that stands for College of Food, Agriculture, and Environmental Sciences) can showcase their research to other students and faculty, as well as a panel of judges. This forum attracts a wide audience of people from many different scientific disciplines and backgrounds. It is a great opportunity to interact with people who may not be familiar with entomology or molecular genetics. At first I was nervous to talk about my research, but it ended up being surprisingly fun to answer people’s questions. I even had a female P. polyturator specimen to show visitors, courtesy of Dr. Luciana Musetti and the Triplehorn Insect Collection.

Designing and presenting the poster was a great experience for me. I quickly learned that designing a poster is much different from writing a traditional scientific paper. Both formats are separated into similar sections such as an introduction, methods, results, and conclusions. A poster, however, has space constraints and must be attractive to a general audience. Large blocks of text typical of papers are unattractive and intimidating on posters. Overall, readability is the most important trait of a good research poster. The font should be large and easy to read, and everything should be spatially organized in a logical manner.

author and research advisor with poster

The author and her research advisor, Dr. Norman Johnson, stand in front of the research poster at the CFAES Research Forum.

author explaining research

The author explains her research to entomologist Dr. Megan Meuti

Presenting my poster at this research forum was a great preparatory experience for the upcoming Denman Undergraduate Research Forum. Because most of the research within CFAES was focused on agriculture and food science, I gained experience speaking to people unfamiliar with the topics of my research. The Denman will feature an even more diverse array of students and faculty, from business to chemistry to art, so I will need to be prepared to explain my research to those completely unfamiliar with entomology or molecular work. I will also be revising my poster using suggestions from the judges of the CFAES forum.

If you would like to learn more about my research project, as well as the research of other hardworking undergraduates, stop by the Denman in the Archie Griffin Grand Ballroom of the Ohio Union on Tuesday, April 3. I will be displaying my poster from 3:00 pm to 5:00 pm, and I would love to see you there!

About the Author: Hannah McKenzie is an undergraduate entomology major at the Ohio State University. She currently works as a curatorial assiatnt at the Triplehorn Insect Collection and is greatly enjoying her undergraduate research project on Pelecinus wasps.

Researching Pelecinids – Part 5

On August 10, I traveled to Zaleski State Forest with Dr. Norman Johnson and Dr. Luciana Musetti to collect Pelecinus polyturator specimens for my research. The area we visited has come to be known as Pelecinus Cove due to the relative abundance of Pelecinus wasps in the area.

image of Pelecinus Cove

The author searching for Pelecinids at Pelecinus Cove, photo by Dr. Luciana Musetti

Upon arriving at Pelecinus Cove, I could clearly see why Pelecinids are attracted to the locality. Adult Phyllophaga beetles tend to lay their eggs in the soil near trees. Pelecinids then parasitize the larvae that hatch from these eggs. However, females are not able to navigate their abdomen through the soil to oviposit if the ground is covered in grass or other dense vegetation. Therefore, the ideal location for a Pelecinus wasp would be a wooded area with mostly open ground. Pelecinus Cove definitely fits that description.

Equipped with insect nets and killing jars, we trekked through tall weeds and swarms of mosquitoes in search of the large parasitoids. Several minutes into our expedition, we noticed that not many insects were present other than a few moths and a lot of mosquitoes. We used our nets and feet to stir up the weeds in hopes of also stirring up resting Pelecinids. Unfortunately, even after an hour and a half of searching, there were no Pelecinids to be found.

Although the location itself was ideal, we speculate that other conditions were not. Ohio received a lot of rain in the weeks prior to our collecting trip, and the ground was very damp. This may have flooded out some of the Phyllophaga grubs or deterred the Pelecinus females from ovipositing. The number of grubs available to parasitize is a major limiting factor of how many Pelecinus females successfully reproduce, so if fewer Phyllophaga grubs were accessible due to damp soil, then fewer Pelecinids would be expected to stay in the area. The weather was also very cool, humid, and overcast. A little more sunshine and warmth may have enticed the Pelecinids, as well as other insects, to come out of hiding.

Despite the disappointing outcome of the collecting trip, fresh specimens are not completely unavailable. Prior to the collecting trip, I found a P. polyturator female in one of the Malaise trap samples. This exciting find also suggests that there are more wasps in the area that could be collected, so I am keeping my hopes up for more fresh specimens in the future.

image of gel electrophoresis

The author learning protocols for gel electrophoresis

Due to my autumn class schedule that begins next week, today is my last day working and researching at the Triplehorn Insect Collection for the summer. Looking back, I realize just how much I learned over the summer through my research experiences, including the value of DNA barcoding, how to extract DNA and amplify genes, how to collect from a Malaise trap, how to photograph large insects, and the fascinating behaviors of P. polyturator.

Although I won’t be participating in active, ongoing research this autumn, that doesn’t mean there won’t be more opportunity for learning and progress. I still plan to revisit my research regularly, read more about Wolbachia, continue editing my research proposal for the Honors project, and learn how to trim and read DNA sequences. When I return to my regular research schedule (hopefully in the spring), I will be well-prepared to continue where I left off. Thank you for following my research journey this far, and I hope that everyone reading has developed a new appreciation for the intriguing parasitoids of the genus Pelecinus.

About the Author: Hannah McKenzie is an undergraduate entomology major at the Ohio State University. She currently works at the Triplehorn Insect Collection and is participating in undergraduate research on Pelecinus wasps.

Researching Pelecinids – Part 4

image of author extracting DNA

The author learning DNA extraction protocols

Over the last several weeks, I have been hard at work with my research on Pelecinids. I continued to collect weekly samples from the Triplehorn Insect Collection’s malaise traps, and I began to practice keying various families and superfamilies of Hymenoptera. I also learned the protocols for DNA extraction, PCR, and gel electrophoresis from graduate student Huayan Chen.

Not every aspect of research, however, is an active endeavor taking place in a lab or in the field. Research also requires a considerable amount of time for reading, writing, planning, and patience.

I am now in the process of shaping my research into an honors project that will span multiple semesters rather than this summer alone. Honors research is a much more demanding ambition than a single semester of research, but it is also a very rewarding experience that will push me to delve deeper into my research topic. I plan to expand the scope of my current research on Pelecinus DNA by also looking for DNA from Wolbachia, a bacteria that can influence sexual differentiation in insects. Huayan and I have already found that Wolbachia is present in several of our wasp specimens. I am also interested in the possibility of geographical parthenogenesis in P. polyturator females.

My current focus is on composing an honors project proposal. The purpose of the proposal is to establish how I plan to conduct my research, what materials I will need, and what goals I aim to meet. The proposal also includes an abstract, which is vital for exhibiting my research to the scientific community, and a literature review, which is important for ensuring the quality and relevance of my project.

The literature review has proven to be the most challenging portion of the proposal. It involves finding and reading the research of others, then synthesizing the information into a concise review and citing the sources in a list of references. Because there is such a vast amount of previously conducted research available to read, picking out the relevant studies and piecing the information together can be a time-consuming process. While searching for material to read, it isn’t uncommon for me to feel a little overwhelmed.

Despite the challenge, the literature review is one of the most important components of any research project for many reasons. First, it prevents the unnecessary repetition of collecting and interpreting data that another study already collected and interpreted in an identical manner. Second, it provides helpful context for the researcher. By reading about work that others have done on Pelecinus, Wolbachia, and parthenogenesis in Hymenoptera, I will be better equipped to design a procedure that is effective and efficient, or modify that procedure should something not work. Third, I can interpret data from other studies in relation to my own research findings, and vice versa, leading to a more comprehensive understanding of my final results.

image of P. polyturator

P. polyturator female

Although summer semester is coming to a close, I still have several research-related plans before I head back to classes this autumn. I will collect fresh P. polyturator specimens from Zaleski State Forest this August, which is when adults are most prominent in Ohio. These specimens will then be used for DNA extraction, along with several frozen or mounted specimens already in the collection. We also received a generous donation of West Virginian and Canadian P. polyturator specimens, which will be very useful for sampling DNA from a wider range of localities. I will also continue collecting from the malaise traps, keying Hymenoptera families and superfamilies, and reading relevant materials on DNA barcoding.

About the Author: Hannah McKenzie is an undergraduate entomology major at the Ohio State University. She currently works at the Triplehorn Insect Collection and is participating in undergraduate research on Pelecinus wasps.

Researching Pelecinids – Part 3

A misconception that I often hear regarding research is that it only takes place inside a laboratory. Although procedures such as PCR and DNA barcoding do take place inside the lab, there is also an in-the-field component that is vital to producing quality molecular research. In order to analyze the DNA of specimens, it is first necessary to collect those specimens. While I research pelecinids in the molecular lab, I will simultaneously learn about the process an insect collection goes through to collect, curate, and document new specimens that can then be made available for research.

image of malaise trap

A malaise trap located at Waterman Farm

One of the most effective means for collecting large quantities of insects in a short amount of time is the malaise trap. These large traps consist of a black mesh base and a white mesh tent. At the peak of the tent is an empty plastic bottle attached to a second bottle full of ethanol. Insects flying close to the ground become caught in the mesh and instinctively climb upward to escape, leading them into the empty jar. The ethanol vapors then knock out the insects and cause them to fall into the ethanol bottle (Vogel, 2017). Not only does the ethanol preserve the specimens, it also makes collection very simple. All one must do is detach the bottle of ethanol and empty it into a storage container such as a Whirl-Pak, then refill the bottle with fresh ethanol and reattach it for further trapping.

The Triplehorn Insect Collection sets up three Malaise traps during the warmer months, one near the Carmack parking lots and two at Waterman Farm. Each trap is set up in a different environment, so each trap collects a different variety of insects. For example, the Carmack trap is located near a pond and collects damselflies, whereas the Waterman Farm traps do not typically collect damselflies because they are located in dryer areas.

Although the traps are mostly intended for flying insects, other organisms such as spiders and ants often climb up the mesh and meet a similar fate. Pelecinid wasps will occasionally find their way into the traps, but it is not a very common occurrence. I will have the opportunity to collect from more Pelecinus-rich habitats later this summer.

I learned how to collect from these malaise traps from Huayan Chen, one of the graduate entomology students conducting research at the collection. In addition to learning how to empty and refill the ethanol bottles, I also learned that I need to cut back the weeds that grow around the traps and how to tie the knot that secures the bottles (a feat which took an embarrassingly excessive number of attempts for me to master). As part of my research experience, it is now my responsibility to collect the specimens from each trap once every week for the next several weeks. Each sample, which must be labelled with the collection date and locality, then gets stored in one of the collection’s freezers.

These samples will eventually get sorted into separate vials for each insect Order. What happens next depends on the goals of the collection. Some vials may be stored in the freezer for future use. Others will be further sorted to Superfamily or Family. They can then be further identified, mounted into the collection and databased, or used in molecular work.

I will use the Hymenoptera from these samples to learn the process of adding specimens to the collection. My plan is to further sort some of these specimens by Superfamily or Family (or even genus!), mount and label them, and enter them into the HOL database. In addition to teaching me the full collection process, this will also provide me with a better understanding of Hymenopetera morphology and identification that will be invaluable for the rest of my research.

Vogel, Gretchen. (2017). Where have all the insects gone? Science, 356 (6338), 576-579.


About the Author: Hannah McKenzie is an undergraduate entomology major at the Ohio State University. She currently works at the Triplehorn Insect Collection and is participating in undergraduate research on Pelecinus wasps.

Researching Pelecinids – Part 2

One of the most useful resources the Triplehorn Insect Collection has to offer is its extensive HOL database, an online taxonomic initiative that documents collected specimens of various insect groups. This database already contains records for over 845 thousand Hymenoptera specimens, and it continues to grow every week through the hard work of countless contributors.

One way to contribute to HOL is to photograph and upload images of collected specimens, further enriching the available data on specific taxa. Prior to the start of my research on pelecinids, there were zero images of the genus Pelecinus available in the database. To remedy this, I had the opportunity to work with Jordan Reynolds, an undergraduate student in Art & Technology who is currently working on specimen photography for the collection. Over the last several weeks, Jordan taught me about the technologies and techniques required for taking high-quality images for the database.

My goal in photographing a selection of the collection’s Pelecinus polyturator specimens is three-fold. First, I will obtain a more in-depth understanding of the external anatomy of these intriguing insects. Second, I can compare the morphologies of males and females of different sizes and localities. Third, I will contribute previously unavailable data to the HOL database.

Photographing collection specimens is not as simple as taking a picture with a camera. Because the specimens are relatively small, and because we require intricate detail in the images, we instead use a method known as focus stacking. With this method, the camera gradually moves along a track and takes multiple images at different distances. The focused areas of each image are then “stacked” together by a computer program so that the entirety of the final image is clear. Because the P. polyturator specimens required a large depth of field, we sometimes had to photograph and stack up to 100 images to create a sufficiently focused final image.

In addition to taking individual specimen images, we also took several comparison images. These provide a quick side-by-side comparison between two different specimens. Some of the images we took compare males and females of similar localities. Other images showcase females of similar localities and their dramatic variance in size.

comparison image of P. polyturator females

Comparison image illustrating the size variation in P. polyturator females

Of course, no system is perfect, and we faced many challenges along the way. Most of the parasitic wasps that get photographed by the collection are very small in size (no more than a few millimeters long). Therefore, the collection’s microscopic camera that is traditionally used for parasitoid photography is designed to take highly detailed images of very small specimens. P. polyturator females are unusual in that they are very large parasitoids that can exceed 40 mm in length. Because of this, many of the specimens we wanted to photograph could not fit under the microscopic camera. We instead opted to use a DSLR camera, which is used for macrophotography of larger specimens such as beetles and butterflies. Using this nontraditional method of parasitoid photography, we were able to produce high-quality images of the larger specimens by exploiting various imaging techniques.

Another challenge we faced was the handling of the specimens. All mounted insects in a collection must be handled with great care because they are dry and brittle. Pelecinids must be handled with extra caution because their long, string-like antennae and fragile abdomens will break with even the slightest bump.

We ended up with many beautiful images of these wasp specimens. The complete collection of images has been uploaded to HOL and is available on the page dedicated to P. polyturator. I would like to thank Jordan for all of his help with this photography endeavor. Without the countless hours he spent performing behind-the-scenes image processing, the final images could never have turned out so well.

About the Author: Hannah McKenzie is an undergraduate entomology major at the Ohio State University. She currently works at the Triplehorn Insect Collection and is participating in undergraduate research on Pelecinus wasps.