In the hallway outside my lab, about 800 species of Bembidiina, together in one tree. :)
I started learning how to do stained glass pieces over the summer, and for my first piece I decided to honor our Discovering Insect Species class by doing the head of an undescribed species of Bembidion (Trepanedoris) that we discovered. I finished the piece on Tuesday; here it is:
Today we reached a milestone: our 100th flavor in our approximately weekly Guess the Potato Chip Flavor event in my lab. Today we decided to celebrate the event not only with our 100th flavor, but also with five other flavors to round out the meal. Those five other flavors were some current Lay’s flavors: West Coast Truffle Fries, Southern Biscuits and Gravy, Greektown Gyro, New York Reuben, and Pico de Gallo. Over the last couple of years we have had quite the variety: Happiness Butter (from Japan, by a South Korean company), Butter Chicken (Lay’s, from Canada), Slow-cooked Ribs (Old Dutch, from Canada), and many others. Some were excellent, some.. not so. The two that stayed in the bowl the longest, by far, were Sea Salt Pomegranate and Cappuccino. We try to stick to potato chips, but we do occasionally branch out into other types (such as corn chips) if there are compelling flavors or if we have no novel potato chip flavors.
But the 100th flavor… now that was a fantastic one. My graduate student James Pflug acquired it online, from the UK, and, well, here are two pictures:
They have edible gold stars on them!
The flavor: Winter Berries and Prosecco with Edible Gold Stars and a touch of Fizz, by Marks and Spencer. Berries and sparkling wine with gold stars. Wow. I can’t imagine how we can top that.
Here’s the full list of what we have tried.
In the Discovering Insect Species course, the eleven of us (students Alex, Ana, Danielle, Elle, Julia, Mamo, Shannon, Tom, and Trevin, plus John and me) formed a great team, and we learned a lot together. Over the course of the term, we became… the Trepaneleven!
As with any group of superheroes, talents vary from hero to hero. In order to recognize the amazing abilities and achievements of the Trepaneleven, we held an award ceremony, at which awards were awarded.
And the winners are:
Julia excelled at finding the best species delimitation using a pre-release version of the Mesquite package Leaves (which attempts to delimit species using gene trees). Her award was a Leaves Champion trophy.
Elle’s interest in insect taxonomy and illustration made her the obvious choice for this award. Her award consisted of a magnifying glass (with built in vial!), some watercolor paper, and some Pigma pens.
Danielle taught us the wonders of sautéing without oil during our Malheur field trip (as, well, the oil was inadvertently left in Corvallis). She was also excellent at leading us to ice cream, which the judges very much appreciated. Her award was a trophy topped with crossing items: a skillet and an ice cream cone.
As someone who has described new species in her native Brazil, the country that probably contains more undescribed species than any other, Ana was a natural for this award. Her project (an interactive key to Bembidion (Trepanedoris)) also swayed the judges. Her award was a copy of E.O. Wilson’s book, The Diversity of Life.
Let’s just say you had to be there, in multiple places at multiple times. With Alex’s interest in forensic entomology, a zombie survival guide and a bug collecting kit seemed the appropriate items to honor his achievement.
Mamo discovered, at Malheur NWR, the habitat of the undescribed species that we first found at Klamath Marsh NWR. This allowed us to collect what will become the type series of the species. It was the most significant habitat discovery during the course, and for this the judges will always be thankful. As important, though, was Mamo’s evangelism for the wonders of Spam sushi, of which we became rather fond on our road trips. Her award consisted of a book on the history of Spam.
Shannon not only discovered a new species in her parents’ backyard (it is still the only known locality), she was also overly adept at collecting little harpalines (thinking they might be Bembidion). She loves words and their histories, and so one way we honored her contributions was with a copy of Brown’s Composition of Scientific Words. On the Malheur trip, her neck decoration one day, a tick, caused such a reaction that we immortalized that tick in a very small clear box, and presented that to her as well.
Tom knew everything about aquatic invertebrates (at least, that was part of the class myth). He also was excellent at manning a walkie-talkie so that we could communicate between vehicles as we drove our convoy to and from the field sites; his British accent and humor (or, rather, humour) were a real plus.
Tom was also adept at spreading parts of our aspirators around the countryside, and so his trophy was affixed with one such piece.
Trevin did an excellent job of following instructions. He also coveted a National Wildlife Refuge sign (containing instructions) that we were given by one of the wildlife refuge managers. In hopes that Trevin would continue to follow instructions, the judges were pleased to give him this award.
When last I blogged, the Discovering Insect Species course was in full swing; we were excitedly waiting for the DNA sequence results from our first big field trip, which was to Klamath Marsh National Wildlife Refuge. I’m sorry I didn’t post regular blogs during the remainder of the course, but so many exciting things happened, so quickly, that I never caught my breath enough to write down what was going on. This overview post will be followed by some blog posts about particular aspects of the course, including the discoveries made in the class, some thoughts on teaching gene tree / species delimitation modeling to students, and the class projects, and the Awards Ceremony.
Cutting to the chase, the course was awesome, and tremendously successful. It was the most rewarding experience of my career as a teacher, and I think the students got a huge amount out of it too. We did indeed discover two new species during the course (as promised in the name of the course!), two species I had never seen before, which very much added to the excitement – more on that later.
The course was quite costly, both financially (I spent about $8000 on it) and in terms of my time (and John’s, who served as a TA), but I hope this single experience has a significant impact on the students who participated.
Below is a general overview of the events in the course. There are many details missing here, and I will cover a few of those in later blogs.
I incorporated the 28S sequences from the beetles the students caught in Corvallis and at Klamath Marsh into the existing matrix of 28S sequences, and passed the matrix around to the class.
The class then did a RAxML analysis to get the gene trees, and they each had to see where their four specimens ended up, and whether or not the relationships were as they predicted.
And then I incorporated all of the remaining sequences, including from a large number of specimens from all around western North America, and did analyses for all four genes. This was all done in class on the big screen, and it was very exciting for all of us to see the results live. The most exciting discovery, by far, was that the two specimens we collected at Klamath Marsh that I initially thought (while in the field) were Bembidion “Lost Lake” were not that species at all, but instead another species I had never seen before!
I got the email from FedEx saying that our package of four plates of 384 PCR samples had arrived at the University of Arizona, where they will be sequenced. These plates contain the DNA from multiple genes from over 120 Trepanedoris that we have not sequenced before; the data we get back will tell us a huge amount about Trepanedoris diversity for our Discovering Insect Diversity class.
We send all of our PCR samples to the University of Arizona Genetics Core to be sequenced. They are relatively inexpensive, and do a great job. In particular Stacy Sotak has been terrific, and has helped us over the years with all of my Sanger sequencing needs. In the rare times that there have been problems, Stacy has really worked well with us to help troubleshoot. I worked with Stacy for some of my time at the University of Arizona, and since I moved to Oregon State University she has continued to be a fantastic (if now more distant) help to our research.
I asked Stacy if she could take pictures of the processing of this set of four plates, so that the class could see how the magic happens of going from PCR product to DNA sequences. I’m excited to see some of the equipment used, as I haven’t actually ever seen it myself.
The plates arrived in good shape to the U of A; here they are coming out of the shipping box!
The PCR products then needed cleaning; here are the first two plates (Bemb213 and Bemb214) sitting in the cleaning system:
Update 1: The DNA in the plates have been quantified. Here they are getting ready to be quantified:
And here is the fluorometer that quantifies the DNA:
Update 2 (1 May, about 10:30 am): The sequencing is done! Here is a picture from Stacy showing the machine that does most of the magic, the sequencer itself:
The instrument is open, and on the right you can see six of the eight plates loaded into it.
You might wonder why there are eight plates rather than four – after all, there are only four plates of PCR products. We sequence each sample twice (once in one direction, once in the other direction) so that we can confirm the results. That’s why we had two primer plates for each PCR sample plates. This means we sequence each original plate twice, which translates into eight sequencing plates in total.
Here’s what the computer attached to the sequencer is showing as the sequencing is happening:
When it was all done, Stacy did some data processing, pressed some buttons, and I got the following emails, which always cause my heart to race in excited anticipation, as they are telling me that the sequences are ready to download:
Alas, I will have to wait to download them. I would normally spend today processing the data and doing analyses, and lose myself in the thrill of all of the cool stories the sequences will tell. However, I have promised myself that I will not do this until class on Tuesday – I want the students to be fully part of the process, and see the results the same time I do. For the next four days, I will need to remind myself that patience is a virtue.
[Thanks so much, Stacy, for taking these pictures for the blog – they are much appreciated by the students!]
In an earlier post, I outlined the steps we took to extract and amplify DNA from the beetles we collected in the Discovering Insect Species field trip to Klamath Marsh. As mentioned, the students extracted DNA from 36 beetles, and amplified two genes for each. Mamo and Danielle came in and amplified some additional Trepanedoris for 28S, and also amplified the MSP gene (Muscle-Specific Protein 300) from 22 Trepanedoris specimens, as part of an exploratory study. We have never looked at MSP before in Trepanedoris, so we are most curious as to what the data will reveal.
Our next step was to get those PCR products sequenced. However, before I write about that, some background.
Here are some of the western locations from which we have Trepanedoris DNA sequences already:
The 36 beetles prepared by the students will add a few key localities (red stars):
Over the last few years we have been accumulating other Trepanedoris, and getting them ready for sequencing, but haven’t sent the PCR products off to be sequenced (we have been saving them for this course). We’ve extracted and obtained PCR products from an additional 83 specimens from around North America, especially in the USA. These 83 specimens (blue squares) provide a much denser sampling, especially in California:
All told, we have over 400 PCR samples from seven genes ready to be sequenced from over 120 Trepanedoris specimens that have never been sequenced before. We chose 384 of those PCR samples for sequencing, and got them ready in four batches of 96:
John then transferred each batch to its own 96-well, clear plastic plate:
Each plate was given its own name: Bemb213, Bemb214, Bemb215, and Bemb216. He then sealed up each plate with foil tape, and put it in the freezer. These are called the “template” plates as they contain the DNA that will serve as a template during sequencing. For each of the four template plates he then needed to make two additional plates, containing primers (short stretches of DNA) used in the sequencing reaction:
Here is plate Bemb213 containing beetle PCR product (the template), covered with foil, and sandwiched between the two primer plates also covered with foil:
John wrapped up that trio, and put it onto dry ice in a cooler.
He did the same for the other three plates, put the cooler into a box, wrapped everything up, and off it went to be FedExed to the University of Arizona, were we get our PCR products sequenced. These went off on Monday.
I am absurdly excited to see what these 300+ sequences reveal about Trepanedoris diversity. Apparently we will get the data back on Friday, but I will hold off on downloading them until class next Tuesday, so that all of the students can be there as we see the results for the first time.
I’ll report back as progress happens!
Shannon, a student in the Discovering Insect Species course, went down to Eugene last weekend and looked for Trepanedoris. To say that she did well would be an understatement. She caught all four species that I had seen from the Willamette Valley of Oregon (Bembidion acutifrons, B. elizabethae, B. fortestriatum, and B. siticum), and more.
The real prize was a male of a species I had never seen from this area. Here’s the live beetle:
At first glance I thought it was an undescribed species, but after looking at it more closely I now think that it is an extremely small specimen of Bembidion canadianum. I have only ever seen one specimen of Bembidion canadianum from Oregon, collected in 1955 from east of the Cascades, around Upper Klamath Lake. The specimen is housed in the OSAC.
The specimen Shannon caught was a male with a rather swollen abdomen, and it walked very slowly. I’ve seen this before, and it usually points to some unpleasantness (at least for the beetle) inside its body cavity, in the form of nematodes. And, sure enough, when I opened up the abdomen to expose the tissues to ethanol so that the DNA would be well-preserved, there were a lot in there:
Here’s what part of the abdomen looked like; all of the little white tubes here are nematodes:
I’ve seen many nematode-filled Bembidion over the years, but this one was unusual, in that it had nematodes of different sizes: a few big ones, and many small ones. Normally all the nematodes are of more or less the same size. Another thing that is unusual about this beetle is that it still had what appeared to be fairly healthy testes and accessory glands; a nematode-filled Bembidion normally has very little of its own tissue remaining.
Although interesting, the nematodes will cause some problems in extracting beetle DNA from the specimen – as it will be hard to avoid extracting nematode DNA instead!
Over the last couple of weeks in our Discovering Insect Species course we have been processing samples from our Klamath Marsh trip, and worked with the DNA of the beetles. Here’s a bit of what we have done.
We start out with a bit of a beetle in a tube. This is what it looks like:
We then went through all of the steps required to dissolve the soft tissue in that sample, and from the soup that results extract the DNA by itself into a clean tube. Here are pictures of a couple of those steps:
In the end, we get beetle DNA in water (plus some salts). It looks just like normal water, but there is beetle DNA in there:
The students extracted DNA from a total of 36 specimens, most of which they had collected on our Klamath Marsh field trip.
Once all of the DNA in the beetle was extracted, we then wanted to pick a few genes, and make many copies of those genes so that they could be sequenced. The first gene we picked was 28S.
Everyone combined various chemicals in some small tubes. These chemicals included nucleotides and an enzyme, Taq polymerase, which copies DNA. We also added short stretches of DNA called primers that match part of the sequences for the 28S gene in the beetles, and that will providing a starting point for the reaction. The end result will be that the enzyme will produce many copies of the beetle’s DNA between the two primer regions on the chromosomes. This reaction is called the Polymerase Chain Reaction, or PCR, and is a standard way of making many copies of (“amplifying”) DNA.
Here we are getting small tubes set up for PCR:
The tubes are then put onto a machine called a thermal cycler, which once closed and started will go through various cycles of changing temperatures:
In the end you get, for each sample, a little tube that contains zillions of copies of the piece of the 28S gene between the primers:
At least that’s what we would hope would happen. To confirm that the DNA piece was amplified for each sample, John then mixed a small amount of the liquid from each PCR tube with a dye that will attach to DNA and fluoresce under UV light, and ran the combination on a gel, thus allowing us to see whether DNA was amplified:
Below is a part of the gel showing the 28S PCR products from the amplification done by Tom and Shannon (on the left) and Mamo and Julia (on the right):
The first eight bands are all good, and indicate that we successfully amplified the 28S gene for those beetles. The ninth spot is dark as that is the negative control – we did everything for that PCR as we did for other PCRs, but no DNA was purposely added. If there had been a band there, it would suggest some DNA accidentally got into the chemicals, which would be bad. The next eight spots are the same, but for a different set of beetles, and the last spot is another negative control for that second reaction.
Here’s John explaining that the 28S reactions were quite successful!
We also did PCRs for the COI gene during the same sessions. Two genes (28S and COI) for 36 beetles combined mean that the students will have produced a total of 72 DNA sequences. The next step is getting the PCR products ready to be sequenced, which I will report on in a future blog post…
There are a number of subgroups within Bembidion subgenus Trepanedoris whose structure of gene flow and species boundaries are not understood. The morphological data indicates several forms within these subgroups, but whether this variation is indicative of separate species is not yet clear. One such subgroup, and a main focus of the Discovering Insect Species course, is the Bembidion acutifrons subgroup. In this subgroup, Bembidion canadianum is fairly distinctive (more on that later), but the rest of the subgroup is a bit more confusing.
Of particular interest in our class’s research is the variation in what is now called Bembidion acutifrons. There are two rather distinctive forms of Bembidion acutifrons: an eastern form with darker and generally larger adults, and very shiny males, which look like this:
This shiny form is what we were finding at Klamath Marsh. It is also the form found at the type locality of Bembidion acutifrons, Alamosa, Colorado. Here’s a picture of Alamosa National Wildlife Refuge, where we found shiny B. acutifrons in 2013:
However, west of the Cascades in Oregon and Washington is a form that is generally smaller, often paler, and with males that are heavily microsculptured and thus dull. The following two pictures shows a comparison of the left elytron between a shiny male (above) and a dull male (below).
We haven’t compared the genitalia of these two forms yet, and we have very limited DNA data. So far we only have sequences from one specimen from Saskatchewan (a shiny male) and one from Corvallis, Oregon (a dull male). These two specimens are less than 1% different in the genes COI, CAD, and topoisomerase I, but they do differ by three bases in the sequenced portion of 28S.
We have additional specimens from Colorado, Utah, and Klamath Marsh, OR, of the shiny form ready to be sequenced, and two more specimens of the dull form from Corvallis. We are rather eager to see what these additional data might say! We also need to start comparing the internal sac sclerites of the genitalia.