A nematode-filled Bembidion canadianum

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:

V100900.Alive2

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:

V100900.Open

Here’s what part of the abdomen looked like; all of the little white tubes here are nematodes:

V100900.Open.Abdomen

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!

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Discovering Insect Species: preparing beetle DNA

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.

Extracting the DNA

We start out with a bit of a beetle in a tube.  This is what it looks like:

A bit of a Bembidion in a tube.  This doesn't happen to be a Trepanedoris, but it would look just about the same if it were.

A bit of a Bembidion in a tube. This doesn’t happen to be a Trepanedoris, but it would look just about the same if it were.

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:

JuliaDNAExtraction ElleDNAExtraction

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:

finalDNAextraction

The students extracted DNA from a total of 36 specimens, most of which they had collected on our Klamath Marsh field trip.

Amplifying a gene we want to study

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:

preparing 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:

ThermalCycler

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:

PCR product

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:

JohnLoadingGel

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):

gel

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!

JohnExplainingGel

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…

Posted in Revising Bembidiina, Uncategorized, Z499 (Discovering Insect Species) | Tagged , , , | 1 Comment

The Bembidion acutifrons story

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:

V100898.Habitus.Scale2.Cleaned

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:

13.092

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).

Left elytron of a shiny male, from Klamath Marsh, OR

Left elytron of a shiny male, from Klamath Marsh, OR

Left elytron of a dull male, from Corvallis, OR

Left elytron of a dull male, from Corvallis, OR

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.

Posted in Fieldwork, Revising Bembidiina, Taxonomic Process, Z499 (Discovering Insect Species) | Tagged , | 3 Comments

The oak tree grows pretty close to where the acorns dropped

Here’s my mom, at age 82, collecting some of what will be the type series of a new species of Bembidion from Jasper National Park in Canada.  This was in 2011, during a great trip she and I made around Saskatchewan and Alberta.  We collected during the day, and stayed at bed and breakfasts or nice hotels in the evening.

LouiseCollecting

 

 

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