PCR: Please Chug Rapidly

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Gel of the Day: FAIL

My new primers didn't work. At all.

Bleah.

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Gel of the Day: A Horny Little Bug That's Pissing Me Off

Just for the record, I would like it known that the 3-4mm long diaperine beetles lumped together into the species Neomida bicornis are stinky stupid-heads and I don't want to play with them anymore.

What can I say after that deeply professional and scientific appraisal? Well, I can offer up this Gel of the Day as evidence of my contentions:

The first two lanes are 28S and 5.8S nuclear ribosomal subunits and the third is cox1 from Neomida bicornis. The last three are the same genes from a positive control, Bolitotherus cornutus. Everything else is, of course, the same. The same aliquots of primers, the same Taq polymerase, the same dNTPs, the same thermocycler protocol.

Now, if this were the first time I'd gotten a result like this, it would be no problem. And, in fact, it wasn't a problem four attempts ago. This gel is my fifth attempt at getting these sequences! I've tried tweaking everything I can think of with my existing protocol, but nothing changes the result. My next step will be to use a forensic DNA extraction and amplification kit, which I've been trying not to do because of the expense involved.

If not for the fact that I have some evidence that N. bicornis is more than one species, I would have given up on this by now. The thing is, it's worth it to me because part of my research has to do with the evolution of host specificity and I've collected this beetle from four different fungi now. Not only is the coloration different in these populations, but there are more subtle morphological differences as well (for example, the proportions of the tarsomeres of the metatarsi). I've also used the large subunit sequences from two populations — one from Ganoderma lucidum in Florida and one from Pycnoporus cinnibarinus in Massachusetts — in a molecular phylogenetic analysis and guess what... they turn out to fall into two separate clades with 99% bootstrap support in a neighbor-joining tree after 500 replications. In other words, they're taxonomically classified as a single species, but considering how slowly this sequence evolves it's highly unlikely that they really are. I need to get the more rapidly-evolving cox1 sequence to clinch this, along with analyzing a few more populations and I can't get the damned thing.

What a tease.

I mean, just look...

If I had hair, I'd be pulling it out right now. I can take or leave the small subunit, but I must have the cox1 sequence.

OK, that's it, "Neomida bicornis," if that is your real name. You've pushed me too far. Next time you see me on the street, you'd better run. I'm gonna be packing Raid.

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Wachusett Wednesday Cancelled: Fall Semester, Florida Expedition and Shifting Priorities

Due to a backlog of lab work in the face of the upcoming semester and the fact that I'll be doing field work in Florida all next week, there will be no Wachusett Wednesday this week. As perfect as the weather is and as much as I will miss this part of my weekly routine, I must put my priorities elsewhere over the next few days.

It's hard to believe that summer has come and gone so quickly, but the fact is that this is the last week before the fall semester begins. I will once again be teaching two lab sections for Introductory Biology this semester. As much as I enjoy teaching (although honesty compels me to admit that I'm not too fond of grading), this essentially means that there will be some combination of a reduction of my research time and late nights at the lab. The classroom portion of my teaching assistantship alone entails six hours each week. Add in grading, office hours, lab setup and miscellaneous issues that arise and a day disappears from my schedule. Additionally, I will be auditing an entomology course at another local university this semester; that adds up to about another six hours each week. It's going to be another very busy semester. With this in mind, I'm trying to spend the rest of this week tying up a bunch of loose ends.

A week from today, of course, I leave for the North Florida field expedition. The loss of my Wednesday on the mountain this week helps to relieve the pressure that a full week out of town will create. On the other hand, all of the planning for that trip has continued to come together very well. The biology faculty at Florida State has been exceedingly accommodating to this alumnus. My colleague and I have been furnished with laboratory space in the new biology building and the head of the university's herbarium has allowed us use of the dehydration equipment for the preservation of specimens. I still need to set up temporary accounts at FSU so that we'll have computer access from the lab.

Florida's DEP and parks management have also been incredibly helpful. In fact, I feel compelled to write a thank you to state biologist Harold Mitchell here. He has been a tremendous help in making the upcoming trip possible and interacting with him has been a pleasure. Moreover, the park managers at both Torreya and Lake Talquin State Parks have likewise been kind enough to provide my colleague and I with free and open access to their land anytime we wish, even going so far as to give us the combinations to open the gates to our sites before regular hours so that we can start as early as we like in order to beat the heat as much as possible in the Big Bend/Panhandle in early September.

Wachusett Wednesday has become one of this blog's most popular recurring features, so to readers who will miss it this week, all I can say is that it's a trade off. Wait until you see what's popping up in Florida this time of year. With the recent and unfortunate stormy weather, I expect that you'll be seeing a bumper crop of amazing and beautiful fungi in this space in the coming week.

On the other hand, Gel of the Day fans (because some people just can't get enough electrophoresis) will get a second, and quite possibly third, installment this week as I attempt to sequence a Ganoderma in the G. lucidum complex whose morphology alone hasn't been enough to provide a positive identification. I also intend to do the same with a sap beetle (Nitidulidae) collected earlier from Laetiporus sulphureus; while not in my core family, I've noticed something interesting about its foot that makes me think that having molecular data from these insects will be informative about one of the evolutionary hypotheses in my study. Markedly reduced fourth tarsomere, anyone? I have so far been unable to adequately resolve the genus of this beetle, though, and genetic sequencing should clarify the picture. Agarose lovers, rejoice!

In the words of Willie Wonka, "So much time, so little to do! Strike that. Reverse it."

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Gel of the Day: What the Heck Happened Here?

Sometimes, a perfectly good gel gets funkified in the process of handling. I think that's what happened with this gel, which was otherwise indicative of a near-perfect PCR:
The specimens in this case were three adult and one larval Bolitotherus cornutus which I collected at the MSA foray in Pennsylvania on August 10. The last eight lanes, 25-32, are from the larva. I haven't successfully sequenced a larva before; maybe my dissection technique is improving. The cox1 bands (31) look a bit weak, but the product should do for sequencing.

If you're familiar with electrophoresis gels or have at least been peeking at previous gels of the day, you're probably asking about now, "Mike, what the heck happened to lanes 21, 22 and 27?"

Well, I'll tell you. In order to take this photo using a UV light, I have to remove the gel from the casting tray, put it in a weigh boat and take it to a dark room upstairs from my lab. In the process of transferring the gel from tray to boat to camera, it gets a little scrape over an edge or two. This time, for whatever reason, that caused the bands in three lanes to get smeared. Nonetheless, there are bands there and since they are duplicates from two different dilutions (as far as specimen and gene go, 21=17, 22=18 and 27=31), it's not a problem.

Also, every fourth well is a control; there shouldn't be anything there. There seems to be a very faint band in lane 4, but it's nothing to worry about. I may just try and sequence it to see what the contamination is, since the position of that band is unlike that of any of the legitimate bands on this gel. I suspect that the culprit is a dirty microtube; if my primers, buffers, etc. had been contaminated, I would expect to see that phantom band in all of the control lanes.

The reason I'm running another batch of B. cornutus is that these are the first specimens I've collected from a site outside of Massachusetts. It's worth looking at them just to see if there are any polymorphisms present in introns in the sequences I'm analyzing.

I also read a very interesting paper today:

Leschen RAB and TR Buckley. 2007. Multistate characters and diet shifts: evolution of Erotylidae (Coleoptera). Systematic Biology 56(1):97-112. DOI: 10.1080/10635150701211844.

It's very much relevant to my own research. In this paper, Leschen and Buckley analyze the evolution of Erotylidae, a family of beetles only distantly related to the family I'm studying, and link their hyperdiversity to a shift to a diet of macrofungi. It sounds rather familiar, no? It's a very interesting read if you're into this sort of thing. It's based on morphology, not molecular biology, and finds a good link between diet and the external reproductive structure (gonocoxa and ovipositor) of Coleopteran females. Mycologists (at least those with an interest in basidiomycetes) and coleopterists alike will undoubtedly find something of interest in the paper. Enjoy.

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Gel of the Day: Half a Forked Fungus Beetle is Better Than None

Not a particularly successful PCR run today. For some reason, the ribosomal small subunit DNA didn't get amplified. I suspect that either I may have used an incorrect primer by mistake or else one of the pair may have gone bad. It happens. Luckily, I still have plenty of stock DNA and can have another crack at it. At least I got the large subunit and one of the cox1 genes from all of the specimens (the last eight wells are just a negative control; it's a good thing there's nothing there). I got nucLSU DNA, so it must be an error on my part that explains why the nucSSU didn't show up.

Oddly, the last two wells were taken from a 1:100 dilution and smeared; the previous two bands from a 1:50 dilution prepared from the same specimen didn't. Smearing usually indicates an excess of product. I'm not sure why that would have happened in this case. It's a duplicate, anyhow, so that's not important, just a bit strange.

The first sixteen wells in this gel are local specimens, collected at Wachusett Mountain. The third set of eight are from one of the specimens I picked up during the MSA foray in Pennsylvania last week. I have three more of those to sequence, too, once I get everything straight with these three.

As for tomorrow's field work, any B. cornutus I find will be shipped off live to Louisiana State for investigation of fungal symbionts in their gut. I don't really need to sequence anymore of them for the time being. Hopefully, I'll get lucky and find species other than this one and Diaperis maculata tomorrow. Those two seem to be dominant around here, but I'm going to try looking in a couple of different niches and see what turns up.

The North Florida field trip is two weeks from tomorrow. I know there will be more diversity down there, but I'd still like to come up with a few more local tenebs before winter sets in.

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Gel of the Day: Meat the Beetles!

This Gel of the Day comes with a dedication to the unnamed UC Santa Cruz researcher whose car was blown up on Saturday by fruitcakes who want to liberate Drosophila.

The Fab Four in this case are four specimens of Diaperis maculata; John, Paul and George were collected together from a decomposing Piptoporus betulinus fruiting body. Ringo joined them later, having been recovered from a Ganoderma applanatum sporocarp. In each of the bosex, the first four lanes represent a 1:50 dilution and the second four a 1:100 dilution of the initial DNA solution. Lanes 1, 5, 9, 13, 17, 21, 25 and 29 are the nucLSU; 2, 6, 10, 14, 18, 22, 26 and 30 are nucSSU; 3, 7, 11, 15, 19, 23, 27 and 31 are cox1 amplified using the general Coleopteran PAT/JER primer set; 4, 8, 12, 16, 20, 24, 28 and 32 were an attempt to amplify cox1 using a custom-designed primer set that works on the other Tenebrionidae on which I've used it but apparently not on D. maculata. This is the same result I got last when I tried a competitive test of the cox1 primer sets on several species which also included another member of the same subfamily.

This might be a useful phylogenetic signal; it could very well indicate a mutation in the binding site for the new primer set which is conserved in all the other Tenebs for which I could find sequences (I could find none for D. maculata when I designed the primers). Moreover, an alignment from my previous attempt came up with an insertion and two transitions for D. maculata at a binding site that wasn't present in the other tenebs. I can't tell yet whether this is a species-specific situation or if the mutations are present in all members of the genus. I'm hoping to collect specimens of D. maculata' cousin, D. nigronotata, in Florida next month. If I can get sequences out of it, I will be able to say that much more about the evolution of genus Diaperis and perhaps about tribe Diaperini as well. That is, if Big Coleopterists don't Expel me over raising the slight possibility that the tribe might not be monophyletic and if no wackadoos with IEDs decide to set me on fire in my sleep.

Have I mentioned lately how cool it is to find something that nobody (as far as I can find in the literature) has seen before? This is the good stuff right here. This is why I get up in the morning and slave over a hot thermocycler.

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Gel of the Day: Sarcodon what?

You might recall that I've twice now collected what I suspect to be a rarely-collected fungus, Sarcodon underwoodii, in the past few weeks. The first collection was made inside Worcester city limits on July 11 and the second time from the Wachusett State Reservation on July 17. I should mention that on my last trip to Wachusett all of the prospective S. underwoodii was gone and the more common S. imbricatum has replaced it entirely. The latter fungus has quite different coloration, the background being a red-brown instead of S. underwoodii's buff.

I've been trying to get enough DNA out of my collections to sequence and so make a positive identification so that I can let the people who manage the land on which I've made these collections know that they have an unusual species present. I tried earlier this week and got nothing. The context of the fungus is loaded with pigments that interfere with DNA amplification. I took a second pass at the problem, though, and let my samples digest in proteinase K for more than two hours during extraction. That seems to have done the trick. I've got PCR product galore and expect to sequence the nuclear ribosomal large subunit and ITS early next week. Here's the gel:


Lanes 3, 5 and 6 are from the fruiting body collected on July 11 and 7 through 12 are from the Wachusett specimen.

By middle of next week, I should know whether both of these really are S. underwoodii and increase what's known about the ecology of the two areas from which I collected them just a tiny bit.

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Gel of the Day: Moderate Success

Sound the trumpets! I actually got some PCR products out of today's experiment with COI primers.

It looks from the results like both primer sets work, although my new Tenebrionidae-specific primers (PATten and JERten) amplifies a smaller fragment than the traditional Coleoptera COI primers. Also, it seems that the traditional primer set worked for one species but not for another in the same tribe (Diaperini), and the new primers worked for the other member of that tribe (both are Diaperini; the new primers worked on Pentaphyllus but not Diaperis and the old ones on Diaperis but not Pentaphyllus). Why? I dunno. I still have my suspicions about how monophyletic tribes, and even subfamilies, really are in this family, and maybe that's part of what I'm seeing in these results. I may know more once I try aligning these sequences and especially when I add this data together with the large and small subunit stuff.

Otherwise, both primer sets worked on everything except one miss by the new set on a single Bolitotherus cornutus, but it worked on the other specimens of the same species, so I really have no idea with that one.

Still, here's the gel. There are a couple of faint bands in the lower half that may be hard to see in the reduced photo, but they're in there. Hopefully I'll still be able to get sequences out of those:

I used several different dilutions (1:100, 1:10, 1:5) and got some products from all of them. It all comes down to using a lower annealing temperature for the COI stuff. From here on in, I should be able to get those sequences reliably.

Now I'm just hanging around and waiting for my sequencing reactions to finish up, which they should do in another 2.5 hours. Thankfully, that Del Tenney DVD I have is a double feature; I can watch Curse of the Living Corpse while I'm waiting. I'll be here late on a Sunday, but I should be ready to go when tomorrow's sequencer run starts up at 11:00 AM. Go, me.

If anybody wants me, I'll be in the conference room.

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Gel of the Day: Mystery Mushroom

One of my lablings pointed out a cluster of mushrooms growing near the loading dock attached to our building the other day. Despite macroscopic and microscopic examination, we've been unable to identify it satisfactorily even to genus. The fact that it wasn't producing enough spores to obtain a print didn't help, and the few spores (fewer than two dozen) that I could find on the lamellae all had the same shape but their size and color varied. Some are colorless (hyaline), some are brown.

The gel above shows PCR products amplified from a small bit of the mushroom after drying. The first two bands are ITS and the second two are large subunit rDNA. Now that I have DNA, I can get the sequence and match it to known sequences. If anyone has sequenced this fungus before, we can identify it via molecular biology. If there's no exact match, the game still isn't over. Thanks to evolution, the sequences will still be closer to some fungi than others. If need be, I can do a quick alignment of similar sequences and then use phylogenetic software to determine to what the specimen is closest. With that much information, there's a very good chance it will be identified to species.

Next time someone tells you that they don't know anything practical that's based on evolutionary principles, ask them how they would have solved a problem like this if the fungus were eating the beams that support their house.

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Gel of the Day: Ambitious but Only Slightly Successful

I was feeling a bit ambitious today; my new COI primers arrived. They didn't work, but I did try them. Amplifying COI is turning out to be a bit tricky. I'm going to have to do some digging on this problem.

The mystery COIs from last time are still more or less a mystery... but they are at least Coleoptera and they are cytochrome oxidases. That much is good. There's nothing with better than 86% identity to them on GenBank, though, including what is supposed to be a Penthe species; I have my doubts as to whether that sequence is actually from that genus.

Anyhow, today's gel:


007 is my putative Pentaphyllus. I tried to amplify lsu and ssu from it. Didn't work. Neither did the same genes from Penthe pimelia (004). 009 and 010 are a pair of Bolitotherus cornutus; I got lsu and ssu from them but no COI. Why? I don't know. For some reason, I can't get any ribosomal sequences out of 007 and 004, only COI... and I can't get COI out of B. cornutus, even though they should all have the same conserved sequences. 015 is a new addition, Diaperis maculatus, a teneb from the subfamily Diaperinae. I only got the ribosomal sequences out of that one, too. Why? I don't know.

I'm neither entirely happy nor entirely unhappy with these products. I at least get to add some more data to my set, and that's something, anyhow. I'll clean and sequence these on Monday and chuck 'em into my alignment. I do wish I had gotten the Penthe and Pentaphyllus rDNA, though. It would have cleared up a couple of questions I still have about these two insects. I may be able to do another extraction from Penthe; it's a big bug and there's still enough left of my one specimen to try one more time. My Pentaphyllus stock is used up, though. They're tiny insects, 3-4 mm long, and I can only use heads and pronota for extraction to avoid contamination by gut symbionts. It's a bit like trying to suck the juice out of a pinhead.

Think I'll go have a nice Scotch now.

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Gel of the Day: ...and Then a Miracle Happens

If you're the kind of person who reads this blog then you have probably seen the cartoon with two physicists standing in front of a chalkboard. In the middle of a bunch of equations are the words "and then a miracle occurs" and one of the physicist is saying "I think you should be more explicit in step 2."

Today I had a similar thing happen with one of my gels:


The first two are DNA from taxa that didn't come out last time I tried. The last group of four is a positive control. I used stock DNA and the same primers for all. Now, here's the really weird part: lanes 4 and 8 shouldn't have a product in them at all. No bands should be there. They're the products of a cytochrome oxidase I primer that I discovered today should not work. The primer I was given is for the wrong beetle family; the one I'm studying is too divergent. I was just investigating the problem this morning, in fact, right after I put these samples in the thermocycler. I wound up designing two new Tenebrionidae-specific COI primers and sending an email off to two of my committee members telling them that I'd done so.

So, of course, the primers that have never worked before, and shouldn't have worked this time, are the only ones that produced bands in the first two taxa... but NOT in the positive control. The only band I got in that one is for the small subunit. I should have one for the large subunit as well. It worked last time using the same dilution and primers.

I would just like to say "Argh." Really, what else can I say?

Now, it may be that I used the wrong primers. I've been very out of sorts after not getting enough sleep last night and I could have screwed up. Still, the way I load my tubes, I should have gotten the same result in the positive control (#001) if that were the case. But I didn't.

Tomorrow, I'll clean the PCR products and get them ready for sequencing using the old COI primers. My bet is that I'll get back nothing. And then I'll just start all over again. Maybe someone tampered with the annealing temperature on the thermocycler or something.

I dunno.

I need a drink.

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Gel of the Day: Epic Fail!

I hate it when I don't get PCR products on a Friday afternoon. I especially hate it when one of the attempts was extraction from a species of which I have but a single specimen. Blah.


#007 is Pentaphyllus sp. and #004 is Penthe pimelia. The latter was going to be an outgroup useful for rooting trees. What I've got is pretty useless, though, because it's nothing. As for Pentaphyllus, it looks like I got my small subunit product but nothing else. I'll take it and try the whole thing again over the weekend, I suppose. Or on Monday. I'm feeling a bit frustrated with the whole thing at the moment. I've got the COI and wingless dilutions down to 1:5, too.

Stupid bugs. How am I going to work out your phylogeny if you won't give me nice big globs of amplifiable DNA?

By the way, something about this blog and why I write about stuff like this. It occurred to me some time ago that there are a lot of people out there who have no idea what it's like doing research and who buy into the whole "big science" bullderdinky (like it? I just made it up.) There's a tendency to think that science is like what gets shown on TV and movies. Everything is gleaming and shiny and digital, hunches always pan out and the experiment always works. In real life that just ain't the way it is.

There are lots of blogs out there that review papers and deal with big issues in science. I like the idea of putting forth what life is like, how the successes and failures feel, and generally getting the point across that we science nerds are just like everybody else and that if there's anything like a "big science" conspiracy it has entirely passed by me and everyone I know.

Today? Epic fail. Tomorrow? Maybe not so epic fail. Maybe even success. In the meantime, I still need to go grocery shopping and, if I'm feeling up to it, do the dishes. See how that works?

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Gel of the Day

Some of my readers seem to enjoy looking at pictures of gels. Here's the gel of the day. It shows PCR product from the lsu rDNA and ITS regions of unidentified polypore 061808-C, which looks a bit like Polyporus admirabilis but was growing on coniferous wood instead of a fruit tree wound:


Hyphoid Logic: The Excitement Never Ends and the Fungus is Just Beginning!™

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