A Few Irreligious Thoughts to Ponder

You've probably heard by now about a certain presumptuous jackass of a columnist named Jim Griffith who writes for the Newnan, Georgia Times-Herald. Griffith has achieved 15 minutes of Internet infamy by penning a column entitled "A few religious thoughts to ponder." In it, he explains how terrible Thanksgiving is for atheists:

Thanksgiving must be a terrible time for atheists. They have no God to thank.

They do not have the privilege of gathering with family and friends to express gratitude by saying: "Praise God from whom all blessings flow." An atheist on his deathbed faces serious uncertainties. Gazing upward, he pleads: "Oh God, if there is a God, please save my soul -- if I have one."

The column was found out and replied to by a series of atheists objecting to such nonsense and, after less than two days, the newspaper saw fit to close the comments so that no more respondents could rake Griffith over the coals for being, as I said, a presumptuous jackass. I've checked other items in the newspaper, including other columns by Griffith, and none of them have had their comments closed after many days or weeks. Just this one. Apparently, the Newnan Times-Herald didn't want too many atheists to have the chance to debunk Griffith's garbage by mentioning how they celebrate Thanksgiving — at least not in a forum that locals were likely to see. It just won't do for a newspaper to report inadvertently that atheists celebrate the holiday in much the same way that non-atheists do and that the atheists are, after all, human beings with families, affections and gratitude. At least, it won't do for the small-time newspaper to reveal these truths to the residents of Newnan, GA.

As a result, those of us who didn't have the chance to respond to Jim Griffith's "few thoughts," which I suspect are a very few, at the paper's website during the 36 hours in which the opportunity to do so was available will have to do it elsewhere. I'll do it here.

I have a lot of things for which to be thankful this year, and not a single iota of it requires or has anything to do with any deities whatsoever.

Like many people across America, regardless of their beliefs, I will be joining my family to celebrate the holiday. LL and I will be leaving within the next couple of hours to head to north central Pennsylvania, just as we did last year. You may not see it readily, but there's a lot of gratitude embodied in that last sentence.

First off, I'm thankful for having gotten through another year during which a blindly religious president mismanaged the country in which I live into numerous hardships. I survived that year despite the existence of religious radicals in other countries who would like to see people like me dead... and when I say "people like me," I mean Americans. They want to inflict pain upon us because their religious fundamentalism calls for violence against people with whom they disagree. I'm thankful that this faith-stuffed anti-intellectual will soon be a former president and that his replacement might actually turn out to be a person of intelligence and ability who could wind up improving things, even if only a little. 2008 hasn't been a very good year for my country and I'm glad it's almost over. I'm thankful to the 7,000,000 people, too, who may well have changed the course of history during the last election. We shall see what really happens in the months and years ahead, but I'm thankful for at least having the hope that it won't be the same as what's gone before.

I'm thankful that I'm getting to spend another Thanksgiving with my sister, niece and brother-in-law. My sister and I had no contact at all for more than a decade, and this was in no small part due to religious belief. My parents found much to hate about the world and about differences of opinion and about people who are different from them thanks to their faith, you see, and they disowned first me in part because of that faith. They told my sister that I was a drug addict, that I was in jail and probably dead, to make as sure as they could that she wouldn't try to find me. When I finally tracked her down, I learned that she had been similarly disowned for the grievous sin of falling in love with someone of a different faith than that of our parents — even though neither my sister nor he practiced any religion at all. When they got married, nobody from my family came to the wedding other than myself. I gave the bride away. Tomorrow, we'll celebrate Thanksgiving together, along with my rather precocious niece and the rest of our non-traditional clan, none of whom are particularly religious and several of whom are outright non-believers. How could I not be grateful for this? The only involvement of a deity here, at least the imagining of one, was to drive people apart.

LL is coming with me, of course. This year, we celebrated our twelfth anniversary together. LL comes from a country in which religious differences led to a generation-long civil war in which members of her family and friends of her family died in terrible and inhumane ways. Her cousin, for example, was detained by a Syrian-backed militia, hauled away to Syria, and tortured for years. She spent long stretches of time huddled with her family in an underground parking garage hoping to avoid being shot or blown up by Syrians and Israelis and their proxies — all on the basis of ancient religious divisions. Survive it she did, though, and this year she became a US citizen and voted in an election for the first time in her entire life. I am thankful that LL is here with me, relatively safe and about to enjoy the holiday. I'm thankful that she survived a long and terrible war, that she made it to the US, and that she's stuck by me all these years, through the best of times and the not-so-good.

I'm thankful to the university I attend and to the people there who give of their time and knowledge as I grind away at earning a graduate degree. There was a time in my life, not so long ago, during which I never thought that I could get this far. I worked hard and here I am, but the opportunity had to be offered by others, too. I have that opportunity and am grateful for it.

I'm thankful that Tycho, a lizard who has practically been family, has survived another year, despite last year's unwarranted death sentence. He's sitting near my shoulder as I write this, probably wondering when breakfast is coming. Alas, the weather is too cold for Tycho to make the trip to Pennsylvania with us this year. He's going to be rather bored with no humans to entertain him for the four days we'll be gone. Tycho has worked out a lot of things in his long lizard lifetime, but how to work the TV remote isn't one of them. I expect that he'll spend much of his time asleep. In any case, he's exceeded the average lifetime of a member of his species in captivity by nearly a third now and is still in good health. Thank you, Tycho, for sticking around for so long. There will be lobster roaches and macaroni and cheese when we return. I suppose someone could argue that some deity is allowing Tycho to go on living. You know, Lizard-Jesus or HerpAllah or something. That would be a very childish person, but I'm sure someone who will read this will have the thought cross their mind.

Which brings me to another bunch for which I'm thankful, and that is the many people who have fought and are still fighting to reverse the ever-threatening flood of irrationality that might otherwise engulf us all and snuff out the light of the Enlightenment. They keep us progressing, sometimes at great cost. They're the ones who hope to remove the motivations for these religious wars and faith-based familial decay, who object to and dissect and defeat all the magical thinking that is only a hair's breadth away from complete ascendancy at times. I'm grateful to the teachers, the scientists, the historians, the living human memory-banks who remember the past and dedicate themselves to our not repeating it.

There is more — much more — but I have to get ready now to hit the road. It's a long drive from here to there. All things considered, I'm thankful to be making that six hour schlep today.

And you know what, Jim Griffith of the Newnan, Georgia Times-Herald? I'm even grateful to you today. You've given me one more reminder of how low we humans can sink if we decide to measure the worth of others with yardsticks designed and built of our own narrow views. You're a good cautionary example of just how wrong we can be, and thus how wrong whole societies can go, when our eyes get so full of our own self-esteem that we place ourselves in judgment of the rest of the world based on nothing but the desire to be thankful to anything other than that little fraction of humanity it is our privilege, and sometimes our burden, to actually know.

Like the majority of Americans, I'll be celebrating Thanksgiving with a ridiculously large meal on Thursday evening. Like those millions of other Americans, there's something else I'll be doing early on Friday morning. It's then that I will be thinking most of Mr. Griffith.

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Gel of the Day: FAIL

My new primers didn't work. At all.

Bleah.

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Yet More Grading

Have I mentioned lately how much I dislike grading?

Having just finished the term papers for the lab section I teach, I'm now grading exams. I drew what is apparently a more difficult couple of problems this time around. I don't personally think they're too tough; it's just a two-parter asking students to write out the genotypes of a couple of fruit flies and then use a Punnet square to predict the genotypes, phenotypes and genders of their offspring.

There are a lot of very strange fruitflies being born from this mating, though. Extra chromosomes keep cropping up in some, whereas others are males lacking a Y chromosome. Still others are lacking genes for one of the two traits involved.

I dislike grading because I don't like to be a bearer of bad news, and there are a lot of students who are going to be getting some bad news when these exams are handed back in a few days. Of course, I also dislike grading because it keeps me out of the lab. I've had to order some new primers and they won't come until tomorrow, so that's not as big a deal at the moment. My goal, though, is to get these exam responses done with today so that I can at least do a little bit of bench work tomorrow in between my other commitments for the day.

I plan on once more going after the cox1 gene from Neomida bicornis with the new primers. That, however, is a story for another day. For now... its back to grading.

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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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Well, Hello There

And this is what I look like while I'm dissecting beetles. My adviser thinks it's a "good look for me." Am I a geek yet?

Yeah, yeah.

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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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A Last Excellent Day at MSA

Today was my last day at the MSA conference. There's still one more day left, but for a variety of reasons I have to get up early tomorrow and make the long drive back to Worcester. Not the least of these is the fact that a petty, bureaucratic,politically-motivated dean at Florida State University has taken it upon himself to put up a massive and ridiculous roadblock in the way of LL's quest for a PhD. I won't get into the details; she'll write about it when and if she's ready. All I'll say is that I've never heard of such a moronic, pig-headed thing being done to someone who has been one of the few bright lights for a department that is now being dissolved largely because of the incompetence of another pig-headed bureaucrat in academic clothing. With people like these deans at the helm, I would have to advise anyone considering a PhD program at Florida State to put it as low on their list of possibilities as they can possibly can. If it were a boat, it would have the word "Titanic" painted at the prow.

Other than learning of LL being screwed over yet again, though, today was the kind of day that makes me regret having to leave a day early. Two lablings gave excellent presentations on their research as did my advisor. I take a good deal of pride in being part of this group even if I haven't begun contributing as much as I hope to... soon.

I had lunch today with Joey Spatafora and my advisor and so had the chance to listen in and/or talk about everything from plans for AFTOL2 to Wall-E and The Fantastic Planet. I learned that Joey reads this blog when he recognized me upon our introduction as Mike O'Risal. So did one of the postdocs from his lab. In fact, no fewer than four people I met today already knew me by my nom d'blog. I had best be careful not to mention my rampant crack addiction and penchant for devouring live kittens here. Someone might be looking.

Today was also the day of the Karling Lecture, delivered by Nick Talbot of the University of Exeter. The topic was "Investigating the biology of plant infection by the rice blast fungus Magnaporthe grisea using functional genomics." The rice blast fungus is a problem right up there with the potential of Puccinia graminis Ug99, wiping out up to 30% of the world's rice crops in any given year. Dr. Talbot's work on the mechanisms by which the fungus initiates and spreads infection seems to hold tremendous promise in addressing the problem. Some of the proteomics involved are well beyond the scope of my understanding; I'm not a proteins person by any stretch. Still, even I can grasp that if one can come to an understanding of how the subcellular mechanics of infection work, there's real hope of coming up with a solution.

After the Karling lecture, I met up with Meredith Blackwell and handed her three dead beetles. That might be perceived as an insult in some circles, but not in this one. I'll be sending some live specimens her way in the near future, too, and hopefully we'll be able to exchange some data and materials. I'm particularly looking forward to continued contact with the people I've met from her lab. Even though I don't think I'll be part of the next round of AFTOL research, others in my lab will be and her lab will also be involved, and we're all working on bugs and fungus in one way or another.

I had dinner at a little Indian place called Kaarma just around the corner from my hotel. I was joined by labling RiGa, Dan Durall from the University of British Columbia Okanagan, and Kentaro Hosaka from the National Museum of Nature and Science of Japan. It was a very nice cap to what has been an inspiring few days for me. If you should find yourself in State College, PA, I recommend checking out Kaarma.

I have indeed come away from this conference feeling inspired. I've got some new ideas and am itching to try them out, particularly when I get back from Florida laden with new specimens. Stephanie Gross and I talked today about there being a coleoptera/fungi symbiosis session at next year's MSA meeting. There are a bunch of us out there, and all we need to do is be in the same place at the same time with material to present. The place next year will be Snowbird in Utah and the time will be July 25-30, 2009. Now all I need is the material.

I'll get back to work on that on Thursday.

For now, my priorities are sleep and then the long drive back to Worcester. Sequences await.

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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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Amplify Naked: In the Lab on a Sunday

I'm here at the lab this afternoon. See?

You can tell it's a lab because there are microscopes and other sciencey-looking things in the picture.

It being Sunday, I'm the only one in the lab, which is a bit lonely but also very nice. During the week, we have an undergrad here on a summer project in which he's working under the supervision of a post-doc to sequence a whole bunch of Boletales specimens from another university to make sure that they're actually what they're supposed to be. Most type specimens were collected before the days of molecular analysis and so in many cases nobody is as sure of their identities as they could be. It's a worthwhile project, but with everybody else doing their molecular thing, too, the clean lab is often at capacity. I have a sizable experiment I'm working on, testing a couple of primer sets for my so far-elusive cytochrome oxidase subunit 1 genes under a new protocol on the thermocycler. It's not that complicated a project, really, but getting it all set up requires a bit more physical room than is usually available. I'm just now waiting for the cycler to finish before I run a gel and hope that I actually get some bands. Then I'll be able to see if one of the primer set works better than the other, if both sets work well and the difficulty has been all about the annealing temperature, or if the whole thing just isn't going to work for some reason. I suspect the combination of a new, family-specific primer set and a different thermocycler regimen will solve the problem. We'll see.

Because there's nobody else here today, though, I also got to watch an old Del Tenney drive-in flick after getting the thermocycler set up. I watched the amazingly goofy Horror of Party Beach in a conference room. There's something particularly gratifying about kicking one's feet up and watching a cheezy flick about radioactive jellyfish-zombie-monsters that eat only nubile young beach party girls and the scientists trying to devise ways to destroy them when one is sitting in a biological science center and waiting for PCR products to be amplified. You can always tell the scientist in a 1960's rubber suit horror flick; he's the guy in the white coat, smoking a pipe and saying things like, "The organs of these beings are too decomposed to retain oxygen so they've been replaced by sea anemones. They're giant protozoa!" Errrr... yeah. I hope I don't give an answer like that when I take my quals.

I did want to get this experiment run today for another reason, too. With LL coming home, I won't be here most of next week. If I get everything ready for the sequencer by the end of the day today, I can have it on a plate tomorrow and get my sequences back on Tuesday. Or I can have it ready for the plate on Tuesday and then download them and toss them into an alignment next Monday. Either way works, really.

34 more minutes left on the thermocycler now. You know, if I'd have known I'd be the only person here today, I'd have amplified naked.

Nobody touch that line. It's going on a t-shirt.

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Florida Expedition Planning Update

Planning for September's North Florida field work has moved forward in the past couple of days. The most important development has been the securing of permission for the first field site, Eleanor Klapp-Phipps Park. I had the good fortune of connecting by phone with Chuck Goodheart, City of Tallahassee Parks and Recreation Manager, in order to discuss what we'll be doing. Chuck was enthusiastically supportive and the outcome of our conversation was that we've got permission to collect in the park and will, in turn, provide Parks and Recreation with a list of all of the fungi and Coleoptera that we identify to add to the department's knowledge of what lives on their land. As I mentioned to Chuck, it's the same arrangement I have with Massachusetts DCR. I'm more than happy to provide useful information to the people tasked with caring for natural areas. That's a big part of why I've chosen my career path in the first place.

Just across Meridian Road is Overstreet Park, part of Alfred B. Maclay Gardens State Park. I'm also trying to get a permit to collect specimens on that property, and that's turning out to be a bit more involved. I called the state DEP Division of Parks and Recreation to find out what I needed to do and the person I got on the phone told me that I should call the park managers and gave me a couple of phone numbers for Maclay and Torreya. I first called the number at Torreya and got voicemail, so I left a message.

Then I dialed the number for Maclay and got a human being. Thinking that I'd been given a phone number for someone who was responsible for making decisions about park usage, I informed that person about what our field work would entail. I received a flat rejection, "You can't bring anything into or take anything out of the park." I was surprised that this sort of rule would be applied to legitimate research, particularly after having been told something else by the state office, and said so. The person on the other end of the line, an older gentleman by the sound of it, simply repeated his previous reply word for word. It occurred to me at this point that I had probably been given the phone number for general inquiries rather than that for a park manager, and so I asked if I could speak to someone who made decisions about the park. The fellow I was speaking to told me that I could write a letter "if I wanted to" to manager Beth Weidner, and he gave me the address at the park office. Does anyone still write letters for this sort of thing? I asked him if Ms. Weidner had a phone number and email address at which I could contact her, but he said that he couldn't give me those. I'd just have to write a letter. Ummmm, OK... end of conversation, then.

I searched online and found an email address for Weidner in a state DEP domain, so I sent an email to that address. It bounced. Bad luck, that.

I was just about to try a phone number I'd found when my phone rang. It was the park manager at Torreya returning my call. He was more than happy to help me out and said it would be "no problem" to collect fungi and beetles for research purposes, but that he wasn't the one to issue a permit. That's the job of the District Biological Team Manager, John Bente. He gave phone numbers and email addresses for both Bente and his second-in-command. I've left a voicemail for Bente — he was out in the field at the time I called — and I hope to speak with him today. Assuming that Overstreet and Torreya are part of the same district, then, it would appear that Beth Weidner wasn't the right person to contact regarding our permit, anyhow. I'll take up Overstreet with Dr. Bente when we talk about Torreya.

Today, I'll dash off an email to my undergraduate research advisor at Florida State to ask about the use of lab space while we're down there. We're going to ship our gear to and from Florida rather than trying to check it in on the plane. While it's not hazardous material, I have no idea how a combination of dried fungi and solutions of phloxine B and 10% ammonia might interact with airport security. I'd rather not have someone contracted by Homeland Security going through my collections and such and asking lots of questions about the stuff. UPS will be our friend in this.

If all goes well, we should have everything lined up for the trip before the end of the month. Then it's off to the land of sweat and pygmy rattlers and incredible biodiversity. I can't wait to see what sorts of polypores prefer gopherwood and whether there are any endemic fungal or Coleopteran taxa in those unique little ravine ecosystems.

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Reasons to Be Cheerful: DNA, Expeditions and Mycological Society Meeting

It's the little things in life sometimes that bring the greatest pleasures... at least when one revels in being a geek.

The first thing I need to do today is to clean up some DNA I have waiting in the freezer. Among them are the rDNA sequences from Diaperis maculata. Once I've cleaned the DNA I'll be able to load it onto a plate and will get the sequences back and be able to add them to my alignments. I'll know a tiny bit more about the evolution of fungus-associated Coleoptera. This is enough to make me look forward to a Monday. It's the little things... and D. maculata is only 5-6 mm long. That qualifies as "little."

I also have to start work on securing permits for the collecting expedition in September. Two months should be enough lead time, hypothetically, but one never knows with bureaucracies. We just need a couple of good sites to work at, though. I'll probably put feelers out on more than we'll actually use. That way if the process gets too long and we get turned down for a couple of sites at the last minute, we'll still have a couple of back-ups. Better to have it and not need it than to need it and not have it.

Speaking of science-related travel, it's only a month until I hit the road for Penn State and the annual Mycological Society of America meeting. It's the largest gathering of professional and amateur mycologists in the nation and promises to be a great time. I'm looking forward to the whole program, but I have to admit that I'm particularly tickled by the opportunity to collect in Pennsylvania, a state in which I've never forayed before. I'm even hauling my industrial-strength dehydrator on the trip so that folks can preserve specimens. The abstracts look like there will be some very interesting talks on fungal evolution and informatics tools. Folks from my lab are doing four of the presentations, I think. I'm not one of them yet (I've just started gathering data!) There are also a couple of talks by David Maddison that look like they'll be particularly interesting stuff. Days of full immersion... I can hardly wait.

Are any readers planning to attend MSA this year? If so, I'd love to meet up for a drink or two.

If you're new to this stuff and this is the first you're hearing about it and you'd like to attend, you can still register for the meeting. Even if you're a novice, you couldn't ask for a better opportunity to learn about fungi. Please consider joining us! If you're a newbie and can make it in time for the foray on August 10, I'd be more than happy to hang out with you in the field and help you get better acquainted with the various chitinous life-forms you'll encounter. I'd consider it a privilege to do so, in fact.

Today, and the next couple of months, are going to be great. Wonder-Geek Powers Activate!

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Worcester-versary

Today marks exactly one year since LL and I arrived in Worcester, having made the journey here from Tallahassee. Just over a year ago, I wrote my last entry as a resident of Florida, and just under a year ago wrote the first one from Massachusetts.

Looking back on our first year in Worcester, I can't say that I've had any regrets about coming here. There are things I miss about Florida; there are things I miss about California, too. Still, I think it was a good move for us. Having grown up in the Northeast, the culture here is more comfortable for me than anywhere else in the country in which I've lived, even more so than the SF Bay Area. With all the challenges and frustrations of grad school, I've been fortunate enough to land in a lab with a bunch of good people who are more than willing to share their knowledge with me. LL has easily adjusted to life here as well; she has even remarked about our first winter here that it wasn't too bad. She's teaching at a college and will be working on an advisory committee to the city council soon, too. Both of our circumstances have worked out well in most ways.

We're marking the close of our first year in Worcester in somewhat inappropriate ways, which is only appropriate for the two of us. LL is still in Lebanon where her recuperation, I'm glad to report, is progressing unusually well. She's already up and about. We spoke via Skype yesterday afternoon; she'd been out clothes shopping only two days after her surgery. I'm putting together plans for a return trip to Florida, as I've already written.

I'll be spending this morning doing field work right in the city. I'll be joining labling Andy Wilson for an outing at Broadmeadow Brook to collect fungi and beetles, which I'll be taking back to the lab for identification and vouchering.

Tonight, there will be fireworks. Isn't that a nice way for Massachusetts to mark the anniversary? People think New Englanders are a bit cold and unfriendly, but my experience has been nothing like that. How many others can look forward to pyrotechnic displays in celebration of their first anniversary in a new home?

Thanks, Worcester, for a good first year. LL and I are looking forward to the beginning of an even better second one.

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Florida Expedition: September 3-10

Guess who's coming to Florida?

A colleague from Harvard and I will be in North Florida to sample fungi and beetles from the area between September 3 and 10. The magnanimous Diego/James F/todabrilla has consented to put us up at his place for the week, insuring us of good company as well as saving two itinerant and impoverished graduate students the cost of a hotel. With our flight from Boston to Tampa now confirmed and lodging assured, I'll next be turning to securing the permits required to do our collecting legally. Flying into Tampa means that we'll have to make a three hour drive to Tallahassee, but it also saved us a ton of money on the cost of travel.

While our days will be filled with collecting, identifying and preserving specimens, I'm looking forward to seeing some old acquaintances in the evenings as well. I'll need to check into whether I can get access to some resources at Florida State (like a couple of microscopes), and in any case I expect to visit the campus and see a couple of my professors as well as attending a meeting of the Union of Freethinking Students. I'm certain I'll also pass at least one evening at Aladdin's Sheesha Café.

North Florida is one of the country's hottest biodiversity hotspots, renowned for its unique ecosystems and endemic species. Even though I spent five years investigating them on my own, it will be a whole new experience seeing them through eyes that have learned a great deal more about fungi and Coleoptera over the course of the past year. I must admit that it will be good to see the many venomous snakes that make field work in the area that much more exciting, too.

I could do without the Confederate flags, of course, but one doesn't see too many in the swamps and forests, anyhow.

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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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I Can Haz PCR Produkt

In rather happier and more personal news, I finally got PCR product out of Bolitotherus cornutus, my first successful attempt at doing so.

The gel shown here demonstrates that I successfully amplified DNA from my samples. This was extracted from the pronota of two B. cornutus, one male and one female. Not all the amplifications worked, though. The lanes labeled 1, 5, 9 and 13 are a segment of the large ribosomal subunit rDNA and those labeles 2, 6, 10 and 14 are the small subunit sequences. Lanes labeled 8 or below are using a 1:100 dilution and those labeled 9 or higher a 1:500 DNA dilution. The lower dilution clearly works better; I get at least two of my four products.

Two other sequences, the wingless gene and cytochrome oxidase 1 (COI) didn't amplify at all. That's probably because there are only a small number of copies of these present in the total genome, unlike the much-duplicated ribosomal sequences. I'll try a lower dilution, maybe just 1:20, and have another go at those later.

Mind you, I haven't actually sequenced these yet. This only shows that I amplified ribosomal DNA from something. While the primers I used are supposed to be beetle-specific, there's still a possibility that I'm looking at DNA that came from something that lives inside the beetle. I won't know that until I get back sequences and try aligning them with known ones.

That's the less likely scenario, though. This is just a small step forward for me, but I'm feeling pretty good about it at the moment. As for today, I expect to be spending more time in the field than in the lab. I'll get to cleaning and readying my products either late today or on tomorrow.

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Tricky Insect Will Tell Me Its Secrets

I had been planning on going into the field with two of my lablings today but the weatherman convinced me otherwise with dire warnings of hail and damaging winds — neither of which has materialized. We'll go out on Wednesday instead. Sigh.

On my previous trip to Wachusett on June 11, I found a beetle in a decaying cluster of Pholiota squarrosoides that I thought at the time was an Eleodes. I was wrong.

One of the things I'm trying to learn is to be able to look at a beetle in the field and at least identify it to family. Being able to say whether or not something was in Tenebrionidae would save me a lot of useless collecting. The beetle family that contains the one I found, though, doesn't make that any easier (and Tenebrionidae are already so variable that it's not an easy task to pick one out without recourse to counting tarsal and antenna segments). It's a mock teneb, so close morphologically to true Tenebrionidae that its family is referred to colloquially as the "false darkling beetles." The family it belongs to is Tetratomidae and the species is Penthe pimelia, sometimes called the velvety bark beetle. It's not really a bark beetle; it's another fungus-eater and sometime dweller that usually specializes on polypores. In this case, though, it happened to be working on an agaricoid when it was captured and dropped into 1.5 ml of 200-proof ethanol.

This beetle looks so much like a teneb that I wonder why it isn't classed with them. It has the same number of joints in its foot, the same number of antenomeres. As variable as tenebs are it's surprising that taxonomists relying exclusively on morphological characters put it in a different family.

Only a few nucleotide sequences have been obtained from this beetle to date, if indeed they are from the same species as the specimen I have. I'm going to start work on getting more sequences out of my voucher specimen tomorrow and see if I can't add to the 16S mtRNA, 18S nucRNA and COI partials. I'm going to go after another chunk of the LSU, the SSU and the wingless gene, too. After that I'll see for myself whether or not it might be a teneb (eventually, anyhow). Even if t isn't, it should make a perfect outgroup for the rest of Tenebrionidae I use in this study.

Right now, I'm just killing time waiting to see if I got a product out of Bolitotherus cornutus. The gel is running. If I can sequence it, it will be the first time (at least as far as GenBank is concerned) that anyone has done so. Fingers (tarsi?) crossed.

EDIT (4:09 PM) - No PCR products from B. cornutus. Crap. I'll have to start over again tomorrow.

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Is It Over Yet? Better Not Be.

It's nearly 4:00 PM. Here in Central Massachusetts, the weather couldn't be better. Low 80's, great breeze, sunny... not a sign that the world is ending anywhere.

In fact, it had better not end or I'm going to be royally PO'd. I've been waiting forever to get sequences out of Bolitotherus cornutus. I spent this morning rehydrating oligonucleotides and dissecting the wing muscles out of two specimens. I got everything set up, went to the freezer room to get some liquid nitrogen... and we're out. I need to do my extraction with the stuff or else muscle tissue sticks to the mortar and I can never get it ground finely enough and so won't get the largest gob of genetic material possible, proteinase K or no.

I really had my heart set on at least getting started on this today. I'm told that someone will be here tomorrow to replenish the liquid nitrogen supply. Sometime. Probably.

Because of this, I am officially postponing the end of the world definitely. There will be no mass die-off of humanity until I have sequenced these beetles! Any deity that attempts to bring on the Apocalypse before that event will have to answer to me.

There are no accessions for divine beings on GenBank. If forced to, I will not hesitate to add the first one. You got me, gods, angels, demons and devils? I'm a frustrated lab rat. You do not want to mess with me.

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