Tuesday, January 30, 2007

Histology 101 Now In Progress, Among Others

The students working with me on the telescope shiner project are doing their first histological preparations this week, using scarlet shiners left over from the parasite project. The lab has a protocol for dehydrating and fixing the tissue so it can be mounted in paraffin, sliced and stained. The basic direction is: soak in Bouin's Fixative, two multiple-hour soakings in water, 70% ethanol, two round of 95% ethanol soakings, two rounds of 100% ethanol soakings, xylene, and tolulene/paraffin. It can be done in about 16 continuous hours of attention, or broken down into two days of paying attention to moving tissues from solution to solution. This is a technology that hasn't much changed since the 1930s. I think they're getting it, it's really nothing complicated but more similar to following a recipe to bake a cake.

I heard from Jo in the Alabama State Lands Division the other day. She called to say that they've granted me a permit to do scientific research at the Walls of Jericho, i.e. monthly collections of 30 telescope shiners. We're legal to go out this Saturday and seine telescopes. It will be something of a challenge, the temperature will be slightly above freezing but with sun if the forecast is to be believed. But we have at least 5 pairs of waders between 6 of us, so we'll be OK. We'll meet Nick at the vehicle entrance to the tract at 8:30 and he'll give me a key to all of the gates on the road into state property.

Yesterday I ran my first PCR in 2 years. Tomorrow we hope to run the results on a gel and see if they worked. I ran 2 DNA samples of Fundulus heteroclitus from Marion, Massachusetts, that I found in my freezer that I had neglected before, along with 3 samples from Nantucket and 1 sample from Virginia Beach. The last four had produced good amplification bands before but the sequences were screwed up, and I hope the Marion samples amplify. I extracted them in 1996 while I was still working out of UMass/Boston, and they've been frozen since. Spectrophotometric analysis at the time showed them to contain lots of DNA and relatively little protein (a PCR inhibitor) so in principle they should work well. I'll know by this time tomorrow...

Thursday, January 25, 2007

The New mtDNA Sequencing Crusade

I talked with Kris today and we toted up where we stand with his project of assessing Fundulus heteroclitus population structure using the mitochondrial cyt-b gene. We have six sequences from Charleston, SC; one from Virginia Beach; two from Chincoteague, VA; four from Falmouth, MA; and one from Nantucket, MA. A good framework but not enough to make a solid analysis. So we're going to re-do some of the PCRs (DNA amplification) of samples I still have and get them sequenced through a different vendor. We'll re-do samples from Boston Harbor, Chincoteague, Nantucket, and Virginia Beach. I also re-discovered two high-quality extractions I did in 1996 from two fish from Marion, MA, on Buzzards Bay. In particular we need northern sequences, and those would certainly qualify. What we've found to date is consistent with my hypothesis that Nantucket fish will be different from mainland populations as the result of Nantucket's isolation due to rising sea levels 8,000 years ago. But, of course, more data equal more confidence. So that's our mission. I hope to do the first PCR next Wednesday.

Jennifer is sending an e-mail to Trevor Pitcher in Ontario asking for his methods for analyzing color intensity in male guppies in a recent publication. His article is one of the inspirations for our work with male scarlet shiners, but getting the software to tell us precisely what we want to know is confusing. So if you know Trevor, remind him to tell us what he did.

Tomorrow my small class for telescope shiner work will hopefully start their first histological preparations. We'll rendezvous with Rachel at 3 and get the basic rundown, and I'll have the students work with some scarlet shiners left over from the gill parasite project.

Monday, January 22, 2007

I Think I'm Coming & Going

The struggle continues on many fronts, which I've found is the only way to get things done. I spent much of yesterday afternoon carefully reading the reviewers' comments on my tentatively accepted Southeastern Naturalist shiner reproduction article. One reviewer in particular had very good suggestions and critiques that will, as the saying goes, greately improve the manuscript. I'd been consciously holding back in some of my comments in the manuscript's Discussion section, and this reviewer urged me to flesh out my analysis of what we found. The big change I'll have to make in the manuscript is cutting out most discussion of my set of developing oocyte measurements. I think they're good, but the trouble is that for some months I only had a single female's oocyte count, making my reporting of results an unfortunate example of pseudoreplication. Yikes! But all three reviewers thought the rest of the data stand by themselves for a good article, so I'll edit it as such. I also talked today to my editor, Todd in Mississippi, to advise him of what I hope to do. He was very supportive for which I'm grateful, a lot of journal editing can be a very tense affair.

My proposed project to extract and quantify carotenoid pigments from scarlet shiners is on track. Abby, an undergraduate, is working with Jim, a colleague's husband, to set up the protocols for this process. If it works, which it should, we could probably do this measurement for lots of fish which is a study in its own right. It's for projects like this that biologists need a grounding in organic chemistry and biochemistry, as dry as they can be. These fields offer some very useful tools if you have both the chops and the imagination.

Friday, January 19, 2007

Burrhead & Silverstripe Shiner Manuscript Accepted By Southeastern Naturalist, Alright!

I heard back from an editor at the journal Southeastern Naturalist today, and they've provisionally accepted our manuscript on the reproductive biology of burrhead and silverstripe shiners. It's a huge relief, and also, of course, very happy(!). I have to do some editorial work on the manuscript to satisfy the three reviewers, especially on my statistical analysis of what we found about reproductive effort. But that's good, I admit that my treatment didn't closely follow earlier work and will in all likelihood be improved by the reviewers' suggestions. I started this project three years ago, so finally putting it to bed is a serious sense of accomplishment. I'd like to thank my co-authors Kevin Butler, Amy Woodall and Bob Muller for their work on this project. I'm also happy to publish in Southeastern Naturalist, the home of a wide range of ol' fashioned field ecology research around the former Confederacy (and I just renewed my subscription).

Thursday, January 18, 2007

Winter Blahs, So Let's Go Fool With Some Fish

I'm entering the phase in my Vertebrate Zoology class where I blitzkrieg through ancestral and modern fishes as examples of the base of vertebrate diversity. Does the phrase Meckel's cartilage ring a bell with you? Do you count gill slits in fish illustrations to check for anatomical accuracy? I sincerely hope that you answered yes to both.

Yesterday I tried to extract DNA from fish for the first time in 2 years. We could use some new mtDNA sequences for the mummichog population structure study, and the longnose/striped killifish study, so I'm training an undergraduate in extraction and hopefully also in PCR. I pulled out four longnoses from St. Joe Beach in Florida for a demonstration of the technique, since these are large fish easy to cut off white muscle strips for extraction. It went great at first; but I screwed up the extractions by somehow emulsifying the protein purification stage where hydrophilic and hydrophobic phases are used to separate out undesirable proteins. Instead of two layers in the tubes, we just had uniform murky fluid. Damn! The last time I did that was 3 years ago when I first showed Brace how to do this. I think we added too much tissue to each extraction tube. Next time, next week, we'll use just half as much. But it was fun to do some real biology work again.

Where this is all going is to come up with some more mtDNA cyt-b sequences for Kris' work. We don't have quite enough sequences from northern populations of Fundulus heteroclitus to really ice our analysis. I realize that I have some Queens, NY fish (!) that I never used so I'd like to get them sequenced. And even worse, when we had our first batch of PCR amplification products sequenced by a commercial service they screwed up many of our sequences. At first one of their employees told us to purify the DNA in one fashion, and just as we sent them our purified samples another employee basically told us that he couldn't believe we were so stupid as to do it that way. Well, at least some of the sequences came back in usable form. So I've found another vendor who is willing to do the work for $7/sample rather than a flat rate of $256 for 96 samples like the original vendor. Since I want to run no more than 10 sequences, paying for it myself, this is a good deal I hope. And this all made possible by my discovery of an unused tube of Taq polymerase in my freezer, so we won't have to shell out the $50 for more.

On Monday afternoon I hope to get out to Limestone Creek to collect black darters to send to Thomas Near at Yale for genetic analysis. At least two students are willing to go with me for this. The forecast is for a high temperature of 49 deg. F that day which will hopefully be tolerable. I have three sets of insulated waders, so we should be set. I'm in need of a collecting trip, even falling in the creek could be fun!

Saturday, January 13, 2007

It's Been A Busy Week, But No New Data Yet

School began this week and I've been going flat out. I've taken on a lot of students for various research type classes which is both good and bad, it's good to have the help but I have to be organized and plan things out in advance so that they can meaningfully participate. Two students will be placed on DNA extraction of some Fundulus collections I have, both for the heteroclitus project and also for the similis/majalis project. One student will help to work on a carotenoid extraction project primarily with scarlet shiners; Jim Anderson has agreed to help establish protocols and with any luck the University's HPLC is both functional and available. Since Jim's a trained equipment tech I have faith he could make it work, I hope so! And finally the biggest group of students will be helping to carry out a study of the reproductive biology of telescope shiners (Notropis telescopus) in Hurricane Creek in the Walls of Jericho tract. The potential is good for both a lot of good work, and for participants to get lost or confused; I'll be doing the polite Mussolini Headkick, keeping the trains running on time. At least I better!

I corresponded with Thomas Near at Yale today after a conversation that started on the NANFA Forum. He's doing a darter DNA study with a particular interest in Tennessee Snubnoses, Etheostoma simoterum. We've arranged it so that I'll send him several collections of darters from around north 'bama, both putative simoterum and also duryi. He needs the data for his work, and I'm curious to see that my ID's are accurate. So on our first Hurricane Creek trip in February I hope to snag around 15 snubnoses to send him. I think I've found both and duryi and simoterum there in the past, so hopefully genetic information will help to clear this up. You'd think it be easy to tell two species apart by physical characteristics like having a snout frenum or not, but life's not so simple. I'll keep you posted, as always.

Saturday, January 06, 2007

Our "Status Of The Flame Chub In Alabama" Summary

What follows is the current "Discussion" section of my developing paper on flame chub distribution across north Alabama. I still have to flesh it out somewhat, but this is the gist of our findings on the current status of the flame chub, Hemitremia flammea, in north Alabama:

A species like the flame chub that is a “narrow endemic” by definition has a patchy distribution. This makes it more difficult to determine if such a species is in decline. The results of this survey show that the flame chub is in decline in Alabama, being found in 18 of 50 historic sites sampled. It is of course possible, maybe likely, that flame chubs were missed at one or more sites. Even if this was true for 7 sites, that would still leave flame chubs missing from a full half of the sampled historic sites.

There is a strong geographical pattern to the remaining flame chub populations in Alabama. There are two primary strongholds for the flame chub in north Alabama. The first is the central and eastern parts of the Cypress Creek system in Lauderdale County. Burcham Creek and Bruton Branch in the western part of this system seem to have lost flame chubs from human alterations of stream flow. The other flame chub stronghold is in the Flint River system in Madison County, comprising Mountain Fork of the Flint River and Acuff Spring. Flame chubs were easily seined in Mountain Fork just below a lowhead dam. Acuff Spring was the only sampled site where schools of flame chub were easily observed. But this site is under various stresses. The spring is on land that belongs to and is managed by a housing development. Recently the management has treated the spring run with herbicide to kill “unsightly” native aquatic vegetation, and a friend of the developer has repeatedly released koi into the spring run to “improve” it (Casper Cox, personal communication).

Other sites yielding flame chubs were erratically scattered with no easily discernible pattern. The only historic site in Marshall County, Eudy Cave, seems to have a healthy flame chub population and is relatively pristine in spite of being in the middle of a cattle farm. The one site of three visited in Morgan County that yielded a single flame chub, Dutton Creek, is also surrounded by cattle pasture with cows having access to the creek both above and below our sampling access.

Surprisingly, flame chubs were found in only one of five historic sites visited in the upper Paint Rock River system of Jackson County. Streams in this area show few obvious signs of any abuse, and support populations of other vulnerable fishes such as the blotchside logperch, Percina burtoni and palezone shiner, Notropis albizonatus. Two historic sites were sampled without success on Larkin Fork in June 2005. One of these sites produced a single flame chub as recently as 2001, and at the other nearby historic site downstream 8 flame chubs were collected in 1980. This population may have been in decline to the point where it’s now difficult to find any flame chubs.

Based on the dramatic reduction of historic sites in Alabama yielding even a single flame chub in this survey, it seems to be time to adjust the NatureServe conservation status of the flame chub in Alabama from a rank of S3, Vulnerable, to S2, Imperiled. The S2 rank would reflect an observed steep decline in populations across Alabama over the last 40 years. The global rank of the flame chub is currently G3, Vulnerable. Because Alabama is currently about half of the range of the flame chub, this ranking may be too optimistic and G2, Imperiled, may be more accurate. A similar survey needs to be done in Tennessee, the other half of the current range of the flame chub. For the same reasons, an argument can be made to change the IUCN category for the flame chub back to Rare from Data Deficient.

Monday, January 01, 2007

A 32-Day-Old Scarlet Shiner

Here's a shot of a 32-day-old scarlet shiner. This one is 6.45 mm long, total length. Notice that the "dot" pattern of chromatophores is starting to get darker and more pronounced. I think the mouth is 0.14 mm across based on a measurement in the program Motic. Still no signs of any fins besides the caudal.