Now I Have A 4WD Vehicle

I flew up to Baltimore on Friday and drove back with a 1994 4WD Toyota truck. Toyota designed these older trucks explicity for off-road use, and I'll be putting it (and me) to the test this Saturday when we return to Hurricane Creek for our March collection of telescope shiners. Passenger capacity is probably five in the cab, at least for carrying people in the 4 miles on what passes for an access road through the gates. All I have to do is get the truck registered and insured this week, with a probable visit to my mechanic. It should be fun!

Tomorrow I hope to get with Kris back on the track of PCR of the mummichog mtDNA. We have a bunch of it ready to go; maybe we can do purification of one or two samples and have them sequenced in the sequencer down the hall (really!) and hope that it works. Yeah, I'm at rish for being a gene jockey at this rate.

Telescope Shiners, Length vs Mass

We have the first monthly set of length vs weight measurements for telescope shiners, from fish collected in Hurricane Creek February 3. This figure pools males and females, since it wasn't possible to sex them from external examination.

This r-squared value of 0.81 seems to be typical of local shiners in north 'bama. I suspect that as the season goes on we'll see a divergence between males and females, as females become proportionally heavier relative to length.

Female Telescope Shiners Show Ovarian Development... Yeah, It's Important!

At least I hope it's important. I sat down with five students yesterday and we started our dissections of the telescopes we caught at the Walls of Jericho on Feb. 3 to look for evidence of gonadal development. We started with four of the largest individuals, since I had a feeling that the larger ones would be females and I'm more interested in them. Sure enough, three of the four we looked at were female and all showed some evidence of gonadal activation. We found evidence of intermediate oocyte development, cortical alveolar or early exogenous vitellogenesis. It wasn't difficult to find the ovaries in the abdominal cavity and remove them under a dissecting microscope. The three had gonadosomatic index (GSI) values of 1.2, 2.9 and 3.2 which is about as high as the burrhead and silverstripe shiner March values from Borden Creek three years ago. I'm surprised that the development was that advanced, even though they weren't nearly ready to spawn. The male we looked at wasn't tuberculated, and we couldn't find his testes, either; I assigned his sex purely because we couldn't find ovaries which were so obvious in the three females. We'll continue with this "quest for ovaries" next week. We also measured standard length for all the fish since I have my digital calipers back from Enrique. Pooling all of the length-weight data together, the correlation coefficient between length and weight was 0.9. I guess that's not surprising, the correlation will probably weaken between sexes once females fill up with eggs.

On the mtDNA front with our mummichogs (Fundulus heteroclitus), Kris has been on a roll with PCR's this week. One run produced good DNA amplification in six of six samples, and another run was five for six. So we now have 17 - 19 successful amplifications ready for purification and sequencing. These include individuals from Martha's Vineyard, Nantucket, Boston Harbor, Chincoteague, Virginia Beach, and maybe Marion, MA. If the sequences all come out clean we'd probably have enough for our final analysis of population structure and history for this species. I think we'll get greedy and run some more.....

Late Winter Research; Good Thing We Have Fish!

It's been a mad-dog week for me workwise, since I've given two exams in my Biology 2 classes which of course means I have to grade them in some good time. That's what I largely did today. But things are still percolating on the side for research.

Kris has been working on the PCRing of more mummichog DNA. We have 6-8 samples ready for sequencing, and now we have two more rounds of product waiting for good gels to be run for assessment. We ran a gel today with 6 samples on it, but the student I let load the gel apparently injected the PCR product too deep in the gel and after ethidium bromide staining the lanes formed a strange fan pattern sitting on an arc of stained DNA. It wasn't very informative, and Kris will re-do it tomorrow. But even so I think I saw two or three real bands in the confusion.

The work on scarlet shiners could take a sharp turn for quick resolution. We realized that rather than do an antibody staining of histologically prepared brain tissue samples to visualize NMDA receptors, we could run western blots of brain tissues and come up with the same information. This is easier for us because Enrique and Rebecca have western experience, and the Bishop lab we're working in is set up to run westerns without any undue effort. So hopefully we can get these blots run in just a few weeks, and see if our hypothesis is right that males have a high, invariable number of these neuronal receptors while females vary hugely with age and reproductive condition. These receptors are key parts of fish brain circuitry for successful reproduction.

Next post I should be able to display our most current map of flame chub distribution in Alabama based on our field work. Kevin has come up with a good design for the map. Now all I have to do is to finish writing my article summarizing our research. Really, I will!

Telescope Shiners In Process

I met with Andrew, Valerie and Loren this afternoon for several hours to start work with the telescope shiners we collected last Saturday. Each fish is individually "named" with a month code (B for February, the second month), T (for telescope) and numbers (o1 - however many), so for instance a fish is BT03. Each fish is in its very own 24 ml glass screw-cap tube in 10% phosphate-buffered formaldehyde with its code in punch-tape stuck on the outside. My digital calipers have gone missing, so we settled for going up to the Bishop lab and weighing each fish on her good balance. The very biggest telescope was about 2 grams, many of them just under 1 g. We record the mass in grams to three decimal places after quickly rolling the fish in a paper towel to absorb excess formaldehyde. We'll get the standard length (SL) by next Friday, and probably eviscerate a few to see if there's any appreciable gonadal development.

Kris ran another PCR with some of our Fundulus heteroclitus DNA. At least two of the samples showed amplification bands on the gel, one from Boston Harbor and one from Chincoteague, VA. We'll run some more on Monday, trying some more Boston Harbor samples. So now we have six (maybe 8) successful amplifications in the last week, not bad for occasional work(!).

We Made It To The Walls Of Jericho, And... We Have PCR Amplification!

We made it in and out of the Walls of Jericho on Saturday. Seven of us met Nick Sharp at 8:30, and we were all able to fit in and on his truck for the bumpy ride in. Andrew and I sat in the back of the truck, while the other five students squeezed in with Nick. It was the first time I've worn my wool cap and insulated gloves in years, gear that I used to wear daily in Boston before moving here. Good thing too, since it was about freezing as we bumped in. But the water temperature at Hurricane Creek was 9 deg. C, not too bad whilst wearing insulated waders. Several of the students had never seined before, but soon got into the groove of the darter dance to chase fish into the net. We quickly got our quota of 30 telescope shiners, none obviously gravid or in breeding condition; but that's the point to collect them before they go into condition. We only netted black and stripetail darters, somewhat disappointing, but we weren't really working the creek for darters. The blacks were just coming into breeding colors. One in particular was a huge male with his belly turning flame orange. For reasons unclear to me the biggest black darters I've ever seen are in this creek. So we have material to work with over the next several weeks. We hope to return to Hurricane Creek on Saturday, March 3.

On another front, it took three gels to prove it but the PCR DNA amplifications Kris did last week with six sample of heteroclitus DNA worked in at least four of the six samples. These are important samples, too; three of them are Nantucket fish, and one is from Virginia Beach. Now I'm confident that our PCR protocols are still good, that the Taq enzyme I have still works (the key enzyme that drives this reaction), and that we can amplify some more samples for further DNA sequencing. The next batch we do will probably be fish from Savin Hill Cove in Boston Harbor, adjacent to the University of Massachusetts in Boston. They amplified before, but the sequencing service screwed up the sequences for all eight that we sent. With that we should have a pretty good story for Kris to analyze for his thesis.