Black Darter Abstract

I felt the motivation this afternoon to look at the black darter reproduction manuscript, which I haven't fooled with since Jan. 4. I made some minor edits, but it looked better than I feared it might. So, now I feel compelled to submit it to a journal for publication. The J. of Fish Biology seems to be a good candidate, so now I'll format the article for them. One of the odd things about science publishing is that every journal has its own very picayune style requirements, and editors can get very pissy if you overlook one of their stylistic requirements. They have a very explicit, detailed style sheet on-line so I'll slog through that. Following is the Abstract for the submission, titled "Reproductive Development in the Blacksided Snubnose Darter, Etheostoma duryi," by B. Stallsmith & R. Beddingfield. (I don't think I've published this before here...)

Reproductive development in the darter fish Etheostoma duryi (subfamily Etheostomatinae) was studied in two north Alabama populations. Sites chosen for comparison were urban Town Creek in Limestone County, and rural Limestone Creek in Madison County. Because of the small body size of this species the study uses a histological approach never before used in population studies of the Etheostomatinae. Microscopic and macroscopic methods were utilized to study gonadal development and investment. Reproductive investment, as measured by gonadosomatic index, relative gonad mass and the proportionality coefficient, increased in both sites (and for both sexes) toward the time of peak spawning. Total number of oocytes differed significantly between populations, possibly attributable to differing body sizes. Clutch size and mass were not significantly different between sites. Reproductive maturation occurs from January until the peak in late March and April at both sites.

Effects Of 11-KT On Growing Scarlet Shiners

Brittany, James and Alexandra have finished measuring the brains of young scarlet shiners exposed to the hormone 11-KT for 6 months. The short answer is, 11-KT seems to stimulate the growth of significantly larger brains compared to wild similar-sized individuals from the same creek. In particular, the ratio of brain weight/body weight is higher in 11-KT exposed fish. We tested this hypothesis with a Mann-Whitney U-test, and the test rejected a similarity between the two data sets with a p<0.001. So 11-KT seems to be very good at stimulating overall brain growth. Our data don't show that any one brain region is bigger than others as a result of 11-KT, just the brain in its entirety is bigger. And also our experiment seems to show the efficacy of adding the 11-KT to aquarium water for fish to take up across their gills, rather than injecting the fish with 11-KT. A juvenile scarlet shiner is so small (~22 mm) that I think the mortality rate from injection would be high.

It Was A Slow Week, Except For The Really Busy Parts

I was swept away by various teaching and departmental business things this past week. I talked to Kris way back last Monday, and should see him tomorrow. We're closing in on ways to describe the mummichog DNA data. The stippled studfish DNA purification got stuck on hold for reasons beyond easy description; come soon? I hope so!

And for those of you who were wondering, Amphioxus is in the phylum Chordata (OK, it's an inside joke from Vertebrate Zoology...).

We Started The Stippled Studfish DNA Purification Today

I met with Kris, Travis and Andrew today. We had to make sure that we had all of the necessary reagents for the Qiagen kit purification process (we did, after begging some PB reagent from the Podila lab), and Kris explained his slight tricks with the generally good Qiagen documentation of purification. We did a test run on 6 of our samples in the Bishop lab, which does indeed have an Eppendorf centrifuge with variable speed control (a strange luxury). We weren't able to do a concentration test since no one was in the Podila lab late in the afternoon, cutting us off from the Nanodrop spec. If we get decent results, it's probably about 1.5 hours worth of work to purify our remaining samples. James is going to Tuscaloosa on Friday, so hopefully we can send these samples down with him and he can hand them off to people he knows in Phil Harris' lab.

I heard from Stan Sung yesterday. He and his pals are planning another Alabama trip in April. I might do a day trip with them down to Tallapoosa County to look at stippled studfish. If so, I hope to collect some Tallapoosa shiners for my aquaria. We'll see.

Another Run At Stippled Studfish DNA

I've arranged with the DNA laboratory at Tuscaloosa to sequence our stippled studfish DNA. We've lost faith in several commercial labs. So, we're going to send Qiagen-purified, twice-PCR'd DNA, 38 samples in all I think. The final prep work will be done next week, I hope. Yesterday Travis ran about 30 re-PCR reactions, so we're about set with that. I hope that Andrew & James can get the Qiagen purification done with Kris' max-yield tricks. Travis pointed out that the counter-top centrifuge I have is rated at 15,000 RPM, with no speed control, while the Qiagen kit calls for 12,000 RPM. And in truth I'm not certain what speed that centrifuge really runs at, since it's fairly old with nothing like regular maintenance(!). So we're going to use a centrifuge in the Bishop lab with a variable speed control, and see if that helps our final DNA concentration. As always, I hope it works.

And congratulations to Ben Keck, Solomon David and Josh Perkin for receiving funding for their proposals submitted to the NANFA Conservation Research Grant program this year. Each will get $750. Ben is working with greenfin darters in the upper Tennessee system, Solomon is working with spotted gar in Michigan, and Josh is working with a disjunct, relict population of ironcolor shiners in Texas.

How Does A Cape Cod Mummichog Differ From A South Carolina Mummichog?

After talking to Kris today for about an hour, I realize that we can specifically answer that question -- looking at about a quarter of the cytochrome-b gene in the mitochondrion, they differ at exactly 5 DNA sequence points, i.e. having a T instead of a C, etc. The fish in our research from Maine, Cape Cod and Nantucket all share these 5 differences while fish from Chincoteague and Virginia Beach, VA; Charleston, SC; and Sapelo Island, GA, have something different. Since DNA typically codes for an amino acid sequence composing a protein, we looked at these five sequence differences and none of them code for a different amino acid in the protein. So they're all apparently neutral mutations, not surprising since the protein cytochrome-b is pretty conserved among vertebrates. The interesting thing about these shared mutations is how old they must be. New England was mostly covered with ice until about 12,000 years ago, at which point coastal fishes like mummichogs were able to recolonize the ice-free coast. But Nantucket was cut off from the mainland by rising sea level about 8,000 years ago, blocking mummichog gene flow with the mainland. The easiest explanation is that the New England fish share a common ancestor after New England was recolonized, but only before sea level rose. So these five mutations date to between 12 and 8 thousand years ago. The furthest north mummichog populations at the time of glacial retreat available to recolonize the northern Atlantic coast would have been at the mouth of what's now Chesapeake Bay, and the two DNA sequences we have from that area don't share any of these five mutations. So, we seem to be looking at some kind of founder effect underlying current mummichog populations in New England, where all existing individuals share ancestry from a very small number of fish sometime between glacial retreat and significant sea level rise. I'm thrilled enough at this as a good story that I'm tempted to say, "That's cool, that's trash" but really I should say, "That's cool, that's real biology." So I just did.

And of course it's Darwin's 200th birthday this Thursday, Feb. 12. I should offer drinks all around, but this medium doesn't support that...

A Pleasant Jaunt To Limestone Creek On Friday

We were lucky to be able to take advantage of the beautiful early spring weather on Friday afternoon and go to Limestone Creek at the Mooresville Road site in Limestone County. It was sunny, about 60 deg. F, with light winds. We were interested in collecting about 20 scarlet shiners primarily for gut content examination. The creek was a little high, as usual this time of year, and around the riffles area which is an old ford the current was probably running about 20 knots in knee-deep water. We found the scarlets we wanted, but they were all in one small area that was protected by a low bar from the fastest current. Scarlets like flowing water but not white-water level like much of the creek that day. It was the best physical workout I had all week, wading upstream against a current that strong. We also found lots of black darters, with the males coloring up and many females plump with eggs. A large female rainbow darter was almost deformed she was so gravid with eggs (we let her go, along with all of the other darters). So spring is starting in north 'bama. James, Brittany and Alexandra were all able to go out with me. We've begun a fairly ambitious gut content monitoring project with scarlet shiners in Limestone Creek, and telescope shiners in Estill Fork. This also includes collecting length/weight data, and probably looking at some more brains too.

Kris has some interesting data on our mummichog DNA project. We're finding that there's a little more diversity in the mitochondrial cyt-b gene in northern populations (Cape Cod and Nantucket) than there is in southern populations (Charleston, SC, and Sapelo Island, GA). This is contrary to what's usually found for reasons having to do with the northern populations are recent, only about 12,000 years old, since that's when the glaciers finally melted in New England. And DNA sequences from Chincoteague and Virginia Beach, VA, are markedly different but intermediate to the northern and southern populations. We're still coming up with a final phylogentetic tree. The Neighbor-Joining algorithm using 1000 bootstrap resamplings shows the best, once we clean it up I hope to post it here. And Kris just called and told me that he's found a complete cyt-b sequence in a recent paper that's from a Maine fish, so we have one more northern sequence to plug into our tree. I'd expect it to be pretty similar to the Cape Cod & Nantucket sequences, but we'll see.

Drift Net In Action At Estill Fork On Saturday

Saturday's trip went well for the primary objective of collecting a decent number of telescope shiners for gut content examination, length/weight data and probably some brain analysis. But we also had a chance to test a drift net I've had for a few years that I built myself (with help from my wife in sewing the bag). The purpose of such a net is to collect passively drifting small animals and maybe algaes. The net is staked into the bottom of the stream so that water is flowing through it, and then wait some period of time before collecting what might have been trapped. Mine is 12" x 18", with a PVC pipe frame holding a bag made from organza silk, which has a very fine mesh. The challenge is how to stake it to the bottom. I've had metal tent stakes in the past that worked, but I lost them in our building move a year ago. So we tried bamboo poles. Unfortunately I couldn't force them into the streambed, so I talked Taito and Brittany into holding the net in place for about 15 minutes as a field test. And it worked!

Below is a photo looking downstream to the area we netted for telescope shiners. The flowing pool gives way to about 100 m of deep riffles with some scourholes in dense packed cobble and gravel.

Here are Taito and Brittany holding the net in place. The top of the net is just above the water level so that (hopefully) water is flowing into the bag, and not being blocked by any kind of turbidity right in front of the net.

Here's a closer view of the bag in action. Water was indeed flowing through it. We quickly saw leaves, sticks and few insects trapped inside the bag. And some protein foam started to build up at the top of the bag.

So I'm happy with the net's results. We also trapped a surprising amount of sand that seemed to be entrained in the current. I didn't keep the collection for further examination, since I think it's still too early for truly interesting drift such as ichthyoplankton to be present. But maybe next trip in early March, especially if I have the wit to go get some decent metal stakes so we can stake it out and take a break for an hour or so before checking the catch.

It's True, It Was A Beautiful Day At Estill Fork

Brittany and Taito joined me for a telescope shiner collecting trip to Estill Fork yesterday. It was sunny and mild, in the low 50's F. The water was fairly high, clear, and flowing fast and cold at 7 deg. C. We were able to net about 50 telescope shiners in about 1.5 hours, definitely the mark of a good trip. We also saw lots of darters, since they usually share the same microhabitat of riffles and scourholes as the telescopes. The rainbow and bluesided males especially were coloring up, although the male snubnoses were well advanced too. Once again I'm leaning to thinking that both black darters and what used to be tennessee darters are present in these tributaries to the Paint Rock River. I know that I saw a coloring-up male black darter yesterday.

I'll have to post pictures later, including my drift net deployed; it works!