I Started The 11-KT Experiment

I just finished dosing aquariums in my lab with different levels of 11-Ketotestosterone to observe what, if any, effect it will have on young scarlet shiners over the next 6-12 months. I have six 10-gallon tanks, each with a hanging filter but no filter pad so as not to trap 11-KT, and 3-5 young fish in each tank. Two of the tanks have received 35 nanograms of 11-KT, two tanks received 15 nanograms, and two tanks received nothing as controls (except 2 ml of ethanol, since the 11-KT is supplied suspended in ethanol). The effects we're looking for will be differential growth in the brain, either the overall size of the brain or the growth of individual structures such as the optic tectum and telencephalon. Paul just finished his project on 14 wild-caught young scarlet shiner brains by taking digital images of the brains, and making measurements on the brains for overall size and the size of individual brain regions. That will probably inform how we analyze our results.

I finally curated some recent stream collections from the stippled studfish project. This involves ID'ing all of the fishes we kept, and as usual I notice things in the lab that weren't apparent in the field. For instance, we found a burrhead shiner at Channahatchee Creek in Elmore County, which isn't really surprising but I just wasn't looking for it as we were seining fish. We also found a silverstripe shiner there, so there are my old friends from an earlier project. Most of what we've kept from these Tallapoosa system creeks are pretty shiners, Lythrurus bellus. Those shiners could be an interesting project just because they're so common around the Tallapoosa, and I haven't found much information on them. I already have a pretty good collection of them!

The Next Bifax Trip Will Go Back To Tallapoosa County

First of all, let me congratulate myself, this is my 200th post on this blog over about 2 years. It just keeps chugging along...

I've sat down and drawn up a list of the dates and sites we've gone down to the Tallapoosa River area looking for stippled studfish. We've visited the sites of 30 of 48 collections in our records, which is at 13 individual sites. This includes almost all of the non-river sites so the project is going well. There remain two sites in Tallapoosa County we haven't visited yet, Sweetwater Creek and a branch of Elkahatchee Creek. There's also Emuckfaw Creek where we found no stippleds in February. I looked up the records today and there have been 7 collections made there over the years, although the largest was of five fish. So I'd like to revisit the place in warm weather when we won't have to worry about stepping into some of the deeper holes in the creek. Tentatively this trip will be on Friday, June 20.

I have an electronic version of Rachel's thesis from last year on black darter reproduction at two sites. I spent an hour this afternoon boiling down some of her introduction for the introduction of a journal article. Here's what I came up with:

The reproductive schedule and effort of a species is a crucial feature of its life history, and is often poorly known in relatively common species. The purpose of this investigation was to compare reproductive traits between two populations of a percid fish, Etheostoma duryi (Henshell), the Blacksided Snubnose Darter, in north Alabama and to closely-related members of the subfamily Etheostomatinae, where such information is available.

E. duryi belongs to the subgenus Nanostoma (Page 1983), which was sometimes previously referred to as Ulocentra. This species is a common inhabitant of runs and pools in streams having a moderate current and a limestone rock substrate throughout the central Tennessee River system in Georgia, Alabama, Tennessee and, probably, Mississippi (Etnier and Starnes 1993).

Page (1983) suggests that spring is the season during which most darters spawn, with southern latitudes typically spawning from February until late April. However, since bodies of water in states having more southern latitudes often have less seasonal variation in water temperature, the darters living there may exhibit a somewhat different spawning pattern. Hubbs (1961a) notes that species spawning earlier in the season were more cold-temperature tolerant than were species spawning later. There is noted latitudinal, geographical, (Hubbs 1958; Hubbs 1961b; Marsh 1984; Parrish et al., 1991), and temporal (Joachim et al., 2003; Heins and Machado 1993) variation in reproductive development within some darter species. As the spawning season approaches, female darters produce increasingly higher percentages of mature ova (often indicated by an increase in size and transparency) (Page and Mayden 1981). The testes of the males become larger and white in color with a spongy texture. There is typically no migration to the spawning grounds because they usually spawn in a certain area within their microhabitat. Nanostoma species are known to attach their eggs to some substrate and subsequently abandon the eggs. Since Nanostoma darters are egg-attachers that abandon their eggs following attachment, one can predict that the females will produce a large number of small eggs.

This study contributes to the natural history of darters. The choice of sites for the study, in that one was in a rural area and one was in an urban area, allows for comparison of environmental impact on darter reproduction. Reproductive traits examined in this study include ovarian and testicular development, the maturation of ova and spermatocytes as the breeding season progresses, and the size and number of ripening ova.This study is unique for two reasons. First, the methodology utilized was new. The study uses a histological approach, which has not been used in darter population studies. Because of the small body size (♂: 32.33-52.95 mm, 0.5-2.6 g; ♀: 28.15-50.06 mm, 0.3-2.2 g) some of the usual methods for measurement could not be employed. Second, most previous studies typically used just one stream for measurement, while data were collected from two streams for this study. One additional aspect of this study that has not been examined much in darters, and has never been studied in E. duryi is the determination of what, if any, effects the presence of the apicomplexan parasite Sarcocystis encapsulated in the tissues of the gastrointestinal region has on their reproduction.

(The above is copyrighted material, 2008, Rachel Bedingfield and Bruce Stallsmith.)

A Bifax Trip To Elmore County, Alabama

I went out yesterday with Travis and Steven to visit three historic Fundulus bifax sites in Elmore County, Alabama. This is as far downstream along the Tallapoosa River that the species has ever been found. All three of these sites are in the Channahatchee Creek system.

The trip didn't start so auspiciously, as at the first site the creek was a series of swampy pools due to beaver dams. The water was turbid and deep, neither one good for bifax. We poked around and decided not to sample since I figured it was neither safe nor effective.

The second site, down the same road -- Highway 63, Kowliga Street (as in the famous Hank Williams song about the wooden Indian) -- looked somewhat better from the road, and we were able to walk down a dirt path to move downstream. The substrate was sand with large patches of soft, puffy sediment that was easily mobilized by walking through it. We quickly caught a bunch of shiners, mostly pretty shiners, but that was it. We didn't see any fish that could be bifax, and usually you can see this fish before you catch it. Several small ditches drained fields in the area where goats and cattle were grazing, and I think these animals were the source of much of the puffy sediment we found in the creek. This wouldn't be good for bifax since their eggs are deposited in the sand, and require no sediment sitting on them to allow enough oxygen uptake for growth and hatching. Two fish collections made at this site over 20 years ago caught 10 or more bifax. I have a bad feeling the species really is gone from this site, at least for now.

The third site was the lower part of Channahatchee Creek not far from where it runs into Lake Martin, the impoundment of the Tallapoosa River. This site at Highway 229 has a new, high bridge with a utility access road alongside it for the power line there. The creek is much wider than the other two sites, and has a mostly sandy substrate with a loading of puffy sediment. It took some effort to get down to the water because of the over-engineering of the bridge. We waded upstream and started to sample likely looking habitat, especially areas alongside sandbars with less current. Finally, about 200 meters upstream, I found the ideal looking sandbar and there was a single bifax hanging in the shallows. Steven and Travis quickly set the seine and scooped up the fish. And that was our only bifax for the day. I might have seen a few more about another 100 meters upstream, near the ruins of an old bridge, but we couldn't corner them. So home we went.

As of now we've found bifax at seven sites in four creek systems, one each in Randolph, Coosa, Tallapoosa and Elmore counties. And there is still a healthy population in Sofkahatchee Creek in Elmore County since Joe Scanlan collected them there last year, an anomalous population in the Coosa River drainage rather than Tallapoosa. The Channahatchee population seems iffy, although it should probably be resampled. I still hope to survey streams in the Tallapoosa drainage where bifax has never been reported, but I suspect that there's likely to four solid creek populations and not too much more. By late fall I should have a better idea, and of course I'll be happy to tell you.

We Found A Flame Chub In A New Location Today

I went out on a creek survey with Allison, Jennifer and Dewey today for the Flint River Conservation Association slackwater darter project. The first place we went to is Brier Fork of the Flint River along a large farm property owned by Alabama A&M University north of Huntsville. We netted huge numbers of scarlet shiners (many in peak breeding color), along with some striped shiners and stonerollers and two species of darter, rainbow and black. The big find was a single flame chub. I went out to several sites along Brier Fork two years ago and didn't find a single flame chub. But today we hit the right microhabitat, a shallow backwater off of the main flow, and there he was. We also found no slackwater darters. This area was right in general, with low hardwood bottomland. But the creek showed clear evidence of erosional sedimentation such as puffs of clay from the substrate as you walk along, and the banks were obviously badly affected by flood pulses.

Then we headed north and sampled Banyan Swamp Creek. It really is a tupelo swamp, but the water flowing through it is clear and mostly over sand and cobble. The site is even better as a slackwater site than Brier Fork with backwaters and pockets of leaf litter. We quickly caught a large warmouth sunfish along with lots of smaller bluegills and green sunfish. Again, we found no slackwater darters, but we did find stripetail darters which if I remember correctly are stream associates of slackwaters. And, we found no black or rainbow darters. As an odd rule, if you find darters with red body color like blacks or rainbows, you won't find slackwaters in that stream. But stripetails have no red, and we hoped we'd find a slackwater. No such luck. The good news is that this stream is probably a good candidate for reintroducing captive-bred slackwaters.

Tomorrow it's back to the Tallapoosa drainage in Elmore County, AL. The sites we're visiting are all off of paved state highways, so I hope we can avoid any adventures with my truck getting stuck in a wall of mud.

Trips For The Week

I'm going out tomorrow with Allison from Alabama A&M, and Jennifer from UAH who's working with her, to scout possible habitat for slackwater darters in the Flint River of Madison County, Alabama. Their project of reviewing GIS images for remaining habitat preferred by the fish--easily flooded hardwood bottomland where the fish spawn during winter floods--has been disappearing with creeping development in the area. I think that Allison and her collaborators have identified some potential habitat, so we'll go out and see if slackwaters are there. We don't want to keep any fish, since they're Federally listed, but just see if they're present.

And on Friday we're doing a trip to Elmore County, Alabama, to look for stippled studfish at three historic sites near the Tallapoosa River. I think that Steven, Travis and Paul are going. Unfortunately we won't meet Joe Scanlan, since he's going to the annual convention of the American Killifish Association in Syracuse, NY. Two of these sites produced >10 stippleds in earlier collections, and one is described as having a bottom of sand and fine gravel, so I suspect we'll find stippleds. But we'll see.

I did an inventory of what stippled DNA extractions and amplifications we have. This week I realized that with a new PCR setup, of new Taq polymerase and dNTPs in particular, our PCR product looks much better. The latest run worked five for five. We may have to re-run some of our earlier work. But, at the moment, we have four amplified products from Cornhouse Creek site #2, one from Cornhouse Creek site #1, four from Hillabee Creek near Alexander City and one from upper Hillabee Creek. We also now have successful amplifcations from three southern studfish, three northern studfish, and a pretty shiner that we kept by mistake and decided to PCR as a form of control. So progress is made, and we can go on to run the 15 or so samples that still need to be run for a first time.

Photos With Stan Sung & Thomas Ganley, From April

I received a CD from Stan Sung with photos from his trip through GA & AL in April with other guys from CT & CA. His fish photos are beautiful, I'm posting his photo of a male scarlet shiner below. The scarlet shiner shot, and the other two shots below, are all from Limestone Creek in Madison County, Alabama, on Wednesday, April 23. All photos by Stan Sung.

Below is a photo of me on the left pulling a seine with Thomas Ganley. This was the stretch of Limestone Creek where we found lots of scarlet and striped shiners.
Below is a group shot at the creek. From L-R: from UAH, Sandi, Daniel; Stan; and me.
Apparently this trip will be the focus of a story in an upcoming issue of Tropical Fish Hobbyist (TFH). TFH was the second magazine I subscribed to in 6th grade, after getting a subscription to Mad Magazine (what else? and, what me worry?).

We have a trip to the Tallapoosa River system next Friday, May 23, It looks like Steven and Travis can both go, hopefully Joe Scanlan can meet us there.

More Amplified Fundulus mtDNA

I ran a gel on Tuesday with Travis of some PCR product that Andrew had done before he took off for Costa Rica. This included three Fundulus stellifer and three F. catenatus individuals, near relatives of F. bifax. Luckily, two of the catenatus samples showed bands in the right place, so we cut the bands out of the gel for future purification. I realized afterwards that we haven't been able to assay the purity and concentration of our DNA extractions from these fish, so it's good that even two of them turned out well. Kris and Travis re-ran PCRs on the remaining four samples yesterday using the new polymerase material we received from New England Biolabs, and hopefully we'll run a gel by Monday.

I also wrote to Anna George at the Tennessee Aquarium today, asking if we could beg a few individuals of F. julisia from their breeding colony of this bifax relative for DNA extraction. Hopefully she's the right person to ask, and also hopefully they're willing to do this.

Does anyone read the magazine Seed? I love it for its somewhat goofy geewhiz boosterism of science as a key part of culture. The latest issue has a short blurb on page 36 about the new scientific field, "Neuroecology". I'd made that word up last January as the course title for a Special Topics class I hosted for a student, since he was working on examining scarlet shiner brains within our larger research project on sexual function and phenotypes among scarlet shiners. I guess I wasn't the only one to come up with the idea; the Seed blurb defines neuroecology as, "...the study of adaptive variation in the brain and in cognition." I can go with that. Our work to date has focused on what makes alpha males the way they are; the central puzzle is, do differences in brain size and structure affect the expression of sex hormones and gonadal growth, or do the expression of sex hormones and gonadal growth affect differences in brain size and structure? If you study this question in wild-caught individuals, you're definitely doing neuroecology. And I never even took a neuroscience class...

Back From The Gulf Coast

I just got back from a Gulf Coast five day trip, visiting the Dauphin Island Sea Lab in Alabama, and going on to New Orleans. The Program Committee meeting at the Sea Lab was more interesting than usual because the place is growing. Two new faculty members and a new Executive Director will be in place by September. There will also now be the possibility for lecture courses by Sea Lab faculty to be simulcast for graduate students at other Alabama institutions to view for course credit. These courses would include subjects we can't offer such as geochemistry and benthic ecology. Not all of my students at UAH would benefit from this, but some would. I'll have to work on arranging this with our Graduate Studies dean.

Ruth and I were able to go to a crawfish boil thrown by the Sea Lab late Friday afternoon. Several senior faculty members actually did the boil off of a pickup truck behind the new Marine Sciences building, and some graduate students sold their left-over beer for fifty cents a can (I'm not sure what it was left-over from...). About 75 people were there on a beautiful early summer afternoon. This is the kind of activity that UAH really needs, to bring together a variety of people at a fun social event. The only funny thing was observing how many grad students had goofy tattoos. Maybe I'm a conservative in my old age, but before you get a tattoo think about it for more than 30 seconds, fer chrissakes.

New Orleans was just for fun, hanging out buying trinkets, eating well, and drinking beer in the street (legally!). The Quarter has several good used book stores and I bought two relevant books: Females of the Species by Maryann Kevles from twenty years ago, and Floods, Famines and Emperors by Brian Fagan. Fagan's book seems to be similar to the now-famous Jared Diamond book about the collapse of human societies from environmental damage, and Kevles' book is a good introduction to the concept of female choice being key in the evolutionary process of sexual selection. Much of my Vertebrate Reproduction course works this concept, and I might hand out a photocopied chapter of Kevles' book as a good, broad introduction to the subject. I almost bought a pristine condition copy of the book Fishes of the Northern Gulf of Mexico, still with its ID wall chart, but $50 seemed to be a little steep. Used copies can be had through Amazon's network for as little as $4 plus shipping which is what I think I'll do.

Fun With Creek And Choctaw Place-Names In Alabama

Ruth and I spent yesterday in Chattanooga on various errands. Chatt is about the same size as Huntsville, but has an interesting downtown, something sorely lacking in Huntsville. There's an interesting used book store on Frazier Ave. called A Novel Idea Bookstore which always has a book or two that I buy. One book they had that I didn't buy in spite of being interested is a slim volume, Indian Place Names In Alabama (if I remember correctly). The price was $20, more than I felt I could pay. But, I did look up a few stream names and found their meanings:

Hillabee--means "quick" in the Creek language.
Tallapoosa--means "grinding (or ground) rock" in Choctaw.
Emuckfaw--means "metallic ornament" in Creek.
Coosa--means "river reed" in Creek.
Sipsey--means "tulip poplar tree" in Choctaw.

People often forget that much of modern Alabama was held as autonomous areas by various Indian nations until the 1810's, and the Indians were forcibly removed by Andrew Jackson in the infamous "Trail of Tears" in 1834. I like much about Andrew Jackson, but not that part of his career.

James informed me that his DNA extractions of our recent stippled studfish collections went well, with decent looking pellets (DNA) at the end. So, we're on to more PCR!

What Concentration Of 11-KT Would YOU Expose Your Scarlet Shiners To?

I'm currently in possession of 20 vials each containing 10 ng of 11-ketotestosterone suspended in 1 ml of ethanol. The point is to expose some juvenile scarlet shiners I have in 10-gallon tanks to different levels of 11-KT, and see how it affects their brain growth over the next 6-12 months. 10 ng is a vanishingly small amount of anything, even a potent androgen steroid. I ran the calculations, and figure that if I add 6 vials to each of two 10-gallon tanks, the resulting concentration of 11-KT will be 5.2 x 10(-11) M; a second treatment, of two vials per tank, results in 1.7 x 10(-11) M, and two tanks would receive no 11-KT. The theory is that the 11-KT will be taken up across the gills, and thus be a large increase in existing levels of circulating 11-KT. Will this work? We have to cogitate more on this...

James extracted DNA from representative samples of the fish we collected last Saturday, and apparently got good pellets from all of them. We're building a believable library of stippled studfish DNA, hopefully at least most of it will produce good sequences for comparison. A pattern of population is beginning to form in my mind, of separate creeks connected by the Tallapoosa River. Will these populations be genetically distinct, or is there just one somewhat scattered population in this species? I'll leave you with that tease.