A Good Day At Sipsey Fork, And Back To The Lab

Today is drop-dead beautiful, low humidity and a blazing sun with air temperature of about 90 F. We went out to the canoe ramp at Sipsey Fork and found the lowest water I've ever seen there. We also found unbelievable numbers of alabama and blacktail shiners, like I've never seen before, and finally found a sweet spot for silverstripe shiners 500-600 meters upstream, up above the Highway 33 bridge, at a riffle system. We wound up with 17 silverstripes, from medium to large size. We didn't find any burrheads which was disappointing because I've found a few in this stretch before. If we're serious about burrheads I'll have to get permission to go back to Borden Creek in the Wilderness because I know we can find them there.

We got most of the fish back to the lab alive, or very freshly dead, so we immediately set out to remove the brains and suspend them in a lysis buffer, this time with a protease inhibitor. I'm proud of the four students for sticking to it without a lunch break. This involved measuring a fish's standard length, body mass, removing the brain, weighing the brain, adding the right amount of lysis buffer based on brain weight, shredding the brain/buffer mix with a probe point, and making sure all of the information was correctly recorded before all of the buffered brains went in to the -80 C freezer. This is what sabbatical is for, I guess. Hopefully on Thursday/Friday we'll do a second round of western blots, with some of these brains, some telescope and scarlet shiners from the fall, and a few repeats of the last round for comparison.

The only sour note was finding the remnants of what looked like a redneck picnic along the beach at the canoe ramp. They even left a fishing pole, tackle box, and a cartoonish large leaf knife. What a bunch of morons; either that, or they were abducted by a UFO with no warning, which might be a preferable ending.

The Telescope Manuscript Is Back In Play

I mailed off the revised telescope shiner manuscript to the editor at American Midland Naturalist today. It's a lot better than it was, and now includes color photos of our four defined developmental stages for both oocytes and ovaries. I hope they like it.

Andrew is on a roll getting gill flukes off of scarlet shiners, and getting good photos of them. We're now pretty confident that what we're seeing is an undescribed species based on both haptor (hooks) arrangement and shape, and also the shape of the reproductive apparatus. Now all we have to do is draw them. But first we'll send off some photos to Don Cloutman and get his opinion before we get carried away. The logical next step from this would be to look at other Lythrurus species and see what kind of flukes they have. But that's a job for another day.

I confirmed today that we still have reasonable samples of our purified DNA extracts from stippled studfish. The question is whether it's really good stuff, or are we running around in hopeless circles trying to get it sequenced. I don't think we're retarded but it's vexing reconstructing our work on this project. I'd love to hear from someone who has a good DNA sequencing operation but they seem to be hard to find of late.

Black Darter Article Accepted For Review By SE Naturalist

I just received an email from Anne, an editor at Southeastern Naturalist, that they have accepted the manuscript on black darter reproduction for review. That’s a good sign from them in my experience, not a guaranteed final acceptance but they think it’s a contender.

 

On other fronts, I’ve kicked the telescope shiner manuscript into near-final shape for resubmission. My major editorial work in the last two days has been discussing the importance of stream flow as a major regulator of stream temperature and also physical environment in general, thus influencing reproductive success of shiners. I’ve found a recent body of work I wasn’t familiar with on this subject especially on an endangered shiner in Texas, the smalleye shiner in the Brazos River. Bart Durham and his doctoral advisor, G.R. Wilde, at Texas Tech have published several works on how impoundments and general alterations of the Brazos have negatively affected the reproductive success of the smalleye shiner and other native species largely by restricting stream flow in the summer. Many of the same concepts are applicable to local stream fishes, although not as directly. For instance, Hurricane Creek is a wonderful environment for telescope shiners precisely because it hasn’t been altered in any big way. So that’s the gist of my new additions to the manuscript.

Back To Sipsey Fork

I hope to go to Sipsey Fork in the Bankhead National Forest next Tuesday to collect silverstripe and burrhead shiners. Both species will be examined for any Dactylogyrus gill flukes, and I'd like to run western blots for NMDA proteins in silverstripe brains since they're a close relative of telescope shiners. So it will be another day of driving out, catching the fish, and coming back to the lab to remove fresh brains into a lysis buffer preliminary to western blots. The working hypothesis is that silverstripes should be like telescopes in terms of having a random sex pattern for NMDA proteins, unlike a strongly sexually dimorphic species like scarlet or tennessee shiners.

We even have official permission from the wildlife biologists of the Forest Service. I called up the office looking for Tom Counts, who wasn't in, but his assistant Allison has graciously emailed me a letter authorizing our visit and seining for shiners. We'll be going to the canoe ramp off of Highway 33, which is reasonably easy access in a drop-dead beautiful stretch of river with eroded limestone cliffs. I'll post pictures, with any luck.

Telescope And Scarlet Shiner Brains Are Different?

Here's the summary of what we found last week with our western blots to compare levels of the brain protein NMDA in telescope shiners who aren't strongly sexually dimorphic (the first group below, NTF) and scarlet shiners who are strongly sexually dimorphic (the second group, LFF). The Pixels column shows the relative density of antibodies sticking to an NMDA subunit on the final western blot; the more NMDA, the more antibodies, the higher the number of pixels in a scan of the western blot sheet. Both groups are ordered in rank from lowest to highest pixel count, with an individual's GSI (gonadosomatic index, relative size of gonads) and most importantly, sex.

Fish Pixels GSI  Sex
NTF4 83790 3.3    F
NTF5 87593 5.1    F
NTF6 89159 3.4    M
NTF7 95164 7.6    F
NTF3 96783 6.2    F
NTF2 99341 10.4   F
NTF8 108922 1.5   M
NTF10 114232 8.2  F
NTF1 133115 3.1   M
NTF9 140939 11.3  F
   
Fish Pixels GSI Sex
LFF3 83610 24.0  F
LFF1 89193 4.2   F
LFF6 91365 5.4   F
LFF7 93240 10.1  F
LFF8 96498 8.3   F
LFF5 99356 7.1   F
LFF2 103807 1.4  M
LFF10 125540 3.1 M
LFF4 139951 1.8  M

The big news is that the three male scarlet shiners in our sample of nine scarlets have the highest pixel counts, i.e. the most NMDA present. The telescope shiners show no clear sex pattern, with the highest pixel individual being a female, and the three males scattered throughout the sample of ten individuals. Interestingly, both species have individuals in an almost identical pixel range of ~83,000 to ~140,000.

We have two possible conclusion about the scarlet shiner brains: either the NMDA content (and function) is more strongly shaped by an individual's sexual status than telescope shiner brains, or an individual's sexual status is shaped by NMDA content (and function). Remember, the NMDA receptors are necessary for learning and sexual activity, among other things. In this group of North American cyprinid fishes, brains may show phenotypic plasticity as well as more obvious phenotypic traits such as standard length and breeding coloration. We'd like to further confirm this relationship, for instance by examining the brains of a relative of the telescope shiner, the silverstripe shiner, which is also not markedly sexually dimorphic, and a relative of the scarlet shiner such as the pretty shiner. There may well be a phylogenetic component to this, it's still too early to make sweeping statements.

It's Good, It's Bad, It's Happening

The Good: We spent Tuesday, Wednesday and Thursday running western blots on the brains of 10 telescope shiners, 9 scarlet shiners and 1 tennessee shiner that we collected a few weeks back. We're trying to quantify the presence of the major subunit of the protein NMDA, which helps to form pores in neuronal membranes enabling learning and sexual activity. The blots came out great, with sharply defined bands. I don't have the complete rundown yet, but we found a strong correlation between NDMA and GSI in female telescopes, and the 3 males in the sample all had higher values than the females. The tennessee shiner male, which was bright orange when we captured him, had a very high number for NMDA, too, which would be consistent with our expectations for breeding condition males. The tennessees are in the subgenus Hydrophlox, with most of the species showing sharp sexual dimorphism in spawning coloration and behavior. I've wanted to extend our work to this subgenus because they should all show this phenotypic plasticity in the brain. And, one of the species in the subgenus, the rough shiner, doesn't show pronounced sexual dimorphism which would be an interesting comparison phylogenetically.

The bad new is that the journal NeuroReport slammed our submitted manuscript on brain size and plasticity in scarlet shiners. It's the second nastiest review I've seen. I was taken aback by some of the things the reviewer said, to the point where I doubt that he read the entire manuscript or maybe isn't competent in English. Yeah, I know, that's what they all say, but it's some wild stuff. When the dust settles I may post some of the more lurid comments. We're all in shock, since this is by far the worst review she's ever received in her career. I think we suffer from trying to connect neuroscience with evolutionary ecology, this could be a hard sell. We may beg them to be allowed to resubmit, we'll see.

Also in the good news department, we've found and isolated Dactylogyrus gill flukes from scarlet shiners. No one has ever described fluke species from scarlets, and these flukes are distinctly different from others we've seen on telescope and striped shiners, so we probably have an undescribed species to write up at some point.

Back To The Telescope Shiners Manuscript

Starting last Thursday I was able to focus more and more on the telescope shiners reproduction timing manuscript. I've come to the realization that the reviewers/editors didn't really reject the manuscript as much as reject it in its current form, partially because the editor's letter talks about "if you want to submit revisions" do it in such & such a way. So I'm incorporating various suggestions which truly improve the manuscript. You really have to step back from your ego in this kind of work.

Tomorrow we'll spend the whole day setting up and running western blots to assay NMDA quantities in telescope and scarlet shiner brains from last fall. Stephanie in the Bishop lab will be our host. The big challenge for me is to dig out our brain preps from one of the big -80 C freezers.

I started a discussion on the NANFA Forum about silver shiners, Notropis photogenis. Don Cloutman at Bemidji State in MN asked if we could get a few for him locally for gill fluke examination, and I realize that the species may well be extirpated from north 'bama. Either way we can probably find some nearby in Tennessee, going out with Dave Neely and/or Casper Cox. I think it's the ultimate "shiny shiner" species. At least I know where to find highland shiners for the same project over in Lauderdale County.

Papers In Play.

It's been a distracting week, so I apologize for not writing for a few days. My wife has been sick so I've had to help her do various rabbit rescue errands more than is typical. But, things are happening.

I received reviewers' comments on the telescope shiner article on Monday from American Midland Naturalist. The editor said it's unacceptable as is, but I know that we can improve it in a range of ways to make it acceptable to everyone. I made some stupid omissions and errors in the manuscript, in part because I was trying to write very concisely. So for a change I underwrote a manuscript instead of overwriting. For instance, our graph of monthly GSI values runs from February through September, but we only report on oocyte condition and size for February through July. This is because in August and September the gonads are so regressed that no meaningful measurements are possible. But, I neglected to explicitly say this in even the one good sentence that it would need, so both reviewers rightfully wondered what was going on. Gad! So finally, today, I finally started to work on fixing various things, with help from Andrew.

One thing one reviewer in particular wanted was color images of our variously described oocyte stages, and ovary maturity stages. We have them up the wazoo, so it's relatively minor for us to find good images in two series and send them off. As usual, you have to leave your ego behind when reading reviewers' comments, I've moved beyond the stage of feeling retarded with knowledge that we can make this manuscript much better.

I also uploaded the scarlet shiners "brains" article to NeuroReport on Tuesday. That article has plenty of digital images included showing Western blots of NMDA receptor proteins. I think (as usual) it's a good piece of work. The editors at this journal go for fast decisions on submitted material, so I think we'll know soon.

And finally, to complete the trifecta, on Monday I mailed off the Stallsmith & Bedingfield black darter article to Southeastern Naturalist. They accept email submissions of manuscripts now, but their limit is 10 megs of material; because we have a bunch of digital images saved as high resolution .pdf's, the manuscript is over 12 megs in size so I burned it to a CD and mailed it off to the office in Maine (yeah, SENat's editorial office is in Maine!). And as usual, I hope they're favorably impressed.

Paper Publishing Prospects

I heard from the Editor at the Journal of Fish Biology last week. They declined our black darters paper on the grounds that it's too local (relative to England, sure...) and uses standard histological techniques. The latter I'd quibble with since we developed a method for doing histology with very small fish, but OK. At least they didn't say the paper sucks. So we're on to Plan B -- I've already started to reformat it for Southeastern Naturalist. SE Nat is a good regional journal, and many of the readers know what a darter is in the first place. So it's not such a loss to work on publishing work for an audience more likely to appreciate it.

The first priority, though, is to get out our scarlet shiners brains paper to NeuroReport. I just now finished some heavy grooming of the figures for it. Once I pump up the figure captions it should be just about ready to upload to the Editor in the next several days. It's turned out to be a good paper after several rounds of rewrites. We're sticking with the title, "Sexual Dimorphism in a Teleost Central Nervous System: Are Dominant Males Smarter?"

And I await a decision on the telescope shiners paper from American Midland Naturalist.

Here's a picture of Estill Fork on May 29. The emergent grasses have started to grow in a big way, which of course is where all of the fish hide.

Fish Brains, Gut Contents, Gill Flukes, We Have Them All

This week has gone by like a blur. We returned to Estill Fork on Tuesday and easily caught about 10 more each telescope and scarlet shiners, and that afternoon removed fresh brains, mashed them up in a lysis buffer, and froze them in the -80 freezer. Someone neglected to advise us that it would be better to have included protease inhibitor with the brains, but because we froze them immediately it shouldn't matter and the NMDA protein we're interested in is probably OK.

We've been in contact with Cloutman from Bemidji State University about his work with Dactylogyrus gill flukes on the shiners. I think he's in shock that someone else is interested in these flukes, and he's given us some good advice for which I'm grateful. Andrew and the Special Topics students (especially Selina and Alan) have worked on looking for more flukes in some preserved scarlet shiners, since there's a good chance they're an undescribed species. It's a dicey kind of work, looking for a small well-defined object in the midst of feathery gill filaments. The flukes we've found in the telescope shiners are almost certainly D. spatulus, which has been found in several local shiners including the striped shiners. And Andrew just informed me that he's found small, yolky eggs in the gut of a male telescope shiner we collected in January. They're <1 mm in diameter, so my initial guess is that they're snail eggs. But we'll see.