Coupled with morphology and molecular genetics, flow cytometry may make sturgeon species identification more accurate.

Examples of histograms, scatter plots and countour plots used in analyzing high resolution genetic content of sturgeon blood cells.

Patty Herman draws blood from a suspected hybrid sturgeon. STEP student Jeremiah Smith assists.

It all started with a simple request to our facility. Would you send blood from any sturgeon you catch? The answer was equally simple for our FWS crew – of course! Now for the complicated part…Why?

Dr. Jill Jenkins, with U.S. Geological Survey in Lafayette, LA, is testing a method for the ability to determine if a sturgeon belongs to a single species (i.e. shovelnose or pallid) or if it is some combination of the two (hybrid). Fisheries biologists, particularly in the middle and lower Mississippi River Basins, could use a quick, definitive way to distinguish these animals, due to their similarity of appearance, especially when they are juveniles. Dr. Jenkins is conducting high resolution DNA analysis of sturgeon blood to determine if these species really do have differences in their genomic content. Simply put, it is how much DNA is in their cells. Because fish, bird, amphibian, and reptilian red blood cells have a nucleus, the DNA is easily stained and measurable with a flow cytometer – the type of instrument used in hospitals for assessing your blood. So now, for the next logical question: What is this instrumentation and how does it work?

Flow cytometry is analogous to a scanner checkout at a store, where items are placed on the conveyor belt, passed singly over the scanner, and their relative dollar amounts recorded. With flow cytometry, where cells are passed one-at-a-time in front of an interrogating laser beam, the tiny amounts of light scattered generate information that is sensitively assessed and recorded. So, nuclear DNA stained with fluorescent dyes (flourochromes) intercepts the laser light, the dye particles are excited proportional to the amount of DNA present, and this is measured by optical detectors responsible for converting the emitted light into amplified electrical pulses. A pinpoint of blood will provide tens of thousands of subsamples from one animal, attesting to the statistical power of the data collected with this instrument.

The results are graphed to aid in the interpretation of the data, where DNA fluorescence can be plotted in a histogram. All experimental blood samples are run alongside a control or standard that has a known genomic size and specific amount of DNA fluorescence. Because each animal species has a specific amount of nuclear DNA, or genomic content, this will appear as a single, distinct “peak” on the histogram. Therefore, a double peak could indicate a mosaic, or hybrid fish.

Who is involved? Dr. Jenkins, supported by the USGS Science Support Partnership Program, has partnered with USFWS offices in the Great Plains, Interior Highlands and Coastal Plain Management Units of the Pallid Sturgeon Recovery Plan. She is asking us, as well as partners in the upper and lower Mississippi River Basins, to provide arterial blood samples from 10 each of pallid, shovelnose and hybrid sturgeon. Typically done by our crew, morphometric and meristic characters are measured for each fish, a Character Index value is calculated, pictures are taken of the ventral side of the head, and a caudal fin tissue is taken for future molecular genetic analysis by Dr. Ed Heist, Southern Illinois University – Carbondale. For Dr. Jenkins, a thimbleful of blood is collected into anticoagulant and shipped overnight for processing within 48 hours. The utility of three preservatives is being researched, with the hope that blood could be stored before shipping for flow cytometric analysis. This would alleviate logistics for field collections, reduce shipping costs by shipping samples collected over a season, and allow the long-term storage of blood for future flow cytometric analysis. These laboratory data are being compared with the available photographs and data on morphometrics by Dr. Jan Dean, Natchitoches National Fish Hatchery.

What have we learned? Currently, this technique, the very best to use for DNA content analyses, is working fine. Can the species be distinguished yet? Not yet, as this is the time for acquiring the data. Thus far, Dr. Jenkins has detected differences among animals, and has discovered unique DNA profiles that will provide discussion topics for geneticists. If distinct DNA content exists between pallids and shovelnose sturgeon, this technology will find it. Along with professional field crew determinations, Dr. Jenkins envisions the intersection of current technologies as the best way to diagnose species. Ultimately, the intention is to add these additional data per fish to compare and contrast species identification by the methods, then in the future, compare these results with available molecular genetics data.

What does this mean for pallid sturgeon? Implications for the pallid sturgeon may be far ranging. Distinguishing between pallid sturgeon and hybrid sturgeon can often be difficult to even the most seasoned field biologist. Although we use morphometric and meristic characters to differentiate in the field, these measures can be problematic. Molecular methods currently employed to genetically distinguish between species is slow, expensive and is not indisputable. Using high resolution DNA analysis by flow cytometry, in comparison, is inexpensive and fast and would be a great additional layer of data to incorporate into the suite of tests already being utilized. Accurate species identification is imperative for endangered species long-term recovery and management. Coupled with spatial data, the flow cytometric technology would be useful in determining distinct geographical regions for wild pallid sturgeon and hybrids throughout their ranges. Because hybrid sturgeon are found only in the middle and lower Mississippi River Basins, genetic data is of particular importance. Additionally, high resolution DNA analysis by flow cytometry would also be important for spawning and propagation of pallid sturgeon. This technique could be implemented to insure that broodstock pallids are of unquestionable heritage and the resulting progeny will not dilute the gene pool when stocked into the Missouri River.

Just halfway through the project, these results are proving to be intriguing. Such data have never been generated before with sturgeon, and the profiles are unique in the animal kingdom. This project is unique in that it is building lasting partnerships and using state-of-the-art management techniques in an effort to manage and conserve an interjurisdictional species.

Co-authored by Patty Herman, USFWS Columbia National Fish & Wildlife Conservation Office, Columbia, MO and Jill Jenkins, USGS National Wetlands Research Center, Lafayette, LA