休眠復活オオミジンコ: プロトコルおよびアプリケーション

The goal of this experiment is to resurrect dormant stages of Daphnia magna species. This is a powerful method that dramatically advances data collection to generate long-term data in natural systems. This method enables the study of eco-evolutionary processes which occur over extended periods of time in nature, within the time frame of a research project.

The main advantage of this technique is that it provides unprecedented power in dissecting the processes and mechanisms of evolution if applied to populations that have experienced changes in selection pressure over time. Also, the methodology of resurrection for planktonic species have been frequently applied. So far they have been propagated among laboratories only by a directed knowledge transfer.

Visual demonstration of this method will allow non-experts to apply resurrection ecology by following a step-by-step guide, promoting the application of a powerful technology to facilitate the study of evolution. After obtaining a core tube of sample sediment from a lake or pond, use a flat metal tool to slice the core into horizontal one centimeter layers. Then, collect the sediment layers in separate bags.

The big bang core is designed to reduce disturbance of the sediment layers during sampling. When other cores are used, the outer ring of each sediment layer might be removed with a cookie cutter sort of blade to limit contamination among layers. Finally, store the sediment samples in a sealed cardboard box, in a dark room at four degrees Celsius.

First, weigh each sediment layer with a precision scale. Then, sieve each layer with two stacked geological sieves. The first sieve has a larger mesh size to remove larger substances from the sample sediment.

The second sieve has a finer mesh, which separates ephippia and other small particles from the remainder of the sample sediment. Move aliquots of the sediment collected by the finer mesh sieve to a white background tray. To facilitate separation of ephippia, add up to 200 milliliters of borehole water to the white tray to re-suspend the sediment fraction.

Inspect the sediment in the white tray for ephippia. Ephippia will be approximately one millimeter in size, and dark brown in color with a distinctive shape. Additional water may be added to the tray to dilute the sediment, and make visual inspection of ephippia easier.

Eye spotting of ephippia in sediment requires prior taxonomic knowledge of the species, as ephippia morphology is species specific. Use microdissection forceps to isolate individual ephippia from the tray, and transfer them to a Petri dish with 10 milliliters of borehole water. First, use microdissection forceps to transfer a single ephippia from the Petri dish to a glass slide under a stereo microscope.

Then, select a magnification that shows the entire chitin case in high definition. Use the microdissection forceps to hold the opposite ends of the ephippia. Then, apply gentle pressure by pushing the forceps towards each other along the ridge of the chitin case.

When the chitin case opens, stabilize the ephippia with one pair of forceps. With the second pair of forceps, gently expose the dormant embryo. Then, use the forceps to carefully remove the soft inner membrane surrounding the embryo.

To avoid breaking the egg cells, blunt end forceps may be used when separating the inner membrane from the embryos. Decapsulation using micro dissection forceps under a sterile microscope requires practice and steady hands. Once mastered, it can be achieved within a few hours.

Use a Pasteur pipette to transfer the dormant eggs to a clean Petri dish with 10 milliliters of borehole water. Dormant eggs from the same sediment layer can be collected in the same Petri dish. Finally, induce hatching by exposing the eggs to full spectrum, long day, photoperiod light and high temperature.

Under these conditions, properly decapsulated eggs should hatch between 48 hours and four weeks. To establish isoclonal lines, use a Pasteur pipette to transfer an individual Daphnia magna to separate jars. Resurrected Daphnia magna from the sedimentary archive of Lake Ring are used to assess an evolutionary response to warming.

Hatching success from the sedimentary archive ranged from 11 to 58%Hatchlings from three temporal subpopulations were exposed to experimental conditions representing the current temperature of Lake Ring, 18 degrees Celsius, and the temperature forecasted for the upcoming 100 years, 24 degrees Celsius. Growth rate, fecundity, size at maturity, and age at maturity showed plastic response to temperature. Whereas mortality was unresponsive.

Evolutionary differences between subpopulations are only observed via ANOVA in population growth rate, which significantly increased in two of the three subpopulations, as it can be seen from reaction norm points. Hatchlings from the three subpopulations were competed in experimental mesocosms kept at 24 degrees Celsius. After four weeks of selection, frequency of subpopulations does not change significantly from the start of the experiment.

After watching this video, you should have a good understanding of how to revive dormant Daphnia magna. While attempting this procedure, it's important to remember to clean instruments between steps to avoid cross-contamination among layers of sediment. It is advisable to handle soil and sediment samples in a dedicated lab space.

Resurrecting dormant populations enables researchers to study historical and modern populations in the same setting, effectively reconstructing evolution through time. The application of next generation sequencing technologies to revive the dormant stages provides unprecedented power in dissecting processes and mechanisms of evolution in populations that have experienced changes in selection pressure over time.