A Simple Approach to Manipulate Dissolved Oxygen for Animal Behavior Observations

The overall goal of this method is to effectively and incrementally manipulate dissolved oxygen or DO conditions in a laboratory setting. To allow the observation of aquatic animal behavioral responses. The neat thing about this method is it can have application for a number of animal behavior experiments.

For both vertebrates and invertebrates. The main advantage to this technique is it can be both implemented for classroom teaching or for research purposes. At a selected field site, within an hour's transit of the laboratory, used stander kick net procedures to collect at least 35 stone flies.

From the stream, collect four liters of stream water per aquarium. Plus an additional three liters per experiment. As well as several rocks with a maximum diameter of two centimeters.

Back at the lab, distribute the rocks into aquariums placed in a refrigerator, set the the stream site temperature. And add four liters of stream water to each aquarium. Then, add 20 to 30 stoneflies to each aquarium.

And place a bubbling stone attached to an aquarium bubbler, into each tank to continuously add room air to the water. After two days, connect a standard wall vacuum tube to the side arm of a two liter side arm flask. And fill the flask with one point nine liters of 12 degrees Celsius stream water.

Place the flask and tubing on a tray large enough to hold an ice bath without obscuring the view of the interior of the side arm flask. And fill the tray with ice. Insert a DO probe into one three millimeter hole of a custom prepared rubber stopper.

And a two millimeter diameter copper pipe equipped with a three millimeter male hose barb coupler through another a three millimeter hole. Until the length of the pipe from the bottom of the stopper is within 10 centimeters of the bottom of the flask. Connect a zero point seven meter, thin walled polyethylene, three millimeter diameter gas tube to the coupler.

Check for a secure seal between the stopper and the flask as well as a snug fit between the pipe and the probe wire within the stopper. It is critical that stoppers and tubing are tightly sealed to maintain an equal pressure environment in the vessel throughout the experiment and to prevent nitrogen leakage from the vessel. Place the polyethylene tube into a one liter flask containing zero point four liters of tap water and tape the tubing to keep the ends submerged throughout the experiment.

Then, connect the three millimeter diameter gas line from the vacuum flask to an aquarium room air bubbler. And turn on the bubbler to oxygenate the water in the two liter flask with the ambient oxygen. Use the DO probe to monitor the water temperature and DO concentration for about five minutes.

Until an equilibrium of the DO is established within the chamber. Before adding the stoneflies, place three to four rocks into the two liter flask so that the insects have a sub straight conducive to push ups. Then began a trial manipulation of the DO by disconnecting the gas tube from the bubbler and attaching it the nitrogen gas line.

Start bubbling the nitrogen at 20 cubic feet per hour for approximately 40 to 60 seconds. Ceasing the flow of nitrogen immediately upon reaching the target concentration. Use the aquarium room air bubbler to return to the target concentration if the DOD creases below the target.

If the DO is unstable during the testing, check that the water volume is still at one point nine liters and that no water has bubbled out. The water temperature is stable, and the seals on all of the fittings are closed tightly. After re equilibriating the set up, add an equal number of stoneflies as there as observers to the flask.

And reseal the flask with a rubber stopper. Bubble the water as just demonstrated until a target concentration of 10 milligrams per liter is reached. Then, record the starting water temperature.

And allow the stoneflies to attach to the rock sub straight in the flask. Each observer will then count and record the number of push ups displayed by an individual stonefly over three minutes. Then, manipulate the DO to the next experimental level and repeat the three minute observation.

In this figure, the average number of push ups performed within a DO level, and within each of the six trials was pulled to plot the push ups against the DO level. For these experiments, only the DO concentration significantly influenced the number of push ups performed by stoneflies. With no other variable or interaction, proving a significant predicator of the push up number.

Once mastered, this technique can be reproduced to meet the specific objectives set forth by the researcher. While attempting this procedure, it is important to avoid changes in temperature, as this could result in changes in DO.With this procedure, other methods, like those that would change PH, can used to answer addition questions about the changes of PH and DO on aquatic organisms. After watching this video, you should have the ability to perform manipulation of dissolved oxygen in a laboratory setting for a number of animal behavior experiments.