The Authors would first like to acknowledge all students from the freshman Biology 121- Ecology Module lab at Juniata College for their help in generating data used in this study. We would also like to thank Dr. Randy Bennett, Chris Walls, Sherry Isenberg, and Taylor Cox for their assistance in acquiring materials necessary to develop this methodology. Additionally, we would like to thank Dr. Norris Muth and Dr. John Unger for their advice on methodological development and Dr. Jill Keeney and the Biology department for their support of this endeavor. We would also like to thank the anonymous reviewers that have helped to shape and focus this manuscript.&#160; Last but not least, I&#39;d like to thank Hudson Grant for his help with the initial stonefly collection for use in development of this technique

Note: This experiment did not use vertebrates and therefore did not require approval by Juniata College&#39;s Institute for Animal Care and Use Committee. However for individuals adapting this method for use with vertebrates, IACUC approval should be sought.
1. Field Sample Collection
<ol>
	<li>Determine and evaluate potential field sites for the ability to collect, store, and transport stoneflies quickly to minimize time in transit with a maximum recommended time in transit of 1 hr.</li>
	<li>Pe...

<ol>
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Section 6.2 DISSOLVED OXYGEN.</a> National Field Manual for the Collection of Water-Quality Data. , (2013).</li><li>Hem, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Study and Interpretation of the Chemical Characteristics of Natural. , (1985).</li><li>Burggren, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=34;Air+Gulping&amp;#34;+Improves+Blood+Oxygen+Transport+during+Aquatic+Hypoxia+in+the+Goldfish+Carassius+auratus.">34;Air Gulping&amp;#34; Improves Blood Oxygen Transport during Aquatic Hypoxia in the Goldfish Carassius auratus.</a> Physiol Zool. 55 (4), 327-334 (2015).</li><li>Frederic, H., Mathieu, J., Garlin, D., Freminet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral,+Ventilatory,+and+Metabolic+Responses+to+Severe+Hypoxia+and+Subsequent+Recovery+of+the+Hypogean+Niphargus+rhenorhodanensis+and+the+Epigean+Gammarus+fossarum+(Crustacea:+Amphipoda).">Behavioral, Ventilatory, and Metabolic Responses to Severe Hypoxia and Subsequent Recovery of the Hypogean Niphargus rhenorhodanensis and the Epigean Gammarus fossarum (Crustacea: Amphipoda).</a> Physiol Zool. 68 (2), 223-244 (2015).</li><li>Ultsch, G., Duke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+Exchange+and+Habitat+Selection+in+the+Aquatic+Salamanders+Necturus+maculosus+and+Cryptobranchus+alleganiensis.">Gas Exchange and Habitat Selection in the Aquatic Salamanders Necturus maculosus and Cryptobranchus alleganiensis.</a> Oecologia. 83 (2), 250-258 (1990).</li></ol>

The authors declare that they have no competing financial interests.

Critical steps 
This procedure provides a simple and efficient way to manipulate DO in a laboratory setting to perform behavioral studies on aquatic organisms. We found there to be several critical steps/items to be aware of when performing this experiment that directly related to the outcomes. Within a trial, it is critical to maintain the chamber pressure to avoid changes in the partial pressure of gasses above the water, and subsequent DO fluctuations. Following the steps outlined in the &#34;testing the stabilit...

Dissolved oxygen (DO) is a key physiochemical parameter important in mediating a number of biological and ecological processes within aquatic ecosystems. Exposures to acute and chronic sub-lethal hypoxia reduce growth rates in certain aquatic insects and reduce the survival of insects exposed1. This protocol was developed to provide a controlled method to manipulate DO levels in stream water to observe the effects on animal behavior. Since all aerobic aquatic organisms&#39; survival depends on the oxygen concentration in order to live and reproduce, changes in the concentration of DO are often reflected in behavioral changes by organisms. More mobile aquatic invertebrates and fish have been observed to respond to low oxygen concentrations (hypoxic) by seeking locales with higher DO2,3. For less mobile aquatic organisms, behavioral adaptations to increase intake of DO may be the only viable option. The aquatic macroinvertebrate order of Plecoptera (stonefly) has been noted to perform &#34;push-up&#34; movements to increase the flow of water, and uptake of oxygen, across their external gills4-6. These adaptive behaviors have been observed in natural environments and in laboratory experiments.
Laboratory manipulation of DO in water opens up significant opportunities for animal behavior studies, but significant gaps in methodological deployment exist. For example, one study used large aquaria to evaluate the physiological response time of Largemouth bass (Micropterus salmoides) to hypoxic environments following gassing with nitrogen, but scant detail is given for the methodology7. Another study performed on Zebra fish (Danio rerio) described using nitrogen gas and a porous stone to deliver gas to water and reduce the DO of the water8. For chemistry-based applications, methods for degasification of solvents utilize specialized apparatus9-11 to remove oxygen from solvents, but would not be suitable for animal behavior studies. While these studies employ methods to remove oxygen from water, no descriptive method could be identified that would allow for evaluation of animal behavior in response to DO changes.
This method described hereafter is an attempt to fully describe a protocol for manipulation of DO of water by using nitrogen gas. Further, this method was developed towards observing relationships between stonefly behavior (pushups) and DO that was employed in a freshman-level biology laboratory. One of the main benefits of this method is that it can easily be performed within a laboratory with common glassware and materials accessible to most secondary and higher education institutions. The protocol is also easily adaptable, allowing for individuals to scale the procedure to meet the objectives set forth for research or teaching applications.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Filter flask 2 L</td>
			<td style="width:163px;">Pyrex</td>
			<td style="width:119px;">5340</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rubber Stopper size 6</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z164534</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nalgene 180 Clear Plastic Tubing</td>
			<td style="width:163px;">Thermo Scienfitic</td>
			<td>8001-1216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Whisper 60 air pump</td>
			<td style="width:163px;">Tetra</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Standard flexible Air line tubing</td>
			<td style="width:163px;">Penn Plax</td>
			<td>ST25</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">0.25 inch Copper tubing</td>
			<td style="width:163px;">Lowes Home Improvement</td>
			<td align="right">23050</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Male hose barb</td>
			<td style="width:163px;">Grainger</td>
			<td style="width:119px;">5LWH1</td>
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			<td height="21" style="height:21px;width:216px;">Female Connector</td>
			<td style="width:163px;">Grainger</td>
			<td style="width:119px;">20YZ22</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Heavy Duty Dissolved Oxygen Meter</td>
			<td style="width:163px;">Extech</td>
			<td style="width:119px;">407510</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:216px;">Nitrogen gas</td>
			<td style="width:163px;">Matheson TRIGAS</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Radnor AF150-580 Regulator</td>
			<td style="width:163px;">Airgas</td>
			<td style="width:119px;">RAD64003036</td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple approach to manipulate dissolved oxygen for animal behavior observations

The ability to manipulate dissolved oxygen (DO) in a laboratory setting has significant application to investigate a number of ecological and organismal behavior questions. The protocol described here provides a simple, reproducible, and controlled method to manipulate DO to study behavioral response in aquatic organisms resulting from hypoxic and anoxic conditions. While performing degasification of water with nitrogen is commonly used in laboratory settings, no explicit method for ecological (aquatic) application exists in the literature, and this protocol is the first to describe a protocol to degasify water to observe organismal response. This technique and protocol were developed for direct application for aquatic macroinvertebrates; however, small fish, amphibians, and other aquatic vertebrates could be easily substituted. It allows for easy manipulation of DO levels ranging from 2 mg/L to 11 mg/L with stability for up to a 5 min animal-observation period. Beyond a 5 min observation period water temperatures began to rise, and at 10 min DO levels became too unstable to maintain. The protocol is scalable to the study organism, reproducible, and reliable, allowing for rapid implementation into introductory teaching labs and high-level research applications. The expected results of this technique should relate dissolved oxygen changes to behavioral responses of organisms.

Six trials of the described setup were performed by 24 freshmen undergraduate students in a teaching laboratory setting to quantify the number of push-ups stoneflies perform in response to different DO concentration in water. The average number of push-ups performed within a DO level and within each trial was pooled to plot push-ups against the DO level in Figure 2. An ANOVA was performed initially utilizing DO concentration, sequential order of trials, temperature, as we...

Watch this Scientific Journal Video about A Simple Approach to Manipulate Dissolved Oxygen for Animal Behavior Observations at JoVE.com

A Simple Approach to Manipulate Dissolved Oxygen for Animal Behavior Observations

This article describes a simple and reproducible protocol to manipulate dissolved oxygen conditions in a laboratory setting for animal behavior studies. This protocol may be used in both teaching and research laboratory settings to evaluate organismal response of macroinvertebrates, fishes, or amphibians to changes in dissolved oxygen concentration.

The ability to manipulate dissolved oxygen (DO) in a laboratory setting has significant application to investigate a number of ecological and organismal ...

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.

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9.2K visualizações.  Juniata College. 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, w...