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A Simple Approach to Manipulate Dissolved Oxygen for Animal Behavior Observations

Published: June 28th, 2016



1Department of Biology, Juniata College

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 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.

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' 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 aquati....

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Note: This experiment did not use vertebrates and therefore did not require approval by Juniata College'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

  1. 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.
  2. Pe.......

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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.......

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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 "testing the stabilit.......

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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.  Last but....

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Name Company Catalog Number Comments
Filter flask 2 L Pyrex 5340
Rubber Stopper size 6 Sigma-Aldrich Z164534
Nalgene 180 Clear Plastic Tubing Thermo Scienfitic 8001-1216
Whisper 60 air pump Tetra N/A
Standard flexible Air line tubing Penn Plax ST25
0.25 inch Copper tubing Lowes Home Improvement 23050
Male hose barb Grainger 5LWH1
Female Connector Grainger 20YZ22
Heavy Duty Dissolved Oxygen Meter Extech 407510
Nitrogen gas Matheson TRIGAS N/A
Radnor AF150-580 Regulator Airgas RAD64003036

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