A Simple Approach to Manipulate Dissolved Oxygen for Animal Behavior Observations

Summary

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.

Abstract

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.

Introduction

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 aquatic invertebrates and fish have been observed to respond to low oxygen concentrations (hypoxic) by seeking locales with higher DO^2,3. 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 "push-up" movements to increase the flow of water, and uptake of oxygen, across their external gills^4-6. 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 apparatus^9-11 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.

Protocol

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. Perform kick-net sampling at the selected field site following standard kick-net procedures enough times to collect at least 35 stoneflies¹².
3. Collect 50 L of stream water and rocks with a maximum diameter of 2 cm from streams.
4. Place aquariums in a refrigerator set to the temperature of the stream site. Distribute rocks collected at the stream site into aquariums and fill with 4 L of stream water per aquarium. Place 20-30 collected stoneflies per aquarium and place a bubbling stone attached to an aquarium bubbler into each tank and turn on bubblers to continuously add room air to the water.
5. Allow the stoneflies to adjust to the new environment in the aquariums for a 48 hr period.

Figure 1. Set up for dissolved oxygen manipulation. (A) 1) Fitting for copper pipe to male hose barb 2) Location of stopper seal to examine for ensuring well sealing flask. (B) 1) 2 L side-arm flask filled with 1.9 L of water 2) Gas tube and air bubbler (blue) for use in nitrogen bubbling and room air bubbling, respectively 3) Nitrogen tank and gauged values 4) 2 L flask filled with 0.4 L of water with vacuum tube submerged 5) Dissolved oxygen meter. Please click here to view a larger version of this figure.

2. Experimental Set up

1. On a bench top, connect a standard walled vacuum tube to the side arm of a 2 L side-arm flask as is shown (1 in Figure 1B).
2. Fill the flask with 1.9 L of stream water from 3 L plastic containers holding collected stream water in refrigerator set to 12 °C.
3. Place the flask and tubing on a tray large enough to hold an ice bath around the side-arm flask without obscuring the view of the flask interior and fill the tray with ice.
4. Drill two 3 mm diameter holes in a rubber stopper to allow the passage of 1) a copper pipe to deliver the gas to the vessel and 2) the probe of a DO meter into the 2 L side-arm flask (1 in Figure 1B).
5. Make a lateral incision from the edge of the stopper to one of the holes to allow seating of the wire of the DO probe into the stopper.
6. Connect a coupler with a 3 mm male hose barb to a piece of 2 mm diameter copper pipe (1 in Figure 1A). Ensure that this pipe is long enough to reach to within 10 cm of the bottom of the flask while reaching through the stopper.
7. Place the pipe with coupler though the second hole in the stopper until the length from the bottom of the stopper is enough to reach to within 10 cm of the bottom of the flask.
8. Connect a 0.75 m length, thin walled polyethylene gas tube with a diameter of 3 mm to the coupler on the pipe.
9. Slide both the DO probe and copper pipe into the flask and seal the flask with the stopper.
10. 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.
11. Fill a 1 L flask with 0.4 L of tap water and place adjacent to the tray with the ice bath and vacuum flask.
12. Submerge the polyethylene tube coming from the large vacuum flask into the water of the 1 L flask. Secure the tube with tape such that it will remain submerged through the experiment.
13. Connect the 3 mm diameter gas line from the vacuum flask to an aquarium room-air bubbler. Begin to bubble the water in the 2 L flask by plugging in the aquarium bubbler, which introduces room air and oxygen to the water.
14. Monitor the DO concentration and temperature of the water with the DO meter for 5 min or until equilibrium of DO is established within the chamber such that little change in DO is occurring.

3. Testing the Stability of the Experimental Set Up

1. Test each setup for DO stability prior to the addition of stoneflies.
2. Add three or four rocks to the 2 L flask so that stoneflies have substrate conducive for pushups.
3. Begin a trial manipulation of DO by disconnecting the gas tube from the bubbler and attaching it to the nitrogen gas line.
4. Start bubbling nitrogen at 20 cubic feet per hr (CFH) for approximately 40 sec to 1 min.
5. Once the DO has dropped to within 0.5 mg/L of the target concentration, reduce the flow to 15 CFH and allow the concentration to decrease to the target.
6. Cease flow of nitrogen immediately once the target concentration is reached.
7. Use the aquarium room-air bubbler to return the concentration to the target concentration if the DO decreases below the target.
8. If the DO is unstable during the testing of a set-up then check the water volume is still at 1.9 L and no water has bubbled out, water temperature is stable and not changing, and seals on all fittings appear to be tight and sealed.
9. Once three trials have been performed and the experimenter has confidence in the ability to control DO, attach the gas line to the bubbler and bubble to equilibrium again.
10. Bubble to equilibrium by attaching the 3 mm diameter gas line to the aquarium bubbler and starting the addition of room air to the water until the concentration of oxygen in the water does not increase or change for 3 min.
11. Once at equilibrium, stop bubbling and unseal the flask.

4. Stonefly Push-up Experiment

1. Divide the total number of stoneflies by the number of observers to determine the number of trials to perform.
2. Determine different DO levels between 2 and 10 mg/L to evaluate the behavioral response of stoneflies (number of pushups).
3. Set up one flask per trial and add an equal number of stoneflies as there are observers to the flask (4 stoneflies within this design), place the probe and pipe back into the flask, then reseal the flask with the rubber stopper.
   Note: An initial DO concentration of 10 mg/L was chosen as the first observation point since it was the DO concentration of the stream from where the stoneflies were sampled.
4. Once the water is at 10 mg/L by bubbling following steps 2.10-2.11, record the starting water temperature and allow the stoneflies to attach to the rock substrate in the flask.
5. Assign only one observer to watch a single stonefly to ensure accurate counting of push-up behavior, which is the up and down body movement exhibited by the stonefly.
6. Count and record the number of push-ups observed over the course of a 3 min observation period.
7. Manipulate DO to the next experimental DO level and repeat 3 min observation period for the additional experimental levels.
   Note: Within this experimental design, three different DO levels were evaluated.

5. Statistical Analysis

1. To perform statistical analysis use average number of push-ups across the four stoneflies across a group for a given DO trial.
2. Use the free R statistical computing software¹² to perform an Analysis of Variance (ANOVA) on the number of push-ups and the DO concentrations using the order of each experimental trial (DO level) and temperature as covariates. Analyzed DO as discrete levels of a single factor.
3. Use an Anderson-Darling normality test on residuals to check for normality¹³.
4. Perform a linear regression on the data by plotting the mean number of push-ups against DO concentrations.

Results

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...

Discussion

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...

Materials

List of materials used in this article

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
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
Radnor AF150-580 Regulator	Airgas	RAD64003036