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>Perform kick-net sampling at the selected field site following standard kick-net procedures enough times to collect at least 35 stoneflies12.</li>
	<li>Collect 50 L of stream water and rocks with a maximum diameter of 2 cm from streams.</li>
	<li>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.</li>
	<li>Allow the stoneflies to adjust to the new environment in the aquariums for a 48 hr period.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/54430/54430fig1.jpg" /> 
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. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54430/54430fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Experimental Set up
<ol>
	<li>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).</li>
	<li>Fill the flask with 1.9 L of stream water from 3 L plastic containers holding collected stream water in refrigerator set to 12 &#176;C.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Connect a 0.75 m length, thin walled polyethylene gas tube with a diameter of 3 mm to the coupler on the pipe.</li>
	<li>Slide both the DO probe and copper pipe into the flask and seal the flask with the stopper.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Testing the Stability of the Experimental Set Up
<ol>
	<li>Test each setup for DO stability prior to the addition of stoneflies.</li>
	<li>Add three or four rocks to the 2 L flask so that stoneflies have substrate conducive for pushups.</li>
	<li>Begin a trial manipulation of DO by disconnecting the gas tube from the bubbler and attaching it to the nitrogen gas line.</li>
	<li>Start bubbling nitrogen at 20 cubic feet per hr (CFH) for approximately 40 sec to 1 min.</li>
	<li>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.</li>
	<li>Cease flow of nitrogen immediately once the target concentration is reached.</li>
	<li>Use the aquarium room-air bubbler to return the concentration to the target concentration if the DO decreases below the target.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Once at equilibrium, stop bubbling and unseal the flask.</li>
</ol>
4. Stonefly Push-up Experiment
<ol>
	<li>Divide the total number of stoneflies by the number of observers to determine the number of trials to perform.</li>
	<li>Determine different DO levels between 2 and 10 mg/L to evaluate the behavioral response of stoneflies (number of pushups).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Count and record the number of push-ups observed over the course of a 3 min observation period.</li>
	<li>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.</li>
</ol>
5. Statistical Analysis
<ol>
	<li>To perform statistical analysis use average number of push-ups across the four stoneflies across a group for a given DO trial.</li>
	<li>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.</li>
	<li>Use an Anderson-Darling normality test on residuals to check for normality13.</li>
	<li>Perform a linear regression on the data by plotting the mean number of push-ups against DO concentrations.</li>
</ol>

<ol>
	<li>Hoback, W., Stanley, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insects+in+hypoxia.">Insects in hypoxia.</a> J. Insect Physiol. 47 (6), 533-542 (2001).</li><li>Craig, J., Crowder, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-induced+habitat+shifts+and+energetic+consequences+in+Atlantic+croaker+and+brown+shrimp+on+the+Gulf+of+Mexico+shelf.">Hypoxia-induced habitat shifts and energetic consequences in Atlantic croaker and brown shrimp on the Gulf of Mexico shelf.</a> Mar Ecol-Prog Ser. 294, 79-94 (2005).</li><li>Gaulke, G., Wolfe, J., Bradley, D., Moskus, P., Wahl, D., Suski, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+and+Physiological+Responses+of+Largemouth+Bass+to+Rain-Induced+Reductions+in+Dissolved+Oxygen+in+an+Urban+System.">Behavioral and Physiological Responses of Largemouth Bass to Rain-Induced Reductions in Dissolved Oxygen in an Urban System.</a> T Am Fish Soc. 144 (5), 927-941 (2015).</li><li>Genkai-Kato, M., Nozaki, K., Mitsuhashi, H., Kohmatsu, Y., Miyasaka, H., Nakanishi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Push-up+response+of+stonefly+larvae+in+low-oxygen+conditions.">Push-up response of stonefly larvae in low-oxygen conditions.</a> Ecol Res. 15 (2), 175-179 (2000).</li><li>McCafferty, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Aquatic Entomology: The Fishermen's and Ecologists' Illustrated Guide to Insects and Their Relatives. , (1983).</li><li>Chapman, L., Schneider, K., Apodaca, C., Chapman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+ecology+of+macroinvertebrates+in+a+swamp-river+system+of+east+Africa.">Respiratory ecology of macroinvertebrates in a swamp-river system of east Africa.</a> Biotropica. 36 (4), 572-585 (2004).</li><li>Suski, C., Killen, S., Kieffer, J., Tufts, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+environmental+temperature+and+oxygen+concentration+on+the+recovery+of+largemouth+bass+from+exercise+implications+for+live+-+release+angling+tournaments.">The influence of environmental temperature and oxygen concentration on the recovery of largemouth bass from exercise implications for live - release angling tournaments.</a> J Fish Biol. 68, 120-136 (2006).</li><li>Abdallah, S., Thomas, B., Jonz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aquatic+surface+respiration+and+swimming+behaviour+in+adult+and+developing+zebrafish+exposed+to+hypoxia.">Aquatic surface respiration and swimming behaviour in adult and developing zebrafish exposed to hypoxia.</a> J Exp Biol. 218 (11), 1777-1786 (2015).</li><li>Gassmann, H., Chen, C., Vermot, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+and+apparatus+for+degassing+viscous+liquids+and+removing+gas+bubbles+suspended+therein.">Method and apparatus for degassing viscous liquids and removing gas bubbles suspended therein.</a> US patent. , (1974).</li><li>Berndt, M., Schomburg, W., Rummler, Z., Peters, R., Hempel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apparatus+for+degassing+liquids.">Apparatus for degassing liquids.</a> US patent. , (2001).</li><li>Sims, C., Gerner, Y., Hamberg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vacuum+degassing.">Vacuum degassing.</a> US patent. , (2002).</li><li>Barbour, M., Gerritsen, J., Snyder, B., Stribling, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Report+number+EPA+841-B-99-002.">Report number EPA 841-B-99-002.</a> Rapid bioassessment protocols for use in streams and wadeable rivers. , (1999).</li><li>Anderson, T., Darling, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Test+of+Goodness+of+Fit.">A Test of Goodness of Fit.</a> J Am Stat Assoc. 49 (268), 765-769 (1954).</li><li>Rounds, S., Wilde, F., Ritz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+A6+Field+Measurements.+Section+6.2+DISSOLVED+OXYGEN.">Chapter A6 Field Measurements. 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.

<table border="0" cellpadding="0" cellspacing="0" width="665">
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		<col />
		<col />
	</colgroup>
	<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Male hose barb</td>
			<td style="width:163px;">Grainger</td>
			<td style="width:119px;">5LWH1</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		<tr height="21">
			<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...

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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 ...

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9.2K Views.  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.

Video: A Simple Approach to Manipulate Dissolved Oxygen for Animal Behavior Observations

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is not an appropriate measure for its availability; bioavailability rather depends on the dynamic interplay of potential mass transfer (flux) of a compound to a microbial cell and the capacity of the latter to degrade the compound. In water-unsaturated parts of the soil, mycelia have been shown to overcome bioavailability limitations by actively transporting and mobilizing organic compounds over the range of centimeters. Whereas the extent of mycelia-based transport can be quantified easily by chemical means, verification of the contaminant-bioavailability to bacterial cells requires a biological method. Addressing this constraint, we chose the PAH fluorene (FLU) as a model compound and developed a water unsaturated model microcosm linking a spatially separated FLU point source and the FLU degrading bioreporter bacterium Burkholderia sartisoli RP037-mChe by a mycelial network of Pythium ultimum. Since the bioreporter expresses eGFP in response of the PAH flux to the cell, bacterial FLU exposure and degradation could be monitored directly in the microcosms via confocal laser scanning microscopy (CLSM). CLSM and image analyses revealed a significant increase of the eGFP expression in the presence of P. ultimum compared to controls without mycelia or FLU thus indicating FLU bioavailability to bacteria after mycelia-mediated transport. CLSM results were supported by chemical analyses in identical microcosms. The developed microcosm proved suitable to investigate contaminant bioavailability and to concomitantly visualize the involved bacteria-mycelial interactions.

A whole cell bioreporter assay with Burkholderia sartisoli RP037-mChe was developed to detect fractions of an organic contaminant (i.e., fluorene) available for bacterial degradation after active transport by mycelia bridging air-filled pores in a water unsaturated model system.

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is ...

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

How to Create Conditioned Taste Aversion for Grazing Ground Covers in Woody Crops with Small Ruminants

Conditioned taste aversion (CTA) is a learning behavior process where animals are trained to reject certain feed after gastrointestinal discomfort has been produced. Lithium chloride (LiCl) is the preferred agent used in livestock to induce CTA because it specifically stimulates the vomit center. In addition, LiCl is commercially available, and easy to prepare and administer using a drenching gun. Nevertheless, some factors have to be considered to obtain an effective long-lasting CTA, which allows small ruminants to graze during the cropping season. A key aspect is to use animals with no previous contact with the target plant (the plant chosen to be avoided; new feed). Due to their native neophobic feeding behavior, small ruminants can easily associate the negative feedback effects with the new feed, resulting in a strong and persistent CTA. The recommended doses are 200 and 225 mg LiCl/kg body weight (BW) for goats and sheep, respectively. To induce CTA, 100 g of the target plant should be individually offered for at least 30 min, and LiCl administered thereafter if the intake is greater than 10 g. Each time the animal eats the target plant without negative consequences, the CTA becomes weaker. Consequently, to minimize the risk of target plant consumption, it is essential to have sufficient palatable ground cover available. The presence of an alternative feed (of quality and quantity) prevents the accidental consumption of the target plant. A close monitoring of the flock is recommended to remove and re-dose any animal consuming more than 4 bites or 10 g of the target plant. At the beginning of each grazing season, check the CTA status of each animal before moving them to the crop.

Feeding behavior preferences in livestock can be modified to implement a grazing management plan in woody crops. Here, we present a protocol to show a lithium chloride dose after eating a new plant that induces conditioned taste aversion.

Conditioned taste aversion (CTA) is a learning behavior process where animals are trained to reject certain feed after gastrointestinal discomfort has ...

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in nutrient and gas cycles. After cores with intact sediment-water interfaces are collected, they are submerged in incubation tanks and kept under aerobic conditions at in situ temperatures. To initiate a time course of overlying water chemistry, cores are sealed without bubbles using a top cap with a suspended stirrer. Time courses of 4-7 sample points are used to determine the rate of sediment water exchange. Artificial illumination simulates day-time conditions for shallow photosynthetic sediments, and in conjunction with dark incubations can provide net exchanges on a daily basis. The net measurement of N2 is made possible by sampling a time course of dissolved gas concentrations, with high precision mass spectrometric analysis of N2:Ar ratios providing a means to measure N2 concentrations. We have successfully applied this approach to lakes, reservoirs, estuaries, wetlands and storm water ponds, and with care, this approach provides valuable information on biogeochemical balances in aquatic ecosystems.

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

Here, we present small core incubations for the measurement of sediment-water gas and solute exchange. These will provide reliable measurements of sediment-water exchange that assess the role of sediment in influencing biological and biogeochemical processes in aquatic ecosystems.

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in ...

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and detritus, and excreting particulate and dissolved compounds, they serve as important agents for benthic-pelagic coupling. Accurately measuring the compounds removed and excreted by suspension feeders (such as sponges, ascidians, polychaetes, bivalves) is crucial for the study of their physiology, metabolism, and feeding ecology, and is fundamental to determine the ecological relevance of the nutrient fluxes mediated by these organisms. However, the assessment of the rate by which suspension feeders process particulate and dissolved compounds in nature is restricted by the limitations of the currently available methodologies. Our goal was to develop a simple, reliable, and non-intrusive method that would allow clean and controlled water sampling from a specific point, such as the excurrent aperture of benthic suspension feeders, in situ. Our method allows simultaneous sampling of inhaled and exhaled water of the studied organism by using minute tubes installed on a custom-built manipulator device and carefully positioned inside the exhalant orifice of the sampled organism. Piercing a septum on the collecting vessel with a syringe needle attached to the distal end of each tube allows the external pressure to slowly force the sampled water into the vessel through the sampling tube. The slow and controlled sampling rate allows integrating the inherent patchiness in the water while ensuring contamination free sampling. We provide recommendations for the most suitable filtering devices, collection vessel, and storing procedures for the analyses of different particulate and dissolved compounds. The VacuSIP system offers a reliable method for the quantification of undisturbed suspension feeder metabolism in natural conditions that is cheap and easy to learn and apply to assess the physiology and functional role of filter feeders in different ecosystems.

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

We introduce the VacuSIP, a simple, non-intrusive, and reliable method for clean and accurate point sampling of water. The system was developed and evaluated for the simultaneous collection of the water inhaled and exhaled by benthic suspension feeders in situ, to cleanly measure removal and excretion of particulate and dissolved compounds.

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and ...

Understanding Dissolved Organic Matter Biogeochemistry Through In Situ Nutrient Manipulations in Stream Ecosystems

Dissolved organic matter (DOM) is a highly diverse mixture of molecules providing one of the largest sources of energy and nutrients to stream ecosystems. Yet the in situ study of DOM is difficult as the molecular complexity of the DOM pool cannot be easily reproduced for experimental purposes. Nutrient additions to streams however, have been shown to repeatedly alter the in situ and ambient DOM pool. Here we demonstrate an easily replicable field-based method for manipulating the ambient pool of DOM at the ecosystem scale. During nutrient pulse experiments changes in the concentration of both dissolved organic carbon and dissolved organic nitrogen can be examined across a wide-range of nutrient concentrations. This method allows researchers to examine the controls on the DOM pool and make inferences regarding the role and function that certain fractions of the DOM pool play within ecosystems. We advocate the use of this method as a technique to help develop a deeper understanding of DOM biogeochemistry and how it interacts with nutrients. With further development this method may help elucidate the dynamics of DOM in other ecosystems.

Understanding Dissolved Organic Matter Biogeochemistry Through In Situ Nutrient Manipulations in Stream Ecosystems

Dissolved organic matter provides an important source of energy and nutrients to stream ecosystems. Here we demonstrate a field-based method to manipulate the ambient pool of dissolved organic matter in situ through easily replicable nutrient pulses.

Dissolved organic matter (DOM) is a highly diverse mixture of molecules providing one of the largest sources of energy and nutrients to stream ecosystems. ...

Basic Research in Plasma Medicine - A Throughput Approach from Liquids to Cells

In plasma medicine, ionized gases with temperatures close to that of vertebrate systems are applied to cells and tissues. Cold plasmas generate reactive species known to redox regulate biological processes in health and disease. Pre-clinical and clinical evidence points to beneficial effects of plasma treatment in the healing of chronic ulcer of the skin. Other emerging topics, such as plasma cancer treatment, are receiving increasing attention. Plasma medical research requires interdisciplinary expertise in physics, chemistry, and biomedicine. One goal of plasma research is to characterize plasma-treated cells in a variety of specific applications. This includes, for example, cell count and viability, cellular oxidation, mitochondrial activity, cytotoxicity and mode of cell death, cell cycle analysis, cell surface marker expression, and cytokine release. This study describes the essential equipment and workflows required for such research in plasma biomedicine. It describes the proper operation of an atmospheric pressure argon plasma jet, specifically monitoring its basic emission spectra and feed gas settings to modulate reactive species output. Using a high-precision xyz-table and computer software, the jet is hovered in millisecond-precision over the cavities of 96-well plates in micrometer-precision for maximal reproducibility. Downstream assays for liquid analysis of redox-active molecules are shown, and target cells are plasma-treated. Specifically, melanoma cells are analyzed in an efficient sequence of different consecutive assays but using the same cells: measurement of metabolic activity, total cell area, and surface marker expression of calreticulin, a molecule important for the immunogenic cell death of cancer cells. These assays retrieve content-rich biological information about plasma effects from a single plate. Altogether, this study describes the essential steps and protocols for plasma medical research.

A high-throughput cold physical plasma treatment protocol of liquids and cells is shown. It involves setting up different feed gas compositions for plasma ignition, measuring the plasma&#39;s emission spectra, and the subsequent analysis of liquids and cellular activity after plasma treatment.

In plasma medicine, ionized gases with temperatures close to that of vertebrate systems are applied to cells and tissues. Cold plasmas generate reactive ...

A Simple Planting Technique for Re-establishing Trees Where Frequent Inundation Occurs

Many of the Everglades tree islands have lost elevation over the past century and most of their trees have died such that they are now covered with herbaceous plants. This protocol describes a simple, cost-effective tree planting technique needed for restoring degraded Everglade tree islands. The design is patterned after a natural Everglades process that creates floating peat islands, which allows tree survival and growth in flooded conditions and often leads to the development of tree islands. Commercially available peat bags were used as the medium for the growth and establishment of potted native tree saplings. The pop-up configuration floated initially and provided additional elevation to minimize inundation, with a single native tree species sapling and a single tree fertilizer spike. During a 3 year study involving 105 pop-ups, most plants survived (80%) and many thrived. Determining whether this technique can establish trees on a degraded tree island will require longer studies and extensive field tests.

This protocol describes a simple, cost-effective tree planting technique for restoring degraded Everglades tree islands that experience inundation. The design creates an island (pop-up) which floats initially and adds elevation to promote tree survival and growth under flooded conditions.

Many of the Everglades tree islands have lost elevation over the past century and most of their trees have died such that they are now covered with ...

Lab-Scale Model to Evaluate Odor and Gas Concentrations Emitted by Deep Bedded Pack Manure

A lab-scaled simulated bedded pack model was developed to study air quality and nutrient composition of deep-bedded packs used in cattle mono-slope facilities. This protocol has been used to effectively evaluate many different bedding materials, environmental variables (temperature, humidity), and potential mitigation treatments that can improve air quality in commercial deep-bedded mono-slope facilities. The model is dynamic and allows researchers to easily collect many chemical and physical measurements from the bedded pack. Weekly measurements, collected over the course of six to seven weeks, allows sufficient time to see changes in air quality measurements over time as the bedded pack matures. The data collected from the simulated bedded packs is within the range of concentrations previously measured in commercial deep-bedded mono-slope facilities. Past studies have demonstrated that 8 - 10 experimental units per treatment are sufficient to detect statistical differences among the simulated bedded packs. The bedded packs are easy to maintain, requiring less than 10 minutes of labor per bedded packs per week to add urine, feces, and bedding. Sample collection using the gas sampling system requires 20 - 30 minutes per bedded pack, depending on the measurements that are being collected. The use of lab-scaled bedded packs allows the researcher to control variables such as temperature, humidity, and bedding source that are difficult or impossible to control in a research or commercial facility. While not a perfect simulation of &#34;real-world&#34; conditions, the simulated bedded packs serve as a good model for researchers to use to examine treatment differences among bedded packs. Several lab-scale studies can be conducted to eliminate possible treatments before trying them in a research or commercial-sized facility.

A protocol has been developed to measure gases, odors, and nutrient composition in lab-scaled bedded manure packs, which can be used to study ways to improve air quality in commercial cattle facilities using deep-bedded manure packs.

A lab-scaled simulated bedded pack model was developed to study air quality and nutrient composition of deep-bedded packs used in cattle mono-slope ...

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more streamlined herbivory protocols. Perturbations of chewing insects are usually studied in whole plant systems. While this whole organism strategy is popular, it is not necessary if similar observations can be replicated in a single detached leaf. The assumption is that basic elements required for signal transduction are present within the leaf itself. In the case of early events in signal transduction, cells need only to receive the signal from the perturbation and transmit that signal to neighboring cells which are assayed for gene expression.
The proposed method simply changes the timing of the detachment. In whole plant experiments, larvae are confined to a single leaf which is eventually detached from the plant and assayed for gene expression. If the order of excision is reversed, from last in whole plant studies, to first in the detached study, the feeding experiment is simplified.
Solanum tuberosum var. Kennebec is propagated by nodal transfer in a simple tissue culture medium and transferred to soil for further growth if desired. Leaves are excised from the parent plant and relocated to Petri dishes where the feeding assay is conducted with the larval stages of M. sexta. Damaged leaf tissue is assayed for the expression of relatively early events in signal transduction. Gene expression analysis identified infestation-specific Cys2-His2 (C2H2) transcription factors, confirming the success of using detached leaves in early response studies. The method is easier to perform than whole plant infestations and uses less space.

The presented method creates natural herbivore damaged plant tissue through the application of Manduca sexta larvae to detached leaves of potato. The plant tissue is assayed for expression of six transcription factor homologs involved in early responses to insect herbivory.

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more ...

A Simple Approach to Manipulate Dissolved Oxygen for Animal Behavior Observations

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A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

How to Create Conditioned Taste Aversion for Grazing Ground Covers in Woody Crops with Small Ruminants

The Benthic Exchange of O₂, N₂ and Dissolved Nutrients Using Small Core Incubations

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

Understanding Dissolved Organic Matter Biogeochemistry Through In Situ Nutrient Manipulations in Stream Ecosystems

Basic Research in Plasma Medicine - A Throughput Approach from Liquids to Cells

A Simple Planting Technique for Re-establishing Trees Where Frequent Inundation Occurs

Lab-Scale Model to Evaluate Odor and Gas Concentrations Emitted by Deep Bedded Pack Manure

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta