Name: a strain gauge monitor (sgm) for continuous valve gape measurements in bivalve molluscs in response to laboratory induced diel-cycling hypoxia and ph
Uploaded: 2018-08-01T14:00:00
Description: Watch this Scientific Journal Video about A Strain Gauge Monitor SGM for Continuous Valve Gape Measurements in Bivalve Molluscs in Response to Laboratory Induced Diel-cycling Hypoxia and pH at JoVE.co

We thank Melinda Forseth for taking photos of oysters and measuring their gape width in ImageJ. We thank Denise Breitburg for access to the aquaria with diel cycling hypoxia and cyclical pH conditions. We thank the Smithsonian Environmental Research Center, Edgewater, Maryland, for space for the experiments. The hypoxia experiments were funded by a National Oceanic and Atmospheric Administration - Center for Sponsored Coastal Ocean Research grant No. NA10NOS4780138 and the Smithsonian Hunterdon Fund to Denise Breitburg. The valve gape measurements during the hypoxia experiments were funded by a Faculty Enhancement Grant by Washington College to Elka T. Porter.

1. Construction of the Wheatstone Bridge for the Valve Gape Apparatus
NOTE: The strain gauge is nominally 1000 &#937;, so to fully balance and null the bridge, all components should be 1000 &#937;.
<ol>
	<li>As in Figure 1, solder two 1 k&#937; precision resistors to each other and then to a ~976 &#937; resistor and a 100 &#937; 10 turn potentiometer. The typical strain gauge range is a few &#937; off of the 1000 &#937; nominal, so only this resistance needs to ...

Typical studies focus on the continuous, extended time periods of low oxygen conditions and the response, often measured as the survival, of animals. However, at present, our understanding of the behavioral responses of animals to diel-cycling hypoxia is minimal63. Thus, more studies should focus on the behavior of organisms in response to diel-cycling hypoxia which occurs regularly over the summer in many estuaries7,8.
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Hypoxia, i.e., dissolved oxygen concentrations [DO] sufficiently low to negatively affect biological and ecological processes but often functionally defined as [DO] of &#60; 2 mg/L1, and anoxia (functionally defined as [DO] of 0.0-0.2 mg/L) are occurring more frequently and severely in the world&#39;s coastal waters, estuaries and the deep ocean2,3 and are often exacerbated by increasing eutrophication4,5. With an increasing areal extent of hypoxia and anoxia, macrofauna are negatively affected and lose habitat extent and habitat quality. Climate change is predicted to worsen hypoxia and anoxia6.
In many stratified, nutrient-enriched estuaries such as Chesapeake Bay, USA, seasonally persistent hypoxia can prevail and can occur year after year2. In addition, diel-cycling of hypoxia is frequent in estuaries such as the Chesapeake Bay and other locations and occurs late during the night or the early morning hours in the summer7,8.
Most studies have focused on the effect of continuous exposure of organisms to low [DO] and on their tolerance to hypoxia and anoxia9,10,11,12,13,14. Moreover, studies have looked at the large-scale shift in species distributions, abundances, and species composition in response to extended low [DO]4,15. Often species that are very sensitive to low [DO], die in masses,16 shifting the remaining species to a younger, smaller-sized, short-lived fauna as, for example, found on the Louisiana-Texas Shelf ecosystem4.
Behavioral changes typically precede community collapse17 and studies have reported on behavioral responses of organisms to extended low [DO]4,16,17,18,19,20,21,22,23,24,25. These studies, however, do not focus on the responses of organisms to diel-cycling exposures of hypoxia and the fluctuating nature of [DO] availability in estuaries.
Diel-cycling hypoxia in shallow estuaries has received increasing awareness as studies monitor [DO] more frequently over the course of days with sondes in estuaries16,26. Water can remain hypoxic for hours at the end of the night or early morning hours in the summer when there is no oxygen-generating photosynthesis during the night but high oxygen-consuming aerobic respiration7,16. It was also found that the tides affected the diel cycling of low DO conditions with the most extreme minima observed when low tides coincided with the end of the night27. Only after several hours of hypoxia does [DO] come back to normoxia7,16,28 in the daily cycle.
To determine the behavioral response of C. virginica to diel-cycling hypoxia and pH we monitored the opening and closing of the valves of oysters exposed to laboratory induced diel cycling of [DO] and cyclical pH. Gape responses of bivalves have been used to detect adverse environmental conditions. Valve closures of bivalves in response to contaminants29,30,31, toxic algae32,33,34, thermal pollution35,36,37, food quantity decrease38,39,40, feeding regime39,41, emersion37,42, photoperiod43,44, pH45,46, and combined pH and dissolved oxygen47 have been measured. Gape techniques have, for example, included direct observations48,49,13, continuous measurements using reed switches and magnets (Dreissena monitor)50, or fiber-optic sensors51 that require clear water. In addition, magnet and magnetic-field strength Hall sensors have been used to study mussel gape angle52,53,54,55, and a high-frequency electromagnetic induction system that can measure the varying distance between two electric coils that are glued on the valves has been used56,57,58,59. A high voltage source is required for the electromagnetic induction system and power has to be delivered to both sides of the shell52. This system is also commercially available as the &#34;MOSSELMONITOR&#34; (http://mosselmonitor.nl/).
On a tight research budget, we constructed an inexpensive strain gauge monitor (SGM) to continuously measure oyster gape over laboratory-induced diel cycling of [DO] and pH, under low visibility conditions. Our system is also much simpler than competing systems, allowing many animals to be instrumented during an experiment. We wanted to determine the behavioral responses of C. virginica to diel cycling severe ([DO] = 0.6 mg/L) hypoxia with control pH (pH = 7.8) and cycling pH (pH = 7.8-7.0), respectively, and gape responses to mild ([DO] = 1.7 mg/L) hypoxia. Moreover, we wanted to determine if oysters are able to rapidly respond to changes in [DO] over the diel cycle and how they respond when normoxia returns after a hypoxic event. Perhaps oysters are optimally adapted to the rapidly fluctuating environment that is found in many estuaries16,27 where they live. While more complex valve gape monitors are available, the SGM offers an inexpensive technique that allows continuous measurements of valve gape in waters even in low visibility conditions.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57404/57404fig1.jpg" /> 
Figure 1.&#160;Wheatstone bridge for the valve gape apparatus. <a href="/files/ftp_upload/57404/57404fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The strain gauge sensors used for monitoring bivalve gape are resistive films in a meander pattern on a polyimide backing. Small amounts of strain modulate the resistance of the sensor. The bivalve flexes the strain gage when it gapes causing a change in the sensor&#39;s resistance. We employed a nulling, balanced, Wheatstone bridge for each bivalve channel as shown in Figure 1 to measure the change in sensor resistance. The Wheatstone bridge is nulled by the potentiometer allowing a fairly high gain to be employed by the datalogger. A Wheatstone bridge is a standard method for accurately measuring an unknown resistance using a ratio to a known resistance standard and a voltmeter. The history of this very old technique is discussed in Ekelof (2001)60. We integrated 12 channels, each with its own Wheatstone bridge and nulling potentiometer, into the Strain Gauge Monitor (SGM) unit.

<table border="0" cellpadding="0" cellspacing="0" style="width:805px;" width="805">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Campbell CR 10x data logger</td>
			<td style="width:268px;">Campbell Scientific, Logan, Utah</td>
			<td style="width:139px;"></td>
			<td style="width:183px;">Or other data logger. At Campbell the CR 10X has been replaced with the CR 1000</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:216px;">Campbell CR 10x multiplexer</td>
			<td>Campbell Scientific, Logan, Utah</td>
			<td style="width:139px;"></td>
			<td style="width:183px;">Data logger needs to have space for 12 channels</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">Dsub connector male crimp pins</td>
			<td style="width:268px;">TE Connectivity</td>
			<td>205089-1</td>
			<td style="width:183px;">pins for gape sensor leads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PCA tape</td>
			<td>Micro Measurements Corp, NC</td>
			<td style="width:139px;"></td>
			<td style="width:183px;">To seal the strain gauge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Duro Quick Gel</td>
			<td>Ace Hardware</td>
			<td style="width:139px;"></td>
			<td style="width:183px;">Superglue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SG13/1000-LY43 or LY41</td>
			<td>Omega Engineering Inc., Stanford, CT</td>
			<td style="width:139px;"></td>
			<td style="width:183px;">Strain gauges</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">32 AWG (7/40) teflon Alpha wires</td>
			<td style="width:268px;">AlphaWire, Elizabeth, NJ</td>
			<td style="width:139px;">2840/7</td>
			<td style="width:183px;">Sensor cables, different colors are available</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1/16&#34; heat shrink tubing</td>
			<td style="width:268px;">Qualtek</td>
			<td>B01A3QKKO6</td>
			<td style="width:183px;">To seal the leads of the sensor cable</td>
		</tr>
		<tr height="210">
			<td height="210" style="height:210px;width:216px;">Weller WES51 Analog Soldering Station</td>
			<td style="width:268px;">Amazon</td>
			<td style="width:139px;"></td>
			<td style="width:183px;">Lots of soldering, need a good soldering iron. https://www.amazon.com/Weller-WES51-Analog-Soldering-Station/dp/B000BRC2XU/ref=sr_1_23?s=hi&#38;ie=UTF8&#38;qid=1505654295 
			&#38;sr=1-23&#38;keywords=soldering+iron</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rosin Soldering Flux Paste</td>
			<td style="width:268px;">Amazon</td>
			<td style="width:139px;"></td>
			<td style="width:183px;">Needed for soldering</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">60-40 Tin Lead Rosin Core Solder Wire</td>
			<td style="width:268px;">Amazon</td>
			<td style="width:139px;"></td>
			<td style="width:183px;">Needed for soldering</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aquarium sealant</td>
			<td style="width:268px;">Home Depot</td>
			<td style="width:139px;"></td>
			<td style="width:183px;">Attach sensors to bivalve</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PC Laptop</td>
			<td style="width:268px;"></td>
			<td style="width:139px;"></td>
			<td style="width:183px;">Any old PC to run Campbell gape program</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">heat gun</td>
			<td>Amazon</td>
			<td></td>
			<td style="width:183px;">shrink shrink tubing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Drill</td>
			<td>Hardware store, Amazon</td>
			<td></td>
			<td style="width:183px;">for twisting wires to make sensor cables</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">AC to DC power module</td>
			<td>Acopian</td>
			<td>DB15-30</td>
			<td style="width:183px;">Wheatstone bridge power supply</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poteniometer</td>
			<td>Clarostat</td>
			<td>733A</td>
			<td style="width:183px;">Wheatsone bridge nulling</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">isolating BNC connector</td>
			<td>Sterren Electronics</td>
			<td>&#34;200-148</td>
			<td style="width:183px;">Wheatstone bridge output for multimeter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Fused AC receptical panel module</td>
			<td>Adam technologies</td>
			<td>IEC-GS-1-200</td>
			<td style="width:183px;">Wheatstone bridge power supply connector</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">976 ohm 1% resistor</td>
			<td>Vishay Dale</td>
			<td>CMF50976R00FHEB</td>
			<td style="width:183px;">Wheatstone bridge resistor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">1 kohm 1% resistor</td>
			<td>Vishay Dale</td>
			<td>CMF501K0000FHEB</td>
			<td style="width:183px;">Wheatstone bridge resistor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Potentiometer scale dial</td>
			<td>Kilo International</td>
			<td align="right">462</td>
			<td style="width:183px;">10 turn dial for nulling potentiometer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">DB25 male panel connector</td>
			<td>TE connectivity</td>
			<td>1757819-8</td>
			<td style="width:183px;">Data logger connector on Wheatstone bridge</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">DB25 female panel connector</td>
			<td>TE connectivity</td>
			<td>1757819-8</td>
			<td style="width:183px;">Sensor connector to Wheatstone bridge</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">perforated circuit board</td>
			<td>Vector electronics</td>
			<td>64P44WE</td>
			<td style="width:183px;">circuit board for mounting of bridge components</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">enclosure</td>
			<td>Hammond Manufacturing</td>
			<td>1444-29</td>
			<td style="width:183px;">Enclosure for sensor readout electronics</td>
		</tr>
	</tbody>
</table>

a strain gauge monitor (sgm) for continuous valve gape measurements in bivalve molluscs in response to laboratory induced diel-cycling hypoxia and ph

An inexpensive, laboratory-based, strain gauge valve gape monitor (SGM) was developed to monitor the valve gape behavior of bivalve molluscs in response to diel-cycling hypoxia. A Wheatstone bridge was connected to strain gauges that were attached to the shells of oysters (Crassostrea virginica). The recorded signals allowed for the opening and closing of the bivalves to be recorded continuously over two-day periods of experimentally-induced diel-cycling hypoxia and diel-cycling changes in pH. Here, we describe a protocol for developing an inexpensive strain gauge monitor and describe, in an example laboratory experiment, how we used it to measure the valve gape behavior of Eastern oysters (C. virginica), in response to diel-cycling hypoxia and cyclical changes in pH. Valve gape was measured on oysters subjected to cyclical severe hypoxic (0.6 mg/L) dissolved oxygen conditions with and without cyclical changes in pH, cyclical mild hypoxic (1.7 mg/L) conditions and normoxic (7.3 mg/L) conditions. We demonstrate that when oysters encounter repeated diel cycles, they rapidly close their shells in response to severe hypoxia and close with a time lag to mild hypoxia. When normoxia is restored, they rapidly open again. Oysters did not respond to cyclical pH conditions superimposed on diel cycling severe hypoxia. At reduced oxygen conditions, more than one third of the oysters closed simultaneously. We demonstrate that oysters respond to diel-cycling hypoxia, which must be considered when assessing the behavior of bivalves to dissolved oxygen. The valve SGM can be used to assess responses of bivalve molluscs to changes in dissolved oxygen or contaminants. Sealing techniques to better seal the valve gape strain gauges from sea water need further improvement to increase the longevity of the sensors.

Oysters exposed to uninterrupted normoxic estuarine water (no hypoxia during the low plateau phase of the daily cycle) were open most of the time and only briefly closed infrequently (Figure 5). When they closed varied from oyster to oyster. This pattern has also been found by Loosanoff and Nomejko 194644 and Higgins 198039. Oysters also did not respond to the difference in dark and light phases.
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Watch this Scientific Journal Video about A Strain Gauge Monitor SGM for Continuous Valve Gape Measurements in Bivalve Molluscs in Response to Laboratory Induced Diel-cycling Hypoxia and pH at JoVE.co

A Strain Gauge Monitor SGM for Continuous Valve Gape Measurements in Bivalve Molluscs in Response to Laboratory Induced Diel-cycling Hypoxia and pH

Understanding the behavioral responses of bivalve suspension-feeders to environmental variables, such as dissolved oxygen, can explain some ecosystem processes. We have developed an inexpensive, laboratory-based, strain gauge monitor (SGM) to measure valve gape responses of oysters, Crassostrea virginica, to diel-cycling hypoxia and cyclical pH.

A Strain Gauge Monitor (SGM) for Continuous Valve Gape Measurements in Bivalve Molluscs in Response to Laboratory Induced Diel-cycling Hypoxia and pH

An inexpensive, laboratory-based, strain gauge valve gape monitor (SGM) was developed to monitor the valve gape behavior of bivalve molluscs in response ...

This method can help answer key questions. For example, how do oysters respond behaviorally to diel-cycling hypoxia and cyclical pH. How do they respond when diel-cycling hypoxia happens?Do they close up? And after normoxia is restored, do they open back up? How do they respond?The main advantage of this technique is that it measures the opening and the closing of the oysters in a very precise manner and is also a very affordable technique. Though this method can provide insight into the valve gape behavior of oysters in response to diel-cycling hypoxia, it can also be used for other species and it can be used to measure, for example, the adverse behavior of bivalves to contaminants or toxic algae. Plan to prepare 12 sensors;one for each channel on the Wheatstone bridge.First, construct valve gape sensor cables. Twist two 32-gauge Teflon-coated wires of specific colors. Cut a length of twice the length of the desired gape sensor cable.Make it twice the length from the aquarium to the SGM, plus a little extra. On one end, secure the wires to a power drill. Two people must hold either end of the wire which make the cable.Now, the holders twist the cable using the drill on, while keeping the cable under tension. Next, cut the cable on the other end so there are four leads, two on each end. Then, strip two centimeters of insulation off each wire.Next, tin the exposed wire. On one end, solder D-sub connector male crimp pins on the two leads. Then, insulate the leads with shrink tubing.Next, solder the other two leads to the two contacts of a 1000-Ohm strain gauge that has 13.5 by 5.5 millimeter grids. Then, add a sensor identifying tag to the sensor cable. Now, seal the strain gauges using PCT two-way tape.Use a 15 millimeter length of tape on the long ends and two millimeter lengths on the sides of the sensor. Next, apply Quick Gel to the sensor leads and to the attached open ends of the cables. Do not allow the Quick Gel to flow anywhere else.Now, attach a second layer of PCT 2-A to the bottom and the top of the sensor. Periodically, while sealing the sensor, check the resistance to make certain it remains in range. To improve the seal, add thin strips of PCT 2-A to the edges, over the edges where the tape meets.The strain gauges corrode quickly in sea water and thus must be very well sealed. The quality of the seal affects the useful life of the sensor. If any improvements can be made for this technique, it is how to better seal the strain gauges.Generally, this experiment is performed on a dozen oysters. For demonstration purposes, only one will be manipulated. Begin with removing the oysters from the diel-cycling hypoxia aquaria before they enter the low plateau phase.Then, clean and dry their shells. Next, use nail polish to write identifying numbers on each oyster. Then attach strain gauges to their valves using aquarium sealant.Now, position the gauge sensor with the leads on the flat valve about midway along the shell and leave a loop of the strain gauge across the valves so as not to impede the opening of the valves. Next, glue the free end of the sensor onto the left valve. Now, check the resistance of the mounted sensors.They must remain within the range of the readout electronics. Observe how the bending of a strain gauge caused by the oyster's opening and closing affects the voltage readings. Generally, 12 oysters are attached to 12 sensors for one experiment.After 30 minutes, the aquarium sealant should be cured. Then, return the bivalves to an oxygenated holding bucket with flow-through water for at least five hours to allow the aquarium sealant to leach before submersion in the experimental aquaria. Later, transfer the oysters in their aquaria during the ramp-up phase to the super-saturation plateau.Run all sensor cables to the SGM. Then, fill the 12 channels of the SGM with 12 sensor cables. Next, zero the gape signal for each individual sensor after using the potentiometer dials on the SGM.At high solidities, plan to correct the drift in the offset of each sensor periodically so it stays in the range of the SGM and the Wheatstone bridge. When recovering the oysters from the aquaria to end the experiment, manually trigger each oyster to close. This produces a closing signal to know the direction and the magnitude of the closing signal.During analysis, define the oyster as open if the sensor valve is larger than one half of the closed value and define the oyster as closed if the sensor value is smaller than one half of the closing value. Oysters exposed to uninterrupted normoxic estuarine water without hypoxia during the low plateau phase of the daily cycle were open most of the time. Most oysters exposed to severe hypoxia during the low plateau phase closed soon after their target-dissolved concentrations were reached.Some closed even sooner. Being at a controlled or cyclical pH had no effect. Under mild hypoxia, closings often occurred late during the low plateau phase, instead of at the point when the target-dissolved oxygen was reached.During the normoxia phase after the low plateau, oysters that experienced severe hypoxia during the low plateau were open most of the time and they often opened while dissolved oxygen levels were ramping up, even before the normoxia plateau had been reached. After watching this video, you should have a good idea how to make gape sensors and how to attach them to bivalves and how to measure behavioral responses of bivalves to diel-cycling hypoxia and cyclical pH. The apparatus is simple, low cost and easy to assemble.After its development, this valve gape technique can also be used on other bivalve species and it can be used to measure the adverse behavior of bivalves to either contaminants or to toxic algae.

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Environment

Optimize Flue Gas Settings to Promote Microalgae Growth in Photobioreactors via Computer Simulations

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently than plants3, but also synthesize advanced biofuels2-4. Generally, atmospheric CO2 is not a sufficient source for supporting maximal algal growth5. On the other hand, the high concentrations of CO2 in industrial exhaust gases have adverse effects on algal physiology. Consequently, both cultivation conditions (such as nutrients and light) and the control of the flue gas flow into the photo-bioreactors are important to develop an efficient &ldquo;flue gas to algae&rdquo; system. Researchers have proposed different photobioreactor configurations4,6 and cultivation strategies7,8 with flue gas. Here, we present a protocol that demonstrates how to use models to predict the microalgal growth in response to flue gas settings. We perform both experimental illustration and model simulations to determine the favorable conditions for algal growth with flue gas. We develop a Monod-based model coupled with mass transfer and light intensity equations to simulate the microalgal growth in a homogenous photo-bioreactor. The model simulation compares algal growth and flue gas consumptions under different flue-gas settings. The model illustrates: 1) how algal growth is influenced by different volumetric mass transfer coefficients of CO2; 2) how we can find optimal CO2 concentration for algal growth via the dynamic optimization approach (DOA); 3) how we can design a rectangular on-off flue gas pulse to promote algal biomass growth and to reduce the usage of flue gas. On the experimental side, we present a protocol for growing Chlorella under the flue gas (generated by natural gas combustion). The experimental results qualitatively validate the model predictions that the high frequency flue gas pulses can significantly improve algal cultivation.

Flue gas from power plants is a cheap CO2 source for algal growth. We have built prototype &#34;flue gas to algal cultivation&#34; systems and described how to scale up the algal cultivation process. We have demonstrated the use of a mass-transfer bio-reaction model to simulate and to design the optimal operation of flue gas for the growth of Chlorella sp. in algal photo-bioreactors.

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently ...

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O or&#160;2H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation1-4. From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage5-7. The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing 13C and 15N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components.
Here, we present the construction and operation of a continuous 13C and 15N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii Kaw demonstrate the system&#39;s ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%13C and 6.7 atom%15N uniform plant label, or material that is differentially labeled by up to 1.29 atom%13C and 0.56 atom%15N in its metabolic and structural components (hot water extractable and hot water residual components, respectively). Challenges lie in maintaining proper temperature, humidity, CO2 concentration,&#160;and light levels in an airtight 13C-CO2 atmosphere for successful plant production. This chamber description represents a useful research tool to effectively produce uniformly or differentially multi-isotope labeled plant material for use in experiments on ecosystem biogeochemical cycling.

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

This method explains how to build and operate a continuous 13C and 15N isotope labeling chamber for uniform or differential plant tissue labeling. Representative results from metabolic and structural labeling of Andropogon gerardii are discussed.

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes ...

Methods of Soil Resampling to Monitor Changes in the Chemical Concentrations of Forest Soils

Recent soils research has shown that important chemical soil characteristics can change in less than a decade, often the result of broad environmental changes. Repeated sampling to monitor these changes in forest soils is a relatively new practice that is not well documented in the literature and has only recently been broadly embraced by the scientific community. The objective of this protocol is therefore to synthesize the latest information on methods of soil resampling in a format that can be used to design and implement a soil monitoring program. Successful monitoring of forest soils requires that a study unit be defined within an area of forested land that can be characterized with replicate sampling locations. A resampling interval of 5 years is recommended, but if monitoring is done to evaluate a specific environmental driver, the rate of change expected in that driver should be taken into consideration. Here, we show that the sampling of the profile can be done by horizon where boundaries can be clearly identified and horizons are sufficiently thick to remove soil without contamination from horizons above or below. Otherwise, sampling can be done by depth interval. Archiving of sample for future reanalysis is a key step in avoiding analytical bias and providing the opportunity for additional analyses as new questions arise.

Repeated soil sampling has recently been shown to be an effective way to monitor forest soil change over years and decades. To support its use, a protocol is presented that synthesizes the latest information on soil resampling methods to aid in the design and implementation of successful soil monitoring programs.

Recent soils research has shown that important chemical soil characteristics can change in less than a decade, often the result of broad environmental ...

Continuous Hydrologic and Water Quality Monitoring of Vernal Ponds

Vernal ponds, also referred to as vernal pools, provide critical ecosystem services and habitat for a variety of threatened and endangered species. However, they are vulnerable parts of the landscapes that are often poorly understood and understudied. Land use and management practices, as well as climate change are thought to be a contribution to the global amphibian decline. However, more research is needed to understand the extent of these impacts. Here, we present methodology for characterizing a vernal pond's morphology and detail a monitoring station that can be used to collect water quantity and quality data over the duration of a vernal pond's hydroperiod. We provide methodology for how to conduct field surveys to characterize the morphology and develop stage-storage curves for a vernal pond. Additionally, we provide methodology for monitoring the water level, temperature, pH, oxidation-reduction potential, dissolved oxygen, and electrical conductivity of water in a vernal pond, as well as monitoring rainfall data. This information can be used to better quantify the ecosystem services that vernal ponds provide and the impacts of anthropogenic activities on their ability to provide these services.

Understanding the ecosystem services and processes provided by vernal ponds and the impacts of anthropogenic activities on their ability to provide these services requires intensive hydrologic monitoring. This sampling protocol using in-situ monitoring equipment was developed to understand the impact of anthropogenic activities on water levels and quality.

Vernal ponds, also referred to as vernal pools, provide critical ecosystem services and habitat for a variety of threatened and endangered species. ...

Adherence of Bacteria to Plant Surfaces Measured in the Laboratory

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell counts. Plant materials used include roots, sprouts, leaves, and cut fruits. The methods described are inexpensive, easy, and suitable for small sample sizes. Binding is measured in the laboratory and a variety of incubation media and conditions can be used. The effect of inhibitors can be determined. Situations that promote and inhibit binding can also be assessed. In some cases it is possible to distinguish whether various conditions alter binding primarily due to their effects on the plant or on the bacteria.

An easy method for measuring and characterizing bacterial adhesion to plants, particularly roots and sprouts, is described in this article.

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell ...

The Calibration and Use of Capacitance Sensors to Monitor Stem Water Content in Trees

Water transport and storage through the soil-plant-atmosphere continuum is critical to the terrestrial water cycle, and has become a major research focus area. Biomass capacitance plays an integral role in the avoidance of hydraulic impairment to transpiration. However, high temporal resolution measurements of dynamic changes in the hydraulic capacitance of large trees are rare. Here, we present procedures for the calibration and use of capacitance sensors, typically used to monitor soil water content, to measure the volumetric water content in trees in the field. Frequency domain reflectometry-style observations are sensitive to the density of the media being studied. Therefore, it is necessary to perform species-specific calibrations to convert from the sensor-reported values of dielectric permittivity to volumetric water content. Calibration is performed on a harvested branch or stem cut into segments that are dried or re-hydrated to produce a full range of water contents used to generate a best-fit regression with sensor observations. Sensors are inserted into calibration segments or installed in trees after pre-drilling holes to a tolerance fit using a fabricated template to ensure proper drill alignment. Special care is taken to ensure that sensor tines make good contact with the surrounding media, while allowing them to be inserted without excessive force. Volumetric water content dynamics observed via the presented methodology align with sap flow measurements recorded using thermal dissipation techniques and environmental forcing data. Biomass water content data can be used to observe the onset of water stress, drought response and recovery, and has the potential to be applied to the calibration and evaluation of new plant-level hydrodynamics models, as well as to the partitioning of remotely sensed moisture products into above- and belowground components.

The hydraulic capacitance of biomass is a key component of the vegetation water budget, which serves as a buffer against short and long-term drought stresses. Here, we present a protocol for the calibration and use of soil moisture capacitance sensors to monitor water content in the stems of large trees.

Water transport and storage through the soil-plant-atmosphere continuum is critical to the terrestrial water cycle, and has become a major research focus ...

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

Design and Use of an Apparatus for Quantifying Bivalve Suspension Feeding at Sea

As shellfish aquaculture moves from coastal embayments and estuaries to offshore locations, the need to quantify ecosystem interactions of farmed bivalves (i.e., mussels, oysters, and clams) presents new challenges. Quantitative data on the feeding behavior of suspension-feeding mollusks is necessary to determine important ecosystem interactions of offshore shellfish farms, including their carrying capacity, the competition with the zooplankton community, the availability of trophic resources at different depths, and the deposition to the benthos. The biodeposition method is used to quantify feeding variables in suspension-feeding bivalves in a natural setting and represents a more realistic proxy than laboratory experiments. This method, however, relies upon a stable platform to satisfy the requirements that water flow rates supplied to the shellfish remain constant and the bivalves are undisturbed. A flow-through device and process for using the biodeposition method to quantify the feeding of bivalve mollusks were modified from a land-based format for shipboard use by building a two-dimensional gimbal table around the device. Planimeter data reveal a minimal pitch and yaw of the chambers containing the test shellfish despite boat motion, the flow rates within the chambers remain constant, and operators are able to collect the biodeposits (feces and pseudofeces) with sufficient consistency to obtain accurate measurements of the bivalve clearance, filtration, selection, ingestion, rejection, and absorption at offshore shellfish aquaculture sites.

A flow-through device for using the biodeposition method to quantify filtration and feeding behavior of bivalve mollusks was modified for shipboard use. A two-dimensional gimbal table built around the device isolates the apparatus from boat motion, thereby allowing the accurate quantification of bivalve filtration variables at offshore shellfish aquaculture sites.

As shellfish aquaculture moves from coastal embayments and estuaries to offshore locations, the need to quantify ecosystem interactions of farmed bivalves ...

PARbars: Cheap, Easy to Build Ceptometers for Continuous Measurement of Light Interception in Plant Canopies

Ceptometry is a technique used to measure the transmittance of photosynthetically active radiation through a plant canopy using multiple light sensors connected in parallel on a long bar. Ceptometry is often used to infer properties of canopy structure and light interception, notably leaf area index (LAI) and effective plant area index (PAIeff). Due to the high cost of commercially available ceptometers, the number of measurements that can be taken is often limited in space and time. This limits the usefulness of ceptometry for studying genetic variability in light interception, and precludes thorough analysis of, and correction for, biases that can skew measurements depending on the time of day. We developed continuously logging ceptometers (called PARbars) that can be produced for USD $75 each and yield high quality data comparable to commercially available alternatives. Here we provide detailed instruction on how to build and calibrate PARbars, how to deploy them in the field and how to estimate PAI from collected transmittance data. We provide representative results from wheat canopies and discuss further considerations that should be made when using PARbars.

Here, we present detailed instructions on how to build and calibrate research quality ceptometers (light sensors that integrate light intensity across many sensors arrayed linearly along a horizontal bar).

Ceptometry is a technique used to measure the transmittance of photosynthetically active radiation through a plant canopy using multiple light sensors ...