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 be matched. Use a fixed 976 &#937; resistor and an adjustable resistor (10 turn potentiometer) for this purpose.</li>
	<li>Bring one side of the variable arm and one side of the 1 k&#937; resistor to a connector to attach to the bivalve strain gauge as shown in Figure 1.</li>
	<li>Attach the top and the bottom of the bridge to a power supply to provide bias current to the bridge as shown in Figure 1.</li>
	<li>Attach the midpoints of the bridge to the preamplifier and data-recorder, as shown in Figure 1. This will record the bivalve response as its gape movement modulates the resistance of the strain gauge.</li>
	<li>After the bivalve strain gauge is attached, null the bridge by adjusting the 10 turn potentiometer until the voltage at the data recorder input is zero. Use a separate BNC connector and a hand held voltmeter for this purpose. 
	NOTE: Nulling the output means that very high gains can be used with the data recorder without saturating the input, allowing very precise voltages (gape response) to be measured. 
	NOTE: Our multichannel setup uses discrete components hand soldered on a standard piece of electronics &#34;perf-board&#34;. This could easily be formalized on a very inexpensive circuit board with little additional effort.</li>
</ol>
2. General Setting for Diel-Cycling Hypoxia and Cyclical pH
<ol>
	<li>Keep oysters with known low disease prevalence61 in 75-L aquaria under a 14 h light: 10 h dark photoperiod, 7 days per week48, and expose them to cyclical changes in [DO] mimicking diel-cycling hypoxia as found in nature7,8. During 14 h of light, simulate the light levels on a sunny day at 2-m depth in the Rhode River, Maryland61.</li>
	<li>Set up diel-cycling hypoxia and cyclical pH in the laboratory for long-term experiments as described in detail by Burrell et al. (2015)49. Manipulate [DO] and pH in each aquarium and hold at pre-determined levels at all times. To avoid valve closure due to food limitation, add phytoplankton62 to the aquaria. Compensate for any potential DO uptake from algal decomposition by regulating [DO] in each aquarium.</li>
</ol>
3. Bivalve Acclimation
<ol>
	<li>Acclimate oysters to diel-cycling hypoxia for about one week before performing gape experiments.</li>
</ol>
4. Setting Up Diel-Cycling Hypoxia Plateau Stages
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57404/57404fig2.jpg" /> 
Figure 2. [DO] was manipulated experimentally every day to generate diel cycling hypoxia. Here, 2 consecutive diel cycles are shown over which gape measurements were taken. Each diel cycle contains four [DO] phases: a = supersaturation (SS), b = normoxia after supersaturation, c = low plateau (LP), d = normoxia after low plateau. For the Low Plateau, severe hypoxia ([DO] = 0.6 mg/L) with control or cycling pH is indicated with a thick black line, mild hypoxia ([DO] = 1.7 mg/L) with cycling pH with a thin grey line and normoxia ([DO] = 7.3 mg/L) with a dashed line. Grey boxes indicate 10-h dark intervals over two days. <a href="/files/ftp_upload/57404/57404fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>To create diel cycling hypoxia, adjust [DO] over the course of a day in four consecutive phases where [DO] is held constant: 2 h supersaturation, 6 h normoxia after supersaturation, 4 h low plateau (normoxia in the case of the controls) and 2 h normoxia after the low plateau phases.</li>
	<li>During the low plateau phase, keep the normoxic treatment constant at a [DO] of 7.3 mg/L, the mildly hypoxic treatment constant at a [DO] of 1.7 mg/L, and the severe hypoxia treatments constant at a [DO] of 0.6 mg/L.</li>
	<li>For severe hypoxia, run two pH treatments during the low plateau, i.e., severe hypoxia with control pH in which the pH is kept at 7.8 and severe hypoxia with cyclical pH where the pH is cycled from 7.8 to 7.0 during the low plateau. Run the mildly hypoxic treatment with cyclical pH where pH is cycled from 7.8 to 7.0. Run a second mildly hypoxic treatment with constant pH if desired.</li>
</ol>
5. Ramping Up/Down of [DO] Between Plateau Stages
<ol>
	<li>Schedule the ramp up or down of [DO] between the four constant phases to take 2 h each, except for the ramp down from normoxia after supersaturation to the low plateau phase, which takes 3 h. Often, hypoxia has been found to occur late at night or early morning hours in the summer7,8; thus, keep the low plateau phase in the dark as well as the periods ramping down to it or up from it, for a total length of darkness of 10 h.</li>
	<li>Repeat diel-cycling hypoxia 4-5 days a week61.</li>
	<li>Add oysters with sensors in the ramp-up phase before supersaturation and remove them, after closing them, two days later so they experience two complete diel cycles (Figure 2) with sensors attached.</li>
</ol>
6. Making the Sensor Cable
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57404/57404fig3.jpg" /> 
Figure 3. Bivalve SGM strain gauge valve sensor. (a) Strain gauge sensor, (b) strain gauge attached to an oyster showing a loop of the strain gauge between both valves, (c) strain gauge sensor mounted on an oyster. <a href="/files/ftp_upload/57404/57404fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Construct valve gape sensor cables by twisting two 32 AWG (7/40) wires (see Table of Materials) of desired color (Figure 3a). Cut a length of wire twice the length of the desired gape sensor cable, i.e., twice the length from the aquarium to the SGM plus a little extra.</li>
	<li>Have two people stand across from each other, each holding one end of the cable.</li>
	<li>Slide the handle of a pair of scissors over the cable so that in the middle of the cable a pair of scissors is held by the cable. Keep the cable under tension at all times (it cannot go slack).</li>
	<li>Have both people twist the cable in the opposite direction keeping the cable under tension. As the cable starts to twist, the pair of scissors in the middle (which provides weight) will sink down, and both ends of the cable can twist around each other.</li>
	<li>Put the two ends of the cable together and hold them high up, not allowing the scissors with the cable to touch the ground or go slack. After the cable is finished twisting, cut the cable on the other end so there are 4 cable leads total, two on each end.</li>
	<li>Alternatively, to speed this step up, have one person hold a drill with the wires clamped in on one end, keep tension on the wires, and twist the wires by turning the drill on.</li>
	<li>Strip 2 cm of insulation from the wires at each end of the twisted pair.</li>
</ol>
7. Soldering the Sensor
<ol>
	<li>Tin the exposed cable.</li>
	<li>Solder Dsub connector male crimp pins on the two leads on one end of the sensor cable by pretinning the pins and inserting the cables.</li>
	<li>Then, insulate the leads by putting shrink tubing over each lead. Choose shrink tubing that is not much larger in diameter than the cable leads. Shrink the heat shrink with a heat gun. The shrink tubing should now be tightly wrapped around the leads. 
	NOTE: These two leads get plugged into the Wheatstone bridge during experiments.</li>
	<li>Solder the remaining end of the sensor cable to the two contacts of a 1000 &#937; strain gauge with dimensions of 13.5 mm * 5.5 mm grids on the remaining two leads (Figure 3a).</li>
	<li>Add a sensor identifying tag to the sensor cable (Figure 3a).</li>
</ol>
8. Sealing the Sensor
Note: The strain gauges corrode quickly in seawater and thus must be very well sealed. The quality of the seal affects the useful life of the sensor.
<ol>
	<li>Seal the strain gauges by attaching them to PCT-2A&#160;tape leaving 15 mm of tape at the long ends and 2 mm on the sides of the sensor (Figure 3a). After attaching PCT-2A tape to the underside of the strain gauge, add quick gel on the sensor leads and the attached open ends of the cables.</li>
	<li>Attach a second piece of PCT-2A tape of the same size as the bottom piece to the top side of the sensor and attach both pieces of tape with the strain gauge in the middle together. Do not allow the quick gel to flow anywhere other than over the leads and the sensor cables.</li>
	<li>To improve the seal, add thin strips of PCT-2A&#160;tape to the edges where the two pieces of tape surrounding the sensor meet. Do this for the three exposed edges of the sensors.</li>
	<li>Repeat steps 6.1 to 8.4 eleven times to construct 12 sensor cables with sensors, one for each channel on the Wheatstone bridge. 
	Note:&#160;Successful sealing of the strain gauges was the key variable affecting bivalve gape sensor survival in seawater and for minimizing occasional &#34;jumps&#34; in sensor output. Earlier trials without the sealing technique described above resulted in early sensor corrosion and subsequent failure, or rapid drift beyond the measurement range. Future improvements should include techniques to improve the seal.</li>
</ol>
9. Attaching the Valve Gape Sensor to a Bivalve (Figure 3b,c)
<ol>
	<li>Remove oyster from diel-cycling hypoxia aquarium before the low plateau phase.</li>
	<li>With nail polish write an identifying number on the clean, dry shell of each C. virginica.</li>
	<li>Attach the sealed strain gauges to the clean and dried valves of live C. virginica (Figure 3b, c) using aquarium sealant to allow the sensor to be removed after the run. Attach the gape sensor with the leads on the right (flat) valve of oyster about mid-way on the shell (Figure 3c) and leave a loop of the strain gauge (Figure 3b) across the valves so as not to impede the opening of the valves (Figure 3b). Glue the remaining free end of the sensor on the left valve. 
	NOTE: Observations confirm that oysters open their valves normally with the sensors in place.</li>
	<li>It does not matter which way around the sensor is glued on, however, the direction of the signal will be altered if some leads are glued on the left valve and some on the right valve. The direction of the signal becomes clear from the forced closing of the bivalve at the end of a run. For all bivalves, gluing the strain gauge part with the sensor leads to the same valve side for a consistent direction of change in the gape response signal.</li>
	<li>During each step of the process of sealing sensors and attaching them to the oysters, check the sensor resistance periodically to make sure it stays within the range of the readout electronics and to check for potential sensor damage during processing.</li>
	<li>Attach 12 oysters to 12 sensors to fill all 12 channels of the SGM.</li>
	<li>Add oysters to the different aquaria just before the supersaturation plateau phase.</li>
</ol>
10. Setup of Datalogger and Multiplexer for the Valve Gape Apparatus
<ol>
	<li>Attach a sensor to the bivalve and then connect the cable to the SGM with individual Wheatstone bridges (Figure 1) for each sensor (a total of 12 bridges) and connected to a datalogger.</li>
	<li>Set the data logger sample period to read once every 2.5 s and then averaged for 15 s. 
	NOTE: The 62k sample memory on the datalogger used here allowed 12 h of unattended operation. Larger dataloggers and faster sampling rates can be selected, if desired.</li>
	<li>Zero the gape signal for each individual sensor after attaching the sensor to a bivalve using the potentiometer dials on the SGM.</li>
	<li>When necessary, as in higher salinities, correct each sensor periodically for drift in sensor response so that the sensor remains in the range of the SGM (-25 to +25 mV) output of the Wheatstone bridge. The SGM range was chosen for maximum sensitivity between the Wheatstone bridge and the input of the datalogger.</li>
</ol>
11. Deployment of Bivalves with Gape Sensors in Diel-Cycling Hypoxia
<ol>
	<li>After the aquarium sealant has cured (ca 30 min) return the bivalves to an oxygenated holding bucket with flow-through water for at least 5 h to allow the aquarium sealant to leach before submersion in the experiment.</li>
	<li>Place oysters in the aquariums during the ramp-up phase to the supersaturation (SS) plateau (Figure 2).</li>
</ol>
12. Recovery of Bivalves With Gape Sensors
<ol>
	<li>When recovering the oyster from the aquarium with diel-cycling hypoxiaat the end of the two day period, use your fingers to trigger the oyster to close and produce a closing signal in the sensor voltage. This way, the direction and the magnitude of the closing signal can be recorded</li>
	<li>Define the oyster as open if the sensor value is larger than &#189; of the closed value. Define the oyster as closed if the sensor value is smaller than &#189; the closing value. 
	NOTE: Eastern oysters do not open much at the intermediate range.</li>
</ol>
13. Linearization of Raw Valve Gape Data
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57404/57404fig4.jpg" /> 
Figure 4.Linearization of the raw gape data of an oyster. (a) 14 h light: 10 h dark phases, (b) [DO] over a two-day period with periods of severe hypoxia ([DO] 0.6 mg/L), (c) Raw gape data time series of one oyster. Linear drift of the data is caused by corrosion of the sensor and has to be removed in post-processing, (d) gape data time series after post-processing, (e) approximate real time. To determine the magnitude and the direction of the closing spike, the oyster is triggered to close at the end of a run. Magnitudes vary between different sensors. <a href="/files/ftp_upload/57404/57404fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Visually identify oyster gape activity, and use the closed (or open, whichever one is longer) phases of the bivalves to determine instrument drift. Use IF statements in spreadsheet software to mark the closed values with one and open values with zero (e.g., =IF (Cell &#62; 5, 1 (true), 0 (false))).</li>
	<li>Remove the shortest phases and run a linear regression on the remaining raw data. Use the regression equation to linearize the experimental data (Figure 4). Linearization has to be done for each sensor individually. 
	Note: Sensor drift is linear over short (minutes) and long (days) time scales and the amount of drift varies from sensor to sensor. Drift is typically negligible over short time spans such as an hour, in which only occasionally linear drift has been noted. Sensor drift necessitates that calibrations of valve gape and sensor gape have to be done for each sensor separately and that a linearization has to be done for each sensor in post-processing of the data.</li>
</ol>
14. Calibration of Sensors: Actual Gape Width Versus Sensor mV
In a side experiment, sensor voltage can be calibrated to the actual gape width of oysters.
<ol>
	<li>In an aquarium, set up an oyster with a gape sensor. Initiate gape recording. Trigger the oyster to close by touching it. Soon after, the oyster will open up again.</li>
	<li>As the oyster is opening, take photographs and write down the sensor voltage of the gape apparatus each time a photograph is taken. Keep track of which photograph goes with which gape apparatus reading. Place a reference scale in the photograph, at the same level of the valves.</li>
	<li>Analyze the distance the oyster is open in each photograph (mm) using an open source image processing program such as ImageJ.</li>
	<li>Compare actual gape width to sensor voltage and run a regression analysis. This gives the calibration for the actual opening status and further sensor measurements can then be related to actual gape width of the bivalve. 
	Note: While this shows gape width is linearly related to sensor voltage, in actuality, this process takes too long and was not performed for the hypoxia gape experiments. For the hypoxia gape experiments it was important to remove the oysters for the least amount of time necessary from their aquaria to equip them with sensors and return them to the aquaria. For some experiments, the gape width measurement may be important and the calibration techniques above should be followed.</li>
</ol>

We don't have anything to disclose.

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.
<p class="jove_...

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">
	<colgroup>
		<col />
		<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.
<p class="j...

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 ...

a-strain-gauge-monitor-sgm-for-continuous-valve-gape-measurements

Research

JoVE Journal

Environment

8.9K Views.  University of Baltimore. 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.

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

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 ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

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 ...