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">
	<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.
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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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