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 />
	</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 ...

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