This study was supported by the Ministry of Education, Youth and Sports of the Czech Republic-projects "CENAKVA" No. CZ.1.05/2.1.00/01.0024 and "CENAKVA II'' No. LO1205 under the National Sustainability Program I, by the Grant Agency of the University of South Bohemia in &#268;esk&#233; Bud&#283;jovice (012/2016/Z), and by the Grant Agency of the Czech Republic (No. 16-06498S)

1. Crayfish Selection
<ol>
	<li>In order to successfully apply the current approach to crayfish, select the respective adult specimens with sufficient carapace sizes (which is a carapace length of at least 30 mm) for sensor attachment, visually examine it for the absence of diseases, and check whether it lifts both chelae when it is touched. The above-mentioned parameters indicate an eligible state of crayfish health. 
	NOTE: If several crayfish are expected to be used in the trial and are exposed to the same conditions, the experimental group should be formed based on several parameters: (i) similar weight and length; (ii) comparable heart rate; (iii) pronounced nocturnal activity; (iv) regular food consumption; (v) inter-molting period17. Sometimes, it is hard to define whether a crayfish is near to molting by the heart rate measurements or visual or tactile examinations only; therefore, the analyses of the crayfish&#39;s hemolymph total protein content can be helpful. Protein content is expected to be higher when the crayfish is closer to molting than in the inter-molting state18.</li>
</ol>
2. Recording of Crayfish Cardiac Activity and Behavior
<ol>
	<li>In order to noninvasively measure crayfish heart rates, preliminarily prepare the sensor for this procedure. Before this, put a crayfish into the tank with water and let it acclimate there for a few days as the preparation of the sensor19 will also take a few days.
	<ol>
		<li>Axially couple an IR light-emitting diode (LED) with a phototransistor. Attach the optical sensor circuit onto a board; it will require a power supply of 5 V. For the LED connection, place a 200 &#937; resistor on the IR sensor board; in order to connect the phototransistor, place a 220 &#937; resistor on the board.</li>
		<li>When attached to the crayfish, the sensor output is modulated by the amount of hemolymph filling the crayfish cardiac muscle and scatters an incident light from the LED. In order to avoid reciprocal interference of the illuminated IR light by the LED and the reflected IR light from the crayfish heart, which is received by the phototransistor, place a small wall (0.5 x 1.5 x 4 mm, thickness x height x width) made of black antistatic plastic between the LED and the phototransistor.</li>
		<li>Place the LED in a waterproof package, and cover the surface of the sensor with the waterproof dielectric gel from the side adjacent to the carapace for the protection of the electronic components from potential damage (Figure 1). Let the gel dry for 3 days in order to gain its best protective properties.</li>
		<li>For an analog signal, attach thin flexible cables (about 3 m long) to the sensor and connect to the analogue-to-digital converter (ADC); from this, a digitized signal will be transferred to a personal computer over a USB interface, at which point the information about the crayfish cardiac activity is saved, analyzed in real-time with special software (see Table of Materials), and stored for further detailed analyses.</li>
	</ol>
	</li>
	<li>As soon as the sensor is prepared, attach it to the crayfish. In order to do this, switch the computer on and run the software. Determine the number of crayfish to be fixed to the sensors and recorded heart rate to be saved to the date file.</li>
	<li>Remove the crayfish from the water and wipe its dorsal carapace side with a paper towel. Wrap the chelae and abdomen of the crayfish in the paper towel in order to avoid any damages by human hand and to eliminate additional stress on the crayfish caused by warm human hands. 
	NOTE: Do not use a previous cooling of the crayfish on ice or in the freezer for its immobilization before manipulations with the sensor attachment. The difference in temperatures leads to crayfish dorsal surface weeping which, in turn, leads to unreliable sensor fastening and quick adhesive detachment from the crayfish&#39;s carapace.</li>
	<li>Prepare a surface (i.e., take a small flat piece of plastic or tear a piece of sticky tape and fix it to a table) and a stick for mixing the glue. Press out two small drops (of a diameter of about 0.5 cm) from tubes A and B containing epoxy glue and quickly mix them.</li>
	<li>Attach the sensor to the crayfish dorsal carapace and try to find the place in which the cardiac signal amplitude would be maximal. Hold the crayfish with the sensor in one hand and, using the other free hand, put a drop of mixed glue on each of the four auxiliary wires located on the sensor (fix them in between steps 2.1.1 and 2.1.4.). Do not move the sensor at least 5 min until the glue hardens (the glue hardening depends on the ambient temperature and humidity). 
	NOTE: When fixing the sensor to the crayfish carapace, examine thoroughly the whole cardiac area from the carapace side in order to define the area with the best (maximal) cardiac signal amplitude. That will help the software to provide more precise heart rate calculations.</li>
	<li>Touch the glue using a free hand, and if it is not sticky, put the unwrapped crayfish with the attached sensor (Figure 2) to the box without water for few more minutes until the glue is completely dry. 
	NOTE: An optimal temperature for crayfish and glue manipulation varies from 18 to 22 &#176;C. At these temperatures, the glue hardens within 5 to 7 min and is completely dry within 8 to 10 min. At lower temperatures, the stress in the crayfish is less pronounced; however, the glue needs more time to harden, about 15 and 20 min under 15 &#176;C and 10 &#176;C, respectively. At higher temperatures, particularly above 25 &#176;C, the glue hardens within 3 min, but the crayfish undergoes much more stress; therefore, try to minimize the exposure of the crustacean to extreme conditions without water.</li>
	<li>Before moving the crayfish back into the tank, dip its cephalothorax into the water several times with short intervals of a few seconds in order to allow a discharge of the air that has accumulated in the gills, and leave the crayfish in the water for approximately 1 h to remove any excess chemicals. After this process is complete, release the crayfish into the water and allow it to acclimate for one to two weeks under experimental conditions, depending on observed physiological indices. Optimal water exchange during the acclimation periods is every other day. 
	NOTE: Characteristics of crayfish that have acclimated and are in a healthy state include pronounced circadian cardiac and locomotor activities, regular food consumption, and spending most daylight in a specialized shelter (if provided).</li>
</ol>
3. Camera and Software Setup
<ol>
	<li>Start the software; the video camera will automatically switch on.</li>
	<li>Select an option of movement detection, thoroughly detect area of each tank on the screen&#160;and the software will start tracking the behavior and linking it with the cardiac activity recordings. 
	NOTE: A crayfish motion detection module consists of a video camera that tracks crayfish behavior from below the tank and the software that combines the behavior with cardiac activity. The data from the module are used to facilitate more precise cardiac activity data processing by eliminating periods in which the crayfish demonstrates high locomotive activity. Sudden crayfish movements (i.e., an escape reaction or feeding initiation) can result in fluctuations or short-time spikes in cardiac signals that may reduce the precision of cardiac interval calculations.</li>
</ol>

<ol>
	<li>Bownik, A., Soko&#322;owska, N., &#346;laska, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+apomorphine,+a+dopamine+agonist,+on+Daphnia+magna:+Imaging+of+swimming+track+density+as+a+novel+tool+in+the+assessment+of+swimming+activity.">Effects of apomorphine, a dopamine agonist, on Daphnia magna: Imaging of swimming track density as a novel tool in the assessment of swimming activity.</a> Science of the Total Environment. 635, 249-258 (2018).</li><li>Jeong, T. Y., Yoon, D., Kim, S., Kim, H. Y., Kim, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mode+of+action+characterization+for+adverse+effect+of+propranolol+in+Daphnia+magna.+based+on+behavior+and+physiology+monitoring+and+metabolite+profiling.">Mode of action characterization for adverse effect of propranolol in Daphnia magna. based on behavior and physiology monitoring and metabolite profiling.</a> Environmental Pollution. 233, 99-108 (2018).</li><li>do Nascimento, M. T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+water+quality,+toxicity+and+estrogenic+activity+in+a+nearshore+marine+environment+in+Rio+de+Janeiro,+Southeastern+Brazil.">Determination of water quality, toxicity and estrogenic activity in a nearshore marine environment in Rio de Janeiro, Southeastern Brazil.</a> Ecotoxicology and Environmental Safety. 149, 197-202 (2018).</li><li>Xiao, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water+quality+monitoring+using+abnormal+tail-beat+frequency+of+crucian+carp.">Water quality monitoring using abnormal tail-beat frequency of crucian carp.</a> Ecotoxicology and Environmental Safety. 111, 185-191 (2015).</li><li>Aagaard, A., Andersen, B. B., Depledge, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+monitoring+of+physiological+and+behavioral+activity+in+marine+organisms+using+non-invasive,+computer+aided+techniques.">Simultaneous monitoring of physiological and behavioral activity in marine organisms using non-invasive, computer aided techniques.</a> Marine Ecology Progress Series. 73 (2), 277-282 (1991).</li><li>Bloxham, M. J., Worsfold, P. J., Depledge, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+biological+and+chemical+monitoring:+in+situ.+physiological+responses+of+freshwater+crayfish+to+fluctuations+in+environmental+ammonia+concentrations.">Integrated biological and chemical monitoring: in situ. physiological responses of freshwater crayfish to fluctuations in environmental ammonia concentrations.</a> Ecotoxicology. 8 (3), 225-237 (1999).</li><li>Depledge, M. H., Andersen, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computer-aided+physiological+monitoring+system+for+continuous,+long-term+recording+of+cardiac+activity+in+selected+invertebrates.">A computer-aided physiological monitoring system for continuous, long-term recording of cardiac activity in selected invertebrates.</a> Comparative Biochemistry and Physiology. A, Comparative Physiology. 96 (4), 473-477 (1990).</li><li>Depledge, M. H., Galloway, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Healthy+animals,+healthy+ecosystems.">Healthy animals, healthy ecosystems.</a> Frontiers in Ecology and the Environment. 3 (5), 251-258 (2005).</li><li>Holdich, D. M., Reynolds, J. D., Souty-Grosset, C., Sibley, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+ever+increasing+threat+to+European+crayfish+from+non-indigenous+crayfish+species.">A review of the ever increasing threat to European crayfish from non-indigenous crayfish species.</a> Knowledge and Management of Aquatic Ecosystems. 11, 394-395 (2009).</li><li>Vogt, G., Holdich, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+anatomy.">Functional anatomy.</a> Biology of freshwater crayfish. , 53-151 (2002).</li><li>Bierbower, S. M., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measures+of+heart+and+ventilatory+rates+in+freely+moving+crayfish.">Measures of heart and ventilatory rates in freely moving crayfish.</a> Journal of Visualized Experiments. (32), e1594 (2009).</li><li>Li, H., Listerman, L. R., Doshi, D., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+rate+in+blind+cave+crayfish+during+environmental+disturbances+and+social+interactions.">Heart rate in blind cave crayfish during environmental disturbances and social interactions.</a> Comparative Biochemistry and Physiology Part A: Molecular &amp; Integrative Physiology. 127 (1), 55-70 (2000).</li><li>Listerman, L. R., Deskins, J., Bradacs, H., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+rate+within+male+crayfish:+social+interactions+and+effects+of+5-HT.">Heart rate within male crayfish: social interactions and effects of 5-HT.</a> Comparative Biochemistry and Physiology Part A: Molecular &amp; Integrative Physiology. 125 (2), 251-263 (2000).</li><li>Burnett, N. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+noninvasive+method+for+measuring+heartbeat+of+intertidal+animals.">An improved noninvasive method for measuring heartbeat of intertidal animals.</a> Limnology and Oceanography: Methods. 11 (2), 91-100 (2013).</li><li>Fedotov, V. P., Kholodkevich, S. V., Strochilo, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+contractile+activity+of+the+crayfish+heart+with+the+aid+of+a+new+non-invasive+technique.">Study of contractile activity of the crayfish heart with the aid of a new non-invasive technique.</a> Journal of Evolutionary Biochemistry and Physiology. 36 (3), 288-293 (2000).</li><li>Kholodkevich, S. V., Ivanov, A. V., Kurakin, A. S., Kornienko, E. L., Fedotov, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real+time+biomonitoring+of+surface+water+toxicity+level+at+water+supply+stations.">Real time biomonitoring of surface water toxicity level at water supply stations.</a> Environmental Bioindicators. 3 (1), 23-34 (2008).</li><li>Kuznetsova, T. V., Sladkova, S. V., Kholodkevich, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+functional+state+of+crayfish+Pontastacus+leptodactylus+in+normal+and+toxic+environment+by+characteristics+of+their+cardiac+activity+and+hemolymph+biochemical+parameters.">Evaluation of functional state of crayfish Pontastacus leptodactylus in normal and toxic environment by characteristics of their cardiac activity and hemolymph biochemical parameters.</a> Journal of Evolutionary Biochemistry and Physiology. 46 (3), 241-250 (2010).</li><li>Sladkova, S. V., Kholodkevich, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+protein+in+hemolymph+of+crawfish+Pontastacus+leptodactylus+as+a+parameter+of+the+functional+state+of+animals+and+a+biomarker+of+quality+of+habitat.">Total protein in hemolymph of crawfish Pontastacus leptodactylus as a parameter of the functional state of animals and a biomarker of quality of habitat.</a> Journal of Evolutionary Biochemistry and Physiology. 47 (2), 160-167 (2011).</li><li>Pautsina, A., Kuklina, I., &#352;tys, D., C&#237;sa&#345;, P., Koz&#225;k, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+crayfish+cardiac+activity+monitoring+system.">Noninvasive crayfish cardiac activity monitoring system.</a> Limnology and Oceanography: Methods. 12 (10), 670-679 (2014).</li><li>C&#237;sa&#345;, P., Saberioon, M., Koz&#225;k, P., Pautsina, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+contactless+system+for+crayfish+heartbeat+monitoring:+Undisturbed+crayfish+as+bio-indicator.">Fully contactless system for crayfish heartbeat monitoring: Undisturbed crayfish as bio-indicator.</a> Sensors and Actuators B: Chemical. 255, 29-34 (2018).</li></ol>

The authors have nothing to disclose.

It has been widely suggested that the measurement of certain physiological parameters (such as heart or ventilation rate or both) is a more reliable method for recording crayfish reactions than the evaluation of behavioral responses that do not always occur immediately11. However, it is evident that the most efficient approach for assessing real crayfish reactions to environmental changes is the combination of cardiac activity and behavior recordings since that makes it possible to see the reason(...

The subject of aquatic organisms&#39; applications, both as model organisms for various laboratory investigations1,2 and as tools for monitoring industrial and natural/environmental water quality3,4, appears to be well studied. Nevertheless, this topic is still of noteworthy interest for humans, irrespective of whether they belong to the scientific community or to other occupations. In spite of the existence of a number of advanced methods for monitoring certain parameters (so-called &#34;biomarkers&#34;)5,6,7,8, the most important requirements for selecting an indicator consist of three simple factors: (i) simplicity, (ii) reliability, and (iii) general availability.
Crayfish, as an essential representative of freshwater fauna, distinguishes itself because it is found worldwide, is widespread, and, in most cases9, has a sufficiently large and hard carapace suitable for manipulation. This crustacean belongs to the group of higher invertebrates that provide sufficient development of vital physiological systems and respective organs while, at the same time, maintaining a relatively simple organization10.
Methods based on the assessment of the range of crayfishes&#39; biological and/or behavioral parameters, as described in the scientific literature, have significantly contributed to the development of biomonitoring and crayfish studies in general. Most of the currently available invasive methods for crayfish heart rate measurements are based on electrocardiogram recordings that require a complex and precise surgical procedure11,12,13; such manipulations can cause significant stress to and may require prolonged adaptation by the crayfish. Also, it is not known how long a crayfish can carry such electrodes and whether it will successfully molt while carrying such an attachment. The described noninvasive methods are based on plethysmographic recordings, which are complicated by hardware complexity and require a conditioning circuit for signal filtering14 and an amplification or precise and expensive optic components15,16.
In this study, we described an approach that contributes to existing results and offers new alternatives for improving current crayfish heart rate measurement procedures. Among the advantages, there are (i) a fast and noninvasive attachment that does not require a prolonged physiological adaptation; (ii) crayfishes&#39; capability to carry the sensor within a period of a few months from molting to molting; (iii) the software capable of monitoring real-time cardiac and behavioral activities and the evaluation of data obtained concurrently from multiple crayfish; (iv) a low manufacturing price and simplicity. The biomonitoring system that we describe permits the noninvasive and continuous monitoring of crayfish cardiac and locomotor activities based on changes in crayfishes&#39; etho-physiological characteristics. This system can easily be applied in laboratory examinations of the crayfish cardiac physiology and/or ethology, in addition to industrial implementations for controlling water quality at water treatment and supply facilities.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IR LED diode</td>
			<td style="width:180px;">KINGBRIGHT ELECTRONIC</td>
			<td style="width:155px;">KP-3216F3C</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phototransistor</td>
			<td style="width:180px;">EVERLIGHT</td>
			<td>ELPT15-21C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Resistor</td>
			<td style="width:180px;">ROYAL OHM</td>
			<td style="width:155px;">0805S8J0201T5E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Resistor</td>
			<td style="width:180px;">ROYAL OHM</td>
			<td style="width:155px;">0805S8F2200T5E</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Capacitor</td>
			<td style="width:180px;">KEMET</td>
			<td style="width:155px;">C0805C334K5RACTU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cable</td>
			<td style="width:180px;">TECHNOKABEL</td>
			<td style="width:155px;">FTP KAT.5E 4X2X0,14C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Connector</td>
			<td style="width:180px;">HARTING</td>
			<td style="width:155px;">21348100380005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Connector</td>
			<td style="width:180px;">HARTING</td>
			<td style="width:155px;">21348000380005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dielectric gel</td>
			<td style="width:180px;">KRAYDEN</td>
			<td style="width:155px;">Sylgard 535</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Analogue-to-digital convertor</td>
			<td style="width:180px;">TEDIA</td>
			<td style="width:155px;">UDAQ-1416CA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glue</td>
			<td style="width:180px;">KUPSITO.SK</td>
			<td>7338723044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Kinect video camera</td>
			<td style="width:180px;">ABCSTORE.CZ</td>
			<td style="width:155px;">GT3-00002</td>
			<td></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Analysis software</td>
			<td style="width:180px;">University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, Institute of Complex Systems</td>
			<td style="width:155px;"></td>
			<td style="width:167px;">Link to the software: www.frov.jcu.cz/crayfishmonitoring 
			User name: frov 
			Password: CF2018</td>
		</tr>
	</tbody>
</table>

continuous noninvasive measuring of crayfish cardiac and behavioral activities

A crayfish is a pivotal aquatic organism that serves both as a practical biological model for behavioral and physiological studies of invertebrates and as a useful biological indicator of water quality. Even though crayfish cannot directly specify the substances that cause water quality deterioration, they can immediately (within a few seconds) warn humans of water quality deterioration via acute changes in their cardiac and behavioral activities.
In this study, we present a noninvasive method that is simple enough to be implemented under various conditions due to a combination of simplicity and reliability in one model.
This approach, in which the biological organisms are implemented into environmental evaluation processes, provides a reliable and timely alarm for warning of and preventing acute water deterioration in an ambient environment. Therefore, this noninvasive system based on crayfish physiological and ethological parameter recordings was investigated for the detection of changes in an aquatic environment. This system is now applied at a local brewery for controlling&#160;quality of the water used for beverage production, but it can be used at any water treatment and supply facility for continuous, real-time water quality evaluation and for regular laboratory investigations of crayfish cardiac physiology and behavior.

As a result, we obtained a combination of crayfish cardiac and behavioral activities, recorded and saved in a txt-format file (Figure 3). Besides the number of experimental crayfish, the date, and the sampling rate, the file consists of three columns: (1) the continual time in hh:mm:ss format; (2) the heart rate automatically calculated in beats per minute; (3) the locomotion registered as absence (0) or presence (1) of any movement. When the crayfish was ina...

Watch this Scientific Journal Video about Continuous Noninvasive Measuring of Crayfish Cardiac and Behavioral Activities at JoVE.com

Continuous Noninvasive Measuring of Crayfish Cardiac and Behavioral Activities

This article presents a noninvasive biomonitoring system for the continuous recording and analyses of crayfish cardiac and locomotor activities. This system consists of a near-infrared optical sensor, a video-tracking module, and software for evaluating crayfish heartbeats that reflects its physiological condition and characterizes crayfish behavior during heartbeat fluctuations.

A crayfish is a pivotal aquatic organism that serves both as a practical biological model for behavioral and physiological studies of invertebrates and as ...

continuous-noninvasive-measuring-crayfish-cardiac-behavioral

Universidad de Cartagena UDEC

Research

JoVE Journal

Behavior

5.9K Views.  University of South Bohemia in České Budĕjovice. This article presents a noninvasive biomonitoring system for the continuous recording and analyses of crayfish cardiac and locomotor activities. This system consists of a near-infrared optical sensor, a video-tracking module, and software for evaluating crayfish heartbeats that reflects its physiological condition and characterizes crayfish behavior during heartbeat fluctuations.

Video: Continuous Noninvasive Measuring of Crayfish Cardiac and Behavioral Activities

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station (INBEST)

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of chronic CNS disorders, there is an increasing demand for murine models with high validity at the behavioral level. However, multiple pathogenic mechanisms and complex functional deficits often impose challenges to reliably measure and interpret behavior of chronically sick mice. Therefore, the assessment of peripheral pathology and a behavioral profile at several time points using a battery of tests are required. Video-tracking, behavioral spectroscopy, and remote acquisition of physiological measures are emerging technologies that allow for comprehensive, accurate, and unbiased behavioral analysis in a home-base-like setting. This report describes a refined phenotyping protocol, which includes a custom-made monitoring apparatus (Integrated Behavioral Station, INBEST) that focuses on prolonged measurements of basic functional outputs, such as spontaneous activity, food/water intake and motivated behavior in a relatively stress-free environment. Technical and conceptual improvements in INBEST design may further promote reproducibility and standardization of behavioral studies.

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station INBEST

Prolonged and comprehensive monitoring of mice in a home-cage environment provides a deeper understanding of aberrant behavior in murine models of brain diseases. This paper describes the Integrated Behavioral Station (INBEST) as the key component of contemporary behavioral analysis.

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of ...

Measuring Neural and Behavioral Activity During Ongoing Computerized Social Interactions: An Examination of Event-Related Brain Potentials

Social exclusion is a complex social phenomenon with powerful negative consequences. Given the impact of social exclusion on mental and emotional health, an understanding of how perceptions of social exclusion develop over the course of a social interaction is important for advancing treatments aimed at lessening the harmful costs of being excluded. To date, most scientific examinations of social exclusion have looked at exclusion after a social interaction has been completed. While this has been very helpful in developing an understanding of what happens to a person following exclusion, it has not helped to clarify the moment-to-moment dynamics of the process of social exclusion. Accordingly, the current protocol was developed to obtain an improved understanding of social exclusion by examining the patterns of event-related brain activation that are present during social interactions. This protocol allows greater precision and sensitivity in detailing the social processes that lead people to feel as though they have been excluded from a social interaction. Importantly, the current protocol can be adapted to include research projects that vary the nature of exclusionary social interactions by altering how frequently participants are included, how long the periods of exclusion will last in each interaction, and when exclusion will take place during the social interactions. Further, the current protocol can be used to examine variables and constructs beyond those related to social exclusion. This capability to address a variety of applications across psychology by obtaining both neural and behavioral data during ongoing social interactions suggests the present protocol could be at the core of a developing area of scientific inquiry related to social interactions.

Research on social exclusion has grown tremendously in recent years. As the field expands, it is imperative to develop sophisticated methodologies allowing for the simultaneous measurement of neural and behavioral outcomes during social exclusion. This protocol utilizes event-related brain potentials to record ongoing neural activity during computerized social interactions.

Social exclusion is a complex social phenomenon with powerful negative consequences. Given the impact of social exclusion on mental and emotional health, ...

A Novel Experimental and Analytical Approach to the Multimodal Neural Decoding of Intent During Social Interaction in Freely-behaving Human Infants

Understanding typical and atypical development remains one of the fundamental questions in developmental human neuroscience. Traditionally, experimental paradigms and analysis tools have been limited to constrained laboratory tasks and contexts due to technical limitations imposed by the available set of measuring and analysis techniques and the age of the subjects. These limitations severely limit the study of developmental neural dynamics and associated neural networks engaged in cognition, perception and action in infants performing &#8220;in action and in context&#8221;. This protocol presents a novel approach to study infants and young children as they freely organize their own behavior, and its consequences in a complex, partly unpredictable and highly dynamic environment. The proposed methodology integrates synchronized high-density active scalp electroencephalography (EEG), inertial measurement units (IMUs), video recording and behavioral analysis to capture brain activity and movement non-invasively in freely-behaving infants. This setup allows for the study of neural network dynamics in the developing brain, in action and context, as these networks are recruited during goal-oriented, exploration and social interaction tasks.

This protocol presents a novel methodology for the neural decoding of intent from freely-behaving infants during unscripted social interaction with an actor. Neural activity is acquired using non-invasive high-density active scalp electroencephalography (EEG). Kinematic data is collected with inertial measurement units and supplemented with synchronized video recording.

Understanding typical and atypical development remains one of the fundamental questions in developmental human neuroscience. Traditionally, experimental ...

The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article describes a rodent version of the PVT&#8212;termed the &#34;rPVT&#34;&#8212;that measures similar aspects of attention (i.e., performance accuracy, motor speed, premature responding, and lapses in attention). Data are presented that demonstrate both the short- and long-term usefulness of the rPVT when employed with laboratory rats. Rats easily learn the rPVT, and learning to perform the basic procedure takes less than two weeks of training. Once acquired, rat performances in the rPVT show a high degree of similarity to these same performance measures in the human PVT, including similarities in, lapses in attention, reaction times, vigilance decrements across session time (i.e., the human &#34;time-on-task&#34; effects), and the response-stimulus interval (RSI) effect described for humans. Thus the rPVT can be an extremely valuable tool for assessing the effects of a wide range of variables on sustained attention quite similar to human PVT performances, and thus can be useful for developing novel treatments for neurobehavioral dysfunctions.

The Rodent Psychomotor Vigilance Test rPVT: A Method for Assessing Neurobehavioral Performance in Rats and Mice

A rat version of the human Psychomotor Vigilance Test (PVT) is described that measures aspects of attention similar to those measured with the human PVT, including aspects of human vigilance such as performance accuracy, motor speed, premature responding, and lapses in attention.

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article ...

Using Wavelet Entropy to Demonstrate how Mindfulness Practice Increases Coordination between Irregular Cerebral and Cardiac Activities

In both the East and West, traditional teachings say that the mind and heart are somehow closely correlated, especially during spiritual practice. One difficulty in proving this objectively is that the natures of brain and heart activities are quite different. In this paper, we propose a methodology that uses wavelet entropy to measure the chaotic levels of both electroencephalogram (EEG) and electrocardiogram (ECG) data and show how this may be used to explore the potential coordination between the mind and heart under different experimental conditions. Furthermore, Statistical Parametric Mapping (SPM) was used to identify the brain regions in which the EEG wavelet entropy was the most affected by the experimental conditions. As an illustration, the EEG and ECG were recorded under two different conditions (normal rest and mindful breathing) at the beginning of an 8-week standard Mindfulness-based Stress Reduction (MBSR) training course (pretest) and after the course (posttest). Using the proposed method, the results consistently showed that the wavelet entropy of the brain EEG decreased during the MBSR mindful breathing state as compared to that during the closed-eye resting state. Similarly, a lower wavelet entropy of heartrate was found during MBSR mindful breathing. However, no difference in wavelet entropy during MBSR mindful breathing was found between the pretest and posttest. No correlation was observed between the entropy of brain waves and the entropy of heartrate during normal rest in all participants, whereas a significant correlation was observed during MBSR mindful breathing. Additionally, the most well-correlated brain regions were located in the central areas of the brain. This study provides a methodology for the establishment of evidence that mindfulness practice (i.e., mindful breathing) may increase the coordination between mind and heart activities.

This manuscript describes how to use the wavelet entropy index to analyze high-density electroencephalography (EEG) and electrocardiography (ECG) data. We show that the irregularity of cerebral and cardiac activities became more coordinated during mindfulness-based stress reduction practice.

In both the East and West, traditional teachings say that the mind and heart are somehow closely correlated, especially during spiritual practice. One ...

Basic Methods for the Study of Reproductive Ecology of Fish in Aquaria

Captive-rearing observations are valuable for revealing aspects of fish behavior and ecology when continuous field investigations are impossible. Here, a series of basic techniques are described to enable observations of the reproductive behavior of a wild-caught gobiid fish, as a model, kept in an aquarium. The method focuses on three steps: collection, transport, and observations of reproductive ecology of a substrate spawner. Essential aspects of live fish collection and transport are (1) preventing injury to the fish, and (2) careful acclimation to the aquarium. Preventing harm through injuries such as scratches or a sudden change of water pressure is imperative when collecting live fish, as any physical damage is likely to negatively affect the survival and later behavior of the fish. Careful acclimation to aquaria decreases the incidence death and mitigates the shock of transport. Observations during captive rearing include (1) the identification of individual fish and (2) monitoring spawned eggs without negative effects to the fish or eggs, thereby enabling detailed investigation of the study species' reproductive ecology. The subcutaneous injection of a visible implant elastomer (VIE) tag is a precise method for the subsequent identification of individual fish, and it can be used with a wide size range of fish, with minimal influence on their survival and behavior. If the study species is a substrate spawner that deposits adhesive eggs, an artificial nest site constructed from polyvinyl chloride (PVC) pipe with the addition of a removable waterproof sheet will facilitate counting and monitoring the eggs, lessening the investigator's influence on the nest-holding and egg-guarding behavior of the fish. Although this basic method entails techniques that are seldom mentioned in detail in research articles, they are fundamental for undertaking experiments that require the captive rearing of a wild fish.

A series of basic methods to enable the study of the reproductive ecology of fish kept in aquaria are described. These are useful protocols for collecting fish using SCUBA, transporting live fish, and observing the reproductive behavior of wild-caught fish kept in aquaria.

Captive-rearing observations are valuable for revealing aspects of fish behavior and ecology when continuous field investigations are impossible. Here, a ...

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer scale. However, current methods mostly rely on tethered cable recordings or only use unidirectional systems, allowing either recording or stimulation of neural activity, but not at the same time or same target. Here, a new wireless, bidirectional device for simultaneous multichannel recording and stimulation of neural activity in freely behaving rats is described. The system operates through a single portable head stage that both transmits recorded activity and can be targeted in real-time for brain stimulation using a telemetry-based multichannel software. The head stage is equipped with a preamplifier and a rechargeable battery, allowing stable long-term recordings or stimulation for up to 1 h. Importantly, the head stage is compact, weighs 12 g (including battery) and thus has minimal impact on the animal&#180;s behavioral repertoire, making the method applicable to a broad set of behavioral tasks. Moreover, the method has the major advantage that the effect of brain stimulation on neural activity and behavior can be measured simultaneously, providing a tool to assess the causal relationships between specific brain activation patterns and behavior. This feature makes the method particularly valuable for the field of deep brain stimulation, allowing precise assessment, monitoring, and adjustment of stimulation parameters during long-term behavioral experiments. The applicability of the system has been validated using the inferior colliculus as a model structure.

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

A wireless, bidirectional system for multi-channel neural recordings and stimulation in freely behaving rats is introduced. The system is light and compact, thus having minimal impact on the animal&#180;s behavioral repertoire. Moreover, this bidirectional system provides a sophisticated tool to assess causal relationships between brain activation patterns and behavior.

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer ...

Noninvasive, High-throughput Determination of Sleep Duration in Rodents

Traditionally, sleep is monitored by an electroencephalogram (EEG). EEG studies in rodents require surgical implantation of the electrodes followed by a long recovery period. To perform an EEG recording, the animal is connected to a receiver, creating an unnatural tether to the head-mount. EEG monitoring is time consuming, carries risk to the animal, and is not a completely natural setting for the measurement of sleep. Alternative methods to detect sleep, particularly in a high-throughput fashion, would greatly advance the field of sleep research. Here, we describe a validated method for detecting sleep via activity-based home-cage monitoring. Previous studies have shown that sleep assessed via this method has a high degree of agreement with sleep defined by traditional EEG-based measures. Whereas this method is validated for total sleep time, it is important to note that sleep bout duration should be assessed by an EEG which has better temporal resolution. The EEG can also differentiate rapid eye movement (REM) and non-REM sleep, giving more detail about the exact nature of sleep. Nevertheless, activity-based sleep determination can be used to analyze multiple days of undisturbed sleep and to assess sleep as a response to an acute event (like stress). Here, we show the power of this system to detect the response of mice to daily intraperitoneal injections.

We describe a high-throughput method of measuring sleep by means of activity-based home-cage monitoring. This method offers advantages over traditional EEG-based methods. It is well validated for the determination of total sleep duration and can be a powerful tool to monitor sleep in rodent models of human disease.

Traditionally, sleep is monitored by an electroencephalogram (EEG). EEG studies in rodents require surgical implantation of the electrodes followed by a ...

An Automated T-maze Based Apparatus and Protocol for Analyzing Delay- and Effort-based Decision Making in  Free Moving Rodents

Many neurological and psychiatric patients demonstrate difficulties and/or deficits in decision making. Rodent models are helpful to produce a deeper understanding of the neurobiological causes underlying the decision-making problems. A cost-benefit based T-maze task is used for measuring decision making in which rodents choose between a high reward arm (HRA) and a low reward arm (LRA). There are two paradigms of the T-maze decision-making task, one in which the cost is a time delay and the other in which it is physical effort. Both paradigms require a tedious and labor-intensive management of experimental animals, multiple doors, pellet reward, and arm choice recordings. In the current work, we invented an apparatus based on traditional T-maze with full automation for pellet delivery, door management and choice recordings. This automated setup can be used for the evaluation of both delay- and effort-based decision making in rodents. With the protocol described here, our lab investigated the decision-making phenotypes of multiple genetically modified mice. In the representative data, we showed that the mice with ablated medial habenular showed aversions of both delay and effort and tended to choose the immediate and effortless reward. This protocol helps to decrease the variability caused by experimenter intervention and to enhance experiment efficiency. In addition, chronic silicon probe or microelectrode recording, fiber-optic imaging and/or manipulation of neural activity can be easily applied during the decision-making task using the setup described here.

This article introduces an automated T-maze apparatus that we invented, and a protocol based on this apparatus for analyzing delay-based decision making and effort-based decision making in free moving rodents.

Many neurological and psychiatric patients demonstrate difficulties and/or deficits in decision making. Rodent models are helpful to produce a deeper ...

Noninvasive, In-pen Approach Test for Laboratory-housed Pigs

Traumatic brain injury (TBI) incidences have increased in both civilian and military populations, and many researchers are adopting a porcine model for TBI. Unlike rodent models for TBI, there are few behavioral tests that have been standardized. A larger animal requires more invasive handling in test areas than rodents, which potentially adds stress and variation to the animals&#39; responses. Here, the human approach test (HAT) is described, which was developed to be performed in front of laboratory pigs&#39; home pen. It is noninvasive, but flexible enough that it allows for differences in housing set-ups.
During the HAT, three behavioral ethograms were developed and then a formula was applied to create an approach index (AI). Results indicate that the HAT and its index, AI, are sensitive enough to detect mild and temporary alterations in pigs&#39; behavior after a mild TBI (mTBI). In addition, although specific behavior outcomes are housing-dependent, the use of an AI reduces variation and allows for consistent measurements across laboratories. This test is reliable and valid; HAT can be used across many laboratories and for various types of porcine models of injury, sickness, and distress. This test was developed for an optimized manual timestamping method such that the observer consistently spends no more than 9 min on each sample.

This protocol describes a new behavioral test&#8212;the human approach test in the pigs&#39; home pen&#8212;to detect functional deficits in laboratory pigs after subconcussive traumatic brain injury.

Traumatic brain injury (TBI) incidences have increased in both civilian and military populations, and many researchers are adopting a porcine model for ...

Continuous Noninvasive Measuring of Crayfish Cardiac and Behavioral Activities

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Chapters in this video

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station (INBEST)

Measuring Neural and Behavioral Activity During Ongoing Computerized Social Interactions: An Examination of Event-Related Brain Potentials

A Novel Experimental and Analytical Approach to the Multimodal Neural Decoding of Intent During Social Interaction in Freely-behaving Human Infants

The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice

Using Wavelet Entropy to Demonstrate how Mindfulness Practice Increases Coordination between Irregular Cerebral and Cardiac Activities

Basic Methods for the Study of Reproductive Ecology of Fish in Aquaria

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

Noninvasive, High-throughput Determination of Sleep Duration in Rodents

An Automated T-maze Based Apparatus and Protocol for Analyzing Delay- and Effort-based Decision Making in Free Moving Rodents

Noninvasive, In-pen Approach Test for Laboratory-housed Pigs