Name: continuous noninvasive measuring of crayfish cardiac and behavioral activities
Uploaded: 2019-02-06T14:00:00
Description: Watch this Scientific Journal Video about Continuous Noninvasive Measuring of Crayfish Cardiac and Behavioral Activities at JoVE.com

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...

<ol>
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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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			<td height="21" style="height:21px;width:216px;">Capacitor</td>
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			<td height="21" style="height:21px;width:216px;">Connector</td>
			<td style="width:180px;">HARTING</td>
			<td style="width:155px;">21348100380005</td>
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			<td height="21" style="height:21px;width:216px;">Connector</td>
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			<td style="width:155px;">21348000380005</td>
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			<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>
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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 ...

This method can help answer key questions in the environmental and ecotoxicalogical fields involving the relationship between cardiac and locomotor activities on the various acute and prolonged stressful conditions. The main advantages of this technique are that crayfish don't require complex prior manipulations or prolonged adaptation and are capable of bearing sensors for a few month between methods. The implications of this technique extend toward etiological, physiological, ecotoxicological, and industrial applications as crayfish aided noninvasive biomonitoring can be implemented in laboratories and water treatment and supply facilities.Though this method can provide inside insight into crayfish cardiac, physiology, and behavior and water quality control, it can potentially be applied to other large freshwater and marine crustaceans. Visual demonstration of this method is critical for understanding the link between noninvasive sensor using near infrared light for evaluation in crayfish heart and movement detection modules based on image processing. A few days before beginning the procedure, select adult specimens with a carapace size of at least 30 millimeters.And examine each specimen for the absence of disease and whether they lift both cheli when touched. Then allow the healthy crayfish to acclimate in a tank of settled tap water until the day of the procedure. As soon as the sensors have been prepared, open the sensor software on the computer and enter the number of crayfish to be fixed to the sensors and displayed on the screen.Set the number of crayfish heart rates to be visualized. And use a paper towel to dry the dorsal carapace of the first animal. Wrap the cheli and abdomen of the crayfish in the paper towel to avoid damage and to eliminate additional stress to the animal.And attach the censor to the dorsal carapace in a position that facilitates a maximal cardiac signal amplitude. Holding the crayfish with the sensor in one hand, use the other hand to add a drop of freshly mixed epoxy glue onto each of the four auxiliary wires located on the sensor. Then allow the glue to dry without moving the sensor for at least five minutes.When the glue is no longer sticky to the touch, place the unwrapped crayfish and sensor into a box without water for a few more minutes until the glue is completely dry. Before moving the crayfish back into the tank, immerse the cephalothorax into the tank water for a few seconds several times to discharge the air that has accumulated in the gills. And leave the crayfish in the holding water tank for approximately one hour to remove any excess chemicals.Then, release the crayfish into the experimental water tanks for one to two weeks of acclimation and under the appropriate experimental conditions depending on the observed physiological indices. As soon as the crayfish are placed in the experimental tanks, start the software. The video camera will automatically switch on.Then select the movement detection option and locate each tank on the screen to start tracking the behavior and linking the behavior of the crayfish with the cardiac activity recordings. The recorded crayfish cardiac and behavioral activities can be save in a TXT format file. It is crucial to include the locomotion of the crayfish in the analysis as this activity can cause changes in the heart rate.For example, in this experiment ten seconds after the start of the experiment a food odor was delivered into the tank via a peristaltic pump. At 14 seconds the crayfish recognized the stimulus and it's heart rate slightly decreased due to the so called orienting response. After 20 seconds, the heart rate increased resulting in a decrease in cardiac intervals.At 26 seconds the crayfish moved toward the stimulus source and both the physiological excitation caused by the food odor and the locomotion initiation resulted in a substantial heart rate increase. At 37 seconds there was evidence of abrupt crayfish motion that could have substantially contributed to the heart rate growth during the reactions to the stimulus. Indeed, a disturbed crayfish typically demonstrates an increase in heart rate that is often associated with occasional locomotion.However, even motionless crayfish can demonstrate a high heart rate that also indicates a pronounced stress. The heart rate of an undisturbed crayfish is characterized by a monotonic amplitude of the heart beat curve and by approximately equal cardiac intervals between each cardiac peak. While attempting this procedure it's important to remember that a quick and thorough sensor attachment will cause less stress to the experimental animals enabling the acquisition of precise physiological characteristics.More advanced monitoring methods include the use of fully contactless crayfish monitoring which allows the heart beat frequency to be determined using only the combination of a near infrared sensor and a sensitive camera. After its development, this approach paved the way for researchers in the fields of behavior, physiology, reproduction, and androgyne of unrestricted crayfish and other large aquatic invertebrates to explore environmental and anthropogenic impact on bioindicator organisms. For the noninvasive biomonitoring, has a very practical application at the local variety in the Czech Republic as a real time water quality early monitoring system.While the stability of the water state is continuously assessed to the dynamics of crayfish ecophysiological characteristics.

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