The study was supported by the Basic Research Founding of the Physics Department and the Biology Department of the Naples University Federico II. The authors thank Dr. Claudia Angelini (Institute of Applied Calculus, Consiglio Nazionale delle Ricerche [CNR], Italy) for the statistical support. The authors thank Martina Scanu and Silvia Frassinet for their technical help with collecting the data, and the departmental technicians F. Cassese, G. Passeggio, and R. Rocco for their skillful assistance in the design and realization of the experimental setup. We thank Laura Gentile for helping conducting the experiment during the video shooting. We thank Diana Rose Udel from the University of Miami for shooting the interview statements of Alessandro Cresci.

The following protocol has been approved by the Institutional Animal Care and Use Committee of the University of Naples Federico II, Naples, Italy (2015).
1. Animal Maintenance
<ol>
	<li>Use tanks of at least 200 L to host a shoal of at least 50 individuals of both sexes in each tank. 
	NOTE: The density of the fish in the tank has to be one animal per 2 L or lower. Under these conditions, zebrafish will display normal shoaling behavior.</li>
	<li>Set the maintenance co...

<ol>
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W., 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=Animal+orientation+strategies+for+movement+in+flows.">Animal orientation strategies for movement in flows.</a> Current Biology. 21 (20), R861-R870 (2011).</li><li>Montgomery, J. C., Baker, C. F., Carton, 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=The+lateral+line+can+mediate+rheotaxis+in+fish.">The lateral line can mediate rheotaxis in fish.</a> Nature. 389 (6654), 960-963 (1997).</li><li>Baker, C. F., Montgomery, J. 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The authors have nothing to disclose.

The protocol described in this study allows scientists to quantify complex orientation responses of aquatic species resulting from the integration between two external cues (water current and geomagnetic field) and one internal factor of the animal, such as personality. The overall concept is to create an experimental design that allows scientists to separate individuals of different personality and investigate their orientation behavior while controlling separately or simultaneously the external environmental cues.
...

In the present study, we describe a lab-based behavioral protocol which has the scope of investigating the role of fish personality on the orientation response of shoaling fish to external orientation cues, such as water currents and magnetic fields.
The orienting decisions of animals result from weighing various sensory information. The decision process is influenced by the ability of the animal to navigate (e.g., the capacity to select and keep a direction), its internal state (e.g., feeding or reproductive needs), its ability to move (e.g., locomotion biomechanics), and several additional external factors (e.g., time of day, interaction with conspecifics)1.
The role of the internal state or animal personality in the orientation behavior is often poorly understood or not explored2. Additional challenges arise in the study of the orientation of social aquatic species, which often perform coordinated and polarized group movement behavior3.
Water currents play a key role in the orientation process of fish. Fish orient to water currents through an unconditioned response called rheotaxis4, which can be positive (i.e., upstream oriented) or negative (i.e. downstream oriented) and is used for several activities, ranging from foraging to the minimization of energetic expenditure5,6. Moreover, a growing body of literature reports that many fish species use the geomagnetic field for orientation and navigation7,8,9.
The study of rheotaxis and swimming performance in the fish is usually conducted in flow chambers (flume), where fish are exposed to the stepwise increase of the flow speed, from low to high speeds, often until exhaustion (called critical speed)10,11. On the other hand, previous studies investigated the role of the magnetic field in the orientation through the observation of the swimming behavior of the animals in arenas with still water12,13. Here, we describe a laboratory technique that allows researchers to study the behavior of fish while manipulating both the water currents and the magnetic field. This method was utilized for the first time on shoaling zebrafish (Danio rerio) in our previous study, leading to the conclusion that the manipulation of the surrounding magnetic field determines the rheotactic threshold (i.e., the minimal water speed at which shoaling fish orient upstream)14. This method is based on the use of a flume chamber with slow flows combined with a setup designed to control the magnetic field in the flume, within the range of the earth&#8217;s magnetic field intensity.
The swimming tunnel utilized to observe the behavior of zebrafish is outlined in Figure 1. The tunnel (made of a nonre&#64258;ecting acrylic cylinder with a 7 cm diameter and 15 cm in length) is connected to a setup for the control of the flow rate14. With this setup, the range of flow rates in the tunnel varies between 0 and 9 cm/s.
To manipulate the magnetic field in the swimming tunnel, we use two methodological approaches: the first is one-dimensional and the second is three-dimensional. For any application, these methods manipulate the geomagnetic field to obtain specific magnetic conditions in a defined volume of water&#8212;thus, all the values of magnetic field intensity reported in this study include the geomagnetic field.
Concerning the one-dimensional approach15, the magnetic field is manipulated along the water flow direction (defined as the x-axis) using a solenoid wrapped around the swimming tunnel. This is connected to a power unit, and it generates uniform static magnetic fields (Figure 2A). Similarly, in the case of the three-dimensional approach, the geomagnetic field in the volume containing the swimming tunnel is modified using coils of electric wires. However, to control the magnetic field in three dimensions, the coils have the design of three orthogonal Helmholtz pairs (Figure 2B). Each Helmholtz pair is composed of two circular coils oriented along the three orthogonal space directions (x, y, and z) and equipped with a three-axial magnetometer working in closed-loop conditions. The magnetometer works with field intensities comparable with the earth&#8217;s natural field, and it is located close to the geometrical center of the coils set (where the swimming tunnel is located).
We implement the techniques described above to test the hypothesis that the personality traits of the fish composing a shoal influence the way they respond to magnetic fields16. We test the hypothesis that individuals with proactive and reactive personality17,18 respond differently when exposed to water flows and magnetic fields. To test this, we first sort zebrafish using an established methodology to assign and group individuals that are proactive or reactive17,19,20,21. Then, we evaluate the rheotactic behavior of zebrafish swimming in shoals composed of only reactive individuals or composed of only proactive individuals in the magnetic flume tank, which we present as sample data.
The sorting method is based on the different tendency of the proactive and reactive individuals to explore novel environments21. Specifically, we use a tank divided into a bright and a dark side17,19,20,21 (Figure 3). Animals are acclimated to the dark side. When access to the bright side is open, proactive individuals tend to quickly exit the dark half of the tank to explore the new environment, while the reactive fish do not leave the dark tank.

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		<tr height="46">
			<td height="46" style="height:47px;width:216px;">9500 G meter</td>
			<td style="width:196px;">FWBell</td>
			<td style="width:141px;">N/A</td>
			<td style="width:389px;">Gaussmeter, DC-10 kHz; probe resolution:&#160; 0.01 &#956;T&#160;</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:216px;">AD5755-1</td>
			<td style="width:196px;">Analog Devices</td>
			<td style="width:141px;">EVAL-AD5755SDZ</td>
			<td style="width:389px;">Quad Channel, 16-bit, Digital to Analog Converter</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:216px;">ALR3003D</td>
			<td style="width:196px;">ELC</td>
			<td style="width:141px;">3760244880031</td>
			<td style="width:389px;">DC Double Regulated power supply</td>
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		<tr height="46">
			<td height="46" style="height:47px;width:216px;">BeagleBone Black</td>
			<td style="width:196px;">Beagleboard.org</td>
			<td style="width:141px;">N/A</td>
			<td style="width:389px;">Single Board Computer</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:216px;">Coil driver</td>
			<td style="width:196px;">Home made</td>
			<td style="width:141px;">N/A</td>
			<td style="width:389px;">Amplifier based on commercial OP (OPA544 by TI)</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:216px;">Helmholtz pairs</td>
			<td style="width:196px;">Home made</td>
			<td style="width:141px;">N/A</td>
			<td style="width:389px;">Coils made with standard AWG-14 wire</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:216px;">HMC588L</td>
			<td style="width:196px;">Honeywell</td>
			<td style="width:141px;">900405 Rev E</td>
			<td style="width:389px;">Digital three-axis magnetometer</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:216px;">MO99-2506</td>
			<td style="width:196px;">FWBell</td>
			<td style="width:141px;">129966</td>
			<td style="width:389px;">Single axis magnetic probe</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:216px;">Swimming apparatus</td>
			<td style="width:196px;">M2M Engineering Custom Scientific Equipment</td>
			<td style="width:141px;">N/A</td>
			<td style="width:389px;">Swimming apparatus composed by peristaltic pump and SMC Flow switch flowmeter with digital feedback</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:216px;">TECO 278</td>
			<td style="width:196px;">TECO</td>
			<td style="width:141px;">N/A</td>
			<td style="width:389px;">Thermo-cryostat&#160;</td>
		</tr>
	</tbody>
</table>

assessing the influence of personality on sensitivity to magnetic fields in zebrafish

To orient themselves in their environment, animals integrate a wide array of external cues, which interact with several internal factors, such as personality. Here, we describe a behavioral protocol designed for the study of the influence of zebrafish personality on their orientation response to multiple external environmental cues, specifically water currents and magnetic fields. This protocol aims to understand whether proactive or reactive zebrafish display different rheotactic thresholds (i.e., the flow speed at which the fish start swimming upstream) when the surrounding magnetic field changes its direction. To identify zebrafish with the same personality, fish are introduced in the dark half of a tank connected with a narrow opening to a bright half. Only proactive fish explore the novel, bright environment. Reactive fish do not exit the dark half of the tank. A swimming tunnel with low flow rates is used to determine the rheotactic threshold. We describe two setups to control the magnetic field in the tunnel, in the range of the earth&#8217;s magnetic field intensity: one that controls the magnetic field along the flow direction (one dimension) and one that allows a three-axial control of the magnetic field. Fish are filmed while experiencing a stepwise increase of the flow speed in the tunnel under different magnetic fields. Data on the orientation behavior are collected through a video-tracking procedure and applied to a logistic model to allow the determination of the rheotactic threshold. We report representative results collected from shoaling zebrafish. Specifically, these demonstrate that only reactive, prudent fish show variations of the rheotactic threshold when the magnetic field varies in its direction, while proactive fish do not respond to magnetic field changes. This methodology can be applied to the study of magnetic sensitivity and rheotactic behavior of many aquatic species, both displaying solitary or shoaling swimming strategies.

As sample data we present results obtained controlling the magnetic field along the water flow direction on proactive and reactive shoaling zebrafish16 using the setup shown in Figure 2A (see section 3 of the protocol). These results show how the described protocol can highlight differences in responses to the magnetic field in fish with different personalities. The overall concept of these trials relies on the finding that the directi...

Watch this Scientific Journal Video about Assessing the Influence of Personality on Sensitivity to Magnetic Fields in Zebrafish at JoVE.com

Assessing the Influence of Personality on Sensitivity to Magnetic Fields in Zebrafish

We describe a behavioral protocol designed to assess how zebrafish&#8217;s personalities influence their response to water currents and weak magnetic fields. Fishes with the same personalities are separated based on their explorative behavior. Then, their rheotactic orientation behavior in a swimming tunnel with a low flow rate and under different magnetic conditions is observed.

To orient themselves in their environment, animals integrate a wide array of external cues, which interact with several internal factors, such as ...

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6.5K Views.  University of Miami. Questo protocollo consente di esplorare come lo stato interno o la personalità di un pesce zebra influenzi la risposta di orientamento ai segnali provenienti dall'ambiente come correnti d'acqua o campi magnetici. Questa tecnica viene utilizzata per osservare come sia i fattori interni che gli spunti ambientali influiscono sull'orientamento del pesce zebra. Tuttavia, può anche essere applicato a un'ampia varietà di progetti sperimentali.Questo metodo potrebbe fornire informazioni sul...