Name: boldness, aggression, and shoaling assays for zebrafish behavioral syndromes
Uploaded: 2016-08-29T13:00:00
Description: Watch this Scientific Journal Video about Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes at JoVE.com

This work was supported by a Howard Hughes Medical Institute Education Grant and an internal grant from the Saint Joseph's University chapter of Sigma Xi. We would also like to thank the three anonymous reviewers who helped strengthen the protocol and interpretations.

The following methodologies for the housing, care, and study of zebrafish have been approved by the Saint Joseph&#39;s University IACUC.
1. Zebrafish Housing and Care
<ol>
	<li>Obtain the subject zebrafish from a local supplier, or from wild-caught populations. Please note that housing the zebrafish is subject to IACUC guidelines and additional permits are required for housing wild-caught populations.</li>
	<li>Identify and separate zebrafish according to sex. Alternatively, skip this step o...

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggression+and+vasotocin+are+associated+with+dominant-subordinate+relationships+in+zebrafish.">Aggression and vasotocin are associated with dominant-subordinate relationships in zebrafish.</a> Behav Brain Res. 167 (1), 94-102 (2006).</li><li>McRobert, S., Bradner, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+body+coloration+on+shoaling+preferences+in+fish.">The influence of body coloration on shoaling preferences in fish.</a> Anim Behav. 56 (3), 611-615 (1998).</li><li>Ruhl, N., McRobert, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+sex+and+shoal+size+on+shoaling+behaviour+in+Danio+rerio.">The effect of sex and shoal size on shoaling behaviour in Danio rerio.</a> J Fish Biol. 67 (5), 1318-1326 (2005).</li><li>Snekser, J., Ruhl, N., Bauer, K., McRobert, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+sex+and+phenotype+on+shoaling+decisions+in+zebrafish.">The influence of sex and phenotype on shoaling decisions in zebrafish.</a> J Comp Psychol. 23, 70-81 (2010).</li><li>Budaev, 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=Using+Principal+Components+and+Factor+Analysis+in+Animal+Behaviour+Research:+Caveats+and+Guidelines.">Using Principal Components and Factor Analysis in Animal Behaviour Research: Caveats and Guidelines.</a> Ethology. 116 (5), 472-480 (2010).</li><li>Paciorek, T., McRobert, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Daily+variation+in+the+shoaling+behavior+of+zebrafish+Danio+rerio.">Daily variation in the shoaling behavior of zebrafish Danio rerio.</a> Curr Zool. 58 (1), 129-137 (2012).</li><li>Wright, D., Krause, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+measures+of+shoaling+tendency+in+zebrafish+(Danio+rerio)+and+other+small+teleost+fishes.">Repeated measures of shoaling tendency in zebrafish (Danio rerio) and other small teleost fishes.</a> Nat Protoc. 1 (4), 1828-1831 (2006).</li><li>Cachat, J., Stewart, A., 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=Measuring+behavioral+and+endocrine+responses+to+novelty+stress+in+adult+zebrafish.">Measuring behavioral and endocrine responses to novelty stress in adult zebrafish.</a> Nat Protoc. 5 (11), 1786-1799 (2010).</li><li>Croft, D. 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A., Wedderburn, R. W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generalized+Linear+Models.">Generalized Linear Models.</a> J Roy Stat Soc A Sta. 135, 370-384 (1972).</li><li>Iman, R. L., Conover, W. 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+distribution-free+approach+to+inducing+rank+correlation+among+input+variables.">A distribution-free approach to inducing rank correlation among input variables.</a> Commun Stat Simulat. 11 (3), 311-334 (1982).</li><li>Fleiss, J. L., Cohen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Equivalence+of+Weighted+Kappa+and+the+Intraclass+Correlation+Coefficient+as+Measures+of+Reliability.">The Equivalence of Weighted Kappa and the Intraclass Correlation Coefficient as Measures of Reliability.</a> Educ Psychol Meas. 33 (3), 613-619 (1973).</li><li>Ruhl, N., McRobert, S. P., Currie, W. J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shoaling+preferences+and+the+effects+of+sex+ratio+on+spawning+and+aggression+in+small+laboratory+populations+of+zebrafish+(Danio+rerio).">Shoaling preferences and the effects of sex ratio on spawning and aggression in small laboratory populations of zebrafish (Danio rerio).</a> Lab Animal. 38 (8), 264-269 (2009).</li><li>Bell, A. 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The protocol will determine if there are consistent associations in boldness, aggression, and shoaling behaviors in zebrafish. If there are consistent associations in a given population between any of these behaviors, then a behavioral syndrome is present. By studying a population&#39;s natural behavioral syndrome, researchers can have a more complete understanding of its behavioral dynamic, population structure, and possibly evolutionary history3. Furthermore, manipulating the environment that affects these b...

There is a growing body of literature investigating the associations between distinct behaviors within individual animals from a given population. These associations are termed behavioral syndromes, and the measurements typically include boldness, aggression, exploratory behavior, and sociability1-5. Behavioral syndromes are valuable for both direct and indirect reasons. Directly, knowledge of behavioral syndromes can provide a more complete view of evolutionary theory, population structure, and population dynamics3. Indirectly, knowledge regarding behavioral associations may inform fields that quantify behavior such as pharmacology6 , toxicology7, behavioral genetics8,9, and endocrinology10. Because of these direct and indirect benefits, an increased knowledge of behavioral syndromes is especially valuable in commonly used model organisms such as the zebrafish. Studies using zebrafish are found in a variety of disciplines, including the analysis of behavioral syndromes11-13. To advance knowledge in behavioral syndrome research, and because other disciplines also use behavioral measurements for hypothesis testing, reliable and succinct descriptions of behavior are required for valid analyses and interpretations and standardized protocols will facilitate inter-study comparisons within species. Our protocol was developed to measure a boldness-aggression-shoaling behavioral syndrome in a population of lab-reared zebrafish14. However, the basis of the protocol (tracking individual fish, ensuring proper randomization, and appropriate analyses) can be easily modified for a variety of alternative behavioral measures. Additionally, boldness, aggression, or shoaling assays can be run individually for the testing of distinct hypotheses. Therefore, while it is our goal to describe how to conduct a behavioral syndrome study and the protocol for successful individual level behavioral measurement, each facet of this procedure can stand alone.
The literature on behavioral syndromes spans several taxonomic groups, from arthropods to humans4 and, in order to measure a behavioral syndrome, at least two behavioral contexts must be quantified. Unfortunately, there is often little consistency in the assays that are used to quantify the behavioral measurement across the axes of behavior. For example, in fish, boldness may be measured using T-maze assays, open-field assays, or introduction of a novel or foreign stimulus15. Aggression studies in fish might involve dyad interactions, video stimulus assays, or clay model assays12,16,17. Likewise, analysis of shoaling behavior, which typically involves the measurement of shoalmate preference, may be performed in different types of tanks, with different methods to determine association time21-23. In this protocol a specific subset of the overall behavioral assay repertoire is presented. Specifically, this protocol presents a methodology to track individuals through boldness, aggression, and shoaling assays in a way that facilitates comparisons within individuals to determine whether the comparisons are consistent across all individuals within a population. We have performed this protocol with zebrafish and convict cichlids (Amatitlania nigrofasciata) in previous studies14,18, and it will work with any similarly sized freshwater fish.
Boldness assays are conducted in a 1.5 L trapezoidal tank that has a horizontal line delineating equal sized areas in the upper and lower portions of the tank. Quantified behaviors include the number of transitions by the test fish between the upper and lower portion of the tank, the time spent in each portion, the number of darts, and the latency to enter the upper portion. The aggression assay is performed in a 19L rectangular tank that includes a 3 inch x 5 inch mirror inclined at about 22&#176; situated in the lower left corner of the tank19. Quantified behaviors include the total amount of time spent by the target fish interacting with the mirror20, along with specific aggressive indicators &#8211; number of bites, lateral displays, darts between the test fish and its reflection. For these specific indicators, bites are defined as quick lunges toward the mirror with an open mouth, lateral displays are defined as the flaring of lateral, pectoral, anal and dorsal fins in the direction of the mirror, and darts are any erratic movements that are not directed toward the mirror. Lastly, the shoaling assay quantifies the behavior of a test fish in center chamber of a tri-chambered tank. The side chambers of the tank are either empty, or contain a &#34;target shoal&#34; of fish, and the time the test fish spends near each side chamber is measured21-23. A single composite score, referred to as Strength of Shoaling (SoS), is calculated for each individual test fish, specific to the stimuli, and can be used in downstream analyses14. All behaviors are scored by a single viewer, or multiple viewers using free behavioral quantification software known as JWatcher24.
Testing the presence of a behavioral syndrome is primarily a statistical endeavor, and it is advisable to follow the guidelines as presented by Budeav 201025. Specifically, it is recommended to perform a principal components analysis (PCA) on centered and normed data in which the inputs are the vectors of an individual&#39;s behaviors in assays with multiple behavioral measurements (i.e., boldness and aggression). The PCA, performed on a correlation matrix, reduces the dimensionality of the behavioral measurements, and thus extracts the most important knowledge that explains a majority of the variation. The extracted components can then be interpreted based on high factor loadings for the individual behavior of interest and regression scores can be extracted for each individual on the basis of the explanatory components. These regression scores can then be compared to the SoS measurement and other various non-behavioral measurements such as fish size or sex.
This workflow has been implemented in a study of zebrafish behavioral syndromes in which a sex specific behavioral syndrome that exists between boldness and shoaling14 was discovered. In this situation, bolder zebrafish males are more likely to associate with a larger, more aggressive species (D. albolineatus), but this association is lost in females. This workflow was also implemented in a study of juvenile convict cichlid kin (Amatitlania nigrofasciata)18 in which a behavioral syndrome was not discovered, potentially indicating behavioral plasticity of the species. Therefore, the following protocol is presented with a goal of delineating the nature of three specific assays (boldness, aggression, and shoaling) in the framework of studying an individual level behavioral syndrome.

<table border="0" cellpadding="0" cellspacing="0" width="783">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Zebrafish Rack System</td>
			<td style="width:224px;">Aquaneering Inc</td>
			<td style="width:119px;">Cat. # ZS550</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pet Valu Tropical Fish Food, 224.0g</td>
			<td>Pet Valu</td>
			<td>Cat. # 31700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Premium Grade Brine Shrimp Eggs, 16 oz</td>
			<td>Brine Shrimp Direct</td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">1.5 L Trapezoidal Tank</td>
			<td>Pentair Aquatic Ecosystems</td>
			<td>Cat. # itsts-a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">19L rectangular tank</td>
			<td>That Fish Place</td>
			<td align="right">211932</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">76L rectangular tank</td>
			<td>That Fish Place</td>
			<td align="right">212180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hitachi KP-D20A CCD Camera</td>
			<td>Prescott&#39;s, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon AF Nikkor 35-105mm f/305~4.5s MACRO lens</td>
			<td>Nikon Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ArtMinds Square Mirror, Value Pack 3&#34;x3&#34;</td>
			<td>Michaels</td>
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boldness, aggression, and shoaling assays for zebrafish behavioral syndromes

A behavioral syndrome exists when specific behaviors interact under different contexts. Zebrafish have been test subjects in recent studies and it is important to standardize protocols to ensure proper analyses and interpretations. In our previous studies, we have measured boldness by monitoring a series of behaviors (time near surface, latency in transitions, number of transitions, and darts) in a 1.5 L trapezoidal tank. Likewise, we quantified aggression by observing bites, lateral displays, darts, and time near an inclined mirror in a rectangular 19 L tank. By dividing a 76 L tank into thirds, we also examined shoaling preferences. The shoaling assay is a highly customizable assay and can be tailored for specific hypotheses. However, protocols for this assay also must be standardized, yet flexible enough for customization. In previous studies, end chambers were either empty, contained 5 or 10 zebrafish, or 5 pearl danios (D. albolineatus). In the following manuscript, we present a detailed protocol and representative data that accompany successful applications of the protocol, which will allow for replication of behavioral syndrome experiments.

Depending on the nature of the study, and specific protocol employed, several distinct results are possible in a behavioral syndromes experiment. The following tables and figures, where indicated, are adapted from our previous study published in the journal Behavioural Processes14 and the journal Zebrafish17. When the proposal (as described above) is carried out in its entirety, two sets of results, &#39;within assay correlations&#39; and &#39;between assay correlati...

Watch this Scientific Journal Video about Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes at JoVE.com

Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes

This manuscript describes the setup, implementation, and analysis of boldness, aggression, and shoaling in zebrafish and testing for the presence of a behavioral syndrome. A standardized approach for behavioral quantification will allow for easier comparison across studies. Modifications to this protocol are possible as each assay can be run individually.

A behavioral syndrome exists when specific behaviors interact under different contexts. Zebrafish have been test subjects in recent studies and it is ...

The overall goal of this protocol is to standardized the methodology used to observe, analyze and interpret boldness, aggression, shoaling behavioral syndromes in Zebrafish. Directly, this method can answer questions in evolutionary theory, population structure and population dynamics. When combined with interdisciplinary techniques it can be applied to fields such as pharmacology, toxicology, behavioral genetics and endocrinology.The main advantage of this technique is that the boldness, aggression, shoaling combination encompasses many well studied, easily quantifiable behaviors in Zebrafish. And each assay can be modified to answer alternative experimental questions. Begin by randomly separating the Zebrafish into the appropriate ratio of focal fish to stimulus fish.Store all fish in Zebrafish housing racks under a strict 12 to 12 hour light/dark cycle. Feed flake food to all focal and stimulus fish everyday. And only feed brine shrimp once a week on non-research days.Next, set up three assay tanks in the laboratory. One for aggression, boldness and shoaling in locations that minimize the possibility for adverse external stimuli. And allow enough room for a video camera to capture the entire tank.Set up the video camera, so that it has enough clarity to view the tank labels, as well as to be to distinguish subtle behaviors of the focal fish. Keep the camera maintained in the same position with a full view of the tank during all behavioral assay recordings, in order to minimize unintended differences in behavioral scoring as a result of differential recordings. Next, define a labeling scheme to track the date, fish number and assay number for all behavioral assays.Label the outside of the tank with tape in view of the camera. Record this information in a lab notebook for quick reference and future quality control. Finally, set up an acclimation tank that will be used between all assays, as well as directly after the individuals are removed from the housing rack.Begin by obtaining a 19 liter rectangular tank, measuring 30 by 15 by 10 centimeters. Outfit the tank with a external opaque partitions surrounding three sides of the aggression tank, leaving the front exposed for viewing to reduce undesirable outside stimuli. Divide the tank with external markings into four equal rectangular quadrants.Use silicone caulk to fix a mirror within the left lower quadrant, inclined at 22.5 degrees, forming a right triangle against the far left side of the tank. Have fresh, aged water, pre-warmed to 27.5 to 28.5 degrees celsius, ready to add to the aggression tank in order to ensure that the temperature of the water in the acclimation tank and aggression tank are within two degrees celsius of the housing rack. Cup a focal fish from the housing rack and transfer it into a separate acclimation tank where it will acclimate for 10 minutes.Confirm that the video camera is focused on the aggression assay tank and press record. Then, immediately transfer the focal fish via cupping from acclimation tank to the aggression tank. Record the aggression tank for 10 minutes and 30 seconds.Then, cup the focal fish from the aggression tank back into the acclimation tank. When watching the recording after the assay is complete, wait 30 seconds after the fish has been introduced to the tank as an additional acclimation period before scoring behaviors. Finally, score a single aggression assay at least twice until the assay is scored with high agreement between iterations.To set up the boldness assay, first obtain a small 1.5 liter trapezoidal tank, measuring 15 centimeters tall with a 26.5 centimeter top and 22.5 centimeter bottom, and a 6 centimeter width. Wrap opaque paper around three sides of the tank, and leave the front exposed for viewing. Place a single thin piece of tape external to the tank to demarcate the top from the bottom portion of the tank.Next, ensure that temperature of the water in the boldness tank is within two degrees celsius of the housing rack. Transfer the focal fish from the housing rack to the acclimation tank and let it acclimate for 10 minutes. Ensure that the video camera is focused on the boldness assay and press record on the device.Immediately transfer the focal fish from the acclimation tank to the boldness tank and record for eight minutes and 30 seconds. When watching the recording after the assay is complete, allow for an additional 30 second acclimation period after the fish is introduced to the tank before scoring. Finally, score a single boldness assay at least twice, until the assay is scored with high agreement between iterations.To set up the shoaling assay, obtain a 76 liter tank that has three compartments divided by two glass dividers sealed with silicone caulk. Externally, use tape to mark two preference zones that are six point three five centimeters from each of the glass dividers that designate each stimulus zone. Apply external opaque partitions around three sides of the shoaling tank and leave the front exposed for viewing.Next, select stimulus shoals to acclimate for at least 12 hours before testing and have them remain in the shoaling tank throughout the course of the experiment. Transfer the focal fish from the housing rack into a separate acclimation tank via cupping where it will acclimate for 10 minutes. Then transfer, by cupping, the focal fish from the acclimation tank to the central compartment of the shoaling tank.Allow for a 10 minute acclimation period before a 10 minute live scoring observation period, in which shoaling fish are defined as being within the preference zone in the chamber housing the target shoal. Next, quantify the time spend in each preference zone, as well as the time spent in the central volume. Finally, after the assays are completed, transfer the fish into a separate terminal tank that will serve as the final destination tank for all used focal individuals.This protocol looked at consistent associations in aggression, boldness and shoaling behaviors of Zebrafish to determine if a behavioral syndrome was present. Here, component one represents aggression behaviors, while component two is associated with boldness behaviors. Since aggression and boldness behaviors are not represented by the same component, it can be concluded that there is an absence of boldness aggression behavioral syndrome in Zebrafish.Once mastered, the entire protocol of one focal fish can be done in 70 minutes. While attempting this procedure, it's important to remember to familiarize yourself and your team with each target behavior in order to minimize observer bias. Scoring the same video recording multiple times will also increase confidence in your results.Following this procedure, other methods in pharmacological, genetic or physiological fields can be performed in order to answer additional questions about what internal and external factors affect behavioral syndromes. After watching this video, you should have a good understanding of how to observe, analyze and interpret boldness, aggression, shoaling behavioral syndromes in Zebrafish. Thanks for watching and good luck with your experiments!

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