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 or replace with a different method of separation based on the study hypothesis. 
	NOTE: Sex characteristics are difficult to determine in zebrafish, particularly in females. Refer to these guidelines10 or useful online manuals to distinguish sex.</li>
	<li>Randomly separate the populations further into the appropriate ratio of focal fish to stimulus fish. Note: Focal fish are the test subjects followed through the series of behavioral assays, and stimulus fish are the individuals that will occupy the stimulus chambers in the aggression and shoaling assays. 
	NOTE: Traditionally, between 1 and 5 stimulus fish per focal fish in shoaling assays and a 1:1 stimulus:focal fish ratio in aggression assays14,17 are used. However, the researcher needs to specifically design the study with the hypothesis in mind (i.e., should stimulus fish be kept constant to maintain consistency in focal fish behavior, or should stimulus fish be rotated between each assay to ensure proper randomization).</li>
	<li>Store all fish in zebrafish housing racks (27.5-28.5 &#176;C) under a strict 12:12 light/dark cycle. Feed all focal and stimulus fish flake food daily and brine shrimp weekly, on non-research days.</li>
	<li>Be sure that all testing is done between ZT1 and ZT5 (zeitgeber time) to avoid circadian rhythm effects26.</li>
</ol>
2. Randomization and Tank Setup
<ol>
	<li>Randomize all individuals a priori to a specific sequence of behavioral assays. Set up a consistent randomization scheme using an online random sequence generator27 or a random sequence generator from a statistical software package (R).
	<ol>
		<li>Label each assay from 1 to 3 and keep this assignment consistent. Assign a random sequence to each individual and map the sequence according to the behavioral assay label. For example, for three total assays (1 mapped to Shoaling, 2 mapped to Boldness, and 3 mapped to Aggression), a given individual may have a randomized sequence of &#39;2,3,1&#39;, which will require them to perform the specific behavioral assays in the specified sequence.</li>
	</ol>
	</li>
	<li>Set up the assay tanks (boldness, aggression, and shoaling) in the laboratory in places that minimize the possibility for adverse external stimuli and be sure to have enough room for a video camcorder to capture the entire tank. Ensure that the camcorder has enough clarity to view tank labels as well as be able to distinguish subtle behaviors of the focal fish and setup the camcorder in this position.</li>
	<li>Record all behavioral assays with the camera maintained in the same position with a full view of the specified tank to minimize unintended differences in behavioral scoring as a result of differential recordings. 
	NOTE: Avoid placing tanks in heavy trafficked areas and by doors as movement and sound will affect zebrafish behavior.</li>
	<li>Establish unique JWatcher protocols24 for scoring each behavioral assay. Refer to the JWatcher manual to learn how to create focal master files to assign behaviors to keystrokes and focal analysis files to summarize data collected. 
	NOTE: JWatcher is behavioral scoring software that allows for reliable and repeatable customized behavioral scoring24.</li>
	<li>Define a labeling scheme to track the date, fish number, and assay number for all behavioral assays (e.g., &#39;F5-B-123115&#39; implying Fish 5, Boldness assay, on December 31st, 2015) and label the outside of the tank with tape in view and in focus of the camera. Keep and record this information in a lab notebook for quick reference and future quality control.</li>
	<li>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. 
	NOTE: The acclimation tanks and terminal tanks are small aquariums (40 cm x 20 cm x 25 cm) with gravel and a filter that closely resemble the housing rack.</li>
	<li>After the assays are all completed, transfer the individual into a separate terminal tank that will serve as the final destination tank for all used focal individuals. Use a tank that is large enough to house a group of individuals for the remainder of their lives (e.g., 25 gallon). 
	NOTE: It is very important to maintain consistent and similar water temperatures and conditions across all tanks. This includes housing, acclimation, and all testing tanks.</li>
</ol>
3. Conducting the Aggression Assay
<ol>
	<li>Conduct the aggression assay in a 19 L rectangular tank measuring 30 cm x 15 cm x 10 cm. Outfit the tank in 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 and fix a mirror permanently (with silicone caulk) within the lower left quadrant, inclined at 22.5&#176; forming a right triangle against the far left side of the tank.</li>
	<li>Ensure that the temperature of the water in the acclimation tank and aggression tank is within 2 &#176;C of the housing rack. To do so, have fresh, aged water pre-warmed to 27.5-28.5 &#176;C ready to add to the aggression tank. Please note that it is important to change the water in between each assay to eliminate unwarranted olfactory cues.</li>
	<li>Transfer, by cupping, a focal fish from the housing rack to a separate acclimation tank where it will acclimate for 10 min. 
	NOTE: It is important to reduce stimulus introduced to the fish through improper handling. Transfer individuals as previously described27.
	<ol>
		<li>Specifically, manually and carefully place a small, transparent plastic cup into the tank of interest. Without disrupting the fish as much as transferring by using a net, slowly scoop up the fish into the cup.</li>
	</ol>
	</li>
	<li>Ensure that the video camcorder is ready to record by confirming that the aggression assay tank (see step 3.1) is in focus and immediately before transferring the individual into the aggression tank, press record on the device. Transfer focal fish via cupping from acclimation tank to aggression tank. 
	NOTE: It is important to record prior to the fish entering the aggression tank to track the exact time of entry.</li>
	<li>Record the aggression tank for 10 min and 30 sec and then cup the focal fish from the aggression tank back into the acclimation tank.</li>
	<li>When watching the recording, wait 30 sec after the fish has been introduced to the tank as an additional acclimation period before scoring behaviors.</li>
	<li>Using the JWatcher scoring system defined for aggressive behaviors, quantify the following behaviors for 10 min: Time spent near mirror, number of mirror approaches, number of darts, number of attempted bites (opening mouth in direction of mirror), and number of lateral displays (dorsal, pectoral, anal, and caudal fin erection)9.</li>
	<li>Score a single aggression assay at least twice, until the assay is scored with high agreement between iterations.</li>
</ol>
4. Conducting the Boldness Assay
<ol>
	<li>Conduct the boldness assay in a small 1.5 L trapezoidal tank, measuring 15 cm tall x 26.5 cm top x 22.5 cm bottom x 6 cm width. Wrap opaque paper around all sides of the tank but 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.</li>
	<li>Ensure that the temperature of the water in the boldness tank is within 2 &#176;C of the housing rack (see step 3.2)</li>
	<li>Transfer the focal fish from the housing rack to the acclimation tank (see step 3.3) and let acclimate for 10 min.</li>
	<li>Ensure that the camcorder is ready to record the boldness assay and, once ready to transfer the focal fish, press record on the device. Transfer the focal fish from the acclimation tank to the boldness tank and record for 8 min and 30 sec.</li>
	<li>When watching the recording, allow for an additional 30 sec acclimation period after the fish is introduced to the tank before scoring.</li>
	<li>Using JWatcher software, quantify boldness behaviors for 8 min. Specifically, quantify the following behaviors: Time near surface, latency to enter upper portion, number of transitions into the upper portion, and number of erratic movements (darts)29.</li>
	<li>Score a single boldness assay at least twice, until the assay is scored with high agreement between iterations.</li>
</ol>
5. Conducting the Shoaling Assay
<ol>
	<li>Conduct the shoaling assay in a 76 L tank that has three compartments divided by two glass dividers sealed with silicone caulk (the resulting three compartments consist of two stimulus zones and one focal compartment). Externally, mark two preference zones designated 6.35 cm from each of the stimulus zones&#39; glass divider, indicative of two body lengths30. Apply opaque partitions externally around three sides of the shoaling tank, but leave the front exposed for viewing. 
	NOTE: This preference zone can be customized to test for specific hypotheses for different sized species.</li>
	<li>Depending on the nature of the study, select stimulus shoals to acclimate for at least 12 hr before testing and remain in the shoaling tank throughout the course of the experiment. 
	NOTE: Stimulus shoals are collections of individual fish that are used to assess how the focal fish behaves in the presence or absence of these shoals. Different stimulus shoal combinations can be selected to test a number of diverse shoaling hypotheses. Be sure that the stimulus shoals are rotated between each stimulus zone to avoid side bias and are also allowed to acclimate for at least 12 hr before testing.</li>
	<li>Transfer the focal fish into the acclimation tank as previously described (see step 3.3)</li>
	<li>Transfer, by cupping, the focal fish from the intermediate acclimation tank to the central compartment in the shoaling tank.</li>
	<li>Allow for a 10 min acclimation period before a 10 min live-scoring observation period. Alternatively, record the shoaling assays and score later using JWatcher. Fish are determined to be shoaling when they are within the preference zone in the chamber housing the target shoal.</li>
	<li>Quantify time spent in each preference zone and time spent in the center volume.</li>
	<li>As an additional descriptive measure, calculate &#39;strength of shoaling&#39; (SoS). Assign a value between -1 and +1 where the magnitude describes how often an individual fish shoals.</li>
	<li>Calculate the ratio of time spent shoaling divided by the area of the shoaling zones and subtract the ratio of time spent not shoaling and the area of the non-shoaling zones. Place this quantity over the sum of time spent in each zone divided by the volume of each zone individually. More information about this calculation can be found in a previous study14.</li>
</ol>
6. Data Quality Control
<ol>
	<li>Analyze data using traditional data analysis tools31-32.</li>
	<li>Test for homogeneity of variance using nonparametric Levene&#39;s test for all behaviors. If a behavior is non-homogeneous perform a log-transformation33.</li>
	<li>Confirm that there are no sequence effects (i.e., that the randomization order did not affect the individuals&#39; behavior) using a Kruskal-Wallis ANOVA on the homogeneous data34.</li>
	<li>Confirm that there are no side biases for the shoaling assay by creating univariate general linear models on SoS indices with side as a fixed factor35. 
	NOTE: If there are little to no issues with the data, downstream analyses can be conducted. If there are issues, reconcile with additional transformations, rationalize with biologically plausible explanations, and/or increase sample sizes.</li>
</ol>
7. Analyze Data
<ol>
	<li>Calculate two-tailed Spearman rank correlations36 for all behaviors separately within the boldness and aggression assays.</li>
	<li>Calculate intraclass correlation coefficients (ICC) as a two-way mixed model with consistency for behavioral assays that were conducted more than one time for a given individual36. In this setting, the ICC is a measurement of how consistently a fish behaved in a given assay measured more than one time.</li>
	<li>Preprocess boldness and aggression behaviors by centering and norming all behaviors to prepare for input to a principal components analysis (PCA)25. 
	NOTE: Preprocessing the measurements by centering and norming is a vital step. Without this preprocessing step behaviors that occur with more frequency and more variably will excessively influence the interpretations of the extracted components. The process of centering and norming will remove this bias.</li>
	<li>Perform a PCA with a correlation matrix to determine if boldness and aggression are linked together as a behavioral syndrome. Interpret behavioral loadings of each component significant at &#62;0.6. See Budaev 201024 for a comprehensive guide for reporting PCA for behavioral studies. A scree plot will be informative on which eigenvectors should be kept for interpretation.</li>
	<li>Using statistical software package such as R or SPSS31,32, extract scores for each component for each individual. The scores represent how much the given individual&#39;s behavior can be explained by the particular component.</li>
	<li>Calculate Spearman rank correlations between factor regression scores and the strength of shoaling36. 
	NOTE: A behavioral syndrome is considered present if boldness and aggression behaviors load on the same component or, if any component with significant interpretable loadings correlates with strength of shoaling. Also, note that strength of shoaling is not included in the PCA to allow for better interpretations of the more complex behavioral quantifications (boldness and aggression). The PCA will orthogonally collapse boldness and aggression features into eigenvectors to maximize the amount of variation explained and is well suited for these complex measurements. Interpreting the resulting eigenvectors and regressing our strength of shoaling measurements on each of them gives a much more intuitive interpretation of how each of the complex behaviors (boldness and aggression) relate to a more well-defined binary shoaling measurement (i.e., the fish is shoaling or not shoaling).</li>
</ol>

<ol><li>Huntingford, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+relationship+between+anti-predator+behaviour+and+aggression+among+conspecifics+in+the+three-spined+stickleback%2C+Gasterosteus+aculeatus%5BTitle%5D)+AND+%22Anim+Behav%22%5BJournal%5D)">The relationship between anti-predator behaviour and aggression among conspecifics in the three-spined stickleback, Gasterosteus aculeatus.</a> Anim Behav. 24, 245-260 (1976).</li>
<li>Réale, D., Reader, S. M., Sol, D., McDougall, P. T., Dingemanse, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrating+animal+temperament+within+ecology+and+evolution%5BTitle%5D)+AND+%22Biol+Rev%22%5BJournal%5D)">Integrating animal temperament within ecology and evolution.</a> Biol Rev. 82 (2), 291-318 (2007).</li>
<li>Sih, A., Bell, A., Johnson, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Behavioral+syndromes%3A+an+ecological+and+evolutionary+overview%5BTitle%5D)+AND+%22Trends+Ecol+Evol%22%5BJournal%5D)">Behavioral syndromes: an ecological and evolutionary overview.</a> Trends Ecol Evol. 19 (7), 372-378 (2004).</li>
<li>Conrad, J. L., Weinersmith, K. L., Brodin, T., Saltz, J. B., Sih, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Behavioural+syndromes+in+fishes%3A+a+review+with+implications+for+ecology+and+fisheries+management%5BTitle%5D)+AND+%22J+Fish+Biol%22%5BJournal%5D)">Behavioural syndromes in fishes: a review with implications for ecology and fisheries management.</a> J Fish Biol. 78 (2), 395-435 (2011).</li>
<li>Wolf, M., Weissing, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+personalities%3A+consequences+for+ecology+and+evolution%5BTitle%5D)+AND+%22Trends+Ecol+Evol%22%5BJournal%5D)">Animal personalities: consequences for ecology and evolution.</a> Trends Ecol Evol. 27 (8), 452-461 (2012).</li>
<li>Langheinrich, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish%3A+a+new+model+on+the+pharmaceutical+catwalk%5BTitle%5D)+AND+%22BioEssays%22%5BJournal%5D)">Zebrafish: a new model on the pharmaceutical catwalk.</a> BioEssays. 25 (9), 904-912 (2003).</li>
<li>Dai, Y. -J., Jia, Y. -F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+as+a+model+system+to+study+toxicology%3A+Zebrafish+toxicology+monitoring%5BTitle%5D)+AND+%22Environ+Toxicol+Chem%22%5BJournal%5D)">Zebrafish as a model system to study toxicology: Zebrafish toxicology monitoring.</a> Environ Toxicol Chem. 33 (1), 11-17 (2014).</li>
<li>Norton, W., Bally-Cuif, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adult+zebrafish+as+a+model+organism+for+behavioural+genetics%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">Adult zebrafish as a model organism for behavioural genetics.</a> Neuroscience. 11 (1), 90(2010).</li>
<li>Gerlai, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebra+fish%3A+an+uncharted+behavior+genetic+model%5BTitle%5D)+AND+%22Behav+Genet%22%5BJournal%5D)">Zebra fish: an uncharted behavior genetic model.</a> Behav Genet. 33 (5), 461-468 (2003).</li>
<li>Dzieweczynski, T. L., Campbell, B. A., Marks, J. M., Logan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acute+exposure+to+17%CE%B1-ethinylestradiol+alters+boldness+behavioral+syndrome+in+female+Siamese+fighting+fish%5BTitle%5D)+AND+%22Horm+Behav%22%5BJournal%5D)">Acute exposure to 17α-ethinylestradiol alters boldness behavioral syndrome in female Siamese fighting fish.</a> Horm Behav. 66 (4), 577-584 (2014).</li>
<li>Miklòsi, Á, Andrew, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Zebrafish+as+a+Model+for+Behavioral+Studies%5BTitle%5D)+AND+%22Zebrafish%22%5BJournal%5D)">The Zebrafish as a Model for Behavioral Studies.</a> Zebrafish. 3 (2), 227-234 (2006).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Zebrafish+protocols+for+neurobehavioral+research%22">Zebrafish protocols for neurobehavioral research</a>. , Humana Press. New York. (2012).</li>
<li>Moretz, J. A., Martins, E. P., Robison, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Behavioral+syndromes+and+the+evolution+of+correlated+behavior+in+zebrafish%5BTitle%5D)+AND+%22Behav+Ecol%22%5BJournal%5D)">Behavioral syndromes and the evolution of correlated behavior in zebrafish.</a> Behav Ecol. 18 (3), 556-562 (2007).</li>
<li>Way, G. P., Kiesel, A. L., Ruhl, N., Snekser, J. L., McRobert, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sex+differences+in+a+shoaling-boldness+behavioral+syndrome%2C+but+no+link+with+aggression%5BTitle%5D)+AND+%22Behav+Process%22%5BJournal%5D)">Sex differences in a shoaling-boldness behavioral syndrome, but no link with aggression.</a> Behav Process. 113, 7-12 (2015).</li>
<li>Toms, C. N., Echevarria, D. J., Jouandot, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+methodological+review+of+personality-related+studies+in+fish%3A+Focus+on+the+shy-bold+axis+of+behavior%5BTitle%5D)+AND+%22J+Comp+Psychol%22%5BJournal%5D)">A methodological review of personality-related studies in fish: Focus on the shy-bold axis of behavior.</a> J Comp Psychol. 23, 1-25 (2010).</li>
<li>Rowland, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Studying+visual+cues+in+fish+behavior%3A+A+review+of+ethological+techniques%5BTitle%5D)+AND+%22Environ+Biol+Fish%22%5BJournal%5D)">Studying visual cues in fish behavior: A review of ethological techniques.</a> Environ Biol Fish. 56 (3), 285-305 (1999).</li>
<li>Way, G. P., Ruhl, N., Snekser, J. L., Kiesel, A. L., McRobert, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Comparison+of+Methodologies+to+Test+Aggression+in+Zebrafish%5BTitle%5D)+AND+%22Zebrafish%22%5BJournal%5D)">A Comparison of Methodologies to Test Aggression in Zebrafish.</a> Zebrafish. 12 (2), 144-151 (2015).</li>
<li>Moss, S., Tittaferrante, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interactions+between+aggression%2C+boldness+and+shoaling+within+a+brood+of+convict+cichlids+%28Amatitlania+nigrofasciatus%29%5BTitle%5D)+AND+%22Behav+Process%22%5BJournal%5D)">Interactions between aggression, boldness and shoaling within a brood of convict cichlids (Amatitlania nigrofasciatus).</a> Behav Process. 121, 63-69 (2015).</li>
<li>Ruhl, N., Kiesel, A. L., McRobert, S. P., Snekser, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Behavioural+syndromes+and+shoaling%3A+connections+between+aggression%2C+boldness+and+social+behaviour+in+three+different+Danios%5BTitle%5D)+AND+%22Behaviour%22%5BJournal%5D)">Behavioural syndromes and shoaling: connections between aggression, boldness and social behaviour in three different Danios.</a> Behaviour. 149 (10-12), 1155-1175 (2012).</li>
<li>Larson, E. T., O'Malley, D. M., Melloni, R. H. Jr <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aggression+and+vasotocin+are+associated+with+dominant-subordinate+relationships+in+zebrafish%5BTitle%5D)+AND+%22Behav+Brain+Res%22%5BJournal%5D)">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/pubmed?term=((The+influence+of+body+coloration+on+shoaling+preferences+in+fish%5BTitle%5D)+AND+%22Anim+Behav%22%5BJournal%5D)">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/pubmed?term=((The+effect+of+sex+and+shoal+size+on+shoaling+behaviour+in+Danio+rerio%5BTitle%5D)+AND+%22J+Fish+Biol%22%5BJournal%5D)">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/pubmed?term=((The+influence+of+sex+and+phenotype+on+shoaling+decisions+in+zebrafish%5BTitle%5D)+AND+%22J+Comp+Psychol%22%5BJournal%5D)">The influence of sex and phenotype on shoaling decisions in zebrafish.</a> J Comp Psychol. 23, 70-81 (2010).</li>
<li>Blumstein, D. T., Evans, C. S., Daniel, J. C. JWatcher. , Available from:
 <a target="_blank" href="http://www.jwatcher.ucla.edu/">http://www.jwatcher.ucla.edu/</a> (2006).</li>
<li>Budaev, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+Principal+Components+and+Factor+Analysis+in+Animal+Behaviour+Research%3A+Caveats+and+Guidelines%5BTitle%5D)+AND+%22Ethology%22%5BJournal%5D)">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/pubmed?term=((Daily+variation+in+the+shoaling+behavior+of+zebrafish+Danio+rerio%5BTitle%5D)+AND+%22Curr+Zool%22%5BJournal%5D)">Daily variation in the shoaling behavior of zebrafish Danio rerio.</a> Curr Zool. 58 (1), 129-137 (2012).</li>
<li>Haahr, M. Sequence randomiser. , Available from:
 <a target="_blank" href="http://www.random.org/lists/">http://www.random.org/lists/</a> (1998).</li>
<li>Wright, D., Krause, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Repeated+measures+of+shoaling+tendency+in+zebrafish+%28Danio+rerio%29+and+other+small+teleost+fishes%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">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/pubmed?term=((Measuring+behavioral+and+endocrine+responses+to+novelty+stress+in+adult+zebrafish%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Measuring behavioral and endocrine responses to novelty stress in adult zebrafish.</a> Nat Protoc. 5 (11), 1786-1799 (2010).</li>
<li>Croft, D. P., Krause, J., Couzin, I. D., Pitcher, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((When+fish+shoals+meet%3A+outcomes+for+evolution+and+fisheries%5BTitle%5D)+AND+%22Fish+Fish%22%5BJournal%5D)">When fish shoals meet: outcomes for evolution and fisheries.</a> Fish Fish. 4, 138-146 (2003).</li>
<li>IBM Corp. IBM SPSS Statistics for Mac OS. , IBM Corp. Armank, NY. Available from:
 <a target="_blank" href="http://www.ibm.com/analytics/us/en/technology/spss/spss.html">http://www.ibm.com/analytics/us/en/technology/spss/spss.html</a> (2011).</li>
<li>R: A language and environment for statistical computing. , R Foundation for Statistical Computing. Vienna, Austria. Available from:
 <a target="_blank" href="http://www.R-project.org">http://www.R-project.org</a> (2013).</li>
<li>Brown, M. B., Forsythe, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Robust+Tests+for+the+Equality+of+Variances%5BTitle%5D)+AND+%22J+Am+Stat+Assoc%22%5BJournal%5D)">Robust Tests for the Equality of Variances.</a> J Am Stat Assoc. 69 (346), (1974).</li>
<li>Kruskal, W. H., Wallis, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+Ranks+in+One-Criterion+Variance+Analysis%5BTitle%5D)+AND+%22J+Am+Stat+Assoc%22%5BJournal%5D)">Use of Ranks in One-Criterion Variance Analysis.</a> J Am Stat Assoc. 47 (260), 583-621 (1952).</li>
<li>Nelder, J. A., Wedderburn, R. W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generalized+Linear+Models%5BTitle%5D)+AND+%22J+Roy+Stat+Soc+A+Sta%22%5BJournal%5D)">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/pubmed?term=((A+distribution-free+approach+to+inducing+rank+correlation+among+input+variables%5BTitle%5D)+AND+%22Commun+Stat+Simulat%22%5BJournal%5D)">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/pubmed?term=((The+Equivalence+of+Weighted+Kappa+and+the+Intraclass+Correlation+Coefficient+as+Measures+of+Reliability%5BTitle%5D)+AND+%22Educ+Psychol+Meas%22%5BJournal%5D)">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/pubmed?term=((Shoaling+preferences+and+the+effects+of+sex+ratio+on+spawning+and+aggression+in+small+laboratory+populations+of+zebrafish+%28Danio+rerio%29%5BTitle%5D)+AND+%22Lab+Animal%22%5BJournal%5D)">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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Behavioural+differences+between+individuals+and+two+populations+of+stickleback+%28Gasterosteus+aculeatus%29%5BTitle%5D)+AND+%22J+Evolution+Biol%22%5BJournal%5D)">Behavioural differences between individuals and two populations of stickleback (Gasterosteus aculeatus).</a> J Evolution Biol. 18 (2), 464-473 (2005).</li>
<li>Wilson, A. D. M., Godin, J. -G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Boldness+and+behavioral+syndromes+in+the+bluegill+sunfish%2C+Lepomis+macrochirus%5BTitle%5D)+AND+%22Behav+Ecol%22%5BJournal%5D)">Boldness and behavioral syndromes in the bluegill sunfish, Lepomis macrochirus.</a> Behav Ecol. 20 (2), 231-237 (2009).</li>
<li>Brodin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Behavioral+syndrome+over+the+boundaries+of+life--carryovers+from+larvae+to+adult+damselfly%5BTitle%5D)+AND+%22Behav+Ecol%22%5BJournal%5D)">Behavioral syndrome over the boundaries of life--carryovers from larvae to adult damselfly.</a> Behav Ecol. 20 (1), 30-37 (2008).</li>
<li>Noldus, L. P., Spink, A. J., Tegelenbosch, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((EthoVision%3A+a+versatile+video+tracking+system+for+automation+of+behavioral+experiments%5BTitle%5D)+AND+%22Behav+Res+Methods+Instrum+Comput%22%5BJournal%5D)">EthoVision: a versatile video tracking system for automation of behavioral experiments.</a> Behav Res Methods Instrum Comput. 33 (3), 398-414 (2001).</li>
<li>Cote, J., Fogarty, S., Weinersmith, K., Brodin, T., Sih, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Personality+traits+and+dispersal+tendency+in+the+invasive+mosquitofish+%28Gambusia+affinis%29%5BTitle%5D)+AND+%22Proc+R+Soc+B%22%5BJournal%5D)">Personality traits and dispersal tendency in the invasive mosquitofish (Gambusia affinis).</a> Proc R Soc B. 277 (1687), 1571-1579 (2010).</li>
<li>Fraser, D. F., Gilliam, J. F., Daley, M. J., Le, A. N., Skalski, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Explaining+leptokurtic+movement+distributions%3A+intrapopulation+variation+in+boldness+and+exploration%5BTitle%5D)+AND+%22Am+Nat%22%5BJournal%5D)">Explaining leptokurtic movement distributions: intrapopulation variation in boldness and exploration.</a> Am Nat. 158 (2), 124-135 (2001).</li>
<li>Dingemanse, N. J., Wright, J., Kazem, A. J. N., Thomas, D. K., Hickling, R., Dawnay, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Behavioural+syndromes+differ+predictably+between+12+populations+of+three-spined+stickleback%5BTitle%5D)+AND+%22J+Anim+Ecol%22%5BJournal%5D)">Behavioural syndromes differ predictably between 12 populations of three-spined stickleback.</a> J Anim Ecol. 76 (6), 1128-1138 (2007).</li>
</ol>

The authors (GW and SPM) have no competing financial interests or conflicts of interest.

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 behavioral syndromes, like in pharmacology6, toxicology7, behavioral genetics8,9, and endocrinology10 studies, can shed light on how different factors might affect behavioral associations. The specific composition of the aggression-boldness-shoaling behavioral syndrome found in this protocol encompasses common behaviors well studied in the model organism zebrafish11-13. While this methodology can be applied to any fish species, the wide application for using zebrafish may help with the interdisciplinary standardization of this protocol. Each assay described above can be more easily controlled and manipulated than other comparable techniques. For example, the aggression assay uses the inclined mirror to elicit a wider array of aggression responses rather than using a live or clay model, video stimulus, or vertical mirror image stimulus17. The behaviors measured in boldness assay are more easily quantified than in a t-maze or large open field test15. Lastly, the shoaling assay described can easily track an individual, which is essential for studying behavioral syndromes at the individual level, and can allow for unlimited combinations of target shoals to ask novel experimental questions. While this protocol can be altered to accommodate a variety of behavioral questions, the standardization of it may lead to further insight on how DNA, cellular mechanisms, cellular development, and introduced chemicals can affect individual and population behavioral syndromes.
In order to be confident that the observed associations are true, the protocol must be followed carefully. For the most part, the protocol is modifiable according to the specific hypothesis; however, there are several steps that must be performed to ensure confidence in the results. First, it is important that the individuals are housed in an appropriate tank, at optimal conditions (see protocol) and are fed properly. The water conditions should be consistent in housing racks, acclimation tanks, and test tanks. If the water is not consistent, the fish will take longer to acclimate to the conditions, and the behavioral measurements will not be properly captured. It is also important that water is recycled between boldness and aggression tanks so that artificial odors are not carried over between assays. Second, it is of utmost importance that all behaviors are scored properly. To ensure this, all assays are videotaped to allow for multiple viewings, and for several training opportunities. A single trained scorer can accurately measure fish behavior, but training does take some time. If a careful training period is ignored, then the confidence of the results of the behavioral study is low. A careful study with low sample sizes performed by a well-trained observer is more powerful than a study with high sample size performed by a poorly trained observer. Lastly, quality control of the collected data is also critical. The steps that are taken to randomize individuals, and remove biases should result in useable, reliable data, and data transformations are recommended for preprocessing according to the specific statistical test (see protocol). If these steps are not carefully done, the output of the analyses may not be reliably interpretable.
As previously mentioned, the technique is heavily modifiable according to the specific test hypotheses. There exist other behavioral measurements that can be investigated to be related in a behavioral syndrome among a number of diverse fish species39-41. While the tenets of the protocol remain the same, the specific questions can be easily altered to allow for diverse hypothesis testing. For example, a researcher may use the aforementioned protocol to test for the presence of an aggression and exploratory activity behavioral syndrome in convict cichlids (Amatitlania nigrofasciata). The question is related, but the study organism is different from the one described by the protocol. However, the general steps remain largely the same. Individuals must be randomized and tracked through a series of assays, with acclimation periods in between assays, the water must be consistent and fresh, and the scientist should be properly trained. One major difference is the behaviors that are being measured are likely to change according to the specific assay, and the behavioral associations may change according to the specific species and original environment.
The limitations of the protocol are linked with some of the traditional uncertainties of behavioral studies. Specifically, if the assays are scored unreliably then it is likely the interpretations of the findings are erroneous, and it would be difficult to identify this error. To overcome this limitation, it is possible that two well-trained scorers observe behaviors. To assess their reliability, calculate intraclass correlation coefficients on measurements applied on the same fish, and, if necessary, adjust for differences. Alternatively, if available, automatic tracking software such as Ethovision can be implemented and validated by a well-trained observer to increase throughput and accuracy42. Furthermore, there are different possible interpretations regarding the naming and scoring of the &#34;boldness&#34; behaviors. Other studies have termed the behavior described43 and others have described the behavior as &#34;exploratory&#34;44,45. In our work the behavior was described as &#34;boldness&#34; as it was measuring behavior in an environment unfamiliar to the focal individual. However, while the term may be subject to alternative interpretations, this does not affect the protocol or the analysis. Additionally, while we expect measurements within boldness and aggression assays to be strongly correlated within a population, there is likely to be some instances of low to no correlation of some behaviors. This limitation is overcome by the strength of the PCA, because it keys in on important sources of variability and, even if measurements are not correlated, the analysis will extract behavioral variation consistent in the collected data. Lastly, as is the case with all scientific methods, if the protocol is universally adopted and is consistently carried out by several labs, and there is some unforeseen, unmeasured confounding introduced by the protocol, then this potential deleterious element persists in the literature, and it becomes difficult to dismantle. Pharmacologically confirming that a representative set of assays elicit the intended behavioral response requires a more thorough understanding of behavioral, neurological and hormonal responses. Behavioral syndromes can help explain the basis of behavior, but this laboratory limitation may be able to be more regularly addressed in future studies. Nevertheless, results have been provided that validates the use of this protocol in zebrafish, and, with proper modifications, the protocol can be extended to a variety of hypotheses in a number of different fish species. By carefully following a detailed protocol for the housing, selection and testing of various behaviorally parameters will enable researchers to make more specific comparisons across a wide array of studies.

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><tbody><tr><td>Zebrafish Rack System</td><td>Aquaneering Inc</td><td>Cat. # ZS550</td><td></td></tr><tr><td>Pet Valu Tropical Fish Food, 224.0 g</td><td>Pet Valu</td><td>Cat. # 31700</td><td></td></tr><tr><td>Premium Grade Brine Shrimp Eggs, 16 oz</td><td>Brine Shrimp Direct</td><td></td><td></td></tr><tr><td>1.5 L Trapezoidal Tank</td><td>Pentair Aquatic Ecosystems</td><td>Cat. # itsts-a</td><td></td></tr><tr><td>19 L rectangular tank</td><td>That Fish Place</td><td>211932</td><td></td></tr><tr><td>76 L rectangular tank</td><td>That Fish Place</td><td>212180</td><td></td></tr><tr><td>Hitachi KP-D20A CCD Camera</td><td>Prescott&#039;s, Inc.</td><td></td><td></td></tr><tr><td>Nikon AF Nikkor 35-105 mm f/305~4.5s MACRO lens</td><td>Nikon Corporation</td><td></td><td></td></tr><tr><td>ArtMinds Square Mirror, Value Pack 3&quot; x 3&quot;</td><td>Michaels</td><td>Cat. # 10334162</td><td></td></tr><tr><td>Jwatcher</td><td></td><td></td><td></td></tr><tr><td>SPSS Statistics Base</td><td>IBM</td><td></td><td></td></tr><tr><td>R</td><td>The R Foundation</td><td></td><td></td></tr></tbody></table>

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 correlations,&#39; are expected (Figure 1).
The first set of results describes within assay consistency. Specifically, the results describe how behaviors are correlated with each other within the boldness assay and the within the aggression assay separately. Tables 1 and 2 describe what these correlations should look like for the boldness and aggression assays and the data presented are adapted from a previous study14. For the boldness assay, it is expected that number of transitions and time spent in the upper portion of the tank are positively correlated, but latency to enter the upper portion is negatively correlated. Representative results for individuals measured on the boldness axis of behavior in the boldness tank (see step 4.1) are given in Table 1. For the aggression assay, it is expected that bites, lateral displays, and time spent near the mirror are all correlated. Representative results for individuals measured on the aggression axis of behavior in the aggression tank (see step 3.1) are given in Table 2. Darts are not usually correlated with aggressive behavior although, in some cases, nonaggressive fish tend to dart (hence a negative correlation). Lastly, a Strength of Shoaling (SoS) measurement for the shoaling assay provides an individual-level measure of shoaling tendency. Shoaling is a highly repeatable behavior across a variety of shoaling assays when measured using SoS (ICC = 0.641)14 and several of our previous studies have confirmed shoaling behavior in zebrafish23,38. Since all individuals will be measured for each of the behaviors, we can calculate Spearman correlations across these behaviors and expect some behaviors to be correlated, as identified for zebrafish in the representative results.
The second portion of the results investigates the presence of a behavioral syndrome. The results of the integrative analysis of all behaviors quantified in the boldness and aggression behavioral assays are summarized using a Principal Components Analysis (PCA). There should be two to four interpretable components based on high loadings for each behavior. If a single eigenvector that explains a good portion of the variation includes behaviors from both the boldness and aggression assays, then a behavioral syndrome has been observed. However, if behaviors from the boldness and aggression assays do not overlap on a single eigenvector, then the study does not describe a behavioral syndrome. Representative results are presented for the absence of a boldness-aggression behavioral syndrome (Figure 2). In this example, component 1 is most strongly representative of aggression behaviors while component 2 is most strongly associated with boldness behaviors. Because aggression and boldness behaviors are not represented by the same component, it can be concluded that there is the absence of a boldness-aggression behavioral syndrome (boldness vectors are roughly orthogonal to aggression vectors). Also, these behaviors are not influenced by sex because the distribution pattern is the same between males and females. To observe if boldness or aggression behaviors are associated with shoaling, the extracted regression scores for each fish are correlated with the fish&#39;s SoS measurement. It is important to ensure that any correlating components are actually describing an interpretable set of behaviors. If there are high absolute value Spearman correlations between any component and the shoaling measurement, then a shoaling behavioral syndrome is present. Regardless of the specific results, it is important to provide feasible biological interpretations to all observations. Representative results are provided for a description of a boldness-shoaling behavioral syndrome (Table 3)14. While the data presented represent typical results for zebrafish, they should not be interpreted as measurements of data quality. There are several reasons why the data will look different across different experiments such as species or population differences.
<img alt="Figure 1" src="/files/ftp_upload/54049/54049fig1.jpg" /> 
Figure 1.&#160;Proposed workflow for a boldness, aggression, and shoaling behavioral syndromes experiment. Typical measurements are listed for each assay. An outline of how to analyze the assays individually and then how to integrate the analyses is also listed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54049/54049fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54049/54049fig2.jpg" /> 
Figure 2.&#160;Representative results of a PCA indicating that a boldness-aggression behavioral syndrome is absent. The red vectors indicate behaviors which contribute to the component scores (left and bottom axes) and the points represent individual scores along each component (right and top axes). Males are represented by blue points and females are represented by green points. Note that &#39;erratic behavior&#39; refer to &#39;darts&#39; and &#39;aggression rate&#39; can be computed by adding the number of bites and lateral displays divided by the total time interacting with the mirror. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54049/54049fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Transitions</td>
			<td>Latency</td>
			<td>Darts</td>
		</tr>
		<tr>
			<td></td>
			<td>&#7465;</td>
			<td>p</td>
			<td>&#7465;</td>
			<td>p</td>
			<td>&#7465;</td>
			<td>p</td>
		</tr>
		<tr>
			<td>Surface Time</td>
			<td>0.65</td>
			<td>&#60;0.001*</td>
			<td>-0.68</td>
			<td>&#60;0.001*</td>
			<td>0.42</td>
			<td>0.007*</td>
		</tr>
		<tr>
			<td>Transitions</td>
			<td></td>
			<td></td>
			<td>-0.40</td>
			<td>0.012*</td>
			<td>0.33</td>
			<td>0.041*</td>
		</tr>
		<tr>
			<td>Latency</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>-0.47</td>
			<td>0.002</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>*&#945; &#60;0.05</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Boldness Spearman rank correlations in a novel tank assay. This table had been modified from Way et al., 2015.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2">Lateral Displays</td>
			<td colspan="2">Darts</td>
			<td colspan="2">Mirror Time</td>
		</tr>
		<tr>
			<td></td>
			<td>&#7465;</td>
			<td>p</td>
			<td>&#7465;</td>
			<td>p</td>
			<td>&#7465;</td>
			<td>p</td>
		</tr>
		<tr>
			<td>Bites</td>
			<td>0.69</td>
			<td>&#60;0.001*</td>
			<td>-0.10</td>
			<td>0.547</td>
			<td>0.63</td>
			<td>&#60;0.001*</td>
		</tr>
		<tr>
			<td colspan="2">Lateral displays</td>
			<td></td>
			<td>-0.06</td>
			<td>0.735</td>
			<td>0.66</td>
			<td>&#60;0.001*</td>
		</tr>
		<tr>
			<td>Darts</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>-0.18</td>
			<td>0.285</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>*&#945; &#60;0.05</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Aggression Spearman rank correlations in a novel tank assay. This table had been modified from Way et al., 2015.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>&#7465;</td>
			<td>p</td>
		</tr>
		<tr>
			<td colspan="2">PCA Components</td>
			<td></td>
		</tr>
		<tr>
			<td>Boldness</td>
			<td>0.339</td>
			<td>0.035*</td>
		</tr>
		<tr>
			<td>Aggression</td>
			<td>0.075</td>
			<td>0.65</td>
		</tr>
		<tr>
			<td>Dart</td>
			<td>-0.012</td>
			<td>0.944</td>
		</tr>
	</tbody>
</table>
Table 3: Using the PCA components to confirm the presence of a shoaling and boldness behavioral syndrome present in this population. This table had been modified from Way et al., 2015.

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 ...

boldness-aggression-shoaling-assays-for-zebrafish-behavioral

Nanyang Technological University

Research

JoVE Journal

Medicine

10.3K Views.  Saint Joseph’s University. 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.

Video: Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

Biology

Behavioral Approaches to Studying Innate Stress in Zebrafish

Responding appropriately to stressful stimuli is essential for survival of an organism. Extensive research has been done on a wide spectrum of stress-related diseases and psychiatric disorders, yet further studies into the genetic and neuronal regulation of stress are still required to develop better therapeutics. The zebrafish provides a powerful genetic model to investigate the neural underpinnings of stress, as there exists a large collection of mutant and transgenic lines. Moreover, pharmacology can easily be applied to zebrafish, as most drugs can be added directly to water. We describe here the use of the 'novel tank test' as a method to study innate stress responses in zebrafish, and demonstrate how potential anxiolytic drugs can be validated using the assay. The method can easily be coupled with zebrafish lines harboring genetic mutations, or those in which transgenic approaches for manipulating precise neural circuits are used. The assay can also be used in other fish models. Together, the described protocol should facilitate the adoption of this simple assay to other laboratories.

This manuscript describes a simple method to measure stress behaviorally in adult zebrafish. The approach takes advantage of the innate tendency that zebrafish prefer the bottom half of a tank when in a stressful state. We also describe methods for coupling the assay with pharmacology.

Responding appropriately to stressful stimuli is essential for survival of an organism. Extensive research has been done on a wide spectrum of ...

Behavior

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system measures spontaneous behaviors and the response to an aversive stimulus, which is shown to the larvae via a PowerPoint presentation. The recorded images are analyzed with an ImageJ macro, which automatically splits the color channels, subtracts the background, and applies a threshold to identify individual larvae placement in the lanes. We can then import the coordinates into an Excel sheet to quantify swim speed, preference for edge or side of the lane, resting behavior, thigmotaxis, distance between larvae, and avoidance behavior. Subtle changes in behavior are easily detected using our system, making it useful for behavioral analyses after exposure to environmental toxicants or pharmaceuticals.

Our laboratory developed a novel high-throughput automated imaging system that is useful for the detection of several different behaviors in 7-day-old zebrafish larvae. The system can be used for detecting subtle changes in behavior after the larvae have been exposed to environmental toxicants or pharmaceuticals.

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system ...

Neuroscience

The Three-Chamber Choice Behavioral Task using Zebrafish as a Model System

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or cognitive impairment. We have modified and developed a three-chamber choice cognitive task similar to that used with rodents for use with hyperglycemic zebrafish. The testing chamber consists of a centrally located starting chamber and two choice compartments on either side, with a shoal of conspecifics used as the reward. We provide data showing that once acquired, zebrafish remember the task at least 8 weeks later. Our data indicate that zebrafish respond robustly to this reward, and we have identified cognitive deficits in hyperglycemic fish after 4 weeks of treatment. This behavioral assay may also be applicable to other studies related to cognition and memory.

We present a behavioral chamber designed to assess cognitive performance. We provide data showing that once acquired, zebrafish remember the task 8 weeks later. We also show that hyperglycemic zebrafish have altered cognitive performance, indicating that this paradigm is applicable to studies assessing cognition and memory.

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or ...

Modeling Fetal Alcohol Spectrum Disorders in Zebrafish to Characterize the Impact of an Adverse Embryonic Environment on Adult Social Behavior

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, and cognitive abnormalities are associated with FASD. Social difficulties are common behavioral abnormalities associated with FASD and often result in serious health issues. Animal models are critical to understanding the mechanisms responsible for ethanol-induced social defects. Zebrafish are social vertebrates that produce externally fertilized transparent eggs; these characteristics provide researchers with a precise yet simple procedure for creating the FASD phenotype and an innate behavior that can be leveraged to model the social deficits associated with FASD. Thus, zebrafish are ideal for characterizing the social deficits of FASD. The goal of the current protocol is to provide the user with a simple behavioral assay that can be used to characterize the consequences of a negative environment early during development and the effects it can have on social behavior in adulthood. The protocol can be used to characterize the effect mutations or teratogens have on adult social behavior. The protocol outlined here demonstrates how to characterize the social behavior of individual fish during a 20-min social assay. Furthermore, the data obtained using the current protocol provides evidence that the protocol can be used to characterize the effects of embryonic ethanol-induced social defects in adult zebrafish.

The goal of the current protocol is to outline the steps necessary to establish and use a social preference assay for adult zebrafish and demonstrate that it can be used to characterize ethanol-induced social defects.

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, ...

Behavioral And Physiological Analysis In A Zebrafish Model Of Epilepsy

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research have uncovered a large spectrum of genes implicated in various forms of epilepsy, highlighting the heterogeneous nature of this disorder. Appropriate animal models are essential for investigating the pathological mechanisms triggered by genetic mutations implicated in epilepsy and for developing specialized, targeted therapies. In recent years, zebrafish has emerged as a valuable vertebrate organism for modeling epilepsies, with the use of both genetic manipulation and exposure to known epileptogenic drugs, such as pentylenetetrazole (PTZ), to identify novel anti-epileptic therapeutics. Deleterious mutations in the mTOR regulator DEPDC5 have been associated with various forms of focal epilepsies and knock-down of the zebrafish orthologue causes hyperactivity associated with spontaneous seizure-like episodes, as well as enhanced electrographic activity and characteristic turn wheel swimming. Here, we described the method involved in generating the DEPDC5 loss-of-function model and illustrate the protocol for assessing motor activity at 28 and 48 h post fertilization (hpf), as well as a method for recording field activity in the zebrafish optic tectum. An illustration of the effect of the epileptogenic drug PTZ on neuronal activity over time is also provided.

Here, we present a protocol for the development and the characterization of a zebrafish model of epilepsy resulting from the transient inhibition of the DEPDC5 gene.

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research ...

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

Behavioral Test Battery to Assess Neurotoxic Effects in Adult Zebrafish

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior.
We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

Author Spotlight: Assessment of Environmental and Drug-Induced Behavioral Changes in Adult Zebrafish 

Here, we present a comprehensive behavioral test battery, including the novel tank, Shoaling, and social preference tests, to effectively determine the potential neurotoxic effects of chemicals (e.g., methamphetamine and glyphosate) on adult zebrafish using a single tank. This method is relevant to neurotoxicity and environmental research.

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. ...

Zebrafish Avoidance and Thigmotaxis Assay: A High-Throughput Method to Examine Larval Behavior in Response to Aversive Visual Stimulus

Source: Richendrfer, H., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/50622/" target="_blank">Automated High-throughput Behavioral Analyses in Zebrafish Larvae</a>. &#160;J. Vis. Exp.&#160;(2013)
The video describes the avoidance and thigmotaxis assay of zebrafish larvae. The video examines the behavior of zebrafish larvae in response to visual aversive stimulus.

Source: Richendrfer, H., et al. Automated High-throughput Behavioral Analyses in Zebrafish Larvae. &#160;J. Vis. Exp.&#160;(2013)
The video describes the ...

Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes

Samenvatting

Meer video's verkennen