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Representative Results






Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes

Published: August 29th, 2016



1Department of Biology, Saint Joseph’s University, 2Genomics and Computational Biology, University of Pennsylvania

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 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.

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 ,....

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The following methodologies for the housing, care, and study of zebrafish have been approved by the Saint Joseph's University IACUC.

1. Zebrafish Housing and Care

  1. 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.
  2. Identify and separate zebrafish according to sex. Alternatively, skip this step o.......

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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, 'within assay correlations' and 'between assay correlati.......

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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'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.......

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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.


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Name Company Catalog Number Comments
Zebrafish Rack System Aquaneering Inc Cat. # ZS550
Pet Valu Tropical Fish Food, 224.0g Pet Valu Cat. # 31700
Premium Grade Brine Shrimp Eggs, 16 oz Brine Shrimp Direct
1.5 L Trapezoidal Tank Pentair Aquatic Ecosystems Cat. # itsts-a
19L rectangular tank That Fish Place 211932
76L rectangular tank That Fish Place 212180
Hitachi KP-D20A CCD Camera Prescott's, Inc.
Nikon AF Nikkor 35-105mm f/305~4.5s MACRO lens Nikon Corporation
ArtMinds Square Mirror, Value Pack 3"x3" Michaels Cat. # 10334162
SPSS Statistics Base IBM
R The R Foundation

  1. Huntingford, F. The relationship between anti-predator behaviour and aggression among conspecifics in the three-spined stickleback, Gasterosteus aculeatus. Anim Behav. 24, 245-260 (1976).
  2. Réale, D., Reader, S. M., Sol, D., McDougall, P. T., Dingemanse, N. J. Integrating animal temperament within ecology and evolution. Biol Rev. 82 (2), 291-318 (2007).
  3. Sih, A., Bell, A., Johnson, J. C. Behavioral syndromes: an ecological and evolutionary overview. Trends Ecol Evol. 19 (7), 372-378 (2004).
  4. Conrad, J. L., Weinersmith, K. L., Brodin, T., Saltz, J. B., Sih, A. Behavioural syndromes in fishes: a review with implications for ecology and fisheries management. J Fish Biol. 78 (2), 395-435 (2011).
  5. Wolf, M., Weissing, F. J. Animal personalities: consequences for ecology and evolution. Trends Ecol Evol. 27 (8), 452-461 (2012).
  6. Langheinrich, U. Zebrafish: a new model on the pharmaceutical catwalk. BioEssays. 25 (9), 904-912 (2003).
  7. Dai, Y. -. J., Jia, Y. -. F., et al. Zebrafish as a model system to study toxicology: Zebrafish toxicology monitoring. Environ Toxicol Chem. 33 (1), 11-17 (2014).
  8. Norton, W., Bally-Cuif, L. Adult zebrafish as a model organism for behavioural genetics. Neuroscience. 11 (1), 90 (2010).
  9. Gerlai, R. Zebra fish: an uncharted behavior genetic model. Behav Genet. 33 (5), 461-468 (2003).
  10. Dzieweczynski, T. L., Campbell, B. A., Marks, J. M., Logan, B. Acute exposure to 17α-ethinylestradiol alters boldness behavioral syndrome in female Siamese fighting fish. Horm Behav. 66 (4), 577-584 (2014).
  11. Miklòsi, &. #. 1. 9. 3. ;., Andrew, R. J. The Zebrafish as a Model for Behavioral Studies. Zebrafish. 3 (2), 227-234 (2006).
  12. . . Zebrafish protocols for neurobehavioral research. , (2012).
  13. Moretz, J. A., Martins, E. P., Robison, B. D. Behavioral syndromes and the evolution of correlated behavior in zebrafish. Behav Ecol. 18 (3), 556-562 (2007).
  14. Way, G. P., Kiesel, A. L., Ruhl, N., Snekser, J. L., McRobert, S. P. Sex differences in a shoaling-boldness behavioral syndrome, but no link with aggression. Behav Process. 113, 7-12 (2015).
  15. Toms, C. N., Echevarria, D. J., Jouandot, D. J. A methodological review of personality-related studies in fish: Focus on the shy-bold axis of behavior. J Comp Psychol. 23, 1-25 (2010).
  16. Rowland, W. J. Studying visual cues in fish behavior: A review of ethological techniques. Environ Biol Fish. 56 (3), 285-305 (1999).
  17. Way, G. P., Ruhl, N., Snekser, J. L., Kiesel, A. L., McRobert, S. P. A Comparison of Methodologies to Test Aggression in Zebrafish. Zebrafish. 12 (2), 144-151 (2015).
  18. Moss, S., Tittaferrante, S., et al. Interactions between aggression, boldness and shoaling within a brood of convict cichlids (Amatitlania nigrofasciatus). Behav Process. 121, 63-69 (2015).
  19. Ruhl, N., Kiesel, A. L., McRobert, S. P., Snekser, J. L. Behavioural syndromes and shoaling: connections between aggression, boldness and social behaviour in three different Danios. Behaviour. 149 (10-12), 1155-1175 (2012).
  20. Larson, E. T., O'Malley, D. M., Melloni, R. H. Aggression and vasotocin are associated with dominant-subordinate relationships in zebrafish. Behav Brain Res. 167 (1), 94-102 (2006).
  21. McRobert, S., Bradner, The influence of body coloration on shoaling preferences in fish. Anim Behav. 56 (3), 611-615 (1998).
  22. Ruhl, N., McRobert, S. The effect of sex and shoal size on shoaling behaviour in Danio rerio. J Fish Biol. 67 (5), 1318-1326 (2005).
  23. Snekser, J., Ruhl, N., Bauer, K., McRobert, S. The influence of sex and phenotype on shoaling decisions in zebrafish. J Comp Psychol. 23, 70-81 (2010).
  24. Budaev, S. V. Using Principal Components and Factor Analysis in Animal Behaviour Research: Caveats and Guidelines. Ethology. 116 (5), 472-480 (2010).
  25. Paciorek, T., McRobert, S. Daily variation in the shoaling behavior of zebrafish Danio rerio. Curr Zool. 58 (1), 129-137 (2012).
  26. Wright, D., Krause, J. Repeated measures of shoaling tendency in zebrafish (Danio rerio) and other small teleost fishes. Nat Protoc. 1 (4), 1828-1831 (2006).
  27. Cachat, J., Stewart, A., et al. Measuring behavioral and endocrine responses to novelty stress in adult zebrafish. Nat Protoc. 5 (11), 1786-1799 (2010).
  28. Croft, D. P., Krause, J., Couzin, I. D., Pitcher, T. L. When fish shoals meet: outcomes for evolution and fisheries. Fish Fish. 4, 138-146 (2003).
  29. Brown, M. B., Forsythe, A. B. Robust Tests for the Equality of Variances. J Am Stat Assoc. 69 (346), (1974).
  30. Kruskal, W. H., Wallis, W. A. Use of Ranks in One-Criterion Variance Analysis. J Am Stat Assoc. 47 (260), 583-621 (1952).
  31. Nelder, J. A., Wedderburn, R. W. M. Generalized Linear Models. J Roy Stat Soc A Sta. 135, 370-384 (1972).
  32. Iman, R. L., Conover, W. J. A distribution-free approach to inducing rank correlation among input variables. Commun Stat Simulat. 11 (3), 311-334 (1982).
  33. Fleiss, J. L., Cohen, J. The Equivalence of Weighted Kappa and the Intraclass Correlation Coefficient as Measures of Reliability. Educ Psychol Meas. 33 (3), 613-619 (1973).
  34. Ruhl, N., McRobert, S. P., Currie, W. J. S. Shoaling preferences and the effects of sex ratio on spawning and aggression in small laboratory populations of zebrafish (Danio rerio). Lab Animal. 38 (8), 264-269 (2009).
  35. Bell, A. M. Behavioural differences between individuals and two populations of stickleback (Gasterosteus aculeatus). J Evolution Biol. 18 (2), 464-473 (2005).
  36. Wilson, A. D. M., Godin, J. -. G. J. Boldness and behavioral syndromes in the bluegill sunfish, Lepomis macrochirus. Behav Ecol. 20 (2), 231-237 (2009).
  37. Brodin, T. Behavioral syndrome over the boundaries of life--carryovers from larvae to adult damselfly. Behav Ecol. 20 (1), 30-37 (2008).
  38. Noldus, L. P., Spink, A. J., Tegelenbosch, R. A. EthoVision: a versatile video tracking system for automation of behavioral experiments. Behav Res Methods Instrum Comput. 33 (3), 398-414 (2001).
  39. Cote, J., Fogarty, S., Weinersmith, K., Brodin, T., Sih, A. Personality traits and dispersal tendency in the invasive mosquitofish (Gambusia affinis). Proc R Soc B. 277 (1687), 1571-1579 (2010).
  40. Fraser, D. F., Gilliam, J. F., Daley, M. J., Le, A. N., Skalski, G. T. Explaining leptokurtic movement distributions: intrapopulation variation in boldness and exploration. Am Nat. 158 (2), 124-135 (2001).
  41. Dingemanse, N. J., Wright, J., Kazem, A. J. N., Thomas, D. K., Hickling, R., Dawnay, N. Behavioural syndromes differ predictably between 12 populations of three-spined stickleback. J Anim Ecol. 76 (6), 1128-1138 (2007).

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