Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Impact of test chamber design on spontaneous behavioral responses of model crustacean zooplankton Artemia franciscana


The use of small aquatic model organisms to investigate the behavioral effects of chemical exposure is becoming an integral component of aquatic ecotoxicology research and neuroactive drug discovery. Despite the increasing use of invertebrates for behavioral phenotyping in toxicological studies and chemical risk assessments, little is known regarding the potential for environmental factors—such as geometry, size, opacity and depth of test chambers—to modulate common behavioral responses. In this work, we demonstrate that test chamber geometry, size, opacity and depth can affect spontaneous, unstimulated behavioral responses of euryhaline crustacean Artemia franciscana first instar larval stages. We found that in the absence of any obvious directional cues, A. franciscana exhibited a strong innate wall preference behavior. Using different test chamber sizes and geometries, we found both increased wall preference and lowered overall distance traveled by the test shrimp in a smaller chamber with sharper-angled vertices. It was also determined through quantifiable changes in the chambers’ color that the A. franciscana early larval stages can perceive, differentiate and react to differences in color or perhaps rather to light transmittance of the test chambers. The interaction between innate edge preference and positive phototaxis could be consistently altered with a novel photic stimulus system. We also observed a strong initial preference for depth in A. franciscana first instar larval stages, which diminished through the acclimatization. We postulate that the impact of test chamber designs on neurobehavioral baseline responses warrants further investigation, in particular considering the increased interest in behavioral eco-neurotoxicology applications.

This is a preview of subscription content, access via your institution

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Impact of test chamber size and geometry on behavioral endpoints.
Fig. 2: The color of test chamber sidewalls induces directional alterations in spontaneous behaviors.
Fig. 3: A crosstalk between positive phototaxis and wall-preference behaviors.
Fig. 4: Influence of test chamber depth on innate behavioral preferences.
Fig. 5: Influence of test chamber depth on innate behavioral preferences with removal of edge preference through horizontal filming.

Data availability

Data from all experiments are available upon request.


  1. Bai, Y., Henry, J. & Wlodkowic, D. Chemosensory avoidance behaviors of marine amphipods Allorchestes compressa revealed using a millifluidic perfusion technology. Biomicrofluidics 14, 014110 (2020).

    Article  CAS  Google Scholar 

  2. Bownik, A. Daphnia swimming behaviour as a biomarker in toxicity assessment: a review. Sci. Total Environ. 601–602, 194–205 (2017).

    Article  Google Scholar 

  3. Libralato, G., Prato, E., Migliore, L., Cicero, A. M. & Manfra, L. A review of toxicity testing protocols and endpoints with Artemia spp. Ecol. Indic. 69, 35–49 (2016).

    Article  CAS  Google Scholar 

  4. Henry, J. & Wlodkowic, D. Towards high-throughput chemobehavioural phenomics in neuropsychiatric drug discovery. Mar. Drugs 17, 340 (2019).

    Article  CAS  Google Scholar 

  5. Morgana, S., Estévez-Calvar, N., Gambardella, C., Faimali, M. & Garaventa, F. A short-term swimming speed alteration test with nauplii of Artemia franciscana. Ecotoxicol. Environ. Saf. 147, 558–564 (2018).

    Article  CAS  Google Scholar 

  6. Bartolomé, M. C. & Sánchez-Fortún, S. Acute toxicity and inhibition of phototaxis induced by benzalkonium chloride in Artemia franciscana larvae. Bull. Environ. Contam. Toxicol. 75, 1208–1213 (2005).

    Article  Google Scholar 

  7. Hellou, J. Behavioural ecotoxicology, an “early warning” signal to assess environmental quality. Environ. Sci. Pollut. Res. Int. 18, 1–11 (2011).

    Article  CAS  Google Scholar 

  8. Campana, O. & Wlodkowic, D. Ecotoxicology goes on a chip: embracing miniaturized bioanalysis in aquatic risk assessment. Environ. Sci. Technol. 52, 932–946 (2018).

    Article  CAS  Google Scholar 

  9. De Esch, C., Slieker, R., Wolterbeek, A., Woutersen, R. & de Groot, D. Zebrafish as potential model for developmental neurotoxicity testing. A mini review. Neurotoxicol. Teratol. 34, 545–553 (2012).

    Article  Google Scholar 

  10. Blackiston, D., Shomrat, T., Nicolas, C. L., Granata, C. & Levin, M. A second-generation device for automated training and quantitative behavior analyses of molecularly-tractable model organisms. PLoS ONE 5, e14370 (2010).

    Article  Google Scholar 

  11. Franco-Restrepo, J. E., Forero, D. A. & Vargas, R. A. A review of freely available, open-source software for the automated analysis of the behavior of adult. zebrafish. Zebrafish 16, 223–232 (2019).

    PubMed  Google Scholar 

  12. Henry, J., Rodriguez, A. & Wlodkowic, D. Impact of digital video analytics on accuracy of chemobehavioural phenotyping in aquatic toxicology. PeerJ 7, e7367 (2019).

    Article  Google Scholar 

  13. Henry, J. & Wlodkowic, D. High-throughput animal tracking in chemobehavioral phenotyping: current limitations and future perspectives. Behav. Processes 180, 104226 (2020).

    Article  Google Scholar 

  14. Garcia, G. R., Noyes, P. D. & Tanguay, R. L. Advancements in zebrafish applications for 21st century toxicology. Pharmacol. Ther. 161, 11–21 (2016).

    Article  CAS  Google Scholar 

  15. Rennekamp, A. J. & Peterson, R. T. 15 years of zebrafish chemical screening. Curr. Opin. Chem. Biol. 24, 58–70 (2015).

    Article  CAS  Google Scholar 

  16. Cartlidge, R. & Wlodkowic, D. Caging of planktonic rotifers in microfluidic environment for sub-lethal aquatic toxicity tests. Biomicrofluidics 12, 044111 (2018).

    Article  Google Scholar 

  17. Kohler, S. A., Parker, M. O. & Ford, A. T. Shape and size of the arenas affect amphipod behaviours: implications for ecotoxicology. PeerJ 6, e5271 (2018).

    Article  Google Scholar 

  18. Kohler, S. A., Parker, M. O. & Ford, A. T. Species-specific behaviours in amphipods highlight the need for understanding baseline behaviours in ecotoxicology. Aquat. Toxicol. 202, 173–180 (2018).

    Article  CAS  Google Scholar 

  19. Kohler, S. A., Parker, M. O. & Ford, A. T. High-throughput screening of psychotropic compounds: impacts on swimming behaviours in Artemia franciscana. Toxics 9, 64 (2021).

    Article  Google Scholar 

  20. Inoue, T., Hoshino, H., Yamashita, T., Shimoyama, S. & Agata, K. Planarian shows decision-making behavior in response to multiple stimuli by integrative brain function. Zoolog. Lett. 1, 7 (2015).

    Article  Google Scholar 

  21. Truong, L. et al. Multidimensional in vivo hazard assessment using zebrafish. Toxicol. Sci. 137, 212–233 (2014).

    Article  CAS  Google Scholar 

  22. Zhang, S., Hagstrom, D., Hayes, P., Graham, A. & Collins, E.-M. S. Multi-behavioral endpoint testing of an 87-chemical compound library in freshwater planarians. Toxicol. Sci. 167, 26–44 (2019).

    Article  CAS  Google Scholar 

  23. Akiyama, Y., Agata, K. & Inoue, T. Spontaneous behaviors and wall-curvature lead to apparent wall preference in planarian. PLoS ONE 10, e0142214 (2015).

    Article  Google Scholar 

  24. Blaser, R. E. & Rosemberg, D. B. Measures of anxiety in zebrafish (Danio rerio): dissociation of black/white preference and novel tank test. PLoS ONE 7, e36931 (2012).

    Article  CAS  Google Scholar 

  25. Harro, J. Animals, anxiety, and anxiety disorders: how to measure anxiety in rodents and why. Behav. Brain Res. 352, 81–93 (2018).

    Article  Google Scholar 

  26. Faimali, M. et al. Old model organisms and new behavioral end-points: swimming alteration as an ecotoxicological response. Mar. Environ. Res. 128, 36–45 (2017).

    Article  CAS  Google Scholar 

  27. Rashid, M. T. et al. Artemia swarm dynamics and path tracking. Nonlinear Dyn. 68, 555–563 (2012).

    Article  Google Scholar 

  28. Forward, R. B. & Rittschof, D. Brine shrimp larval photoresponses involved in diel vertical migration: activation by fish mucus and modified amino sugars. Limnol. Oceanogr. 44, 1904–1916 (1999).

    Article  CAS  Google Scholar 

  29. Gerhardt, A. Aquatic behavioral ecotoxicology—prospects and limitations. Hum. Ecol. Risk Assess. 13, 481–491 (2007).

    Article  Google Scholar 

  30. Ford, A. T. et al. The role of behavioral ecotoxicology in environmental protection. Environ. Sci. Technol. 55, 5620–5628 (2021).

    Article  CAS  Google Scholar 

  31. Bolker, B. M. et al. Generalized linear mixed models: a practical guide for ecology and evolution. Trends Ecol. Evol. 24, 127–135 (2009).

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations



J.H. and D.Wlodkowic developed and designed the experimental concepts. J.H., D.Williams, Y.B. and A.L. performed the experiments and collected and analyzed the data. J.H. wrote the manuscript. Y.B., A.F. and D.Wlodkowic reviewed and edited the manuscript.

Corresponding author

Correspondence to Donald Wlodkowic.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Lab Animal thanks Maria Violetta Brundo, Jake Martin and Joseph Covi for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Table 1. Detailed statistical results from a GLMM.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Henry, J., Bai, Y., Williams, D. et al. Impact of test chamber design on spontaneous behavioral responses of model crustacean zooplankton Artemia franciscana. Lab Anim 51, 81–88 (2022).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing