Skip to main content
Advertisement

Main menu

  • Home
  • Articles
    • Accepted manuscripts
    • Issue in progress
    • Latest complete issue
    • Issue archive
    • Archive by article type
    • Special issues
    • Subject collections
    • Interviews
    • Sign up for alerts
  • About us
    • About JEB
    • Editors and Board
    • Editor biographies
    • Travelling Fellowships
    • Grants and funding
    • Journal Meetings
    • Workshops
    • The Company of Biologists
    • Journal news
  • For authors
    • Submit a manuscript
    • Aims and scope
    • Presubmission enquiries
    • Article types
    • Manuscript preparation
    • Cover suggestions
    • Editorial process
    • Promoting your paper
    • Open Access
    • Outstanding paper prize
    • Biology Open transfer
  • Journal info
    • Journal policies
    • Rights and permissions
    • Media policies
    • Reviewer guide
    • Sign up for alerts
  • Contacts
    • Contact JEB
    • Subscriptions
    • Advertising
    • Feedback
    • For library administrators
  • COB
    • About The Company of Biologists
    • Development
    • Journal of Cell Science
    • Journal of Experimental Biology
    • Disease Models & Mechanisms
    • Biology Open

User menu

  • Log in

Search

  • Advanced search
Journal of Experimental Biology
  • COB
    • About The Company of Biologists
    • Development
    • Journal of Cell Science
    • Journal of Experimental Biology
    • Disease Models & Mechanisms
    • Biology Open

supporting biologistsinspiring biology

Journal of Experimental Biology

  • Log in
Advanced search

RSS  Twitter  Facebook  YouTube  

  • Home
  • Articles
    • Accepted manuscripts
    • Issue in progress
    • Latest complete issue
    • Issue archive
    • Archive by article type
    • Special issues
    • Subject collections
    • Interviews
    • Sign up for alerts
  • About us
    • About JEB
    • Editors and Board
    • Editor biographies
    • Travelling Fellowships
    • Grants and funding
    • Journal Meetings
    • Workshops
    • The Company of Biologists
    • Journal news
  • For authors
    • Submit a manuscript
    • Aims and scope
    • Presubmission enquiries
    • Article types
    • Manuscript preparation
    • Cover suggestions
    • Editorial process
    • Promoting your paper
    • Open Access
    • Outstanding paper prize
    • Biology Open transfer
  • Journal info
    • Journal policies
    • Rights and permissions
    • Media policies
    • Reviewer guide
    • Sign up for alerts
  • Contacts
    • Contact JEB
    • Subscriptions
    • Advertising
    • Feedback
    • For library administrators
CORRESPONDENCE
Responses of larval zebrafish to low pH immersion assay. Comment on Lopez-Luna et al.
B. K. Diggles, R. Arlinghaus, H. I. Browman, S. J. Cooke, I. G. Cowx, A. O. Kasumyan, B. Key, J. D. Rose, W. Sawynok, A. Schwab, A. B. Skiftesvik, E. D. Stevens, C. A. Watson, C. D. L. Wynne
Journal of Experimental Biology 2017 220: 3191-3192; doi: 10.1242/jeb.162834
B. K. Diggles
1DigsFish Services, Banksia Beach, QLD 4507, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for B. K. Diggles
  • For correspondence: ben@digsfish.com
R. Arlinghaus
2Department of Biology and Ecology of Fishes, Leibniz-Institute of Freshwater Ecology and Inland Fisheries & Humboldt-Universität zu Berlin, Berlin 12587, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
H. I. Browman
3Institute of Marine Research, Austevoll Research Station, Marine Ecosystem Acoustics Group, Sauganeset 16, 5392 Storebø, Norway
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
S. J. Cooke
4Department of Biology, Carleton University, Ottawa, K1S 5B6, Canada
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
I. G. Cowx
5Hull International Fisheries Institute, University of Hull, Hull HU6 7RX, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
A. O. Kasumyan
6Department of Ichthyology, Faculty of Biology, Moscow State University, Moscow 119991, Russian Federation
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
B. Key
7School of Biomedical Sciences, University of Queensland, St Lucia, QLD 4072, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
J. D. Rose
8Department of Zoology and Physiology, University of Wyoming, Laramie, WY 82071, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
W. Sawynok
9Infofish Australia, Frenchville, QLD 4701, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
A. Schwab
10Biglen, Bern 3507, Switzerland
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
A. B. Skiftesvik
3Institute of Marine Research, Austevoll Research Station, Marine Ecosystem Acoustics Group, Sauganeset 16, 5392 Storebø, Norway
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
E. D. Stevens
11Biomedical Sciences, Atlantic Veterinary College, Charlottetown, PE, C1A 4P3, Canada
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
C. A. Watson
12Tropical Aquaculture Laboratory, University of Florida, Gainesville, FL 33547, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
C. D. L. Wynne
13Department of Psychology, Arizona State University, Tempe, AZ 85287, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Info & metrics
  • PDF
Loading

Lopez-Luna et al. (2017) observed behavioral responses of larval zebrafish (Danio rerio) exposed for 10 min to pH 2.6–3.6 when acetic acid (0.01–0.25%) or citric acid (0.1–5%) was added to the tank water in the presence or absence of aspirin (1–2.5 mg l−1), morphine sulfate (1–48 mg l−1), lidocaine (1–5 mg l−1) and flunixin (8–20 mg l−1). Fish exposed to 0.1–0.25% acetic acid were less active than controls while those exposed to citric acid and 0.01% acetic acid were more active. Administration of high doses of aspirin, morphine and lidocaine for 30 min before exposure prevented the reduction in activity induced by 0.1–0.25% acetic acid.

These behavioral responses were interpreted as evidence that acetic acid immersion provided a noxious stimulus (i.e. activated nociceptors) that was reliable for use as a model system for the study of analgesic substances. We identify methodological weaknesses and inconsistencies in the interpretation of results, and emphasize that activation of nociceptors was assumed, not demonstrated. As a result of several processes and interactions that were not accounted for or discussed, we warn their conclusions are unfounded.

A critical omission was the failure to report water conductivity, hardness and alkalinity data. These determine the magnitude of acute osmoregulatory effects that occur in fish exposed to highly acidic water (Wood, 1989). Trials by other researchers using water with different conductivity, hardness or alkalinity profiles could, therefore, generate significantly different results. Immersion of fish in low pH water also introduces several unavoidable and uncontrolled interactions that prevent unequivocal interpretation of the behavioral changes observed.

For example, sudden exposure of fish to water of pH <4 results in gill dysfunction, iono-regulatory failure and pathological lesions of the gill epithelium (Wood, 1989). These reduce respiratory efficiency, initiating compensatory behavioral responses such as surface respiration (Kramer, 1987), which appears synonymous with ‘top-dwelling behavior’ reported by Currie (2014) in adult zebrafish immersed in 0.03% acetic acid (pH 3.9–4.0). Notably, aquatic surface respiration can occur in a variety of natural circumstances in the absence of nociception (Kramer, 1987), so this behavior is insufficient evidence that nociception is occurring.

In contrast to Currie (2014) and Steenbergen and Bardine (2014), Lopez-Luna et al. (2017) considered reduced (not increased) activity as evidence of ‘alleged pain behavior’ in zebrafish exposed to 0.1–0.25% acetic acid. Steenbergen and Bardine (2014) interpreted increased activity and cyclooxygenase-2 gene expression as evidence of nociception in larval zebrafish immersed in 0.0025–0.025% acetic acid. However, cyclooxygenase-2 expression is a non-specific marker of several physiological processes (Wang et al., 2016), meaning its expression is also insufficient evidence of nociception. A critical observation is that larval zebrafish in the study by Lopez-Luna et al. (2017) continued to exhibit increased activity when exposed to pH 2.6 in the 5 mg l−1 citric acid experiment. Because of the strong likelihood of acute pathological damage to gills, eyes and other tissues at such low pH (Daye and Garside, 1976), the absence of ‘alleged pain behavior’ in the citric acid treatment calls into question whether nociception was occurring at all. Furthermore, the fact that both increased and decreased activity are being interpreted by different researchers as evidence that nociception is occurring in larval zebrafish exposed to acetic acid casts doubt upon the construct validity of the assay.

The authors noted that at pH 3.3, exposure to 0.25% acetic acid had the opposite effect on behavior (less activity) compared with 0.1% citric acid (more activity). They stated this indicated ‘another mechanism affecting the response of the nociceptors other than the pH’, but did not elucidate further. Because of the immersion design, we contend those other mechanisms do not have anything to do with nociception. Rather, an alternative and more parsimonious explanation is the behavioral changes were due to detection by, or interference with, chemosensors (Kasumyan, 2001).

Chemosensory systems are active, and chemosensory cells are fully developed and functional in zebrafish before 5 days post-hatching (Kotrschal et al., 1997). Dose-dependent behavioral responses to different chemicals are common and could explain the behavioral differences found between citric acid, acetic acid and, importantly, also the pharmaceuticals used. Indeed, citric acid was identified as a potent gustatory feeding stimulant in zebrafish (Kasumyan and Doving, 2003). Furthermore, acute exposure to pH <4.0 can cause pathological alteration of the olfactory epithelium (Daye and Garside, 1976) and low pH interferes with chemoreceptors responsible for both olfaction (Tierney et al., 2010) and gustation (Kasumyan, 2001). Acute exposure to low pH can extinguish or change behavioral responses to odors, including attraction to previously repulsive chemicals (Royce-Malmgren and Watson, 1987). Because the chemicals studied drop pH and activate chemoreceptors, this interaction makes it difficult to determine what mechanism(s) was driving fish behavior.

Currie (2014) reported bottom-seeking behavior consistent with chemosensory avoidance responses in adult zebrafish exposed to 0.5–3 mg l−1 morphine or 0.03% acetic acid via the water. Increased locomotor activity in zebrafish was also reported by Lopez-Luna et al. (2017) as a ‘side-effect’ of morphine administration. They tried to circumvent these behavioral artefacts using a 30 min ‘acclimation period’ prior to exposure to the acid treatments. The pathological effects of immersion in high concentrations of drugs such as morphine or aspirin are largely unknown, though exposure to anti-inflammatory drugs (e.g. Diclofenac) causes damage to gill epithelia at extremely low concentrations (ca. 1 µg l−1). Immersion in high concentrations of pharmaceuticals for 30 min prior to treatment therefore may have significant unintended effects on chemosensory receptors and gill function, making subsequent behavioral responses and interactions with other chemicals unpredictable and/or hopelessly confounded.

Immersion trials therefore provide no advantage over the injection methods previously used, which, while having their own problems (Rose et al., 2014), are more likely to target specific tissues and induce nociception, all while being more economical with the use of reagents. Injection inflicts fewer negative effects on the welfare of wild fishes whereas chemicals used in tank immersion enter waste water and, ultimately, the environment as organic contaminants (Tierney et al., 2010).

The strong possibility that the authors measured behavioral changes due to factors other than nociception cannot be excluded. It is, therefore, premature for Lopez-Luna et al. (2017) and others (Steenbergen and Bardine, 2014) to claim zebrafish larval immersion models have utility for nociception research.

  • © 2017. Published by The Company of Biologists Ltd

References

  1. ↵
    1. Currie, A. D.
    (2014). Toward a novel model of pain in zebrafish: exposure to water containing dilute concentrations of acetic acid. Psychology Honors Projects, paper 33, Macalester College. http://digitalcommons.macalester.edu/psychology_honors/3.
  2. ↵
    1. Daye, P. G. and
    2. Garside, E. T.
    (1976). Histopathologic changes in surficial tissues of brook trout, Salvelinus fontinalis (Mitchill), exposed to acute and chronic levels of pH. Can. J. Zool. 54, 2140-2155. doi:10.1139/z76-248
    OpenUrlCrossRefPubMed
  3. ↵
    1. Kasumyan, A. O.
    (2001). Effects of chemical pollutants on foraging behavior and sensitivity of fish to food stimuli. J. Ichthyol. 41, 76-87.
    OpenUrl
  4. ↵
    1. Kasumyan, A. O. and
    2. Doving, K. B.
    (2003). Taste preferences in fish. Fish Fish. 4, 289-347. doi:10.1046/j.1467-2979.2003.00121.x
    OpenUrlCrossRef
  5. ↵
    1. Kotrschal, K.,
    2. Krautgartner, W. and
    3. Hansen, A.
    (1997). Ontogeny of the solitary chemosensory cells in the zebrafish, Danio rerio. Chem. Senses 22, 111-118. doi:10.1093/chemse/22.2.111
    OpenUrlCrossRefPubMedWeb of Science
  6. ↵
    1. Kramer, D. L.
    (1987). Dissolved oxygen and fish behavior. Environ. Biol. Fishes 18, 81-92. doi:10.1007/BF00002597
    OpenUrlCrossRef
  7. ↵
    1. Lopez-Luna, J.,
    2. Al-Jubouri, Q.,
    3. Al-Nuaimy, W. and
    4. Sneddon, L. U.
    (2017). Reduction in activity by noxious chemical stimulation is ameliorated by immersion in analgesic drugs in zebrafish. J. Exp. Biol. 220, 1451-1458. doi:10.1242/jeb.146969
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Rose, J. D.,
    2. Arlinghaus, R.,
    3. Cooke, S. J.,
    4. Diggles, B. K.,
    5. Sawynok, W.,
    6. Stevens, E. D. and
    7. Wynne, C. D.
    (2014). Can fish really feel pain? Fish Fish. 15, 97-133.
    OpenUrl
  9. ↵
    1. Royce-Malmgren, C. H. and
    2. Watson, W. H.
    (1987). Modification of olfactory-related behavior in juvenile Atlantic salmon by changes in pH. J Chem Ecol 13, 533-546. doi:10.1007/BF01880097
    OpenUrlCrossRef
  10. ↵
    1. Steenbergen, P. J. and
    2. Bardine, N.
    (2014). Antinociceptive effects of buprenorphine in zebrafish larvae: An alternative for rodent models to study pain and nociception? Appl. Anim. Beh. Sci. 152, 92-99. doi:10.1016/j.applanim.2013.12.001
    OpenUrlCrossRef
  11. ↵
    1. Tierney, K. B.,
    2. Baldwin, D. H.,
    3. Hara, T. J.,
    4. Ross, P. S.,
    5. Scholz, N. L. and
    6. Kennedy, C. J.
    (2010). Olfactory toxicity in fishes. Aquat. Toxicol. 96, 2-26. doi:10.1016/j.aquatox.2009.09.019
    OpenUrlCrossRefPubMed
  12. ↵
    1. Wang, T.,
    2. Mai, K. and
    3. Ai, Q.
    (2016). A review of cyclooxygenase-2 role in fish. Austin J. Nutr. Metab. 3, 1037.
    OpenUrl
  13. ↵
    1. Wood, C. M.
    (1989). The physiological problems of fish in acid waters. In Acid Toxicity and Aquatic Animals (ed. R. Morris, E. W. Taylor, D. J. Brown and J. A. Brown), pp. 125-152. Society for Experimental Biology Seminar Series 34. Cambridge: Cambridge University Press.
Previous ArticleNext Article
Back to top
Previous ArticleNext Article

This Issue

 Download PDF

Email

Thank you for your interest in spreading the word on Journal of Experimental Biology.

NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

Enter multiple addresses on separate lines or separate them with commas.
Responses of larval zebrafish to low pH immersion assay. Comment on Lopez-Luna et al.
(Your Name) has sent you a message from Journal of Experimental Biology
(Your Name) thought you would like to see the Journal of Experimental Biology web site.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
CORRESPONDENCE
Responses of larval zebrafish to low pH immersion assay. Comment on Lopez-Luna et al.
B. K. Diggles, R. Arlinghaus, H. I. Browman, S. J. Cooke, I. G. Cowx, A. O. Kasumyan, B. Key, J. D. Rose, W. Sawynok, A. Schwab, A. B. Skiftesvik, E. D. Stevens, C. A. Watson, C. D. L. Wynne
Journal of Experimental Biology 2017 220: 3191-3192; doi: 10.1242/jeb.162834
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
Citation Tools
CORRESPONDENCE
Responses of larval zebrafish to low pH immersion assay. Comment on Lopez-Luna et al.
B. K. Diggles, R. Arlinghaus, H. I. Browman, S. J. Cooke, I. G. Cowx, A. O. Kasumyan, B. Key, J. D. Rose, W. Sawynok, A. Schwab, A. B. Skiftesvik, E. D. Stevens, C. A. Watson, C. D. L. Wynne
Journal of Experimental Biology 2017 220: 3191-3192; doi: 10.1242/jeb.162834

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Alerts

Please log in to add an alert for this article.

Sign in to email alerts with your email address

Article navigation

  • Top
  • Article
    • References
  • Info & metrics
  • PDF

Related articles

Cited by...

More in this TOC section

  • Response to ‘No evidence for hibernation in rockwrens’
  • No evidence for hibernation in rockwrens
  • Response to: Lipid content of whale blubber cannot be measured using biopsies
Show more CORRESPONDENCE

Similar articles

Other journals from The Company of Biologists

Development

Journal of Cell Science

Disease Models & Mechanisms

Biology Open

Advertisement

Predicting the Future: Species Survival in a Changing World

Read our new special issue exploring the significant role of experimental biology in assessing and predicting the susceptibility or resilience of species to future, human-induced environmental change.


Adam Hardy wins the 2020 Journal of Experimental Biology Outstanding Paper Prize

Congratulations to winner Adam Hardy for his work showing that goby fins are as touch sensitive as primate fingertips. Read Adam’s paper and find out more about the 12 papers nominated for the award.


Stark trade-offs and elegant solutions in arthropod visual systems

Many elegant eye specializations that evolved in response to visual challenges continue to be discovered. A new Review by Meece et al. summarises exciting solutions evolved by insects and other arthropods in response to specific visual challenges.


Head bobbing gives pigeons a sense of perspective

Pigeons might look goofy with their head-bobbing walk, but it turns out that the ungainly head manoeuvre allows the birds to judge distance.

Articles

  • Accepted manuscripts
  • Issue in progress
  • Latest complete issue
  • Issue archive
  • Archive by article type
  • Special issues
  • Subject collections
  • Interviews
  • Sign up for alerts

About us

  • About JEB
  • Editors and Board
  • Editor biographies
  • Travelling Fellowships
  • Grants and funding
  • Journal Meetings
  • Workshops
  • The Company of Biologists
  • Journal news

For Authors

  • Submit a manuscript
  • Aims and scope
  • Presubmission enquiries
  • Article types
  • Manuscript preparation
  • Cover suggestions
  • Editorial process
  • Promoting your paper
  • Open Access
  • Outstanding paper prize
  • Biology Open transfer

Journal Info

  • Journal policies
  • Rights and permissions
  • Media policies
  • Reviewer guide
  • Sign up for alerts

Contact

  • Contact JEB
  • Subscriptions
  • Advertising
  • Feedback

 Twitter   YouTube   LinkedIn

© 2021   The Company of Biologists Ltd   Registered Charity 277992