Responses of larval zebrafish to low pH immersion assay. Comment on Lopez-Luna et al.

Abstract

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 . 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 . 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 a...

Fish sentience denial: Muddying the waters

“…That is why we are happy to submit this persistent peer disagreement to open peer commentary in this journal. One important point on which we are in complete agreement with Diggles et al (2017), however, is that more funding is needed in the area of fish welfare to provide robust empirical evidence we can use to inform the humane and ethical treatment of fish.…”

“…Some authors even feel the need to refer to "humans and animals," as if humans were not themselves animals. In some countries this speciesism has led to obstacles and scepticism when it comes to the protection of aquatic animals in particular (e.g., Stevens et al 2016;Diggles et al 2017;Key et al 2017).…”

“…This response was then ameliorated by a range of analgesic or pain-relieving drugs. Diggles et al (2017) do not contest that adult zebrafish experience pain, but they do criticise the use of 5dpf larvae to replace adults despite these findings, which demonstrate that the use of larval zebrafish could replace current protocols using adults as recommended by the concept of the 3Rs in the European legislation and in the guidelines for animal care in other countries. Fish larvae are not protected in some countries until they are free-feeding forms, and in the case of zebrafish, this is from 5dpf onwards (Strähle et al 2012).…”

Time to (finally) acknowledge that fish have emotionality and pain

“…Fish behavior is complex and emotionality-driven (Kalueff et al, 2012). Some skepticism remains, however, as to whether zebrafish are sentient (Diggles et al, 2017;Key, 2015Key, , 2016Rose, 2002Rose, , 2007Rose, , 2016Rose et al, 2014), and whether, from a bioethical perspective, they should be protected (similarly to the way mammals are). A recent study (Sneddon et al, 2018) rigorously challenges such views through a thorough discussion of nociception and analgesia studies in zebrafish (Danio rerio).…”

“…In summary, whereas the denial of fish nociception and emotionality (Diggles et al, 2017;Key, 2015Key, , 2016Rose, 2016;Rose et al, 2014) continues to ignore the rapidly growing body of positive empirical evidence (Kalueff et al, 2012;Lopez-Luna, Al-Jubouri, Al-Nuaimy, & Sneddon, 2017;Sneddon, 2002Sneddon, , 2003Sneddon, , 2009Sneddon et al, 2018;Stewart et al, 2015), the field should also adapt and reshape in response. Specifically, it can turn away from discussing dated and unsupported views and move full speed ahead to focusing on what really matters: studying the mechanisms of fish pain and emotionality and their translational relevance to human conditions in order to build a reliable, reproducible and ethically sound line of research using these sentient aquatic species.…”