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

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.

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