Zebrafish in their natural habitat are inhabiting slow-moving or standing water bodies, the edges of streams and ditches adjacent to rice-fields. In order to avoid predatory birds and other predators, zebrafish shoals preferably hide under floating plants and they retain this behavior under laboratory conditions, where both males and females prefer the tank compartment with floating plants over a barren one (Schroeder et al.
2014). The high rate of planktonic organisms in their diet indicates that zebrafish feed mainly in the water column. However, terrestrial insects are also consumed, suggesting surface feeding as described above. In a shoaling species such as zebrafish, fish perform better in groups and stress restricts learning ability in isolated individuals (Spence et al.
2008). In this pilot study, groups (shoals) of 4 adult zebrafish could successfully adapt to the relatively small volume of test tanks so that 24 h of habituation period (day 1) was sufficient, allowing training with two identical objects and testing the novel object recognition behavior on day 2 and day 3. The experimental design described hereby was similar to that of rodents where the NOR task procedure consists of the following three phases: habituation, familiarization and test phase (Antunes and Biala
2012). Zebrafish kept and tested in these conditions and within shoals could easily overcome anxiety of novel and barren tank and as being kept without feeding their desire for nourishment surpassed fear in accordance with De Lombaert et al. (
2017). Vigorous mouth-object contacts could be detected either on day 2 during training or on day 3 during NOR tests (Fig.
3). Aluminum foil balls and plastic pearls provoked similar exploratory behavior no significant differences in the numbers of mouth-object contacts on day 2 during training (Fig.
4). When either floating plastic pearl or floating aluminum foil ball was used as novel object, significantly higher number of mouth-object contacts were recorded on day 3 during NOR test (Fig.
5). Therefore, according to the results described above, the type of floating object had no significant effect on the intensity of exploratory behavior so that any innate preference can be ruled out in each case. Since zebrafish in their natural habitat are often feeding close to water surface, consequently smaller floating and non-stationary objects as potential preys attract exploring fish even more than submerged stationary underwater objects. Such stationary objects may provoke neophobia in zebrafish depending on shape and size as reported by May et al. (
2016) who suspected that fish perceived larger objects as predators. Zebrafish tested in this pilot study showed neophilia in each experiment according to the results described above, whereas smaller floating objects, close to prey size, were used as novel objects. Sudden changes in refraction inducing bright and twinkling light signals together with intense red (as suggested by Spence and Smith
2008; Avdesh et al.
2012) and other attractive colors (mimicked hereby using floating crumpled aluminum foil balls, and pink plastic hollow pearls) are observed by fish feeding close to surface and presumably considered as insects or similar floating preys. We suggest further extensive research in order to determine the optimal shapes, size, surface properties and colors of floating novel objects to be used in NOR studies on zebrafish.