In laboratory experiments, variously colored strains of animals, including those with albino phenotypes, are commonly used. The melanocortin theory suggests, however, that coloration phenotypes alter animal physiology and behavior (Ducrest et al., 2008; Santostefano et al., 2019). Albino animals exhibit physiological limitations that separate their behavior during the diurnal cycle from that of pigmented conspecifics. Ocular anomalies related to retinal degradation are most frequently mentioned (Oetting, 2000; Prusky et al., 2002). A lack of melanin can lead to an underdeveloped central part of the retina adapted for high-acuity vision (fovea centralis) and a reduction in rod number (Jeffery, 1997). Physiological changes are followed by reduced visual acuity (Balkema and Dräger, 1991; Buhusi et al., 2005), impaired perception of movement (Hupfeld and Hoffmann, 2006) and higher sensitivity to light (van Abeelen and Kroes, 1967; Kehas et al., 2005). The eyes of albino rodents show reduced adaptation to light, often leading to photoreceptor degradation (Prusky et al., 2002; Refinetti, 2007; Marc et al., 2008), which in turn can cause loss of vision (Buhusi et al., 2005) and, eventually, photophobia and acrophobia (van Abeelen and Kroes, 1967; Owen et al., 1970). Albino animals are less active (Defries, 1969) with prevailing nocturnal activity (Stryjek et al., 2013), and deteriorated spatial orientation is probably the reason why, e.g., albino fish, show a reduced escape response (Ren et al., 2002).

Light is an important zeitgeber that controls diurnal rhythms through the melanocortin system (Sánchez-Vázquez et al., 1997; Oliveira et al., 2013). Light information passes through the retina to the pineal organ and initiates the secretion of melatonin hormone, which is released to blood in low concentrations during the day and high concentrations during the night, thereby controlling the daily patterns of behavior (López-Olmeda et al., 2009; Oliveira et al., 2009; Sánchez-Vázquez et al., 2019). Fish specifically respond to light alternation by showing diurnal, nocturnal or crepuscular daily patterns of behavior (Madrid et al., 2001). The locomotor activity associated with light oscillation may be altered, however, due to food intake (Sánchez-Vázquez et al., 1995, 1997), the number of individuals (Boujard, 1995; Vera et al., 2011) or social relationships (Alanärä et al., 2001; Brännäs, 2008), and individuals can switch their activity from diurnal to nocturnal and vice versa or show dual activity (Alanärä and Brännäs, 1997; Vera et al., 2009; Slavík and Horký, 2012).

Light conditions are associated with the level of aggressiveness (Britz and Pienaar, 1992; Almazán-Rueda et al., 2004). Increasing melatonin secretion during low light intensities attenuates the activity and aggressiveness of organisms (Almazán-Rueda et al., 2004; Carvalho et al., 2012). Biological interpretation of a decrease in aggressiveness during the dark phase suggests that, e.g., fish, do not see each other, as well as prey, and hence, stimuli to be aggressive are waned (Valdimarsson and Metcalfe, 2001; Lopes et al., 2018). Agonistic interactions are accompanied by stress that can be observed as changes in blood plasma parameters (Pottinger and Carrick, 2001; Sloman et al., 2001), e.g., cortisol hormone concentration (Pickering, 1992; Øverli et al., 2004) and brain antioxidant activity (Costantini et al., 2011; Chowdhury and Saikia, 2020). For example, a lower level of cortisol is reported in dominant and more aggressive fish than in subordinate and less aggressive fish (Koolhaas et al., 1999; Sloman et al., 2001; Øverli et al., 2004). Agonistic behavior is a metabolically demanding process (Haller, 1995; Copeland et al., 2011) requiring mobilization of glucose from storage glycogen, leading to its increase in the blood (Fernandez et al., 1994); hence, it is accompanied by hepatic glycogen reduction and an increase in the glucose level in the plasma (Haller, 1991; Neat et al., 1998) that is higher for subdominant individuals and losers (Elofsson et al., 2000; Copeland et al., 2011). The cortisol level also corresponds with the light/dark (L/D) cycle and feeding schedule, while the glucose level response is weaker (Polakof et al., 2007; López-Olmeda et al., 2009; Oliveira et al., 2013). The activity of antioxidants in fish brains is reported as an oxidative stress, which is caused by an imbalance between harmful free radicals and antioxidants neutralizing these radicals (Chowdhury and Saikia, 2020). Oxidative stress can occur after acute stress as a response to increased glucocorticoids, which may induce oxidative stress by increasing the metabolic rate and thus free radicals (Costantini et al., 2011). The rise in antioxidant activity is associated with changes in light (Sreejith et al., 2007; Kim et al., 2016) and the level of aggressiveness (Almeida et al., 2009). In summary, the L/D cycle influences fish behavior, which can be recorded as changes in locomotor activity and aggressiveness accompanied by changes in physiological parameters in blood plasma and antioxidant activity.

Animals, including fish, with a higher level of melanin are more aggressive and dominant (Horth, 2003; Kittilsen et al., 2009) and vice versa; i.e., less pigmented individuals are more sensitive to stress and are often subdominant (Höglund et al., 2000; Kittilsen et al., 2012). In fish, individuals with an albino phenotype show lower aggressiveness, often being ostracized by pigmented conspecifics (Slavík et al., 2015, 2016b), and display higher sensitivity to stress (Slavík et al., 2022) than those with the pigmented phenotype. Data on the behavioral response of albino animals to light alterations during the L/D cycle are rare, and specific information about albino fish is not available.

In our study, we observed the locomotor activity and aggressiveness of albino and pigmented European catfish, Silurus glanis (Linnaeus, 1758), during the L/D cycle. We assumed that the locomotor activity of the albino phenotype would occur predominantly during the dark phase of a day and the frequency of aggressiveness would be higher during the light phase, corresponding to increasing avoidance of light and shelter availability. Blood plasma, brain, gill, and liver samples were collected to evaluate stress parameters accompanying aggressive behavior. Our study aimed to compare the behaviors of albino and pigmented phenotypes of catfish in terms of expected differences in aggressiveness during the L/D cycle. We issue a word of caution regarding the use of experimental albino strains of animals for the simulation of behavioral responses of animals in the wild.

The diel rhythms of the behavioral and biochemical indices generally varied in pigmented and albino catfish (Tables 1, 2). The locomotor activity in the artificial stream without shelters showed no rhythmicity in albino catfish, while pigmented catfish showed a peak during the nocturnal phase of the day (Figure 2). When subjected to limited shelter availability in the experimental arena, pigmented and albino catfish showed similar diel rhythmicities in locomotor activity (Figures 3A,B), aggressiveness (Figures 3C,D), and shelter reassignment (Figures 3E,F). The peak of locomotor activity occurred at night. High-level locomotor activity was also maintained during dawn together with the peak of aggressiveness and shelter reassignment. A nocturnal peak in locomotor activity indicates natural activity patterns, while a dawn peak reflects competition for limited resources (shelter) with the aim of avoiding daylight conditions. The avoidance of daylight was more pronounced in albino catfish, as the presence of a shelter induced their switch to diel rhythmicity, and their stress from daylight was apparent from the peaks in blood cortisol (Figure 4A), glucose (Figure 4B), and lactate (Figure 4C) levels. In contrast, pigmented catfish showed no diel rhythmicity in blood cortisol, glucose, or lactate level. Analogously, significant diel rhythmicity was detected in 11 of the 15 measured blood biochemical indices in albino catfish, while pigmented catfish showed rhythmicity in none of them (Table 1). Among the 4 antioxidants and 1 oxidative stress indicator detected in 3 tissues (brain, gill, and liver), with 15 indices in all, albino catfish showed significant diel rhythmicity in 8 of them, whereas pigmented catfish showed rhythmicity in 5 of them (Table 2). Peaks of antioxidants in the brain (SOD – Figure 5A; CAT – Figure 5B) or liver (SOD, GST, and GSH) of albino catfish were detected during the day, suggesting their relation to daylight stress. On the other hand, antioxidants (GST and GSH) in the gills of albino catfish peaked during dawn, suggesting their relation to the increase of aggressiveness and shelter reassignment. The antioxidants in the liver (SOD) and brain (CAT – Figure 5C) of pigmented catfish peaked at night, suggesting their relation to increased activity, while the antioxidants in gills peaked during the day (SOD) or at dusk (CAT). The oxidative stress indicator TBARS peaked during dusk in both catfish groups; however, in albino catfish, a significant trend was found in the liver, while in pigmented catfish, a significant trend was found in the gills.

A complex model was fitted with the aim of evaluating the links between behavioral and biochemical indices. Time rhythmicity was modeled using a sine wave and confirmed a parallel course of agonistic interactions in both catfish groups (P < 0.001; Figure 6), with the peak during dawn associated with a 4.08-fold (conf. int. 3.53–4.7) increase. The effect of SOD concentration in the brain was significant only in pigmented catfish (P < 0.01), in which a one unit increase in SOD (i.e., 10^–2 nmol NBT/min/mg protein) was related to a 7% decrease in agonistic interactions. The effect of glucose was significant, but opposite effects were observed in pigmented (P < 0.001) and albino (P < 0.001) catfish. An increase in glucose by one unit (i.e., 10^–1 mmol/L) was associated with a 14% decrease in agonistic interactions in albino catfish, while in pigmented catfish, agonistic interactions increased by 342%. The effects of blood cortisol, lactate, and catalase in the brain were significant (P < 0.001) but not influenced by catfish pigmentation. Thus, an increase in cortisol by one unit (i.e., 10^–1 ng/ml) caused an increase in agonistic interactions of 177%, while an increase in lactate or catalase in the brain by one unit (i.e., 10^–1 ng/ml; resp. 10^–2 μmol H₂O₂/min/mg protein) was related to a 16% (LAC) or 39% (CAT) decrease in agonistic interactions in both catfish groups.

Albinism is associated with higher light sensitivity and photoreceptor degradation (Prusky et al., 2002; Vihtelic et al., 2006) with behavioral repercussions (Ren et al., 2002). For example, increased sensitivity to sunlight was reported for albino Caribbean Sea urchins, Tripneustes ventricosus (Lamarck, 1816), which tend to cover themselves with plants more than pigmented individuals (Kehas et al., 2005). In albino rodents, avoidance of light and lower locomotor activity during daylight are generally known behavioral characteristics (Fuller, 1967; Defries, 1969; Stryjek et al., 2013), but information about the physiological processes underlying photophobia is rare, e.g., in the albino cave tetra Astyanax mexicanus (De Filippi, 1853), higher sensitivity to stress is associated with mutation of the oca2 gene and catecholamine synthesis (Kvetnansky et al., 2009; Bilandžija et al., 2013). In our study, we observed differences in behavior between albino and pigmented European catfish, S. glanis, during the L/D cycle influenced by the complexity of the environment. These differences were accompanied by changes in physiological parameters in blood plasma and the brain, gill, and liver. In nature, the locomotor activity of pigmented S. glanis follows the L/D cycle, being mostly nocturnal; however, it is highly variable across seasons (Slavík et al., 2007). Moreover, the activity of S. glanis is highly individualistic; some individuals show nocturnal activity, some show daylight activity, and some do not prefer any phase of the L/D cycle (Slavík and Horký, 2012), as commonly reported for other fish species (Madrid et al., 2001; Vera et al., 2009; Sánchez-Vázquez et al., 2019). Under experimental conditions, the locomotor activity and food intake of S. glanis during the L/D cycle are affected by the social environment; for example, individually bred fish are arrhythmic, while grouped fish are rhythmic (Boujard, 1995).

Our results suggested that melanin absence is associated with the typical behavioral performance of the albino phenotype during the L/D cycle and showed more stress in the albino phenotype under light conditions than in pigmented conspecifics. Rare albino individuals observed in nature display different behavioral and physiological responses under experimental laboratory conditions compared to those of pigmented individuals, which show a lower stress response and behavioral patterns approaching those observed during ecological studies in natural conditions.

The datasets generated for this study can be found in Dryad repository (Valchářová et al., 2022).

The animal study was reviewed and approved by the Ministry of Agriculture of the Czech Republic (63479/2016-MZE-17214).

OS and PH conceptualized the research. PH, JV, MM, and TV managed the data curation. JV and AS conducted the biochemical analyses. PH, IH, MM, and MP conducted the statistical analyses. OS, JV, AS, PH, and TV conducted the investigation and sampling in the laboratory. OS, PH, JV, and AS developed the methodological approach. TV, OS, and PH wrote the manuscript with input from all authors.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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