Heat‐Stress Alters the Effects of Antibiotics on Fish Behavior

ABSTRACT Antibiotics are a common contaminant of freshwaters, yet surprisingly understudied compared to other classes of drugs. Even trace concentrations of antibiotics can alter the gut microbiome, in turn affecting multiple systems including physiology, biochemistry, and behavior. Concurrently, climate change is leading to higher average temperatures and more frequent temperature spikes. The microbiome of poikilotherms is also highly sensitive to temperature. Little is known of how these two stressors interact. In this study we investigated the effects of amoxicillin, an antibiotic that is widely used in human and veterinary medicine, on the swimming behavior and predatory avoidance responses of zebrafish at two temperatures, 28°C (normal) and 32°C (heat‐stressed). Additionally, we investigated the effects of the two stressors on the gut microbiome and heat shock protein expression. Both temperature and amoxicillin affected key behaviors including boldness, sociability, and swimming speed, with fish exposed to low‐dose amoxicillin having higher swimming velocity suggesting a possible hormesis response. Amoxicillin exposure also increased fish boldness, while temperature had the opposite effect. A high temperature also reduced fish sociability. Both stressors also altered zebrafish responses to a conspecific alarm cue and induced heat shock protein gene expression. Heat stress and amoxicillin both altered the fish gut microbiome at the genus level with heat stress alone increasing several genera (e.g., Nordella , Nocardia , Reyranella ), while heat stress plus high amoxicillin (20 μg/L) further increased others (e.g., Bradyrhizobium , Legionella , Xanthobacter ). Overall alpha diversity (Shannon) changed little, but community composition (ANOSIM) shifted most clearly under high amoxicillin and under heat combined with amoxicillin, indicating dose‐ and temperature‐dependent restructuring of the gut microbiome. Overall, the microbiome is emerging as an important regulator of physiology and behavior that is vulnerable to multiple stressors.