Wolbachia has subtle effects on thermal preference in highly inbred Drosophila melanogaster which vary with life stage and environmental conditions.

Temperature fluctuations are challenging for ectotherms which are not able to regulate body temperature by physiological means and thus have to adjust their thermal environment via behavior. However, little is yet known about whether microbial symbionts influence thermal preference (T) in ectotherms by modulating their physiology. Several recent studies have demonstrated substantial effects of Wolbachia infections on host T in different Drosophila species. These data indicate that the direction and strength of thermal preference variation is strongly dependent on host and symbiont genotypes and highly variable among studies. By employing highly controlled experiments, we investigated the impact of several environmental factors including humidity, food quality, light exposure, and experimental setup that may influence T measurements in adult Drosophila melanogaster flies. Additionally, we assessed the effects of Wolbachia infection on T of Drosophila at different developmental stages, which has not been done before. We find only subtle effects of Wolbachia on host T which are strongly affected by experimental variation in adult, but not during juvenile life stages. Our in-depth analyses show that environmental variation has a substantial influence on T which demonstrates the necessity of careful experimental design and cautious interpretations of T measurements together with a thorough description of the methods and equipment used to conduct behavioral studies.