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 (Tp) in ectotherms by modulating their physiology. Several recent studies have demonstrated substantial effects of Wolbachia infections on host Tp in different Drosophila species. These data indicate that the direction and strength of thermal preference variation is strongly depend 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 Tp measurements in adult flies. Additionally, we assessed the effects of Wolbachia infection on Tp of Drosophila at different developmental stages, which has not been done before. We find no effects of Wolbachia on host Tp at any life stage. Instead, we show that environmental variation has a substantial influence on Tp measurements which demonstrates the necessity of careful experimental design and cautious interpretations of Tp measurements together with a thorough description of the methods and equipment used to conduct behavioral studies.

Temperature plays a fundamental role for the fitness of all organisms. In particular, it strongly affects metabolism and reproduction in ectotherms that have limited physiological capabilities to regulate their body temperature. Ectotherms thus have to maintain thermal homeostasis by behavioral adjustments. The influence of temperature variation on the physiology and behavior of ectotherms is well studied but we still know little about the influence of symbiotic interactions on thermal preference (Tp) of the host. The Wolbachia-Drosophila host-symbiont system represents an ideal model for addressing these questions. A growing number of studies demonstrated that different Wolbachia types can influence Tp in different Drosophila species, but these results may be confounded by the use of long-term Drosophila lab-strains that may not be representative for natural fly populations. To account for this, we investigated the effect of Wolbachia on Tp in wild-type D. melanogaster flies recently collected from nature. Consistent with previous data, we found reduced Tp compared to an uninfected control in one of two fly strains infected with the wMelCS Wolbachia type. Additionally, we, for the first time, found that Wolbachia titer variation influences thermal preference of the host fly. These data indicate that the interaction of Wolbachia and Drosophila resulting in behavioral variation is complex and strongly influenced by the genetic background of host and symbiont. Our results further emphasize the necessity for more in-depth studies to better understand the evolutionary significance of Tp variation influenced by Wolbachia in natural Drosophila populations.