Fruit flies exploit behavioral fever as a defense strategy against parasitic insects.

Behavioral fever, a thermoregulatory response in which ectothermic animals seek warmer environments to elevate body temperature and combat parasite infections, is well documented against microparasites. However, its role and mechanisms against macroparasites remain largely unknown. Here, we show that Drosophila hosts use behavioral fever to defend against Leptopilina parasitoid wasps. This thermal preference increases wasp mortality and enhances host survival. We find that behavioral fever is mediated by up-regulation of Heat shock protein 70 (Hsp70) genes in infected hosts as Hsp70 loss abolishes behavioral fever, whereas its overexpression induces heat-seeking behavior. We further find that behavioral fever up-regulates immune genes in infected hosts, including 12 antimicrobial peptide (AMP) genes, which disrupt the gut microbiota homeostasis of parasitoid wasps and, in turn, lead to substantial wasp mortality. Our findings elucidate the detailed mechanisms of behavioral fever in Drosophila hosts, advancing our understanding of ectothermic animal defenses against macroparasites.