Supplementary material from "Seasonal plasticity in daily timing of flight activity in Anopheles stephensi is driven by temperature modulation of dawn entrainment" Version 2

The Asian malaria vector Anopheles stephensi is invading Africa, requiring adaptation to novel climates and ecosystems. In part, this may be facilitated by An. stephensi’s poorly understood seasonal behavioural plasticity in flight timing, leading to earlier biting activity in cold Asian winters and later biting times in the warm summer. Changes in behavioural timing could be directly imposed by seasonal variation in ambient light and temperature levels, or result from altered entrainment of intrinsically expressed circadian rhythms by these factors. We demonstrate that An. stephensi entrained flight activity timing is phase-locked to dawn and is not affected by constant ambient temperature, which cannot explain earlier biting activity in colder winters with later dawn. Instead, we show that where night temperatures are the colder part of daily temperature cycle, the entrained phase-angle between dawn and flight activity is altered, whereby increasingly colder, winter-like nights progressively advance flight activity onset. We propose that seasonal timing plasticity optimises behaviour to warmer daytime in winters, and colder nights in summer, providing protection against both heat-desiccation and cold-immobility. The adaptive advantage of this plasticity could be relevant to the successful invasion and survival of An. stephensi in African climates, and changing climate worldwide.