Environmental and Genetic Conditions Shaping Monarch Butterfly Migration Biology.

Monarch butterflies in North America migrate south each autumn, but the mechanisms initiating their migratory flight remain unclear. We investigated environmental, developmental, and genetic factors shaping directional flight using three flight simulators: two at ground level (with and without wind blockage) and a novel balloon-based system that lifted butterflies 30 meters into the air. We tested summer and autumn-generation monarchs, as well as summer monarchs reared under autumn-like conditions to assess developmental influences. Autumn-generation monarchs exhibited significant southwestern flight orientation, observed only in the balloon simulator, highlighting the importance of high-altitude flight. Summer monarchs reared under autumn-like conditions displayed southward orientation, larger wings, and partial reproductive diapause, indicating that seasonal cues alone can induce migratory traits. In contrast, a lab-reared monarch lineage maintained in captivity since 2016 showed reduced wing size and impaired orientation, even when raised outdoors in autumn, suggesting a loss of migratory traits without migration. Unexpectedly, many butterflies in the balloon simulator oriented upwind, suggesting wind may serve as a directional cue. These findings reveal key environmental and genetic influences on monarch migration and establish the balloon flight simulator as a valuable tool for studying migratory behaviour, with implications for conservation in a changing climate.