Should I dive or should I float? Behavioural plasticity of Aedes mariae pupae under predation threat

Background: The pupal stage in holometabolous insects is a critical transition between larval and adult forms, during which feeding ceases and survival depends on stored energy reserves. Mosquito pupae exhibit active diving behaviour in response to threats, which is energetically costly due to their positive buoyancy. Whether pupae are able to adjust diving behaviour according to environmental conditions, balancing predator avoidance and energy expenditure, remains poorly understood. Here, we investigated how water salinity affects the diving behaviour of Aedes mariae pupae, a species inhabiting Mediterranean rock pools characterised by highly variable salinity conditions. Methods: Pupae were maintained and tested in two salinity conditions: low (50%) and high (150%). Diving behaviour was recorded following an automated mechanical stimulus, and we measured: (i) time spent underwater, (ii) pupal activity (i.e. the number of abdominal movements during the immersion and the ratio of movements to time spent underwater) and (iii) the proportion of time spent by a pupa at different depths along the height of the water column (space use). Results: We found that pupae in high-salinity conditions spent 20.6% less time underwater than those in low salinity. They also performed fewer abdominal movements during dives but showed no significant differences in movements per unit time. Analysis of space use showed that pupae in high salinity spent more time in the upper part of the water column and less time in the middle and lower parts. Conclusions: Ae. mariae pupae modify their diving behaviour in response to different salinity conditions, adopting energy-efficient responses to external stimuli that promote survival in variable habitats. These findings highlight the importance of pupal behavioural flexibility for overall fitness and underscore the need to investigate pupal behavioural plasticity, which remains largely unexplored.