Spiroplasma and heat hardening can buffer insect male fertility loss at high temperatures

Insects' upper thermal limits for survival, activity, and fertility have been used to assess vulnerability to climate change, yet heritable endosymbionts - present in over 70% of insect species - are often overlooked. While emerging research suggests some endosymbionts can increase thermal tolerance, their effects on upper lethal and fertility thermal limits has rarely been investigated. Additionally, short-term exposure to sub-lethal high temperatures (heat hardening) can increase insect heat tolerance, but its impact on male fertility is unclear and potential interactions with endosymbionts has not been explored. Here, we investigate whether the endosymbiont Spiroplasma poulsonii and heat hardening influence upper survival and fertility thermal limits in a native widespread host of Spiroplasma (Drosophila hydei) and a novel host (Drosophila birchii), a rainforest-restricted species with low heat tolerance. Heat hardening generally improved survival and fertility following a heat shock. The presence of Spiroplasma increased survival and fertility of D. hydei males following heat-shock, while in D. birchii, it did not enhance survival but protected male fertility after heat-shock and modulated hardening responses at sub-lethal temperatures. While protective effects varied across species, sex, and trait, both Spiroplasma and hardening significantly buffered male fitness loss during heat shock in both host species and halved heat exposure risk under current and predicted climate change in the rainforest restricted D. birchii. These findings highlight that, beyond generating novel phenotypic variation, endosymbionts can interact with plastic responses to heat stress, emphasising the importance of accounting for endosymbiont-mediated effects on thermal tolerance when assessing insect vulnerability to climate change and exploring strategies to manipulate insect thermal tolerance.