Unintentionally evolved fruit flies show how insects survive CO₂ knockout

It's a cute resurrection party trick: give a fruit fly (Drosophila melanogaster) a puff of CO₂ and the insect topples over apparently dead, only to revive minutes later as it emerges from a coma triggered by the anaesthetic. So, when evolutionary biologist Adam Chippindale, from Queen's University, Canada, heard that Chengfeng Xiao and Meldrum Robertson from down the corridor were curious about the physiological mechanisms underpinning the insects’ powers of recovery from anoxia (oxygen deprivation), he realised that he already had populations of the animals ideally suited to investigate the phenomenon.

‘Back in the 1980s, my PhD supervisor, Michael Rose, began a famous experiment’, says Chippindale. Curious about the effects of aging, Rose, at the University of California, Irvine, USA, began mixing aged insects together in the hope of evolving populations of fruit fly super-agers. However, he also kept other populations of flies descended from the same ancestors in small vials – which had to be mixed together manually every 2 weeks to breed – to ensure that the changes that occurred in the elderly populations were truly down to evolved longevity. To sort male and female vial-housed flies from each other before setting up their blind dates, Rose knocked out the insects with CO₂. Chippindale realised that these other ‘control’ populations had inadvertently been performing an evolutionary experiment of their own for the last four decades. They had been naturally selected to withstand the effects of CO₂ and oxygen deprivation, which was exactly what Xiao and Robertson were curious about. Chippindale also had alternative populations of flies that had simply been allowed to live naturally with no interference, for comparison with the CO₂-anaesthetised populations. ‘We decided it would be fun to see if the different kinds of populations had evolved to have different responses to anoxia’, says Chippindale.

Faced with the immense task of screening almost 2000 flies – some from the populations that had been knocked out for almost four decades and others from the populations that had never had a whiff of CO₂ – Xiao designed and built an arena that would allow him, Kaylen Brzezinski and Niki Bayat Fard to keep track of up to 128 flies simultaneously. Ten seconds after delivering a puff of CO₂ to the fruit flies, Xiao and Brzezinski saw the animals have a mini seizure before collapsing in a coma. However, the fruit flies from the populations that had been treated with CO₂ for decades recovered much faster; they began waking within ∼4 min, whereas the insects that had never been anaesthetised showed no sign of recovery for 7–8 min. Investigating further, the team noticed that females from the CO₂-treated populations were back on their feet 30% faster than males from the same populations. And when Bayat checked how the flies fared when knocked out with an alternative anaesthetic – nitrogen – the evolved populations recovered even faster, beginning to come to about 3 min after inhaling the gas.

Rose and Chippindale's accidental evolutionary experiment has conveniently provided Xiao and Robertson with an ideal ‘animal of choice’ in which to investigate how insects and other animals withstand the effects of plummeting oxygen levels. And Xiao is optimistic that he has already identified one gene that could help. ‘We have reported that the classic eye colour gene white in Drosophila speeds up the recovery from nitrogen anoxia’, Xiao explains and he is keen to discover whether the same gene contributes to the CO₂ resilience of Chippindale and Rose's unintentionally evolved fruit flies.