To become an adult fruit fly, a Drosophila larva will have to undergo complete metamorphosis. However, before they are able to begin this process they first need to reach a critical mass during their final larval instar. Once attained, a well-studied hormonal cascade is initiated, culminating in a peak of the hormone ecdysone just prior to the transition to the pupal stage where they undergo metamorphosis. Although the hormones' roles are well characterised, Viviane Callier wondered: ‘What are the physiological cues that an insect uses to initiate metamorphosis? How do they physiologically know that they are big enough to become adults?’ Callier, a post-doc in Jon Harrison's lab at Arizona State University, USA, suspected oxygen might be involved, as she explains: ‘Larvae do not have lungs; instead they breathe through tubes called tracheae. During the growth phase, oxygen demand is going to increase but the supply structures are largely fixed. So, the hypothesis was that as larvae are growing they start to sense that they're running out of oxygen, and that might be the physiological cue that tells them: okay, I'm going to be running out of oxygen soon, I need to moult and make a bigger tracheal system to continue growing.’ So, Callier decided to investigate with help of other lab members and collaborators (p. 4334).
To test the theory, the team reared larvae just up until their transition to the final larval instar under normal oxygen conditions. The larvae were then divided into three groups: one group remained in a chamber with normal oxygen conditions (about 21% oxygen), another group was put in hypoxic conditions (10% oxygen) and the third group was housed in a hyperoxic environment (30% oxygen). The team then measured the critical weight for each group as well as growth rates and time taken to form the pupa after reaching critical weight. ‘The idea was, again, very simple; if larvae are running out of air as they grow through their instar, then if you give them less oxygen they should run out of air at a smaller size and therefore moult [into a pupa] when they're smaller. If you give them more oxygen, that should relieve a constraint on the oxygen supply and they should therefore moult larger and later’, explains Callier.
As expected, the team did indeed find that the critical weight had decreased in hypoxic larvae: ‘They initiate metamorphosis at a smaller size, so they do pupate smaller’, says Callier. ‘But they actually take much longer to pupate, so their third instar is actually extended.’ What's more, the team found that hyperoxic larvae didn't grow bigger as anticipated, and in fact, they showed slower growth rates at the beginning of the instar. When the team measured ecdysone levels they saw basal levels were raised in both hypoxic and hyperoxic larvae, which they suspect might be responsible for slowing growth rates down.
On the whole, Callier concludes, ‘We found that oxygen is important for determining the timing and the size at which metamorphosis is initiated, but it's not a single cue and there's probably no single physiological cue for body size regulation. There are multiple inputs, multiple cues and those are going to differ from one species to another.’
