Most people don't really pay attention to the air they breathe. However, if you go up a mountain, where the oxygen levels are lower, you'll notice as you huff and puff while taking a normal walk around town. Changes in the amounts of the different gases that make up the air we breathe can have noticeable effects in other animals as well, especially insects. ‘Low concentrations of carbon dioxide (CO2) are often used as an attractant or repellent’, says Anna Cressman of Pennsylvania State University, USA; however, high concentrations can cause behavioural and physiological changes, and can even result in unconsciousness. Frequently, researchers use 100% CO2 to anaesthetize insects (called CO2 narcosis), but the side-effects of exposure to high levels of CO2 are often not considered. Working with Etya Amsalem, also of Pennsylvania State University, Cressman asked what is causing these side-effects in bumblebees (Bombus impatiens) – the lack of oxygen, or the high level of CO2 itself.Carbon dioxide is usually found at levels of around 0.04% in normal air, so the duo pumped either 100% CO2, 50% CO2, normal air with low oxygen, or 100% nitrogen gas (which means that there is no oxygen) into a chamber containing bees for 1 min, recording how many of the bees passed out, and then filmed the insects to monitor how they behaved once they awoke, as well as checking how the different gases affected bees with different roles, queens and workers, in the colony.Sure enough, when the team compared the effects of pure CO2 and 100% nitrogen, the bees’ responses were quite different. Even though both gases knocked the bees out, after recovering from the sedation, the bees that had inhaled 100% CO2 had different side-effects from the bees anaesthetised with pure nitrogen. The CO2-anaesthetised bees pumped their abdomens more and the queens took longer to regain consciousness. In addition, when Cressman compared the effects of the different gases on worker and queen bees, the workers produced smaller eggs after several doses of 100% CO2, whereas the queens produced larger eggs after a single dose of the pure gas. Cressman also compared the impact of the two gases on the expression of six specific genes – involved in egg production and protection from low oxygen levels – and she discovered that the genes reacted differently.The responses of the queen bees and workers to pure CO2 also differed: the queens seemed to be more vulnerable to the gas than the workers, only requiring one dose, to begin showing side-effects during their recovery, in contrast to the workers, which required multiple doses of the gas before the side-effects became apparent. ‘The fact that the physiological changes are different suggests that CO2 is not simply depriving the bees of oxygen but does something else and is likely to have a more direct impact on cells’, Amsalem concludes.So, inhaling CO2 can affect bees with different roles in the colony in different ways, and researchers should be aware of the potential complications that this may cause when using the gas to anaesthetize insects in their experiments.