Examining the individual and additive effects of cold storage and CO₂ narcosis on queen survival and reproduction in bumble bees

Diapause is a pre-programmed arrest of development allowing insects to survive in unfavorable environments. In adult insects, diapause termination is often followed by a reallocation of macronutrients and a transition to reproduction, and in some insects, this transition can be achieved using narcosis with CO₂. However, whether CO₂ narcosis and diapause act in concert to affect reproduction remains unknown. Here, we investigated the separated and combined effects of diapause and CO₂ on female reproduction in queens of the common eastern bumble bee Bombus impatiens. Queens were treated with CO₂ following a cold storage period (zero days, two weeks, two and four months) and were compared with untreated queens at the same timepoints for survival, colony initiation, egg-laying latency, and offspring production. We found that both CO₂ and a period of at least two months in cold storage induced a transition to egg laying in gynes, and as expected, survival decreased with cold storage length. When CO₂ and cold storage were combined, CO₂ narcosis positively affected egg laying in the earlier timepoints but its impact diminished following a longer cold storage. These data suggest that the impacts of CO₂ narcosis and cold storage are partially additive, and application of CO₂ is effective only after a short cold storage. It further demonstrates that CO₂ has complex effects on insect reproduction that are independent from diapause.