Examining the individual and additive effects of cold storage and CO 2 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 CO . However, whether CO narcosis and diapause act in concert to affect reproduction remains unknown. Here, we investigated the separated and combined effects of diapause and CO on female reproduction in queens of the common eastern bumble bee Bombus impatiens. Queens were treated with CO 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 CO 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 CO and cold storage were combined, CO 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 CO narcosis and cold storage are partially additive, and application of CO is effective only after a short cold storage. It further demonstrates that CO has complex effects on insect reproduction that are independent from diapause.