Virgin queen behaviour and controlled mating in the stingless bee Tetragonula carbonaria (Meliponini)

Improving knowledge of virgin queen behaviour and mating biology in stingless bees (Meliponini) is an important step towards artificial selection programmes for managed colonies. Here we study the Australian stingless bee Tetragonula carbonaria to (1) describe the behaviour of queens from eclosion until egg-laying, and (2) assess whether young queens can be reared and mated under constrained conditions. We extracted mature virgin queen cells of T. carbonaria from large colonies and reared them in queen maturation boxes containing a limited number of workers (“micro-colonies”). Queen behaviour followed a typical pattern with three phases: a period of high activity and wing-flapping on top of the brood (5 ± 2 days of age), attempts to leave the colony for the nuptial flight (12 ± 3 days of age), and oviposition (20 ± 6 days of age). In total, 71% (68 of 95) of queen cells hatched in our maturation boxes and 57% (39 of 68) survived to mating age, with some observed to be actively killed by workers within the first week of life (8 of 68; 11%). For a subset of our queens, we performed controlled matings (either with or without CO₂ narcosis) by presenting constrained females to males in mating aggregations, allowing one male to mate (deposit mating plug) and then returning queens to their box. In the absence of CO₂ narcosis, 30% of queens mated this way were subsequently found to have sperm in their spermathecae, indicative of a successful mating (5 of 17). The remaining 70% however contained no sperm, despite the presence of the male mating plug. Amongst queens that were CO₂ narcotized before and during the controlled mating, none had sperm in their spermathecae (0 of 12), though most went on to activate their ovaries following the mating and lay male (haploid) eggs. Together, these trials indicate that controlled matings with T. carbonaria are possible, but suggest that queens may need to actively accept mating for successful sperm transfer.