Effects of nutritional deprivation on development and behavior in the subsocial bee Ceratina calcarata (Hymenoptera: Xylocopinae)

By manipulating resources or dispersal opportunities, mothers can force offspring to remain at the nest to help raise siblings, creating a division of labor. In the subsocial bee Ceratina calcarata, mothers manipulate the quantity and quality of pollen provided to the first female offspring, producing a dwarf eldest daughter that is physically smaller and behaviorally subordinate. This daughter forages for her siblings and forgoes her own reproduction. To understand how the mother’s manipulation of pollen affects the physiology and behavior of her offspring, we manipulated the amount of pollen provided to offspring and measured the effects of pollen quantity on offspring development, adult body size and behavior. We found that by experimentally manipulating pollen quantities we could recreate the dwarf eldest daughter phenotype, demonstrating how nutrient deficiency alone can lead to the development of a worker-like daughter. Specifically, by reducing the pollen and nutrition to offspring, we significantly reduced adult body size and lipid stores, creating significantly less aggressive, subordinate individuals. Worker behavior in an otherwise solitary bee begins to explain how maternal manipulation of resources could lead to the development of social organization and reproductive hierarchies, a major step in the transition to highly social behaviors.

Lipid quantification To quantify body lipids from lab-reared adult bees raised on manipulated pollen quantities, we followed methods previously published for honey bees (Toth and Robinson, 2005) and solitary bees (Richards and Packer, 1994; O’Neill et al., 2015). Briefly, bees previously frozen at −80°C (see ‘Behavioral assays’, below) were placed in a homogenizer tube and ground with a glass rod. Extraction was carried out by addition of 5 ml of 2:1 chloroform:methanol solvent overnight. To purify the sample, we poured the solvent and sample through glass wool and rinsed with 2 ml of 2:1 chloroform: methanol. The sample was quantified using a Spectramax 250 spectrophotometer and compared with a standard curve of 0, 10, 50, 100 and 500 µl of cholesterol in petroleum ether.

To quantify the behavioral effects of nutritional addition or reduction, we used circle tube assays to observe behavioral interactions between age and size-matched treatment and control individuals. Pairs were observed for 20 min and all interactions were documented. Interactions, when bees were within one body length of one another, were classified into four categories previously published by Rehan and Richards (2013): aggression, avoidance, tolerance and following. Briefly, aggressive behaviors include nudging, biting or C-posturing, when the bee curls its abdomen under the thorax, displaying both mandibles and sting. Avoidance behaviors include backing away from or reversing 18 deg to move away from the other bee. Passing, and antenna-toantenna or head-to-head contact were considered tolerant behaviors. Following has been classified as a cooperative behavior in some communal species (McConnell-Garner and Kukuk, 1997; Boesi and Polidori, 2011), a subordinate behavior in some eusocial species (Breed et al., 1978; Michener, 1990) and as a dominant behavior in other species (West-Eberhard, 1979). Because of the uncertainty of the meaning of this interaction, following behaviors were classified separately. After behavioral trials, all bees were killed at −80°C for later lipid quantification. To account for differences in paired circle tube assays, we compared the relative differences in behavioral frequencies. All differences between pair members were calculated as (value for treatment bee, pollen added or removed)−(value for control bee).