DOI: 10.6084/m9.figshare.5596621
title: Effects of nutritional deprivation on development and behavior in the subsocial bee Ceratina calcarata (Hymenoptera: Xylocopinae)

description: <b>Abstract</b>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 <i>Ceratina calcarata</i>, 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.<br><br><b>Pollen manipulation- dataset pollen consumed and head width and development data</b>Early stage larvae and eggs were removed from nests, weighed using a Mettler analytical balance (accuracy 0.01 mg) and randomly assigned to a control group or one of two treatment groups. The pollen of the control group remained unmanipulated. For the treatment groups, approximately 1/3 of the pollen ball was removed from one group and added to the other group. Both treatment groups were reweighed and offspring and pollen ball were placed in PCR tubes in the incubator at 25°C with 50% humidity until reaching their final molt. Offspring from later developmental stages were raised in the incubator as additional controls. Every other day, the development of the offspring was assessed. The head width and sex were recorded for all brood that reached adulthood. Sex was determined by counting the number of metasomal terga; females have six segments, while males have seven (Rehan and Richards, 2010a). To quantify the exact amount of pollen consumed, we calculated the amount of pollen provided minus the amount of pollen remaining when offspring began pupation.<br><b>Lipid quantification- dataset lipid quanitifications</b>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.<br><br><b>Behavioral assays- dataset circle tube assays</b>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).<b><br></b>