Single-cell dissection of aggression in honeybee colonies

RNA-Sequencing performed on 177 honey bee whole-brains, divided into "soldier" and "forager" groups from Puerto Rican honey bee colonies. Overall design: Sample phenotyping and collection: Thirteen colonies from eight locations were sampled across Puerto Rico. All colonies except the one residing there were transported to the University of Puerto Rico's Gurabo Agricultural Field Station. Colonies were set up so that no one colony was less than 1 meter apart from another. Following transportation, colonies were allowed a period of at least two weeks to acclimate to the new location before undergoing our phenotyping assay and sample collection. Phenotype was measured using two assays applied to each colony individually. In the first assay we closed the colony entrance and collected returning foraging bees (naïve foragers, NFr group), flash freezing them with liquid nitrogen as they were aspirated from the colony entrance using a modified collection vacuum The entrance was then re-opened, and immediately after, a string of black suede patches was placed 21 cm from the entrance. Once in place we disrupted the colony by rhythmically striking the top cover. This elicited a defensive response from bees which streamed out of the colony to sting the black suede patches. For two minutes from the beginning of the disruption, we aspirated and flash-froze emerging bees in the act of stinging (aggressors, Agg group). At the end of the two minutes we rapidly collected the suede patches, placing them in a sealable plastic bag and removing them from the area. A researcher then stepped ~3 meters from the colony, and rapidly collected and flash froze bees that followed and remained hovering (pursuers, Pur group). Quickly after we restricted the entrance to the colony and used talcum powder to dust bees that continued to emerge for 30 seconds more. The colony was then left alone for 30 minutes, after which the entrance was again sealed, and we collected those bees returning to the colony that had obvious signs of foraging (pollen loads) and retained the talcum powder (returning foragers, For group). This last group were bees present during disturbance, i.e. perceived the disrupting stimulus, but did not respond with a defensive action. The method simultaneously provided the individual level phenotype and one of the two components of the colony phenotype. Colony phenotype from this assay was measured as the number of stings counted across the set of black suede patches. Only two colonies were sampled each day using this protocol, observing that the two colonies sampled were no closer than 3 meters from each other. Our scheduling also assured that colonies adjacent to the those phenotyped were themselves undisturbed for at least 48 hours when possible. Upon completion of collection, samples were transported in liquid nitrogen to the University of Puerto Rico, Rio Piedras, and transferred to a -80 °C freezer. The second phenotyping assay took place two weeks after the last colony was phenotyped by our first method. The process examined colony aggression using a behavioral ranking assay which assigns intensity to four discrete behaviors for each colony then sums the total. This approach is a variation of a standard practice in assessing colony-level defensive behavior. A Kendall's tau non-parametric correlation test between the scores showed a significant positive correlation between the metrics (Kendall's tau, n = 13, z = 0.38, p-value = 0.044. Our final phenotype score was derived by combining these ranks using a multi-dimensional scaling (MDS) analysis. The MDS provided two dimensions, one highly correlated with colony defense in both scores (Dimension 1 vs. Mean of Rank Scores, Kendall's Tau, n = 13, z = 4.76, p-value < 0.001; Dimension 2 vs. Mean of Rank Scores, Kendall's Tau, n = 13, z = -0.24, p-value = 0.807), and the second dimension correlated with consistency between the scores (Dimension 1 vs. Mean of Rank Scores, Kendall's Tau, n = 13, z = 0.18, p-value = 0.855; Dimension 2 vs. Mean of Rank Scores, Kendall's Tau, n = 13, z = -4.71, p-value < 0.001). Immediately after phenotyping a colony in this way, we collected and flash-froze 12 queens from all 13 colonies. Two of these queens came from one colony that was poised to split (colony number 6). At the end of the study, samples were individually assessed and sorted into microcentrifuge tubes while on dry ice in preparation for shipping. Samples were later transported to the facilities at the Carl R. Woese Institute for Genomic Biology inside a dry-shipper (CXR500 Cryogenic Shipper, Taylor-Wharton America, Baytown, TX). Sample selection for sequencing: Of the thirteen sampled colonies, we excluded three from consideration for sequencing. For two of these, colonies number 5 and 13, we were unable to secure the queen, and thus had no representative parental genotype. The remaining colony number 8, showed signs of swarming at the point of queen collection, likely to have occurred at some point after the first phenotyping effort. Due to uncertainty in sample and indeed colony phenotype, we decided not to consider this colony. Colony 6 was not excluded, as both queens were collected, and the mother-queen was identified through ovary dissections. For the remaining ten colonies we retained over one hundred individuals for each of the four behavioral groups. A total of twenty Agg and For individuals were selected for sequencing along with their corresponding queens. Honey bees from both these groups would have experienced the colony disturbance but responded in markedly different ways.