Search Behavior of Individual Foragers Involves Neurotransmitter Systems Characteristic for Social Scouting

Abstract

In honey bees search behavior occurs as social and solitary behavior. In the context of foraging, searching for food sources is performed by behavioral specialized foragers, the scouts. When the scouts have found a new food source, they recruit other foragers (recruits). These recruits never search for a new food source on their own. However, when the food source is experimentally removed, they start searching for that food source. Our study provides a detailed description of this solitary search behavior and the variation of this behavior among individual foragers. Furthermore, mass spectrometric measurement showed that the initiation and performance of this solitary search behavior is associated with changes in glutamate, GABA, histamine, aspartate, and the catecholaminergic system in the optic lobes and central brain area. These findings strikingly correspond with the results of an earlier study that showed that scouts and recruits differ in the expression of glutamate and GABA receptors. Together, the results of both studies provide first clear support for the hypothesis that behavioral specialization in honey bees is based on adjusting modulatory systems involved in solitary behavior to increase the probability or frequency of that behavior.

Keywords: GABA; glutamate; histamine; honey bee; mass spectrometry—LC-MS/MS; octopamine.

Figure 1

Removal of the feeder led foragers to perform search flights. (A) Experimental design to study the dynamics and persistence of search behavior and after foragers were confronted with the absence of a known feeder. Individually marked foragers are allowed to visit the feeder at 300 m distance from the hive for an initial 1.5 h. The feeder is removed, and the outbound flight activity of the marked foragers is monitored at the hive entrance for the following 2 h. (B,C) Search flights following feeder removal for two colonies. (D,E) Increase in hive-to-hive duration and (F,G) duration of hive stays before and after feeder removal. (H) Hierarchical clustering of foragers based on search behavior sequence. The maximum average silhouette width 0.58 gave a five-cluster solution with agglomerative coefficient 0.93. (I,J) Search behavior sequences for foragers along with cluster information for two colonies.

Figure 2

Search behavior is correlated with reduced GABA and glutamate titers in the central brain. (A) Experimental design to collect foragers for mass spectrometric analysis of brain neurotransmitter titers. Individually marked foragers are allowed to visit the feeder at 300 m distance from the hive for an initial 1.5 h (foraging phase). The feeder is removed for 1 h (search phase) and reinstalled for 1 h (revisit phase). Foragers were captured at hive entrance during each behavior phase (marked red on experiment timeline). (B,C) Glutamate and GABA levels decrease in the central brain (CB) after bees experience a loss of their expected feeder. (D,E) A detailed look at the dynamics of change indicates that glutamate levels gradually but linearly decrease over increasing search trips (decrease by 112 ng per search flight, p-value = 0.001), but GABA levels only decrease post the first experience and stay that way. (F) Histamine (G) Aspartate (decrease by 87 ng per search flight, p-value = 0.031). The neurotransmitter values are scaled by the MS batch. *p < 0.05; ***p < 0.001.

Figure 3

Reinitiation of foraging increases levels of Glutamate and GABA in the optic lobes. (A,B) Glutamate and GABA levels increase after bees start revisiting the feeder. There are no changes due to the experience of feeder loss. (C,D) A detailed look at the dynamics show that the number of search trips do not affect the modulator levels, but only the experience of the feeder does. (E–O) Replacement of the feeder causes abrupt and global changes in multiple modulators in the OL. The neurotransmitter values are scaled by the MS batch. *p <0.05; **p <0.01; ***p <0.001.

Figure 4

Phenotypes of the collected foragers. (A) Hierarchical clustering of foragers collected during search phase based on search behavior sequences. The maximum average silhouette width 0.52 gave a three-cluster solution with agglomerative coefficient 0.94. (B) Search behavior sequences for the foragers from the collection experiments along with cluster information. Only bees used for cluster analysis of neurotransmitter titers are shown.

Figure 5

Search intensity negatively correlates with OA and positively correlates with DOPA and HA titers in the CB. (A,B) DOPA and Histamine levels show an increase with increased search trips. (C) Octopamine levels decrease with increased search trips. (D,E) Foragers most motivated to continue search flights showed decreased levels of aspartate in the optic lobes. In addition, bees that performed the FS trip but are phenotypically different in their hive stay times show differences in aspartate levels, though non-significant. Bees without detailed behavior data were added to the phenotype analysis by comparing available relevant behaviors. *p <0.05; ***p <0.001.

Figure 6

Neuromodulators vary significantly between different colonies. PCA of modulator titers in the CB (A) and OL (B) show >50% variance explained by colony membership. (C–O) Individual modulators vary significantly between the different colonies. In general, the 2017 colony shows a lower amount of most transmitters than the 2018 and 2019 colonies. Only the differences between the 2017 and the other 2 years are shown. *p <0.05; **p <0.01; ***p <0.001.