Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.)

Summary

In this protocol we show how to condition harnessed honey bees to tactile stimuli and introduce a 2D motion capture technique for analyzing the kinematics of fine-scale antennal sampling pattern.

Abstract

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability^{1, 2}. The worker bees spend the first half of their life inside the dark hive, where they are nursing the larvae or building the regular hexagonal combs for food (e.g. pollen or nectar) and brood³. The antennae are extraordinary multisensory feelers and play a pivotal role in various tactile mediated tasks⁴, including hive building⁵ and pattern recognition⁶. Later in life, each single bee leaves the hive to forage for food. Then a bee has to learn to discriminate profitable food sources, memorize their location, and communicate it to its nest mates⁷. Bees use different floral signals like colors or odors^{7, 8}, but also tactile cues from the petal surface⁹ to form multisensory memories of the food source. Under laboratory conditions, bees can be trained in an appetitive learning paradigm to discriminate tactile object features, such as edges or grooves with their antennae^{10, 11, 12, 13}. This learning paradigm is closely related to the classical olfactory conditioning of the proboscis extension response (PER) in harnessed bees¹⁴. The advantage of the tactile learning paradigm in the laboratory is the possibility of combining behavioral experiments on learning with various physiological measurements, including the analysis of the antennal movement pattern.

Protocol

1. Preparing the Bees

1. Nectar or Pollen foragers are caught in the field either from a sucrose feeder or directly from the hive entrance while returning from a foraging trip. Each single bee is captured into a glass vial that is closed with a foam plug and taken immediately into the laboratory for further handling.
2. In the laboratory, the captured bees are briefly cooled in the refrigerator at 4 °C until they show first signs of immobility.
3. Each single immobilized bee is mounted in a small metal tube with adhesive tape between head and thorax and over the abdomen. Care should be taken that the proboscis and antennae are freely mov....

Results

In the following experiment we studied how tactile learning affects antennal sampling behavior in honey bees. For this, we monitored the movement of the antennal tip in naïve and conditioned bees before and during presentation of a tactile stimulus.

First, the spontaneous antennal movement of a group of pollen foragers (N = 42) was recorded for 1 min. One half of the bees (N = 21) was then conditioned by pairing five times a tactile stimulus with a 30% sucrose reward. This was the conditioned .......

Discussion

Preparation of bees: Collecting and fixing the bees should be done quickly, in order to keep the stress level of the animal low. Stress has an effect on the PER-responsiveness and, therefore, could have an indirect effect on the learning performance in bees^{19, 20}. The stress level can be decreased by placing the glass vials with the bees directly on ice immediately after collection to immobilize them quickly. It has to be taken into account that bees need more time to recover the longer the an.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Pollen foragers, Apis mellifera L.			Private hives
Glass vials (22 x 45 mm)	Carl Roth GmbH Co	X655.1
Brass tubes, 10 x 30 mm			Self-produced
Brass cube, 3 x 5 mm			Self-produced
Tipp-Ex (solvent-free)	Tipp-Ex GmbH Co KG
Sucrose solution (30%)			Household suger diluted in water
Ethanol (70%)	Carl Roth GmbH Co	9065.3
Syringe (1 ml) and needle (0.6 x 25)	Carl Roth GmbH Co	59.1
Stop Watch	Carl Roth GmbH Co	L423.1
Micromanipulator	Märzhäuser MM33	00-42-101-0000
Digital video camera	Basler A602f-2
Macro lens for camera	TechSpec VZM 200
Matlab R2009b	The MathWorks