Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

Summary

This manuscript describes several protocols for administering pharmacological agents to honey bees, including simple noninvasive methods for free-flying bees, as well as more invasive variants that allow precise localized treatment of restrained bees.

Abstract

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize and to study. Therefore, bees have long been a favored model amongst neurobiologists and neuroethologists for studying the neural basis of social and natural behavior. It is important, however, that the experimental techniques used to study bees do not interfere with the behaviors being studied. Because of this, it has been necessary to develop a range of techniques for pharmacological manipulation of honey bees. In this paper we demonstrate methods for treating restrained or free-flying honey bees with a wide range of pharmacological agents. These include both noninvasive methods such as oral and topical treatments, as well as more invasive methods that allow for precise drug delivery in either systemic or localized fashion. Finally, we discuss the advantages and disadvantages of each method and describe common hurdles and how to best overcome them. We conclude with a discussion on the importance of adapting the experimental method to the biological questions rather than the other way around.

Introduction

Since Karl von Frisch elucidated their dance language¹, honey bees have remained a popular study species for researchers in animal behavior and neurobiology. In recent years a myriad of new disciplines have emerged at the intersection of these two fields, and several other disciplines (e.g., molecular biology, genomics, and computer science) have arisen alongside them. This has led to rapid development of new theories and models for understanding how behavior results from activity within nervous systems. Because of the unique lifestyle, rich behavioral repertoire, and ease of experimental and pharmacological manipulation, bees have remained at the ....

Protocol

1. Drug Administration for Harnessed Bees

1. Oral treatment
   1. Prepare 1.5 M sucrose solution by mixing 257 g of sucrose with 500 ml of water (it is easier to dissolve this amount of sucrose in boiling water). Store sucrose solution at 4 °C until use.
      NOTE: Sucrose solution provides a very hospitable environment for certain microorganisms, and thus easily becomes contaminated and unpalatable to bees. Bulk sucrose solution can be aliquoted and stored at -20 °C until use.
   2. Decide on an appropriate drug dose (how to achieve it, is addressed in the discussion section below), and prepare a solution such that th....

Results

A selection of representative results for the methods described above are shown, primarily to demonstrate that the methods allow pharmacological agents to reach the brain and affect honey bee behavior.

Specific effects on brain processes can be easily obtained following thorax injection.

Because pharmacological agents injected through the thorax may act on multiple target.......

Discussion

The methods outlined above allow simple, effective and robust treatment of either free-flying or harnessed honey bees. These methods are compatible with many experimental paradigms and biological questions (Table 1). All of the free-flying methods can easily be applied to harnessed bees. The reverse is less successful, however, since temporary restraint and invasive treatment methods can often compromise bees' flying ability.

The methods have been presented from a brain-ce.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sucrose	Sigma-Aldrich	S8501	Any supplier will do
Sodium Chloride	Sigma-Aldrich	S7653
Potassium Chloride	Sigma-Aldrich	P9333
Magnesium Chloride hexahydrate	Sigma-Aldrich	M2670
Calcium Chloride dihydrate	Sigma-Aldrich	C8106
Dextrose monohydrate	Sigma-Aldrich	49159
Phosphate Buffer Saline (PBS)	Sigma-Aldrich	P4417
Protection Wax	Dentaurum	124-305-00
HEPES	Sigma-Aldrich	H3375
dimethylformamide	Sigma-Aldrich	D4551
95% Ethanol	Sigma-Aldrich	493511
Glass capillary	WPI	1B100F-3
23 G NanoFil needle	WPI	NF33BV-2
Very fine forsceps	Dumont	0208-55-PO
Electrode puller	SRI	2001
FemtoJet Microinjector	Eppendorf	5247 000.01
Eicosane	Sigma-Aldrich	219274
manual micromanipulator	Brinkmann Instrumentenbau	MM-33
electronic micromanipulator	Luigs & Neumann Feinmechanik + Elektortechnik	Junior unit XYZ
stereomicroscope	Leica	M80
soldering iron	Weller	WESD51
Dextran, Alexa Fluor 546, 10,000 MW	ThermoFisher Scientific	D-22911
Dextran, Alexa Fluor 568, 10,000 MW	ThermoFisher Scientific	D-22912
small Petri dish	Sigma-Aldrich	P5481
mineral oil	Sigma-Aldrich	M5904
50 ml Centrifuge tube	ThermoFisher Scientific	339652
forceps	Australian Entomological Supplies
Blade holder and breaker	Australian Entomological Supplies	E130
Feather double edged razor blade	ThermoFisher Scientific	50-949-135
Nichrome wire			Any supplier will do
Electrical wires			Any supplier will do
Model paint	Tamiya USA	Depends on colour
Repeating dispenser	Hamilton company	PB-600-1
Glass syringe	WPI	NANOFIL
flourescence viewing system	Nightsea	SFR-GR
graticule	ProSciTech	S8014-24
microcapillary with holder	Drummond	1-000-0010
Liquid silicone			Any supplier will do
Thermocouple	Digitech	QM-1324
Micropipette	Eppendorf