This project was funded by ARC grant DP0986021 and NHMRC grant 585442. ABB is supported by an ARC Future Fellowship (FT140100452). JAP is supported by an iMQRES scholarship awarded by Macquarie University and by a DAAD-Doktorandenstipendium awarded by the German Academic Exchange Service. JMD is supported by CNRS and University Paul Sabatier.

1. Drug Administration for Harnessed Bees
<ol>
	<li>Oral treatment
	<ol>
		<li>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 &#176;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 &#176;C until use.</li>
		<li>Decide on an appropriate drug dose (how to achieve it, is addressed in the discussion section below), and prepare a solution such that the preferred drug dose is dissolved in 20 &#181;L sucrose solution (e.g., to deliver 20 &#181;g, dilute drug at a ratio of 1 mg/ml). Harness bees according to Felsenberg et al. (2011)16. Do this step at least 12 hr before drug treatment to ensure that bees are no longer stressed out from harnessing when the drug solution is presented. 
		NOTE: For more consistent results, it is best to starve bees (by placing harnessed bees in an incubator at 34&#160;&#176;C and 70% humidity) O/N.</li>
		<li>Using a micropipette, touch a drop of 1.5 M sucrose water to the antenna of a harnessed bee. When the proboscis is extended, touch a 20 &#181;l droplet of 1.5 M sucrose containing the drug directly to the proboscis of the bee. Make sure the bee consumes everything. As vehicle control use 1.5 M sucrose solution without added drugs. 
		NOTE: The amount of sucrose solution might need to be adjusted based on experimental plans. If appetitive conditioning is intended, feeding the bees just prior to training will interfere with bees&#39; responsiveness.</li>
		<li>Discard or set aside bees that do not consume all of the sucrose. 
		NOTE: If a large number of bees fail to drink the sucrose solution, the feeding schedule might need to be adjusted.</li>
	</ol>
	</li>
	<li>Injection into the thorax
	<ol>
		<li>Prepare drug in honey bee Ringer17as follows:
		<ol>
			<li>Mix and autoclave 7.45 g NaCl, 0.448 g KCl, 0.812 g MgCl2,&#160;0.735 g CaCl2,&#160;54.72 g sucrose, 4.95 g D-glucose, and 2.48 g HEPES in 1,000 ml of water. Be careful when storing Ringer as it is easily contaminated. Aliquot and store Ringer at -20 &#176;C until use.</li>
			<li>Dissolve the drug in Ringer solution and then dilute so that the desired amount is present in 5 &#181;l. As an example, if bees are to be treated with 5 &#181;g of a drug, 1 g can initially be dissolved in 1 ml in Ringer, before being diluted 1:1,000 in Ringer for a final solution of 1 &#181;g/&#181;l. 
			&#8203;NOTE: Alternatively, commercially available PBS (Phosphate-buffered Salin) can be used instead of Ringer solution.</li>
		</ol>
		</li>
		<li>Make a microscalpel by breaking off the corner of a double-edged razor blade with a blade holder. Attach the blade fragment to a blade holder so that it makes a nice blade with a sharp end point.</li>
		<li>Under a stereomicroscope, carefully use the microscalpel to cut a 2 mm hole just above the scutellum, next to the posterior wing process of a bee&#39;s thorax. Avoid cutting too deep as this might injure flight muscles, and be careful to avoid the wing hinges. Ideally, only cut three sides, so that the flap of cuticle can later be folded back to close the site of injury.</li>
		<li>Using a micropipette, deposit 5 &#181;lL Ringer (or PBS) containing the drug on top of the hole in the thorax. Carefully monitor under microscope to ensure the entire drop is absorbed into the hemolymph. Use Ringer (or PBS) as a vehicle control.</li>
		<li>If possible, move the cuticle flap back over the hole. After 5-10 hr, it will reattach and seal. 
		NOTE: As an alternative to this technique, inject 1 &#181;l directly into the thorax using a glass syringe, after opening a small hole in the middle of the frenum (transverse line in the posterior region of the scutellum) with a syringe needle (diameter: 0.6 mm, G: 23). This circumvents the need to first cut the thorax with a scalpel and the injection site is smaller, but this method will leave the injection site exposed.</li>
	</ol>
	</li>
	<li>Ocellus injection 
	&#8203;NOTE: This is a method suitable for delivering molecules throughout the head capsule, into the hemolymph.
	<ol>
		<li>Prepare drugs as in 1.2.1, but adjust drug concentrations such that the desired dose will be contained in 1 &#181;l of Ringer or PBS (less volume can be absorbed through the ocellus hole than through the thorax).</li>
		<li>Prepare a microscalpel as in 1.2.2. Under a stereomicroscope, lock the head of a harnessed bee in place by filling the neck crevice with wax. Use low-temperature melting wax (e.g., dental wax) in order to avoid damaging antennal olfactory receptors or other cells that may be important for assessing behavior (e.g., olfactory learning). Then carefully remove the lens of the median ocellus by inserting the tip of the microscalpel under the lens and gently break the lens free from the head capsule. 
		&#8203;NOTE: It is also possible to place wax carefully over the antennae to prevent movement.</li>
		<li>Carefully pipette drug onto the ocellus hole. Wait until all is taken into the head capsule. Remove dental wax from the antennae and allow the bee to rest for a while before continuing the experimental procedure. Use Ringer (or PBS) as a vehicle control.</li>
	</ol>
	</li>
	<li>Injection into the ocellar tract 
	NOTE: The ocellar tract contains large fibers, connecting to most regions of the central brain18. This treatment method enables applying compounds to the brain only, but not targeting specific subregions of the brain.
	<ol>
		<li>Prepare bee as in 1.3.2. and remove the lens of the median ocellus with the tip of a microscalpel as in 1.3.3. 
		&#8203;NOTE: This can be done up to 2 hr before the injection. Based on our experience, fed bees are better able to cope with this surgery than starved bees</li>
		<li>Fill a 10 &#181;l glass syringe equipped with a small gauge (e.g., 33, diameter: 210 &#181;m) needle with drug solution prepared as in 1.3.1.</li>
		<li>Using a manual micromanipulator, insert the syringe tip through the ocellar retina into the head capsule to a depth of 50 &#181;m and inject 250 nl of solution.</li>
		<li>After use, rinse the syringe&#160;3 times with distilled water, then 3 times with 75% ethanol.</li>
	</ol>
	</li>
	<li>Microinjection into particular brain structures 
	&#8203;NOTE: In addition to the systematic treatments mentioned above, it is possible to perform microinjections into particular brain structures. This allows for pharmacological manipulation of one or more brain regions, while leaving others unaffected. This works best with brain regions that are easy to recognize from the anterior brain surface (e.g., antennal lobes, mushroom body calyces or vertical lobes, or the optic lobes), but other regions have been targeted. Please note that the orientation (anterior/posterior, dorsal/ventral) refers to the body axis, rather, than to the neuraxis19.
	<ol>
		<li>Prepare the drug in Ringer or PBS in the same manner as in 1.2.1, adding a fluorescent (e.g., 0.5 mg/ml Dextran, Alexa 546 or 568 fluor) or nonfluorescent dye (e.g., 1 mM methylene blue). 
		NOTE: The addition of a fluorescent dye will allow verification of the injection location after the experiment is over (using confocal microscopy, following brain dissection), whereas nonfluorescent dyes allow direct monitoring during the experiment.</li>
		<li>To make glass pipettes for injection, insert glass capillaries of the correct diameter into holder clamps of an electrode puller (1.0 mm for the standard holder included for the microinjector mentioned in the materials list). Adjust pull and heat settings to produce an approximately 0.5 cm long tip (settings will be different for every puller, even if the same model is used). 
		NOTE: Ideally, the two pipettes pulled from one glass should have the same length and shape, so that both can be used.</li>
		<li>Under a stereomicroscope, break the tips to obtain an outer diameter of about 10-15 &#181;m, based on visual estimation using a scale on a graticule inserted into the ocular. The steps on the scale are defined by the manufacturer and can be corrected for the magnification used.</li>
		<li>Then, fill the glass pipettes with the solution to inject. If glass capillaries with filaments are used, fill the pipette by placing the back side into the drug solution, otherwise fill tip using microloader tips.</li>
		<li>Insert the filled glass pipette into the capillary holder of a microinjector, which is controlled by a manual or electronic micromanipulator.</li>
		<li>Calibrate the microinjector to inject the desired volume (0.5-2 nl, depending on the size of the brain structure targeted). For this, inject directly into a small Petri dish containing mineral oil and measure the diameter of the droplet with the graticule. Change settings until the desired volume is reached.</li>
		<li>Fix the head of a harnessed bee using soft dental wax as in 1.3.2, before cutting an opening into the anterior part of the head capsule, using a microscalpel, with three cuts: one just below the median ocellus (ventral), one at the border of the right or left eye and one above the antenna stems (dorsal). Use a piece of dental wax to hold the opened flap in place.</li>
		<li>Carefully push glands and trachea lying on top of the brain aside using fine forceps, then make a small rupture into the neurilemma (very thin membrane around the brain) above the targeted brain structure. 
		NOTE: If many bees are to be treated at once, this procedure can be performed earlier; however, be careful to not leave bees in this state too long (no more than 30 min), as their brains might desiccate.</li>
		<li>Insert the tip into the desired brain region, and adjust depth perpendicular to the brain surface (e.g., 60 &#181;m for mushroom body calyces). Inject the preset volume. For lateral brain regions, inject bilaterally (i.e. do one injection to each hemisphere). If a nonfluorescent dye is used, ensure the injection occurred in the right region upon observation while injecting. If a fluorescent dye is used do the same under fluorescent light using a stereomicroscope with a fluorescence viewing system.</li>
		<li>Afterwards, place the open flap back over the bee&#39;s head. Melt a crystal of eicosane, which is approximately 1 mm in diameter, using a thin wire wrapped around the tip of a micro soldering iron (melting temperature is 35-37&#176;C) and seal the cuts. This will greatly reduce mortality.</li>
		<li>Release the bee from the harness for behavioral analysis (but see discussion), or keep in the harness for experiments on restrained bees &#8211; e.g., proboscis extension reflex (PER) testing20.</li>
		<li>If a fluorescent dye was used, ensure that the injection hit the area of interest after the experiment is over using a confocal laser scanning microscope (Figure. 1). 
		NOTE: This is particularly useful when targeting deeper brain areas (where it would be hard to see nonfluorescent dye during the injection phase).</li>
	</ol>
	</li>
</ol>
2. Drug Administration Methods for Free-flying Bees
<ol>
	<li>Oral treatment
	<ol>
		<li>Prepare drug in the same way as in steps 1.1.1-1.1.2. Add drug solution to a feeder and place in refrigerator for storage. 
		NOTE: Any feeder will do, such as an upside-down bottle cap or a jar inverted on tissue paper.</li>
		<li>Train bees to a gravity feeder containing 1 M or 0.5 M sucrose solution by placing a feeder close to the hive. Once bees start foraging at the feeder, gradually move it further away until it is at a comfortable distance to avoid being stung (minimum 5 m).</li>
		<li>Paint-mark bees in order to keep track of individual honey bees. Make a list of all color combinations that will be used. When a bee lands at the feeder, carefully mark its abdomen with two colors, and make a note on the list that the combination is taken.</li>
		<li>Swap the gravity feeder for a feeder containing the drug/sucrose solution. Take note of the marked bees that visit the feeder. Catch any unmarked bee visiting the feeder as bees are prolific recruiters, and the numbers of bees visiting the drugged feeder can quickly get out of control. This is especially problematic if the same experiment is to be performed on successive days, as na&#239;ve bees might no longer be na&#239;ve. 
		NOTE: As an alternative to training individual bees to a feeder, previous authors have successfully fed drug-laced sucrose water to an entire hive21-23.</li>
	</ol>
	</li>
	<li>Topical treatment 
	&#8203;NOTE: The objective is to dissolve the compound of interest in a solvent that can penetrate the waxy insect cuticle. Different solvents can be used for this purpose. The most commonly used include acetone, dimethylformamide (DMF) and dimethylsulphoxide (DMSO).
	<ol>
		<li>&#8203;Evaluate which solvent works best for the compound at hand. If a strong phenotype is expected from an overdose (e.g., paralysis or death), treat bees (step 2.2.2) with a high dose (e.g., 20 &#181;g cocaine7) dissolved in each of the different solvents and carefully monitor time until paralysis or death.</li>
		<li>Using a 1 &#181;L microcapillary (or a microsyringe, which can be fitted on an appropriate repeating dispenser) and microcapillary holder, draw 1 &#181;l of the drug solution (e.g., 3 &#181;g/&#181;l of cocaine) into the capillary. Expel the drop, and carefully paint it onto the thorax of a marked bee. Cover as large of an area a possible with the solution, rather than leaving a solid drop, as the bee is then likely to groom it off. Be careful not to allow the compound to contact the wing hinges, or this can draw it off the thorax and along the wings where it will evaporate without being absorbed into the hemolymph. 
		NOTE: Depending on the research goal, this method can also be used to administer drugs to the bee&#39;s abdomen. However, drugs reach the CNS quicker and in larger quantities when applied to the thorax24.This method works equally well with harnessed as with free-flying bees.</li>
	</ol>
	</li>
	<li>Volatilized treatment
	<ol>
		<li>Dissolve drugs (previously this method has been used to deliver cocaine to honey bees10) in 100 % ethanol. To ensure solubility, do not use a hydrochloride or other salt forms of the drug if possible. When making a dilution prepare it so that the amount to be delivered to a bee is present in 100 &#181;l. Use pure ethanol as a vehicle control.</li>
		<li>To create a filament, use the same procedure as McClung and Hirsh25.
		<ol>
			<li>Briefly explained: wind up nichrome wire tightly around a nail and attach to two electrical wires (one on each end of the filament). Remove the nail. The remaining nichrome coil is referred to as the filament.</li>
			<li>Thread the two wires through carefully drilled holes in the lid of a 50 ml centrifuge tube, which should be resistant to the temperature chosen. Glue the wires in place with liquid silicone. 
			&#8203;NOTE: This will make the tube airtight. This is essential to avoid secondary exposure to the experimenter and ensure that bees are treated with the appropriate dose.</li>
		</ol>
		</li>
		<li>Attach the wires leading to the filament to a power source. Using a thermocouple to measure the temperature of the filament, experiment with different voltage/current combinations until one that results in an appropriate temperature profile for the drug in question, ideally, one that allows for 10 sec of heating or less. This is very important, refer to relevant literature (e.g.,&#160;in order for cocaine to volatilize it needs to be heated to at least 200 &#176;C, but at temperatures over 350 &#176;C it is broken down into secondary compounds26).</li>
		<li>Carefully pipette 100 &#181;l of drug containing ethanol solution onto the filament. Spread the liquid over as much filament surface as possible as this will increase evaporation efficiency. Leave the filament exposed at room temperature until all the ethanol has evaporated. 
		&#8203;NOTE: If the ethanol is not sufficiently evaporated, bees will be treated with both the drug of choice and ethanol. Bees are extremely sensitive to ethanol, and some drugs have synergistic interactions with ethanol, which will bias experimental results.</li>
		<li>Once the ethanol has completely evaporated (drug precipitate can usually be seen on the dry filament under a microscope), catch a free-flying bee in a 50 ml tube. Carefully close the lid containing the filament.</li>
		<li>Turn on the power for 10 sec, turn the power off and wait another 50 sec (to allow the volatilized compound to cool and thereby condense or deposit). Release the bee. 
		NOTE: While this treatment method works excellently for free-flying bees, it can be used just as effectively with harnessed bees. Simply attach the harnessed bee inside a 50 ml tube. Reload the filament as described in 2.3.4 between bees. For higher throughput, several filaments can be used in parallel.</li>
	</ol>
	</li>
</ol>

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E., Maleszka, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+injection,+feeding+and+topical+application+methods+for+treatment+of+honeybees+with+octopamine.">Comparing injection, feeding and topical application methods for treatment of honeybees with octopamine.</a> J. Insect Physiol. 53 (2), 187-194 (2007).</li><li>McClung, C., Hirsh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotypic+behavioral+responses+to+free-base+cocaine+and+the+development+of+behavioral+sensitization+in+Drosophila.">Stereotypic behavioral responses to free-base cocaine and the development of behavioral sensitization in Drosophila.</a> Curr. Biol. 8 (2), 109-112 (1998).</li><li>Martin, B. R., Lue, L. P., Boni, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrolysis+and+volatilization+of+cocaine.">Pyrolysis and volatilization of cocaine.</a> J. Anal. Toxicol. 13 (3), 158-162 (1989).</li><li>Lefer, D., Perisse, E., Hourcade, B., Sandoz, J. -. C., Devaud, J. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+waves+of+transcription+are+required+for+long-term+memory+in+the+honeybee.">Two waves of transcription are required for long-term memory in the honeybee.</a> Learn. Mem. 20 (1), 29-33 (2012).</li><li>Urlacher, E., Soustelle, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Honey+Bee+Allatostatins+Target+Galanin/Somatostatin-Like+Receptors+and+Modulate+Learning:+A+Conserved+Function?.">Honey Bee Allatostatins Target Galanin/Somatostatin-Like Receptors and Modulate Learning: A Conserved Function?.</a> PLoS One. 11 (1), e0146248 (2016).</li><li>Stollhoff, N., Menzel, R., Eisenhardt, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+recovery+from+extinction+depends+on+the+reconsolidation+of+the+acquisition+memory+in+an+appetitive+learning+paradigm+in+the+honeybee+(Apis+mellifera).">Spontaneous recovery from extinction depends on the reconsolidation of the acquisition memory in an appetitive learning paradigm in the honeybee (Apis mellifera).</a> J. Neurosci. 25 (18), 4485-4492 (2005).</li><li>Barron, A. B., Maleszka, R., Helliwell, P. G., Robinson, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cocaine+on+honey+bee+dance+behaviour.">Effects of cocaine on honey bee dance behaviour.</a> J. Exp. Biol. 212 (2), 163-168 (2009).</li><li>Devaud, J. -. M., Papouin, T., Carcaud, J., Sandoz, J. -. C., Gr&#252;newald, B., Giurfa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+substrate+for+higher-order+learning+in+an+insect:+Mushroom+bodies+are+necessary+for+configural+discriminations.">Neural substrate for higher-order learning in an insect: Mushroom bodies are necessary for configural discriminations.</a> Proc. Natl. Acad. Sci. , 1-9 (2015).</li><li>Vergoz, V., Roussel, E., Sandoz, J. -. C., Giurfa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aversive+learning+in+honeybees+revealed+by+the+olfactory+conditioning+of+the+sting+extension+reflex.">Aversive learning in honeybees revealed by the olfactory conditioning of the sting extension reflex.</a> PLoS One. 2 (3), e288 (2007).</li><li>Henry, M., B&#233;guin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+common+pesticide+decreases+foraging+success+and+survival+in+honey+bees.">A common pesticide decreases foraging success and survival in honey bees.</a> Science. 336 (6079), 348-350 (2012).</li><li>S&#248;vik, E., Perry, C. J., LaMora, A., Barron, A. B., Ben-Shahar, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+impact+of+manganese+on+honeybee+foraging.">Negative impact of manganese on honeybee foraging.</a> Biol. Lett. 11 (3), 20140989 (2015).</li><li>Farooqui, T., Vaessin, H., Smith, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Octopamine+receptors+in+the+honeybee+(Apis+mellifera)+brain+and+their+disruption+by+RNA-mediated+interference.">Octopamine receptors in the honeybee (Apis mellifera) brain and their disruption by RNA-mediated interference.</a> J. Insect Physiol. 50 (8), 701-713 (2004).</li><li>Guo, X., Su, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recipe+for+a+Busy+Bee:+MicroRNAs+in+Honey+Bee+Caste+Determination.">Recipe for a Busy Bee: MicroRNAs in Honey Bee Caste Determination.</a> PLoS One. 8 (12), e81661 (2013).</li><li>Cristino, A. S., Barchuk, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroligin-associated+microRNA-932+targets+actin+and+regulates+memory+in+the+honeybee.">Neuroligin-associated microRNA-932 targets actin and regulates memory in the honeybee.</a> Nat. Commun. 5, 5529 (2014).</li><li>Vargaftig, B. B., Coignet, J. L., de Vos, C. J., Grijsen, H., Bonta, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mianserin+hydrochloride:+Peripheral+and+central+effects+in+relation+to+antagonism+against+5-hydroxytryptamine+and+tryptamine.">Mianserin hydrochloride: Peripheral and central effects in relation to antagonism against 5-hydroxytryptamine and tryptamine.</a> Eur. J. Pharmacol. 16 (3), 336-346 (1971).</li><li>Beggs, K. T., Tyndall, J. D. A., Mercer, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Honey+bee+dopamine+and+octopamine+receptors+linked+to+intracellular+calcium+signaling+have+a+close+phylogenetic+and+pharmacological+relationship.">Honey bee dopamine and octopamine receptors linked to intracellular calcium signaling have a close phylogenetic and pharmacological relationship.</a> PLoS One. 6 (11), (2011).</li><li>Matsumoto, Y., Menzel, R., Sandoz, J. -. C., Giurfa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+olfactory+classical+conditioning+of+the+proboscis+extension+response+in+honey+bees:+a+step+toward+standardized+procedures.">Revisiting olfactory classical conditioning of the proboscis extension response in honey bees: a step toward standardized procedures.</a> J. Neurosci. Methods. 211 (1), 159-167 (2012).</li></ol>

The authors have nothing to disclose.

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&#39; flying ability.
The methods have been presented from a brain-ce...

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 forefront of this revolution.
Honey bees are being used to study basic neurobiological questions such as those underlying learning and memory2,3, decision making4, olfactory5,&#160;or visual processing6. In recent years, the honey bee has even been used as a model for studying topics generally reserved for medical research, such as the effects of addictive drugs7-11, sleep12, ageing13, or the mechanisms underlying anaesthesia14.
Unlike for the classical genetic model organisms (e.g.,&#160;D. melanogaster, C. elegans, M. musculus), there are very few genetic tools available for manipulating neural functions in honey bees, although this is currently changing15. Instead, honey bee studies have primarily relied on pharmacological manipulations. This has been very successful; however, the diversity of bee research is such that a range of methods for pharmacological administration are needed. Research with honey bees addresses highly diverse questions, is studied by researchers from different disciplines and backgrounds, and uses a variety of experimental approaches. Many research questions require bees to either be free-flying,&#160;freely interacting in their colony, or both. This can make it difficult to keep track of individual experimental animals, and makes restraint or cannulation unfeasible.
To accommodate the diversity of honey bee research, a variety of drug delivery methods are needed, allowing for robust and flexible administration while ensuring that the pharmacokinetic and pharmacodynamic profiles, invasiveness of the method, and its reliability, suit the paradigm in question. Because of these diverse needs, most research groups have developed their own unique drug administration methods. So far, this has been a strength of the bee research community;&#160;it has led to the development of arrays of methods allowing for administration of the same drug in different circumstances. Our goal here is not to develop a single standardized method for pharmacological manipulations of bees, but rather to highlight methods that have proven to be particularly successful, and help researchers adopt these. We discuss the basic principles of how they work, as well as their advantages and disadvantages.

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

neuropharmacological manipulation of restrained and free-flying honey bees, apis mellifera

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&#160;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.

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...

Watch this Scientific Journal Video about Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera at JoVE.com

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

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.

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

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize ...

neuropharmacological-manipulation-restrained-free-flying-honey-bees

Research

JoVE Journal

Neuroscience

9.9K Views.  Volda University College.  Dit manuscript beschrijft meerdere protocollen voor het toedienen van farmacologische middelen aan de bijen, waaronder eenvoudige niet-invasieve methoden voor vrij vliegende bijen, evenals meer invasieve varianten die precies gelokaliseerde behandeling van ingehouden bijen mogelijk. 

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

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees (Apis mellifera)

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the survival of a honeybee colony depends on the exploitation of food sources, forager bees learn and memorize variable flower sites as well as their profitability. Forager bees can be easily trained in natural settings where they forage at a feeding site and learn the related signals such as odor or color. Appetitive associative learning can also be studied under controlled conditions in the laboratory by conditioning the proboscis extension response (PER) of individually harnessed honeybees3,4. This learning paradigm enables the study of the neuronal and molecular mechanisms that underlie learning and memory formation in a simple and highly reliable way5-12. A behavioral pharmacology approach is used to study molecular mechanisms. Drugs are injected systemically to interfere with the function of specific molecules during or after learning and memory formation13-16.
Here we demonstrate how to train harnessed honeybees in PER conditioning and how to apply drugs systemically by injection into the bee flight muscle.

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees Apis mellifera

We demonstrate how to implement a behavioral pharmacology method in an appetitive olfactory conditioning paradigm in honeybees (Apis mellifera) by systemic application of drugs. This method allows investigation of the mechanisms underlying learning and memory formation in a simple and reliable way.

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the ...

Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees

The European honey bee, Apis mellifera L., is an economically and agriculturally important pollinator that generates billions of dollars annually. Honey bee colony numbers have been declining in the United States and many European countries since 1947. A number of factors play a role in this decline, including the unintentional exposure of honey bees to pesticides. The development of new methods and regulations are warranted to reduce pesticide exposures to these pollinators. One approach is the use of repellent chemistries that deter honey bees from a recently pesticide-treated crop. Here, we describe a protocol to discern the deterrence of honey bees exposed to select repellent chemistries. Honey bee foragers are collected and starved overnight in an incubator 15 h prior to testing. Individual honey bees are placed into Petri dishes that have either a sugar-agarose cube (control treatment) or sugar-agarose-compound cube (repellent treatment) placed into the middle of the dish. The Petri dish serves as the arena that is placed under a camera in a light box to record the honey bee locomotor activities using video tracking software. A total of 8 control and 8 repellent treatments were analyzed for a 10 min period with each treatment was duplicated with new honey bees. Here, we demonstrate that honey bees are deterred from the sugar-agarose cubes with a compound treatment whereas honey bees are attracted to the sugar-agarose cubes without an added compound.

The loss of honey bee colonies presents a challenge to crop pollination services. Current pollinator protection practices warrant an alternative approach to minimize the contact of honey bees to harmful pesticides using repellent chemistries. Here, we provide detailed methods for a visual tracking protocol to screen deterrents for bees.

The European honey bee, Apis mellifera L., is an economically and agriculturally important pollinator that generates billions of dollars annually. Honey ...

Environment

Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom body (MB) is a higher brain center proposed to be the neural substrate of complex honeybee behaviors. Although previous studies identified genes and proteins that are differentially expressed in the MBs and other brain regions, the activities of the proteins in each region are not yet fully understood. To reveal the functions of these proteins in the brain, pharmacologic analysis is a feasible approach, but it is first necessary to confirm that pharmacologic manipulations indeed alter the protein activity in these brain regions.
We previously identified a higher expression of genes encoding phospholipase C (PLC) in the MBs than in other brain regions, and pharmacologically assessed the involvement of PLC in honeybee behavior. In that study, we biochemically tested two pharmacologic agents and confirmed that they decreased PLC activity in the MBs and other brain regions. Here, we present a detailed description of how to detect PLC activity in honeybee brain homogenate. In this assay system, homogenates derived from different brain regions are reacted with a synthetic fluorogenic substrate, and fluorescence resulting from PLC activity is quantified and compared between brain regions. We also describe our evaluation of the inhibitory effects of certain drugs on PLC activity using the same system. Although this system is likely affected by other endogenous fluorescence compounds and/or the absorbance of the assay components and tissues, the measurement of PLC activity using this system is safer and easier than that using the traditional assay, which requires radiolabeled substrates. The simple procedure and manipulations allow us to examine PLC activity in the brains and other tissues of honeybees involved in different social tasks.

To test the inhibitory effects of pharmacologic agents on phospholipase C (PLC) in different regions of the honeybee brain, we present a biochemical assay to measure PLC activity in those regions. This assay could be useful for comparing PLC activity among tissues, as well as among bees exhibiting different behaviors.

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom ...

Biochemistry

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to measure odor evoked activity in the antennal lobe1. From then on, it has been continuously refined and applied to different neuropiles in the bee brain. Here, we describe the preparation currently used in the lab to measure activity in mushroom body neurons using a dextran coupled calcium-sensitive dye (Fura-2). We retrogradely stain mushroom body neurons by injecting dye into their axons or soma region. We focus on reducing the invasiveness, to achieve a preparation in which it is still possible to train the bee using PER conditioning. We are able to monitor and quantify the behavioral response by recording electro-myograms from the muscle which controls the PER (M17)2.
After the physiological experiment the imaged structures are investigated in greater detail using confocal scanning microscopy to address the identity of the neurons.

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

Bees can be conditioned in an appetitive olfactory learning paradigm (PER-conditioning). Using odors as stimuli, we established a method in which behavior is recorded while simultaneously Calcium Imaging is used to measure odor evoked activity in mushroom body neurons in vivo.

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to ...

Biology

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

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 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 odors7, 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 antennae10, 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.

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

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.

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker ...

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.	
	RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.	
	The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.
We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.

The Proboscis Extension Response (PER) conditioning protocol, developed for the honey bee (Apis mellifera), provides an ecologically-relevant and easily quantifiable means for studying several different mechanisms of learning in many insect species.

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). ...

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees (Bombus terrestris)

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from flowering plants. Only a few studies have described the gustatory responses of bees toward toxins in food, and these experiments have mainly used the proboscis extension response on restrained honey bees. Here, a new behavioral assay is presented for measuring the feeding responses of freely-moving, individual worker bumble bees to nutrients and toxins. This assay measures the amount of solution ingested by each bumble bee and identifies how tastants in food influence the microstructure of the feeding behavior.
The solutions are presented in a microcapillary tube to individual bumble bees that have been previously starved for 2-4 hr. The behavior is captured on digital video. The fine structure of the feeding behavior is analyzed by continuously scoring the position of the proboscis (mouthparts) from video recordings using event logging software. The position of the proboscis is defined by three different behavioral categories: (1) proboscis is extended and in contact with the solution, (2) proboscis is extended but not in contact with the solution and (3) proboscis is stowed under the head. Furthermore the speed of the proboscis retracting away from the solution is also estimated.
In the present assay the volume of solution consumed, the number of feeding bouts, the duration of the feeding bouts and the speed of the proboscis retraction after the first contact is used to evaluate the phagostimulatory or the deterrent activity of the compounds tested.
This new taste assay will allow researchers to measure how compounds found in nectar influence the feeding behavior of bees and will also be useful to pollination biologists, toxicologists and neuroethologists studying the bumble bee&#39;s taste system.

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees Bombus terrestris

A novel behavioral assay is described for investigating the short term gustatory responses of the mouthparts of freely-moving bumble bees (Bombus terrestris) toward nutrients and toxins in solution.

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from ...

Preparation of Single-cohort Colonies and Hormone Treatment of Worker Honeybees to Analyze Physiology Associated with Role and/or Endocrine System

Honeybee workers are engaged in various tasks related to maintaining colony activity. The tasks of the workers change according to their age (age-related division of labor). Young workers are engaged in nursing the brood (nurse bees), while older workers are engaged in foraging for nectar and pollen (foragers). The physiology of the workers changes in association with this role shift. For example, the main function of the hypopharyngeal glands (HPGs) changes from the secretion of major royal jelly proteins (MRJPs) to the secretion of carbohydrate-metabolizing enzymes. Because worker tasks change as the workers age in typical colonies, it is difficult to discriminate the physiological changes that occur with aging from those that occur with the role shift. To study the physiological changes in worker tissues, including the HPGs, in association with the role shift, it would be useful to manipulate the honeybee colony population by preparing single-cohort colonies in which workers of almost the same age perform different tasks. Here we describe a detailed protocol for preparing single-cohort colonies for this analysis. Six to eight days after single-cohort colony preparation, precocious foragers that perform foraging tasks earlier than usual appear in the colony. Representative results indicated role-associated changes in HPG gene expression, suggesting role-associated HPG function. In addition to manipulating the colony population, analysis of the endocrine system is important for investigating role-associated physiology. Here, we also describe a detailed protocol for treating workers with 20-hydroxyecdysone (20E), an active form of ecdysone, and methoprene, a juvenile hormone analogue. The survival rate of treated bees was sufficient to examine gene expression in the HPGs. Gene expression changes were observed in response to 20E- and/or methoprene-treatment, suggesting that hormone treatments induce physiological changes of the HPGs. The protocol for hormone treatment described here is appropriate for examining hormonal effects on worker physiology.

Here we describe our detailed protocol for the preparation of single-cohort honeybee colonies&#160;&#8211; a useful tool for analyzing the role-associated worker physiology. We also describe detailed protocols for treating workers with juvenile hormone and ecdysone to evaluate the involvement of these hormones in the regulation of worker behavior and/or physiology.

Honeybee workers are engaged in various tasks related to maintaining colony activity. The tasks of the workers change according to their age (age-related ...

SwarmSight: Real-time Tracking of Insect Antenna Movements and Proboscis Extension Reflex Using a Common Preparation and Conventional Hardware

Many scientifically and agriculturally important insects use antennae to detect the presence of volatile chemical compounds and extend their proboscis during feeding. The ability to rapidly obtain high-resolution measurements of natural antenna and proboscis movements and assess how they change in response to chemical, developmental, and genetic manipulations can aid the understanding of insect behavior. By extending our previous work on assessing aggregate insect swarm or animal group movements from natural and laboratory videos using the video analysis software SwarmSight, we developed a novel, free, and open-source software module, SwarmSight Appendage Tracking (SwarmSight.org) for frame-by-frame tracking of insect antenna and proboscis positions from conventional web camera videos using conventional computers. The software processes frames about 120 times faster than humans, performs at better than human accuracy, and, using 30 frames per second (fps) videos, can capture antennal dynamics up to 15 Hz. The software was used to track the antennal response of honey bees to two odors and found significant mean antennal retractions away from the odor source about 1 s after odor presentation. We observed antenna position density heat map cluster formation and cluster and mean angle dependence on odor concentration.

This protocol describes steps for using the novel software, SwarmSight, for frame-by-frame tracking of insect antenna and proboscis positions from conventional web camera videos using conventional computers. The free, open-source software processes frames about 120 times faster than humans and performs at better than human accuracy.

Many scientifically and agriculturally important insects use antennae to detect the presence of volatile chemical compounds and extend their proboscis ...

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

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