This work was supported by a Grant-in-Aid for Scientific Research (B) and a Grant-in Aid for Scientific Research on Innovative Areas 'Systems Molecular Ethology' from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. T.U. was the recipient of a Grant-Aid from the Japan Society for the Promotion of Science for Young Scientists.

1. Preparation of Single-cohort Colonies
<ol>
	<li>Prepare three honeybee colonies to create two single-cohort colonies and to obtain a sufficient number of newly emerged workers.
	<ol>
		<li>Check that some pupae in the capped peripheral cells in the combs have brown eyes and a pigmented cuticle by opening the capped combs using tweezers. If these pupae exist in peripheral comb cells, most of pupae in the whole combs will emerge in approximately 1-3 days.</li>
		<li>Subsequently, collect the combs containing these pupae after all adherent adult bees have been removed with a brush, and mix combs from the three colonies to minimize variability among colonies.</li>
	</ol>
	</li>
	<li>Place the collected combs in an empty hive box and incubate at 33 &#176;C under humid conditions (&#62;60% relative humidity). Collect approximately 6,000 newly emerged workers over 3 days (3,000 workers per colony) from the collected combs.</li>
	<li>Determine the quantity of workers based on the weight of five newly emerged workers randomly collected from unmanipulated colonies.
	<ol>
		<li>Weigh five newly emerged workers randomly collected from unmanipulated colonies using weighing machines. 
		Note: The weight of five newly emerged workers is generally 500-600 mg.</li>
		<li>Estimate the quantity of collected workers by dividing the weight of the collected workers by the average weight of five newly emerged workers.</li>
	</ol>
	</li>
	<li>Apply paint marks to the thoraces of approximately 1,800 workers (900 workers per colony) using water-based poster paint markers. 
	Note: Washable marker which is used to apply the paint is listed in the Table of Materials.</li>
	<li>Introduce a queen and approximately 3,000 of the 0-2 day old workers to a new hive box that contains two combs, one comb containing honey and pollen as preserved foods and another empty comb for egg-laying by the queen. Collect the comb containing honey and pollen from another unmanipulated colony after all adherent bees are removed. 
	Note: The use of nuclear hive box (small sized hive box) is recommended.</li>
	<li>Collect nurse bees and precocious foragers with paint marks 6 to 8 days after creating single-cohort colonies.
	<ol>
		<li>To collect nurse bees, pick up workers that put their heads into comb cells to take care of the brood. Use tweezers to collect the nurse bees from combs that contain many brood and adult workers. 
		Note: If HPGs of collected workers are developed when dissected as described in step 1.7, these workers are defined as nurse bees. Holding the thoraces by tweezers is recommended to pick up workers from the combs.</li>
		<li>To collect foragers, use an insect net to capture workers that return to their hives with pollen loads on their hind legs. 
		Note: If HPGs of captured workers appear shrunken when dissected as described in step 1.7, those workers are defined as foragers.</li>
	</ol>
	</li>
	<li>Classify the HPG into three groups, &#39;Developed&#39;, &#39;Intermediate&#39;, and &#39;Shrunken&#39; by the dissection under a stereomicroscope.
	<ol>
		<li>Anesthetize 10-15 bees in an insect cage in a refrigerator for 10-15 min until bees cannot fly or walk. Then, move bees onto ice and complete the anesthesia on ice for 5 min. 
		Note: It takes about total 15-20 min to achieve anesthesia. If the anesthesia time needs to be shortened, the number of bees in a cage should be reduced to 1-3 bees per cage.</li>
		<li>Dissect the head from the body with scissors and tweezers. Fix the head on dental wax placed on a Petri dish with pins. Insert two pins into the bases of the antennae to fix the head. Soak fixed heads in insect saline (130 mM NaCl, 5 mM KCl, 1 mM CaCl2).</li>
		<li>Remove the anterior aspect of the cuticle of the head using fine tweezers and a surgical knife under a binocular microscope. 
		Note: The HPGs can be easily found in the head after removal of cuticle. The HPG is a botryoid organ that exists around the brain in the head.</li>
		<li>Remove the tracheal tissue which is a white membranous tissue under the cuticle. Then, dissect the HPGs from the head by slowly pulling them with tweezers.</li>
		<li>Classify HPGs with large and circular acini as &#39;Developed&#39;. Classify HPGs with small and distorted acini as &#39;Shrunken&#39;. Classify HPGs corresponding to neither &#39;Developed&#39; nor &#39;Shrunken&#39; as &#39;Intermediate&#39;. 
		Note: Representative photographs that show these three classes of HPG states are indicated in Figure 2.</li>
		<li>Subject &#39;Developed&#39; and &#39;Shrunken&#39; HPGs to RNA extraction as &#39;nurse bee HPGs&#39; and &#39;forager HPGs&#39;, respectively, and compare gene expression in the HPGs between nurse bees and foragers (section 4).</li>
	</ol>
	</li>
</ol>
2. Injection of 20E
<ol>
	<li>Collect nurse bees from typical colonies as described in the procedure 1.6.1. 
	Note: HPGs cannot be examined in these bees as the bees will be reared.</li>
	<li>Anesthetize the bees at 4 &#176;C in a refrigerator (not on ice). 
	Note: It takes about 30 min to achieve anesthesia. If the anesthesia time is shortened, the number of bees in the cage should be reduced to 1-3 bees per cage.</li>
	<li>Using tweezers, immobilize the bees on dental wax placed on a Petri dish.</li>
	<li>Inject 1 &#181;l of 20E solution into the anterior aspect of the head using an injection tip made from a calibrated capillary micropipette using a glass needle puller.
	<ol>
		<li>Dissolve 20E in ethanol-insect saline (130 mM NaCl, 5 mM KCl, 1 mM CaCl2) mixture (1:4) at 2.5 &#181;g/&#181;l.</li>
		<li>Connect the injection tip to a rubber tube, and connect a yellow micropipette tip to the opposite side of the tube.</li>
		<li>Place 1 &#181;l of 20E solution on parafilm using a micropipette, hold the yellow micropipette tip in the mouth, and draw the solution into the injection tip.</li>
		<li>Insert the injection tip into the base of the antennae, and inject the solution by blowing through the yellow micropipette tip. 
		Note: The protocol shown in this step is the representative injection method, but the use of the automated injection machine is recommended for safety as necessary20.</li>
	</ol>
	</li>
	<li>Rear the injected bees in insect cages at 33-36 &#176;C (the ideal temperature is 35 &#176;C) under dark and humid conditions (&#62;60 % relative humidity) for 1 or 3 days. Supply honey-water mixture (1:1) to the bees. If needed, count surviving bees after injection.</li>
</ol>
3. Application of Methoprene
<ol>
	<li>Collect the combs that contain pupae from typical colonies after all adherent adult bees are removed with a brush. Incubate the combs at 33-36 &#176;C (the ideal temperature is 35 &#176;C) in humid conditions (&#62;60% relative humidity) for up to 1 day.</li>
	<li>Collect newly emerged bees from the collected combs and apply paint marks to their thoraces using poster paint as described in the protocol for preparing single-cohort colonies. Return the marked bees to their colonies.</li>
	<li>After 6 days, collect painted workers (6 day old) from the colonies. Pick up painted workers from the combs by holding their thoraces with tweezers.</li>
	<li>Anesthetize the bees at 4 &#176;C in a refrigerator (not on ice) as in the procedure 2.2. Using tweezers, immobilize the bees on dental wax placed on a Petri dish and apply 1-5 &#181;l of methoprene solution (50-250 &#181;g/&#181;l) dissolved in acetone to their heads using a micropipette. Use a filter tip that is resistant to acetone. 
	Note: Acetone may have the harmful effects on bees leading to death. If the high mortality of bees is observed, the reduction of the volume of acetone to 1 &#181;l at the minimum is recommended.</li>
	<li>Rear the bees in insect cages at 33-36 &#176;C (the ideal temperature is 35 &#176;C) for 7 days under dark and humid conditions (&#62;60% relative humidity). Supply honey-water mixture (1:1) to the bees. If needed, count surviving bees after topical application. 
	Note: In this article, surviving bees are counted 7 days after application (Table 3).</li>
</ol>
4. RNA Extraction and Quantitative RT-PCR
<ol>
	<li>Anesthetize bees and dissect HPGs as described in steps 1.7.1 to 1.7.4.</li>
	<li>Collect the dissected HPGs in a microcentrifuge tube on dry ice and store at -80 &#176;C until use.</li>
	<li>Add 400 &#181;l of the reagent for protein denaturation. 
	Note: This reagent is commercially available (see the Table of Materials).</li>
	<li>Homogenize the HPG tissues using a hand held electric mixer with a homogenization pestle that fits inside a microcentrifuge tube. Homogenize at a rotation speed of approximately 300 rpm for 1 min on ice to break and lyse tissue cells. 
	Note: The hand held electric mixer is commercially available (see the Table of Materials).</li>
	<li>Add 80 &#181;l chloroform and mix well. Incubate for 5 min at RT.</li>
	<li>Centrifuge the microcentrifuge tubes at 14,170 x g for 15 min at 4 &#176;C. Transfer the aqueous phase to a new tube. Add an equal volume of isopropanol and 1 &#181;l glycogen (5 mg/ml). Incubate at -20 &#176;C for at least 20 min.</li>
	<li>Centrifuge the microcentrifuge tubes at 14,170 x g for 10 min at 4 &#176;C and remove the supernatant.</li>
	<li>Add 400 &#181;l of 75% ethanol to the pellet and mix well, centrifuge the microcentrifuge tubes at 14,170 x g for 15 min at 4 &#176;C, and remove the supernatant. Repeat these procedures once more.</li>
	<li>Air-dry the pellet for 5-10 min and then dissolve it in 10 &#181;l RNase-free water. 
	Note: Complete drying of the pellet may cause insolubilization of the RNA pellet in water. Thus, the pellet which is slightly wet is appropriate for dissolving.</li>
	<li>Measure the RNA concentration using a spectrophotometer such as NanoDrop or similar.</li>
	<li>Treat 500 ng of total RNA with 2.0 U of DNase I by incubating at 37 &#176;C for 30 min to degrade any contaminating genomic DNA.</li>
	<li>Reverse-transcribe 200 ng of DNase I-treated RNA and perform real time PCR using commercially available kits (see the list in the Table of Materials and Reagents) and gene-specific primers (mrjp2; 5&#39;-AAATGGTCGCTCAAAATGACAGA-3&#39;, and 5&#39;-ATTCATCCTTTACAGGTTTGTTGC-3&#39;, Hbg3; 5&#39;-TACCTGGCTTCGTGTCAAC-3&#39; and 5&#39;-ATCTTCGGTTTCCCTAGAGAATG-3&#39;). Perform the PCR as follows: [95 &#176;C x 30 sec + (95&#176;C x 5 sec + 60&#176;C x 15 sec + 72 &#176;C x 20 sec) x 45-55 cycles].</li>
	<li>Normalize the amount of transcript with that of elongation factor 1&#945;-F2 (EF1&#945;&#727;F2) or ribosomal protein 49 (rp49)9,16,21,22. These genes are reliable control genes for analyzing gene expression in the HPGs using qRT-PCR.</li>
	<li>Perform two-tailed t-test to detect the significant differences between two experiment groups (nurse bees vs. precocious foragers, 20E-treated bees vs. control bees, and methoprene-treated bees vs. control bees) using statistical software. 
	Note: If F-test assumes the homogeneity of variance, Student&#39;s t-test can be used. If not, Welch&#39;s t-test can be used.</li>
</ol>

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The authors have nothing to disclose.

Preparation of single-cohort colonies
Here we described the protocol used in our previous study16 to prepare single-cohort colonies for analysis of HPG physiology associated with the shift in the role of worker bees. Nurse bees and precocious foragers that satisfied the criteria described in procedures 1.6-1.7 and Figure 2 were observed in the single-cohort colonies (Table 1). The photographs in Figure 2 would be useful in classific...

The European honeybee, Apis mellifera, is a eusocial insect with a highly organized society1. Worker honeybees (labor caste) are engaged in various tasks to maintain colony activity, and these tasks change according to the worker honeybee&#39;s age after eclosion, which is referred to as age-related division of labor2-4. Young workers (&#60;13 days old) take care of the brood in the hive by secreting royal jelly (nurse bees), while older workers (&#62;15 days old) collect nectar and pollen outside of the hive (foragers)2-4. The physiology of the workers changes in association with this role shift. For example, the function of the hypopharyngeal glands (HPGs), paired exocrine glands located in the head, changes in association with the role shift from nursing to foraging2,5. Nurse bee HPGs mainly synthesize major royal jelly proteins, which are major components of bee milk. On the other hand, forager HPGs mainly synthesize carbohydrate-metabolizing enzymes, such as &#945;-glucosidase III, to process nectar into honey by converting sucrose into glucose and fructose. Our previous studies revealed that the expression of mrjp2, which encodes a major royal jelly protein, and Hbg3, which encodes &#945;-glucosidase III, changes during the role shift6-9.
To determine whether the physiological changes in the worker tissues, including the HPGs, is associated with the role shift or with the age of the workers, it would be useful to manipulate the population composition of a honeybee colony, such as to prepare single-cohort colonies in which workers of almost the same age perform different tasks10,11. Robinson et al. (1989) described a method for establishing a single-cohort colony10. Single-cohort colonies initially comprise a queen and 0-2 day old workers. Several days after establishing the colonies, workers of almost the same age assume different tasks. Some workers perform nursing tasks as in typical colonies, whereas other workers perform foraging tasks earlier than usual and are thus called precocious foragers. Gene expression comparisons between nurse bees and precocious foragers would provide useful information about the role-associated physiology of worker tissues12-16. Here, we describe a detailed protocol for preparing single-cohort colonies for analysis of the role- and/or age-associated physiology of HPGs16. We also briefly describe how to examine the gene expression of mrjp2 and Hbg3 by quantitative reverse transcription-polymerase chain reaction (RT-PCR) to evaluate HPG physiology.
In addition to the analysis of worker physiology in single-cohort colonies, examination of the endocrine system is important for analyzing the regulatory mechanisms of role-associated worker physiology. Juvenile hormone (JH), which is known as the &#39;status quo&#39; hormone in insect larvae, accelerates the shift in the role from nursing to foraging in worker honeybees11. Furthermore, ecdysone, which is known as the molting hormone during metamorphosis, might be involved in the role shift as genes encoding ecdysone signaling molecules are expressed in the mushroom bodies, a higher center of the worker brain17-19. Therefore, we also describe the detailed protocol used in our previous study16 to treat workers with 20E, which is an active form of ecdysone, and methoprene, a JH analogue, for analysis of the effect of the endocrine system on HPG physiology (expression of mrjp2 and Hbg3).

<table><tbody><tr><td>UNIPOSCA</td><td>Mitsubishi pencil</td><td>PC-5M</td><td>Marker pen for the application of marks to bees&amp;nbsp;&amp;nbsp;</td></tr><tr><td>20-hydroxyecdysone</td><td>Sigma Aldrich</td><td>H5142</td><td></td></tr><tr><td>Methoprene</td><td>Sigma Aldrich</td><td>33375</td><td></td></tr><tr><td>Breeding case insect</td><td>IRIS OHYAMA</td><td>CP-SS</td><td></td></tr><tr><td>Electromotion mixier&amp;nbsp;</td><td>ISO</td><td>23M-R25</td><td>homogenization of tissue</td></tr><tr><td>TRIZol Reagent</td><td>Invitrogen</td><td>15596-026</td><td>the reagent for total RNA extraction</td></tr><tr><td>DNase I&amp;nbsp;</td><td>Takara</td><td>2270A</td><td></td></tr><tr><td>PrimeScript RT reagent kit</td><td>Takara</td><td>RR037A</td><td>the reagent for reverse transcription</td></tr><tr><td>SYBR Premix ExTaq II</td><td>Takara</td><td>RR820A</td><td>the reagent for real-time PCR</td></tr><tr><td>LightCycle 1.2 Instrument</td><td>Roche</td><td>12011468001</td><td>the instrument for real-time PCR</td></tr><tr><td>LightCycle Capillaries (20&amp;mu;l)</td><td>Roche</td><td>4929292001</td><td>the material for real-time PCR</td></tr></tbody></table>

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.

An overview of the protocol for preparing single-cohort colonies is illustrated in Figure 1A. Time-course of experiments from preparing single-cohort colonies to sample collection is shown in Figure 1B. Workers that satisfied the behavioral criteria for nursing behavior or foraging behavior were collected from single-cohort colonies, and HPG development was estimated in these workers. Table 1 shows the classification of HPG development in...

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

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 ...

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15.8K Views.  The University of Tokyo.  Hier beschrijven wij onze gedetailleerde protocol voor de voorbereiding van de single-cohort bijenvolken - een nuttig instrument voor het analyseren van de role-geassocieerde werknemer fysiologie. We beschrijven ook gedetailleerde protocollen voor de behandeling van werknemers met juveniele hormoon en ecdyson de betrokkenheid van deze hormonen in de regulatie van de werknemer gedrag en / of fysiologie te evalueren. 

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

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 ...

Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered &#34;bee-safe,&#34; there is mounting evidence that fungicide residues in pollen are associated with bee declines (for both honey and bumble bee species). While the mechanisms remain relatively unknown, researchers have speculated that bee-microbe symbioses are involved. Microbes play a pivotal role in the preservation and/or processing of pollen, which serves as nutrition for larval bees. By altering the microbial community, it is likely that fungicides disrupt these microbe-mediated services, and thereby compromise bee health. This manuscript describes the protocols used to investigate the indirect mechanism(s) by which fungicides may be causing colony decline. Cage experiments exposing bees to fungicide-treated flowers have already provided the first evidence that fungicides cause profound colony losses in a native bumble bee (Bombus impatiens). Using field-relevant doses of fungicides, a series of experiments have been developed to provide a finer description of microbial community dynamics of fungicide-exposed pollen. Shifts in the structural composition of fungal and bacterial assemblages within the pollen microbiome are investigated by next-generation sequencing and metagenomic analysis. Experiments developed herein have been designed to provide a mechanistic understanding of how fungicides affect the microbiome of pollen-provisions. Ultimately, these findings should shed light on the indirect pathway through which fungicides may be causing colony declines.

Microbial consortia within bumble bee hives enrich and preserve pollen for bee larvae. Using next generation sequencing, along with laboratory and field-based experiments, this manuscript describes protocols used to test the hypothesis that fungicide residues alter the pollen microbiome, and colony demographics, ultimately leading to colony loss.

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered ...

Environment

Evaluating the Effect of Environmental Chemicals on Honey Bee Development from the Individual to Colony Level

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be brought back to the beehive after the bees have foraged on flowers that have been sprayed with pesticides. Pesticide contaminated food can be exchanged between workers which then feed larvae and therefore can potentially affect the development of honey bees. Thus, residual pesticides in the environment can become a chronic damaging factor to honey bee populations and gradually lead to colony collapse. In the presented protocol, honey bee feeding methods are described and applied to either an individual honey bee or to a colony. Here, the insect growth regulator (IGR) pyriproxyfen (PPN), which is widely used to control pest insects and is harmful to the development of honey bee larvae and pupae, is used as the pesticide. The presenting procedure can be applied to other potentially harmful chemicals or honeybee pathogens for further studies.

Herein we present a method to feed pesticide contaminated food to both an individual honey bee and a beehive colony. The procedure evaluates the pesticide effect on individual honey bees by in vivo feeding of basic larval diet and also on the natural condition of beehive colony.

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be ...

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide regulation studies. The risk of exposure to fungicide residues is likely to be especially high if the spray occurs on, or near host plants while the bees are collecting pollen to provision their nests. For species of Osmia that consume pollen from a select group of plants (oligolecty), the inability to use pollen from non-host plants can increase their risk factor for fungicide-related toxicity. This manuscript describes protocols used to successfully rear oligolectic mason bees, Osmia ribifloris sensu lato, from egg to prepupal stage within cell culture plates under standardized laboratory conditions. The in vitro-reared bees are subsequently used to investigate the effects of fungicide exposure and pollen source on bee fitness. Based on a 2 &#215; 2 fully crossed factorial design, the experiment examines the main and interactive effects of fungicide exposure and pollen source on larval fitness, quantified by prepupal biomass, larval developmental time, and survivorship. A major advantage of this technique is that using in vitro-reared bees reduces natural background variability and allows the simultaneous manipulation of multiple experimental parameters. The described protocol presents a versatile tool for hypotheses testing involving the suite of factors affecting bee health. For conservation efforts to be met with significant, lasting success, such insights into the complex interplay of physiological and environmental factors driving bee declines will prove to be critical.

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

Fungicide sprays on flowering plants may expose solitary bees to high concentrations of pollen-borne fungicide residues. Using laboratory-based experiments involving in vitro-reared bee larvae, this study investigates the interactive effects of consuming fungicide-treated pollen derived from host and non-host plants.

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide ...

Measuring Hypopharyngeal Gland Acinus Size in Honey Bee (Apis mellifera) Workers

The nurse hypopharyngeal glands produce the protein fraction of the worker and royal jelly that is fed to developing larvae and queens. These paired glands that are located in the head of the bee are highly sensitive to the quantity and quality of pollen and pollen substitutes that the nurse bee consumes. The glands get smaller when nurses are fed deficient diets and are large when they are fed complete diets. Because nurse hypopharyngeal gland size is a robust indicator of nurse nutrition, it is essential that those studying honey bee nutrition know how to measure these glands. Here, we provide detailed methods for dissecting, staining, imaging, and measuring nurse bee hypopharyngeal glands. We present comparisons of unstained and stained tissue and data that were used to study the impact of pollen on gland size. This method has been used to test how diet impacts hypopharyngeal gland size but has further use for understanding the role of these glands in hive health.

Measuring Hypopharyngeal Gland Acinus Size in Honey Bee Apis mellifera Workers

Hypopharyngeal gland acinus size is a robust measure of nurse honey bee nutrition. Here, we provide a detailed protocol for dissecting, staining, imaging, and measuring nurse bee hypopharyngeal gland acini.

The nurse hypopharyngeal glands produce the protein fraction of the worker and royal jelly that is fed to developing larvae and queens. These paired ...

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). ...

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.

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.

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

Obtaining Specimens with Slowed, Accelerated and Reversed Aging in the Honey Bee Model

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best established model to study how plasticity in lifespan and aging is explained by social factors.
The worker caste of honey bees includes nurse bees, which tend the brood, and forager bees, which collect nectar and pollen. Previous work has shown that brain functions and flight performance senesce more rapidly in foragers than in nurses. However, brain functions can recover, when foragers revert back to nursing tasks. Such patterns of accelerated and reversed functional senescence are linked to changed metabolic resource levels, to alterations in protein abundance and to immune function. Vitellogenin, a yolk protein with adapted functions in hormonal control and cellular defense, may serve as a major regulatory element in a network that controls the different aging dynamics in workers.
Here we describe how the emergence of nurses and foragers can be monitored, and manipulated, including the reversal from typically short-lived foragers into longer-lived nurses. Our representative results show how individuals with similar chronological age differentiate into foragers and nurse bees under experimental conditions. We exemplify how behavioral reversal from foragers back to nurses can be validated. Last, we show how different cellular senescence can be assessed by measuring the accumulation of lipofuscin, a universal biomarker of senescence.
For studying mechanisms that may link social influences and aging plasticity, this protocol provides a standardized tool set to acquire relevant sample material, and to improve data comparability among future studies.

In honey bee workers, aging depends on social behaviors rather than on chronological age. Here we show how worker-types with very different aging patterns can be obtained and analyzed for cellular senescence.

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best ...

Monitoring Colony-level Effects of Sublethal Pesticide Exposure on Honey Bees

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment methods. Here we describe methods to measure the quantities of adult bees, brood, and food resources by weighing hives and hive parts, by photographing frames, and by installing hives on scales and with internal sensors. Data from these periodic evaluations are then combined with running average and daily detrended data on hive weight and internal hive temperature. The resulting datasets have been used to detect colony-level effects of imidacloprid applied in a sugar syrup as low as 5 parts per billion. The methods are objective, require little training, and provide permanent records in the form of sensor output and photographs.

Sublethal doses of pesticides may affect colonies in ways that are difficult to detect using only periodic or visual methods. Methods to measure total adult bee mass, brood, and food resources by weighing hives and hive parts, photographing frames, and installing sensors, are provided. Data analysis is also addressed.

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment ...

Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees (Apis mellifera)

Honey bees are of great ecological and agricultural importance around the world but are also subject to a variety of pressures that negatively affect bee health, including exposure to viral pathogens. Such viruses can cause a wide variety of devastating effects and can often be challenging to study due to multiple factors that make it difficult to separate the effects of experimental treatments from preexisting background infection. Here we present a method to mass produce large quantities of virus particles along with a high throughput bioassay to test viral infection and effects. Necessitated by the current lack of a continuous, virus-free honey bee cell line, viral particles are amplified in vivo using honey bee pupae, which are extracted from the hive in large volumes using minimally stressful methodology. These virus particles can then be used in honey bee cage bioassays to test inocula viability, as well as various other virus infection dynamics, including interactions with nutrition, pesticides, and other pathogens. A major advantage of using such particles is that it greatly reduces the chances of introducing unknown variables in subsequent experimentation when compared to current alternatives, such as infection via infected bee hemolymph or homogenate, though care should still be taken when sourcing the bees, to minimize background virus contamination. The cage assays are not a substitute for large-scale, field-realistic experiments testing virus infection effects at a colony level, but instead function as a method to establish baseline viral responses that, in combination with the semi-pure virus particles, can serve as important tools to examine various dimensions of honey bee-virus physiological interactions.

Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees Apis mellifera

Here we describe two protocols: first to propagate, extract, purify, and quantify large quantities of honey bee non-enveloped virus particles, including a method for removing honey bee pupae and second to test the effects of viral infection using a highly repeatable, high-throughput cage bioassay.

Honey bees are of great ecological and agricultural importance around the world but are also subject to a variety of pressures that negatively affect bee ...

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

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