This work was supported by internal funds from the USDA-ARS (Project Number: 2022-21000-017-00-D). The ARS/USDA is an equal opportunity employer and provider.

1. Dissecting and Staining the Hypopharyngeal Glands from Nurse Workers
<ol>
	<li>Make a wax dissection plate by melting cool setting wax into a small (60 mm x 15 mm) or large (100 mm x 15 mm) glass Petri dish. Cool the wax completely before using the plate for dissections.</li>
	<li>For each bee to be processed, prepare 20 &#181;L of a 1:20 working Giemsa solution (1:20 v/v of prepared Giemsa stain in phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4)). 
	CAUTION: Giemsa stain contains methanol and is flammable. It is toxic if swallowed, inhaled, or if it comes in contact with skin. It should be disposed of according to the local institution&#8217;s requirements. 
	NOTE: Always make a fresh working solution just for the current batch of samples because Giemsa stain quickly degrades once it is diluted. Discard the diluted stain if a precipitate develops.</li>
	<li>Pipet 20 &#181;L Giemsa stain into the wells of the microscope slides and 50&#8211;100 &#181;L saline onto the slide adjacent to the well.</li>
	<li>Detach the head from a bee, using the 10 mm micro-spring scissors, and embed the head front side up into the wax dissection plate using forceps and a wax-carving pen. Pin the head down into the wax plate for additional stability by inserting pins in the eyes and one in the mouth.</li>
	<li>Using a sharp breakable razor blade fixed into a pin vise, make a small (~2&#8211;3 mm) incision between the eyes and mandibles on each side of the face plate. Gently run the micro scissors under the face plate and cut the antennal nerve that runs between the antennae and the brain.</li>
	<li>Using fine forceps, grab the face plate by the mouth, flip it up, and pin it to the wax plate with fine point forceps and a pin. If needed, remove the face plate completely.</li>
	<li>Pipet 20 &#181;L PBS onto the cut open section of the head. 
	NOTE: The glands may float up at this point or one must search for them. The glands look like a string of pearls and are located on top of the brain if intact.</li>
	<li>Use super fine point forceps to gently remove one of the hypopharyngeal glands (the gland may break, requiring it to be removed in segments), and immediately transfer the gland to the Giemsa stain on the microscopy slide.</li>
	<li>Allow the gland to incubate in the Giemsa solution for 5 min, and then use forceps to transfer the gland to the pool of saline on the same slide. If needed, cut the gland into smaller pieces with the micro scissors. 
	NOTE: This helps to make the glands lie in a flatter plane, thus making it easier to obtain clear images of the gland acini.</li>
</ol>
2. Measuring the hypopharyngeal Gland Acini
<ol>
	<li>Turn on the microscope and open the measurement program. Turn on the light source for the microscope if it is not already on. Set the microscope magnification to 10X.</li>
	<li>Find the glands and focus the magnified image in the eyepiece, without the computer. Increase the magnification to 60&#8211;80X and focus the image in the eyepiece.</li>
	<li>Under the &#39;Acquire&#39; tab, adjust and focus the glands on the live image on the computer.</li>
	<li>Type image name/sample description into the &#39;Image Name&#39; box and click &#39;Acquire Image&#39; to take an image of the glands for further measurements.</li>
	<li>Select the &#39;Analysis&#39; tab. Select the &#39;Area tool&#39; (highlighted in Supplemental Figure 1A) and unselect &#39;Value&#39; under &#39;Display Labels&#39;, which will also unselect &#39;Unit&#39;. Set all other settings to default (Supplemental Figure 1A). 
	Note: The software automatically calibrates the area measurements according to the level of magnification so that the area obtained from a lower magnification is the same as that obtained from a higher magnification.</li>
	<li>Measure each acinus by continuously clicking or continuously holding down the left mouse button while tracing the acinus perimeter as carefully as possible. Measure at least 10 acini per bee. 
	NOTE: More may be needed depending on the experiment (see the Discussion). It may require multiple images/sections of the gland to find enough clear, properly oriented acini for measurement.</li>
	<li>Once all of the acini from a single image have been measured, select &#39;Create Report&#39;. Ensure that options are selected as shown in Supplemental Figure 1B and click &#39;Export&#39;.</li>
	<li>Save the report as desired. If a file has already been created for the set of samples and additional measurements are being added, ensure that the report file is not open so that the new data are added to the existing file.</li>
	<li>When finished, turn off the microscope camera and light source. Wipe the liquid and glands off the microscopy slides and safely dispose of any used material. Rinse the slides with distilled water and wipe clean with ethanol (70% v/v in water) for reuse.</li>
</ol>

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M., Walton, A., Schwan, M. R., Anderson, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+markers+of+sub-optimal+nutrition+in+developing+Apis+mellifera+nurse+workers.">Transcriptional markers of sub-optimal nutrition in developing Apis mellifera nurse workers.</a> BMC Genomics. 15, 134 (2014).</li><li>Sagili, R. R., Pankiw, T., Zhu-Salzman, K. <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+soybean+trypsin+inhibitor+on+hypopharyngeal+gland+protein+content,+total+midgut+protease+activity+and+survival+of+the+honey+bee+(Apis+mellifera+L).">Effects of soybean trypsin inhibitor on hypopharyngeal gland protein content, total midgut protease activity and survival of the honey bee (Apis mellifera L).</a> Journal of Insect Physiology. 51 (9), 953-957 (2005).</li><li>Sagili, R. R., Pankiw, T. <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+protein-constrained+brood+food+on+honey+bee+(Apis+mellifera+L.)+pollen+foraging+and+colony+growth.">Effects of protein-constrained brood food on honey bee (Apis mellifera L.) pollen foraging and colony growth.</a> Behavioral Ecology and Sociobiology. 61 (9), 1471-1478 (2007).</li><li>Hrassnigg, N., Crailsheim, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+hypopharyngeal+gland+development+to+the+brood+status+of+honeybee+(Apis+mellifera+L.)+colonies.">Adaptation of hypopharyngeal gland development to the brood status of honeybee (Apis mellifera L.) colonies.</a> Journal of Insect Physiology. 44 (10), 929-939 (1998).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9, 671 (2012).</li><li>Jack, C. J., Uppala, S. S., Lucas, H. M., Sagili, R. R. <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+pollen+dilution+on+infection+of+Nosema+ceranae+in+honey+bees.">Effects of pollen dilution on infection of Nosema ceranae in honey bees.</a> Journal of Insect Physiology. 87, 12-19 (2016).</li><li>Crailsheim, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protein+balance+of+the+honey+bee+worker.">The protein balance of the honey bee worker.</a> Apidologie. 21, 417-429 (1990).</li></ol>

The authors have nothing to disclose.

Hypopharyngeal gland size is sensitive to the amount of protein and pollen in the diet and is a critical marker of nourishment in young adult bees. Here, we demonstrate an inexpensive and reproducible way to dissect and measure this tissue. These tissues can be difficult to dissect, but with practice, one can obtain increasingly cleaner dissections with the tissue relatively intact. The main advantage of the method presented here is that the tissue is stained, which enables the researcher to clearly visualize the borders of each gland acinus. Without a stain, the borders of these acini are difficult to visualize and focus under the microscope which lessens the researcher&#39;s ability to obtain an accurate acinus measurement. Despite the ease of staining and obtaining a clear image for the acini, several critical points should be considered in order to obtain accurate measurements; these are discussed below.
Large glands come from well-fed nurse bees that are approximately 7&#8211;10 days of age9,20. It is always easier, especially when learning to dissect these tissues, to first practice dissecting large glands before moving onto smaller glands as they can be small, fragile, and difficult to dissect. Fresh tissue also yields the best dissections. If one must freeze the bee for a period of time prior to the dissection, know that the longer that a bee is frozen, the more fragile the tissue becomes. This can make dissections problematic. With practice and sharp forceps, the researcher will eventually overcome these issues. We have not noticed a difference in staining between frozen and fresh tissue.
Fresh stain is necessary in order to obtain proper staining of the glands. Older stain can clump, leaving the stain unable to properly permeate the tissues. It is important that fresh stain be prepared in small batches, approximately every hour. This will ensure proper contrast between the tissue and the background under the microscope, which leads to sharp images with defined acinus edges. It is also helpful to work with a range of stain dilutions if the stain does not permeate the tissue properly. We found that a 1:20 fresh working stock of stain works best, but other dilutions can also work if one desires a darker or lighter stain.
We used a camera attached to the dissecting microscope and commercially available software to measure and record the acini data. The camera and software are somewhat costly and may therefore not be available to all labs. Although it is necessary to have a camera attached to the dissecting microscope in order to obtain the image and measure the acini there are several lower cost options, including free software18, that can be used.
Here, we show the basic steps for staining and measuring the hypopharyngeal gland acini, but stress that it up to the researcher to decide how many acini to measure, whether to measure acini on one or both glands, and whether to measure multiple areas of each gland. For example, in order to detect more subtle differences among experimental treatments, one might need to measure more than 10 acini per gland and more than 10 bees per treatment. We have not noticed any acini size differences based upon their locations on the gland. We have also not noticed any differences in sizes of acini that are located on the left or right glands. If the researcher suspects any size differences, multiple areas of the hypopharyngeal gland and perhaps both glands should be measured.
Researchers should be able to successfully obtain accurate measurements of hypopharyngeal gland size using the protocol described here for dissecting and staining gland tissue and measuring acini. With practice, these measurements can be obtained rather quickly, allowing the researcher to process multiple samples in one sitting. We expect that more researchers will employ the methods outlined here as they seek to further understand the factors that influence hypopharyngeal gland size, and how these glands relate to colony health and individual behavior.

Honey bees are essential for agriculture because they pollinate a variety of crops that are consumed by humans and animals. Much attention has been paid to the decline of honey bee populations as colony losses hover around 30-40% each year in the United States1 and 10&#8211;15% in Europe2,3. Multiple factors, including reduced access to high quality forage, likely act together to negatively impact honey bee health. Monoculture, drought, unsustainable beekeeping practices, and other factors decrease the diversity and amount of natural pollens available to colonies4,5. Because honey bees derive almost all of their dietary proteins and lipids from pollen, reduced access to pollen can severely limit individual and colony health.
The hypopharyngeal glands are secretory structures located in the bee&#39;s head between the eyes and the brain6. Under normal circumstances, the developmental and functional trajectory of the glands mirror that of the bee they are located in. At approximately 5&#8211;10 days of age, the bee performs nursing behaviors in the hive. At this same time, the hypopharyngeal glands reach their peak size and secretory capacity, producing the major protein fraction of the brood food or jelly fed to developing larvae and other adults, such as the queen. At this peak size the glands resemble a bunch of grapes where each grape is a discrete lobe structure known as the acinus (plural: acini). As the worker bee ages and takes on different tasks in the hive, the hypopharyngeal glands shrink and take on different functions, like breaking down sugars in nectar7,8. The hypopharyngeal glands are therefore correlated with the age of the bee and their age-associated task.
Nurse hypopharyngeal gland size is sensitive to the quantity and quality of protein in their diet9,10,11. When nurse bees are well-nourished, their glands are large. Whereas, the glands are small when the bee is deprived of pollen, particularly in the first week of adult development. In order to determine the nutritional status of a nurse bee, researchers typically measure the hypopharyngeal glands, either by directly measuring gland acinus size11,12,13,14 or protein content15,16 or by measuring the protein content11 or fresh weight17 of the entire head where they are located. Each method has its own pros and cons. We prefer the resolution obtained from measuring the gland acini, though this method can be challenging in two major ways. The first challenge is to properly identify and dissect the gland. The second is obtaining an accurate measure of each acinus. Under a dissecting light microscope, the glands appear clear or milky white and the borders of the acini can be difficult to define. Having tools to better define the edge of the acini, and to increase the likelihood of obtaining accurate gland measurements is beneficial to anyone studying honey bee nutrition.
Here, we show interested researchers how to dissect, stain, image, and measure hypopharyngeal glands so that accurate measurements of acinus size can be achieved. The method we describe offers researchers an easy, accurate, and replicable method for achieving multiple gland measurements in a relatively short period of time once the experimenter is sufficiently practiced. One could confidently measure the glands of almost 10 individuals in just over an hour. We offer details on both the method and materials needed to obtain these measurements. The most important aspects of the methods outlined below are the proper dissection and staining of the glands. Although we capture the magnified images and measure the acini with commercial software, the methods we present can easily be adapted to other platforms18.

<table><tbody><tr><td>Cool setting wax</td><td>Grobet USA</td><td>21.450</td><td></td></tr><tr><td>Glass petri dish, small</td><td>VWR</td><td>89000-310</td><td></td></tr><tr><td>Glass petri dish, large</td><td>VWR</td><td>89000-314</td><td></td></tr><tr><td>Super Max Wax Pen</td><td>Eurotool</td><td>PEN-520.00</td><td></td></tr><tr><td>Breakable razor blades</td><td>Electron Microscopy Sciences</td><td>72004</td><td></td></tr><tr><td>pin vise</td><td>BioQuip</td><td>4845</td><td></td></tr><tr><td>2A-SA flat/rounded tip forceps</td><td>Rubis/BioQuip</td><td>4522</td><td></td></tr><tr><td>Fine point forceps</td><td>Rubis/BioQuip</td><td>4523</td><td></td></tr><tr><td>5A-SA super fine point forceps</td><td>Rubis/BioQuip</td><td>4525</td><td></td></tr><tr><td>10 mm micro spring scissors</td><td>BioQuip</td><td>4715</td><td></td></tr><tr><td>3 mm micro spring Vannas scissors</td><td>Roboz</td><td>RS-5610</td><td></td></tr><tr><td>Glass Depression Slides, Single Cavity</td><td>GSC International</td><td>4-13057-DZ-12</td><td></td></tr><tr><td>PBS tablets</td><td>VWR</td><td>97062-730</td><td></td></tr><tr><td>Giemsa stain, modified solution</td><td>Sigma Aldrich</td><td>32884</td><td></td></tr><tr><td>Insect pins</td><td>ENTO SPHINX S.R.O.</td><td>02.02</td><td></td></tr><tr><td>Leica Applications Suite measurement software</td><td>Leica Microsystems</td><td></td><td>any measurement software, including free software, can be used</td></tr></tbody></table>

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.

Hypopharyngeal glands were dissected from nurse workers and visualized with and without stain at 60&#8211;80X magnification (Figure 1). In unstained tissue, it is difficult to find proper contrast to fully focus and define the edges of the acini. In the stained tissue, the edges of the acini are sharp because of the improved contrast between the stained tissue and the white background.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58261/58261fig1.jpg" /> 
Figure 1: Unstained (A, B) or stained (C, D) hypopharyngeal glands from nurse-aged workers. Note that while the glands are visible in both cases, the stained glands are more well-defined and therefore easier to measure. Also note that the somewhat curled and coiled morphology of the whole gland results in different focal planes. The gland can be sectioned to prevent this from occurring. Scale bars = 500 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58261/58261fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Honey bee workers were collected at &#8804;18 h after emergence and were assigned to two different dietary regimes: a diet of spring pollen naturally present in Tucson, Arizona, USA mixed with honey or just honey with no pollen. In order to restrict the bees to these diets while in the hive, push-in wire cages were used &#8212; as described in earlier studies13,14 &#8212; confining the bees at a density of approximately one bee per square centimeter. Each dietary treatment was repeated in three hives (N = 3). Bees exposed to either dietary regime were collected at 5 d and 8 d of age for analysis of their hypopharyngeal glands. Using the dissection, staining, and measuring methods outlined above, the hypopharyngeal gland acini of these bees were measured and compared (Figure 2). In each of the three hives, three bees were measured for each age x diet treatment combination. Ten acini were measured for each bee. The acini measurements were averaged for each bee to obtain an average acinus size per bee. These values were then averaged to obtain a value for the age x treatment combination for that hive. ANOVA analysis on the acinus measurements showed that diet (F1,8= 2.65, p = 0.001), age (F1,8 = 10.03, p = 0.013), and the interaction between diet x age (F1,8= 0.02, p = 0.020) had significant effects. We observed that the hypopharyngeal glands grew over time in the bees that were fed pollen, as determined by a Tukey&#39;s HSD. This pattern has been demonstrated previously9,11,19. The size of the gland acini did not differ between the 5 d and 8 days old bees when the bees were deprived of pollen.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58261/58261fig2.jpg" /> 
Figure 2: Hypopharyngeal gland sizes of well-fed nurses and those deprived of pollen. Workers were fed a diet of pollen and honey (gray bars) or a diet of honey alone (white bars) for 5 or 8 d. During this time, bees were inside of the hive and caged over honey or honey and pollen. Their hypopharyngeal gland acini were measured as described above. Error bars represent standard error for the mean acinus size across the three colonies (N = 3) tested. Three bees were measured from each colony for each treatment age x diet treatment combination. Bars connected by an asterisk are significantly different from each other according to a Tukey&#39;s HSD (&#945; = 0.05). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58261/58261fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Bees can also be maintained in cages separate from the hive and fed defined diets. Honey bee workers were collected at &#8804; 18 h after emergence and were assigned to acrylic glass cages (100 bees per cage) with one of four dietary regimes: a diet containing no pollen or a diet containing one of three bee-collected pollens: &#34;almond&#34; pollen from an almond orchard monoculture, &#34;desert&#34; pollen from the Sonoran Desert containing a mix of desert plants, or &#34;SE&#34; pollen from colonies located in the southeastern United States, as described in Corby-Harris et al.12. Sucrose (50% w/v), water, and pollen (where appropriate) were provided ad libitum. Five cages were constructed for each of the four dietary treatments, resulting in a total of 20 cages. At 8 d of age, the hypopharyngeal glands of ten bees were collected and measured. Ten acini were measured for each individual. The average acinus size was calculated for each bee and these values were used to obtain an average acinus size for each cage. We observed that hypopharyngeal gland size is sensitive to both the presence of pollen in the diet (pollen versus no pollen: t1 = 5.64, p &#60;0.0001) and the type of pollen provided (almond vs. desert vs. SE pollen: F2,148 = 8.06, p = 0.0005; Figure 3). Bees fed desert or almond pollen had equivalent gland sizes. Bees fed SE pollen had glands that were smaller than bees fed almond or desert pollen.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58261/58261fig3.jpg" /> 
Figure 3: Hypopharyngeal gland sizes of nurse-aged workers fed three different types of pollen or deprived of pollen. Nurse bees were placed in cages after emergence and were fed a diet consisting of sucrose alone (no pollen) or sucrose and one of three pollens (almond, desert, or SE pollen) until 8 d of age. Error bars represent standard error for the mean acinus size in bees sampled from five cages (N = 5). Ten bees were sampled and measured in each cage to obtain an average acinus size for the cage and to calculate the variation among cages. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58261/58261fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="S1" src="/files/ftp_upload/58261/S1.jpg" /> 
Supplementary File 1: Screen shots of measurement software while measuring the gland acini (A) and creating the report (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58261/S1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Measuring Hypopharyngeal Gland Acinus Size in Honey Bee Apis mellifera Workers at JoVE.com

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.

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 ...

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Nanyang Technological University

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11.0K Views.  USDA-ARS, Carl Hayden Bee Research Center. 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.

Video: Measuring Hypopharyngeal Gland Acinus Size in Honey Bee Apis mellifera Workers

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 ...

Neuroscience

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 ...

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 ...

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 ...

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 ...

Biology

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 ...

Assessing Agrochemical Risk to Mated Honey Bee Queens

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not accurately capture the effects of agrochemical exposure on honey bee queens. As the sole producer of fertilized eggs inside a honeybee colony, the queen is arguably the most important single member of a functioning colony unit. Therefore, understanding how agrochemicals affect queen health and productivity should be considered a critical aspect of pesticide risk assessment. Here, an adapted method is presented to expose honey bee queens and worker queen attendants to agrochemical stressors administered through a worker diet, followed by tracking egg production in the laboratory and assessing first instar eclosion using a specialized cage, referred to as a Queen Monitoring Cage. To illustrate the method&#39;s intended use, results of an experiment in which worker queen attendants were fed diet containing sublethal doses of imidacloprid and effects on queens were monitored are described.

This protocol was developed to enhance the understanding of how agrochemicals affect honey bee (Apis mellifera) reproduction by establishing methods to expose honey bee queens and their worker caretakers to agrochemicals in a controlled, laboratory setting and carefully monitoring their relevant responses.

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not ...

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

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

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

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

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

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

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

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

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

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

Assessing Agrochemical Risk to Mated Honey Bee Queens