Name: measuring hypopharyngeal gland acinus size in honey bee (apis mellifera) workers
Uploaded: 2018-09-14T14:00:00
Description: Watch this Scientific Journal Video about Measuring Hypopharyngeal Gland Acinus Size in Honey Bee Apis mellifera Workers at JoVE.com

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 KH2PO<...

<ol>
	<li>Kulhanek, K., 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+national+survey+of+managed+honey+bee+2015-2016+annual+colony+losses+in+the+USA.">A national survey of managed honey bee 2015-2016 annual colony losses in the USA.</a> Journal of Apicultural Research. 56 (4), 328-340 (2017).</li><li>Jacques, A., 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+pan-European+epidemiological+study+reveals+honey+bee+colony+survival+depends+on+beekeeper+education+and+disease+control.">A pan-European epidemiological study reveals honey bee colony survival depends on beekeeper education and disease control.</a> PLoS One. 12 (3), e0172591 (2017).</li><li>Zee, R. v. d., 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=Results+of+international+standardised+beekeeper+surveys+of+colony+losses+for+winter+2012-2013:+analysis+of+winter+loss+rates+and+mixed+effects+modelling+of+risk+factors+for+winter+loss.">Results of international standardised beekeeper surveys of colony losses for winter 2012-2013: analysis of winter loss rates and mixed effects modelling of risk factors for winter loss.</a> Journal of Apicultural Research. 53 (1), 19-34 (2014).</li><li>Decourtye, A., Mader, E., Desneux, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Landscape+enhancement+of+floral+resources+for+honey+bees+in+agro-ecosystems.">Landscape enhancement of floral resources for honey bees in agro-ecosystems.</a> Apidologie. 41 (3), 264-277 (2010).</li><li>Vaudo, A. D., Tooker, J. F., Grozinger, C. M., Patch, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bee+nutrition+and+floral+resource+restoration.">Bee nutrition and floral resource restoration.</a> Current Opinion in Insect Science. 10, 133-141 (2015).</li><li>Snodgrass, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anatomy of the Honey Bee. , (1984).</li><li>Winston, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Biology of the Honey Bee. , (1987).</li><li>Johnson, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Division+of+labor+in+honeybees:+form,+function,+and+proximate+mechanisms.">Division of labor in honeybees: form, function, and proximate mechanisms.</a> Behavioral Ecology and Sociobiology. 64 (3), 305-316 (2010).</li><li>Crailsheim, K., Stolberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+diet,+age+and+colony+condition+upon+intestinal+proteolytic+activity+and+size+of+the+hypopharyngeal+glands+in+the+honeybee+(Apis+mellifera+L).">Influence of diet, age and colony condition upon intestinal proteolytic activity and size of the hypopharyngeal glands in the honeybee (Apis mellifera L).</a> Journal of Insect Physiology. 35 (8), 595-602 (1989).</li><li>Pernal, S. F., Currie, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pollen+quality+of+fresh+and+1-year-old+single+pollen+diets+for+worker+honey+bees+(Apis+mellifera+L).">Pollen quality of fresh and 1-year-old single pollen diets for worker honey bees (Apis mellifera L).</a> Apidologie. 31 (3), 387-409 (2000).</li><li>DeGrandi-Hoffman, G., Chen, Y., Huang, E., Huang, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+diet+on+protein+concentration,+hypopharyngeal+gland+development+and+virus+load+in+worker+honey+bees+(Apis+mellifera+L).">The effect of diet on protein concentration, hypopharyngeal gland development and virus load in worker honey bees (Apis mellifera L).</a> Journal of Insect Physiology. 56 (9), 1184-1191 (2010).</li><li>Corby-Harris, V., Snyder, L., Meador, C., Ayotte, T. <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+(Apis+mellifera)+nurses+do+not+consume+pollens+based+on+their+nutritional+quality.">Honey bee (Apis mellifera) nurses do not consume pollens based on their nutritional quality.</a> PLoS One. 13 (1), e0191050 (2018).</li><li>Corby-Harris, V., 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=Transcriptional,+translational,+and+physiological+signatures+of+undernourished+honey+bees+(Apis+mellifera)+suggest+a+role+for+hormonal+factors+in+hypopharyngeal+gland+degradation.">Transcriptional, translational, and physiological signatures of undernourished honey bees (Apis mellifera) suggest a role for hormonal factors in hypopharyngeal gland degradation.</a> Journal of Insect Physiology. 85, 65-75 (2016).</li><li>Corby-Harris, V., Jones, B. 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>

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...

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="Fig...

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 ...

This method can help answer key questions in the honey bee nutrition field, about the link between poor nutrition and hypopharyngeal gland growth and development. The main advantage of this technique is that one can use it to easily stain, visualize, and measure bee hypopharyngeal glands. Generally, individuals new to this method will struggle because the hypopharyngeal glands can be difficult to locate and dissect without visual guidance.Before beginning the procedure, pour melted cool setting wax in a glass Petri dish. Add 20 microliters of freshly prepared Giemsa stain into one well, and 50 to 100 microliters of saline into an adjacent well of one microscope slide, per nurse worker bee to be processed. When the wax has set, use 10 millimeter micro spring scissors to remove the head from the first bee and use forceps, a dissecting microscope, and a wax carving pen to embed the head front-side-up in the wax dissection plate.Insert pins in the eyes and mouth to secure the head to the plate, and use a sharp, breakable razor blade fixed into a pin vise to make a two to three millimeter 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 antenna and the brain. Using fine forceps, grab the face plate by the mouth, and pin the tissue face-side-down to the wax with fine point forceps and a pin.Next, pipette 20 microliters of PBS onto the open section of the head. The hypopharyngeal glands look like a string of pearls and are located on top of the brain if intact. Using super fine point forceps, gently harvest one of the glands for immediate immersion in the Giemsa stain on the microscopy slide.After a five minute incubation, transfer the gland to the pool of saline on the same slide, cutting the gland into smaller pieces under the microscope with the micro scissors as necessary. To measure the glands, open the measurement program for the light microscope, and select the 10 times magnification. Place the slide onto the microscope stage and manually locate the glands through the eye piece.Increase the magnification to 60 to 80 times and manually adjust the focus of the glands at the new magnification. Further adjust the focus of the glands on the live image on the monitor and enter the image name and sample description into image name box. Click Acquire Image to obtain an image of the glands and under the Analysis tab, select the Area tool and un-select Value under Display tabs.Unit will also be un-selected. Set all of the other settings to default. To measure the acini, continuously click or hold down the left mouse button while tracing one acinus perimeter at a time as carefully as possible.Once all of the acini from a single image have been measured select Create Report, confirming that the appropriate options are selected, and click Export. Then save the report in a suitable format and clean up the work area and materials as appropriate. It is difficult to find a proper contrast and defined edges of the acini in unstained tissues.In stained hypopharyngeal glands, the edges of the acini are sharp because of the improved contrast between the stained tissue and the white background. The hypopharyngeal glands grow over time in bees that are fed honey mixed with pollen, compared to bees that are fed honey alone. Hypopharyngeal gland size is also sensitive to the type of pollen provided.For example, bees fed desert or almond pollen demonstrate glands of an equivalent size, while bees fed southeastern pollen diets exhibit glands that are larger than honey alone fed bees, but smaller than glands from bees fed either almond or desert pollen. While attempting this procedure, it's important to remember to carefully dissect the glands and to not over-or under-stain them. Don't forget that working with Giemsa stain can be hazardous and that precautions, such as keeping the stain away from flames, using proper disposal, and wearing gloves and eye protection should always be taken while performing this procedure.

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