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>

<ol>
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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>

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

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. Radioisotope tracing is a major technique used to measure such fluxes, both within plants, and between plants and their environments. Flux data obtained with radiotracer protocols can help elucidate the capacity, mechanism, regulation, and energetics of transport systems for specific mineral nutrients or toxicants, and can provide insight into compartmentation and turnover rates of subcellular mineral and metabolite pools. Here, we describe two major radioisotope protocols used in plant biology: direct influx (DI) and compartmental analysis by tracer efflux (CATE). We focus on flux measurement of potassium (K+) as a nutrient, and ammonia/ammonium (NH3/NH4+) as a toxicant, in intact seedlings of the model species barley (Hordeum vulgare L.). These protocols can be readily adapted to other experimental systems (e.g., different species, excised plant material, and other nutrients/toxicants). Advantages and limitations of these protocols are discussed.

In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. ...

A Push-pull Protocol to Reduce Colonization of Bird Nest Boxes by Honey Bees

Introduction of the invasive Africanized honey bee (AHB) into the Neotropics is a serious problem for many cavity nesting birds, specifically parrots. These bees select cavities that are suitable nest sites for birds, resulting in competition. The difficulty of removing bees and their defensive behavior makes a prevention protocol necessary. Here, we describe a push-pull integrated pest management protocol to deter bees from inhabiting bird boxes by applying a bird safe insecticide, permethrin, to repel bees from nest boxes, while simultaneously attracting them to pheromone-baited swarm traps. Shown here is an example experiment using Barn Owl nest boxes. This protocol successfully reduced colonization of Barn Owl nest boxes by Africanized honey bees. This protocol is flexible, allowing adjustments to accommodate a wide range of bird species and habitats. This protocol could benefit conservation efforts where AHB are located.

Preventing colonization of bird nest boxes by invasive Africanized honey bees is important for conservation efforts of nest-site limited birds. We provide an integrated pest management approach to &#34;push&#34; bees away from nest boxes with a repellant insecticide, permethrin, and &#34;pull&#34; them toward pheromone baited swarm traps.

Introduction of the invasive Africanized honey bee (AHB) into the Neotropics is a serious problem for many cavity nesting birds, specifically parrots. ...

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 ...

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 ...

Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees

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

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

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

An Ultra-clean Multilayer Apparatus for Collecting Size Fractionated Marine Plankton and Suspended Particles

The distributions of many trace elements in the ocean are strongly associated with the growth, death, and re-mineralization of marine plankton and those of suspended/sinking particles. Here, we present an all plastic (Polypropylene and Polycarbonate), multi-layer filtration system for collection of suspended particulate matter (SPM) at sea. This ultra-clean sampling device has been designed and developed specifically for trace element studies. Meticulous selection of all non-metallic materials and utilization of an in-line flow-through procedure minimizes any possible metal contamination during sampling. This system has been successfully tested and tweaked for determining trace metals (e.g., Fe, Al, Mn, Cd, Cu, Ni) on particles of varying size in coastal and open ocean waters. Results from the South China Sea at the South East Asia Time-Series (SEATS) station indicate that diurnal variations and spatial distribution of plankton in the euphotic zone can be easily resolved and recognized. Chemical analysis of size-fractionated particles in surface waters of the Taiwan Strait suggests that the larger particles (&#62;153 &#181;m) were mostly biologically derived, while the smaller particles (10 - 63 &#181;m) were mostly composed of inorganic matter. Apart from Cd, the concentrations of metals (Fe, Al, Mn, Cu, Ni) decreased with increasing size.

Plankton and suspended particles play a major role in the biogeochemical cycles in the ocean. Here, we provide an ultra-clean, low stress method for the collection of various sizes of particles and plankton at sea with the capability of handling large volumes of seawater.

The distributions of many trace elements in the ocean are strongly associated with the growth, death, and re-mineralization of marine plankton and those ...

Experimental Study of the Relationship Between Particle Size and Methane Sorption Capacity in Shale

The amount of adsorbed shale gas is a key parameter used in shale gas resource evaluation and target area selection, and it is also an important standard for evaluating the mining value of shale gas. Currently, studies on the correlation between particle size and methane adsorption are controversial. In this study, an isothermal adsorption apparatus, the gravimetric sorption analyzer, is used to test the adsorption capacity of different particle sizes in shale to determine the relationship between the particle size and the adsorption capacity of shale. Thegravimetric method requires fewer parameters and produces better results in terms of accuracy and consistency than methods like the volumetric method. Gravimetric measurements are performed in four steps: a blank measurement, preprocessing, a buoyancy measurement, and adsorption and desorption measurements. Gravimetric measurement is presently considered to be a more scientific and accurate method of measuring the amount of adsorption; however, it is time-consuming and requires a strict measuring technique. A Magnetic Suspension Balance (MSB) is the key to verify the accuracy and consistency of this method. Our results show that adsorption capacity and particle size are correlated, but not a linear correlation, and the adsorptions in particles sieved into 40 - 60 and 60 - 80 meshes tend to be larger. We propose that the maximum adsorption corresponding to the particle size is approximately 250 &#181;m (60 mesh) in the shale gas fracturing.

We use an isothermal adsorption apparatus, the gravimetric sorption analyzer, to test the adsorption capacity of different particle sizes of shale, in order to find out the relationship between particle size and the adsorption capacity of shale.

The amount of adsorbed shale gas is a key parameter used in shale gas resource evaluation and target area selection, and it is also an important standard ...

Combined Size and Density Fractionation of Soils for Investigations of Organo-Mineral Interactions

Combined size and density fractionation (CSDF) is a method used to physically separate soil into fractions differing in particle size and mineralogy. CSDF relies on sequential density separation and sedimentation steps to isolate (1) the free light fraction (uncomplexed organic matter), (2) the occluded light fraction (uncomplexed organic matter trapped in soil aggregates) and (3) a variable number of heavy fractions (soil minerals and their associated organic matter) differing in composition. Provided that the parameters of the CSDF (dispersion energy, density cut-offs, sedimentation time) are properly selected, the method yields heavy fractions of relatively homogeneous mineral composition. Each of these fractions is expected to have a different complexing ability towards organic matter, rendering this a useful method to isolate and study the nature of organo-mineral interactions. Combining density and particle size separation brings an improved resolution compared to simple size or density fractionation methods, allowing the separation of heavy components according to both mineralogy and size (related to surface area) criteria. As is the case for all physical fractionation methods, it may be considered as less disruptive or aggressive than chemically-based extraction methods. However, CSDF is a time-consuming method and furthermore, the quantity of material obtained in some fractions can be limiting for subsequent analysis. Following CSDF, the fractions may be analyzed for mineralogical composition, soil organic carbon concentration and organic matter chemistry. The method provides quantitative information about organic carbon distribution within a soil sample and brings light to the sorptive capacity of the different, naturally-occurring mineral phases, thus providing mechanistic information about the preferential nature of organo-mineral interactions in soils (i.e., which minerals, what type of organic matter).

Combined size and density fractionation (CSDF) is a method to physically separate soil into fractions differing in texture (particle size) and mineralogy (density). The purpose is to isolate fractions with different reactivities towards soil organic matter (SOM), in order to better understand organo-mineral interactions and SOM dynamics.

Combined size and density fractionation (CSDF) is a method used to physically separate soil into fractions differing in particle size and mineralogy. CSDF ...

Measuring the Shape and Size of Activated Sludge Particles Immobilized in Agar with an Open Source Software Pipeline

Experimental bioreactors, such as those treating wastewater, contain particles whose size and shape are important parameters. For example, the size and shape of activated sludge flocs can indicate the conditions at the microscale, and also directly affect how well the sludge settles in a clarifier.
Particle size and shape are both misleadingly &#39;simple&#39; measurements. Many subtle issues, often unaddressed in informal protocols, can arise when sampling, imaging, and analyzing particles. Sampling methods may be biased or not provide enough statistical power. The samples themselves may be poorly preserved or undergo alteration during immobilization. Images may not be of sufficient quality; overlapping particles, depth of field, magnification level, and various noise can all produce poor results. Poorly specified analysis can introduce bias, such as that produced by manual image thresholding and segmentation.
Affordability and throughput are desirable alongside reproducibility. An affordable, high throughput method can enable more frequent particle measurement, producing many images containing thousands of particles. A method that uses inexpensive reagents, a common dissecting microscope, and freely-available open source analysis software allows repeatable, accessible, reproducible, and partially-automated experimental results. Further, the product of such a method can be well-formatted, well-defined, and easily understood by data analysis software, easing both within-lab analyses and data sharing between labs.
We present a protocol that details the steps needed to produce such a product, including: sampling, sample preparation and immobilization in agar, digital image acquisition, digital image analysis, and examples of experiment-specific figure generation from the analysis results. We have also included an open-source data analysis pipeline to support this protocol.

The size and shape of particles in activated sludge are important parameters that are measured using varying methods. Inaccuracies arise from non-representative sampling, suboptimal images, and subjective analysis parameters. To minimize these errors and ease measurement, we present a protocol specifying every step, including an open source software pipeline.

Experimental bioreactors, such as those treating wastewater, contain particles whose size and shape are important parameters. For example, the size and ...

Modeling the Size Spectrum for Macroinvertebrates and Fishes in Stream Ecosystems

The size spectrum is an inverse, allometric scaling relationship between average body mass (M) and the density (D) of individuals within an ecological community or food web. Importantly, the size spectrum assumes that individual size, rather than species&#8217; behavioral or life history characteristics, is the primary determinant of abundance within an ecosystem. Thus, unlike traditional allometric relationships that focus on species-level data (e.g., mean species&#8217; body size vs. population density), size spectra analyses are &#8216;ataxic&#8217; &#8211; individual specimens are identified only by their size, without consideration of taxonomic identity. Size spectra models are efficient representations of traditional, complex food webs and can be used in descriptive as well as predictive contexts (e.g., predicting responses of large consumers to changes in basal resources). Empirical studies from diverse aquatic ecosystems have also reported moderate to high levels of similarity in size spectra slopes, suggesting that common processes may regulate the abundances of small and large organisms in very different settings. This is a protocol to model the community-level size spectrum in wadable streams. The protocol consists of three main steps. First, collect quantitative benthic fish and invertebrate samples that can be used to estimate local densities. Second, standardize the fish and invertebrate data by converting all individuals to ataxic units (i.e., individuals identified by size, irrespective of taxonomic identity), and summing individuals within log2 size bins. Third, use linear regression to model the relationship between ataxic M and D estimates. Detailed instructions are provided herein to complete each of these steps, including custom software to facilitate D estimation and size spectra modeling.

This is a protocol to model the size spectrum (scaling relationship between individual mass and population density) for combined fish and invertebrate data from wadable streams and rivers. Methods include: field techniques to collect quantitative fish and invertebrate samples; lab methods to standardize the field data; and statistical data analysis.

The size spectrum is an inverse, allometric scaling relationship between average body mass (M) and the density (D) of individuals within an ecological ...