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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 10–15% in Europe^2,3. 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 colonies^4,5. 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'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 5–10 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 nectar^7,8. 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 diet^9,10,11. 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 size^11,12,13,14 or protein content^15,16 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.

Protokół

1. Dissecting and Staining the Hypopharyngeal Glands from Nurse Workers

1. 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.
2. For each bee to be processed, prepare 20 µL 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’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.
3. Pipet 20 µL Giemsa stain into the wells of the microscope slides and 50–100 µL saline onto the slide adjacent to the well.
4. 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.
5. Using a sharp breakable razor blade fixed into a pin vise, make a small (~2–3 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.
6. 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.
7. Pipet 20 µL 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.
8. 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.
9. 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.

2. Measuring the hypopharyngeal Gland Acini

1. 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.
2. Find the glands and focus the magnified image in the eyepiece, without the computer. Increase the magnification to 60–80X and focus the image in the eyepiece.
3. Under the 'Acquire' tab, adjust and focus the glands on the live image on the computer.
4. Type image name/sample description into the 'Image Name' box and click 'Acquire Image' to take an image of the glands for further measurements.
5. Select the 'Analysis' tab. Select the 'Area tool' (highlighted in Supplemental Figure 1A) and unselect 'Value' under 'Display Labels', which will also unselect 'Unit'. 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.
6. 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.
7. Once all of the acini from a single image have been measured, select 'Create Report'. Ensure that options are selected as shown in Supplemental Figure 1B and click 'Export'.
8. 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.
9. 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.

Wyniki

Hypopharyngeal glands were dissected from nurse workers and visualized with and without stain at 60–80X 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.

Dyskusje

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...

Materiały

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