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

Published: September 14th, 2018

DOI:

10.3791/58261

1USDA-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.

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.

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 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

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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 KH2PO<.......

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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.

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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.......

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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.

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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

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