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

Summary

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.

Abstract

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.

Introduction

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 colonies⁴

Protocol

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....

Results

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.

Discussion

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.......

Materials

List of materials used in this article

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