I gratefully acknowledge the support of the Slovenian Research Agency, grant no. P4-133.

1. Basic histology for honeybee research
<ol>
	<li>Dissection of honeybee tissue 
	NOTE: For the dissection of worker bees, use a dissecting microscope with an LED light source. The most useful magnification is ~20x.
	<ol>
		<li>Manipulation and dissection
		<ol>
			<li>Carefully take a worker bee with forceps and put it on ice (or into the freezer at -20 &#176;C) for 2 min to immobilize it22. Pin the bee on the Petri dish diagonally through the uppermost back portion of the thorax twice, from left to right and from right to left.</li>
			<li>Pour insect saline to cover the body. Place the Petri dish under the microscope, focus, and adjust.</li>
			<li>Prepare the instruments (see the Table of Materials).</li>
		</ol>
		</li>
		<li>Dissection of midgut
		<ol>
			<li>Start with the abdomen by inserting one point of the scissors under the tergite A5 (Figure 1) in the center of the right side of the bee body. Cut to the tergite A2.</li>
			<li>Keep the inner blade of the scissors parallel with the side of the body to avoid damaging the internal organs. Turn the scissors left and make one cut; turn right and make another cut. Gently open the left part of the abdomen and pin it. Repeat on the other side.</li>
			<li>Using forceps with one hand, gently pull the honeybee stomach upward, and with scissors in the other hand, cut at the very end of the esophagus. Pull the stomach and midgut away from the abdomen and cut at the rectum. Use a pipette with insect saline solution and remove any feces or parts of the tissue.</li>
		</ol>
		</li>
		<li>Dissection of HPGs
		<ol>
			<li>Immobilize a worker bee on ice as described in step 1.1.1. Cut the head off and place it on the smaller plate with the antennae facing up. Secure the head with two pins: one through the left compound eye and the second through the right compound eye.</li>
			<li>Make a cut across the first compound eye on the inner side of the pins, continue to the labrum, and then make another cut on the other side across the second compound eye (Figure 2).</li>
			<li>Cut off the antennae. Lift off the mask and cut where still attached. Take the forceps and carefully remove the glands together with the brain and part of the compound eyes.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Fixation, dehydration, and paraffin embedding 
	NOTE: Wear protective gloves.
	<ol>
		<li>Place the tissue in penicillin bottles, filled 3/4 with 10% formalin. Keep in a refrigerator at 4 &#176;C.</li>
		<li>After 24 h, dehydrate the tissue in a series of alcohols: 70%, 80%, 90%, 100%, for 1 h each, 100% 2-propanol for 1 h, 100% 2-propanol for 12 h, and finally 100% 2-propanol for 1 h.</li>
		<li>Place the tissue in histocassettes; mark and place them in the glass chambers with 2-propanol and paraffin (1:1) in an incubator at 60 &#176;C for 24 h.</li>
		<li>Move the histocassettes to another chamber with paraffin (I.) for another 24 h. Repeat the procedure with fresh paraffin twice more (II. and III.), both for 24 h.</li>
		<li>Finally, prepare the mounting station and start embedding the tissue into wax.
		<ol>
			<li>Open each histocassette and remove the cover. Fill the mold with wax and carefully put the tissue with warm forceps in the middle of the mold.</li>
			<li>Place the histocassette on the mold and slightly cover it with wax. Immediately place the mold on the cold surface of the mounting station for a few seconds, then place it on the cold plate for a few minutes until the wax hardens and separates from the mold together and the histocassette.</li>
		</ol>
		</li>
		<li>Store the finished samples in a box, away from dust and heat.</li>
		<li>Cut 4 &#181;m thin sections on a microtome: first, two sections attached to each other and then one separately. Transfer the sections with forceps and let them float on distilled water (42 &#176;C), then collect them on clean slides by placing two sections together on the left side of the objective glass and the third one on the right side, remaining distinctly separate. Leave the marked slides overnight on the heating device and finally store them in a box dedicated for histology samples.</li>
	</ol>
	</li>
	<li>Dewaxing and rehydration 
	NOTE: Wear protective gloves.
	<ol>
		<li>Prepare nine Coplin jars and put the sections into a series of clearing agents (I., II., III.) for 5 min each.</li>
		<li>Put into 2-propanol, ethanol 96% (I., II.), alcohol 90% and 80%, and distilled water for 3 min each.</li>
	</ol>
	</li>
	<li>Dyeing with hematoxylin and eosin 
	NOTE: Wear protective gloves.
	<ol>
		<li>Prepare six Coplin jars.</li>
		<li>For hematoxylin and eosin (H&#38;E) staining, put the dewaxed, rehydrated sections in hematoxylin for 5 min, then carefully place them under the running tap water for 2 min. Then put them into distilled water for 1 min and eosin for 4 min (for eosin, the Coplin jar is not necessary).</li>
		<li>Place the slides in ethanol 96% for 1 min, then 2-propanol for 2 min, and finally into the clearing agent for 2 min.</li>
		<li>Add mounting medium and a cover glass and let them dry. Observe under a light microscope.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64141/64141fig01.jpg" /> 
Figure 1: Dorsal view of honeybee body. A1-A7 tergites. The detailed instructions on honeybee dissection can be found in Carreck et al.24. <a href="https://www.jove.com/files/ftp_upload/64141/64141fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64141/64141fig02v2.jpg" /> 
Figure 2: Dorsal view of HPGs, parts of compound eyes attached to the brain (not visible). A young worker bee aged 5 to 6 days has plump and creamy white HPGs. The acini are located on the brain and fill the head area with branches reaching the back of the brain. In foraging bees, these glands are greatly shrunken and leave only thin thread-like remains. For this reason, it is better to remove glands together with the brain to make it easier in further procedures to avoid losing the tissue. Scale bar = 500 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64141/64141fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Cell death detection in tissue sections
<ol>
	<li>Apoptosis detection kit (Assay A) 
	NOTE: Follow the manufacturer&#39;s protocol (see the Table of Materials).
	<ol>
		<li>Prepare the Coplin jars.</li>
		<li>After dewaxing and rehydration (see step 1.3), immerse the slides in 0.85% NaCl solution, and then in phosphate-buffered saline (PBS) (5 min).</li>
		<li>Put the slides in 4% paraformaldehyde 2 x 15 min.</li>
		<li>Place the slides flat in the container and add 100 &#181;L of a Proteinase K (20 &#181;g/mL) solution, then leave them for 10-30 min.</li>
		<li>Place the slides in PBS (5 min).</li>
		<li>Place the slides in 4% paraformaldehyde in PBS (5 min).</li>
		<li>Immerse the slides in PBS (2 x 5 min).</li>
		<li>Place the slides flat in the container, add 100 &#181;L of equilibration buffer, and leave them for 5-10 min.</li>
		<li>Add 100 &#181;L of TdT reaction mix. Put paper towels inside the container, around the slides, moisten the towels with water, and then cover with plastic wrap. Incubate slides for 60 min at 37 &#176;C.</li>
		<li>Place the slides back in the staining rack and immerse in 2x saline-sodium citrate (SSC) for 15 min.</li>
		<li>Immerse the slides 3 x 5 min in PBS, then in 0.3% hydrogen peroxide for 3-5 min, and then in PBS again, 3 x 5 min.</li>
		<li>Again, place the slides flat in the container, add 100 &#181;L of Streptavidin HRP (horseradish peroxidase), and leave for 30 min (cover with plastic wrap).</li>
		<li>Immerse the slides 3 x 5 min in PBS.</li>
		<li>Place the slides flat in the container and add 100 &#181;L of 3,3&#39;-diaminobenzidine (DAB) solution. Look for a light brown background.</li>
		<li>Return the slides to the rack and wash them several times in water (double-distilled).</li>
		<li>Mount the slides under glass coverslips in mounting medium and leave flat to dry.</li>
		<li>Observe under a light microscope.</li>
	</ol>
	</li>
	<li>Apoptosis detection kit (Assay B) 
	NOTE: Follow the manufacturer&#39;s protocol (see the Table of Materials).
	<ol>
		<li>Prepare Coplin jars.</li>
		<li>Prepare Proteinase K (20 &#181;g/mL diluted in PBS).</li>
		<li>After dewaxing and rehydrating the sections (step 1.3), place the slides in PBS for 5 min.</li>
		<li>Place the slides flat in the container and add Proteinase K (20 &#181;g/mL, 60 &#181;L per 5 cm&#178; specimen).</li>
		<li>Wash the slides 2 x 2 min in distilled water.</li>
		<li>Quench in endogenous peroxidase (in 3% hydrogen peroxidase) at room temperature.</li>
		<li>Rinse the slides 2 x 5 min with PBS or water.</li>
		<li>Place the slides flat in the container and apply equilibration buffer (75 &#181;L/5 cm2) for 10 s at room temperature.</li>
		<li>Carefully wipe around the tissue.</li>
		<li>Add the TdT enzyme (terminal deoxynucleotidyl transferase) to each section and incubate in a humidified chamber for 1 h at 37 &#176;C. Put paper towels inside the tray, around the slides, moisten the towels with water, and cover them with plastic wrap.</li>
		<li>After incubation, put the specimens in the rack and leave them in stop/wash buffer (10 min).</li>
		<li>Warm the anti-digoxigenin conjugate to room temperature.</li>
		<li>Wash the slides in PBS (3 x 1 min).</li>
		<li>Carefully wipe off around the tissue.</li>
		<li>Add two drops of Anti-Digoxigenin-Peroxidase conjugate (65 &#181;L/5 cm&#178;) to the sections and incubate for 30 min in a humidified container.</li>
		<li>After washing in PBS 4 x 2 min, prepare working-strength peroxidase substrate, and gently tap off excess liquid and aspirate around the section.</li>
		<li>Cover the sections with peroxidase substrate (75 &#181;L/5 cm&#178;) and stain for 5 min. Place a slide under the microscope and determine the optimal staining time.</li>
		<li>Wash the slides in a staining rack in distilled water (3 x 1 min).</li>
		<li>Incubate the slides in distilled water for 5 min.</li>
		<li>Counterstain using hematoxylin for 2 min.</li>
		<li>Place the slide under running tap water for 3 min.</li>
		<li>Wash the slide in distilled water.</li>
		<li>Mount the slides under glass coverslips in mounting medium and leave flat to dry.</li>
		<li>Observe under a light microscope.</li>
	</ol>
	</li>
	<li>Apoptosis detection kit (Assay C) 
	NOTE: Follow the manufacturer&#39;s protocol (see the Table of Materials).
	<ol>
		<li>Prepare the Coplin jars.</li>
		<li>Dewax and rehydrate the tissue sections (see step 1.3).</li>
		<li>Incubate the tissue with Proteinase K (15-30 min at 37 &#176;C).</li>
		<li>Place the slides back on the rack and rinse 2x in PBS.</li>
		<li>Cover with 50 &#181;L of &#39;TUNEL reaction mixture&#39;. Place wet paper towels inside the container, cover them with plastic wrap, and leave them for 60 min at 37 &#176;C.</li>
		<li>Rinse 3x with PBS.</li>
		<li>Place the slides in the container and dry the area around the tissue sample.</li>
		<li>Add 50 &#181;L of Converter-AP to the sample and incubate in a humidified container for 30 min at 37 &#176;C.</li>
		<li>Rinse 3x in PBS.</li>
		<li>Add 50-100 &#181;L of substrate solution and leave for 10 min in the dark. 
		NOTE: Observe the staining under a light microscope.</li>
		<li>Rinse the slides 3x with PBS.</li>
		<li>Counterstain by transferring sections into hematoxylin for 2 min and then carefully rinse in running tap water for 5 min.</li>
		<li>Mount the slides under glass coverslips in an aqueous mounting medium and leave them flat to dry.</li>
		<li>Observe under a light microscope. Assess the affected (positive) cells by counting 70 to 100 cells in each sample of the midgut or HPGs under a light microscope.</li>
	</ol>
	</li>
</ol>

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D., Ciocca, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+detection+of+apoptotic+cells+using+a+modified+in+situ+TUNEL+technique.">Improved detection of apoptotic cells using a modified in situ TUNEL technique.</a> Journal of Histochemistry and Cytochemistry. 47 (6), 837-839 (1999).</li><li>Gregorc, A., Bowen, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmed+cell+death+in+the+honeybee+(Apis+mellifera+L.)+larvae+midgut.">Programmed cell death in the honeybee (Apis mellifera L.) larvae midgut.</a> Cell Biology International. 21 (3), 151-158 (1997).</li><li>Gregorc, A., Poga&#269;nik, A., Bowen, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+in+honey+bee+(Apis+mellifera)+larvae+treated+with+oxalic+or+formic+acid.">Cell death in honey bee (Apis mellifera) larvae treated with oxalic or formic acid.</a> Apidologie. 35 (5), 453-460 (2004).</li></ol>

The author has no conflicts of interest.

In living organisms, cell death is defined as apoptosis or necrosis25&#160;and can be accompanied by autophagy26. The difference between apoptotic and necrotic cells is that apoptosis is a form of programmed cell death and appears in normal cells, whereas necrosis occurs due to lethal conditions (e.g., accident, disease)27,28. Apoptosis can be detected using assay kits based on the TUNEL technique (detection ofDNA f...

Honeybees (Apis mellifera L.) are, among other wild pollinators, the most important pollinators of agricultural plants. Over thousands of years, the changing environment has influenced bees to adapt their morphology, physiology, behavior, and tolerance to several pathogens and parasites. Therefore, honeybees have developed a highly diverse range of species and subspecies around the globe1. These results are consistent with previous findings, that there is genetic variation in the honeybee&#39;s digestive tract structure, but also suggest that alterations of the midgut are due to environmental factors2,3.
The digestive tract of the honeybee has three main parts: foregut, midgut (ventriculus), and hindgut4. The ventriculus is an essential organ for the digestion of pollen and nectar/honey; in the hindgut, osmotic control takes place through absorption of water and ions2. The hypopharyngeal glands (HPGs) of honeybee workers are located in the head and synthesize and secrete royal jelly components to feed the brood, the queen, and members of the colony. Their size changes with age and tasks and depends on proper nutrition (quality pollen). Nurse workers aged 6 to 18 days perform brood rearing, and the size of HPGs increases5,6. In forager bees, the HPGs degenerate and only secrete enzymes that are important to convert the complex sugars into simple ones (&#945;-glucosidases, leucine arylamidase, invertase) in honey7.
Honeybees are exposed to several biotic and abiotic stressors8, and the digestive tract can be affected by several negative stimulants. The first barrier that protects the organism from pathogens is the peritrophic membrane in the midgut, which consists of intestinal mucosa to protect against pathogens4. The development and function of HPGs depend on diet, age, and colony condition9, and are affected by insecticides, acaricides10, and pathogens11,12,13. Acaricide residues in the hive due to varroa control treatment and pesticides from the environment affect forager bees and nurse bees14,15. The greatest threat to honeybee colonies is the mite Varroa destructor, both as a vector of viruses contributing to colony losses16&#160;and as a consumer of the host&#39;s fat body (an important vital organ in honeybees), which consequently affects the individual&#39;s body and colony functions17.
However, intensive farmland habitats can provide a short-term food supply for honeybees. Therefore, agri-environmental schemes should enhance the availability of honey flowers in agricultural landscapes18. To assess the morphology of different subspecies6,19,20,21&#160;or sublethal effects of these factors at the cell or tissue levels, especially midgut and HPGs, histological and immunohistochemical methods are practical and sufficiently accurate to be used in histology research in honeybees.

<table><tbody><tr><td>2-Propanol</td><td></td><td></td><td></td></tr><tr><td>ApopTag Peroxidase kit (ApopTag Peroxidase In Situ Apoptosis Detection)</td><td>Sigma-Aldrich</td><td>S7100</td><td>Assay B, https://www.sigmaaldrich.com/SI/en/product/mm/s7100?gclid=CjwKCA&lt;br/&gt; jw7vuUBhBUEiwAEdu2pPanI9SE&lt;br/&gt; j81ZTl-nLHEoxXAv7ViKwPA_QRx&lt;br/&gt; H7fciMRNcYwR7lbPQbhoCqcQQA&lt;br/&gt; vD_BwE; Positive controls included in S7101</td></tr><tr><td>Covers</td><td></td><td></td><td></td></tr><tr><td>DeadEnd Colorimetric TUNEL system</td><td>Promega</td><td>G7360</td><td>Assay A, https://worldwide.promega.com/products/cell-health-assays/apoptosis-assays/deadend-colorimetric-tunel-system/?catNum=G7360</td></tr><tr><td>Dissecting microscope&amp;nbsp; (for bee dissection)</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Distilled water</td><td></td><td></td><td></td></tr><tr><td>Embedding cassette</td><td></td><td></td><td></td></tr><tr><td>EnVision System alkaline phosphatase kit</td><td>Dako</td><td></td><td></td></tr><tr><td>Eosin Y Solution</td><td>Sigma-Aldrich</td><td></td><td>alcoholic</td></tr><tr><td>Ethanol</td><td></td><td></td><td>95% (or less pure), 90%, 80%</td></tr><tr><td>Faramount mounting medium, aqueous</td><td>Dako</td><td></td><td>mounting medium</td></tr><tr><td>Flattening table</td><td>Leica</td><td>HI1220</td><td></td></tr><tr><td>Forceps&amp;nbsp; (for bee dissection)</td><td>Fine science tools</td><td>11294-00</td><td>Standard #4</td></tr><tr><td>Formalin 10%</td><td></td><td></td><td>Formaldehyde</td></tr><tr><td>Hematoxylin</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>HistoChoice Clearing Agent</td><td>Sigma-Aldrich</td><td></td><td>clearing agent</td></tr><tr><td>Hydrogen peroxidase 3%</td><td></td><td></td><td></td></tr><tr><td>Incubator</td><td>BioRad</td><td></td><td></td></tr><tr><td>Insect pins&amp;nbsp; (for bee dissection)</td><td>Entosphinx</td><td>44594</td><td>Insect pins stainless steel &amp;ndash; white, size 2</td></tr><tr><td>ISCDDK, AP (In Situ Cell Death Detecteion Kit, Alkaline Phosphatase)</td><td>Roche</td><td>11684809910</td><td>Assay C, https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/362/737/11684809910b&lt;br/&gt; ul.pdf</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>Lab clock</td><td></td><td></td><td></td></tr><tr><td>Light microscope</td><td>Leica</td><td></td><td></td></tr><tr><td>Microscope slides</td><td></td><td></td><td>Box with the slides must be preserved in a plastic wrap to prevent dust</td></tr><tr><td>Microtome</td><td>Leica</td><td></td><td></td></tr><tr><td>Modular tissue embedding station</td><td>Leica</td><td></td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>NaCl</td><td></td><td></td><td></td></tr><tr><td>Paraformaldehyde 4%</td><td></td><td></td><td></td></tr><tr><td>Paraplast</td><td>Leica</td><td></td><td></td></tr><tr><td>Pasteur pipettes</td><td></td><td></td><td>1.5 mL; 3 mL</td></tr><tr><td>PBS</td><td></td><td></td><td></td></tr><tr><td>Petri dish&amp;nbsp; (for bee dissection)</td><td></td><td></td><td>Filled with condensation silicon&amp;nbsp; (Xantoprene L blue and Universal liquid plus activator)</td></tr><tr><td>Proteinase K</td><td>Merck</td><td>21627</td><td></td></tr><tr><td>Ringers&#039; solution&amp;nbsp; (for bee dissection)</td><td></td><td></td><td>7.5 g NaCL, 2.38 g Na&lt;sub&gt;2&lt;/sub&gt;HPO4, 2.72 g KH&lt;sub&gt;2&lt;/sub&gt;PO4, 1 L distilled water</td></tr><tr><td>Scissors&amp;nbsp; (for bee dissection)</td><td>Fine science tools</td><td>1406-09, 14061-09</td><td>Straight and curved, 9 cm</td></tr><tr><td>Universal liquid plus activator&amp;nbsp; (for bee dissection)</td><td>Kulzer</td><td></td><td></td></tr><tr><td>Watchmaker&amp;rsquo;s forceps (for bee dissection)</td><td>Fine science tools</td><td>91100-12</td><td></td></tr><tr><td>Water bath</td><td>Leica</td><td></td><td></td></tr><tr><td>Watercolor brush</td><td></td><td></td><td>2x</td></tr><tr><td>Xantoprene L blue&amp;nbsp; (for bee dissection)</td><td>Kulzer</td><td></td><td></td></tr></tbody></table>

histology basics and cell death detection in honeybee tissue

Honeybees (Apis mellifera L.) inside the hive (nurse workers and other hive bees) and outside the hive (foragers) are exposed to climate and weather changes, various pesticides, pathogens, and malnutrition, mainly entering through the mouth and primarily affecting the digestive tracts of adult bees. To understand and prevent the effects of such external and internal stressors on honeybees, one useful research method is the immunohistochemical method. A basic protocol is described to prepare the midgut (ventriculus) and hypopharyngeal glands (HPGs) of adult bees for histological analysis. A detailed methodology is described to assess the level of cell damage and distinguish necrosis from programmed cell death (apoptosis) as a natural process of tissue regeneration. The results of adult honeybee treatment with oxalic acid and pesticides (insecticide and acaricide) and the determination of cell death in the ventriculus and HPGs are presented. The pros and cons of the methodology are also discussed.

Cell death detection in the midgut 
Newly emerged worker bees (Apis mellifera carnica) from the experimental apiary at the Agricultural Institute of Slovenia in Ljubljana were individually treated with 3% oxalic acid (OA)23. OA is frequently used in beekeeping for Varroa destructor control. After the treatment, the worker bees (three from each group) were immobilized on ice. The midgut was dissected and fixed it in 10% formalin. The tissue was then dehydrate...

Watch this Scientific Journal Video about Histology Basics and Cell Death Detection in Honeybee Tissue at JoVE.com

Histology Basics and Cell Death Detection in Honeybee Tissue

Immunohistochemical methods are useful in honeybee research to detect and assess the level of apoptosis and necrosis in the midgut and hypopharyngeal glands of adult bees.

Honeybees (Apis mellifera L.) inside the hive (nurse workers and other hive bees) and outside the hive (foragers) are exposed to climate and weather ...

histology-basics-and-cell-death-detection-in-honeybee-tissue

Research

JoVE Journal

Biology

2.2K Views.  Agricultural Institute of Slovenia. Dit protocol is nuttig in veel biologische en toxicologische studies van honingbijen behandeld met pesticiden. Het helpt bij het detecteren van de cellulaire reacties van honingbijenweefsel van acariciden die worden gebruikt in bijenkolonies voor de behandeling tegen varroamijten. Het is een krachtig hulpmiddel voor de detectie van sub-little effecten op weefsel in combinatie met gedrag van bijen of andere nuttige insecten.Neem om te beginnen voorzichtig een werkbij met een tang en zet...

Video: Histology Basics and Cell Death Detection in Honeybee Tissue

Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom body (MB) is a higher brain center proposed to be the neural substrate of complex honeybee behaviors. Although previous studies identified genes and proteins that are differentially expressed in the MBs and other brain regions, the activities of the proteins in each region are not yet fully understood. To reveal the functions of these proteins in the brain, pharmacologic analysis is a feasible approach, but it is first necessary to confirm that pharmacologic manipulations indeed alter the protein activity in these brain regions.
We previously identified a higher expression of genes encoding phospholipase C (PLC) in the MBs than in other brain regions, and pharmacologically assessed the involvement of PLC in honeybee behavior. In that study, we biochemically tested two pharmacologic agents and confirmed that they decreased PLC activity in the MBs and other brain regions. Here, we present a detailed description of how to detect PLC activity in honeybee brain homogenate. In this assay system, homogenates derived from different brain regions are reacted with a synthetic fluorogenic substrate, and fluorescence resulting from PLC activity is quantified and compared between brain regions. We also describe our evaluation of the inhibitory effects of certain drugs on PLC activity using the same system. Although this system is likely affected by other endogenous fluorescence compounds and/or the absorbance of the assay components and tissues, the measurement of PLC activity using this system is safer and easier than that using the traditional assay, which requires radiolabeled substrates. The simple procedure and manipulations allow us to examine PLC activity in the brains and other tissues of honeybees involved in different social tasks.

To test the inhibitory effects of pharmacologic agents on phospholipase C (PLC) in different regions of the honeybee brain, we present a biochemical assay to measure PLC activity in those regions. This assay could be useful for comparing PLC activity among tissues, as well as among bees exhibiting different behaviors.

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom ...

Biochemistry

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.

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.

The nurse hypopharyngeal glands produce the protein fraction of the worker and royal jelly that is fed to developing larvae and queens. These paired ...

Environment

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to measure odor evoked activity in the antennal lobe1. From then on, it has been continuously refined and applied to different neuropiles in the bee brain. Here, we describe the preparation currently used in the lab to measure activity in mushroom body neurons using a dextran coupled calcium-sensitive dye (Fura-2). We retrogradely stain mushroom body neurons by injecting dye into their axons or soma region. We focus on reducing the invasiveness, to achieve a preparation in which it is still possible to train the bee using PER conditioning. We are able to monitor and quantify the behavioral response by recording electro-myograms from the muscle which controls the PER (M17)2.
After the physiological experiment the imaged structures are investigated in greater detail using confocal scanning microscopy to address the identity of the neurons.

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

Bees can be conditioned in an appetitive olfactory learning paradigm (PER-conditioning). Using odors as stimuli, we established a method in which behavior is recorded while simultaneously Calcium Imaging is used to measure odor evoked activity in mushroom body neurons in vivo.

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to ...

Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells

Interest in the biological roles of cell membrane&#8211;derived vesicles in cell&#8211;cell communication has increased in recent years. Microparticles (MPs) are one such type of vesicles, ranging in diameter from 0.1 &#956;m to 1 &#956;m, and typically shed from the plasma membrane of eukaryotic cells undergoing activation or apoptosis. Here we describe the generation of T lymphocyte&#8211;derived microparticles (LMPs) from apoptotic CEM T cells stimulated with actinomycin D. LMPs are isolated through a multistep differential centrifugation process and characterized using flow cytometry. This protocol also presents an in situ cell death detection method for demonstrating the proapoptotic effect of LMPs on bronchial epithelial cells derived from mouse primary respiratory bronchial tissue explants. Methods described herein provide a reproducible procedure for isolating abundant quantities of LMPs from apoptotic lymphocytes in vitro. LMPs derived in this manner can be used to evaluate the characteristics of various disease models, and for pharmacology and toxicology testing. Given that the airway epithelium offers a protective physical and functional barrier between the external environment and underlying tissue, use of bronchial tissue explants rather than immortalized epithelial cell lines provides an effective model for investigations requiring airway tract tissue.

Cell membrane&#8211;shed microparticles (MPs) are active biological vesicles that can be isolated and their pathophysiological effects investigated in various models. Here we describe a method for generating MPs derived from T lymphocytes (LMPs) and for demonstrating their proapoptotic effect on airway epithelial cells.

Interest in the biological roles of cell membrane&#8211;derived vesicles in cell&#8211;cell communication has increased in recent years. Microparticles ...

Immunology and Infection

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and inflammatory intractable diseases. We herein developed an experimental method to investigate phagocytosis quantitatively using the fruit fly Drosophila, in which the gene network controlling engulfment reactions is evolutionally conserved from mammals. In order to accurately detect and count engulfing and un-engulfing phagocytes using whole animals, Drosophila embryos were homogenized to obtain dispersed cells including phagocytes and apoptotic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay in which it is possible to observe all phagocytes and apoptotic cells in whole embryos and precisely quantify the level of phagocytosis. We confirmed that this method reproduces those of previous studies that identified the genes required for the phagocytosis of apoptotic cells. This method allows the engulfment of dead cells to be analyzed, and when combined with the powerful genetics of Drosophila, will reveal the complex phagocytic reactions comprised of the migration, recognition, engulfment, and degradation of apoptotic cells by phagocytes.

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

We herein describe a phagocytosis assay using the dispersed embryonic cells of Drosophila. It enables us to easily and precisely quantify in vivo phagocytosis levels, and to identify new molecules required for the phagocytosis of apoptotic cells.

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and ...

Mass Histology to Quantify Neurodegeneration in Drosophila

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health worldwide. Although mammalian models have provided important insights into the underlying mechanisms of pathogenicity, the complexity of mammalian systems together with their high costs are limiting their use. Therefore, the simple but well-established Drosophila model-system&#160;provides an alternative for investigating the molecular pathways that are affected in these diseases. Besides behavioral deficits, neurodegenerative diseases are characterized by histological phenotypes such as neuronal death and axonopathy. To quantify neuronal degeneration and to determine how it is affected by genetic and environmental factors, we use a histological approach that is based on measuring the vacuoles in adult fly brains. To minimize the effects of systematic error and to directly compare sections from control and experimental flies in one preparation, we use the &#39;collar&#39; method for paraffin sections. Neurodegeneration is then assessed by measuring the size and/or number of vacuoles that have developed in the fly brain. This can either be done by focusing on a specific region of interest or by analyzing the entire brain by obtaining serial sections that span the complete head. Therefore, this method allows one to measure not only severe degeneration but also relatively mild phenotypes that are only detectable in a few sections, as occurs during normal aging.

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophila is widely used as a model system to study neurodegeneration. This protocol describes a method by which degeneration, as determined by vacuole formation in the brain, can be quantified. It also minimizes effects due to the experimental procedure by processing and sectioning control and experimental flies as one sample.

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

Neuroscience

Anti-RDL and Anti-mGlutR1 Receptors Antibody Testing in Honeybee Brain Sections using CRISPR-Cas9

Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a gene editing technique widely used in studies of gene function. We use this method in this study to check for the specificity of antibodies developed against the insect GABAA receptor subunit Resistance to Dieldrin (RDL) and a metabotropic glutamate receptor mGlutR1 (mGluRA). The antibodies were generated in rabbits against the conjugated peptides specific to fruit flies (Drosophila melanogaster) as well to honeybees (Apis mellifera). We used these antibodies in honeybee brain sections to study the distribution of the receptors in honeybee brains. The antibodies were affinity purified against the peptide and tested with immunoblotting and the classical method of preadsorption with peptide conjugates to show that the antibodies are specific to the corresponding peptide conjugates against which they were raised. Here we developed the CRISPR-Cas9 technique to test for the reduction of protein targets in the brain 48 h after CRISPR-Cas9 injection with guide RNAs designed for the corresponding receptor. The CRISPR-Cas9 method can also be used in behavioral analyses in the adult bees when one or multiple genes need to be modified.

Presented here is a protocol to use the CRISPR-Cas9 system for reducing the production of a protein in the adult honeybee brain to test antibody specificity.

Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a gene editing technique widely used in studies of ...

Obtaining Specimens with Slowed, Accelerated and Reversed Aging in the Honey Bee Model

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best established model to study how plasticity in lifespan and aging is explained by social factors.
The worker caste of honey bees includes nurse bees, which tend the brood, and forager bees, which collect nectar and pollen. Previous work has shown that brain functions and flight performance senesce more rapidly in foragers than in nurses. However, brain functions can recover, when foragers revert back to nursing tasks. Such patterns of accelerated and reversed functional senescence are linked to changed metabolic resource levels, to alterations in protein abundance and to immune function. Vitellogenin, a yolk protein with adapted functions in hormonal control and cellular defense, may serve as a major regulatory element in a network that controls the different aging dynamics in workers.
Here we describe how the emergence of nurses and foragers can be monitored, and manipulated, including the reversal from typically short-lived foragers into longer-lived nurses. Our representative results show how individuals with similar chronological age differentiate into foragers and nurse bees under experimental conditions. We exemplify how behavioral reversal from foragers back to nurses can be validated. Last, we show how different cellular senescence can be assessed by measuring the accumulation of lipofuscin, a universal biomarker of senescence.
For studying mechanisms that may link social influences and aging plasticity, this protocol provides a standardized tool set to acquire relevant sample material, and to improve data comparability among future studies.

In honey bee workers, aging depends on social behaviors rather than on chronological age. Here we show how worker-types with very different aging patterns can be obtained and analyzed for cellular senescence.

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best ...

Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination services, as well as an important model social insect used to study memory and learning, aging, and more. Here we describe a detailed protocol for the dissection of the dorsal abdominal wall of a bee in order to visualize its dorsal vessel, which serves the role of the heart in the insect. A successful dissection will expose a functional heart that, under the proper conditions, can maintain a steady heartbeat for an extended period of time. This allows the investigator to manipulate heart rate through the application of cardiomodulatory compounds to the dorsal vessel. By using either a digital microscope or a microscope equipped with a digital camera, the investigator can make video recordings of the dorsal vessel before and after treatment with test compounds. The videos can then be scored at a time convenient to the user in order to determine changes in heart rate, as well as changes in the pattern of heartbeats, following treatment. The advantages of this protocol are that it is relatively inexpensive to set up, easy to learn, requires little space or equipment, and takes very little time to conduct.

The abdominal dorsal vessel of the honey bee and other insects serves as the functional equivalent of the mammalian heart and plays an important role in nutrient transport, waste removal, immune function, and more. Here we describe a protocol for the visualization and pharmacological manipulation of bee heart rate.

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination ...

An Introduction to Cell Death

Necrosis, apoptosis, and autophagic cell death are all manners in which cells can die, and these mechanisms can be induced by different stimuli, such as cell injury, low nutrient levels, or signaling proteins. Whereas necrosis is considered to be an &#8220;accidental&#8221; or unexpected form of cell death, evidence exists that apoptosis and autophagy are both programmed and &#8220;planned&#8221; by cells.In this introductory video, JoVE highlights key discoveries pertaining to cell death, including recent work done in worms that helped identify genes involved in apoptosis. We then explore questions asked by scientists studying cell death, some of which look at different death pathways and their interactions. Finally, several methods to assess cell death are discussed, and we note how researchers are applying these techniques in their experiments today.

Cell Death: Apoptosis, Autophagy and Necrosis

Necrosis, apoptosis, and autophagic cell death are all manners in which cells can die, and these mechanisms can be induced by different stimuli, such as ...

Histology Basics and Cell Death Detection in Honeybee Tissue

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