Histology Basics and Cell Death Detection in Honeybee Tissue

This protocol is useful in many biological and toxicological studies of honeybees treated with pesticides. It helps to detect the cellular responses of honeybee tissue of acaricides that are used in bee colonies for the treatment against Varroa mites. It is a powerful tool for the detection of sub-little effects on tissue in combination with behavior of bees or other beneficial insects.

To begin, carefully take a worker bee with forceps and put it on ice or freezer at minus 20 degrees Celsius for two minutes to immobilize it. 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. Poor insect saline to cover the body.

Place the Petri dish under the stereomicroscope, focus, and adjust. To dissect the midgut, start with the abdomen by inserting one point of the scissors under the tergite A5 in the center of the right side of the bee body. Cut to the tergite A2.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 cut. Gently open the left part of the abdomen and pin it. 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. For dissection of hypopharyngeal glands, immobilize a worker bee on ice as described before.

Cut the head off and place it on a smaller plate with the antenna facing up. Secure the head with two pins, one through the left compound eye and the second through the right compound eye. 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.

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.

For hematoxylin and eosin staining, put the dewaxed, rehydrated sections in hematoxylin for five minutes. Then, carefully place them under running tap water for two minutes. Then, put them into distilled water for one minute and eosin for four minutes.

Place the slides 96%ethanol for one minute then 2-propanol for two minutes, and finally into the clearing agent for two minutes. Add mounting medium and a cover glass and let them dry. Observe under a light microscope.

Prepare Coplin jars. Prepare proteinase-K. After dewaxing and rehydrating the sections, place the slides in PBS for five minutes.

Place the slides flat in the container and add proteinase-K. Wash the slides in distilled water. Quench in endogenous peroxidase at room temperature.

Rinse the slides with PBS or water. Place the slides flat in the container and apply equilibration buffer for 10 seconds at room temperature. After wiping around the tissue, add the terminal deoxynucleotidyl transferase enzyme to each section, and incubate in a humidified chamber for one hour at 37 degrees Celsius.

Before incubation, put moistened paper towels inside the tray around the slides and cover them with plastic wrap. After incubation, put the specimens on the rack and leave them in stop-wash buffer. After washing the slides in PBS and wiping around the tissues, add two drops of warm anti-digoxigenin peroxidase conjugate to the sections and incubate for 30 minutes in a humidified container.

After washing in PBS, prepare working strength-peroxidase substrate, cover the sections with peroxidase substrate, and stain for five minutes. Place the slide under the microscope and determine the optimal staining time. After washing the slides in a staining rack and distilled water, incubate the slides in distilled water for five minutes.

Counter-stain using hematoxylin for two minutes. Wash the slides by placing them under running tap water for three minutes and then in distilled water. Mount the slides under glass cover slips and mounting medium and leave flat to dry.

Observe under a light microscope. The percentage of affected cells was calculated using a light microscope. The results indicated that the treatment with oxalic acid significantly affected the cells in the midgut.

Immunostaining showed positive red or brown reaction products in the apoptotic nuclei of hypopharyngeal glands. Positive reaction product after imidacloprid or coumaphos treatment was determined in the majority of glandular cells. When lifting of the mask of the bee head, take care not to damage or pull off the parts of the hypopharyngeal glands.

This technique detects subclinical changes in bees and is also suitable for the detection of other environmental effects on bees and some other living organisms.