Related Topic: Honey Bee Parasites, Pests and Predators - New slide set, viewable on Web

The female Acarapis woodi, or honey bee tracheal mite, is 143-174 µm long and the male 125-1 36 µm. The body is oval; widest between the second and third pair of legs; and whitish or pearly white with shining, smooth cuticle. A few long hairs are present on the body and legs. This mite has an elongate, beaklike gnathosoma with long, bladelike styles (mouthparts) for feeding on the host.

The population of Acarapis woodi may vary seasonally. During the period of maximum bee population, the number of bees with mites is reduced. The likelihood of detecting tracheal mites is highest in the fall. In sampling for this mite, one should try to collect either moribund bees that may be crawling near the hive entrance or bees at the entrance as they are leaving or returning to the hive. These bees should be placed in 70% ethyl or methyl alcohol as soon as they are collected. One should not collect bees that have been dead for an unknown period because they are less than ideal for the diagnosis of tracheal mites.

No one symptom characterizes this disease. An affected bee could have disjointed wings and be unable to fly, or have a distended abdomen, or both. Absence of these symptoms does not necessarily indicate freedom from mites. Positive diagnosis can be made only by microscopic examination of the tracheae; since only Acarapis woodi is found in the bee tracheae, this is an important diagnostic feature.

A healthy trachea appears cream color or white. The trachea of a severely infested bee has brown or black blotches with crustlike lesions and is obstructed by many mites in different stages of development. The trachea must be examined carefully for the presence of mites. The trachea may not always be discolored when mites are present, and a cloudy or discolored trachea does not always contain mites.

Methods for diagnosing Acarapis woodi are listed below. Each of these methods has its advantages and disadvantages.

Method 1
Pin the bee on its back and remove the head and first pair of legs by pushing them off with a scalpel or razor blade in a downward and forward motion. Using a dissecting microscope, remove the first ring of the thorax (tergite of prothorax) with forceps. This exposes the tracheal trunks in the mesothorax. When the infestation is light, it is necessary to remove the trachea. Place the trachea in a drop of lactic acid on a glass slide for clearing, and cover with a cover glass for examination at X 40-100 on a compound microscope.

Method 2
Grasp the bee between your thumb and forefinger and remove the head and first pair of legs. Then with a scapel, razor blade, or fine pair of scissors, cut a thin transverse section from the anterior face of the thorax in such a way as to obtain a disk. Place the disk on a microscope slide and add a few drops of lactic acid. This makes the material more transparent and also helps to separate the muscle. With the aid of dissecting microscope, carefully separate the muscles, remove the trachea, and examine the preparations as in method 1. This is recommended for quick examination of a few bees.

Method 3
Cut a few thoracic disks as described in method 2, place them on a slide, and add a few drops of 10% potassium hydroxide (KOH). Heat the slide gently for 1-2 minutes (do not boil), cover with a cover glass, crush the disks lightly, and examine microscopically. This procedure is advantageous when the bees have been dead for some time.

Method 4
Prepare transverse-section disks from the thoraces of 50 honey bees as described in method 2, place them in 5% KOH, and incubate at 37?C for 16-24 hours. The KOH dissolves the muscle and fat tissue, leaving the trachea exposed. Then examine the disk-trachea suspension under a dissecting microscope. Remove suspicious tracheae from the disks and examine the tracheae microscopically (X 40-100). This procedure is recommended for large samples of bees.

Method 5
Remove the heads, abdomens, wings, and legs from 20-200 thoraces and place them in a homogenizing jar with 25 mL of water. Homogenize three times for several seconds at 10,000 rpm, using just enough water to rinse the inside of the jar. Then strain the suspension through a 0.8-mesh sieve and rinse with water. The final volume of the filtrate should be about 50 mL. Centrifuge the filtrate at about 1,500 g for 5 minutes and discard the supernatant. Then add a few drops of lactic acid to the preparation, and allow it to stand for 10 minutes. Finally, place the sediment on a slide for examination. In this method, a microscope with an oil immersion objective is required to correctly identify Acarapis woodi because other mites associated with honey bees are morphologically similar. This technique is described by Cohn et al. (1979).

Method 6
In the flotation method (Camazine 1985), bees cannot be stored or killed in alcohol. For cleanest preparations, remove the head, wings, legs, and abdomen (saving only the thoraxes) of recently killed bees. This removal is easily done using one's fingers when the bees are frozen. Place 25-100 bees in a household blender with enough water added to cover the blades. Blend the preparation for no more than 15 seconds, just until the thoraces are broken apart. (Blending for longer periods will pulverize the tracheae.) Pour the resulting mixture into a series of test tubes (2-3 cm in diameter). Most of the denser muscle fibers and cuticular fragments fall to the bottom while the tracheae and air sacs float, forming a thin whitish layer on the surface of the water. Suction off this layer with a pipette, place on one or more slides, and cover with cover slips. Examine the slides under a compound microscope at X 100-250. Examine the slide in a systematic manner for darkened, blotchy, and discolored tracheae and for undamaged tracheae that may also contain mites and eggs.

Method 7
In the modified methylene blue staining technique (Peng and Nasr 1985), prepare transverse-section disks from the thoraces of 50 bees as described in method 2. Place the disks in a beaker of 8% KOH solution, and heat to boiling with continuous gentle stirring of the disks. Remove the solution from the heat and continue stirring until the soft tissues inside the disks are dissolved and cleared (about 10 minutes). Excessive stirring and heating will damage the specimens and subsequently reduce the color intensity of the mites. Recover the disks from the KOH by filtration through a perforated Tissue-Tek processing capsule. After filtration, cover the processing capsule with a lid, place in a beaker, and wash with tap water to remove the remaining KOH. After washing, transfer the processing capsule to a modified methylene blue staining solution (prepared by first dissolving 1% aqueous methylene blue and then adding sodium chloride to make a 0.85% sodium chloride solution). Immerse the capsule in that solution for 5 minutes and then in distilled water for 2-5 minutes; finally, rinse the capsule with 70% ethyl alcohol. Examine the disks for stained mites within the tracheae under a dissecting microscope at X 10-25.

Method 8
Differentiation of live mites from dead mites (Eischen et al. 1986) is the method of choice for evaluating chemicals used to control tracheal mites. Anesthetize live bees with carbon dioxide and remove the abdomens with a scalpel to prevent being stung during examination. Remove the head and first pair of legs of each bee by holding the bee on its back and gently pushing this section off with a downward and forward motion. Place each bee, held in this position, under a dissecting microscope, and remove the first ring of the thorax with fine forceps. This exposes the tracheal attachment to the thoracic wall, which is often the only location of mites in a light infestation. Remove tracheae that appear abnormal with tweezers and transfer to a glass slide containing a thin film of glycerol. Then dissect the tracheae using a pair of fine needle probes. Mites are considered dead if they do not move; also, dead mites often appear discolored and desiccated. Living mites have a translucent gray or pearl color and move within a few seconds after dissection.

Method 9
For serodiagnosis, Ragsdale and Furgala (1987) produced an antiserum against extracts of Acarapis woodi-infested tracheae to be used as the primary antibody in a direct enzyme-linked immunosorbent assay (ELISA). Ragsdale and Kjer (1989) improved the ELISA technique, making it as reliable as dissection for the detection of A. woodi. Their ELISA is accurate, sensitive, reproducible, cost effective, rapid, and easy to use.

Related Topic: Honey Bee Parasites, Pests and Predators - New slide set, viewable on Web