David A. Cushman
http://www.dave-cushman.net/bee/acarine_diagnosis.html

Diagnosis Of Acarine (tracheal mite) Infestation In Honey Bees

Identification methods
 The mites can to be observed inside the tracheae or removed from them to be observed independantly using a microscope or high power hand lens.

The thoraces of suspect bees can be dissected to expose the trachea. Each trachea is examined using a microscope, the mites can be seen through the transparent wall of the trachea.

Larger samples of suspect bees can be ground up or homogenised in water, followed by coarse filtration of the suspension, and centrifugation. The deposit is treated with undiluted lactic acid for 10 minutes and mounted for microscopic examination. This method is not discussed further here.

Acarine mites can be stained so that they can be observed in stronger contrast within the bee trachea.

The honey bee problem known as Acariosis or Acarine, often called a disease, is actually an infestation of adult bees of Apis mellifera and other Apis species, caused by the microscopic Tarsonemid mite Acarapis woodii. In USA they are known as tracheal mites.

The Acarine mite is approximately 150 µm in size, and it is an internal parasite of the respiratory system of honey bees. The mites enter, live, and reproduce mainly in the large prothoracic tracheae, feeding on the haemolymph of their host. Sometimes, particularly in strong infestations, they may also found in the head, thoracic and abdominal air sacs.
 
The symptoms found in infected bees depend on the number of parasites within the trachea and are due to physical injuries and to physiological disorders that are caused by to the obstruction of the airways, lesions in the walls of the trachea, and to a minor extent depletion of haemolymph. As the number of mites increases, the walls of the trachea change from white/translucent to opaque and discoloured with irregular blotchy dark patches.

The infestation spreads by direct contact, only newly hatched bees less than 10 days old are susceptible. Mite reproduction occurs within the tracheae of adult bees, where female acarine mites may lay between eight and twenty eggs. There are two to four times as many females as there are males. Development takes 11 or 12 days for males and 14 or 15 days for females. Light infestations occur near to the spiracle opening and heavier infestations reach deeper into the tracheal network.

There are no reliable external clinical signs for the diagnosis of acariosis as the signs of affliction are not specific and the bees behave in much the same way as bees that are affected by various diseases. They crawl around on the ground in front of the hive and climb blades of grass, unable to fly. Dysentery and/or signs of 'K' wing may be present.


Identification of the problem
Some claim that Acarine infestation can only be detected under laboratory conditions using microscopic examination or an enzyme-linked immunosorbent assay (ELISA). There is no reliable method for detection of very low levels of infection.

The number of bees sampled determines the detection threshold of the method. It has been shown that a 2% rate of infection can be detected by sampling 50 bees, while a 1% rate of infection is detected using 100 bees (confidence limit is 80% for a colony of average size in spring). Because of the high level of manual work involved, it is suitable to examine 50 bees. Sequential sampling is a neat trick to reduce the overall workload


Sequential Testing
Take a sample of 50 bees kill them then and examine them one by one. You plot the result on the graph depending whether they are positive or negative. The graph is basically cumulative positive versus No. of bees examined.

Once the plot gets above the upper line the sample has an infection level above the probability selected (in the case of diagram it is 20%), if the plot lies between the two lines, then further bees need to be examined, and if the plot dips below the lower line then the level of infestation is below the lower limit set for action. (Original material for the diagrams, supplied by Ruary Rudd.)


Dissection to expose Acarine infestation
A random sample of 50 bees is collected from the suspect colony. These should be mainly bees crawling and unable to fly, found within about 3 metres of the front of the hive, rather than random collection from within the colony. The sample bees may be living, dying, or dead. Live bees must first be killed with ethyl alcohol or in a deep freeze at -20°C

Each bee should be impaled, using a double needle placed at an angle away from the head through the thorax between the second and third pairs of legs (as shown at right). The bee should ventral side up on an angled cork base, the angle is not critical, but is usually between 45° and 60°. Using a single edged razor blade, cut off the head and first pair of legs, the cut should be made from behind the first pair of legs to the back of the bee's head, indicated by the red line on the drawing, the severed head and front pair of legs can then be removed using tweezers.
 
Fine tipped tweezers can be used to peel away the collar (shown red at right) in order to expose the tracheae more fully. Pull upwards with a circular motion, following the ring of the collar. It will peel off easily, usually in one piece. The collar itself can be saved for later preparation as a microscope slide specimen, if required, by immersing in 70% isopropyl alcohol.
    
As mites enter through the spiracle check the outer end of the trachea first. Light infestations may be difficult to see, heavy infestations are easily visible as shadows or lumpy dark objects in trachea that can be clear to dark brown. Old and/or heavy infestations will render the trachea orange, brown or black.


Alternative Method
The trachea can be isolated by cutting a disc through the thorax in front of the middle pair of legs and the base of the forewings using a razorblade. These thin disks can then be further treated to clear muscle tissue by macerating in a 10% solution of potassium or sodium hydroxide boiling for a few minutes or by leaving them to stand overnight at room temperature until the soft internal tissues are dissolved and cleared leaving the chitinous parts intact. Using heat is more relaible as you can constantly monitor progress. Wash the sections in tap water to remove the alkali.

Examine the main pair of trachea using a dissecting microcope or a stereo microscope that gives an erect image. A magnification of x18 or x20 is suitable, or the trachea can be cut from the chitin ring and transferred to a glass slide, add glycerin or water and observe at whatever magnification you choose.

At low magnifications the mites are visible through the transparent tracheal wall, but are small, indistinct oval bodies, with higher magnifications the mites can be recognised.


Preferential Staining
The mites and trachea can be stained specifically, rendering them more easily visible under the microscope.

Remove the head and forelegs, create thoracic discs 1 mm to 1.5 mm thick, then clear the sections using a 10% solution of potassium or sodium hydroxide and wash as described above.

Staining and mount the sections... Cationic stains are the most suitable and specific as they stain the mites intensely than they stain the trachea containing them. A solution of 1% aqueous methylene blue is the most suitable staining agent and it can be prepared by dissolving the methylene blue first and then adding sodium chloride to make a 0.85% or 0.9% NaCl solution. To actually stain the specimens, immerse in 1% aqueous methylene blue solution for 5 minutes differentiate sections in distilled water for 2 to 5 minutes, rinse the sections in 70% alcohol (ethyl or isopropyl). Examine the disks under a dissecting microscope at anyy aproppriate magnification (10x up to 30x).


References
Bancroft J.D. & Stevens A. (1982). Theory and Practice of Histological Techniques. Churchill Livingstone, Edinburgh, UK.

Colin M.A., Faucon J.P., Gianfert A. & Sarrazin C. (1979). A new technique for the diagnosis of Acarine infestation in honey bees. Journal of Apicultural Research, 18, 222-224.

Grant G., Nelson D., Olsen P. & Rice W.A. (1993). The ELISA detection of tracheal mites in whole honey bee samples. American Bee Journal, 133, 652-655.

Peng Y. & Nasr M.E. (1985). Detection of honey bee tracheal mites Acarapis woodi by simple staining techniques. Journal of Invertebrate Pathology, 46, 325-331.

Mozes-Koch R. & Gerson U. (1997). Guanine visualization, a new method for diagnosing tracheal mite infestation of honey bees. Apidologie, 28, 3-9.

Wolfgang Ritter (1996). Diagnostik und BekÄmpfung der Bienenkrankheiten (Diagnosis and control of bee diseases). Gustav Fischer Verlag, Jena, Stuttgart, Germany.

FAKHIMZADEH, K. 2001. Detection of major mite pests of Apis mellifera and development of non-chemical control of varroasis.   Doctoral Dissertation, University of Helsinki, Department of Applied Biology, Publication no: 3, Helsinki, 46 pp. + appendix articles. ISBN 951-45-9914-4.

Sammataro D. & Needham G.R. (1996). Host-seeking behaviour of tracheal mites (Acari: Tarsonemidae) on honey bees (Hymenoptera: Apidae). Experimental and Applied Acarology, 20, 121-136



Treatment of Honey Bees for Acarine Mite (Tracheal Mite) Infestation

Various Treatments that are (or have been) recommended for treating the Acarine or tracheal mite that infests honey bees, are listed on this page and the links that lead from it.

Folbex VA
Folbex VA used to be the preferred treatment for Acarine in UK, but has not been available for many years as it would be too costly to obtain a veterinary licence. However there is a linked page on Folbex VA which gives the details of how it was used when it was available.

Oil of Wintergreen
Also known as Methyl Salicylate, formula... C8H8O3
It is distilled from various species of plants that stay green in winter, hence the name. It should be noted that there are different species used in UK to those in many US texts.     

Somewhere between 1920 and 1925, Dr. J. Rennie, recommended wintergreen in preference to the original Frow treatment for treatment of Isle of Wight Disease (whatever it really was).

Between 1920s and 1940s it was common in UK for a wicked bottle of wintergreen oil to be kept in the bottom of the hives as a matter of routine, This was purported to control Acarine infestations, but I have seen no specific trial results on the effectiveness.

Manley used a small bottle (about 25 ml) of Oil of Wintergreen with a wick, or a small flat shoe polish tin with the lid perforated with holes of 6 mm to 9 mm. The tins were filled with cotton wool, with a disc of felt as the top surface, the cotton wool being completely saturated with the wintergreen. He kept such evaporators in all hives and mating nucs all year round.

The wick used has been reported differently in different texts, but such a wick is only a means to provide a large surface for evaporation and should not be critical. Three forms are mentioned... The first being cotton pyjama cord, the second being lamp wick (used in the flat tins by some), but the third requires a little explanation... It uses a soft cotton 'string' that was sold for knitting into dishcloths, six strands of this were laid parallel and loosely knotted close to one end, the long strands were fed into the small bottle and knot rested in or on the mouth and the short strands were fanned out to do the majority of the evaporating.

Menthol
In the United States Acarine is a big problem, they refer to the Acarine mite by the term 'tracheal mite', which of course, reflects it's method of infestation.

Bee treatment in the US requires approval by their food and drug administration department. Menthol crystals are the only US approved chemical treatment for Acarine or tracheal mites.
Menthol is a crystalline alcohol, formula... C10H20O It is obtained from oil of peppermint and is used in medicines, cooling salves and smoking materials.     

Menthol crystals are used as a fumigant, the crystals sublime into the gaseous state as well as going through a liquid phase, but the process is very temperature sensitive. Vapour is released above 21°C and as the gas is heavier than air the crystals should be above the brood nest, however above a temperature of 27°C the vapour release rate becomes so high that the fumes may drive out the bees from the hive and so above this temperature the crystals should be placed on the hive floor (bottom board). If an Open Mesh Floor or screened bottom board is used the varroa monitoring board should be in position to close off the bottom of the hive even if no monitoring is actually taking place at the time (there may be some varroa mite lethality involved with menthol, but we are discussing tracheal mites here)

The evaporation rate of menthol not only depends on temperature, but on the physical size of the crystals and the method of containment, the dosage recommended by the Connecticut Agricultural Experiment Station is 50 gm made up into a flat 180 mm square packet formed from a plastic mesh of such screen size as to stop any crystals from falling out of the packet.

Duration of treatment should be between fifteen and twenty five days with outdoor temperature around 20°C.
The Blue Shop Towel Method for Tracheal Mite Control in Honey Bees

The timing of Treatment will depend on weather and nectar flows in your area, you should not use menthol when the colony has supers on. Generally an autumn treatment is most effective although you must have adequate temperatures.

Various formulations that mix the menthol with grease or vegetable oils in an attempt to obtain a continuous release of the gas into the hive have been tried, but the one mentioned below that uses paper towels has been given much promotion on the internet.

Shop towel method
The developer of this method, Allen Dick, has detailed the blue shop towel method on his diary pages.

Frow treatment
This method is no longer practiced, owing to the dangerous constituents and risk of cancer and so it's mention here is purely for historical interest.

The ingredients varied over time and with availability of components... Nitro benzene being involved in all formulations and generally the major constituent. Safrol, Ligrion, Petrol (gasoline) and Methyl Salicylate were all used as ingredients in different ratios and at different times.

Frow treatment was usually administered by giving small doses of about one millilitre on to a felt pad, daily or on alternate days, for a period six to ten days, then removing the pad after a further week to ten days.

Use of Sulphur
Flowers of sulphur (powdered Sulfur) has been recommended in some books and can be both sprinkled on bees or the bees may be fumigated by a smoker that has been lit and is going well having a teaspoonful of flowers of sulphur added to it. The bees are fumigated, late in the evening after flying has finished, in a closed hive that has its entrance blocked for about twenty minutes.

Part of the action of such fumigation will be due to sulphur dioxide but there is likely to be some Sublimation of Sulphur that occurs in the hot, oxygen starved, atmosphere that exists inside the smoker. The fine particles of sulphur that result will have a good chance of physical contact with an Acarine mite within the trachea of an infested bee.

Grease patties
The vegetable greases and oils used with menthol to produce a finely divided homogeneous mixture have been found to have a direct effect on the tracheal mite themselves. The vegetable shortening or oil interferes with ability of tracheal mites to transfer from one bee to another.

The recipe being one part by weight of vegetable lard (shortening) to two parts of sugar (finely granulated). Or if vegetable oil is used, then the ratio is one part by weight of vegetable oil to three parts of sugar (in this case 'icing' or powdered sugar may make the finished patties hold together better).

For delivery of this mixture into the hives, form the patty similar to a hamburger about 100 mm in diameter by about 9 mm thick and sandwich it between wax paper discs. To use, place the patty centrally on top bars of the frames within the brood box and peel off the upper wax paper disc.

Grease patties can be used in early spring and/or the autumn. As there is no 'active chemical ingredient' there are many claims that the grease or oil will not contaminate honey supplies, but I am not sure that this is totally true, nor am I convinced that there will not be contamination of the beeswax in the combs.



    
The Blue Shop Towel Method for Tracheal Mite Control in Honey Bees
    
Dipping 1/3 roll of towel (core removed) into a 50/50 mix of Crisco and menthol.  Keep the mixture just above the melting point. Removing the saturated roll of towel.  The roll is hot and it drips.  There are strong fumes.  Be careful.  Work outdoors if possible. Bagging a finished roll of towels. Menthol evaporates if not enclosed.  Use ASAP, and store towels in a cool place until needed 1/3 towel placed at back of hive to avoid driving bees away from the brood. The pink is an extender patty. Feeder in foreground  

Makes 60 rolls of 55 sheets each. 
Treats 3,300 hives once or 1,650 hives twice

Melt roughly equal amounts (by weight) of Crisco vegetable shortening and menthol crystals in a pot.  Do it outside or under a fume hood.  Be aware: the mixture is very flammable.  Avoid excess heat, since menthol is very volatile -- and too expensive to boil off for no good reason.
           *  1 box Crisco is 20 kg.  That is 20,000 g (or 44 lbs)
           *  1 drum of menthol is 25 kg.  That is 25,000 g (or 55 lbs)
           *  1 ice cream pail of menthol is about 4 lbs.
           *  Melt 1/2 box of Crisco and 5-1/2 pails menthol at a time, using a washtub and torch or burner.

Cut 20 rolls of blue shop towels into thirds to make 60 rolls about the size of toilet paper rolls .

Remove the cardboard inner core.  Soak the rolls of  towel in the menthol/oil mixture until fully saturated.

Drain a bit and place the rolls into ZipLoc bags.  Store them in a cool place until needed.
            *   Result: 60 (1/3 size) rolls x 2 batches
            *   60 x 55 sheets per roll = 3,300 sheets each with 6g menthol and 6g Crisco           

Take them to the field and place one of the 1/3 sheets on the top bars of each hive at the front or back, not right over the cluster.  Daytime temperatures should not exceed 70 degrees F or be much under 50.  Results will vary with the hive and entrance design, location and whether the bees are still wrapped or not.  Menthol will evaporate if left in the sun or a hot place.  Keep unused rolls sealed and in a cool place.  Warning: Too much menthol will drive bees off the brood or kill brood.

Repeat in ten days or two weeks

That's it.  Forget about tracheal mites for a year or more. (Do check a bit in the fall, though)

We only got around to one treatment and found a year later that we did not find tracheal mites
 in a cursory exam that should have turned up any serious problems.

We treat in spring before the weather gets too hot.

It's best to check your bees twice a year, though -- spring and fall.
Don't just assume you have control. Be sure.

Note: Samples taken after treatment will test positive, since dead mites remain inside
 the adult bees until the bees die of old age.  Therefore subsequent testing to prove
efficacy must wait at least six weeks after the treatment.