Original article

Guanine visualization: a new method for diagnosing tracheal infestation of honey

R Mozes-Koch U Gerson

Department of Entomology, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel

(Received 17 June 1996; accepted 27 January 1997)

Summary — A new method for diagnosing tracheal mite infestation in honey bees is described. The method is based on the visualization of guanine, a purine which is the main end product of nitrogen metabolism in , but in excretions occurs at most in trace quantities. Thoraces of bees are homogenized in acidic or basic solutions, run on TLC plates and scanned by UV. The new assay was compared to the direct thoracic disk method and to the indirect ELISA method, and their relative advantages and disadvantages are discussed. Apis mellifera / woodi / diagnosis / guanine

INTRODUCTION wings and/or a distended abdomen, absence of these symptoms does not mean absence of The tracheal mite, Acarapis mites, which are located mainly within the woodi (Rennie), a serious pest of honey bees prothoracic tracheal system of their host (Eischen et al, 1989; Komeili and Ambrose, bees. Several methods have been proposed 1990), has recently been found in Israel and implemented for positive tracheal mite (Gerson et al, 1994). Efforts towards its identification. These methods, reviewed by management, including various cultural and Shimanuki and Knox (1991), roughly fall chemical methods, are usually hindered by into either direct or indirect categories. The difficulties in evaluating their effects. These former includes bee dissection and homog- difficulties stem from the fact that no sin- enization, and staining of tracheae, by which gle symptom fully characterizes the pests’ the mites are actually seen. The indirect effect (Shimanuki and Knox, 1991). method uses the ELISA technique (in which Although affected bees may have disjointed infestation is determined by a colorimetric

* Correspondence and reprints Tel: (972) 8 9481220; fax: (972) 8 9466768; e-mail: [email protected] reaction in microplates precoated with the until they were assayed. These samples were tracheal mite antigen) (Ragsdale and Kjer, used to compare essay methods (see below). 1989; Grant et al, 1993; Grant and Nelson, in press). Guanine visualization Herein we describe a new, indirect method, in which a purine compound, gua- Thoraces of the second, 70-bee subset were sep- nine, was used to determine the of presence arated into two equally-sized groups. Those of A woodi. Guanine (2-amino-6-oxypurine) the first group were placed in 0.5 mL 0.1 HCl is the main end product of nitrogen (acid solution), those of the second in 0.1 M metabolism in many , including NaOH (basic solution). All thoraces were indi- and mites (Anderson, 1966; Bron- vidually homogenized by an Ultra-Turrax tissue followed et al, 1989), but is almost absent from homogenizer (2 min), by teflon-glass swijk (2 and ultrasonic cell dis- bee excretions 1992, homogenization min) honey (Atmowidjojo, ruption (2 min) and finally centrifugation cited Erickson et Its occur- by al, 1994). (12 000 g for 20 min, at 4 °C). The supernatant rence in honey bees would therefore indi- was collected for guanine determination via TLC. cate that they are infested with mites. Gua- Two types of plates were compared for this pur- nine is also excreted by Varroa jacohsoni pose: Cellulose F254 (Merck) and Polygram® Oudemans (Erickson et al, 1994), another Cell 300 UV254, (Macherey-Nagel, Doren, Ger- acarine attacking the honey bee, but this many). Supernatant samples of 10 μL were loaded 3 cm from the edge, with 2 cm between mite is an external parasite, not present samples. Guanine (Sigma Ultra, 10 μL of within the bees’ bodies. 0.4 μg/μL) was loaded as a standard. An eluent of 95% ethanol/acetic acid/water (70:25:5) was most suitable for samples extracted in an acid whereas a mixture of 5% w/v ammo- MATERIALS AND METHODS solution, nium sulfate/13 M ammonia/1-propanol (60:30:10) was best for basic extracts. Guanine Bees spots were visualized by a UV lamp and com- pared to spots obtained by the above standards. Ultraviolet of Older honey bees (recognized by canying pollen) spectral analyses (200-300 nm) the were tested means of an Uvi- were collected from frames within commercial, homogenate by con-810 and were non-treated colonies determined to spectrophotometer compared previously to markers in basic and acidic solutions. be affected by tracheal mite. Older bees are known to be infested by larger numbers of tra- A preliminary evaluation of the method was cheal mites (Pettis and Wilson, 1996). Several conducted by dissecting the insects in one of the hundred worker bees were thus obtained during 100-bee samples from group A. Their tracheae the spring of 1996 over a period of several weeks were ranked in four categories of infestation (0: (group A). All bees were kept at -20 °C until uninfested, meaning both tracheae were quite assayed. A random sample of ca 300 bees from silvery; 1: incipient, one trachea with vague dark this group was then divided into four subsets. blemishes; 2: medium, one trachea clearly dark; One included 35 and another 70 bees, and the 3: high, tracheae brown to black). The examined two others were of 100 each, kept for the tracheae were then individually homogenized ACAREX® and ELISA tests (see below). The and visualized for their guanine content on TLC thoraces of the first 35-bee subset were dissected, plates. The outcome was evaluated according to the thoracic disks were cleared in NaOH (Shi- the degree of concordance or overlap (estimated manuki and Knox, 1991) and examined micro- by χ2 tests) between the results of the dissection scopically. and guanine method. Thirteen additional samples (each of ca 200 Guanine presence has been used to evaluate older worker bees) (group B) were collected in the contribution of mites to house dust aller- the spring of 1996 from other commerial (treated genicity; a commercial test kit is available for and non-treated) apiaries located throughout this purpose (Bronswijk et al, 1989; Twiggs et al, Israel. These bees were likewise kept at -20 °C 1992). We tested this product (ACAREX®, Aller- gopharma, Joachim Ganzer KG, Reinbeck, Ger- different method. Bees in one group (n = 35) many), following the manufacturer’s instruc- were individually dissected, and mite presence in tions, for its sensitivity to tracheal mite presence. their thoraces established after clearing (Shi- About a dozen heavily infested bees from group manuki and Knox, 1991). Mite infestation in A were individually homogenized in the fluid bees of the second group (n = 35) was deter- provided with the kit, and enclosed test stripes mined by the guanine method, whereas infesta- were dipped into various stock guanine prepa- tion of bees in the third group (n = 100) was rations and into the bee homogenate. The resul- assayed by ELISA. All data were converted to tant colors were compared to color standards percentages. provided by the manufacturer.

ELISA RESULTS

A competitive monoclonal-antibody ELISA Best guanine extraction was obtained with (enzyme-linked immunosorbent assay) kit which the basic solution. The Macherey-Nagel included with tracheal microplates precoated plates were more sensitive and required less mite antigen (Grant and Nelson, in press) was running time than the Merck plates. used. A binding competition is set up on the as as 2-4 were plates between the fixed, precoated antigen and Amounts of guanine low μg visualized this had an the free sample antigen, for a mite-specific mon- by procedure; they Rf- oclonal antibody. An enzyme-labeled anti- value of 0.35 (fig 1). immunoglobin conjugate-substrate system is The evaluation was used to detect the specific antibody which binds preliminary promis- in to the uninfested the precoated antigen. Tracheal mites in the sam- ing regard (category 0), ple inhibit the binding of the monoclonal anti- medium infested (2) and highly infested (3) body to the tracheal mite-precoated antigen. groups (fig 2). A good concordance, or fit, Degree of inhibition is proportional to the loga- between the two methods was obtained in rithm of the amount of mite antigen in the sam- ple. The resultant colors are measured by an ELISA colorimeter and the obtained values are then interpolated from a standard graph (% ELISA inhibition on tracheal mite infestation), sent with the ’ELISA kit for tracheal mite detec- tion’, supplied by Grant and Nelson (Beaver- lodge, AB, Canada). Less than six infested bees in a 100-bee sample (6%), or more than 60 infested bees in such a sample (60%) could not be evaluated precisely by this method. Procedure and interpretation of results followed the proto- col supplied by Grant and Nelson. Presence of the mite antigen was tested in the second 100-bee sample from group A. They were homogenized in 500 mL of 0.05% Tween 80 (Sigma), in phos- phate-buffered saline (PBST), with a commer- cial blender. The homogenized mixture was allowed to settle for 2 min and a liquid sample of 5 mL was immediately assayed, or frozen at -20 °C, for replicate assays. Each ELISA test was replicated three times or more.

Comparison between methods

The 200-bee samples (group B) were individually subdivided into three batches, each assayed by a categories 0, 2 and 3 (P < 0.05; χ2 tests), turer). A strong reaction was seen with suggesting that the guanine method pro- 1.5 mg guanine. These amounts are far vided results comparable to the dissections. greater (by a factor of 100) than the gua- The data in category 1 (incipient infesta- nine concentrations detected on TLC plates tion), on the other hand, were dissimilar by UV, as noted above, which negated the (P > 0.05, χ2 test), indicating lack of con- further use of ACAREX® for tracheal mite cordance between methods. In this category detection purposes. some slight blemishes may be due to causes A comparison between the three meth- other than mites, and very early mite attack ods (table I) indicated that qualitatively the not result in blemishes. We conclude may results of the three were consistent. Sam- that the method is as as dis- guanine good ples in which bees were infested (at or above sections for detecting A woodi in bees whose 6%, the lowest level detectable by ELISA), tracheae are infested. clearly tracheal mite presence was confirmed by The ACAREX® test was neither able to all methods. Similarly, very low to no infes- detect the presence of guanine in a basic tation was also uniformly detected. How- (NaOH extract) homogenate from infected ever, none of the methods were in consistent thoraces nor from whole homogenated bees. harmony with those of the other two. There With a prepared guanine solution the color was acceptable agreement between the dis- scale indicated no result when 0.1 mg gua- sections (representing the standard method) nine or less was in the stock solutions, and and the ELISA results in samples 1 and 8 a weak result with a 0.23 mg guanine solu- (9 and 6%, and 14 and 15%, respectively). tion (definitions according to the manufac- Samples 6, 7 and I 1 had reasonable con- cordance between dissections and guanine and may not detect mites if they are very (80 and 71 %, 62 and 59% and 71 and 69%, scarce or present only in other tracheae or in respectively); the latter method and ELISA the head sacs. Other direct methods require provided similar results with sample 2 (28 additional (including microscopical) equip- and 30%); an acceptable fit was obtained ment and/or more elaborate preparations, with all three methods in regard to samples such as staining. and 10 37 and and 34 and 9 (36, 33%, 40, The ELISA method, while accurate and 30%, whereas 12 respectively) sample gave sensitive, is probably most suitable for con- different data and Sam- very (62, 48 37%). comitantly assaying many bulk apiary sam- 5 and 13 consistent sim- ples 3, 4, provided ples. However, it requires special proce- ilar of no or low infestation. results, very dures, some technical expertise, may react to other species of Acarapis which could be present on the bees, and will deliver precise DISCUSSION results only at infestation levels between 6-60%. Thus it is not suitable for work with As noted by Shimanuki and Knox (1991), individual bees. Initially this method could each of nine methods listed for diagnosing read only infestations of more than 90 mites tracheal mite presence had advantages and per 100 bees, based on the assumption of disadvantages. The common prothoracic an average of 15 mites per parasitized bee disk method is labor intensive, monotonous (Grant et al, 1993). Grant and Nelson (in press) recently reported a lower detectable acariens mais n’est présent qu’à l’état de level of 21 mites per 100 bees. traces dans les excrétions des abeilles, a été utilisée comme test dia- The guanine method, although simpler biologique pour gnostiquer l’infestation des trachées de because it detects mite presence by specific l’abeille par l’acarien Acarapis woodi (Ren- excretions, and avoids the extensive prepa- Les abeilles infestées ont été rations needed for immunological methods, nie). homogé- néisées dans des solutions acides ou is likewise unable to differentiate between Les cellules ont été déchirées aux A woodi and other bee-colonizing Acarapis basiques. ultrasons puis centrifugées. Le surnageant spp. The results regarding incipient infes- a été étalé sur des chromato- tation (category 1, fig 2) indicate that the plaques pour graphie en couche mince avec les marqueurs new method could not detect very low tra- de et visualisé en lumière cheal mite numbers. This drawback is shared guanine appropriés UV Dans un test la all other methods. Another (fig 1). préliminaire, by diagnostic des résultats de la méthode disadvantage of the guanine method is that comparaison classique (dissection de 100 abeilles) et de la it is very time-consuming when individual méthode de visualisation de la guanine sur bees are to be examined, as each thorax must les mêmes abeilles individuellement, be homogenized separately. However, the prises a montré les deux méthodes donnaient guanine method has the advantage that it que des résultats identiques quel que soit le taux might be used as a quantitative tool in diag- d’infestation ou nosing infestations in individual bees (and (nul, moyen fort) (fig 2). La méthode de la guanine n’a pas pu mettre hives), as well as a qualitative tool in bulk en évidence de nette les infestations following whole bee façon apiary samples, débutantes. Les valeurs d’ACAREX®, homogenization. Such qualitative diagnoses pro- duit commercial mis au détecter would be and less labor intensive point pour quicker la d’acariens de la than other available indirect methods. présence poussière domestique par lecture de la guanine, étaient trop grossières pour diagnostiquer l’acarien des trachées. On a comparé trois types ACKNOWLEDGMENTS d’essais (la méthode du disque thoracique, la méthode ELISA et la méthode de la gua- This was Grant No IS-2508- study supported by nine) sur 13 échantillons de 170 acariens 95 from the United States — Israel (Binational) chacun La concordance entre Research and Fund (tableau I). Agricultural Development les bonne sur le mais (BARD). We wish to thank DL Nelson and résultats, plan qualitatif GA Grant of the Northern Agricultural Research seulement moyenne sur le plan quantitatif, Centre, Beaverlodge, AB, Canada, for supply- indique que la méthode de la guanine pour- ing the ELISA kit and many helpful suggestions rait être utilisée pour établir la présence d’A for its use. WA Bruce, Bee Research Labora- woodi. Les avantages et inconvénients de and tory, USDA-ARS, Beltsville, MD, USA, cette nouvelle méthode sont discutés. D Sammataro, Extension Bee Laboratory, The Ohio State University, Wooster, OH, USA, read the manuscript and offered useful comments. Apis mellifera / Acarapis woodi / diagnos- tic / guanine

Résumé — Visualisation de la guanine : nouvelle méthode pour diagnostiquer Zusammenfassung — Guanin-Visuali- l’acariose des trachées de l’abeille domes- sierung: Eine neue Methode der Diagnose tique. La présence de guanine (2-amino-6- eines Tracheenmilbenbefalls bei Honig- oxypurine), purine qui est le principal pro- bienen. Der Nachweis von Guanin wurde duit final du métabolisme azoté chez les als Biotest zum Erkennen eines Tracheen- milbenbefalls von Honigbienen (Acarapis REFERENCES woodi (Rennie)) genutzt. Das Purin Guanin (2-Amino-6-Oxypurin) ist das Hauptend- Anderson JF (1966) The excreta of spiders. Comp des Stickstoffwechsels bei Milben, Biochem Physiol 17, 973-982 produkt AH (1992) behavior and kommt aber höchstens in in den Aus- Atmowidjojo Comparative Spuren physiology of feral and domestic honeybees, Apis scheidungen der Bienen vor. Befallene Bie- mellifera L. Thesis, University of Arizona, AZ, nen wurden in sauren oder basischen Lösun- USA gen homogenisiert, die Zellen durch Bronswijk JEMH van, Bischoff E, Schirmacher W, Ultraschall zerstört und zentrifugiert. Der Kniest FM (1989) Evaluating mite () aller- genicity of house dust by guanine quuntification. Überstand wurde auf TLC Platten zusam- J Med Entomol 26, 55-59 men mit geeigneten Guanin-Markern auf- Eischen FA, Cardoso-Tamez D, Wilson WT, Dietz A getrennt und mit einer UV-Lampe sichtbar (1989) Honey production of honey bee colonies gemacht (Abb 1). In einem vorläufigen Test infested with Acarapis woodi (Rennie). Apidolo- wurde das Ergebnis einer klassischen Di- gie 20, 1-8 Erickson EH, Cohen AC, Cameron BE (1994) Mite agnose des Tracheenmilbenbefalls durch excreta: a new diagnostic for varroasis. BeeScience Präparation mit dem einer Guanin-Visuali- 3, 76-78 sation an 100 individuellen Bienen gegen- Gerson U, Dag A, Efrat H, Slabezki Y, Stern Y (1994) übergestellt und ergab vergleichbare Ergeb- Tracheal mite, Acarapis woodi, comes to Israel. nisse für unbefallene, mittelstark und stark Am Bee J 134, 486 befallene Bienen (Abb 2). Ein beginnender Grant GA, Nelson DL The ELISA detection of honey Befall konnte mit der Guaninmethode aller- bee tracheal mite, Acarapis woodi. In: Acarology IX; Proc (Mitchell R, Horn DJ, Needham GR, nicht klar unterschieden werden. Die dings Welbourn WC, eds), Ohio Biological Survey, Bestimmung mit ACAREX®, einem kom- Columbus, OH, USA (in press) merziellen Guanintest zum Nachweis von Grant GA, Nelson DL, Olsen PE, Rice WA (1993) Hausstaubmilben, war für eine Diagnose The ELISA detection of tracheal mites in whole honey bee samples. Am Bee J 133, 652-655 der Tracheenmilben zu unempfindlich. Die Komeili AB, Ambrose JT (1990) Biology, ecology Eignung von drei verschiedene Testmetho- and damage of tracheal mites on honey bees (Apis den zum Erkennen eines Tracheenmilben- mellifera). Am Bee J 130, 253-257 befalls (Thorax-Schnittpräparatmethode, Pettis JS, Wilson WT (1996) Life history of the honey ELISA und Guanin-Visualisierung) wurden bee tracheal mite Acarapis woodi (Acari: Tarson- anhand von 13 Proben mit jeweils 170 Bie- emidae). Ann Entomol Soc Am 89, 368-374 KM of tracheal nen verglichen (Tabelle I). Die gute quali- Ragsdale DW, Kjer (1989) Diagnosis mite (Acarapis woodi) of honey bees tative, nur allerdings mäßige quantitative using a monoclonal based enzyme-linked die der Übereinstimmung belegt Eignung immunosorbent assay. Am Bee J 129, 550-553 Guaninmethode zum Erkennen eines Tra- Shimanuki H, Knox DA (1991) Diagnosis of Honey cheenmilbenbefalls. Die Vorteile und Nach- Bee Diseases. USDA Agricultural Handbook No teile der Methode werden diskutiert. AH-690 Twiggs JT, Gray RL, Marx JJ Jr (1992) Correlation of the Acarex™ test for guanine contents of vac- uum dust with the amount of mite allergen present woodi Apis mellifera / Acarapis / Diagnose/ as determined by RAST inhibition assay. Immun Guanin Allerg Prac 14, 334-339