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

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Guanine visualization: a new method for diagnosing

tracheal mite infestation of honey bees

R Mozes-Koch, U Gerson

To cite this version:

Original

article

Guanine visualization:

a new

method

for

diagnosing

tracheal

mite infestation

of

_honey

bees

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 mites, but in bee 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

/

_Acarapis

woodi /

_diagnosis

/

_guanine

INTRODUCTION

The

_honey

bee tracheal

mite,

Acarapis

woodi

_(Rennie),

a serious _pest of

_honey

bees

(Eischen

et

al, 1989;

Komeili and

Ambrose,

1990),

has

_recently

been found in Israel

(Gerson

et

_{al, 1994).}

Efforts towards its

management,

including

various cultural and

chemical

_methods,

are

_usually

hindered

_by

difficulties in

_evaluating

their effects. These difficulties stem from the fact that no

sin-gle

symptom

fully

characterizes the

_pests’

effect

_(Shimanuki

and

_Knox,

_1991).

Although

affected bees _may have

_disjointed

wings

and/or a distended

abdomen,

absence of these

_symptoms

does not mean absence of

mites,

which are located

_mainly

within the

prothoracic

tracheal _system of their host

bees. Several methods have been

_proposed

and

_implemented

for

_positive

tracheal mite identification. These

methods,

reviewed

_by

Shimanuki and Knox

_(1991),

_roughly

fall

into either direct or indirect

categories.

The

former includes bee dissection and

homog-enization,

and

_staining

of

tracheae,

by

which the mites are

_actually

seen. The indirect

method uses the ELISA

_technique

_(in

which

infestation is determined

_by

a colorimetric

*

Correspondence

and

_reprints

reaction in

_{microplates precoated}

with the tracheal mite

_{antigen) (Ragsdale}

and

_Kjer,

1989;

Grant et

_{al, 1993;}

Grant and Nelson,

in

_press).

Herein we describe a new, indirect

method,

in which a

_{purine compound,}

gua-nine,

was used to determine _{the presence} of

A woodi. Guanine

_{(2-amino-6-oxypurine)}

is the main end

_product

of

_nitrogen

metabolism _{in many}

_arachnids,

_including

spiders

and mites

_{(Anderson, 1966;}

Bron-swijk

et

_{al, 1989),}

but is almost absent from

honey

bee excretions

_{(Atmowidjojo,}

1992,

cited

_by

Erickson et al,

1994).

Its occur-rence in

_honey

bees would therefore

indi-cate that

_they

are infested with mites. Gua-nine is also excreted

_{by Varroa jacohsoni}

Oudemans

_(Erickson

et al,

1994),

another

acarine

_attacking

the

_honey

bee, but this mite is an external

_parasite,

not _present

within the bees’ bodies.

MATERIALS AND METHODS

Bees

Older

_honey

bees

_{(recognized by canying pollen)}

were collected from frames within commercial, non-treated colonies

_previously

determined to be affected

_by

tracheal mite. Older bees are known to be infested

_{by larger}

numbers of tra-cheal mites _(Pettis and Wilson, 1996). Several hundred worker bees were thus obtained

_during

the

_spring

of 1996 over a

_period

of several weeks

(group A). All bees were

_kept

at -20 °C until

assayed.

A random

_sample

of ca 300 bees from this group was then divided into four subsets. One included 35 and another 70 bees, and the two others were of 100 each,

_kept

for the

ACAREX® and ELISA tests (see below). The thoraces of the first 35-bee subset were _dissected, the thoracic disks were cleared in NaOH

(Shi-manuki and Knox, 1991) and examined

micro-scopically.

Thirteen additional

_samples

(each of ca 200 older worker _bees)

_(group

_B) were collected in

the

_spring

of 1996 from other commerial _(treated and non-treated)

apiaries

located

_throughout

Israel. These bees were likewise

_kept

at -20 °C

until

_they

were

_assayed.

These

_samples

were used to _{compare essay methods} (see below).

Guanine visualization

Thoraces of the second, 70-bee subset were sep-arated into two

_{equally-sized}

_{groups. Those of} the _{first group} were

_placed

in 0.5 mL 0.1 HCl

(acid solution), those of the second in 0.1 M NaOH (basic solution). All thoraces were

indi-vidually homogenized by

an Ultra-Turrax tissue

homogenizer

(2 min), followed

_{by teflon-glass}

homogenization

(2 min) and ultrasonic cell

dis-ruption

(2 min) and

_{finally centrifugation}

(12 000 g for 20 min, at 4 °C). The _supernatant

was collected for

_guanine

determination via TLC.

Two _types of

_plates

were

_compared

for this

pur-pose: Cellulose

_F

₂₅₄

(Merck) and

Polygram®

Cell 300

_UV

₂₅₄

_,

_{(Macherey-Nagel,}

Doren,

Ger-many). Supernatant

samples

of 10 _μL were loaded 3 cm from the

_edge,

with 2 cm between

samples.

Guanine

_(Sigma

_Ultra, 10

_μL

of

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

solution, whereas a mixture of 5% w/v ammo-nium sulfate/13 M

_{ammonia/1-propanol}

(60:30:10) was best for basic extracts. Guanine spots were visualized by a UV

_lamp

and

com-pared

to _spots obtained

_by

the above standards. Ultraviolet

_{spectral analyses}

(200-300 nm) of the

_homogenate

were tested

_by

means of an

Uvi-con-810

_{spectrophotometer}

and were

_compared

to markers in basic and acidic solutions. A

_preliminary

evaluation of the method was conducted

_{by dissecting}

the insects in one of the 100-bee

_samples

_{from group A. Their tracheae}

were ranked in four

_categories

of infestation _(0: uninfested,

meaning

both tracheae were

_quite

silvery;

1:

_incipient,

one _{trachea with vague dark}

blemishes; 2: medium, one trachea

_clearly

dark;

3:

_high,

tracheae brown to black). The examined tracheae were then

_individually

_homogenized and visualized for their

_guanine

content on TLC

plates.

The outcome was evaluated

_according

to the

_degree

of concordance or

_overlap

_(estimated

by

χ

2

tests) between the results of the dissection and

_guanine

method.

Guanine presence has been used to evaluate the contribution of mites to house dust

aller-genicity;

a commercial test kit is available for this purpose

(Bronswijk

et al, 1989;

Twiggs

et al,

Aller-gopharma,

Joachim Ganzer _{KG, Reinbeck,}

Ger-many),

following

the manufacturer’s

instruc-tions, for its

_sensitivity

to _{tracheal mite presence.} About a dozen

_heavily

_{infested bees from group} A were

_{individually homogenized}

in the fluid

provided

with the kit, and enclosed test

_stripes

were

_dipped

into various stock _guanine prepa-rations and into the bee

_homogenate.

The

resul-tant colors were

_compared

to color standards

provided

by the manufacturer.

ELISA

A

_{competitive monoclonal-antibody}

ELISA

(enzyme-linked

immunosorbent _assay) kit which included

_{microplates precoated}

with tracheal mite

_antigen

(Grant and Nelson, in _press) was

used. A

_{binding competition}

is set _up on the

plates

between the _fixed,

_{precoated antigen}

and the free

_{sample antigen,}

for a

_{mite-specific}

mon-oclonal

_antibody.

An

_{enzyme-labeled}

anti-immunoglobin conjugate-substrate

system is used to detect the

_{specific antibody}

which binds the

_{precoated antigen.}

Tracheal mites in the

sam-ple

inhibit the

_binding

of the monoclonal

anti-body

to the tracheal

_{mite-precoated antigen.}

Degree

of inhibition is

_proportional

to the

loga-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

different method. Bees in one _group (n = ₃₅₎

were

_individually

_dissected, and mite presence in

their thoraces established after

_clearing

(Shi-manuki and Knox, 1991). Mite infestation in bees of the second group (n = ₃₅₎ was deter-mined

_by

the

_guanine

method, whereas infesta-tion of bees in the third group (n = _{100) was}

assayed by

ELISA. All data were converted to

percentages.

RESULTS

Best

_guanine

extraction was obtained with the basic solution. The

_{Macherey-Nagel}

plates

were more sensitive and

_required

less

running

time than the Merck

_plates.

Amounts of

_guanine

as low as 2-4 μg were visualized

_by

this

_{procedure; they}

had an

_R

_f

-value of 0.35 (fig

1).

The

_preliminary

evaluation was

promis-ing

in

_regard

to the uninfested

_{(category 0),}

medium infested

₍₂₎

and

_highly

infested

₍₃₎

groups

(fig

2).

A

_good

concordance, or fit,

categories

0,

2 and 3

(P

< 0.05;

_&chi;

₂

tests),

suggesting

that the

_guanine

_method

pro-vided results

_comparable

to the dissections. The data in

_category

_{1 (incipient}

infesta-tion),

on the other

_hand,

were dissimilar

(P

>

_0.05,

&chi;

2

_test),

_indicating

lack of

con-cordance between methods. In this

_category

some

_slight

blemishes _may be due to causes

other than

mites,

and very

early

mite attack may not result in blemishes. We conclude that the

_guanine

method is as

_good

as

dis-sections for

_detecting

A woodi in bees whose tracheae are

_clearly

infested.

The

ACAREX&reg;

test was neither able to

detect the _{presence of}

_guanine

in a basic

(NaOH

extract)

homogenate

from infected thoraces nor from whole

_homogenated

bees.

With a

_prepared

_guanine

solution the color scale indicated no result when 0.1

_mg

gua-nine or less was in the stock

_solutions,

and a weak result with a _{0.23 mg}

_guanine

solu-tion

_(definitions

_according

to the

manufac-turer).

A _strong reaction was seen with 1.5 mg

guanine.

These amounts are far

greater

(by

a factor of

₁₀₀₎

than the

gua-nine concentrations detected on TLC

_plates

by

UV,

as noted

above,

which

_negated

the further use of

ACAREX&reg;

for tracheal mite detection _purposes.

A

_comparison

between the three

meth-ods

_{(table I)}

indicated that

_{qualitatively}

the results of the three were consistent.

Sam-ples

in which bees were infested

_(at

or above

6%, the lowest level detectable

_by

_ELISA),

tracheal mite _presence was confirmed

_by

all methods.

_Similarly,

_{very low} to no infes-tation was also

_uniformly

detected. How-ever, none of the methods were in consistent

harmony

with those of the other two. There

was

_acceptable

_{agreement between} the

dis-sections

_{(representing}

the standard

_method)

and the ELISA results in

_samples

1 and 8

(9

and

6%,

and 14 and

_15%,

_{respectively).}

con-cordance between dissections and

_guanine

(80

and

71 %, 62

and 59% and 71 and

69%,

respectively);

the latter method and ELISA

provided

similar results with

_sample

2

₍₂₈

and

_30%);

an

_acceptable

fit was obtained

with all three methods in

_regard

to

_samples

9 and 10

(36,

37 and

_33%,

and

_40,

34 and

30%,

respectively)

whereas

_sample

12 gave very different data

(62, 48

and

37%).

Sam-ples

3, 4,

5 and 13

_provided

consistent sim-ilar

results,

of no or _{very low} infestation.

DISCUSSION

As noted

_by

Shimanuki and Knox

_(1991),

each of nine methods listed for

_diagnosing

tracheal mite _presence had

_advantages

and

disadvantages.

The common

_prothoracic

disk method is labor

intensive,

monotonous

and _may not detect mites if

_they

are _very scarce or _present

_only

in other tracheae or in the head sacs. Other direct methods

require

additional

_(including

_{microscopical)}

equip-ment and/or more elaborate

preparations,

such as

_staining.

The ELISA

method,

while accurate and

sensitive, is

probably

most suitable for

con-comitantly assaying

many bulk

apiary

sam-ples.

However,

it

_requires

_special

proce-dures, some technical

expertise,

_may react to

other

_species

_{of Acarapis}

which could be

present

on the

bees,

and will deliver

precise

results

_only

at infestation levels between 6-60%. Thus it is not suitable for work with individual bees.

_Initially

this method could

read

_only

infestations of more than 90 mites per 100

bees,

based on the

assumption

of an _average of 15 _{mites per}

_parasitized

bee

press) recently reported

a lower detectable

level of 21 mites _per 100 bees.

The

_guanine

method,

although

simpler

because it detects mite _presence

_{by specific}

excretions,

and avoids the extensive

prepa-rations needed for

_{immunological}

_methods,

is likewise unable to differentiate between A woodi and other

_{bee-colonizing Acarapis}

spp. The results

regarding incipient

infes-tation

_(category

1,

fig

2)

indicate that the new method could not _{detect very low}

tra-cheal mite numbers. This drawback is shared

by

all other

_diagnostic

methods. Another

disadvantage

of the

_guanine

method is that it _{is very}

_{time-consuming}

when individual bees are to be

examined,

as each thorax must

be

_{homogenized separately.}

However,

the

guanine

method has the

_advantage

that it

might

be used as a

_quantitative

tool in

diag-nosing

infestations in individual bees

_(and

hives),

as well as a

_qualitative

tool in bulk

apiary samples,

following

whole bee

homogenization.

Such

_{qualitative diagnoses}

would be

_quicker

and less labor intensive than other available indirect methods.

ACKNOWLEDGMENTS

This

study

was

_{supported by}

Grant No IS-2508-95 from the United States &mdash; Israel (Binational)

Agricultural

Research and

_Development

Fund

(BARD). We wish to thank DL Nelson and GA Grant of the Northern

_Agricultural

Research

Centre,

Beaverlodge,

AB, Canada, for

supply-ing

the ELISA kit and many

helpful suggestions

for its use. WA Bruce, Bee Research

Labora-tory, USDA-ARS, Beltsville, MD, USA, and D Sammataro, Extension Bee

_Laboratory,

The Ohio State

_University,

Wooster, OH, USA, read the

_manuscript

and offered useful comments.

Résumé &mdash; Visualisation de la

_{guanine :}

nouvelle _{méthode pour}

_{diagnostiquer}

l’acariose des trachées de l’abeille

domes-tique.

La

_présence

de

_guanine

(2-amino-6-oxypurine), purine qui

est le

_principal

pro-duit final du métabolisme azoté chez les

acariens mais n’est

_{présent qu’à}

l’état de

traces dans les excrétions des

abeilles,

a été

utilisée comme test

_biologique

_pour

dia-gnostiquer

l’infestation des trachées de l’abeille

_{par l’acarien}

_Acarapis

woodi

(Ren-nie).

Les abeilles infestées ont été

homogé-néisées dans des solutions acides ou

basiques.

Les cellules ont été déchirées aux

ultrasons

_{puis centrifugées.}

Le _surnageant a été étalé sur des

_plaques

_pour

chromato-graphie

en couche mince avec les marqueurs de

_guanine

_appropriés

et visualisé en lumière

UV

_(fig

_1).

Dans un test

_{préliminaire,}

la

comparaison

des résultats de la méthode

classique

(dissection

de 100

_abeilles)

et de la méthode de visualisation de la

_guanine

sur

les mêmes abeilles

_prises

individuellement,

a _{montré que les deux méthodes} donnaient des résultats

_{identiques quel}

_que soit le taux

d’infestation

_(nul,

_moyen ou

_fort)

_(fig

_2).

La méthode de la

_guanine

n’a _{pas pu} mettre

en évidence de

_façon

nette les infestations débutantes. Les valeurs

d’ACAREX&reg;,

pro-duit commercial mis au

_point

_pour détecter

la

_présence

d’acariens de la

_poussière

domestique

par lecture de la

guanine,

étaient

trop

grossières

pour

diagnostiquer

l’acarien

des trachées. On a

_comparé

trois

_types

d’essais

_(la

méthode du

_{disque thoracique, la}

méthode ELISA et _{la méthode de la}

gua-nine)

sur 13 échantillons de 170 acariens

chacun

_{(tableau I).}

La concordance entre

les

résultats,

bonne sur le

_{plan qualitatif}

mais

seulement moyenne sur le

_{plan quantitatif,}

indique

que la méthode de la

guanine

pour-rait être utilisée _{pour établir} la

_présence

d’A

woodi. Les

_avantages

et inconvénients de

cette nouvelle méthode sont discutés.

Apis

mellifera / Acarapis

woodi /

diagnos-tic /

_guanine

Tracheen-milbenbefalls von

_Honigbienen

_(Acarapis

woodi

_(Rennie))

_genutzt. Das Purin Guanin

(2-Amino-6-Oxypurin)

ist das

Hauptend-produkt

des Stickstoffwechsels bei

_Milben,

kommt aber höchstens in

_Spuren

in den

Aus-scheidungen

der Bienen vor. Befallene Bie-nen wurden in sauren oder basischen Lösun-gen

homogenisiert,

die Zellen durch Ultraschall zerstört und

_{zentrifugiert.}

Der

Überstand

wurde auf TLC Platten zusam-men mit

_geeigneten

Guanin-Markern

auf-getrennt

und mit einer

_UV-Lampe

sichtbar

gemacht

(Abb 1). In

einem

_vorläufigen

Test wurde das

_Ergebnis

einer klassischen Di-agnose des Tracheenmilbenbefalls durch

Präparation

mit dem einer

Guanin-Visuali-sation an 100 individuellen Bienen

gegen-übergestellt

und

_{ergab vergleichbare}

Ergeb-nisse für unbefallene, mittelstark und stark

befallene Bienen

_{(Abb 2).}

Ein

_beginnender

Befall konnte mit der Guaninmethode

aller-dings

nicht klar unterschieden werden. Die

Bestimmung

mit

_ACAREX&reg;,

einem kom-merziellen Guanintest zum Nachweis von

Hausstaubmilben,

war für eine

_Diagnose

der Tracheenmilben zu

_{unempfindlich.}

Die

Eignung

von drei verschiedene Testmetho-den zum Erkennen eines Tracheenmilben-befalls

_{(Thorax-Schnittpräparatmethode,}

ELISA und

_{Guanin-Visualisierung)}

wurden

anhand von 13 Proben

_{mit jeweils}

170

Bie-nen

_verglichen

_{(Tabelle I).}

Die

_gute

quali-tative,

allerdings

nur

_{mä&szlig;ige quantitative}

Übereinstimmung

belegt

die

_Eignung

der

Guaninmethode zum Erkennen eines

Tra-cheenmilbenbefalls. Die Vorteile und

Nach-teile der Methode werden diskutiert.

Apis mellifera / Acarapis

woodi

_{/ Diagnose/}

Guanin

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