Original article

Toluidine blue staining as a rapid measurefor initiation of oocyte growth and fertility

in Varroa jacobsoniOud.

Claudia GARRIDOa, Peter ROSENKRANZa*, Matthias STÜRMERa, b,Ralph RÜBSAMc, Jürgen BÜNINGc

a Universität Hohenheim, Landesanstalt für Bienenkunde, August-von-Hartmann-Strasse 13,70599 Stuttgart, Germany

b Bayerische Landesanstalt für Bienenzucht, Burgbergstrasse 70, 91054 Erlangen, Germanyc Universität Erlangen-Nürnberg, Institut für Zoologie I, 91054 Erlangen, Germany

(Received 17 February 2000; revised 21 April 2000; accepted 19 May 2000)

Abstract – A whole mount method was used to determine the moment of activation of oocyte growthin Varroa jacobsonifemales. Ovaries of the mites were dissected and stained with toluidine blue. Thecoloration of the terminal oocyte indicates the uptake of euplasmatic and/or yolk material and, there-fore, the initiation of the reproductive phase. In phoretic mites from adult bees, no staining of the ovarycould be detected. V. jacobsonifemales artificially introduced into freshly capped brood cells andremoved for dissection 6 h later already showed clear blue staining of the terminal oocyte. Theovaries of V. jacobsonifemales introduced 14 h after capping of the brood cell, however, remaineduncolored after incubation in toluidine blue. We conclude that, in phoretic mites, oogenesis is arrestedat a previtellogenic phase. Immediately after invasion of the brood cell, reproduction is activatedby an as yet unknown host factor. This factor is present in freshly capped brood cells but not inbrood cells 14 h after capping. Our new method offers new possibilities for the exact determinationof oocyte growth and, therefore, for the study of V. jacobsonifertility in different host and parasitepopulations.

Varroa jacobsoni/ host-parasite relationship / vitellogenesis / oogenesis / fertility / reproduction

Apidologie 31 (2000) 559–566 559© INRA/DIB-AGIB/EDP Sciences

* Correspondence and reprintsE-mail: bienero@uni-hohenheim.de

C. Garrido et al.560

1. INTRODUCTION

The fertility of Varroa jacobsonimites(Acari, Varroidae) reproducing in workerbrood cells has been shown to affect moststrongly the population dynamics of the par-asite in honey bee colonies [11]. In the orig-inal host species, Apis cerana Fabr., femalemites do not reproduce in worker brood butthey have high fertility in drone brood,which they prefer over worker brood [2, 6,14, 21]. In the secondary host Apis melli-fera L., reduced fertility of the mites is cor-related with increased tolerance to varroosis,as seen in the Africanized honey bees ofBrazil [7, 15, 17].

The signals triggering the activation ofoocyte maturation of the parasite after inva-sion of the brood cell are as yet unknown. Inphoretic mites, oogenesis is arrested at anearly stage of egg cell development. Oocytegrowth in reproductive V. jacobsonifemalesdepends on the consumption of haemolymphfrom freshly sealed larvae [10, 25]. Thesehaemolymph meals were assumed to initiatelyrate organ function and, later on, to lead tovitellogenesis. Artificial introduction ofmites into sealed brood cells containing aprepupa, however, failed to induce mitereproduction [4, 20]. The supposed stimu-lation by host derived hormone [12] or othertrophogenous factors [20] has not been con-firmed [19, 21]. Recent studies indicate astage-specific semi-chemical stimulation ofV. jacobsonireproduction [26]: the polarfraction of host larval cuticle induced eggmaturation, though only if extracted fromlarvae of the fifth instar.

In order to evaluate the moment of effec-tive stimulation and subsequent growth ofV. jacobsoni’s terminal oocyte, histologi-cal examination with time-consuming serialsections were required [22, 23]. Here wepresent a method for rapid toluidine bluestaining of ovary whole mount dissections.By means of this technique, we provide asensitive method for monitoring the initia-tion of oocyte growth in V. jacobsoni.

2. MATERIALS AND METHODS

2.1. Artificial introductionof V. jacobsonifemales

Studies were performed at the apiary ofthe University of Hohenheim with Apis mel-lifera carnica. Phoretic mites were takenfrom living nurse bees on combs with openbrood. A brood comb of another, lightlyinfested honey bee colony with recentlysealed worker brood cells was used for arti-ficial infestation. A small gap in the cellsealing was opened with an insect needleand carefully closed again after introduc-tion of one V. jacobsonifemale. All miteswere introduced within 30 minutes afterremoval from the adult bees. They were sep-arated into two groups.

2.1.1. “Early introduction”

V. jacobsonifemales were introducedinto recently sealed brood cells (0–2 hours).Using this method, 80 to 90 percent ofV. jacobsonifemales laid eggs, similar tothe percentage in naturally invaded cells atour apiary [16, 20].

2.1.2. “Delayed introduction”

V. jacobsonifemales were introducedinto brood cells that had been sealed for14–16 hours. Less than 10% of these mitesare able to lay eggs [Rosenkranz andStürmer, unpublished data]. Therefore, thesemites represent a group of “artificially infer-tile” V. jacobsonifemales.

In our test colony, naturally infestedworker brood cells were checked 7–9 dayspost capping for V. jacobsoni reproductionto ensure that, in the V. jacobsonipopulationwe used for our experiments, rates of mitereproduction were normal.

2.2. Removal of mitesand record of body weight

After artificial introduction, V. jacobsonifemales were removed at 6-hour intervals

Initiation of oocyte growth in V. jacobsoni

3. RESULTS

3.1. Reproduction in naturally infestedbrood cells

From 15 worker brood cells of our testcolony naturally infested with a singleV. jacobsonifemale, 14 mites laid eggs,indicating that the respective V. jacobsonipopulation had a normal reproductive capac-ity.

3.2. Change in V. jacobsoniweight

Phoretic mites weighed 325 µg (± 26 µg).Body weight increased to 411 µg (± 30 µg,“early introduction”) and 407 µg (± 27 µg,“delayed introduction”) 24 hours after intro-duction to a cell. During this first day of thereproductive cycle, there was no significantdifference (p > 0.05, Friedmann-Test) inweight change between the two groups ofV. jacobsonimites (Fig. 1).

3.3. Oocyte growth

We analyzed ovaries of 18 phoretic mitescollected from nurse bees and 41 ovariesfrom brood mites. In phoretic mites, thesomewhat larger terminal oocyte, which is

(6, 12, 18 and 24 hours) from brood cellsand immediately weighed on a Mettler finebalance (0.1 mg accuracy) in groups of5 mites to record average body weight.Phoretic mites were weighed in the sameway. For each data point, at least 20 groupsof mites were weighed.

2.3. Dissection, whole mount staining,and microscopic analysisof V. jacobsoniovaries

V. jacobsonifemales sampled from adultbees or from brood cells were opened byremoving the ventral shield. The ovarytogether with spermatheca and lyrate organwere carefully transferred into PBS buffer(phosphate buffered saline, pH 7.2–7.4).After 30 minutes fixation in formaline (4%),the reproductive tracts were washed threetimes with buffer and incubated for 30 min-utes in toluidine blue (0.005%). After furtherwashing in PBS (three times 15 minutes)the coloring of the oocytes in the ovary wasanalyzed with a Zeiss photomicroscope400 ×). In this study, we distinguishedbetween colored and non-colored oocytesto determine whether there had been incor-poration of euplasmatic material and/or yolkproteins into the oocyte; this would indicatean activation of the oocyte.

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Figure 1. Increase in bodyweight of V. jacobsonifemales after introductioninto capped brood cells.Mites introduced immedi-ately after capping of thebrood cells had an identicalweight increase as mitesintroduced 14 hours aftercapping.

C. Garrido et al.

expected to become the first egg, had a paleand non-specific staining after incubationin toluidine blue. It did not differ from thesurrounding tissue of the ovary or thesmaller oocytes (Fig. 2a). Six hours aftercell sealing, a clear blue staining of the ter-minal oocyte was already clearly visible(Fig. 2b, Tab. I). The intensity of the bluecolor, however, still varied among the18 specimens. After 12 and 18 hours, thestaining of the primary oocyte was moreintense and without the variation seen inoocytes sampled at 6 hours (Fig. 2c). There-fore, toluidine blue staining of the biggestoocyte, indicating uptake of euplasmaticand/or yolk material, could be verified for all41 ovaries from brood mites (Tab. I).

From the 10 V. jacobsonifemales intro-duced 14–16 h after capping of the broodcells, we could find a stained oocyte in onlya single ovary, meaning that 90% of theV. jacobsonimites of this group had notcommenced oocyte maturation (Tab. I).

4. DISCUSSION

By the use of our new method, we wereable to determine for the first time the exacttiming of activation of the terminal oocyte in

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Figure 2. The terminal oocyte of V. jacobsoniovaries dissected and stained according to ourwhole mount technique. In phoretic mites (a) theterminal oocyte is already larger than the otherones, but no incorporation of euplasmatic mate-rial or yolk protein is visible. 6 hours after cellcapping (b) the first incorporation is clearly vis-ible by blue staining. 18 hours after cell capping(c) the oocyte is completely colored.

a

b

c

Table I. Oocyte growth in dissected ovaries.V. jacobsonifemales were introduced into freshlycapped brood cells and removed at 6 hours inter-vals.

Time after Number of Number ofcell capping ovaries examined actived ovaries

phoretic 18 06 h 18 1812 h 11 1118 h 7 724 h 5 524 h* 10 1

* V. jacobsonifemales were introduced 14–16 hoursafter cell capping and removed 24 hours later for dis-section.

Initiation of oocyte growth in V. jacobsoni

[16]. The lower percentage of fertile mitesunder natural conditions may be explained byan arrest of oogenesis in already activatedovaries, probably due to oosorption [22].

Surprisingly, V. jacobsonifemales whichwere assumed to remain infertile (“delayedintroduction”) increase their body weightduring the first 48 hours after artificial intro-duction to a brood cell, and to a similar mag-nitude as fertile mites (“early introduction”).This feeding behavior indicates the generalreadiness of phoretic V. jacobsonifemales toenter the reproductive phase which, how-ever, requires the action of an initiation fac-tor. At least, oocyte activation does notdepend on a phagostimulus or on the quan-titative uptake of larval haemolymph, as hasbeen assumed previously [20].

Our preliminary results support thehypothesis that the assumed factor for oocyteactivation is only present in freshly cappedbrood cells. Whenever V. jacobsonifemalesare introduced into brood cells which havebeen capped for 14 h, they do not initiateoocyte growth at all. The nature of this hypo-thetical activation factor remains unknownand, since the rejection of the JH hypothesis[12, 19, 21], no other specific host-derivedcompound with a distinct peak shortly aftercell capping has yet been identified. Thevery quick physiological reaction of mitessupports the conclusions of Trouillier andMilani [26] that V. jacobsonireproduction isstimulated by a chemotactic signal.

There is still the important question ofwhether the high proportion of infertileV. jacobsonifemales in worker brood ofApis ceranaand Africanized honey bees[14, 15], for instance, might be explainedby reactions to a specific host signal.V. jacobsoniinfertility may reflect varia-tion of an host-dependent factor as well as areaction of certain mite genotypes towardthis signal [3, 5, 8].

Therefore, we need more data on the ini-tiation of oocyte maturation, particularlyin situations where a high percentage ofinfertile mites exists. Our new method offers

the V. jacobsoniovary. Whole mount prepa-rations were used to examine the initiation ofoocyte growth in large samples of individualV. jacobsonimites. This approach can beapplied even in semi-field conditions. Ofcourse, statements about the quantity or thespecificity of the material incorporated intothe oocyte are not possible. For details ofthe course of oogenesis, time consumingsectioning and immunocytochemical tech-niques are still necessary [1, 22, 24].

In all the phoretic mites examined bytoluidine blue staining, no coloration of theovary was detected. Even the previouslyenlarged oocyte which is about to enter thefirst gonocycle [23] did not reveal specificstaining. This confirms the view thatphoretic mites remain in a previtellogenicstage [9, 23]. In our study, oocyte colorationwas independent of the status of the opistho-soma of the phoretic mites (“swollen” or“not swollen” as described formerly [4]).Six hours after cell capping, the terminaloocytes already were clearly blue-coloredindicating activated oocyte growth. This issignificantly earlier than the assumed start ofvitellogenesis, previously thought to be ini-tiated 15 hours after cell capping [22, 23].Probably, during this initial phase of oocytegrowth, euplasmatic components suppliedby the nursing lyrate organ [22] are incor-porated into the oocyte rather than yolk pro-teins. Under the selection pressure of therelatively short postcapping period of thehost [13, 18], the early start of oocyte growthmay be an adaptive strategy to ensure that atleast two adult descendants, one male andone female, can reach sexual maturity beforethe host bee emerges.

During the further course of oogenesis,additional control mechanisms may influ-ence V. jacobsonireproductive success. Inall V. jacobsonifemales which were intro-duced into recently sealed brood cells(n = 41), we confirmed activation of theovary. Year long records at Hohenheimrevealed percentages of fertile mites inworker brood of between only 80 and 90%

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the possibility for exact determination ofthe oocyte growth of V. jacobsonifemalesand allows extensive examination in popu-lations with different degrees of host-para-site adaptation.

ACKNOWLEDGEMENTS

We are grateful to Prof. Wolf Engels for crit-ical discussion and to Dr. Robert Paxton for read-ing of the manuscript.

Résumé – Coloration au bleu de toluidinepour une détermination rapide du débutde la croissance des ovocytes et de la fer-tilité chez Varroa jacobsoniOud. La ferti-lité réduite des acariens Varroa jacobsonidans les cellules de couvain d’ouvrières(Apis melliferaL.) est l’un des facteurs clésliés à la tolérance à la varroose, comme celaa été montré chez l’hôte d’origine ApisceranaFabr. et chez les abeilles africani-sées du Brésil. Les signaux qui déclenchentl’activation de la reproduction de l’acarienaprès l’invasion de la cellule de couvain res-tent à ce jour inconnus. Nous avons utiliséune nouvelle méthode de coloration in totopour déterminer avec précision le début dela croissance des ovocytes.Des femelles phorétiques de V. jacobsoniont été prélevées sur des abeilles adultes etintroduites artificiellement dans des cellulesde couvain fraîchement operculées et dansdes cellules de couvain operculées depuis14–16 h. Toutes les 6 h, les acariens ont étéretirés des cellules et pesés sur une balancede précision. Immédiatement après la pesée,les ovaires ainsi que la spermathèque etl’organe en forme de lyre, ont été soigneu-sement disséqués, fixés dans la formaline à4 % et colorés durant 30 min au bleu detoluidine à 0,005 %. Les ovocytes ont étélavés dans un tampon PBS et leur colora-tion a été analysée au microscope. La colo-ration de l’ovocyte dépend de l’incorpora-tion de vitellus ou d’autres constituantseuplasmatiques et marque donc l’activationde l’ovaire.

Tous les acariens présents sur le couvainont vu leur poids augmenter d’environ325 µg à plus de 400 µg durant les 24 h quiont suivi l’introduction (Fig. 1), quel quesoit le moment de l’introduction. Ce com-portement alimentaire indique une disposi-tion générale des femelles phorétiques deV. jacobsonià entrer dans la phase de repro-duction. Mais chez tous les acariens phoré-tiques examinés (n = 18) aucune colorationau bleu de toluidine n’a été détectée (Tab. I ;Fig. 2a). Ceci confirme les observations anté-rieures selon lesquelles l’ovaire des acariensphorétiques est bloqué à un stade de prévi-tellogenèse. Pourtant, dans les ovaires desacariens présents sur le couvain, une colo-ration bleu clair de l’ovocyte terminal étaitdéjà visible 6 h après l’introduction artifi-cielle des acariens dans les cellules fraîche-ment operculées (n = 41 ; Tab. I ; Fig. 2b).Sur les 10 femelles de V. jacobsoniintro-duites 14 à 16 h après l’operculation, nousn’avons trouvé un ovocyte coloré que dansun seul ovaire (Tab. I), c’est-à-dire que, chez90 % des acariens de ce groupe, la matura-tion des ovocytes n’avait pas commencé.En conclusion, la reproduction de V. jacob-sonidoit être stimulée par un signal qui n’estprésent que dans les cellules fraîchementoperculées. Si les femelles de V. jacobsonisont introduites dans des cellules opercu-lées depuis 14 h, la croissance des ovocytesn’est pas du tout démarrée, ce qui indiquel’absence de signal d’activation à ce stade del’hôte. La nature du signal et les mécanismesde stimulation de la reproduction deV. jacobsonirestent inconnus. Notre nou-velle méthode offre la possibilité de déter-miner avec précision la croissance des ovo-cytes dans de grands échantillons defemelles de V. jacobsoni. C’est donc un outilapproprié pour des études en conditionssemi-naturelles de la fertilité de V. jacob-soni dans des populations qui présententdivers degrés de tolérance à la varroose.

Varroa jacobsoni/ relation hôte parasite /vitellogenèse / ovogenèse / fertilité /reproduction

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Initiation of oocyte growth in V. jacobsoni

Abb. 2a). Dies bestätigt frühere Untersu-chungen, nach denen das Ovar von phoreti-schen Milben in einem prävitellogenen Sta-dium arretiert ist. In den Ovarien derBrutmilben wurde dagegen bereits 6 Stun-den nach dem Einsetzen in frisch ver-deckelte Brutzellen eine eindeutige blaueFärbung der terminalen Oocyte festgestellt(n = 41, Tab. I, Abb. 2b). Bei den 10 V.jacobsoni-Weibchen, die 14–16 Stunden

nach der Zellverdeckelung in die Brutzel-len eingesetzt wurden, konnte lediglich ineinem Ovar eine gefärbte Oocyte nachge-wiesen werden (Tab. I). Dies bedeutet, dassaus dieser Gruppe bei 90 % der Milbenkeine Aktivierung der Oocyten erfolgt war.Wir schlieβen daraus, dass die V. jacobsoni-Reproduktion durch ein Signal stimuliertwird, das ausschlieβlich in frisch ver-deckelten Brutzellen vorhanden ist. Da dieverspätet eingesetzten V. jacobsoni-Weib-chen nicht mit der Reifung der Oocytenbeginnen, ist dieses Aktivierungssignal zudiesem Zeitpunkt offensichtlich schon nichtmehr vorhanden. Unsere neue Methodeerlaubt die exakte und rasche Bestimmungder Aktivierung der Oocyten bei einzelnenV. jacobsoni-Weibchen. Sie ist daher her-vorragend geeignet zur Untersuchung derV. jacobsoni-Fertilität in Populationen mitunterschiedlichem Toleranzniveau.

Varroa jacobsoni / Wirt-Parasit-Beziehung / Vitellogenese / Oogenese /Fertilität / Reproduktion

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Zusammenfassung – Färbung mit Tolui-dinblau als rasche Methode zur Beurtei-lung der Aktivierung von Oocyten-wachstum und Fertilität bei VarroajacobsoniOud. Eine reduzierte Fertilitätder V. jacobsoni-Weibchen in Arbeiterin-nenbrut ist einer der Schlüsselfaktoren füreine Toleranz gegenüber der Varroose. Dieswurde insbesondere durch Untersuchungenbeim Ursprungswirt Apis ceranaund beiden Afrikanisierten Honigbienen Brasiliensbestätigt. Die Signale für die Aktivierungund Steuerung der V. jacobsoni-Reproduk-tion sind aber nach wie vor unbekannt. Wirverwendeten eine neue “whole mount”-Technik, um den exakten Zeitpunkt derOocyten-Aktivierung zu bestimmen. Phoretische Milben wurden von Adultbienenabgesammelt und in frisch verdeckelte Brut-zellen und in Brutzellen, die bereits seit14–16 Stunden verdeckelt waren, eingesetzt.In Abständen von 6 Stunden nach dem Ein-setzen wurden die Milben aus den Brutzel-len entnommen und auf einer Analysen-waage das Körpergewicht bestimmt. Direktnach dem Wiegen wurden die Ovarienzusammen mit der Spermatheka und demlyraförmigen Organ herauspräpariert, in For-malin (4 %) fixiert und für 30 Minuten inToluidinblau (0,005 %) gefärbt. Nach demWaschen in PBS-Puffer wurde die Färbungder Oocyten lichtmikroskopisch bestimmt.Die Färbung der Oocyte ist mit dem Ein-bau von Dotter oder anderen euplasmati-schen Bestandteilen korreliert und ist daherein klarer Hinweis für die Aktivierung desOvars. Bei allen Brutmilben nahm das Körperge-wicht während der ersten 24 Stunden nachEinsetzen in die Brutzellen von ca. 320 µgauf über 400 µg zu (Abb. 1). Diese Zunahmewar unabhängig vom Zeitpunkt des Einset-zens der Milben. Die rasche und umfang-reiche Nahrungsaufnahme lässt auf einegenerelle Bereitschaft der phoretischenV. jacobsoni-Weibchen zur Reproduktionschlieβen. Allerdings wurde bei keiner der18 untersuchten phoretischen Milben eineToluidinblau-Färbung festgestellt (Tab. I,

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