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The resistance of Varroa jacobsoni Oud to pyrethroids:a laboratory assay

N Milani

To cite this version:N Milani. The resistance of Varroa jacobsoni Oud to pyrethroids: a laboratory assay. Apidologie,Springer Verlag, 1995, 26 (5), pp.415-429. �hal-00891305�

Original article

The resistance of Varroa jacobsoni Oudto pyrethroids: a laboratory assay

N Milani

Dipartimento di Biologia applicata alla Difesa delle Piante,Udine University, Via delle Scienze, 208, I 33100 Udine, Italy

(Received 19 April 1995; accepted 12 July 1995)

Summary — A bioassay has been developed to assess the susceptibility of Varroa mites to fluvalinate,flumethrin and acrinathrin. No significant differences were found among groups of mites from thesame origin but from different brood stages (capped larvae, pupae with white eyes, pupae with dark eyesand white or pale bodies), while a rather variable response was observed when mites from adult beeswere assayed. The LC50 of mites from areas where treatments with fluvalinate are no longer effectivewas about 25-50 times higher than that of susceptible mites; an even larger increase in the LC95 was

found. The LC50 of flumethrin and acrinathrin on mites surviving Apistan treatments increased 10-60times.

pyrethroid / Varroa jacobsoni / resistance / laboratory assay

INTRODUCTION

Since 1992, a reduction in the effective-ness of Apistan against Varroa jacobsoniOud in a large, rapidly expanding area ofnorthern Italy has been reported (Loglioand Plebani, 1992; Astuti et al, 1995; Lode-sani et al, 1995). In many cases this wasrevealed by a serious weakening or col-lapse of colonies still heavily infested afterthe treatment and resulted in dramatic beelosses.

The effectiveness of Apistan can bechecked by using another highly effective,chemically unrelated acaricide in the

absence of capped brood. However, thistechnique is time consuming and is moreeasily applied (at least under the climaticconditions of northern Italy and most Euro-pean regions) in late summer or in earlyautumn. At this time of the year, the results

can be influenced by the increased reinfes-tation rate (Greatti et al, 1992) and, in addi-tion, many colonies are already weakenedand cannot recover.

The development of a laboratory test toassay the susceptibility of the Varroa miteto fluvalinate would help clarify to whatextent failures in the control of V jacobsoniusing Apistan are due to the spread of

strains resistant to fluvalinate and possiblyother pyrethroids. A reliable laboratoryassay is essential to study some aspectsof the resistance of V jacobsoni topyrethroids (heritability, biochemical mech-anisms, fitness of the resistant strains andpossible reversion).

For this reason, we developed a bioassayto measure the susceptibility of the Varroamite to pyrethroids, refining the previouslyused technique (Milani, 1994; Milani et al,1995). The method chosen takes intoaccount the mechanism of action of Apis-tan, Bayvarol and other varroacide prod-ucts (eg, Gabon PA-92; Vesely, 1993) for-mulated in plastic strips: the active ingredientcontained in the strips contaminates thecuticular lipids of the bees and is progres-sively taken up by the mite by indirect con-tact. Thus, the action of the acaricide is moreeasily counteracted by detoxifying mecha-nisms before the lethal dose is built up (DBassand, personal communication), than ifthe same amount of active ingredient wereapplied all at once (Ritter and Roth, 1988;Abed and Ducos de Lahitte, 1993).

The technique was tested on strains ofVarroa mites believed to be susceptible;these came from apiaries that had neverbeen treated with pyrethroids or from areaswhere Apistan had been used for severalyears but with no reduction of effectiveness.Strains of mites surviving Apistan treat-ments, and thus supposed to be resistant,were also used.

MATERIALS AND METHODS

Pyrethroids tested

More extensive investigations were carried outon τ-fluvalinate (Sandoz), using the standard sup-plied by the manufacturer (rs-flu-101990, 92.3%purity). Assays were carried out also usingflumethrin (Bayer), standard supplied by the

manufacturer (920107dor01, 87.8%) andacrinathrin (Hoechst), kindly supplied by Prof VVesel&jadnr;, VUV&jadnr;, Dol, Czech Republic; the con-centration of the latter sample was assumed to be100%.

Origin of the mites

Mites were sampled from infested colonies inthe following localities (fig 1): a) Udine (Friuli,north-eastern Italy), where Apistan has beenused by most beekeepers since 1989, but noreduction in effectiveness has been reported sofar (Greatti, unpublished data); b) Tirano (Lom-bardy, northern Italy), where treatment with Apis-tan had been unsatisfactory; a single colony,heavily infested a few months after treatmentwith Apistan, was brought to Udine in spring 1994and kept in a flightroom; c) Lunz-am-See andRandegg (Niederösterreich), from apiaries nevertreated with pyrethroids (the former has beenkept in an isolated area, without any chemicaltreatment since 1986) (Pechhacker, personalcommunication); d) Como (Lombardy), Chi-avenna (Lombardy) and Varallo Pombia (Pied-mont, but right on the border with Lombardy)from apiaries previously treated with Apistan upto 4 d before or being still treated and heavilyinfested.

The mites from colonies belonging to the sameapiary were pooled, owing to the continuousexchange of mites that takes places betweencolonies of the same apiary (Sakofski andKoeniger, 1988).

Collection of the mites

Infested combs (or pieces of comb) were broughtto the laboratory and adult Varroa females weretaken from capped brood by opening and inspect-ing individual cells; the mites were kept in a glassPetri dish with a paper towel in the base for 1-3 h,on bee larvae, until a sufficient number of miteswas collected.

In the case of the colonies kept in Udine (eitherfree-flying or in the flightroom: a and b above),the combs were brought to the laboratory im-mediately before the experiment. In the remainingcases (c and d), the combs were brought to thelaboratory in an insulating box and kept in anincubator at 34.5 °C, 70% RH until needed (for notmore than 2 d).

As a rule, Varroa mites from brood of differentages were assayed separately. In the case of theUdine colonies (a) and the colony kept in theflightroom (b), the age of the larvae was deter-mined by marking the cells at the capping; thefollowing groups were considered: mites from lar-vae 0-15 h after capping (I5); from pupae withwhite eyes (pw), 96-120 h after capping; frompupae with dark eyes (pd), 168-192 h after cap-ping. This was not possible when the mites weretaken from colonies kept elsewhere (c and d), sothe approximate age of the brood was inferredon the basis of the morphology and pigmenta-tion of the larva or the pupa; the following groupswere considered: mites from spinning larvae (I5)and stretched larvae (sl), from pupae with whiteeyes (pw) and with dark eyes and white or palebody (pd). The use of the same name does notimply that the age was necessarily within the timeintervals indicated for the Udine mites. Varroa

mites from pupae with pigmented body (9 or moredays after capping) or from adult bees about toemerge were not used, because newly moultedadult mites can be present in the cells at that timeand sometimes cannot be easily distinguishedfrom the parent mite; their response might not beuniform, as hardening of the cuticle is still in

progress at that time.

Varroa mites from different brood stages werepooled only when their number was too small tocarry out separate assays and previous resultshad indicated that no difference among them wasto be expected.

Varroa mites were also taken from adult bees

from the Udine apiary, both by dusting the bees

with flour and by picking up infested bees byhand, when they were seen on the combs.

Mites found on dead or clearly diseased beelarvae, and the few mites which seemed weakor otherwise abnormal, were discarded.

The assays were carried out between Juneand the beginning of November 1994; about 5 500mites were assayed with fluvalinate, 1 500 with

flumethrin and 1 000 with acrinathrin.

The bioassay

Shallow capsules, made of 2 glass disks (62 mmdiameter) and 1 or 2 stainless steel rings (56 mminner diameter, 3-5 mm total height) were pre-pared. The interior of these capsules (includingthe rings) was coated with paraffin wax (Merck7151, melting point 46-48 °C) containing a knownconcentration of the active ingredient. Four gramsof paraffin wax was melted in a Petri dish kept ina water bath heated to 60°C and then the requiredamount of pyrethroid, dissolved in 2 ml hexane(Sigma H9379), was added. Hexane only wasadded to the control. The mixture was stirred for1 min and the hexane was allowed to evaporatefor at least 10 min. The steel rings were immersedinto the molten paraffin wax and one side of theglass disks was coated by lowering the disk ontothe molten paraffin; in a series of 14 capsules,the total weight of paraffin was in the range1.6-2.0 g. The concentration used are reported intable I.

The capsules were then kept open for at least24 h at room temperature to allow hexaneresidues to evaporate. The capsules were usedfor 1 month after they had been prepared; whennot in use, they were kept at room temperature(23-29°C). Some assays, however, were carriedout using capsules prepared about 2 months ear-lier, to assess the effects of ageing.

Ten or 15 Varroa females were introducedinto each capsule; after 6 h they were transferredto a clean glass Petri dish (60 mm diameter) withrespectively 2 or 3 worker larvae taken from cells0-24 h after capping.

The mites were observed under a dissectingmicroscope 6 (when transferred to the Petri dish),24 and 48 h after the introduction into the cap-sule and classified in the following categories:i) mobile mites: when they could move when puton their legs and stimulated if necessary, though

sometimes they were affected by the treatment toa varying degree and their movements were moreor less uncoordinated; ii) paralysed mites: whenthey could move one or more appendages, butthey could not progress; and iii) dead mites: whenthey did not react to stimulation repeated 3 times.

Mites lost or accidentally killed (eg, crushedbetween the steel rings) were not included in thecounts. The mites inside the capsules coated withparaffin and in the Petri dishes were kept in anincubator at 32.5 °C and 70% RH.

In an experiment aimed at assessing the influ-ence of the temperature, assays were carried outat 20 and 26°C, 70% RH, on mites from the Udineapiary.

As a rule, the assay was repeated 2 or 3 timeson mites from a given origin and a given broodstage, until 30 mites per concentration wereassayed. However, assays were not repeated atconcentrations higher than those which wereexpected to give 100% mortality, or when mor-tality not exceeding that of the controls wasexpected at a higher concentration, since thesedata give little information. In a few experiments,when the number of mites was limited, more miteswere assayed at concentrations around themedian lethal concentration (Finney, 1971).

Further details on the bioassay can beobtained from the author.

The assays whose results are reported hereare summarised in table II.

Precautions to avoid contamination

Precautions were taken to avoid contamination,due to the extremely large range of concentra-tions used (up to 1:50 000).

In each test, controls and capsules with lowerconcentrations were examined first. A sheet of

paper was spread out on the bench and changedafter processing each batch of cells. A differentbrush for each active ingredient and concentrationwas used to manipulate mites.

All the equipment that had come into contactwith pyrethroids was washed separately; if it could

withstand the treatment, it was immersed in con-

centrated KOH or NaOH (∼10%) for some hoursat least, before washing at 75°C in an automaticwasher using an alkaline detergent. The prepa-ration of the capsules was carried out in a sepa-rate room.

Statistical analysis

The data were analysed using the probit trans-formation; the natural mortality rate was takeninto account using the iterative maximum likeli-hood approach, according to Finney (1949). As astarting estimate of the natural mortality (whichis to some extent arbitrary), both the proportion ofmites dead in the control capsules in that exper-iment (m0) and the weighted mean of m0 and of

the proportion of mites dead in the controls ofthat series of experiments (with weights √(n0) andto √(nt/1 0), no and nt being the number of mitesassayed in the controls of that test and in thewhole series of tests) were used. The latter of-ten allowed a faster convergence of the iterative

computation, especially when n0 was small. Threeiterations were computed, although the first usu-ally gave a satisfactory approximation, except fora single case, when 6 iterations were necessary.The fiducial limits were calculated according toFinney (1971), including a heterogeneity factorwhen appropriate. The heterogeneity was com-

puted using the χ2 statistics. In a few cases, largeχ2 resulted from contributions of groups with smallor very small expectations, leading to an over-estimation of the heterogeneity. In these cases,the χ2 was recalculated combining the groupswith small expectations, until at least an expec-tation of 4 (or more) dead or surviving mites wasreached.

Experimental values plotted in the graphs arecorrected for the natural mortality.

RESULTS

Methodological aspects

The conditions of the mites at different times,the influence of the sampling technique, theeffects of temperature and the ageing of thecapsules were investigated using mainly flu-

valinate. Only results obtained with this aiare reported here.

Conditions of the mites at different times

At 6 h, when the Varroa mites were trans-ferred to Petri dishes, a large proportion,usually increasing with the concentration,was affected to a varying degree. The pro-portion of dead mites was quite variableeven within a given strain. The classifica-tion of surviving mites into the categories’mobile’ and ’paralysed’ was difficult and ina few cases partly subjective, since thesecategories are the extremes of a continuumof conditions. The conditions of the mites

(table III) was much clearer at 24 h andespecially at 48 h, when most mites hadeither recovered or were dead, often after

having regurgitated a conspicuous droplet offluid. At 48 h, most mobile mites did notshow any sign of intoxication, and the fewparalysed individuals were usually mori-bund. At 20 and 26°C the proportion ofparalysed mites at 48 h was higher, with amaximum of 20 and 24% respectively at 50ppm. The relationship between the propor-tion of dead mites at 6 and 48 h was veryloose. In contrast, the sum of dead and

paralysed mites at 6 h (or at 24 h) was closeto the number of mites dead at 48 h in sev-

eral samples, but there were variations inboth directions (fig 2).

The mortality in the controls (’natural mor-tality’) of experiments using fluvalinate wasrather low when mites were taken from

brood, varying from an average of 1.6% at6 h, 4.4% at 24 h, to 5.8% at 48 h; 10% mor-tality was exceeded only in 2 capsules, indifferent experiments. Preliminary resultsindicated a faster increase after 2 d, andthus the observations were limited to the48 h period. The natural mortality was rathervariable, and generally higher, up to 71%in a capsule, when mites were taken fromadult bees.

For these reasons, the mortality of themites at 48 h will be reported and discussedin the next sections.

Comparison of Varroa mites from diffe-rent brood stages and from adult bees

A good fit using the probit regression wasfound in nearly all the cases and the resultsof the replications were consistent. Exceptwhen otherwise noted, no heterogeneitywas found using the χ2 test.

The susceptibility of mites from differentbrood stages (larva just after capping, pupawith white eyes, pupa with dark eyes andwhite or pale body) was similar in all theVarroa strains tested. The differences in themedian lethal concentration (LC50) betweengroups of mites of the same strain but dif-ferent brood stages were rather small and

with broadly overlapping confidence inter-vals (table IV).

In contrast, the response of the mitestaken from adult bees was rather variable.

The mites fallen following dusting adult beeswith wheat flour at the end of September(when brood was still present in the

colonies) showed a somewhat higher sus-

ceptibility. However, this result left somedoubt whether selection of somehow weakerVarroa females, ie more easily removedwhen adult bees are dusted, had takenplace. The small number of mites assayedin the control gives little information. How-ever, when mites were collected later in theseason (October 17) taking infested beesfrom the combs, the mortality was evenhigher: in the control it reached 17% by 24 h,with a highly significant difference from thatof mites taken from brood. This trend wasconfirmed by the successive assays (onOctober 27 and November 4), when about50% of the mites died in the control within 24h. In the period of time during which theassays were carried out, the amount ofbrood in the colonies rapidly decreased and(especially in the latest assays) more Varroafemales were found between the abdomi-nal sternites rather than on other parts ofthe bodies of the honeybees. Many of thesemites were thinner than those taken frombrood. The proportion of dead mites couldnot be corrected for the natural mortalityusing the so-called Abbott’s formula (Tat-tersfield and Morris, 1924), since the mor-tality caused by fluvalinate might not beindependent of natural mortality, and thedata were not analysed using the probitregression.

Effect of the temperature

The assays carried out with Varroa mitesfrom Udine at 20 and 26°C gave results sim-ilar to those carried out at 32.5°C; the mor-tality curve differs only slightly and not sig-nificantly from that obtained at 32.5°C, witha somewhat increased LC50 (20.2 ppm at20°C, 24.7 ppm at 26°C). However, the χ2test reveals a significant heterogeneitybetween the replications, at both tempera-tures. In assays carried out at these tem-

peratures an unusually large number ofparalysed mites (table III) was counted at48 h, especially at concentrations ranging

from 10 to 50 ppm. If the sum of the paral-ysed and dead mites is considered instead,there is no heterogeneity between the repli-cations and the agreement with the resultsat 32.5°C is even better (the concentrationat which 50% of the mites are dead or paral-ysed are 15.0, 17.8 and 15.1 ppm at 20, 26and 32.5°C respectively).

Effect of ageing of the capsules

The results obtained in successive assayscarried out on the same strain within 1month of the preparation of the capsulesare not statistically different, though in somecases a slight increase in the mortality ofmites was observed as the age of the cap-sules increased. The mortality obtained inthe assay carried out on the Como strainwith capsules prepared 75 d earlier washigher than that obtained using capsulesprepared 2 weeks earlier; a significant,6-fold decrease of the LC50 was observedand a highly significant heterogeneity (χ2 =

46, 16 df) between the 2 series of data wasfound. Two further tests carried out on the

susceptible strain from Udine and thus on alimited range of concentrations confirmedthis result, using freshly prepared capsulesand capsules prepared 50 d before. About a2-fold reduction of the LC50 in the latter anda highly significant heterogeneity betweenthe series of data were observed.

Comparison of the susceptibilityto pyrethroid acaricides of Varroamites from different origins

Fluvalinate

The susceptibility to fluvalinate of mites fromUdine and the 2 Austrian localities (com-bining the data on mites from different broodstages) are quite similar (fig 3), and no sig-nificant differences were found. The LC50lies between 15.9 and 18.5 ppm (table V),

with broadly overlapping fiducial intervals.The probit curves fit very closely the exper-imental data.

The mites from Lombardy (Tirano andComo) showed a more variable response(fig 3), making the fit less close, even if the

heterogeneity χ2 is not significant, and anincreased LC50 (about 24 and 54 timeshigher, respectively, than that of the Udine

strain). Moreover, the regression lines devi-ate considerably from parallelism and thusthe LC95 (though its estimate is affected by alarge error) is even more increased (about440 and 1 100 times higher, respectively,than that of the Udine strain; table V); 100%kill was not obtained in the assays even at

the highest concentration tested (50 000ppm).

Flumethrin

The overall pattern of the mite mortalityobserved in assays carried out with

flumethrin was very similar to that obtained

using fluvalinate, except that, at the sameconcentration, flumethrin is more activeagainst Varroa than fluvalinate.

The percentage of paralysed mitesdecreased from 44% at 6 h, to 8% at 24 hand 1 % at 48 h. No mortality in the controlswas observed at 6 h, while it was 1 % at 24 hand reached 3.1 % at 48 h.

The LC50 of mites from Udine and Lunz-am-See are very similar (fig 4 and table VI).The LC50 of mites from Chiavenna and

Varallo Pombia, which had survived an Apis-tan treatment, was 30-60 times higher. TheLC95 was also greatly increased.

Acrinathrin

The overall pattern of the mite mortality wasvery similar to that observed using the otherpyrethroids. The proportion of paralysedmites decreased from 35% at 6 h to 15%

at 24 h and 3.4% at 48 h. No mortality wasobserved in the controls at 6 and 24 h, whileit reached 1.2% at 48 h.

The LC50 was intermediate betweenthose of fluvalinate and flumethrin. Mites

from Udine and Lunz-am-See do not differ

significantly, while the LC50 of mites from

several origins in Lombardia is much higher(fig 5, table VII). The LC50 of Como and Var-

allo Pombia mites and heterogeneitybetween the 2 series of data were detected

using the χ2 test and do not significantly dif-fer; the samples were rather small. On the

other hand, the curve for the Chiavennamites is very different and the LC50 is exag-gerated. A closer inspection of the datashowed that in this sample an unusuallylarge number of mites occurred at 48 h atthe highest concentrations tested (4 out of10 mites at 500 ppm) and thus the mortalityalone underestimates the toxic effects ofthe active ingredient. If paralysed mites areadded to dead mites, this anomaly disap-pears.

The mortality curves of the susceptibleand the resistant strains tend to be more

parallel for flumethrin and acrinathrin than forfluvalinate. In fact, there is no significantdeparture from parallelism between someof these curves.

DISCUSSION

The observations carried out at 48 h

ensured more reliable results, since the pro-portion of paralysed mites was usually neg-ligible, and no uncertainty in the classifica-tion of the mites could arise. Caution in the

interpretation of the data is necessary in thefew cases when a large proportion of mitesshows symptoms of unrecoverable intoxi-cation but is still alive at 48 h. This can lead

to an underestimate of the toxic effects ifthe mortality alone is considered. At 6 h,the number of dead mites gave little infor-

mation, while the sum of the number of deadand paralysed mites was closer to the num-ber of mites dead at 48 h.

The temperature at which the assayswere carried out did not play a critical role,at least with susceptible mites, except that at20 and 26°C a large number of mites arestill paralysed at 48 h. This result is some-what unexpected, because the toxicity ofseveral pyrethroids is affected by the tem-peratures (Eesa and Moursy, 1993).

The capsules coated with paraffin canbe used for 1 month after their preparation;they must be discarded later. A possible

explanation for the increase in the mite mor-tality observed using capsules about 2months old is that the surface of the paraf-fin wax was progressively enriched in flu-valinate during ageing.

No significant differences were foundamong mites from different brood stages(excluding pupae with dark bodies, whennewly moulted adult mites may be present).For this reason, the results of assays carriedout on Varroa females from different brood

stages could be combined, and in somelater tests, when the number of mites wastoo low to assay them separately, mitestaken from different stages could be pooled.

In contrast, a different and rather variable

response and an increased natural mortalitywere observed when mites from adult bees

were assayed. Thus mites taken from adultbees should not be used, at least at the

beginning of autumn. The different

responses of the mites from adult bees couldbe due to a different physiological condition;Bruce and Needham (1995) noted that Var-roa mites removed from the bees during thewinter weigh less, lose water faster and thusdie earlier. No mites taken from adult bees in

spring or summer were assayed and furtherexperiments in these seasons, in colonieswith and without brood, are necessary.

The technique could detect differencesin the susceptibility of V jacobsoni to somepyrethroids. The LC50 of mites from areaswhere treatments with fluvalinate are no

longer effective was about 25-50 timeshigher than that of areas where the mitesare susceptible; an even larger increase inthe LC95 was found. This can explain thefailures in the control of Varroa using Apis-tan. The similarity in the LC50 of mites fromthe Austrian localities where no pyrethroidshave been used and from Udine shows thatno selection of resistant mites (or even par-tially resistant mites) has taken place so farin Udine, despite the fact that Apistan hadbeen used as almost the sole varroacide

product for 5 years.

It is not known to what extent the largervariability of the resistant strain is due tosome mixing with susceptible mites, but itcannot be entirely explained on the basisof this factor, since the Como colonies hadbeen treated with Apistan until a few daysbefore the brood from which the mites werecollected was taken. This further treatmentcould account for the slight, insignificant in-crease in the tolerance towards fluvalinateobserved in the latter strain. While satis-

factory results could be obtained assaying30 mites or even less at 3-5 concentrations,plus the control with susceptible strains, inthe case of resistant strains a larger numberof mites per concentration might be neededto obtain a comparable accuracy.

The increase in the LC50 of flumethrinand acrinathrin on mites surviving Apistantreatments indicates the presence of cross-resistance between fluvalinate and these

pyrethroids, although it has not been possi-ble to assay the same strain both with flu-valinate and these pyrethroids, due to thelack of a sufficient number of mites from suit-able brood stages. The presence of cross-resistance is not unexpected, due to the sim-ilarity in the molecules of the ai and inparticular of their alcohol moieties (cf AWFarnham in Denholm and Rowland, 1992).The small size of the samples, the scarcity ofinformation on possible effects of impuritiesat high concentrations of the ai and espe-cially the lack of comparisons on the samemite strain do not allow us to attach too much

significance to the different shapes of themortality curves for the resistant strains.

These results highlight the risk of basingthe strategies for the control of Varroa onchemical treatments alone.

ACKNOWLEDGMENTS

The author wishes to acknowledge G DellaVedova and M Greatti for their collaboration inthe collection of the data. Thanks also to the col-

leagues, the beekeepers and the beekeeping

associations that made assays on strains from

regions outside Udine possible: Associazioneapicoltori di Sondrio; R Büchler, Kirchhain; HPechhacker, Lunz-am-See; M Spreafico, Milan; AWallner, Randegg; V Vesel&jadnr;, Dol, for sending asample of acrinathrin; and to the companies San-doz Ltd and Bayer AG which made available theai fluvalinate and flumethrin. J Rogers revisedthe English text.

Research carried out with the financial con-tribution of the Italian MURST, research project’Controllo e miglioramento delle produzioni apis-tiche’, coordinator R Prota, University of Sassari.

Résumé — Résistance de Varroa jacob-soni Oud aux pyréthroïdes : mise aupoint d’un test. On a mis au point uneméthode pour étudier la sensibilité de l’aca-rien Varroa au fluvalinate, à la fluméthrine età l’acrinathrine. Les acariens ont été préle-vés à partir de couvain operculé (jusqu’austade de nymphe aux yeux noirs et au corpsclair), et, dans le cas des Varroa d’Udine,également sur des ouvrières adultes. Descapsules formées de 2 disques en verre (de62 mm de diamètre) et d’un anneau en acierinox (de 56 mm de diamètre interne et 3-5mm de haut) ont été recouvertes intérieu-rement avec de la paraffine contenant uneconcentration connue du composé actif. Lesacariens ont été conservés dans ces cap-sules pendant 6 h et transférés ensuite dansune boite de Pétri propre (60 mm de dia-mètre) où ils ont pu se nourrir sur des larvesd’abeille ; ils ont été observés 6 h, 24 h et 48h après leur introduction dans les capsules,et classés en 3 catégories : mobiles, para-lysés (quand ils pouvaient bouger les appen-dices sans pouvoir se déplacer), morts. Lesobservations réalisées à 48 h (quand la pro-portion d’acariens paralysés est négligeable)donnent les résultats les plus fiables. Lesacariens ont été échantillonnés dans diffé-

rentes localités : Randegg et Lunz-am-See(Autriche), dans des ruchers jamais traitésavec des pyréthroïdes ; Udine (nord-est del’Italie), où le fluvalinate a été largement uti-lisé depuis 1989 et était encore efficace ; 4

localités de Lombardie (nord de l’Italie) oùApistan n’est plus efficace. Aucune diffé-rence significative n’a été trouvée entre desacariens provenant de la même localité maisissus de stades de couvain différents (larvesoperculées, nymphes aux yeux blancs,nymphes aux yeux noirs et au corps blanc),alors que des réponses variables ont étéobtenues avec des acariens provenant d’ou-vrières adultes. La LC50 des Varroa prove-nant de régions où le fluvalinate n’est plusefficace était environ 25-50 fois plus éle-vée que celle provenant de régions où lesacariens sont encore sensibles ; une mêmeaugmentation existe également pour la LC95(fig 3 et tableau V). La LC50 des Varroa

d’Udine et d’Autriche n’était pas différente,montrant ainsi qu’aucune sélection d’aca-riens résistants ne s’est mis en place àUdine. Une augmentation de la LC50 de lafluméthrine et de l’acrinathrine a été obser-vée chez les acariens survivants au traite-ment à l’Apistan (figs 4 et 5).

pyréthroïdes / Varroa jacobsoni / résis-tance / test de laboratoire

Zusammenfassung — Resistenz von Var-roa jacobsoni Oudemans gegen Pyro-throide: ein Labortest. Um die Empfind-lichkeit der Varroa Milben gegenüberFluvalinat, Flumethrin und Acrinathrin abzu-schätzen, wurde ein neuer Labortest ent-wickelt. Die Milben wurden aus verdeckel-ter Brut (Entwicklungsstadien bis zu hellenPuppen mit dunklen Augen) gewonnen undvon Adulten gesammelt. Es wurden Ver-suchskapseln hergestellt, die aus 2 Glas-scheiben bestanden (d = 62 mm), die durcheinen Halter aus rostfreiem Stahl (innererDurchmesser d = 56 mm, Höhe 3-5 mm) ineinem Abstand von 3-5 mm gehalten wur-den. Sie wurden auf der Innenseite mit Par-affinwachs überzogen, das den jeweiligenWirkstoff in bekannten Konzentrationen ent-hielt. Die Milben wurden 6 Stunden in diese

Kapseln gesperrt und danach in eine sau-

bere Petrischale mit frischen Bienenlarven

gesetzt, von denen sie Hämolymphe sau-gen konnten. Sechs, 24 und 48 Stundennach Einsetzen in die Kapseln wurden siekontrolliert und in 3 Kategorien eingeteilt:bewegliche, unbewegliche (wenn sie nochKörperanhänge bewegten, ohne daß eine

Fortbewegung erfolgte) und tote Milben. DieBeobachtungen nach 48 Stunden, bei denender Anteil der unbeweglichen Milben ver-nachlässigt werden kann, ergaben die zuver-lässigsten Werte. Die Milben wurden an ver-schiedenen Orten gesammelt (Abb 1): inRandegg und Lunz am See (Niederöster-reich) von Ständen, die nie mit Pyrethroidenbehandelt wurden; in Udine (nordöstlichesItalien), wo seit 1989 regelmäßig Fluvalinatangewendet wurde, und an 4 Stellen in derLombardei (Norditalien), wo Apistan keineausreichende Wirkung mehr hat. ZwischenMilben vom gleichen Ort, die von verschie-denen Brutstadien (verdeckelten Maden,Puppen mit weißen Augen, puppen mit dun-klen Augen und weißem oder leicht pigmen-tiertem Körper) stammten, zeigten sich keinestatistischen Unterschiede. Die Ergebnissemit Milben von adulten Bienen waren dage-gen sehr variabel. Die LC50 der Milben ausGebieten mit unwirksamer Apistan-Behand-lung war 25-50 mal höher als die LC50 der

Milben aus Gebieten, in denen die Milbennoch empfindlich sind. Bei der LC95 war der

Anstieg noch höher (Abb 3 und Tabelle V).Die LC50 der Milben von Udine und Öster-reich unterschieden sich nicht. In Udine sind

also noch keine Resistenzen selektiert wor-den. Eine höhere LC50 bei Flumethrin undAcrinathrin wurde bei Milben beobachtet, dieApistan Behandlungen überleben (Abb 4, 5).

Pyrethroide / Varroa jacobsoni / Resi-stenz / Labortest

REFERENCES

Abed T, Ducos de Lahitte J (1993) Détermination de laDL50 de l’amitraze et du coumaphos sur Varroa jacob-

soni Oud au moyen des acaricides Anti-varroa (Scher-ing) et Perizin (Bayer). Apidologie 24, 121-128

Astuti M, Spreafico M, Colombo M (1995) Indaginesull’efficacia degli interventi di controllo di Varroajacobsoni attuati nel 1993 in Lombardia. Apilombar-dia, Minoprio (Como) 8-9 October 1994. Selezioneveterinaria (in press)

Bruce WA, Needham GL (1995) Effects of temperatureand water vapour activity on the survival of Varroajacobsoni (Acari: Varroidae). Proc IX Intern CongrAcarol, 17-22 July, Columbus, Ohio (in press)

Denholm I and Rowland MW (1992) Tactics for manag-ing pesticide resistance in arthropods: theory andpractice. Annu Rev Entomol 37, 91-112

Eesa NM, Moursy LE (1993) Temperature relationshipsin pyrethroid toxicity to the 2-spotted spider mite,Tetranychus urticae Koch. Exp Appl Acarol 17, 617-620

Finney DJ (1949) The estimation of the parameters of tol-erance distributions. Biometrika 36, 139-256

Finney DJ (1971) Probit Analysis. 3rd ed, CambridgeUniversity Press, Cambridge, MA, USA

Greatti M, Milani N, Nazzi F (1992) Reinfestation of anacaricide-treated apiary by Varroa jacobsoni. ExpAppl Acarol 16, 279-286

Lodesani M, Colombo M, Spreafico M (1995) Ineffec-tiveness of Apistan® treatment against the mite Var-

roa jacobsoni Oud in several districts of Lombardy(Italy). Apidologie 26, 67-72

Loglio G, Plebani G (1992) Valutazione dell’efficaciadell’Apistan. Apic Mod 83, 95-98

Milani N (1994) Possible presence of fluvalinate-resistantstrains of Varroa jacobsoni in northern Italy. In: NewPerspectives on Varroa (A Matheson ed), Proc InternMeet, &jadnr;e&jadnr; Prague, 8-11 Nov 1993, IBRA, Cardiff,87

Milani N, Greatti M, Della Vedova G (1995) Resistenzadi Varroa jacobsoni al fluvalinate: un metodo di lab-oratorio per l’individuazione di ceppi resistenti. Api-lombardia, Minoprio (Como) 8-9 October 1994.Selezione veterinaria (in press)

Ritter W, Roth H (1988) Experiments with mite resis-tance to varroacidal substances in the laboratory.In: European Research on Varroatosis Control (RCavalloro, ed), Balkema, Rotterdam, 157-160

Sakofski F, Koeniger N (1988) Natural transfer of Varroajacobsoni among honeybees colonies in autumn. In:European Research on Varroatosis Control (R Cav-alloro, ed), Balkema, Rotterdam, 81-84

Tattersfield F, Morris HM (1924) An apparatus for test-ing the toxic values of contact insecticides undercontrolled conditions. Bull Entomol Res 14, 223-233

Vesel&jadnr; V (1993) Acrinathrin gegen Varroa jacobsoni.Apidologie 24, 499-500