Original article

Honey bee hygienic behaviorand defense against Varroa jacobsoni

Marla Spivak

Department of Entomology, 219 Hodson Hall, University of Minnesota, Saint Paul, MN 55108, USA

(Received 17 May 1996; accepted 14 August 1996)

Summary— Hygienic and non-hygienic colonies from ’Starline’ stock of Apis mellifera were tested fortheir ability to remove pupae infested with Varroa mites. The hygienic and non-hygienic lines wereselected and bred on the basis of their removal response to freeze-killed brood. A Jenter Box® was usedto test whether they would remove experimentally infested pupae following methods of Boecking andDrescher (1992). In 1994, the hygienic colonies removed significantly more pupae infested with one miteper cell than the non-hygienic colonies. In 1995, there was no significant difference between thehygienic and non-hygienic colonies when one or two mites were introduced per pupa due to variationin response among hygienic colonies. There was no significant difference between the rate of removalof infested pupae from the Jenter Box and from natural wax comb by the hygienic colonies. The num-ber of mites damaged by grooming ranged from 6.0 to 42.3% among all colonies. The reproductive suc-cess of the mites not removed from the cells by the bees was low in both hygienic and non-hygieniccolonies.

Apis mellifera / Varroa jacobsoni / hygienic behavior / grooming / mite resistance / breeding

INTRODUCTION

The ectoparasitic mite, Varroa jacobsoniOudemans, is the most destructive pest of

honey bees in the US and Europe. Becauseof the risks and disadvantages of usingchemical treatments in mite-infestedcolonies (eg, Lodesani et al, 1992, 1995),it is important to determine if honey beeshave any heritable defense mechanisms

against the mite which may be readily incor-porated into breeding programs.

A balanced host-parasite relationshiphas evolved between V jacobsoni and its

natural host, Apis cerana, in Asia. Mostfemale Varroa are not able to reproduce

successfully in worker brood of A cerana,therefore, the reproduction of the mites islimited to the seasonal cycle of drone pro-duction within the colony (Boecking and Rit-ter, 1994; reviewed in Büchler, 1994). Inaddition, A cerana has two behavioraldefenses which help maintain the numbersof mites within tolerable limits. Thesedefenses are grooming and removal(hygienic) behaviors.

In grooming, adult bees detect andremove phoretic mites from themselves

(auto-grooming) or from nestmates (allo-grooming) (Peng et al, 1987a). In the pro-cess, the legs of the mite may be cut off orthe cuticle of the idiosoma may be dam-

aged by the bees’ mandibles, causing thedamaged mite to fall to the bottom of thecolony (Ruttner and Hänel, 1992). Suc-cessful grooming of mites has been demon-strated in A cerana (Peng et al, 1987a;Büchler et al, 1992; Fries et al, 1996). A mel-lifera of European origin also exhibits groom-ing behavior but to a lesser extent thanA cerana (Peng et al, 1987a; Büchler et al,1992; Büchler, 1994; Fries et al, 1996).

Removal behavior involves the ability ofsome bees to detect, uncap, and removeinfested worker pupae from the cells. A cer-ana efficiently removes infested brood (Penget al, 1987b; Rath and Drescher, 1990;Rosenkranz et al, 1993). The removal ofinfested pupae interrupts the reproduction ofthe fertile mites inside sealed brood cells.In addition, the immature mites are killedwhich decreases the average number of off-

spring per mother mite (Rath and Drescher,1990; Fries et al, 1994).A mellifera of European origin removes

infested worker pupae, but to a limited extent

compared to A cerana (reviewed in Boeckinget al, 1993; Boecking and Ritter, 1994; Büch-ler, 1994). Africanized honey bees in Brazilremove significantly more mite-infestedpupae than European bees in the same loca-tion (Guerra et al, submitted for publication).Boecking and Drescher (1992) found thatthe type of comb affected the rate of removal;more infested pupae were removed from

plastic comb than from natural wax comb.They also found a positive correlation (r =0.74) between the removal of brood infestedwith two mites per cell and the removal offreeze-killed brood, a commonly usedmethod to assay bee colonies for hygienicbehavior (see Methods).

Hygienic behavior is considered the pri-mary mechanism of resistance to at leasttwo diseases of larval and pupal honey bees:American foulbrood caused by the bacterium

Bacillus larvae (Rothenbuhler, 1964) andchalkbrood caused by the fungus,Ascosphaera apis (Gilliam et al, 1983, 1988).Hygienic bees have the ability to detect,uncap, and remove diseased brood from thenest before the causative organisms reachthe sporulating stage (Woodrow and Holst,1942). Rothenbuhler (1964) postulated thathygienic behavior is controlled by two inde-pendently assorting, recessive genes - onefor uncapping and one for removing diseasedbrood from the nest. In a re-evaluation ofthe two-locus model for hygienic behavior,however, Moritz (1988) found the model tobe an oversimplification. He thought a multi-locus model or other more complex patternsof inheritance determined the expression ofthe phenotype. Rapid hygienic behavioroccurs at a relatively low frequency in mosthoney bee populations thus far studied (Spi-vak and Gilliam, 1993).

A two-way selection program for hygienicbehavior was initiated at the University ofMinnesota in 1992 with the original goal ofselecting colonies resistant to chalkbrood.Lines of hygienic and non-hygienic colonieswere selected on the basis of their removal

response to freeze-killed brood, an assayused by previous researchers to selectcolonies for this purpose (Gilliam et al, 1983,1988). The present experiment testedwhether the hygienic colonies from thebreeding program would also remove pupaeinfested with Varroa. A comparison wasmade between the rate of removal ofinfested pupae within plastic-based cellsand within natural wax comb. In addition,the degree of grooming behavior of thesame colonies was determined.

METHODS

Experimental bee colonies

The hygienic and non-hygienic lines used in theexperiment were bred from ’Starline’ stock,derived from Italian A mellifera ligustica. The

degree of hygienic behavior in the colonies wasdetermined by a freeze-killed brood assay inwhich the amount of time was recorded for beesto detect, uncap, and remove a 6 x 5.5 cm combsection containing freeze-killed pupae. Each sec-tion of pupae, containing approximately 100pupae per side of the comb, was cut out andfrozen at -20 °C for 24 h before it was placed inthe colony to be tested. Previous experimentshave shown that neither hygienic nor non-hygienicbees removed live pupae from similar size sec-tions of comb which had been cut out and

replaced (Spivak and Gilliam, 1993; Spivak,unpublished observations). Therefore, the timetaken to remove freeze-killed brood by thecolonies was considered a measure of the bees’

ability to remove diseased or abnormal brood.Colonies that removed the freeze-killed broodwithin 48 h in two consecutive trials were con-sidered hygienic; colonies that took longer than1 week to remove the dead brood in both trialswere considered non-hygienic (Taber and Gilliam,1987; Spivak and Gilliam, 1993).

To establish and maintain the lines, queenbees were raised from colonies that displayedthe most rapid and the least rapid removal rates.Each daughter queen was inseminated with4-6 μL of mixed semen drawn from multipledrones from either hygienic or non-hygieniccolonies, with the exception of three hygienicqueens which were raised in the spring of 1995and were each inseminated with the semen of a

single drone. All colonies were wintered outdoorsand then tested again the following spring usingthe freeze-killed assay. The colonies with single-drone inseminated queens were assayed withfreeze-killed brood in August 1995 when all thebees in the colonies were progeny of the intro-duced queens. Only the most hygienic and leasthygienic colonies based on the second freeze-killed brood assays were used in the experimentsto test whether the colonies would remove broodinfested with Varroa.

In 1994, the experiments included fourhygienic and three non-hygienic colonies, and in1995 they included seven hygienic and four non-hygienic colonies. All colonies were treated withtwo Apistan® (fluvalinate) strips per colony theprevious fall and were sampled for Varroa in thespring. No mites were detected in any of thecolonies in the spring of 1994 or 1995 before theexperiments began. However, as the season pro-gressed, all colonies became reinfested with Var-roa due to natural causes (drifting and robbingbetween colonies from surrounding apiaries). All

colonies were maintained in standard Langstrothequipment and had approximately 8-12 frames ofbrood when they were tested for removal of Var-roa mites.

Removal of infested pupae

A Jenter Box® (Brushy Mountain Bee Farm, Mora-vian Fall, NC, USA) was used to test whether theselected hygienic and non-hygienic colonies ofbees would remove pupae experimentally infestedwith Varroa mites (following methods of Boeck-ing and Drescher, 1992). This box containsapproximately 300 plastic-based worker cells andfits into a standard brood frame. Ninety of the cellswithin the box have false bottoms fitted with remov-able plugs which allow one access to individuallarvae or pupae through the base of the cell.

The inseminated queens in each experimen-tal colony were confined until they had laid eggsin most of the cells of the Jenter Box (6-24 h).Eight or nine days later, Varroa mites were intro-duced through the plugs in the cells containingfifth-instar larvae. The cells containing these lar-vae had been sealed with wax within the last6-8 h, before the fifth instar larvae had spun acocoon and begun pupation. All mites were col-lected off adult workers and drones from one

highly infested colony located in an apiary over 5km away. The reproductive status of the mitesat the time of collection and introduction was not

known; however, care was taken to introducemites that were fully pigmented. The mites wereintroduced into the cells using a fine, camel-hairpaint brush following the methods of Boeckingand Drescher (1992).

In 1994, one Varroa mite per cell was intro-duced into 10-20 cells containing fifth-instar lar-vae. Another group of cells serving as controlshad the plugs removed and replaced with no miteintroducton. The infested and control cells weremarked on a transparent sheet of plastic (follow-ing Infantidis, 1983), and were inspected on days1, 2, 4, 7, and 10 after infestation to determine ifthe bees had detected and removed the infestedbrood. In 1995, the same procedures were fol-lowed with the additional treatment of two mites

per cell. On the tenth day of the experiment in1995, or one day before the pupae were due toeclose as adults, all cells containing infestedpupae that were not removed by the bees wereopened to determine the reproduction of theremaining mites.

To test whether the hygienic colonies removedmore infested pupae from the plastic-based cellsof the Jenter Box than from natural wax cells,separate trials were conducted in which fifth-instarlarvae in wax cells were infested with one or twomites per cell. The wax cappings of recentlycapped brood cells containing fifth-instar larvaewere partially opened using a fine scalpel (fol-lowing de Ruijter, 1987). After inserting the miteor mites with a paint brush, the cell cappings werecarefully closed again. Control cells were openedand closed with no mite introduction. The infestedbrood cells and the control cells were marked ona transparent plastic sheet and were inspectedas before.

Grooming and mite counts

To determine natural mite mortality, a ’stickyboard’ (Dewill Inc, Varroa Mite Detector Insert,Elmhurst, IL, USA) covered with mesh screenwas placed on the bottom board of the samecolonies for a 10 day period in 1994 (in July), andfor two 10 day periods in 1995 (July and August).The number of mites that fell to the bottom andadhered to the board was counted under a micro-

scope. Of the total number of fallen mites, thepercent that were damaged when adult beesgroomed the mites off each other was countedonly in 1995. The screen was elevated from thesticky board by a wooden frame to ensure thatthe bees could not reach any fallen mites. There-

fore, all damaged mites collected on the stickyboards would have been damaged by the beesprior to falling on the board.

Statistical analysis

The differences in the results of the freeze-killedbrood assays between the hygienic and non-hygienic colonies were analyzed using a Stu-dent’s t-test for each year (Wilkinson, 1990-1992).The mean percentages of mite-infested and con-trol pupae removed from the Jenter Box on day 10 of the experiment were analyzed using a split-plot two-way ANOVA on arcsine-transformed datafor each year. The error term for bee type wascolony (bee-type), and for the treatment effectwas the residual error (SAS Institute, Release6.10, 1995). The same analyses were used tocompare the amount of infested brood removed

from natural wax comb in both years. Paired t-

tests were used to compare the response of the

hygienic colonies to infested brood in the plasticcells of the Jenter Box versus natural wax comb.

Separate t-tests were used to compare theamount of infested pupae removed by day 10 foreach treatment (one mite per cell, two mites percell, and the controls) (Wilkinson, 1990-1992).

RESULTS

Freeze-killed brood assays

The results of the freeze-killed brood assaysconducted before the mites were introducedinto the colonies in 1994 and 1995 are pre-sented in figure 1. In both years, the hygieniccolonies removed significantly more deadbrood than the non-hygienic colonies within48 h (P = 0.001 both years). In 1995, therewas no difference between the rate ofremoval by colonies containing queensinseminated with the sperm of one or of

many drones; therefore, the results from allhygienic colonies were pooled for theremainder of the analyses.

Removal of mite-infested pupae

The results of the assay for the ability of thehygienic and non-hygienic colonies to detect,uncap, and remove mite-infested pupaefrom the cells within the Jenter Box are given

in figure 2. The mean percent infestedpupae removed by day 10 and results ofstatistical analysis are given in table I. In

1994, the effects of bee type (hygienic vsnon-hygienic) and of treatment were signif-icant. The four hygienic colonies removed

significantly more pupae infested with onemite per cell by day 10 than the three non-hygienic colonies and the controls from bothbee types. The same assay in 1995 yieldeddifferent results. There was no significanteffect of bee type; the seven hygieniccolonies did not remove significantly moreinfested pupae than the non-hygieniccolonies. However, there was a significanttreatment effect; significantly more pupaethat were infested with two mites per cell

were removed than cells infested with one

mite per cell and the control cells (Tukeystest for mean separation: P < 0.05). Thebees removed the infested pupae at anytime during the 10 day interval (fig 2), and

thus the detection of infested pupa did not

appear to be limited to any particular stagein the development of the immature bee orin the reproductive stages of the mite.

There was considerable variation in the

amount of infested brood removed by theseven hygienic colonies in 1995. Four of thehygienic colonies, one of which contained asingle-drone inseminated queen, removed< 15% of the infested pupae when one mite

per cell was introduced. The remaining threeremoved 45.5 ± 6.46% and 69.6 ± 26.69% of

the pupae when one and two mites per cell

were introduced, respectively.The removal of infested brood from nat-

ural wax comb by the hygienic and non-

hygienic colonies is shown in table II and

figure 3. In both years, there was a highdegree of variability in the response of thehygienic colonies and no statistical differ-ence between the hygienic and non-hygieniccolonies was found. There was a significanttreatment effect in 1994 between theremoval of brood infested with one mite percell and the controls, and in 1995 betweenthe removal of brood infested with two mites

per cell and the controls (Tukey’s mean sep-aration < 0.05).

The hygienic colonies did not removesignificantly more infested or control pupaefrom the plastic-based cells of the JenterBox than from natural wax comb by day 10 (all paired t-tests > 0.05).

Mite reproductive success

The reproductive success of the mites intro-duced into cells in the Jenter Box and natu-ral wax comb in 1995 is shown in table III.

Only the cells into which one mite was intro-duced were counted, as the mites’ repro-ductive success decreases as more mitesinfest each cell (Moosbeckhofer et al, 1988;Fuchs and Langenbach, 1989; Eguaras etal, 1994; Fuchs, 1994). Fewer mites repro-

duced on pupae within cells of the JenterBox than in natural wax cells in both the

hygienic and non-hygienic colonies. Of themites that successfully reproduced on pupaein wax cells, a higher percentage wasobserved in the non-hygienic colonies(37.0%) than in the hygienic colonies (27.1 %).When an introduced mite did not repro-

duce, it often deposited feces on the pupaerather than in the normal location on the cellwall. In the hygienic colonies, 87.1% of thenon-reproductive mites introduced into theJenter Box cells, and 50.1 % introduced intonatural wax cells deposited feces on thepupae. In the non-hygienic colonies, 82.1and 86.4% of non-reproductive mitesdeposited feces on the pupae in cells of theJenter Box and natural wax, respectively.Only in two cases (hygienic and non-hygieniccolonies combined) did a successfully repro-ducing mite deposit feces on the pupae; bothof these occurred within the Jenter Box.

Grooming behavior

The percentage of mites that were damagedas a result of grooming in 1995 ranged from6.0 to 42.3% in the seven hygienic colonies,and from 14.9 to 31.4% in the four non-

hygienic colonies. The differences betweenthe colony types were not significant (t-test: T= 0.591; df = 9; P = 0.57). Of the threecolonies headed by single-drone inseminatedqueens, one displayed the lowest number ofdamaged mites, and another the highestnumber. Most of the damage was to themites’ legs, and less often to the idiosoma.

Colony infestations

The numbers of mites that fell from natural

mortality to the sticky boards during theexperiment are given in table IV. In 1994,few mites but large numbers of chalkbroodmummies were collected on the stickyboards in two of the non-hygienic colonies.The third non-hygienic colony was highlyinfested with mites, but had a lower chalk-brood infestation, resulting in large standarddeviations around the means (table IV).Inspection of the brood in these non-hygieniccolonies indicated that the mites died alongwith the pupae that were infested with chalk-brood. Many of the chalkbrood mummiesand dead mites remained within sealedcells. However, the bees removed some ofthe chalkbrood mummies from the cells, andthe mummies which were not carried fromthe nest fell and adhered to sticky boards.No chalkbrood mummies were found in the

hygienic colonies.In 1995, sticky boards were placed in the

colonies for 10 day intervals once in Julyand again in August to monitor natural mitefall. No chalkbrood infection was observedin either the hygienic or non-hygieniccolonies, and no mummies were collectedon the sticky boards. The number of mitesincreased dramatically in one of the singledrone-inseminated hygienic coloniesbetween July and August; concomitantly,the number of adult bees in the colonyincreased abnormally. It was suspected thatan infested swarm entered this colony. Themarked inseminated queen, however, was

not superseded. Over three times as manymites fell to the bottom boards in Augustthan in July in the hygienic colonies (exclud-ing the hygienic colony mentioned above).Over six times as many mites fell in Augustthan in July in the non-hygienic colonies.

DISCUSSION

Boecking and Drescher (1992) found a cor-relation between the removal of freeze-killedbrood and removal of pupae experimentallyinfested with two mites per cell. They sug-gested that using the freeze-killed broodassay could facilitate the selection ofcolonies that would remove brood infestedwith Varroa. In the present study, colonieswere selected first on the basis of theirremoval of freeze-killed brood and weretested subsequently for their removal ofinfested pupae. In 1994, the four hygieniccolonies that removed freeze-killed broodwithin 48 h removed an average of 69.2% ofthe pupae infested with just one mite byday 10. In 1995, however, of the sevenhygienic colonies that consistently removedfreeze-killed brood within 48 h, only threeremoved an average of 45.5% of the pupaeinfested with one mite by day 10; the remain-ing four removed an average of 9.2% byday 10. (Preliminary data from 1996 indi-cated that some of the same hygieniccolonies, headed by the same queens, thatremoved low numbers of infested pupae in1995 removed significantly higher numbersin 1996. These results will be published at alater date.) The cues the bees used to detectand remove frozen pupae are not neces-

sarily the same as those used to detect andremove mite-infested pupae. Because the

non-hygienic colonies generally did notdetect and remove significant amounts ofinfested pupae, the results of this experi-ment indicate that the freeze-killed brood

assay is a useful screening procedure in

selecting colonies for their ability to removepupae infested with Varroa.

Hygienic behavior is genetically deter-mined. Previous studies, however, haveshown that there is high degree of variabil-ity in the expression of the behavior. Forexample, lack of incoming nectar has beenshown to reduce the hygienic response(Momot and Rothenbuhler, 1971). Weak-ened colonies (those with small populations)also display a reduced hygienic response(Boecking and Drescher, 1993; Spivak andGilliam, 1993). The tests in 1994 and 1995were conducted in July and August whenall experimental colonies were collectinglarge amounts of nectar and pollen and werestrong and populous.

Research on A cerana indicated thatmites introduced from foreign colonies(either intra- or inter-specific colonies) wereremoved more rapidly by the bees thanmites collected from their own colony, butthis was not the case with A mellifera

(Rosenkranz et al, 1993). In the presentexperiment, all mites were collected fromone ’foreign’ colony of A mellifera each year,which controlled for the possibility that themites could have acquired distinctive odorsfrom the colonies in which they developed.

Boecking and Drescher (1992) reportedthat the type of comb influenced theremoval response of the colonies. Pupaeinfested with mites in the Jenter Box with

plastic cell bases, and in fully plastic (’ANP’)comb were removed more quickly thanwere pupae in natural wax comb. The sametrend was evident in the present study, butno statistically significant differences weredetected between the removal of infested

pupae from the Jenter Box and wax combin either year.

The hygienic colonies removed morepupae from control cells than the non-

hygienic colonies in both years (table I),although this trend was not statistically dif-ferent. The removal and replacement of thecell plug in the Jenter Box may have dis-

rupted the larva, providing a stimulus for thehygienic bees to uncap, inspect, and pos-sibly remove the larva. Also, some pupaein controls cells may have been removed

by the hygienic bees if they were infestednaturally (Boecking and Drescher, 1991).

It is not known how the bees determinethat a particular larva or pupa is infestedwith Varroa (Boecking and Drescher, 1992;Boecking and Drescher, 1994). Moreover, itis unclear how the type of comb (plastic orwax) might influence the bees’ ability todetect mites within a wax-capped cell. It has

been speculated that the bees use mechan-ical and olfactory cues to detect mites underthe wax cell capping of the pupa. For exam-ple, Tewarson et al (1992) reported thatA cerana indica bees release the mites frominfested cells without removing the pupaein some cases. These researchers specu-lated that a foreign object, such as a miteunder a capped cell, may alter the behaviorof the larva or pupa, which may be signaledby sound. Workers cue into the sound andopen the cell. If the object has an alienscent, the pupa may be eaten or removed.If there is no alien scent, the cell may be

recapped allowing the pupa to emerge nor-mally (Tewarson et al, 1992). Rosenkranzet al (1993) demonstrated that the removalof mites by A cerana depends on the alienscent adhering to the mite, but the reponseof A mellifera to the odor of mites collectedfrom different colonies was very low. Pre-

liminary experiments indicated that hygieniccolonies did not detect and remove an

appreciable amount of pupae inoculatedwith frozen mites, mites preserved in alco-hol, or dead mites collected from hive debris(Boecking and Drescher, 1994; M Spivak,unpublished data) which implies that theodor of the mite itself may not be an impor-tant cue to A mellifera.

Bees may have a threshold response tothe cues elicited by the mite or by theinfested brood. If the colony is highlyinfested, the bees may cease to respond to

the cues and not remove the infested pupaewhich could explain why some hygieniccolonies did not remove higher numbers ofinfested pupae in 1995. Also, it may not be

advantageous for the bees to remove allinfested worker pupae which could sub-

stantially reduce the adult population of thecolony.

The mite counts in this study only esti-mate how many mites were in the phoreticstage, and do not include the number ofmites reproducing within brood cells. Thenumber of mites which fell to the stickyboards during the experiment may indicatemortality of newly emerged mites (Boot et al,1995). To determine to what extent hygienicbehavior actually reduces the mite load in acolony, it would be necessary to comparemite levels and mite reproductive success incolonies that were not manipulated and intowhich no mites were experimentally intro-duced.

Other factors in combination with hygienicbehavior contribute to overall defense

against Varroa. Grooming behavior isanother defense, but there is no reason toexpect that bees with genetic tendency togroom mites from adult bees would also be

hygienic. The results in 1995 demonstratedthat there was no difference between the

hygienic and non-hygienic lines in the num-ber of mites that were damaged by groom-ing. However, the hygienic colony that dam-aged the highest number of mites (42.3%)was also one that removed a relatively highnumber of infested pupae (52.6 and 58.8%when one and two mites per cell were intro-

duced, respectively). It is possible, there-fore, to select colonies that display bothbehaviors in breeding programs for beesthat display defenses against Varroa.

Another factor which contributes to

colony resistance to Varroa is the apparentinability of the mites to reproduce on workerpupae in A cerana and some A melliferacolonies (Camazine 1986; Ruttner andHänel, 1992; Anderson, 1994; Fuchs, 1994;

Rosenkranz and Engels, 1994). In the pre-sent experiment, a low percentage of intro-duced mites reproduced successfully on theworker pupae, but this low fertility most likelywas not due to colony resistance. Rather,the low reproductive success may haveresulted from the handling of the mites dur-ing the experimental procedure, or the intro-duced mites may not have been reproduc-tively mature. For example, previous studieshave shown that young mother mites maynot always reproduce in the first cell theyenter, but may do so in subsequent cells(de Ruijter, 1987). Young mites that werenot allowed sufficient time in the phoreticstage on adult bees before they were intro-duced may have delayed oviposition andlay fewer eggs (Beetsma and Zonneveld1992; but see Boot et al, 1995). Also, thefertility of the mites decreased when theywere introduced 24 h or more after the lar-

val cell was sealed (Beetsma and Zonn-eveld, 1992). In all cases in the presentexperiment, however, care was taken tointroduce the mites within 24 h of the cells

being sealed; thus, the timing of mite intro-duction probably did not contribute to theobserved lack of fertility.

It was noted that mites which did not

reproduce on worker pupae deposited feceson the pupa itself rather than in the normal

location on the upper cell wall. The place-ment of feces on the upper cell wall appar-

ently serves as a ’rendezvous site’ for themother and her immature offspring withinthe cell (Donzé and Guerin, 1994) and soplays an important role in the life-cycle ofthe mite. In most cases, the non-reproduc-tive mites deposited feces on the abdomenof the pupae (see also Ruttner et al, 1984,p 47), but in other cases, the feces wasfound scattered over the entire body of thepupa. The placement of the feces is proba-bly a symptom of the mite not being ready orable to reproduce. However, this phe-nomenon warrants further investigation.

In conclusion, many factors seem to reg-ulate the expression of hygienic behaviorof honey bees and the removal of pupaeinfested with Varroa. Hygienic behavior hasthe potential to limit the population growth ofVarroa in three ways: 1) the immature mitesare killed when the pupa is removed, whichdecreases the average number of offspringper reproducing mite; 2) the phoretic periodof adult female mites is extended of those

mites that survive removal of the pupae;and 3) the mortality of the adult mitesincreases if they are damaged by the adultbees through grooming when they escapethrough the opened cell. However, theextent to which the behavior actuallyreduces the mite load within a colonyremains to be studied.

ACKNOWLEDGMENTS

I thank O Boecking for demonstrating his methodsfor introducing mites into the Jenter Box to me,and for his valuable comments on the experi-ments and manuscript. M Gilliam, G Richardson,and I Burns also provided helpful suggestions onthe manuscript. Thanks to J Breyfogle, G Reuter,R Melton, and T Bruce for their help collectingdata. The research was funded by beekeepingassociations from Minnesota, Wisconsin, NorthDakota, South Dakota and California, with match-

ing funds from the Minnesota Agricultural Uti-lization Research Institute (AURI PRO-130) andthe Almond Board of California. Special thanksto the Minnesota Honey Producers Association fortheir generous and continued support of this pro-ject.

Résumé &mdash; Comportement hygiéniquede l’abeille mellifère (Apis mellifera) etdéfense contre Varroa jacobsoni. On atesté la capacité de colonies présentant uncomportement hygiénique (colonies «hyg»)et de colonies ne le présentant pas (colo-nies «non-hyg»), issues de la souche «Star-line» d’Apis mellifera, à éliminer lesnymphes infestées par l’acarien Varroa

jacobsoni. Les lignées «hyg» et «non-hyg»ont été sélectionnées et élevées sur la base

de leur réponse à éliminer du couvain tuépar le froid. Une boîte Jenter&reg; a été utili-sée pour tester l’élimination des nymphesinfestées expérimentalement selon lesméthodes de Boecking et Drescher (1992).Deux supports ont été utilisés : la boîte Jen-ter&reg;, qui comporte des cellules avec unebase en plastique, et le rayon en cire natu-relle. On a comparé le taux d’éliminationdes nymphes infestées en fonction du sup-port. On a aussi déterminé le succès repro-ducteur des varroas, le degré de compor-tement de toilettage et l’estimation de lacharge des varroas dans les colonies. En1994, au 10e jour après l’infestation, lesquatre colonies «hyg» avaient éliminé signi-ficativement plus de nymphes infestées parun acarien/cellule (69,2 ± 16,41) que lescolonies «non-hyg» (10,0 ± 10,0) et queles témoins des deux groupes. En 1995,les sept colonies «hyg» n’ont pas éliminésignificativement plus de nymphes infes-tées (24,7 ± 20,06) que les quatre colonies«non-hyg» (11,3 ± 6,29), quand un varroaétait introduit par cellule. Quatre de cescolonies «hyg» ont éliminé &le; 15 % desnymphes infestées et les trois restantes45,5 % ± 6,46. Néanmoins, il y a eu plusde nymphes éliminées que dans lestémoins quand il y avait deux acariens parcellule. Au 10e jour, chez les colonies«hyg», il n’y a pas eu de différence signifi-cative entre les deux types de supportsdans l’élimination des nymphes infestéeset des nymphes témoins (tous les tests t

par paires > 0,05). Le succès reproducteurdes varroas, qui n’étaient pas éliminés avecles nymphes par les abeilles, a été bas. En1995, sur l’ensemble des varroas introduits,le pourcentage de varroas avec une des-cendance a été de 17,2 % (cellules de laboîte Jenter) et de 27,1 % (cellules en cirenaturelle) chez les colonies «hyg» et, res-pectivement, de 7,3 % et 37,0 % chez lescolonies «non-hyg» (tableau III). Quand unvarroa introduit ne se reproduisait pas, il

déposait souvent des fécès sur les

nymphes plutôt que sur la paroi de la cellulecomme il le fait normalement. Le pourcen-tage de varroas lésés par le toilettage, cal-culé d’après le nombre de varroas qui tom-baient pendant l’expérience sur les

planchers collants, a varié entre 6,0 et 42,3% selon les colonies. Il n’y a pas eu de dif-férence entre les colonies «hyg» et les«non-hyg» (p > 0,05). Il n’y a pas eu de dif-férence significative entre le nombre devarroas récoltés sur les planchers collantspendant l’expérience (tableau IV). D’autresexpériences sont nécessaires pour déter-miner dans quelles mesure le comporte-ment hygiénique diminue la charge desacariens dans une colonie.

Apis mellifera / Varroa jacobsoni / com-portement hygiénique / sélection

Zusammenfassung &mdash; Hygienisches Ver-halten der Honigbiene und Abwehr vonVarroa jacobsoni. Hygienische und nicht-hygienische Völker der Linie ’Starline’ (Apismellifera) wurden darauf untersucht, wiehäufig sie von Varroa befallene Puppenentfernen. Die Zucht der hygienischen undnicht-hygienischen Stämme erfolgte aufGrundlage der Entfernung von durch Tief-gefrieren abgetöteter Brut. Um das Entfer-nen künstlich infizierter Puppen zu beob-achten, wurde eine Jenterwabe

entsprechend der Methode von Boeckingund Drescher (1992) benutzt. Die Rate derEntfernung von befallenen Puppen aus denPlastikzellen der Jenterwabe wurde mit deraus Zellen von natürlichen Wachswaben

verglichen. Der Fortpflanzungserfolg derMilben und der Grad des Putzverhaltenswurde bestimmt. Zusätzlich wurden Schät-

zungen über die Anzahl der Milben im Volk

durchgeführt. Im Jahr 1994 entfernten dievier hygienischen Völker bis zum 10. Tagnach der Infektion signifikant häufiger vonmehr als einer Milbe befallene Puppen(69,2% ± 16,41) als die 3 nicht-hygieni-

schen Völker (10,00 ± 10,00) und als dieKontrollvölker für beide Gruppen. Jedochentfernten die 7 hygienischen Völker 1995nicht signifikant mehr befallene Puppen alsdie 4 nicht-hygienischen Völker, wenn nureine Milbe pro Zelle eingesetzt wurde(hygienisch 24,7 ± 20,6; nicht-hygienisch11,3 ± 6,29). Vier dieser hygienischen Völ-ker entfernten &le; 15% der befallenen Pup-pen und die restlichen 3 Völker 45,5% ±6,46. Es wurden jedoch gegenüber denKontrollen signifikant mehr Puppen ent-fernt, die mit 2 Milben infiziert waren. In denhygienischen Völkern wurden bis zum 10.Tag nicht signifikant mehr befallene bzw.Kontrollpuppen aus den Plastikzellen derJenterwabe entfernt als aus Zellen dernatürlichen Wachswaben (alle gepaartent-Teste > 0,05%). Der Reproduktionserfolgder Milben, die nicht mit den Puppen ent-fernt wurden, war niedrig. 1995 betrug derProzentsatz reproduzierender Milben, bezo-gen auf die insgesamt eingesetzten Milbenin den hygienischen Völkern, 17,2% (Jen-terwabe) und 27,1% (Wachswabe). In dennicht-hygienischen Völkern waren es 7,3%(Jenterwabe) und 37,0% (Wachswabe).Wenn eine eingesetzte Milbe keine Nach-kommen hatte, setzte sie häufig die Fäka-lien auf der Puppe statt wie normalerweisean der Zellwand ab. Der Protzentsatz derdurch Putzen verletzte Milben wurde ausder Anzahl der Milben bestimmt, diewährend des Experiments auf die klebri-gen Bodeneinlagen fielen. Er schwanktezwischen 6,0% und 42,3% bei allen Völ-ken. Zwischen dem Prozentsatz der ver-letzten Milben der hygienischen und dernicht-hygienischen Völker bestand keinUnterschied (P > 0,05%). Es gab währendder Versuche keine signifikanten Unter-schiede in der Anzahl der Milben, die aufder Bodeneinlage gesammelt wurden. Für die Bestimmung des Grades, in dem hygie-nisches Verhalten die Milbenanzahl ineinem Volk reduziert, sind weitere Versuchenotwendig.

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