Experimental and Applied Acarology 27: 195–207, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

Effect of acaricide resistance on reproductive abilityof the honey bee mite Varroa destructor

STEPHEN J. MARTIN1,∗, PATTI J. ELZEN2 and WILLIAM R. RUBINK2

1Laboratory of Apiculture and Social Insects, Department of Animal and Plant Sciences,Sheffield University, Western Bank, Sheffield, S10 2TN, UK2USDA, Honey Bee Unit, 2413 E. Highway 83, Weslaco, TX 78596, USA

(Received 3 May 2001; accepted in revised form 18 October 2002)

Abstract. The reproduction of pyrethroid-resistant Varroa destructor mite, a brood parasite ofhoney bees, was observed in Weslaco, Texas, and the results compared with known susceptiblemite populations from other studies. Seven Apis mellifera colonies that had mite populationsresistant to the acaricide Apistan® were used. Pyrethroid-resistance was confirmed whenonly 17% rather than 90% of mites confined in dishes containing Apistan® died after 12 hof exposure. The average number of eggs laid by resistant mites invading worker and dronecells was 4.4 and 5.4 respectively. This is similar to the number of eggs laid by susceptiblemites in worker (4.4–4.8) or drone (4.7–5.5) cells. Also the average number of fertilisedV. destructor female mites produced by resistant mites in worker (1.0) and drone (2.1) cellswere similar to the number produced by susceptible mites in worker (0.9) and drone (1.9–2.2)cells. In addition, no major differences between the resistant and susceptible mite populationswere observed in either worker or drone cells when six different reproductive categoriesand offspring mortality rates were compared. Therefore, it appears that there is little or noreproductive fitness cost associated with pyrethroid resistance in V. destructor in Texas.

Key words: Varroa destructor, Apis mellifera, pesticide resistance, mite reproduction,Africanised bees

Introduction

The ecto-parasite mite Varroa destructor (Anderson and Trueman, 2000), isthe most serious worldwide pest of the honey bee Apis mellifera L. As it isalmost impossible to eradicate the mite, even from small isolated populations(Sampson and Martin, 1999), beekeepers have become reliant on variousmethods of mite control. Two of the most common acaricides used, Apistan®and Bayvarol®, are based on the synthetic pyrethroids, tau-fluvalinate andflumethrin. Therefore, it was of great concern when in 1991, 10 years afterthe mite first arrived in Sicily, Italy (Barbattini, 1981), resistance to theseproducts was reported (Loglio and Plebani, 1992). These resistant mites

*Author for correspondence (Tel.: (0)114-2220149; Fax: (0)114-2220002; E-mail:s.j.martin@sheffield.ac.uk)

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spread throughout Europe (Trouiller, 1998), reaching Belgium in 1996(Bruneau et al., 1998) and the UK by 2001 (Thompson et al., 2002). InAugust 1997 the first pyrethroid-resistant mites were reported in the USA(Baxter et al., 1998). Currently, the only other reports of pyrethroid-resistanceare from mite populations in Argentina (Fernandez and Omar, 1997) andIsrael (Mozes-Koch et al., 2000).

Pesticide resistance is common among mites, especially those that repro-duce arrhenotokously (Croft and Van de Baan, 1988). In Varroa, two differentmechanisms of resistance have so far been suggested. In Europe and Israel,resistant mites have increased levels of detoxicating enzymes such as mono-oxygenases (Hillesheime et al., 1996; Mozes-Koch et al., 2000), while in theUSA, mutations that change the voltage-sensitive sodium channels, which arethe target sites of pyrethroids, have been found (Wang et al., 2000). If thesetwo different mechanisms of resistance are confirmed, it would suggest thatpyrethroid resistance has arisen at least twice.

Often resistant populations of arthropods demonstrate lower fitness thantheir susceptible counterparts (Denholm and Rowland, 1992; Georghiou andTaylor, 1986). Possessing an unusual characteristic which makes an indi-vidual resistant can result in less efficient metabolic or reproductive capacitythan average. For example, some resistant strains of mosquitoes had only onequarter of the reproductive potential of susceptible strains in the absence ofthe insecticide (Georghiou and Taylor, 1977), although this probably repre-sents an extreme case. Conversely the disadvantage associated with resistancecan be small (reviewed by Roush and Daly, 1990) and in some cases zero.

Watkins (1996) suggested that resistant Varroa mites do have a lower fit-ness compared to susceptible mites but no details or data were given. Also theloss of resistance in mite populations over time (reversion) has been reported,where levels of pyrethroid resistance decreased by an order of magnitudeover a three year period in Italy (Milani, 1999; Milani and Della Vedova,2002) and in under two years in Florida, USA (Elzen et al., 2001). Theobvious explanation for this reversion is a lower reproductive ability of theresistant mite population. Therefore, the aim of this study is to comparethe reproductive ability of resistant mites with susceptible populations duringa single reproductive cycle.

Materials and Methods

Honey bee colonies

This study was conducted during February and March, 2001 at the USDAHoney Bee Research Unit in Weslaco, Texas, using seven European

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A. mellifera ligustica colonies which had been moved onto the site withinthe previous two years. Due to the presence of Africanised bees in the area(Rubink et al., 1996) it was suspected that some colonies may have becomehybridised. As Varroa reproduction may be affected by the smaller cells builtby Africanised bees (Message and Goncalves, 1995; Medina and Martin,1999; Martin and Kryger, 2002), the internal diameter of 10 worker cellsfrom each colony were measured. In addition, to determine the degree ofhybridisation, samples of 22 adult bees from each colony were collected andrun individually against two isozymes (Nunamaker et al., 1984; Spivak et al.,1988). The presence of both malate dehydrogenase and hexokinase isozymesconfirmed representative individuals from the study hives as Africanised.

Mite resistance

Because the use of Apistan® had failed to control the Varroa populations inthe bee yard, it was assumed that the mite population was pyrethroid-resistant.To confirm this, six groups of 10 adult female mites were removed from thesealed brood cells of the seven study colonies and placed in petri-dishes con-taining bee pupae and moist tissue paper. Into each of three petri-dishes wasplaced a 20 mm × 20 mm section of a normal Apistan® strip which contains10% tau-fluvalinate. Three dishes (controls) received no Apistan®. The miteswere held at 27◦C and survival was checked after 12 h. Death was determinedby the lack of any movement shown by the mite after physical stimulation(Elzen et al., 1999).

Mite reproduction

Each day the age of the sealed brood within each colony was determinedby their eye and body colour (Martin, 1994) and any suitable frames wereremoved and brought back to the laboratory. Only cells containing pupaesealed for 200 h or longer (at least at the yellow thorax stage) were carefullyopened and bee stage recorded. When mites were present their sex, develop-mental stage and state (live or dead) were recorded. In addition, all femaledeuteronymphs were classified into five groups depending on their size usingthe photograph in Ifantidis (1983) as a guide. This information allowed themite family to be reconstructed in birth order and so permitted the offspringmortality rates to be determined.

We determined three different aspects of mite reproduction:

1. The mean number of eggs laid by mites invading worker and drone cells.To allow a more accurate comparison between studies only mites thatlaid two or more eggs were compared. Mites that produce no eggs

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(non-reproductive) or one egg (single male) are considered as distinctreproductive categories (see below).

2. All mites that had invaded worker or drone cells were analysed by placingthe mother mites into one of the following six categories: (1) motherdead; (2) mother alive, no offspring; (3) mother plus only male offspring;(4) mother plus live mature male and female offspring so mating is as-sumed; (5) mother plus live female offspring and dead male offspring,these female offspring remain unfertilised since they cannot mate (Harrisand Harbo, 1999); (6) mother plus no live female offspring. The averagenumber of mature females (unfertilised, category 5 and fertilised, cat-egory 4) and fertilised female offspring (only category 4) produced perinvading mother mite (all categories) were also calculated from the rawdata.

3. The mortality of the mite offspring in worker and drone cells was cal-culated by comparing the number of live and dead offspring at eachposition in birth order, that is, first offspring, second offspring, etc. Thenthe average number of surviving females (unfertilised and fertilised) werecalculated using only the levels of offspring mortality.

These data were then compared with four studies (Martin, 1994, 1995;Medina and Martin, 1999; Martin and Kryger, 2002) where the same datafor reproductive parameters of susceptible mites were obtained by using theexact same methodologies and criteria as in this study.

Results

Honey bee colonies

All the honey bees analysed from four of the study colonies were entirelyEuropean, while the remaining three colonies showed levels of Africanisationof 45%, 45% and 82%. No difference in the mean diameter of worker cellsbetween the European (X̄ = 5.35 ± 0.05 mm, n = 4 colonies) and those whichhad become hybridised (X̄ = 5.37 ± 0.05 mm, n = 3 colonies) were found.Clearly the cells in the colonies that were becoming Africanised were stillthose originally built by the European bees.

Mite resistance

After 12 h, only five of the 30 mites (17%) had died in dishes containing theApistan strip compared to one out of 30 mites in the control dishes. Typicallywith Apistan-susceptible mites, 90% of the mites, that is, 27 out of 30, aredead after 12 h (Elzen, personal observation). Therefore, about 83% of the

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Table 1. Comparison of the mean (± SD) number of eggs laid by Varroa destructor mitesinvading drone and worker cells, between this and four other studies. All mites which laidtwo or more eggs are included

Worker cells Drone cells

X̄ n X̄ n

Resistant mites, Africanised bees in 4.39 ± 0.97 41 5.35 ± 1.00 144

Texas (this study)

Resistant mites, European bees in Texas 4.42 ± 0.84 141

(this study)

European/Buckfast bees in UK (Martin, 4.82 ± 0.77 136

1994)

European/Buckfast bees in UK (Martin, 5.45 ± 0.95 814

1995)

Apis mellifera scutellata bees in South 4.36 ± 0.88 72 4.73 ± 1.16 48

African (Martin and Kryger, 2002)

Africanised bees in Mexico (Medina and 4.81 ± 0.70 929

Martin, 1999)

mite population showed pyrethroid resistance, which confirms earlier obser-vations of the failure of Apistan® to control mite populations within our beeyard and other nearby colonies.

Mite reproduction

From the four European colonies, 2800 worker cells were opened and 170 mitefamilies reconstructed. From the three hybridised colonies, the inspection of1700 worker and 560 drone cells revealed 60 and 173 mites families respec-tively. There was no significant difference (P > 0.1, comparison of means)between either the number of eggs laid (Table 1) or the final number of fer-tilised Varroa females produced (Table 2) in the worker cells of the Europeanor hybridised colonies.

There was little difference in the number of eggs laid by resistant andsusceptible mites invading either drone or worker cells (Table 1).

The comparison of the reproductive fate of resistant and susceptible mitesagain revealed very little differences in any of the six reproductive categories(Table 2). Comparison of offspring mortality levels (Table 3) between resis-tant and susceptible mites also revealed little difference in either worker ordrone cells.

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Table 2. Comparison of six reproductive categories of V. destructor between the resistant (R) and susceptible (S) strains invading honey bee workerand drone cells. Numbers are % of total. Also the estimated number of mature (unfertilised and fertilised) and fertilised females produced per invadingmother mite is given. Only mites from cells estimated to have been sealed for 200 h or longer were analysed

Worker cells Drone cells

R-mites R-mites S-mites R-mites S-mites

European bees Africanised bees Africanised bees

(Texas; (Texas; UKa S. Africac Mexicod (Texas; UKb S. Africac

n = 168) n = 60) (n = 135) (n = 118) (n = 1031) n = 153) (n = 834) (n = 98)

Reproductive categories

Mother dead 4 10 2 6 2 7 5 6

No offspring 8 15 10 13 12 5 3 2

Only male offspring 8 7 9 15 11 9 14 20

Fertilised female 53 53 63 51 43 63 63 59

offspring

Unfertilised female 23 8 12 11 19 13 10 9

offspring due to

male death

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.

Unfertilised female 4 7 4 4 13 3 5 4

offspring due to

other causes

Number of females produced/mother

Mature females 1.30 1.17 1.0 1.0 0.9 2.58 2–2.2 2.5

Fertilised females 1.03 1.02 0.9 0.9 0.7 2.09 1.9–2.1 2.2

a Recalculated from Martin (1994).b Recalculated from Martin (1995).c Martin and Kryger (2002).d Medina and Martin (1999).

202Table 3. Comparison of offspring mortality rates of resistant (R) and susceptible (S) stains of V. destructor invading honey bee worker and drone cells.Numbers are % of total. Also the estimated survival of the five female offspring into mature (unfertilised and fertilised) or fertilised females are given.Only mites from cells sealed for 200 h or longer where analysed

Worker cells Drone cells

R-mites R-mites S-mites R-mites S-mites

European bees Africanised bees Africanised bees

(Texas) (Texas) (Texas)UKa S. Africac Mexicod UKb S. Africac

Birth order

Male 41 22 20 28 43 23 10 16

1st female 7 11 6 4 32 6 2 6

2nd female 19 21 62 19 57 6 7 5

3rd female 34 35 87 85 72 11 16 6

4th female 100 100 100 100 100 22 24 15

5th female 100 100 100 100 100 39 37 33

Number of surviving females

Estimated 2.38 2.33 1.45 1.92 1.39 4.16 4.14 4.35

female survival

Estimated 1.45 1.82 1.16 1.40 0.79 3.2 3.73 3.65

fertilised female survival

a Martin (1994).b Martin (1995).c Martin and Kryger (2002).d Medina and Martin (1999).

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All these factors indicate that there was little or no difference betweenreproduction of resistant mites in this study and susceptible populations ofthe V. destructor mites from previous studies.

Discussion

In this study, Varroa reproduction was not apparently affected by the partialAfricanisation that some European bee colonies had undergone. This findingwas similar to that reported by DeGuzman et al. (2001), but different to otherstudies which reported lower reproductive rates of the mite in fully African-ised bee colonies (Medina and Martin, 1999; Vandame et al., 2000). Thesedifferences may be in part due to the cell size, which in this study was a sizeconsistent with those built by European bees (5.3 mm) and not the smallercells (4.6–5.0 mm) built by Africanised bees (Message and Gonclaves, 1995;Spivak et al., 1988). This may also help explain the observations that despitethe partial Africanisation of European colonies in southern Texas, they stillcollapse within 1–2 years if no Varroa-control measures are taken. This alongwith the presence of many deformed bees in collapsing colonies stronglysuggest Varroa as the main cause (Martin, 2001). However, in the local feralpopulation where the Africanised bees build smaller cells, their populationshave persisted for several years or are increasing, despite the presence ofthe mite (Rubink, 1994; Rubink et al., 1995, and unpublished data). Thesesmaller cells were also present in the Africanised bee colonies studied byMedina and Martin (1999) and Vandame et al. (2000).

Although we used three different methods to compare the reproductiveability of resistant and susceptible mite populations, few differences betweenthe mite populations could be detected. In an ideal world, the reproduc-tive rates of pyrethroid-resistant and pyrethroid-susceptible mites should havebeen studied in the same colony. However, this would be a very difficult taskto accomplish so it is important to view the findings of this study in light ofthe mite’s natural history.

Firstly, although honey bees are found over a wide range of climatic con-ditions, the environmental conditions within the brood cells where Varroareproduces are very constant (Winston, 1987). This helps to explain why,when the same methodologies are used, the reproduction of susceptibleVarroa mites are very similar despite the studies been carried out in differentcountries on different races of A. mellifera (Table 1). However, since mitereproduction is in small integers (e.g., 2, 3, 4, etc. offspring), variance termswill be large, so small differences in fecundity would be difficult to detect,especially when comparing different mite populations.

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Secondly, Varroa evolved to reproduce within the drone brood ofA. cerana where high mite offspring survival results in nearly all eggs reach-ing adulthood (Boot et al., 1997). However, Varroa has not adapted wellto its new host A. mellifera, and only around 30 or 80% of eggs laid inworker or drone cells respectively reach adulthood. This high failure rate inA. mellifera worker brood is mainly caused by its shorter developmental time.So, although the first female egg has a 90+% chance of reaching adult-hood, the survival of subsequent female eggs falls rapidly (Table 3). As most(≈85%) mites reproduce in worker cells (Martin, 1998), any small reductionin egg number associated with pyrethroid resistance will have little or no ef-fect on the overall growth of the mite population. It is possible that resistancecould have affected the general egg or offspring viability but this did notappear to be the case (Tables 2 and 3).

If there is loss of reproductive fitness associated with pyrethroid resistancein this population of V. destructor, it is not very great. In Italy no or only asmall disadvantage associated with fluvalinate resistance in V. destructor wasfound which is consistent with observations on other insects when resistanceis due to mono-oxygenases (Milani and Della Vedova, 2002). Thus, the re-version previously observed in Florida, USA, and possibly Italy, is unlikelyto have been caused by different reproductive rates between the resistantand susceptible populations during a single reproductive cycle. It is possiblethat the influx of susceptible mites into the resistant population could havecaused the reversion. Milani (1995) suggested that the large variability ofresistant mite strains may be due to mixing with susceptible mites. Also,the initial distribution of resistant mites is patchy and usually in areas wheremigratory beekeeping is more active (Milani, 1995). However, in the Floridastudy, although mite-mixing cannot be ruled out due to the large amount ofmigratory beekeeping in this area, it is unlikely due to the widespread use ofpyrethroids.

In addition, inbreeding (brother–sister mating) which normally occurs inVarroa will greatly reduce the gene transfer between any resistant and sus-ceptible populations, and so slow down the rate of reversion. This study doesnot rule out other costs to resistant mites such as the long term mite survivalbut these are very difficult to study. However, if the resistance mechanismsof Varroa in the USA are due to point mutations (Wang et al., 2000) themetabolic load on resistant mites could be very small and so differences inmite reproduction would not be expected, as this study found.

It is possible that different populations of Varroa have different mech-anisms of resistance which confer different levels of fitness costs. So it isimportant to study both the mechanism of mite resistance along with thereversion phenomena which currently cannot be explained, as these may opennew avenues of Varroa control.

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Acknowledgements

We wish to thank the efforts of Jesus Maldonado, Noe Buenrostro,Roy Medrano, Raul Rivera and two anonymous reviewers. Collaboration wasmade possible via a fellowship to S.J. Martin under the OECD Co-operativeResearch Program: Biological Resource Management for Sustainable Agri-culture Systems.

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