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Issue
Apidologie
Volume 41, Number 2, March-April 2010
Page(s) 181 - 193
DOI http://dx.doi.org/10.1051/apido/2009068
Published online 27 November 2009

© INRA/DIB-AGIB/EDP Sciences, 2009

1. INTRODUCTION

The varroa mite, Varroa destructor (Acari: Varroidae), is a well-adapted parasite of the Eastern honeybee (Apis cerana) in northern regions of mainland Asia. It also dramatically expanded its distribution when it shifted host to the Western honeybee (A. mellifera) in the last century. Prior to and long after the shift, V. destructor was mistaken for V. jacobsoni, a morphologically similar species first described from A. cerana in Indonesia (Oudemans, 1904). It was not until 2000, following a comprehensive molecular and morphological studies of so-called V. jacobsoni mites on A. cerana throughout Asia, that it was recognized as a distinct species (Anderson and Trueman, 2000). In that particular study, 18 different mite haplotypes [mites with distinct mitochondrial (mt) DNA sequences] were discovered, nine of which were members of V. jacobsoni, six of V. destructor, and the identities of three others were unresolved. Each haplotype was named after the country or island in which it was first found, e.g., the China 1 (C1) haplotype was found in China. One more haplotype of V. destructor has subsequently been found on A. cerana in China using the same marker (China 2 or C2) (Zhou et al., 2004).

Of the eight known haplotypes of V. destructor on A. cerana, only Japan 1 (J1) and Korea 1 (K1) have successfully colonized A. mellifera (Anderson and Trueman, 2000; Solignac et al., 2005). J1 first shifted from A. cerana to A. mellifera in Japan last century following the introduction of A. mellifera (Sakai and Okada, 1973). It then spread on A. mellifera into Thailand and Brazil (Anderson and Trueman, 2000) and then into North America (de Guzman et al., 1999). According to de Guzman et al. (1997) and Oldroyd (1999), K1 first switched from A. cerana to A. mellifera near Vladivostok (north of the Korean Peninsula) following the introduction of A. mellifera from Ukraine during the late 1950s (Crane, 1978). It thereafter spread on A. mellifera into Europe in the 1960s (Crane, 1978), then around the world, arriving in North America in 1987 (de Guzman et al., 1997; Oldroyd, 1999; Sammataro et al., 2000). On A. mellifera, J1 and K1 are partially isolated clones (Solignac et al., 2005) for reasons that are not understood.

Here, we sought to gain further insights into the invasion of A. mellifera by V. destructor by identifying and genotyping isolates of V. destructor infesting both A. cerana and A. mellifera in regions where the J1 and K1 host shifts first occurred as well as in a broader area of V. destructor’s natural geographic range in Asia. The aim of our study was to obtain insights into the following questions: (i) how variable is V. destructor on its primary host (A. cerana) and its new host (A. mellifera) in Asia where the J1 and K1 host shifts occurred? and (ii) are J1 and K1 on A. mellifera derived from different mite populations, or do they simply reflect haplotypes randomly sampled in a single large Varroa population?

2. MATERIALS AND METHODS

Female Varroa mites were collected from 21 A. cerana and 11 A. mellifera colonies in several Asian countries where V. destructor is known to occur alone (China, Japan, Korea, far East Russia, Taiwan and North Vietnam) or sympatric with V. jacobsoni (Thailand) (Tab. I). Several mites per colony were collected alive from the bee brood and preserved in 95% ethanol until needed for DNA analysis. In most cases, a single bee colony was sampled at one locality, the exceptions being where A. mellifera and A. cerana colonies were hived next to each other in the same apiary, as in Nanchang (China), where 8 colonies were sampled (5 A. cerana and 3 A. mellifera), or where V. destructor and V. jacobsoni exist sympatrically as in Chang Mai, Thailand where 5 colonies were sampled. In all cases, three mites were molecularly analyzed from each colony sampled, except in the case where 10 mites were analyzed from a single A. mellifera colony from Chang Mai (Thailand).

Table I

Origin, collection details and identity of Varroa destructor (VD) and V. jacobsoni (VJ) isolates examined from Apis cerana (Ac) and Apis mellifera (Am) (shaded) collected in Asia. Mites were assigned to a known (e.g. V1) or new haplogroups based on partial nucleotide sequence (458 bp) of their mtDNA cox1 gene (Anderson and Trueman, 2000). Haplotypes were also further defined (e.g. V1-2) based on concatenated nucleotide sequences of fragments of their mtDNA cox1, cox3, atp6 and cytb gene sequences (2700 bp) obtained in this study.

Total DNA was extracted from single mites using the DNeasy tissue Kit (Qiagen, USA), following manufacturer’s instructions as indicated for the spin-column protocol from Cultured Animal Cells with the following slight modifications: (i) all volumes were reduced to half; (ii) incubation was at 70 °C for 3 h; (iii) final elution was in 50 μL of DNase-free water. Four fragments from either the cox1, cytochrome oxidase III (cox3), ATP synthase 6 (atp6) and cytochrome b (cytb) mitochondrial genes were amplified by the polymerase chain reaction (PCR) from each V. destructor mite whereas only fragments of the cox1, cox3, atp6 genes (same sequences as above), were amplified from V. jacobsoni mites (Tab. I). The PCR primers (Tab. II) were developed from published sequence of the complete mtDNA genome of V. destructor (GenBank Accession No. AJ493124.1; Evans and Lopez, 2002; Navajas et al., 2002). A 458 base pair (bp) fragment of the cox1 gene was also amplified for each mite using C1/C1R primers defined on sequences published by (Anderson and Fuchs, 1998). Reactions were carried out in 25 μL of PCR solution containing 2.5 μL of 10X buffer, 1U Taq polymerase (Qiagen), 0.25 mM of each dNTP, 0.5 μM of each oligonucleotide primer, 2.5 mM of MgCl2 and 2 μL of template DNA. Samples were denatured at 94 °C for 4 min and then PCR was done for 35 cycles of 30 s denaturation at 94 °C, 30 s annealing (see Tab. II for annealing temperature for each fragment) and 1 min extension at 72 °C. PCR products were cleaned-up using the ExoSAP-IT® reagent and subsequently directly sequenced in two directions using the BigDye® Terminator method (Parkin Elmer, Foster City, CA, USA) in an ABI PRISM 377 automated DNA sequencer (Applied Biosystems Inc). Sequences obtained using the C1/C1R primers were used to assign mites to a particular cox1 haplotype for direct comparison with haplotypes found in previous studies (Anderson, 2000; Zhou et al., 2004; Solignac et al., 2005), whereas sequences obtained using the other primers allowed the detection of variation within the haplotypes defined using cytochrome oxidase I (cox1) sequences (458 bp).

Table II

DNA amplified fragments to study variation in Varroa destructor samples. Amplified gene fragment, product size base pairs (bp) and annealing temperatures (Ta) are indicated. PCR primers defined based on the complete mitochondrial genome sequence of V. destructor (Navajas et al., 2002).

Sequences were assembled using the STADEN package 1.6 (http://staden.sourceforge.net/) and edited with BioEdit V.5.0.6 software (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) (Hall, 1999). Sequence alignments were initially performed using ClustalW version 1.8 (Thompson et al., 1994) and optimized by visual inspection. Sequences obtained from individual mites were concatenated into a single data set to increase the power of haplotype relatedness reconstruction. Concatenated sequences of the cox1, cox3, atp6 and cytb genes of each V. destructor haplotype (2700 nucleotides) was aligned by eye without the need for numerical algorithms. Concatenated sequences of fragments of the cox1, cox3 and atp6 genes from V. jacobsoni haplotype contained 1635 nucleotides (cytb PCR primers failed to amplify V. jacobsoni DNA). Identical sequences were grouped and defined as one haplotype. Standard sequence diversity indices (Nei, 1987) were computed including A (number of alleles = variable haplotypes), S (number of segregating sites = variable nucleotide positions), and π (nucleotide diversity) using the DnaSP 4.10.4 package (http://www.ub.es/dnasp/) (Rozas et al., 2003). Intraspecific haplotype genealogies based on the 2700 bp V. destructor sequences were reconstructed. Gene genealogies were first inferred by computing haplotype network methods using the TCS program v2.1 (Clement et al., 2000), which essentially gave the same information as a phylogenetic reconstruction based on the Neighbour-Joining methods as implemented in the MEGA Package version 3.1 (Kumar et al., 2004). Only the resulting phylogenetic tree is presented here.

3. RESULTS

3.1. Mite variation based on a 458 bp fragment of the mitochondrial cox1 gene

Seven different sequences were identified among DNA fragments amplified from a total of 103 Varroa mites collected from 32 colonies using the previously defined C1/C1R primers. Five sequences were identical to either the published China 1 (C1), China 2 (C2), Japan 1 (J1), Korea 1 (K1) or Vietnam 1 (V1) haplotypes of V. destructor (Anderson and Trueman, 2000; Zhou et al., 2004) (GenBank accession numbers: AF106900, AY372063, AF106897, AF106899 and AF106901, respectively). A sixth sequence was identical to a Laos 1 (L1) haplotype of V. jacobsoni, which was first described by Zhou et al. (2004). A new sequence was also identified and is hereafter referred to as the China 3 (C3) haplotype of V. destructor. Only V. destructor haplotypes were detected in geographical regions (China, Japan, Korea, Russia, Taiwan and Northern Vietnam) where V. destructor had been previously found to occur alone (Tab. I, Fig. 1). Both V. destructor and V. jacobsoni haplotypes were found in Thailand where both mites had been previously found sympatrically (Fig. 1). In all cases, the three mites studied per colony had identical mitochondrial sequences as did the 10 mites analyzed from an A. mellifera colony near Chang Mai (Thailand).

thumbnail Figure 1

Geographical distribution of Varroa destructor mitochondrial haplotypes on Apis cerana and A. mellifera (in bold). All haplotypes were identified as V. destructor except two L1 haplotypes, which belong to V. jacobsoni (circled) from Thailand. The V. destructor haplotypes found on A. mellifera elsewhere are K1-1 and J1-1 (Solignac et al., 2005). Solid line indicates approximate range of V. destructor and dotted line is that of V. jacobsoni, after Anderson and Trueman (2000) and data provided by D.L. Anderson.

3.2. Mite variation based on concatenated mtDNA sequences

The concatenated sequences revealed greater genetic variation among mites (18 haplotypes; Tab. III) than was revealed by the sequences of the 458 bp cox1 fragment alone (7 haplotypes, see previous section). Hence, mites with identical cox1 sequences were regarded as members of the same ‘haplogroup’, of which there were 7 and, mites of the same haplogroup that showed variation within their concatenated sequences were regarded as variants of a particular haplogroup.

Table III

Nucleotide positions of aligned mitochondrial sequences of four genes (cox1, atp6, cox3 and cytb) that distinguish haplotypes of Varroa destructor. Dots indicate identical nucleotide as in the first sequence. Figures correspond to position numbers in the complete mitochondrial sequence of V. destructor (AJ493124.1). Mite samples were collected on either Apis mellifera (shaded haplotype code) or Apis cerana.

Among mites collected from A. mellifera in regions of northeast Asia where only V. destructor, not V. jacobsoni, has been previously reported, 3 variants were found of the K1 haplogroup (K1-1, K1-2, and K1-4) and 2 variants of the J1 haplogroup (J1-1 and J1-6). Among mites collected from A. cerana in this same region, 1 variant was found of the K1 haplogroup (K1-3), 3 of the J1 haplogroup (J1-2, J1-3 and J1-4), 2 of the V1 haplogroup (V1-1 and V1-2) and 1 variant each of the C2 and C3 haplogroups (C2-1 and C3-1). Hence, only variants of V. destructor were found on A. mellifera and A. cerana in these regions. Also, only variants of the K1 and J1 haplogroups were found on A. mellifera and none of those variants were found on A. cerana. Among mites collected from Thailand, where V. destructor and V. jacobsoni coexist, two variants were found of the V1 haplogroup on A. cerana (V1-3 and V1-4) and two of the L1 haplogroup (L1-1 and L1-2) also on A. cerana. A variant of the J1 haplogroup (J1-5) was found on A. mellifera. Hence, only V. destructor was found on A. mellifera in Thailand but both V. destructor and V. jacobsoni were found on A. cerana. Again, none of the mite variants found on A. cerana was found on A. mellifera and vice versa.

Table IV

Estimates of diversity and divergence of mitochondrial sequences of Varroa samples: (A) molecular diversity indices calculated for samples of Varroa destructor collected from Apis cerana and Apis mellifera colonies; (B) divergence between samples of V. destructor collected from A. mellifera and A. cerana.

The concatenated sequences of the K1-1 and J1-1 variants described are those of V. destructor collected from Taichung (Taiwan) and Tokyo (Japan), respectively (Tab. I), which had been previously identified using microsatellites as K1 and J1 types by Solignac et al. (2005).

GenBank accession numbers for the sequences of the 4 partial mitochondrial genes described here are given in Table I and their origin of detection in Asia is shown in Figure 1.

3.3. Nucleotide diversity

Standard sequence diversity indices (π) were computed from the mitochondrial concatenated sequences (2700 bp) for V. destructor collected on both A. mellifera and A. cerana (Tab. IV). The indice (π) for the 11 V. destructor haplotypes collected from A. cerana was π = 0.0078, whereas for the 7 V. destructor haplotypes carried by A. mellifera it was π = 0.0047, and for V. destructor isolates collected from both A. cerana and A. mellifera it was π = 0.0075. Sequences of only 2 individuals of V. jacobsoni were analyzed in this study and indices of diversity were not estimated. The two haplotypes (L1-1 and L1-2) differ by 2 point mutations in the cox3 sequence.

3.4. Phylogenetic relationships

Sixty-six (66) nucleotides of the concatenated V. destructor sequences were polymorphic. An unrooted neighbour-joining tree of all V. destructor sequences showed two well-supported clades (Fig. 2), the first of which grouped variants of the Korea and Japan haplogroups and, the second, variants of the China and Vietnam haplogroups. A common lineage grouped all variants of the V1 haplotype together with the two variants of the C1 haplotype (95% bootstraps score), the two other China haplotypes (C2 and C3) remaining apart from this grouping.

thumbnail Figure 2

Phylogenetic relationships among haplotypes belonging to six different haplogroups (K1, J1, C1, C2, C3, V1) of Varroa destructor collected from two hosts, Apis cerana (Ac) and A. mellifera (Am) in Asia. Each haplotype is preceded by Am or Ac according to the host where the mite was sampled. Haplotypes were defined by sequences of 2700 nucleotides long of the mitochondrial genome spanning 4 genes (cox1, cox3, atp6 and cytb).

4. DISCUSSION

Our study shows that the original K1 and J1 haplotypes of V. destructor on A. mellifera worldwide were derived from two distinct mite populations (represented by our K1 and J1 haplogroups, respectively) that infest A. cerana in north-east Asia. Both populations contain much more genetic variation than previously realised and newly discovered haplotypes in each population have successfully colonized A. mellifera in Asia, but have not yet spread from that region. These new haplotypes now represent new potential threats to A. mellifera outside of Asia.

4.1. Sensitivity of mitochondrial markers for detecting Varroa variation

Most variation detected to date among varroa isolates has been found using a single mtDNA marker. This marker, developed by Anderson and Fuchs (1998), was widely used by Anderson and Trueman (2000) to clarify the level of genetic variation among varroa mites on A. cerana throughout Asia and to help resolve varroa taxonomy. It has also been used in several more recent studies on varroa variation (Koeniger et al., 2002; Zhou et al., 2004; Solignac et al., 2005; Warrit et al., 2006). The marker detects sequence variation in a 458 DNA base-pair region of the mt cox1 gene. It can readily resolve an individual mite to the species level, as variation in this gene region is less than 2% among mites of the same species and more than 6% among mites of different species (Anderson and Trueman, 2000).

Here, we employed new longer mitochondrial sequences to determine how useful the cox1 marker is for detecting varroa variation. It was not surprising that concatenated sequence data from the new sequences were far more sensitive at detecting variation than the cox1 marker alone. However, it became obvious that, when used alone, the cox1 marker was able to define particular populations of mites (‘haplogroups’), whose members (haplotypes) were identified using the new markers. Hence, the combined use of the cox1 marker and the new markers allows for detection of genetic variation both within and between different varroa populations. The sensitivity of the new mt sequences at detecting genetic variation falls between that of the less sensitive cox1 marker and the more sensitive microsatellites used by Solignac et al. (2005).

4.2. Genetic variation among varroa mites on A. cerana in north-east Asia

We uncovered significant genetic diversity among varroa mites on A. cerana in northeast Asia (Tab. I; Tab. III). Although only 1 new haplogroup (C3 in Tab. I) was detected, 10 haplotypes were uncovered within haplogroups in which no genetic variation was previously known. Thus, we showed that varroa mites are more genetically variable on A. cerana in Asia than previously recognized.

Our results also showed that some closely-related varroa haplotypes on A. cerana had similar and quite restricted geographical distributions. For example, C1-1 and C1-2 in Figure 2 were both collected from the same province of Guangdong in China. On the other hand, members of some haplogroups such as those found in Thailand, China and Vietnam, were widely distributed. It is possible that the artificial movement of A. cerana in Asia by humans may have influenced such wide distributions. Hence, a better way of determining natural distributions may come from examining the associations of particular mite haplogroups with particular genotypes of A. cerana, as previous studies have noted that the biogeography of varroa haplotypes in Asia seems to correlate with the biogeography of A. cerana haplotypes (Anderson and Trueman, 2000; Warrit et al., 2006). Interestingly, none of the varroa haplotypes detected here on A. cerana was found on A. mellifera and vice-versa.

4.3. Genetic variation among varroa mites on A. mellifera in north-east Asia

As well as redefining the taxonomy of varroa, Anderson and Trueman (2000) also showed that K1 and J1 haplotypes of V. destructor were the only varroa mites to have successfully invaded and colonized A. mellifera globally. Solignac et al. (2005) verified those findings and further showed that there was almost no genetic variation between K1 and J1 (our K1-1 and J1-1) on A. mellifera for reasons that were not clear.

Here, V. destructor was the only varroa species detected on A. mellifera in north-east Asia and all haplotypes found were members of the K1 and J1 haplogroups. However, four (4) new haplotypes not previously reported on A. mellifera were also found (Tab. I; Tab. III). A new K1-2 haplotype was found on A. mellifera in Vietnam, a K1-2 and a K1-4 in China, a J1-5 in Thailand, and a J1-6 in Japan. The presence of these haplotypes in A. mellifera colonies at these locations is not unequivocal evidence that they have successfully colonized the colonies. Only mites that are able to reproduce on A. mellifera brood could successfully colonize and we do not have that information on the new haplotypes. The presence of varroa mites in an A. mellifera colony in Asia could simply indicate that the mites invaded the colonies from a local A. cerana colony without colonizing, as has been shown to occur for the V1-1 haplotype of V. destructor in Vietnam, a Java haplotype of V. jacobsoni in Indonesia and for three unique varroa genotypes in the Philippines (Anderson, 1994; Fuchs et al., 2000). However, there is convincing evidence from our studies that at least 2 of the new haplotypes (J1-5 and K1-2) have successfully colonized A. mellifera in Asia. The most convincing evidence is the presence of J1-5 in A. mellifera colonies in Thailand. No member of the J1 haplogroup has ever been reported on A. cerana outside of Japan and certainly not in Thailand. J1-1 has been reported outside of Japan on A. mellifera, but it shows almost no genetic variation (Solignac et al., 2005). It has been known that a member of the J1 haplogroup has been present on A. mellifera in Thailand since 1997 (Anderson and Trueman, 2000). This same member was reported there again in 2001 (Warrit et al., 2006). Here we have shown that that member is J1-5, not J1-1. The only way J1-5 could have survived for 4 years in Thailand is if it could colonize A. mellifera. In our study we collected 10 mites from several different pupae in a single A. mellifera colony in Thailand and each was J1-5, which is way above the expected detection threshold if those mites were simply casual invaders of the colony (Anderson, 1994). Hence, it is clear that J1-5 has colonized A. mellifera in Asia. Our observations corroborate anecdotal evidence that A. mellifera was imported into Thailand from Japan via Taiwan as part of a bee extension program (Anderson, unpublished data).

The presence of K1-2 on A. mellifera in Vietnam and China provides less convincing evidence, but still substantial support, that haplotypes other than K1-1 and J1-1 have colonized A. mellifera in Asia. The detection of this haplotype in A. mellifera colonies was above the detection threshold expected for a casual invader and, interesting, like all other varroa haplotypes that have successfully invaded A. mellifera, we failed to detect K1-2 on A. cerana.

4.4. Insights into the geographical origins and invasive biologyof V. destructor

Although there was no clear geographical structure of most mitochondrial haplotypes of V. destructor in Asia, most haplotypes of the J1 haplogroup (5 out of the 6) were found in Japan, confirming previous claims that this region was the origin of the J type (de Guzman et al., 1997; Anderson and Trueman, 2000). As for J1-1, evidence indicates it shifted to A. mellifera in Japan and was subsequently transported to Brazil during the 1970’s (de Jong and Gonçalves, 1981; De Jong et al., 1982).

Whether or not all haplotypes of the K1 haplogroup colonizing A. mellifera originated from a discrete geographic region of Korea, as suggested by Warrit et al. (2006), needs further investigation. Here, we found K1-1 only on A. mellifera in Korea (Seoul) and Russia (Vladivostok), which lends support the hypothesis that K1-1 first colonized A. mellifera in Far-Eastern Russia (Danka et al., 1995) following a host-shift somewhere near Korea. Evidence suggests that the host-shift was made possible because mites of a single genetic linage (K1-1) were able to invade an A. mellifera colony from a colony of their indigenous A. cerana and were then able to lay eggs and produce offspring on the A. mellifera brood (Solignac et al., 2005). Our inability to find K1-1 on A. cerana in Asia lends further support to this hypothesis. After shifting to A. mellifera, K1-1 was then moved out of Asia into Europe, from where it spread globally.

By discovering new haplotypes of V. destructor colonizing A. mellifera in Asia, our studies suggest that the colonization of A. mellifera in Asia by new haplotypes is dependent on the length of exposure of those haplotypes to A. mellifera. Perhaps longer exposure eventually results in one type being selected for their ability to reproduce on A. mellifera. Given this scenario, we predict that other types of V. destructor, including those that are currently known to be non-reproductive on A. mellifera, will eventually colonize A. mellifera in Asia. Indeed, it is possible that such types have existed before in some parts of Asia, but may have completely exterminated A. mellifera and hence themselves.

In contrast to the V. destructor polymorphism unravelled in Asia, the V. destructor colonizing A. mellifera worldwide (K1-1 and J1-1) shows a complete lack of genetic diversity. These results are consistent with the hypothesis of a bottleneck that occurred at the very beginning of the invasion of the Western honeybee by V. destructor as suggested by (Solignac et al., 2005). This scenario may have resulted from two independent events, one for each of the J and K types belonging to two distinct monophyletic haplogroups (Fig. 2). Due to varroa’s rapid growth rate (about 12-fold increase in mite numbers yearly) (Martin, 1998), the situation is hardly compatible with the erosion of variability of V. destructor after colonizing A. mellifera colonies. A strong founder effect was also confirmed by the fact that J1-5 in Thailand, K1-2 in Vietnam and China, and J1-6 in Japan, which reproduce on A. mellifera, have been only found in Asia.

One of the main conclusions that have emerged from wider studies on invasive species is that the successful establishment of an exotic species into a new range is quite improbable and that many failed attempts occur before a successful invasion develops (Nivak, 2007). It has been often hypothesized that a selection of ad hoc genotypes operates at the beginning of an invasive process (Facon et al., 2006), and it can be speculated that the genotypes having invaded A. mellifera worldwide were pre-adapted to this new host. It is now known that some variation exists in the ability of varroa to reproduce on A. cerana, depending on the bee subspecies (Fuchs et al., 2000). Likewise, Warrit et al. (2006) suggest that populations of varroa on different host populations are genetically differentiated and may be adapted to specific characteristics of their local host bee populations. Understanding these adaptive characteristics may eventually lead to new ways of controlling invasive haplotypes on A. mellifera.

4.5. Implications for world apiculture

New V. destructor haplotypes that colonize A. mellifera may show slight differences in their life cycles, which cause them to become more virulent than the current K1-1 and J1-1. Indeed, J1-1 is assumed to be far less virulent to A. mellifera than K1-1 (de Guzman and Rinderer, 1999) and is thought to be displacing J1-1 in places where the two are sympatric (de Guzman et al., 1997; Anderson, 2000; Garrido et al., 2003). These observations caution against the free movement of honeybees and signal the need for strict and proper quarantine for the trade of live honeybees between countries.

Acknowledgments

We thank Prof. Z. Zeng and Prof. Wongsiri for providing sampling facilities in respectively China, and Thailand and S. Cros-Arteil and C. Moreau for help in sequencing. A part of the sequences in this work are from the graduate diploma, University Montpellier II, defended by J. Clement in 2004. Funding was provided by the European grant FEOGA (Fonds Europeen d’Orientation et de Garantie Agricole) ID B24000059 to MN and YLC, and a USDA-SCP grant to ZYH. Data used in this work were (partly) produced through molecular genetic analysis technical facilities of the IFR119 Montpellier Environnement Biodiversité.

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All Tables

Table I

Origin, collection details and identity of Varroa destructor (VD) and V. jacobsoni (VJ) isolates examined from Apis cerana (Ac) and Apis mellifera (Am) (shaded) collected in Asia. Mites were assigned to a known (e.g. V1) or new haplogroups based on partial nucleotide sequence (458 bp) of their mtDNA cox1 gene (Anderson and Trueman, 2000). Haplotypes were also further defined (e.g. V1-2) based on concatenated nucleotide sequences of fragments of their mtDNA cox1, cox3, atp6 and cytb gene sequences (2700 bp) obtained in this study.

Table II

DNA amplified fragments to study variation in Varroa destructor samples. Amplified gene fragment, product size base pairs (bp) and annealing temperatures (Ta) are indicated. PCR primers defined based on the complete mitochondrial genome sequence of V. destructor (Navajas et al., 2002).

Table III

Nucleotide positions of aligned mitochondrial sequences of four genes (cox1, atp6, cox3 and cytb) that distinguish haplotypes of Varroa destructor. Dots indicate identical nucleotide as in the first sequence. Figures correspond to position numbers in the complete mitochondrial sequence of V. destructor (AJ493124.1). Mite samples were collected on either Apis mellifera (shaded haplotype code) or Apis cerana.

Table IV

Estimates of diversity and divergence of mitochondrial sequences of Varroa samples: (A) molecular diversity indices calculated for samples of Varroa destructor collected from Apis cerana and Apis mellifera colonies; (B) divergence between samples of V. destructor collected from A. mellifera and A. cerana.

All Figures

thumbnail Figure 1

Geographical distribution of Varroa destructor mitochondrial haplotypes on Apis cerana and A. mellifera (in bold). All haplotypes were identified as V. destructor except two L1 haplotypes, which belong to V. jacobsoni (circled) from Thailand. The V. destructor haplotypes found on A. mellifera elsewhere are K1-1 and J1-1 (Solignac et al., 2005). Solid line indicates approximate range of V. destructor and dotted line is that of V. jacobsoni, after Anderson and Trueman (2000) and data provided by D.L. Anderson.

In the text
thumbnail Figure 2

Phylogenetic relationships among haplotypes belonging to six different haplogroups (K1, J1, C1, C2, C3, V1) of Varroa destructor collected from two hosts, Apis cerana (Ac) and A. mellifera (Am) in Asia. Each haplotype is preceded by Am or Ac according to the host where the mite was sampled. Haplotypes were defined by sequences of 2700 nucleotides long of the mitochondrial genome spanning 4 genes (cox1, cox3, atp6 and cytb).

In the text