The loci in Scaffolds 300 and 1635 had considerable variability and identified three and four different haplotypes, respectively. This variation was equivalent to what we found using a variable part of the EF1α gene, whereas no differences were found in the ITS sequences among
the 12 A. apis isolates. When all five intergenic loci and EF1α sequences were combined into one analysis, seven different haplotypes were identified among the 12 A. apis isolates (Fig. 3). These seven haplotypes could also be distinguished from each other using a combined data set of the three most variable loci (Scaffold 300, Scaffold 1635 and EF1α), or in any combined data sets where these three loci were included. We describe five new polymorphic intergenic loci and a variable part of the gene encoding EF1α that can be used to differentiate haplotypes of A. apis. Sequence analysis using 12 A. apis isolates, ten originating from Denmark and two from North America, Galunisertib demonstrated a high level of intraspecific variation at these loci. We detected no differences in the sequences of the ITS region among our A. apis isolates, which is congruent with the result reported by
Anderson et al. (1998), who used 23 A. apis isolates with origins that were even more widespread than our samples. The genetic heterogeneity among our ten Danish and the two North American A. apis isolates was surprisingly high, and within this small sample size, seven different haplotypes were detected. All seven could be differentiated by combining PLX-4720 manufacturer the three most variable loci: EF1α, Scaffold 300, and Scaffold 1635. In a study conducted including 84 South American and
two Japanese A. apis isolates, only five distinct types were found using a repetitive element PCR fingerprinting method with BOX, REP, and ERIC as random primers (Reynaldi et al., 2003). This could reflect a founder effect because honey bees are not native to America. The first scarce introduction of honey bees to this continent took place during the 4th Colon trip in 1536 at Santo Domingo MYO10 Island, and around a century later, a few colonies were introduced to South America, Uruguay and Brazil (Bierzychudek, 1979). The differences in the observed heterogeneity between South American and Danish isolates could, however, also reflect that our method is more effective at identifying haplotypes. Repetitive element DNA fingerprinting is a quick and cheap method, but the fragment patterns can be difficult to reproduce between laboratories (Deplano et al., 2000). Furthermore, such fingerprinting methods cannot handle complex biomasses in a cultivable independent manner, but requires in vitro isolation of the target organism. Our method should be more repeatable because of high primer specificity and could be applied directly to DNA extracted from field samples of diseased larvae, and similarly, direct processing of field samples is also possible with the microsatellite primers recently developed for A.