4.2. Molecular methods and systematics

Molecular technology was first used in Tropilaelaps research in the 1990s to examine genetic variation between so-called T. clareae and T. koenigerum (Tangjingjai et al., 2003). It has since proved very useful to examine genetic variation within the genus and to help re-define known species and describe new species and new types within species (Anderson and Morgan, 2007).

DNA sequence obtained from a 538 base-pair fragment of Tropilaelaps mtDNA cox1 gene, as well as from the entire nuclear ITS1-5.8s-ITS2 genes, provides a useful means for identifying mites to the species level. The cox1 sequence is also useful for looking at genetic variation within a species.

Methods for extracting, amplifying and sequencing Tropilaelaps DNA are the same as those described for Varroa in Dietemann et al., (2013). The DNA primers used to amplify the cox1 and ITS1-5.8s-ITS2 gene regions are shown in Table 2.

Once a DNA sequence is obtained from a particular mite specimen, it is compared to other sequences of the same region that have been deposited in the GenBank database (Dietemann et al., 2013). The ITS region shows no genetic variation within a particular species of Tropilaelaps, whereas cox1 sequence shows from 1-4% variation within species and from 11-15% between species. Mites of particular species that vary in the cox1 gene sequence are referred to as ‘haplotypes’. The concept of ‘haplogroup’, as described for Varroa mites in Dietemann et al., (2013), has not been adopted for Tropilaelaps mites, as there is much more variation in the cox1 gene of Tropilaelaps than in Varroa (Anderson and Trueman, 2000).

Tropilaelaps mites can also be identified to the species level by digesting amplified fragments of their cox1 gene with a combination of the FauI BsrI BstYI and SwaI restriction enzymes, without the need for sequencing the fragments. Products produced from these digestions are visualized as bands in 2% agarose gels (Anderson and Morgan 2007). The results of using these restriction enzymes to digest the cox1 gene fragment of the 4 known species are shown in Fig. 7. In summary, Fau1 only digests coxI fragments obtained from T. koenigerum, BsrI only digests fragments from T. koenigerum and T. mercedesae (2 bands are produced from each species, but the smaller band produced from T. mercedesae is larger than that of T. koenigerum), BstYI only digests fragments from T. clareae, while SwaI only digests fragments from T. thaii.

As cox1 gene sequence can resolve Tropilaelaps mites to the species level it is useful in phylogenetic studies. Methods used to carry out phylogenic analyses on Tropilaelaps cox1 gene sequence are the same as those used for V. destructor and other species, described in Dietemann et al., (2013). A phylogenetic tree of all the currently known and published Tropilaelaps haplotypes is shown in Fig. 8.

Table 2. Forward (F) and reverse (R) primer sequences (and their names) used in Tropilaelaps research to amplify fragments (base pairs) of specific genes.

Gene region

Fragment size (bp)

 Primer sequences (5’-3’)

Primer name


















Fig 7. Mites can be identified by digesting fragments of their cox1 gene with FauI, BsrI, BstYI and SwaI restriction enzymes (labeled a–d respectively). The numbers 1-4 represent: T. koenigerum, T. mercedesae, T. clareae and Thaii respectively. M = 100 bp DNA Ladder. Photo: Denis Anderson.



Fig. 8. A phylogenetic tree of all the currently known and published haplotypes of Tropilaelaps. Photo Denis Anderson and John Roberts.