The main focus of this paper (Siddal et al. 2007) was to use various molecular techniques (discussed in detail later) as a means of identifying what Hirudo species are available commercially as medicinal leeches. While at least five species of medicinal leeches have been recognized, they are almost indistinguishable morphologically. Although historically there has been extensive use of leeches medicinally, only Hirudo medicinalis has been approved by the United States Food and Drug Administration (US FDA). The goals for determining the various species of Hirudo available commercially are as follows: maintaining accordance with US FDA regulations on these commercially available organisms; ensuring correct identification of H. medicinalis as a model organism for research; and extending conservation efforts to other species of Hirudo unknowingly used for medicinal purposes.
Thirteen individuals of both H. medicinalis and H. verbana were collected and identified by dorsal color patterning. Along with wild individuals, commercially available specimens were collected from four separate suppliers and from two labs where H. medicinalis is used as a model organism. Molecular data was provided via DNA barcoding and nuclear microsatellites. Microsatellite data can be used to distinguish between individuals of the same species and show genetic variablility between closely related taxa.
Wild and commercially available individuals were sampled with nine loci (HcA10, HvH08, Hm8, Hm10, Hm12, Hv351, HvT379, Hm1, and Hm2) to help in determination their species placement. Amplification of these loci was performed via polymerase chain reactions (PCRs) with various primer sets. Microsatellite data was also subjected to computational analysis of population structure in STRUCTURE. An analysis of molecular variance (AMOVA) was conducted with ARLEQUIN. Annealing temperatures varied across the different primers. DNA barcoding data was collected. DNA barcoding is another method to distinguish between individuals of closely related taxa, but instead of mapping across multiple sites in the genome like microsatellites, barcoding primers amplify one specific region of interest. In this case, researchers amplified cox1 in all specimens subjected to microsatellite analysis. Sequence data from the DNA barcoding was analyzed with PAUP* to distinguish the placement of commercially available Hirudo species in relation to wild H. medicinalis and H. verbana.
Species segregation of H. medicinalis and H. verbana was seen in four (Hm8, Hm12, HvA10, and HvH07) of the nine microsatellite data. Various degrees of allelic variability between and within species of wild individuals were demonstrated in the other five microsatellite loci. Population structure results placed individuals in species categories (with <98.5% confidence) concurrent with assignments based of dorsal coloring. The AMOVA conducted on...