Development and Characterization of 18 Microsatellite Markers for the Asian Buffalo Leech Hirudinaria manillensis (Lesson, 1842) and Cross-Species Amplification with Its Congeners

Abstract

The Asian buffalo leech Hirudinaria manillensis is an obligatory ectoparasite freshwater leech, feeding on blood of vertebrates. Its natural populations have been threatened by habitat destruction and overexploitation for traditional medicine. In this study, we developed 18 species-specific microsatellite markers for H. manillensis using a next-generation sequencing approach. Microsatellite polymorphisms were screened in 30 individuals collected from four localities in Thailand. Number of alleles per locus was relatively high, ranging from 5 to 18, with an average of 9.94. The observed and expected heterozygosity values ranged from 0.200 to 0.900, and 0.567 to 0.932, respectively. The polymorphism information content (PIC) varied from 0.509 to 0.910. Ten out of the 18 loci showed significant deviation from Hardy-Weinberg Equilibrium. This might be caused by the Wahlund effect and small sample size. Null allele frequencies ranged between 0.000 and 0.396. There was no significant linkage disequilibrium between pairs of loci after a sequential Bonferroni correction. These markers were also successfully cross-species amplified in all of its congeners (H. javanica, H. bpling and H. thailandica), with the amplification success rate between 56–89% of the loci used. These highly polymorphic loci will be useful for further population genetics study of H. manillensis and its congeners.