Leech Speciation and Detection of Hirudin Gene among the Leech Population in Asejire Oyo State, Nigeria

Abstract Leeches are segmented, parasitic and sanguivorous worms of the family Hirudinae and phylum Annelida. Various ailments have been treated with them since ancient times. Recent evidence has established the presence of over 100 medicinally useful bioactive substances in their saliva. Based on the therapeutic properties of the substances, they have been used in different areas of medical care and most prominently in Plastic and Reconstructive Surgery. Wounds and injuries account for more than a third of hospital visits and as much as 9% annual mortality in Africa. Nigeria with her abundant leech population however still accounts for the bulk of the population living with conditions treatable with leech therapy. Hence, this study aimed at confirming the presence of Hirudin and identifying the species of Leeches collected from Asejire, Ibadan. Fifty Leeches were collected from Asejire, Ibadan, Oyo State. Ethical approval (AD 13/479/4346 B ) was obtained from the Oyo State ethical board, Identification was morphologically done using the Borda and Siddall classification criteria. DNA extraction and amplification for Hirudin gene was similarly done using PCR. The mitochondrial cytochrome oxidase subunit 1 (COI) and nuclear internal transcribed spacer-2 (ITS2) genes were sequenced using Sanger sequencing. Multiple Sequence Alignments were done using the MUSCLE alignment method and phylogenetic trees were constructed via MEGA-x software using the Kimura-2 parameter model and Maximum Likelihood method. The ITS2, COI and Hirudin genes were characterized at 517bp, 658bp and 226bp respectively. A BLAST search showed the COI gene in our local Leech species is most closely related to the Aliolimnatis michaelsenis with 94.50%, nucleotide similarity, and the ITS2 is similar to Poecilobdella (Hirudinaria) manillensis with a 97.18% nucleotide similarity. This study confirms the presence of Hirudin in the Leech species found in our locality thus their feasibility for use in Leech therapy. Taken together, the phylogenetic inferences showed the Leech Species in our population is a unique previously unidentified species. Further study is required to determine other active compounds in our unique species and how it can be harnessed for medical use.