A Taxonomic Study comparing the two types of  Medicinal Leeches available in Iraq


Islam Khalid1, Najah Sobhi Nayyef2, Marwan M. Merkhan3*

1,3College of Pharmacy, University of Mosul, Mosul, Iraq.

2College of Science, University of Mosul, Mosul, Iraq.

3College of Pharmacy, Ninevah University, Mosul, Iraq.

*Corresponding Author E-mail: marwanmerkhan@uomosul.edu.iq



Medical leeches are ectoparasites invertebrates which are widely used in medical and surgical treatments because it was found since the ancient time that many diseases can be cured by medicinal leeches, then it was discovered that leech saliva contains anti-inflammatory, analgesic, antibacterial, anticoagulant, and even anticancer enzymes, also they found that medicinal leeches have a great role in treating diabetic ulcers. Tow samples of leeches were collected from two regions in Iraq from September to the end of December 2020. These samples were examined a live by dissecting microscope and then they were examined after fixing them in formaldehyde 5%. The samples of leeches that were used to analyze their genetic DNA were fixed and preserved in absolute ethanol. The classification process was done through a molecular diagnosis of samples of medical leeches by using polymerase chain reaction PCR and technique tracking nitrogenous bases in the nucleic acid chain, and the molecular diagnosis depending on Cytochrome oxidase subunit 1 (CO1), and then the result of PCR of medical leech samples was sent with the primers of the resulting bands and the gene sequences were read depending on the Genetic Analyser Device 3130 supplied by Japanese Hitachi company. Then the gene sequences were matched with gene sequences documented in the National Center Biotechnology Information NCBI. The results were analyzed depending on the BLAST program through which two types of medical leeches were detected Hirudo medicinalis and Hirudo verbena.


KEYWORDS: Leeches, Hirudo medicinalis, Hirudo verbena, PCR.




The French naturalist Lamarck published many papers on the classification of the lower animals and was the first scientist to introduce the term ‘‘biology’’. Additionally, Lamarck suggested the idea of organorganismic evolution. In1818 Lamarck coined the word Hirudinea and classified leech species known at that time1,2,3. The medicinal leeches are famous blood-sucking ectoparasite invertebrates that are used all over the world to treat many medical and surgical diseases. Its medical use was known in the Roman Empire, Galenus the famous physician mentioned this medical parasite in the second century.


In the 18th and early 19th century, 30-40 million leeches/year were used in France alone,75 million leeches/year were caught in Russia during the first half of the 19th century. Through nearly 2000 years of documented history, the medicinal leeches have received considerable attention in some aspects of human health, its habit of feeding (hematophagous) has been used for medical and surgical diseases for centuries2. It was found that medicinal leeches improve drainage of tissues after surgical implantation and stimulate venous circulation to work.


In such cases, hirudotherapy is very important in correcting venous congestion. It was discovered that the substances which are present in leech saliva play a great role in preventing thrombosis of blood and improving blood circulation4. Over time there has been an advance in the medical use of leeches both as a source of pharmacological substance or directly for treating diseases or in plastic surgery and dermatology5. The present study is aimed to determine the status and relationship between two morphologically different types of medical leeches. This study depends on molecular biology and is based on DNA sequences of a protein-coding mitochondrial gene (CO1), the mitochondrial 12S rRNA gene, and a variable part of the nuclear ribosomal gene cluster1,6.



Samples of medicinal leeches were collected from Khormal village in Sulaymania governorate and from Shaqlawa district in Erbil governorate. Each group of leeches sample was kept in a glass container which was filled up to its two-thirds with chlorine-free water, the glass container was covered with porous cloth for ventilation and the piece of cloth was attached to the mouth of the container with a rubber band, the water in the container was changed every 3-6 days depending on the amount of the leeches in the container7,8.


Primers were blasted using NCBI information bank and the species selected to be specific for leeches using accession number and the selection of the primer (table 1) from the bank were based on selected criteria of specific primer melting temperature and size9,10.


Table 1. Reverse and forward sequence of designed primer from NCBI blast.








Then the nucleic acids were extracted from these leeches to determine the species of the leeches in the above-mentioned regions. The extraction of the nucleic acid from medical leeches was done depending on the kit supplied by the Geneaid factory (Figure 1). Then the bands resulting from the reaction of PCR with agarose gel were extracted and purified to perform nucleotide sequence test depending on the analysis device supplied by Geneaid company. Molecular diagnosis of medical leeches parasites was done depending on cytochrome C Oxidase subunit 1(CO1). After that, the reaction tubes were put in a thermocycler device for multiplicative reaction (Figure 2). The thermal cycle on the sequencing machine was performed using a standard thermal cycle (Table 2)


Table 2. Standard thermal cycle used for melting the primer and sequencing on sequencing machine.

Cycle number






6 min.


Initial denaturation



1.30 min.




1.30 min.




2 min.





5 min.


Final extension


DNA sequencing analysis is the cornerstone in detecting and determining the new races of living organisms. In recent years the results of the DNA sequencing technique were very accurate in identifying the genetic mutations. The results of the reaction of PCR of leech samples with the primers of the resulting band were sent and then the genetic sequences were read depending on the genetic Analyzer Device supplied by the Japanese Hitachi company. The gene sequences were matched with gene sequences documented in the National Center of Biotechnology Information NCBI and the results were analyzed depending on the Blast program.



The technique of identifying the sequence of nucleotides of nucleic acid (DNA sequencing) is considered the base for determining the new races of living organisms and the area highly preserved for micro-organisms. Usually, the result of polymerase chain reaction (PCR) is used to identify the sequences of amplified segments to determine the genetic differences in them (Figure 2).



Figure 1. PCR result for isolated DNA of Leeches sample confirmation.


In recent years the results obtained from the DNA sequencing technique were very accurate in identifying the genetic mutations (Figure 2).


Figure 2. DNA sequencing results of Leeches samples.

On the other hand, if the result of PCR was containing more than one band then the required segment of gel agarose is purified and isolated, but if the result of PCR was specific for only one band then it is possible to depend on it directly in determining the sequences. The result of polymerase chain reaction PCR for medicinal leech samples was obtained depending on the 18r DNA primer and on the result of the 710bp reaction. Then the sequences of genes were matched with the documented gene sequences in the National Center of Biotechnology Information NCBI (Table 3).


Table 3. The results were analyzed depending on the NCBI-Blast program.


Hirudo medicinalis

Hirudo verbena

Sequence ID

KU 672396.1

MG 973413.1



(No. of Matches=1)


(No. of Matches=1)

Range (Gene Bank graphics)

8499 – 9142

1 - 815 


Based on the origin of the taxonomy of leeches the present study was reached the decision of the taxonomy as supplied in table 4.


Table 4. Taxonomy origin of leeches11.

Taxonomy Origin

Hirudo medicinalis

Hirudo verbena























Hirudo medicinalis

Hirudo verbena



The medicinal leeches which belong to the genus Hirudo are very famous invertebrates and were studied in detail both biologically and physiologically because they are widely used in medicine and surgery11,12,13. Additionally, they were studied from a historical, ecological, and genetical point of view14. Large numbers of medicinal leeches were obtained from lands and water ponds in the eighteenth and the beginning of the nineteenth century, but at the end of that period, the leeches became rare in many countries15.


The classical studies of taxonomy used to be based on the general shape, color, pattern, length of the body, the system of annulation, and the number of segments before and after the clitellum and the color of the dorsal and ventral surfaces11. This was done in the past years and before the advances in biology and before the appearance of molecular biology. The taxonomy or classification of any organism was based on the morphology of the organism and that is called classical taxonomy. Other types are based on the phylogenic relationship of the organism, which refers to the relative times in the past that species shared common ancestors, this is called modern taxonomy.


In classical taxonomy, only a few individuals of the organisms are studied, while in modern taxonomy a large number of individuals were studied. The taxonomy or classification of the organisms which depends on the molecular biology of the organism is more accurate than the taxonomy which depends on the morphology of the organisms16. The main external feature of the leeches in the two regions from which we collected the leeches was as mentioned below.


One group of these samples had similar colors and patterns of the dorsal and ventral surfaces. The basic external characters of these leeches were (i) The dorsal part had an articular pattern with two paramedian broad pale orange stripes; (ii) The ventral surface was greenish to yellow; (iii) Presence of a pair of black ventrolateral stripes.


According to these features and characters and after matching them with the information and photos of other leeches, then these leeches were suspected to be Hirudo verbena13. The external features of the group of the leeches in the other samples were as follows, (i) The dorsal surface was dark brown or black; (ii) The ventral surface was spotted; (iii) There were six longitudinal red or brown stripes in the dorsal surface.


When matching this information and characters with the information and photos of the leeches, we suspected that those were Hirudo medicinalis, and this suspicion should be confirmed or refuted by molecular diagnosis. They noticed later that the anatomical pictures and the above-mentioned characters are not reliable in determining the species1,6. This and some phylogenetic uncertainties triggered first molecular studies1,17.



The information which was obtained with the help of the National Center Biotechnology Information NCBI was analyzed by the BLAST program and by different methods, the results were taxonomic structures which proved that Hirudo medicinalis and Hirudo verbana are true distinct species. These two results confirmed the diagnosis which depended on the morphological features.



The authors are very grateful for the college of Science/University of Mosul.



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Received on 11.05.2021            Modified on 01.06.2021

Accepted on 15.06.2021           © RJPT All right reserved

Research J. Pharm.and Tech 2022; 15(3):1119-1122.

DOI: 10.52711/0974-360X.2022.00187