INTRODUCTION:

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 time^1,2,3. 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 18^th and early 19^th 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 19^th 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 cluster^1,6.

MATERIALS AND METHODS:

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 container^7,8.

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 size^9,10.

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

Sequence	Primer
5′- GGTCAACAAATCATAAAGATATTGG-3′	Forward
5′- TAAACTTCAGGGTGACCAAAAAATCA-3′	Revers

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	Time	Temperature	Stage	No.
1	6 min.	95	Initial denaturation	1
35	1.30 min.	95	denaturation	2
	1.30 min.	50	Annealing	3
	2 min.	72	Extension	4
1	5 min.	72	Final extension	5

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.

RESULTS:

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.

Parameters	*Hirudo medicinalis*	*Hirudo verbena*
Sequence ID	KU 672396.1	MG 973413.1
Length	14729 (No. of Matches=1)	1761 (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*
Kingdom	Animilia	Animilia
Phylum	Annelida	Annelida
Class	Clitellate	Clitellate
Sub-class	Hirudinea	Hirudinea
Order	Arhynchobdellida	Hirudinida
Family	Hirudinidae	Hirudinidae
Genus	Hirudo	Hirudo
Species	Hirudo medicinalis	Hirudo verbena

DISCUSSION:

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 surgery^11,12,13. 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 species^1,6. This and some phylogenetic uncertainties triggered first molecular studies^1,17.

CONCLUSION:

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.

ACKNOWLEDGMENT:

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

REFERENCES:

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Received on 11.05.2021 Modified on 01.06.2021

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

DOI: 10.52711/0974-360X.2022.00187