Biology

Of the 650 species of leech listed, Hirudo medicinalis is one of the most extensively studied. This annelid from the subclass Hirudinea has 2 suckers, one housing the mouth and its 3 jaws.

It lives mainly in the stagnant waters of marshes and ponds. When it comes to reproduction, the animal is a hermaphrodite. Inseminated leeches lay their eggs during the spring and summer months, outside the water, with cocoons containing as many as thirty young. Leeches mainly feed on the blood of mammals and can survive for long periods without eating.

When it bites its prey, the leech then feeds on the blood and at the same time injects numerous substances with a variety of properties.

Aware of the power of the leech, over the course of time people have associated with it virtues that sometimes border on the highly fanciful.

Background

In 1554, the naturalist Guillaume RONDELET presented what was probably one of the first descriptions of the different species of leeches in his “Natural History of Fishes”. In 1735, LINNAEUS produced the classification of Hirudo medicinalis and Hirudo sanguisuga in his work “Systema Naturae”. But it was in the 19^th century that numerous monographs were dedicated to leeches: VITET (1809, Treaty of Medical Leeches), MOQUIN-TANDON (1827, Monograph of the Hirudinea family), MARTIN (1847, Paper on the issue of leeches) and FERMOND (1854, Monograph of the medical leech).

At the beginning of the 20^th century, the French pharmacopoeia gave a detailed description of the pigmentary characteristics of the medical leech.

Etymology

The Latin word Hirudo is thought to come from hoero which means “I adhere to”, and sanguisuga from the word sanguis – blood – and suga –I suck.

The term Bdella comes from the Greek and gave its name to the order of Arhynchobdellida.

In the Bible, is appears under the name aluka and in some Arabic accounts under the name aleca. In English, the primitive meaning of leech is doctor. In the year 900, laece meant “doctor” or “leech”, probably demonstrating its use in medicine.

Classification

Of the 650 species of leech that exist, Hirudo medicinalis is one of the most extensively studied annelids and the most widely used species in the therapeutic setting. Its scientific name reflects its importance in medicine.

It belongs to :

• Branch of Annelids
• Class of Clitellata (have neither parapodia, nor setae)
• Order of Arhynchobdellida (presence of jaws but no proboscis)
• Sub-Class of Hirudinea
• Genus Hirudo

Morphology

The leech Hirudo medicinalis is a segmented worm that is dorso-ventrally flattened. The length of its body varies depending on whether it is observed extended or contracted in its “olive” form.

European medicinal leeches come in a large number of varieties.

Depending on their geographic origin, leeches have different pigmentations making it possible to differentiate between different varieties.

• The Hungarian leech comes from Hungarian and Yugoslavian marshlands. Its dorsal surface is brightly coloured with various shades: dark green, brick red, yellow and black.The median band of its dorsal surface is dark green, demarcated on each side by an evenly serrated, thin black line. Its ventral surface is green.
• French leeches originate from the Landes and Camargue regions.
• The Landes leech has a median emerald green band demarcated on each site by a thin, straight black line on its dorsal surface. Its ventral surface is light green and smooth to the touch, with two black stripes running along each edge .
• The Camargue leech has a yellowish-green longitudinal median band demarcated on each site by a black line on its dorsal surface. Its ventral surface is yellowish green and smooth to the touch with a straight black stripe.
• “Mongrel” leeches: inferior quality in which are found other leech species (Haemopis, Nephelis, Aulastomes)

Anatomy

The body of medicinal leeches is covered by a unilaminar epithelium composed of epithelial, sensory and mucous glandular cells. This epithelium is covered by a thin cuticle that is renewed every time the leech moults. This thin, transparent moult, revealing the segmented structure of leeches, can be readily observed in the storage jars of the animals.

Under the integument, the dermis is composed of muscle fibres organised into an external circular layer and a continuous internal longitudinal layer, giving leeches a very high level of contractility. As a result, the length of the leech varies greatly between its contracted “olive” form and its extended form when it is moving through water or on a support.

The coelom is very small and occupied by parenchymatous tissue: the coelenchyme. It consists of 2 longitudinal sinuses (one dorsal, one ventral) joined by thin transversal sinuses, where a red plasmatic fluid circulates.

The locomotor system

Each end of its body has a sucker :

• the anterior or oral sucker is the smallest.

This sucker, which is perforated by the mouth, has a conformation enabling it to exert suction on supports. Its musculature is not very powerful since it consists of a single circular fibre and a few longitudinal muscles.

• the posterior sucker is bigger

This much larger sucker is disc-shaped and non-perforated. It is essential for locomotion.

The digestive system

This is located in the dorsal position, apart from the mouth, which has a ventral position. It consists of 4 parts :

• The mouth, which opens at the base of the anterior sucker, has 3 half moon-shaped jaws, the free edge of which have numerous calcite teeth making the incision. These jaws cut through the skin of the leech’s prey, leaving a characteristic Y-shaped bite.
• The pharynx, equipped with powerful muscles, is used to suck and swallow blood. It evaginates into the wound to absorb the blood. Its wall contains numerous salivary glands.
• The stomach has 11 pairs of gastric caeca, with the longest of these in the posterior position. These structures, which are equipped with small valves, make it possible to hold the blood ingested, enabling the leech to store very large quantities of blood for long periods, thereby promoting the leech’s capacity to fast.
• The middle intestine is the active digestive zone and the short posterior intestine or rectum ends with the anus.

The respiratory system

There is no differentiated respiratory system. Leeches breathe directly through the epidermis.

The circulatory system

In Hirudo medicinalis, the circulatory system is composed of 4 blood vessels, including a ventral vessel attached to the nervous system, a dorsal vessel and 2 contractile lateral vessels joined by a network of fine capillaries. This system is devoid of a heart. The blood of Hirudo medicinalis contains leukocytes. It is coloured red by a chromoprotein.

The nervous system

The nervous system of the medicinal leech is formed by 2 cerebroid ganglions joined by a peri-oesophageal collar to a sub-oesophageal nerve mass, innervating the oral sucker, and composed by coalescence of the ganglions of the first 5 metameres (segments). The ventral nervous system, located within the ventral sinus, consists of 19 pairs of ganglions, with one per metamere.

The excretory system

Metamerised (segmented) and hypertrophic, the excretory system is composed of 17 pairs of nephridia opening at the integument by nephridiopores. Each nephridium represents an autonomous unit and includes a horseshoe-shaped gland composed of excretory tissue, extended by a short excretory duct leading to a bladder in the ventral position ending at the ventral pore.

The reproductive system

Leeches are hermaphrodites and therefore have both a male genital organ and a female genital organ.

The male genital system is formed of 9 pairs of testicles positioned on either side of the ventral nervous system (12^th and 20^th metamere). At the level of the tenth segment, the sperm ducts, which transport spermatozoids, form the epididymis then become symmetrical again to form an ejaculatory duct within the long filiform penis. The base of the penis has a prostate containing spermatozoids. The male pore is located between the 24^th and 25^th ring.

The female genital system (11^th metamere) consists of 2 globular ovaries, 2 oviducts, a uterus, the secretions of which form the shell of the eggs, and a vagina. The female pore is located between the 29^th and the 30^th ring.

Lifestyle/Behaviour

The medicinal leech is a blood-sucking aquatic animal. It lives in mostly stagnant (marshes, ponds) fresh water but can also be found in streams.

In natural pools, it moves around its environment either by swimming, using undulating movements, or by moving along the substrate using suckers. On the ground, it moves by affixing the posterior sucker and then extending its body to attach the anterior sucker. It then detaches the posterior disc and contracts on its point of support to move the posterior part of the body towards the anterior sucker before re-applying the posterior sucker.

In the natural environment, leeches are alerted to the presence of potential prey by water vibrations. They then swim towards the main source of the vibrations.

Although they have 5 pairs of eyes, vision does not appear to be the major component in the detection of prey. The eyes, or ocelli, simply appear to give them light sensitivity; when placed in a lit aquarium, they instinctively seek out dark places.

The other major stimulus for leeches is the temperature of the potential prey. A source of heat, such as a balloon filled with hot water, placed in the water of the pool attracts leeches to its surface. They probably detect warm-blooded vertebrates more easily therefore.

Touch, through their integument and suckers, is also an important component in contact with potential prey. In contact with certain substances, leeches have the capacity to retract, reducing their length by up to two thirds.

Hence the sensitivity of leeches to certain chemical substances has been identified. These substances include quinine, saccharin, chloral hydrate or odorous solvents. Some skin odours actually prevent them biting.

Leeches also appear to be sensitive to sounds.

Feeding

Medicinal leeches are reputed to be a strictly haematophagous (blood-sucking) species. Their preferred food is mammal blood. But young individuals can initially feed on insect larvae and small molluscs. They may also feed on amphibian eggs, amphibians themselves and fish but the weight gain of the leech appears to be relatively minimal in this case.

Wildlife living in natural pools do not only represent a potential source of food, since some insects actually represent a threat to leeches, either because they are capable of injuring them or because they feed on them. The leeches most at risk are probably the young ones. The predators are many: mayfly larvae with their pointed spine-formed jaws, Anisoptera larvae (such as dragonflies) which have spurs on their abdominal extremity and a mask with hooks, and Dysticidae larvae and adults with jaws equipped with hooks.

Once attached to their prey by their two suckers, the leech makes a three-cleft incision in the integument or skin using its 3 toothed jaws. The secretory content of the salivary cells is released when they bite. The mucus facilitates lubrication of the site and hydration of the blood and the hirudin injected promotes ingestion of the blood meal by preventing coagulation. The blood is sucked by means of rhythmic contractions of the posterior region of the pharynx.

Once it is replete, around 20 to 40 minutes later, the leech detaches itself from its host. At this point it is swollen with blood and moves with difficulty. It can ingest between 3 and 10 times its own weight in blood, i.e. an amount of 5 to 10 ml of blood for an average-sized leech.

Digestion of the blood meal is made possible by the presence of bacterial symbiotes, Aeromonas hydrophila and Pseudomonas hirudinis living in the digestive tract of the annelid. In the days following the blood meal, the blood contained in the diverticula of the stomach is concentrated by around 40% by the excretory system. The blood then undergoes very slow changes, in particular by progressive lysis of the erythrocytes involving 2 successive mechanisms: haemolysis and proteolysis. The haemoglobin is split into globin and haem; the globin is used by the leech’s metabolism and the haem is split into excreted iron and protoporphyrin. The digestion progress is extremely slow and takes several months. Once sated, leeches do not bite.

In the natural environment, access to prey is not always regular but the method of digestion of leeches makes them adapted to this lifestyle since they present an extraordinary resistance to fasting, being capable of fasting for 6 to 8 months at a time. Some leeches can even survive a fasting period of 18 months.

At Ricarimpex, for obvious health and safety reasons, blood feeding is performed in the laboratory only under controlled conditions; mammal blood – although the preferred food of leeches – has been replaced by poultry blood. The use of this avoids the risk of disease transmission, which is much higher between mammals. In any case, it is essential to use a controlled blood source (veterinary controls on slaughtered animals and analysis of the blood).

Reproduction

Since they have both male and female reproductive systems, leeches are hermaphrodites. However, self-fertilisation is not possible and mating of two individuals is therefore necessary for reproduction. Copulation takes place head-to-tail, with the penis of one of the individuals being inserted into the vagina of the other, where sperm is deposited.

As egg-laying approaches, leeches present bulging in the anterior part of the body, accompanied by colouring of the area, in a shade ranging from green to yellow, or even orange. Eggs are laid at the soonest around 1 month after fertilisation, outside water. The clitellum secretes a mucous ring, which the leech moves out of, leaving behind it a sort of mucous sleeve, which closes at both ends. From an initial “egg white” consistency, the cocoon rapidly takes on a relatively firm consistency with a shell that becomes spongy under the effect of humidity. The cocoon containing the fertilised eggs contains a nutritional albuminous liquid ensuring the development of the young leeches before they emerge into the external environment.

The ovoid cocoons, the size of which ranges from 2 to 3 cm in length and 1 to 1.5 cm in width, are deposited on the ground. After a minimum period of 3 weeks, the young perforate one end of the cocoon and reach the water as quickly as they can.

Reproduction begins in the spring time and finishes at the end of autumn. In clement climates, the reproductive period may be extended.

Active Substances

In the classic injury scenario (open wound, cut), coagulation or clot formation involves 2 processes that occur simultaneously to fill the vascular gap: platelet aggregation (primary haemostatis) and conversion of soluble fibrinogen into insoluble fibrinogen, as a result of a cascade of enzymatic reactions (plasma coagulation).

When leeches are feeding, it is essential for them to maintain the blood in a liquid state. It is necessary to prevent coagulation at the bite site, obstruction of the deep vessels and also to prevent an increase in blood mass in their digestive tract.

To ensure this, leeches secrete saliva containing a number of active substances, such as anticoagulants, platelet aggregation inhibitors, proteinase inhibitors, etc.

The salivary glands are composed of 3 cell populations that cover 2 sorts of secretions :

• A mucous secretion, the main role of which is mechanical, to lubricate the jaws but also to ensure hydration of the stored blood.
• A protein secretion ensuring diffusion, vasodilation, anticoagulation and digestion thanks to numerous active substances.

Anticoagulants

Hirudin

This is a peptide secreted by the leech’s salivary glands that is injected into the wound during sucking to prevent the blood coagulating.

It acts on two levels :

1. It inactivates thrombin by taking the place of its natural substrate : fibrinogen.
2. It also acts on factor Xa, which catalyses the conversion of prothrombin into thrombin. Hirudin has the capacity to significantly accelerate release of factor Xa from the epithelial cells. Under the action of hirudin, factor Xa is therefore dissolved in the plasma, where it is subject to the action of its inhibitors.

There are several variants of hirudin :

HV1, which comes from the body of the leech and has no antithrombin activity.

HV2, which comes from the head and has an antithrombin activity. Its molecular weight is 6.95 KDa.

This is the most studied active substances in leech extracts and has been produced using a recombinant process. However, recombinant hirudin is less active than natural hirudin. The major difference between recombinant hirudins and natural hirudin is the desulphated form of the tyrosine residue in position 63; these “desulphatohirudins” are 10 times less active than natural hirudin.

Platelet aggregation inhibitors

In plasma, platelets can aggregate under the influence of numerous substances, such as ADP, epinephrine, thrombin and collagen.

Leech saliva proves to be an effective platelet aggregation inhibitor. This property might explain the fact that leeches are capable of separating the “coagulation time” and the “bleeding time”. The characteristic anticoagulant effect of a leech bite is due to the hirudin secreted while the animal is feeding, although it has been demonstrated that hirudin was degraded after around 15 minutes, whereas bleeding persisted for several hours.

The bleeding is thought to be a result of inhibition of platelet functions.

Calin

This protein interferes directly with the platelet-collagen interaction but also with Von Willebrand factor and collagen. These 2 effects might contribute to inhibition of platelet adhesion.

Apyrase

This is a phosphohydrolase that hydrolyses ATP and ADP. It is a potent anti-platelet aggregant.

Collagenase

This enzyme splits the collagen chain. And collagen is involved in activation of platelet aggregation.

A prostaglandin

This substance acts like prostacyclin and its analogues and has an effect on platelet aggregation by preventing the attachment and diffusion of platelets on collagen and activating the adenyl-cyclase of the platelet membranes, thereby generating an anti-aggregant substance.

Proteinase inhibitors

Bdellin

This enzyme is an inhibitor of trypsin and chymotrypsin. Its action obstructs the action of hirudin on blood coagulation. There are 2 types: bdellin A and bdellin B.

Eglin

This is a lysosomial and bacterial proteinase inhibitor released during certain inflammatory processes, like chymotrypsin, elastase produced by human neutrophils, cathepsin G and other enzymes made by human/granulocytes. This enzyme can play a preventive role in pulmonary emphysema. The elastase/antielastase balance plays a critical role in maintaining the integrity of human pulmonary alveolar structures.

Kallikrein inhibitor

This is an inhibitor of the coagulation factors, kallikrein and factor XIIa, which play a role in the intrinsic coagulation process.

Proteinases

Destabilase

This acts like an isopeptidase i.e. it liquefies soluble fibrin by lysing the εいぷしろん-(γがんま glutamyl)-lysine bonds of fibrin stabilised by factor XIIIa in the presence of Ca2+. This enzyme therefore gives leeches the ability to lyse clots; this is therefore no longer simply an anticoagulant process but a fibrinolytic supply process.

Lipase and esterase

The salivary secretions of Hirudo medicinalis have a lipolytic capacity in order to play a digestive role with respect to the ingested blood. Two enzymes are responsible for this capacity: a lipase and a cholesterol-esterase.

Hyaluronidase

Leech extract presents a diffusion factor. The enzyme responsible for this activity is a strict endo-βべーた-glucuronidase with hyaluronic acid as the only substrate. This enzyme, hyaluronidase, degrades hyaluronic acid, thereby increasing the diffusion of all the active substances inoculated by the annelid’s bite.

A vasodilator substance

This is a substance similar to histamine that is thought to play a vasodilator role during sucking.

An anaesthetising substance

The fact that leech bites are practically painless suggests the presence of anaesthetising substances, although this has not been demonstrated.