Light Microscopy and Ultrastructure of Body Wall in Leech Haemadipsa zeylanica

Abstract

Background Information: 

Leeches are widely distributed worldwide in a diversity of habitats, such as freshwater, seas, desert, and oases. Since literature does not have an elaborate study on histology of leeches, thus emphasis is given to study the histology.

Aim: 

The aim of this study is to examine the histology of free-living leeches Haemadipsazeylanica under light microscopy and extended to ultrastructural study under transmission electron microscopy.

Materials and Methods: 

Tissue was fixed in 10% formalin, dehydrated with alcohol, infiltrated and embedded in wax, and thin section was cut using a rotary microtome. Sections were stained with hematoxylin and eosin.

Results: 

The epithelial layer of cells in H. zeylanica had a thin layer of cuticles covering it. In light microscopy, the epidermis has three types of epidermal cells, namely, glandular cells (Type I, Type II, and Type III), supporting cells (Type IV), and pigment cells (Type V). When the three glandular cells of the body wall of H. zeylanica epidermis were examined under an electron microscope, three types of distinct electron-dense granules were identified. Type I cells with granules, Type II cells with course granules close to the dermis were identified as pear-shaped secretory cells, and Type III cells with electron-dense granules.

INTRODUCTION

Leeches are parasitic Annelida organisms found on land, in freshwater, and in the sea. There are about 700 species of leeches now recognized, with around 100 being marine, 90 being terrestrial, and the remainder being freshwater. Taxonomy and ecological diversity of leeches have been studied insufficiently and unsystematically. To investigate their existence, the biological description and ecology of different leeches, from various habitats, preliminary information is essential. They live on stones, submerged wood, beneath the rocks and aquatic vegetation in ponds, streams, and rivers, and clinging to vegetation. In recent years, some leech populations have declined dramatically due to overexploitation for fishing bait and medicinal purposes (particularly in Europe and Asia), and due to pollution. Leeches are an important part of aquatic biota not only as an element at the trophic level but also as parasites of other hydrobionts. Literature on description of the biology and ecology of six Hirudinea species is lacking.^[1] Even though Hirudinea species had significant importance in the field of medicine. Its interest increased in recent years. Due to its possible relationship to the transmission of bacterial and viral infections, it is considered to be pathogenic to organisms. Moreover, hemorrhage and inflammation associated with leech attachment sites weaken the host undoubtedly and may predispose hosts to bacterial infections. Many species of leeches are ectoparasites of invertebrates and vertebrates.

In several coastal habitats of tropical and subtropical oceans, Hirudinean of the family Piscicolidae are well known as marine leeches StibarobdellamooreiandStibarobdellamacrothela on sharks and rays have been recorded in Brazil,^[2,3] some other species of leeches Hemiclepsismarginata,^[4]Cryptobranchusmastacembelus,^[5]Limnatisnilotica,^[6]Cystobranchusmammillatus, and Fadejew obdella quinqueannulata,^[7] from Iraq, two new Helobdella species HelobdellastagnalisandHorkeliacalifornica (Annelida, Hirudinida, Glossiphoniidae) from the Intermountain region of the United States^[8] have been documented. Mandal CK, Chandra M, and Ghosh GC have considerably contributed toward the taxonomy of leeches of India.^[9–11] The majority of the new species of leeches were described by^[12,13] new Rhynchobdellida leech and^[14] three new species from West Bengal, Paraclepsisgardensi, Placobdellaharasundarai (Hirudinea: Glossiphonidae), and Haemadipsaanaigundiensis sp. (Hirudinea: Haemadipsidae).^[15–17] Two species Haemadipsaanaigundiensis sp. and Haemadipsakodairensis recorded from Tamil Nadu have been described.^[18,19] A new species Paraclepsisjorapariensis sp. from, Jharkhand, India was described.^[20]

There have been studies on the histology of the body wall of Erpobdellaoctoculata and Haemopissanguisuga.^[21] Significant studies have been carried out on the ultrastructure of transient nephridia in E. octoculata^[22] and midgut in juvenile and adult Piscicolageometra was investigated in relation to digestive activities.^[23] The leech fauna of India is poorly documented, except very few reports on their existence. Moreover, leech species H. zeylanica from the mountainous terrain of Western Ghats of Matheran region which has dense evergreen forest receiving heavy rainfall of an average of 30–80 inches has not been detailed. Since there is scarcity of literature on histology of leeches, and moreover, this aspect has not been explored in H. zeylanica before, thus the present article focuses on the histology of H. zeylanica light microscopy and transmission electron microscopy (TEM).

MATERIALS AND METHODS

Leeches were collected during the monsoons during July 2017–September 2017 from the Matheran Raigad district (18.98°N, 73.27°E). They were found attached to pebbles and stones. Samples were picked up by forceps and transferred to sample bottles in live condition. Acquired permission from National Biodiversity Board, Nagpur for handling specimens for biodiversity. No human subject were used for trial.

Leeches were treated gradually with 10% alcohol to inhibit movement, relax the specimen, and identification of specimens were carried out based on coloration and morphological characteristics which were compared with the key and literature as referenced.^[9,10,24]

Light microscopy

Tissue was fixed in 10% formalin for 48 h, thoroughly washed in water and dehydrated with alcohol, infiltrated and embedded in wax, and ribbons were cut at a thickness of 5 microns using a rotary microtome. Sections were stained with hematoxylin and eosin.

Transmission electron microscopy

For TEM, the tissue was fixed in 3% Glutaraldehyde, washed three times in 0.1M sodium cacodylate buffer, and fixed in 1% Osmium tetroxide. Dehydrated through grades of ethanol and embedded in Araldite mixture blocks. Ultrathin sectioning was cut using Transmission Electron Microscope, JEM 1400 Plus, JEOL (Japan) at 120 kV, Electron Microscope Facility, ACTREC.

RESULTS

In a relaxed state, Haemadipsazeylanica measured 2.7 cm in length and 0.6 cm in width. The anterior sucker is 2 mm in diameter, whereas the posterior sucker is 4 mm in diameter. An arch of five pairs of eyes can be seen anterodorsally. On the second somite, the first pair of eyes is mid-dorsal, followed by the second pair on the third somite, the third pair on the fourth somite, the fourth pair on the first and second annuli of the fifth segment, and the fifth pair on the second annuli of the sixth somite [Figure 1].

Figure 1: Anterior end bearing five pairs of eyes in Haemadipsa zeylanica

Light microscopy

A transverse section of H. zeylanica’s body wall region was examined under microscope which reveals the inner epidermis made up of a single layer of thin connective tissue, where the pigment cells are located and lined by the thin cuticle. Whereas the dermis comprises comparatively thin outer layer of circular muscle and oblique muscles, dense inner layer of longitudinal muscles. Between the muscles, the cluster of cells known as botryoidal tissue was observed. Several slender, thread-like radial muscles extending from the body wall to the crop region of H. zeylanica, which is medially located, were observed, along with numerous folds [Figure 2a]. Three types of cells were observed. Glandular cells were differentially granulated and vacuolated with duct and without duct, one type of supporting cells, and one type of pigment cells [Figure 2b]. Depending on the presence of granules, the cells were classified as follows.

Figure 2: (a) Microphotograph showing cross-section of body wall through crop in Haemadipsa zeylanica circular muscle (cm), oblique muscle (om), botryoid tissue (bo), microvillia (mv), haemocolic cavity (hc), sinusoid (d) (HE staining) (magnification ×400). (b) Microphotograph showing cross-section of body wall through crop in Haemadipsa zeylanica pear-shaped cell (p), glandular gland (g), cuticle (cu), pigment cell (pc), large glandular gland (Lg), epithelial cell (ep) (HE staining) (magnification ×150)

Type I (large glandular cells)

Large oval-shaped glandular cells are found slightly beneath the epithelial surface. Their apical regions extend to the epithelial surface, whereas their basal regions are found in the coelomic lacunae’s superficial epithelial layer [Figure 2b].

Type II cells (pear-shaped cells)

These glandular cells have a large base with a thin, long, and narrow duct connecting it to the surface. Granulation generated large, vacuoles in the basal regions [Figure 2b].

Type III cells

These small glandular cells appeared morphologically identical to Type II cells which are asymmetric granules with dense appearance [Figure 2b].

Type IV cells (supporting cells)

These are elongated, oval cells with round basal nuclei, interspersed between the epidermal layer [Figure 2b].

Type V (pigment cells)

Is an additional cell type which is similar to supporting cells and contains a deeply-stained substance [Figure 2b].

Ultrastructure

Ultrathin sections of the body wall under TEM illustrate different types of granules inside glandular cells. The zonula adherens connect adjacent glandular cells [Figure 3a], Nucleus is heterochromatin and small folds in the basal membrane represent the basal lamina [Figure 4], which in some invade the perinuclear area. Golgi complexes, rough endoplasmic reticulum, and mitochondrial cisterns are abundant in the area near the nucleus. Free ribosomes are abundant throughout the cytoplasm [Figures 5 and 6]. Microvilli were observed in the cuticle and inner lining of crop [Figures 3 and 7]. The interior regions of the body wall, particularly around the muscles, are home to Types II and III cells. The glandular cells in H. zeylanica were differentiated based on variations in granulation [Figure 3b].

Figure 3: Electron microphotograph of body wall showing microvilli (mv) (magnification ×10000). (a) Electron microphotograph of showing Type-I, Type-III, Type-IV cells, and zonula adherens (za) (magnification ×1500). (b) Electron microphotograph of body wall showing Type-I with electron-dense granule (*), and less electron-dense granules and with vacuoles (arrow) (magnification ×3000). (c) Electron microphotograph showing small glandular (sg) cells with electron dense granules (*), Type II pear-shaped cells (p) with duct (arrow) and without duct (magnification ×3000). (d) Electron microphotograph showing a pear-shaped cell with large vacuoles (arrow) with less granulation (magnification ×5000). (e) Electron microphotograph showing a pear-shaped glandular cell with duct (arrow) with less dense granules and vacuoles (magnification ×5000)

Figure 4: Electron photomicrograph showing basal lamina (bl) (magnification ×10000)

Figure 5: Electron photomicrograph showing a glandular cell with nucleus (n), mitochondria (m), rough endoplasmic reticulum (rer), and free ribosomes (r) (magnification ×10000)

Figure 6: Electron microphotograph showing microvilli (mv) in the inner lining of the crop region and supporting cell (su) with small and large granules (magnification ×8000)

Figure 7: Electron photomicrograph showing supporting cell with small and large granules (arrow), glandular cell (g) in between the muscle (mu) with electron-dense granules (magnification ×5000)

Type I (Glandular cells)

The electron microscope displays huge glandular cells with electron-dense giant granules and a membrane that appears to be generated by the fusion of tiny granules [Figure 3b].

Type II (Pear-shaped cells)

Are electrons dense, with small-to-large granules, and are connected to the surface by a duct. Between the basal areas, single and sparsely dispersed tiny cells were detected [Figure 3c]. The small pear-shaped glandular cell appears to be of two types one with electron-dense granular cell and vacuoles and the second with less granulation. Large vacuoles with less granulation were observed [Figure 3d and e].

Type III cells

These glandular cells are small which are mostly of equal electron-dense granules [Figure 3a and 6,7].

Type IV cells (supporting cells)

Vesicles are asymmetric, electron-dense, lucid, and vacuolated [Figures 3a, 7]. They are more prominent in the muscle tissue.

DISCUSSION

In the present investigation, H. zeylanica has five pairs of eyes which can be seen anterodorsally,^[24] the cuticle covering the epidermis was thin layer. Our findings are consistent with observation in two species of leeches, E. octoculata and H. sanguisuga.^[21] However, it differed from those described in lumbricoides worm clitellates as thick layer epidermis.^[23] In H. zeylanica, the epidermis has a variety of cells beneath it, epidermal supporting cells, small and large glandular cells, pear-shaped cells, and pigment cells. Similar findings were reported in the epidermis of an aquatic leech who discovered four types of secretory cells in the epidermis of an aquatic leech.^[21,25,26] Three types of glandular cells and three types of nongranular cells were reported in the epidermis of lumbricid worms (Annelida, Clitellata).^[23] Furthermore, the dermis was discovered to have multiple body wall muscles, which are critical for aquatic leeches’ motility and swimming activity.^[27] The present study circular muscles, oblique muscles, and longitudinal muscles were reported in H. zeylanica, similar findings were reported by earlier author.^{[8,21,28–30]}

In the present finding glandular cells, supporting cells, and pigment cells were reported which are in accordance with the findings made by earlier authors.^[8,21,26] Small glandular cells were found throughout the leech epidermis, including Type I large glandular cells, which were distinguished from other glandular cells by the presence of dense granules. Type II cells or pear-shaped with long tubule neck that reaches the integument’s surface and contains course granules.^{[8,21,26,31,32]} Type III cells were granular cells found behind smaller cells and contain mostly two types of granules. Larger granules appeared to be produced by the merger of smaller granules in empty cells.^[21,33] Type IV supporting cells reveal asymmetric vesicles with dense granules, lucid, and vacuolated. In the present finding, it was observed that the Type IV cells were prominent in between the muscle tissue.^[8,23] Other cell types appear to have minimal roles to play.^[21,26]H. zeylanica’s revealed five different types of epidermal cells in the body wall which are concurrent to those reported in E. octoculata and H. sanguisuga,^[21]W. pigra^[30] and S. moorei.^[2]

Further investigation under TEM showed that the glandular cells of H. zeylanica’sepidermis revealed three unique types of electron-dense granules: Type I cells with electron-dense granulation and less electron-dense, Type II pear-shaped cells with coarse granules with duct and without duct with large vacuoles, Type III small glandular cell with large and small electron-dense granules, and Type IV supporting cells small dense granulation and vacuolated. Similar findings have been made in the epidermis of Oligochaeta worms, as well as E. octoculata, H. sanguisuga, and aquatic leeches.^[21,23] In H. zeylanica’s, microvilli were observed in the cuticle and inner lining of the crop.^[23]

CONCLUSION

Thus, in the present study Haemadipsa revealed five types of epidermal cells under light microscopy. The glandular cells of Haemadipsa epidermis when examined with an electron microscope showed three types of distinct electron-dense granules: Type I cells characterized by membrane-bound granules, Type II containing coarse granules, and Type III and Type IV having electron-dense granules. Histological studies can be carried out to compare leeches from different habitats and further molecular characterization can be done to study the phylogeny.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.