MORPHOLOGICAL CHARACTERIZATION OF HEMOCYTES AND HISTOLOGICAL STRUCTURE OF THE HEMOPOIETIC TISSUE "WHITE BODY" OF THE CUTTLEFISH "SEPIA OFFICINALIS" (CEPHALOPOD:SEPIIDAE)

Safaa M. Ali 1 , Torkia A. Mohammed 1 , Ahmed M. Mandour 2 and Asmaa R. Abd EL-Malek 1 . 1. Departmentof Zoology, Faculty of Science, Assiut University, Egypt. 2. Department of Parasitology, Faculty of Medicine, Assiut University, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 04 October 2018 Final Accepted: 06 November 2018 Published: December 2018

380 shape), different positions (central or peripheral) and has one, two or three lobes (Fig. 2). The multinucleated cells have two or three nuclei with rounded or triangular shape (Fig. 3).

Granulocytes with basophilic cytoplasm and acidophilic granules (1% of total cells counted):
In this type the acidophilic granules are grouped in a peripheral position in the cytoplasm. It may have one bilobed peripheral nucleus (Fig. 5a, b) or two central nuclei (Fig.5c).

Agranulocytes (10% of total cells counted) have two sub-types:
Agranulocytes may have acidophilic or basophilic cytoplasm as follow: Agranulocytes with acidophilic cytoplasm (7% of total cells counted): they may be uninucleated cells (rounded, oval, Irregular or club-shaped cells with rounded or oval nucleus) (Fig. 6) or multinucleated cells with two or three nuclei (Fig.7).
Agranulocytes with basophilic cytoplasm (3% of total cells counted): they have different shape (rounded or oval). Their nuclei may be rounded or oval (Fig. 8).
One or more vacuoles may be present in the agranulocytes (Fig. 10a,b). Some hemocytes may contain or secrete refractile material (Fig. 10 c-f) or may contain inclusion bodies (Fig. 10g, h). Cell fragments were recorded as in figures 6f and 8a.
The previous results revealed that the predominant hemocyte type is the granulocytes that have acidophilic granules and cytoplasm especially those with kidney shape nucleus.

Histological structure of the white body (the hematopoietic organ):
The white body is amultilobed organ, with white color, located in the orbital pit behind the eye (Fig.11) Examination of serial sections of the white body by the light microscope showed that it consists of several lobes which are separated anteriorly and gradually interconnected posteriorly (Fig.12). The white body is enclosed in a thin layer of connective tissue capsule which is composed of collagenous fibers (Fig. 13a). Histologically there are two types of lobes. The first type consists of sex layers (Fig. 13b); the first layer is supported by a net of connective tissue fibers which enclosed the hemocytes within it (Fig. 13c, d). The second and the fourth layers are composed of compact layer of connective tissue (Fig. 13e, f). The third and fifth layers are composed of patches of supportive connective tissue fibers where hemocytes are attached (Fig. 13b, e, f). The sixth layer lines the lobe and is composed of connective tissue fibers which have a reticular appearance (Fig. 13b).
The second type is composed of clusters of hemocytes and few connective tissue fibers (Fig. 14a, b).
Examination of the hemocytes that located in the white body by the transmission electron microscope showed that hemocytes may be granulated or agranulated and may have oval, rounded or kidney shaped nucleus. The hemocyte that contain oval shape nucleus is the predominant type, while hemocytes with kidney shaped nucleus are very rare in the white body (Fig. 14).        In the present study the main criteria which used for the classification of hemocytes are: the presence or absence of cytoplasmic granules, the ability of the cytoplasm and granules (if the latter is present) to be stained by acidophilic or basophilic stains, the shape of the cell and the nucleus and the ability to form pseudopodia. 3. In the present work hemocytes of Sepia officinalis were studied and classified into three main types: Hyalinocytes, granulocytes and agranulocytes. Granulocytes with kidney shape nucleus are the predominant cell type. Hyalinocytes may be granulated but differ from granulocytes in that they have a very small amount of 390 cytoplasm. Both granules and cytoplasm may be acidophilic or basophilic. Cell fragments were recorded in the present study. 4. Malek and Cheng (1974) reported that molluscanhemocytes can be distinguished into agranular cells and large granular cells. The latter can be classified according to their granules into granulocytes with acidophilic, basophilic, refractile granules or the three types of granules may be present in the same cell. Also in the present study some hemocytes which contain or secrete refractile material were recorded. The first cell type was large and had many granules. They reported that this cell type was rounded, with an abundant cytoplasm, u-shaped, eccentric nucleus, and has many pseudopodia; the second cell type was lesser in number, size, and granulation, had fewer pseudopodia, irregular in shape and had a rounded nucleus. 11. However, three hemocyte types were recorded in the study of the blood cells of Octopus vulgaris; hyalinocytes, granulocytes and haemoblast-like cells (Salazar et al., 2015). 12. The present study agrees with Salazar et al. (2015) in the presence of more than one hemocytes type which differs in their shape, presence or absence of cytoplasmic inclusions and shape of their nuclei but differ from that of Castellanos-Martinez et al. (2014) in that the number of cells that produce pseudopodia is very small. 13. Troncone et al. (2014) identified three hemocyte types in octopus; granulocytes, hyalinocytes and haemoblastlike cells. The granulocytes are able to extend pseudopodia. Hyalinocytes are round to ovoid in shape and might contain granules in their cytoplasm. Haemoblast-like cells have a small size. 14. Phagocytosis is the most important defense mechanism in invertebrates. Hemocytes of mollusks are known to efficiently phagocytose foreign material such as bacteria and cell debris (Malek and Cheng 1974). In the present study few phagocytic cells were recorded. 15. In the present work, two types of pseudopodia: blunt pseudopodia and filopodia could be formed in agranulocytes. Also one or more vacuoles may be present in this type of hemocytes. This result agrees with Helal and Abd El-Maksoud (1999) who mentioned that infected Limnaea natalensis snails possess hemocytes with many inclusions and produce many long, often branched, pseudopodia. 16. The result of Sminia (1981) revealed that granulocytes and hyalinocytes are phagocytic, but granulocytes were found to be much more active. Malek and Cheng (1974) revealed that all molluscan haemolymph cells are capable of pseudopodia formation. 17. In squid and octopus, the white body is considered to be an immune organ mainly due to the fact that blood cells, or hemocytes, are known to be present in high numbers and in different developmental stages. Hence, the white body has been described as the site of hematopoiesis in cephalopods (Salazar et al., 2015). 18. The present study and Claes (1996) found that the white bodies of Sepia officinalis are located in the optic sinuses. They are white in color. Salazar et al,.

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(2015) described the white body as a multilobed organ and also the present study and the study of Cowden and Curtis (1974) showed that each white body is consisted of several interconnected lobes. 19. Bolognari (1951) revealed that the white bodies of many European species of cephalopod are composed of two lobes. Claes (1996) found that the white bodies of Sepia officinalis are composed of two main lobes of different size. Both lobes are divided into several secondary lobes and a large number of small lobules were recorded they give a glandular appearance to the organ. 391 20. In the present study two types of lobes with different histological structure were recorded. One of these lobes consists of histologically different layers which suggested that each layer perform different function as explained by Claes (1996). 21. In the present work the white body enveloped by a thin fibrous connective tissue capsule. This result agrees with Bolognari (1951) as he reported that the white bodies of several cephalopod species are covered with a thin layer of connective tissue. 22. The present work studied the different types of circulating hemocytes and also deals with the histological structure of each layer of the hematopoietic organ (white body) of Sepia officinalis. 23. Two major haemocyte developmental stages have been identified in the white body of O. vulgaris, O.
Haemocytoblasts are found in the reticulum of the white body's lobes, and give rise to leukoblasts, which are characterized by a reduced cytoplasmic volume and nuclear size compared to their progenitors (Ford 1992).