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Morphological and Structural Characteristics of the Hemocytes of the Anodonta cygnea



S. Jamili, L. Salimi, A. Motalebi and M. Rostami-Beshman
 
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ABSTRACT

The Anodont (Anodonta cygnea) constitutes one of the most important bivalves along of Anzali Lagoon. In last decade, Anodont have suffered a high degree of mortality. Introduce of the morphological characterization of hemocytes is a prerequisite to further exploring the causes of death in bivalves, therefore in this study, the circulating hemocytes of the Anodonta cygnea in Anzali Lagoon were identified. At first, two types of hemocytes were recognized, granulocytes and agranulocytes, were identified based on the existence of cytoplasmic granules under light microscopy. The hemocytes were then stained and the granulocytes subclassifed into eosinophilic and basophilic granulocytes and an intermix. The eosinophilic granulocytes were distinctive small and large granules. Agranulocytes could be subdivided into hyalinocytes and blast-like cells and another cell type, vesicular cells, was observed as unclassified cells. Results were compared with similar researches about other bivalves observations.

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  How to cite this article:

S. Jamili, L. Salimi, A. Motalebi and M. Rostami-Beshman, 2009. Morphological and Structural Characteristics of the Hemocytes of the Anodonta cygnea. Research Journal of Environmental Sciences, 3: 218-224.

DOI: 10.3923/rjes.2009.218.224

URL: https://scialert.net/abstract/?doi=rjes.2009.218.224
 

INTRODUCTION

Hemocytes of bivalve molluscs play an important and central role in the internal defense and are known to be involved in other processes like wound and shell repair, nutrient digestion, transport and excretion (Cheng, 1981). There have been many studies on the morphology, structure, function and classification of hemocytes in bivalves. The most important reviews of the various morphofunctional aspects of the hemocytes of the whole Mollusca phylum are those of Cheng (1981) and Hine (1999), who identified 2 fundamental hemocyte types in bivalve hemolymph: granulocytes and hyalinocytes (or agranulocytes). The presence of these two types was confirmed in Mya arenaria (Huffman and Tripp, 1982), Mytilus edulis (Pipe et al., 1997), Mytilus galloprovincialis (Cajaraville and Pal, 1995; Carballal et al., 1997), Mercenaria mercenaria (Tripp, 1992), Crassostrea virginica (Ford et al., 1994) and Ruditapes decussatus (Lopez et al., 1997). However, the classification schedules were so varied that three, four or even more morphologically different populations have been proposed by authors for various bivalve species (Moore and Lowe, 1977; Chang et al., 2005; Hine 1999; Nakayama et al., 1997). Three types of agranulocytes have been identified: blast-like cells, basophilic macrophage-like cells and hyalinocytes (Hine, 1999). On the other hand, granulocytes may be subdivided into neutrophils, acidophils and basophils (Cheng, 1981; McCormick-Ray and Howard, 1991; Lopez et al., 1997). Nevertheless, different opinions concerning these classifications of hemocytes in bivalves exist due to different techniques used. For example, although the hemocytes of the oyster have been studied for a long period of time, types of subpopulations and their morphofunctions are still controversial (Sun and Wu, 2004; Chang et al., 2005; Bachere et al., 1988; Adema et al., 1994).

Image for - Morphological and Structural Characteristics of the Hemocytes of 
        the Anodonta cygnea
Fig. 1: Anzali Lagoon one of international Lagoonis in North of Iran

The Anodont (Anodonta cygnea) constitutes one of the most important bivalves along the of Anzali Lagoon. In last decade, Anodont have suffered a high degree of mortality. Several factors have been implicated as possible causes of this mass mortality, such as variations in temperature and salinity, industrial and pesticide pollution and so on. Since bivalve hemocytes play an important role in homeostatic functions and defense mechanisms, the morphological characterization of hemocytes is a prerequisite to further exploring the causes of death in bivalves (Cheng, 1981; Fisher, 1985). The number and constitutes of hemocytes, is believed to be an essential parameter for characterizing the immune capacity in bivalves. Hence, morphological characterization and subtyping of Anodont hemocytes were established via light and in the present study.

Anzali Lagoon (37 °28` N, 49`°25` E) is one of the international Lagoon in Iran and this Lagoon is one of the most important habitant for fishes, bivalves and birds in the South of the Caspian Sea (Fig. 1).

MATERIALS AND METHODS

Experimental Animals
The experimental bivalves (Anodonta cygnea) used in this study were collected from the Inland Waters Aquaculture Research Center, Anzali Lagoon in the North of Iran in August 2007. Before the experiments took place, all of the bivalves were kept in aerated tanks for 14 to 20 day, where fresh water temperature was maintained at 25±1°C.

Hemolymph Collection
Hemolymph was collected from the posterior adductor muscle sinus of Anodont (Fig. 2), using a sterile syringe with a 25-gauge needle (Lowe and Pipe, 1994).

Observation of Live Hemocytes
The collected hemolymph was placed into 6 well plates and permitted to settle at room temperature for 10~20 min. Live hemocytes attached themselves to the surface, therefore nonadherent cells were carefully washed off with sterile freshwater. The adhering hemocytes were further examined under an light microscope (Nikon).

Light Microscopy
Hemolymph from the experimental animals was placed onto glass slides and cells were allowed to settle for 10~20 min at room temperature. After light washing, the adherent cells were fixed with ethanol (96%). The slides were then carefully washed with water and stained with May-Grünwald Giemsa stain. Following this, observations were carried out with an light microscope

Image for - Morphological and Structural Characteristics of the Hemocytes of 
        the Anodonta cygnea
Fig. 2: Hemolymph was collected from the posterior adductor muscle sinus of Anodont

RESULTS AND DISCUSSION

Light Microscopy
In the stained hemocyte monolayers from bivalves, the amount of agranulocytes of the total hemocyte were very much for granulocytes and granulocytes were further subclassifed into eosinophilic and basophilic granulocytes (Fig. 3).

Eosinophilic granulocytes were observed; moreover, two distinctive sizes of granules were recognized. Small eosinophilic granulocytes contained many small granules which stained light pink. The large eosinophilic granulocytes had larger deeply pink stained granules.

Agranulocytes generally devoid of granulations, were divided into hyalinocytes and blast-like cells (Fig. 4). Hyalinocytes were larger with slightly basophilic cytoplasm. Flattened hyalinocytes, spread by extending pseudopodia, were observed in the smears. Blastlike cells were relatively smaller, round and basophilic in appearance and possessed a high nucleus: cytoplasm ratio.

Another cell type, vesicular cells, was observed. These cells had eccentric nuclei and prominent ectoplasm, similar to those of the granulocytes, but they had vacuolated cytoplasm and lacked cytoplasmic granules, which made these cells distinctive (Fig. 5).

Morphological criteria were considered generally in order to characterize hemocytes in bivalves, however, the existing nomenclature of bivalve hemocytes is inconsistent, being dependent on the observer and the technique used (Chang et al., 2005; Sun and Wu, 2004; Ford et al., 1994; Cheng, 1975, 1981; Moore and Lowe, 1977; Rasmussen et al., 1985; Zhang et al., 2005). Cheng (1981) presented a morphological scheme based on numbers of cytoplasmic granules, dividing cells into two types: granulocytes, cells containing granules which rang from very few to numerous and agranulocytes, cells containing few or no granules.

In this study, we identified main hemocyte type as well in Anodonta cygena, under light microscopy: granulocytes and agranulocytes. In addition, vesicular cells were observed as unclassified cells. The granulocytes were characterized by the abundant content of granules, presenting a noncentral small nucleus, spreading with pseudopodia related with the phagocytic abilities. According to the cell size, agranulocytes appeared to have two types: small hyalinocytes and large hyalinocytes. The large hyalinocytes were morphologically characterized by the relatively large and central nuclei surrounded by the small volume of cytoplasm and by few or no cytoplasmic granules. The small hyalinocytes have the least cell diameter and large and central nuclei surrounded by the small volume of cytoplasm with the absence of cytoplasmic granules. Many authors suggested that the hyalinocytes were non-different cells; however, hyalinocytes were classed into 2 types, small and large hyalinocytes, by Xue et al. (2000), which supported our classification. Some studies suggested that it was possible to distinguish acidophilic and basophilic granulocytes according to the staining affinities of the cytoplasmic granules of the granulocytes (Cheng, 1981; Nakayama et al., 1997; Lopez et al., 1997; Park et al., 2002; Zhang et al., 2005).

Image for - Morphological and Structural Characteristics of the Hemocytes of 
        the Anodonta cygnea
Fig. 3: Light micrographs of hemocytes stained with May-Grünwald Giemsa stain. (a) large eosinophilic granulocyte (b) small eosinophilic granulocyte and (c, d) basophilic granulocyte, Scale = 100

Image for - Morphological and Structural Characteristics of the Hemocytes of 
        the Anodonta cygnea
Fig. 4: (a) Agranulocyte and (b, c) hyalinocytes and blast-like cells, Scale = 100

Image for - Morphological and Structural Characteristics of the Hemocytes of 
        the Anodonta cygnea
Fig. 5: (a, b) Vesicular cell , Scale = 100

In addition, the multinucleate granulocytes were observed on smears. These cells have been observed in other mollusks (Park et al., 2002; Cheng, 1981; Adema et al., 1994; Zhang et al., 2005) with light microscopy. They are considered to be the result of a fusion of granulocytes in some pathological conditions such as postmortem changes or rejection of grafts. However, the origin and progress of their forming are not clear.

In most bivalves, such as the oyster Crassostrea virginica (McCormick-Ray et al., 1991), the mussels Mytilus edulis (Noel et al., 1994; Pipe et al., 1997) and Mytilus galloprovincialis (Carballal et al., 1997) and the clams Ruditapes decussatus (Lopez et al., 1997) and Tapes philippinarum (Cima et al., 2000) and Meretrix lusoria and Crassostrea gigas (chang et al., 2005) granulocytes were separated into two subclasses, basophilic and eosinophilic granulocytes, based on their granular affinity to specific dyes.

This observations of Anodonta cygena confirm a higher amount of basophilic than of eosinophilic granulocytes. Although, Cheng (1981) suggested that the granules in immature granulocytes may be basophilic, becoming acidophilic when mature, basophilic granulocytes have not been observed in some bivalves, such as the clam Tridacna crocea (Nakayama et al., 1997) or the mussel Perna perna (Barracco et al., 1999). The tinctorial properties of the granules may result from different amounts of hydrolysis enzymes (Cajaraville et al., 1995; Xue et al., 2000). Another cell type with lucent vacuoles, named the vesicular cell, was found in Anodonta cygena. The cytoplasm of vesicular hemocytes frequently containing endocytotic vacuoles was observed in the stained monolayers. In view of several morphological similarities, vesicular hemocytes and granulocytes may be related, but these cells have not yet been classified. To date, vesicular hemocytes have only been reported from oysters (Cajaraville et al., 1995; Adema et al., 1994), cockles (Russell-Pinto et al., 1994) and possibly arcid clams (Holden et al., 1994) and zebra mussels (Giamberini et al., 1996). In present species, Anodonta cygena, vesicular hemocytes observed. Agranulocytes were divided into two groups: blast-like cells and hyalinocytes.

Blast-like agranular hemocytes have been reported from many other bivalve mollusks (Hine, 1999; Zhang et al., 2005), but not all (Nakayama et al., 1997). They are assumed to be hemocyte progenitors based on their basophilic cytoplasm, suggesting the presence of free ribosomes and immaturity (Hine, 1999). The same morphological hemocytes were also reported in T. philippinarum as hemoblasts. Due to their morphology and positive reaction to the anti-CD34 antibody known to identify hempoietic cells in mammals, these cells are suggested to possibly represent stem cells in bivalves (Cima et al., 2000). In oysters, different observations about agranulocytes have been reported (Bachere et al., 1988; Adema et al., 1994). Herein, only one agranulocyte cell type, hyalinocytes. Actually not all hemocyte types occur in each bivalve species. Nomenclature, morphofunctions, as well as differentiation pathways of hemocytes in bivalve species still remain controversial. Inasmuch Anodonta cygena is a shellfish species with high dispersion around of world, we believe that the morphological characterization of Anodont hemocytes established in the present study can provide further insights into the immune defense of the this bivalve, as well as the influences of environmental factors on their hemogram.

ACKNOWLEDGMENTS

We would like to express our sincere thanks to Mis. Felor Mazhar, Azad University Laboratory, (North Tehran Branch) and Mr. Ahmad Ghane and Mr. Sayad Rahim, Inland Water Research Center-Bandar Anzali, for their assistance in this study.

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