STUDIES ON THE HEMOCYTES OF ACHATINA FULICA

Indian Journal of Multidisciplinary Research Ind. J. Multi. Res. 2010. Vol. 6 (2) : 207 - 214 05 ISSN - 0973 - 2225 STUDIES ON THE HEMOCYTES OF ACHATINA FULICA KARUTHAPANDI. M* Department of Advanced Zoology and Biotechnology, St. John's College, Palayamkottai, Tamilnadu. 627 002. *Present address: GTB-JRF, Institute of Forest Genetics and Tree Breeding, Coimbatore - 641 002. India. E mail: kpandi83@gmail.com (Received 25 January 2010, Accepted 30 March 2010) ABSTRACT Morphological characters of hemocytes Achatina fuilca were studied under light microscope to provide descriptions of cell types, their size distribution. Two types of hemocytes were identified they are agranulocytes (diameter 9.25±1.1µm) which represent about 20% of the total cell population and granulocytes represent about 80% of the total cell population. It's also sub typed into granulocyte I, granulocyte II and granulocyte III, (diameter is 7.08±1.7µm, 16.87±2.5µm and 12.50±1.8µm). The subtypes were classified based on the size, shape, neucelocytoplasmic ratio and staining characteristics. Keywords: Achatina fuilca, hemocytes, agranulocytes and granulocytes INTRODUCTION Molluscan hemocytes are not only known to play major role in immunological defense reactions (Cheng, 1975) and wound healing (Sparks, 1972), but they are also thought to be involved in the absorption and digestion of food in the digestive gland and in the removal of residual materials from this organ and other parts of the body (Wagge, 1955). Blood cells play a major part in the internal defense system (Tripp, 1970; Harris and Cheng, 1975; Sminia, 1977). The hemocytes have been shown to morphologically and functionally heterogeneous (Sminia and van der knaap, 1986; Amen et al., 1991; Mohandas et al., 1992). The blood cells are broadly classified into hyalinocytes or agranulocytes (George and Ferguson, 1950; Muller, 1956; Cheng, 1969). Sminia (1981) classified the blood cells, based on the size of the cell, nucleus to cytoplasm ratio, the shape of the nucleus, formation of pseudopodia and the presence or absence of cytoplasm granules. Cheng and Rifken (1970) and Cheng (1975) found that granulocytes were the most predominant phagocytes in terms of number and host defense. On other hand, Farley 1986 for Crassostrea virginica and Ruddell (1969) for C.gigas reported that agranulocytes may also play an importantant role. IND. J. MULTI. RES. Volume - 6 ( No. 2), 2010 207 Most workers agree that granular hemocytes can be readily recognized (Takatsaki, 1934; Liebman, 1946; Galtsoff, 1964; Narain, 1973; Cheng, 1975, Renwarantz et al., 1979; Fisher et al., 1986; Auffret, 1988). The granulocytes were sub typed into granulocyte I, granulocyte II and granulocyte III by Mahilini and Rajendran (2007) in Trachea vittata, Indoplanorbis exustus and Pila globosa. The internal defense system of mollusks is mostly represented by circulating elements of the hemolymph (hemocytes) Delgudo et al., 2001. Mollusk hemocytes are varying their number and morphology depending on environmental conditions and physiological condition of the animal (Anna, 2003). Based on light microscopic and electron microscopic studies the hemocytes are classified into agranulocytes and granulocytes (Yonow and Renwrantz, 1986). The granulocytes were sub-dived into granulocytes I, granulocytes II, and granulocyte III. Hyalinocytes had thin rim of cytoplasm surrounding the round nucleus. Granulocyte I had nucleus that occupies the whole portion of the cell and literally no cytoplasm and they were basophilic. They were smallest cell of the all hemocytes. Granulocyte II had nucleus in the center and basophilic. Granulocyte III had abundant acidophilic cytoplasm with eccentrically located nucleus. Similar classification proposed by Cheng and Guida (1980) in Bulinus truncates rohlfsi and in Trachea vittata, Indoplanorbis exustus and Pila globosa by Mahilini (2000). In gastropods the hemolymph constitutes 97% of granulocytes of total hemolymph cells. Alterations may occur in the morphology, number and types of the hemocytes during their defense against biotic and abiotic challenges (Suresh and Mohandas et al., 1990). McCormick-Ray and Howard (1991) related the changes in hemocyte types to physiological need in Crassostrea virginica. Cheng (1988) has reported that significant fluctuation in different cell counts could influence the efficiency of mollscan community. The present study is aimed to gather enough information about the defence mechanism of Achatina fulica and its level of hemocytes and it's Morphology during the environmental changes. MATERIALS AND METHODS The giant African snail Achatina fulica were collected from the banana field of Chenallpattai, Tirunelveli district. The collections were made during rainy season on the month of October 2007. At that time all the animals were active in condition. Forty five snails were collected which were approximately uniform in size, brought to the laboratory and released into the terrarium. The temperature (26±1°C) and a relative humidity (78±2%) were maintained in the terrarium. The animals were fed regularly with leaves of papaya (Carica papaya) and shoe flower (Hibiscus rosasinensis). Collection of hemolymph For the collection of hemolymph from A.fulica, each snail was cleaned externally with water and then isopropanol, and the hemolymph was collected by shell puncture (Renwarantz et al., 1981). The hemolymph was kept in an ice bath immediately after collection. IND. J. MULTI. RES. Volume - 6 ( No. 2), 2010 208 Hemocyte count Enumeration of total hemocyte was done with haemocytometer with improved Neubaur's ruling. Differential hemocytes count, a minimum of 100 hemocytes were classified and counted from each smear. They were identified at 10x X 40x magnification and when necessary 10x X 100x magnification using different stains such as Giemsa stain, gention violet and Methylen blue. Nucleocytoplasmic ratio of the total hemocytes and individual hemocyte nucleus of the cell using micrometer. RESULTS Hemocyte morphology The hemocytes were broadly classified into two categories, such as agranulocytes and granulocytes based on the staining characteristics. Agranulocytes are otherwise called as hyalinocytes. The nucleus is round and thin rim of cytoplasm which is basophilic. Here, the nucleus to cytoplasm ratio is higher. Hyalinocytes remain spherical when they adhered to glass. Granulocytes are round cells with granules and globular inclusions, granulocytes are grouped into granulocytes I (basophilic) which are smaller cells. The larger granulocytes cells have been distinguished into granulocyte II (basophilic) and granulocytes III (acidophilic), which have an intensely pink stained cytoplasm. Morphometry of Hemocyte The morphometry of hemocytes are presented in the Table 1. Diameter of agranulocyte is 9.25±1.1 µm and the nucleus is 6.5±1.2 µm. Granulocyte I has the diameter of cell 7.08±1.7 µm and nucleus 6.75±1.1 µm. Granulocyte II has the diameter of cell 16.87±2.5 µm and nucleus 12.85±3.2 µm. The diameter of entire granulocytes III is 12.50±1.8 µm, while nucleus has 11.17±0.57 µm. Nucliocytoplasmic Ratio Table 1 shows the nucleocytoplasmic ratio of hemocytes of Achatina fulica. The nucliocytoplasmic ratio of agranulocyte is 73±3.6%. Granulocytes I showed the highest nucliocytoplasmic ratio (95±2.3%) than the other cells. The nucliocytoplasmic ratio of granulocytes II (85±1.5%) was higher then that of granulocytes III (55±1.8%). Total Hemocyte count The observation of THC of active and aestivated snails of Achatina fulica is shown in Table.2. The mean number of THC cells was 8552±64 cells/mm3 during active condition. In the aestivated condition the THC was 7675±167 cells/mm3 of circulating hemolymph. IND. J. MULTI. RES. Volume - 6 ( No. 2), 2010 209 Differential hemocyte count Differential hemocyte counts in active and aestivated Achatina fulica are shown in Table 2. Two categories of hemocytes were observed in the present study, they are agranulocytes and granulocytes. The percentage of agranulocyte is 21±1.6%, granulocyte I is 40±0.01%, granulocyte II is 25±1.6% and granulocyte III is 14±3.2% during active condition. The percentage of agranulocyte is 20.8±0.23%, granulocyte I is 38±0.81%, granulocyte II is 30.5±0.04% and granulocyte III is 10.5±1.22% during aestivated condition. Even through much difference could not be observed in agranulocytes of both active and aestivated snails, changes in the hemocytes were noticeable in granulocyte I, granulocyte II and granulocyte III. Table. 1: Morphometry the of Hemocytes (µm) and Nucleo Cytoplasmic Ratio (%) of Achatina fulica Active condition Cell Types A granulocyte Diameter of entire cell (µm) 9.25 ±1.1 Nucleus (µm) 6.5 ±1.2 Nucleo Cytoplasmic Ratio (%) 73±3.6 Granulocyte I 7.08± 1.7 6.75± 1.1 95±2.3 Granulocyte II 16.87± 2.5 12.85± 3.2 85±1.5 Granulocyte III 12.50± 1.8 6.87 ±1.0 55±5.5 Table. 2: Differential Hemocyte Counts and Total Hemocytes Counts of Achatina fulica Active condition Cell types Total Hemocytes Count (cell / mm3 ) Agranulocyte Differential 21 ± 1.6 20.8 ± 0 Hemocytes Granulocyte I 40 ± 0.01 38.0 ± 0 Counts Granulocyte II 25 ± 1.6 30.5 ± 0 (%) Granulocyte III 14 ± 3.2 10.5 ± 1 8552 DISCUSSION The hemolymph of Achatina fulica contains two categories of hemocytes agranulocytes and granulocytes. The granulocytes were sub categorized into granulocyte I, granulocyte II and granulocyte III. The blood cell types identified in the present study corresponds to the findings of Cheng and Harris (1976) in Biomphalaria glabrata, Yoshino (1976) in Cerithidea cyonowalifornia, Yonow and Renwarantz (1986) in Aceton tornatilis; IND. J. MULTI. RES. Volume - 6 ( No. 2), 2010 Aestiva condit 210 7675 in B.tenagophyla two populations of hemocytes, hyalinocytes (large nucleus and basophilic cytoplasm) and granulocytic cells (large cell, small nucleus and high cytoplasmic count) were revealed by Barraco et al., (1993). Mahilini (2000) categorized 3 types of Granulocyte I, II and III in the three gastropod snail species Trachea vittata, Indoplanorbis exhustus and Pila globosa. There are two morphologically distinguishable types of hemocytes in Biomphalaria glabrata, granulocytes and hyalinocytes. Living granulocytes are initially spherical, each measuring 9.45±1.78µm in diameter and with nucleus which measuring 3.4±0.77µm in diameter. Hyalinocytes is 3.25±0.83 µm and its nucleus measures 1.47±0.47µm in diameter (Cheng and Auld, 1977). In Acteon tornatilis the hemolymph contains hyalinocytes (diameter 6.0+0.8 µm) which stain acidophilic and hemocytes with basophilic granulocytes (4-19 µm in diameter) (Yonow et al., 1986). The whole the land snail hemocytes were larger than the hemocytes of the aquatic snail (Adema et al., 1992). Average dimensions (length of hemocytes obtained terrestrial snail were 44.3±10 µm. Hemocytes of all snail species consisted chiefly of large, spreading cell with a high cytoplasm to nucleus ratio (Adema et al., 1992). Only few round small cells with high nucleus to cytoplasm ratio were observed in Achatina fulica. The number of circulating hemocytes varies between and within species, and depends on environmental factors (Adema et al., 1992). The number of hemocytes in the hemolymph is influenced by the snail's physiological condition (Dikkeboom et al., 1985 and Granath et al., 1984). A similar report was reported by Sminia (1981) in Biomphalaria glabrata. The current study revealed that the total hemocyte count of active snail A.fulica was higher than those of the snails which were aestivated up to 50 days. The plausible reason for the decrease in total hemocytes count of the aestivated A.fulica may be due to food and environmental condition and another reason may be due to the poor ability to elude, wide variation in environmental extremes as shown by Livingston and de Zwann, (1983).The number of hemocytes is also under the effect of the animal's age, temperature and infections (Noda and Loker, 1989; Ottaviani, 1989) and water content in the tissues (Zbikowske, 1998). The differential hemocyte count on Achatina fulica revealed that there is no much variation in the percentage of agranulocytes, granulocyte I and granulocyte III of active and aestivated snail where as the granulocytes II of the aestivated Achatina fulica showed higher percentage than the active snails. This may be correlated to the functional differences between agranulocytes and granulocytes (McCormik-Ray and Howard, 1991). This variation may be due to several causes, including seasonal stimulation to fulfill physiological needs and environmental stresses (McCormik-Ray and Howard, 1991). On the whole the proportion of agranulocytes and granulocytes in Achatina fulica in the total blood cell population was 80% and 20%, respectively in both seasons, and was close to the proportion of granulocytes IND. J. MULTI. RES. 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