In vitro Studies on Cellular Mediated Immune Response in Haemocytes of Crab— Episesarma tetragonum  

Jeyachandran Sivakamavalli , Perumal Rajakumaran , Baskaralingam Vaseeharan
Crustacean Molecular Biology and Genomics Lab, Department of Animal Health and Management, Alagappa University, Karaikudi 630 004, Tamil Nadu, India
Author    Correspondence author
International Journal of Molecular Zoology, 2013, Vol. 3, No. 7   doi: 10.5376/ijmz.2013.03.0007
Received: 26 Aug., 2013    Accepted: 27 Sep., 2013    Published: 17 Oct., 2013
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Sivakamavalli et al., 2013, In vitro Studies on Cellular Mediated Immune Response in Haemocytes of Crab—Episesarma tetragonum, Int'l J. Mol. Zoo., Vol.3, No.7, 24-31 (doi:10.5376/ijmz. 2013. 03.0007)


Studying the biological interaction with the invertebrate immune system is attractive for the advancement of a basic knowledge on invertebrate and vertebrate general immunity, because it offers various possible alternatives for disease management. The invertebrate immune system involves encapsulation, nodule formation and phagocytosis process by haemocytes (cellular mediated). In the present study, we investigated the morphology of the haemocytes of Episesarma tetragonum, their role in cellular mediated immune responses such as encapsulation, phagocytosis and viability assays. Encapsulation response of haemocytes is tested with the CM cellulose, Sepharose CL-6B and DEAE Sepharose 6CLB beads. DEAE Sepharose 6CLB beads showed (80%) good cellular response. Phagocytic activity of haemocytes showed pathogen engulfing activity against the microbial patterns (N-acteyl muramic acid (NAM), N-acetyl glutamic acid (NAG), lipopolysaccharide, β-1,3-glucans, chitins, laminarins, zymosan and peptidoglycans) present on the surface of the pathogens. Phagocytic activities of the haemocytes were decreased when increasing the incubation time. Viability of haemocytes was decreased when the time intervals of absorption of tryphan blue were increased. The present investigation enlightens the role of hemocytes in the cellular mediated immune responses of crustacean crab including all invertebrates.

Episesarma tetragonum; Cell mediated responses; Viability; Phagocytosis

Research on an innate immune system of crustaceans is greatly motivated by economical requirements, because of their culture is limited by the development of infectious diseases. Lack of poorly develop immune system in all invertebrates, haemocytes could not able to produce the immunoglobulins as like vertebrates immune system. When these defense mechanisms fail to protect the bacteria, viruses, fungi, protozoa and their products, disease develops and a negative impact takes place in the crab culture system. Innate immune reactions are the first line of defense in vertebrates and invertebrates (Piao et al.,2005), that are initiated with the recognition of invading organism as foreign (Piao et al.,2007). In invertebrates circulating cells (coelomocytes or hemocytes) represent the primary effect or component in immune response (Ratcliffe et al., 1981; Coombe et al., 1984; Beck et al., 1994). Crustacean immune system (Sindermann et al., 1971; Ey et al., 1991; Cerenius et al., 1995) consists of humoral and cellular immune components, intimate interactions between the two systems facilitates the expression of highly effective host immune responses.

Prophenoloxidase (proPO) and its active formphenoloxidase play an important role in melanization of pathogens (Nappi et al., 2001, Sivakamavalli and Vaseehaaran, 2013), cuticle sclerotization (Sugumaran, 1991). The cell mediated immunity is triggered by Pattern Recognition Proteins (PRPs) that recognizes the surface of microorganisms (Medzhitov, 2000a, b). In the case of cellular responses include encapsulation, phagocytosis, nodule formation, cytotoxicity and exocytosis of immune reactive substances (Coombe et al., 1984; Becket et al., 1994). In encapsulation, haemocytes enclose the foreign body and cytotoxic products are releases as free radicals to destroy the invader (Ratcliffe et al., 1987). It occurs by the involvement of the individual cells and connective fibres (Cheng et al., 1970). In molluscan, helminthus species were destroyed by encapsulation process (Cheng et al., 1967). Lysozyme enzyme mechanism and encapsulation were correlated defence systems in mollusc (Cheng et al., 1983).
Hemocytes are the primary effectors of cellular immunity in crustaceans (Gargioni et al., 1998; Giulianini et al., 2007; Vafopoulou et al., 2007). Phagocytes mediate their innate immunological response by releasing products that damage or kill the invading or ingested microorganisms. In addition, phagocytes in vertebrates have been shown to kill or limit the replication of pathogenic microbes by using reactive nitrogen intermediates (RNIs) as well (Va´zquez-Torres et al., 2001). In the case of invertebrates too, ROI and RNI generation has been shown in hemocytes (Sierra et al., 2005; Dikkeboom et al., 1998; Anderson et al., 1992; Arumugam et al., 2000; Bergin et al., 2005; Cheng et al., 2006), but not as extensively characterized as in crustacean crab. The immune system and immune responses plays a major role in the protective mechanism of the invertebrates. Understanding of cellular mediated immune response gave a congregate evidence for the mechanism involved in immune system of invertebrates and used for comparative studies. Hence, the present study was designed to determine the role of cellular mediated immunity and its immune responses in the crab Episesarma tetragonum.
1 Materials and Methods
1.1 Experimental animals
Crab E. tetragonum (100 - 200 g) (Figure 1) were collected from the estuarine area of Katumavadi, Tamil Nadu, India. The animals were acclimated for 10 days before experiments were carried out. Crabs were fed daily with fish or shrimp meat at 10% of body weight. Haemolymph (10 mL) was withdrawn by inserting a syringe into the sinus at the base of right chelate leg into a 50 mL polyethylene tube containing 10 mL of precooled (4?) anticoagulant (10% trisodium citrate, 13 mM citric acid, 50 mM EDTA) (Sivakama valli and Vaseeharan, 2012). The diluted haemolymph was centrifuged at 500 × g at 4? for 20 min. Cell-free haemolymph was aspirated from the haemocyte pellet, which was then resuspended in 1 mL FSW (Fresh Water Saline). The resulting cell suspensions and cell-free haemolymph samples were divided equally in separate tubes and stored on ice for subsequent use in assays of phenoloxidase activity and melanin formation. Haemolymph of crabs does not coagulate, nearly all the hemocytes remain free and refractile without any sign of degranulation for at least 30 min. Conversely, small aggregates of hemocytes were occasionally noticed.

Figure 1 Encapsulation response of E. tetragonum haemocytes with the chromatographic DEAE sepharose CL6B beads
1.2 Separation of plasma and haemocytes
Pooled haemolymph samples were immediately centrifuged (4000 g, 6 min, 4?) and the resulting supernatants were used as plasma. The pellet (haemocytes) was gently resuspended and washed once in iso osmotic TBS-I (50 mM Tris; 400 mM NaCl; pH 7.5; 840 mOsm) by centrifugation (4000 g, 5 min, 4?). The haemocytes were finally suspended in 2 mL Tris - HCl buffer (250 mM Tris, pH 6.5).
1.3 Preparation of haemocyte lysate
Immediately after withdrawal, the crab haemolymph was centrifuged at 300 gfor 3–5 min at 4?. The supernatant (plasma) was separated and the cell pellet suspended in 10 mM sodium cacodylate buffer, containing 0.40 M NaCl, (10 mM CaCl2 and 20 mM MgCl2 (Perazzolo and Barracco, 1997). The cell pellet was washed twice in the same buffer by centrifugation and then suspended in equal volume of the buffer. The mixture was homogenizedwith a glass piston and centrifuged at 35,000 ×gfor 20 min at 4 ? (Söderhäll and Smith, 1983). The precipitate was discarded and the resulting supernatant represented the crab haemocyte lysate supernatant (HLS).
1.4 Preparation of haemocyte conditioned medium (HCM)
Haemolymph sample (500 µL) was spread on a glass slide to prepare the haemocyte monolayer. Incubate the haemocytes for 10-15 min. Haemocytes were settle and some are attached with the slides. The supernantent was carefully removed by the aspiration and the haemocyte monolayer was immediately rinsed with TBS (50 mM Tris; 400 mM NaCl; pH 7.5; 840 mOsm) and acetate buffer (sodium acetate, 250 mM, acetic acid, 25mM).Slides were covered with the lid and incubate undisturbed at 22?. After 1 hr, collect the supernatants from the slide and suspending the beads for the encapsulation.
1.5 Total haemocyte count (THC)
Haemolymph was mixed with equal volume of marine anticoagulant (MAC, 0.1 M glucose, 15 mM trisodium citrate, 13 mM citric acid, 50 mM EDTA, 0.45 M sodium chloride, pH 7.5) to reduce the haemocyte aggregation. This haemocyte suspension was then fixed with 100 µL paraformaldehyde (4% w/v). The number of haemocytes per ml was estimated individually (60 animals) in males and females separately using an Improved Neubauer haemocytometer. As described like WBC counting.
1.6 Sepharose beads preparation
Three different types of beads were used for the encapsulation reaction. Sepharose CL-6B (neutral), CM Sepharose (negatively charged) and DEAE (Diethylaminoethyl cellulose) Sepharose (Posiitvely charged) beads were suspended in the 0.9% saline and prepare the HCM. When suspending the beads with the saline the size of the beads was increased due to the swelling. Mix the beads thoroughly and allowed to settle the beads to get the larger size for 20-25 min. Subsequently, beads were recovered from the suspension and dissolved in TBS. Obviously beads get the diameter from 100-150 µm, that was seen under the Nikon light microscope and it was stored at 4? until use. A 75µL aliquot of DEAE sepharose beads were suspended and allowed to settle, then the supernatant was removed and these beads were resuspended in plasma, HLS and HCM.
1.7 Haemocytes isolation
Haemolymph of 300-400 µL was diluted with the TBS and centrifuged at 4000 × g for 5 min and discard the supernatant, remaining concentrated haemocytes were resuspended in TBS. This suspension was used for all encapsulation assays in in vitro.
1.8 Encapsulation assay
V-bottomed microtiter plates were used for the encapsulation assay (Nunc Roskilde plates, Denmark). Twenty five microlitres of plasma samples were serially diluted two -fold in TBS/Ca2+ pH 7.4 buffers. To each well, 25 µL of sepharose beads (6 mg/ml in TBS/Ca2+ pH 7.4) and 25 µL of haemocyte lysate supernatant were added, mixed, and incubated for 45 min ~ 1 h at RT gentle mixing at 15 min interval. Finally, 25 µL of this mixture was taken for the microscopic studies. The entire volume of the suspension was spreaded on the glass slide and incubated at moist chamber for 20 min, 22? for the beads settlement. Controls were made without sample plasma, sepharose beads, or HLS, replacing them with TBS/Ca2+ pH 7.4 buffers. Encapsulated beads by the haemocytes E. tetragonum were seen under the microscope (Nikon, Japan) and encapsulated beads were calculated. To compare the encapsulation activity in plasma, HCM and HLS, simultaneously use the beads which are undiluted plasma, HCM and HLS with TBS buffer.
1.9 Viability test
Haemolymph sample (500 µL) was spread on a glass slide to prepare the haemocyte monolayer. Incubate the haemocytes for 5-10 min with the tryphan blue dye exclusion test.
1.10 Phagocytosis assay
Haemocytes were kept with FSW for 30 min, to enhance the cells adhesion to the slides, after the incubation time FSW was removed from the culture chambers and replaced with equal volumes of a suspension of yeast (S. cerevisiae; yeast:haemocyte ratio ¼ 10:1) and laminarin (3.5 × 107/mL) in FSW. Two different target particles were chosen to evaluate the influence of particle dimension (6 mm and 3 mm in yeast and laminarin, respectively) on phagocytic capability of haemocytes. Haemcyte monolayers were incubated for 60 min at 25?, washed with FSW to eliminate uningested particles and fixed by of 1% glutaraldehyde, 1% sucrose in FSW at 4? for 30 min. It was washed in phosphate buffered saline, pH 7.2, (PBS: 1.37 M NaCl, 0.03 M KCl, 0.015 M KH2PO4, 0.065 M Na2HPO4), stained with 10% Giemsa for 5 min, mounted on glass slides, and then observed under a light microscope. Two hundred cells per slide were counted, and the phagocytic index was expressed as the percentage of cells containing ingested particles.
PR=(Phagocytic haemocytes/Total haemocytes)×100
1.11 Statistical analysis
Difference between control and test sample was calculated by the statistical significance using Students t-test.
2 Results
2.1 Total haemocyte count (THC)
The total haemocyte count per mm3 of haemolymph (THC) in E. tetragonum was first estimated, individually, in three groups of ten animals. In the first group, the THC was determined immediately after the removal of the animals from the culture station; the second and third groups were analyzed after 3–4 and 7–8 days in the laboratory, respectively. This was done to determine whether maintenance under laboratory conditions could influence the haemocyte numbers. The results indicated that the THC did not significantly vary (Student t-test, P>0·05) in Groups 1 and 2 (THC=5.08 ± 0.14_107 cells/mL and 4.85, THC=7.90±0.07 and 6.28 cells/mm3 in each group respectively), but did so in the third group (THC=11.09±0.5 and 10.82 cells). Therefore, all analyses on haemocyte numbers, THC, were performed on animals which were kept in the laboratory for a maximum of 4 days.
2.2 Encapsulation response in in vitro
Crab haemocytes has the ability to encapsulate the chromatographic beads such as DEAE Sepharose CL-6B, CM sepharose CL-6B. Sepharose CL-6B was tested with the haemocytes of the crab E. tetragonum. Haemocytes were suspended with the TBS buffer and tested; it showed the passive response in CM sepharose. Interestingly, DEAE sepharose 6CLB beads showed good cellular response against this bead type tested (Figure 2). The washed DEAE beads were incubated with the plasma, then intensively observed the encapsulation reaction. The encapsulation response was more significant (80%), in control shows 62% when comparing the plasma encapsulation with other HLS, HCM (Table 1, Table 2 and Table 3). Similarly, washed haemocytes were suspended in DEAE beads, incubated with the HLS, the intensively encapsulation was observed. The encapsulation response was more significant in HLS (47%), when compare to plasma the cellular response was less, in control showed 75% cellular response. Washed beads were harvested in HCM medium and the cellular response 75% observed, but in control showed 62% response. Among the three different medium tested, plasma showed the highest immune response when compared with other HCM, HLS (Table 3 and Table 4).

Figure 2 Phagocytic responses of E. tetragonum haemocytes

Table 1 Encapsulation response of E. tetragonum haemocytes against different chromatographic beads

Table 2 Encapsulation response of crab E. tetragonum haemo- cytes against DEAE sepharose 6CLB beads

Table 3 Encapsulation response of HLS against DEAE sepharose 6CLB beads

Table 4 Encapsulation response of HCM against DEAE sepharose 6CLB beads

2.3 Phagocytosis
Phagocytosis results showed that haemocytes of E. tetragonum were able to engulf the yeast cells or laminarin. In the present study, haemocytes involved in the phagocytic activity with the two target particles yeast and laminarin. Results showed that engulfing target particles with the respective incubation time, when increase the incubation time haemocyte loose the phagocytic activity (Figure 3).

Figure 3 Total haemocytc count of crab E. tetragonum
2.4 Viability test
Viability test results depicts that during the spreading of haemocytes with the addition of tryphan blue, both nuclei and cytoplasmic contents of the haemocytes became more clearly visible, which enabled the easy identification of three distinct haemocyte morphotypes based on the presence and abundance of cytoplasmic granules. The three haemocyte types identifiable were hyaline or agranular cells with relatively a few or no granules, semi granules with moderate number of granules and granular cells with abundant granules of varying size. Moreover, the viability of the haemocytes was decreased when increases the incubation time along with tryphan blue (Table 5).

Table 5 Viability of E. tetragonum haemocytes
3 Discussion
Haemocytes were classified based on different criteria although the classification of crustacean haemocytes remains contentious (Hose et al., 1987; 1990a). In decapods blood cells are represented by unified terms (Hose and Martin, 1989; Tsing et al., 1989). In invertebrates, haemocytes mediated cellular immune response were differed, when changes occurred in the environment (Pipe et al., 1995). In the present study we reports E. tetragonum immune responses were studied based on the environment where animals collected. Results showed that E. tetragonum has a great inter-individual variability in circulating haemocyte number occurred, and the resulting mean haemocyte number was 8.02 (×106) /mL haemolymph. This report coincide with THC values reported in other crustacean species, such as Cancer pagurus (2.55 × 107/mL) (Vogan et al., 2002), Potamon fluviatilis (10.53 × 105/mL) (Yavuzcan et al., 2002), Penaeus paulensis (4.5 × 105/mL), P. monodon (21.0 × 106/mL) (Owens et al., 1997), M. acanthurus and M. rosenbergii (2.55 × 107/mL, respectively) (Gargioni et al., 1998). Although the significance of this marked variation in THC remains unclear, influences of moult cycle (Tsing et al. 1989), diet (Wang et al., 2005) harvesting (Jussila et al., 1997), diseases (Smith et al. 1980; Smith et al. 1983; Martin et al. 1993) and environmental contaminants (Smith et al. 1995) annot be excluded. Haemocytes of invertebrates plays the vital role in defense mechanisms; the haemocytes are recognizing the pathogens and interact with the foreign substances by endocytosis, encapsulation and phagocytosis (Nowak et al., 1976, Sivakamavalli et al., 2012). In the present study, E. tetragonum haemocytes circled and engulfed the HB RBC cells. Previous reports suggest that hyalinocytes, from M. rosenbergii, M. acanthurus and P. monodon, were able to engulf yeast particles (Gargioni et al. 1998). Carcinus maenas haemocytes also involved in the bacteria engulfing activity (Powell and Rowley, 2008). However, other crustaceans such as freshwater prawn species, (Sahoo et al., 2007) observed that large ovoid haemocytes were mostly phagocytic, and only engulf the chicken erythrocytes (Vázquez et al., 1997). In decapod crustaceans phagocytosis was mediated by the semi granular and granular haemocytes (Smith et al., 1983; Söderhäll et al., 1983; Hose et al., 1990). M. rosenbergii haemocytes have the ability to eliminate the bacteria through phagocytosis (Sung et al., 2000). We observed that the basal phagocytic index (evaluated using non-opsonised yeast or HB RBC and laminarin) was very low in the haemocytes of E. tetragonum. Moreover, E. tetragonum haemocytes showed the uptake of very few bacteria, indicates that close association between haemocytes and bacteria. Those studies indicated a relationship between target particles used in phagocytosis assays and phagocytic capability of crustacean haemocytes. In the present study, we chose HB RBC cells and laminarin as target particles, as they have different size. However, no differences in phagocytic index were observed when haemocytes from E. tetragonum were incubated with yeast or HB RBC and laminarin. Similarly, in American lobster Homarus americanus, activated haemocytes showed increased phagocytosis of opsonised sheep red blood cells (Goldenberg et al., 1984). E. tetragonum haemocytes showed the passive phagocytic response with the microbes. In freshwater crustaceans, semigranular hemocytes of Astacus astacus and Pascifastacus leniusculus have been shown to engulf bacteria or yeast cells pre-treated with or without plasma/haemocyte lysate supernatant thereby showing the process of opsono-phagocytosis (Cerenius et al., 1994; Smith et al., 1983). Further, the ability of the hemocytes of M. rosenbergii to show phagocytosis of microbe Enterococcus (Cheng et al., 2002) as well as a variety of fixed erythrocytes from mammals and birds (Vazquez et al., 1997; Sierra et al., 2005) have been reported. In crab E. tetragonum haemocytes showed the encapsulation reaction with the DEAE sepharose CL6B beads, this result was matched with already reported molluscan Perna viridis and insects (Dunphy et al., 1980; Vinson,et al., 1974). This results suggests that the presence of negative charges on the haemocytes may be helps the initial interaction with the beads. Moreover, based on the surface charge of the haemocytes, encapsulation response and interaction was altered in insects (Ratner and Vinson., 1983, Soderhall et al., 1984; Pech et al., 1996; Lavine et al., 2001), crustaceans (Persson and Soderhall, 1987) and molluscs (Sminia et al., 1974). The plasma of crab E. tetragonum has the encapsulation activity towards the positively charged beads, it indicates the haemocytes has the negative charge on its surface. In in vitro encapsulation, haemocytes interacted with the surface of the positively charged beads (Dunphy et al., 1980), negatively charged beads (Götz et al., 1986), neutral beads (Pech et al., 1994), positive as well as negative charge beads (Lackie et al., 1983). Encapsulation stimulating factors observed in plasma of few insects (Pech et al., 1996; Davies et al., 1988; Lackie et al., 1988; McKenzie et al., 1992).
4 Conclusion
In conclusion, E. tetragonum haemocytes are not very active phagocytic cells, and only hyalinocytes are able to phagocytose yeast cells or laminarin (Bache`re et al., 1995). In addition, it was demonstrated that haemocytes of E. tetragonum have showed modest opsonic activity against the microbes. Haemocytes of E. tetragonum were more active in terms of phagocytic activity engulfing the HB RBC cells, suggesting the greater capacity of haemocytes to neutralise foreign materials by phagocytises. Based on the encapsulation results, it is one of the most important immune response in E. tetragonum haemocytes, and showed the activity against DEAE sepharose CL6B beads, based on the surface charges of the molecule and haemocytes. This study will support the understanding of the cellular mediated immune responses of crab E. tetragonum and more studies are needed to analyze the immune functions of crustaceans to control the diseases.
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