Effect of temperature on phagocytosis activity in garden snails Helix aspersa.
Key words: phagocytosis, HLC, GLC, H. aspersa, stress, yeast
Invertebrates are widely distributed and can be found in almost any kind of habitat. Their dispersal and survival depend on successful defenses mechanism against various kinds of microorganisms and parasites, especially that they live in the same environmental conditions .
Invertebrates lack immunoglobulin antibodies and their associated adaptive immune responses; instead, they have highly effective innate immune reactions that defend them against a broad spectrum of microorganisms . However, previous study has shown that a specific memory that consider as acquired immunity in defense systems of invertebrates .
Invertebrate innate immune system is composed of both cellular and humoral immune responses. hemocytes-mediated immune responses such as phagocytosis, nodule formation and melanotic encapsulation are major cellular responses [5, 6]
Hemocytes from bivalve mollusks have most often been divided into two types, hyalinocytes and granulocytes, both of which are phagocytic . These two cell types have been found in many bivalve species [7, 8]. In many of these species, granulocytes have been further subclassified as eosinophilic, basophilic and neutrophilic cells . Granulocytes have the ability to phagocytose microbial pathogens and they contain a mixture of hydrolytic enzymes that contribute to intracellular killing. They are often more active phagocytes than hyalinocytes. Hyalinocytes are smaller than granulocytes. Some contain few granules and are usually more morphologically heterogeneous than granulocytes. Hyalinocytes have been divided into two types, small hyalinocytes, with large nuclei and scanty cytoplasm, and large hyalinocytes with low nucleus: cytoplasm ratios .
Phagocytic blood cells are major component of innate defense in both invertebrates and vertebrates, where they are also critical for linking innate to adaptive immunity. Moreover, phagocytosis seems relevant for one of the most promising candidates for specific immunity in insects and crustaceans, the alternatively spliced Down syndrome cell adhesion molecule (Dscam).previous study demonstrate that, enhanced phagocytosis resulting from prior exposure to endotoxin or live pathogenic bacteria in invertebrates .
Both the density and functions of mollusc hemocytes can be affected by changes in environmental conditions. Different types of stressors, including pathogens, parasites and xenobiotic, have been identified as hemocytes effectors. Hemocytes response patterns may depend on the nature of stressor [12, 13].
Hemocytes also serve a role in the molluscan stress response involving endocrine molecules shared with the vertebrates [14, 15]. Recent studies have shown that molluscs possess a primitive form of neuroendocrine response to stress involving catecholamine and neuropeptides such as adrenocorticotropic hormone .
The objective of this study to investigated the effect of different temperature on phagocytosis activity in Helix aspersa
2. Materials and method
Adult specimens of Helix aspersa were obtained from the northern area of AL-Karak city.it is governorate lies 140 km to the south of Amman on the King's Highway. It is situated on a hilltop about 1000 meters above sea level and is surrounded on three sides by a valley and from Karak can be seen wonder view of the Dead Sea, and the geographical coordinates to karak Governorate are 31[degrees] 11' 5" North, 35[degrees] 42' 17" East. The snails were kept in plastic container, at room temperature. The water and soil was changed every week and the snails were fed lettuce twice weekly.
Treated snails were kept and incubated in plastic container at different temperature (25C[degrees], 35 C[degrees], 40 C[degrees], and 15 C[degrees]) for two days.
2.2. Hemolymph sampling and counts
Hemolymph was collected directly from the heart using 22-gauge needles fitted to 3-ml syringes When the snail retracts into its shell, a drop of hemolymph was extruded through the hemal pore, and can be collected using a micropipette. Hemolymph (10 [micro]l) was directly dropped in hemocytometer. Total number of living hemocytes and the number of hemocytes in each cell population identified according to, Van der Knaap  using Optika B350 (Italy) light microscopy.
2.3. Cells analysis and morphology
2.3.1. Live cell analysis
The analysis of live hemocytes was carried out by placing a 30 [micro]l of whole hemolymph on acid alcohol cleaned microscope slides and allowing the cells to adhere for 10 min. The slides were then covered with a glass cover slip and viewed at high magnification with an Inverted phase microscope (Leica, Germany)
2.3.2. Staining method
After adhesion to slides, hemocytes were fixed for 20 min in methanol (80%) and then immersed in Giemsa stain for 30 min before being washed in distilled water and air dried. Slides were observed by Optika B350 (Italy) light microscopy.
2.4. Phagocytic activity
Phagocytic activity was assessed using baker's yeast, Saccharomyces cerevisiae (Sigma Aldrich), as target cells. Five milligrams of yeast was suspended in 5ml distilled water and mixed with an equal volume of Congo red. The suspension was autoclaved at 120 Co for 15 min before being washed twice by centrifugation at 1300g for 5 min and resuspended in 10 ml distilled water .To measure phagocytosis; 100 ml of whole hemolymph (adjusted to 1 x [10.sup.6] hemocytes [ml.sup.-1]) was placed on glass coverslips (22 x 22 mm) and incubated in a moist chamber. Hemocytes were allowed to settle and adhere for 20 min at room temperature (25 Co). The supernatants were then removed and the adherent hemocytes were rinsed 5 times with distilled water. The coverslips were overlaid with 100 ml of Congo red stained yeast (0.7 x [10.sup.6] [ml.sup.-1]) and incubated for 30 min at room temperature. The coverslips were washed 10 times with distilled water to remove nonphagocytosed yeast cells. They were then inverted over clean microscope slides and sealed with nail polish. A minimum of 100 hemocytes on each coverslip were examined and the number of hemocytes that had phagocytosed one or more yeast was recorded so that the percentage of phagocytic cells could be calculated .
2.5. Statistical analysis
All data were presented as means[+ or -]standard errors. All experiments were conducted three times. Data are shown for one representative experiment from each trial. In each experiment, different groups of five snails were tested at each time point. Data were analyzed using the Microsoft Excel package. Statistical analysis was performed by Student's t-test to determine the significance of differences between mean values. Differences were considered to be significant if P<0.05
3.1. Hemocyte morphology
Two major hemocytes types were identified by light microscopy according to presence or absence of granules in their cytoplasm, hyalinocytes-like cells (HLC) and granulocytes-like cells (GLC). HCL were the first type of hemocytes identify in H. aspersa (Fig. 1B) these cells comprised 57.6 [+ or -]2.6% of the total hemocytes population. They varied in size within a range of 6-10[micro]m, with an average diameter of 9 [+ or -] 0.9 Lm. HLC had an amoeboid movement and they formed filopodia. HLC were also characterized by a cytoplasm containing no granules. Some HLC maintained a round shape when attached to glass slides and spread only slightly, forming some small filopodia. GLC were the second type of hemocytes identified in H. aspersa (Fig.1A). These cells comprised 42.4 [+ or -] 2.6% of the total hemocytes population. They varied in size from 5-12 Lim with an average diameter of 9.6 [+ or -] 1.4 Lim. GLC adhered to glass slides within 15-20 min and they were highly ameboid after they adhered. They also had the ability to form many long filopodia, and were characterized by numerous cytoplasmic granules.
3.2. Phagocytosis activity
H.aspersa hemocytes were capable of ingesting yeast cells (Fig.2A). The phagocytic activity was measured at different temperature (Fig.2B), and a significant decreased in activity of H.aspersa held at 35 C[degrees] (19.2 [+ or -] 1.95) as compared to those treated at 25 C[degrees] as control 50.8 [+ or -] 2.3, (P < 0.05),and the activity represent more decreasing at 40 C[degrees] (3.6 [+ or -] 0.4) (P < 0.05) than the activity at 35 C[degrees] as compared to the activity at 25C[degrees]. When the H.aspersa kept in low temperature (15C[degrees]), the phagocytic activity dropped to (4.4 [+ or -] 0.6) with compared to those at 25C[degrees]. very little difference in phagocytic activity was found between H.aspersa kept at 40 C[degrees] and those kept at 15 C[degrees]. The average number of yeast cells which was detected in haemocyte of H.aspersa kept at 25C[degrees] is (4cells [+ or -] 0.4), 35C[degrees] (2cells [+ or -] 0.4), 40 Co (1.2 cells [+ or -] 0.2), and 15 Co (1.2 cells [+ or -] 0.2) (Fig.2 C).
Hemocyte classification in invertebrates is often a controversial issue. Simply because there is no universal procedure to classify hemocytes . In bivalves mollusks, hemocytes have been classified in many species, such as R. philippinarum, M. lusoria, C.gigas, C. farreri, T. crocea, D. polymorpha, and M. galloprovincialis [18, 19]. Hemocytes classification depends mainly on host defense, including the types of granules and enzymes found within cells .
Blood cells are differentiated into two types: granulocytes and hyalinocytes, [7, 8]. The results of the present study confirm that there are two types of hemocytes (type I, type II), this result are similar to that reported in helix aspersa maxima into(type I, type II). 
Similar finding has been reported earlier in P. corneus [21, 22]. Haemocytes type I in H. aspersa morphologically similar to granulocytes, and haemocytes II to hyalinocytes therefor it is possible to suggest that hemocyte type I is Granulocyte like Cell (GLC), and hemocyte type II is Hyalinocyte like Cell (HLC).
Several studies have demonstrated that defence mechanisms in mollusks are susceptible to changes in environmental factors, such as temperature, salinity, dissolved oxygen, food availability and presence of pollutants [23, 24, 31, 32, 33]. In particular, increases in temperature have been shown to induce mortality in bivalves, probably due to fact that high temperature induced suppression to the immune system in these animals [25, 34]. Furthermore, the data obtained of the present study demonstrated that H.aspersa hemocytes are sensitive to temperature and there is a good correlation between temperature and phagocytic activity in this species.
Phagocytosis is a temperature-dependent process in bivalve molluscs, so it is the main mechanism against foreign materials in snail's internal defence system . In the present study, a significant decrease of phagocytic activity was found in snails kept at (15 C[degrees], 35C[degrees], 40 C[degrees]) with respect to those kept at 25 C[degrees]. Similar finding has been reported in C. virginica when exposed to a temperature variety for 1 week . Conversely, Carballal MJ  found that the percentage of phagocytic haemocytes from M. galloprovincialis was lower at 10 Co than at 20 C[degrees] and 30 C[degrees].  observed no differences between phagocytosis at 15 C[degrees] and 21 C[degrees] in both, R. decussatus, and M. galloprovincialis. In the present study, the efficiency of hemocyte for phagocyted yeast cell decreased at 15 C[degrees] to (1.2 cells), 35 C[degrees] to (2 cells), and 40 C[degrees] to (1.2 cells) respectively when compared with 25 C[degrees] (4 cells), a previous study  indicate that the max number zymosan particles was phagocyted by hemocytes cells is (3 particles) at room temperature.
In conclusion, the influence of temperature on activity of phagocytosis has been investigated, and showed that both high temperature and low temperature have a clear effect on phagocytosis. As the temperature increased, the activity is clearly reduced. A similar pattern has been recorded with low temperature. Two types of cells (GLC, HLC) seem to have role in phagocytosis in the present investigation.
[FIGURE 1 OMITTED]
[FIGURE 2A OMITTED]
I thank prof. Khalil Altaif and Dr. Saleem Adaileh for her help, and biology department in AL-Hussein bin Talal University for providing the chemicals and devices and for her help.
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Ahmad. K. Ma'aqbeh
Department of Biology Al-Hussein bin Talal University Ma'an, Jordan
Ahmad. K. Ma'aqbeh, Effect of temperature on phagocytosis activity in garden snails Helix aspersa; Adv. Environ. Biol., C(): CC-CC, 2010
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|Title Annotation:||Original Article|
|Author:||Ma'aqbeh, Ahmad. K.|
|Publication:||Advances in Environmental Biology|
|Date:||May 1, 2010|
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