The Role of Starvation on Selective Immunological Parameters in Land Snail Helix Aspersa

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The role of starvation on selective immunological parameters in land snail Helix aspersa

Atika AL-Rawadeh

Department of Biological Sciences, College of Science , AL Hussein Bin Talal University

Atika AL-Rawadeh,The role of starvation on selective immunological parameters in land snail Helix aspersa Adv. Environ. Biol., C(C): CC-CC, 2010

ABSTRACT

Helix aspersa, (garden snail), is very common and widespread species in mediterranean region and western Europe. This species is edible in many places around the world and has many applications in traditional medicine.The role of starvation on various factors related to immunological defence in Helix aspersa were tested in controlled laboratory experiments. The mmunological and biochemical measurements were performed on 30 snails. A various parameters were assessed, including total hemocyte counts, phenoloxidase (PO) and phagocytosis. The phenoloxidase activity and total hemocyte frequencies decreased significantly after three weeks of starvation. However, the present results also showed that granulocyts are more affected by starvation than hyalinocytes, and the a ctivitity of phagocytsis depressed after the same period of starvation. These data indicate that starvation compromises immunological activity, and Accordingly affect immune responses. So that good nutrition play a major role in maintaining immunological parameters in Helix aspersa

Key words: Helix aspersa, starvation ,phenoloxidase ,phagocytion.

Introduction of criteria based on combinations of morphology, cytochemistry and function. Hemocytes from snails Helix aspersa, (garden snail), is a species of land mollusks have most often been divided into two snail, pulmonate gastropod, and is very common and types, hyalinocytes and granulocytes [10,11,12]. widespread in mediterranean region and western Granulocytes are the most observed cells with high Europe [1]. This species is edible in many places phagocytic ability. Hyalinocytes, unlike granulocytes, around the world, such as France, Italy, Greece. In lack Iilopodia, include few cytoplasmic granules and addition to that, it has also been used for centuries in have low phagocytic ability. [10]. Phenoloxidase is a traditional medicine [2]. key enzyme involved in the immunlogical defence of Invertebrates do not possess an acquired invertebrates. It is synthesized as inactive immunity, however, phenomena of specificity has prophenoloxidase, which is activated to phenoloxidase recently been demonstrated in invertebrates [3]. by serine proteases [7,13-15]. Invertebrates immune systems involve innate There are many environmental stress factors, like cellular and humoral responses asimilar to those that salinity and temperature, which reduce immunological found in vertebrates [4,5,6]. Cellular defenses include activity among invertebrates, and increased hemocyte- or coelomocyte-mediated responses such Susceptibility to pathogens [16-21]. Starvation is asphagocytosis, nodulation and encapsulation [7,8]. other environmental stressor that has an effect on Also they have humoral effectors including reactive many molluscs, such as Megalobulimus oblongus oxygen species, antimicrobial peptides, and where it increased haemolymph glucose levels and coagulation and melanization factors [6,8,9]. glycogen concentration in hepatopancreas, mantle and Hemocytes have been classified using a variety muscle [22].

Corresponding Author Atika AL-Rawadeh , Department of Biological Sciences, Collegeof Science , AL Hussein Bin Talal University E-mail: [email protected] Adv. Environ. Biol., 4(2): 265-270, 2010 266

Starvation also caused a reduction in the µl of whole hemolymph were added per well synthesis of galactogen and glycogen in the albumen followed by the addition of 100 µl of L-DOPA (4 gland and the mantle of thepond snail Lymnaea mg ml-1 in PBS), containing 1 mM MBTH. The stagnalis, [23]. absorbance of the reaction mixture was measured at In H. aspersa, starvation caused 60% reduction 490 nm immediately after the addition of substrates in body weight and significantly decreased the using a microplate spectrophotometer (SpectroUV.Vis number of digestive cells [24]. Starvation also Auto.UV-2602). A second reading was made after the decreased the egg laying and the activity of the plates had been incubated for one hour at room albumen glands in Bulinus truncates [25]. Also temperature. Enzyme activities are expressed as the starvation decreases the content of ammonia in change in optical density at 490 nm (OD492). Bradybaena similaris snail [26]. The aim of the present study is to evaluate the Phenoloxidase Activity Cytology effects of starvation on immunological parameters (phagocytosis, phenoloxidase (PO) activity , Thirty µl of whole hemolymph was immediately hemocyte frequencies) in H. aspersa. placed on acid alcohol washed microscope slides coated with poly-l-lysine and left to adhere for 10 Materials and methods minutes. The hemocytes were then stained for phenoloxidase activity. Experimental Animals and Design. The phenoloxidase stain was prepared in PBS and contained 5μ M L-DOPA and 5 mM MBTH. H. asperse were collected from Alkarak province The adherent hemocytes were overlaid with 30 µl of is located in south of Jordan (near to The Ded sea). the stain and allowed to stand for 10 minutes. They They were transported to Ma’an province (about 180 were then covered with a clean cover slip and sealed km from the capture site) and maintained in the with nail polish. Slides were then incubated for a laboratory under suitable conditions (10 per plastic further 30 min at room temperature. A light box, with wet filter paper in the bottom, and compound microscope was used to examine randomly temperature (25°C ±1). They were maintained for selected fields of view on the slide. A total of 200 two weeks with continuously regular feeding (twice hemocytes were examined so that the frequency of weekly). All the other conditions were controlled phenoloxidase-positive hemocytes (red stained) according to Armelle et al method [27]. Before relative to unstained hemocytes could be determined. starting the starvation period, the size and weight of each adult snail has been determined. A total of 60 Phagocytosis Assay adult snails were randomly chosen and divided into two equal groups: control (not starved) and Phagocytic activity was assessed In vitro using experimental (starved ) snails. The experimental Saccharomyces cerevisiae (Yeast, Sigma Aldrich), as snails were starved for 3 weeks. target cells. Five mg yeast were suspended in 5 ml PBS and mixed with an equal volume of filtered Haemolymph Collection and Preparation Congo red (Sigma Aldrich; 0.8% in PBS). The suspension was autoclaved at 120º C for 15 After three weeks of treatment, 10 snails from minutes before being washed twice by centrifugation each group were removed and washed with distil at 1,300g for 5 minutes and resuspended in 10 ml water. Hemolymph was then withdrawn by cardiac PBS. Forty µl of whole hemolymph were placed on puncture, directly through a notch in the shell. acid alcohol washed microscope slides coated with Hemolymph samples were immediately placed in poly-l-lysine and left to adhere for 20 minutes in a polypropylene tubes and held on ice. moist chamber at room temperature (25º C). The supernatants were then removed and the Phenoloxidase Assays adherent hemocytes were rinsed twice with PBS. The slides were overlaid with 100 µl of Congo red Phenoloxidase activity in whole haemolymph stained yeast (0.7 x 106 ml-¹) and incubated for was determined spectrophotometrically according to further 30 min at room temperature. The slides were Peters and Raftos [28]. Assays were performed by washed 4 times with PBS to remove non- recording the diphenolase activity with the substrate, phagocytosed yeast cells. They were then covered L-3,4-dihydroxyphenylalanine (L-DOPA, ICN, Irvine, with a clean cover slip and sealed with nail polish. CA, USA). The chromogen, 3-methyl-2- A minimum of 200 hemocytes were examined and benzothiazolinone hydrazone (MBTH, Sigma Aldrich) the number of hemocytes that had phagocytosed one was added to the substrate. Assays were carried out or more yeast was recorded so that the percentage of in 96 well flat bottom microtiter plates. One hundred phagocytic cells could be calculated. Adv. Environ. Biol., 4(2): 265-270, 2010 267

Total and Differential Hemocyte Counts Total and Differential Hemocyte Counts

The total hemocyte frequencies in hemolymph The starvation of snails caused a significant was determined by useing a Neubauer decrease in the total number of hemocytes in whole hemocytometer. Hemocyte monolayers were used to hemolymph ascompared to normal snails. Fig. 3Aand calculate differential hemocyte frequencies for shows that the number of hemocytes in starved snails granulocytes and hyalinocytes. Monolayers were decreased significantly (P < 0.05) after 3 weeks of prepared by allowing hemocytes to attach to acid starvation. Also there was a significant decrease in alcohol cleaned slides for 25 min at room the frequency of the granulocytes temperature. Microscope was then used to Fig. 3B in hemolymph after the starvation, in differentiate between hemocytes types according to comparison with control snails. This decreased the presence or absence of cytoplasmic granules. significant at(P < 0.05). However, the decrease in the frequency of the hyalinocytes was not significant at Statistical Analysis (P < 0.05).

All experiments were conducted three times. One way analysis of variance (ANOVA) was used to determine the significance of differences between mean values. Differences were considered to be significant if P < 0.05.

Results

Phenoloxidase Activity: Hemolymph phenoloxidase activities decreased after the 3 weeks of starvation. Fig. 1 shows that Fig. 3A: The effect of starvation on the whole hemolymph phenoloxidase activities totalnumber of hemocytes(THC). (monophenolase and diphenolase) were reduced significantly (P < 0.05) after starvation.

Fig 3B: The effect of starvation on the total number Fig. 1: The effect of starvation on the PO activity of granulocytes and hyalinocytes

The Frequency of Phenoloxidase-positive: Phagocytic Activity and Starvation:

Fig.2 demonstrates that the frequency of Figure 4 reveals that starvation decreased the Phenoloxidase activity -positive hemocytes decreased phagocytic ability of hemocytes.After 3 weeks of significantly (P < 0.05) after 3 weeks of starvation starvation, the frequency of phagocytic hemocytes fall, significantly (P < 0.05)as compared to control snails.

Fig 2:The effect of starvation on the number of the PO positive cells Fig. 4: The effect of starvation on the phagocytic ability Adv. Environ. Biol., 4(2): 265-270, 2010 268

Discussion Acknowledgements

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