Printer Friendly

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

Introduction

Helix aspersa, (garden snail), is a species of land snail, pulmonate gastropod, and is very common and widespread in mediterranean region and western Europe [1]. This species is edible in many places around the world, such as France, Italy, Greece. In addition to that, it has also been used for centuries in traditional medicine [2].

Invertebrates do not possess an acquired immunity, however, phenomena of specificity has recently been demonstrated in invertebrates [3].

Invertebrates immune systems involve innate cellular and humoral responses asimilar to those that found in vertebrates [4,5,6]. Cellular defenses include hemocyte- or coelomocyte-mediated responses such asphagocytosis, nodulation and encapsulation [7,8]. Also they have humoral effectors including reactive oxygen species, antimicrobial peptides, and coagulation and melanization factors [6,8,9].

Hemocytes have been classified using a variety of criteria based on combinations of morphology, cytochemistry and function. Hemocytes from snails mollusks have most often been divided into two types, hyalinocytes and granulocytes [10,11,12]. Granulocytes are the most observed cells with high phagocytic ability. Hyalinocytes, unlike granulocytes, lack Iilopodia, include few cytoplasmic granules and have low phagocytic ability. [10]. Phenoloxidase is a key enzyme involved in the immunlogical defence of invertebrates. It is synthesized as inactive prophenoloxidase, which is activated to phenoloxidase by serine proteases [7,13-15].

There are many environmental stress factors, like salinity and temperature, which reduce immunological activity among invertebrates, and increased Susceptibility to pathogens [16-21]. Starvation is other environmental stressor that has an effect on many molluscs, such as Megalobulimus oblongus where it increased haemolymph glucose levels and glycogen concentration in hepatopancreas, mantle and muscle [22].

Starvation also caused a reduction in the synthesis of galactogen and glycogen in the albumen gland and the mantle of thepond snail Lymnaea stagnalis, [23].

In H. aspersa, starvation caused 60% reduction in body weight and significantly decreased the number of digestive cells [24]. Starvation also decreased the egg laying and the activity of the albumen glands in Bulinus truncates [25]. Also starvation decreases the content of ammonia in Bradybaena similaris snail [26].

The aim of the present study is to evaluate the effects of starvation on immunological parameters (phagocytosis, phenoloxidase (PO) activity , hemocyte frequencies) in H. aspersa.

Materials and methods

Experimental Animals and Design.

H. asperse were collected from Alkarak province is located in south of Jordan (near to The Ded sea). They were transported to Ma'an province (about 180 km from the capture site) and maintained in the laboratory under suitable conditions (10 per plastic box, with wet filter paper in the bottom, and temperature (25[degrees]C [+ or -]1). They were maintained for two weeks with continuously regular feeding (twice weekly). All the other conditions were controlled according to Armelle et al method [27]. Before starting the starvation period, the size and weight of each adult snail has been determined. A total of 60 adult snails were randomly chosen and divided into two equal groups: control (not starved) and experimental (starved ) snails. The experimental snails were starved for 3 weeks.

Haemolymph Collection and Preparation

After three weeks of treatment, 10 snails from each group were removed and washed with distil water. Hemolymph was then withdrawn by cardiac puncture, directly through a notch in the shell. Hemolymph samples were immediately placed in polypropylene tubes and held on ice.

Phenoloxidase Assays

Phenoloxidase activity in whole haemolymph was determined spectrophotometrically according to Peters and Raftos [28]. Assays were performed by recording the diphenolase activity with the substrate, L-3,4-dihydroxyphenylalanine (L-DOPA, ICN, Irvine, CA, USA). The chromogen, 3-methyl-2benzothiazolinone hydrazone (MBTH, Sigma Aldrich) was added to the substrate. Assays were carried out in 96 well flat bottom microtiter plates. One hundred [micro]l of whole hemolymph were added per well followed by the addition of 100 [micro]l of L-DOPA (4 mg ml-1 in PBS), containing 1 mM MBTH. The absorbance of the reaction mixture was measured at 490 nm immediately after the addition of substrates using a microplate spectrophotometer (SpectroUV.Vis Auto.UV-2602). A second reading was made after the plates had been incubated for one hour at room temperature. Enzyme activities are expressed as the change in optical density at 490 nm ([OD.sub.492]).

Phenoloxidase Activity Cytology

Thirty Ll of whole hemolymph was immediately placed on acid alcohol washed microscope slides coated with poly-l-lysine and left to adhere for 10 minutes. The hemocytes were then stained for phenoloxidase activity.

The phenoloxidase stain was prepared in PBS and contained 5[micro] M L-DOPA and 5 mM MBTH. The adherent hemocytes were overlaid with 30 [micro]l of the stain and allowed to stand for 10 minutes. They were then covered with a clean cover slip and sealed with nail polish. Slides were then incubated for a further 30 min at room temperature. A light compound microscope was used to examine randomly selected fields of view on the slide. A total of 200 hemocytes were examined so that the frequency of phenoloxidase-positive hemocytes (red stained) relative to unstained hemocytes could be determined.

Phagocytosis Assay

Phagocytic activity was assessed In vitro using Saccharomyces cerevisiae (Yeast, Sigma Aldrich), as target cells. Five mg yeast were suspended in 5 ml PBS and mixed with an equal volume of filtered Congo red (Sigma Aldrich; 0.8% in PBS).

The suspension was autoclaved at 120[degrees] C for 15 minutes before being washed twice by centrifugation at 1,300g for 5 minutes and resuspended in 10 ml PBS. Forty Ll of whole hemolymph were placed on acid alcohol washed microscope slides coated with poly-l-lysine and left to adhere for 20 minutes in a moist chamber at room temperature (25[degrees]C).

The supernatants were then removed and the adherent hemocytes were rinsed twice with PBS. The slides were overlaid with 100 [micro]l of Congo red stained yeast (0.7 x [10.sup.6] [ml.sup.-1]) and incubated for further 30 min at room temperature. The slides were washed 4 times with PBS to remove nonphagocytosed yeast cells. They were then covered with a clean cover slip and sealed with nail polish. A minimum of 200 hemocytes 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.

Total and Differential Hemocyte Counts

The total hemocyte frequencies in hemolymph was determined by useing a Neubauer hemocytometer. Hemocyte monolayers were used to calculate differential hemocyte frequencies for granulocytes and hyalinocytes. Monolayers were prepared by allowing hemocytes to attach to acid alcohol cleaned slides for 25 min at room temperature. Microscope was then used to differentiate between hemocytes types according to the presence or absence of cytoplasmic granules.

Statistical Analysis

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 whole hemolymph phenoloxidase activities (monophenolase and diphenolase) were reduced significantly (P < 0.05) after starvation.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Total and Differential Hemocyte Counts

The starvation of snails caused a significant decrease in the total number of hemocytes in whole hemolymph ascompared to normal snails. Fig. 3Aand shows that the number of hemocytes in starved snails decreased significantly (P < 0.05) after 3 weeks of starvation. Also there was a significant decrease in the frequency of the granulocytes

Fig. 3B in hemolymph after the starvation, in comparison with control snails. This decreased significant at(P < 0.05). However, the decrease in the frequency of the hyalinocytes was not significant at (P < 0.05).

[FIGURE 3A OMITTED]

[FIGURE 3B OMITTED]

[FIGURE 4 OMITTED]

Discussion

Land snails can enter dormancy under many unfavorable conditions, including low humidity [29], low concentration of nutrition [30,31]. and high temperatures [32]. They may remain dormant for many months or even years and during these long period of starvation, all the stored materials (glucose, lipid and protein) are metabolized and consumed [33]. The reduction of all of these sources of nutrients in invertebrates caused a reduction in the body weight, changes in the histologic structure of digestive gland, oxygen consumption rate, and changes in the structure and composition of the shell. [34,35,24].

Before discussing the role of starvation on immune system of invertebrate, some consideration must be given to the question of whether stress factors played any significant part in determining immunity in invertebrate . The biochemical and immunological changes recorded here werein consistent with previous studies on that some stress factors like altered temperature, hypoxia, pollutants, altered salinity and mechanical disturbance, have negative impacts on invertebrate immune system leading to increased disease susceptibility [36-40]. Furthermore the results of the present exeperiment reveal that short term starvation (three weeks) inhibited all the immunological parameters tested. After 3 weeks of starvation, phenoloxidase activity (PO) decreased significantly compared to the control snalis. This is inagreement with those of Butt et al [41], who found that starvation caused a significant decreases in PO, in Saccostrea glomerata. However, Yang et al [42], found that starvation causeda decrease in the melanotic encapsulation rate but not in the PO activity in the Epirrit aautumuata. In addition to phenoloxidase activity, total hemocyte frequencies also decreased significantly after the sameperiod of starvation. However, the results showed that granulocyts are more affected by starvation than hyalinocytes. These results arepartially in agreement with those in the Sydney rock oyster [41] , and Zhikong scallop (Chlamys farreri) [43]. Starvation also decreased significantly the phagocytic activity. Many studies had showed that in invertebrates the granulocytes are more phagocytic than hyalinocytes [44]. The decrease in the phagocytic ability can be explained by the significant decrease in the granulocytes after the three weeks starvation period.These results support the earlier findings reported in Chlamys farreri and in Saccostrea glomerata [41,43].

Finally, the results of this study showed that good nutrition play a major role in maintaining immunological parameters in Helix aspersa.

Acknowledgements

Many thanks tot he head department of biological sciences Dr Saleem Aladaileh,

Prof. KhalilAL-taif , the technical staff and Dept. of biology

References

[1.] Kerney, M.P. & R.A.D. Cameron, 1979. A field guide to the land snails of Britain and north west Europe. William Collins Sons and Co. Ltd., London

[2.] Murphy, B., 2001. Breeding and Growing Snails Commercially in Australia, A report for the Rural Industries Research and Development Corporation, Kingston.

[3.] Hauton, C. and V.J. Smith, 2007. Adaptive immunity in invertebrates: a straw house without a mechanistic foundation, Bioessays, 29: 1138-1146.

[4.] Ratcliffe, NA., A.F. Rowley, S.W. Fitzgerald & C.P. Rhodes, 1985. Invertebrate Immunity: basic concepts and recent advances. Int Rev Cytol, 97: 183-350.

[5.] Cerenius, L., K. Soderhall, 2004. The prophenoloxidase-activating system in invertebrates. Immunol Rev., 198(1): 116-126.

[6.] Philippe, R., 1999. Defense mechanisms and disease prevention in farmed marine invertebrates Aquaculture, 172(1-2): 125-145.

[7.] Jiravanichpaisal, P., BL. Lee, K. Soderhall, 2006. Cell-mediated immunity in arthropods: Hematopoiesis, coagulation, melanization and opsonization. Immunobiol., 211(4): 213-236.

[8.] Lavine, M.D., M.R. Strand, 2002. Insect hemocytes and their role in immunity. Insect Biochem Mol Biol., 32(10): 1295-1309.

[9.] Muta, T., S. Iwanaga, 1996. The role of hemolymph coagulation in innate immunity. Curr Opin Immunol., 8(1): 41-47.

[10.] Thomas, C., Cheng. Vincent, G. Guida, 1980. Hemocytes of Bulinus truncatus rohlfsi (Mollusca: Gastropoda),Journal of Invertebrate Pathology, 35 (2): 158-167.

[11.] Dikkeboom, Ronald., M.G.H. Jolanda, Elly C Tijnagel, Wil P.W. Mulder, van der Knaap, 1987. Hemocytes of the pond snail Lymnaea stagnalis generate reactive forms of oxygen Journal of Invertebrate Pathology, 49(3): 321-331.

[12.] Adema, M., R.A. Harris, E.C. van DeutekomMulder, 1992. A comparative study of hemocytes from six different snails: Morphology and functional aspects Journal of Invertebrate Pathology, 59 (1): 24-32.

[13.] Soderhall, K. and L. Cerenius, 1998. Role of the prophenoloxidase-activating system in invertebrate immunity, Curr Opin Immunol ., 10: 23-28.

[14.] Ashida, M., PT. Brey, 1998. Recent advances in research on the insect prophenoloxidase cascade. In: PT. Brey, D. Hultmrk, editors. Molecular mechanisms of immune responses in insects. springer, pp: 135-172.

[15.] Aspan, A., J. Sturtevant, V.J. Smith, K. Soderhall, 1990. Purification and characterization of a prophenoloxidase activating enzyme from crayfish blood cells. Insect Biochem., 20(7): 709-718.

[16.] Chou, H.Y., H.J. Li and C.F. Lo, 1994. Pathogenicity of a birnavirus to hard clam (Meretix lusoria) and effect of temperature stress on its virulence. Fish Pathol., 29: 171-175.

[17.] Lacoste, A., F. Jalabert, S.K. Malham, A. Cueff, SA. Poulet, 2001. Stress and stress-induced neuroendocrine changes increase the susceptibility of juvenile oysters (Crassostrea gigas) to Vibrio splendidus. Appl Environ Microbiol., 67(5): 2304-2309.

[18.] Sung, H.H., Y.L. Yang, Y.L. Song, 1996. Enhancement of microbicidal activity in the tiger shrimp Penaeus monodon via immunostimulation. J Crustac Biol., 16(2): 278284.

[19.] Martello, L.B., C.S. Friedman, R.S. Tjeerdema, 2000. Combined effects of pentachlorophenol and salinity stress on phagocytic and chemotactic function in two species of abalone. Aquat Toxicol., 49(3): 213-225.

[20.] Truscott, R., K.N. White, 1990. The influence of metal and temperature stress on the immune system of crabs. Funct Ecol., 4: 455-461.

[21.] Gagnaire, B., H. Frouin, K. Moreau, H. Thomas-Guyon, T. Renault, 2006. Effects of temperature and salinity on haemocyte activities of the Pacific oyster, Crassostrea gigas (Thunberg). Fish Shellfish Immunol., 20(4): 536-547.

[22.] Isabel, Cristina Rossi., S.M. Roselis, da Silva, 1993. Effects of starvation and a carbohydraterich diet on glycogen metabolism in a gastropod mollusc, Megalobulimus oblongus Comparative Biochemistry and Physiology Part A: Physiology, 106(4): 831-836.

[23.] Veldhuijzen, J.P., 1975. Cuperus, Roelck. Effects of Starvation, Low Temperature and the Dorsal Body Hormone On the in Vitro synthesis of galactogen and glycogen in the albumen gland and the mantle of the pond snail (Lymnaea stagnalis. Netherlands Journal of Zoology, 26(1): 119-135(17).

[24.] Porcel, D., J.D. Bueno, A. Almendros, 1996. Alterations in the digestive gland and shell of the snail Helix aspersa Muller (gastropoda, pulmonata) after prolonged starvation Comparative Biochemistry and Physiology Part A: Physiology, 115(1): 11-17.

[25.] Bayomy, M.F., R.van. Elk, J. Joosse, 1987. Effects of starvation and refeeding on egg laying and the synthetic activity of the albumen gland in Bulinus truncatus, a snail vector of urinary schistosomiasis. Comp Biochem Physiol A Comp Physiol., 87(3): 607-12 (ISSN: 0300-9629

[26.] Claudia, R.S., de. Lira, M. Edna Gomes, 2000. Generoso M. Chagas; Jairo Pinheiro. Influence of the starvation on the total proteins and ammonia contents in the hemolymph of Bradybaena similaris (Ferussac) (Gastropoda). Rev. Bras. Zool. 17: 4 Curitiba dic.

[27.] Armelle, Ansart., Vernon. Philippe, Maryvonne Charrier, Jacques Daguzan, 2002. The effect of antibiotic treatment on the supercooling ability of the land snail Helix aspersa (Gastropoda: Pulmonata) Cryobiology, 44(2): 189-192.

[28.] Peters, R., D.A. Raftos, 2003. The role of phenoloxidase suppression in QX disease outbreaks among Sydney rock oysters (Saccostrea glomerata). Aquaculture, 223(1-4): 29-39.

[29.] Lazaridou.Dimitriadou, M. and D.S. Saunders, 1986. The influence of humidity, photoperiod and temperature on the dormacy activity of Helix lucorum L. (Gastropoda; Pulmonata). J. Mollusc. Stud., (52): 180-189.

[30.] Little, C., In: (20th Ed. ed.), 1983. The Colonisation of Land: Origins and Adaptations of Terrestrial Animals, Cambridge University Press, Cambridge, 33-61. 39.

[31.] Riddle, W.A., 1986. Physiological ecology of land snails and slugs. In: (20th Ed. ed.),W.D. Russell-Hunter, Editor.The Mollusca. 6.Academic Press, New York, 431-461.

[32.] Jacqueline Bride, Remy Bonnefoy-Claudet, Lucien Gomot, 1993. Effect of temperature on haemolymphatic glucose and albumen gland polysaccharides during dormancy in the snail Helix aspersa maxima Comparative Biochemistry and Physiology Part A: Physiology, 106(4): 701-705.

[33.] Dormancy, Encyclopedia of Ecology, 2008. 952-957 P.C. Withers, C.E. Cooper

[34.] Aardt, W.J., K.N. van, de Kock, K. Naude, 2003. The respiratory properties of Biomphalaria glabrata exposed to Schistosoma mansoni infection, starvation, CO, and choices of different oxygen concentrations Experimental Parasitology, 103(3-4): 93-101.

[35.] Hesham, M. Sharaf., 2009. Histochemical changes of carbohydrate and protein contents in the digestive gland cells of the land snail Monacha cartusiana following starvation Saudi Journal of Biological Sciences, 16(1): 51-55.

[36.] Butt, D., K. Shaddick, D. Raftos, 2006. The effect of low salinity on phenoloxidase activity in the Sydney rock oyster, Saccostrea glomerata. Aquaculture., 251(2-4): 159-166.

[37.] Hanna Ericson, Gunnar Thorsen, Linda Kumblad, 2010. Physiological effects of diclofenac, ibuprofen and propranolol on Baltic Sea blue mussels Aquatic Toxicology, 99(2-15): 223-231.

[38.] Fernandez, B., J.A. Campillo, C. MartinezGomez, J. Benedicto, 2010. Antioxidant responses in gills of mussel (Mytilus galloprovincialis) as biomarkers of environmental stress along the Spanish Mediterranean coast Aquatic Toxicology, 99(2): 186-197.

[39.] Saleem Aladaileh, V. Sham, Nair. David, A. Raftos., 2008. Effects of noradrenaline on immunological activity in Sydney rock oysters Developmental & Comparative Immunology, 32 (6): 627-636.

[40.] Hauton, C., L.E. Hawkins, S. Hutchinson, 2000. The effects of salinityon the interaction between a pathogen (Listonella anguillarum) and components of a host (Ostrea edulis) immune system. Comp. Biochem. Physiol., 127: 203-212.

[41.] Butt, D., S. Aladaileh, W. O'Connor, D. Raftos, 2007. Effect of starvation on biological factors related to immunological defence in the Sydney rock oyster (Saccostrea glomerata).Science DirectAquaculture, 264: 82-9.

[42.] Yang, S., T.Ruuhola and M.J. Rantata, 2007. Impact of starvation on immune defense and other life history traits of an out breakinggeometrid, Epirria autumnata: a possible causal trigger for the crash phasefor population cycle. Finnish Zoological and Botanical Publishing Board, 44: 89-96.

[43.] Biao, Xu., Chen. Muyan, Yang. Hongsheng, Zhao. Sanjun., 2008. Starvation-induced changes of hemocyte parameters in the Zhikong scallop Chlamys farreri. Journal of Shellfish Research, 6: 420.

[44.] Saleem Aladaileh, V. Sham Nair, Debra Birch, A. David Raftos, 2007. Sydney rock oyster (Saccostrea glomerata) hemocytes: Morphology and function Journal of Invertebrate Pathology, 96(1): 48-63.

Corresponding Author

Atika AL-Rawadeh , Department of Biological Sciences, Collegeof Science , AL Hussein Bin Talal University E-mail: atikaalrawadeh@yahoo.com

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
COPYRIGHT 2010 American-Eurasian Network for Scientific Information
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2010 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:Original Article
Author:Al-Rawadeh, Atika
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:1USA
Date:May 1, 2010
Words:3029
Previous Article:Sustainable control of Striga hermonthica in maize (Zea Mays L.) by the use of Parkia biglobosa based products and post-emergence herbicides.
Next Article:Effect of temperature on phagocytosis activity in garden snails Helix aspersa.
Topics:

Terms of use | Copyright © 2017 Farlex, Inc. | Feedback | For webmasters