INTRODUCTION
Inorganic ions play an important role in the live of mollusks. Calcium and sodium play an essential role in the nerve-muscle transmission; Ca (HCO₃)₂ functions as a buffer in the haemolymph¹. The phagocytic activity of hemocytes and lectins cooperating in defense reactions depends on the presence of calcium ions in hemolymph². Mishkin and Jokinen³ reported that environmental calcium has a positively effects on the fecundity and cercarial production of B. glabrata infected with S. mansoni. Moreover, large amount of calcium are used in the reproduction of the snails⁴. Magnesium plays a fundamental role in the regulation of many cellular functions such as protein synthesis and enzyme activation. Amongst the enzymes in which Mg²⁺ acts as an essential co-factor are those concerned with glycolysis, respiration and membrane transport processes, e.g. Na⁺ and Ca²⁺ pumps⁵. Na⁺/K⁺-ATPase has a critical vital function in the maintenance of plasma membrane potential difference in all animal cells, pumping Na⁺ and K⁺ against their concentration gradients to maintain high sodium levels outside cells and high potassium inside. The pump consumes a great deal of energy; for example, in resting endotherms it is responsible for 5-40% of total ATP consumption, depending on cell type⁶.

The disturbance occurred in the metallic ion concentrations in the snails infected with trematode a larva was considered as one of the causes of alterations occurred in the biological activities and the increase in the mortality of the infected snails^7,8,9,10.

Alteration in the calcium content was in the focus of interest of many authors due to its importance in the life of mollusks and the hypothesis of hypercalcification; that is, the increase in the calcium content of the shell of their snail hosts due to larval trematodes induce was discussed by several investigators^{9,10,11,12,13,14,15,16}.

The present study aimed to study the changes in the concentration calcium, potassium, magnesium and sodium in soft parts and shells of Biomphalraia alexandrina naturally infected with Schistosoma mansoni or Echinostoma liei and to examine the hypothesis of hypercalcification in shells of infected snails.

MATERIALS AND METHODS
Biomphalaria alexandrina snails were collected from Nile River at Kafer Alsheikh and Menofia Provinces, Egypt and transferred to Medical Malacology laboratory at Theodor Bilharz Research Institute (TBRI), Egypt. Snails were examined immediately for trematode infection by exposing them to artificial light to induce cercarial shedding. Shedding snails were isolated and kept at -20°C until used in analysis. The remaining snails were crushed and examined under binocular microscope; snails free from any trematode larval stages were isolated and kept at -20°C until used for comparison with infected snails from the same field. Non-infected, lab bred B. alexandrina snails were obtained from Medical Malacology laboratory at TBRI and used for comparison with non-infected, field collected snails. The diameter of the shells used in the present study was ranged from 5-8 mm.

Soft parts were separated from shells and both were rinsed, at least, three times with deionized water. Excess water was removed from the specimens by using filter papers. Three pools of soft parts and shells from: snails shedding S. mansoni cercariae, snails shedding E. liei cercariae, non-infected, field collected snails and non-infected, lab bred snails were prepared for analysis. Wet-weighted samples were digested in 10 mL of concentrated nitric acid by boiling to dryness. The residue from each digested sample was diluted to 25 mL with deionized water in a volumetric flask.

Elemental analysis by flame atomic absorption spectrometry using Perkin-Elmer model 3100 AAS was performed to determine the concentration of heavy metals Al, Cd, Cu, Fe, Mn, Pb and Zn in the soft parts and shells of the snails. The flam wavelength and sample aspiration rate were optimized according to the manufacturer’s recommendations and four aqueous standards having analytic concentrations within the linear response range of the instrument and containing the same concentration of nitric acid as the samples were used for calibration. Each sample, standard and blank, was analyzed using three 10 sec integrations. The reagent blank was prepared and its value was subtracted to give the final concentration. The final element concentration(C) was calculated according to the following equation:

where, F is the standard factor calculated from the standard curve, V is the volume of sample and wt is the wet weight of sample. Data of potassium, magnesium and sodium are expressed in micrograms of element per gram of wet tissue and data of calcium are expressed in percentage of weight of wet tissue. Results were subjected to one-way ANOVA test followed by post hock Duncan test using SPSS program version 8 to determine the significant of data.

RESULTS
In comparison with lab-bred snails, the Ca content in non-infected snails collected from Kafer Alsheikh province was insignificantly higher; in contrast it was significantly lower in snails collected from Menofia province. Generally, Ca concentration was significantly higher in shell than soft parts regardless the status of snails. In comparison with non-infected, field collected snails, the infection with E. liei leads to increase of Ca content in the shells of snails collected from Kafer Alsheikh and Menofia. However, the infection with S. mansoni leads to insignificant decrease of the same element in the shell if compared with non-infected, field collected snails (Table 1).

The concentrations of K and Mg were significantly higher in soft parts than shells in all tested snails. However, the total concentration of the two elements was significantly higher in non-infected, lab-bred than in non-infected, field-collected ones. Na concentration was significantly higher in the shell than soft part in all snails examined. In non-infected snails, the total concentration of Na was higher in field collected snails than in lab-bred snails (Table 2).

In comparison with non-infected, field collected snails, infection with E. liei leads to significantly increase in the concentrations of K, Mg and Na elements in snails collected from Kafer Alsheikh and Menofia provinces. On other hand, in snails infected with S. mansoni, K⁺ and Na⁺ concentrations were significantly lower when compared with non-infected, field collected snails and Mg concentration was significantly higher when compared with the same snails.

Table 1: Alteration in the concentration of Ca ion (%) in non-infected and trematode-infected Biomphalaria alexandrina

Table 2: 2: Alteration in the concentration of K, Mg and Na ions in non-infected and trematode-infected Biomphalaria alexandrina

DISCUSSION
In the present investigation, the calcium content of non-infected snails collected from Kafer Alsheikh province was significantly higher than that in lab-bred, non-infected snails and the contrast is true for snails collected from Menofia province. Such changes may be due to the calcium content in the water from which snails collected, such suggestion was supported by Zblkowska¹⁴ who found no differences in the calcium carbonate concentrations in Lymnaea stagnalis naturally infected with digenean larvae except in a case where the infected snails came from a lack with a low calcium concentration; such author suggested that the calcium content of water was responsible for calcium carbonate concentration in the snails rather than the presence of digenean larvae within it. Moreover, Young and Harris¹⁷ reported that reduction of calcium concentration in water can reduce the occurrence of the snails in aquatic system.

In the present work, calcium content was significantly higher in the shells than in the soft parts of the snails, regardless infected or non-infected. This observation was correlated with that of White et al.¹⁵ who mentioned that under conditions of variable Ca concentrations in the water and trematode parasitism, pulmonate snails are able to maintain a high concentration of CaCO₃ in their shells.

Hypocalcification observed in the shells of S. mansoni-infected B. alexandrina snails in the present study was correlated with the observation of White et al.¹⁵ on their study on the effect of S. mansoni on Heliosoma trivolvis, B. glabrata and Physa sp. and with the observation of Mostafa¹⁶ on his study on B. alexandrina and Bulinus truncatus snails shedding S. mansoni and S. haematobium cercariae respectively. In addition, Mostafa⁹ observed a hypocalcifiction in L. natalensis infected with Fasciola gigantic. The cercariae of S. mansoni sequester large amounts of calcium in their pre-acetabular glands and such sequestration probably occurs at expense of calcium in the shell and haemolymph of the snail¹⁸. Therefore, we can suggest that hypocalcification observed in the shells of S. mansoni-infected B. alexandrina snails in the present study may be due to the cercariae within that snails utilized large amount of calcium which may be compensated by calcium from the shell and in contrast the hypercalcification noted in the shells of E. liei-infected B. alexandrina snails may be due to the E. liei cercariae within that snails utilized small amount of calcium. Davies and Erasmus¹⁹ reported that B. glabrata containing the larval stages of S. mansoni at 40 days post infection show disintegration of the calcareous corpuscles in Type-A calcium cells and erosion of the inner surface of the shell.

The hypercalcification observed in the shells of snails infected with E. liei was in agreement with several authors. Mazuran et al.⁴ reported that large amounts of calcium were used in reproduction of snails; the inhibition of reproduction caused by the parasites could affect calcium distribution, leading to its deposition in the shells and mantle¹².

Thus we can report that the hypercalcification hypothesis was not upheld in all of the snail-larval digenean system studied here. The present findings have not validated the generalization of hypercalcification hypothesis of snail shells due to infection by parasites.

In the present investigation, infection of B. alexandrina snails with E. liei lead to increase in the concentration of K, Mg and Na in the soft parts of snails collected from both Kafer Alsheikh and Menofia provinces; this was in agreement with Ong et al.²⁰ in their study of the effects of S. mansoni infection on B. glabrata snails. However, the concentration of Mg and Na in the soft parts of B. alexandrina infected with S. mansoni was increased agreement with Ong et al.²⁰ but the concentration of K was decreased in snails collected from Kafer Alsheikh. In addition Layman et al.²¹ reported that Na was present in the digestive gland-gonad complex at higher concentration in infected snails relative to the uninfected snails. However, these results are in contrast to the findings of Evans et al.²² and Bergey et al.²³ in which parasitism lowered the amounts of certain elements in infected hosts. On other hand, the alteration pattern of the same element within the same snails from the same habitat may differ according to the type of the digenean infection. This was correlated with the observation of Hassan²⁴ in here study on the effects of digenean infection on the metallic ions of Lanistes carinatus snails collected from River Nile at Sohage province; she found that the K ion was increased in digestive gland of snails infected with xiphidiocercariae and was decreased in digestive gland of snails infected with gymnocephalus cercariae. Thus we can concluded that there was no fixed pattern of inorganic ions alterations in snails infected with digenean trematode.

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