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
snails7,8,9,10.
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 investigators9,10,11,12,13,14,15,16.
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 manufacturers
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.
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Table 1: Alteration in the concentration
of Ca ion (%) in non-infected and trematode-infected Biomphalaria
alexandrina |
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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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