Measurement of Selected Enzymatic Activities in Solanum nigrum-Treated Biomphalaria arabica Snails
Sooad Al- Daihan
In the present study, glucose, acid and alkaline phosphatases
(ACP and ALP), α-amylase and lipase were measured for the first time
in tissue homogenates of Biomphalaria arabica snails, molluscan
intermediate host for Schistosoma mansoni in Saudi Arabia. Also,
the effect of sublethal concentrations (LC25) of dry powdered
Solanum nigrum leaf was tested as plant molluscicide against this
snail species. The tested enzymes were altered in molluscicide-treated
snails compared to control. While ALP and amylase were slightly affected,
ACP and lipase were significantly altered. Glucose as an important energy
source for a successful schistosome-snail relationship was significantly
reduced in molluscicide-treated snails. In conclusion, sublethal concentration
of the molluscicide showed potent effect in disturbing snail biochemistry
which may render them physiologically unsuitable for the developing of
schistosome parasite. This could be considered as a promising strategy
to control the disease.
Schistosomiasis, also known as bilharziasis is a major parasitic disease
of humans. It is thought to infect about 200 to 300 million people across
Africa, South America, the Caribbean, the Middle East, China and Southeast
Asia. The major causes of the disease are Schistosoma mansoni,
Schistosoma japonicum and Schistosoma haematobium. Transmission
of the schistosomes to humans is via contact with fresh water that contains
the parasite`s intermediate snail host and that has been contaminated
by urine or faecal material from infected individuals. As intermediate
hosts, molluscs play a major role in the transmission of schistosomes;
they are the sites of an intense multiplication of parasites. Thus, snail
control strategies are considered a priority for the reduction of schistosomiasis
(Lardans and Dissous, 1998). The high cost of synthetic molluscicides,
used in the control of the intermediate snail hosts of schistosomiasis,
has resulted in renewed interest in plant molluscicides. The prohibitive
costs (time and financial) of random surveys for activity necessitated
the development of an objective selection procedure. Species were ranked
on cumulative scores for: (a) coincidence of the endemic areas of the
plant, snail host and disease; (b) ethnomedicinal value which would provide
greater incentive for cultivation and (c) molluscicidal activity (if known,
a minimum LC90 of < or = 100 ppm) (Clark et al.,
1997). A simple chemical will probably not be applicable for all situations;
hence we must continue to investigate other control options and develop
an integrated approach to schistosome-snail control. At present, the only
viable molluscicide in terms of efficacy and complete evaluation is bayluscide
but it is highly toxic to fish (LC50 0.03-0.23 mg L-1)
(United States Environmental Protection Agency, 1999).
The development of new, specific and naturally occurring plant molluscicide
was favoured by the World Health Organization (WHO). Many plant species
have been evaluated as sources of naturally chemicals to control schistosome
snails (Silva et al., 2007). The study of plants exhibiting snail
toxicity has been encouraged with the aim of finding alternatives for
use in the fight against snail vectors. The aim of studies on plant molluscicides
is to complement methods for controlling snails acting as intermediate
hosts of schistosomes. Attenuation of cercariae released from molluscicide-treated
snails leading to reduced infectivity in mice could be achieved in concentrations
below those exerting acute toxicity (i.e., sublethal concentrations) (El-Ansary
et al., 2003; Abdel Kader et al., 2005).
Biological markers have been defined as xenobioticaly-induced variations
in cellular or biochemical components in any biological system (Icen et
al., 2005). Carbohydrate metabolizing enzymes (e.g., α-amylase),
lipid degrading enzymes (e.g., lipase) and phosphatases (acid and alkaline
phosphatases) were recorded among target enzymes which should be disturbed
and may provide more accurate information on the molluscicide-induced
stress on molluscs. Impairment of these enzymes could be effective in
affecting the feeding and reproductive competence at the mollusc population
level (fertility, fecundity and reproductive rate).The effect of sublethal
concentrations of different synthetic and natural molluscicide agents
on reproduction and biochemical aspects of schistosome snail hosts is
recently encouraged (Abdel Kader et al., 2005). These information
initiated our interest to measure ACP, ALP, amylase, lipase and glucose
in tissue homogenates of B. arabica, the snail hosts to S. mansoni
in Saudi Arabia. The present study was extended to evaluate the effect
of sublethal concentration (LC25) of Solanum nigrum
as a potent plant molluscicide on these biochemical parameters.
MATERIALS AND METHODS
Snail collection and maintenance: Specimens of B. arabica
were collected from a farm near Riyadh on June 2005. The snails were left
in the lab for 45 days and were examined to be sure that they were free
from parasitic infection. They were fed with lettuce leaves ad lib.
A sample of the snails was randomly chosen and dissected.
Preparation of tissue homogenate: One gram of snail soft tissue
was homogenized in 5 mL distilled water and then centrifuged at 3000 rpm,
the supernatant was used for the biochemical analyses (Nabih et
Molluscicide-treatment: This was performed according to the toxicity
study of S. nigrum plant on B. arabica snails previously
done by El-Ansary and Al-Daihan (2007). Four groups 10 snails each were
exposed to 3 ppm concentration of S. nigrum (LC25) dissolved
in dechlorinated water in 1 L capacity tank. Snails were fed fresh lettuce
leaves ad lib during the 24 h contact period. Dead snails were
discarded and the remaining snails were used for the biochemical analysis.
Untreated control groups were established.
Biochemical analysis Measurement of glucose: Glucose was measured
according to the method of John and Turner (1975) using a diagnostic kit
a product of United Diagnostic Industry (UDI), Riyadh, KSA.
Enzyme assays Measurement of phosphatases: ACP and ALP were measured
according to the method of Bowers and McComb (1966), using a kinetic diagnostic
kit, a product of United Diagnostic Industry (UDI), Riyadh, KSA.
Measurement of lipase: Lipase was measured according to the modified
method of Vogel and Zieve (1963) turbidimetric method using a diagnostic
kit a product of United Diagnostic Industry (UDI), Riyadh, KSA.
Measurement of amylase: Amylase was measured according to the
method of Wallenfels et al. (1978) using a diagnostic kit a product
of United Diagnostic Industry (UDI), Riyadh, KSA.
Statistical analysis: The statistical analysis of the data was
carried out using Student t-test (GraphPad Prism computer program).
RESULTS AND DISCUSSION
Table 1 shows the enzymatic activities of ALP, ACP,
amylase, lipase and glucose in control and S. nigrum-treated B.
arabica snails. Values are expressed as mean±SD of four different
measurements. It can be easily seen that lipase was the most affected
enzyme shows a highly significant lower activity in molluscicide-treated
snails. ACP shows less significant reduced activity compared to control.
On the other hand glucose, ALP and amylase were non-significantly affected.
Figure 1 shows the percentage changes induced by the
molluscicide in the measured parameters.
Ingested molluscicides are only partly transported down the digestive
tract in the food mass and large proportion is quickly resorbed by the
cells of the oesophagus (Triebskorn, 1995). After being released from
these cells into the haemolymph, molluscicides molecules are distributed
in the body quickly transported to peripheral tissues and organs.
In the present study, the molluscicidal activity of S. nigrum against
B. arabica snails could be confirmed by the previous reports of Silva
et al. (2005, 2006), Bekkouche et al. (2000) and Mkoji et
al. (1989) who recorded a potent molluscicidal activity of this plant against
B. glabrata, B. pfeifferi, Bulinus globosus and Lymnea
||Enzymatic activities of ALP, ACP, amylase, lipase and
glucose in control and S. nigrum-treated B. arabica
Percentage changes induced by the molluscicide in the
ALP, ACP, amylase, lipase and glucose
ACP and ALP have been shown to be influenced by molluscicide treatment,
where the enzymes of this group can catalyses the breakdown of ester bonds
in the orthophosphate esters under acidic and alkaline conditions, respectively.
In B. glabrata, molluscan snail host to S. mansoni, ACP
and ALP were detected histochemically among the enzymes that are important
for the encapsulation reaction formed around schistosme sporocysts (McKerrow
et al., 1985). A significant difference in the distribution and
abundance of acid phosphatase as a lysosomal enzyme marker in blood cells
(hemocytes) of schistosome-susceptible (PR albino M-line) and a resistant
(10-R2) strain of B. glabrata during the course of infection with
S. mansoni was recorded (Granath and Yoshino, 1983; Sasaki et
al., 2005). They reported that, a significant increase of ACP 2 week
PE, indicating a possible response to tissue damage resulting from migrating
daughter sporocysts. On the other hand, alkaline phosphatase does not
show any significant different in activity between normal and infected
snails (Dardenne et al., 1979). In the present study, both enzymes
were inhibited but only ACP showed statistically significant inhibition
(p<0.008). This is in good agreement with the previous reports of Ahmed
and Refaat (2004) who observed the inhibition of these enzymes in animals
treated with naturally occurring molluscicides. Moreover, present results
could be supported by the previous results of El-Ansary et al.
(2000) and who recorded a potent molluscicidal activity of S. nigrum
and recorded the impairment of hexokinase, glucose isomerase and pyruvate
kinase in S. nigrum-treated B. alexandrina snails. In their
study they recorded that LC10 concentrations of S. nigrum
reduced considerably the infection rate of B. alexandrina
with S. mansoni to be 34% compared to an infection rate of 80%
in control, non-treated snails. Longer prepatent period and remarkable
decrease in cercarial production. This confirms that sublethal concentration
of S. nigrum is effective in disturbing the metabolism of the snail
host to be less compatible to S. mansoni parasite (El-Ansary et
Nabih et al. (1998) reported that B. alexandrina and Bulinus
truncatus as mo lluscan hosts to S. mansoni and S. haematobium,
respectively have higher α-amylase activity and lower Km value to
starch when compared to Lymnea truncatula and Physa acuta
as non-target to schistosome parasites. They recorded that highly active
amylase is of critical importance for the success of host-parasite relationship.
Highly active amylase could easily provide the developing parasite with
glucose as important nutrient needed by the developing intramolluscan
parasite. In the present study, lower activity of α-amylase was observed
in S. nigrum-treated snails, which could be easily correlated to
the recorded lower glucose level. Importance of glucose for the development
of schistosome larvae in B. arabica as a molluscan hosts to S.
mansoni in Yemen was previously recorded (El-Sheikh and Nagi, 1991)
who reported that Schistosome infection caused a marked decrease in tissue
glycogen and protein of B. arabica and remarkable lower glucose
content in the hemolymph of infected snails compared to control.
P31 NMR studies demonstrated that the levels of free phospholipids,
particularly phosphatidylcholine, in the digestive gland of the snail,
B. glabrata, were dramatically reduced when the host was infected
with S. mansoni. It was speculated that absorption of host phosphatides
may be an important source of membrane phospholipid precursors and fatty
acids for developing sporocysts and cercariae (Thompson et al.,
1991). Regarding the effect of molluscicide treatment on the enzymatic
activity of lipase, it can easily be noticed that lipase was the most
affected enzyme showing very high significant inhibition (p<0.0002).
The remarkable inhibition of lipase in S. nigrum-treated snails
could easily render the snail host physiologically unsuitable for the
development of schistosome parasite since it is well known that schistosomes
are unable to synthesize fatty acids de novo but absorbed it from
their snail host tissues and haemolymph.
The obtained data could be supported by the previous reports of Ahmed
and Ramzy (1997) who recorded that B. alexandrina snails exposed
to sublethal concentration of S. nigrum shed significantly
fewer cercariae than unexposed snails. Moreover it could clarify the previous
study of El-Ansary et al. (2003) which reported that attenuated
cercariae released from S. nigrum-metabolically disturbed snails
has reduced pathogenicity to the mammalian hosts. Reduction of cercarial
number released from S. nigrum treated snails (Ahmed and Ramzy,
1997) could be attributed to the significant lower ACP activity. Lower
ACP activity could induce the cellular immune response of the host resulting
in the killing of larger number of intramolluscan schistosome larvae,
which lead to shedding of fewer cercariae. Moreover, lower lipase activity
could lead to the production of cercariae with disturbed lipid profile
which could be easily correlated to the lower pathogenicity to the mammalian
hosts previously reported by El-Ansary et al. (2003). In view of
the present results, S. nigrum could become an affordable and effective
component of an integrated approach to schistosomiasis control in Saudi
The authors are grateful for the Deanship of Scientific Research, King
Saud University, Kingdom of Saudi Arabia for Supporting this work as an
Applied National Research Project Grant DSR-AR-2 (35).
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