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375
J. Egypt. Ger. Soc. Zool. ISSN 1110-5321
Http:www.egsz.com The 19th
International Conference
Email: [email protected]. 30 April-2 May, 2011
Vol. (63A): Comparative physiology, 375-397 Faculty of Science,
Beni-Sueif University
Rec. 28/5/2011 July 2011
PHYSIOLOGICAL CHANGES IN THE BROWN GARDEN
SNAIL, EOBANIA VERMICULATA INDUCED BY SUBLETHAL
DOSES OF TWO BOTANICAL MOLLUSCICIDES
Samir Mohamed H. Beltagi, Mohammed Salah. A. Al-Shinnawy,
Nabawy Abdel-Rahman I. Elkattan and Hany Nady Yousef
Department of Biological and Geological Sciences, Faculty of
Education, Ain Shams University
Keywords: Thymol; Nicotine; Snails; Eobania vermiculata;
Haemolymph, Digestive gland.
ABSTRACT
The present work was carried out to study the physiological
responses of Eobania vermiculata snails, one of the agricultural pests in
Egypt, to sublethal doses (LD25 and LD50) of two potent botanical
molluscicides (Thymol and Nicotine) after 1, 7 and 15 days post
exposure as a recovery period using the topical application technique.
Treated snails showed common signs of toxicity as excessive
production of mucus along with specific symptoms as haemolysis in the
case of snails treated with LD50 Thymol or Nicotine in addition to
paralysis of the foot in the case of Nicotine-treated snails. Snails treated
with LD50 Thymol exhibited a significant decrease in the heart rate;
while the rest treatments caused an elevation in the heart rate along the
three experimental periods. A general significant increase in levels of
some biochemical parameters (total proteins, total lipids, total
cholesterol and glucose levels) of the haemolymph were detected in most
cases. Both examined materials caused a marked enhancement in the
activity of acid phosphatase (ACP), aspartate aminotransferase (ASAT)
and alanine aminotransferase (ALAT) enzymes in the haemolymph;
while the activity of alkaline phosphatase (ALP) exhibited a significant
suppression. In addition, the examined biochemical parameters (total
soluble proteins, total lipids and glycogen content) in the digestive gland
exhibited marked reduction in response to both examined materials at the
three post exposure periods.
Conclusion, It was concluded that application of both tested
materials interfere with the snails' physiology and the recovery period
Physiological changes in the brown garden snail, Eobania vermiculata induced by
sublethal doses of two botanical molluscicides
376
did not enable the treated snails to eliminate the adverse effects of their
application; so Thymol and Nicotine may be of great value in controlling
the terrestrial snails but further studies are needed to evaluate their
efficacy as safe and economic molluscicides in the field.
INTRODUCTION The terrestrial snail Eobania
vermiculata, family Helicidae, is a
cosmopolitan agricultural pest. In
Egyptian fields, this snail causes a
great damage to all plant parts of
different vegetations including
orchard trees, vegetable crops as
well as ornamental plants (El-
Okda, 1979 and Mahrous et al.,
2002).
For avoiding the deleterious
effects induced by application of
synthetic molluscicides, much
effort has been focused on plant
materials for potential use as
commercial pesticides in the hope
that they might provide economic,
locally produced, biodegradable,
environmentally safe and effective
control agents (Hussein et al.,
1994; Hollingsworth et al., 2003
and Gabr et al., 2006).
After dermal application, the
primary targets for molluscicides
are the epithelial cells of skin
including the mucus cells
(Triebskorn et al., 1998). The
gastropod skin is known to be
involved in the acquisition and
resorption of various molecules
and free ions from the
environment (Henderson, 1970;
Zylstra, 1971; Machin, 1977;
Ryder and Bowen, 1977 and
Bullock et al., 1992). Chemicals
passing through the skin reach the
haemolymph and are thus
transported throughout the body
(Henderson and Triebskon, 2002).
Mode of action studies aim to
discover which molluscan systems
are affected by molluscicides.
These systems include activity at
the cellular level, uptake into the
snail, distribution, metabolism and
excretion (Duncan, 1987).
Therefore, Physiological and
biochemical responses of snails
are very important means for
understanding the mode of action
of molluscicides.
The objectives of the present
study were to elucidate the
physiological and biochemical
alterations in the haemolymph and
digestive gland of the brown
garden snail, E. vermiculata in
response to sublethal doses of two
potent botanical molluscicides
(Thymol and Nicotine) using the
topical application technique.
MATERIALS AND METHODS
Collection and adaptation of
snails
Adult specimens (20-30 mm
shell diameter) of the land snail,
E. vermiculata were collected
from Al-Montazah Park,
Alexandria governorate during
spring 2010. The collected snails
were transferred in cloth sacs to
the laboratory and kept in aerated
cages (40×30×30cm, with 100
individuals per cage) for two
weeks to acclimatize with the
laboratory conditions (26-30 °C
and 62±2 RH). Snails were fed
Beltagi, S.M.H., et al.
- 377 -
fresh lettuce leaves three times a
week.
Bioassays
Laboratory bioassays were
previously carried out in our
laboratory (Beltagi et al., 2010)
for evaluating the efficacy of
certain plant materials including
Nicotine (C10H14N2, MW 162.23,
purity 99%), Caffeine (C8H10N4O2,
MW 194.19, purity 99%), Thymol
(2-[(CH3)2CH] C6H3-5-(CH3)OH,
MW 150.22, purity 99.5%),
Menthol (C10H20O, MW 156.27,
purity 99%) and Camphor
(C10H16O, MW 152.23, purity
95%) as molluscicides against the
brown garden snail, E. vermiculata
using the topical application
method. The obtained results
(Table 1) proved that Nicotine and
Thymol were the most promising
from the molluscicidal point of
view with LD50 of 204.02 and
551.20μg/snail for the two
materials, respectively. All Plant
materials used in the present study
were purchased from Sigma-
Aldrich Company.
Experimental design
The experimental animals
were divided into five groups,
with 500 snails for each:
Group I (Control snails): Each
snail of this group was treated,
using the topical application, with
a single dose of 30µl of DMSO.
Group II (LD50 Thymol-treated
snails): Animals of this group
were topically treated with a
single dose of 30µl of LD50
Thymol.
Group III (LD25 Thymol-treated
snails): This group was topically
treated with a single dose of 30µl
of LD25 Thymol.
Group IV (LD50 Nicotine-
treated snails): In this group a
single dose of 30µl of LD50
Nicotine was topically applied on
the body surface of each snail.
Group V (LD25 Nicotine-treated
snails): Animals of this group
were received a single dose of
30µl of LD25 Nicotine.
Snails of the five groups were
supplied with lettuce leaves three
times a week. Few milliliters of
water were added daily into each
box to provide suitable humidity
for snail activity.
Clinical signs of toxicity: Behavioral signs of toxicity
for each experimental group were
noted directly post exposure.
Estimation of heart rate:
At the experimental time
periods, 1, 7 and 15 days post
treatment, 10 survived snails were
taken randomly from each
experimental group. As heart rate
is inversely proportional to size
(Lee and Cheng, 1971), care was
taken to choose snails of the same
size for this experiment. The
snails wiped dry with a cloth and
a small window was made in the
shell above the heart then the time
required to complete 10
successive ventricular
contractions was recorded. The
mean of three records was
calculated for each snail then the
mean reading for all snails of each
experimental group was
Physiological changes in the brown garden snail, Eobania vermiculata induced by
sublethal doses of two botanical molluscicides
- 378 -
calculated. The times recorded
were then converted to
beats/minute.
Collection of haemolymph and
preparing tissue homogenate
At the experimental time
periods, 30 survived snails were
taken from each experimental
group, and then were divided into
6 groups each of 5 snails. The
selected snails of each group were
then allowed for sampling 6
replicates (n=6). Each snail was
wiped dry with a cloth then a
small window was made in the
shell above the pulmonary cavity
using incisor. The pulmonary vein
passing through the wall of the
cavity was cut, so the
haemolymph released freely. The
released haemolymph from the
five snails of each replicate
(ranging from 1.5-2 ml) was
collected in a tube embedded in
crushed ice and centrifuged at 0 ◦C in high speed (10000 rpm) for
five min. in order to remove the
haemocytes and cell debris. The
resulting supernatant was freezed
at -20 o
C until used in the analyses
described below.
The digestive glands were
dissected out from the five snails
of each replicate and then divided
into two portions. The first
portion was weighed and then
homogenized at 0 ◦C using
bidistilled water (0.5g. tissue/5ml
bidistilled water) using a mortar
and pestle embedded in crushed
ice. The homogenate was
centrifuged at cold for 10 min. at
10000 rpm and the resulting
supernatant was then used for
analysis of proteins and lipids.
The other portion of digestive
glands was weighed and
homogenized in 5%
trichloroacetic acid (0.5g.
tissue/5ml 5% TCA) at 0 ◦C to be
used for glycogen determination.
Biochemical measurements
Total soluble proteins in
haemolymph and digestive gland
were determined according to the
method of Biuret as described by
Kachmar (1970a) using bovine
serum albumin as standard; while
total lipids were measured
according to the method of Fring
et al. (1972). Total cholesterol of
haemolymph was estimated by the
CHOD-PAP enzymatic
colorimetric method of Richmond
(1973) using reagent kits
purchased from the Egyptian
company for Biotechnology.
Glycogen concentration in the
digestive gland was estimated
using the anthrone reagent
according to the method of
Carroll et al. (1956); while
glucose level in haemolymph was
determined by the method of
Trinder (1969) using reagent kits
manufactured by Audit
Diagnostics, Business &
Technology Park, Carrigtwohill,
Co. Cork (Ireland).
Aminotransferases (ASAT and
ALAT) activities in haemolymph
were estimated by the method of
Reitman and Frankel (1957) using
reagent kits purchased from the
Egyptian company for
Biotechnology. Alkaline
phosphatase (ALP) activity in
haemolymph was estimated
Beltagi, S.M.H., et al.
- 379 -
according to the method of DGKC
(1972) using reagent kits
manufactured by Audit
Diagnostics, Business &
Technology Park, Carrigtwohill,
Co. Cork (Ireland). Acid
phosphatase (ACP) activity in
haemolymph was measured
following the method of Kind and
King (1954).
Statistical analysis
Biochemical data were
expressed as Means±SE and
Student's t test was applied to
locate significant (P<0.05)
differences between treated and
control groups using SPSS 17.0
on Windows® platform Version
2002.
RESULTS
Clinical signs of toxicity
Fig. (1) shows some of the
behavioral symptoms appeared on
the treated snails following the
exposure to the applied doses of
the examined materials. Most of
the treated snails appeared
inactive and remained retracted
within their shells. Others were
partially extended but their
response to the mechanical
stimulation was slow. Also,
excessive production of mucus
was obvious in all cases.
Haemolysis was recorded in
members of group II and IV.
Paralysis of the foot was recorded
in some snails treated with
Nicotine of both sublethal doses.
Heart rate
Data represented in Fig. (2)
indicated that, except for
specimens treated with LD50
Thymol, all groups exhibited
elevations in their heart rate. This
elevation was statistically
significant (p<0.05) along the
three periods post exposure. On
the other hand, the reduction in
heart rate recorded in the case of
LD50 Thymol-treated group was
statistically significant (p<0.05) at
the first and second time periods
post exposure; while it was
insignificant (p>0.05) at the last
time interval post exposure. Also,
the data represented in Fig. (2)
indicated that the deviation of the
treated groups from the
corresponding controls was least
pronounced at the end of the
recovery period (15 days post
treatment) but these snails
remained unable to return to their
normal state.
Biochemical changes of the
haemolymph
The data of the biochemical
analyses indicated (total proteins,
total lipids, total cholesterol and
glucose levels) in the
haemolymph of E. vermiculata
snails exposed to the sublethal
doses of Thymol and Nicotine
along the three time intervals post
exposure are shown in Table (2).
There was a very highly
significant increase (p<0.001) in
the mean values of total proteins
among snails treated with either
Thymol or Nicotine when
compared with the corresponding
controls. The maximum increase
was observed after 1 day post
exposure; while it was the least at
the end of the period of recovery
in all cases.
Physiological changes in the brown garden snail, Eobania vermiculata induced by
sublethal doses of two botanical molluscicides
- 380 -
Snails treated with Nicotine,
either low or high sublethal doses,
showed marked increases in the
mean values of total lipids relative
to the corresponding controls
along the three periods post
exposure; while the snails treated
with Thymol showed fluctuation
in their content of lipids as they
recorded an increase after 1 day
post exposure, then the values
decreased in a very highly
significant manner (p<0.001) after
7 days post exposure and returned
back to increase significantly at
the end of the recovery period.
General elevation in the
values of total cholesterol was
recorded along the three periods
post exposure for both examined
materials but this elevation was
statistically insignificant (p>0.05)
in most cases as compared with
the corresponding controls.
The level of glucose in
haemolymph of snails treated with
LD50 Thymol was increased along
the experimental periods but this
elevation was statistically
insignificant at the first
experimental period. In addition,
snails treated with LD25 Thymol
showed a very highly significant
(p<0.001) elevation in their levels
of glucose after 1 day post
exposure but this level was
dropped insignificantly at the next
experimental periods. On the
other hand, snails treated with
Nicotine showed a very highly
significant increase (p<0.001) in
their levels of glucose in
comparison with the
corresponding controls along the
three periods post exposure. This
elevation was most pronounced at
the last period post exposure
reaching 94.56% and 141.93%
higher than the corresponding
controls for the low and high
sublethal doses of Nicotine
respectively..
Table (3) shows the change in
enzymatic activities (ASAT,
ALAT, ACP and ALP) in
haemolymph of E. vermiculata
snails in response to sublethal
doses of Thymol and Nicotine
along three time intervals post
exposure.
From the recorded results it
seems obvious that, treatment of
snails with both the high and low
sublethal doses of the two applied
plant molluscicides enhanced
markedly the activities of ASAT,
ALAT and ACP enzymes in
comparison with the equivalent
untreated snails along the three
periods post exposure. The levels
of these enzymes were decreased
gradually, in most cases, with the
succession of time post exposure
achieving their lowest values at
the end of the recovery period for
all cases. Regarding the activity of
ALP enzyme, all treated snails
exhibited marked suppression as
compared with the corresponding
controls along the three
experimental periods. The
recorded reduction was most
pronounced after 1 day post
exposure then decreased gradually
until recording its lowest values at
the end of the recovery period.
Beltagi, S.M.H., et al.
- 381 -
Biochemical changes of the
digestive gland
The data of biochemical
parameters (total soluble proteins,
total lipids and glycogen content)
in the digestive gland of E.
vermiculata snails treated with
sublethal doses of Thymol and
Nicotine along the three time
intervals post exposure were
shown in table (4).
The recorded data revealed
that there was a very highly
significant reduction (p<0.001) in
the mean values of total proteins,
total lipids and glycogen content
in the digestive gland of the snails
exposed to either Thymol or
Nicotine when compared with the
equivalent controls at the three
post exposure periods.
DISCUSSION
Increased mucus production
followed by increased mucus
secretion is one of the first
reactions of gastropods to many
kinds of stressors, including
chemical irritation caused by
molluscicidal chemicals (Godan,
1983; Triebskorn and Ebert,
1989; Triebskorn et al., 1998).
This phenomenon was recorded in
snails treated with both the tested
materials. One effect of the
produced mucus is to form a
protective barrier preventing
direct contact between the toxin
and the epithelial cells of the skin,
so reducing the toxicity of the
chemicals (Port and Port, 1986;
Triebskorn and Ebert, 1989). At
the same time, the mucus may
also dilute the chemical substance.
Haemolysis induced by treatment
of snails with the high sublethal
dose of either Nicotine or Thymol
may be attributed to rupture of the
external membranes. This
hypothesis was previously
postulated by Harry et al. (1957).
In addition, paralysis of the foot
induced by Nicotine may be an
indicator for the effect of this
substance on the nervous system
of snails. Terrestrial gastropods
poisoned by carbamates, such as
methiocarb, showed similar
response as they soon become
immobilized and muscle tonus is
lost (Godan, 1983). These
chemicals were known to act as
nerve poisons by the inhibition of
cholinesterase (Matsumura, 1985;
Eldefrawi and Eldefrawi 1990).
Heart beat rate plays a crucial
role in a number of physiological
processes and the animal can
manipulate many aspects of its
physiology by simply changing its
heart rate (Joosse and Geraerts
1983). There is a general tendency
for heart rate to increase under
conditions that increase metabolic
activity. This heart response is
advantageous to the animal since
during increased metabolic
activity the blood must deliver
more oxygen to the tissues. This
can be achieved through an
increase in cardiac output
resulting from an increase in heart
rate and/or stroke volume (Smith,
1987). The present study revealed
that the low sublethal dose of both
examined materials in addition to
the high sublethal dose of
Nicotine induced an increase in
the heart rate; while the high
sublethal dose of Thymol exerted
Physiological changes in the brown garden snail, Eobania vermiculata induced by
sublethal doses of two botanical molluscicides
- 382 -
significant cardioinhibitory effect.
Activity of the heart of pulmonate
mollusks can be modulated by the
nervous system via release of
different neurotransmitters or
neurohormones (Hill and Welsh,
1966). The cardioexcitatory
effects in gastropods have been
shown for many substances of
neural origin, serotonin and
FMRFamide are best examples;
while acetylcholine exerts an
inhibitory effect on the heart
activity (Zhuravlev et al., 1991).
Increase or decrease of the heart
rate may be attributed to release
of neural peptides or hormonal
agents that stimulate or inhibit the
heart muscle or may be due to a
direct action of the test materials
on the pacemaker of heart muscle
by accelerating or inhibiting of the
myogenic self-excitation process.
The cardioexcitatory effect
obtained in the current study
comply with that of Romero and
Hoffmann (1996) who recorded an
increase in the heart rate of
Megalobulimus sanctipauli in
response to an increase in
temperature. On the other hand,
The cardioinhibitory response of
snails to the high sublethal dose of
Thymol fits well with the
response of Biomphalaria
glabrata snails to copper
concentrations (Cheng and
Sullivan, 1973); Heltsoma duryi
snails in response to lethal
concentrations of three
molluscicidal plants: Warburgia
salutaris, Gardenia thunbergia
and Apodytes dimidiate (Clark
and Appleton, 1996) and
Biomphalaria havanensis after
treatment with lethal doses of
plants related to Agavaceae
family (Garces and Lopez, 1996).
Biochemical parameters are
sensitive indices to changes due to
xenobiotics and can constitute
important diagnostic tool in
toxicological studies (Radwan et
al, 2008). Carbohydrates are the
primary and immediate source of
energy (Lehninger, 1978). It is
evident from the present results
that both the examined plant-
derived molluscicides caused a
significant reduction in the
glycogen content of the digestive
gland accompanied with elevation
of the glucose level in
haemolymph. Decrease of the
tissue glycogen may be due to the
increased rate of glycogenolysis
under stress conditions induced by
the action of pesticides, thus
increased the haemolymph
glucose to meet the increased
energy demand (Arasta et al.,
1996). These results are also in
accordance with those of Tripathi
and Singh (2002); Tiwari and
Singh (2005); Mello-Silva et al.
(2006); Wang et al. (2006);
Radwan et al. (2008) and Bakry
(2009).
Proteins are mainly involved
in the architecture of the cell.
During chronic periods of stress
they are also a source of energy
(Umminger, 1977). Under stress
conditions, the snails need more
energy to detoxify the toxicants
and to overcome the induced
stress. Since snails have a limited
amount of carbohydrates, the next
alternative source of energy to
meet the increased energy demand
Beltagi, S.M.H., et al.
- 383 -
is proteins (Lehninger, 1978). In
the current study, the significant
decrease in the protein content of
the digestive gland may be due to
their degradation to release
energy. Tripathi and Singh
(2003); Singh et al. (2005) and
Radwan et al. (2008) have also
reported decline in protein content
in tissue of snails exposed to
sublethal concentrations of
pesticides. On the other hand, the
rise in protein content of the
haemolymph could be related to
acceleration of the rate of
gluconeogenesis in response to
intoxication caused by the applied
materials. The degraded tissue
proteins reach the haemolymph to
share in synthesis of glucose that
is used for energy production.
These results are in accordance
with those of Mello-Silva et al.
(2006). Histological alterations,
including degeneration and tissue
death, were recorded by many
authors in response to intoxication
from products with molluscicidal
activity (Bode et al.,1996; Lajtner
et al., 1996 and Hamed et
al.,2007). These alterations could
lead to cell lysis, resulting in the
release of a large quantity of
proteins and lipids, mainly from
the cells of the digestive gland,
which may explain the diminution
in the level of total lipids in the
digestive gland and its elevation
in the haemolymph of snails under
stress induced by the applied
materials used in the current
study. The increased rate of
lipolysis reflects imbalance
between the rate of synthesis and
the rate of release from the
parenchymal cells into the
systematic circulation towards the
catabolism for energy
requirements. These results are in
agreement with those reported by
Rambabu and Rao (1994); Rawi
et al. (1996) and Radwan et al.
(2008). However, other
mechanisms for the depletion of
lipids and proteins such as the
formation of lipoproteins which
are utilized for repair of damaged
cell and tissue organelles can not
be ruled out (Rambabu and Rao,
1994).
Cholesterol supplies affects
the growth, reproduction and
survival of land snails (Wacker,
2005). Slight increase in the
cholesterol level of the
haemolymph was recorded in
most cases. This action may be
attributed to the effect of the
tested materials on lipid
metabolism in correlation with
energy production. Lustrino et al.
(2010) postulated that the increase
in haemolymph cholesterol may
be a consequence of two
processes: i) increasing of
cholesterol biosynthesis and/or ii)
remodeling of cell membranes
with release of its cholesterol
molecules. If it is assumed that
there is elimination of cholesterol
molecules in cell membranes, this
fact will lead to increased fluidity
of the membrane, consequently
causing an increase in the rate of
metabolic processes involving
membrane proteins, such as those
that work in the transport chain of
electrons in the mitochondria,
even changing the cell
Physiological changes in the brown garden snail, Eobania vermiculata induced by
sublethal doses of two botanical molluscicides
- 384 -
permeability (Narayanan and
Venkateswararao, 1980).
The aminotransferases
constitute a group of enzymes
which catalyze the
interconversion of amino acids
and α-ketoacids by transfer of
amino groups (Kachmar, 1970b).
They have an important role in
linking of the amino acids and
carbohydrate metabolism, being
an essential group of enzymes in
the gluconeogenesis pathway.
Beyond this, the
aminotransferases are good
indicators of tissue lesions.
Elevation of the activities of
ASAT and ALAT enzymes
recorded in the current
investigation may be due to the
necessity of enhanced
deamination for the process of
gluconeogenesis especially under
conditions of impaired
carbohydrate metabolism and/or
diffusion of these enzymes from
their intracellular sites to the
haemolymph that implies tissue
lesions induced by the applied
plant molluscicides. Asada and
Galambos (1963) suggested that
the evaluation of the transaminase
activities gave good indications
for the parenchymal cell damage
and toxic biochemical effects.
Also, induced transamination has
been evidenced by Tiwari and
Singh (2005) in different tissues
of the freshwater snail Lymnaea
acuminata after sublethal
exposure to the Euphorbia
tirucalli latex extract. Similar
results were obtained by Radwan
et al. (1993) using Chlorfluazuron
against the terrestrial snails Helix
aspersa.
Acid phosphatase is a
lysosomal enzyme and plays an
important role in catabolism,
pathological necrosis, autolysis
and phagocytosis (Abu-Donia,
1978). In the present work, both
the examined molluscicides
caused a significant enhancement
in the activity of ACP enzyme.
These compounds may cause
destabilization of the lysosomal
membrane and the consequent
release of the enzyme into the
hemolymph or can trigger
hypersynthesis of acid
phosphatase which is
subsequently released into the
hemolymph (Suresh and
Mohandas, 1990). Similar results
have been obtained by Cheng and
Butler (1979).
Alkaline Phosphatase has
critical roles on protein synthesis
(Pilo et al., 1972) and shell
formation (Timmermans, 1969). It
plays an important role in
spermatogenesis (Pavlikova and
Repas, 1975). In the current study,
exposure of E. vermiculata snails
to both sublethal doses of Thymol
and Nicotine showed a marked
inhibition in the activity of ALP
enzyme. Reduction of ALP
activity may be related to the
cessation of protein synthesis due
to the effect of the toxin on the
general metabolism of the animal
(Henderson and Triebskorn,
2002). The present results
complied with those recorded by
Sheng-Xia et al. (2007); Jaiswal
et al. (2008) and Gawish et al.
(2009).
Beltagi, S.M.H., et al.
- 385 -
Conclusion, depending on
the current study, it could
concluded that application of both
the tested plant pesticides
interfere with the snails'
physiology, biochemistry and the
recovery period did not enable the
treated snails to eliminate the
adverse effects of the applied
materials; so Thymol and
Nicotine may be of great value in
the control of terrestrial snails but
further studies are needed to
evaluate the efficacy of these
materials as safe and economic
molluscicides in the field..
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- 391 -
Table (1): Molluscicidal activity of selected botanical materials against
E. vermiculata snails using the topical application method.
Botanical
material
LD25a
(µg/snail) after
48hr.
LD50a
(µg/snail) after
48hr.
Confidence limits
for LD50b
Nicotine
Thymol
Caffeine
Menthol
Camphor
116.17
377.63
524.30
673.75
893.56
204.02
551.20
932.37
1252.17
1354.07
173.32-233.47
491.65-609.33
822.19-1055.62
1065.13-1466.92
1210.68-1509.42
a Means based on 4 replicates (n = 4), 10 animals each.
b Lower and upper Limits for LD50 at 95% confidence limit.
Physiological changes in the brown garden snail, Eobania vermiculata induced by
sublethal doses of two botanical molluscicides
- 392 -
Table (2): In vitro effects of low and high sublethal doses of Thymol and
Nicotine on total proteins, total lipids, total cholesterol and glucose level
in the haemolymph of E. vermiculata snails at three post exposure time
intervals.
Note: Means based on of six replicates±SE (n=6), 5 animals each.
Values in parentheses indicate percent of change with control taken as
100%. *significant (P<0.05),
**highly significant (P<0.01),
***very highly
significant (P<0.001)
Measurement Time
interva
l (days)
Paramete
r Nicotine Thymol Control
LD25 LD50 LD25 LD50
14.94±0.11**
*
(+238.40)
12.60±0.27**
*
(+185.44)
10.57±0.38**
*
(+139.52)
17.54±0.49**
*
(+297.36)
4.41±0.12
1
Total
proteins
(mg/ml)
15.24±0.30**
*
(+198.00)
12.02±0.24**
*
(+135.15)
12.10±0.34**
*
(+136.67)
9.47±0.34***
(+85.22)
5.11±0.13
7
8.58±0.47***
(+74.77)
10.22±0.18**
*
(+108.05)
10.14±0.24**
*
(+106.54)
8.12±0.23***
(+65.28)
4.91±0.19
15
2.43±0.11***
(+28.83)
2.29±0.06*
(+21.52)
3.12±0.13***
(+65.69)
2.76±0.11**
(+46.80)
1.88±0.09
1
Total
lipids
(mg/ml)
2.29±0.08*
(+14.33)
2.22±0.08
(+11.17)
0.51±0.03***
(-74.40)
0.28±0.02***
(-85.77)
2.00±0.09
7
2.15±0.07*
(+21.97)
2.48±0.06***
(+40.15)
2.29±0.06*
(+29.65)
2.23±0.06*
(+26.19)
1.77±0.08
15
50.95±1.11
(+4.95)
51.75±1.76
(+6.60)
57.56±1.08**
*
(+18.56)
53.65±1.60
(+10.52)
48.55±1.80
1
Total
cholestero
l
(mg/dl)
50.25±2.14
(+1.21)
51.15±2.06
(+3.02)
60.26±2.30*
(+21.37)
53.05±1.64
(+6.85)
49.65±2.04
7
49.75±1.12*
(+14.52)
60.56±1.63**
(+39.40)
46.75±1.35
(+7.60)
49.65±1.92
(+14.29)
43.44±1.55
15
85.23±3.16**
*
(+91.48)
71.36±4.29**
*
(+60.31)
90.07±1.87**
*
(+102.35)
48.25±0.97
(+8.41)
44.51±0.99
1
Glucose
level
(mg/dl)
70.26±1.84**
*
(+92.88)
62.57±0.58**
*
(+71.78)
35.28±2.43
(-3.15)
46.41±1.26**
*
(+27.40)
36.43±1.42
7
55.34±0.79**
*
(+94.56)
68.81±1.55**
*
(+141.93)
26.75±1.04
(-5.96)
40.62±0.93**
*
(+42.81)
28.44±1.71
15
Beltagi, S.M.H., et al.
- 393 -
Table (3): In vitro effects of low and high sublethal doses of Thymol
and Nicotine on certain enzyme activities (ASAT, ALAT, ACP and
ALP) in the haemolymph of E. vermiculata snails at three post exposure
time intervals.
Note: Means based on of six replicates±SE (n=6), 5 animals each.
Values in parentheses indicate percent of change with control taken as
100%. *significant (P<0.05),
**highly significant (P<0.01),
***very highly
significant (P<0.001)
Enzyme activity (U/L) Time
interval
(days)
Parameter Nicotine Thymol
Control LD25 LD50 LD25 LD50
32.07±0.63***
(+51.12)
48.77±1.00***
(+129.82)
41.60±0.89***
(+96.02)
50.48±1.69***
(+137.90)
21.22±0.86
1
ASAT
34.10±1.06***
(+71.21)
37.67±1.25***
(+89.12)
36.58±0.72***
(+83.68)
43.75±1.63***
(+119.64)
19.92±0.73
7
27.76±0.57***
(+38.49)
30.09±1.05***
(+50.14)
29.87±0.87***
(+49.01)
33.04±1.27***
(+64.85)
20.04±0.45
15
5.99±0.26***
(+40.85)
7.00±0.27***
(+64.39)
6.25±0.16***
(+46.96)
7.29±0.21***
(+71.29)
4.26±0.05
1
ALAT 5.15±0.27
*
(20.92)
5.36±0.30*
(+26.01)
5.29±0.28*
(+24.36)
6.36±0.39**
(+49.51)
4.26±0.05
7
4.53±0.22
(+3.00)
4.60±0.19
(+4.63)
4.94±0.13
(+12.41)
5.15±0.13
(+17.26)
4.39±0.21
15
5.32±0.19***
(+145.28)
7.12±0.40***
(+227.95)
5.56±0.26***
(+155.91)
4.67±0.89*
(+114.96)
2.17±0.08
1
ACP 6.88±0.22
***
(+232.64)
5.97±0.23***
(+188.84)
5.31±0.20***
(+156.61)
3.47±0.18**
(+67.77)
2.07±0.13
7
3.29±0.11***
(+32.76)
4.24±0.11***
(+71.03)
3.12±0.10**
(+25.86)
3.08±0.13*
(+24.14)
2.48±0.14
15
25.43±5.95***
(-55.97)
30.17±10.70*
(-47.75)
29.25±0.60***
(-49.34)
32.32±1.80***
(-44.03)
57.74±2.87
1
ALP 30.17±10.70
*
(-49.74)
37.37±1.36***
(-37.76)
40.59±1.35***
(-32.40)
35.23±1.94***
(-41.33)
60.04±2.05
7
39.52±1.29***
(-34.35)
41.66±1.29***
(-30.79)
46.26±1.02***
(-23.16)
42.12±1.17***
(-30.03)
60.19±1.69
15
Physiological changes in the brown garden snail, Eobania vermiculata induced by
sublethal doses of two botanical molluscicides
- 394 -
Table (4): In vitro effects of low and high sublethal doses of Thymol
and Nicotine on total proteins, total lipids and glycogen content of the
digestive gland of E. vermiculata snails at three post exposure time
intervals.
Note: Means based on of six replicates±SE (n=6), 5 animals each.
Values in parentheses indicate percent of change with control taken as
100%. *significant (P<0.05),
**highly significant (P<0.01),
***very highly
significant (P<0.001)
Measurement ( mg/g wet tissue) Time
interval
(days)
Parameter Nicotine Thymol Control
LD25 LD50 LD25 LD50
51.86±0.90***
(-27.42)
58.98±1.03***
(-17.46)
47.17±1.60***
(-33.99)
56.04±1.30***
(-21.57)
71.46±1.20
1
Total
proteins
52.65±0.55***
(-23.24)
56.56±0.67***
(-17.54)
46.66±0.64***
(-31.98)
58.45±0.77***
(-14.78)
68.59±0.80
7
56.92±1.02***
(-18.98)
60.05±0.32*
(-14.53)
49.90±0.80***
(-28.97)
58.82±0.47***
(-16.28)
70.26±0.46
15
13.16±0.40**
(-25.11)
14.05±0.45**
(-20.06)
12.59±0.58***
(-28.34)
14.18±0.49***
(-19.29)
17.57±0.57
1
Total lipids 13.01±0.36
***
(-22.56)
14.16±0.31***
(-15.72)
11.75±0.13***
(-30.08)
13.84±0.38***
(-17.61)
16.80±0.24
7
14.12±0.32***
(-17.63)
13.83±0.29***
(-19.35)
12.88±0.23***
(-24.84)
15.07±0.39**
(-12.07)
17.14±0.24
15
0.95±0.02***
(-72.97)
1.16±0.05***
(-67.01)
0.84±0.03***
(-76.02)
1.27±0.06***
(-63.66)
3.51±0.10
1
Glycogen
content
0.91±0.04***
(-72.69)
1.07±0.03***
(-68.03)
0.76±0.02***
(-77.21)
1.49±0.06***
(-55.67)
3.35±0.05
7
1.07±0.006***
(-69.63)
1.18±0.06***
(-66.79)
1.00±0.04***
(-71.78)
1.11±0.04***
(-68.73)
3.54±0.09
15
Beltagi, S.M.H., et al.
- 395 -
Fig. (1): Patterns of response shown by E. vermiculata snails
following molluscicides exposure.
a- Specimens treated with LD50 Thymol showing highly mucoid
secretion (arrows) and haemolysis (arrow head).
b- Specimens treated with LD25 Thymol showing excessive secretion of
mucus (arrows)
c- Snails treated with LD50 Nicotine showing paralysis of the foot
(arrows) and haemolysis (arrow head).
d- Snails treated with LD25 Nicotine showing paralysis of the foot
(arrows), highly mucoid secretion (star) and specimen attracted inside
the shell (arrow head).
Physiological changes in the brown garden snail, Eobania vermiculata induced by
sublethal doses of two botanical molluscicides
- 396 -
Fig
. (2): In
vitro
effects of lo
w an
d h
igh
subleth
al do
ses of T
hy
mo
l and
Nico
tine o
n h
eart rate (beat/m
in) o
f E. verm
icula
ta sn
ails at three p
ost
exp
osu
re time in
tervals. T
he co
lum
ns sh
ow
the m
ean an
d th
e vertical
bars sh
ow
the stan
dard
error o
f the m
ean.
- 10
20
30
40
50
60
70
80
90
17
15
Po
st ex
po
sure
time
(da
ys)
Heart rate (beat/min)
contro
l
LD
50 T
hym
ol
LD
25 T
hym
ol
LD
50 N
icotin
e
LD
25 N
icotin
ev
Beltagi, S.M.H., et al.
- 397 -
أوباوا ،قوقع الحدائق البىالىاججة عه معاملة بوكمائةالحغرات الفسولوجة وال
بجرعات جحث ممحة الثىه مه مبدات الرخوات الىباجة فرمكوالجا
- محمد صالح عبد الحمد عبدهللا الشىاوى - سمر محمد حسه بلحاجي
وسف واد هاو -القطان إبراهم عبد الرحمه وبو
جايعة ع شس -كهة انحربة -قسى انعهىو انبىنىجة وانجىنىجة
أىباةا يرييىتجةانهقىقة بىكائةةجى إجراء انبحد انحان ندراسة انحغرات انفسىنىجة وان
تذة ية (LD50, LD25) كاسحجابة نهعايهة بجرعةات جحةث يحةة )أحد اتيات انسراعة ي يصر(
، 7، 1)ية انعايهةة عقب يحرات زيةة يتحهفةة )انيىج وانرىل( دات انرخىات انباجة انفعانة يب
باسحتداو غرقة انحطبق انىظع. وذنك ىو( 11
أظهرت انقىاق انعايهة بعط أعراض انسة انشحركة كئيراز انتاغ بصةىر كرفةة بااظةاية
بانة انعايهةة عةة عهة حةد كانححهةم انةديىي ية حانةة انقىاقةبعةط اتعةراض انةس نيةم يجى إن
LD50 انيةىج.، بااظاية إن حدوخ شهم عصب بانقدو ي انقىاقة انعايهةة بةا يىج أو ذىل
ية ةايعى اتفاظةا( ية انرةىل LD50ظهرت انقىاق انعايهة بانجرعة جحث انحة انرجفعة )أوقد
يعةةدل ظةةربات انقهةةب عهةة يةةدار يةة ارجفاعةةاانجىعةةات بةةاقأظهةةرت بةةا ،هةةبيعةةدل ظةةربات انق
بىكائةةةانجةةى جسةةجم زةةا ذات تنةةة إحصةةائة يةة قةةى انيىةةات . انةةر خسيةةة انحابعةةة انيحةةرات
انجهىكةةىز( ونهيىنسةةحرول انححةةىي انيهةة –نهةةدنى انححةةىي انيهةة –نهبةةروج )انححةةىي انيهةة
ية ايهحىظة احةدخ جحسةأيعايهة انقىاق بي ي انا ج انتحبةرج يعظى انحاتت. يىنف نهه
( ACPإنة إةسى انفىسةفاجس انحايعة )بااظةاية (ASAT - ALATسات انقم اتي )إشاغ
نةىحع أ يقةد ،ن ذنةكبااظاية إ(. ALP) انقاعدإسى انفىسفاجس حدخ اتفاض يهحىظ نشاغ با
قةةد سةةجهث ،انجهيةةىج( نهغةةد انهعةةة –انةةدنى انيهةةة – )انبةةروج انيهةة بىكائةةةانيىةةات ان
انحانةة انسية انةر خ انحابعة هعايهة بانا ج انتحبرج عه يدار يحرات نسحجابة ا ايهحىظ اتفاظا
هعايهة.ن
حةةدوخ خهةةم بانعهةةات انفسةةىنىجة نهقىاقةة يةةا ح جسةةبباانتحبرجةةانا جةةا أ االسننحىحا مكننه
انحتهص ية انحةاذرات انسةهبة نهةىا انتحبةر ححة هاةة يحةر اتسحشةفاء، ي حنى جج انعايهة وانح
إت أ جطبةق نةاج ، األرظةةيى نهرىل وانيىج ور يعال ي ييايحة انقىاقة أ وي ذى ي
ن انسد ي اندراسات .إحاجة ية واقحصا ة يآت انا ج ي انحقم كبدا
اندون انحاس عشرؤجر ان
عة انصرة اتناة نعهى انحىاانجنجهة
2011 2 ياى 30 -ابرم
(A انراند وانسحىانعد )
2011 ىنى
ISSN 1110-5321
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