physiological changes in the brown garden snail,...

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

mailto:[email protected]

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



- 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


- 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.



- 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.


- 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



- 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


- 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



- 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).


- 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.



- 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


- 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.





***very highly


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



- 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.





***very highly


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


- 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).



- 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


- 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

Http: www.egsz.com

Email: [email protected].

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physiological changes in the brown garden snail,...

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