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Chapter 6

BIOCHEMISTRY

168

Chapter 6

Biochemistry

6.1 Introduction

The metabolic needs are dependent on nutritional and reproductive state and

some phylogenetic factors. For maximizing the power of reproduction, individuals

naturally devote their more energy for the reproduction. Studies have reported that

much of the energy resources of molluscs are stored in the digestive gland and foot

muscle (Sastry and Blake, 1971; Pazoc, et al., 1997 and Berthelin, et al., 2000).

Earlier studies have also shown that the digestive gland is implicated not only in

nutrient storage but also in the transfer of assimilated food to body tissues in molluscs

(Sastry and Blake, 1971).

Most toxic substances exert their effects on basis level in the organism by

reacting with enzymes or by affecting membrane and other functional compartments

of the cell (Patel and Patel, 1985). The hepatopancreas has been found to be the major

site of heavy metal accumulation in the molluscs (Berger and Dallinger, 1989; Nott

and Nicolaidou, 1989 and Presing, et al., 1993). Alteration in enzyme activity can be

correlated with molluscicide induced changes in the ultrastructure of the cells

(Triebskorn, 1991). Ultrastructural response in the hepatopancreas can be used as

biological markers to detect different levels of biological response to toxic conditions

(Rubio, et al., 1993). Molluscicides greatly affect the metabolic activities of the snail

intermediate host (Rawi, et al., 1995). Biochemical parameters in organism exposed

to toxic contaminant have been used as biomarkers and can constitute an important

diagnostic tool to assess the exposure and effects of xenobiotics (Forbes, et al., 1997

and McLoughlin, et al., 2000), in addition to, they act on glucose level, lipid and

protein content in snail tissues and haemolymph (Abdel, 1999; Mantawy and

Mahmoud, 2002; El-Moghazy and Abdel-Aziz, 2005 and Bakry, 2009). Changes in

the physiological state of snail can affect the biochemical values of the haemolymph

(Akinloye and Olorode, 2000). Molluscicides act on different enzymes, chiefly those

of respiration and carbohydrate metabolism (Sakran and Bakry, 2005 and Sharaf,

2006). Biochemical parameters are sensitive index to monitor changes due to

xenobiotics and can constitute important diagnostic tool in toxicological studies

(Radwan, et al., 2008).

169

Lipids and glucose are rich energy source which can be stored as glycogen and

glycerol in the body for later use. The concentration of protein is relatively higher

than other metabolites. Land snails remain dormant for many months or even years,

until favorable conditions allow them to resume vital processes of nutrition and

reproduction (Herreid, 1977; Riddle, 1986 and Whitman and Storey, 1990). During

these prolong periods of starvation previously stored materials are metabolized;

glucose, a major source of energy, is the first of such materials to be used (De Silva

and Zancan, 1994 and Rossi and De Silva, 1993;). When this metabolite is depleted,

the animal mobilizes lipid reserves (Barclay, et al., 1983 and Piretti, et al., 1988),

finally protein reserves are used as a last resort (Barclay, et al., 1983).

6.1.1 Significance of Albumin (ALB)

Bakry, (2009) found decrease in albumin content of the haemolymph of

Biomaphalaria alexandrina infected with Schistosoma mansoni. The drop in albumin

content may also reflect damage in the hepatic parenchyma which is considered the

site of albumin origin (El-Husseini, et al., 1986 and Rawi, et al., 1995). Till date

meager data was found related to albumin in molluscs.

6.1.2 Significance of Alkaline phophatase (ALP)

Alkaline and acid phosphatases are two phosphomonoesterases that catalyse

transphosphorylation and hydrolysis on numerous orthophosphate esters.

Alkaline phosphatase breaks down ester compounds of orthophosphate acids

under alkaline condition between pH 9.2 and 9.8 (Adolph and Lorenz, 1981). Alkaline

and acid phosphatase proved to be good sensitive tools in the detection of any

variations in the physiological process of living organisms (Tolpa, et al., 1991).

Stimulation or inhibition of these enzymes will thus result in disturbances in

metabolism. Alkaline phosphatase is brush border enzyme, which catalyses

dephosphorylation of many molecules including nucleotides, proteins and alkaloids at

alkaline pH. It is well known that phosphatases are involved in carbohydrate

metabolism, growth and differentiation, protein synthesis, synthesis of certain

enzymes, secretory activities and transport of phosphorylated intermediates across the

cell membranes. Hydrolysis of phosphoesters, phosphate transferase activity, protein

phosphatase activity, phosphate transport, modulation of organic cation transport and

involvement in cell proliferation have been suggested as possible function of alkaline

170

phosphatase (Sarrouilhe, et al., 1992). The enhanced activity of alkaline phophatase in

the hepatopancreas might be due to activation of intracellular energy consuming

process because the alkaline phosphatase facilitates breakdown of ATP to ADP and

inorganic phosphate, thereby making free energy available for metabolic processes

(Botham and Mayes, 2003). Alkaline and acid phosphatase are expressed in a constant

pattern and help to identify the different zone of the adult shell forming tissue

(Marxen, et al., 2003). Alkaline and acid phosphatases were recorded among the

target enzymes which should be distrurbed 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) (Hasheesh, et

al., 2011).

6.1.3 Significance of Glucose (GLU)

All living cells contain carbohydrate in different forms. These compounds are

perhaps best defined as polyhydroxy aldehydes or ketones and their derivatives. The

storage of carbohydrate in invertebrate and vertebrate body is important as they have

reserves of calories (energy) for the body tissues. According to Clark (1975) the

carbohydrates are considered to be the first organic reserve degraded under toxic

stress condition imposed on animal. There is a causal connection between synthesis of

glucose or glycerol and catabolism of glycogen (Storey, 1981; Steiner, 2000 and Li, et

al., 2002). Carbohydrate is stored largely as glycogen and specialized galactogen and

is transported and available from the blood as glucose, glycogen is distributed

generally throughout the tissues i.e. hepatopancreas, foot and mantle. In order to

promote energy production, gastropods categorize primarily carbohydrates, which are

stored in certain tissues as glycogen and transported in the haemolymph as glucose

(Livingstone and deZwaan, 1983). Glycogen level is one of the parameters that

reflects the energetic and reserves status of organisms. Moreover, glycogen is used

rapidly when organisms are under stress and levels of this energy reserve have been

suggested as useful biomarker of general stress (Hugget, et al., 1992 and Vasseur and

Cossu-Leguille, 2003). Carbohydrates are generally used as energy supply

particularly in case of stress. It is well known that the sugar serve as energy reserve

for the metabolic process. Carbohydrates are considered to be the first among the

organic nutrients degraded in response to stress conditions imposed on an animal.

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6.1.4 Significance of Total Protein (TP)

The proteins are the basic structural units present in all the biological systems.

The physio-biochemical activities consist the life of the cell are catalyzed by the

enzymes, which are almost proteins. Proteins form one of the most important and

most complex groups of biological materials comprising the chief nitrogenous

constituents of the body tissues.

The concentration of free amino acids in the tissues and extracellular fluid

compartments of mollusc varies with the diet, season, temperature, reproduction and

developmental stage and environmental stress related to desiccation, anaerobiosis,

osmotic pressure, pollution and parasitism (Bishop, et al., 1983). Similarly, snails

have been reported to be a good source of protein (Amusan and Omidiji, 1988 and

Akinnusi, 2002). South (1992) observed that protein is the most abundant solute in the

snail’s haemolymph. Meenakshi, (1995) observed the total remarkable increase in

total free amino acids may also represent the proteolysis and a derangement of protein

and release of amino acids from the liver to blood. Singh, et al. (1996) reported that

the quantity of proteins depends on the rate of synthesis or its degradation. It is

affected due to impaired incorporation of amino acids into polypeptide chain. Lipids

and glucose are rich energy sources which can be stored as glycerol and glycogen in

the body for later use. Depletion in protein level is due to eithersuppressed

incorporation of protein synthesis or increased breakdown of proteins in to amino

acids, which diffused out of cells (Gaur, 2011).

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6.1.5 Significance of Uric Acid (UA)

The nitrogenous products of degradation under physiological conditions of

stress may be altered that would cause in the snail an intoxication by the excess of

these products. Uric acid is produced and stored by species of Pomacea and Pila, both

during period of activity and of aestivation (Meenakshi, 1995 and Little, 1968; 1981)

and much has been written on the possible role of uric acid in nitrogen excretion in

these contrasting physiological states (Lal and Saxena, 1952 and Little, 1968). Uric

acid being a typical storage excretory product is often determined in molluscs in

homogenates of the whole body or in certain organs such as hepatopancreas, kidney

or foot (Becker and Schmale, 1975). The land snail P. canaliculata invest a large

amount of nitrogen in the eggs perivitelline fluid, in the form of at least three

perivitelline glycolipoproteins (Heras and Pollero, 2002; Dreon, et al., 2006 and

Heras, et al., 2007), only a very low concentration of uric acid was found in the

albumen gland and none was found in newly deposited eggs of P. canaliculata.

Active Pomacea canaliculata does not excrete uric acid to any detectable extent, this

purine is found in the circulation and that it accumulates in crystalloids within

specialized cells of the midgut gland (Cueto, et al., 2005 and Vega, et al., 2007).

Giraud-Billoud, et al. (2008) found that uric acid concentration in the albumen gland

and in the newly deposited eggs of P. canaliculata.

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6.2 Material and Methods

6.2.1 Collection of test animal

As given in Chapter No. 5

6.2.2 Tested molluscicides

Two molluscicides Thiamethoxam and Diafenthiuron belonging to two

different chemical groups were tested.

6.2.3 Experimental Design

Five times less concentration of LC50 concentration (0.10ppm and 0.12ppm) of

both Thiamethoxam (0.51ppm) and Diafenthiuron (0.64ppm) were taken for treating

the test animal by spraying it on a food given to snail after starving it for 24hrs. This

treatment was carried up to 7 days and then natural untreated food was provided to

recover them up to 7 days again. Tissue samples were taken for biochemical studies

after 7 days and 14 days to observe the treated and recovery effect of snails.

6.2.4 Sample preparation

After 7 days of treatment, shells of tested snails were removed by making a

cut around the whorls in a continuous manner starting at the shell opening and the

broken fragments of the shell were carefully removed. Foot and hepatopancreas

tissues were dissected out and homogenized in distilled water (50mg/ml). The

homogenates were centrifuged at 8000 rpm for 15 min. at 5ºC in refrigerated

centrifuge. The deposits were discarded and the supernatants were kept in a deep

freezer till use to determine the activities of ALB, ALP, GLU, TP and UA.

Prepared homogenate was then processed as per the procedure according to

the respective parameters. Readings were taken by using the Quantiamate

turbidimetry chemistry, Auto Analyzer for biochemical tests of Tulip Diagnostics (P)

Ltd. Goa, India.

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Fig. 6.1 Auto Analyzer

6.2.5 Biochemical studies

Following parameters were studied to observe the effect of Thiamethoxam and

Diafenthiuron on the pest snail M.indica.

6.2.5.1 Albumin (ALB) (BCG method), Bromocresol Green, End Point Assay (Liquid

Gold), Span Diagnostics Ltd. India.

Procedure-

Pipette into tube marked Blank Standard Test

Sample - - 10µl

Reagent 2 - 10µl -

Reagent 1 1000µl 1000 µl 1000 µl

Mixed well and incubated at room temperature (15-300C) for 1 minute.

Programmed the analyzer on 24 No. as per assay parameter.

1. Blanked the analyzer with reagent blank.

2. Measured absorbance of standard followed by the test.

3. Result calculated as per given calculation formula.

Calculation

Albumin (g/dl) =Absorbance of test

Absorbance of Standard × 4

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Globulins= Total Protein - Albumin.

Conversion factor

Albumin concentration in g/L= Albumin concentration in g/dl × 10.

6.2.5.2 Alkaline Phosphatase (ALP) Test Kit (pNPP-AMP (IFCC)), Kinetic assay

(Autospan), Span Diagnostics Ltd., India.

Procedure-

Pipette into tube marked Test

Sample 20µl

Working ALP Reagent 1000 µl

Mixed well and aspirated immediately for measurement.


1. Blanked the analyzer with purified water.

2. Read absorbance after 30 seconds. Repeated readings after every 30

seconds i.e. up to 120 seconds at 405nm wavelength.

3. Determined the mean absorbance change per minute (Δ A/minute).

Calculation:

ALP activity (U/L) = Δ A/minute × Kinetic factor

Where,

Δ A/minute = change in absorbance per minute

Kinetic factor (K) = 2712

Kinetic factor is calculated by using following formula

K =1M ×

TVST ×

1P × 10

M= Molar extinction coefficient of p-Nitrophenol and is equal to 18.8×103 lit

at 405nm

TV= Sample volume + Working reagent volume

P= Optical path length

106= Constant

6.2.5.3 Glucose (GLU) (Enzymatic, GOD/POD method), End point assay and Kinetic

assay (Autospan), Span Diagnostics Ltd. India.

Procedure-

176


Sample - - 10µl

Reagent 3 - 10µl -

Working Glucose Reagent 1000µl 1000 µl 1000 µl

Mixed well and incubated at 370C for 10 minutes.





Calculation

For end point mode

Sample Glucose (mg/dl) =Absorbance of test


For fixed rate kinetic mode

Sample Glucose (mg/dl) =AT2 − AT1AS2 − AS1 × 100

Where,

AT1: Initial O.D. of Test

AT2: Final O.D. of Test

AS1: Initial O.D. of Standard

AS2: Final O.D. of Standard

Conversion factor

Glucose concentration in mmol/L= Glucose concentration in mg/dl × 0.05551

6.2.5.4 Total Protein (TP) Test Kit (Biuret method), Modified Biuret, End point assay

(Liquid Gold), Span Diagnostics Ltd. India.

Procedure-


Sample - - 10µl

Reagent 2 - 10µl -


177

Mixed well and incubated at 370C for 5 minutes.





Calculation

Total Protein Concentration(g/dl) =Absorbance of test

Absorbance of Standard × 6.5

Globulins= Total Protein- Albumin

Conversion factor

Total protein concentration in g/L= Total protein concentration in g/dl × 10.

6.2.5.5 Uric acid Test Kit (UA) (Uricase/POD method), End Point Assay (Liquid

Gold), Span Diagnostics Ltd., India.

Procedure-


Sample - - 20µl

Reagent 2 - 20µl -


Mixed well and incubate at 370C for 5 minutes.





Calculation

Sample Uric acid (mg/dl) =Absorbance of test


Conversion factor

Uric acid concentration in mmol/L= Uric acid concentration in mg/dl × 0.059

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6.3 Observations

As per Chapter No. 5.

6.4 Results

The significant change in the content of ALB, ALP, GLU, TP and UA were

observed and shown in the Table 6.1 and Chart 6.1-6.10.

Tissue Pesticide Set ALB ALP GLU TP UA

Foot

Control 15.6±0.47 5.42±0.07 0.19±0.003 0.25±0.004 0.16±0.01

Thaim-

ethoxam

Treated 10.4±0.22

**

4.24±0.06

***

0.11±0.005

***

0.19±0.007

***

0.21±0.01

***

Recovery 12.5±0.28

*

4.56±0.16

**

0.13±0.003

***

0.21±0.005

***

0.20±0.01

***

Diafen-

thiuton

Treated 11.1±0.40

**

4.62±0.08

**

0.12±0.006

***

0.20±0.003

***

0.19±0.02

***

Recovery 12.6±0.55

*

4.94±0.09

***

0.15±0.002

***

0.23±0.005

***

0.18±0.008

***

Hepato.

Control 16.3±0.33 6.26±0.10 0.23±0.002 0.26±0.013 0.23±0.003

Thaim-

ethoxam

Treated 12.6±0.20

***

4.92±0.40

*

0.15±0.013

***

0.18±0.004

***

0.26±0.008

***

Recovery 14.04±0.38

***

5.24±0.08

**

0.19±0.003

***

0.21±0.003

***

0.24 ±0.01

***

Diafen-

thiuton

Treated 13.7±0.28

**

5.68±0.10

**

0.17±0.009

***

0.20±0.008

***

0.25±0.009

***

Recovery 14.1±0.3

**

5.84±0.05

**

0.20±0.002

***

0.22±0.005

***

0.23±0.007

***

Table 6.1: Change in the content of parameters in foot and hepatopancreas.

Note: Thiamethoxam (LC50/5) and Diafenthiuron (LC50/5) were used and *, **, *** =

significantly different from control at p<0.05, p<0.01 and p<0.001, respectively.

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0

5

10

15

20

Control Treated Recovery Treated Recovery

Con

cent

ratio

ns in

mg/

g

Thiamethoxam Diafenthiuron

Graph 6.1: Changes in Albumin Content of Foot

02468

1012141618


Con

cent

ratio

ns in

mg/

g


Graph 6.2: Changes in Albumin Content of Hepatopancreas

180

0123456

Control Treated Recovery Treated RecoveryCon

cent

ratio

ns in

mg/

g


Graph 6.3: Changes in Alkaline Phosphatase Content of Foot

01234567


Con

cent

ratio

ns in

mg/

g


Graph 6.4: Changes in Alkaline Phosphatase Content of Hepatopancreas

181

0

0.05

0.1

0.15

0.2

0.25


Con

cent

ratio

ns in

mg/

g

Thiamethtoxam Diafenthiuron

Graph 6.5: Changes in Glucose Content of Foot

0

0.05

0.1

0.15

0.2

0.25


Con

cent

ratio

ns in

mg/

g


Graph 6.6: Changes in Glucose Content of Hepatopancreas

182

0

0.05

0.1

0.15

0.2

0.25

0.3


Con

cent

ratio

ns in

mg/

g


Graph 6.7: Changes in Total protein Content of Foot

0

0.05

0.1

0.15

0.2

0.25

0.3


Con

cent

ratio

ns in

mg/

g

Thiamenthoxam Diafenthiuron

Graph 6.8: Changes in Total protein Content of Hepatopancreas

183

0

0.05

0.1

0.15

0.2

0.25


Con

cent

ratio

ns in

mg/

g


Graph 6.9: Changes in Uric acid Content of Foot

0

0.05

0.1

0.15

0.2

0.25

0.3


Con

cent

ratio

ns in

mg/

g


Graph 6.10 Changes in Uric acid Content of Hepatopancreas

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6.5 Discussion

6.5.1 Albumin (ALB)

Esmaeil (2009) found decrease in total proteins and albumin in B. alexandrina

snails treated with the fungicide Topas to degeneration of the snail’s ovotestis and

digestive glands cellular structure. Mohamed, et al. (2012) observed reduction of total

proteins and albumin concentration in tissues of snails treated with the pesticides

Basudin and Selecron and the phytoalkaloid Colchicine due to structural damages of

their internal organs (Tolpa, et al., 1997).

In present study the significant decrease in the content of albumin (ALB) was

noted in the foot and hepatopancreas at 10.4±0.22 mg/g (P<0.01), 11.1±0.40 mg/g

(P<0.01) Chart 6.1 and 12.6±0.20 mg/g (P<0.001), 13.7±0.28 mg/g (P<0.01) Chart

6.2 compared to control 15.6±0.47 mg/g and 16.3±0.33 mg/g and significant changes

are observed after 7 days of treatment at 12.5±0.28 mg/g (P<0.05), 12.6±0.55 mg/g

(P<0.05) Chart 6.1 and 14.04±0.38 mg/g (P<0.001), 14.1±0.30 mg/g (P<0.01) Chart

6.2 treated with both Thiamethoxam and Diafenthiuron. This result correlates with

findings of Esmaeil (2009) and Mohamed, et al. (2012).

6.5.2 Alkaline phosphatase (ALP)

Saxena and Malhendru, (2004 and 2005) found that alkaline phosphatase

activity decreased with increasing dichlorvos activity and duration of exposure. The

mucous cells had acid and alkaline phosphatase activities and the mucous released by

the activity of both the enzymes in the mucus cells (Ning, et al., 2005). Sangita, et al.

(2006) observed that the combinations of few plant derivatives were more effective

against acid and alkaline phosphatase in the snail tissue as compared to treated alone.

Jaiswal, et al. (2010) studied the effect of molluscicidal components of Myristica

fragrans trimyristin and myristicin activity on Lymnaea acuminata which showed

singnificant decrease in the acid and alkaline phosphatase and acetylcholinesterase

activities in the nervous tissue of treated snails. Akinpelu, et al. (2012) observed the

effect of sub-lethal concentration of saponin on Lanistes lybicus and found that

decrease in activity of muscle alkaline phosphatase but increased enzyme activity in

hepatopancreas. Increase in protein concentration with increasing saponin

concentration.

185

In present findings the significant decrease in the content of Alkaline

phosphatase (ALP) was noted in the foot and hepatopancreas at 4.24±0.06 mg/g

(P<0.001), 4.62±0.08 mg/g (P<0.01) Chart 6.3 and 4.92±0.40 mg/g (P<0.05),

5.68±0.10 mg/g (P<0.01) Chart 6.4 compared to control 5.42±0.07 mg/g and

6.26±0.10 mg/g and significant changes are observed after 7 days of treatment while

feeding normally at 4.56±0.16 mg/g (P<0.01), 4.94±0.09 mg/g (P<0.001) Chart 6.3

and 5.24±0.08 mg/g (P<0.01), 5.84±0.05 mg/g (P<0.01) Chart 6.4 treated with both

Thiamethoxam and Diafenthiuron. These results are in agreement with the results of

Saxena and Malhendru (2004 and 2005); Jaiswal, et al. (2010) and Akinpelu, et al.

(2012).

6.5.3 Glucose (GLU)

Effects in snails under starvation and trematode infected with reduction in the

glucose level in the hemolymph and depletion of the carbohydrates reserves (Cheng

and Lee, 1971; Friedle, 1971; Lee and Cheng, 1972; Manohar and Rao, 1976; Teng,

et al., 1979 and Joosse and Van Elk, 1986). Ishak, et al. (1975) reported depletion of

glycogen in infected snails and the reason suggested was the low energy efficiency of

anaerobic metabolism leading to fast depletion of carbohydrate store.

Pinheiro and Amato (1994) found that in the snail B. similaris infected with E.

coelomaticum the glucose content in the hemolymph was reduced in more than 60%

and the deposits of glycogen were reduced in 90% at the end of pre-patent period.

Pinheiro (1996) observed that under starvation B. similaris has reduction more than

80% in its carbohydrate reserves of cephalopedal mass, digestive gland and albumen

gland. Chemical stress causes rapid depletion of stored carbohydrates primarily in

liver and other tissues (Jyothi and Narayan, 2000). Hamlet, et al. (2012) observed the

effect of Thiamethoxam on hepatopancreas carbohydrate level of H. aspersa, the

insecticide formulation was 0, 25, 50, 100, 200 mg/L after six weeks of treatment

biochemical study showed that the content of total carbohydrate was not significantly

reduced at 25 and 50 mg/L of Thiamethoxam but very significant reduction in total

carbohydrate level was observed in H. aspersa exposed at 100 mg/L and highly

significant depletion in the concentration at 200 mg/L as compared to control value.

In present findings the significant depletion in the content of Glucose (GLU)

was noted in the foot and hepatopancreas at 0.11±0.005 mg/g (P<0.001), 0.12±0.006

mg/g (P<0.001) Chart 6.5 and 0.15±0.13 mg/g (P<0.001), 0.17±0.009 mg/g (P<0.001)

186

Chart 6.6 compared to control 0.19±0.003 mg/g and 0.23±0.002 mg/g and significant

changes are observed after 7 days of treatment while feeding normally at 0.13±0.003

mg/g (P<0.001), 0.15±0.002 mg/g (P<0.001) Chart 6.5 and 0.19±0.003 mg/g

(P<0.001), 0.20±0.002 mg/g (P<0.001) Chart 6.6 treated with both Thiamethoxam

and Diafenthiuron. The present findings agreed with Pinheiro (1996) and Hamlet, et

al. (2012).

6.5.4 Total protein (TP)

Reduction in protein level may be due to the inhibition of alkaline phsphatase

activity, as it plays an important role in protein synthesis (Pilo, et al., 1972) and other

secretary activities (Ibrahim, et al., 1974). The synthesis of protein in any of a tissue

can be affected due to RNA synthesis at the transcription stage and affects the uptake

of amino acids in the polypeptide chain both possibilities may account for the

lowering the protein content in the affected tissues (Tariq, et al., 1977; Shrivastava, et

al., 2010 and Singh, et al., 2010). Similar observations were also noted in Pila

globosa (Rao Ramana and Ramamurthy, 1980). Rao, et al. (1981) reported the

decrease in total proteins in snail tissues exposed to methyl parathion suggest the

possible utilization of these compounds for metabolic purposes. The decrease of

proteins in tissues of mollusc is due to their degradations and possible utilization of

degraded products for metabolic purposes. Enhanced protease activity and decreased

protein level have resulted in marked increase of free amino acids in snail tissue. The

accumulation of free amino acids can also be attributed to their involvement in the

maintenance of an acid-base balance (Moorthy, et al., 1984) and the less use of amino

acids (Rao, et al., 1987). The decline in protein content may be related to impaired

food intake, the increased energy cost of homeostasis, tissue repair and the

detoxification mechanism during stress (Neff, 1985). Rao, et al. (1993) observed

decrease in protein content of fresh water gastropod, Indoplanoribis exustus due to

toxicity. Patil, et al. (1995) reported the depletion protein level in fresh water snail,

Thiara lineate due to pesticidal stress. The quantity of protein depends on the rate of

protein synthesis or its degradation. It also affected due to impaired incorporation of

amino acids into polypeptide chains (Singh, et al., 1996). Reduction in the total

protein level was observed in Eobania vermiculata snails treated with carbamate

pesticides (Radwan, et al., 2008). Kulkarni, et al. (2005) observed significant

decrease in total protein content in foot, hepatopancreas and gills of freshwater

187

mussel, Lamellidens corrianus on exposure to organo chlorine insecticide, lindane.

Yadav, et al. (2007) studied that the animals exposed to chemicals obtain extra energy

requirement from the tissue protein. Singh and Gupta (2007) reported the decrease in

protein level in foot and mantle, while an elevation in hepatopancreas of Pila globosa

under stress of azodyes. Gupta, et al. (2010) observed significant inhibition in protein

content in foot, mantle and gill, while an elevation in hepatopancreas of Lamellidens

marginalis exposed to congored. The depletion of cellular proteins might be caused

by inhibition of amino acid incorporation, breakdown of proteins into amino acids and

diffusion out of the cells. The synthesis of RNA plays an important role in protein

synthesis. The inhibition of RNA synthesis at transcription level may affect the

protein level. The decrease in the RNA concentration may have been a cause of

protein depletion. The increase in protease activity may be the cause of increased

protein degradation (Singh and Singh, 2010). Glycogen is available for instant energy

source while proteins are important organic constituents of the animal cells playing a

vital role in the process of interactions between intra and extracellular media. The

active depletion of these metabolites might be due to their mobilization to liberate

energy during pesticidal stress (Ahirrao and Kulkarni, 2011).

In present findings the significant depletion in the content of Total Protein

(TP) was noted in the foot and hepatopancreas at 0.19±0.007 mg/g (P<0.001),

0.20±0.003 mg/g (P<0.001) Chart 6.7 and 0.18±0.004 mg/g (P<0.001), 0.20±0.008

mg/g (P<0.001) Chart 6.8 compared to control 0.25±0.004 mg/g and 0.26±0.013 mg/g

and significant changes are observed after 7 days of treatment while feeding normally

at 0.21±0.005 mg/g (P<0.001), 0.23±0.005 mg/g (P<0.001) Chart 6.7 and 0.21±0.003

mg/g (P<0.001), 0.22±0.005 mg/g (P<0.001) Chart 6.8 treated with both

Thiamethoxam and Diafenthiuron. These findings correlate with the results of Tariq,

et al. (1977); Rao Ramana and Ramamurthy (1980) Rao, et al. (1993); Patil, et al.

(1995); Kulkarni, et al. (2005); Radwan, et al. (2008) and Ahirrao and Kulkarni,

(2011).

6.5.5 Uric acid (UA)

Becker and Schmale (1975) observed alteration in nitrogenous products of

degradation metabolism in snails in response to the alteration that occurred during

starvation. Bishop, et al. (1983) observed that in terrestrial species of molluscs the

most of nitrogenous products of degradation were excreted as urea or uric acid and

188

other purine compounds. In the terrestrial snails under normal conditions, the urea

excretion is a process more advantageous than uric acid excretion, once that the urea

can be excreted highly diluted avoiding the intoxication of the snail. Becker (1983)

studied the alteration in uric acid and guanine in the tissues of Biomphalaria glabrata

under starvation and Schistosoma mansoni infected and observed an increase of these

purines concentration. Circulating uric acid may serve as a free radical scavenger and

antioxidant (Becker, 1993), particularly during the tissue reoxygenation that occurs

after aestivation in snails (Hermes-Lima, et al., 1998).

In present findings the significant increment in the content of Uric acid (UA)

was noted in the foot and hepatopancreas at 0.21±0.01 mg/g (P<0.001), 0.19±0.02

mg/g (P<0.001) Chart 6.9 and 0.26±0.008 mg/g (P<0.001), 0.25±0.009 mg/g

(P<0.001) Chart 6.10 compared to control 0.16±0.01 mg/g and 0.23±0.003 mg/g and

significant changes are observed after 7 days of treatment while feeding normally at

0.20±0.01 mg/g (P<0.001), 0.18±0.008 mg/g (P<0.001) Chart 6.9 and 0.24±0.01 mg/g

(P<0.001), 0.23±0.007 mg/g (P<0.001) Chart 6.10 treated with both Thiamethoxam

and Diafenthiuron. These results are in agreement with the results of Becker (1983).

6.6 Conclusion

In present study both the pesticides caused an alteration in some biochemical

aspects, which could lead to serious metabolic and cellular damage. The magnitude of

the stress was more in Thiamethoxam than Diafenthiuron. Data in Table 6.1 showed

that pesticide Thiamethoxan (LC50/5) (0.51ppm) and Diafenthiuron (LC50/5) (0.64ppm)

shows significant changes in the ALB, ALP, GLU, TP and UA of foot and

hepatopancreas of the M. indica. Thiamethoxam and Diafenthiuron treated M. indica

shows significant decrease in ALB, ALP, GLU and TP while slightly increase in UA

content.

189

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(*- original not cited)

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