S

II

INTRODUCTION:

Pesticides selectively effective against the insect pest without adversely

affecting its important natural enemies are to be identified for incorporation

in integrated pest management programme (Hassan et al., 199 1). Pesticides

which are used to control the pest of cultivated crops not only affect the

target groups (i.e., the pests), they also affect the non-target groups including

their natural predator such as spiders. Patel et. al., (1 986) reported that

pesticides are highly toxic to spiderlings of cliefracant/ilum metanostoma,

PIiia'!ppus bismati/ca and Argiope nathabliai.

Ridgeway (1 978) classify pesticides into three groups, organo phosphate,

Organochiorine and carbonates. Organophosphate compounds have several

orders of toxicity from insects to mammals (Lehman 1965 and O'Brien 1967). The

brganophosphate are preferred inview of their selective toxicity and easy

biodegradability (0' Brien 1967 ). Organophosphate compounds are

neurotoxins and they kill animals by inhabiting acetylcholine are nerve endings

(Reinsford 1978).

Effects of four pesticides such as tailux, endosulfan, malathion,

monocrotophos were studied on species of spiders like Cyrtophora cicatrosa,

Crossoprisa semiriiigopus, Mrapissa caIcutaeiisis and Stegoa'yphus

93

sarassinorum (Sekar and Shunmugavelu I 992b). They suggested that use

of these chemicals for controlling insect pest warrants more careful

considerations because of their potentially negative influenze on the natural

enemies of these pests. Relative efficacy of DDT and BHC dust formulations

against pod borer was investigated by Patel [1 990].

Singh et al., [1 990] reported the effect of malathion and BHC on the

haemolymph of protein and aminoacid concentrations in Epilachna

vigint/etopunctata. It is a common pest of vegetables in North Bihar. It was

exposed to sublethal doses of malathion and BHC. After 48 hours of exposure,

the haernolyrnph proteins and free aminoacid concentration were analysed

colori metrically. He suggested that after pesticidal treatment there is decline

in haernolymph protein and arninoacids. BHC being more toxic in action was

more potent in adversely affecting the protein synthesis than malathion.

Pandey and Mathur (1 990) studied arninoacid alternations in insecticidal

treated insects. The study reveals the effect of certain insecticides viz.,

endosulfan, methyl parathion and premethrin on haernolymph of the larvae of

Diacrisia oblique and the adults of Hieroglyplius ben/an. This experiment

established inter relationship between aminoacid changes and the behavioural

manifestations in the insecticide treated insects.

El

I.

Suba Rao et al., [1 985] analysed the influence of certain aminoacids in

paddy varieties on the infestive ability of lesser grain borer Rliizopertlia

cloinin/ca. Seven aminoacids considered to be important from insect dietetics

point of view namely lysine, leucine, tryptophan, rnethionine, tyrosine, cysteine

and phenyl alanine from twelve paddy varieties. Out of twelve, six were

related as resistant and the rest as susceptible to the pest. Aminoacids play a

role in host plant resistant against some phytophagous pests [Painter 1969).

Effect of malathion on the free aminoacid in the haemolymph of Dysciercus

koeniii was investigated by Singh [1982]. He concluded that there is a

decline in the concentration of all free amino-acids after malathion treatment.

Insects are characterised by the hi g h level of amino acids in their

haemolymph [Buck 1953; Gilmour 1961 & 1965]. The high concentration

of free amino acids is believed to play an important role in osmoregulation as

suggested by Bishop et al., [1926] and Beadle and Shaw [1950].

Buck [1953] concluded that the free aminoacids may be concerned with

protein synthesis. Wyatt [1961] observed that the free arninoacids may be

concerned with energy production for flight and also in cocoon construction.

Insecticides to affect the level of free aminoacids in the tissues including

haemolymph of insects. Malathion WS chosen as the test insecticide because

9'5.

it does not inhibit the krebs cycle [0 Brien 1957). Mansigh (1964) has

studied the effect of malathion on the haernolymph of Cockroach.

Lipids play a definite role in growth and metamorphosis of insects.

Insects are known to have an ability to store large quantities of fats and use

this substrate as a source of energy for sustained muscle activity. Phospho

lipids play a definite role in biological membranes were observed by Dawson

[1966].

Salarna et al., [1 976] and Patil [1 986] was studied the Impact of pesticides

on the lipid content. Patil [1 986] observed increase in lipid content after

treatment with malathion, endosulfan, carbaryl and copper sulphate.

MATERIALS AND METHODS:

The tropical spiders are cyrtopl7ora cicatrosa and Hersiia sa vigyni

were collected and brought to the laboratory condition for experimentation.

Each spider was kept in separate plastic containers filled with perforated lids.

They were fed daily with mosquitoes, Mtisca c'omestica, P17ylloclr01713

humbert/aila and various small insects.

Qualitative estimation of amino acids and quantitative estimation of

carbohydrate, protein and lipids were carried out in Benzene Hexa chloride

(BHCJ treated and control animals. It is a stomach and contact poison and

has fumigant action. It can rapidly penetrate the insect cuticle like DDT. BHC

is available under various trade names such as BHC 5% or 10% dust and

50% wp. It is a poisoning, increases respiration rate and symptoms of

poisoning are tremor, ataxoa (Unsteadiness), convulsions (violent agitation)

and prostration (Vasantharaj David 1975).

Experiments were carried out in siX groups each consisting 10 spiders.

One group was considered as control others were uniformly dusted with

different concentrations such as 5, 10, 15, 20 and 25 mg of BHC powder.

Mortality rate and survival rate were recorded for spiders at different

concentration. After 12 hrs of treatment the animals were taken for further

bio-chemical analysis.

ESTIMATION OF AMINOACIDS.

The tissue content and gut content of the spiders were homogenized

using the morter and pestile in I ml of 80% ethanol. The homogenate was

centrifuged at 3000 rpm for 10 minutes. The supernatent was collected and

concentrated by evaporation and spotted on suitably cut whatman No. I filter

paper.

The chromatogram was run in the solvent mixture containing Butanol,

acetic acid and water in the ration of 4:1:5 (Solvent 1) and another solvent

91

mixture containing phenol and water in the ratio of 4:1 (solvent 2). The

chromatogram was developed using Ninhydrin reagent.

The various aminoacids present in the control and experimental samples

were determined by referring to the respective Rf values as given in the laboratory

manual in Biochemistry [Jayaraman 198 1].

ESTIMATION OF CARBOHYDRATE:

Total carbohydrate in the sample was determined by following the

procedure given below. The weighted of tissue [1 0mg] was put in the morter

2 ml of 80% methanol was added and the sample was ground well. The

homogenate was centrifuged for 10 minutes at 3000 rpm. I ml of supernatent

was taken and 5 ml of 0.2% Anthrone reagent was added. Test tubes were

covered with cotton and kept in a boiling water bath for 10 minutes. They

were cooled at room temperature and CD was measured at 620 urn using

spectrophotometer.

Respective volume of reagent was added to I ml of distilled water served

as blank. Glucose served as the standard. A standard g raph was drawn by

using standard glucose of different known concentrations. The CD was

compared with standard graph to estimate the carbohydrate present [Roe 1955).

ESTIMATION OF PROTEINS.

10 mg of tissue sample was weighed and was ground using a mortar

and pestle and homogenized with 5 ml of 10% TCA. The homogenate was

centrifuged at 3000 rpm for 10 minutes and the residue was dissolved in 5

ml of 0. IN NaOH. Then 0. 1 ml of the residual solution was pipetted out

and made up to I ml with distilled water 5 ml of solution "C" was added to

that I . I ml solution. After 10 minutes 0.5 ml of Folinciocalteau [I: I]

reagent was added. Finally after 10 minutes the coloured solution was read at

700 nm on a digital systronix spectrophotometer. BSA was added as standard

(Lowry et al., 1951].

REAGENT USED:

10% TCA prepared by dissolving 10 gms of TCA in 100 ml of Dist.

0. 1 N NaOH - 4 gm in 1 000 ml dist. H 2 o

Solution A - 2gm Na 2 Co 3 , 400mg NaOH in 1 000 ml dist. H20.

Solution B - 500 mg CuSo 4 I gmp otassium tartarate in 100 ml

dist. H2

Solution C - 50 ml solution A + 1 ml solution B

ESTIMATION OF LIPIDS:

1 0mg of the tissue sample was taken in the mortar and 2 ml of chloroform

was added and homogenized. This homogenate was centrifuged at 2500 rpm

for 10 minutes. The supernatent was transferred to a test tube and evaporated

to dryness at room temperature. To this 3 ml of distilled water and an equal

volume of freshly prepared 2% K2 Cr207 [Prepared by dissolving 2 g mof K2

Cr20 7 in 1 00 ml of conc - H 2 So,) was added. The intensity of the colour

developed was measured at 625 tim in spectrophotometer. Respective volume

of reagent cfter above was added to I ml of distilled water served as blank.

Cholesterol served as the standard. The OD was compared with the standard

graph to estimate the lipid. [Bragdon 1981].

RISULTS

The rults of these studies are tabulated in tables 34 to 38 and fig 70

to 71. The effect of BHC on the mortalityand survival rate of the two species

of the spider after 12 hours of treatment were observed and tabulated [Table

34] . 5mg of BHC does not seem to affect the spider H. savigyni 50°h

monali occurs at 15 mg concentration of BHC and at 25 mg 80% of

mortality occurs. 5mg 81-IC does not seem to affect the spider C. cicatrosa

100

PLATE 22 :Chromatograms showing estimation of different

aminoacids using solvent I (A&CI and solvent II

B&D in tissue content (A&B) and gut-content

(C&D) of the spider Hers/I/a sav/gyn/.

IC 1f'5

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22

50% mortality occurs at 15 mg concentration of BHC and at 25 mg 90% of

mortality Occurs.

ANALYSIS FOR AMINOACIDS:

Different amino acids were analysed in the tissue and gut contents of the

spiders exposed to different concentration of BHC. It is noted that the

number of amino acids was slightly decreased in the experimental animal while

comparing the control of both the species.

Amino acids present in control and treated animal tissue and gut contents

of H. saviyn! were observed to differ radically [Table 35 & Plate 22]. In

normal tissue of H. savigyni 12 amino acids are present. Out of these

Histidine, glycine, alanine, tyrosine, methionine and leucine were represented

in higher intensities as indicated by the appearance of dark spots. While

serine, lysine, argenine, glutamic acid, threonine and valine were detected in

lower intensities as indicated by the appearance of light spots.

On the contrary treated tissue of this spider contained 11 amino acids.

Out of these serine, aspartic acid, glutamic acid, alanine, tyrosine and Isoleucine

were represented in higher intensities than the rest viz., histidine, glycine,

methionine, valine and isoleucine.

101

PLATE 23 : Chromatograms showing estimation of different

aniinoacids using solvent I (A&C) and solvent II

[B&D] in tissue content (A&B) and gut content[CUD] of the spider cyrtopliora cicatorsa.

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Gut Content of H. sav/gyiii contained 12 amino acids, oLit of these

aspartic acid, glutaniic acid, glycine, tyrosine, valine and isolecucine were

respresented in high intensities as indicated by the appearance of dark spots.

While alanine, threonine, proline, methionine, tryptophan and phenyl alanine

were detected in lower intensities as indicated by the appearance of light

spots. On the contrary gut content of this treated spider shows 10 amino

acids. Out of these aspartic acid, glutaniic acid, glycine, tyrosine were

represented higher intensities than the rest of 6 other arninoacids viz, serine,

arginine, threonine, proline, valine and tryptophan (Plate 22).

Results on the qualitative analysis of amino acids present in control and

treated tissue and gut content of C. cicatrosa were observed to differ radically.

[Table 36, Plate 23] . Normal tissue of C. cicatrosa contained 7 amino

acids, out of these arginine, aspartic acid, glycine, alanine were represented in

higher intensities as indicated by the appearance of dark spots. While serine,

methionine and leucine were detected in lower intensities as indicated by the

appearance of light spots. On the contrary treated tissue of this spider

exhibits the presence of 8 amino acids. Out of these histidine, serine, aspartic

acid, glutamic acid, alanine, tyrosine were represented in higher intensities

than the rest of two other amino acids viz., valine and leucine.

102

Gut content of C cicatrosa reveals the presence of 6 amino acids.

Out of these alanine, methionine and leucine were represented in higher

intensities as indicated by the appearance of dark spots. While proline,

tryptophan, and oleucine were detected in lower intensity as indicated by

the appearance of light spots. On the reveals 10 amino acids out of these

histidine, aspartic acid, glutamic acid, tyrosine were represented by higher

intensities than the rest of six amino acids viz., lysine, alanine, proline,

methionine, leucine and Phenyl alanine (Plate23).

QUANTITATIVE ESTIMATION:

Protein , Carbohydrate and lipids were estimated after 12 hours of

treatment in both the species of spiders [Table 37 & 38). Protein level was

decreased in BHC treated animals. The result shown that the mean value of

protein in untreated animal was 11 .8 ± 0.1 02/gm tissue and it decreased in

the experimental animal to 2.62±0.27 mg/I gm tissue in H. saviyni.

Protein in the tissue of the untreated Ccicatrosa was 13.7±0.1 2mg /

1gm it is depleted in the treated animal [25 mg of BHC) 9.9±0.27 / 1gm

tissue.

The carbohydrate levels were estimated using the anthrone method. It is

found to decrease in the experimental animal [6.82 ± 1 .57mg / gm tissue]

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In the Cicicatrosa, the carbohydrate level is present to decrease in the

experimental animal (9.74±0.54 mg/gm tissue) while comparing that of control

animal (11.4 4±1.45 mg/gm tissue)

Lipids levels were estimated using Bragdon method. Results reveal that

lipid level increases in the experimental animal (7.38±0.348 / 1gm tissue)

than the control animals (6.86±0.42mg/gm tissue) of H.sav/gyni.

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C.cicatrosa [10.3±0.17 MG (1 111 tissue].b

DISCUSSION

Pesticides selectively effective against the pest without adversely affecting

are to be identified for incorporation in the inte g rated pest management

programme [Hassan et al., 199 11.

Effects of sublethal dose of various synthetic insecticides on the

biochemical constituents of the body have been studied by many workers on

different insects (Singh 1986, Bharathi et al., 1987) and on other animals

(Shakoori et al., 1976).

104

The effect of BHC on the mortality rate and survival rate of the two

species of the spiders after 12 hours of treatment were observed. It is

concluded that the higher concentration of BHC (ie. 25mg) adversely affected

the spider.

The present study also incorporates the results of aminoacid alterations

in the insecticide treated trophical spiders H. sav/gyni and C. cicatrosa.

The studies have shown that there is a drastic reduction in the free aminoacid

pool in both the spider species. Some free aminoacids are present in controlled

animals and disappear in the treated animals.

Insects are characterised by the high level of aminoacids in their

haemolymph [Chen 1962; Clements 1963]. The high concentration of free

aminoacids is believed to play an important role in osmoregulation as suggested

by Bishop et al., (1926). Free aminoacids may concerned with protein

synthesize (Buck 1953) and energy production for cocoon construction [Wyatt

196 1]. A great number of aminoacids like alanine, glutamine, glycine and

proline are known to have important roles in the synthesis of cuticle proteins,

chitin and other constituents of the cuticle. Some of the amino aids are

produced under stress conditions.

The possible explanation for the geperal decrease in the concentration

of aminoacids is the increased neuromuscular actively in the treated animals

0

10

which resulted in higher demands for energy. As a result of this physiological

change that appears to be an increase in the rate at which the different free

aminoacids (FAA) enter in tri carboxylic acid cycle (TCA) and get oxidized.

The present findings are in support of the observation made by Ray (1964);

Mansingh (1 964) and Kuikarni (1 969) in their poisoned insects. Thus it is

concluded that the depletion of aminoacid was mainly consequence of the

hi gher metabolic activity of the poisoned insects. The decrease in the level of

aminoacids is due to all imbalance between the rates of anabolism and

catabolism in the poisoned insects. Since g lutamine enters into many metabolic

process (Meister 195 7) it is difficult to explain the increase in its concentration

in the spider tissues after the insecticidal treatment.

It has been reported that protein concentration of haemolymph and

other tissues of the body shows a decline in insecticide treated insects due to

depletion of the reserve proteins as a result of stress and due to the direct

effect on protein synthesizing machinery. In the present investigation, BHC

was more potent in aggressively affecting the protein biosynthesis and

carbohydrate metabolism.

The lipid constitutes all important group of organic substances insect

tissues and act as chief form ill which energy stored. Different researcher like

Lambremount and Blum [1 963) and Fast (1 964) studied the lipid content

El

106

of different insects. They found that many factors such as age, sex, stage of

development and food supply affect the lipid content. Saxena and Vinod

kurnar [1981] observed increased lipid content in Ehrotogonus

• tracliypterus when exposed to difluron, BHC and penfluron. Similar research

were recorded by Saxena and Srivastava [1969] in Periplaneta americana;

Saxena and Aditya (1974) in Poecilocerus pictus. In the present study

H.savigyni and Ccicatrosa treated with BI-IC exhibited increase in lipid

content appears due to condensation of more than one type of acetate during

pesticides intoxications, as the aceto acetyl Co-A units which may form melonic

acid, squalone and cholestrol [Swami et. al., 1983].

107

Investi gation was carried out on Biology of five species of spiders namely

CyrtophOr3 c/ca trosa, A rg/ope puIchella, Argiope catenulata, Hippasa

pisaurina and Hers/I/a sav/gyn/. Biochemical aspects of the toxis effect of

BHC on two species of tropical spiders namely, the orb-weaving spider

[C.cicatrosa] and the two tailed spider [H. savIgynfi.

Cc/catrOSa is found commonly on branches of trees, shrubs, herbs

and fences. A. ilchella is found tornmonly on branches of trees and in the

paddy field. A. catenulata is found only in the paddy field. H.sav/gyn/ is

found commonly on trees like coconut, rubber, tamarind, Jack etc., which are

located near paddy field. H. pisaurina is found on fences near the paddy

field.

C. c/catrosa constructs dome shaped permanent web and the animal

sits motionless in the centre of the web with folded appendages. The web acts

as an extension of the spider sensory system. A. pu/chef/a and A. cateilu/ata

an orb weaving spider and the web is semi permanent with stabilimentum on

all four directions. It is made up of frame, radial spokes and catching spiral.

H. p/saurina is a funnel web spider. it builds a permanent web on the

ground and fences that consists of a silken sheet with an attached funnel that

extends into some features of the habitat such as crevice or a clump of grass.

10

H. saviyni does not construct webs but makes use of silk in activities such

as sperm induction, tOlIStRICtiOn of egg cocoon and at prey time.

In the paddy field, the web traps many insects. These spiders are exploited

to economic advantage for the control of rice bug and various types of beetles,

larval forms in the paddy field.

C. cicatrosa, A. puichella and A. catenulata capture their prey in

the web, the spider wraps the prey and the prey is bundled in a band of silk

before feeding commences. H. pisaurina and H. savigyni are active hunting

spiders. H. sawgyni spins scant webs of irregular threads. As SOOfl as

these spiders come into contact with the prey the long spinnerets are swayed

rapidly and the prey is bundled in a band of silk before feeding commences.

H. pisaurina has a poor vision. So when the prey creates vibrations in the

web, the spider orients towards the prey, which is entangled in the threads of

the web. Then they dra g the prey to the hub by holding it with the chelicerae.

The predatory potential of the spiders on an insect pest of paddy f\e\ds,

it is concluded that the paddy field spider A. pu/chef/a and A. catenu/ata

Consumedsuiiied more prey than the C. cicatrosa, H. pisaurina and H. sa vigyni.

The remains of prey or ganisms present in the web of the spiders concluded

that, during monsoon season the spider feeds more insects than in summer.

109

This is because insects are plenty in all places in monsoon. All these spiders

are polyphagouS. They destroy various types of pests in their wild.

H. sav/gyn/ C. cicatrosa, A. pu/c/let/a these spiders make different

types of special egg Cocoons in which they lay their °°s After their egg

0

laying the female guards the ° cocoons by visit' them periodically. Ill

above three moulting takes place five times durin G IL s growth period for male

and six times • during its growth period for female. Cannibalism takes place

during the moulting stages in which the spiderUngs devour each other.

Experiments were conducted to document the toxic effects of BHC on

the spiders H. savigyni and CcicatrOsa 5mg BHC has less effect oil

spiders of both species. 80-90% mortality for spiders was found to occur at

25 mg BI-IC. chromatography studies were performed to estimate different

amino acids in the tissue and gut contents of the spiders exposed to different

concentration of BHC for a period of 12 hours. It was observed that the

number of amino acids was slightly decreased in the experimental animals

than the control in both the species. Blo - chemical analysis was performed in

both the species of spiders after 12 hours of BHC treatment. The results

reveal that in treated animals protein and carbohydrate levels are decreased

and lipid level is increased while comparing control animals.

110

Spiders can be employed as effective biocontrol agents from the frontiers

of natural enemies to control the pests of paddy fields, groundnut, cotton

and other fields seem to be more effective for sluggish and non flying insect

pests where as, for the Hying insect pests weaving spiders seem to be the key

for control.

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