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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/.
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]
I'
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while comparing control animal [14.2 ± 2.0 mg/gm tissue] of H. saviyni.
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.
Lipid level was found to an increase in the experimental animal [11 .3
0.10m tissue] when compared to the control of animals of/1am±
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.
11