INTRODUCTION
In a unicellular organism,
a single cell performs
all
the
functions of life.There is,therefore,no need for any special
mechanism for coordination
multicellular animals
of the
the cells
various
transform
activities.
into
In
different
tissues and organs of specific functions. In such an animal,
the coordination of activities of
various organs
which be-
comes an unavoidable necessity for the organism concerned is
accomplished by the nervous system.
For a proper understanding
of the
nervous
system
and
an
appreciation of its role in animal, it is important to know,
in general terms,
what an animal
is made up of and how the
activity results and how it is controlled/regulated and utilized.
We know that every organism is a well organised mass
2
of matter and is composed of many different kinds of chemical compounds. This matter is charged with the potentiality
of chemical and physical.~ctivities, and the latent energy
present in it tends to remain in a state of equilibrium
with its environment. The different chemical compounds present in an organism are either stable or labile. The stable
compounds form the integument, the skeleton, the connective
tissue and the supporting frame-work of muscles, glands and
cells. The labile compounds occur in the
secreting cells,
glandular cells and the contractile tissue of muscles
and
the receptive and the conductive parts of nerve tissue. The
molecular structure of each of these labile compounds is unstable though, it manifests its activities after receiving a
stimulus. Such an activity becomes a necessity to an animal
for making suitable adjustments according to changes in its
environment. The transmission of stimulus from stable periphery to internal labile tissues of an organism and the necessary mechanism for controlling and directing the liberated
energy for successful living are furnished
by
the nervous
system and its motor mechanism.
The insects have never attracted the attentions of biochem-
~t
..
~
~~
-
ists to the same extent as either the microorganisms or the
vertebrates have done. In fact, it is probably no great exaggeration to say that a major part of our current knowledge
3
of biochemistry stems from the observations made on either
. '
f
the tissue from rat or
.
·~
'
~.coli.
:.__.,
;
rhe nervous system of insects has, for a long time, been
the favoured material of many neurophysiologists, and much
basic knowledge of nerve function has resulted from studies
)n insects or other invertebrates. It is important to empha·
size that, so far, the same basic mechanisms involving the
same neurotransmitters have been found in all the
systems
studied in the vertebrates or the invertebrates.However,the
)iochemists or more particularly neurochemists seem to have
Largely ignored the nervous system of insects.
lnformation on the biochemistry of these systems
~he
outcome of the works of neurophysiologists
rith elucidating the molecular mechanisms
Much of the
has
been
concerned
underlying
the
>hysiological activities. It should be emphasized that our
;nowlodge in this area is still sparse and is based on ob1ervations made on a very limited number of insect types.
'hua it Js wellkni.gh impossible to present as comprehensive
//
~ picture as one can b?~st of the vertebrate nervous system.
lowever, it is hoped that the very fact that such large gaps
o exist in our knowledge on the biochemistry of insect ner·ous system· will encourage workers to leave their favoured
ield 'rat cerebral cortex' and turn their attention to the
nsect world.
4
Insect neuropharmacology as a discipline is relatively of
recent origin coming as an off-shoot of neurobiology/insect
biology and is interdisciplinary in status.The nervous systern of insects and other invertebrates, in contrast
to the
vertebrate nervous system, offers an investigator the advan·
tage of working with relatively small numbers of experimentally manipulatable and in many cases, identifiable neurons.
Briefly, the nervous system of insects may be considered to
consist of three main units. A dorsal anterior brain,a ventral nerve cord often showing segmental ganglia
from which
nerves run to the peripheral sense organs and muscle systems,
(frequently jointly refarred to as the insect central nervous
system) and a peripheral nervous system.
The insect central
nervous system offers the neurochemist a useful
range
of
attributes. Its economical use of neurons (at least in contrast to the mammalian CNS) and the robustness of its structure have, for a long time, been exploited by the physiologists, with the result that the physiology and the neuroanatomy of the system by now are well known.
The nervous system constitutes the connecting link
between
the various sensillae located at the periphery of the body,
r e s po n s i b 1e for receiving the various stimuli
from the en-
.'t},,.?'
11ironment on tone hand and the effector organs (muscles)
1
on
5
the other. The effector organs respond to the stimuli so
that the normal functioning of the body goes on smoothly in
an organism.
The nervous system of an insect is a modular structure. The
modules are the neurons. In insect this system
consists of
large number of neurons which are produced into long processes, or axons and non-nervous parts, neural lamella and neuroglia which provide support and nutrition to neurons.The conduction of impulse along a nerve axon is an electrochemical
process which is. influenced by the composition of the fluid
bathing the nerves. But the axons do not
form a continuous
system as the successive axons are separated by small gaps.
The nerve cells communicate with each other and with effector
cells such as muscle fibres and gland cells
primarily
by
means of the controlled release of chemical messengers.Within
each neuron a signal carried by the nervous system
passes
from point to point virtually without any alteration,when the
signal passes from neuron to neuron, however it must cross a
gap. It is generally believed that in most cases the transmission across a gap is effected by the release of a chemical
compound called a neurotransmitter.
A neurotransmitter is a
chemical messenger that is secreted
into the
intercellular
cleft at a morphologically differentiated synapse
between a
6
---~---------
neuron and its follower cell and then acts on a receptive region of the membrane of the post synaptic cell. When a nerve
impulse arrives at a nerve terminal, it triggers the
secre-
tion of a neurotransmitter that can travel across the gap and
stimulate the next cell, thereby carrying the impulse forward.
An insight into the mechanism underlying its
release
is
major importance to the understanding of brain function
of
and
the action of toxic substances and drugs on the nervous system.
A general review of the insect nervous system
is
given
Calhoun (1963); Narahashi (1963,1965a); Smith
and
by
Treherne
(1963); Herridge (1965); Treherne (1966) and Boistel (1968).
The chemical pattern of the arthropod central nervous system
has generally been determined in close relation
to possible
neurotransmitter, especially in the insect group (Klemn,l976).
In insect central nervous system one of the primary neurotransmitters is the Ace1ylcho1ine (Corteggiani and Serfaty, 1939;
Roeder, 1939). The nerve cord in Periplaneta contains it some
15 times the average concentration in the
mammalian
central
nervous system (Mikalonis and Brown, 1941; Tobias, 1946).Many
workers have worked on the particular neurotransmitter acetylcholine on the nervous system of different insects and studied
the di.fferent aspects on the different angles
( Lewis
and
Smallman, 1956, Treherne and Smith, 1965, Mansingh and Smallman, 1967, Pichon, 1974, Prescott U g., 1977; Donnellau and
-------------------------------------------~------------------------------------------
~
7
lhtrris, 1977; Borkowska et
Ta keno e t
~
!!!••
1. , 1981; BanerJee
1978; Lunt, 1978; Breer,l981;
tl a 1., 1984a;
Banerjee
and
Choudhuri, 1985a).
This neurotransmitter
acetylc:holine~synthesized
I
of the enzyme choline acetylase
with the help
(choline acetyltransferase)
(ChN;) and hydrolyzed by the enzyme cholinesterase.
Acetyl-
choline, cholinesterase and choline acetylase are all present
in the nervous system of insect (Smallman, 1956).0nly a limited number of insect species have been analyzed for ACh, AChE
and ChAC contents. Both biochemical and histochemical studies
reveal that the activities of ACh and AChE of the insect CNS
are much higher than that of the other
invertebrate species
(Pichon, 1974). The cholinesterase in insects was first
re-
ported by Gautrelet (1938) who found a high titre of ChE
and
ACh in the heads of bees. Uptil now many scientists investigated experimentally the cholinesterase activity
in the ner-
vous system of different insects in various ways
and
gave
different opinions. The workers of earlier days (Corteggiani
and Serfaty, 1939; Roeder, 1939; Roeder and Roeder, 1939;
Mikalonis and Brown, 1941) had already recorded the occurrence of cholinesterase in various insects.
Richards
and
Cutkomp {1945) detected cholinesterase in the brain of workers
of
~~
mellifica. The activity of ChE in the CNS as a whole
8
of cockroach was studied by Stegwee (195l).The highest activity of cholinesterase was noticed in the most active insects
such as Musca, Apis and Periplaneta
(Metcalf~
al., 1955).
The activity of ChE of homogenates of the thoraces and heads
of housefly and of thoracic central nervous system of cock-
roach was determined according to a titrimetric method(Chadwick~
al., 1953,1954; Sternburg and Heiwitt, 1962).
The
cholinesterase activities in the brain of adult worker honey
bee, cecropia silk moth Pieris brassicae, red flour beetle
t
Tribolium castaneum during metamorphosis were
studied
by
,fRockstein, 1956; Heywood, 1965; Shappirio et al., 1967;
Zettler and Brady, 1969.pirespectively.{j According
to
some
workers the acetylcholinesterase activity was associated with
the circadian rhythmicity in the brain of house crickets
(Cyrnborowsky
~ ~1.~,
~'
1970), ·in the nervous system of the cock-
roach, grasshopper (Vijayalakshmi, 1977; Vijayalakshmi
and
<I
;:-_--~..,,
1
century lot of works emanated
Sasira Babu, 1978). In the 19th
_r
from different places on the cholinesterase activity
nervous system of different insects (Bauer, 1976;
1977; Tri.pathi and O'Brien, 1977;
1978al
Pant~
1 981 ; Ho u k e t
el.••
~.
Barker~!.!.··
in
Call et
the
~.,
1978; O'Brien,
1.978; T.ilyabaev, 1979; E:dwards, 1980;Breer,
, 1 981 ; Vo s s , 1 981 ; S i 1v e r and Prescot t , 1 9 8 2 ) •
It lR known that the toxic substances used for the purposes of
insect control are selected q,n the basis of their differential
9
--------toxicity in respect of their mode and mechanism of action .on
the target cells, tissues and organs. These toxic substances
are gr6uped into three categories 1) Neurotoxic
2) Respiro-
toxic and 3) Cytotoxic. Of them the neurotoxic ones
are
of
great importance and widely used. The increased and
indis·
criminate use of the chemical pesticides in plant protection
and in vector control in most parts of the world
since 1945
has resulted in widespread environmental hazard caused by the
and
contamination with more persistent
-------
in
many
..........
cases of acutely toxic effects on non_-_!a_r,get
animals exposed
.
.
\
to highly toxic but less persistent ones. The pesticides are
generally used to protect the crops from the ravages of pests
~f
diverse nature, nevertheless they cause drastic hazards to
1on-target species. But after the second world war,the pesti·
--·--
:ides of organophosphate and carbamate groups have been used
~normously.
~re
So due to their injudicious and unwanted use they
known to cause untold hazards in the environment and ad-
rerse effects on the fishes, birds and mammals.
situation arises when they are contaminated
A dangerous
with the
resources {as reported from the river systems
of the
3tates and U.K.). In some countries the use of
some
water
United
of
the
:ompounds like DDT, Aldrin, Dieldrin have been banned because
>f their unacceptable persistence, bad effects
1nd evidence on possible carcinogenic activity.
on
wildlife
The mode of
10
---------------------------------------------------------------action of the organophosphate and carbamate pesticides in
animals is disruption of nerve impulse transmission by metaboU.tes that "irreversibly" inhibit acetylcholinesterase
(AChE), which modulates levels of the neurotransmitter acetylchoUne (O'Brien, 1.960,1967,1978b).
The intoxication of an organism with any insecticide involves
variety of steps and reactions (Narahashi, 197la). The uptake
of insecticide is the first step to occur,
and a number of
factors such as lipid solubility and vapor pressure of
in-
secticides are related to this process. The insecticide that
has entered the body is then transported to various organs and
.x//
may undergo a variety of biotransformations in which
it
is
either converted into a more potent compound or degraded to
one which is relatively nontoxic. The active form of insecticide eventually reaches its target site
and
exerts effects
characteristic of the insecticide and the tissue concerned.
Since the nervous system constitutes the major,
and in many
cases the sole, target site for the majority of insecticides
which have been developed, an ability to measure their effects
on this system {s critical to establishing the precise mechanism by which they act. The literature
so far published
mainly concerned with the cholinergic transmission
and
is
the
effects of neurotoxic substances (different organophosphorous,
11
chlorinated hydrocarbon, and carbamate compounds) on tke central nervous system of different insect species and their comparison with vertebrate central nervous system (O'Brien, 1960;
Brady and Sternburg, 1966,1967; Hama and Iwata, 1971;
and Parveen, 1974; Devonshire, 1975; Hama, 1976;
Feroz
Narahashi,
1976a; Kasturi Bai and Reddy, 1977; O'Brien, 1978a).
There have been a few demonstration on the behaviour/fate of
different other macromolecules like protein,carbohydrate,lipid,
phospholipid,cholesterol,fatty acids etc.
and enzymes
like
choline acetylase, glutamic oxalacetic transaminase, glutamic
pyruvic transaminase, acid phosphatase, alkaline phosphatase,
5 1 -nucleotidase, catalase, peroxidase, etc. apart from acetylcholine and cholinesterase in the central nervous system of
insects after the
treat~ent
of different neurotoxic substances.
The neurotoxic substances other than organophosphorous, carbamate and chlorinated hydrocarbon compounds;pyrethrum the botanical origin organic insecticide has not been well tested regarding their effects on the central nervous system and therefore, their differences with other neurotoxic substances
still in nebular state (Narahashi, 1976a;
Because the organophosphates and carbamates
O'Brien, 1978a).
produce harmful
effects on the wildlife, there is an urgent need for
native chemical or biological agents for pest control.
---------------'
are
alterThe
12
pyrethroids developed within the past seven years help meet
this need to some extent. The pyrethrum is chosen
for
its
more favourable persistence and also for producing less harmful effects on humans and pets (Casida, 1980).
It hns been known for a long time that the nervous system is
the major target site of pyre thro ids. For example the pyrethroids cause repetitive
diachar~es
and eventual
paralysis
in the nerve fibre and nerve cord of insects(LDwenstein,1942;
Nelah and Gordon, 1947; Nnrahashi, 1962a,b; Burt and Goodchild,
197la,b: Camougis and Davis, 1971). The synapses are possibly
the initial target sites of the
pyr~l;r~ds,
because they are
affected at concentrations considerably
lower
effective on nerve fibres. According to
Roy~
than
those
al.,(l943)when
it is introduced into the body cavity of an insect, the pro---'
~ressive
nature of paralysis of its legs indicates
~yrethrin
- is
~
-
..-"
that
the
carried with the circulation of the body fluid.
rhe pyrethrins represent typical nerve poisons causing succes-
;ively i) excitation, ii) convulsion, iii) paralysis and iv)
ieath in insects. Even when given in small dosages,they cause
t
very rapid "knock down"· action. The insects recover
com-
)letely from the effects of sublethal doses if they are yet to
)ass the initial stage of paralysis (Yamamoto,l970). The pyre•
;hrum causes repetitive discharges of impulses in the arthropod
13
nerve due to interference with axonal sodium and potassium
\
channels, and the blockade of both sodium and potassium conductance is the cause of paralysi• (Yamamoto, 1970). During
~'/
conduction blockade in the CNS caused by the pyrethrum
potassium content significantly decreases with the
in sodium eoncentrati.ons (Banerjee tl
~.,
the
increase
1977a). The enzy-
matic pathway in the central nervous system of Schizodactylus
monstrosus is completely hampered after application of pyrethrum. This is due to the inhibition of acetylcholinesterase
activity and also due to the accumulation of phosphatase enzymes (Banerjee
tl.!l.!..•• 1984a).The role of CNS enzymes,este-
rnses are capable of detoxifying noxious compounds.The peripheral layers of the insect CNS may have to perform a liver
like detoxifying role to meet the needs of the deeper,neuronal layers of the tissue. An early histochemical
Wigglesworth (1956) rlemonstrated that the
insoet porineurium
w1u
study
cytoplasm
of
of the
exceptionally rich in oxidases, dehy-
drogenases and various esterases. Now in view of the above
I
~~ts ono may ask a partinent question : whether there is any
~elatioE among the different macromolecules and enzymes within
____......-....
.
--
the central nervous system following the application of pyrethrum. Information about the toxic effects of pyrethrum on the
biochemical components of the central nervous system
from extensive/comprehensive though earlier works
is
far
suggested
14
·-·------------------
-..--·-·--···--··----.... -·······-...
that the action of pyrethrum was confined to the central
nervous system.
rurther it is known that the mode of action of neurotoxic
substances on the central nervous system in insects is simi·
lar in quality to that of vertebrates (O'Brien, 1978a). The
insect central nervous system and its synaptic zones are also
quite similar to the vertebrate system, i.e., the cholinergic
system (Boistel, 1968). The individual ganglia lack a vascular system and yet possess, in their neural lamella and cells
of the perineurium, mechanical protection and a blood-brain
barrier analogous to that of a mammal (Treherne,l974).Equally
important is the fact that even when isolated
in vitro, the
ganglion possess high level of spontaneous activity which may
be quite easily monitored as action potentials from one of the
major nerves. Since smoking has been a common human habit from
long back, interest has often centered round
its effects
this habit on human health. In India the use of
tobacco
of
is
fairly widespread. Its different mooe of consumption include,
in addition to smoking, chewing it raw or use as snuff. Nicotine, the pyridine alkaloid compound of tobacco causes a dangerous situation in human society due to the habitual smoking
of man. Nicotine does not possess any theraputic
application
though, yet its high toxicity and highest content in tobacco
···--·----·--------------------------
15
adduce to it a considerable measure of medical importance,
Accordingly, the pharmacological action of nicotine requires
due attention. Nicotine has long been of interest to pharmacologists as because its toxicity varies a
great deal
with
the mode and form of application. In small doses it exerts a
stimulating effect on autonomic ganglia. It
activates
nicotinic cholinergic receptors on ganglion cells.
the
Through
circulBtion maximum quantity of nicotine accumulates in the
brain causing paralysis of the central nervous system
(Tsujlmoto et al., 1955J Hansson and Schmiterlow, 1962).
--
Since there exist a number of good reasons to apply the knowledRe nf pharmacology of insects to the
vertebrate
nervous
system, recently n number of researchers worked in this area
with particular attention to acetylcholine and acetylcholinesterase and their behaviour under different experimental conditions in insects (Matsumura and Hayashi, 1967;
Dowdall~
al.,
1976; Hung Ho and Sudderuddin, 1976; Haubrich and Chippendale,
1977; Murphy, 1980 and Hildebrand, 1982).
The electrophysiological data indicate that the nicotine
receptor is the primary target of nicotine
in
(Volle and Koelle, 1965) as well as in insects
ACh
vertebrates
(Roeder
and
Roeder, 1939; Flattum and Shankland, 197l).The chronic administration of nicotine, in a dose which mimics that obtained by
16
heavy smokers, causes biochemical changes in an autonomic,
cholinergic neuron
(Dahlstrom~
al., 1980). It has led to
the assumption that nicotine is not active in vitro but requires a system whereby it may be broken down into various
other components which serve as the active principle
(Banerjee et al., 1982).
--
No report is there on the biochemical changes in the central
lnervous system of human beings after application of nicotine
I
though in vertebrates there exist some information on this
aspect (Gudbjarnason, 1968; Sershen and Lajtha, 1979a,b;
Sankaran
~ ~.,
1981; Sershen
~ ~.,
1981).
Due
to
the
accumulation of nicotine in brain the nerve impulse transmission pathway is completely blocked.
The
inhibition
nerve impulse transmitting enzyme acetylcholinesterase
of
in
the CNS is regarded aa a significant parameter to assess the
complex toxicogenetic effects of this toxicant.Now the question may be posed as to whether or not there exist any substn.nc:a which plays an important role to revive the AChE: act i-
vity nnd acts as
B
putative neurotransmitter during inhibi-
t ion of AChE.
As it is known that the mode of action of neurotoxic substances on the central nervous system in insects is similar
in quality to that of vertebrates. So one of the objectives
17
of the present investigation is to examine whether this is
true or not. Nicotine is chosen here because it is known as
hazardous chemical pollutant (smoking hazard)in human society and for that matter it becomes an academic necessity
to
find out the exact nature of the pharmacology of the insect's
central nervous system. Precisely more, attempts are made to
ascertain the effects of toxic stress, i.e., pyrethrum
with
sublethal doses and nicotine with sublethal doses on the biochemistry of central nervous system of orthopteran
female insect, Sch_izodactylus monstrosus Drury.
male and
In
recent
years the biologists have used the term "stress" for any environmental condition potentially unfavourable to living organisms. 1be insects form the most advanced group among the invertebrates from the evolutionary view point and particularly
the insect, §..
~nstrosus
which is used here
naterial, among the insects is highly potent,
as
the
robust
studyand
vigorous.
l•
!!!Q~J.:ro f!~
is common 1y found in the sandy beds,
the area
\djacent to the wall irrigated crop fields nearer to the river
Jamodar. By their mode of feeding and
~ndowed
burrowing habit being
with the well developed mandibles
and characteristic
[orelegs they reportedly cause some damage to the potato crop
lnd other vegetables. In India, however, it does not
appear
lA
to have assumed the status of a pest of either potato or
other vegetables.
The experiments are also conducted to show the comparative
effects of different time exposures after treatment.
This
study is further extended to find out the probable correlations between the synthesis and the activity of major enzymes and macromolecules in the nervous system of this insect.
fhe biochemical components such as total sugars, glycogen,
trehalose, glucose, total lipids, cholesterol,
free fatty
acids (FFA), phospholipids, total protein, total free amino
acids (FAA), acetylcholine (ACh) and some cellular enzymes
such as general esterase, acetylcholinesterase(AChS),choline
1cetyltransferase (ChAC), alkaline phosphatase, acid phosphatase, glutamic oxalacetic transaminase
(GOT~glutamic
pyruvic
;ransaminase (GPT), 5'-nucleotidase (5'-ND), catalase, perlxidase and the sodium (Na+) and potassium ions (K+) of the
>rain and ventral nerve cord with ganglia have been analysed.
")
io this study is expected to explain clearly
'~"'t.: --~-~
':>
t
the probable
:orrelations existing in between these biochemical components,
1etween the activity of enzymes and different macromolecules,
ons etc. This study will also reveal the
differences
in
ensitivity or potentiality to the neurotoxic action by the
ifferent tissues.
-·---------------·------------------