Effect of Azadirachtin on Insects
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Humans have always been in direct competition with a myriad of insects, pests from our ancestral beginning. This competition for food with insects intensified when humans began to cultivate plants converting the natural ecosystem to an agroecosystem. Also insects serve as vectors of various diseases caused by bacterial, filarial nematode, protozoans and viruses. Therefore control of insects posed a major concern for the development of the economy. In 1939, the discovery of insecticidal properties of DDT (Dichlorodiphenyl trichloroethane) by Paul H. Mueller changed the scenario of pest management. During World War 2 DDT was extensively used to prevent epidemics of several insect vectored diseases such as yellow fever, typhus elephantiasis and malaria. This drew attention to the possibilities of more synthetic insecticides and as a result the use of pesticides in various arenas soared from 1940-60, complete reliance on pesticides intensive pest management was leading agriculture on a “pesticide treadmill”. The overreliance on synthetic pesticides from late 1940s to mid-1960 was referred to as “Dark Ages” of pest control. The cheapness and effectiveness of synthetic insecticides threw natural compounds into shade. But very soon other shades also began to appear. In 1962, the appearance of book “Silent Spring” (by Rachel Carison) showed that pesticide residues were building up in ecosystem with detrimental effects on wildlife and beneficial insects. Due to extensive and intensive use, misuse and abuse of insecticides the following problems were becoming prominent and intolerable (ecological backlashes):
- Development of insecticide resistance- many insects started developing resistance against pesticides which increased the cost of management.
- Due to killing of natural controlling agents, the phenomenon of pest resurgence became more evident.
- Also minor pest were achieving the status of major pest i. e. secondary pest outbreak due to significant decline in its natural enemy (predators and parasites).
- Ecological imbalance due to poisoning of all the realms of environment.
- Increase in the concentration of hydrochlorinated insecticide in food chain.
- Intolerable residues on the food made the food obtained after such treatment uneconomical as it became unfit for consumption and unfit for exports due to high toxic residues.
- Killing and harmful effects on Non target organisms became more prominent (like birds, fishes and other wildlife).
Overviewing these effects, there was an utmost need for the development of environmentally sound management practices. This lead to the idea of Integrated Pest Management (IPM) . A panel of experts put the concept of IPM in 1968. IPM as defined by FAO is a system which in consideration with the present environment and pest population dynamics, integrates all the sustainable techniques of pest management as compatible a manner as possible and maintain the population of pest below the level which can cause economic damage (i. e. below economic injury level). The approach is to minimize the dependence on insecticides and maximize the use of ecofriendly methods so as to cause minimum damage to the environment. Botanical pesticides, thus is an very important component of IPM as
- They are easily degradable.
- Don't affect non target organisms, natural controlling agents such as predator, parasites.
- Don't form residues
- And has no harmful effect on humans as they are very specific in action.
Botanical pesticides refer to the use of chemical or organic compound produced by plants, plant products, which have harmful effects on the growth, development and survival of insect pests. Plants are a rich source of such organic compounds.
The practice of utilizing the derivatives of plant i. e. botanical pesticides in agriculture dates at least two millennia back in ancient China, Egypt, Greece and India. Even in North America and Europe, the documented use of botanicals extend back more than 150 years before the discovery of major class of synthetic chemical insecticides (OP, carbamates and pyretheroids) in mid 1930s to 1950. It is very clear from the recent history that the chemical insecticides have essentially relegated the botanical pesticides from an important role in agriculture to a trivial position in the market among various crop protection strategies.
The total number of 20 phylochemicals is estimated to be 500000, so far only 10000 of these have been isolated. At present four major types of botanicals are being used for the control of insects. These include:
- Neem (Azadirachtin).
- Essential oils.
Others are in limited use like Ryania, Nicotine, Sabdella. Whereas Nicotine, Rotenene, Natural Pyrethrins constitute the outstanding example of older botanicals, extracts and compounds from the Neem tree (A. indica) have emerged as the most prominent phytochemical pesticides in recent years. Among the various biologically active compounds that can be extracted from the Neem tree like- triterpenoid, phenolic compounds, carotenoids, steroids, ketones; the tetranortriterpenoid azadirachtin has been the most extensively studied pesticide as 1) it is relatively abundant in Neem kernels. 2) has biological activity on a wide range of insects.
PLANT SECONDARY METABOLITES
Plants produce a large, diverse array of organic compounds that appears to have no function in growth and development. These substances are known as SECONDARY METABOLITES or secondary products or natural products. Secondary metabolites differ from Primary metabolites (amino acids, nucleotides, sugars, acyl lipids) as:
- They have no direct roles in photosynthesis, respiration, protein synthesis etc
- They have restricted distribution in plant kingdom.
In a seminal paper Fraenkel stressed the role of secondary metabolites as defense system against insects, pests and other natural enemies.
Though they play no role in growth and metabolism they play important ecological role in plants:
- They protect plants against being eaten by herbivore and being infected by microbial pathogen.
- They serve as attractants for pollinators and seed dispersing animals and as agents of plant-plant competition.
Because of their ecological role, plant secondary metabolites are classified as ALLELOCHEMICALS, a term coined by WHITTAKER. An allelochmical is defined as a non nutritional chemical produced by an individual of one species that affects growth, health, behavior, population ecology of another species. Plants produce an astonishing array of Secondary metabolites. Even a single plant species may produce an extensive pharmacopeia of recondite chemicals. Periwinkle for example contains about more than 100 monoterpenoid indole alkaloids. It has been estimated that plant kingdom synthesizes hundreds of thousands of different secondary metabolites. The no of identified compounds now exceeds 10000.
Secondary metabolites as plant defense is result of co evolution between plants and herbivores
Plant secondary metabolites can be divided into three chemically distinct groups:
- NITROGEN CONTAINING COMPOUND
The terpenes constitute the largest class of secondary products . the diverse substances of this class are generally insoluble in water. They are biosynthesized from acetyl coA. Terpenes are classified by no of five carbon units they contain as:
- Monoterpenes: Contain 2 five carbon skeleton
- Sesquiterpenes: Contain 3 five carbon skeleton
- Diterpenes: Contain 4 five carbon skeleton
- Triterpenes: 30 carbons
- Tetraterpenes: 40 carbons
- Polyterpenoids: (C5)n,where n>8
Some terpenes have role in growth and development
Terpenes defend against herbivore in many plants. Terpenes are toxins and feeding deterrentsto many plant feeding insects, thus they appear to play important defensive role in plant kingdom and protection of agricultural crops. Examples of important Terpenes:
- PYRETHROIDS: These are monoterpenoid that occurs in leaves and flowers of Chrysanthemum species show very striking insecticidal activity. Both natural and synthetic pyrethroids are popular ingredients in commercial insecticide because of their low persistence in the environment. Pyrethrum is the predominant botanical in use accounting for 80% of global botanical insecticide.
- ESSENTIAL OILS: These are the mixture of monoterpene and sesquiterpene that lends a characteristics odor to the foliage . e. g Menttholin Peppermint oil and Limonenein lemon oil are monoterpenes. Essential oils have well known insect repellent properties. They are frequently found in glandular hairs and serve to advertize the toxicity of plant repelling potential. Phytophagus insects even they take a trial bite.
- VOLATILE TERPENES: In corn & wild tobacco certain monoterpenes and sesquiterpenes are produced and emitted only after insect feeding has already begun. These substances prevent oviposition and kill plant feeding insects and so help in controlling further damage. These also attract natural enemies of plant feeding insects so promise a sound means of pest control.
- LIMNOIDS: These are a group of nonvolatile Triterpene. Among these the most powerful deterrent to insects feeding known is Azadirachtin. It is a complex limnoid from Neem tree which is feeding deterrent to some insects at as low as 50ppm and it exerts a variety of toxic effect. It has considerable potential as a commercial insect control because of its low toxicity to mammals.
Plants produce a variety of secondary products that contain a phenol group, these are called phenolic compounds. Plants phenolics are a chemically heterogeneous group of nearly 10000 compounds . many of these serve as defense compounds against herbivores.
The release of phenolics into soil limits the growth of other plants.
LIGNIN a highly branched polymer of phenylpropanoid group has significant protective function in plants. Its physical toughness deters feeding by insects and chemical durability makes it relatively indigestible.
The flavoids are one of the largest classes of plants phenolics e. g. anthocyanins, flavones etc.
Anthocyanins are colored flavonoids that attract insects to flower and fruits by providing visual and olfactory signal.
Flavonoids protect against damage by UV light.
Tannins deter feeding by herbivores and it also act as feeding repellents to a great diversity of insects
NITROGEN CONTAINING COMPOUND
A large variety of plant secondary metabolites have nitrogen in their structure. This category includes well known defense against phytophagus insects as alkaloids and cyanogenic glycosides,glucosinolates.
ALKALOIDS: These are a large family of more than 15000 nitrogen containing secondary metabolites with a heterocyclic ring. Several different types including nicotine and its relative are derived from ornithine . Most alkaloids now function as defenseagainst their predators because of their toxicity and deterrence capability. Alkaloids increase in response to initial damage fortifying against further damage e. g. wild tobacco produces higher level of nicotinefollowing damage by tobacco caterpillars.
CYANOGENIC GLYCOSIDES: These are not toxic themselves but are readily broken down to give off volatile poisons; well known poisonous gas Hydrogen cyanide. When the leaf is damaged due to insects feeding on it, the cell content of different tissue mix and HCN is formed. HCN is a fast acting toxin that inhibits metalloprotiens such as iron containing cytochrome oxidase; a key enzyme of mitochondrial respiration, thus affecting physiology of insects. Thus presence of cyanogenic glycosides deters feeding by insects.
GLUCOSINOLATES: A Class of plant glycosides that break down to release volatile defensive substances, also called Mustard oil glycosides. Found principally in the Brassicaceae and related plant families, where glucosinolates give off compounds responsible for smell and taste of vegetables like cabbage, cauliflower, mustards etc. These compounds function in DEFENCE as toxin and feeding repellent. But certain insects are adapted for feeding on glucosinolate containing plants without ill effects. For example glucosinolates serve as stimulant for Cabbage butterfly for feeding and egg laying and isothiocyanates serve as volatile attractants.
PLANT PROTIENS: Certain plant protein also interfere with insect digestion, for example plants produce LECTINS,defensive proteins that bind to epithelial cell lining digestive tract and interfere with nutrient absorption. The best known anti digestive proteins in plants are protein inhibitors found in legumes, tomatoes, and other plants. After entering herbivores digestive tract they interfere with protein digestion, as a result insects suffer reduced rates of growth and development.
THE NEEM TREE, PROPERTIES AND DISTRIBUTION
The Neem tree also known by names like Indian Lilac, Margosa tree is an evergreen fastgrowing tree belonging to the order “Rutales” and family “Meliaceae”. The genus Azadirachta indica was described by A. juss in 1830.
Neem tree is indigenous to Indian Subcontinent from where is has spread to many Asian and African countries such as Pakistan, Bangladesh, Mynamar, Sri Lanka, Thailand, Indonesia, Malaysia, Singapore, Iran, Yemen, Australia, New Guinea, Nigeria, Fizi, Tanzania, Madagascar, USA, Latin America, Germany, France, Portugal, Spain and UK. It is now grown in most tropical and sub-tropical parts of the worls.
The origin of A. indica is not very clear. Some say that is has originated from Burma whereas others point it to south India. It is considered that it has originated from south-eastern and southern Asia.
In Indonesia Neem exists in low lying Northern and Eastern parts of java. In Philippines it was introduced from India, Africa. Ketkar (1967) reported about 14 million trees in India. There are more than 20 million trees available in entire India. In Africa Neem was introduced from India and is concentrated in a belt stretching across the African continent from Somalia to Mauretania. In America Neem trees are prominent in Haiti, Surinam and propagation has started in Brazil, Puerto Rico, Cuba and Nicaragua. Neem trees also grow in our neighbouring countries, Middle East, Saudi Arabia and Yemen.
Neem tree is a fast growing sclerophyllous tree.
- It grows well in humid to semi-humid climate.
- It thrives well at altitudes upto 700-800m above the sea level.
- Neem trees are hardy and are able to grow in severe drought condition also. They thrive well in regions with less than 500 mm annual rainfall and upto 2500 mm annual rainfall.
- Neem tree exist in poor, shallow, sandy and stony soil. It also grows in black cotton soil in India.
- Neem tree can flourish in warm to very hot climates. It grows well between 21-320c temperatures but it can tolerate upto 500c during summer.
- Ph value between 6. 2-7 seems to the best for the growth of Neem tree.
- It is a fast-growing tree, reaching a height of 4-7 m during the first 3 years and 5-11m during the following 5 years.
- It begins to bear fruit within 3-5 years and becomes fully productive in the 10th year, when it may yield up to 50 kg fruit per tree per year.
- The Neem tree produces its fruits, which are the main source for its production of pesticides, on drooping panicles, usually about once a year, although two fruiting periods per year occur in certain areas (e. g. West Africa).
- A mature Neem tree produces annually 30-50 kg of fruit, but this may depend upon rainfall and soil conditions. More conservative estimates range around 20 kg per tree; 40 kg of fresh fruit yield about 24 kg of dry fruit.
- Neem has the reputation of possessing a large number of biological activities which include insecticidal, nematicidal, bactericidal, and anti-fungal. It has attracted world-wide attention due to its wide ranging capacity as a biocide.
PESTICIDAL CONSTITUENT OF NEEM
Neem tree is the only tree in which every part of tree produces biologically active products which has various properties such as antifeedant, deterrent, growth regulation, oviposition alteration, insecticidal properties, fungicidal properties,etc.
Though bark, heartwood, leaves, fruits of it produce these substances in various concentrations but it is the fruits specifically seeds which are of major importance. Neem seed kernels contain the highest amount of the active compound. 40-50 kg of fruit can yield about 5 kg of kernels (10% of fruit). Each seed contains about 1-3 kernels.
Till date more than 140 active principles have been identified in different parts of the tree. Insecticidal properties of Neem is due to the presence of a class of Limnoids which include compounds like Azadirachtin, Melantriol, Salanin, Mimbines, Salannol and various sulfur containing compounds. Among these Azadirachtin is the most active and predominant insecticidal compound concentrated mainly in the seed kernels. The Azadirachtin occurs in seeds at the concentration of about 0. 1-0. 9%. It is estimated that 20-30 kg of Neem seeds are required per hectare if 2g of Azadirachtin per kg of sed is obtained. The highest yield of Azadirachtin obtained till date was about 10g/kg of seed.
AZADIRACHTINAND ITS STRUCTURE
Azadirachtin is a highly oxidized limnoid chemically being a tetranortriterpenoid and is the main component responsible for both anti-feedant and toxic effects in Azadirachtin. Butterworth and Morgan were the first to isolate Azadirachtin in 1968 from Neem seed. Morgan established correct molecular formula of Azadirachitn (C55H44O16). In 1971 they developed a simplified method to isolate azadirachitn by doing solvent partitioning followed by column and preparative thin layer chromatography. However its structure was determined in 1975 by Nakanishi's team through the application of new NMR methods. There were some inaccuracies in the given model. Then again renewed efforts were made by the group of Ley, Kraus, Nakanishi and they gave the correct structure by using X-ray crystallography.
A. indica produces a plethora of triterpenoids, the biosynthesis of which culminates in azadirachtin. The biosynthesis of azadirachtin starts with a steroid precursor - tetracyclic triterpene “tirucallol”. Opening of C-ring followed by processing via two main levels of structural complexity i. e. furan ring formation leads to Azadirachtin.
VARIOUS PESTICIDAL FORMULATIONS
Neem insecticides which are obtained from Neem seeds contain various arelated triterpenoids in addition to the Azadirachtin. However their efficacy is related directly to the content of Azadirachtin. These compounds do possess biological activity and they add to its effects. Pure Azadirachtin was shown to be effective in the fields (Mordue et al, 1997) but the natural mixtures of azadirachtin in Neem insecticides may usefully mitigate against the development of resistance compared to azadirachtin alone (Feng and Isman, 1995).
The complex nature of azadirachtin and other sophisticated Neem constituents prevent their mass production by synthesis in the foreseeable future. The pesticidal Neem products used in practice include dried leaves, whole seed, decorticated seed, seed kernels, Neem oil, and Neem cake, remaining after extraction or extrusion of the oil from the seeds. Several Indian companies or institutions produce commercially Neem-based insecticidal formulations, such as "RD-9 Repelin" and "Wellgro", for spraying against cutworms and other insect pests in tobacco growing areas; "Nimbosol" and "Biosol" for control of whiteflies; and the products "Neemrich" and "Neemark, the latter also as an azadirachtin-enriched granular Neem formulation. In the U. S. A. , the EPA hasgranted registration to "Margosan-O", an azadirachtin-enriched, concentrated Neem seed kernel extract formulation, for use on non food crops and ornamentals. Margosan-0 was developed by R. Larson of Vikwood Botanicals Inc. at Sheboygan, WI, in collaboration with the USDA Agricultural Research Center at Beltsville, MD. The rights to this product, which contains 0. 3% azadirachtin and 14%Neem oil (the 0 in the name of the product stands for oil), and has an oral toxicity in excess of 5,000 mg/kg in rats. Margosan-0 has been evaluated successfully against an extensive series of insects in the U. S. A. and Canada, Lyriornizu leafminers on ornamentals and tomatoes, cotton bugs, cockroaches and mosquitoes. Margosan-0 demonstrated highest activity against Ostriniu nubilalis , and against leafhoppers, against two species of local cotton pests, Enrias insulana and Spodoptera littoralis. Recently in the U. S. A. a further Neem formulation, developed. under the auspices of the Natural Products Institute, Salt Lake City, UT is ”Azatin”(Agridyne Technologies, Salt Lake City, UT). Also, Safer Ltd. , a Canadian manufacturer specializing in environmentally safe pest control formulations, developed insecticides based on Neem. Safer, however, has been acquired recently by Ringer Corp. , Minneapolis, MN, which distributes Margosan-0 in the home garden market under the tradenames of “Bioneem” and “Neemesis”.
Contrary to registration practices in use until now, no precise chemical descriptions of all the ingredients of Margosan-0 were required, but rather, demonstration of the biological activity and innocuousness of the whole mixture to no target organisms was used in the registration process. Hopefully such specially tailored toxicity studies will be used to judge and register Neem and similar natural products in the future. A recent report claims that the EPA has approved a Neem-based biological pesticide developed by an Indian company for use on a wide range of food crops.
MODES OF ACTION
Major modes of action of azadirachtin are:
- Powerful IGR.
- Feeding Deterrant.
- Oviposition Deterrant.
These are the three modes of action of azadirachtin which make azadirachtin much sought after biopesticide in today's agriculture industry.
IGR: Azadirachtin acts as a powerful growth regulator for insects and this IGR effect is the most pronounced mode of action of Azadirachtin.
Normally IGR effect the hormonal system of insects, preventing the insects from developing into normal mature insects. This IGR property of Azadirachtin doesnot leads to immediate death of insects, pests.
Azadirachtin as an IGR:
The IGR property of Azadirachtin arise due to the fact that:
Azadirachtin is structurally analogous to natural hormone Ecdysone. As Ecdysone regulates the development of insect, any disruption in its balance leads to improper development.
Also Azadirachtin interferes with the production and reception of Ecdysone at the time of insects' growth and moulting. Thus Azadirachtin in this manner block the moulting cycle resulting in the death of the insect, pest.
The main action of Azadirachtin appears to be at the release site of PTTH. The mode of action of Azadirachtin as IGR is thus an Indirect Physiological Effect. It is exerted via the endocrine system. The copora cardiaca is supposed to be the target for the Azadirachtin as is affects the PTTH, Eclosin Hormone, Bursicin Hormone release. PTTH release is inhibited rather than Ecdysine from Prothorasic gland. Thus the Azadirachtin affects the neurosecretory cells of Brain. Various experiments show that Azadirachtin doesn't directly act on Prothorasic Glands.
- In the in vitro culture of Prothorasic (H. virescens) gland showed that the PTTH induced release of the Ecdysine was medium (Bidmon et al, 1987, Barnby and Klocke, 1990). Also it was not blocked in PTTH simulated cultured glands from M. sexta pupa penetrated with Azadirachtin in last larval instar (Pener et al, 1988). However receptivity of Prothorasic gland to PTTH was affected in H. virescens.
- Neurosecretory proteins stained with paraldehyde in L. migratolia females when was compared with similar aged azadirachtin treated females there was an accumaulation of stainable material in corpora cardiaca of brain neurosecretory system in treated insects. Thus is appears that azadirachtin blocks release of neurosecretory material from corpora cardiac.
It can thus be concluded that Azadirachtin does block the release of peptide hormones from brain neurosecretory cell corpora cardiac complex.
Azadirachtin also exhibit IGR effect by altering the titre of Juvenile Hormone (JH). Azadirachtin affects the release of allotropins into corpora dillata hence block the synthesis and release of the Juvenile Hormone. This block leads to a rapid decrease in whole body JH titres, which is maintained for several days. Experiments prove that in M. sexta larvae, azadirachtin infection on day 0 (1. 0-10 µg/ larva) results in induction of supernumerary moults (Sch et al, 1985; Beckage et al, 1988) presumably due to an inhibition and subsequent delay in JH titre. In adult female L. migratolia also azadirachtin treatment causes a rapid decrease in juvenile hormone titres with associated disturbances in oogenesis (Rembold, 1984; Rembold et al, 1987).
Thus, on a conclusionary note, the effect of azadirachtin is both dose and time dependent. It prevents both apolysis and ecdysis and thus can cause death before the moults, during the moults or delays of moult to form permanent larvae.
Feeding Deterrance: Feeding behavior is both dependent on chemical senses stimulated due to contact chemoreceptors on trasi, mouthparts and oral activity and integration of the sensory code with the CNS. Azadirachtin acts as feeding deterrant. Inhibition of the feeding behavior occurs:
There are receptors present on and around mouthparts of insects which normally respond to Phagostimulants. So azadirachtin may act by blocking the input from these receptors.
Also there are present specific “deterrent cells” in insects which prevent insect from feeding. Azadirachtin acts to stimulate these “deterrent cells” leading to feeding deterrence. Many experiments were done in this regard.
Using different concentration of sucrose and azadirachtin, either singly or together, the neurophysiological responses from medial and lateral sensillia styloconica of maxillae showed different group of receptors are receptive to sucrose (sugar cells) or azadirachtin (deterrent cell) in S. exempta and M. brassicae in most of the cases, the rate of firing of sugar sensitive cells were reduced in presence of both chemicals (Simmonds and Blaney, 1984). Such an interaction was also found in P. brassicae. This leads to a reduced or complete inhibition of feeding.
Direct mode of action: Incorporation of azadirachtin results in direct toxic effect after ingestion. Azadirachtin prevents the secretion of Proteolytic enzymes and thus significantly impair ability of insects to digest and absorb nitrogenous food. When azadirachtin is ingested it can result in the disfunctioning of gut, as a result of which midgut epithelial cells become round. Swelling of cells and organells occur with some vacuolization and cell burst resulting in necrosis (as observed in S. gregarea and L. migratolia Naseruddin and Mordue (Luntz), 1993a; Cottee, 1984). There is also reduction in the regenerative cells and increase in the connective tissue layer with some invading heomocytes. This would lead to disruption of enzyme secretion and nutrient absorption.
Also the antifeedant effect can be attributed to the action of azadirachtin on the peristaltic movement of gut wall. The gut of treated insects lack tone, midgut to hindgut junction becomes flaccid and co-ordinated peristalsis is lacking which leads to antifeeding behavior.
EFFECT OF AZADIRACHTIN ON INSECTS
Effects on Feeding
Azadirachtin is a classical example of a natural plant defence chemical affecting feeding. Antifeedancy is the major insecticidal effect of Azadirachtin. Antifeedant effect in insect pest on application of Azadirachtin is divided into two main categories:
- Primary Antifeedancy: It refers to the deterrence of feeding in insects. Primary Antifeedancy is also called Gustatory antifeedancy. It can be defined as the inability to ingest resulting from the perception of antifeedant at a sensory level (Schmutterer 1985). Insects fail to eat treated crops and as starvation ensued results in the death of insects.
- Secondary Antifeedant effect: It refers to the non-feeding after the ingestion of treated plant. Secondary antifeedancy is also called Non-Gustatory antifeedancy. It can be defined as the reduction in food consumption and digestive efficiency subsequent to and as a consequence of ingestion, application or injection of antifeedant (Schmutterer, 1985).
Experiments conducted in the past in this regard by various persons:
The first detailed experiment was conducted in S. gregaria (desert locusts) in India. Insects from different orders show marked difference in their response to azadirachtin. (Table 1)
- Lepidopteras showed extreme sensitivity to azadirachtin and depending upon species, effective anti-feedance was observed from less than 1 to 50 ppm.
- Hemiptera (Homoptera), Coleoptera are less sensitive to azadirachtin with 100 % antifeedancy observed at 100-600 ppm.
- However, in Orthoptera wide range of sensitivity has been observed.
Reed and Pierce in 1981 tested the repellant effect of Neem extract to striped cucumber beetle (A. vittateim), by cutting leaves and dipping them in extract solution and placing them in a dish with untreated leaf pieces. When 5 fasting beetles were placed in a dish, 0. 1 % azadirachtin gave protection for atleast three days.
The intake of food by various homopteran insects Nilaparvata lugens, Nephotettix virescens was significantly reduced on rice plants sprayed with 1-50% emulsion of Neem oil. ( ). In green rice leafhopper, N. virescens feeding on the phloem of neem oil treated plants (1. 25-10%) was significantly less than of solvent treated control plants, whereas xylem feeding increased. Hemipteran insects feeding on tobacco seedlings which had been systemically treated with 500 ppm azadirachtin, were shown initially to feed normally but, after termination of the initial feed, the interval prior to the next subsequent feed was significantly increased and feeding activity thereafter was suppressed (Nisbetet al. 1993). When azadirachtun was impregnated on discs at a concentration of 0. 1-10 ppm, S. littoralis(African cotton leafworm),Spodoptera frugiperda(J. E. Smith) (fall armyworm),Heliothis virescens(F. ) (Tobacco budworm) andHelicoverpa armigera(Hüb. ) (Old world bollworm) showed significant behavior response and are prevented from feeding on the discs dependent on species (Blaney et al. 1990, Simmonds et al. 1990, Mordue (Luntz) et al. 1998)
Insects from different Orders differ markedly in their behavior responses to azadirachtin (Table 1). Lepidoptera are extremely sensitive to azadirachtin and show effective antifeedancies from <1-50 ppm, depending upon species. Coleoptera, Hemiptera and Homoptera are less sensitive to azadirachtin behaviorally with up to 100% antifeedancy being achieved at 100-600 ppm although there are some aphid species which also show behavioral sensitivity e. g. strawberry aphid. The Orthoptera show an enormous range in sensitivity fromS. gregaria(a polyphagous species which has chemoreceptor finely tuned to many plant secondary compounds) toLocusta migratoria(L. ) (a graminaceous species which does not have chemoreceptors tunes to feeding deterrents) to the extreme insensitivity ofMelanoplus sanguinipes(Fab. ), the North American Plains grasshopper which is an evolutionary sense has never encounteredA. indicaand has no chemoreceptors responding to azadirachtin. Such 'primary' (or gustatory) antifeedancy - 'the inability to ingest resulting from the perception of the antifeedant at a sensory level' (Schmutterer 1985), is responsible for crop protection in several species of Lepidoptera and S. gregaria. Desert locusts (S. gregaria) are very sensitive to azadirachtin and fail to feed on sugar impregnated discs when the compound is present at concentrations of 0. 01 ppm and above (Mordue (Luntz)et al. 1996). Azadirachtin sprayed onto barley seedlings infested withS. gregarianymphs protect plants at low doses (2 ppm) (Nasiruddin & Mordue (Luntz) 1993). S. littoralis(African cotton leafworm),Spodoptera frugiperda(J. E. Smith) (fall armyworm),Heliothis virescens(F. ) (tobacco budworm) andHelicoverpa armigera(Hüb. ) (old world bollworm) also respond behaviorally to low concentrations of azadirachtin and are prevented from feeding on discs impregnated with the compound at concentrations of 0. 1 - 10 ppm dependent upon species (Blaneyet al. 1990, Simmondset al. 1990, Mordue (Luntz)et al. 1998).
The antifeedant effects observed in these species are highly correlated with the sensory response of chemoreceptors on the insect mouthparts (Mordue (Luntz)et al. 1998). Feeding behavior depend upon both neural input from the insects' chemical senses (taste receptor on tarsi, mouthparts and oral cavity) and central nervous integration of this 'sensory code'. Azadirachtin stimulates specific 'deterrent' cells in chemoreceptors and also blocks the firing of 'sugar' receptor cells, which normally stimulate feeding (Blaneyet al. 1990, Simmondset al. 1990, Mordue (Luntz)et al. 1999). This results in the starvation and death of these species by feeding deterrency alone.
In most other species of phytophagous insect however crop protection results from a combination of antifeedancy and physiological effects resulting from ingestion of azadirachtin. These physiological effects include 'secundary'antifeedancy whereby feeding is reduced post-ingestively. These "secondary" antifeedant effects include 'a reduction in food consumption and digestive efficiency subsequent to, and as a consequence of, ingestion, application or injection of the antifeedant' (Schmutterer 1985).
Such secondary antifeedant effects result from the disturbance of hormonal and/or other physiological system e. g. movement of food through the gut, inhibitions of digestive enzyme production, effects on the stomatogastric nervous system etc. Mordue (Luntz)et al. 1985, Koul & Isman 1991, Timmins & Reynolds 1992, Trumm & Dorn 2000). For example locusts injected with azadirachtin, which by-passes the taste receptors, show a reduced ingestion of food as seen by faecal pellet production (Nasiruddin & Mordue (luntz) 1993). Also aphids which had fed on artificial diets containing much lower concentration of azadirachtin (25 ppm) exhibited no signs of primary antifeedant effects during an initial 24h period of access to the diets but their feeding rate fell dramatically in the subsequent 24h period (Nisbetet al. 1994).
A consequence of interrupted feeding activity can be an effect on the ability of insects to transmit pathogens. Aphids require extended feeding periods to acquire persistently-transmitted luteoviruses (e. g. potato leafroll virus, PLRV) from plants. Treatment of PLRV-infected tobacco plants with azadirachtin reduced sustained feeding byMyzus persicae(Sulzer) (peach-potato aphid) and reduced the ability of aphids to acquire and transmit PLRV. However, azadirachtin does not always reduce the spread of plant virus diseases by aphids. Treatment of uninfected seedlings with the same concentrations of azadirachtin (500 ppm) failed to prevent them from becoming infected when viruliferous aphids fed on them (Nisbetet al. 1996a). The successful infection of a plant with luteoviruses is dependent upon the transfer of aphid saliva to the plant, a process which may be brief by comparison with the time required for virus acquisition by the aphid, and is not overcome by the presence of the antifeedant. Similarly, azadirachtin failed to protect seedlings from infections with a non-persistently transmitted potyvirus (potato virus y) from viruliferous aphids (Nisbet 1992).
Effects on Physiology
The physiological effects of azadirachtin are much more consistent than the antifeedant effects, and result from interference with growth and moulting, interference with reproduction and interference with cellular processes Table 2. In all species tested dose response effects can be seen as reduced growth, increased mortalities, abnormal moults and delayed moults. These effects are related to disruption of endocrine system controlling growth and moulting. The moulting effects are due to a disruption in the synthesis and release of ecdysteroids (moulting hormone) and other classes of hormones and this can be demonstrated by accurately timed injections of azadirachtin into the haemoulymph of vthinstar nymphs ofL. migratoria(Mordue (Luntz)et al. 1986). Measurements of haemolymph ecdysteroid levels by radioimmunoassay (RAY) revealed the normal peak of hormone release at day 8 of an 11-day instar. In those insects injected with azadirachtin before hormone release, ecdysteroid release is blocked entirely and the insects die before the moult after an extended instar; in those injected at the start of ecdysone release, the peak is delayed and its decline slow down. This prevents the release of eclosion hormone which controls the motor programme of eclosion or moulting and these insects also die before the moult. Finally, if injected at the peak of ecdysone release moult initiation proceeds but the insects die during ecdyses unable to swallow enough air to extricate themselves from the old cuticle (Plates 1 a - c).
The physiological effects of azadirachtin can be categorized in two ways:
- Indirect Effects- Exerted via the endocrine system. The neurosecretory system of the brain affected by azadirachtin, which causes a blockage of the release of morphogenetic peptide hormones e. g. PTTH (prothoracicotropic hormone) and allatostatins. These control the function of the prothoracic glands and the corpora allata respectively. Moulting hormone (â-hydroxyecdysone) from the prothoracic glands in turn controls new cuticle formation and ecdyses (the act of extrication from the old cuticle) whereas juvenile hormone (JH) from the corpora allata controls the formation of juvenile stages at each moult. In the adult both hormones can be involved in the control of yolk deposition in the eggs. Any disruption in these cascade events by azadirachtin results in the many various but well-defined effects seen as moult disruption, moulting defects and sterility effects.
- Direct Effects- On cells and tissues. Azadirachtin is taken up into cells and causes inhibition of both cells pision and protein synthesis. Such effects are seen in flaccid paralysis of muscles, midgut cells necrosis and loss of nidi (regenerative cells) of the gut and lack of midgut enzyme production.
The sum total of the physiological effects of azadirachtin is consistent throughout species when compared to antifeedant effects. An ED50of around 1 mg/g body weight is seen though the many insects species tested (Mordue (Luntz) & Blackwell 1993).
Effects on Reproduction
When the primary antifeedant properties do not operate due to low sensitivity of chemoreceptors or are circumvented by injection or applying the compound topically, azadirachtin can be shown to cause profound effects on the reproductive process of both male and female insects. For example, inL. migratoriaazadirachtin inhibits both oogenesis and ovarian ecdystereroid synthesis so preventing oviposition (Rembold & Sieber 1981). Aphids are insensitive to the primary antifeedant effects of azadirachtin below 100 ppm, although secondary antifeedant effects are observed (Nisbetet al. 1994). When female aphids are fed on diets containing low concentrations of azadirachtin (5ppm), their fecundity decreases dramatically within 48h of feeding and, if they were fed in diets containing more than 10 ppm azadirachtin any nymphs which were produced were non-viable (Mordue (Luntz)et al. 1996).
Male reproduction is also affected by azadirachtin. Injection of maleO. fasciatuswith 0. 125 mg per insect severely reduces male potency as seen by an 80% reduction in the fecundity of normal females when mated with treated males (Dorn 1986). Testes dimensions of male desert locusts injected with low concentrations of azadirachtin during their development were significantly reduced and the meiotic processes which are responsible for the production of mature sperm in adult males were interrupted. Blockage of cell pisions was shown to occur prior to metaphase (Lintonet al. 1997). Metaphase is the stage of cell pision at which microtubules form the spindle apparatus prior to the physical separation of homologous pairs of chromosomes to opposite at this stage in cell pision suggest that cell microtubular events may have been affected by azadirachtin (Mordue (Luntz), Mordue & Nibet-unpublished).
Understanding the Effects of Azadirachtin on Insects
The primary antifeedant effects Lof azadirachtin on insects are produced by the stimulation of specific deterrent chemoreceptors on the mouthparts together with an interference of the perception of phagostimulants by other chemoreceptors (Mordue (Luntz)et al. 1998). The secondary effects on feeding, developmental and reproductive disruption are caused by effects of the molecule directly on somatic and reproductive tissues and indirectly through the disruption of endocrine processes. Research is now being carried out to understand the effects of azadirachtin at the cellular level in insect tissues.
In mature adult maleS. gregaria, a tritium-labeled azadirachtin derivative, ([22,23-3h2] dihydroazadirachtin), was shown to bind specifically to several tissues but the most intense binding per unit of protein was in preparation from testes. This binding was almost (kd8. 7nM) and essentially irreversible (Nisbetet al. 1995). Localization of the binding by autoradiography revealed preferential binding in the testes follicles, localized on the tails of developing sperm. This binding was therefore associated with one of the sub-cellular components of the developing sperm tail; membrane, axoneme or mitochondrial body (Nisbetet al. 1996b).
Sub-cellular fractionation of Sf9 cells (captured insect cells derived fromS. frugiperda) incubated with [22,23-3H2] dihydroazadirachtin during logarithmic growth phase revealed high affinity specific binding to the nuclear fraction of the cells (Nisbetet al. 1997). A comparison of binding of tritiated dihydroazadirachtin to these two insect tissues shows specific, time-dependent, saturable high affinity binding in both tissues, with many similarities in binding characteristics (Table 3). Preliminary characterization of the binding sites has indicated that it is proteinaceous, heat-labile and may be associated with cellular RNA (Mordue (luntz)et al. 1999). Unsuccessful attempts to solubilise the protein and extract it for identification by ligand binding assays suggests that its 3-dimensional integrity within membranes is essential for its activity.
Azadirachtin prevents the proliferation of Sf9 cellsin vitroand alters both the protein content and abundance in those cells (Fig. 3(Barry, Sternberg & Mordue (Luntz) unpublished), Rembold & Annadurai 1993). It therefore appears from these observations that azadirachtin operates at the cellular level by disrupting protein synthesis and secretion events and, more fundamentally, at the molecular level by altering or preventing the transcription of proteins expressed during and/or translation of proteins expressed during periods of rapid protein synthesis e. g. in piding cells or cells forming new assemblages of organelles or cytoskeleton. Ongoing studies to fully characterize the azadirachtin binding sites are presently being carried out using insect cell lines.
PREVENTION OF AFLATOXIN PRODUCTION
Afurther unexpected, nonentomological finding is based on the work of Bhatnagar and coworkers (see for recent review) at the USDNARS Southern Regional Research Center, New Orleans. Since Neem has also antimicrobial and antifungal properties, Bhatnagar investigated the effect of Neem leaf extract on the growth of two aflatoxigenic (aflatoxin-producing) strains of the fungi Aspergillus fravus and A. parasiticus, as well as on aflatoxin production. Nonvolatile, somewhat heat-labile Neem leaf constituents did not affect fungal growth (i-e. , mycelial weight), but completely inhibited aflatoxin production. This was demonstrated not only in a fungal growth medium in submerged culture, but also under in vivo conditions in developing cotton bolls. Injection of Neem leaf extract into artificial 3 mm deep surface holes on locules of developing bolls (30 days postanthesis) followed by an A. fravus suspension 48 h later, again did not affect fungal growth; however, the seeds from the locules exhibited a higher than 98% inhibition in aflatoxin production. It was demonstrated by in vitro studies that aflatoxin biosynthesis was irreversibly inhibited in the mycelia of both fungi by Neem constituents; removal of mycelia from exposure to the leaf extracts did not restore aflatoxin synthesis. Margosan-0 was equally efficient in inhibiting aflatoxin synthesis in developing cotton bolls.
PREVENTION OF VIRUS TRANSMISSION
Neem seed formulations effectively prevent or reduce transmission of rice tungro, ragged stunt and other virus diseases on rice by the green leafhopper, Nephoffefixv irescens, or the brown planthopper, Nilaparvata Zugetz
Differential Effects in Insects and Non-Target Organisms
In order to fully understand the mechanisms by which azadirachtin operates, the differential effects of azadirachtin must be distinguished:
- In insects, to help decide which are the significant lesions involved in its mode of action.
- In other non-target organisms e. g. vertebrates to make quite certain that the margin for insecticide use is real and defined.
Two examples here related to firstly the effects of azadirachtin on locust excretory mechanisms and secondly to the effects on vertebrate cultured neurons. Studies with tritiated dihydroazadirachtin had indicated that azadirachtin accumulated in high amounts in Malpighian tubules, the excretory organs of insects (Remboldet al. 1988). Such concentrations must be associated with excretion of azadirachtin but also may be associated with its mode of action, it has been shown that azadirachtin reduces both basal and diuretic peptide-stimulated urinary secretions in locusts (Fig. 4), and that the reduction in stimulated urine levels is induced through inhibition of cyclic AMP (cAMP) - regulated processes (Mordue (Luntz), Coast, Mordue & Nisbet unpublished). This reduction however, occurs in the presence of azadirachtin at mM levels only, with the threshold response being close to this, i. e. at levels some 1000 fold less sensitive than more established azadirachtin effects (e. g. Rembold & Annadurai 1993). Azadirachtin treated insects do become slightly bloated whit time post-treatment (Cottee & Modue (Luntz) 1982, Nasiruddin & Mordue (Luntz) 1993) presumably as a result of lesions to the Malpighian tubules, however it is very clear that lack of diuresis by the cAMP secondary messenger cascade is not the main mode of action of azadirachtin.
MAMMALIAN TOXICITY AND EFFECT ON BENEFICIAL ORGANISMS
So far, no indications of mammalian toxicity of Neem, per 0sor by dermal treatment, have been found in the USAand Germany with carefully prepared Neem seed kernel extracts under conditions of rigid test procedures; such preparations of Neem, unless misused, can be considered safe for humans. It should be mentioned, however, that infants in Malaysia and India which had been fed per 0s large doses (5 ml or more) of Neem oil as a home remedy against minor ailments, developed severe symptoms of poisoning within hours of ingestion. These consisted of vomiting, drowsiness, metabolic acidosis and encephalopathy. A4-month-old Indian child fed twice 12 ml Neem oil (on two successive days)”for cough”, died 12 days later. It was assumed that the oil may have been involved in the etiology of “Reye‘ssyndrome” , which, in turn, is probably due to a synergistic effect between aflatoxins contaminating the oil samples used and meliatoxins present in the oil]. This rather specific problem of Neem oil fed to infants in southern and southeastern Asia was reviewed by Jacobson. Other parts of the Neem tree may, however, be toxic to warm-blooded animals, e. g. , Neem leaves to sheep], goats and guinea pigs. Neem has been shown to be outstandingly safe to beneficial organisms. Honeybees, parasitizing insects such as parasitic wasps, and predators such as spiders, earwigs, ants and predaceous mites, are not harmed by azadirachtin and Neem products . This is due to the lack of contact toxicity in most cases, the lack of a direct ovicidal effect, and the absence of toxicity against nonphytophagous adult insects. Neem products are, therefore, very selective, although they have a rather broad spectrum of activity. Azadirachtin-containing insecticides act first as oral (stomach) poisons. In some cases, for instance in the soft-skinned larvae of Leptinotarsa decemlineata,the insects also react after dermal contact with the active principle (Steets, 1975). The death of the target insects is dose-dependent. It usually occurs a few days after application of Neem pesticides, but in extreme cases the larvae may live up to several weeks when they become unable to moult. ' In such “permanent larvae” the imaginal discs and parts of the epidermis are destroyed, for instance in Epilachna varivestis(Schliiter and Schulz, 1984). Azadirachtin, alcoholic and aqueous extracts of Neem seeds and enriched formulations have, according to all tests carried out up to now, no oral or dermal toxicity to mammals. Neem flowers and leaves are eaten as a vegetable in Asia (India, Burma, Thailand). Honeybees under practical conditions are also not endangered (Schmutterer and Holst, 1987). In addition, important natural enemies of pests, such as spiders, earwigs, ants and some parasitic wasps are only slightly or not at all harmed (Hellpap, 1985; Mansour et al. ,1987), in some cases even favoured (Saxena et al. , 1981; Joshi et al. , 1982). This is because of the lack of contact toxicity in most insects, the lack of ovicidal effect and the lack of or low effect against non-phytophagous adult insects. Hence, Neem products are quite selective.
To date, there is no reported case of resistance to Neem, and standard procedures of selection for resistance, e. g. , in Plutella xylostella, the diamondback moth, for 42generations have not led to resistance . This was true even in a deltamethrin-resistant strain. It must be stressed that P. xylostella is a problem insect, which rapidly developed high-level resistance in the field to modern synthetic pesticides, including both photostable pyrethroids and acylureas. Non evolution of resistance to Neem formulations may be due to their being mixtures of various, often related compounds. The latter obviously have several and different modes of action, accounting for manifold activities, such as antifeeding and repellent effects, including oviposition repellency; growth and development regulation; sterilizing effects, inhibition of oviposition and/or reduction of fecundity proper, and, in certain species, disruption of mating. In addition, Neern products sometimes exert interesting physical effects on insects (e. g. ,autotomy induced by Neem oil in adult locusts) and occasionally even conventional-type toxicity.
Neem and Azadirachtin in Insect Control
The complexity of the molecular structure of azadirachtin precluded its synthesis for pesticide use. Extracts of Neem seeds containing azadirachtin together with several structurally related molecules have formed the basis of Neem usage in insect control (Isman 1997). Future approaches may also include the production of azadirachtin for insect control byin vitrotissue cultures of Neem (Allanet al. 1994, 1999). Neem insecticides are effective mainly as insect growth regulates and sterilants, against a broad spectrum of pest insects. Crude Neem extracts have been used at a local, small-farm level for some time in countries where Neem grows indigenously or where plantations have been established. In the major western countries of the world such as the USA and Canada and in Europe few commercial Neem insecticides have reached the market place to date. Progress has been hampered by lack of supplies of Neem kernels of known azadirachtin content, by lack of standardization of formulated products, by cost of the product and by lack of regulatory approval of the complex mixture of compounds found in neem extracts. Until recently these problems had meant that Neem insecticides had not generated much impact on the marketplace. Times, however, may well be changing.
With the resolution of many of the problems of supply and standardization, the full regulatory approval of Neem insecticides by the USA and now in Germany for use on potatoes, apples and tomatoes, much field data is being generated which are establishing Neem insecticides as viable alternatives to more conventional approaches, particularly in integrated pest management system. Now that it is realized that disruption of growth and reproduction rather then antifeedancy are the main characteristic of pest control, Neem is being used in the field at lower concentrations than those originally recommended (>100 ppm ai). Treatment of artificial diet with levels as low as 5 ppm or 0. 25 ppm azadirachtin have been shown to significantly reduce reproduction output inM. persicae(Mordue (Luntz)et al. 1996), and feeding growth and development inS. littoralis(Martinez & van Emden 1999) respectively. The value of low concentrations of Neem in pest control has generated research into combined approaches using both neem and beneficial species. In the laboratory usingM. persicaeand its parasitoidEncarsia formosa5 ppm azadirachtin treatments of leaf discs together withE. Formosaproduce additive effects compared with either approach separately and can entirely prevent nymph production ofM. persicae(Fig. 5) (Sugden, Armb & Mordue(Luntz) unpublished). In the field and in more complex laboratory situations, however, such results are more difficult to demonstrate. It would appear that there is a fine line between the level of azadirachtin required to affect the pest and the level which will not affect the parasitoid or predator (Belmainet al. 2000, Pereraet al. 2000, Raguraman & Singh 2000, Simmondset al. 2000). Such integrated approaches to pest control however are an encouraging way forward for the use of Neem pesticides.
Neem pesticides may also have a useful role to play in resistance management. It has been demonstrated that the effects of Neem in reducing levels of detoxification enzymes (due to its blockage of protein synthesis) may make insecticides more effective in resistant strains of insect (Lowery & Smirle 2000). Also, it has been shown in Bt resistant strains of Leptinotarsa decemlineataSay, the Colorado potato beetle, that 0. 25% Neemix combined withBacillus thuringiensiscan act as a resistance breaking compound (Trisyono & Whalon 2000). In this instance depending upon the resistance mechanism, the Neem effects may be due also to blockage of enzyme production, or to the reduced midgut cell turnover rate (Nasiruddin & Mordue (Luntz) 1993).
Summarizing the results with neem products under field conditions, simple aqueous and alcoholic, as well as enriched, formulated products have a high potential for pest control especially in developing countries where the raw material is present in abundance. However, to succeed, certain strategies must be followed, because the application of neem products differs from that of most synthetic compounds.
They are as follows:
- As Neem products are ultra-violet sensitive stomach insecticides, the target insects must take them up as soon as possible during feeding; the more active material they consume the better. The application of Neem products should therefore coincide with the most active feeding phases of the target insects.
- Neem products must be applied against the most sensitive larval/nymphal instars of the target insects, as there are also remarkable differences in sensitivity during metamorphosis.
- Because of their delayed effect Neem products may be unsuitable if no further damage to treated plants is tolerable and if no insects should be present on plants during marketing. This may sometimes be the case in industrialized countries. Summarizing the many-sided results presented in this paper, it can be said that perhaps unique mode of action of azadirachtin, which means its controlling effect on insects hormones, especially ecdysone, and the favorable toxicological and selective properties of Neem products provide a basis for a new promising way of environmentally sound pest control with biorational pesticides within the framework of integrated pest management. Due to the longer residual and systemic effects pesticides based on Neem are more suitable than most juvenoids
Azadirachtin from Neem effects insects in a variety of different ways: as an antifeedent, insect growth regulator and sterilant. As antifeedant sensitivity varies greatly between insects the overriding efficacy of Neem insecticide use lies in its physiological toxic effects. An understanding of the physiological effects of azadirachtin in Neem has been reached and biochemical approaches have begun to define its mode of action at the cellular level. Further work is however required to fully understand its mode of action. It is now accepted that Neem insecticides have a wide margin of safety for both user and consumer. Increasing knowledge of how to use neem insecticides in the field is proving a solid base from which successful market penetration should be achieved.
Pests and diseases
Neem trees are generally pest-free, due perhaps to the presence of azadirachtin and other insecticidal compounds. However, Neem plantations have been badly damaged by a scale insect (Aonidiella orientalis) in Africa, and to a lesser extent in India (NRC 1992). Certain species of ants, moths and bugs are also known pests of Neem (NRC 1992). Live specimens are susceptible to borers and termites (Hearne 1975).
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