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Potential of azadirachtin-containing pesticides for integrated pest control in developing and industrialized countries
0022-1910/88
J. Insect Physiol.
Vol. 34, No. I, pp. 713-719, 1988 Printed in Great Britain. All rights reserved
$3.00 + 0.00
Copyright 0 1988Pergamon Press plc
POTENTIAL OF AZADIRACHTIN-CONTAINING PESTICIDES FOR INTEGRATED PEST CONTROL IN DEVELOPING AND INDUSTRIALIZED COWTltIES Institut fiir Phytopathologie
H. SCHMUTTERER und Angewandte Zoologie, Justus-Liebig-Universit& D-6300 Giessen, F.R.G.
Ludwigstrasse 23,
Abstract-Azadirachtin and azadirachtin-containing neem (Azadirachta indica)-seed extracts cause various effects in insects. They act as antifeedants, growth regulators and sterilants. The effect upon insect development is most important froin the viewpoint of practical insect pest control. Several hypotheses exist on the mode of action. An interference with the neuroendocrine system controlling ecdysone and juvenile hormone synthesis is suggested, b+ also an inhibition of ecdysone release from the hormone-producing gland (Culliphora). In addition, azadirachtin is a chitin synthesis inhibitor. Good iesults in insect control are obtained with azadirachtin containing seed extracts under field conditions. Aqueous, alcoholic and enriched (azadirachtin-rich) extracts are used. The residual effect usually lasts about 4-8 days, depending on the environmental conditions and the plant species treated. Systemic effects last somewhat longer. Ultra-violet light, rainfall and perhaps high acidity on treated surfaces of plants cause a fast degradation or loss of the active material. Consequently, much higher concentrations of azadirachtin have to be used in the field to obtain results comparable to those in the laboratory. Several problems have to be overcome to develop reliable pesticides from neem-seed extracts. Inspite of the sensitivity of insects of most orders to azadirachtin, neem products are selective as they do not harm important natural enemies of pests. They are also non-toxic to warm-blooded animals. Neem-seed extracts have, therefore, a considerable potential for integrated pest control measures especially in developing but also in industrialized countries, provided certain strategies are considered for their application.
INTRODUCTION Despite extensive attempts after the Second World War using numerous synthetic pesticides to reduce their number, insects remain the main competitors of man for food, especially in developing countries. Insect pests are also still a serious threat to human health, primarily as vectors of tropical diseases, whose importance is even increasing in several regions. Owing to an indiscriminate use of pesticides, various side effects have been observed in man and the environment, and many insect pests have become resistant to one or more pesticides. Consequently, the search for new, preferably environmentally sound insecticides is an important task. After a study of the properties of juvenile hormones from a practical viewpoint, Williams (1967) concluded that these compounds could be very suitable for pest control and that, insects should not be able to develop resistance to these hormones as they are their own products. He also expected insectspecific effects from them and therefore less negative influence on the environment than with broad spectrum pesticides. Founded on Williams’s philosophy, extensive basic and applied research developed during the seventies. At the same time commercial firms synthesized a considerable number of juvenile hormone analogues and mimics from which they expected even stronger negative effects on the metamorphosis and fe&mdity/fertility of insects than from’ the ‘natural hormones. 713
Reviewing the efforts of juvenile hormone research to develop new pesticides, it is notable that scientists grew progressively less enthusiastic than they had been at the onset of this line of research, when the “third generation of pesticides” was announced (Williams, 1967). Indeed, there are still a number of important barriers to the economic use of such compounds in large-scale pest control. As a consequence, only a few juvenile hormone analogues were registered in the U.S.A., for instance methoprene and hydroprene, and used on a limited scale, such as for mosquito and household pest control (Staal et al., 1985). The reasons were, among others, the short residual effects under field conditions, the few and short sensitive phases of the target insects during their metamorphosis, and the costs of production. Regarding mamtialian toxicity and environmental side effects, most juvenile hormone analogues showed positive features. Another line of research, which evolved after the discovery of the antiallotropic preconenes by Bowers et al. (1976), also failed in practice. The effect of the simply structured compounds from the bedding plant Ageratum houstonianum, which selectively destroy the corpora allata and thereby stop the production of juvenile hormone, was satisfactory in only a few important pest species, mainly Heteroptera, and relatively high’doses were needed to achieve this. There are also’ toxicplogical problems. Compared to juvenile hormones, the attempts to dkvelop pesticides based on insect moulting hormones and their analogues were moderate, although ,.
H. SCHMLJTTERER
714
Fig. 1. Structural formula of azadirachtin.
numerous more-or-less effective ecdysteroids were discovered, for instance in plants. The reasons might be, inter alia, some reluctance to use steroid hormones in the environment and the costs of synthesizing such compounds. My personal opinion is, however, that the antihormonal concept of insect control, as represented by the effect of precocenes against juvenile hormone production, is still a very promising means of insect control which deserves special attention. In principal, anti-ecdysteroids should achieve results similar to those of anti-juvenile hormones, as the moulting hormone is essential for all moults and also heavily involved in insect reproduction. Consequently, disturbing the production or mode of action of the moulting hormone should seriously affect the insects’ metamorphosis and fecundity. EFFECTS
OF AZADIRACHTIN
The triterpenoid azadirachtin was isolated from the seeds of the tropical neem tree (Azadirachta indica) by Butterworth and Morgan (1968). Its definite structural formula, which resembles somewhat that of ecdysone, was finally elucidated in 1985 (Kraus et al., 1985a,b; Bilton et al., 1985) [Fig.l]. The most important effects of azadirachtin on insects from the applied viewpoint can be described as follows:
Treatments
C
First, azadirachtin or azadirachtin-containing extracts from neem seeds show obvious antifeedant effects. There are clear differences, however, regarding the strength of these effects, depending on the concentration of the active principle and the species of insect treated. The desert locust, Schistocerca gregaria, for instance, prefers to die from starvation than to feed on treated foodplants. A “primary” antifeedant effect depending on sensory organs on the mouthparts, and a “secondary” antifeedant effect must be differentiated. The latter is observed not only after uptake of contaminated food per OS but also after topical application and injection of the active principle (Schmutterer, 1985). Second, azadirachtin is a potent disruptor of insect development. In my opinion, these effects are the most important for the practical control of insects. They have been observed in Orthoptera, Heteroptera, Homoptera, Hymenoptera, Coleoptera, Lepidoptera and Diptera (Figs 2 and 3). Some ostracods are also sensitive (Grant and Schmutterer, 1987), as well as nematodes. Third, azadirachtin is an effective sterilant. After uptake of the active material, females of some insect pest species, for instance the Colorado potato beetle, Leptinotarsa decemlineata, are sterilized to a high degree, sometimes completely (Steets, 1976; Schmutterer, 1987) (Fig. 4). The lifespan of treated females is prolonged, but their food consumption is very low. Egg fertility, as a rule, is not influenced by azadirachtin. As it is well known, juvenoids and ecdysteroids also strongly affect fecundity and/or egg sterility if applied during sensitive phases of target insects. Fourth, the fitness of insects is often reduced after application of dosages so low that moulting is not disturbed. Adults resulting from such treatments are, for instance, unable to copulate, such as males of Oncopeltus fnsciatus (Dorn, 1986, Dorn et al., 1987), or cannot recognize the male pheromone, such as females of the fiuitfly Ceratitis capitata (Steffens and Schmutterer, 1982). In addition, adults of this fly and those of Phormia terrae-novae lost their flying ability (Bayer, 1986; Wilps, 1987).
AZT-VR-K
H20
-E
Fig. 4. Fecundity of Leptinotarsa decemlineata in 3 months after consumption of azadirachtin-treated food (AZT-VR-K = enriched seed kernel extract, H,O-E = water extract). 10 females were used per group.
Fig. 3. Pupa of Pieris brassicae after uptake by fifth-instar larva.
Fig. 2. Fourth-instar larva of Epilachna varivestis with . I .. black . . spots in the thoraclc region after uptake ot azadirachtm.
715
of azadirachtin
Azadirachtin MECHANISM
OF ACTION
The mode of action of azadirachtin is not yet clear, but various hypotheses exist. Some authors reported a reduction of the ecdysone titre and/or the delay of ecdysone production after application of the active principle. Rembold et al. (1984) suggest interference with the neuroendocrine system controlling ecdysone and juvenile hormone synthesis. A high accumulation of stainable neurosecretory material was found in the corpora cardiaca of Locusta migratoria. Investigations of Dorn et al. (1987) in Oncopeltus fasciatus seem to point in the same direction. The control by azadirachtin of the juvenile hormone titre in females of L. migratoria prevented vitellogenin production and therefore caused sterility (Rembold et al., 1984). Studies of Bidmon et al. (1987) on Calliphora vicina showed an inhibition of ecdysone release from the hormone-producing brain-ring gland. Furthermore, it was suggested that azadirachtin interferes with some transmitters involved in the regulation of ecdysone biosynthesis and/or release. A supernumerary moult to a non-viable sixth-instar larva was observed so far only in Manduca sexta (Haasler, 1984). Garcia and Rembold (1984) found that ecdysone and a juvenile hormone analogue counteracted the ecdysis inhibition induced by azadirachtin in Rhodnius prolixus. For all these reasons azadirachtin could be called an anti-hormonal active compound, at least in a broad sense. Recently it was found by Cassier et al. (1987) that azadirachtin also acts as an inhibitor of chitin biosynthesis. Many positive laboratory results, often with concentrations around l-5 ppm, encouraged a growing number of research workers to carry out field experiments for insect control. There was considerable scepticism in the beginning, because of negative experiences with many synthetic analogues of juvenile hormones, that is, they degraded so quickly under field conditions that they were unsuitable for pest control. Some years after the first discovery of the disrupting effects of azadirachtin (Ruscoe, 1972; Steets, 1975) and of methanolic neem-leaf extracts (Leuschner, 1972) research workers used azadirachtin, and aqueous, alcoholic, and other extracts from neem seeds and leaves. In many cases good to very good pest control was achieved, sometimes comparable to that of synthetic compounds (Dreyer, 1987; Kirsch, 1987). However, field’trials have also shown a number of limitations connected with compounds like azadirachtin. They are as follows: As is typical for natural products, the degradation of azadirachtin in the field takes place faster than in the laboratory, apparently mainly because of the influence of ultraviolet light. The residual effect of neem pesticides under tropical conditions lasts about 5 days, in case of systemic effects some days longer. Juvenoids lose their activity normally already within l-2 days under field conditions. The species of treated plant may also play an important role. To overcome the degrading effect of ultraviolet light to a certain extent, higher concentrations of the active principle are needed in the field than in the laboratory. Temperature seems to play only an indirect role.
111
Higher values may increase the effect, because the target insects are more active under such conditions and an undesirable “primary” antifeedant effect, if there is any, is overcome faster than at lower temperatures. Under field conditions, the residual effect of azadirachtin-containing extracts normally lasts about 4-8 days. Heavy rainfall washes down most of the active material if the interval between application and rain is short, i.e. a few hours. If a systemic effect takes place, indicating the uptake and translocation of the active material by the treated plant, the residual effect is prolonged (Heyde von der et al., 1984). In exceptional cases, for instance after the use of enriched neem seed kernel extracts (AZT-VR-K) for control of the sheep blowfly, Lucilia cuprina, a residual effect in the sheep’s wool of around 3 months was observed in Australia (Sexton, personal communication). ‘The systemic effect mentioned above is important for the control of insects which feed on the vascular tissues of plants. Very small amounts of the active compound seem to be transported in the phloem, which explains the unsatisfactory control of phloemfeeding aphids (Schauer, 1984). Leafhoppers, such as Nephotettix virescens, which usually feed on the phloem, change to xylem-feeding on neem-treated plants (Saxena and Khan, 1985). Neem products usually lack an ovicidal effect, but their residual effect is often long enough to prevent the first moult of larvae that emerge from the treated eggs. TOXICITY
AND RESISTANCE
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, although they have an otherwise broad-spectrum effect. Attempts to select for resistance against neem products in the laboratory were not successful. After
H.
718
,%SMlJTTERER
about 42 generations of the cabbage moth, Plutella which develops resistance to numerous synthetic pesticides, no remarkable decrease of sensitivity to neem products was observed (Viillinger, 1987). This may be because neem pesticides are mixtures of various related compounds with perhaps different modes of action. Azadirachtin itself has several modes of action. Some insect pests did become resistant to synthetic juvenile hormones. xylostella,
CONCLUSIONS
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: 1. 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. 2. 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. 3. 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 favourable 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.
REFERENCES Bayer U. (1986) Auswirkungen von Niemkern-Bxtrakten
auf unterschiedliche Entwicklungsstadien von Ceratitis
capitata unter besonderer Berticksichtigung von Sterilitiit und Fekunditiit der Adulten. Dipl. thesis, University of Giessen, F.R.G. Bidmon H.-J., Kiiuser G., Mdbus P. and Koolman J. (1987) Effect of azadirachtin on blowfly larvae and pupae. Proc. 3rd Znt. Neem Conf (Nairobi, 1986). In press. Bilton J. N., Broughton H. B., Ley S. V., Lidert Z., Morgan E. D:, Rzepa H. S. and Sheppard R. N. (1985) Structural reappraisal of the limonoid insect antifeedant azadirachtin. J. them. Sot., Chem. Commun. 968-971. Bowers W. S., Ohta T., Cleere V. S. and Marsella P. A. (1976) Discovery of anti-juvenile hormones in plants. Science 193, 542-547.
Butterworth J. H. and Morgan E. D. (1968) Isolation of a substance that suppresses feeding in locusts. Chem. Commun. 23-24.
Cassier P., Chalaye D., Besse N. De, Papillon M., Baldaia L. and Forcheron A. (1987) Ecdysteroids and activation of epidermal cells in locust. Poster, VZZZEcdysone Workshop; Marburg 1987, p. 39. Dorn A. ‘(1986) Effects of azadirachtin on reproduction and egg development of the heteropteran Oncopeltus fasciatus Dallas. 2. ungew. Ent. 102, 313-319. Dorn A., Rademacher J. M. and Sehn E. (1987) Effects of azadirachtin on reproductive organs and fertility in the large milkwed bug, Oncopeltus fascia&s. Proc. 3rd Znt. Neem Co& (Nairobi, 1986). In press. Dreyer M. (1987) Field and laboratory trials with simple neem products as protectants against pests of vegetables and field crops in Togo. Proc. 3rd Znt. Neem Conf. (Nairobi, 1986). In print. Garcia E. and Rembold H. (1984) Effects of azadirachtin on ecdysis of Bhodnius prolixus. J. Insect Physiol. 30, 939-941.
Grant I. F. and Schmutterer H. (1987) Effects of aqueous neem seed kernel extracts on ostracod (Class Crustacea) development and population density in lowland rice fields. Proc. 3rd Znt. Neem Conf. (Nairobi, 1986). In press. Haasler C. (1984) Effects of neem seed extract on the post-embryonic development of the tobacco hornworm, Manduca sexta. Proc. 2nd Znt. Neem Conf (Rauischholzhausen, 1983), pp. 321-330. Hellpap C. (1985) Populationsiikologie und biologischbiotechnische Bekiimpfung von Spodoptera spp. in Nitaragua. Ph.D. thesis, Universities of Frankfurt and Giessen, F.R.G. Heyde J. von der, Saxena R. C. and S&mutterer H. (1984) Neem oil and nee.m extracts as potential insecticides for control of hemipterous rice pests. Proc. 2nd Znt. Neem Conf (Rauischhblzhausen, 1983), pp. 377-390. Joshi B. G.. Ramanrasad G. and Sitaramaiah S. (1982) Effect of ‘neem &ed kernel suspension on Telenomus remus, an egg parasite of Spodoptera litura. Phytoparasitica 10, 6163. Kirsch K. (1987) Studies on the efficacy of neem extracts in controlling major insect pests in tobacco and cabbage. Proc. 3rd Znt. Neem Conf. (Nairobi, 1986). In press. Kraus W., Bokel M., Cramer R., Klenk A. and Pijhnl H. (1985a) Constituents of neem and related species. A revised structure of azadirachtin. Abstr. 3rd Znt. Conf. Biolog. Act. Natn. Prod. (Sofia, 1985), Vol. 4, pp. 446-450. Kraus W., Klenk A. and Pohnl H. (1985b) The structure of azadirachtin and 22,23-dihydro-238-methoxyazadirachtin. Tet. Z_ett. 26, 64356438. Leuschner K. (1972) Effect of an unknown plant substance on a shield bug. ‘Naturwissenschaften 59,-217-218. Mansour F. A.. Ascher K. R. S. and Gmari N. (1987) Insecticidal effect of neem seed kernel extracts-from different solvents on the predacious mite Phytoseiulus persimilis, and the phytophagous mite Tetranychus cinnabarinus, as well as the predatory spider Chiracanthium mildei. Proc. 3rd Znt. Neem Conf. (Nairobi, 1986). In print. Rembold H., Forster M., Czoppelt C. H., Rao P. J. and Sieber K. P. (1984) The azadirachtins; a group of insect growth .regulators from the neem tree. Proc. 2hd Znt. Neem Co& (Rauischholzhausen 1983). pp. 153-161. Ruscoe C. N. E. (1972) Growth disruption effects of an insect antifeedant. Nature, New Biol. 236, 159-160. Saxena R. C., Waldbauer G. P., Liquid0 N. J. and Puma B. C. (198 1) Effects of neem seed oii on the rice leaf folder, CnaohaZocrocis medinalis.
Proc.
1st Znt. Neem
Conf
(Ro&h-Egern, 1980), pp. 189-204. Saxena R. S. and Khan Z. R. (1985) Electronically recorded disturbances in feeding behavior’of Nephotett& virescens
Azadirachtin (Homoptera: Cicadellidae) on neem-oil treated rice plants. J. econ. Ent. 78, 222-226. Schauer M. (1984) Effects of variously formulated neem seed extracts on Acyrthosiphon pisum and Aphis fabae. Proc.‘ 1st Znt. Neem Conf (Rottach-Egern, 1980), pp. 141-150. Schliiter U. and Schulz W.-D. (1984) Structural damages caused by neem in Epilachna.varivestis. A summary of histological and ultrastructural data. I. Tissues affected in larvae. Proc. 2nd Znt. Neem Conf (Rauischholzhausen, 1983), pp. 227-236. Schliiter U., Bidmon H.-J. and Grewe S. (1985) Azadirachtin affects growth and endocrine events in larvae of the tobacco homworm, Manduca sexta. J. Insect Physiol. 31, 773-777.
Schmutterer H. (1985) Which insect pests can be controlled by application of neem seed kernel extracts under field conditions? Z. angew. Ent. 100, 468475. Schmutterer H. (1987) Fecundity-reducing and sterilizing effects of neem seed kernel extracts in the Colorado potato beetle, Leptinotarsa decemlineata. Proc. 3rd Znt. keem Conf (Nairobi, 1986). In press. S&mutterer H. and Holst H. (1987) Untersuchungen iiber die Wirkung des angereicherten und formuherten Niemextraktes AZT-VR-K auf die Honigbiene Apis mellifera L. Z. angew. Ent. 103, 208-213.
719
Staal B. G., Henrick C. A., Grant D. L., Moss D. W., Johnston M. J., Rudolph R. R. and Donahue W. D. (1985) Cockroach control with Juvenoids. In Bioregulators for Pest Control (Ed. by Hedin P. A.). ACS Symoosium Series 276, on. 201-218. Steets k. (1975) Die Wirkung von Rohextrakten aus den Meliaceen Azadirachta indica und Melia azedarach auf verschiedene Insektenarten. Z. angew. Em. 77, 306312. Steets R. (1976) Zur Wirkung eines gereinigten Extraktes aus Friichten von Azadirachta indica A. Juss auf LIDtinotarsa decemlineata Say (Coleoptera, Chrysomelidae). Z. angew. Ent. 82, 16%176. Steffens R. J. and Schmutterer H. (1982) The effect of a crude methanolic neem (Azadirachta indica) seed kernel extract on metamorphosis and quality of the Mediterranean fruitfly, Ceratitis capttata Wed. (Diptera, Tephritidae). Z. angew. Ent. 94, 98-103. Viillinger M. (1987) The possible development of resistance against neem seed kernel extract and deltamethrin in Plutella xylostella. Proc. 3rd Znt. Neem Conf. (Nairobi, 1986). In press. Williams C. M. (1967) Third generation pesticides. Sci. Am. 217. 13-17. Wilps’H. (1987) Growth and adult molting of larvae and pupae of the blowtly Phormia terrae-novae in relationship to azadirachtin concentrations. Proc. 3rd Znt. Neem Conf. (Nairobi, 1986). .Ia.press.
J. Insect Physiol.
Vol. 34, No. I, pp. 713-719, 1988 Printed in Great Britain. All rights reserved
$3.00 + 0.00
Copyright 0 1988Pergamon Press plc
POTENTIAL OF AZADIRACHTIN-CONTAINING PESTICIDES FOR INTEGRATED PEST CONTROL IN DEVELOPING AND INDUSTRIALIZED COWTltIES Institut fiir Phytopathologie
H. SCHMUTTERER und Angewandte Zoologie, Justus-Liebig-Universit& D-6300 Giessen, F.R.G.
Ludwigstrasse 23,
Abstract-Azadirachtin and azadirachtin-containing neem (Azadirachta indica)-seed extracts cause various effects in insects. They act as antifeedants, growth regulators and sterilants. The effect upon insect development is most important froin the viewpoint of practical insect pest control. Several hypotheses exist on the mode of action. An interference with the neuroendocrine system controlling ecdysone and juvenile hormone synthesis is suggested, b+ also an inhibition of ecdysone release from the hormone-producing gland (Culliphora). In addition, azadirachtin is a chitin synthesis inhibitor. Good iesults in insect control are obtained with azadirachtin containing seed extracts under field conditions. Aqueous, alcoholic and enriched (azadirachtin-rich) extracts are used. The residual effect usually lasts about 4-8 days, depending on the environmental conditions and the plant species treated. Systemic effects last somewhat longer. Ultra-violet light, rainfall and perhaps high acidity on treated surfaces of plants cause a fast degradation or loss of the active material. Consequently, much higher concentrations of azadirachtin have to be used in the field to obtain results comparable to those in the laboratory. Several problems have to be overcome to develop reliable pesticides from neem-seed extracts. Inspite of the sensitivity of insects of most orders to azadirachtin, neem products are selective as they do not harm important natural enemies of pests. They are also non-toxic to warm-blooded animals. Neem-seed extracts have, therefore, a considerable potential for integrated pest control measures especially in developing but also in industrialized countries, provided certain strategies are considered for their application.
INTRODUCTION Despite extensive attempts after the Second World War using numerous synthetic pesticides to reduce their number, insects remain the main competitors of man for food, especially in developing countries. Insect pests are also still a serious threat to human health, primarily as vectors of tropical diseases, whose importance is even increasing in several regions. Owing to an indiscriminate use of pesticides, various side effects have been observed in man and the environment, and many insect pests have become resistant to one or more pesticides. Consequently, the search for new, preferably environmentally sound insecticides is an important task. After a study of the properties of juvenile hormones from a practical viewpoint, Williams (1967) concluded that these compounds could be very suitable for pest control and that, insects should not be able to develop resistance to these hormones as they are their own products. He also expected insectspecific effects from them and therefore less negative influence on the environment than with broad spectrum pesticides. Founded on Williams’s philosophy, extensive basic and applied research developed during the seventies. At the same time commercial firms synthesized a considerable number of juvenile hormone analogues and mimics from which they expected even stronger negative effects on the metamorphosis and fe&mdity/fertility of insects than from’ the ‘natural hormones. 713
Reviewing the efforts of juvenile hormone research to develop new pesticides, it is notable that scientists grew progressively less enthusiastic than they had been at the onset of this line of research, when the “third generation of pesticides” was announced (Williams, 1967). Indeed, there are still a number of important barriers to the economic use of such compounds in large-scale pest control. As a consequence, only a few juvenile hormone analogues were registered in the U.S.A., for instance methoprene and hydroprene, and used on a limited scale, such as for mosquito and household pest control (Staal et al., 1985). The reasons were, among others, the short residual effects under field conditions, the few and short sensitive phases of the target insects during their metamorphosis, and the costs of production. Regarding mamtialian toxicity and environmental side effects, most juvenile hormone analogues showed positive features. Another line of research, which evolved after the discovery of the antiallotropic preconenes by Bowers et al. (1976), also failed in practice. The effect of the simply structured compounds from the bedding plant Ageratum houstonianum, which selectively destroy the corpora allata and thereby stop the production of juvenile hormone, was satisfactory in only a few important pest species, mainly Heteroptera, and relatively high’doses were needed to achieve this. There are also’ toxicplogical problems. Compared to juvenile hormones, the attempts to dkvelop pesticides based on insect moulting hormones and their analogues were moderate, although ,.
H. SCHMLJTTERER
714
Fig. 1. Structural formula of azadirachtin.
numerous more-or-less effective ecdysteroids were discovered, for instance in plants. The reasons might be, inter alia, some reluctance to use steroid hormones in the environment and the costs of synthesizing such compounds. My personal opinion is, however, that the antihormonal concept of insect control, as represented by the effect of precocenes against juvenile hormone production, is still a very promising means of insect control which deserves special attention. In principal, anti-ecdysteroids should achieve results similar to those of anti-juvenile hormones, as the moulting hormone is essential for all moults and also heavily involved in insect reproduction. Consequently, disturbing the production or mode of action of the moulting hormone should seriously affect the insects’ metamorphosis and fecundity. EFFECTS
OF AZADIRACHTIN
The triterpenoid azadirachtin was isolated from the seeds of the tropical neem tree (Azadirachta indica) by Butterworth and Morgan (1968). Its definite structural formula, which resembles somewhat that of ecdysone, was finally elucidated in 1985 (Kraus et al., 1985a,b; Bilton et al., 1985) [Fig.l]. The most important effects of azadirachtin on insects from the applied viewpoint can be described as follows:
Treatments
C
First, azadirachtin or azadirachtin-containing extracts from neem seeds show obvious antifeedant effects. There are clear differences, however, regarding the strength of these effects, depending on the concentration of the active principle and the species of insect treated. The desert locust, Schistocerca gregaria, for instance, prefers to die from starvation than to feed on treated foodplants. A “primary” antifeedant effect depending on sensory organs on the mouthparts, and a “secondary” antifeedant effect must be differentiated. The latter is observed not only after uptake of contaminated food per OS but also after topical application and injection of the active principle (Schmutterer, 1985). Second, azadirachtin is a potent disruptor of insect development. In my opinion, these effects are the most important for the practical control of insects. They have been observed in Orthoptera, Heteroptera, Homoptera, Hymenoptera, Coleoptera, Lepidoptera and Diptera (Figs 2 and 3). Some ostracods are also sensitive (Grant and Schmutterer, 1987), as well as nematodes. Third, azadirachtin is an effective sterilant. After uptake of the active material, females of some insect pest species, for instance the Colorado potato beetle, Leptinotarsa decemlineata, are sterilized to a high degree, sometimes completely (Steets, 1976; Schmutterer, 1987) (Fig. 4). The lifespan of treated females is prolonged, but their food consumption is very low. Egg fertility, as a rule, is not influenced by azadirachtin. As it is well known, juvenoids and ecdysteroids also strongly affect fecundity and/or egg sterility if applied during sensitive phases of target insects. Fourth, the fitness of insects is often reduced after application of dosages so low that moulting is not disturbed. Adults resulting from such treatments are, for instance, unable to copulate, such as males of Oncopeltus fnsciatus (Dorn, 1986, Dorn et al., 1987), or cannot recognize the male pheromone, such as females of the fiuitfly Ceratitis capitata (Steffens and Schmutterer, 1982). In addition, adults of this fly and those of Phormia terrae-novae lost their flying ability (Bayer, 1986; Wilps, 1987).
AZT-VR-K
H20
-E
Fig. 4. Fecundity of Leptinotarsa decemlineata in 3 months after consumption of azadirachtin-treated food (AZT-VR-K = enriched seed kernel extract, H,O-E = water extract). 10 females were used per group.
Fig. 3. Pupa of Pieris brassicae after uptake by fifth-instar larva.
Fig. 2. Fourth-instar larva of Epilachna varivestis with . I .. black . . spots in the thoraclc region after uptake ot azadirachtm.
715
of azadirachtin
Azadirachtin MECHANISM
OF ACTION
The mode of action of azadirachtin is not yet clear, but various hypotheses exist. Some authors reported a reduction of the ecdysone titre and/or the delay of ecdysone production after application of the active principle. Rembold et al. (1984) suggest interference with the neuroendocrine system controlling ecdysone and juvenile hormone synthesis. A high accumulation of stainable neurosecretory material was found in the corpora cardiaca of Locusta migratoria. Investigations of Dorn et al. (1987) in Oncopeltus fasciatus seem to point in the same direction. The control by azadirachtin of the juvenile hormone titre in females of L. migratoria prevented vitellogenin production and therefore caused sterility (Rembold et al., 1984). Studies of Bidmon et al. (1987) on Calliphora vicina showed an inhibition of ecdysone release from the hormone-producing brain-ring gland. Furthermore, it was suggested that azadirachtin interferes with some transmitters involved in the regulation of ecdysone biosynthesis and/or release. A supernumerary moult to a non-viable sixth-instar larva was observed so far only in Manduca sexta (Haasler, 1984). Garcia and Rembold (1984) found that ecdysone and a juvenile hormone analogue counteracted the ecdysis inhibition induced by azadirachtin in Rhodnius prolixus. For all these reasons azadirachtin could be called an anti-hormonal active compound, at least in a broad sense. Recently it was found by Cassier et al. (1987) that azadirachtin also acts as an inhibitor of chitin biosynthesis. Many positive laboratory results, often with concentrations around l-5 ppm, encouraged a growing number of research workers to carry out field experiments for insect control. There was considerable scepticism in the beginning, because of negative experiences with many synthetic analogues of juvenile hormones, that is, they degraded so quickly under field conditions that they were unsuitable for pest control. Some years after the first discovery of the disrupting effects of azadirachtin (Ruscoe, 1972; Steets, 1975) and of methanolic neem-leaf extracts (Leuschner, 1972) research workers used azadirachtin, and aqueous, alcoholic, and other extracts from neem seeds and leaves. In many cases good to very good pest control was achieved, sometimes comparable to that of synthetic compounds (Dreyer, 1987; Kirsch, 1987). However, field’trials have also shown a number of limitations connected with compounds like azadirachtin. They are as follows: As is typical for natural products, the degradation of azadirachtin in the field takes place faster than in the laboratory, apparently mainly because of the influence of ultraviolet light. The residual effect of neem pesticides under tropical conditions lasts about 5 days, in case of systemic effects some days longer. Juvenoids lose their activity normally already within l-2 days under field conditions. The species of treated plant may also play an important role. To overcome the degrading effect of ultraviolet light to a certain extent, higher concentrations of the active principle are needed in the field than in the laboratory. Temperature seems to play only an indirect role.
111
Higher values may increase the effect, because the target insects are more active under such conditions and an undesirable “primary” antifeedant effect, if there is any, is overcome faster than at lower temperatures. Under field conditions, the residual effect of azadirachtin-containing extracts normally lasts about 4-8 days. Heavy rainfall washes down most of the active material if the interval between application and rain is short, i.e. a few hours. If a systemic effect takes place, indicating the uptake and translocation of the active material by the treated plant, the residual effect is prolonged (Heyde von der et al., 1984). In exceptional cases, for instance after the use of enriched neem seed kernel extracts (AZT-VR-K) for control of the sheep blowfly, Lucilia cuprina, a residual effect in the sheep’s wool of around 3 months was observed in Australia (Sexton, personal communication). ‘The systemic effect mentioned above is important for the control of insects which feed on the vascular tissues of plants. Very small amounts of the active compound seem to be transported in the phloem, which explains the unsatisfactory control of phloemfeeding aphids (Schauer, 1984). Leafhoppers, such as Nephotettix virescens, which usually feed on the phloem, change to xylem-feeding on neem-treated plants (Saxena and Khan, 1985). Neem products usually lack an ovicidal effect, but their residual effect is often long enough to prevent the first moult of larvae that emerge from the treated eggs. TOXICITY
AND RESISTANCE
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, although they have an otherwise broad-spectrum effect. Attempts to select for resistance against neem products in the laboratory were not successful. After
H.
718
,%SMlJTTERER
about 42 generations of the cabbage moth, Plutella which develops resistance to numerous synthetic pesticides, no remarkable decrease of sensitivity to neem products was observed (Viillinger, 1987). This may be because neem pesticides are mixtures of various related compounds with perhaps different modes of action. Azadirachtin itself has several modes of action. Some insect pests did become resistant to synthetic juvenile hormones. xylostella,
CONCLUSIONS
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: 1. 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. 2. 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. 3. 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 favourable 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.
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Staal B. G., Henrick C. A., Grant D. L., Moss D. W., Johnston M. J., Rudolph R. R. and Donahue W. D. (1985) Cockroach control with Juvenoids. In Bioregulators for Pest Control (Ed. by Hedin P. A.). ACS Symoosium Series 276, on. 201-218. Steets k. (1975) Die Wirkung von Rohextrakten aus den Meliaceen Azadirachta indica und Melia azedarach auf verschiedene Insektenarten. Z. angew. Em. 77, 306312. Steets R. (1976) Zur Wirkung eines gereinigten Extraktes aus Friichten von Azadirachta indica A. Juss auf LIDtinotarsa decemlineata Say (Coleoptera, Chrysomelidae). Z. angew. Ent. 82, 16%176. Steffens R. J. and Schmutterer H. (1982) The effect of a crude methanolic neem (Azadirachta indica) seed kernel extract on metamorphosis and quality of the Mediterranean fruitfly, Ceratitis capttata Wed. (Diptera, Tephritidae). Z. angew. Ent. 94, 98-103. Viillinger M. (1987) The possible development of resistance against neem seed kernel extract and deltamethrin in Plutella xylostella. Proc. 3rd Znt. Neem Conf. (Nairobi, 1986). In press. Williams C. M. (1967) Third generation pesticides. Sci. Am. 217. 13-17. Wilps’H. (1987) Growth and adult molting of larvae and pupae of the blowtly Phormia terrae-novae in relationship to azadirachtin concentrations. Proc. 3rd Znt. Neem Conf. (Nairobi, 1986). .Ia.press.