Activity and Biological Effects of Neem Products Against Arthropods of Medical and Veterinary Importance

Home , Neem oil, Tetranortriterpenoid

Journal of the American Mosquito Control Association' 15(2):133-152, 1999 Copyright O 1999 by the American Mosquito Control Association, Inc.

ACTIVITY AND BIOLOGICAL EFFECTS OF NEEM PRODUCTS AGAINST ARTHROPODS OF MEDICAL AND VETERINARY IMPORTANCE

MIR S. MULLA ENN TIANYUN SU

Depdrtment of Entomobgy, University of Califomia, Riverside, CA 92521-O314

ABSTRACT. Botanical insecticides are relatively safe and degradable, and are readily available sources of biopesticides. The most prominent phytochemical pesticides in recent years are those derived from neem trees, which have been studiedlxtensively in the fields of entomology and phytochemistry, and have uses for medicinal and cosmetic purposes. The neem products have been obtained from several species of neem trees in the family Meliaceae. Sii spicies in this family have been the subject of botanical pesticide research. They are ATadirachta indica A. Jrtss, Azadirachta excelsa Jack, Azadirachta siamens Valeton, Melia azedarach L., Melia toosendan Sieb. and Z1cc., and Melia volkensii Giirke. The Meliaceae, especially A. indica (Indian neem tree), contains at least 35 biologically active principles. Azadirachtin is the predominant insecticidal active ingredient in the seed, leaves, and other parts of the neem tree. Azadirachtin and other compounds in neem products exhibit various modes of action against insects such as antifeedancy, growth regulation, fecundity suppression and sterilization, oviposition repellency or attractancy, changes in biological fitness, and blocking development of vector-borne pathogens. Some of these bioactivity parameters of neem products have been investigated at least in some ipecies of insects of medical and veterinary importance, such as mosquitoes, flies, triatomines, cockroaches, fllas, lice, and others. Here we review, synthesize, and analyze published information on the activity, modes of action, and other biological effects of neem products against arthropods of medical and veterinary importance' The amount of information on the activity, use, and application of neem products for the control of disease vectors and human and animal pests is limited. Additional research is needed to determine the potential useful- ness of neem products in vector control programs.

KEY WORDS Meliaceae, neem, azadirachtin, insecticides, mosquitoes, flies, triatomines, cockroaches, fleas, lice, pest control

INTRODUCTION (Mulla 1997). Interest in botanical insecticides started in the early 1930s and continued to the l950s The extensive and widespread use of synthetic in- (Campbell et al. 1933; Jacobson 1958), but the insecticides during the past half century, since the dis- terest in their development and use was phased out covery of DDT during World War II, for the control when synthetic insecticides appeared on the scene. of household, agricultural, and sylvan pests, as well Interest in the development of natural products has as human disease vectors has caused some concerns been revived during the last 2 decades. Thus far regarding the toxicity and environmental impact of about 10,000 secondary metabolites or phytochem- some of these agents. Some inherent features and use icals have been isolated, but their total number is patterns of the conventional synthetic insecticides estimated to be much higher, close to 500,000 that lead to these concerns are toxicity to mammals (Ascher 1993). More than 2,000 plant species re- including livestock, fish, birds, and beneficial organ- portedly possess chemicals with pest control prop- isms; human poisoning, especially in Third World erties (Ahmed et al. 1984), and among these about countries; adverse effects on the environment, caus- 344 species ofplants have been shown to have some ing contamination of soil, water, and air; resurgence degree of activity against mosquitoes (Sukumar et of insect pest populations because of the emergence al. 1991). The most prominent phytochemical pes- and widespread occurrence of physiologic resistance ticides studied in recent years are those based on the to conventional insecticides; the high cost of the de- neem prducts, which have been researched exten- velopment of new synthetic insecticides, which puts sively for their phytochemistry and exploitation in the new agents out of the reach of pest control pro- pest control programs. This paper presents a concise grams in Third World countries; and not meeting the review of recent advances in research on the activity, modern criteria of use in integrated trrestmanagement efficacy, and potential uses ofneem products for the programs. control of arthropods of medical and veterinary im- Because of these problems and concerns, the portance. search for new, environmentally safe, target-specific insecticides is being conducted all over the world. OVERVIEW OF NEEM PRODUCTS To find new modes of action and to develop active agents based on natural plant products, efforts are Neem tree being made to isolate, screen, and develop phyto- Six species in the family Meliaceae have been chemicals possessing pesticidal activity. These cat- studied for pesticidal properties in different parts of egories of pesticides are now known as biopesticides the world. They are Azadirachta indica A. Juss

133 t34 JounNer- op tHe AN4BRrcnNMosguno CoNTRor- AssocrnrtoN VoL. 15,No.2

(syn. Melia azadirachta, Antelaea azadirachta\. have been reported to contain AZ (Morgan and commonly known as the neem tree, Margosa tree, Thornton 1973). The detailed chemistry of AZ cart or Indian lttlac1'Aaadirachta excelsa Jack (syn. A. be found in Ley et al. (1993). integrifoliola), called the Philippine neem tree or In addition to AZ, a number of other active in- marrango; Azadirachta siamens Valeton, or the Si- gredients have also been isolated and identified from amese neem tree or Thai neem tree: Melia azedar- different parts of the neem tree. Compounds such as ach L., commonly known as Persian lilac, china- salannin, salannol, salannolacetate, 3-deacetylsalan- berry, Australian bead tree, Chinese umbrella tree. nin, azadiradion, l4-epoxyazadiradion, gedunin, or pride of India; Melia toosendan Sieb. andZrtcc.; nimbinen, deacetylnimbinen, 23-dihydro-23p-meth- and Melia volkensii Giirke. Varieties of these oxyazadirachtin, 3-tigloylazadirachtol, and l-tigloyl- named species occur, some of which have been as- 3-acetyl- 1 1-methoxyazadirachtin were isolated from signed species status in some countries. Generally, oil extracted from neem seed kernels (Schmutterer the neem tree is referred to A. indica. However. 1990). Vilasinin derivatives, meliantriol, azadiradi- herewith we use neem products for all of the bio- one, l4-epoxyazadiradione, 6-0-acetylnimbandiol, 3- active components originating from this group of deacetylsalannin, and deacetylazadirachtinol were plants in the family Meliaceae. Plants in the family also recovered from neem seed oil (Schmufterer Meliaceae, especially A. indica, contain at least 35 1990), whereas nimocinolide and isonimocinolide biologically active principles (Rao and Parmar were recovered from fresh leaves (Siddiqui et al. 1984) and these products have many industrial, me- 1986). Some other compounds, such as alkanes, dicinal, and pesticidal uses. Various components of were isolated from dried leaves (Chavan 1984, the tree have potential uses in toiletries, pharma- Chavan and Nikam 1988), and nonterpenoidal con- ceuticals, the manufacture of agricultural imple- stituents, isocoumarins, coumarins, and saturated hy- ments and furniture, cattle and poultry feeds, nitri- drocarbons were recovered from fresh, uncrushed fication of soils for various agricultural crops, and twigs (Siddiqui et al. 1988). Although the bark, pest control (Koul et al. l99O). heartwood, leaves, fruit, and seeds of neem have been investigated chemically for their main biocidal components, the renewable parts (seeds and leaves) Active ingredients received major research attention. A number of bioactive components have been isolated from various parts of the neem ffee. These Mode of action chemical compounds have different designations, among which azadirachtin (AZ) (C..ItoO,) is the Neem products are capable of producing multiple major component. The insecticidal properties of effects in insects such as antifeedancy, growth reg- products from the neem tree were flrst reported by ulation, fecundity suppression and sterilization, ovi- Chopra (1928). Forty years later, AZ, a steroidlike position repellency or attractancy, and changes in tetranotriterpenoid (limonoid), was isolated by But- biological fitness. These aspects are discussed in terworth and Morgan (1968) from A. indica. How- detail in recently published comprehensive reviews ever, it was not until 1987 that the chemical struc- (Ascher 1993; Mordue and Blackwell 1993; ture of AZ was completely elucidated by Kraus et Schmutterer 1988, 1990). Most of the information al. (1987). Azadirachtin is the predominant insec- reviewed in these papers was gathered with regard ticidal active ingredient in the seeds of the neem to phytophagous insects. Some of these phenomena tree. The AZ content in neem oil was highly cor- are briefly reviewed and discussed below. related with its bioactivity against test insects (Is- Antiftedancy.' Chemical inhibition of feeding has man et al. 1990). A marked difference has been been studied in detail for a few phytophagous in- reported in the yield of AZ from neem seeds from sects. The mechanism of feeding inhibition could different geographical origins (Schmutterer and Ze- be a blockage of the input from chemoreceptors bitz 1984, Chirathamjaree et al. 1997), or even in normally responding to phagostimulants, which can different seasons in the same geographical area be reversed by increasing the amount of phagosti- (Sidhu and Behl 1996). Azadiachtin analogs, de- mulants, or stimulation of specific deterrent cells or pending on the minor variations in chemical struc- broad spectrum receptors, or through both of these ture, were given different designations. Azadirach- mechanisms (Chapman 1974, Schoonhoven 1982). tin-A is the major component of the total AZ Azadirachtin and AZ-containing extracts from the (Rembold 1989). Azadirachtin-B (3-tigloylazadi- neem tree show distinct antifeedant activity, pri- rachtol) is present at concentrations of up to l5Vo marily through chemoreception (primary antifee- of the total AZ. Other AZ analogues such as AZ- dancy), but also through reduction in food intake C through AZ-G occttt at much lower concentra- due to toxic effects after consumption (secondary tions. Recently, 2 new tetr:rnortriterpenoids, 1 1-epi- antifeedancy) in lethal quantities. However, clear azadirachtin H (Ramji et al. 1996) and AZ-K differences exist in the magnitude of these effects, (Govindachari et al. 1992), have been isolated from depending on the concentration and formulation of neem seeds. In addition to A. indica, other plants the active principle, the methods of application of in the family Meliaceae, such as M. azedarach, also the neem products, and the species of test insects. JuNe 1999 EFFEcrs oF NEEM Pnoousrs 135

By applying the neem products in different ways, ness of many insect species was substantially re- antifeedant activity of neem products has been duced even at neem concentrations and dosages be- found in Orthoptera, Isoptera, Hemiptera, Coleop- low those interfering with the molting process. tera, Lepidoptera, and Diptera (Mordue and Black- Changes in biological fitness included reduced life- well 1993). span (Wilps 1989), high mortality (Dorn et al. Growth regulation: The growth regulatory ef- 1987), loss of flying ability (Wilps 1989), low ab- fects of AZ and other neem-related products are of sorption of nutrients (Wilps 1989), immunodepres- considerable theoretical and practical interest. sion (Azambuja et al. 1991, Azambuja and Garcia Tfeatment of insects by injection, oral ingestion, or 1992), enzyme inhibition (Naqvi 1987), and disrup- topical application of AZ caused larval growth in- tion of biological rhythms (Smietanko and Engel- hibition, malformation, and mortality. These are ef- mann 1989). This means that AZ has subtle effects fects that are similar to those observed in treating on a variety of tissues and cells, especially those insects with insect growth regulators (IGRs). This with rapid mitosis, for example, epidermal cells, activity has been proven in Orthoptera, Hemiptera, midgut epithelial cells, ovary, and testis, which Lepidoptera and Diptera (Ascher 1993; Mordue and form part of the overall toxic syndrome of poison- Blackwell 1993; Schmutterer 1988, 1990). A major ing. action of AZ is to modify hemolymph ecdysteriod and juvenile hormone titers (significant reduction or Commercial formulations and persistence delays) by inhibiting the release of morphogenetic peptide, prothoracicotropic hormone (PTTH), and The pesticidal neem products used in practice in- allatotropins from the brain-corpus cardiacum clude those from leaves. whole seed. decorticated complex (Ascher 1993). Extracts from various parts seed, seed kernels, neem oil, and neem cake after of the neem tree had different degrees of IGR type extraction or extrusion of the oil from the seeds. of activity. For example, with the diamond-back Large-scale chemical synthesis for pesticidal use is moth (Plutella rylostella L.) the order of growth- precluded by the extremely high cost of synthesis inhibiting potency of neem extracts was seeds > of the complex structure of AZ and other bioactive leaves ) bark/twig ) wood (Tan and Sudderuddin neem constituents. Therefore, it is only practical to 1978). extract neem bioactive agents from the renewable Fecundity suppression and sterilization.' The in- parts of the tree and to manufacture various pesti- terruption of insect reproduction is also an impor- cidal formulations. The commercial formulations of tant feature of AZ compounds. Because ecdysteroid neem products produced by Indian companies in- is one of the hormones regulating vitellogenesis, clude RD-9 Repelin (ITC Ltd., Andhra Pradesh, In- arld AZ can modify hemolymph ecdysteriod by india), Neemgard (Gharda Chemicals, Bombay, In- hibiting the release of PTTH and allatotropins from dia), and Neemark (West Coast Herbochem, the brain-corpus cardiacum complex, adverse ef- Bombay, India). These products are used for the fects on ovarian development, fecundity, and fertil- control of a variety of pests. In the USA, an AZ- ity (egg viability) occur in Orthoptera, Hemiptera, enriched, concentrated neem seed kernel extract Heteroptera, Coleoptera, Lepidoptera, Diptera, and formulation, Margosan-O (Vikwood Ltd., Sheboy- Hymenoptera (Ascher 1993; Mordue and Blackwell gan, WI, USA), has been granted registration by 1993; Schmutterer 1988, 1990). In addition, the sex the U.S. Environmental Protection Agency for the behavior of the males and females in mating and control of pests in nonfood crops and ornamentals. response to sexual pheromones (Dorn et al. 1987), Other formulations of neem, Azatin WP4.5, Azatin and spermatogenesis in males (Shimizu 1988) were EC4.5. Azad WP10. Azad EC45. and Neernix affected by AZ treatment. EC0.25 by the Thermotrilogy Co. (Columbia, MD); Oviposition repellency: When oviposition sites Bioneem and Neemesis by Ringer Corp. (Minne- were treated with AZ or other neem-related prod- apolis, MN); Neemazal by Trifolio M GmBH (D- ucts, oviposition repellency, deterrency, or inhibi- 6335 Lahnau 2, Germany); and Safer's ENI by tion occurred in Coleoptera, Lepidoptera, and Dip- Safer Ltd. (Victoria, BC, Canada) are also avail- tera (Schmutterer 1988, 1990: Dhar et al. 1996). able. Other companies in Europe are involved in Blocking the development of vector-borne path- formulating and distributing pesticidal formulations ogens: This aspect of the mode of action has been based on neem products. studied in the infection of triatomid bugs by try- Like other natural products, the degradation of panosomes. Complete blocking of the development AZ under field conditions takes place faster than in of Trypanosoma cruzi in triatomine vectors was ob- the laboratory because of the effects of untraviolet served after a single treatment with AZ (see details (UV) light, temperature, pH, and microbial activity below; Garcia 1988; Garcia et al. 1989a, 1989b. (Sta* and Walter 1995, Sundaram 1996, Szeto and 1991, 1992: Rembold and Garcia 1989; Azambuja Wan 1996). The degradation of AZ at a basic pH and Garcia 1992; Gonzalez and Garcia L992). To is faster than at acidic pH (Szeto and Wan 1996). date, no similar studies have been reported on other For increasing the effiCacy and longevity of AZ, vector-pathogen systems. degradation was shown to be lower with the addi- Changes in biological fitness: The biological fit- tion of UV absorbers and protectants such as 2,4- 136 JounN,rt-or rns AN{snrcaNMosquno CoNrnol AssocrertoN VoL. 15, No. 2 dihydroxybenzophenone to AZ-A at a rate of 1:l tomines, cockroaches, fleas, lice, sandflies. and (w/w) (Sundaram 1996). Under field conditions the ticks. The actions of neem products against these residual effect of neem pesticides lasts about 4-8 arthropods were considered to include but not be days, and in the case of systemic effects some days limited to antifeedancy or landing repellency, longer. growth regulation, fecundity reduction, reduced fitness, and blocking of the development of vector- borne pathogens. Information Effects on nontarget organisms on these aspects of neem products is synthesized and summarized in Because of a relatively weak contact mode of Table 1. Information for each group of arthropods action in insects and lack of activity against pred- is presented and discussed in more detail below. ators and parasitoids, neem products have been shown to be very safe to beneficial organisms such Mosquitoes as honeybees, natural enemies of pests, and so forth. Most of the investigations related to the potential Neem products are relatively safe to mammals of neem products for the control of insects of med- and humans according to tests carried out to date. ical and veterinary importance were conducted on Neem flowers and leaves are eaten as vegetables in mosquitoes during the past 20 years, where the Asian countries (India, Myenmar, Thailand). More- neem preparations used were extracts prepared in over, some neem products obtained from the fruit, the laboratory from different parts of the neem tree. bark, or leaves have played a prominent role in In- The major interest was focused on the IGR type of dian traditional medicine for many centuries. Neem activity ofneem preparations in both the laboratory products are also incorporated into a number oftoi- and the field, even though a few investigations were letry and pharmaceutical products in India, Paki- also conducted to study the repellent activity of stan, and Germany. However, neem products are neem oil on adult mosquitoes, and the effects on not risk free. Adverse effects of neem components reproductive capacity of mosquitoes. were noted in nontarget aquatic invertebrates (Stark Activity against immature stages: Laboratory 1997), fish (Tangtong and Wattanasirmkit 1997), tests. Laboratory tests with different neem prepa- some mammals (Ali and Salih 1982, Ali 1987), and rations against the immature stages of mosquitoes humans (Reye's syndrome) (Sinniah and Baskaran have been mostly carried out on the following 7 1981; Sinniah et al. 1982,1986). Finally, aflatoxins species, Anopheles arabiensis Patton, Anopheles were usually present in neem extracts if the fungus culicifacies Glles, Anopheles stephensi Liston, Cu- Aspergillus was present during the storage of neem lex pipiens molestus Forskal, Culex quinquefascia- products (Sinniah et al. 1982,1983, 1986, Hansen /ns Say, Aedes aegypri (L.), and Aedes togoi Theo- et al. 1994). Therefore, caution must be exercised bald. The IGR activity of neem and related products to control the level of these toxins to insure safe are discussed below. use. In genereLl,AZ products are considered to have The neem tree has received much attention as a a good margin of safety to fish, wildlife, and other source of active agents for mosquito larval control. nontarget biom. More than 2 decades ago small-scale tests of neem products against An. stephensi were conducted (see Zebitz 1984), which was rhe lst attempt in this Development of resistance field. Extracts from A. indica seeds such as neem No cases of resistance to neem products have oil and neem seed kernel extracts (NSKEs) provid- been reported. Laboratory selection experiments for ed a good source of pesticidal ingredients. Attri and "neem resistance to neem products it P. rylostella did not Prasad (1980) tested the oil extractive," a result in derrelopment of resistance (Vdllinger waste from neem oil refining, against larvae of this 1987), which may be because the products were species. The complete failure of lst-stage larvae to mixtures of various related compounds with differ- develop to the adult stage occurred at the concen- ent modes of action. Development of a high level tration of O.OOSVoof neem oil. But the material was of resistance to chemicals or products that possess also toxic to mosquito fish (Gambusia sp.) and tad- numerous modes of action is somewhat difficult. poles at t his concentration. Continuous exposure of 4th-stage larvae of Ae. aegypti in water treated with crude or Az-effiched NSKEs resulted in no- RESEARCH ON NEEM PRODUCTS FOR ticeable growth disrupting effects. An extreme pro- THE CONTROL OF ARTHROPODS OF longation of the larval period was attained when MEDICAL AND VETERINARY lst-stage larvae were continously exposed in treat- IMPORTANCE ed water until adult emergence. The effectiveness Relatively little information is available regard- of the extracts increased with decreasing polarity of ing the effects of neem products on arthropods of the solvents used for extraction. The time necessary medical and veterinary importance. Studies, testing. for lethal action of NSKEs was similar to that re- and evaluation started in the early 1970s. Neem ported for some synthetic IGRS (zebitz 1984). products were tested against mosquitoes, flies, tria- Singh (1984) found that aqueous extracts from de- Jurn 1999 EFFEcrs oF NEEM PRoDUcrs t37

Table 1. Effects of neem products on feeding, reproduction, and survivorship of arthropods of medical and veterinary imPortance. I

Species Stages Type of action Neem components References Antifeedancy or repellency Anopheles culici- Adult Repellency Neem oil Sharma et al. (1993a) facies Aiopheles and Adult Repellency Neem oil Sharma et al. (1993a, 1993b), Sharma Cukx spp. and Ansari (1994) Culzx quinquefas- Larvae Antifeedancy AZ formulations Su and Mulla (1998a) ciatus Cx. tarsalis Larvae Antifeedancy AZ formulations Su and Mulla (1998a) Musca domestica Adult Antifeedancy Ethanol extract of Warthen et al. (1978) seeds Phormia terraeno- Larvae Antifeedancy AZ Wilps (1987) vae Rhodnius prolixus Nymph Antifeedancy AZ Garcia et al. (1984), Garcia and Rem- bold (1984) Phlebotomus ar- Adult Repellency Neem oil Sharma and Dhiman (1993) gentipes Insect growth regulation An. arabiensis Larvae IGR Fruit kemel exhact' Mwangi and Mukiama (1988) An. culicifacies Larvae IGR AZ-rich fractions Rao et al. (1988) An. stephensi Larvae IGR NSKEs, AZ, oil ex- (see Z.ebtlz 1984), Kumar and Dutta tracts (1987), Zebitz (1987) Cr. spp. Larvae IGR Lipid/AZ-rich frac- Rao (1987), Rao and Reuben (1989), tions, neem cake Rao et al. (1992,1995) Cx. pipiens Larvae IGR AZT-VR-K-Er Tnbttz (1987) Cx. p. molestus Larvae IGR Acetone extracts4 Al-Sharook et al. (1991) Cx. quinquefascia- Larvae IGR Petroleum ether, Chavan et al. (1979), Attri and Prasad tus chloroform, alco- (1980), Chavan (1984), Singh (1984), hol leaves extracts, Zebitz (7987), Chavan and Nikam alkanes,neem oil (1988), Rao et al. (1988), Sinniah et extract, NSKEs, al. (1994), Amorose (1995), Irungu AZ, AZ-richfrac- and Mwangi (1995), Sagar and Sehgal tions, defatted (1996), Mulla et al. (1997) cake, AZ-based formulations. fruit extracts2 Cx. vishnui sub- Larvae IGR AZ-ricVneem oil for- Rao (1997) group mulations Ae. aegypti Larave IGR Neem oil, NSKEs, Zebitz (1984, 1987), Hellpap and Zebitz AZ, nimocinolide (1986), Naqvi (1987), Naqvi et al. and isonimocinoli- (1991), Sinniah et al. (1994), Mwangi de of fresh winter and Rembold (1987, 1988) leaves, extracts of fruits' Ae. togoi Larvae IGR NSKEs, AZ Hellpap and Zebitz (1986), Zebitz (l987) Calliphora vicina Larvae IGR AZ Bidmon et al. (1987) Haematobia irri- Larvae IGR AZ, seed kernel Wilps (1995), Miller and Chamberlain tans, Stomorys (1989) calcitrans, and M. domestica Lucilia cuprina Larvae IGR AZ Meurant et al. (1994) M. autumnalis Larvae IGR AZ Gaaboub and Hayes (1984a) P. terraenovae Larvae IGR AZ Wilps (1987) R. prolixus Nymph IGR AZ, AZ-A, AZ-8,7- Garcia and Rembold (1984), Garcia et acetyl AZ-A al. (1984, 1986, 1987, l99oa, 1990b, l99r) Triatoma vitticeps' Nymph IGR AZ-A Garcia et al- (1989a) Blana orientalis6 Nymph IGR Seed extract Adler and Uebel (1985) B. germanica Nymph IGR AZ Adler and Uebel (1985), Prabhakaran and Kamble (1996) Periplaneta ameri- Nymph IGR AZ Qadri and Narsaiah (1978) cana Ctenocephatides Larvae IGR NSKEs Kilonzo (1991) felis and Xenopsylla brasiliensis 138 JounNnr- oF rne Ar\4nntcen Mosquno Coxrnol AssocrenoN VoL. 15, No. 2

Thble l. Continued.

Species Stages Type of action Neem components References X. cheopis Larvae IGR AZ Chamberlainet al. (1988) Bovicola ovis Larvae IGR AZ Heath et al. (1995) Reproduction suppression and ovicidal activity An. culicifacies Adult Antioviposition, GC Neem oil, volatiles Dhar et al. impaired, vitellogenesis impaired An. stephensi Adult Antioviposition, GC Neem oil, volatiles Dhar et al. (1996) impaired, vitellogenesis impaired Cx. quinquefascia- Adult Oviposition repellen- Neem oil, AZ formu- T.ebitz (1987), Irungu and Mwangi tus cy lations, fruit ex- (1995), Su and Mulla (1998c) tract2 Cx. quinquefuscia- Eggs Ovicide Seed extract, AZ for- Zebitz (1987), Su and Mulla (1998a) tus mulations Cx- tarsalis Adult Ovi-modification AZ formulations Su and Mulla (1998b) Cx. tarsalis Eggs Ovicide AZ formulations Su and Mulla (1998c) Ae. aegypti Adult Oocyte growth re- AZ Ludlum and Sieber (1988) duction Glossina morsitans Adult Fecundity reduction Neem oil, NSKEs Kaaya (see Wilps 1995) morsitans, G. m. centralis L. cuprina Adult Antioviposition Petroleum ether, ex- Rice et al. (1985) tracts of ripe fruits L. sericata Adult Antioviposition Neem oil Hobson (1940) M- autumnalis Adult Fecundity reduction AZ Gaaboub and Hayes (1984a) P. tenaenovae Eggs Ovicide NSKEs P. terraenovae Adult Fecundity reduction AZ, NSKEs wilps (1995), wilps (1989) S. calcitrans Eggs Ovicide AZ Grll (19'72) (see Wilps 1995) R. prolixus Adult Fecundity reduction AZ, AZ-A Garcia et al. (1987, l99oa), Feder et al. (1988), Moreira et al. (1994) Amblyomma amer- Adult Fecundity reduction AZ Kaufman (1988), Lindsay and Kaufman icanum (r 988) Blocking the development of trypanosomes R. prolixus Nymph Block development AZ Garcia (1988), Garcia et al. (1989a, of Typanosoma 1989b), Rembold and Garcia (1989), cruzi Azambuja and Garcia et al. (7992) R. prolixus, T. in- Nymph Block development AZ Gonza\ez and Garcia (1992) festans, Dipetalo- of T. cruzi gaster maximus Change biofitness G. m. morsitans, Adult Increased mortality Neem oil, NSKEs seeWilps (1995) G. m. centralis, G. pallidipes M. domestica Adult Enzyme inhibition Petroleum ether frac- Naqvi (1987) tion of fruits M. domestica Adult Splitting of activity AZ Smietanko and Engelmann (1989) rhythm P. teraenovae Adult Changes in metabo- NSKEs wilps (1989) lism B. germanica Nymphs Enzyme inhibition Neutral fraction of Naqvi (1987) leaves R. prolixus Nymph Immune depression AZ Azambuja et al. (1991), Azambuja and Garcia (1992)

I AZ, azadirachlin; IGR, insect growth regulationl NSKEs, neem seed kernel extracts; GC, gonotrophic cycle. 2 From Melia volkensii. I AZT-VR-K-E was prepared from neem seed kemels in a Soxhlet apparatus using an zeotropic mixture of lerr-butylmethyl ether and methanol. Fatty components were separated by petrol ether extraction and cmling. a From M. volkensii and Melia azedarach. 5Including the following species: Triatomo vitticeps, T. pseudorcculata, T. maculata, T. brasiliensis, T. lecticulqris, T. mtogrossensis, T- infestans, Rhodnius prolixus, R. neglectus, R. robustus, Panstrongylus megistus, nd P. herrera. 6 Including the following species: Blatta orientalis, B. germanica, Byrsotiafumigata, Gromphadorhina protentosa, P. americana, and Supella longipalpa. JuNe 1999 Errecrs or Nser\aPnonucrs r39 oiled neem seed kernels caused 10070 mortality in tant effect was larger than expected and showed a Cx. quinquefasciatus larvae at 62.5, 125,250, and synergistic action (Hellpap and 7ob1tz 1986). ZE- 50O ppm after 8, 6,4, arrd2 days of exposure, re- bitz (1987) investigated the IGR effects of NSKEs spectively. The reduced concentration of 31.2 ppm and crude and pure AZ on mosquito larvae and still yielded 85Vo mortality within 12 days of ex- their joint activities with methoprene, a juvenile posure. Another comparable study conducted re- hormone analog (JHA). The susceptibility of young cently by Sagar and Sehgal (1996) also confirmed 4th-stage larvae of 4 species of mosquitoes to the activity of the aqueous extracts of deoiled neem NSKEs was studied. Their susceptibility in decreas- seed kernel, which was more active than those of ing order was Ae. togoi, An. stephensi, Cx. quin- karanja (Pongamia glabra Vent) seed kernel quefasciatus, and Ae. aegyptL Azadirachtin and against the same species. Four new AZ-rich frac- NSKEs exerted a pronounced IGR type of effect tions prepared from neem seeds with different ex- during preimaginal development. Joint action of traction procedures, designated as nemidin, vemi- methoprene and NSKEs resulted in pronounced din, vepcidin, and nemol, were tested for larvicidal synergistic effects. The results pointed out a strong activity against the early 4th-stage larvae of An. interference of AZ with hormonal balance, most culicifacies and Cx. quinquefasciatus. Among probably with ecdysteriod titer. The petroleum ether these, nemidin, which was found to have a high extracts of various parts of neem tree also had syn- content of AZ, showed high larvicidal activity ergistic effects with synthetic chemical insecticides, against both test species as shown by low median such as phenthoate and fenthion (Kalyanasundaram lethal concentration (LCro) values of t.lZ and 3.9 and Babu 1982). ppm, respectively. However, the other fractions In addition to A. indica seeds, the leaves of this showed moderate larvicidal activity only. Nemidin species also contain some mosquitocidal compo- produced rectal prolapse in Cr. quinquefasciatus nents. Tests by Chavan et al. (1979) showed that larvae but not in An. culicifacies (Rao et al. 1988). extracts of neem leaves with organic solvent were Sinniah et al. (1994) tested neem oil extract from toxic to 4th-stage larvae of Cx. quinquefasciatus. crushed seeds against Ae. aegypti and. Cx. quinque- Dried leaves were extracted with petroleum ether, fasciatus. At concentrations of O.O2Vo (relatively ether, chloroform, and ethanol, and the solvents high concentration) or more, lNVo mortality was were removed from the extracts in a water bath, attained irr 24 h for these 2 species. At a concen- yielding residues of the active compounds. The res- tration of O.OOlVo,59 and 63Vo mortality, and at a idues were redissolved in a solvent and bioassayed concentration of O.ONSVo, 2 and 22Vo mortality against larvae of this species. At tEo concentration, was obtained in 96 h for these 2 species, respec- lNVo mor1lality of mosquito larvae occured within tively. Amorose (1995) tested the larvicidal effica- 24 h when petroleum ether and ether were used as cy ofneem oil and defatted neem cake on Cx. quin- exffaction solvents. However, under identical con- quefasciatus. Against 3rd- and 4th-stage larvae, ditions, the residues from extracts with chloroform LC,os were 0.99 and 1.20 ppm for neem oil, and and ethanol at lVo only yielded 65 and 6OVo mor- 0.55 and O.72 ppm for the deoiled neem cake. tality, respectively, within the same period of ex- Third-stage larvae were more susceptible than were posure. The residues from extracts with petroleum 4th-stage larvae. The growth-modifying effects of ether and ether also exhibited good residual activity the methanolic extract from neem seeds in Ae, ae- (up to 144 h) at the concentrations of I and,0.2Vo, gypti often was not correlated with the AZ content, respectively (which are very high by current stan- which implied the existence of other principles ex- dards). Residues from petroleum ether extract was hibiting synergistic or antagonistic effects on the further purified with a neutral alumina colunm and active ingredients contained in neem seed extract by eluting with different solvent systems. Benzene (Schmutterer andZebitz 1984). Most likely, differ- eluate showed promising results and yielded a ma- ent components from the crude extracts of neem, terial that crystallized and gave IOOVo mortality at not AZ alone, act together against mosquito larvae. l0 ppm, which also had residual activity producing As to the interaction between the seed extracts lOOTo mortality for 5 days. When used at the very of A. indica and microbial and chemical insecti- high concentration of 100 ppm, IOOVo mortality cides, when 4th-stage larvae of Ae. togoi were ex- was obtained for 8 days (Chavan 1984). Chavan posed in water treated with NSKEs, Bacillus thu- and Nikam (1988) discovered that the alkanes sep- ringiensis israelensis, or mixtures of both at arated from petroleum ether extracts of neem leaves different concentrations until adult emergence, the were effective larvicides against Cx. quinquefascia- dead larvae to dead pupae ratio was shifted to a /as. The concentration of 100 ppm yielded 1007o higher number of dead pupae in the combination mortality; l0 ppm caused 6OVo mor1lality in 24 h. mixtures compared with the ratios obtained after Naqvi (1987) and Naqvi et al. (1991) tested 2 new application of each material alone. In most of the compounds and their parent fractions obtained from combinations, the resultant effect of the 2 substanc- fresh winter neem leaves against the larvae of Ae. es was the same as the value expected on the basis aegypti. The LC.os were 0.58 ppm for a neutral of the single material, which amounts to an additive fraction of winter neem leaves extracts containing action. However, in some combinations the resul- mostly nimocinolide and iso-nimocinolide and 140 JounNaL oF THE AMERTcANMoseurro Con-rnol AssocrATtoN VoL. 15,No.2 some other tetranotriterpennoids in small quantity, tion (Mwangi and Rembold 1987, 1988). A fraction 0.625 ppm for nimocinolide (CrrH.uOr, melting of the fruit kernel extract from M. volkcnsii was point 160'C), and O.74 ppm for its isomer iso-ni- tested against the larvae of An. arabiensis. The mocinolide (C2sH36O7,melting point 165"C). Thus, LCro in 48 h was 5.4 pglml. At low concentrations, it can be concluded that the fresh leaves have more this fraction had growth-inhibiting activity, pro- active ingredient than the dried leaves as used by longing the duration of larval instars, where lethal Chavan (1984). effects were exhibited during ecdysis. Further frac- Upon the availability of experimental formula- tionation of this fraction yielded 7 bands in thin- tions of AZ, Mulla et al. (1997\ tested the larvicidal layer chromatography, among which 2 of the most activity of Azad WP10, Neemix F;CO.25, and Nee- lipophilic bands showed acute toxic effects against mazad EC4.5 (Thermotrilogy Co.) against Cx. the larvae, the next 2 bands had growth inhibiting quinquefas c iatus. Tlre susceptibilities of 2nd-stage effects, and the 3 trailing bands had no activity at larvae and early and late 4th-stage larvae of this all. More work is needed to isolate and identify species to the test materials were essentially the these principles. The acute toxicity and growth-in- same. Larvicidal activity was very much a function hibiting effects of the bioactive principles were de- of the formulation; the wettable powder (WP) was stroyed by heat during the drying of the fruit 10 times more active than the emulsifiable concen- (Mwangi and Mukiama 1988). The larvicidal ef- trate (EC). The cumulative mortality (larvae, pupae, fects of acetone extracts from the seeds of M. volk- and adults) was greater than 807o when 4th instars ensii and M. azedarach were compared with pure were treated at 0.1-l ppm AZ of different formu- AZ-A for their morphogenetic effects against Cr. lations. Neem products are believed to affect the pipiens molestus. The insecticidal activities of the feeding activity of mosquito larvae, which is an ad- crude extracts were significantly different. Acetone ditional advantage of AZ formulations when used extract of M. volkensii was equally toxic for both as larvicides. Antifeedancy of 3 neem formulations larvae and pupae, with an LC.n of 30 pglml. Ace- (Azatin WP4.5, Azad EC4.5, and Neemix 8C4.5, tone extract of M. azedarach was exclusively lar- Thermotrilogy Co.) against mosquito larvae was vicidal with an LC.o of 40 pglml, and had no in- found in Culex tarsalis Coquillett and Cx. quinque- hibitory effect on the pupal stage. Like the crude fasciatus. A significant level of antifeeding activity extract from M. volkensii, pure AZ-A was also was indicated at 5 and l0 ppm AZ for all test for- equally toxic to both larvae and pupae, with an mulations. Within the test concentration range of LCro of l-5 pg/ml. The bioactive compounds from AZ (l-l0 ppm), 5 ppm was the minimum effective M. volkensii were thermostable (different from the concentration for antifeedancy expression in most results reported by Mwangi and Mukiama 1988) cases. The Cx. tarsalis larvae were more suscepti- and partially soluble in water (Al-Sharook et al. ble than were Cx. quinquefasciafas larvae to Alad 1991). A chromatographically enriched fraction EC4.5 in tenns of antifeedancy effects. Formula- from dry fruits of M. volkensii was purified from a tion-related differences in antifeedancy activity crude methanolic extract by cold precipitation and were noted when the concentration of AZ was in- elution of the precipitate dissolved in a hexane- creased to 10 ppm. At this AZ concentration, in Cx. ethyl acetate solvent system through a silica gel tarsalis, AzadEC4.5 and Neemix EC4.5 were more column. Larvae of Cx. quinquefasciatus were effective in causing antifeedancy than was Azatin reared in water containing the fraction at concen- WP4.5. ln Cx. quinquefasciatus, Azatin WP4.5 and trations of between 5 and 200 ppm. The LCro for Neemix EC4.5 were more effective than Azad this fraction was 34.72 pglml in 48 h. Second-stage EC4.5 (Su and Mulla 1998a). larvae were more susceptible to fraction B when In addition to products of A. indica, several other compared to 4th-stage larvae. All 4th-stage larvae species of Meliaceae have been investigated in the that survived the treatment molted into short-lived laboratory against mosquitoes during the past few larval-pupal intermediates (Irungu and Mwangi years. Kumar and Dutta (1987) tested the larvicidal 1995). activity of oil extracts of 10 species of plants in 8 genera against An. stephensi. Melia azadirachta FIELD TRIALS ranked 4th with an LCro of 88.5 ppm, with the range of 63.2-113.0 ppm for all tested plants. Ex- A few semifield or field tests of neem materials tracts from dry fruits of M. volkensii exhibited such as NSKEs and neem cake have been carried growth-inhibiting activity against the larvae of Ae. out in the breeding sites of culicine mosquitoes, and aegypti. At a concentration of 2 p"glml in wate! the the efficacy was reported to be promising. In a sem- active fraction prolonged the duration of the larval ifield trial using drums, Zebitz (1987) showed the stage. Mortality in treated larvae was high, espe- final concentration of 10-20 ppm of NSKEs to be cially during the molting and melanization process- effective in diminishing populations of preimaginal es. The LCro for larval mortality was 50 pglml in stages of Culex pipiens L. for at least 4 wk. Eval- 48 h. The active compound was not identical to AZ, uation of samples of larvae taken from treated and and had greater acute toxic and growth inhibiting untreated drums revealed that lst- and 2nd-stage effects ofi Ae. aegypti than the AZ-containing frac- larvae died during molting to the following larval Eppscrs oF NEEM Pnooucrs instar; most 3rd- and 4th-stage larvae died as pu- promise as an environmentally benign method for pae. rice-field mosquito control that could be self sus- Rao and his colleagues conducted field studies taining and implemented by farmers (Rao et al. using neem cake powder to control immature rice 1995). Recently, in India, application of AZ-ich field-breeding mosquitoes. Based on their studies and neem oil-based formulations either alone or on the larvicidal activity of neem cake powder in coated over urea at the time of transplantation in simulation trays planted with rice (Rao 1987), a rice paddies was shown to significantly reduce the dosage of 500 kg/ha (a very high dosage) was used density of the Culex vishnui subgroup, including in a 6,000 m2 fleld. In these tests 81 and 43Vo re- C ulex tritaenioy hync hus Glles, Culex vis hnui'fheo- ductions of late larval instars and pupae of culicine bald, Culex pseudovishnui Colless, and Culex bi- mosquitoes were attained, respectively. Subse- taeniorhynchas Giles. The neem oil-based formu- quently, Rao and Reuben (1989) carried out trials lations were also effective in reducing the pupal using urea coated with neem powder in rice fields density of Anopheles subpictus Grassi and Anoph- but failed to control immature populations of culi- eles vagus Donitz when combined with intermittent cines or to enhance rice yield because of the intrin- irrigation (Rao 1997). sic poor quality of the test materials. Therefore, Effects on adult mosquitoes: Landing and blood- Rao et al. (1992) developed a laboratory bioassay feeding repellency were tested in the laboratory and to screen neem cake powder against the larvae of field against anopheline and culicine mosquitoes, Cx. quinquefasciatus. Only samples of neem caus- but the conclusions were variable. Additionally, the ing more thar' 9OVobioassay mortality at a 2qo con- neem products might exert detrimental effects on centration within 48 h were used in field trials. the reproductive events of mosquitoes, which has When good-quality neem cake powders were ap- been tested in An. culicifocies, An- stephensi, Cx. plied at the dosage of 500 kgftta, either alone or tarsalis, Cx. quinquefosciatus, and Ae. aegypti. coated over urea, a striking reduction occurred in Landing and bloodfeeding repellency. The land- the abundance oflate larval instars and pupae. Only ing and bloodfeeding repellency effect against mos- 14 pupae were obtained over a period of 13 wk in quitoes was investigated under laboratory and field plots treated with neem cake powder, and 4 pupae conditions. Sharma et al. (1993a) reported the re- were obtained in plots treated with neem coated sults of field studies on the repellent action of neem urea, compared with 101 pupae in control plots. In oil against An. culicifacies and other anopheline another field trial, neem cake-coated urea was test- mosquitoes. Even at concentrations as low as 0.5 ed at 500 kg/ka and 250 kg/ha in combination with and l7o, strong repellent action was observed when water management practices. Both rates of appli- the material was applied to the skin. At a concen- cation yielded similar results. Larval abundance in tration of 2Vo, no anophelines bit and the protection plots under water management alone did not differ provided was lOOTo during a l2-h period. Further- significantly from the controls, but was signiflcantly more, burning neem oil in kerosene may provide reduced when water management was combined personal protection from mosquitoes (Sharma and with neem products. Two stable formulations, Ansari 1994). Kerosene lamps containing neem oil Neemrich-I (lipid rich) and Neemrich-Il (AZ rich), were burned in a living room, and mosquitoes rest- gave good suppression of immature culicines. All ing on the walls or attached to human bait were the treatments with neem also gave higher grain collected inside rooms from 180O to 060O h. Neem yield than the control. oil (0.01-1.OVo) mixed in kerosene reduced biting Considering that neem cake powder is bulky and of human volunteers and catches of mosquitoes deteriorates under improper storage conditions, resting on the walls in the rooms. Protection was which made large-scale use impractical, Rao et al. more pronounced for anopheline than for culicine (1995) field-tested relatively stable lipid-rich frac- mosquitoes. A l7o neem oil-kerosene mixture may tions of neem, which were as effective as good- provide economical personal protection from mos- quality crude neem products in the control of cu- quito bites. Considering the effective repellent ac- licine vectors of Japanese encephalitis, and tion of neem oil on mosquitoes, neem oil could be produced a slight trut significant reduction in pop- a good substitute for pyrethroids in some mosquito- ulations of anopheline pupae. Neem-based formu- repellent products such as coils and mats (Sharma lations coated over urea significantly increased et al. 1993b). However, in cage tests with female grain yield, but neem products used alone did not. Ae. aegypti, neem oil showed no repellent potency Combination of neem-coated urea and water man- (Zebitz 1987). Azadirachtin fed in bloodmeals to agement by intermittent irrigation had a greater ef- adult female Ae. aegypti through an artificial mem- fect on grain yield than that of water management brane did not cause feeding inhibition over a wide alone. The neem fractions are relatively cost-effec- dose range (0-2OO ng/female) (Ludlum and Sieber tive, and the combined water management and 1988), Different formulations and methods of usage neem-coated urea strategy is acceptable to the seem to yield different results. Equally important is farmers, who are already aware of benefits of the the species of mosquitoes, which may respond dif- use of neem-coated urea, and of water manage- ferently to neem or other related products. ment. Therefore, this technology has considerable Effects on reproduction. The application ofneem 142 JounNel or rnr Augntcnu Moseurro CoNrnol AssocrATroN Vor. 15,No.2 products and their uptake through cuticle or inges- pensions at concentrations of 0.5, l, 5, and 10 ppm tion caused detrimental effects on the reproductive AZ, oviposition attractancy was indicated at 1, 5, events in adult mosquitoes. The gonotrophic cycle and 10 ppm AZ for the WP, and repellency was of female An. stephensi and An. culicifacies was found at 5 and 10 ppm AZ for the EC in Cx. tar- impaired by exposure to volatile compounds con- salis. These 2 formulations did not have any effects tained in a neem extract. Vitellogenesis was im- on the oviposition behavior of Cx. quinquefascia- paired irreversibly by long-term exposure to neem tus. ln multiple tests for longevity, the aged sus- odor and some extracts. The effect of volatiles pensions of the WP at 0.5 and I ppm AZ were more seemed to be regulated by absorption through the attractant to Cx. tarsali,s. The attractancy of the sus- cuticle, although passage through the spiracles pensions of the WP was lost in 14- and 21-day-old (Dhar could not be excluded et al. 1996). In Ae. preparations of 0.5 and I ppm AZ, respectively. aegypti, a high dose of ingested AZ falled to inhibit The repellency of the EC suspension at 5 pprn AZ or delay oviposition. However, significant transient against Cx. tarsalis was lost in >1-day-old suspen- retardation of oocyte growth was observed for up sions. In Cx. quinquefosciatus, the oviposition re- to 72 h after feeding. Immature oocytes were ob- pellency of both the WP and the EC suspensions at served in86% of AZ-fed females decapitated 10 h l0 ppm AZ was lost in >4-day-old suspensions. after a blood meal, whereas 96Vo of decapitated Among other species of Meliaceae, gthe component control females contained maturing oocytes (Lud- from M. volkensii also has the property of ovipo- lum and Sieber 1988). This suggests that AZ delays sition deterrency against gravid mosquitoes. A the release of one or more factors from the brain chromatographically enriched fraction from that regulate oogenesis. Adult females were pro- dry posed to overcome the effect of AZ by rapid me- fruits of M. volkensii was purified from a crude tabolism rather than by excretion of the compound, methanolic extract by cold precipitation and elution because as soon as 2 h after ingestion of art AZ- of the precipitate dissolved in a hexane-ethyl ace- enriched blood meal, only O.lVo of ingested AZwas tate solvent system through a silica gel column. recovered from excreta and 5Vo was recovered from This extract was found to be an oviposition deter- the body (Ludlum and Sieber 1988). rent at a concentration of 20 ppm and greater Effects on oviposition behavior. A few studies against Cx. quinquefasciatus (Irungu and Mwangi have indicated that neem products act as an ovi- 195). position repellent or deterrent or attractant against Effects on egg viability: Egg rafts of Cx. quin- mosquitoes. For instance, neem oil containing 40O quefasciatus oviposited on water treated with 1tg AZJg was an oviposition repellent against Cx. NSKEs at concentrations of 2.5, 5,7.5, 10, and 2O quinquefasciatrs at various test concentrations. The ppm had reduced hatching ability (Zebitz 1987). dilutions of 0.125, O.25, O.5, and l7o yielded ovi- Recently, Su and Mulla (1998b) conducted a deposition activity indexes (OAIs) of -O.2727, tailed study on ovicidal activity of various formu- -0.4762, -0.9176, and -1, respectively (Zebitz lations of AZ against Cx. tarsalis and Cx. quinque- 1987); here the minus values indicated oviposition fasciatus. The formulations tested were Azad repellency. In another study, a brief exposure (90 WPIO, Azad EC4.5, and technically pure AZ. T\e min) to the volatiles from broken neem seed ker- ovicidal activity of the test neem products was in- nels, neem oil, and neem volatile fraction sup- fluenced by concentration of AZ, age of the egg pressed rather than inhibited oviposition in gravid rafts, and age of the neem preparations. Other fac- An. stephensi ard An. culicifacies. Complete inhi- tors such as formulation and mosquito species were bition of oviposition was caused by exposure of also involved in the degree of ovicidal activity. mosquitoes to odors from neem oil and one fraction When the egg rafts were deposited directly in fresh containing volatile components for I-7 days (Dhar neem suspension and left there for 4 h before trans- et al. 1996). All these neem products tested con- fer to untreated water, I ppm AZ produced almost tained numerous compounds rather than a single I$OVo mortality in eggs. When 0-, 4-,8-, l2-, and material. It will be interesting to determine which 24-h-old egg rafts were exposed to 10 ppm neem specific compounds induce the observed effects. suspensions for 36 h, ovicidal activity was attained Recently, the effects of experimental AZ formula- in the egg rafts deposited directly (0 h old) in the tions Azad WP10 and Azad EC4.5 on oviposition behavior of the mosquitoes Cx. tarsalis and Cx. neem suspension. On aging, depending on the for- quinquefasciat s were investigated by Su and Mul- mulations and mosquito species, the neem suspen- la (1999). In paired tests of distilled water vs. neem sions at 1 ppm completely lost ovicidal activity suspension, the WP showed significant oviposition within 7-20 days. The egg rafts of Cx. quinquefas- in ciatus were slightly more susceptible to the EC and attractancy Cx. tarsalis at the concentrations of 'lhe 0.5, 1, 5, and 10 ppm AZ. The repellency of the the technical AZ than those of Cx. tarsalis. EC was noted at higher concentrations of 5 or 10 formulated neem products were more persistent and ppm AZ. ln Cx. quinquefasciatus, repellency was slightly more effective than technical AZ. In Cx. only found at 10 ppm AZ of both formulations. In tarsalis, the WP had greater ovicidal activity than multiple tests using distilled water and neem sus- the EC. JuNr 1999 EFFEcrsoF NEEMPnooucrs 143

Flies weighing 15-20 mg resulting from starvation without neem treatment completed eclosion successfully A variety of neem products have been evaluated (Wilps 1987). Injection of AZ into larvae of the and found to have different types of activities blow fly led to a number of abnormal biological against both immatures and adults of several spe- effects. In a dose- and stage-dependent manner, in- cies of flies. The antifeedancy effect was shown in jected AZ caused a delay in pupariation of larvae Musca domestica L. and Phormia terraenovae L. when injected in the first one half of the last larval Insect growth regulator activity was found in Cal- instar (but no effect when injected into late instars), lip ho ra vic ina Robineau-Desvoidy, H aemato bia ir- reduction of pupal weight, and inhibition of adult ritans L., Lucilia cuprina Wied., Musca autumnalis emergence. Adults emerging from AZ-treated lar- De Geer, M. domestica, P. terraenovae, arld Sto- vae were smaller and showed various malforma- morys calcitrans L. Reproduction suppression oc- tions (Bidmon et al. 1987). curred in Glossina morsitans centralis Machado. G. Azadarachtin was also found to impact the im- m. morsitans Westwood. and M. autumnalis. and mature stages of the horn fly H. irritans, the stable oviposition inhibition occuned in L. cuprina and fly S. calcitrans, and the house fly M. domestica. Lucilia sericata Meigen. Ovicidal activity was seen When an ethanolic extract of ground up seed (2.7 in S. calcitrans and P. terraenovae, and flnally, re- mg AZJg) was blended into cow mankure, the LC.o duced biological fitness occurred in G. m. centralis, and LCeo for the larvae of horn flies were 0.096 and G. m. morsitans, Glossina pallidipes Austen, M. 0.133 ppm AZ, respectively. For the EC formula- domestica. and P. terraenovae. tion of Margosan-O (3 mg AZnD, the LCro and Activity against immature stages: Azadirachtin LCnn were 0.151 ppm and 0.268 ppm AZ in ma- and other related neem products act as IGRs to in- nure, respectively, for the larvae of this species. For hibit the development of the immature stages of larvae of stable flies. the EC formulation had an muscoid flies, even at very low concentrations. LC,n of 7.7 ppm and an LC* of 18.7 ppm AZ in When 3rd-stage larvae of the face fly M. autum- manure. Against the larvae of house flies, the LC.o nalis were exposed in 0.OOO01pglml and O.OOOO39 and LC* were 10.5 and 2O.2 ppm AZ in manure, pglml suspensions of AZ in aqueous acetone for 2O respectively. From this bioassay, among muscoid min,2.75 and ll.5vo of the larvae died, respective- flies, horn flies clearly are more susceptible than ly, and 21.4 and 52.6% of inhibition of adult for- stable flies and house flies to the test EC formula- mation was recorded, respectively (Gaaboub and tion. When the ethanolic extract was administered Hayes 1984b). Exposures for 10 or 20 min to the daily per os in gelatin capsules at the rate of0.023- AZ aqueous acetone suspensions, ranging from 0.045 mg AZlkg body weight, horn fly develop- O.fiDOl to 0.1 pglml, caused about 20-83.5Vo or ment in manure produced by treated cattle was al- 22.5-97.6Vo inhibition of adult formation, respec- most completely inhibited. The same level of tively. Doses required to inhibit the emergence of efficacy was attained under identical conditions 5OVoof the adults (ICro) were very low, O.OO241t"g/ when the EC formulation was given at >0.03 mg ml and 0.000039 pClrnl, for the l0- and 20-min AVkgbody weight or ground up seeds at >lO mg/ exposures, respectively. The effects on pupae (pro- kg body weight. However, when the ground up portion of undeveloped individuals) and adults (in- seeds were mixed with the feed and fed daily at the complete eclosion, attachment to puparia, or in- high rate of 100-400 mg seeds/kg body weight, ability to fly) were dose-dependent (Gaaboub and inhibition of development of stable flies in the ma- Hayes 1984a). nure was less than 507o (Miller and Chamberlain The addition of AZ to the culture medium of 1989). larvae of the blow fly P. tercaenovae l;.ad an anti- Regarding the mechanism of action of AZ on the feedant effect and resulted in a lower growth rate. development and growth of immature stages of Intake of this substance, at approximately 3.5 1rg/g synanthropic and synzootic flies, the key factor is fresh weight, produced larvae weighing one half as the disruption of endocrinologic events by the treat- much as controls. A higher intake of AZ was cor- ment with AZ. For example, in C. vicina, the treat- related with a further decrease in weight and an ment of larvae with AZby injection had a negative increase in mortality. Depending on the AZ quan- dose-dependent effect on the ecdysteroid content of tities consumed, the ratio of the amount of food larvae and pupae. Azadirachtin caused a delay in converted into body weight to the amount of food the appearance of ecdysteroid, which triggers pu- ingested decreased from 64Vo in controls to I6Vo in parium formation, in the larvae. The peak of the the treatment when the larvae consumed AZ at 15 hormone titer in AZ-treated larvae was lower but pglg fresh weight. Accordingly, the body weight of lasted longer. The negative effects of AZ on the the resulting pupae was reduced from 65 mg in the production of ecdysteroid (biosynthesis and re- control to 15 mg in treatment. Pupae resulting from lease) were further demonstrated in vitro with iso- t}re AZ treatment weighing <40 mg failed to com- lated brain-ring gland complex. Azadirachtin not plete development and molt to adults. The failure only diminished the rate of production of the ec- in morphogenesis in tl;ie AZ-treated animals was dysteroid but also decreased the rate of its metab- not due to the low body weight, because pupae olism. The hydroxylation of ecdysteroid at C-20 by t44 JounNnl oF THE AMERTcIN Moseurro CoNrnol AssoctATtoN Vor-. 15, No. 2 isolated fat body in vitro was decreased in a dose- various concentrations of aqueous extract and AZ- dependent manner. The changes in the concentra- enriched NSKEs between the 2nd and 4th day after tion of ecdysteriods observed in the blow fly larvae eclosion, the intake of either extract shortened life- and pupae may be the result of interference of AZ span by 4 days, reduced food ingestion and egg with several sites of the hormone-producing sys- deposition, and caused a complete loss of flying tem. Because ecdysteroids regulate eclosion, the ability. Measurement of concentrations of low mo- persisting concentration, rather than the normal lecular weight carbohydrates and glycogen in he- peaking of ecdysteroids in pupae, likely is the cause molymph showed that NSKEs-fed flies lost the of inhibition of adult eclosion (Bidmon et al. 1987). ability to alter carbohydrate concentration and mo- Evidence to further support this point is also avail- bilize glycogen content of the abdomen. However, able from the structural changes in endocrine the anabolism of glycogen was not affected (Wilps glands induced. by AZ treatment. Meurant et al. 1989). When female P. temaenovae were injected (1994) investigated the effect of AZ-A on the ul- AZ at I p.glfly, egg production was reduced by trastructure of the prothoracic gland, the corpus al- 85Vo, weight of ovaries was reduced 4OVo, andhe- latum, and the corpus cardiacum in the blow fly Z. molymph and ovary ecdysteroids were reduced 60 cuprina. When early 3rd-stage larvae were fed on and 4OVo,respectively (Wilps 1995). Research con- 50 ppm AZ-A (high concentration) in the culture ducted by Kaaya indicated that when neem oil or medium until pupation, all the endocrine glands AZ-riched NSKE was applied topically to female within the ring complex exhibited ultrastructural G. morsitans morsitans or G. m. centralis, delayed growth changes during the period of exposure. The effects larval and abortions occurred. Increased mortality in treated on the nucleus were the most noticeable, and in- females was observed in the above 2 species as well as in G. pallidipes (see cluded the crenulation of nuclear shape, clumping Wilps 1995). of heterochromatin, and pyknosis. The degenerative Neem products acted as an oviposition deterrent changes in the nuclei seemed to precede changes against some species of muscoid flies. Three per- within the cytoplasm. It was concluded that the de- cent emulsions of Margosan oil sprayed on the back generation of all 3 endocrine glands within the ring of sheep (60O mVsheep) inhibited oviposition activ- complex would contribute to a generalized disrup- ity of L. sericata for 8 days (Hobson 1940). Crude tion of neuroendocrine function and thus unsuc- neem oil (CN) and 3OVo methanolic, dewaxed sol- cessful molting (Meurant et al. 1994). vent extract (AZI) acted as a powerful antiovipos- Effects on adult Azadiachtin and other flies: ition agent for L. cuprina. The AZT threshold was neem products have "Ihe been shown to influence the O.OO24.OO5EI. with total deterrence at O.O2Vo. feeding, reproduction, oviposition, and metabolism, CN threshold was O.24.5Vo, with pure neem oil of adult muscoid flies. Feeding by adult M. domes- giving 9IVo deterrence. Tlre AZt extract is l0O- tica was deterred when one of the neem compo- 1,00O times more potent than CN, probably because nents. salannin. isolated from an ethanol extraci of its AZ content had been greatly increased (Rice et neem seeds was incorporated in sucrose at the con- al. 1985). centration of O.lVo (Warthen et al. 1978). In addition to the effects discussed above, the Residual or delayed effects of larval treatment circadian rhythm of locomotor activity was also af- on adult reproduction occurred in M. autumnalis. If fected after AZ was injected into adult M. domes- the 3rd-stage larvae were exposed to O.OOOOIp,g/ tica. TIre period was increased by 0.4 h in 83Vo of ml and O.0OOO39pg/ml solutions of AZ in aqueous the Az-treated flies, whereas in flies injected with acetone for 20 min, egg production of the resulting the solvent of AZ, ethanol, the period increased by adults was reduced by 67.2 and 85.9%orespectively, 0.3 h in 22Vo only.In 46Vo of the AZ-injected flies, when treated males mated with treated females. Re- a splitting of the activity rhythm into 2 components duction in egg hatching was also observed when with the same period was induced, whereas only untreated males or females mated with treated in- one of the ethanol controls (2Vo) showed splitting dividuals of the opposite sex. Egg hatchability was after injection (Smietanko and Engelmann 1989). decreased more than 30 and 6OVo by the lower Effects on egg viability: The ovicidal activity of (0.00001 pC/rnl) and higher (0.000039 pglml) treat- 807o was obtained when the eggs of S. calcitrans ment concentrations, respectively (Gaaboub and were transferred to the filter paper treated with Hayes 1984b). O.OlVo AZ (Wilps 1995). An oviposition substrate When administered to adults topically or orally, treated with NSKEs (l.7Vo AZ) at the rate 50 pVg neem products also showed toxicity such as mor- substrate exerted significant ovicidal activity no mattet tality, reduced biological fitness, and suppressed re- against the eggs of P. terraenovae, the eggs were laid on the treated substrate production. The LC.o of a mixture of triterpenoids whether or transferred to it thereafter (Wilps 1986, 1987;' from the petroleum ether-soluble neutral fraction of Wilps, unpublished data; see Wilps 1995). ripe neem fruits against adult M. domestica (topical application, 24 h), was 1..4 ltglfly.Inhibition of cholinesterase, acid phosphatase, and alkaline phospha- Triatomine bugs tase were 38, 45, artd 48Vo, respectively (Naqvi A number of studies have been carried out on 198D. When adults P. terraenovae were fed with triatomine bugs. Neem active agents had a variety JUNE 1999 EFFEcrs oF NEEM Pnooucrs 145

of effects such as antifeedancy, growth regulation, seemed to have blocked a structural element of AZ- immune depression, and blockage of the develop- A that was important either for absorption through ment of trypanosome parasites. Labeled [22, 23- the gut or for the antimolting effect itself. Garcia 3Hrldihydroazadirachtin A administered orally in a et al. (1986) established a dose-response relation- blood meal to Rhodnius prolixus Stal, was absorbed ship of AZ-A using molting inhibition and mortal- and transported in the hemolymph, retained, and ity as effective parameters. Azadirachtin-A, if in- then excreted in unmetabolized form through the jected l-3 days after bloodfeeding, inhibited the Malpighian tubules. The highest level of elimina- molting process. However, if injected during or af- tion of dihydroazadirachtin A was found during the ter the onset of epidemal mitosis, ecdysis was not first 12-24 h after feeding. Thereafter, a relatively affected. A single dose of AZ-A was able to block constant quantity of dihydroazadirachtin A was re- the onset of mitosis in the epidermis, which was covered from the head, visceral structures, and rest associated with the molting cycle and was triggered ofthe body 5 and 10 days after ingestion. The high- by molting hormone. Ecdysteriod titers were too est amount per milligram of tissue was found in the low for an induction of ecdysis in the AZ-A-treated Malpighian tubules and midgut, the lowest level nymphs (Garcia et al. 1987, 1991). The inhibition was in the head and the rest of the body (Garcia et of molting was fully reversed by ecdysone therapy al. 1989c). During the course of feeding, absorp- (Garcia et al. 1987). Long-term experiments with 4 tion, transportation, retention, and excretion, AZ feedings on Az-free blood after ingestion of single exerted pronounced effects on feeding behavior, de- dose of AZ were performed with 4th-stage nymphs velopment, reproduction, and development of try- and adult females. Only in the nymphs treated with panosomes, of which the triatomine bugs are a low-dose AZ at O.Ol and 0.1 pg/rnl blood, was de- transmission vector. velopment partially restored; after a single 1.0 pgl Antiftedancy.' Azadirachtin-induced antifeedan- ml blood treatment, about 5O7o of the treated cy was found in a few studies with triatomine bugs. nymphs were still alive 120 days posttreatment Given through a blood meal, the median effective without any adult emergence (Garcia et al. 1990a). dose (ED.o) for the antifeedant effect ranged from The susceptibility of triatomine bugs to AZ 25 to 3O pg/ml of blood for the 3 compounds AZ- varies from species to species. The EDro for molting A, AZ-8, and 7-acetyl-AZ. Adenosine triphosphate inhibition by injected AZ-Ain 4th-stage nymphs of as a phagostimulant to R. prolixrzs (Friend and all the test triatomines, Tiatoma vitticeps Std, fn- Smittr 1977), reversed the antifeedant action of AZ- atoma pseudomaculata Correa and Spinola, Triar- A when applied orally. Therefore, AZ-A may block oma maculata Erichson, Triatoma brasiliensis Nei- the input of phagostimulant receptors (Garcia et al. va, Triatoma lecticularis StaL Trtatoma mato- 1984). This mechanism also held true for the anti- grossensis Leite and Barbosa, Triatoma infestans feedant action of precocene (Azambuj a et al. 1982). Klug, R. prolixus, Rhodnius neglectus Lent, Rhod- However, Garcia and Rembold (1984) suggested nius robustus Larrousse, Panstrongylus megistus that feeding inhibition was an indirect effect due to Burmeister, and Panstrongylus herrera ranged from interference of AZ with the endocrine system rather lO to 25 nglnymph (Garcia et al. 1989b). This than through the inhibition of chemoreceptors. Ob- mode of administration, although of physiologic in- viously, more investigations are needed to conflrm terest, has no practical application. The contact the antifeeding mechanism. Probably both primary mode of entry will be more relevant to control ob- and secondary antifeedancy are involved n Az-lro,- jectives than injection or feeding per os. duced antifeedancy effects. Regarding the growth regulation mechanism of Growth regulntion: The pronounced IGR type of action of AZ, Garcia et d. (1990b) proposed the activity of AZ against triatomine bugs has been inhibition of PTTH synthesis and release in the well documented. Garcia and Rembold (1984) stud- treated animals. Azadirachtin-A (1.0 pglmf), if fed ied the effects of AZ administered through a blood to last-stage nymphs of R. prolixus through a blood meal on the development of 4th-stage nlrrnphs of meal, affected PTTH production, as shown by a R. prolixus. A dose-response relationship of AZ reduction or absence of ecdysteroid titers in decap- was established using ecdysis inhibition as the ef- itation experiments. The host nymphs, when decap- fective parameter. The EDro was 0.0(X)4 pglrnl itated 5 days after feeding, showed a steady decline blood for ecdysis inhibition effects. Ecdysone given of ecdysteroid titers in hemolymph, whereas un- orally at 5.0 pglrnl blood and JHA applied topically treated nymphs at this stage maintained their molt- at 7O pglinsect counteracted the AZ-induced inhi- ing hormone titer. Head transplantations from un- bition of ecdysis. Subsequently, Garcia et al. (1984) treated donors 4-5 days after feeding to the tested the IGR activity of AZ-A, AZ-B, andT-ace- headless nymphs sustained hormone production for tyl AZ-4. The EDro for molting inhibition was 0.04, about 18 h. Heads from AZ-treated donors were 0.05, and 0.45 pglml blood, respectively. The dos- unable to sustain a constant ecdysteroid level in this age for molting inhibition was much lower than that case, and levels declined immediately after trans- for antifeedant action (see above). The structure plantation. In converse experiments, head trans- change from AZ to 7-acetyl-AZ resulted in a great plantation from untreated donors 5 days after feed- loss of molting inhibition activity. This acetylation ing stimulated the production of ecdysteroids in 146 Jourulal op rHn AvenrclN Moseurro CoNrnol Assocrerrorq VoL. 15,No.2

AZ-treated recipients (40 days after AZ-A treat- (Azambuja et al. 1991, Azambuja and Garcia men0 for about 1E h. Therefore, it is suggested that 1992).However, unlike otherirffnune reactions, no the synthesis and release of PTTH was deficient in evidence was found of interference of AZ with the t}re Az-treated animals. prophenoloxidase-activating system, because mel- Reproduction suppression: Azadirachtin, like anin production was not reduced when this system precocene, is an effective inhibitor of reproduction was stimulated by trypsin or by the presence of in R. prolixu,x drastically affecting the oogenesis bacteria in the hemolymph (Azambuja et al. 1991). process and egg deposition. Precocene-induced The immune response was suggested to be deficient sterilization was reversed by application ofjuvenile in the AZ-treated insects. hormone III; however, the effect of AZ on repro- Blocking the development of trypanosomes: Gar- duction was not reversed. Ecdysteriod titers in cia and his colleagues studied the effects of neem nymphs and adult females were decreased by the treatment on the development of vector-borne path- treatments. In vitro analysis suggested that preco- ogens in triatomine bugs. Development of trypano- cene and AZ may act directly on the prothorax and somes in triatomine bugs has been blocked in a ovaries, which produce ecdysteriod (Garcia et al. number of species. 1987). Females treated per os had a lower survival Tfypanosomes are ingested by triatomine bugs, and egg deposition rate than the controls; this ac- and the trypomastigotes transform and differentiate tivity was dosage-dependent (Garcia et al. 1990a). into metacyclic trypomastigotes as the blood meal In a more detailed study (Feder et al. 1988), AZ-A moves through the midgut, which eventually ac- given orally through bloodfeeding to R. prolixus cumulates in the rectum, from where the trypomas- caused a reduction in oocyte growth and conse- tigotes are transmitted to vertebrate hosts. Not quently in egg production in a dose-dependent much is known about the interaction of the parasite manner. However, egg viability was not affected. A with its invertebrate host and the factors triggering significant correlation between these effects and the parasite development and differentiation. One such titers of both vitellogenin in the hemolymph and factor may be insect hormone titers, which change vitellin in the ovaries was observed. Ecdysteroid during the development and reproduction of the in- titers in hemolymph and ovaries, as determined by sect host (Garcia 1988). When 4th-stage nymphs radioimmunoassay, were decreased by this treat- were treated with precocene (2O pg ethoxypreco- ment. In vitro analysis suggested t}llat AZ may di- cene IVml of blood meal) or AZ (l 1t"g AZ-Nml rectly interfere with the production of ecdysteroid blood), the development of Trypanosoma cruzi in ovaries. Chagas in R. prolixus increased in precocene-treat- In a more detailed study, the administration of ed insects and decreased in AZ-treated ones. The AZ in bloodmeal (1 pglrnl) inhibited phospholipid effect of AZ could be partially reversed by ecdy- transfer to the ovaries in R. prolixzs, but it did not sone given to the bugs (Garcia 1988). In several alter tghe availability of yolk protein in the hemo- more detailed studies, the administration of a single lymph. The lipid composition of lipophorin, its dose of AZ (l.O 1t"gAZ A/ml blood meal of R. concentration in the hemolymph, and its density prolixus) was able to block the development of T. were nonnal in AZ-A-treated females. Partial in- cruzi if given through the blood meal at different hibition of phospholipid transfer was observed intervals, together with, before, or after parasite in- when lipophorin from AZ-A-treated females was fection. The same held true with different triatom- injected into normal females, or when lipophorin ine species and different strains of T. cruzi (Garcia from normal females was injected into AZ-A-treat- et al. 1989a. 1989b; Rembold and Garcia 1989; ed females. The 2 effects were additive, so that the Azambuja and Garcia 1992). A single dose of AZ- transfer of phospholipids from the lipophorin of A was enough to induce permanent resistance of AZ-A-fteated females to the oocytes of AZ-A-treat- the insect host against infection by T. cruzi (Garcia ed females was nearly eliminated. In combination, et al. 1991, Azambuja and Garcia 1992) and to these effects of AZ-A on phospholipid transfer to block ecdysis for a long time (Garcia et al. 1991). the oocytes limited the capacity of AZ-A-treated Gonzalez and Garcia (1992) carried out a long- females to produce eggs (Moreira et al. 1994). term comparative study on the effects of AZ on the Immune depression: The immune system medi- course of T. cruzi infection in different triatomine ated by the hemocytes themselves or the molecules vector species. In R. prolixus, the development of produced by them has been well documented in a T. cruzi clone Dm28c decreased in a dose-depen- number of insects. Azadirachtin (1.0 p,g/ml blood), dent manner, and the EDro of this inhibitory effect if fed to last-stage nymphs of R. prolixus, affected was 0.25 1tg AZml blood. Using this insect, an the immune reactivity, as shown by a significant experiment showed that treatment by AZ at 1.0 pgl reduction in numbers of hemocytes and nodule for- ml blood completely blocked the development of mation following challenge with Enterobacter clo- T. cruzi from 4 subsequent infective meals taken acae, a reduction in ability to produce antibacterial during the 120 days posttreatment. Similarly, com- activities in the hemolymph when injected with plete elimination of 7. cruzi in feces and urine was bacteria, and a decrease in the ability to destroy also observed over a period of 5O days after infec- infection caused by inoculation with E. cloacae tion in insects treated witlr AZ. Fifth-stage nymphs EFFECTSon Neept Pnooucrs t47 of R. prolixus, T. infestans, and Dipetalogaster ously consuming treated Lab-Chow pellets during maximus Uhlet infected with different strains of 7. a'24-wk observation period. Topical application of cruzi displayed drastic inhibition of trypanosome Margason-O to the abdomen of last-stage nymphs development when treated with AZ (Gonzalez and of B. orientalis at 2 pVnymph, or injection of this Garcia 1992). material at 0.5 pVnymph, resulted in reduction of Studies have documented that AZ does not affect growth and increased mortality. However, placing T. cruzi growth in axenic media, Az-A-treated in- lst-stage Blatta orientalis nymphs on a surface fected blood remains infectious, the compound treated with the neem extract had no notable effect does not interfere with ?" cruzi infection in mam- (Adler and Uebel 1985). The LDro of a neutral frac- malian hosts, and ecdysone and juvenile hormone tion of winter neem leaves (mixture of triterpe- in vitro do not interfere directly with the develop- noids) against 4th-stage nymphs of B. germanica ment of T. cruzi. Thus, the direct effect of these was 0.5 pglindividual. This treatment caused inhi- compounds on T. cruzi could be excluded. There- bition of cholinesterase (39Vo), of acid phosphatase fore, it is suggested that the extensive physiologic (62Vo), and of alkaline phosphotase (74Vo) (Naqvi and biochemical changes in the neuroendocrine 1987). system and gut have been suggested to create a mi- Recently, Prabhakaran and Kamble (1996) stud- croenvfuonment that was no longer suitable for the ied the toxicity of technical grade AZ (ll.46%o AI) survival and development of trypanosomes, and re- against insecticide-resistant and -susceptible strains sulted in failure of infection in vector (Garcia 1988, of B. gerrnanicaby itjection and diet incorporation. Garcia et al. t992). Further studies are needed as The effect of AZ was dose-dependent. The LDro to how neem or other products might be used in and LDno for 3 strains ranged between 2.49 and blocking trypanosome growth and reproduction in 2.56 1tg per insect and 6.49 and 6.92 pg per insect, triatomine vectors in a practical mtrnner. respectively. Incorporation of AZ in food pellets resulted in median lethal times (LTrus) of 22--23 days for different strains. No significant differences Cockroaches were observed in toxicity of AZ to either insecti- Antifeedancy, IGR type of activity, and reduced cide-resistant or -susceptible strains. The body biological fitness have been shown in several spe- weight of test animals varied with time depending cies of cockroaches through oral or topical admin- upon the dosage administered. The cockroaches in- istration of AZ or conrmercial formulations. Qadri jected with 2 arrd 3 1tg of AZ showed a continuous and Narsaiah (1978) studied the effects of AZ on weight reduction. The toxicity of AZ to the cock- the molting processes of last-stage nymphs of Peri- roach may be associated with altered feeding be- planeta americana L. The 24-hLDso of AZ for last- havior and disruption of hormonal events. stage male and female nymphs was 1.5 mg/g body mg/g body weight de- weight. Azadirachtin at O.75 Fleas layed molting from the 23rdto the 38th day without a toxic effect. A positive correlation existed be- Fairly good results were achieved in flea control tween the concentration of Az and histopathologic by neem products. Fourth-stage larvae of labora- changes in hemocytes and levels of hemolymph tory-reared Ctenoc ephalides /e/ls Bouche and Xen- proteins, cholesterol, and nucleic acids. Disruptive opsylla brasiliensis Baker were exposed to 50Vo and vacuolated changes were characterized both in NSKEs incorporated into the culture medium for cytoplasmic and nuclear regions of hemocytes. Re- periods varying from 30 min to 24 h. Monality of ductions as much as 2O-25Vo were observed in the larvae was observed for exposure periods of 2 h hemocyte population 24 h after injection; a 25- and longer in both species. At 24 h of exposure, 4OVo reduction in albumin and globulin levels oc- 95.3 and 94JVo of C. felis and X. brasiliensis, te- cvfied I-24 days after injection; a l5-45%o reduc- spectively, were killed. The LTros were approxi- tion was found in cholesterol levels: and a 1-lOVo mately 6 h and 10.5 h for C. felis and X. brasilien- reduction in RNA and DNA levels in hemolymph sis, respectively. It was generally concluded that occurred 1-6 days after injection. Margosan-O, a NSKEs were potentially larvicidal against the 2 flea commercial preparation of 4O% neem seed extract species and that NSKEs could be effectively used in 95Vo ethanol (34O p"g AZ/rml), was tested for its for controlling the insects by properly dusting the effects as a repellent, toxicant, or growth inhibitor breeding sites with appropriate concentrations of against 6 species of cockroach: Blatta orientalis L., the extract (Kilonzo 1991). Chamberlain et al. Blattella germanica L., Byrsotria fumigata Guerin- (1988) tested 31 different compounds against lar- Meneville, Gromphadorhina protentosa Schaum, vae of the oriental rat flea, XenopsylLa cheopis P. americana, and Supella longipalpa F Last-stage Rothschild. These 3l compounds represented 6 nymphs of these species fed on Lab-Chow pellets chemical groups: organophosphates, carbamates, impregnated with neem extract at a rate of 0.5 mU pyrethroids, diphenylureas, dodecadienoates, and pellet had increased mortality and retarded devel- sulfonates, plus a miscellaneous group including opment. All lst-stage nymphs of B. orientalis, B. AZ. The inhibitory effect IE.o dose of AZ was as germanica, ard S. longipalpa died after continu- low as 0.31 ppm. 148 JounNeL oF THE AMERIcet Mosquno Cor.rrRor- Assocrnnot VoL. 15,No.2

Lice by the neem tree is being cultured and promoted in tropical and subtropical areas of the world in re- Azadirachtin was also tested to control the sheep forestation programs as source of wood, fertllizer, biting louse Bovicola ovls Schrank. A variety of cattle feed, oil, shade, medicinals, and a source of insecticidal substances (considered acceptable by environmentally friendly insecticides for worldwide Organic Production Standards) (New Zealand) such use (Koul et al. 1990, Mordue and Blackwell 1993, as AZ, pyrethrum, and soap, were applied to louse- Schmutterer 1995). Azadirachtin and other bioac- infested sheep and their efficacies compared with tive compounds from neem extracts can exert mul- that of a conrmercial formulation of cypermethrin. tiple actions affecting feeding, growth and devel- The sheep treated witlr AZ and pyrethrum had sig- opment, reproduction, or even the development of niflcantly fewer lice than either the control or soap- vector-borne pathogens in their vectors. Neem treated sheep over the 48-day period of the trial. products are degradable in the environment and are Both AZ and pyrethrum were as effective as cy- relatively safe to nontarget organisms. No cases of permethrin. Control (reduction in louse score) of resistance to neem products have been reported. 85.O-lNVo was achieved over the period of the Lack of resistance is due to the complexity of the trial (Heath et al. 1995). contained components and multiple modes of action in insects. Equally important, the neem tree is Sand flies adapted to vast areas in tropical and subtropical countries. This tree and its products provide an The landing and bloodfeeding repellent action of abundant resource for the manufacturing of a va- neem oil applied to human skin was evaluated riety of products having industrial, medicinal, and against sand flies under laboratory and field con- agricultural applications. The costs of planting and ditions. Concentrations of ZVo neem oil mixed in managing neem trees and producing neem insecti- coconut or mustard oil provided l0o7o protection cides are relatively low. against Phlebotomus argentipes Ann and Brun The production and application of neem insecti- ttroughout the night under field conditions. Under cides have several drawbacks. First, even though laboratory conditions, 2 artd 5Vo neem oil provided considerable effort has been made to synthesize the lNVo protection from the bites of Phlebotomus pa- predominant insecticidal component in neem prod- patasi '+ Scopoli up to a mean of 7 h 20 lm:rra 34 ucts, we are still a long way from achieving eco- + min and 7 h 54 min 25 min, respectively (Sharma nomic synthesis, because the chemical structure of and Dhiman 1993). AZ is too complicated. To date, the manufacturing of neem products is still dependent on the natural Ticks resources that are renewable, thus providing for a sustainable source of the bioactive agents at a rel- Little work has been done on the activity of atively modest cost. Fortunately, the research by neem products against hard and soft ticks. In a few Allan et ^1. (1994) and Wewetzer (1998) has shown studies, the relationship between salivary gland de- that callus from leaves of the neem tree produce generation and levels of ecdysteroids as well as ef- natural AZ when grown in defined media. Second, fects of AZ on this relationship were elucidated in neem products, like many other natural products, ixodid ticks. Injection of the ecdysteroid inhibitor show a limited spectrum of persistence under field AZ (up to lOO pglg body weight) into engorged conditions because of UV light, temperature, pH, female Amblyomma americanum L. inhibited the and microbial degradation. The UV absorber, 2,4- onset of oviposition by a few days and the rate of dihydroxybenzophenone added to AZ-A at a con- oviposition during the next 6 days, but did not sig- centration of l:1 (dw) could increase the halflife nificantly inhibit vitellogenesis or reduce total egg of AZ-A (Sundararn 1996). Finally, the delayed ef- production. The salivary gland degeneration prob- fects and relatively low degree of efficacy (if not ably caused by ecdysteroid could not be prevented applied in high doses) of neem products may irri- by the application of AZ, whicll implied that ticks tate users accustomed to synthetic insecticides with possess at least one Az-insensitive ecdysteriod sys- a broad-spectrum of activity and strong contact ef- tem (Kaufman 1988, Lindsay and Kaufman 1988). ficacy. Compared with the amount of effort and application technology of neem products in agricultural CONCLUSIONS pest control, the developmental effort in medical Considerable information has been gathered dur- and veterinary entomology is meager. Regarding ing recent years on the insecticidal properties of the activity and application of neem products, es- botanical pesticides, especially neem products. The pecially of AZ against mosquitoes, flies, and other "village pharmacy" philosophy of use of neem insects of medical and veterinary importance, fur- products in India and elsewhere where they have ther research is needed on the practical use of AZ been used for its antiseptic, medicinal, and insec- products in disease vector control programs. Stud- ticidal properties for many years, has now devel- ies are necessary to determine effects on feeding "neem oped into the technology" approach where- behavior; the mechanism of growth regulation, that JuNB 1999 EFFECTS oF NEEM PRODUCTS 149 is, the interference of AZ and related products with Proc. 3rd Int. Neem Conf. (Nairobi, Kenya, 1986). ecdysone and juvenile hormone levels; effects on GTZ, Eschborn, Germany. Butterworth, J. H. and E. D. Morgan. 1968. Isolation of reproduction including spermatogenesis, mating, a substance that suppresses feeding in locusts. Chem. vitellogenesis, oogenesis, oviposition, and the Commtn.23--24. hatching ability of the eggs; effects on metabolism; Campbell, F, L., W. W. Sullivan and L. N. Smith. 1933. effects on aspects of biological fitness; effects on The relative toxicity of nicotine, anabasine, methyl the development of vector-borne pathogens (which anabasine and lupinine for culicine mosquito larvae. J. only has been studied in triatomine bugs); and the Econ. Entomol. 26:500-5O9. possibility of resistance development. Detailed lab- Chamberlain, W. F, J. Maciejewska and J. J. Matter. 1988. oratory and field studies on a variety of neem prod- Response of the larvae and pupae of the oriental rat flea ucts, especially those that are commercially avail- (Siphonaptera: Pulicidae) to chemicals of different types. J. Econ. Entomol. 8l:142O-1425. able and developed for agricultural pest control, chemical Chapman, R. F 1974. The chemical inhibition of feeding will facilitate the practical use of AZ applications by phytophagous insects: a review. Bull. Entomol. Res. for the control of arthropods of medical and vet- 64:339-363. erinary importance. Chavan, S. R. 1984. Chemistry of alkanes separated from leaves of Azadirachta indica and their larvicidaVinsec- ticidal activity against mosquitoes, pp. 59-65. In: H. RETERENCES CITED Schmutterer and K. R. S. Ascher (eds.). Proc. 2nd Int. Adler, V. E. and E. C. Uebel. 1985. Effects of a formu- Neem Conf. (Rauischholzhausen, Germany, 1983). lation of neem extract on six species of cockroaches GTZ, Eschborn, Germany. (Orthoptera: Blaberidae, Blattidae and Blattellidae). Chavan, S. R. and S. T. Nikam. 1988. Investigation of Phytoparasitica 13:3-8. alkanes from neem leaves and their mosquito larvicidal Ahmed, S., M. Grainge, J. W. Hylin, W. C. Mitchell and activity. Pesticides 22(7):32-33. J. A. Litsinger. 1984. Some promising plant species for Chavan, S. R., P B. 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