ACTIVITY AND BIOLOGICAL EFFECTS OF NEEM PRODUCTS AGAINST ARTHROPODS OF MEDICAL AND VETERINARY IMPORTANCE
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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 start-
ed 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 in-
secticides 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
133t34 JounNer- op tHe AN4BRrcnNMosguno CoNTRor- AssocrnrtoN V o L . 1 5 ,N o . 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 and persistence
Commercial formulations
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 in- dia), 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 fit-
ness, and blocking of the development of vector-
Effects on nontarget borne pathogens. Information on these aspects of
organisms
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
as honeybees, natural enemies of pests, and so Mosquitoes
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 oil extractive," a
resistance to neem products it P. rylostella did not Prasad (1980) tested the
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.OOSVo of 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 brasi-
liensis138 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 Chamberlain et 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, vitello-
genesis impaired
An. stephensi Adult Antioviposition, GC Neem oil, volatiles Dhar et al. (1996)
impaired, vitello-
genesis 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 ( r988)
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 see Wilps (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 and140 JounNaL oF THE AMERTcANMoseurro Con-rnol AssocrATtoN V o L . 1 5 ,N o . 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 larvalEppscrs 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 ofneem142 JounNel or rnr Augntcnu Moseurro CoNrnol AssocrATroN V o r . 1 5 ,N o . 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
could not be excluded (Dhar 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
the release of one or more factors from the brain sition deterrency against gravid mosquitoes. A
that regulate oogenesis. Adult females were pro- chromatographically enriched fraction from 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 de-
position 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-
tions Azad WP10 and Azad EC4.5 on oviposition in the egg rafts deposited directly (0 h old) in the
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-
attractancy in Cx. tarsalis at the concentrations of ciatus were slightly more susceptible to the EC and
'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 with-
out 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 weighingYou can also read
