0022-1910/93 $24.00 + 0.

00
J. Insect Phy siol. Vol. 39, No.11, pp. 903- 924, 1993
Printed in Great Britain. All rights reserved Copyright 0 1993Pergamon Press Ltd

REVIEW

Azadirachtin: an Update
A. J. MORDUE (LUNTZ),**t A. BLACKWELL* zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
Received 12 May 1993 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

Research into the insecticidal effects of azadirachtin, a lhuonoid from the Indian Neem tree,
&a&~ kta in&u, has been ongoing for some 30 years. Its strong antifeedant, insect growth
regulatory and reproductive effects are now well understood and documented although its biochemical
effects at the cellular level are still unknown. Antifeedaucy varies markedly between species with
Lepidoptera being particularly sensitive to azadirachtin. The physiological elfects on growth, moultiug
and reproduction are more consistent between species although cuticle or gut may provide barriers to
bioavailability in some species. The mode of action of azadirachtin lies in (i) effects on deterrent and
other cbemoreceptors resulting in antifeedancy (ii) effects on ecdysteroid and juvenile hormone titres
through a blockage of morphogenetic peptide hormone release (e.g. PTTH, allatotropins) and (iii)
direct effects on most other tissues studied resulting in an overall loss of fitness of the insect. The
complexity of the molecular structure of azadiiachtin has precluded its synthesis for pesticide use
although novel synthesis of the parent molecule is now almost complete and research iato simpler
mimetic substances is ongoing. Applied research has concentrated on a variety of natural formulations
from neem seed kernels which contain azadirachtin together with several structurally related
molecules. This review brings the reader up to date with both pure and applied research in the field
and provides a detailed overview of present thinking into the mode of action of azadirachtin. Wherever
possible comparative approaches have been made between species of the effects of pure azadirachtin
and areas for future research are indicated.

Azadirachtin Neem Pest control Antifeedancy IGR effects Reproduction Mode of action

INTRODUCTION (Rembold, 1989), is the major component of neem seeds.

Azadirachtin is a tetranortriterpenoid plant limonoid
with potent insect antifeedant and growth disrupting
properties. It was isolated from the seeds of Azadirachta
indica by Butterworth and Morgan (1968) and its full
structural determination was completed some 17 years
later. Nakanishi and co-workers presented the first struc-
ture proposal (Zanno et al., 1975), the correct structure
appeared in papers submitted in 1985 by Broughton
et al. and Kraus et al. (Kraus et al., 1985; Broughton
et al., 1986), and the full details were finally described
by all groups in 1987 (Bilton et al., 1987; Kraus et al.,
1987a; Turner et al., 1987). Azadirachtin is a highly
oxidized limonoid with many reactive functional groups
in close proximity to each other. Its biosynthesis is
thought to involve tirucallol, a tetracyclic triterpenoid,
and a series of oxidation and rearrangement reactions
which produce finally, amongst others, the tetranor-
triterpenoids salannin, nimbin and azadirachtin (Ley
et al., 1993). Azadirachtin, also termed Azadirachtin A
*Departmentof Zoology, University of Aberdeen, Tillydrone Avenue,
Aberdeen AB9 2TN, U.K.
tTo whom all correspondence should be addressed.
903
3-Tigloylazadirachtol (azadirachtin B) is present at con-
centrations up to 20% of that of azadirachtin, other
azadirachtins (C-I) occur at much lower concentrations.
For a recent comprehensive review of the chemistry of
azadirachtin see Ley et al. (1993).
A large amount of data is available detailing both
feeding deterrency and the growth disruptive properties
of azadirachtin and neem formulations to numerous
species and stages of insects of many orders: see the three
International Neem Conferences (Eds Schmutterer et al.,
1981; Schmutterer and Ascher, 1984, 1987); two Ameri-
can Conferences (Eds Locke and Lawson, 1990; Ed.
Ahmed, 1993) and other reviews (Jacobson, 1986;
Schmutterer, 1988; Jacobson, 1988; Arnasan et al., 1989;
Warthen, 1989; Schmutterer, 1990; Subrahmanyam,
1990; National Research Council, U.S.A., 1992; Ascher,
1993).
Recent advances in azadirachtin research relate to new
field trial data, using commercial and semi-commercial
preparations of neem, against phytophagous insects and
their associated beneficials, investigations against stored
product pests and also insect vectors of disease. An
increased understanding of the chemistry of azadir-
achtins and their natural and synthetic analogues is
904 REVIEW

leading to important studies on structure: activity re- Introduction). More recent work, relates to new appli-
lationships and a breakthrough in synthesis of the parent cations of azadirachtin in the management of pests of
molecule (Ley zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
et al., 1993). Studies on the mode of action ornamental crops (Price et al., 1990), both contact and
of azadirachtin are lagging behind and while many of its systemic action of the neem-based insecticide Margosan-
physiological effects are now understood its biochemical O@ (0.3% azadirachtin, 14% neem oil) on the spiny
mode of action is still entirely unknown. bollworm, Eurias insuluna and the leafhopper, Assym-
This review, therefore, primarily deals with pure aza- metrasca decedens (Meisner et al., 1990, 1992), signifi-
dirachtin, or neem formulations of known azadirachtin cant IGR effects of low topical doses of Margosan-O@
content, with a view to quantifying and comparing to Spodoptera littoralis larvae (Meisner and Nemny,
across pest species the main effects of the parent 1992), a sexual dimorphism in susceptibility to neem
molecule azadirachtin, and to analyse its possible mode extracts of the Colorado potato beetle, Leptinotarsu
of action. decemlineata (Kaethner, 1992) and varying susceptibility
of nymphal instars of Aphis fabae to Margosan-O@
(Dimetry and Schmidt, 1992).
INSECT CONTROL
The limitations of compounds such as azadirachtin for
The first commercial product of neem, Margosan-O@ field use were reviewed by Schmutterer (1988), and
(W. R. Grace & Co., Cambridge, MA, U.S.A.) was include its inapplicability for crops such as fruit and
registered in the U.S.A. in 1985. Several commercial and vegetables where any pest damage is unacceptable and
semi-commercial preparations are now available includ- its rapid degradation, when topically applied, giving a
ing Azatin (Agridyne Technologies, Salt Lake City, UT, residual effect of usually only 4-8 days. For field appli-
U.S.A.); Bioneem and Neemesis (Ringer Corp., cations, careful consideration is required regarding a
Minneapolis, MN, U.S.A.); Safer’s EN1 (Safer Ltd, compound’s stability in the particular climatic con-
Victoria, B.C., Canada, now incorporated into Ringer ditions and any steps necessary for its stabilization
Corp.); Wellgro and RD-Repelin (ITC Ltd, Andhra should be taken. For example, the antifeedant potency
Pradesh, India); Neemguard (Gharda Chemicals, of azadirachtin exposed to sunlight for 7 days was
Bombay, India); Neemark (West Coast Herbochem, reduced by more than half compared with non-exposed
Bombay, India) and Neemazal (Trifolio M GmBH, azadirachtin against S. frugiperdu first instars, with no
D-6335 Lahnau 2, Germany). Neem seed oil is often a activity remaining after 16 days (Stokes and Redfem,
starting material for such insecticides and its biological 1982). However, even though breakdown of azadirachtin
activity is closely related to its azadirachtin content does occur in U.V. light, the resulting metabolites may
(Isman et al., 1990). However with the different formu- still have biological activity (Barnby et al., 1989a). In
lations of neem used and the many varied modes of recent years, successful control has been achieved in the
application, detailed comparisons of efficacy against field, employing careful selection of particular solvents
different pest species are extremely difficult to make. For for the formulation of field applications. Present formu-
example, limonoid mixtures may be more effective than lations include emulsifiable concentrates (ECs), suspen-
azadirachtin alone; neem oil itself has insecticidal prop- sion concentrates (SCs), ultra low volume (ULV)
erties unrelated to its azadirachtin content and crude formulations (Feuerhake and Schmutterer, 1985) and
formulations contain volatile repellent components. In granular formulations (Saxena, 1989). Systemic appli-
the near future commercial products will consist of neem cations also have been particularly successful, giving
seed formulations containing mixtures of compounds. control for several weeks (Knodel et al., 1986).
However the synthesis of azadirachtin or more impor- Cotton crops have received particular attention (e.g.
tantly simpler mimetics (Ley et al., 1993) and its pro- Phadke et al., 1988; Flint and Parks, 1989), together with
duction by other means such as tissue culture (e.g. corn (Klocke and Bamby, 1989), potato (Kaethner,
Kearney et al., 1993) are avenues which are already been 1992) and more diverse crops such as chrysanthemums
explored and which may yield more easily quantifiable (Knodel et al., 1986) and Mahogany (Howard, 1990).
end products. Laboratory trials have been replicated in the field (e.g.
Kaethner, 1992), with azadirachtin and neem extracts
I. Phytophagous insects often showing superiority to certain commercially avail-
The insecticidal performance of neem products has able pesticides (Kirsch and Schmutterer, 1988) and there
been assessed in terms of both antifeedancy and insect have been promising results with some groups of pests
growth regulatory (IGR) effects. Direct toxicity, involv- which are developing resistance rapidly against conven-
ing both the previous parameters and rapid mortalities tional pesticides, including Diubroticu spp. beetles
has also been measured. The predominant effect in a (Hummel, 1989). Work comparing pure azadirachtin
species often varies with dose. There are numerous with neem preparations from Trifolio-M GmbH against
examples of laboratory and greenhouse studies of the lepidopterous pests of cabbages in Scotland, showed
pest control potential of neem and its major active azadirachtin to be as effective as Neemazal-F at the same
constituent, azadirachtin. Lepidoptera and other phyto- concentration of active ingredient (50 p.p.m., 4OOl/ha)
phagous insects remain the main targets, and the although control was not as great as that achieved by
reader is referred to the relevant reviews for details (see Cypermethrin [Mordue (Luntz) et al., 19931, however, in
 REVIEW 905

Canada equivalent pest control to that achieved by and biting midge larvae and adults in a littoral environ-
pyrethrum has been demonstrated (Isman et zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
al., 1991). ment. Molluscs, but not crustaceans were also signifi-
Hence, azadirachtin as an antifeedant has potential cantly affected (Schriider, 1992). However, ostracod
for pest management but for its full potential to be crustacea did show growth and moulting abnormalities
realized in the field careful formulation is required. after exposure to neem seed kernel extracts (NSKE) in
Incorporation into neem oil gives a degree of protection the laboratory and in rice paddies (Grant and Schmut-
of the azadirachtin molecule (Stokes and Redfern, 1982). terer, 1987).
Alternately, modifications of the azadirachtin molecule
may significantly increase its stability. For example, 4. Other organisms
hydrogenation of the C-22,23 double bond of the di- Azadirachtin, apart from its unique mode of action
hydrofuran ring yields dihydroazadirachtin, a more against insects, can also affect other organisms including
stable compound with greater potential for field use nematodes, fungi, viruses and protozoa.
(Broughton et al., 1986; Yamasaki and Klocke, 1987). A variety of plant parasitic nematodes are reported to
This compound is as active as azadirachtin in feeding be affected by neem products. For example, root dipping
trials with four economically important lepidopteran and seed treatment with azadirachtin, neem leaf extract
species (Blaney et al., 1990) and with Epilachna uarivestis and neem oil prevented larval M eloidogy ne incognita
(Rembold, 1989) and Locusta migratoria (Nasiruddin, from causing root-knot infections in tomato, eggplant
unpublished). It is, however, significantly less active than and okra (Siddiqui and Mashkoor, 1988; Abid and
azadirachtin against the locust Schistocerca gregaria Maqbool, 1991; Pradhan et al., 1991). Of a range of local
[Nasiruddin and Mordue (Luntz), 1993b]. tree seeds evaluated for nematicidal control in India
A. indica (as neem cake incorporated into the soil) gave
2. Stored product pests the best control of parasitic nematodes of subabul
Interesting developments have been made also with (Leucaena leucocephala) (Azmi, 1990). However, this
other pests. Neem has proved to be effective in protect- treatment was only effective against nematodes infesting
ing stored products, particularly grain, whose losses if lentil when combined with synthetic insecticides (Gaur
untreated can be high. Such losses are frequent in and Mishra, 1990). Interestingly, the residual effect of
developing countries due to the inability to apply ex- soil treatments for nematicidal control lasts for a number
pensive chemical pesticides. Effects on stored product of months, with the yield of subsequent crops in the
pests include antifeedancy, oviposition, reduced egg same plots being noticeably increased (Siddiqui and
hatch and emergence, and direct lethality (Morallo- Mashkoor, 1991). Also of significance, in countries such
Rejesus et al., 1990; Ivbijaro, 1990; Naqvi et al., 1990). as India with both winter and summer growing seasons
The protection of neem may persist for a number of and where soil conditions can vary drastically between
months, although this varies between insect species, areas, neem’s efficacy was consistent all-year round, in
perhaps related to the insects’ behaviour (Makanjuola, all soil conditions (Alam, 1991).
1989). Seed damage is not always reduced to the same There are numerous instances of the effects of
degree as with various synthetic insecticides (Sehgal and azadirachtin and neem on fungal pathogens, including
Ujagir, 1990), however, a clear benefit of neem is that the inhibition of spore germination and mycelial growth
subsequent germination of stored seed is not impaired by of Helminthosporium nodulosum and Py ricularia grisea
treatment (Gupta et al., 1989). Most laboratory trials on finger millet, with acetone extracts of neem being
have involved crude neem extracts but promising long more effective than water extracts (Jagannathan and
term effects of Margosan-O’@ have also been demon- Narasimham, 1988). Paddy (Ory za satiua) also suffers
strated against Rhy zopertha dominica, suggesting that it from fungal attack and a neem seed extract significantly
should be evaluated in the field (Jilani and Saxena, reduced the infection of 0. sativa seeds by Trichoconiella
1990). More diverse effects of neem, other than those on padwickii (Shetty et al., 1989). In addition the infection
grain, include the preservation of (and associated in- of barley by a foot-rot pathogen, Sclerotium rolfsii has
creased aesthetic attractiveness and therefore market- been reduced to 8% by neem oil (Singh and Dwivedi,
ability) of dried fish in Nigeria (Okorie et al., 1990). 1990). Finally, certain fruit rots can cause huge econ-
omic losses and their control by neem extracts in the
3. Aquatic arthropods laboratory was very promising (Arya, 1988), advocating
Azadirachtin is potentially useful in the aquatic en- the development of field trials in affected areas. Neem,
vironment, but further work is required to establish its however, is not universally effective against fungi: Khan
stability and half-life in water. Mosquitoes have been the et al. (1988) could show no effects of dried neem
primary subjects of laboratory trials, demonstrating materials on 14 common pathogenic fungi (dermato-
toxic effects of neem seed extracts and pure azadirachtin phytes, yeasts and moulds).
(Zebitz, 1987; Al-Sharook et al., 1991) and of two new In addition to nematicidal and fungicidal properties,
compounds isolated from winter neem leaves; isonimo- neem products can exhibit significant antiviral activity,
cinolide and nimocinolide, which also had IGR effects, e.g. in greenhouse trials against tobacco mosaic virus
resulting in abnormal larvae and pupae (Naqvi et al., (Mishra and Rao, 1988) and cowpea mosaic virus (Singh
1991). A Neemazal-F preparation killed both mosquito- et al., 1988). Nimbin and nimbidin have also been shown
906 REVIEW

to inhibit potato virus X (PVX) (Verma, 1974). Antiviral (Mansour et al., 1986), with one of its prey, Tetrany chus
activity of azadirachtin was not demonstrated, however, cinnabarinus being highly susceptible to neem (Mansour
against potato leafroll virus PLRV or potato virus Y and Ascher, 1983). Another example of azadirachtin’s
(PVY) in tobacco seedlings (Nisbet, 1992). As far as promise for IPM programmes involves the predacious
antiprotozoan activity is concerned azadirachtin inhib- mite Phy toseiulus persimilis which preys only on phyto-
ited microfilarial release from zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
Brugia pahangi without phagous spider mites, including T. cinnabarinus and is
affecting the host’s motility or viability (Barker et al., currently used for biocontrol programmes in green-
1989). A. indica may also have antimalarial activity, the houses and on outdoor crops in the U.S.A. and Israel.
most active constituent identified is gedunin (Khalid P. persimilis is susceptible to many insecticides but
et al., 1989). Bray et al. (1990), however, could detect neem-seed extracts (NSE), in laboratory trials at least,
only weak antiplasmodial activity of gedunin and were up to 58 times more toxic to T. cinnabarinus than
suggested that the claimed effectiveness against malaria, to its predator and had only minimal effects on the
might be due more to the anti-inflammatory and predator’s fecundity (Mansour et al., 1987).
immunomodulating activities of the neem plant. Adverse effects of azadirachtin against beneficial or-
Examples of azadirachtin’s impact on parasites in ganisms cannot be discounted, however, as shown by
insects are numerous and include protozoan species of suppression of ecdysis and 100% mortality of Cotesia
Plasmodium, Entomoeba, Leishmania and Try panosoma congregata, an hymenopteran parasitoid of M anduca
(Phillipson and O’Neil, 1989). Azadirachtin, for sexta, following injection of 10 pg azadirachtin into
example, inhibits Try panosoma infection of triatomid newly ecdysed fourth or fifth instar host larvae; i.e. prior
insects, including Bhodnius prolixus which transmits to the first larval ecdysis of the wasps. Later injections
T. cruzi, causing Chagas’ disease (Rembold and Garcia, of azadirachtin did not, however, -interfere with ecdysis
1989) and with low doses (1 ,ug/ml blood), remaining of second instar parasitoids (Beckage et al., 1988). Also,
functions of the host appear unaffected (Garcia et al., treating the Colorado potato beetle, L. decemlineata
1989b) (see also “Mode of Action”). with NSE resulted in moulting delays and deformities in
its predator, Perilly s bioculatus (Hough-Goldstein and
5. Beneficials Keil, 1991) and NSE reduced the numbers of Encarsia
Ufider laboratory conditions, low doses of azadirach- spp. and Aleurodiphiulus spp. parasitoids of the sweet-
tin (10bnd 20 ppm) did not harm the hymenopteran potato whitefly, Bemisia tabaci, a major pest species for
parasitoid Apanteles glomeratus but its host, final instar which azadirachtin shows significant promise as a con-
Pieris brassicae larvae, showed reduced feeding followed trol agent (Price and Schuster, 1991). However, despite
by a gradual death (Schmutterer, 1992). The parasitoid the above effects on whitefly hymenopteran parasites,
is killed however, when younger P. brassicae larval azadirachtin was non-toxic to Delphastus pusillus, a
instars are treated, as would normally be the case in the coccinellid predator of the same species when either the
field. Hence Schmutterer (1992) suggests that in an IPM plant or B. tabaci eggs were treated (Hoelmer et al.,
programme it would be better to delay the spraying of 1990).
neem extracts until the later larval stages appear, NSE was not completely harmless to bees, but serious
although there is then the risk that the larger caterpillars damage to them in the field appears unlikely if spraying
may inflict unacceptable levels of crop damage. is carried out prior to flowering, thus allowing the use of
The effects of azadirachtin and a neem-seed extract neem on pollen- and nectar-producing plants (Schmut-
(NSE) on tephritid fruit flies and their parasitoids have terer and Holst, 1987).
shown that azadirachtin is selective against fruit flies and The data presented above are derived mostly from
prevents them from either emerging to the adult stage, laboratory- and greenhouse-studies but do indicate that
or else reduces the survival of those adults which do extensive testing of azadirachtin for IPM programmes
emerge. Low doses of NSE (ca 10 ppm a.i.), allow the with some of the world’s primary crops (cotton, cereals,
hymenopteran parasitoids to emerge unharmed and able soybean, etc.), is worthwhile but as always caution must
to mate and seek new fruit fly hosts, so enhancing the be observed. In addition to overcoming some of the
effectiveness of biological control (Stark et al., 1990a,b, problems associated with azadirachtin’s relative instabil-
1992). Considering the economic importance of fruit flies ity in the field, further studies are required to examine
as agricultural pests and the repeated failure of eradica- any long-term effects on the behaviour and fitness of
tion attempts, these findings suggest that azadirachtin natural enemies following azadirachtin treatment, in
may prove valuable as a soil treatment, as one com- terms of their effectiveness as predators and parasitoids
ponent of a fruit fly IPM programme. The biochemical and on the development of their offspring (Hoelmer
mechanisms responsible for the differential suscepti- et al., 1990). zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONML
bilities of parasite and pest are yet to be determined.
Spiders and mites play important roles in the suppres- EFFECTS OF AZADIRACHTIN
sion of insect pests in certain agroecosystems and there
are many examples of neem’s lack of toxicity against 1. Antifeedancy
them after treatment of the crop. These include Chira- Azadirachtin is a classic example of a natural plant
canthium milaki, a useful predator of citrus fruit pests defence chemical affecting feeding, primarily through
 REVIEW 907

chemoreception (primary antifeedancy) but also through
a reduction in food intake due to toxic effects if con-
sumed (secondary antifeedancy, see below). From initial
observations of its antifeedant effects it was a natural
progression to test azadirachtin and azadirachtin-
containing compounds for commercial use in pest con-
trol. A variety of preparations have been applied to
assess azadirachtin’s antifeedant activity, from crude to
refined neem extracts (water or oil extracted) to neem
enriched extracts to pure azadirachtin. This large variety
of formulations has been applied in many different ways
against more than 200 species, e.g. to crops, within
artificial diets, or on simplified feeding discs, or to the
insects as sprays, topical applications, or by injection or
cannulation so hindering direct comparisons of the
susceptibility of different insect species to its antifeedant
effects. Published data, using only comparable protocols,
is presented in Table 1, and reveals a vast range in
sensitivity between species. It is clear, that Lepidoptera
are extremely sensitive to azadirachtin and show effective
antifeedancies from < l-50 ppm, depending upon
species. Coleoptera, Hemiptera and Homoptera are less
sensitive to azadirachtin behaviourally with up to 100%
antifeedancy being achieved at lOMOO ppm, whereas
Orthoptera show an enormous range in sensitivity
from the most sensitive species tested, S. zyxwvutsrqponmlk
gregaria
(effective concentration to reduce feeding by 50%
EC,, = 0.05 ppm) through the moderate sensitivity of zyxwvutsrqpo
L. migratoria (EC,, = 100 ppm) to the extreme insensi-
tivity of M elanoplus sanguinipes (EC,, > 1000 ppm)

TABLE 1. The antifeedant effects of azadirachtin (AZAD) and Margosan-O@ (M-O) (0.3% AZAD) on a selected range
of insect pest species in laboratory and greenhouse triak
AZAD content Antifeedancy
Species Treatment (&pm) (%)* Reference
Lepidoptera (larvae)
Spodoptera frugiperda AZAD (fd) 1.0 92 Blaney el al. (1990)
Heliothis virescens AZAD (fd) 1.0 94 Blaney et al. (1990)
Spodoptera littoralis AZAD (fd) 1.0 95 Blaney et al. (1990)

Choristoneura fumtferana M-O (ad) 0.2 50 Thomas et al. (1992)

Choristoneura fumtferana M-O (ad) 0.3 95 Thomas et al. (1992)
Heliothis virescens AZAD (ad) 0.07 50 Yamasaki and Klocke (1987)
Peridroma plorans AZAD (ad) 0.4 50 Champagne et al. (1989)
Spodoptera littoralis AZAD (ad) 10.0 100 Meisner ef al. (1981)
Earias insulana AZAD (ad) 50.0 100 Meisner et al. (1981)

Spodoptera litura AZAD (t) 50.0 37 Ramachandran zyxwvutsrqponmlkjihgfedcbaZYXWVU

et al. (1989)
Spodoptera frugiperda AZAD (t) 50.0 43 Raffa (1987)
Peridroma saucia AZAD (t) 2.4 50 Isman et al. (1990)
Ostrinia nubilalis AZAD (t) 24.0 50 Arnason ef al. (1985)
Achoea janata AZAD (t) 1.0 54 Ramachandran et al. (1989)
Achoea janata AZAD (t) 10.0 7.5 Ramachandran et al. (1989)

Spodoptera littoralis AZAD (sp) 0.06 50 Pflieger and Muckensturm (1987)

M acalla thy rsisalis M-G (sp) 20.0 41t Howard (1990)
Spodoptera frugiperda AZAD (sp) 600.0 100 Klocke and Bamby (1989)

Pieris brassicae AZAD (syst) 30.0 56 Arpaia and van Loon (1993)

Coleoptera
Epilachna varivestis AZAD (t) 500.0 100 Kraus et al. (1987b)
Diabrotica undecimpunccata
howardi AZAD (sp) 100.0 98 Reed et al. (1982)
Acaly mma vittatum AZAD (sp) 100.0 98 Reed et al. (1982)
Leptinotarsa decemlineata AZAD (sp) 600.0 0 Klocke and Bamby (1989)

Hemiptera
M y zus persicae AZAD (ad) 100.0 80 Nisbet et al. (1992)
Rhopalosiphum padi and
Sitobion avenae AZAD (t) 250.0 501 West and Mordue (Lutz) (1992)
Asymmetrasca decedens M-O (sp) 60.0 50 Meisner et al. (1992)
M y zus persicae AZAD (syst) 300.0 3% Nisbet et al. (1993a)
Rhopalosiphum padi and
Sitobion avenae AZAD (syst) 500.0 183 West and Mordue (Luntz) (1992)
Asymmetrasca decedens M-O (syst) 60.0 100 Meisner et al. (1992)
Rhodnius prolixus AZAD 25.0 50 Garcia and Rembold (1984)
(in blood meal)
Isoptera
Coptotermes ,formosans M-O (fd) 100.0 49 Grace and Yates (1992)
Continued overleaf
908 REVIEW

TABLE zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGF
I-Continued

AZAD content Antifeedancy

Species Treatment @pm) (%)* Reference
Diptera
Liriomy za lrifolii M-O (syst) 10.0 31 Knodel et al. (1986)

Orthoptera
Schistocerca gregaria AZAD (fd) 0.008 50 Haskell and Mordue (Luntz) (1969)
AZAD (fd) 0.005 95 Morgan (1981)
AZAD (fd) 0.01 100 Haskell and Mordue (Luntz) (1969)
AZAD (fd) 0.04 100 Butterworth and Morgan (1971)
AZAD (fd) 0.05 100 Morgan (1981)
AZAD (fd) 0.05 100 Nasiruddin (1993)
AZAD (fd) 0.07 100 Butterworth and Morgan (1968)

Locusta migratoria AZAD (fd) 100.0 50 Cottee et al. (1988)

AZAD (fd) 100.0 50 Pradhan et al. (1962)
AZAD (fd) 50.0 100 Nasiruddin (1993)
Dissosteira Carolina M-O (fd) 150.0 82 Adler and Uebel (1984)

Schistocerca gregaria AZAD (ad) 0.001 100 Mulkern and Mongolkiti (1975)
M elanoplus sanguinipes AZAD (ad) 1000.0 0 Mulkern and Mongolkiti (1975)

Ey prepocnemis plorans AZAD (t) 0.01 44 Ascher et al. (1989)

AZAD (t) 0.1 85 Ascher et al. (1989)
Diapheromera femoraia M-O (t) 150.0 75 Adler and Uebel (1984)
M-O (t) 600.0 91 Adler and Uebel (1984)
Gry llus pennsy lvanicus M-O (t) 150.0 100 Adler and Uebel (1984)

Schisrocerca gregaria AZAD (sp) 1.0 50 Nasiruddin and Mordue

(Luntz) (1993b)
Treatment applied via: fd, filter paper or glass fibre discs; ad, artificial diet; t, topical treatment to leaves/leaf discs; sp, spray
application to crop; syst, systemic application.
*Antifeedancy given in relation to control feeding.
tDecrease in faecal pellet production.
S“Probing” activity.
$Duration of phloem penetration period (N.B. probing on treated plants increased compared with controls).

(Table 1). The need to evaluate EC&s and ECg5s for a
range of phytophagous pests, in terms of insect control,
is realized in the context that spray regimes of < 20 ppm
azadirachtin may be a prerequisite for not affecting
beneficials, as suggested by the current literature (see
“Beneficials”).
A simple and common laboratory bioassay to assess
azadirachtin’s antifeedant potency is the leaf-, filter
paper- or glass fibre-disc feeding test, in either a “choice”
or “no choice” situation (e.g. Adler and Uebel, 1984;
Zehnder and Warthen, 1988; Ascher et al., 1989; Klocke
and Kubo, 1991; Arpaia and van Loon, 1993; Nasirud-
din, 1993). These bioassays provide essential data for
more elaborate trials in both the laboratory and field.
For example, conversion of the protective concentration
for 95% of the crop (PC,,) (0.1 pg/leaf disc) for S.
fiugiperda from leaf-disc trials to an equivalent dose for
crop spraying, gave good protection of corn plants
(Klocke and Kubo, 1991). Disc-feeding trials are also
sensitive enough to reveal differences in susceptibility
between often closely related species. Within the acri-
dids, there are marked susceptibility differences between
Old World species and New World species, the latter
often being far less sensitive to azadirachtin than the
former (Mulkern and Mongolkiti, 1975; Adler and
Uebel, 1984), (Table 1). Differences also exist within the
Old World species with the polyphagous S. gregaria for
example showing far more sensitivity to azadirachtin
and its analogues than the oligophagous zyxwvutsrqponmlkjihgfedcbaZY
L. migratoriu in
feeding experiments (Nasiruddin, 1993).
In addition to those insects already mentioned, rela-
tively high azadirachtin doses are required to inhibit
feeding in the Formosan subterranean termite, Copto-
termes formosama, for which doses of 3 100 ppm azadir-
achtin were required to reduce feeding significantly on
filter paper when compared against the amount of
control filter paper consumed (Grace and Yates, 1992),
and in R. prolixus where there is a 600-fold difference
between the threshold for toxic effects and that for
antifeedancy (Garcia and Rembold, 1984). Such differ-
ences may relate directly to the insensitivity of insect
gustatory chemoreceptors and subsequent central pro-
cesses regulating feeding and result in toxic IGR effects
being manifest before any antifeedant effects.
On systemically-treated plants, high concentrations of
azadirachtin (> 100 ppm) are required to produce pri-
mary antifeedant effects on aphids (Griffiths et al., 1978;
Nisbet et al., 1993a). Azadirachtin concentrations of
 REVIEW 909

more than 250 ppm prevented cereal aphids zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

(Rhopalosi- adults with unplasticized wing lobes seen as crippled
phum padi and Sitobion avenae) from settling on system- wings, (3) nymphs or larvae which die during ecdysis
ically-treated barley seedlings although lower doses unable to complete the moulting process, (4) pupae with
(50 ppm) produced the same effects when applied severe deformities to the head and thoracic appendages,
topically to the leaves. These treatments increased loco- (5) nymphs or larvae which die immediately before the
motor-y behaviour and reduced probing frequency of the moult after a normal instar length, (6) insects which
aphids for up to 4 days /West and Mordue (Luntz), remain as “over-aged” larvae for a greatly extended
19921. The low sensitivity of aphids to the primary period and (7) insects which die within hours of treat-
antifeedant effects of azadirachtin may result from the ment. Such phenomena have now been described
poor phloem-mobility of the compound (Schmutterer, throughout a wide variety of insect taxa including
1985) though concentrations of up to 75 ppm in artifi- Lepidoptera (Arnason et al., 1985; Schliiter et al., 1985;
cial diets do not initially deter aphids from feeding Barnby and Klocke, 1987; Koul et al., 1987) Diptera
(Nisbet et al., 1993b). (Zebitz, 1987; Miller and Chamberlain, 1989), Orthop-
With sufficiently high doses (IO&1000 ppm), however, tera [Sieber and Rembold, 1983; Mordue (Luntz) et al.,
primary antifeedant effects on aphids are significant, as 1985; Rao and Subrahmanyam, 1986; Ascher et al.,
measured by the reduction in honeydew production, 1989; Champagne et al., 19891, Hemiptera (Redfern
even when aphids appear to be settled (Nisbet et al., et al., 1981; Koul, 1984; Garcia and Rembold, 1984;
1992). These results have important implications for Dorn et al., 1986) Coleoptera (Ladd et al., 1984;
reduced plant damage and plant virus transmission, Schliiter, 1985) and Hymenoptera (Rembold et al., 1982,
particularly if the mobility of azadirachtin in phloem is’ 1984).
such that it will be translocated to new plant growth A summary of the IGR effects of azadirachtin applied
after treatment. However, even though the primary directly to the insect shows a remarkable consistency
antifeedant effects of 500 ppm azadirachtin, applied between species (Table 2) unlike the antifeedant effects
systemically to tobacco seedlings, prevented Myzus (Table I), apart from the marked sensitivity of Oncopel-
persicae from acquiring a persistently transmitted virus tus fasciatus which is known to be very susceptible to
(PLRV) they were insufficient to prevent viruliferous endocrine disruption. The effective dose for 50% mor-
aphids from inoculating treated plants with either PLRV tality (ED,,) after injection into the haemolymph varies
or the non-persistently transmitted virus PVY (Nisbet, between 1 and 4,ug/g body weight in all species shown
1992). Control of aphid populations and virus trans- except for 0. fasciatus which has in ED,, of 0.6pg/g
mission then may best be achieved by using concen- body weight, or less. Oral cannulation in lepidopterous
trations of azadirachtin below that which causes primary larvae and topical application in M. sanguinipes are
antifeedancy but which causes reproductive effects in similar to injection of azadirachtin, whereas topical
aphids (e.g. Schauer, 1984; Dimetry and Schmidt, 1992). application in lepidoptera and oral cannulation in
In M. persicae, for example, significantly fewer viable M. sanguinipes and in the locusts S. gregaria and
nymphs were produced by apterous adults which had fed L. migratoria [Cottee and Mordue (Luntz), 19821are less
for 24-52 h on diets containing azadirachtin at a concen- effective than injection showing that the cuticle of lepi-
tration of 25 ppm than on control diets (Nisbet et al., dopterous larvae and the gut of orthopteran nymphs can
1993b). act as barriers to bioavailability.
When feeding does occur in antifeedant trials with The developmental effects of azadirachtin are at-
azadirachtin, significant insect mortality is often tributed to a disruption of endocrine events (see “Mode
recorded, particularly in no-choice trials, as secondary of action-endocrine effects”). Complete moult inhibition
toxic effects of azadirachtin begin to predominate. has been shown to be due either to a total blockage of
Almost 50% mortality was recorded in the red pumpkin haemolymph ecdysteroids or to a delay in the appear-
beetle, Aulacophora foueicollis after 11 days feeding on ance of the last ecdysteroid peak, with or without a
leaf discs treated with (0.5-2%) neem seed extract reduction in peak height, and a slow abnormal decline
(NSE) (Gujar and Mehrotra, 1988) and 73% mortality in the peak [Redfern et al., 1982; Sieber and Rembold,
of Colorado potato beetle, L. decemlineata was recorded 1983; Schhiter et al., zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQP
1985; Mordue (Luntz) et al., 19861.
after 72 h on potato leaves treated with 1.2% NSE In the former case with a blockage or delay of release of
(2.72 mg azadirachtin/g extract) (Zehnder and Warthen, ecdysteroids the moult would not be initiated due to the
1988). lack of haemolymph ecdysteroids and hence the lack of
moulting commitment and cuticle synthesis, whereas in
2. Insect growth regulation the latter case of an abnormally slow decline in the
The growth regulatory effects of azadirachtin on larval ecdysteroid peak, moult inhibition may occur due to a
insects are well known. Treatment of insects or their prevention of release of the neurosecretory peptide,
food with azadirachtin causes growth inhibition, mal- eclosion hormone. Eclosion hormone is released from
formation and mortality in a dose-dependent manner. the brain-corpus cardiacum complex following the rapid
Hence increasing doses of azadirachtin in larval stages decline of ecdysteroid titre in the haemolymph at the end
result in: (1) adults with reduced longevity and fecundity of the instar and triggers various stereotyped behaviours
(see “Reproductive effects”), (2) wingless adults or which serve to extricate the insect from its old skin. The
910 REVIEW

TABLE 2. The insect growth regulatory (IGR) effects of azadirachtin on insects after injection, oral cannulation or topical
application
Azadirachtin
Treatment at concentration Moult
day O-1 of inhibition and
Species instar pg/insect Pglg mortality (%) Reference
Lepidoptera
Heliothis virescens Injection 0.25 (1.7) 15 Simmonds et al. (1990)
Spodoptera fncgiperda Injection 0.25 (1.5) 45 Simmonds et al. (1990)
Spodoptera littoralis Injection 0.25 (I.91 35 Simmonds et al. (1990)
Spodoptera litura Injection 1.1 50 Rao and Subrahmanyam (1987)
Achoea janata Injection 4.1 50 Rao and Subrahmanyam (1987)
Peridroma saucia Injection l-2 50 Koul and Isman (1991)
Bomby x mori Injection l-2 50 Koul et al. (1987)

Spodoptera littoralis Oral cann. 0.25 (1.5) 50 Simmonds et al. (1990)

Spodoptera frugiperda Oral cann. 0.25 (1.9) 50 Simmonds et al. (1990)
Heliothis virescens Oral cann. 1.0 (5) 100 Barnby and Klocke (1987)
Heliothis virescens Oral cann. 0.25 (1.7) 15 Simmonds et al. (1990)

Spodoptera littoralis Topical applic. 0.25 (1.5) 15 Simmonds et al. (1990)

Spodoptera jirugiperda Topical applic. 0.25 (1.9) 20 Simmonds et al. (1990)
Heliothis virescens Topical applic. 0.25 (1.7) 5 Simmonds et al. (1990)

Hemiptera
Rhodnius prolixus Injection 0.0035 (0.2) 50 (moult Garcia et al. (1986)
inhibition only)
Rhodnius prolixus Injection 0.035 (2.0) 50 Garcia et al. (1986)
(mortality only)
Oncopeltus fasciatus Injection 0.015 (0.6) 50 Dorn et al. (1986)
Oncopeltus fasciatus Topical applic. 0.0035 (0.14) 50 Isman et al. (1990)

Orthoptera
Schistocerca gregaria Injection 1.7 50 Rao and Subrahmanyam (1986)
Locusta migratoria Injection 1.3 50 Sieber and Rembold (1983)
Locusta migratoria Injection 2.0 50 Mordue (Luntz) et al. (1985)
M elanoplus sanguinipes Injection 3.2 50 Champagne et al. (1989)

M elanoplus sanguinipes Oral cann. 11.3 50 Champagne et al. (1989)

M elanoplus sanguinipes Topical applic. 4.5 50 Champagne et al. (1989)
Figures in parentheses refer to calculated values.

slow decline of the ecdysteroid titre may thus inhibit the
release of eclosion hormone. Low aberrant ecdysteroid
peaks also result in incomplete resorption of exuvial fluid
and incomplete sclerotization and pigmentation of new
cuticle.
Over-aged nymphs which have a greatly extended
instar may survive for several weeks, e.g. L. migratoria
[Mordue (Luntz) et al., 19851, 0. fascia&s (Dorn et al.,
1986) or several months, e.g. R. prolixus (Garcia and
Rembold, 1984) beyond the normal instar length of
several days, and have been used extensively to investi-
gate the onset of maturation with chronological age.
Such insects have not metamorphosed and thus have not
achieved the imaginal competence for adult physiology
and development (Pener and Shalom, 1987; Pener et al.,
1989; Van der Horst et al., 1989).
L. migratoria over-aged nymphs do, however, achieve
partial adult competence despite the lack of moulting
and metamorphosis as demonstrated by male mating
behaviour (Pener and Shalom, 1987) and the competence
to respond to adipokinetic hormone (AKH) (Pener et al.,
1989). It is known that azadirachtin induced over-aged
nymphs of 0. fasciatus, L. migratoria and S. gregaria
show, after a period of time, some corpus allatum
activity and endogenous juvenile hormone activity
(Dorn et al., 1986; Pener and Shalom, 1987). It may be
that the ethogenesis of adult characteristics is promoted
by juvenile hormone deficiency following azadirachtin
treatment, with their subsequent expression following
the delayed reactivation of the corpora allata (Pener and
Shalom, 1987). Certainly over-aged female nymphs of
L. migratoria and 0. fasciatus produce vitellogenin,
which is a juvenile hormone dependent process, and
show some egg development (Rembold, 1984; Dorn
et al., 1987) (see also “Reproductive effects”). None of
the characteristics so described, however, are expressed
or function at the same levels as in normal adults.
3. Reproduction
Antifeedancy and IGR effects are often the most
overt effects seen following azadirachtin treatment.
However, the interruption of insect reproduction is also
 REVIEW 911

an important and potent effect. Adverse effects on seen by over-aged nymphs of locusts which displayed
ovarian development, fecundity and fertility (egg mating behaviour but at a subnormal level (Pener and
viability) have all been reported (reviewed by Karnavar, Shalom, 1987). In an zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPON
in vitro study of spermiogenesis
1987). Interference with either the synthesis of vitel- in diapausing M amestra brassicae males, 3 ppm azadir-
logenin or its uptake by oiicytes is a linking factor achtin caused spermatocysts to degenerate, even in
between different insect species. For example in zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFE
L. the presence of 20-hydroxyecdsone, suggesting a direct
migratoria azadirachtin inhibits both oiigenesis and effect on the testicular membrane, rendering the tissue
ovarian ecdysteroid synthesis, so preventing any ovipos- incapable of developing spermatocysts (Shimizu, 1988).
ition (Rembold and Sieber, 1981). Female S. exempta Also injection of male 0. fasciatus with 0.125 pg per
which emerged from larvae topically treated with 0.01 insect severely reduced male potency as seen by a 80%
and 0.1 pg azadirachtin, exhibited reduced fecundity but reduction in the fecundity of normal females when mated
not fertility due to a failure of many oiicytes to mature with treated males (Dorn, 1986). zyxwvutsrqponmlkjihgfedcbaZYX
(Tanzubil and McCaffrey, 1990). Through measure-
ments of both fat body and ovarian protein levels of S. MODE OF ACTION
exempta, it was concluded that azadirachtin treatment
caused a lower uptake of fat body proteins by the ovary 1. Chemoreception
in the treated insects. 0. fasciutus injected with 4pg Inhibitory chemicals play a major role in host plant
azadirachtin also exhibited reduced fecundity (Dorn selection by phytophagous insects, with the behavioural
et al., 1987) and in adult female R. prolixus, ingestion of response being a consequence of the balance between
azadirachtin in the blood meal was associated with phagostimulation, inhibition and the internal physio-
reduced vitellogenin levels in both the haemolymph and logical state of the insect. Feeding behaviour depends
ovaries with a dose-dependent reduction in oocyte upon both neural input from the insects’ chemical senses,
growth. Radioimmunoassay of such azadirachtin treated with contact chemoreceptors on the tarsi, mouthparts
R. prolixus revealed lower ecdysteroid titres in both and oral cavity being those mainly involved, and central
haemolymph and ovaries (Feder et al., 1988). nervous integration of this “sensory code”. Despite the
Azadirachtin treatment of larvae or pupae, produces in enormous volume of literature on chemoreception in
those adults which survive, reduced oijgenesis as seen in insects and the sensory coding underlying the neural
the stored grain pest, Trogoderma granarium (Chellayan mechanisms leading to food selection behaviour (e.g.
and Karnaver, 1990). Reduced longevity and fecundity Schoonhoven, 1972, 1982) little work has been related
in the fruit fly Ceratitis capitata (Stark et al., 1990b) and specifically to the mode of action of azadirachtin and
the leaf miner Liriomy za trlfofii (Parkman and Pi- much of interest remains to be done. The work to
enkowski, 1990) was also recorded after sand or soil date has almost exclusively been that of Blaney and
drenches with azadirachtin. Simmonds who have studied in some detail the neuro-
In azadirachtin induced over-aged nymphs of physiological effects of azadirachtin on the chemorecep-
0. fusciutus some ovarian development was demon- tors of locusts and lepidopterous larvae and related these
strated despite ecdysis not having occurred (Dorn et al., effects to feeding responses (Blaney, 1980; Winstanley
1986) and in L. migratoria fat body vitellogenesis took and Blaney, 1978; Simmonds and Blaney, 1984).
place to the extent of haemolymph vitellogenin levels In general, inhibition of feeding behaviour results
being 6-7 times greater than in controls. Such high blood from either blockage of the input from receptors which
levels were thought to be related to substantial oiicyte normally respond to phagostimulants, or from stimu-
resorption, returning vitellogenin to the haemolymph, lation of specific “deterrent” cells or both (Chapman,
with subsequent oocytes being underdeveloped (Shalom 1974; Dethier, 1982). A detailed comparative analysis of
et al., 1988). the neurophysiological effects of azadirachtin on feeding
Hatchability of 0. fasciatus eggs and S. exempta eggs responses in several polyphagous and oligophagous lepi-
topically treated with 1.25640 pg/lOO pl/lOO eggs dopterous larvae revealed differences and complexities
or I pg azadirachtin was not affected (Dorn, 1986; in chemoreceptor responses both between different
Tanzubil and McCaffery, 1990), although in the latter chemoreceptors in the same insect and between species
case the emerging larvae were considerably less viable, (Simmonds and Blaney, 1984). Neurophysiological re-
probably as a consequence of consuming azadirachtin sponses from the medial and lateral sensilla styloconica
with the egg shell, eaten after hatching (Tanzubil and of the maxillae, using different concentrations of sucrose
McCaffery, 1990). Reproductive behaviour of females and azadirachtin (together with an electrolyte) either
may also be affected by azadirachtin treatment due zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCB
to singly or together, revealed in most cases a different
such parameters as a changed and deficient courtship population of receptors responsive either to sucrose (the
signal in the brown planthopper Nilaparvata lugens “sugar” cell) or azadirachtin (the “deterrent” cell). In
(Saxena, 1989) and a prolapse of the anal brush in the medial sensilla of S. exempta and M. brassicae there
S. litura (Rao and Subrahmanyam, 1987). was no apparent interaction between the cells, suggesting
The majority of investigations into the reproductive integration only in the CNS. However, in all other
effects of azadirachtin in insects have concentrated on instances some degree of interaction also occurred at
females. Effects, however, are also apparent in males as the peripheral level so that the rate of firing of the
912 REVIEW

sugar-sensitive cell was reduced in the presence of both Interestingly, Simmonds and Blaney (1984) showed,
chemicals (Simmonds and Blaney, 1984). Such an inter- using azadirachtin containing diets, that induction of
action of azadirachtin or other limonoids with both food preferences could occur whereby “experienced”
“deterrent” and “sugar” cells of the styloconic sensilla polyphagous species in particular showed a decreased
was also found in P. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
brassicae (Schoonhoven and Jermy, firing rate of their sensilla in response to azadirachtin
1977) and Eldana saccharina, the sugar cane stem borer stimulation which was associated with an increased food
(Waladde et al., 1989) and is an example of a very intake. A similar phenomenon was also described at the
common phenomenon in insect chemoreceptors behavioural level for S. gregaria using sucrose paper
(Schoonhoven, 1986). impregnated with azadirachtin (Gill, 1972). Such a plas-
The mode of action of azadirachtin on cells other than ticity at the behavioural and peripheral sensory level has
“deterrent” receptors is not known. The reduced firing important consequences in terms of understanding the
rate in the “sugar” cell is not an injurious effect of strategies of food selection in polyphagous and
azadirachtin on the cell membrane, as the response to oligophagous species and modelling of sensory infor-
other compounds was always normal within 2.5 min of mation using neutral networks is now being attempted
azadirachtin stimulation (Simmonds and Blaney, 1984). (Hanson et al., 1992). Polyphagous insects might profit
Further work is required to show whether the interaction from the ability to adapt quickly to new plants or to
is at the cell membrane or if it is an inter-molecular learn by experience to avoid others, whereas those of
effect. Investigations into the “secondary antifeedant” oligophagous insects, with restricted habits, are pro-
effect (see section on “Gut Physiology”) of azadiractin tected from potential toxins in new plants and have a
would also be of great interest as it may give insight into more behaviourally limiting sensory system, as suggested
central regulating mechanisms of receptor responsive.- by single sensillum work. This phenomenon is also of
ness. It is known that the internal “milieu” can affect great relevance when considering the use of antifeedants
chemosensory responsiveness, particularly after feeding, in insect control as aversion learning and/or food prefer-
which in this instance could involve direct feedback of ence induction in polyphagous species may alter the
haemolymph azadirachtin or indirect involvement of behaviour of the insects and so reduce the efficacy of the
hormones or other haemolymph constituents to alter semiochemicals (Jet-my et al., 1982; Szentesi and
pore size or receptor lymph composition (Blaney et al., Bernays, 1984). Assays have been designed to quantify
1986). behavioural desensitization to antifeedants (Raffa and
In all cases, although there was no single neural Frazier, 1988) although, in the case of azadirachtin, the
response pattern after stimulation of receptors, the toxic effects after ingestion would presumably compen-
associated behavioural response to azadirachtin was sate for any change in antifeedancy, in terms of effective-
a reduced or complete inhibition of feeding. The ness in insect control.
polyphagous species S. littoralis, S. frugiperda, Heliothis Inhibitory stimuli, such as that provided by azadirach-
virescens and H. armigera tended to have a higher rate tin, are also of importance in the choice of oviposition
of firing of sensilla than the oligophagous species sites by phytophagous insects. Neem seed extracts and
S. exempta and M. brassicae. This situation is similar to neem oil have been shown to be effective at deterring
that in the polyphagous S. gregaria where increased oviposition of S. litura (Joshi and Sitaramaiah, 1979;
deterrence is related to an overall increase in the rate of Ayyangar and Rao, 1989) the bruchid beetles Calloso-
change of firing of chemoreceptors, in contrast to the bruchus maculatus, C. analis and C. chinensis (Yadav,
oligophagous L. migratoria. In the latter case deterrence 1985) and the sheep blowfly Lucilia sericata (Rice et al.,
is related to a decreased rate of firing so that a “gating” 1985). However H. armigera were not deterred from
mechanism exists whereby feeding is not initiated if laying eggs on filter paper treated with azadirachtin
sensory input is below a certain threshold (Winstanley (Saxena and Rembold, 1984).
and Blaney, 1978). The tarsal and proboscis chemoreceptors are the main
Polyphagous species have the option to forage receptors involved in oviposition site selection by adult
optimally among plant species and do well on dietary female lepidoptera and both respond to phagostimulants
mixing as seen in the grasshopper Taeniopoda eques and allelochemicals. In larvae there appear to be two
(Bernays, 1992). S. gregaria, most unusually for a populations of cells responding separately but this is not
polyphagous acridid, has chemoreceptors on its the case in adults. Whereas the mouthpart receptors of
maxillary and labial palps and A, receptors of the adult S. littoralis, H. virescens and H. armigera respond
clypeo-labrum with a very sensitive and specific response to sugars and amino acids with a significant correlation
to azadirachtin (Haskell and Schoonhoven, 1969; to behaviour they are less sensitive to compounds such
Blaney, 1980). Such a “labelled line” phenomenon as azadirachtin than the larvae and apart from
together with an “across-fibre patterning” analysis in H. armigera show no correlation of detection of allelo-
central nervous integration for the remaining chemo- chemicals with behavioural response (Blaney and
receptors may account for the particular sensitivity of Simmonds, 1988). It is suggested that the tarsal chemore-
S. gregaria to this plant secondary compound when ceptors, which may act as a preliminary screen for
compared with other compounds (Blaney, 1986; allelochemics, are important in mediating oviposition
Cottee et al., 1988). behaviour (Ma and Schoonhoven, 1973) and that the
 REVIEW 913

proboscis predominantly assesses the nutrient quality of cultured glands from M. sexta pupae pretreated with
saps, nectar and other sources of adult food (Blaney and azadirachtin in the late last larval instar (Pener et al.,
Simmonds, 1988, 1990). There is, however, also some 1988), i.e. PTTH (as brain extract) from control individ-
evidence that although females do select suitable ovipos- uals will still stimulate normal levels of ecdysone release
ition media for their progeny, the oviposition preference from prothoracic glands which have been exposed to
of adults appears to be narrower than the range of azadirachtin. The synthetic capacity of the azadirachtin
substrates suitable for larval development (Jermy and treated prothoracic glands is normal. There is however
Szentesi, 1978). Again, further work is required on the some indication that the receptivity of the prothoracic
interplay between both food selection and oviposition glands to PTTH may have been affected in H. virescens
site selection and the use of azadirachtin based insecti- where prothoracic glands from pretreated larvae were
cides where polyphagous and oligophagous species may unable to respond fully to PTTH-like brain extracts
vary in their behavioural responsiveness and may learn (Barnby and Klocke, 1990) although this is not the case
from experience. in M. sexta (Pener et al., 1990). Removal of endocrine
glands by decapitation or ligation followed by sub-
2. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
Endocrine efects sequent reimplantation have also demonstrated that
(a) Ecdy steroids. A major action of azadirachtin is to release of PTTH activity is deficient in azadirachtin
modify haemolymph ecdysteroid titres. IGR effects of treated R. prolixus and B. mori (Garcia et al., 1990; Koul
azadirachtin manifest as developmental abberations in et al., 1987).
immature insects are caused by significant reductions The main action of azadirachtin thus appears to be at
and/or delays in moulting hormone titres of the haemo- the release sites of PTTH from the corpora cardiaca.
lymph [e.g. Redfern et al., 1982; Sieber and Rembold, Acetylcholine and GABA play a part in inhibiting the
1983; Schliiter et al., 1985; Garcia et al., 1986; Dorn release of ecdysone from BRCs in C. vicina (Kluser
et al., 1986; Mordue (Luntz) et al., 1986; Bidmon et al., et al., 1987). It is interesting to note that the action of
1987; Malczewska et al., 1988; Barnby and Klocke, atropine (an acetyl choline antagonist) and picrotoxin
19901.The effects of azadirachtin are both dose and time (an antagonist of GABA) in stimulating ecdysone release
dependent; prevent both apolysis and ecdysis; can cause from BRCs of C. vicina in vitro is blocked in the presence
death before the moult, during the moult or delays of the of azadirachtin at doses which cause classical IGR effects
moult sometimes to the extent of inducing permanent (10m6 to 10d4M azadirachtin) (Bidmon et al., 1987;
larvae (see “Effects of azadirachtin”). Koolman et al., 1988). It has also recently been shown
In Tenebrio pupae the injection of 1 pg azadirachtin in both Vth instar larvae and adult female L. migratoria
induces a delayed and reduced ecdysteroid peak which and S. gregaria that injection of 1.84 pg/g azadirachtin
inhibits the imaginal moult, despite this insect having no results in a significant elevation of serotonin levels in the
prothoracic glands at this stage (Marco et al., 1990). brain, suboesophageal ganglion and corpora cardiaca
Epidermal cells or oenocytes, as potential sites of ecdy- after a period of 5 days (Banerjee and Rembold, 1992).
steroid production, may well be the target for azadirach- It is not yet known whether serotonin plays a part in the
tin action in this case. In adult female L. migratoria release mechanisms of neurosecretory peptides.
ecdysteroid synthesis by the ovaries, together with The corpus cardiacurn has been suggested to be a
oogenesis, is inhibited by a dose of 1Opg azadirachtin target for azadirachtin in terms of PTTH, eclosion
per insect (Rembold and Sieber, 1981). hormone, bursicon or general tropic hormone release
Such striking effects on ecdysteroid levels have led to [Sieber and Rembold, 1983; Mordue (Luntz) et al., 1986;
much investigation into the effects of azadirachtin on Bidmon et al., 1987; Rembold et al., 19891. Staining
prothoracic gland functioning in insect larvae. In vitro neurosecretory proteins with paraldehyde fuchsin in
experiments whereby ecdysone production was rapidly maturing females of L. migratoria compared
measured by RIA from prothoracic glands incubated in with similar aged azadirachtin treated females revealed
the presence or absence of azadirachtin and/or PTTH an accumulation of stainable material in the corpora
(prothoracicotropic hormone) have shown unequivo- cardiaca and neuropilar storage areas of the brain
cally that azadirachtin does not act directly on the neurosecretory system in treated insects which was as-
prothoracic glands. It is also clear, despite earlier claims, sociated with a lack of ovarian development (Subrah-
that azadirachtin does not bind to ecdysteroid receptors manyam et al., 1989). A similar condition occurs in
at target sites since azadirachtin does not compete with starved insects [Highnam and Mordue (Luntz), 19741
Ponasterone A binding in a competitive RIA (Koolman although such azadirachtin treated insects (3 pgg/g) ate
et al., 1988). sufficient food to maintain their body weight. Auto-
In blowfly larvae, Caliiphora vicina and the lepid- radiography of the brain-corpora cardiaca complex of
opterans, Bombyx mori and H. virescens, in vitro culture locusts treated with [3H]dihydroazadirachtin revealed
of brain-ring gland complexes (BRCs) or prothoracic that label could not pass through the brain neurilemma
glands showed that PTTH-induced release of ecdysone whereas the corpora cardiaca were heavily labelled
was not inhibited by the presence of azadirachtin in the (Subrahmanyam et al., 1989). Thus it would appear that
culture medium (Bidmon et al., 1987; Barnby and azadirachtin blocks release of neurosecretory material
Klocke, 1990) nor was it blocked in PTTH stimulated from the corpora cardiaca with a reduced turnover, seen
914 REVIEW

as a subsequent accumulation of material, within the (b) Juvenile hormone. The effects of azadirachtin upon
system. Such a feedback may also affect the rate of juvenile hormone levels are not so easy to define due to
synthesis of PTTH by brain neurosecretory cells as has the close interrelationships between juvenile hormone,
been suggested by Barnby and Klocke (1990) in ecdysone levels and neurosecretions in the moulting
H. virescens. Here prothoracic glands incubated with process. Malczewska et al. (1988) used chilled Galleria
brain extracts from azadirachtin treated last instar larvae mellonellu larvae to investigate the effects of azadirachtin
produced significantly less ecdysone than prothoracic on juvenile hormone and ecdysteroid titres: chilled lar-
glands incubated with control brain extracts. In vae undergo supernumerary moults, due to increases in
M. sex& however, in similar experiments, the PTTH juvenile hormone titre (Bogus and Cymborowski, 1981).
content of the brain was shown not to be affected by Azadirachtin inhibits, in a dose-dependent manner, such
azadirachtin treatment (Pener zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
et al., 1990). supernumerary moults of last instar larvae, possibly by
It now remains to be finally and unequivocally demon- affecting the release of allatotropins into the CA and
strated that azadirachtin does block the release of hence blocking the synthesis and release of juvenile
peptide hormones from the brain neurosecretory cell- hormone. This block causes a rapid decrease in whole
corpora cardiaca complex; an achievement which will body juvenile hormone titres which is maintained for
only be realized with the synthesis of the relevant several days. In last instar M. sexta larvae azadirachtin
peptides and the development of sensitive bioassays such injection on day 0 (1 .&lo pgg/larva) results in the induc-
as RIA or ELISA systems for the detection of the small tion of supernumerary moults (Schliiter et al., 1985;
quantities of hormones involved. Beckage et al., 1988) presumably due to an inhibition
Azadirachtin does not affect the synthetic capacity of and subsequent delay in juvenile hormone titres so that
the prothoracic glands directly as demonstrated by the the presence of the hormone extends into the critical
conversion rates of radiolabelled ketodiol to ecdysone period for commitment to larval rather than pupal
in vitro in C. vicina (Koolman et al., 1988); however, cuticle. In adult female L. migratoria also, azadirachtin
there is some evidence that ecdysone catabolism is treatment causes a rapid decrease in juvenile hormone
affected. Ecdysone 20-mono-oxygenase from the midgut titres with associated disturbances in oiigenesis
and fat body is the insect cytochrome P-450 dependent (Rembold, 1984; Rembold et al., 1987). It is puzzling,
hydroxylase responsible for the conversion of ecdysone however, that juvenile hormone analogues were unable
to its more active metabolite, 20-hydroxyecdysone. to restore the juvenilizing effects at the moult, of azadir-
Formation of 20-hydroxyecdysone by 20-mono-oxygen- achtin treated chilled Galleria larvae (Malczewska et al.,
ase, is affected within 1 h of exposure to azadirachtin 1988) and that juvenile hormone analogues also counter-
(Bidmon et al., 1987; Smith and Mitchell, 1988). The acted the moult inhibitory effect of azadirachtin in
half-life of ecdysone in vitro is significantly increased by Rhodnius (Garcia and Rembold, 1984). Such counter-
1O-s M azadirachtin in C. vicina in association with a action of moult inhibition with juvenile hormone or
decrease in levels of 20 mono-oxygenase (Bidmon et al., BEPAT (the juvenile hormone esterase inhibitor, S-ben-
1987). Cytochrome P-450 levels, are also significantly zyl-o-ethyl phosphoramidothiolate) treatment were not
decreased in midguts of Vth instar S. gregariu and seen in Peridroma sauciu last instar larvae treated with
L. migrutoria orally cannulated with 25 pg azadirachtin azadirachtin (Koul and Isman, 1991). It is known,
daily for 4 days (Cottee, 1984). In addition, in Drosophila however, that the timing of juvenile hormone levels
melanogaster, Aedes aegy pti and M. sexta 50% inhi- relative to ecdysteroid levels is of critical importance in
bition of 20-mono-oxygenase was brought about by 10m4 both regulating the expression of juvenile characteristics
to 4 x 10e4 M concentrations of azadirachtin, although at the moult and in the maintenance of prothoracic
as the authors point out, these concentrations are an glands until the last larval instar. Gross interference of
order of magnitude higher than that needed to achieve the hormonal milieu of insects, by reimplantation or
moult inhibition and alterations of ecdysteroid titre in injection of glands and hormones, in insects which are
the relevant species (Smith and Mitchell, 1988). already disrupted by azadirachtin treatment, is too crude
Nevertheless, in T. molitor pupae the depletion in a procedure and renders interpretation of the results
haemolymph ecdysteroid levels brought about by difficult due to these many and varied interrelationships.
azadirachtin treatment is mainly due to a lack of That azadirachtin blocks juvenile hormone release is
20-hydroxy-ecdysone, ecdysone levels remaining rela- indicated strongly by the selective destruction of larval
tively unchanged in comparison with controls (Marco crochets or hooked setae of the prolegs in M. sexta
et al., 1990). It remains a possibility then, that ecdysone (Reynolds and Wing, 1986; Beckage et al., 1988). In
catabolism may play a part in the overall toxic syndrome M unduca the crochet epidermis remains competent to
of azadirachtin poisoning. Injections of 20-hydroxy- moult until the juvenile hormone titre declines on day 2
ecdysone into azadirachtin treated insects, however, will of the last larval instar (Riddiford, 198 1). In response to
not restore azadirachtin treated insects to normal the decline, the epidermis loses its ability to make cuticle
development (Pener et al., 1988; Barnby and Klocke, and subsequently dies when next exposed to ecdysteroids
1990), although partial recovery of moulting was seen in even when juvenile hormone is present again. The critical
H. virescens after such treatment (Barnby and Klocke, timing of azadirachtin treatment (prior to the first
1990). ecdysteroid peak) and the inability of such tissue to
 REVIEW 915

synthesise new crochet cuticle as monitored by sub- are direct on the target cells but which are not expressed
sequent implantation into untreated hosts (Reynolds until the next ecdysteroid peak comes along. Indeed it is
and Wing, 1986) strongly suggests a momentary lack of possible to mimic the effects of azadirachtin with colchi-
juvenile hormone at this critical period, perhaps from a tine under certain conditions (Reynolds and Riddiford,
lack of release of neurosecretory allatotropins. personal communications). Using colchicine, Schhiter
The toxic effects of azadirachtin cannot be entirely (1987) was able to demonstrate in IVth instar larvae of
explained by their effects on the endocrine system alone. E. uarivestis that azadirachtin affects mitosis directly.
Azadirachtin has direct effects on a whole variety of The injection of 0.5 pg azadirachtin into 20 h L,
tissues and organs which suggest either a number of Epilachna, a stage at which rapid cell division and
different modes of action or a specific toxic lesion to all growth of wing discs is occurring, results in almost
cells which manifests itself more obviously in some than complete degeneration of the wing discs within 24 h.
others; the major effects on moulting and metamorpho- Injection of 1 fig colchicine at 19 h also reveals degener-
sis with blockage of ecdysteroid and juvenile hormone ated wing discs, with a few cells fixed with chromosomes
release being obvious examples here of far reaching and at metaphase. If injection of colchicine is given at 4 h
massive aberrations caused by specific target action on post azadirachtin treatment followed by fixation at 24 h,
peptide tropic hormone release. very little degeneration of wing discs occurs and very
many cells are seen with chromosomes in the metaphase
3. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
Other phy siological efects arrangement. It is suggestive that cell death in these
Many of the reported manifestations of azadirachtin dividing cells due to azadirachtin treatment is occurring
treatment around the moult such as lack of larval at some stage post metaphase, since cells are reaching
crochets (Haasler, 1984; Reynolds and Wing, 1986), lack that stage as witnessed by the colchine treatment. Also,
of differentiation of tissues such as ommatidia of the eyes in Tetrahy mena thermophila a ciliate protozoan,
at the larval-pupal moult (Schliiter, 1985; Schmutterer,’ Fritzsche and Cleffmann (1987) showed cell proliferation
Nicol and Freisewinkel, personal communication), lack to be inhibited by azadirachtin, with RNA synthesis
of differentiation of imaginal discs, too early a differen- being strongly affected.
tiation of wing discs resulting in uneverted tanned pupal (b) Muscle phy siology . In addition to affecting those
wing discs (Schliiter, 1987) occurrence of black patches tissues known to be controlled by developmental hor-
on the cuticle, etc. may be explained in terms of the mones, azadirachtin has subtle effects on a variety of
balance between both the presence or absence of other tissues which form part of the overall toxic syn-
ecdysone and juvenile hormone and their relevant titres drome of poisoning and which may provide clues as to
in the haemolymph at the critical period related to its cellular mode of action. For example, adult locusts
commitment of epidermal cells for metamorphosis. For treated with azadirachtin become sluggish and show
example, solitarization effects, linked with high juvenile reduced iocomotory and flight activity [Mordue (Luntz),
hormone titres at critical times in the instar, seen as Plane and McBeath, unpublished observation; von Nicol
green haemolymph, brown and green cuticle, lack of and Schmutterer, 1991; Wilps et al., 19921. Such a
black pigment and supernumerary moulting occurs in reduced “tendency” to fly results in a significantly
gregarious S. gregaria and L. migratoria after spraying reduced elevation of blood lipids after flight activity
with neem oil in the early instars (Schmutterer and compared with controls, which cannot be increased by
Freres, 1990; Nicol and Schmutterer, 1991; von previous injections of adipokinetic hormone (AKH)
Freisewinkel and Schmutterer, 1991). Also, cuticular (Wilps et al., 1992). Further experiments are required to
melanization, resulting in characteristic black spots, clarify whether the release of AKH from the glandular
is often seen in azadirachtin treated insects (e.g. lobes of the corpora cardiaca has been inhibited or the
Malczewska et al., 1988). Such blackening, due to an flight muscles themselves have been directly affected by
absence of juvenile hormone and low levels of ecdy- azadirachtin and cannot respond to stimulation by
steroid titre (Hori et al., 1984) are likewise linked to the AKH, or both. The fat body also is known to be affected
low levels of ecdysteroid in azadirachtin treated insects by azadirachtin with necrosis of scattered cells occurring
as they are absent in 5-day azadirachtin treated thorax after treatment (Schliiter, 1985). Insect muscle has been
ligated B. mori (minus prothoracic glands and brain), shown to be affected by azadirachtin: histological studies
but present in whole treated larvae (Koul et al., 1987). of midgut muscle of S. gregaria and L. migratoria show
(a) M itosis. It is more difficult to demonstrate un- that the muscles become swollen and disrupted in a dose
equivocally that azadirachtin is having a direct effect on and time dependent manner after azadirachtin treatment
cells of endocrine target tissues over and above those [Nasiruddin and Mordue (Luntz), 1993a; Cottee, 19841.
indirect effects brought about by perturbations in the Ultrastructurally the mitochondria appear swollen and
hormone levels themselves. It is known that azadirachtin often burst although the arrangement of the myofibrils
inhibits mitosis in germinal discs and other areas of rapid does not appear to be disrupted [Nasiruddin and Mor-
mitosis, e.g. epidermal cells, midgut epithelial cells, due (Luntz), 1993a]. Food passes more slowly through
ovary, testis, etc. However it is not always possible to the gut of locusts treated with azadirachtin [Mordue
remove the endocrine control element in the process. It (Luntz) et al., 19851, treated nymphs which die a~ tne
would appear that azadirachtin must have effects that moult are unable to swallow enough air to crealr :ne
916 REVIEW

correct hydrostatic pressure for ecdysis [Mordue (Luntz) within 4 days of treatment [Mordue (Luntz) et al., 1985;
et zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
al., 19861 and hindguts of such insects in vitro are in Cottee, unpublished]. A separation of such secondary
a more flaccid and extended condition than are controls starvation effects from the insect growth regulatory
(Helliwell, 1991). In viva observations show that guts of effects has been achieved, however, by treating insects
treated insects lack tone, the midgut to hindgut junction after the critical weight for moulting has been achieved.
is flaccid, substantial amounts of fluid are found Thus moult inhibition has been shown to be due to
throughout the length of the gut and co-ordinated azadirachtin treatment only, by using mid-Vth instar
peristalsis may be lacking. Initial preliminary exper- L. migratoria and comparing them with starved controls
iments linking azadirachtin action with an inhibition of (Sieber and Rembold, 1983), by treating 8-day-old
proctolin stimulation of the hindgut [Mordue (Luntz) M. sexta last instar larvae which are fully grown and
and Evans, 19871 have not been substantiated in more post-feeding (Pener et al., 1988) or by injecting locusts
detailed studies. Neurogenic stimulation of the with relatively low doses of azadirachtin (3 pg/g) and
proctodeal nerve or bath application of proctolin to comparing with semi-starved controls (Subrahmanyam
perfused hindguts from azadirachtin treated L. migrato- et al., 1989).
ria results in normal contractions compared with con- More recently, it has been shown with several lepi-
trols. These are also often larger in dimension due to the dopteran species that avoidance of “primary anti-
significantly relaxed state of the muscle prior to stimu- feedancy” can be achieved quite easily by the methods
lation (Helliwell, unpublished). Bath application of described above, since the consumption rate (CR) of
lop6 M azadirachtin, however, in intracellular record- P. saucia (Koul and Isman, 1991) and M. sexta (Tim-
ings from isolated hindgut superior longitudinal muscle mins and Reynolds, 1992) have been shown to be similar
bundles resulted in a blockage of spontaneously induced to controls after topical application or injection of
action potentials often associated with a slow hypopolar- 0.3-3.0 pg/g or 3-15 pg/g azadirachtin respectively. In
ization of the muscle over a period of some 20 min addition, in both cases there was a significantly lower
[Helliwell, 1991; Helliwell and Mordue (Luntz), 19911. growth rate (GR) of the treated insects indicating that
Miniature excitatory post synaptic potentials (mepsps) some physiological parameter of food utilization had
were not affected in this muscle nor in the extensor tibia been affected by the azadirachtin treatment. The dis-
muscle of the hindleg of S. gregaria in the presence of parity in consumption rate and growth rate is not seen
10m6M azadirachtin (Walther, personal communi- in experiments involving azadirachtin treated diets, as
cation). It would appear that azadirachtin may exert its both parameters (CR and GR) decrease with increasing
effect by reducing muscle tone perhaps “simply” by azadirachtin concentration as would be expected from
affecting the firing pattern of “slow” motorneurones, the involvement of chemoreceptors (Barnby and Klocke,
assuming that tone is produced by a basal rate of firing. 1987; Koul and Isman, 1991). Clearly, insect growth
Azadirachtin at 10e6 or lo-‘M does not affect the inhibitory and antifeedant effects are independent of
resting K+ conductance of the jumping muscle of the each other. Measurements of the nutritional indices; EC1
locust as measured with a two-electrode voltage clamp (efficiency of conversion of ingested food to body sub-
(Walther, personal communication). Clearly further stance); ECD (efficiency of conversion of digested food
work is required in this area before the role of azadirach- to body substance); and AD (approximate digestibility)
tin in affecting muscle function is understood. have been made in several lepidopteran larvae given
(c) Gut phy siology . Azadirachtin has a marked anti- azadirachtin treated diet or after azadirachtin injection
feedant effect on most insect species which is regulated or topical application, with wide variations in effect.
through the mouthpart chemoreceptors. The degree of Nevertheless it would appear that EC1 and ECD are
antifeedancy varies from species to species depending often reduced after azadirachtin treatment (Barnby and
upon the sensitivity of deterrent chemo-receptors to the Klocke, 1987; Koul and Isman, 1991; Timmins and
compound so that in all but a few species (e.g. Reynolds, 1992) whereas AD may stay the same (Koul
R. prolixus; M . sanguinipes) the incorporation of azadir- and Isman, 1991; Timmins and Reynolds, 1992) or
achtin into the diet will involve a compounding effect of increase (Barnby and Klocke, 1987). The reduced EC1
both starvation and direct toxic effects after ingestion. It and ECD must result from a reduction in the efficiency
is not easy to study the IGR effects separate from to convert foodstuffs into growth, perhaps by a diversion
antifeedant effects, as over and above the “primary” of energy from production of biomass into detoxifica-
antifeedancy there is often a “secondary” antifeedant tion, i.e. an increase in costs. The fact that AD was not
effect whereby food intake is reduced after the appli- reduced may well reflect a decreased rate of passage of
cation of azadirachtin in ways which bypass the mouth- food through the gut, as for example in L. migratoria
part chemoreceptors (e.g. topically, injection into the [Mordue (Luntz) et al., 19851, since the AD index relies
haemocoel or cannulation via the mouth into the upon a standard throughput rate in the gut. Also
midgut). In both Vth instar S. gregaria and L. migratoria prolongation of food in the gut may increase AD due to
for example food intake decreases proportionately with increased exposure to digestive enzymes (Barnby and
increasing dose of azadirachtin, injected into the haemo- Klocke, 1987).
coei, so that a 2, 3 or 5 pg/g dose reduces feeding, as Despite no change in the overall ability of treated
measured by faeces production, by some 30,40 and 50% M. sexta larvae to digest and absorb nutrients (AD) it
 REVIEW 917

was shown that the ability of such larvae to handle doubt that T. cruzi in its triatomid host is inhibited
nitrogenous food was significantly impaired (Timmins by azadirachtin treatment of the host (Garcia et al.,
and Reynolds, 1992). Nitrogen absorption efficiency, 1989b; Rembold and Garcia, 1989; Garcia et al., 1991;
AD (nitrogen), was reduced as was the ability of treated Gonzalez and Garcia, 1992). Whereas preincubation of
insects to digest protein. Thus in M. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
sexta azadirachtin the parasite with azadirachtin does not affect its infectiv-
prevents the secretion of proteolytic enzymes and in so ity to mice, and maintenance of T. cruzi in LIT medium
doing reduces growth, even though there is no anti- together with azadirachtin does not affect its develop-
feedancy. In contrast, however, haemolytic activity and ment (unpublished results in Rembold and Garcia, 1989)
cathepsin-like protease activity have been shown not to the development of several T. cruzi strains in R. prolixus
be affected by azadirachtin treatment in R. prolixus and other triatomid vector species is completely blocked
(Garcia et al., 1989b). by the addition of azadirachtin to the blood meal
Disfunction of the insect gut following azadirachtin ( 1.Opg/ml) (Gonzalez and Garcia, 1992). Both the devel-
treatment is now well established and recent work on opment of T. cruzi into epimastigotes and infective
S. gregaria and L. migratoria has demonstrated histo- metacyclic trypomastigotes, which accumulate in the
logically that the midgut epithelial cells show signs of insect rectum prior to transmission on defaecation, and
necrosis seen as a rounding-up and swelling of the cells also the elimination of such stages in faeces and urine are
and organelles, with some vacuolization and cell burst- blocked for up to 120 days after a single treatment of
ing l?Iasiruddin and Mordue (Luntz) 1993a; Cottee, azadirachtin. Engorgement on blood containing azadir-
19841. Concomitant with these effects are a reduction in achtin produces permanent larvae with an associated
the regenerative cells of the nidi and an increase in the inhibition of PTTH and ecdysteroid production. The
connective tissue layer together with many invading poor vector capabilities of such treated insects may be
cells/haemocytes, all occurring in a dose and time depen- due to azadirachtin acting either indirectly (through the
dent manner with azadirachtin injection, in addition to endocrine system) or directly on gut physiology to
the muscle effects described earlier [Nasiruddin and render the microenvironment inhospitable for try-
Mordue (Luntz), 1993a]. It is likely that such disrupted panosome survival. Gut enzymes do not appear to be
tissues would function abnormally and enzyme secretion affected after azadirachtin treatment of R. prolixus,
and nutrient absorption would be disrupted. The cause although the rate of shedding of the outer microvillar
of the cell necrosis is unknown but it has been shown membrane is drastically reduced (Garcia et al., 1989).
that Na/K-ATPase activity in vitro using Anopheles Such membranes are found particularly in the midgut
stephensi midgut homogenates as the enzyme source is cells of R. prolixus forming a covering of the apical cell
not affected by up to 14mM azadirachtin (Billingsley, surface; an outer luminal apical membrane (LAM) and
Djamgoz and Russell, personal communication). a complex extracellular membrane layer (ECML) ex-
(d) Immunosuppression /parasite eflects. Insects are tending further into the midgut (Billingsley, 1990). Para-
subject to attack by micro-organisms and parasites site attachment to these membranes by their flagellum is
and have immune reactions employing a variety of important in both differentiation and multiplication and
mechanisms, including haemocytes for phagocytosis Gonzalez and Garcia (1992) suggest that disruption of
or encapsulation, antibacterial polypeptides and the membranes by azadirachtin treatment may well
prophenoloxidases. In R. prolixus, apart from the render the microenvironment incapable of supporting
prophenoloxidase system where melanin production was and establishing infections of trypanosomes, although
not reduced, it was found that azadirachtin at a concen- they do not describe in detail which membranes are
tration of 1.0 ,ug/ml in the blood meal caused a reduced affected by azadirachtin.
immune response within 6 days in Vth instars challenged (e) Biological rhy thms. Other diverse effects of azadir-
with Enterobacter cloaca B12 strain (Azambuja et al., achtin involve such parameters as biological rhythms
199 1). This immune deficiency could in part be due to the where it has been shown that the neural and endocrine
role of ecdysteroids in the regulation of haemocyte regulated circadian rhythms of Leucophaea maderae and
numbers but may also be due to a general inhibition of M usca domestica can be abolished or split by azadirach-
metabolism by azadirachtin whereby the insect may have tin treatment (Han and Engelmann, 1987; Smietanko
a diminished ability to undertake protein synthesis. and Engelmann, 1989). Similarly in crustacea, injection
Protein synthesis in brain, corpus cardiacum, of Margosan-O@ containing 1.2 pugazadirachtin into the
haemolymph and suboesophageal ganglion (SOG) in fiddler crab, Uca pugilator results in splitting of both
S. gregaria has recently been shown to be affected by circadian and circa lunidian rhythms, sometimes with
azadirachtin treatment (Annadurai and Rembold, 1993). the induction of arrhythmia (Palmer, 1990).
Two-dimensional electrophoresis revealed polypeptide
profiles showing mainly disappearance but also induc- 4. Cellular mode of action
tion of proteins within 3 days of treatment (2.5 rug/g Very little is known of the biochemical mode of action
azadirachtin) of adult female S. gregaria. of azadirachtin. Initial studies carried out on the tissue
When considering insects as vectors of disease it is of specific incorporation of azadirachtin using tritium
importance to know whether azadirachtin affects the labelled 22, 23 dihydroazadirachtin show that some
disease organism within its insect vector and there is no 75% of labelled material is rapidly excreted from the
918 REVIEW

haemolymph within 24 h of injection leaving a residue activity a lipophilic region is important, possibly for
which is permanently bound to different tissue com- transport phenomena.
ponents (Rembold et zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
al., 1988; Garcia et al., 1989a; More detailed studies are being carried out to define
Bamby et al., 1989b). Dry mount autoradiography of a clearer molecular picture of the interactions and
tissues from 8-day-old adult female L. migratoria in- recognition phenomena. A bioassay at the molecular
jected with [3H]dihydroazadirachtin 5 days previously level would greatly assist in such a study and the next
revealed specific and dense labelling of the corpora leap forward in azadirachtin research must surely be a
cardiaca and neurilemma of the brain (Subrahmanyam resolution of its molecular mode of action at the cellular
and Rembold, 1989) and of the Malpighian tubules level. The availability of [3H]azadirachtin of high specific
(Rembold et al., 1988). The relative distribution of label activity and the application of biochemical techniques to
per unit weight of tissue showed that the Malpighian look for binding sites should yield results in the near
tubules retained the great majority of the bound material future. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJI
followed by the corpora cardiaca, gut, then ovary and
rest of the body. Autoradiographs showed the neuro- CONCLUSIONS
secretory axons of the corpora cardiaca and the
Much is now known of the biological effects of
Malpighian tubule cells to be heavily labelled. The latter
azadirachtin. The antifeedant effects are undisputed
were specifically labelled in the region of basal infoldings
although sensitivity between species is profound. On the
of the cells and round the nuclear membrane. The cell
other hand the IGR effects are much more consistent
cytoplasm, apical region and tubule lumen were not
between species. The IGR phenomenon of azadirachtin
labelled. Extraction of labelled material from the
is brought about by significant reductions or delays in
Malpighian tubules showed it to be mainly in its original
ecdysteroid titres of the haemolymph due to a blockage
form so suggesting the presence of high affinity binding
of release of PTTH from the brain-corpus cardiacum
sites at the basal infoldings of the Malpighian tubule
complex. These effects work in conjunction with a
excretory cells (Rembold et al., 1988). Such cells play an
blockage in allatotropin and juvenile hormone titres to
important role in the excretion and clearance of allelo-
give both growth and moulting aberrations and repro-
chemicals and other compounds from the haemolymph,
ductive defects following azadirachtin treatment. In
but the significance in this instance is not understood
addition, overall fitness of the insect is reduced, with
since Malpighian tubule function is not greatly affected
most tissues showing some detrimental response to
by azadirachtin treatment either in uiuo or in vitro. Basal
azadirachtin. The cellular mode of action of azadirachtin
levels but not stimulated levels of secretion are reduced
remains unsolved at present but significant progress
after azadirachtin treatment [Nisbet, Mordue (Luntz)
is expected as interest in commercialisation of neem
and Mordue, unpublished observations].
products intensifies.
Tritiated dihydroazadirachtin is initially excreted un-
The “village pharmacy” philosophy of neem in India,
changed in L. migratoria after which time it is gradually
where it is used for its antiseptic, medicinal and insecti-
converted to more polar metabolites, presumably by the
cidal properties, has now developed into the “Neem
gut, as material extracted from the fat body and
Technology” approach whereby the tree is being pro-
Malpighian tubules show mainly parent compound
moted in tropical and subtropical areas of the world for
(Rembold et al., 1984; Rembold et al., 1988). In
reforestation programmes as a source of wood, fertiliser,
H. virescens, however, metabolism of dihydroazadirach-
cattle feed, oil, shade, medicinals and pesticides.
tin is more rapid and polar products are excreted soon
Azadirachtin and neem products are also being actively
after injection (Barnby et al., 1989b).
studied as environmentally sound insecticides for world-
Structure-activity relationships of azadirachtin have
wide use.
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Acknowledgement-We would like to thank W. Mordue for advice and
encouragement.