Effect of Fruit and Leaves of Meliaceae Plants (Azadirachta

indica and Melia azedarach) on the Development of Lutzomyia

longipalpis Larvae (Diptera: Psychodidae: Phlebotominae) Under
Experimental Conditions
Author(s): Cláudia A. Andrade-Coelho , Nataly A. Souza , Cheryl Gouveia ,
Vanderlei C. Silva , Marcelo S. Gonzalez , and Elizabeth F. Rangel
Source: Journal of Medical Entomology, 46(5):1125-1130. 2009.
Published By: Entomological Society of America
DOI: http://dx.doi.org/10.1603/033.046.0520
URL: http://www.bioone.org/doi/full/10.1603/033.046.0520

BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the

biological, ecological, and environmental sciences. BioOne provides a sustainable online
platform for over 170 journals and books published by nonprofit societies, associations,
museums, institutions, and presses.
Your use of this PDF, the BioOne Web site, and all posted and associated content indicates
your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use.
Usage of BioOne content is strictly limited to personal, educational, and non-commercial
use. Commercial inquiries or rights and permissions requests should be directed to the
individual publisher as copyright holder.

BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers,
academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.
 VECTOR CONTROL, PEST MANAGEMENT, RESISTANCE, REPELLENTS

Effect of Fruit and Leaves of Meliaceae Plants (Azadirachta indica and

Melia azedarach) on the Development of Lutzomyia longipalpis Larvae
(Diptera: Psychodidae: Phlebotominae) Under
Experimental Conditions
CLÁUDIA A. ANDRADE-COELHO,1,2 NATALY A. SOUZA,1 CHERYL GOUVEIA,1
VANDERLEI C. SILVA,1 MARCELO S. GONZALEZ,3 AND ELIZABETH F. RANGEL1

J. Med. Entomol. 46(5): 1125Ð1130 (2009)

ABSTRACT This no-choice, laboratory study focuses on the feeding of dried, ground, homogeneous
powdered, unprocessed fruit and leaves of Azadirachta indica and Melia azedarach to Lutzomyia
longipalpis larvae to determine the effects on their mortality and metamorphosis. A. indica and M.
azedarach fruit and leaves signiÞcantly increased larval mortality in comparison to larvae fed the
untreated, standard diet. A. indica fruit and leaves blocked the molting of the larvae to the fourth instar,
resulting in them remaining as third instars until the end of the experiment. M. azedarach fruit also
blocked the molting of larvae, which remained permanently in the fourth instar. Feeding M. azedarach
leaves resulted in greater molt inhibition. All insects in this group stopped their development as
second-instar larvae. No antifeedant effect was detected for any experimental treatment. The results
indicate that nontoxic, unprocessed materials obtained from A. indica and M. azedarach are potent
development inhibitors of L. longipalpis larvae.

KEY WORDS Lutzomyia longipalpis, Azadirachta indica, Melia azedarach, metamorphosis, ecdysis

American visceral leishmaniasis (AVL) has been con-
sidered one of the most important public health prob-
lems in Brazil, and Lutzomyia longipalpis (Lutz and
Neiva) is identiÞed as the main vector in Brazil and the
Americas as a whole (Deane 1956, Lainson and Rangel
2005). According to the suggested procedures for con-
trolling the disease, the main objectives of vector con-
trol strategies are to disrupt the transmission cycle and
reduce humanÐvector contact. Chemical control us-
ing residual action insecticides has been suggested in
the context of collective protection (Ministério da
Saúde 2003). Although insecticides are known for
their effective control in reducing vector-borne dis-
eases, they also display side effects, such as the de-
velopment of insect resistance and toxicity (Boeke et
al. 2004, Senthil Nathan et al. 2006). Thus, there has
been major interest in developing plant-based insec-
ticides and insect growth regulators (IGRs) that can
be tested in integrated vector control programs, be-
cause they are ecologically safe, biodegradable, and
target speciÞc (Isman 2005, Shaalana et al. 2005).
Some species of Meliaceae are known as potential
sources of insecticidal substances. Extracts from seeds
and leaves of Azadirachta indica, A. juss, and Melia
1 Laboratório de Transmissores de Leishmanioses, Fundação Os-
waldo Cruz, Av., 4365 Manguinhos, Rio de Janeiro, RJ 21045-900,
Brazil.
2 Corresponding author, e-mail: rabbit@ioc.Þocruz.br.
3 Departamento de Biologia Geral, Universidade Federal Flumi-
nense, Morro do Valonguinho S/N, Niterói, RJ 24001-970, Brazil.
azedarach L. have shown insecticidal properties
against several medically relevant insects (Schumut-
terer 1990, 1995; Mordue and Blackwell 1993; Boeke et
al. 2004; Garcia and Azambuja 2004). Recent studies
have focused on the effects of azadirachtin, a highly
oxygenated triterpenoid originally extracted from Me-
liaceae plants, on the development and mortality of L.
longipalpis larvae (Butterworth and Morgan 1968, An-
drade-Coelho et al. 2006).
The current research was undertaken to evaluate
the effect of A. indica and M. azedarach extracts (fruit
and leaves) on the larval growth of L. longipalpis under
experimental conditions.
Materials and Methods
Insects. A colony of L. longipalpis sand ßies from the
municipality (county) of Santarém, Pará State, Brazil,
was maintained in the Laboratory of Vectors of Leish-
maniases, of the Oswaldo Cruz Institute (FIOCRUZ)
according to methods and laboratory conditions de-
scribed by Rangel et al. (1985) and Souza et al. (1999).
Feeding Procedure. Larvae were separated imme-
diately after eclosion and placed in petri dishes with
plaster of Paris as a substrate. At least three batches of
30 Þrst-instar larvae each were formed per study group
and maintained as previously described (Rangel et al.
1985). The larval food for the control groups (standard
diet) was prepared using a mixture of liver powder
(Integalmédica; BZ Ind., São Paulo, Brazil), amino

0022-2585/09/1125Ð1130$04.00/0 䉷 2009 Entomological Society of America

1126 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 46, no. 5

Table 1. Effect of fruit and leaves of A. indica on the mortality and development of L. longipalpis larvae

95% CI
Intermoulting period Treatments n Percent mortality ␹2 OR
⫺ ⫹
1Ð7 d (Þrst instar) Control 3.3 ⫺⫺⫺ ⫺⫺⫺ ⫺⫺⫺ ⫺⫺⫺
8Ð14 d (second instar) 11.1 ⫺⫺⫺ ⫺⫺⫺ ⫺⫺⫺ ⫺⫺⫺
15Ð21 d (third instar) 14.4 ⫺⫺⫺ ⫺⫺⫺ ⫺⫺⫺ ⫺⫺⫺
22 d (fourth instar) 20.0 ⫺⫺⫺ ⫺⫺⫺ ⫺⫺⫺ ⫺⫺⫺
Total 90
1Ð7 d (Þrst instar) Fruit 63.3 72.9 50.1 14.7 171.1
8Ð14 d (second instar) 66.7 0.03a 1.1 0.3 4.7
15Ð21 d (third instar) 95.6 77.3 166.8 35.0 795.2
22 d (fourth instar) 100.0 33.7 ⫺⫺⫺ ⫺⫺⫺ ⫺⫺⫺
Total 90
1Ð7 d (Þrst instar) Leaves 75.6 98.3 89.6 25.8 312.0
8Ð14 d (second instar) 100.0 76.0 ⫺⫺⫺ ⫺⫺⫺ ⫺⫺⫺
Total 90

␹2 test of independence, conÞdence intervals (CIs), and odds ratio (OR). Differences between treated and control groups were considered
statistically signiÞcant when OR and 95% CI were ⬎1.0. Mortality values represent the mean percentages of at least three experiments.
a
␹y2 ⫽ according to Yates.

acids (Probiótica; BZ Ind., São Paulo, Brazil), Þsh food
(Vitormônio; BZ Ind., São Paulo, Brazil), and pollen
grains (Herbarium; BZ Ind., Paraná, Brazil). This lar-
val diet (standard diet) is routinely used for the main-
tenance of sandßy colonies) and was offered to larvae
at daily dose of 2 g/cm2 in petri dishes (Souza et
al.1999). Experimental groups were fed different diets
prepared as described below. The food for both con-
trol and experimental groups was offered routinely
every day until the prepupal stage.
Collection and Acquisition of Leaves and Fruit.
Azadirachta indica fruit and leaves were obtained
from the Empresa Natural Rural (São Paulo, Brazil).
M. azedarach fruit and leaves were harvested during
the spring (October) in the afternoon (1700 hours)
from a tree on the FIOCRUZ campus, located in the
city of Rio de Janeiro, Rio de Janeiro State (22⬚30⬘Ð33⬚
S; 42⬚15⬘Ð19⬚ W). Meliaceae specimens were identi-
Þed according to descriptions provided by others
(Linnaeus 1737, Jussie 1830, Pennington and Styles
1975, Tewari 1992) and by comparison with others
specimens in the Herbarium of the General Biology
Department, Fluminense Federal University, Rio de
Janeiro State.
Preparation of Experimental Diets. Fruit and leaves
from both Meliaceae plants were dried at 35⬚C in a
heater (Soc. Fabbe Ltda., São Paulo, Brazil) and trit-
urated in a commercial blender at maximum speed for
5 min in successive intermittent cycles until obtaining
a homogeneous powder and were offered to the larvae
at 2 g/cm2 in the petri dishes as daily dose.
Biological Evaluation. The biological effects on L.
longipalpis larval development were recorded by ob-
servation of feeding, mortality, intermolting periods,
and stasis of ecdysis. Experimental and control groups
were observed every other day. Every 9 d after starting
treatment, insects from each group were separated
into batches to measure average weight, using an an-
alytical scale, accurate to 0.0001 g. Each experiment
was repeated at least three times.
Data Analysis. SigniÞcance of the results was ana-
lyzed using the ␹2 test of independence (␹2), conÞ-
dence intervals (CIs), and odds ratio (OR) according
to BioEstat 4.0 software (Ayres et al. 2005). Differ-
ences between treated and control groups were con-
sidered statistically signiÞcant when ORs and 95% CIs
were ⬎1.0. Probability levels are speciÞed in the text.
All experiments were repeated three times at least.
Results
Background Experiments. Azadirachta indica and
Melia azedarach fruit and leaves did not interfere with
the increase in body weight in L. longipalpis larvae
compared with control groups fed with standard diet,
indicating that none of the experimental treatments
had an antifeeding effect (data not shown).
Effects of A. indica Fruit and Leaves on Mortality.
Mortality in the control group (fed on standard diet)
was 3.3% at day 7. Meanwhile, groups treated with A.
indica fruit or leaves showed day 7 mortality of 53.3
and 55.6%, respectively. Fruit and leaves gradually
increased the mortality to 77.8 and 70.0%, respectively,
at day 14 after starting treatment, whereas only 11.1%
of the larvae in the control group died. All remaining
insects from both the fruit and leaf groups died as
third-instar larvae, whereas only 20.0% of control
group larvae were dead at 22 d after starting treatment.
Throughout the experiment, signiÞcant differences
were observed in the mortality between experimental
and control groups, because the ORs and 95% CIs were
always ⬎1.0 (Table 1).
Effects of A. indica Fruit and Leaves on Ecdysis.
Table 2 shows that all 55.6% surviving larvae in the
control group were in the pupae stage in 42Ð 44 d after
the beginning of the experiment. All of them also
reached the adult stage (data not shown). In the group
fed with fruit, 46.7% of larvae began molting to the
second instar, and only 22.2% of the larvae began
molting from the second to the third. Likewise, in the
group fed with leaves, 44.4% of the larvae molted to the
second instar and 30% from the second to the third.
For both treatments, all surviving larvae remained in
the third instar until the end of the experiment (day
September 2009 ANDRADE-COELHO ET AL.: EFFECT OF MELIACEAE ON L. longipalpis 1127

Table 2. Effects of fruit and leaves of A. indica on molt and Table 4. Effects of fruit and leaves of M. azedarach on molt and
metamorphosis of L. longipalpis metamorphosis of L. longipalpis

Intermolting periord (days after starting treatment) Intermolting period (days after starting treatment)
Groups Groups
7Ð9 14 Ð16 21Ð23 28 Ð30 35Ð37 42Ð 44 7Ð9 14 Ð16 21Ð23 28 Ð30 35Ð37 42Ð 44
Control 1 (100) 2 (96.7) 3 (88.9) 4 (85.6) PP (80.0) P (55.6) Control (100) (97.8) (86.8) (84.9) (79.0) (40.1)
Fruit 1 (100) 2 (46.7) 3 (22.2) 4 (00.0) PP (00.0) P (00.0) Fruit (100) (36.7) (33.3) (4.4) (00.0) (00.0)
Leaves 1 (100) 2 (44.4) 3 (30.3) 4 (00.0) PP (00.0) P (00.0) Leaves (100) (24.4) (00.0) (00.0) (00.0) (00.0)

First-instar larvae (1), second-instar larvae (2), third-instar lar-
vae (3), fourth-instar larvae (4), pre-pupae (PP), and pupae (P).
Numbers in parentheses represents the mean percentage ⫾ SD of
surviving insect instars of at least three experiments. The value 0,0
signiÞes that no insects reached the developmental stage repre-
sented by the set bar.
First-instar larvae (1), second-instar larvae (2), third-instar lar-
vae (3), fourth-instar larvae (4), pre-pupae (PP), and pupae (P).
Numbers in parentheses represent the mean percentage ⫾ SD of
surviving insect instars of at least three experiments. The value 0,
0 signiÞes that no insects reached the developmental stage repre-
sented by the set bar.

42). SigniÞcant differences were observed in molting

between the experimental and control groups, be-
cause the ORs and 95% CIs were always ⬎1.0 through-
out the observation.
Effects of M. azedarach Fruit and Leaves on Mor-
tality. Mortality in the control group (fed on standard
diet) was 3.3% at day 7, whereas groups treated with
M. azederach fruit or leaves showed 7-d mortality of
63.3 and 75.6%, respectively. Fruit gradually increased
the mortality to 66.7 and 95.6% at days 14 and 21,
respectively, after beginning treatment, whereas only
11.1 and 14.4% of larvae from the control group had
died by this time. All remaining insects from the fruit
group died as fourth-instar larvae, whereas only 20.0%
of the control group larvae were dead at 22 d after
beginning treatment. The group fed on leaves showed
the most signiÞcant mortality (75.6% at day 7) among
Þrst-instar larvae compared with the control group. In
this group, all remaining insects died as second-instar
larvae, whereas only 11.1% of control group larvae
were dead at 14 d after beginning treatment. Through-
out the experiment, signiÞcant differences were ob-
served between the mortality of control and experi-
mental groups, because the ORs were always ⬎1.0 and
the 95% CIs were only ⬍1.0 at 8 Ð14 d in the fruit group
(Table 3).
Effects of M. azedarach Fruit and Leaves on Ecdysis.
Considering the surviving larvae, in the control group,
40.1% reached the pupae stage at 42Ð 44 d after be-
ginning treatment. All of them also reached the adult
stage (data not shown). In the group treated with fruit,
only 36.7% began metamorphosis to the second, 33.3%
to the third, and 4.4% to the fourth instar. In this
treatment, all surviving larvae remained in the fourth
instar until the end of the experiment (day 42). The
most signiÞcant blockage of molting was observed in
the group fed on leaves, where only 24.4% of larvae
molted to the second instar, and all surviving larvae
remained in this stage until day 42. SigniÞcant molting
differences were observed between the experimental
and control groups, because the ORs were always ⬎1.0
throughout the observation and the 95% CI was only
⬍1.0 at 8 Ð14 d in the fruit group (Table 4).
Discussion
In the search for environmentally safe insecticides,
numerous studies have investigated the use of plants
to control vector-borne diseases (Schumutterer 1990,
Al-Sharook et al. 1991, Boeke et al. 2004, Garcia and
Azambuja 2004, Wandscheer et al. 2004, Senthil
Nathan et al. 2006). Plants from the Meliaceae family,
especially the neem tree, A. indica, and the chinaberry
tree, M. azedarach, have probably been studied most
extensively because of their effects against insects,
especially those of agricultural importance. However,
a few studies have focused on medically relevant in-

Table 3. Effect of fruit and leaves of M. azedarach on the mortality and development of L. longipalpis larvae

95% CI
Intermoulting period Treatments n Percent mortality ␹2 OR
⫺ ⫹
1Ð7 d (Þrst instar) Control 3.3 ⫺⫺⫺ ⫺⫺⫺ ⫺⫺⫺ ⫺⫺⫺
8Ð14 d (second instar) 11.1 ⫺⫺⫺ ⫺⫺⫺ ⫺⫺⫺ ⫺⫺⫺
15Ð21 d (third instar) 14.4 ⫺⫺⫺ ⫺⫺⫺ ⫺⫺⫺ ⫺⫺⫺
22 d (fourth instar) 20.0 ⫺⫺⫺ ⫺⫺⫺ ⫺⫺⫺ ⫺⫺⫺
Total 90
1Ð7 d (Þrst instar) Fruit 53.3 55.4 33.1 9.8 112.6
8Ð14 d (second instar) 77.8 32.0 12.6 4.7 33.5
15Ð21 d (third instar) 100.0 83.7 ⫺⫺⫺ ⫺⫺⫺ ⫺⫺⫺
Total 90
1Ð7 d (Þrst instar) Leaves 55.6 59.1 36.3 10.7 123.2
8Ð14 d (second instar) 70.0 12.4 5.5 2.0 15.2
15Ð21 d (third instar) 100.0 92.7 ⫺⫺⫺ ⫺⫺⫺ ⫺⫺⫺
Total 90

␹2 test of independence, conÞdence intervals (CIs), and odds ratio (OR). Differences between treated and control groups were considered
statistically signiÞcant when OR and 95% CIs were ⬎1.0. Mortality values represent the mean percentages of at least three experiments.
a
␹y2 ⫽ according to Yates.
1128 JOURNAL OF MEDICAL ENTOMOLOGY
sects. All the parts of these tropical trees contain bitter
compounds (Van der Nat et al. 1991, Chawla et al.1995,
Boeke et al. 2004) that can interfere in hormonal
processes in insects (Schumutterer 1990, Asher 1993)
by eliciting a variety of effects, including antifeedant,
growth retardation, reduced fecundity, molting dis-
orders, morphogenetic defects, behavior changes, and
inhibition of vectorÐparasite interactions (Saxena et
al. 1984; Zebitz 1986; Phillipson and O⬘Neil 1989; Mor-
due and Blackwell 1993; Su and Mulla 1998a, b, 1999;
Gonzalez et al. 1999; Mulla and Su 1999; Juan et al.
2000; Cortez et al. 2002; Carpinella et al. 2003; Garcia
and Azambuja 2004; Senthil Nathan et al. 2005).
Although the present larval no-choice feeding study
was conducted under laboratory conditions, the re-
sults suggest that A. indica and M. azedarach are able
to block immature development (molting and meta-
morphosis) and increase the mortality of L. longipalpis
larvae. M. azedarach leaves fed to larvae resulted in
greater molt inhibition and second-instar mortality
than M. azedarach fruit or A. indica fruit or leaves.
Overaged immature stages with a greatly extended
instar may survive for several weeks but are unable to
achieve metamorphosis and adult physiology and re-
production. This behavior has been a common Þnding
in azadirachtin-treated hemimetabolous insects, for
example, overaged nymphs of Rhodnius prolixus and
Oncopeltus fasciatus (Garcia and Rembold 1984, Mor-
due et al. 1985, Dorn and Rademacher 1986, Penner et
al. 1989, Van der Horst et al. 1989, Mordue and Black-
well 1993). Some of the results presented here are
quite similar to those recently published by Andrade-
Coelho et al. (2006), who showed overaged larvae of
L. longipalpis induced by the treatment with pure
azadirachtin A. However, the effects of fruit and
leaves from both Meliaceae plants proved to be more
intense than those elicited by azadirachtin alone.
Plant allelochemicals can be quite useful in increas-
ing the efÞcacy of biological control agents, because
plants produce a wide variety of compounds that in-
crease their resistance to insect attack (Murugan et al.
1996, Berenbaum and Zangerl 1998, Senthil Nathan et
al. 2005). Regarding variations in chemical compo-
nents, ⬎50 different bioactive compounds, mainly ter-
penoids, liminoids, lignans, and triterpenoids, have
been reported in A. indica and M. azedarach (Schu-
mutterer 1990, 1995; Cabral et al. 1995, 1999, 2000;
Garcia et al. 2000; Sidhu et al. 2003; Siddiqui et al. 2004;
Zhou et al. 2004, 2005).
In the Þeld, delayed phenology of surviving larvae
and reduced pupal weight are common after treat-
ment with neem products (Zebitz 1984, 1986; Schu-
mutterer 1990, 1995). Because no antifeedant effect
was detected for any experimental treatment in this
study, this cannot serve as a basic explanation for
growth inhibition, suggesting that the compounds
present in leaves and fruit are able to synergistically
disrupt the neuroendocrine system of treated L. lon-
gipalpis larvae. Also, the normal increasing of body
weight along the life cycle indicates that the nutri-
tional status of treated larvae is similar to that obtained
in control insects. Moreover, depending on the insect
Vol. 46, no. 5
species, different doses of azadirachtin A could show
different speciÞc effects on the development. For ex-
ample, the dose of azadirachtin A responsible for molt-
ing inhibition in Rhodnius prolixus is 650 times lower
than the dose related to phago-inhibition and also
mortality (Garcia and Azambuja 2004). This is an
interesting observation, because a polyphagous insect
like the L. longipalpis larva may ingest food from a
wide variety of sources (Forattini 1973, Wermelinger
and Zanuncio 2001, Volf et al. 2002, Feliciangeli 2004,
Nery et al. 2004), thus accepting parts of Meliaceae
plants, which can beneÞt insecticide-based control
programs. Because no extractions were performed,
the materials used here can be classiÞed as unproc-
essed and nonconcentrated. Therefore, toxicity is ex-
pected to be less pronounced than in extracts and
more safe for avoiding accidental intoxication of non-
target organisms (Singh 2003, Boeke et al. 2004, Hos-
sain and Poehling 2006). Additionally, the plants
tested in this study are reported to be environmentally
friendly and normally nontoxic to vertebrates (Al-
Sharook et al. 1991). Moreover, the residual effects of
neem-based products are generally restricted to a few
days but are sufÞcient to obtain reasonable pest con-
trol under tropical and subtropical conditions with
easy biodegradation to nontoxic products (Alkofahi et
al. 1989, Schumutterer 1990, Rao et al. 1992, Agyarko
et al. 2006). Because there are so many organic com-
pounds in whole extracts, more reÞned studies with
active ingredients in know concentrations, as recently
performed by our group (Andrade-Coelho et al.
2006), are necessary to establish the participation of
different substances in the effect of A. indica and M.
azedarach on the development of L. longipalpis, but it
is worth mentioning that the use of unprocessed and
nonconcentrated plant material, where no complex
extraction can be performed because of the lack of
solvents and equipment, would be desirable for a con-
trol program under low-resource conditions in trop-
ical countries (Seyoum et al. 2002, Boeke et al. 2004).
The effects of azadirachtin A on the neurosecretory
system of phlebotomines have been previously de-
scribed, showing neuroregulatory evidence, including
growth inhibition and mortality of L. longipalpis larvae
(Andrade-Coelho et al. 2006). The results of this study
further conÞrm the potential negative effect of A.
indica and M. azedarach extracts against the develop-
ment of L. longipalpis populations.
Acknowledgment
We thank P. Cabello for revision of the data analysis in the
manuscript.
References Cited
Agyarko, K., P. K. Kwakye, M. Bonsu, B. A. Osei, N. A.
Donkor, and E. Amanor. 2006. Breakdown of azadirach-
tin A in a tropical soil amended with neem leaves and
animal manures. Pedosphere 16: 230 Ð236.
Alkofahi, A., J. K. Rupprecht, J. E. Anderson, J. L. Mclaughlin,
K. L. Mikolajczak, and B. A. Scott. 1989. Search for new
September 2009 ANDRADE-COELHO ET AL.: EFFECT OF MELIACEAE ON L. longipalpis
pesticides from higher plants, pp. 25Ð 43. In J. T. Arnason,
P.J.R. Philogeny, and P. Morand (eds.), Insecticides of
plant origin. American Chemical Society, Washington,
DC.
Al-Sharook, Z., K. Balan, Y. Jiang, and H. Rembold. 1991.
Insect growth-inhibitors from 2 tropical Meliaceae: effect
of crude seed extracts on mosquito larvae. J. Appl. En-
tomol. 111: 425Ð 430.
Andrade-Coelho, C. A., N. A. Souza, D. Feder, C. E. Silva,
E. G. Souza, P. Azambuja, M. S. Gonzalez, and E. F.
Rangel. 2006. Effects of azadirachtin on the develop-
ment and mortality of Lutzomyia longipalpis larvae (Lutz
& Neiva, 1912) (Diptera: Psychodidae: Phlebotominae).
J. Med. Entomol. 43: 262Ð266.
Asher, K.R.S. 1993. Nonconventional insecticidal effects of
pesticide available from neem tree (Azadirachta indica).
Arch. Insect Biochem. Physiol. 22: 433Ð 449.
Ayres, M.A.M., Jr., D. L. Ayres, and A. M. Santos. 2005.
Bioestat 4.0 Aplicações Estatṍsticas nas Áreas das Ciências
Biológicas e Médicas. Imprensa OÞcial do Estado do Pará,
Brasil.
Berenbaum, M. R., and A. R. Zangerl. 1998. Chemical phe-
notype matching between a plant and its insect herbivore.
Proc. Natl. Acad. Sci. U.S.A. 95: 13743Ð13748.
Boeke, S. J., M. G. Boersma, G. M. Alink, J.J.A. Loon, A. Huis,
M. Dicke, and I.M.C.M. Rietjens. 2004. Safety evalua-
tion of neem (Azadirachta indica) derived pesticides. J.
Ethnopharmacol. 94: 25Ð 41.
Butterworth, J. H., and E. D. Morgan. 1968. Isolation of a
substance that supresses feeding in locusts. Chem. Com-
mun. 35: 23Ð24.
Cabral, M.M.O., E. S. Garcia, and A. Kelecom. 1995. Lig-
nanes from the Brazilian Melia azedarach, and their ac-
tivity in Rhodnius prolixus (Hemiptera, Reduviidae).
Mem. Inst. Oswaldo Cruz 90: 759 Ð763.
Cabral, M.M.O., A. Kelecom, and E. S. Garcia. 1999. Effects
of the lignan, pinoresinol on the moulting cycle of the
bloodsucking bug Rhodnius prolixus and of the milkweed
Oncopeltus fasciatus. Fitoterapia 70: 561Ð567.
Cabral, M.M.O., P. Azambuja, O. R. Gottlieb, and E. S. Gar-
cia. 2000. Effects of some lignans and neolignans on the
development and excretion of Rhodnius prolixus. Fitot-
erapia 71: 1Ð9.
Carpinella, M. C., M. T. Defago, G. Valladares, and S. M.
Palacios. 2003. Antifeedant and insecticide properties of
a limonoid from Melia azedarach (Meliaceae) with po-
tential use for pest management. J. Agric. Food Chem. 51:
369 Ð374.
Chawla, A. A., M. Kumar, and L. Bansal. 1995. Chemical
constituents and biological activity of nemm-review. In-
dian Drugs 32: 57Ð 64.
Cortez, M.G.R., M. S. Gonzalez, M.M.O. Cabral, E. S. Garcia,
and P. Azambuja. 2002. Dynamic development of
Trypanosoma cruzi in Rhodnius prolixus: role of decapi-
tation and ecdysone therapy. Parasitol. Res. 88: 697Ð703.
Deane, L. M. 1956. Leishmaniose visceral no Brasil. Serviço
Nacional de Educação Sanitária, Rio de Janeiro, Brazil.
Dorn, A., and J. M. Rademacher. 1986. Effects of azadirach-
tin on the moulting cycle, endocrine system and ovaries
in last-instar larvae of the milkweed bug, Oncopeltus fas-
ciatus. J. Insect. Physiol. 32:321Ð238.
Feliciangeli, M. D. 2004. Natural breeding places of phle-
botomine sandßies. Med. Vet. Entomol. 18: 71Ð 80.
Forattini, O. P. 1973. Entomologia médica. Edgar Blucher,
São Paulo, Brazil.
Garcia, E. S., and H. Rembold. 1984. Effects of azadirachtin
on ecdysis of Rhodnius prolixus. J. Insect. Physiol. 30:
939 Ð942.
1129
Garcia, E. S., and P. Azambuja. 2004. Lignoids in insects:
chemical probes for the study of ecdysis, excretion and
Trypanosoma cruziÑtriatomine interactions. Toxicon 44:
431Ð 440.
Garcia, E. S., M.M.O. Cabral, G. A. Schaub, O. R. Gottlieb,
and P. Azambuja. 2000. Effects of lignoids on a hema-
tophagous bug, Rhodnius prolixus: feeding, ecdysis and
diuresis. Phytochemistry 55: 611Ð 615.
Gonzalez, M. S., N. F. Nogueira, C. B. Mello, W. de Souza,
G. A. Schaub, P. Azambuja, and E. S. Garcia. 1999. In-
ßuence of brain and azadirachtin on Trypanosoma cruzi
development in the vector, Rhodnius prolixus. Exp. Para-
sitol. 92: 100 Ð108.
Hossain, M. B., and H. M. Poehling. 2006. Non-target effects
of three biorationale insecticides on two endolarval para-
sitoids of Liriomyza sativae (Dipt., Agromyzidae). J. Appl.
Entomol. 130: 360 Ð367.
Isman, M. B. 2005. Botanical insecticides, deterrents, and
repellents in modern agriculture and an increasingly reg-
ulated world. Annu. Rev. Entomol. 51: 45Ð 66.
Juan, A., A. Sans, and M. Riba. 2000. Antifeedant activity of
fruit and seed extract of Melia azedarach and Azadirachta
indica on larvae of Sesamia monagrioides. Phytoparasitica
28: 311Ð319.
Jussie, A. 1830. Mémoire sur le group des Meliacées. Mem.
Mus. Hist. Nat. Paris 19: 220.
Lainson, R., and E. F. Rangel. 2005. Lutzomyia longipalpis
and the eco-epidemiology of American visceral leishman-
iasis, with particular reference to Brazil: a review. Mem.
Inst. Oswaldo Cruz 100: 811Ð 827.
Linnaeus, C. 1737. Critica botanica. Leiden, Med. Doet.
Soc. Ac. Imp. Nat. Cur., Leiden, The Netherlands.
Ministério da Saúde. 2003. Manual de Vigilância e Controle
da Leishmaniose Visceral. Secretaria de Vigilância em
Saúde, Normas e Manuais Técnicos, Núcleo de Comuni-
cação, Brasilia, Brazil.
Mordue, A. J., and A. Blackwell. 1993. Azadirachtin: an up-
date. J. Insect. Physiol. 39: 903Ð924.
Mordue, A. J., P. K. Cottee, and K. A. Evans. 1985. Aza-
dirachtin: its effect on gut motility, growth and moulting
in Locusta. Physiol. Entomol. 10: 431Ð 437.
Mulla, M. S., and T. Su. 1999. Activity and biological effects
of neem products against arthropods of medical and vet-
erinary importance. J. Am. Mosq. Control Assoc. 15: 133Ð
152.
Murugan, K., R. Babu, D. Jeyabalan, N. Senthil Kumar, and
S. Sivara-makrishnan. 1996. Antipupacional effect of
neem oil and neem seed kernel extract against mosquito
larvae Anopheles stephensi (Liston). J. Entomol. Res. 20:
137Ð139.
Nery, L.C.R., E. S. Lorosa, and A.M.R. Franco. 2004. Feed-
ing preference of the sand ßies Lutzomyia umbratilis and
L. spathotrichia (Diptera: Psychodidae, Phlebotominae)
in an urban forest patch in the city of Manaus, Amazonas,
Brazil. Mem. Inst. Oswaldo Cruz 99: 571Ð574.
Penner, M. P., A.T.M. Van den Broek, W.J.A. Van Marrewijk,
J. M. Van Dorn, D. J. Van der Horst, and A.M.T. Beenak-
kers. 1989. Development of imaginal competence to adi-
pokinetic hormone in Locusta: lipid and carbohydrate and
glycogen phosphorylase activity in azadirachtin-induced
over-aged nymphs. Comp. Biochem. Physiol. 94b: 293Ð
298.
Pennington, T. D., and B. T. Styles. 1975. A generic mono-
graphic of the Meliaceae. Blumea 22: 419 Ð 450.
Phillipson, J. D., and M. J. OⴕNeil. 1989. New leads to the
treatment of protozoal infection based on natural product
molecules. Acta Pharm. Nord. 1: 131Ð144.
1130 JOURNAL OF MEDICAL ENTOMOLOGY
Rangel, E. F., N. A. Souza, E. D. Wermelinger, and A. F.
Barbosa. 1985. Estabelecimento de colônia, em labora-
tório, de Lutzomyia intermedia (Lutz & Neiva, 1912)
(Diptera: Psychodidae: Phlebotominae). Mem. Inst. Os-
waldo Cruz 80: 219 Ð226.
Rao, D. R., R. Reuben, M. S. Venugopal, B. A. Nagasampagi,
and H. Schmutterer. 1992. Evaluation of neem, Aza-
dirachta indica, with or without water management, for
the control of culicine mosquito larvae in rice-Þelds. Med.
Vet. Entomol. 6: 318 Ð324.
Saxena, R. C., H. D. Justo, and P. B. Epino. 1984. Evaluation
and utilisation of neem cake against the brown planthop-
per, Nilaparvata lugens. Natural pesticides from the neem
tree and other tropical plants. Proc. 2nd Int. Neem Conf.
Rauischholzhausen 1983: 391Ð 402.
Schumutterer, H. 1990. Properties and potential of natural
of natural pesticides from the neem tree, Azadirachta
indica. Ann. Ver. Entomol. 35: 271Ð297.
Schumutterer, H. 1995. Biological active components, pp.
35Ð93. In Schumutterer H (ed.), The neem tree. VHC
Press, New York.
Senthil Nathan, S., K. Kalaivani, and K. Sehoon. 2005. Ef-
fects of Dysoxylum malabaricum Bedd. (Meliaceae) ex-
tract on the malarial vector Anopheles stephensi Liston
(Diptera: Culicidae) Biores Technol. 97: 2077Ð2083.
Senthil Nathan, S., M. Young Choi, C. Hoon Paik, H. Yul Seo,
J. Duk Kim, and S. M. Kang. 2006. The toxic effects of
neem extract and azadirachtin on the brown planthopper,
Nilaparvata lugens (Stål) (BPH) (Homoptera:Delphaci-
dae). Chemosphere 72: 1393Ð1400.
Seyoum, A., E. W. Kabiru, W. Lwande, G. F. Killeen, A.
Hassanali, and B.G.J. Knols. 2002. Repellency of live
potted plants against Anopheles gambiae from human
baits in semi-Þeld experimental huts. Am. J. Trop. Med.
Hyg. 67: 191Ð195.
Shaalana, E.A.S., D. Canyonb, M.W.F. Younesc, H. Abdel-
Wahaba, and A. H. Mansoura. 2005. A review of botan-
ical phytochemicals with mosquitocidal potential. Envi-
ron. Int. 31: 1149 Ð1166.
Siddiqui, B. S., F. Afshan, T. Gulzar, and M. Hanif. 2004.
Tetracyclic triterpenoids from the leaves of Azadirachta
indica. Phytochemistry 65: 2363Ð2367.
Sidhu, O. P., V. Kumar, and H. M. Behl. 2003. Variability in
neem (Azadirachta indica) with respect to azadirachtin
content. J. Agric. Food Chem. 51: 910 Ð915.
Singh, S. 2003. Effects of aqueous extract of neem seed ker-
nel and azadirachtin on the fecundity and post-embry-
onic development of the melonßy, Bactrocera cucubitae
and the oriental fruit ßy, Bactrocera dorsalis (Diptera:
Tephritidae). J. Appl. Entomol. 127: 540 Ð547.
Souza, N. A., C. A. Andrade-Coêlho, A. F. Barbosa, M. L.
Vilela, and E. F. Rangel. 1999. The new larval diet for
colonizing phlebotomine sand ßies. Mem. Inst. Oswaldo
Cruz 94: 845Ð 847.
Vol. 46, no. 5
Su, T., and M. S. Mulla. 1998a. Antifeedancy of neem prod-
ucts (azadirachtin) against the mosquitoes Culex tarsalis
and Culex quinquefasciatus (Diptera: Culicinae). J. Vector
Ecol. 23: 114 Ð122.
Su, T., and M. S. Mulla. 1998b. Ovicidal activity of neem
products (azadirachtin) against the mosquitoes Culex tar-
salis and Culex quinquefasciatus (Diptera: Culicinae).
J. Am. Mosq. Control Assoc. 14: 204 Ð209.
Su, T., and M. S. Mulla. 1999. Oviposition bioassay responses
of Culex tarsalis and Culex quinquefasciatus to neem prod-
ucts containing azadirachtin. Entomol. Exp. Appl. 91:
337Ð345.
Tewari, D. N. 1992. Monography of neem. Int. Book Dis-
tributors, Dehra Dun, India.
Van der Horst, D. J., J. M. Van Doorn, M. P. Pener, A.T.M.
Van den Broek, W.J.A. Van Marrewijk, and A.M.T.
Beenakkers. 1989. Development of imaginal compe-
tence to adipokinetic hormone in Locusta: lipophorin
conversions in precoxcene-induced adultiforms and in
azadirachtin-induced over-aged nymphs. Comp. Bio-
chem. Physiol. 92b: 133Ð136.
Van der Nat, J. M., W. G. Van der Sluis, K.T.D. de Silva, and
R. P. Labadie. 1991. Ethnopharmacognostical survey of
Azadirachta indica A. Juss (Meliaceae). J. Ethnopharma-
col. 35: 1Ð24.
Volf, P., A. Kiewegova, and A. Nemec. 2002. Bacterial colo-
nisation in thwe gut of Phlebotomus duboscqui (Diptera:
Psychodidae): transtadial passage and the role of female
diet. Folia Parasitol. 49: 73Ð77.
Wandscheer, C. B., E. J. Duque, M.A.N. Silva, Y. Fukuyama,
J. L. Wohlke, J. Adelmann, and J. D. Fontana. 2004.
Larvicidal action of ethanolic extracts from fruit endocrps
of Melia azedarach and Azadirachta indica against the
dengue mosquito Aedes aegypti. Toxicon 44: 829 Ð 835.
Wermelinger, E. D., and J. C. Zanuncio. 2001. Develop-
ment of Lutzomyia intermedia and Lutzomyia longipalpis
(Diptera: Psychodidae: Phlebotominae) larvae in differ-
ent diets. Braz. J. Biol. 61: 405Ð 408.
Zebitz, C.P.W. 1984. Effect of some crude and azadirachtin-
enriched neem (Azadirachta indica) seed kernel extracts
on larvae of Aedes aegypti. Entomol. Exp. Appl. 35: 11Ð16.
Zebitz, C.P.W. 1986. Effects of three neem seed kernel ex-
tracts and azadirachtin on larvae of different mosquito
species. J. Appl. Entomol. 102: 455Ð 463.
Zhou, H., A. Hamakazi, J. D. Fontana, H. Takahashi, T.
Esumi, C. B. Wandscheer, H. Tsujimoto, and Y.
Fukuyama. 2004. New ring C-seco limonoids from Bra-
zilian Melia azedarach and their cytotoxic activity. J. Nat.
Prod. 67: 1544 Ð1547.
Zhou, H., A. Hamakazi, J. D. Fontana, H. Takahashi, C. B.
Wandscheer, and Y. Fukuyama. 2005. Cytotoxic limi-
noids from brazilian Melia azedarach. Chem. Pharm. Bull.
53: 1362Ð1365.
Received 14 December 2007; accepted 30 March 2009.