VECTOR CONTROL, PEST MANAGEMENT, RESISTANCE, REPELLENTS
Potential Use of Neem Leaf Slurry as a Sustainable Dry Season
Management Strategy to Control the Malaria Vector Anopheles
gambiae (Diptera: Culicidae) in West African Villages
KYPHUONG LUONG,1,2 FLORENCE V. DUNKEL,3,4 KERIBA COULIBALY,5
1,6
AND NANCY E. BECKAGE
J. Med. Entomol. 49(6): 1361Ð1369 (2012); DOI: http://dx.doi.org/10.1603/ME12075
ABSTRACT Larval management of the malaria vector, Anopheles gambiae Giles s.s., has been
successful in reducing disease transmission. However, pesticides are not affordable to farmers in
remote villages in Mali, and in other material resource poor countries. Insect resistance to insecticides
and nontarget toxicity pose additional problems. Neem (Azadirachta indica A. Juss) is a tree with many
beneÞcial, insect bioactive compounds, such as azadirachtin. We tested the hypothesis that neem leaf
slurry is a sustainable, natural product, anopheline larvicide. A Þeld study conducted in Sanambele
(Mali) in 2010 demonstrated neem leaf slurry can work with only the available tools and resources
in the village. Laboratory bioassays were conducted with third instar An. gambiae and village methods
were used to prepare the leaf slurry. Experimental concentration ranges were 1,061Ð21,224 mg/L
pulverized neem leaves in distilled water. The 50 and 90% lethal concentrations at 72 h were 8,825
mg/L and 15,212 mg/L, respectively. LC concentrations were higher than for other parts of the neem
tree when compared with previous published studies because leaf slurry preparation was simpliÞed
by omitting removal of Þbrous plant tissue. Using storytelling as a medium of knowledge transfer,
villagers combined available resources to manage anopheline larvae. Preparation of neem leaf slurries
is a sustainable approach which allows villagers to proactively reduce mosquito larval density within
their community as part of an integrated management system.
KEY WORDS Azadirachta indica, Mali, larvicide, bottom-up, integrated pest management (IPM)
Billions of dollars are spent each year to combat malaria, however, the problem continues to persist despite the best efforts and intentions (World Health
Organization 2011). It is the disengagement of villagers, however, that may lead many malaria control
programs to fail. Without villagers taking responsibility for their own health management, malaria control
may diminish when researchers and Þeld workers
leave the site.
Insecticide resistance is a major problem with toxicologically based management methods, the conventional approach to manage the vectors of human malaria (MartinezÐTorres et al. 1998, Hargreaves et al.
2001). Dichlorodiphenyltrichloroethane (DDT) was
the Þrst of the synthetic chemicals used to manage
malaria vectors and other adult mosquitoes, and it has
been succeeded by many other synthetic pesticides
1 Department of Entomology, University of California, Riverside,
CA 92521.
2 Current address: Division of Health Sciences, 310 Leon Johnson
Hall, Montana State University, Bozeman, MT 59717.
3 Department of Plant Sciences and Plant Pathology, 119 Plant
Biosciences, Montana State University, Bozeman, MT 59717.
4 Corresponding author: email address: [email protected].
5 lÕInstitut dÕEconomie Rurale, B.P. 16 Sikasso, Mali.
6 Department of Cell Biology and Neuroscience, University of California, Riverside, CA 92521.
(Davies et al. 2007). However, escalating mosquito
resistance to these pesticides as well as health hazards
for humans and other nontarget species continues to
be a problem. DDT, which has been sprayed to control
adult populations in the past and is now being reintroduced in some African countries because of mosquito resistance to other pesticides, has negative impacts on the environment (Turusov et al. 2002,
OÕShaughnessy 2009, Van de Berg 2009, Tren and Roberts 2010). Moreover, DDT resistance has already
been observed in Anopheles populations in Africa
(Balkew et al. 2006). Resistance to the synthetic larval
growth regulator methoprene, as well as to neurotoxins such as pyrethroids and organophosphates, has
been observed (Hemingway and Ranson 2000, Cornel
et al. 2002, Nazni et al. 2005). Even resistance to the
toxic crystalline proteins in the parasporal body of
Bacillus thuringiensis subsp. israelensis de Barjac (Bti),
the common commercial biopesticidal bacterial agent,
has been observed in mosquito populations (Paul et al.
2005). Biological methods, such as predatory animals,
both Þsh and invertebrates, as well as environmental
manipulation, have been used for mosquito control
since the early 1900s along with natural plant products
(Imbahale et al. 2011; Kweka et al. 2011a,b).
0022-2585/12/1361Ð1369$04.00/0 䉷 2012 Entomological Society of America
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JOURNAL OF MEDICAL ENTOMOLOGY
Table 1.
Vol. 49, no. 6
The larvicidal properties of the various parts of Azadirachta indica against mosquito larvae
Formulation
Species
Instar
Duration
Kernal extract (0.15% wt:vol
azadirachtin)c
An. stephensi
C. quinquefasciatus
Ae. aegypti
An. stephensi
C. quinquefasciatus
Fourth
Fourth
Fourth
Third/fourth
Third/fourth
72 h
72 h
72 h
Until emergence
Until emergence
An. gambiae
An. gambiae
Third
Fourth
Until death
Until death
Kernal extract (Neemarind;
0.15% wt:wt active
ingredients)
Supernatant from wood
chips
LC50a or IE50
(mg/L)a,b
1.6
1.8
1.7
0.35
0.69
180b
400b
Reference
Due et al. (2009)
Vatandoost and Vaziri (2004)
Howard et al. (2009)
a
Data from each publication were converted to mg/L for comparison.
IE50 indicates inhibition of eclosion.
BMR and Company, Pune, India.
d
Biotech International Ltd., New Delhi, India.
b
c
The neem tree Azadirachta indica A. Juss is an
evergreen angiosperm belonging to the mahogany
family (Meliaciae) and is used for a variety of purposes. Brought to Africa to abate desertiÞcation four
decades ago, neem was introduced without the understanding of its toxicological properties (Mortimore
1989, Radcliffe et al. 1990). The neem tree produces
over a 135 active compounds that exhibit a host of
beneÞcial functions including antibacterial, antiviral,
antimalarial, spermicidal, and insecticidal (Mulla and
Su 1999, Biswas 2003). Azadirachtin is the most potent
insecticidal component and well-studied neem tree
compound. Other compounds, such as 22,23-dihydronimocinol and des-furano-6a-hydroazadiradione,
both found in the leaves, have insecticidal activity as
well (Brahmachari 2004). Evolution of resistance to
neem and neem-based compounds, like other pesticides, is a possibility. While there has yet to be reports
of resistance to azadirachtin in mosquito populations,
previous research has indicated that green peach
aphids can develop resistance after forty generations
of exposure (Feng and Isman 1995). The onset of
resistance, however, is likely to be delayed by the
presence of a complex of multiple bioactive compounds found in neem tree tissues (Okumu et al.
2007).
Previous work shows that neem compounds are
sustainable larvicides because they do not require
nonrenewable resources, pollute the environment, or
create resistant populations. Neem oil has shown high
toxicity to Anopheles gambiae Giles s.s. larvae as well
as being an inhibitor of pupal development (Okumu et
al. 2007). Likewise, wood chips from the neem tree are
also toxic to larvae, causing inhibition of pupation, and
reduction of adult eclosion (Howard et al. 2009). Field
applications in Niger suggest that neem seed powder
is a cost-effective biopesticide that can suppress mosquito populations (Gianotti et al. 2008). While there
are many projects that have used different parts of
neem trees, neem tree leaves have received little attention. It has been known for over a decade that dried
neem leaves show mosquito larvicidal activity in the
laboratory although poor reporting of methods and
quantiÞable concentrations caused difÞculties in
translating the information for implementation in the
Þeld (Murugan and Jeyabalan 1999). Interestingly,
previous studies showed that fresh leaves exhibit high
toxicity to Culex quinquefasciatus Say but did not show
any mortality-inducing activity in An. gambiae until
pupation (Obomanu et al. 2006). However, it is still
unknown if an increase in fresh neem leaf concentration compared with those concentrations used by
Obomanu et al. (2006) would result in increased mortality of An. gambiae larvae or pupae. LC50 values
indicate that neem seeds are the most potent source
of insecticidal activity and neem wood chips are least
effective (Table 1). However, neem seeds are not
available in the early larval season and the time consuming preparation can discourage regular usage.
Neem leaves provide a year-round alternative that
requires less labor from villagers.
Production of neem leaf slurry is feasible at a village
level. Villagers already have mortars and pestles used
speciÞcally for medicinal purposes. Neem trees are
abundant in villages in West, Central, and East Africa.
The village of Banizoumbou, Niger, has a mean density
of 85 trees in a 500-m radius (Gianotti et al. 2008). The
village of Sanambele (Mali) has been producing neem
leaf slurries for over a decade to help with insect pest
management in crops for preharvest insect management
(Gamby et al. 2001). Therefore, we hypothesized that
neem leaf slurry can be used as a sustainable anopheline
larvicide that villagers can readily make with the tools
and resources they already possess.
Materials and Methods
Study Site. Field research was conducted in the
village of Sanambele, Mali, located 50 km south of the
Malian capital city of Bamako (Fig. 1). The population
of the village during the time of our research was
⬇1,200. Sanambele has two seasons: dry season (midSeptember to mid-June) and wet season (mid-June to
mid-September). Temperatures can reach as high as
45⬚C (113⬚F) during March with a relative humidity of
9 Ð27%. Approximately 0.4 km east of the village is the
Zangolo River, a channel of the Niger River (Figs. 1C
and 2). The Zangolo River Þlls during the wet season
and empties during the dry season leaving residual
pools for larval mosquito populations and development to adulthood. The pools contained similar population sizes of both Culex and Anopheles larvae in
November 2012
LUONG ET AL.: An. gambiae CONTROL USING NEEM SLURRY
1363
Fig. 1. Location map of Sanambele, Mali. Source: Jamie Robertson. (Online Þgure in color.)
2010, but in 2011, only 1 in 10 mosquito larvae were
Anopheles. Higher larval densities were observed
along the shady areas of the perimeter of the pool and
higher densities observed in more turbid pools. These
pools were also the source of clay for village brick
makers whose processing disrupts larval mosquito development.
Village Neem Tree Survey. The neem tree survey
was conducted in the village of Sanambele to assess
the natural resources available to produce the neem
leaf slurries. The survey was conducted on foot over
three days in March 2010. Names of local neighborhoods in the village were provided by the children of
the village. The map was veriÞed by villagers in 2011.
All trees were classiÞed as small, medium, or large.
Small trees are ⬍8 m high. Medium trees are between
8 m and 12 m high. Large trees are taller than 12 m high
(Fig. 3). Trees that were accessible (not on family
property to avoid privacy issues) were categorized as
without seeds, tiny seeds (seeds ⬍5 mm in diameter),
immature seeds (seeds that are larger than 5 mm in
diameter, but still green in color), or mature seeds
(seeds that are larger than 5 mm in diameter and
yellow in color).
Preliminary Bioassay. Third and fourth instars of
An. gambiae for the preliminary experiment in the
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JOURNAL OF MEDICAL ENTOMOLOGY
Vol. 49, no. 6
Fig. 2. Map of Sanambele as of March 2010 (GPS coordinates: 12.308402, ⫺7.846434). Map indicates all local neighborhoods located in the village. Each dot indicates a neem tree. Neem tree density was higher in the schoolyard and near the
river. All 81 neem trees were ßowering and 22 had seeds. All trees with seeds were located inside family compounds. Dry
season residual pools are larval rearing areas for Anopheles gambiae and for Culex spp. as well as dry season brick making areas.
Source: Jamie Robertson. (Online Þgure in color.)
Þeld were collected by villagers from nearly dried-up
pools in the river channel near the village of Sanambele in March 2010. Neem leaves were also collected
by villagers from the riverbank trees. Using a mortar
and pestle similar to those used in food preparation,
60 g of neem leaves were pulverized and homogenized
by villagers in 40 g of water. Twenty grams of homogenized leaves were transferred to 200 ml water, making a concentration of 1 g slurry per 10 ml water. Two
serial dilutions, 3 g slurry per 200 ml water and 17 g
slurry per 200 ml water, were made with 30 and 170 ml
neem slurry, respectively, and 170 and 30 ml water,
respectively (assume 1 g neem leaf slurry is equivalent
to 1 ml neem leaf slurry). The lower 3 g/200 ml slurry
treatment was used immediately. The 17 g/200 ml
slurry, which was included after the initial experiment
was set up to not waste plant material, stood for 3 h
before treatment.
Experimental containers were created by cross-sectionally cutting empty 1 liter water bottles. The control was 200 ml village well water. The second bottle
had 3 g of slurry in 200 ml of water. The third bottle
contained 200 ml of water and a sprinkle of Bti (because of the lack of equipment in the village to accurately measure minute weight; Mosquito Bits, Summit
Chemical, Inc., Baltimore, MD). This was an unreplicated preliminary experiment conducted in collaboration with villagers. The last bottle contained 17 g
neem leaf slurry in 200 ml of water. Eight to 11 larvae
were added to each container. Mortality was assessed
0.5, 1, 1.5, 2, 4, and 24 h after inoculation. Daytime
temperature in the village was ⬇39 and 24⬚C during
the night. The photocycle for that region in Africa was
a photoperiod of ⬇12:12 (L:D) h.
Experimental Insects for Lab Bioassay. Third instar
An. gambiae mosquitoes obtained were from the laboratory of Dr. William Walton, Department of Entomology, University of CaliforniaÐRiverside. Larvae
were maintained at 27⬚ ⫾ 1⬚C in a Percival I-35VL
incubator with a photoperiod of 16:8 (L:D) h. Scotophase began at 22:00 and ended at 06:00. Larvae were
held in 177-ml waxed Dixie cups. Rearing cups had an
approximate density of 200 larvae per cup. Larvae
were fed with TetraMin (Tetra, Blacksburg, VA) tropical Þsh food, once a day.
Neem Leaf Slurry Preparation. Neem leaf samples
used for laboratory assays were harvested in March
2010 from trees along the riverbank of the village of
Sanambele and held in one gallon zip-lock plastic bags.
Neem leaves were frozen within 3 h of being picked
from the tree and remained frozen at ⫺15⬚C at all
times with the exception of the 36 h transit from Mali
to the United States. Bagged leaves were removed
from the freezer an hour before preparation of neem
leaf slurry. Ten grams wet neem leaves were weighed
and ground with a mortar and pestle. Distilled water
was added to the leaf slurry to make a concentration
of 0.1 g neem leaf per 1 ml water. Emulsions were
November 2012
LUONG ET AL.: An. gambiae CONTROL USING NEEM SLURRY
1365
Fig. 3. A large neem tree located in Sanambele, Mali. (Online Þgure in color.)
allowed to stand for 1 h at room temperature (25⬚C)
before setting up the experimental cups.
Grass Control Preparation. To simulate Þeld methods in which neem leaf slurry would be added to pools
of water, laboratory experiments did not Þlter neem
leaf materials. Grass, therefore, was used as a control
to test whether pulverized plant matter diluted in
water induced mortality. Grass was germinated in soil
using Bare Spot Repair seeds (Pennington Seeds Inc.,
Lebanon OR; 34% Prospect Tall Fescue, 34% Not
Stated Tall Fescue, 28.5% Gult Annual Ryegrass, 1.5%
Other Crop Seeds, 1.7% Inert Matter, 0.3% Weed
Seed) obtained from LoweÕs Home Improvement
(Moreno Valley, CA). Soil was obtained from the
University of CaliforniaÐRiverside Physical Plant.
Seeds were planted at least 10 d before the experiment
and placed in a Percival I-35VL incubator at 27 ⫾ 1⬚C.
Seeds and germinated grass seedlings were watered
every other day. The grass slurry was made from 3 g
grass ground by a mortar and pestle and Þlled to 30 ml
water to make a concentration of 0.1 g grass per 1 ml
water.
Determination of Neem Leaf Moisture Content.
Neem leaves vary in moisture content. Water creating
the moisture in the leaf is not an active compound. To
estimate the actual active material in the leaf, it was
necessary to determine the moisture content of the
leaf. Neem leaves were weighed on a Mettler AE 240
balance before and after drying in a Fisher Isotemp
Incubator model 655D set at 57⬚C for 24 h. Leaves
were weighed and then returned to the incubator at
57⬚C for an additional 24 h for further drying to ensure
evaporation of all moisture. Leaf moisture content was
calculated according to the following formula:
(Weightwet leaves ⫺ Weightdry leaves)
Weightwet leaves
Experimental Yield Determination for Neem Leaf
Slurry. The neem leaf biomass per milliliter neem leaf
slurry was determined by evaporating the liquid in the
neem leaf slurry used in the laboratory bioassay. Glass
test tubes were weighed and 1, 2, 3, or 6 ml slurry were
pipetted into the test tubes. After 72 h drying at 57⬚C,
test tubes were weighed again. Grams neem leaf wet
biomass per ml neem leaf slurry was calculated with
the formula:
(Weightdried slurry⫹glass ⫺ Weightglass)/
(1 ⫺ [% moisture content/100])
# ml neem leaf slurry added to
tube prior to drying process
Laboratory Bioassay. Forty 60 ⫻ 15 mm glass petri
dishes were washed with soap and water, rinsed with
70% ethanol, and air-dried 1 h in a Fisher Isotemp
Incubator model 655D at 57⬚C. Serial dilutions of
neem leaf slurry were 0.03, 0.1, 0.3, and 0.6 g neem leaf
slurry per 15 ml water formulated by adding 0.3, 1, 3,
and 6 ml slurry, respectively, with remaining water to
make a total of 15 ml in each petri dish. Additional
bioassays included 0.2 g neem leaf slurry in 15 ml water
concentration by adding 2 ml neem leaf slurry in 13 ml
water. Each petri dish held six third instar An. gambiae
and each concentration had Þve dishes for a total of 30
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JOURNAL OF MEDICAL ENTOMOLOGY
Vol. 49, no. 6
Table 2. Probit analyses of mortality, LC50 and LC90, in a petri dish environment using a distilled water-based leaf slurry of neem,
Azadirachta indica, against third instar larvae of Anopheles gambiae (n ⴝ 30)
Assay
date
Hours after
inoculation
Slope ⫹ SE
(mg/L)
Pearsons
␹2
LC50 (95% CI)
(mg/L)a,b
LC90 (95% CI)
(mg/L)a,b
17 May
72
7.097 ⫾ 1.639
0.071
26 June
72
7.598 ⫾ 1.259
0.077
6 Aug.
72
4.665 ⫾ 0.747
0.082
8,825 ⫾ 844
(7,279Ð10,802)
9,122 ⫾ 710
(7,867Ð10,842)
9,388 ⫾ 933
(7,644Ð11,517)
15,212 ⫾ 1,566
(12,786Ð19,543)
15,086 ⫾ 1,458
(12,858Ð19,190)
19,101 ⫾ 1,910
(15,522Ð24,750)
a
b
1 mg/L wt:wt is equivalent to 1 ppm. These mg/L values are for the combination of total neem leaf slurry (supernate ⫹ leaf material).
Concentrations ranged between 1,061 and 21,224 mg/L.
larvae per concentration per experiment. Bioassays
were repeated three times: May, June, and August
2010.
Three control groups were set up: water without
TetraMin; 0.07 g TetraMin daily; 3 ml grass slurry plus
12 ml water. Larvae were maintained at 27⬚C in a
Percival I-35VL incubator with a photoperiod of 16:8
L:D h. Behavior and mortality were assessed at 0.25,
0.5, 1, 2, 4, 24, 48, and 72 h postapplication. Assessment
was made with visual observations. If the larva was
immobile, a tactile stimulus was applied to the larval
siphon (one to three probes). A larva was determined
to be “normal” if it displayed an immediate escape
response away (downward) from the stimulus when
its siphon was probed; “moribund” if larva either failed
to ambulate (swim) within 2 s of probing or displayed
abnormal motility (backwards or sideways swimming); “dead” if larva exhibited no ambulatory or peristaltic movement. Behavior and mortality of individuals that molted to the fourth instar and pupa during
the 72-h period were also reported. Pupae were removed from the petri dishes. Normal pupae remained
at the surface of the water and exhibited escape behavior when the surface tension was broken. Abnormal pupae preferred the bottom of the dish even when
unstimulated or had an inverse spatial orientation (upside-down when stationary and swimming).
Statistical Analyses. For laboratory bioassays, LC50
and LC90 values (milligrams pulverized neem leaves/
liter distilled water) with 95% CIs were calculated
using a probit analysis (MiniTab version 16.1.0 survival
probability tool). AbbottÕs formula (Abbott 1925) was
applied to the May 2010 trial to adjust to the 13.3%
mortality in the control containing TetraMin. AbbottÕs
formula was not applied to the experiments in June
and August because control mortality was zero.
Results
Neem Tree Survey. The survey (Fig. 2) indicated
that there were 81 neem trees within and locally surrounding the village of Sanambele. Trees were scattered throughout the village with a majority found in
areas accessible to the public. The highest density of
trees was located near the riverbank slightly outside of
the village (16 trees) and in the school courtyard (24
trees), all of which were small trees. In the village,
there were 51 small trees, 25 medium trees, and 5 large
trees. Tree clusters contain small and medium size
trees. The few large trees in the village stood alone.
During March 2010, the neem trees varied in both the
stages of ßowering and seed production. Only one tree
in the entire village had mature seeds, although many
trees had immature seeds.
Preliminary Bioassay. Percent mortality increased
over time in all experimental groups. There was no
mortality during 24 h in the controls. The higher concentration of neem leaf slurry (17 g neem leaf slurry
in 200 ml water) caused mortality to 75% of the larvae
within 1 h of inoculation while the lower neem concentration (3 g neem leaf slurry in 200 ml water)
showed more mortality between 4 and 24 h. Mortality
in the Bti treatment (18%) Þrst appeared within 2 h of
treatment. At 3 h posttreatment, mortality was: 0% (3
g neem slurry/200 ml local well water treatment); 27%
(Bti); and 75% (17 g neem slurry/200 ml well water).
At 4 h posttreatment, mortality was: 0% (3 g neem
slurry/200 ml local well water treatment); 64% (Bti);
and 75% (17 g neem slurry/200 ml local well water).
At 24 h posttreatment, mortality was: 33% (3 g neem
slurry/200 ml local well water treatment); 73% (Bti);
and 88% (17 g neem slurry/200 ml well water).
Moisture Content and Yield Determination. Neem
leaf moisture content was 68.1 ⫾ 2.72 SD % (n ⫽ 6).
In the experimental yield determination 1 ml of leaf
slurry contained 17.26 ⫾ 1.92 SD mg (n ⫽ 10) of dry
leaf mass. Therefore, each milliliter of pipetted neem
leaf slurry contains only 54.11 ⫾ 6.35 SD mg of neem
leaves (wet weight).
Laboratory Bioassay. LC50 values of the three bioassays were consistent across the three experiments
(Table 2). The 72-h LC50 values were 8,825, 9,122, and
9,388 mg/L for May, June, and August 2010 experiments, respectively. Overlap of SEs indicates that LC50
values are equivalent. The 72-h LC90 values showed
less agreement over the three experiments. Experiments in May and June have LC90 values at 15,212 and
15,086 mg/L respectively, with overlapping standard
error. The August 2010 experiment, however, had a
LC90 value of 19,101 mg/L and was 27% higher than
the other 2 d. One hundred percent mortality was not
reached in the highest concentration in the August
2010 experimental run, unlike the other two previous
runs. Preliminary visual observations with fourth instars indicated that affected pupae had inverse spatial
orientation and died before sclerotization.
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LUONG ET AL.: An. gambiae CONTROL USING NEEM SLURRY
Discussion
Concentrations of active ingredients in the neem
tree vary with the environment, including water availability, hours of sunlight, and ambient temperature
(Singh 2009). Aliero (2003) reported Anopheles species were twice as susceptible to the leaves as the bark
(Table 1). In contrast, Howard et al. (2009) reported
IE50 (inhibition of eclosion) values for neem wood
chippings against third instar An. gambiae to be 180
mg/L, three magnitudes less than our LC50 value.
However, Howard et al. (2009) allowed their experiments to proceed until death or emergence while we
ended our experiments at 72 h. Howard et al. (2009)
used only the supernatant while we used the entire
leaf, thereby mimicking the actual village situation.
Plant parts, such as cells and organelles, contributed to
the weight used to calculate our concentration value.
Dua et al. (2009) and Vantandoost and Vaziri (2004)
both used kernel (seed) extract formulations, which
have highly concentrated active ingredients.
Despite the lower concentration of active components than other plant parts, neem leaf slurries have
distinct beneÞts over using parts such as neem seeds
or kernels (Mordue and Nisbet 2000) that are not
available year round. During March 2010 (dry season),
of the 81 village neem trees only one tree had mature
seeds. As a larvicide, neem only reduces larval populations and future adult populations, not current adult
populations already established (National Academy of
Science 1992). Dry season management is important
in reducing adult populations during the rainy season
and neem leaves provide an alternative when neem
seeds are not available. Equally important is to treat
pools immediately after the Þrst rain (usually June) of
the new wet season because reappearance of aestivating adult female An. gambiae will give rise to a new
generation of larvae (Lehmann et al. 2010). Treatment
must be coupled with a village-based monitoring process for anopheline larvae with an appropriate treatment threshold. We used one An. gambiae larva per
250 ml water sample in one of 10 samples as an appropriate treatment threshold. Neem leaf preparation
requires less labor and time than other neem-based
interventions. Neem leaf slurry can be created from
fresh neem leaves immediately with typical village
equipment, for example, a medicinal mortar and pestle. Neem seed preparation, however, requires harvesting of fruits, removal of pulps, and spreading out
and drying before seeds can be milled by a mortar and
pestle. It would take 3 d per week of labor for one
villager during the wet season that is a busy planting
and cultivating season for villagers (Gianotti et al.
2008).
The village of Sanambele has a large quantity of
neem trees to produce neem leaves for sustainable
usage as a larvicide. There are 81 neem trees in and
nearby the village, similar to the village of Banizoumbou, Niger that initiated successful village-based An.
gambiae larval management with neem kernel powder
(Gianotti et al. 2008). In March 2010, only a few pools
in the dried up riverbed and a few pools as a result of
1367
trapped water from the shower stallsÕ efßuent in the
village were observed. The 81 neem trees in the village
will be able to provide the neem leaves necessary for
mosquito management each year. In March 2009, the
only pool located in the Zangolo riverbed near the
village was ⬇15 ⫻ 6 m. We predict that 17.4 kg of neem
leaf will be required to treat a 15 ⫻ 6 m pool per
treatment. Treatment should be is limited to 0.3 m
along the perimeter of the pool where larval density
was highest. The number of neem trees in the villages
will continuously provide resources well into the future. Leaf quantity required to achieve the LC90 is
estimated at 500 g per tree, ⬍1% of a medium treeÕs
overall leaf biomass. However, 2009 and 2011 dry season pools were larger than pools in other years (2007,
2008, and 2010). Along with the 30 large or medium
trees present in the village, there are 51 small trees for
future use. These small trees will mature to medium
size trees in 10 yr to provide future leaf resources.
Neem trees are a renewable resource that can be easily
planted.
In June 2011 when the treatment threshold was
reached, villagers tested the neem leaf slurry production process. To achieve the LC90 concentration, villagers used 500 g from each of 34 small and medium
trees in Sanambele June 2011. Authors observed 32
villagers (men, women, and children) make the slurry
in 2.25 h. Villagers concluded that if two or more
mortar and pestle sets were used to grind leaves, large
scale production of neem slurry treatment could be
completed in an hour. Previous experiments with a
water-based solution made from ground neem kernel
on sorghum seedlings indicated neem persistence lasts
⬇72 h (Radcliffe et al. 1990). Under laboratory conditions (27⬚C and 12:12 photophase:scotophase), mosquitoes hatch and develop into third instar within 8 Ð9
d. Given the 3-d persistence of neem leaf slurry and
the developmental rate of mosquito larvae, we, therefore, recommended applying neem leaf slurry every
12 d to eliminate all third instars and younger mosquitoes. Other studies indicate the resulting mosquito
population decline may be enhanced by oviposition
deterrence effect of neem leaf slurry (Murugan and
Jeyabalan 1999).
Even though neem leaf slurry shows anopheline
toxicity in a laboratory setting, it is important to consider barriers to adoption at the village level by villagers. In Sanambele, topical application of neem leaf
slurry has been used as a preharvest pest management
strategy since 2000 (Gamby et al. 2001). More important, however, understanding villagersÕ cultural practices is required to effectively transfer knowledge
from Western science. Storytelling in Bambara villages, such as Sanambele, is the most effective medium
to convey knowledge (Ba 1972). We presented the
malaria lifecycle as a story of how the malaria-causing
protozoan travels with humans and mosquitoes as it
changes forms and multiplies. We also showed points
of intervention where the mosquito lifecycle could be
disrupted. Villagers quickly made use of commercial
Bti gifted in 2008 because they already understood the
importance of dry season larval management from the
1368
JOURNAL OF MEDICAL ENTOMOLOGY
mosquito-protozoan lifecycle stories. Another village
cultural practice that must be considered is the use of
standing water to make mud bricks, the primary village
building material. This process creates a conundrum
for malaria management. Brick makers use standing
water in riverbed pools to create their bricks during
the dry season. This process physically removes larval
rearing pools. In the wet season, however, when the
Zangolo River is a swift ßowing channel, the river is
not favorable for brick making or for An. gambiae egg
deposition. Brick makers often create pools of standing water in clay soil near the river to make bricks
during the wet season. These pools become temporary
rearing habitats until used by the brick makers.
Long-term engagement and use of the holistic process differentiates this project from other plant-based
pesticide projects. First deÞned by Savory and ButterÞeld (1999), the holistic process is a formal, deliberate method of determining oneÕs own or oneÕs villageÕs life values, current resources, and sustainable
future resources to achieve or maintain those life values. Neem leaf slurry was a current resource that
helped to achieve the villageÕs life value of freedom
from malaria. Use of the slurry was readily adopted in
village of Sanambele and has the potential for adoption
in neighboring villages. The use of neem leaf slurry
grew out of a holistic process that began in 2005 and
was revisited annually each year since.
Local dialogue between neighboring villages is the
key to anopheline management. Neighboring villagers
can serve as reservoirs for human malaria and mosquitoes in the area could reinfect inhabitants of Sanambele. Infected mosquitoes can traverse the distance
to Sanambele from infected neighbor villages or travel
with villagers moving between villages. The distance
between Sanambele and Bogola, a neighboring village
to Sanambele, is 4 km, while a blood-fed female An.
gambiae can ßy up to 10 km (Kaufmann and Briegel
2003). Likewise, infected villagers from surrounding
villages can unknowingly carry the malaria protozoan
back to Sanambele. Two of the authors visited Bogola
and found that it had a malaria epidemic the previous
yearÕs wet season (2009). Sanambelean villagers decided, without the researchersÕ interventions, to share
the malaria-mosquito lifecycle story with their neighbors. Sanambeleans and their neighbors identiÞed
standing bodies of water, and used Bti to treat larval
habitats the following year. Bti, however, is not sustainable in these remote subsistence villages as it requires cash to purchase. Like most Malian villages,
neem trees are abundant in Bogola. Neem leaf slurry
is a capable substitute that can disseminate through
the villages.
This bottom-up approach to malaria management
could be disseminated from one village to another by
storytelling or other forms of farmer-to-farmer knowledge transfer. We, therefore, accept our hypothesis
that neem leaf slurry, along with knowledge transferred by storytelling, can be a sustainable, long-term,
dry season, village-based management for An. gambiae. We further predict that with village-led dry season larval management communities already under-
Vol. 49, no. 6
standing lifecycles of the malaria protozoan and
mosquito vector, malaria incidence will be reduced.
Success will be measured by the continuance of the
use of neem leaf slurry after researchers leave the site.
Acknowledgments
We thank William Walton and Margaret Wirth (University of CaliforniaÐRiverside [UCR]) for providing the Anopheles gambiae larvae for laboratory bioassays. We would also
like to thank Hawa Coulibaly, Bourama Coulibaly, and Karim
Coulibaly of Sanambele, Mali for their support in the villages
as well as Mickolai Balibay for his assistance in the laboratory
(UCR). Support was provided by the Montana Agricultural
Experiment Station (Dunkel). This publication was made
possible, in part, through support provided by USDA National Institute of Food and Agriculture, Higher Education
Challenge Grant 2007-38411-18609 (Dunkel) and the USAID
through the OfÞce of Higher Education for Development
(HED) under Cooperative Agreement HNE-A-00-9700059-00 (Dunkel). The opinions expressed do not necessarily reßect the views of USDA, USAID, or HED.
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Received 24 March 2012; accepted 1 August 2012.