Larvicidal effects of a neem (Azadirachta indica) oil formulation on the malaria vector Anopheles gambiae
© Okumu et al; licensee BioMed Central Ltd. 2007
Received: 24 January 2007
Accepted: 22 May 2007
Published: 22 May 2007
Larviciding is a key strategy used in many vector control programmes around the world. Costs could be reduced if larvicides could be manufactured locally. The potential of natural products as larvicides against the main African malaria vector, Anopheles gambiae s.s was evaluated.
To assess the larvicidal efficacy of a neem (Azadirachta indica) oil formulation (azadirachtin content of 0.03% w/v) on An. gambiae s.s., larvae were exposed as third and fourth instars to a normal diet supplemented with the neem oil formulations in different concentrations. A control group of larvae was exposed to a corn oil formulation in similar concentrations.
Neem oil had an LC50 value of 11 ppm after 8 days, which was nearly five times more toxic than the corn oil formulation. Adult emergence was inhibited by 50% at a concentration of 6 ppm. Significant reductions on growth indices and pupation, besides prolonged larval periods, were observed at neem oil concentrations above 8 ppm. The corn oil formulation, in contrast, produced no growth disruption within the tested range of concentrations.
Neem oil has good larvicidal properties for An. gambiae s.s. and suppresses successful adult emergence at very low concentrations. Considering the wide distribution and availability of this tree and its products along the East African coast, this may prove a readily available and cheap alternative to conventional larvicides.
Malaria in sub-Saharan Africa can be controlled by attacking its prime vectors, notably Anopheles gambiae s.l. Since the onset of mosquito control activities in the early 1900s, several challenges continue to hinder efforts to effectively control malaria. These include insecticide resistance, limited access to essential resources (human, capital, and equipment) that affect conventional use of control methods, and insect adaptation and altered behavioural traits, such as exophily and exophagy . The need to develop and incorporate new alternative tools for integrated vector management remains key, where methods to reduce adult biting and control of aquatic stages are used in combination. Use of larvicides, which dates back to as early as 1899, when Ronald Ross applied kerosene on anopheline larval breeding sites in Sierra Leone , is an approach with great potential for future malaria vector control . It is worth emphasizing that although larval control is not widely used in the tropics today, in the past the greatest achievements in malaria control were based on the use of larvicides, for example the eradication of An. gambiae from Brasil  and Anopheles arabiensis from Egypt .
At present, mosquito larvicides include organophosphates, insect growth regulators and microbial larvicides. Current research focuses on microbials such as Bacillus thuringiensis var. israelensis (Bti) and Bacillus sphaericus [6, 7] as well as on botanicals with larvicidal, oviposition inhibiting, repellent or insect growth regulatory effects [8, 9]. Such products contain a multitude of active ingredients with different modes of action, which lessens the chance of resistance developing in mosquito populations. The neem plant (Azadirachta indica) and its derived products have shown a variety of insecticidal properties on a broad range of insect species [10, 11].
Neem products have been shown to exhibit a wide range of effects that are potentially useful for malaria control and include antifeedancy , ovicidal activity, fecundity suppression , insect growth regulation [13, 14] and repellency [15–17]. These effects are frequently attributed to the azadirachtin contents of the products [10, 13]. Recent studies have also demonstrated neem-induced effects on vitellogenesis and severe degeneration of follicle cells during oogenesis in mosquitoes . It has been argued that the pesticidal efficacy, environmental safety, and public acceptability of neem and its products for control of crop pests would ensure its adoption into mosquito control programmes [12, 18]. Presently, however, none of the commercially available neem formulations, which include emulsifiable concentrates (ECs), wettable products (WPs), suspension concentrates, ultra low volume (ULV) and granular formulations, are used for this purpose.
Neem-based products are relatively safe towards non-target biota, with only minimal risk of direct adverse effects on aquatic macro invertebrates resulting from contamination of water bodies with neem-based insecticides [19–21]. In addition, the products are less likely to induce resistance due to their multiple modes of action on insects . Research on neem products for the control of arthropods of medical and veterinary importance has been ongoing for some time. Various studies have focused on the culicine species Culex tarsalis and Culex quinquefaciatus [12, 18, 22, 23], besides Aedes aegypti [24–26]. There have also been studies that assessed the larvicidal potential of neem products on anophelines, notably Anopheles culicifacies, An. arabiensis, An. gambiae and Anopheles stephensi. [9, 22, 27, 28]. The current studies aimed to determine the larvicidal potency of an emulsified neem oil formulation (32% neem oil) against An. gambiae s.s., which is one of the most notorious malaria vectors in sub-Saharan Africa.
Materials and methods
The An. gambiae s.s. larvae used in this study were from a colony established in 2001 at the Thomas Odhiambo campus of the International Centre of Insect Physiology and Ecology (ICIPE), Mbita Point, western Kenya. Mosquitoes were reared under semi-natural conditions in a greenhouse, following standard operating procedures for mosquito maintenance [29–31].
Two experimental formulations, both of which were emulsified concentrates, were tested and compared. The test formulation was an emulsified concentrate, containing 32% neem seed oil (an equivalent of 0.03% azadirachtin), an emulsifier (5%) and 63% isopropanol (solvent). The neem oil was extracted from seeds collected in coastal Kenya. A corn oil formulation with similar solvent and emulsifier contents and proportions was used as the control formulation.
The larvicidal effects of the neem oil formulation were tested on An. gambiae s.s. under greenhouse conditions . Baseline tests were initially run in distilled water to determine the range of lethal doses of the formulation . The maximum dosage of the neem formulation to be applied was determined as 32 ppm, as this resulted in high larval mortality within days. In the main experiment, the larvae were reared in 15 × 20 cm plastic trays. Stock solutions of 1,000 ppm (0.1%) of the two experimental formulations were prepared. Six aliquots were prepared from this stock solution to obtain concentrations of 0.5, 2, 4, 8, 16 and 32 ppm respectively. A fresh stock solution was prepared for each replicate experiment. X ppm in this case refers to X parts of the experimental formulation mixed with (1.000.000-X) parts of the ordinary larval breeding medium. Thus each of the six trays had the ordinary larval rearing medium supplemented with the neem formulation at the different concentrations (i.e. 0.5 to 32 ppm). The same method of application was used for the corn oil formulation. Fifty 3rd to 4th instar larvae were introduced carefully into each tray which were then topped up to 1 L. A negative control was run in freshly collected water from Lake Victoria, routinely used to rear larvae of the colony.
The larvae in all the trays were fed every 24 hrs on equal amounts of Tetramin® Baby fish food using a 'dip stick' (approx. 0.015 g). Tetramin® Baby is a powdered diet and spreads evenly across the water surface. Six replicates were run under the same microclimatic conditions. The mortality of the larvae was monitored every 24 hrs. All the pupae were collected, counted and kept in labeled glass vials capped with cotton wool. The solution with which the pupae were collected was also kept in the same vials such that the pupae remained under the same experimental conditions and concentrations as during the larval stages. The pupae were further monitored for 24 to 48 hours when emerging adults were counted and recorded. Larvae were observed during their entire lifespan, in order to monitor the usually delayed effects of neem products [34, 35].
Percentage cumulative pupation and the mean larval periods were calculated for all concentrations of both the corn oil and neem oil formulation. The mean larval periods for each tray were determined using the following formula:
((A*1) + (B*2) +(C*3) + (D*4) .........+ (H*8))/total number of pupae collected
where A, B, C, D......H are the number of pupae that were collected on days 1,2,3,4 to 8 respectively. The logical argument in this formula is that the larvae which pupated after a particular number of days had actually lived that same number of days. The larval period is summed across the third and fourth larval instars. Growth indices of the larvae were determined as the ratio between percent pupation and the mean larval periods .
Percentage emergence inhibition was determined as 100-A where A was the % successful emergence. Emerging adults were grouped age-wise whenever they emerged, and kept in different cages. Adults emerging from each experimental tray were kept separately. This set up was used to study the sublethal effects of the treatments that might have been carried over from the aquatic stage treatments. Adult mosquitoes were continuously provided with water and a 6% glucose solution dispensed from clean cotton wool daily. These mosquitoes were kept at room temperature and a photoperiod of 12 hrs light/dark. No further neem oil or corn oil treatments were administered in this phase of the experiment. Adult mortality was recorded every 24 hrs. The mean longevity of these adults was calculated for both sexes and oil formulations at the different concentrations. Longevity was calculated as the total number of days lived by a single adult mosquito from emergence to death. Adults emerging from the negative control trays (no oil) were used as the control group. The maximum number of days lived by the emerged adults from each group was also recorded.
The mortality of the larvae (300 per concentration) in both the neem and corn oil formulation were corrected using Abbott's formula , each with the data gained from the negative control. Log-probit analysis  was used to determine the median (LC50) and 90% lethal concentration (LC90). Emergence inhibition (EI) as caused by the two formulations was also corrected with Abbott's formula  and the EI50 and EI90 values determined using probit analyses. The aquatic developmental parameters; growth indices, larval periods and pupation were compared, for both the oil formulations and the negative control, by Analysis of Variance (ANOVA) using Tukey's studentized range test (honestly significant difference test). The longevity of the emergent adults was compared by student t-tests. SAS software was used for the analyses.
Larval mortality and emergence inhibition of An. gambiae s.s. after exposure to neem oil and corn oil formulations.
Larval mortality †
Effects of neem oil and corn oil formulations on pupation, larval growth period and aquatic development rate of An. gambiae s.s.
Larval period (days)
Oil Concentration (ppm)
Effect of sub-lethal concentrations of neem and corn oil formulations on mean (± SD) and maximum (between brackets) adult An. gambiae s.s. longevity (in days).
17.6 ± 1.8
19.6 ± 1.7
19.4 ± 2.1
20.3 ± 2.1
20.9 ± 1.9
21.8 ± 1.6
22.1 ± 2.7
27.3 ± 2.4
25.1 ± 3.0
21.6 ± 2.6
24.6 ± 2.9
26.2 ± 2.9
21.2 ± 2.7
29 ± 1.9
The longevity of both male and female adult An. gambiae s.s. whose larvae and pupae had been reared in a diet enriched with the neem oil formulation was significantly lower than the longevity of the adults whose larvae and pupae had been reared in the corn oil enriched diet (P < 0.001). At all the tested concentrations, the maximum number of days lived by the emergent adults was significantly higher after the corn oil formulation treatments than after the neem oil formulation treatment (P < 0.001). Generally the adults emerging from neem oil formulation treated trays had a shorter life span than emergent adults from either corn oil formulation treated or the untreated trays. There were no observable sub-lethal effects of the corn oil formulation.
Neem oil was an effective larvicide against An. gambiae larvae; it was highly toxic to mosquito larvae and inhibited the development of pupae. The high rates of larval mortality observed at higher concentrations (16 and 32 ppm of the neem oil formulation) within 72 hrs after exposure indicate the high toxicity of the product. The oil is also a potent insect growth regulator (IGR) which led to a 97.5% increase in larval development time and 97.1 % decrease in pupation at 32 ppm when compared to the corn oil and, as a result of the two, there was a 2.2 (8 ppm) to 44.5 (32 ppm) decrease in the growth indices of the insects. These aspects, combined with the emergence inhibition activity ensure that the resultant mosquito population reduction is substantial, even where the larvicidal potential is minimal.
As an emulsifiable concentrate, the neem oil formulation had greatly reduced-sized particles and was evenly mixed within the water column with a few suspended particles on the water surface. The spread of these fine particles probably increased the efficacy of the formulation since An. gambiae s.s. are small particle surface feeders. Larval feeding in this species also entails age-dependent and indiscriminate ingestion of any suitably sized particle , especially by the larger third and fourth instar larvae. When ingested, the neem product particles induce antifeedancy in larvae either by altering the insect's chemoreception or by reducing the food intake due to its toxicity . Growth disruption was exhibited in both the pupae and the larvae. The percentage emergence in most cases was less than the percentage pupation, which suggests some pupal mortality, although this was not different from the control. The emergence inhibition (EI) values depicted with the neem oil formulation treatments were much lower than the respective lethal concentration (LC) values, an indication that the growth disruption activity of the neem product extended to pupal stages. This additional effect of neem oil ensures that it reduces the overall population of the insects beyond its larvicidal action.
The observation that the action of neem oil formulation was slow and that the neem oil formulation increased the mosquito larval periods was not unusual. Mortality of first instar culicids larvae collected after application of 30 mg/L Margosan-O (an oil based neem seed extract) was 100% after 15 days exposure in pool water . Singh  found that a concentration of 32.1 ppm of de-oiled neem seed kernel extract yielded 85% mortality in Cx. quinquefaciatus after 12 days of exposure. Considering that our results were obtained by exposing mosquitoes as third and fourth instars, it is likely that treatment of younger instars would lower LC and EI values, thus providing even greater larvicidal potential. A number of studies have also elucidated this trend. For example Boschitz and Grunewald  studying Ae. aegypti showed age-dependent growth disruption; the sensitivity towards NeemAzal (a neem seed kernel powdered extract with 40% azadirachtin content) decreased with increasing larval age.
The reduction in longevity of the emergent adults indicates that the neem oil formulation had sublethal effects carried over from the larval treatments. This observation is of significance with regard to the afrotropical malaria vector An. gambiae s.l., as a reduction in the average adult daily survival rate is key towards lowering its life-time transmission potential [40, 41].
Since the control formulation of corn oil had very minimal effects on the mosquitoes, it is certain that the effects described are due to the neem oil and not the emulsifier or solvent. The limited mortality exhibited by the corn oil formulation could have been caused by its oil effects. An important issue with regard to using neem-based products as larvicides is the rapid decay of its active ingredients such as azadirachtin when exposed to sunlight and pH changes [11, 13]. Therefore, short term and repeated treatments may be necessary in field applications. This will increase application costs of larval control programmes, but will have the advantage of minimal residual activity and possible side effects.
Comparison of the effects of various neem-based products and their azadirachtin contents on various mosquito species.
Larval Sages tested
Neem oil formulation, An emulsified concentrate made from neem seed oil extracts
0.03% azadirachtin content (32% neem oil)
Larval mortality, IGR and inhibition of adult emergence
LC 50 of 10.68 ppm and EI 50 of 6.44 ppm
3rd and 4th instars larvae and Adults
Neem Azal (Neem seed kernel powdered extract)
34% Azadirachtin A and a total limonoids content of 57.6%
Effects of blood feeding, oviposition, and oocyst ultrastructure
10–1000 ppm treatments impair feeding, oogenesis and oviposition
Adults and oocyst
Lucantoni et al 2006
Neem Azal (Neem seed kernel powdered extract)
40% Azadirachtin content
Larval mortality, molting inhibition
Molting inhibition and larval mortality occurred at all instars
2nd, 3rd and 4th instars larvae
Boschitz. And Grunewald 1994
5 ppm-10 ppm AZ induces antifeedancy
Su and Mulla 1998
Neemix EC 4.5
Azad EC 4.5
5 ppm-10 ppm
Azad WP 10: wettable Product Azad EC 4.5: Emulsifiable concentrate
0.001% 10 ppm
0% hatching rate observed with Azad WP 10 and 46.7% hatching rate with Azad EC 4.5
Su and Mulla 1998
Water based pure neem oil emulsion
An. stephensi Cx. quinquefasciatus
Inhibition of Adult emergence
0.1 ml/l of 5% of the neem oil caused 100% emergence inhibition
Imatures (aquatic stages)
Batra et al 1998
Water based pure neem oil emulsion
Inhibition of Adult emergence
0.4 ml/l of 5% of the neem oil caused 100% emergence inhibition
Imatures (aquatic stages)
Batra et al 1998
Pure neem oil made from seed extracts
Cx. quinquefasciatus Ae. aegypti
0.02–0.1% caused 100% larval mortality
4th instar larvae
Sinniah et al 1994
Neem formulation (name in the original paper is in Arabic)
0.6 ppm-1.9 pm 0.00006% – 0.00019%
Ochlerotatus japonicus Cx. pipiens pallens
Larval mortality and inhibition of adult emergence.
LC50 of 0.342 and 0.367 for Ochlerotatus japonicus and Culex pipiens pallens respectively. 1.9 ppm and 0.6 ppm solutions caused 99% and 75% emergence inhibition respectively
4th Instar Larvae
Mikami and Yamashita 2004
Neem trees are found throughout Africa with a myriad of uses in medicine, pest control, reforestation etc. . The oil can be obtained through pressing (crushing) of the seed kernel both through cold pressing or through a process incorporating temperature controls. The oil yield varies from 25–45% . Its use as a mosquito larvicide will require addition of a surfactant and solvent to ensure equal distribution over water surfaces. Initial experimentation with a neem formulation applied from a knapsack sprayer demonstrated the relative ease with which larval control can take place. Manufacturing of these larvicides can be stimulated through local businesses and does not, unlike current larvicides such as Bti or temephos, require importation from outside Africa. With more decentralized and community-based vector control initiatives underway in Africa [45, 46] neem-based larvicides may present an ideal option to increase these efforts.
The neem oil formulation is a highly effective larvicide for anopheline mosquito vector control. Field application for this product may include high pressure knapsack or ultra-low volume (ULV) sprayers ensuring even application. Further studies are necessary to evaluate the optimum dosages for efficient mosquito control under natural field conditions. Non-target effects on other water inhabiting insects, especially mosquito larvae predators also need further investigation.
The International Center of Insect Physiology and Ecology (ICIPE) is acknowledged for providing essential laboratory and green house facilities. We thank Prof. Steve Lindsay and Dr. Sarah Moore for commenting on earlier versions of the manuscript. Our appreciation goes to the Ifakara Health Research and Development Centre, Tanzania, for logistical and financial support during the final stages of the manuscript development. The work was funded through sponsorship from a private donor from the USA and a VIDI grant (no. 864.03.004) awarded by the Dutch Scientific Organization (NWO) to BGJK.
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