Toxic effects of neem products ( Azadirachta indica A . Juss ) on Aedes aegypti Linnaeus 1762 larvae

Université Cheikh Anta Diop de Dakar, Faculté des Sciences et Techniques, Département de Biologie Animale, Laboratoire de Biologie de la Reproduction (L.B.R.), Unité d’Entomologie, de Rickettsiologie, de Bactériologie, et de Virologie (U.E.R.B.V.), Sénégal. Senchim, Groupe Industries Chimiques du Sénégal (Chemical Industry Group of Sénégal), Km 13 Route de Rufisque BP 21236 Dakar Sénégal. Université Cheikh Anta Diop de Dakar, Faculté de Médecine de Pharmacie et d’Odonto-Stomatologie, Laboratoire d’Histologie-Embryologie-Cytogénétique, Sénégal.

Abbreviations: LC, Lethal concentration; Sol, Solvesso; LT, lethal time; F.N.O., formulated neem oil; LT, lethal time ; N.P., neem powder.tionally been affected by an application of synthetic chemical insecticides in solution of oil as emulsion or wettable powder.Nevertheless, repeated use of insecticides leads constantly to the risk of contamination of water used for domestic purposes, animals and humans by pesticides residues.It is also important to note the high cost of chemical pesticides and the development of resistances phenomena of much mosquito species vectors (Sivagnaname and Kalyanasundaram, 2004;Konan et al., 2003;El Hag et al., 1999).
The interest in developing biopesticides with natural origins such as azadirachtin has increased during the recent years because of the drawbacks of synthetic chemical pesticides (Tianyun and Mulla, 1999).Azadirachta indica (neem) belonging to the family of meliaceae, had insecticidal activity (Aliero, 2003).The neem contains several biologically active constituents such as azadirachtin (Naganishi, 1975).The azadirachtin and others related compounds in neem products exhibit various modes of action against insects such as toxics effects (Azmi et al., 1998;El hag et al., 1999;Scott and Kaushick, 2000;Aliero, 2003;Wandscheer et al., 2004;Scott and Kaushick, 2000), antimitotic effects (Salehzadeh et al., 2003), antifeedancy, growth regulation, fecundity suppression and sterilization.In fact, azadirachtin has harmful effects on endocrinien system of an insect of Coleoptera Epilachnus varivestus and caused sterility in Epilachnus varivestus ects females (Schumuthere et al., 1981).Aliero (2003) reported that azadirachtin deteriorated the cuticle of larvae preventing them from moulting.
Based on the encouraging research on azadirachtin and others related compounds for control agricultural pests, it is desirable to investigate the potential usefulness of neem products on mosquito control.In the effort to explore biological effects of neem products, the current research was initiated to investigate the toxicity, histopathologicals and growth retarding effects on last instars larvae of A. aegypti.

Experimental insect
A. aegypti Linnaeus 1762 (Diptera: Culicidae) was taken as experimental insect because of its role as a vector of pathogens of Yellow and Dengue fevers.Larvae of A. aegypti were collected in a plastic bottle 10 x 10 x 7 cm from the vat of culture of mosquitoes (artificials habitats) of the Department of Animal Biology in the Faculty of Sciences in University Cheikh Anta Diop of Dakar.The mosquitoes larvae are fed with bread powder under the laboratory conditions, following a temperature of 24°C +/-1°C, a relative humidity of 80% +/-3% and 11/13 h (light / dark) photoperiod.

Neem products
The test materials were two experimental formulations of neem products (1% Suneem and 1% formulated neem oil), a wettable powder (0.3% neem powder).This neem products as well as their manufacturing process have been provided to us by SENCHIM, an industry of Senegal (Dakar, Senegal) (Figure 1).

Toxic and growth retarding effects tests
The toxics effects are measured using five differents concentrations of each compound selected.These concentrations have varied from 2 -10 mg/l for Formulated Neem Oil and Suneem and from 3 -15 mg/l for Neem Powder.Twenty-five (25) late fourth instars larvae of A. aegypti are introduced in each square plastic bottles 10 x 10 x 7 cm and exposed to various concentrations of neem for either 23 -25°C, with 11 / 13 h (dark / light, photoperiod) and 60 -80% (Relative Humidity), in 500 ml of distilled water.
However, twenty-five (25) larvae of A. aegypti are exposed in 500 ml of distilled water with 0.01 ml of Solvesso (solvent used in formulation of neem products) (solvent control) (i) for treatment with 1% Suneem and 1% formulated neem oil (F.N.O.) or only 500 ml of distilled water in plastic bottle (blank control) (ii) for treatment with neem powder (N.P.).During the experimentation, both larvae are fed with bread powder.The experimentation with the larvae are replicated three times.
Larvae affected by the effect of neem, descended to the bottom of the bottle.Considered like dead are all larvae that do not move or do not answer to an excitation and leaving the ones that can make clear movements.Observations of the mortalities of the larvae are made daily and results noted.
In order to determine the effect of neem in the delay of the larvae growth, the same treatment is applied and the number of nymphs and adults emergence is registered every 24 h.

Data analysis
The results of the bioassay experiments conducted in the laboratory were analyzed as percent mortality and corrected for control mortality with Abbott's formula (Hubert, 1992): The effect of neem products on A. aegypti larvae was analyzed by determining the significant level of mortalities results with ANOVA paired t-test and by comparing toxicity of neem products used in current studies with "Khi-deux" test.

Histopathological effects
The moribund larvae during the treatment and untreated larvae are taken and fixed in Carnoy 2 during 72 h.The dehydratation of tissue is made with two baths of 95% alcohol (ethanol) for 4 or 5 h.The larvae of A. aegypti are placed in two baths of butanol for 4 h.Then the larvae are impregnated by a bath of butyparaffin and included in the pure paraffin.Some blocks are achieved with the bars of Leuckart, and then are built with a scalpel.Some cuts are done with the "Minot" microtom models "Stiasnie".The cuts are glued on a blade.The coloration is made according to the technic of Trichome of Masson Goldner variant described by Martoja and Martoja (1969).At the end of the coloration, the cuts are recovered with a balm of Canada.The blades are dried at the steam-room (60 o C).The observation of the blades is made by the photonic microscope "Motic" connected to a computer, permitting us to get photographs of the midgut cells of the treated and untreated larvae of A. aegypti.Histopathological effects are been studied in anterior, middle and posterior parts of the gut of A. aegypti larvae.

Toxic effects of neem products on A. aegypti
The toxic effects of the neem products on mosquito larvae are presented in Tables 1, 2, and 3.The neem products (1% formulated neem oil, 1% Suneem, and 0.3 % neem powder) extracted from the seed kernel of A. indica have shown larvicidal activity on A. aegypti mosquitoes.
A. aegypti is a hematophagous mosquito and vector of diseases like dengue and yellow fever.We have measured the effect of the neem on four instars larvae of A. aegypti by determining the mortality rates to 50 and 100%.After we have exposed larvae of A. aegypti to various concentrations of the neem products (Suneem, formulated neem oil and neem powder) for 120 h, the mortality of four instars larvae of A. aegypti increased significantly according to the concentrations (ddl = 4; F = -6.708;P = 0.0026 < 5 %) and the time of exposure to Suneem (ddl = 4; F= -1.623; P = 0.017 < 5%) (Tables 1,  2, and 3).After five days of treatment, the average of mortality of A. aegypti larvae is 74.85%.In the control, the highest mortality is reached at 6%. Mortalities of the larvae in all of the concentrations used are very significantly different compared with those obtained in the control (ddl = 4; F = 81, 97; P < 0.001).The fourth instars larvae of A. aegypti are very sensitive to the various concentrations used in 1% Suneem.Suneem was found to be effective against early and late fourth instar larvae of A. aegypti.
The toxic effects of Formulated neem oil tested on A. aegypti larvae are shown in Table 2.The formulated neem oil killed A. aegypti larvae to 50% at the concentration of 8 mg/l (CL 50 ).This larval mortality at 50% occured after a very short time 24 h (TL 50 ).Moreover, A.aegypti larvae had 100% (LC 100 ) mortality in all of the concentrations in this current study at 144 h (TL 100 ).The mortalities of fourth instars larvae of A. aegypti varied significantly with time (ddl = 4; F = -4.305;P = 0.0126 < 5%).This significant variation of the mortality of A. aegypti larvae is in increasing direction following the different concentrations used (ddl = 4; F = -7.522;P = 0.00017 < 5%).The percent mortality produced in all of the concentrations from 24 -144 h was significantly different from that of the control (6%) (ddl = 5; F = -11.58;P = 0.0015 < 5%).Treatment of A. aegypti larvae by The formulated neem oil was found to be very effective against fourth instars larvae of A. aegypti than 1% Suneem.
The LC values (LC 50, 90 and 100) of neem powder on A. aegypti at different intervals of time are given in Table 3.Indeed, no mortality was recorded at 50% in both of concentrations with neem powder.However, a mortality of 46.90% was obtained at 3 mg/l.This mortality occurred after a relative long time of exposure (120 h) of larvae of A. aegypti to the neem powder.Larval mortality at 90% is slightly exceeded with 91.83% at 15 mg/l after 48 h.

Effects on growth and development of A. aegypti
Table 4 show the data obtained for the effects of neem products (Suneem, formulated neem oil and neem powder) on growth and development of A. aegypti larvae.
No adult emergence (0%) was recorded at 10 mg/l for 1% Suneem.However, in this same concentration for formulated neem oil, 5% adults emerged.At 15 mg/l, there was no adult emergence for neem powder.Pupa that emerged at this concentration (15 mg/l), died a few days later.In all treatment with neem powder, no adult emergence was recorded at 50%.The passage of the larvae of A. aegypti from nymphs to adults decreased with treatment of 1% Suneem.In comparison with treatment by formulated neem oil, this passage increas-ed.In all of the treatment, majority of the appeared nymphs died, also showing a very white cuticle.Sometimes no nymphs passed into adults and died at this stage (Example: 0% of adults emergence for 10 mg/l Suneem, 12 mg/l and 15 mg/l Neem powder, respectively).

2A 2B
Figure 2. Longitudinal section part in the midgut (Figure 2b) and in the gastric caecum (Ccg).
(Figure 2A) of control larva of A. aegypti Linneaus 1762.One observes nomals epithelias cells with a nucleus (N) that not affected.These cells were laid on a basal membrane (Bm).Adipose fabric (af) is present.Microvilli (Mv) of bross border (Bb) seem normal.

Histopathological effects
The histopathological effects of neem in gastric caeca, stomach regions were studied.The choice of these regions is justified by the fact that they are directly in contact with toxic element (azadirachtin) of neem compounds.The untreated larvae of A. aegypti Linneaus, 1762 (control) midgut and gastric caecum showed a wellpreserved layer of epithelial cells.The ovoid nuclei are located in the center of the cell.One observes a regularly microvilli border in the midgut and gastric lumens (Figures 2A and B).For the treated larvae of Aedes aegypti, the signs of intoxication began on the level of caecum gastric.The histology of A. aegypti Linneaus 1762 larvae showed on the level of this region morphologicals and serious damages of the epithelial columnar cells (Figure 3).However, muscles, nuclei and microvilli of brush border appeared very normal as well as adiposis fabric (Figure 3).The signs of the intoxication continue at the intestinal level precisely in the middle of the gut larvae.The second signs of intoxication consisted in a perturbation of alimentary flow in the alimentary canal (Figure 4).Some cells appear slightly hypertrophied with a perceptible beginning of vacuolization at the apical level.These vacuoles (arrow in Figure 4) invaded the midgut cells.Sometimes, we noted an enlargment of intercellular spaces (Figure 4).
The third signs of intoxication are perceptible in the posterior part of the gut of A. aegypti larvae.Epithelial cells of the intestine start to burst and we noted a cytoplasmic rejection of cells material that mixed with food column (Figure 4).
On the level of the anterior and posterior intestine, some cells degenerated and showed beaches of lysis (Figure 3).These cells are very advanced in their infection.

DISCUSSION
The neem products show remarkable bioactivity against A. aegypti larvae.Azmi et al. (1998) report LC 50 of Neem   Leaves Extracts (N.L.X.) values against late third instar larvae of Culex fatigans Wild Strain at 390 ppm.In the present finding related in this article, the LC 50 values of neem products were 2 mg/l in 24 h for 1% Suneem, 8 mg/l in 24 h for formulated neem oil (F.N.O.) and 3 mg/l in 96 h for neem powder.This LC 50 of neem products found in this study was lower compared with the results of Azmi et al. (1998).The neem products that Azmi et al. (1998) used in their study are more effective than those used in the present study.This difference can also be due to insects species used, environment of the bioassays and the mode of treatment.Wandscheer et al. (2004) study on different ethanolic extracts of Melia azedarach and A. indica against the dengue mosquitoes A. aegypti Linneaus 1762 in the laboratory observed LC 50 values at 25 and 30°C as 0.166 and 0.152 g%, respectively, for M. azedarach and 0.044 and 0.063 g%, respectively, for A. indica.In this present investigation, the LC 50 for 1% Suneem, formulated neem oil and neem powder were 2, 8 and 3 mg/l.These results appear comparable with those obtained by Wandscheer et al. (2004).However, neem used in this work is slightly more effective than the ethanolic extracts of M. azedarach and A. indica.Naqvi et al. (1991) in determining the toxicity of crude neem extract (NFD) on A. aegypti (PCSIR strain), obtained LC 50 as 0.58 ppm of NFD, 0.625 ppm for nimocinolide and 0.47 ppm for isonimocinolide.In our present results, the 1% Suneem, formulated neem oil and neem powder are slightly more toxic than crude neem extract (NFD), nimocinolide and isonimocinolide.This difference could be due to morphology and physiology of insects species used, change of environment of the bioassays and mode of treatment.Amorose (1995) reports LC 50 values of neem oil and deoiled neem cake against 3 rd and 4 th larval instars of Culex quinquefasciatus Say 1823 at 0.99 and 1.20, 0.55 and 0.72 ppm respectively.The toxicity of 1% Suneem, formulated neem oil and neem powder is more evident compared to the toxicity of neem oil and deoiled neem used by Amorose (1995) against mosquitoes larvae.
El hag et al. (1999) reported that no further larval development of Culex pipiens took place beyond the second instars larvae at concentrations of 400 ppm and above in the Rhazya stricta methanol extract, and only 3.3% successful pupation occurs at 200 ppm.In addition, no adult emergency were observed in any of the two R. stricta extracts except for 3.3% in the ether extract at 400 ppm.However, in our study, no nymphs and adults emergence of A. aegypti Linnaeus 1762 took place at concentration of 10 mg/l for 1% Suneem; all larvae have been killed.Therefore, 1% Suneem is more effective than R. stricta methanol extract.El hag et al. (1999) also reported that all three materials (A.indica, R. stricta, Syzygium aromaticum) tested conferred significant negative influences on larval development to varying degrees, consequently reducing pupation and inhibiting adult emergence.In our study, no nymphs and adults emergence of A. aegypti occured at 10 mg/l for 1% Suneem.Moreover, dead nymphs were covered with a very white cuticle.This whiteness of the cuticle can be due to his non-sclerotisation.
Histopathological effects of neem products are presented in Figures 2, 3, 4 and 5. Neem compounds showed remarkable effects on caecum and gut of A. aegypti larvae.Karch and Coz (1983) and Kallen et al. (1965) reported that the ingestion of Bacillus sphaericus 1593-4 by the larvae of Culex pipiens and by the larvae of Culex tarsalis revealed a perturbation of alimentary flow in the alimentary canal, a hypertrophy or swelling of cells in the posterior part of the gut.Some vacuoles invaded the midgut cells.In an advance stage of infection of the cells, lysosomes appeared in the apical part.A bursting of the cell occurred, followed by a rejection of the cytoplasmic material into the ectoperitrophic space.In our investigation, we reported a morphological and serious damage in epithelial columnar cells and a perturbation of alimentary flow in alimentary canal.In posterior part of gut, cells appear slightly hypertrophied with a perceptible beginning of vacuolisation.Cells start to burst with cytoplasmic rejection of cells material.Our results corroborate the observations of Karch and Coz (1983) and Kallen et al. (1965).We note that a morphological evolution of the intoxication differ from one region to another.Indeed, the cellular damage as well as the degree of intoxication is not the same on all levels in the gastric caeca and the intestinal regions.This fact seems linked to the difference of morphological and physiological cells in oesophagus, gastric and intestine regions.Koua et al. (1998) reported that after the treatment of Anopheles gambiae with aqueous extract of Persea americana, the larvae suffer important damages in the midgut with hypertrophic cells.Then most of the cells lyse, with a rejection of cytoplasmic material towards the lumen gut, between the peritrophic membran and the midgut epithelium.Almost, total cell degeneration is observed.In our investigation, we obtain the same results.Cavados et al. (2004) revealed, in these study of Simulium pertinax larvae treated with Bacillus thuringiensis Serovar israelensis, a serious damage of the epithelial columnar cells.The most characteristic effects were midgut columnar cells vacuolization, microvilli damages, epithelium cell contents passing into Ndione et al. 2853 the midgut lumen and finally the cell death.Our observations are also in agreement with these results.

3Figure 3 .
Figure 3. Longitudinal section part of gastric caecum of A. aegypti Linneaus 1762.This section of larvae treated with neem products showed a morphological lesions of gastric epithelial cells (GEC) with beach lysis (BL).Nuclei (N), muscles (M), adipose fabric (AF) are not affected and seem normal.Microvilli of Brush border (BB) are not affected (B.membr, basal membrane; and C.inf, cells infected).

Figure 5 .
Figure 5. Longitudinal section in posterior part of the gut of A. aegypti Linneaus 1762 showed that some cells appeared to be degenerated (Dég.cell and arrows).Epithelial intestinal cells (Ep.int.cell)showed a beach of lysis (BL).Cells are in an advanced stage of infection (head of arrows).The ventral nerve chain (VNC) appeared to be normal.Bross border (Bb.destr.),microvilli (Mv) and peritrophic membrane (Pmb) are destroyed.Nuclear (N), alimentary flow (Alim.flow) is present in alimentary canal (Alim.canal).Bm = Basal membrane.

Table 4 .
Percentage (%) of successful pupation and adult emergence of 4 th instars larvae of A. aegypti.