Memoire Online - Alternative à la résistance aux pyréthrinoà¯des au Burkina Faso: évaluation des activités insecticides et répulsives-irritantes d'huiles essentielles de plantes locales seules et en combinaison sur les populations de anopheles gambiae et de aedes aegypti (diptera: culicidae)

Huiles essentielles	C1		C3	C4	C5	C6	C7	C8	C9
Cymbopogon nardus (ul)	10	20	30	40	50	60	70	80	90
Ocimum americanum (ul)	90	80	70	60	50	40	30	20	10

NB : A partir d'une dilution en cascade, on obtient les concentrations de 0,1 %, 0,5 % et 1 %,

Annexes 2 :

Essential Oils From Five Local Plants: An Alternative Larvicide for Anopheles gambiae s.l. (Diptera: Culicidae) and Aedes aegypti (Diptera: Culicidae) Control in Western Burkina Faso

Mahamoudou Balbone' ¹, Dieudonne' Diloma Soma², Moussa Namountougou^2,3, Samuel Fogne' Drabo¹, Hamadou Konate' ^2,3, Oumarou Toe³, Koama Bayili², Georges Benson Meda², Roch Kounbobr Dabiré² and Olivier Gnankine¹*

1 Laboratoire d'Entomologie Fondamentale et Applique'e, Unite' de Formation et de Recherche en Sciences de la Vie et de la Terre (UFR-SVT), Universite' Joseph KI-ZERBO, Ouagadougou, Burkina Faso, ² Institut de Recherche en Sciences de la Sante' /Centre Muraz, Département d'Entomologie Médicale, Bobo-Dioulasso, Burkina Faso, ³ Universite Nazi Boni, Institut des Sciences de la Santé, Bobo-Dioulasso, Burkina Faso

Reviewed by: Carlos Manuel Dutok-Sa'nchez, Universidade Federal do Amapa', Brazil Sarita Kumar, University of Delhi, India Paloma Mendonca, University of Vassouras, Brazil

Introduction: Malaria and dengue are two main vector-borne infectious diseases transmitted by Anopheles gambiae and Aedes aegypti, respectively, in tropical and subtropical regions. The concern for environmental safety and the increasing development of resistance to the chemical in main arthropod vectors raises interest in the search for botanicals such as essential oils (EOs) that can be used in vector control.

Specialty section: This article was submitted to Vector Biology, a section of the journal Frontiers in Tropical Diseases

Methods: Larvicidal bioassays were performed according to the WHO standard methods using Ae. aegypti and An. gambiae larvae collected from Bobo-Dioulasso and in the Kou Valley (VK), respectively, two areas located in Houet Province of western Burkina Faso. Kisumu and Bora bora, the susceptible strains of An. gambiae and Ae. aegypti, respectively, were used as controls. OEs extracted from leaves of five aromatic plants, Cymbopogon citratus, Cymbopogon nardus, Eucalyptus camaldulensis, Lippia multiflora, and Ocimum americanum, naturally growing in Burkina Faso were tested. The pyriproxyfen was used as a positive control.

Citation: Balbone' M, Soma DD, Namountougou M, Drabo SF, Konate' H, Toe O, Bayili K, Meda GB, Dabiré RK and Gnankine O (2022) Essential Oils From Five Local Plants: An Alternative Larvicide for Anopheles gambiae s.l. (Diptera: Culicidae) and Aedes aegypti (Diptera: Culicidae) Control in Western Burkina Faso. Front. Trop. Dis. 3:853405. doi: 10.3389/fitd.2022.853405

Results: As a result, the lethal concentrations (LC50) for Ae. aegypti from Bobo-Dioulasso and An. gambiae from VK ranged from 41.9 to 103.8 ppm and 39.5 to 138.1 ppm, respectively. As for _LC90 values, they ranged from 74.6 to 311.3 ppm for Ae. aegypti from Bobo-Dioulasso and from 90.2 to 328.9 ppm for An. gambiae from VK. Among the EOs tested, L. multiflora showed the highest activity against all the strains of An. gambiae and Ae. aegypti larvae. No difference in terms of _LC50 values was found between L. multiflora and pyriproxyfen used as a positive control on An. gambiae larvae. It is not the case for Aedes aegypti populations, where pyriproxyfen remains the most toxic. Overall, An. gambiae populations were the most susceptible to EOs tested as compared to Ae. aegypti populations.

Frontiers in Tropical Diseases | www.frontiersin.org 1 July 2022 | Volume 3 | Article 853405

Conclusion: Our study furthers our knowledge of the larvicidal activity of EOs in the western part of Burkina and opens new avenues in their putative use in vector control strategies.

Keywords: resistance, essential oils, larvicidal activities, lethal concentration, Bobo-Dioulasso

Vector-borne diseases are human diseases caused by parasites, viruses, or bacteria transmitted by vectors. Each year, more than 700,000 deaths occur worldwide due to vector-borne diseases such as dengue, yellow fever, Japanese encephalitis, leishmaniasis, Chagas disease, onchocerciasis, malaria, schistosomiasis, and human African trypanosomiasis (1). Among these diseases, malaria remains the most serious vector-borne disease, affecting between 300 and 500 million people, and 1.4 to 2.6 million deaths annually throughout the world were recorded. More than 40% of the world's population lives in malarious areas (2).

According to the epidemiological bulletin, in the first half of 2018, Burkina Faso recorded 3,501,245 cases of malaria, including 1,002 deaths, for a case-fatality rate of 0.7% (3). The same is true for dengue, where the number of reported cases worldwide peaked in 2019, and all WHO regions were affected (4). In Burkina Faso, an increase in the weekly incidence of dengue cases was noted from week 31 of the year 2017 (5).

Current mosquito control strategies depend primarily on chemical insecticides. The discovery, development, and use of chemicals have reduced the interest in plant products. However, widespread use of these insecticides in public health and agriculture for the control of vector and pest species has favored many concerns such as the increasing physiological resistance in major vector species, environmental pollution, and toxic hazards to human and other non-target organisms due to their broad spectrum of activity (6-9). As a result, there has been an increased interest in developing potential alternative or additional control methods/tools that are effective against the target vector species, which are environmentally safe, biodegradable, and low cost and can be used by individuals and communities in specific situations (10, 11). Therefore, in recent years, various workers have been concentrating their efforts on the search for natural products derived from plants as an alternative to conventional insecticides used in controlling vectors for which resistance was detected (12). Among many natural products, essential oils (EOs) and their constituents have received considerable attention in the search for new pesticides and have been found to possess an insecticidal potential (13).

EOs show several interesting properties. First, they easily penetrate insect cuticles, which increases their bioavailability (14). This property could be of interest if it results in a shortened stay of insects on treated surfaces. Second, EO compounds such as acyclic or monocyclic monoterpenes are small and volatile molecules that might have spatial repellency properties. For example, insect sensilla are specialized for detecting odorants and have been shown to respond to volatile monoterpenes (14). Finally, active compounds in EOs may have a specific mode of action, which makes them good alternatives to the use of pyrethroids.

The EO of Lippia multi!ora is used as a spice, a meal condiment, a drink flavoring (15), and a mouthwash. In addition to insecticidal activities, Abena et al. (16) found analgesic, antipyretic, and anti-inflammatory effects of L. multi!ora oil. Cymbopogon citratus EO has antimicrobial activity against Helicobacter pylori, a bacterium responsible for gastroduodenal diseases (17). Cymbopogon nardus is a medicinal plant because of its analgesic effect and is widely used in gastronomy (18). Also, several studies noticed the insecticidal properties of EO of C. nardus, C. citratus, Ocimum americanum, and Eucalyptus camaldulensis (18-21).

In Burkina Faso, some studies on the insecticidal effects of EOs with mosquitoes in general and mosquito larvae in particular have involved strains restricted to Ouagadougou (21-23). Little is known about the EO susceptibility in Anopheles and Aedes populations in the western part of this country. Moreover, previous studies did not take into account the positive control in the various tests.

Our study aimed at evaluating the larvicidal activities of EOs of five local aromatic plants from Burkina Faso, including C. citratus, C. nardus (DC.) Stapf, E. camaldulensis Dehn, L. multi!ora, and O. americanum (Wild.) A.J. Paton in terms of lethal concentrations _(LC50^and_LC90)upon field populations of Aedes aegypti from Bobo-Dioulasso and Anopheles gambiae from the Valleìe du Kou (VK).

An. gambiae larvae were collected from district number 7 of the Valleìe du Kou (Bama) (11°24'N and 4°24'O), a rice-growing area located 30 km north of Bobo-Dioulasso. Ae. aegypti larvae were collected from tires in various urban settings in Bobo-Dioulasso, the economic capital city of Burkina Faso. Bobo-Dioulasso and Bama are two localities in the province of Houet in western Burkina Faso. The larvae were collected from June to October 2021 and transported to the insectary of the Institute of Research in Health Sciences (IRSS) located in Bobo-Dioulasso where 3 and 4 larval instars were sorted according to Filho (24). Larvae from laboratory reference strains were used as controls. The insectary conditions were 27°C #177; 2°C temperature, 80% #177; 10% relative humidity, and 12-h light and 12-h dark photoperiod.

The five plant species tested, C. citratus, C. nardus (Linn.), E. camaldulensis, L. multi!ora, and O. americanum, were used in

Frontiers in Tropical Diseases | www.frontiersin.org 2 July 2022 | Volume 3 | Article 853405

this current study. They were chosen firstly due to endogenous data provided from informants questioned in areas investigated as well as the data from the literature. These plants were collected from the garden of the Research Institute of Applied Sciences and Technologies (IRSAT). The identification of the plants and extraction of EOs were performed by IRSAT. All the EOs were extracted from the leaves of the test plants by hydrodistillation (HD) using a clevenger-type apparatus and stored in a dark glass bottle at 4°C prior to use. The oils thus obtained were separated from water in the condenser and stored in airtight containers under refrigeration (4°C) till their later use for larval bioassays. The major compounds of these EOs were obtained by gas chromatography-mass spectrometry (GC-MS).

The larval bioassay tests were carried out following the standard WHO larval bioassay test method (25). They were carried out in the laboratory of the Institute of Research in Health Sciences located in Bobo-Dioulasso. Clear plastic cups with capacities of 200 ml each were used for the larvicidal bioassays. An appropriate amount of each EO was dissolved in acetone to prepare 1 ml of stock solution. Fresh stock solutions of each of the above stock solutions were prepared by adding distilled water to produce the required concentrations (50, 100, 150, and 200 ppm). Four replicates were carried out for each test concentration and species of mosquito larvae. Twenty active third and fourth larval instars of An. gambiae and Ae. aegypti in 10 ml of distilled water were transferred into each clear plastic cup that contained 139 ml of distilled water. One milliliter of the mixing solution of EO and acetone was added to each cup that contained 149 ml of distilled water to give a final solution of 150 ml with the desired concentrations. Two replicates of the control were carried out simultaneously with 149 ml of distilled water and 1 ml of acetone. Pyriproxyfen, the reference chemical larvicidal, was used at different concentrations as a positive control.

Analyses were performed by using the statistical software XLSTAT version 2,015.1.01. The LC50, LC90, and the 95% CIs were calculated by probit analysis using the same statistical

software in order to compare the larvicidal potency of the plants and susceptibility of the test mosquito larvae. _LC50 and LC90 values were judged as significantly different between the EOs (p < 0.05) if the CIs did not overlap (14, 26). In all the tests, no control mortality was detected after the 24-h exposure; hence, no correction was required based on Abbot's formula.

The major compounds of the 5 EOs are summarized in Table 1. The EO of C. citratus was found to be rich in geranial (48.1%), neral (35.8%), and myrcene (11%). The EO of C. nardus was predominantly composed of citronellal (41.7%), geraniol (20.8%), and p-elemene (11%). The EO of E. camaldulensis was rich in 1,8-cineole (59.5%) and a-pinene (9.17%). The OE of L. multi!ora was characterized by p-cymene (25.27%), p-caryophyllene (12.7%), and thymol (11.88%). O. americanum EO was characterized by a high percentage of 1,8-cineole (31.22%) followed by camphor (12.73%).

EOs from 5 plant species demonstrated larvicidal activities against susceptible strains and field-collected 3 and 4 larval instars of An. gambiae. Overall, five EOs were the most toxic on 3 and 4 larval instars of An. gambiae susceptible strain. On the susceptible strain larvae of An. gambiae (Kisumu), the _LC50 and LC90 values were from 3.9 to 75.8 ppm and from 29.5 to 193.5 ppm, respectively (Table 2). On field-collected larvae of An. gambiae (VK), the _LC50^and_LC90 values were between 39.5 to 138.1 ppm and from 90.2 to 328.9 ppm, respectively (Table 3).

Regarding _LC50^and_LC90 values, L. multi!ora exhibited the highest larvicidal activity against the 2 strains of An. gambiae (LC50 = 3.9 ppm, LC90 = 29.5 ppm for Kisumu larvae and LC50 = 39.5 ppm and LC90 = 90.2 ppm for VK larvae).

As for O. americanum, it exhibited the weakest larvicidal activity (LC50 = 75.8 ppm; LC90 = 193.5 ppm) for Kisumu larvae, while C.

TABLE 1 | Major compounds of the 5 essential oils tested on larvae of Anopheles gambiae and Aedes aegypti.

Essential oils	Major compounds	Retention time (min)	Percentage (%)
Cymbopogon citratus	Geranial	21.181	48.1
	Neral	21.130	35.8
	Myrcene	12.481	11
Cymbopogon nardus	Citronellal	18.994	41.7
	Geraniol	22.121	20.8
	p-Elemene	24.880	11
Eucalyptus camaldulensis	1,8-Cineol	14.022	59.55
	a-Pinene	10.505	9.17
Lippia multillora	p-Cymene	13.740	25.27
	p-Caryophyllene	25.116	12.70
	Thymol	21.791	11.88
Ocimum americanum	1,8-Cineol	14.022	31.22
	Camphor	17.733	12.73

Frontiers in Tropical Diseases | www.frontiersin.org 3 July 2022 | Volume 3 | Article 853405

TABLE 2 | Median and 90% lethal concentrations and their Confidence Limits (CLs) of plant essential oils against 3 and 4 larval instars of Anopheles gambiae susceptible

strain from Kisumu.
Treatment	LC50 (ppm)	Confidence limit 95%	LC90 (ppm)	Confidence limit 95%	X²(df)	Slope ( #177; SE)
Pyriproxyfen	3.4^a	0.8-5.8	13.6^a	9.5-18.4	16.6	2.2 #177; 0.52
Cymbopogon citratus	49.1^c	42.1-54.8	87.6^c	78.2-102.9	53.6	5.0 #177; 0.69
Cymbopogon nardus	30.7^b	17.9-40.0	74.7^c	63.1-90.8	26.4	3.3 #177; 0.64
Ocimum americanum	75.8^d	65.4-85.5	193.5^d	164.6-244.4	74.8	3.1 #177; 0.36
Lippia multillora	3.9^a	1.5-6.6	29.5^b	22.0-40.8	38.5	1.4 #177; 0.23
Eucalyptus camaldulensis	29.4^b	15.6-40.1	90.3^c	75.3-113.1	27.4	2.6 #177; 0.50

LC (lethal concentration) in ppm (part per million). LC values followed by different letters are significantly different.

citratus showed the weakest larvicidal activity (LC50 = 138.1 ppm; LC90 = 328.9 ppm) on VK larvae.

Furthermore, oils from E. camaldulensis, C. nardus, and C. citratus still showed strong larvicidal activities after L. multi!ora with _LC50 values <50 ppm and _LC90 values <91 ppm on Kisumu larvae. On VK larvae, EOs of C. nardus, O. americanum, and E. camaldulensis showed strong larvicidal activities after L. multi!ora with _LC50 values <140 ppm and _LC90 values <270 ppm.

Only the EO of L. multi!ora gives _LC50^and_LC90 values close to that of the positive control pyriproxyfen against Kisumu and VK larvae.

Based on the overlap of the CIs of the _LC50^and_LC90 values, no significant difference was observed between L. multi!ora and pyriproxyfen on the two An. gambiae strains tested.

All EOs from five (5) plant species exhibited toxicity against susceptible strain larvae and 3 and 4 larval instars of Ae. aegypti field-collected strain. Tables 4 and 5 show the _LC50^and_LC90 values of the EOs of five (5) plants tested against susceptible strains and 3 and 4 larval instars of Ae. aegypti field-collected strain.

As in the case of An. gambiae populations, among the EOs tested, L. multi!ora was the most toxic EO on the two (2) strains of Ae. aegypti tested. Indeed, _LC50^and_LC90 values were 42 and 74.6 ppm, respectively, for L. multi!ora tested on the susceptible strain of Ae. aegypti (Bora bora) and 3 and 4 larval instars of Ae. aegypti field-collected strain from Bobo-Dioulasso and lower for those from other EOs tested where the _LC50^and_LC90 values ranged from 61.5 to 103.8 ppm and 108.8 to 311.3 ppm, respectively.

For the Bora bora strain, _LC50^and_LC90 values were 61.5, 62.4, 91.8, 41.9, and 101.3 ppm and 132, 108.8, 180.9, 68.5, and 188.8 ppm for EOs from C. citratus, C. nardus, E. camaldulensis, L. multi!ora, and O. americanum, respectively (Table 4). These

values were close to those obtained with the field strain collected from Bobo-Dioulasso_(LC50^and_LC90 values were 74.6, 63.5, 79.4, 41.9, and 103.8 ppm and 152.3, 148.5, 226.3, 74.6, and 1,053.6 ppm with EOs from C. citratus, C. nardus, E. camaldulensis, L. multi!ora, and O. americanum, respectively) (Table 5). EO of E. camaldulensis remains the least toxic to both strains of Ae. aegypti regarding the _LC50^and_LC90 values above 100 and 188 ppm, respectively.

Based on the overlap of the CIs of the _LC50^and_LC90 values, there were no significant differences between L. multi!ora and pyriproxyfen at _LC90 with Bora bora. Also, there were no significant differences between C. citratus and C. nardus regarding _LC50^and_LC90 values with the two Ae. aegypti strains tested. Also, there were no significant differences between E. camaldulensis and O. americanum regarding LC90 with the two Ae. aegypti strains tested and _LC50 with Bora bora. It is also the same with C. citratus and C. nardus, in regard to LC50 and _LC90 values found in two Ae. aegypti strains tested.

Up to now, long-lasting insecticidal nets (LLIN) and indoor residual spraying (IRS) remain the two main tools targeting mainly adults An. gambiae and to lesser extent Ae. aegypti vectors. Thus, there is an urgent need to develop innovative tools and techniques including EOs used against eggs, larvae instar, and adults in order to increase the success of vector control (11).

Our study aimed at evaluating the bioefficacy of five EOs on Ae. aegypti and An. gambiae larvae according to the WHO standard methods. Most studies have already performed the larvicidal tests but did not include the chemical control pyriproxyfen, an insect growth regulator (IGR). IGR acts by mimicking one of these hormones or directly perturbating cuticle formation/deposition or lipid biosynthesis.

TABLE 3 | Median and 90% lethal concentrations and their Confidence Limits (CLs) of plant essential oils against 3 and 4 larval instars of Anopheles gambiae from VK,

field strain.
Treatment	LC50 (ppm)	Confidence limit 95%	LC90 (ppm)	Confidence limit 95%	X²(df)	Slope ( #177; SE)
Pyriproxyfen	23.7^a	9.3-35.5	83.3^a	66.6-106.2	11.5	1.4 #177; 0.41
Cymbopogon citratus	138.1^c	124.5-155.3	328.9^c	267.4-451	72.4	3.3 #177; 0.39
Cymbopogon nardus	76.5^b	67.6-84.9	163^b	143.9-193.5	97.5	3.9 #177; 0.39
Ocimum americanum	89.3^b	81.1-97.4	173.0^b	154.2-201.4	114.0	4.4 #177; 0.41
Lippia multillora	39.5^a	28.6-47.8	90.2^a	78.3-108	45.1	3.2 #177; 0.48
Eucalyptus camaldulensis	121.1^c	109.9-133.6	267.3^c	226.6-339.4	87.7	3.7 #177; 0.39

LC (lethal concentration) in ppm (part per million). LC values followed by different letters are significantly different.

Frontiers in Tropical Diseases | www.frontiersin.org 4 July 2022 | Volume 3 | Article 853405

TABLE 4 | Median and 90% lethal concentrations and their Confidence Limits (CLs) of plant essential oils against 3 and 4 larval instars of Aedes aegypti susceptible strain

from the Bora bora.
Treatment	LC50 (ppm)	Confidence limit 95%	LC90 (ppm)	Confidence limit 95%	X²(df)	Slope ( #177; SE)
Pyriproxyfen	9.5^a	6.4-12.3	43.3^a	34.0-61.8	49.7	1.9 #177; 0.27
Cymbopogon citratus	61.5^c	51.4-70.4	132.0^b	119.4-150.1	71.9	3.2 #177; 0.38
Cymbopogon nardus	62.4^c	50.2-72.5	108.8^b	98.4-124.0	74.2	3.09 #177; 0.35
Ocimum americanum	91.8^d	83.3-100.3	180.9^c	160.8-211.5	112.3	4.3 #177; 0.41
Lippia multillora	41.9^b	31.8-48.7	68.5^a	61.3-84.2	39.5	2.2 #177; 0.35
Eucalyptus camaldulensis	101.3^d	73.1-132.7	188.8^c	169.5-217.6	120.1	1.2 #177; 0.31

LC (lethal concentration) in ppm (part per million). LC values followed by different letters are significantly different.

In this current study, all EOs tested exhibited variable insecticide activity against the larvae of An. gambiae and Ae. aegypti. Regarding _LC50^and_LC90 values, the EOs from C. citratus, C. nardus, E. camaldulensis, L. multi!ora, and O. americanum exhibited higher larvicidal activities against third and fourth instar laboratory-reared larvae of An. gambiae and Ae. aegypti after 24 h of exposure, the most toxic of all being that of L. multi!ora. _LC50 of EO from E. camaldulensis was the least toxic against Ae. aegypti, whereas C. citratus remains the least toxic against An. gambiae. These data are not in agreement with those found by Manh et al. (27), who reported that on Ae. aegypti larvae, C. citratus EO (LC50 = 120.6 ppm) was less effective than that of E. camaldulensis (LC50 = 33.7 ppm).

Our results obtained with C. nardus on An. gambiae in this study were below those obtained by Ahouansou et al. (19), who obtained a value of 97.3 ppm for _LC50 on An. gambiae. Our results were similar to those reported by Solon et al. (28) on Ae. aegypti populations. In their study, O. americanum and C. citratus exhibited values of LC50 equaling 67 and 69 ppm, respectively. However, the results with O. americanum were below those reported by Wangrawa et al. (21) on An. gambiae whose _LC50 was 209.84 ppm.

The _LC50^and_LC90 values obtained with the L. multi!ora in this current study were lowest than those reported by Bassole et al. (23). Recently, the same trend was observed by Yameogo et al. (22), who showed also the toxic effect of L. multi!ora against Ae. aegypti populations from Tabtenga, Ouagadougou.

Interestingly, the high toxicity of the L. multi!ora EO could result in the presence of three major components: thymol, p-cymene, and 13-caryophyllene. According to Folashade (29), thymol, germacrene D, p-cymene, thymyl acetate, and sabinene were found in EOs of L. multi!ora in Ghana and seem to be involved in Lippia toxicity. Conversely, the lower larvicidal activities of C. citratus, C. nardus, E. camaldulensis, and O. americanum oils could be explained by the minor effect of piperitone, geraniol, and

1.8-cineole against Ae. aegypti and An. gambiae larvae. This could explain the differences observed in biological activity among the EOs.

Regarding the _LC50^and_LC90 values, the bioef!cacy of L. multi!ora on An. gambiae strains is closer to that of the positive control used in this study, which is pyriproxyfen, the reference larvicide used against larvae. This would open new alternatives for mosquito control.

Globally, in An. gambiae strain populations, the laboratory-reared larvae were found to be the most susceptible to EOs than those from the field population (VK), known for their resistance to pyrethroid (30-32).

As observed in An. gambiae populations, Ae. aegypti larvae were the most susceptible to L. multi!ora after the chemical pyriproxyfen. The _LC50 values found in this study were slightly lower than those observed in Ae. aegypti populations from Ouagadougou (22), explaining the occurrence of the phenotypic resistance to the main chemical.

As shown by Namountougou et al. (33), tests performed with deltamethrin on Ae. aegypti populations collected from Bobo-Dioulasso showed mortality rates reaching 89.62% and 82.72% for 2013 and 2014, respectively. Mortality rates of Ae. aegypti collected from Ouagadougou were 50.7% and 20.7% for 2013 and 2014, respectively. All these values were lower than 90%, suggesting a resistance. It is very urgent to search for an alternative to chemicals. Our data will help to develop innovative strategies based on EOs.

Alternative à la résistance aux pyréthrinoà¯des au Burkina Faso: évaluation des activités insecticides et répulsives-irritantes d'huiles essentielles de plantes locales seules et en combinaison sur les populations de anopheles gambiae et de aedes aegypti (diptera: culicidae)

ANNEXES

Annexe 1

Annexes 2 :