Efficacy of Seed Extracts of Annona Squamosa and Annona Muricata (Annonaceae) for the Control of Aedes Albopictus and Culex Quinquefasciatus (Culicidae)

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Asian Pac J Trop Biomed 2014; 4(10): 798-806 798

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Asian Pacific Journal of Tropical Biomedicine

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Document heading doi:10.12980/APJTB.4.2014C1264 2014 by the Asian Pacific Journal of Tropical Biomedicine. All rights reserved. 襃 Efficacy of seed extracts of Annona squamosa and Annona muricata (Annonaceae) for the control of Aedes albopictus and Culex quinquefasciatus (Culicidae)

1 1 2 Lala Harivelo Raveloson Ravaomanarivo *, Herisolo Andrianiaina Razafindraleva , Fara Nantenaina Raharimalala , Beby 1 3 2,4 Rasoahantaveloniaina , Pierre Hervé Ravelonandro , Patrick Mavingui 1Department of Entomology, Faculty of Sciences, University of Antananarivo, Po Box 906, Antananarivo (101), Madagascar 2International Associated Laboratory, Research and Valorization of Malagasy Biodiversity Antananarivo, Madagascar 3Research Unit on Process and Environmental Engineering, Faculty of Sciences, University of Antananarivo, Po Box 906, Antananarivo (101), Madagascar 4UMR CNRS 5557, USC INRA 1364, Vet Agro Sup, Microbial Ecology, FR41 BioEnvironment and Health, University of Lyon 1, Villeurbanne F-69622, France

PEER REVIEW ABSTRACT

Peer reviewer Objective: Annona squamosa Annona é è muricata To evaluate the potential efficacy of seed extracts of Aedes and albopictus Dr.é éDelatte H l ne, UMR Peuplements Culexused quinquefasciatus as natural insecticides to control adult and larvae of the vectors V g taux et Bio-agresseurs en M’ ilieu andMethods: under laboratory conditions. Tropical, CIRAD-3P7, Chemin de l IRAT, Aqueous and oil extracts of the two plants were prepared from dried seeds. Preliminary 97410 Ligneé Paradis, Saint Pierre, La identifications of the chemical components of each seedAedes extracts albopictus were performedCulex quinquefasciatus using micro- R union, France. Tel: 026249 9235, Fax: reactional and GCP techniques. Larvae and adults of and 026249 9200, E-mail: [email protected] were collected from the breeding sites in coastal and highlands regions of Madagascar. WHO Co-reviewer: Prof. Victor Jeannoda, standardized tests of susceptibility for larvae and imaginal stage of mosquitoes were realized to Antananarivo, Madagascar. Results:determine mortality and LC50 of mosquitoes. C Comments hemical identifications showed that these extracts contain alkaloids and flavonoids compounds that probably confer their biological insecticidal proprieties. CPG analysis showed This is a good research work in also the presence of various fatty acids. On adult mosquitoes, significant insecticidal effects were which authors have demonstrated the observed with both aqueous and oil extracts of the two plantAnnona seeds compared muricata to mortality induced by deltamethrin, an insecticide used as reference. Extracts of induced high mortality insecticideA. squamosa activity ofA. twomuricata Annonaceae Annona squamosa ( and ) seed rate to both species of mosquito compared to extracts of at all concentrations extracts against two mosquito species Conclusions:tested. The LC50 of seed extracts ranged from 1% to 5% for adults and 0.5% to 1% for larvae. of health importance. The activity The seed extracts of these two plants may be used as mosquito controlling agents and offer a new approach to a less costly, practical and environmentally friendly control of vector was assessed based in onvivo biochemicalA. borne diseases. parameterssquamosa A.and muricata tests. and were found to be promising candidate plants for future KEYWORDS insecticide use on mosquitoes. Details on Page 805 Annonaceae, Seed extracts, Biological insecticides, Chikungunya, Rift Valley fever, Vector control

1. Introduction outbreaks of chikungunya and Rift Valley fever[1-3]. The chikungunya virus (CHIKV) was detected in the coastal Since 2006, Madagascar was affected by successive regions of Madagascar such as Toamasina, Antsiranana

*Corresponding author: Lala Harivelo Raveloson Ravaomanarivo, Departement of Article history: Entomology, Faculty of Sciences, University of Antananarivo, Po Box 906, Antananarivo Received 27 Nov 2013 (101), Madagascar. Received in revised form 12 Feb, 2nd revised form 19 Mar, 3rd revised form 6 Apr 2014 Tel: +261320243006 Accepted 17 May 2014 E-mail: [email protected] Available online 27 Aug 2014 07 012 Foundation Project: Supportedô by the grants FRB-CD-AOOI- - and CMIRA Coopera 2011 from Region Rh ne-Alpes 11MIF-MAVINGUI-10851. Lala Harivelo Raveloson Ravaomanarivo et al./Asian Pac J Trop Biomed 2014; 4(10): 798-806 799 squamosa A. squamosa Annona muricata A. muricata and Taolagnaro, whereas the Rift Valley fever virus (RVFV) ( ) and ( ) has emerged in several regions of the highlands, including were used to control adult mosquitoes as well as larval Ae. albopictus Cx. quinquefasciatus Anjozorobe, Manjakandrina, Antananarivo, Ambatondrazaka, stages of the vectors and . Amparafaravola, and the Haute Matsiatra region Annonaceae are empirically known to elicit insecticidal Aedes albopictus Ae. (Fianarantsoa I, II, Ambalavao)[2-5]. ( activities[12,16]. Plant species in this family contain an albopictus ) has been identified as the main vector of CHIKV array of toxic compounds such as acetogenins, alkaloids, and dengue virus in urban areas, because of its distribution flavonoids that confer to these plants their insecticidal A. squamosa A. muricata which coincided to the epidemic areas and its abundance proprieties[12]. Both seeds of and Ae. albopictus during periods of outbreaks[1,6]. were found contain a great amount of acetogenins[16]. This special group ovipositing and breeding in microhabitats located in the of chemical compounds is known as mitochondrial complex A. muricata vicinity of human habitations, including natural and/or I inhibitor[12,17,18]. Some alkaloids were found in . A. muricata artificial pounds, crevices, tree trunk excavations and other And extracts of not only affect the mortality artificial containers such as discarded cans, used tires and rate of pupal and adult stages of mosquitoes but also reduce abandoned buckets[1,6,7]. The most important vectors for the reproductive success of surviving adults by decreasing Aedes Culex Culex RVFV are and mosquitoes[8,9]. is widely fecundity and egg hatchability[19]. A. squamosa distributed in urban areas of Madagascar[4]. Transmission Petroleum ether leaf extracted from leaves of Culex is facilitated by the feeding behavior of which is both were reported to possess an insecticidal and growth endophilic and exophilic. Larval stages are known to breed regulating activities on three mosquito species namely Anopheles stephensi Cx. quinquefasciatus Aedes in large range of polluted waters with lentic facies[6,7]. , and aegypti As no vaccine and efficient therapeutic treatments are [19]. currently available for CHIKV (Alphavirus), dengue virus The aim of this study was to evaluate the actual efficacy A. squamosa A. squamosa (Flavivirus) or RVFV (Bunyavirus), various methods have been of seed extracts of and under applied to control populations of vectors but none of them laboratory conditions in order to assess their potential were able to eradicate these vectors in Madagascar. Among uses as natural insecticides to control adult mosquitoes as Ae. albopictus Cx. the wide range of biological methods, natural enemies well as larval stages of the vectors and quinquefasciatus such as predators of mosqitoes at different developmental . A preliminary screening using various Culex tigripes stages have been used. Aquatic larvae of micro-reactional techniques and also GC analyses were play an important role in regulating populations of vectors also performed to identify the range of chemical groups by consuming the larvae of other species of mosquitoes in of ingredients in the compositions of the aqueous and oil natural pounds[6]. However, it has been demonstrated that extracts of the two seeds. the most efficient method that may control the numbers of larvae, remains the environmental sanitation by removing 2. Materiels and methods any potential breeding sites of the vectors[1], although its application at a large scale is restricted. 2.1. Collection of seeds and preparation of plant extracts Conventional method using chemical insecticides, including organochlorides, pyrethroids mainly the A. squamosa A. muricata deltamethrin and organophosphates such as malathion and and are commonly planted and fenthion were still applied as last resort for vector control[9]. harvested in the East coast of Madagascar. Their fruits These synthetic compounds are not only environmentally produce a large quantity of seeds that can be used as polluting but also have concomitant hazardous effects to insecticides. Fruits of the two species were collected in ° ’ ” ° ’ ” non-target organisms and to human health[6]. Manakara (22 8 45.68 S; 48 0 15.9 E) at the south east region Phenomenon of resistance to the widely used chemical of Madagascar. insecticides were found in many vectors of disease, Prior to chemical extraction, seeds were completely dried Culex quinquefasciatus Cx. particularly in the case of ( under a ventilated hood then grinded to powder. A total quinquefasciatus )[10-13] causing sudden epidemic spreads of 200 g of each grinded seeds of the two plants were then of Rift Valley fever and the resurgence of the dengue and soaked and macerated separately in 1 L of distilled water chikungunya. Resistance to chemical insecticides in other overnight. Each macerated solution was diluted with distilled Ae. albopictus Aedes aegypti vectors such as and was also water to obtain different lower concentrations (0.5%=1 g/ confirmed[9-15]. L, 1%=2 g/L, 5%=10 g/L, 10%=20 g/L and 20%=40 g/L). The A. squamosa To face the increasing emergence of mosquito resistance aqueous extracts were quoted as EAAS for and A. muricata to chemical insecticides, a sound option lays on the use EAAM for . Similar procedure used to obtain Annona of natural products. In this study, seed extracts of aqueous extracts was adopted to obtain oil extracts by Lala Harivelo Raveloson Ravaomanarivo et al./Asian Pac J Trop Biomed 2014; 4(10): 798-806 800 ° ’ ” ° ’ ” soaking 200 g of seed powder separately into 1 L of various Toamasina (18 08 50 S; 49 23 43 E; 10 m), Mahajanga ° ’ ’’ ° ’ ” solvents with different polarity. Three different solvents were (15 45 39.83 S; 46 20 9.38 E; 18 m), and Manakara ° ’ ” ° ’ ” used for extractions: ethanol, dichloromethane, and acetone. (22 8 45.68 S; 48 0 15.9 E; 20 m) as well as in the highland ° ’ ” ° ’ ” The oil extracts were quoted EET1 and EET2 for ethanol regions of Antananarivo (18 52 47 S; 47 34 35 E; 1245 ° ’ ” ° ’ ” extracts, DCM1 and DCM2 for dichloromethane extracts of m), Ambositra (20 54 211 S; 47 24 159 E; 1310 m) and A. squamosa A. muricata ° ’ ” ° ’ ” and respectively; and ACE1 for Fianarantsoa (21 27 34.05 S; 47 6 33.75 E; 1280 m). Adults of A. squamosa Cx. quinquefasciatus acetone extract of . were collected in human habitations and bedrooms of hospitals early in the morning, whereas 2.2. Chemical analysis Ae. albopictus adults were captured with small nets in their natural habitats and shrubs during daytime while flying. Preliminary identifications of the chemical ingredients Investigations of all potential larval pounds and breeding in the composition of the two aqueous extracts from the sites of the two species were also performed and specimens two seeds were performed using various micro-reactional collected as well. techniques to detect specific chemical compound families[20] 2.4. Mosquito rearing (Table 1). In contrast, oil extracts were injected into Shimazu GCP JC-17A (Version 3 equipped with capillary column: Cx. quinquefasciatus Ae. albopictus T TR 30 伊0 32 伊0 25 I racsil° -wax, m . m . m; njection temperature: and caught in the 260 C) for chemical identification of the components of the wild were transported to the insectarium of Department of oil extracts. Entomology and were reared following WHO procedures and laboratory rearing method[11,21]. The egg strips and rafts 2.3. Mosquito collection were immersed in natural water which is collected in ponds nearby and placed into plastic bowls (dim: 6 cm diameter). Mosquitoes were collected in the coastal regions of Synchronous hatched larvae were transferred into same

Table1

Micro-reactional techniques used to detect presence of chemical compound groups[20]. Chemical groups Tests Reagent Observations Indications Alkaloids Dragendorff (NO2)2BI/IK Precipitation Alkaloids Hager Saturated picric acid Precipitation Marme I2c.d./IK Precipitation Mayer HgCl2/IK Precipitation Wagner I2/IK Precipitation Carditonic glycosides Few drops of FeCl3+1 mL iced acetic Rare sugars Keller-Killiani test Appearance of red ring color acid+1 mL of H2SO4 concentrated 3 drops of anhydride acetic+1 drop Crimson, pink, red, Terpenoids, ü H2SO4 violet, aquamarine Steroid nucleus Liberman-Br chard test 1-5 mL Kedde regeant+2 to 3 drops Lactone insaturated pink purple of NaOH ö Anthraquinones B ntrager test 5 mL of NaOH (1 mol/L) Pink Anthraquinones Cyanides Heating in a flask intercalated with a Cyanide Shift of color from dark yellow to red brick picrate sodium filter paper Coumarines Heated for 10 mn and then observed Coumarines Fluorescence under UV lamp Saponines Foaming test Vortexed in water Appearance of 3cm foam layer after 30 mn of resting Saponines Flavonoids Magnesium ribbon+5 to 6 drops of Flavone, flavanol, Red concentrated HCl flavonone, flavanone Shinoda test Leucoanthocyanins 2 mL HCl (2 mol/L)+2 drops of Leucoanthocyanin Red purple isopropanol Tannins and Formaldehyde 2-3 drops of gelatin 1% Precipitate Tannins polyphenols 2-3 drops of gelatin- NaCl Precipitate aquamarine Tannins 2-3 drops FeCl3 Precipitate Polyphenols 5-6 drops bromine - water Precipitate Tannins Catechin 3 drops of formaldehyde 40%+6 Vanillin-HCl Precipitate crimson red drops of HCl 10% 1 mL vanillin reageant+1 drop of Gallic tannin concentrated HCl+5 mL of lime- Precipitate blue gray water Cyanogenic glycosides Colorimetric method Paper sodium picrate+heating Brick red color Cyanide Lala Harivelo Raveloson Ravaomanarivo et al./Asian Pac J Trop Biomed 2014; 4(10): 798-806 801 bowl containing natural water to maintain synchronous After incubation time-period, mortality of larvae in development of colonies. Male mosquitoes were fed on 6% each beaker was recorded. Each experiment comprised a sucrose imbibed cotton pad. The females were blood-fed minimum number of 4 replicates. Immobile and moribund I twice a week on laboratory° mice. nsectarium temperature larvae were counted as dead. was maintained at (25依2) C, relative humidity at (80依2)% and 2.7. Statistical analysis photoperiod 12 h/12 h. 2.5. Bioassays for adults The average values of mortality of mosquitoes were calculated and mortality curves were drawn on log-log WHO standardized method was used to test the graph paper to find out LC50 for each extract. Data were Ae. albopictus Cx. quinquefasciatus susceptibility of and analysed using SPSS. 10 (Version, 2010). Mortality rates of α adults to different concentrations of each seed extracts[21]. different treatments were subjected to ANOVA ( =0.05). When Bioassays were performed using adapted exposure tubes ANOVA results were significant, means were separated using α (WHO tubes, dim: L: 12 cm伊4.5 cm diameter) having two Tukey-Kramer test ( =0.05). adjacent compartments, the exposure compartment and the observation compartment, interconnected by a sliding glass 3. Results separator that allowed mosquitoes to be grouped within one of the two compartments at a time. Filter papers (0.10 mm 3.1. Major families of chemical compounds identified in thick and 6 cm diameter) were impregnated with 5 mL of extracts aqueous extracts with the respective concentrations of 1%, 5%, 10% and 20% or 1 mL of crude oil extracts, and introduced and walled the exposure tube individually. A number Similar families of chemical compounds ranging from of 25 unfed adult mosquitoes were released into a tube flavonoids, leucoanthocyanes, triterpenes, unsaturated corresponding to a replicate. Four replicates were performed. sterols, polyphenols and polysaccharides (Table 2) were Time of exposure to the extract was standardized to one identified in the aqueous extracts. Alkaloids were present A. muricata A. squamosa hour for each replicate then mosquitoes were transferred in aqueous extract of but absent in into the adjacent observation tube and monitored for 24 h. and that was the converse with tannin. Table 2 C ontrols comprised two different treatments; one using filter A. squamosa Main chemical groups found in aqueous seed extracts of paper impregnated with distilled water and another with a A. muricata. 2% and filter paper impregnated with deltamethrin taken as the A. squamosa A. muricata positive reference. During observation, test and control tubes Tests ( ) + were covered with wooden wall box with aeration aperture Mayer Alkaloids - Wagner (Alkaloids) -- and moisten tissues to maintain appropriate humidity. After Dragendorff (Alkaloids) -- 24 h, falling or immobile mosquitoes were counted as dead. Wilstater (Flavones, flavanones, flavanols) ++ - I f mortality rate of mosquitoes of the non-impregnated Modified Wilstater ((Flavones, flavanones) ++ + 20% (4 ) 24 control tube exceeded mosquitoes within the h of Bate-Smith ++ incubation, the whole batch of test was considered as invalid Anthocyanins - then repeated. Lieberman Burchard (Triterpenoids) ++ ++ ( ) + + 2.6. Bioassays for larvae Salkowski Unsaturated sterols Badget-Kedde (laconoid sterols) -- – Keller Killiani (Desoxy-2 sugar) -- T 0 5% 1% 2% Grignard (cyanogenic glycosides) -- hree different concentrations . , and of each ä plant aqueous extract were used for bioassays[22]. For each Bornstr ger (anthraquinones) -- concentration prepared, a batch of two beakers of 100 mL Foam height (saponosides) -- ++ + was used. Two controls were run: one blank control with 100 Gelatin 1% (Polyphenols) S G (T ) ++ mL of water, and one with 100 mL water supplemented with alted elatin aninns - FeCl3 (condensed hydrolysable tannins) ++ - 1 mL of ethanol that was used as the positive reference. For Polysaccharides screening +++ ++ oil extracts, 1 mL acetone was added to 100 mL of natural -: Not detected, +: Present in small amount, ++: Present in large amount. breeding pound water prior to adding 1 mL crude oil seed Ae. extract of each plant. Twenty-five third instar larvae of Various fatty acids ranging from palmitic acid, oleic albopictus Cx. quinquefasciatus A. or were introduced in each acid, linoleic acid were identified in the oil extract of squamosa beaker and kept immersed for 24 h. (Table 3). In addition to the range of major fatty Lala Harivelo Raveloson Ravaomanarivo et al./Asian Pac J Trop Biomed 2014; 4(10): 798-806 802 A. squamosa A. muricata (14依2) ( 依SE) acids found in , the oil extract of in the deltamethrinn P reference tests means contained more specific fatty acids including myristic acid, (F(7,111)=22.18, =118, =0.0001, ANOVA, Tukey-Kramer test). palmitoleic acid, stearic acid, linolenic acid, arachidic acid By plotting average values of mortalities on log-log graph, and others acids with C18, C19 and C20 (Table 3). the LC50 was found to be at the concentration of 1% for EAAM 5% EAAS 3.2. Efficacy of A. squamosa and A. muricata extracts to adult and for Ae.. albopictus I 24 mosquitoes Cx. quinquefasciatus and Ae. albopictus n adults of , mortalities after h exposure to 1% concentration of EAAS and of EAAM were respectively 18依2 24依1 ( 依SE) (T 4) M 3.2.1. Aqueous extracts and means able . ortalities of adult EAAM A. squamosa mosquitoes treated by were similar to mortalities B (23依2) ( y using aqueousCx. extracts quinquefasciatus of , numbers of observed in the deltamethrinA. squamosa reference tests means 24 依SE) (18依2) adult mosquitoes killed after h , whereasn thoseP of were lower were proportional to the concentrations of the extracts: 10 [F(3.36)=44.72, =39, =0.0001, ANOVA, Tukey-Kramer test]. 依2, 13依2, 18依2 and 23依2 (means依SE) for the concentrations The LC50 was found to be lower than the concentration of 1% 1% 5% 10% 20% W EAAS EAAM ,A. muricata, and , respectively. hen aqueous extractsCx. for both and . ofquinquefasciatus were used, mortalities to adults 3.2.2. Oil extracts 18依2 20依2 23依2 23依2 ( 依SE) were , , and means Cx. quinquefasciatus for the concentrations of 1%, 5%, 10% and 20%, respectively Mortalities of adults after 24 h were (Table 4). Mortalities of adult mosquitoes using EAAS and 23依2, 22依3, 19依3, 22依3 and 21依4 (means依SE), respectively for EAAM were significantly superior to mortalities observed DCM1, DCM2, EET1, EET2 and EAC1 (Table 5). Mortalities of Table 3 A. muricata A. squamosa. Fatty acid groups for and A. muricata A. squamosa ° N Fatty acids Relative concentration of the analyzed sample (%) Fatty acids Relative concentration of the analyzed sample (%) 1 Myristic acid 0.07 2 Palmitic acid 16:0 20.93 Palmitic acid 18.37 3 Palmitoleic acid 16:1w7 2.29 4 Stearic acid 18:0 3.90 5 Oleic acid C18:1w9 42.97 Oleic acid C18:1w8 4.06 6 Linoleic acid c18:2w6 29.29 Linoleic acid 38.32 7 C18:3(n-3) or C19:1(n-8) 0.88 Polyinsaturated C18 35.30 8 Linoleic acid C18:3w3 0.20 Linoleic acid c18:3w3 2.51 9 Polyinsaturated C19 0.81 10 Arachidic acid 0.53 11 C20:1(n-7) 0.22 12 C20:4(n-6) 0.52 13 C20:3(n-6) 0.17 14 C21:0 0.06 Table 4 A. squamosa A. muricata. Mortality of adult mosquitoes induced by aqueous seed extracts of and Treatments EAAS EAAM EAAS EAAM EAAS EAAM EAAS EAAM Control Deltamethrine 1% 1% 5% 5% 10% 10% 20% 20% 2% Cx. quinquefasciatus c ab c ab ab a a a d bc Average mortality of (means依SE) 10依2 18依2 13依2 20依2 18依2 23依2 23依2 23依2 1依2 14依2 Number of individuals tested 550 350 200 200 300 200 200 200 475 200 Ae. albopictus a a b a Average mortality of (means依SE) 18依2 24依1 ND ND ND ND ND ND 1依1 23依2 Number of individuals tested 300 200 ND ND ND ND ND ND 200 300 P Different letters indicate significant means ( =0.05, ANOVA, Tukey-Kramer test). ND: not determined. Table 5 A. squamosa A. muricata. Mortality of adult mosquitoes to oil seed extracts of and DCM1 crude DCM2 crude EET1 Crude EET2 Crude EAC1 Crude Deltamethrine Treatments Control 1 mL/pap 1 mL/pap 1 mL/pap 1 mL/pap 1 mL/pap 2% Cx. quinquefasciatus a a ab a ab c b Average mortality of (means依SE) 23依2 22依3 19依3 22依3 21依4 1依3 14依2 Number of individuals tested 200 100 100 100 50 100 200 Ae. albopictus a a a b a Average mortality of (means依SE) 24依1 25依1 24依1 ND ND 1依1 23依2 Number of individuals tested 200 100 25 ND ND 200 200 P Different letters indicate significant means ( =0.05, ANOVA, Tukey-Kramer test). ND: not determined. Lala Harivelo Raveloson Ravaomanarivo et al./Asian Pac J Trop Biomed 2014; 4(10): 798-806 803 Table 6 A. squamosa A. muricata. Mortality of mosquito third instar larvae to different concentration of aqueous seed extracts of and Treatments EAAS 0.5% EAAM 0.5% EAAS 1% EAAM 1% EAAS 2% EAAM 2% Ethanol Control Cx. quinquefasciatus e d bc c b a f f Average mortality of (means依SE) 8依2 12依1 19依1 18依1 22依1 24依1 1依1 1依1 Number of individuals tested 100 250 500 300 200 200 250 200 Ae. albopictus c b b a a d d Average mortality of (means依SE) 15依1 20依2 22依1 24依1 24依1 ND 1依1 1依1 Number of individuals tested 150 100 350 100 250 ND 200 200 P Different letters indicate significant means ( =0.05, ANOVA, Tukey-Kramer test). ND: not determined.

Table 7 A. squamosa A. muricata. Mortality of mosquito third instar larvae to different concentration of oil seed extracts of and Treatments DCM1 1% DCM2 1% EET1 1% EET2 1% Ethanol 1% Control Cx. quinquefasciatus b a c b c c Average mortality of (means依SE) 17依1 24依1 5依2 13依2 1依1 1依1 Number of individuals tested 200 200 100 100 250 200 Ae. albopictus a a d c Average mortality of (means依SE) 23依2 24依1 ND ND 1依1 2依1 Number of individuals tested 100 200 ND ND 100 100 P Different letters indicate significant means ( =0.05, ANOVA, Tukey-Kramer test). ND: not determined.

A. squamosa n P mosquitoes using oil extracts of (DCM1, EET1 and [F(6,47)=284.50, =54, =0.0001, ANOVA, Tukey-Kramer test]. A. muricata EAC1) and (DCM2 and EET2) were significantly The LC50 was found to be lower than 0.5% for both EAAS and superior to mortalities observed in deltamethrin reference EAAM. n P tests (14依2) (means依SE) [F(5,44)=5.89, =49, =0.0003, ANOVA, 3.3.2. Oil extracts Tukey-Kramer test]. Ae. albopictus Cx. quinquefasciatus For adults, mortalities after 24 h were 24依1, Mortalities of third instar larvae of c 25依1, 24依1 (means依SE) respectively to DCM1, DCM2 and EET1 after 24 h were respectively 17依1, and 5依2 (means依SE) for (Table 5). There were no difference in mortalities between the oil extracts DCM1 and EET1, and 24依1 and 13依2 (means依 A. squamosa A. muricata extracts of (DCM1, EET1) and (DCM2) SE) for the oil extracts DCM2 and EET2 respectively (Table 7). n A. muricata to that induced by deltamethrin (23依2) (F(4,21)=56.33, =25; Mortalities of mosquitoes under oil extracts of P =0.0001, ANOVA, Tukey-Kramer test). (DCM2 and EET2) were significantly higher than mortalities observed in the ethanol and the control reference tests (both 3.3. Efficacy of A. squamosa and A. muricata extracts to A. squamosa 1依1) (means依SE) whereas oil extracts of (DCM1 third instar nymph mosquitoes Cx. quinquefasciatus and Ae. and EET1) induced a mild mortality to third instar larvae of albopictus Cx. quinquefasciatus n P (F(7,48)=58.97, =55, =0.0001, ANOVA test, Ae. Tukey-Kramer test). Mortalities of third instar larvae of 3.3.1. Aqueous extracts albopictus A. squamosa A. by using oil extracts of (DCM1) and muricata For the three concentrations 0.5%, 1% and 2%, the (DCM2) were significantly higher than mortalities Cx. quinquefasciatus mortalities of third instar larvae of after observed in the ethanol and the control reference tests (1 n 24 h were respectively 8依2, 19依1 and 22依1 (means依SE) for 依1 and 2依1 respectively) (means依SE) [F(5,24)=650.73, =30, A. squamosa P the aqueous extracts of (EAAS), and 12依1,18依1 =0.0001, ANOVA, Tukey-Kramer test]. A. muricata Ae. albopictus and 24依1 (means依SE) for the aqueous extracts of Overall, the results showed that populations (EAAM), respectively (Table 6). Mortalities of larvae using were more sensitive to the seed extracts than populations of Cx. quinquefasciatus EAAS and EAAM were significantly superior to mortalities . observed in the ethanol and control reference tests (both 1依 n P 1) (means依SE) [(F(7, 88)=320.05, = 95, =0.0001, ANOVA, Tukey- 4. Discussion Kramer test]. By plotting average values on log-log graph, the LC50 was found to be 0.5% for EAAM and 1% for EAAS. For the two concentrations 0.5% and 1% after 24 h of Chemical screening and GC analyses results showed Ae. albopictus exposure, third instar larvae of exhibited a range of potent and bioactive compound groups such c b A. muricata mortalities of 15依1 and 22依1 (means依SE) for the aqueous as flavonoids, and fatty acids in both and A. squamosa A. squamosa extracts of (EAAS), and 20依2 and 24依1 (means aqueous and oil seed extracts. Chemical A. muricata 依SE) for the aqueous extracts of (EAAM), screening was in agreement with the results obtained respectively (Table 6). Mortalities of larvae using EAAS and by different authors who have already identified a broad EAAM were significantly higher than mortalities observed in range of alkaloids, flavonoids and acetogenins compounds A. squamosa A. the ethanol and control reference tests (both 1依1) (means依SE) produced in seeds of [16,17,19] and Lala Harivelo Raveloson Ravaomanarivo et al./Asian Pac J Trop Biomed 2014; 4(10): 798-806 804 muricata A. squamosa [16,23] suggesting their probable role in insecticidal than those of at the same concentration. activities[23]. Alkaloid compounds including nicotine found Similar trend was observed when using both aqueous and Cx. quinquefasciatus in tobacco, have been demonstrated to inhibit the active oil extracts on the larvae of . This site of acetylcholine in many organisms[10,19,24]. These difference of efficacy between the two species is probably A. muricata A. muricata alkaloids were only found in aqueous extracts. due to the alkaloids present in which are not et al A. squamosa Acetogenins were discovered in 1982 by Jolad ., they found in [16]. Aqueous extracts were all efficient are specific of Annonaceae[17,23]. These compounds are at a very low dose (0.5%) and with a proportional increase of derivatives of 32 to 37 carbons long chain fatty acids[12]. The the lethal activity following the gradual increase of extract major acetogenins in seeds are respectively annonacin in concentrations. At the highest dose (2%), all mosquitoes A. muricata A. squamosa [23] and squamocin in [17]. Although tested were killed during the tests. Although extracts typical acetogenins were not detected with the two methods obtained from all organic solvent elicited adulticidal used, an in-depth identification of the factual nature of the activities, dichloromethane extracts of both seed plants were active ingredients belonging to all the chemical families significantly potent (DCM1, DCM2) compared to ethanolic using GC-MS will enable further determination of their extracts, in particular for larval stages. In contrast, only A. muricata precise roles in the insecticidal activities of the extracts. ethanolic extracts (EET2) of elicited adulticidal Cx. quinquefasciatus Numerous types of toxicity result from physical proprieties activities against , with regard to oil of fatty acids: toxicity by inhalation due to volatile organic extracts. compounds, toxicity by contact due to aggregation and For all extracts and at all concentrations tested, the Ae. albopictus formation of thin film at the surface of water which does not populations of caught from five regions Cx. allow respiration of aquatic insects, and by penetration due of Madagascar were more susceptible than those of quinquefasciatus to the amphibolic propriety of certain molecules; Oleic acid . This higher susceptibility might be due to Ae. albopictus C18 and undecylenic acid C11 possess direct insecticidal the intrinsic natural ecology and behavior of activities or may enhance the toxicity of other toxic larvae that usually breed into relatively clean and less compounds[25]. polluted water. This behavior reduced at its minimum Currently, plant-based chemical control may broaden their contact to organic compounds into highly polluted up gradually the existing arsenal of methods in vector waters, which in turn might be among the mechanisms that control. Many plants are used as biocides in different prevented populations of the species to remain susceptible. Piper nigrum Cx. quinquefasciatus parts of the world, naming (black pepper), In contrast, larvae of are known to Nicotiana tabacum Melia azedarach Derris (tobacco), , , breed into polluted water containing a broad variety of Lonchocarpus Tephrosia Azadirachta indica and and (neem) organic compounds namely alkaloids and/or derivative Cx. quinquefasciatus [11,12,24,26]. The latter plant is the most widely used biocide compounds. The repeated contacts of as its insecticidal properties have been known for over 300 larvae to these compounds might have conferred to these years and was applied as a foliar spray and vapor to control larvae and consequently to adults, a range of adaptations to insects, particularly in greenhouses and other protected various chemicals hence their relative moderate sensitivity crop fields. to these plants extracts. In comparison to synthetic deltamethrin, a compound Nevertheless, the use of plant-based pesticides presents usually taken as standard reference by WHO in a number of challenges and advantages[9,26] as follows: susceptibility tests for mosquitoes, a higher insecticidal (i) the active ingredients of many plant-based pesticides Ae. albopictus Cx. effect on both adult and larvae of and are not known, but are essential for the development of quinquefasciatus was observed when using the aqueous specifications. (ii) The active ingredients can vary both in A. squamosa A. muricata and oil seed extracts of and . composition and concentration in the same plant species, This higher efficacy opens the possibility to exploit these in different clones, in different parts of the plants, at extracts as biological insecticides to control the two tested different stages of plant growth and under different climatic mosquito species, and extendedly likely to other mosquito and soil conditions. (iii) The activity may not be due to a vector taxa. Results of bioassays likely were in concordance single ingredient but to a mixture of compounds, which Cx. quinquefasciatus with those obtained on larva of . Thus, may act synergistically. (iv) The development of resistance A. muricata they confirm the insecticidal activities of [16] to pesticides containing a mixture of active ingredients A. squamosa and [12,16,19]. Strikingly, the present study also may occur less readily. (v) Adequate toxicological and eco- brought up additional evidence and support concerning the toxicological data are not available for many plant-based prevalence of resistance to deltamethrin, the insecticide pesticides. (vi) Analytical standards of the active ingredients reference advocated by WHO[10,12]. may not be easily obtainable. A. muricata It is also found that the aqueous extracts of As the two tested plants have a wide geographical Ae. albopictus elicited higher adulticidal activities in distribution in eastern region of Madagascar where malaria Lala Harivelo Raveloson Ravaomanarivo et al./Asian Pac J Trop Biomed 2014; 4(10): 798-806 805 Comments and other virus-borne vectors are endemic[1], integrating and promoting their use as an alternative plant-based Background method in vector control could be advocated, especially Ae. albopictus Cx. quinquefasciatus in the case of Madagascar. If used properly at an efficient and are mosquito vectors Annona dose, these extracts of were revealed to be a of several human diseases all over the world. Among them low cost and powerful environmentally friendly tool by chikungunya and Rift Valley fever diseases have been recently avoiding accumulation and negative impacts on non-target reported for severe outbreaks in several countries, such as the entomofauna. The possibility of newly synthesized bioactive islands of the south west part of the Indian Ocean. Most of the molecules forming all extracts and that from the different methods used today to eradicate those vector populations are organic solvent used thus remains pivotal in the design of not efficient or too costly for the country. Therefore there is efficient and practical plant extract-based methods in vector need of new molecules to improve those control methods. control. A. squamosa A. Research frontiers The aqueous and oil extracts of and muricata both showed insecticidal properties though at The present research work depicts insecticide activity of A. muricata A. squamosa A. muricata varying levels. Extracts of displayed higher aqueous and oil extracts of and A. squamosa insecticidal potential compared to extracts of . (Annonaceae) seeds against 2 mosquito species of health Ae. albopictus Cx. Ae. albopictus Cx. quinquefasciatus Besides, was more susceptible than importance: and by quinquefasciatus to all extracts. Lethal concentrations (LC50) identifications of the chemical components of each seed in vivo of all extracts of the two plants tested ranged from less than extracts and tests of susceptibility for larvae and Ae. albopictus 1% for adult mosquitoes and 0.5% for larvae of , imaginal stage of mosquitoes (determining mortality and CL 50 and 1% to 5% for adult mosquitoes and 0.5% to 1% for larvae of mosquitoes). Cx. quinquefasciatus A. of . Dichloromethane extracts of muricata Related reports showed high lethal effect to larval and adult stages Ae. albopictus Cx. quinquefasciatus of and compared to Annonaceae are empirically known to elicit insecticidal deltamethrin which is an OMS insecticide reference product. activities. Plant species in this family contain an array of Hence, this extract might already be considered having high toxic compounds such as acetogenins, alkaloids, flavonoids insecticidal potential as an alternative agent in the control that confer to these plants their insecticidal proprieties. The of mosquitoes though further studies on identification and folklore medicine has evidence of effectiveness of those chemical characterization of the active ingredients of these extracts as insecticides. two plants are still needed for its complete integration as Innovations and breakthroughs mosquito controlling agents. Annonaceae are empirically known to elicit insecticidal activities however in the present study, authors have looked Conflict of interest statement for their different chemical compounds and demonstrated A. squamosa A. muricata the insecticide activity of and seeds We declare that we have no conflict of interest. against 2 mosquito species of health importance.

Applications Acknowledgements This scientific study is, as mentioned by author, a preliminary work, screening 2 plant species for their We are grateful to the Ministry of Water and Forestry and insecticide activity. Nevertheless their results even preliminary to Madagascar National Parks (formerly ANGAP) for fully are supporting and suggesting these seeds extract for future authorizing collection of wild mosquitoes within parks. We insecticide use. thank Rahanitriniaina Sahondra and Rajaonera Tahina Ernest Peer review for assistance and cooperation, Rajaonarivo Solofoniaina for providing all the plant extracts, and Brown Rakotoarisoa This is a good research work in which authors have A. and Ratsimbazafy Jonah for helpful suggestions on the demonstrated the insecticide activity of two Annonaceae ( squamosa A. muricata manuscript. This work was funded by the grants FRB-CD- and ) seed extracts against 2 mosquito ô AOOI-07-012 and CMIRA Coopera 2011 from Region Rh ne- species of health importance. The activity was assessed based in vivo A. squamosa Alpes 11MIF-MAVINGUI-10851, and was carried out within the on biochemical parameters and tests. and “ é é A. muricata frameworks of GDRI Biodiversit et D veloppement Durable were found to be promising candidate plants for à ” Madagascar . future insecticide use on mosquitoes. Lala Harivelo Raveloson Ravaomanarivo et al./Asian Pac J Trop Biomed 2014; 4(10): 798-806 806 References [13] K amgang B, Marcombe S, Chandre F, Nchoutpouen E, Nwane Aedes aegypti P, Etang J, et al. Insecticide susceptibility of and Aedes albopictus Parasit Vectors 4 [1] R atsitorahina M, Harisoa J, Ratovonjato J, Biacabe S, Reynes in Central Africa. 2011; : 79. JM, Zeller H, et al. Outbreak of dengue and chikungunya fevers, [14] K han HA, Akram W, Shehzad K, Shaalan E. 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