Larvicidalefficacy ofm onoterpenes against the larvae of Anopheles gambiae

Eliningaya J.Kweka,Tam ires Cardoso Lima,Chrian M.Marciale,Dam ião Pergentino de Sousa,Division of Livestock and Human Diseases Vector Control,Tropical Pesticides Research Institute,P.O.Box 0,Arusha, TanzaniaDepartmentofMedical Parasitology and Entomology,Catholic University of Health and Allied Sciences,P.O.Box 6, Mwanza,TanzaniaDepartment of Pharmacy,Federal University of Sergipe,CEP 900-000,São Crist´ovão,Sergipe,BrazilDepartmentof Pharmaceutical Sciences,Federal University of Paraíba,CEP 5805-970,João Pessoa,Paraíba,Brazil



Larvicidalefficacy ofm onoterpenes against the larvae of Anopheles gambiae

Eliningaya J.Kweka1,2*,Tam ires Cardoso Lima3,Chrian M.Marciale1,Dam ião Pergentino de Sousa3,41Division of Livestock and Human Diseases Vector Control,Tropical Pesticides Research Institute,P.O.Box 3024,Arusha, Tanzania
2DepartmentofMedical Parasitology and Entomology,Catholic University of Health and Allied Sciences,P.O.Box 1464, Mwanza,Tanzania
3Department of Pharmacy,Federal University of Sergipe,CEP 49100-000,São Crist´ovão,Sergipe,Brazil
4Departmentof Pharmaceutical Sciences,Federal University of Paraíba,CEP 58051-970,João Pessoa,Paraíba,Brazil

Original article http://dx.doi.org/10.1016/j.apjtb.2016.03.001

ARTICLE INFO

Article history:

Received in revised form 29Oct,2nd

revised form 25 Dec 2015

Accepted 12 Jan 2016

Availableonline10Mar2016

Larvicidal activity

Malaria

Anopheles gambiae s.s.

Essential oils

Monoterpenes

Natural products

Mosquito

ABSTRACT

Ob jective:To evaluate the larvicidal ef fi cacy of eight volatile components of essential oils against3rd instar larvae of Anopheles gambiae s.s.

M ethods:Larvicidal effects of each compound were evaluated in both laboratory and semi-fi eld trials.Stock solution was prepared and serial dilutions were made in six concentrations for each compound.A total of 20 larvae were exposed to larvicides for each replicate and monitored at intervals of 12,24,48 and 72 h.Larvaemonitoring was done on basis of dead and live larvae in all intervals.

Resu lts:A ll assayed compoundswere larvicides and presented varying degrees of larval toxicity,w ith LC50values ranging from 1.28 to 1938.92 mg/L depending on the treatment time(12,24,48 or 72 h).(−)-Perillyl alcohol presented the strongest larvicidal activity towards Anopheles gambiae larvae,w ith LC50values of 73.60,18.36,1.72 and 1.28mg/L after 12,24,48 and 72 h of exposure,respectively.The next strongestwere (−)-isopulegol(LC50=135.10,49.39,34.39 and 20.22mg/L)and(−)-carvone epoxide (LC50=168.86,124.74,80.84 and 23.46 mg/L).A fter 12,24 and 48 h of treatment, hydroxydihydrocarvone was the least toxic compound,w ith LC50values of 1938.92, 1172.18 and 401.03mg/L,respectively.

Conclusions:The data obtained in this study suggest that all evaluated monoterpenes, especially(−)-perillyl alcohol,have remarkable larvicidal effects and may be considered as potential sources for the development of suitable natural larvicides for mosquito management programs.Further small-scale fi eld trials should be conducted.

1.Introduction

Mosquitoes constitute an important group of arthropods for public health.They transm it a w ide range of human diseases such as fi lariasis,malaria,dengue,yellow fever and Japanese encephalitis,causing m illions of deaths worldw ide each year [1,2].Global patterns of climate change and urbanization have increased the threat of humans contracting arthropod-borne viral infections[3].

Malaria is among themost important vector-borne diseases, being endem ic to more than 100 countries worldw ide,particularly in tropicaland subtropical regions[4].The disease is caused by one-celled parasites that are transm itted to humans via the bite of infected anopheline mosquitoes such as Anopheles gambiae s.s.Giles(An.gambiae s.s.),Anopheles arabiensis Patton and Anopheles stephensi Liston[5].In the last 30 years, malaria incidence has increased,due mainly to the emergence of drug and insecticide resistance in parasites and vectors, respectively,aswell as poor socioeconom ic conditions[6].Butin the recent past,malaria vector and parasite populations have declined drastically due to increased investments in intervention,diagnosis and treatment[7,8].

Plants are a rich resource of alternative synthetic compounds for the control ofmosquito larvae.They possess aw ide range of bioactive phytochem icals that are selective,biodegradable,and havem inor or no adverse effects on non-target organisms and the environment,making them potentially appropriate for use in integrated pest management programs.Approximately 2000 species of terrestrial plants have been described for their insecticidal properties[9–11].

Various studies have focused on the use of natural products, especially plant-derived essential oils,as suitable bioactive agentsagainst the larvaeof An.gambiae s.s.and othermosquito species[12–15].Essential oils are complex natural m ixtures of volatile organic compounds,principally mono-and sesquiterpenes,which are considered to be among the best alternatives for the control of disease vectors[16,17].

The present study investigated the larvicidal effects of eight monoterpenes found in volatile oils against themalaria vector mosquito An.gambiae s.s.

2.M aterials and m ethods

2.1.Mosquito larvae

The An.gambiae s.s.larvaeused in laboratory and sem i-fi eld assays were obtained from the insectary of the Tropical Pesticides Research Institute.Only 3rd instar larvae were used,according to World Health Organization protocol[18].Larval rearing in the insectary was carried out according to the protocol developed by Balestrino et al.[19].Larvae were reared at(27.0±2.0)°C,a photoperiod of 12:12 h(light: dark),and(78±2)%relative hum idity.Larvae were fed a diet of TetraM in fi sh food.

2.2.Larval assays in the laboratory

The assayed compounds(−)-perillyl alcohol,(−)-isopulegol, (+)-limonene epoxide,(+)-limonene,terpinen-4-ol,and terpinolene were acquired from Sigma–A ldrich,USA.The(−)-carvone epoxide[20]and(−)-hydroxydihydrocarvone[21]were prepared as previously described.

Larvicidal bioassayswere conducted as described by Mdoe et al.[12,13].A stock solution w as prepared for each test compound by dissolving the com pound in 98 m L normal laboratory larval rearing water and 2 m L dimethylsulfoxide (DMSO)in a 100 m L plastic container.The solution was thoroughly m ixed to get a homogeneous m ixture,and serial dilutions of 200,100,50,25 and 12.5 mg/L were prepared. Each experiment was replicated at least six times w ith two controls:one containing normal laboratory larval rearing water,the other containing an aqueous solution of 1% DMSO to evaluate the effect of the solvent on the larvae.For the larvicidal experiments,each replicate and each control received 20 live 3rd instar larvae.No nutritional supplements were added during the assays.Larval mortality was registered after 12,24,48 and 72 h of exposure. The larvae were considered dead if they did not present movement.

2.3.Larval assays in the semi-fi eld

Sem i-fi eld larvae bioassays were conducted using the same concentrations used in the laboratory assays.Sem i-fi eld environmentstructures used in thisstudy were designed according to previous studies[12,22]and follow ing World Health Organization recommendations[18].Each experiment was carried out in six replicates w ith two controls,one having an aqueous solution of 0.5%DMSO and the other having normal laboratory larval rearing water.For the larvicidal assay,20 live 3rd instar larvae were placed in each assay replicate and in each control.

2.4.Statistical analysis

Scheff´e'smultiple comparison procedure was used to determ ine the statistical signi fi cance of the larvicidal activity of the tested compounds,w ith results expressed as mean±SE.Statistical analysis was performed using SAS.Assessments of surviving larvae were recorded after 12,24,48 and 72 h of exposure.Mortalitywas reported as LC50,the concentration that produced 50%mortality.The 95%con fi dence intervals(CI)for LC50were also recorded.

3.Resu lts

In this study,the larvicidal toxicity of a series of eight monoterpenes(Figure 1)present in volatile oils was evaluated against 3rd stage larval instars of An.gambiae s.s.,one of the most anthropophilic vectors of malaria.Larval mortality rates were registered after 12,24,48 and 72 h of treatment in varying concentrations of the test solutions.The result of each bioassay was reported as the lethal concentration estimated to kill 50%of the treated larvae(LC50),expressed inmg/L.The LC50values foreach compound and treatment time,along w ith 95%CI,were given in Table 1.

Figure 1.Chemical structuresof the evaluated compounds.

A ll the assayed compounds had larvicidal effects and exhibited different degrees of larval toxicity,w ith LC50values varying between 1.28 and 1938.92 mg/L depending on the treatment time(12,24,48 or 72 h).Among the eightmonoterpenes,(−)-perillyl alcohol(1)showed the strongest larvicidal activity towards An.gambiae larvae,w ith LC50valuesof 73.60, 18.36,1.72 and 1.28mg/L after12,24,48 and 72 h of exposure, respectively.The next strongest were(−)-isopulegol(2) (LC50=135.10,49.39,34.39 and 20.22mg/L)and(−)-carvoneepoxide(3)(LC50=168.86,124.74,80.84 and 23.46 mg/L). A fter 12,24 and 48 h of treatment,(−)-hydroxydihydrocarvone (8)was the least toxic compound,w ith LC50values of 1938.92, 1172.18 and 401.03 mg/L,respectively.Terpinolene(7) exhibited the lowest toxicity at 72 h post-treatment,w ith an LC50value of 259.40mg/L.

Table1 LC50(mg/L)and 95%CI of the compounds 1–8.

Larvalmortality rateswere found to be directly proportional to monoterpene concentrations.Sim ilarly,larval mortality increased w ith increasing exposure time,as all assayed compounds showed the highestmortality rates after 72 h of treatment.The most remarkable result was seen w ith (−)-hydroxydihydrocarvone(8),which was 38-fold more bioactive at 72 h post-treatment(LC50=50.95 mg/L)than at 12 h(LC50=1938.92 mg/L).To better understand the relationship between themolecular structure of the assayed monoterpenes and their larval toxicity,speci fi c structural and functional group variations were identi fi ed as possibly contributing to larvicidal activity.In general,the oxygenated monoterpenes exhibited stronger larvicidal effects than the monoterpene hydrocarbon(+)-limonene(5).The position of the carbon–carbon double bond in the p-menthane skeleton appeared to in fl uence larvicidal potency;after 48 and 72 h of treatment time,(+)-limonene(5)(LC50=152.95 and 97.72, respectively)was more bioactive than terpinolene(7) (LC50=343.79 and 259.40,respectively).Sim ilarly,the position of the hydroxyl group(endo-or exocyclic)also altered the toxicity,as seen in themonoterpenoids(−)-perillyl alcohol(1), (−)-isopulegol(2)and terpinen-4-ol(6),which each exhibited different degrees of larval toxicity.Furthermore,replacementof a C–C double bond by an epoxide group did not signi fi cantly affect larvicidal potency,as(+)-limonene epoxide(4)and (+)-limonene(5)showed sim ilar activity.However,the addition of a ketone group in the cyclohexane ring seemed to contribute to larvicidal ef fi cacy,as seen in the relatively high bioactivity of (−)-carvone epoxide(3)compared to(+)-limonene epoxide(4) and(+)-limonene(5).

4.Discussion

The fi ndings of this study have shown that,com poundsw ith different orientations of the active groups in primary structure in fl uence the outcome of larvae mortality differently.These results are interesting,since the evaluated compounds are highly volatile.Several studies in the literature have described the larvicidalactivity ofmonoterpenes against different species of mosquitoes[17,23–27].Perumalsam y et al.reported the larvicidal potential of themonoterpenes camphene,fenchone, terpinolene,γ-terpinene,(+)-and(−)-β-pinene,(+)-and (−)-α-pinene,α-terpineol,myrcene,terpinen-4-ol,(+)-limonene,Δ3-carene,borneol,1,8-cineole,linalool,verbenone and α-phellandrene against three mosquito species,Culex pipiens pallens,Aedes aegypti and Ochlerotatus togoi[28].In another study,Tabanca et al.found that(−)-perillyl alcohol, (−)-perilla aldehyde,(−)-perillic acid and(−)-limonene exhibited high toxicity against 3rd instar larvae of Aedes aegypti,w ith LC50values of 39.1,35.3,56.5 and 29.1mg/L, respectively[29].Liu et al.showed that the monoterpenes (+)-limonene and geraniol,both isolated from the essential oil from the roots of Toddalia asiatica(L.)Lam.,displayed an interesting larvicidal activity against 3rd instar larvae of Aedes albopictus,w ith LC50values of 19.84 and 30.13μg/ m L,respectively[16].

The fi ndings of the current study suggest possibilities for further research on the larvicidalactivity of plant-derived essential oils and their chem ical components.Future studies should focus on developing more stable and effective formulations, investigating themode of the constituents'actions,decreasing costs,and exam ining the effects of these compounds on nontarget organisms and the environment[6,30,31].The compounds are found in essential oils of plants,such as Conyza newii (perillyl alcohol and limonene)[32],Eucalyptus citriodora (isopulegol)[33],Carum carvi(carvone epoxide)[34],Artemisia nilagirica var.septentrionalis(terpinen-4-ol)[35],lemon (limonene-1,2-epoxide)[36],Mangifera indica L.(terpinolene) [37],and Nicotiana tabacum(hydroxydihydrocarvone)[38]. These monoterpenes toxicity against mosquito larvae have shown the prospect of replacing synthetic larvicides which are losing ef fi cacy or been incorporated in integrated vector control management programme.

A ll of the tested monoterpenes exhibited larvicidal activity against An.gambiae s.s.,w ith(−)-perillylalcohol themost toxic after 12,24,48 and 72 h of treatment.These results underscore the importance of evaluating plant essential oils and their chem ical components as effective natural larvicides for controlling Anopheles larvae,especially in areaswhere vectorshave developed resistance or dim inished susceptibility to conventional synthetic insecticides.

Con fl ict of interest statement

Acknow ledgm ents

The authors would like to thank the Tropical Pesticides Research Institute for providing infrastructure and resources toconduct the trials;Conselho Nacional de Desenvolvimento Científi co e Tecnol´ogico,Coordenação de Aperfeiçoamento de Pessoal de Nível Superior,and Fundação de Apoio`a Pesquisa e Inovação Tecnol´ogica do Estado de Sergipe for providing fi nancial support(Grant#475520/2012-2).

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29 Sep 2015

*Corresponding author:Eliningaya J.Kweka,Division of Livestock and Human Diseases Vector Control,Tropical Pesticides Research Institute,P.O.Box 3024, Arusha,Tanzania.

Tel:+255 787745555

E-mail:pat.kw eka@gm ail.com

Foundation Project:Supported by Conselho Nacional de Desenvolvimento Científi co e Tecnol´ogico,Coordenação de Aperfeiçoamento de Pessoal de Nível Superior,and Fundação de Apoio`a Pesquisa e Inovação Tecnol´ogica do Estado de Sergipe(Grant#475520/2012-2).

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