Larvicidal effect of Clinopodium macrostemum essential oil extracted by microwave-assisted hydrodistillation against Culex quinquefasciatus ( Diptera : Culicidae )

Introduction: In the present study, we determined the chemical composition of Clinopodium macrostemum essential oil obtained by microwave-assisted hydrodistillation and its effect on the growth and development of Culex quinquefasciatus mosquito larvae. Methods: The essential oil compounds were identified by gas chromatography-mass spectrometry analysis, and bioassays were conducted to evaluate the influence on the developmental stages of early second instar larvae of Cx. quinquefasciatus until the emergence of adults, using essential oil concentrations of 50, 100, 200, 400, and 800mg/L. Results: The gas chromatographymass spectrometry analyses revealed that the leaf essential oil of C. macrostemum contained 32 compounds and the major chemical compounds identified were linalool (55.4%), nerol (6.4%), caryophyllene (6.25%), menthone (5.8%), geraniol acetate (4.1%), terpineol (3.7%), and pulegone (2.8%). The essential oil yield obtained by microwave-assisted hydrodistillation was 0.8% at 20 min. The treatments showed lethal concentrations (LC50 and LC90) of 22.49 and 833.35mg/L, respectively, after the final measurement of the total number of dead larvae (second, third, and fourth instars), and LC50 and LC90 of 6.62 and 693.35mg/L, respectively, at the end of the experimental period. The essential oil inhibited the growth and development of the mosquito larvae by 32% (relative growth index = 0.68) at 50mg/L, and up to 47% (relative growth index = 0.53) at 800mg/L. Conclusion: The present study demonstrated the larvicidal effect of C. macrostemum essential oil on Cx. quinquefasciatus, which can be attributed to the oxygenate compounds obtained by the extraction method.

In Mexico, the traditional uses of C. macrostemum date back to pre-Hispanic times.Francisco Hernández, a Spanish physician who visited Nova Spain during the mid-16 th century, reported several medicinal uses for this plant among the Mexican natives.Infusions of fresh leaves are used as a remedy for various gastrointestinal disorders, coughs, and to promote digestion 2,5 ; tea infusions are used as drinking water or for relieving a hangover caused by the excessive consumption of alcoholic beverages; the leaves are used for seasoning foods or as milkweed; and the branches or leafy sticks, in the form of bunches, are utilized as adornments in religious and civil festivities 4 .
Several Lamiaceae species have been evaluated for their larvicidal and insecticidal activities.For example, the aqueous extracts from Clinopodium laevigatum Stand.were evaluated against those from Culex quinquefasciatus (Say, 1983); however, they did not show any effectiveness 6 .The essential oil obtained by hydrodistillation from Clinopodium gracile (Benth.)Kuntze had a larvicidal effect on Aedes (Stegomyia) albopictus (Skuse, 1894), which is one of the two main vectors for dengue fever and malaria reported in China 7 , and the essential oil obtained by hydrodistillation from C. chinense (Benth.)Kuntze was reported as having insecticidal activity on Liposcelis bostrychophila (Badonnel, 1931) 8 .However, in the abovementioned studies, the essential oils were obtained with only hydrodistillation as the extraction method.Hydrodistillation at atmospheric pressure is routinely applied for the isolation of essential oils from plant material 9 and, when assisted with microwaves, the combined method offers additional advantages, e.g., reduction in the isolation time, less environmental effects, less energy cost, and lower use of water as a solvent 10,11 .Conversely, the compounds present in the essential oils and their concentrations depend on the extraction method employed 10 ; therefore, it is important to consider different extraction techniques to obtain the constituents and quantities required.
The family Culicidae is native to Mexico and has a great biodiversity containing 203 registered species, specifically 22 species from the subgenus Culex, highlighting Cx. quinquefasciatus as being widely distributed in residential and agricultural areas 12 .This mosquito species is an important vector for the dissemination of arboviruses such as Zika, which has an important influence because it can increase the cases of Guillen-Barré and congenital syndrome 13 , and lymphatic filariasis 14 , with the World Health Organization reporting that 856 million people in 52 countries are threatened by this disease and are in need of prophylactic treatment.The traditional control for this mosquito species is via dichlorodiphenyltrichloroethane (DDT) and pyrethroid insecticides 15 ; however, it has become resistant to a wide range of chemical pesticides 16,17 .Another alternative to control this species is by biopesticides such as Bacillus sphaericus (Neide, 1904); however, a study by Fajar et al. 18 reported only a mild toxicity in this mosquito species and others from genus Culex.The use of essential oils obtained from plants 19 has been reported as an alternative control for Cx.quinqufasciatus and could be environmentally friendly as they are a botanical derivative, for which the composition and quality employed are very important and depend on the nature of the plant material and the extraction method applied 20 .The aim of the present study was to perform a qualitative analysis and characterization of the chemical composition of the essential oil obtained by microwave-assisted hydrodistillation (MAH) from C. macrostemum, and evaluate the larvicidal effect of this essential oil on Cx. quinquefasciatus as an important arbovirus vector.

Plant material
Fresh leaves were collected from plants without flowers that were growing in forest in the Santa Maria Huitepec village, municipality of Totontepec Villa de Morelos, in the Mixe region of Oaxaca state, Mexico, with previous permission from the local authority.A voucher specimen was pressed and deposited in the herbarium of the Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional (OAX) Unidad Oaxaca.

Extraction kinetics
The plant material was dried at average environmental temperature of 28°C and rehydrated 45 min before MAH.The extraction method was performed in an aromatic water recirculation system adapted with a MW1235WB microwave (SAMSUNG, Seoul, South Korea) using a 1:10 (w/v) relationship from 100g of plant material and 1,000mL of water, with a irradiation frequency of 2450 MHz.The essential oil extracted was quantified every 10 min (radiation time) and left to stand for 5 min; the extraction was stopped when three successive quantifications remained constant.Every extraction kinetic was performed in triplicate.

Gas chromatography-mass spectrophotometry identification
Gas chromatography-mass spectrophotometry (GC-MS) analysis was performed using an Agilent 6890N coupled to a JEOL-JMS-GC-MATE III.Samples were analyzed on a fused silica capillary column HP5MS (30m × 0.32mm I.D., film thickness 0.25µm), with helium as the carrier gas, injector and detector temperatures set at 300°C, injected volume of 1µL, oven temperature programmed at 30°C with increases of 8°C/ min up to 300°C, and ionization energy of 70 eV.Identification of the component was based on computer matched NIST 98 and the Dictionary of Natural Products 21 .

Collection of egg rafts and maintenance of mosquito larvae
The egg rafts of Cx. quinquefasciatus were collected from standing water in the facilities of the Research Interdisciplinary Centre for Regional Integral Development (CIIDIR-Oaxaca).The egg rafts were taken to the CIIDIR-Oaxaca bioplant laboratory and individually placed into plastic trays of 47cm × 35cm × 12cm containing 300mL of water for biological development.Mosquito larvae were fed with a sprayed product used to feed fish (Api-barge).

Preparation of the essential oil concentrations for application
From the essential oil stock solution, 0.008mL was solubilized using Tween-20 (0.01% of polysorbate) as the emulsifier agent and diluted in 10mL of distilled water to prepare a serial dilution of test concentrations.Serial dilutions at concentrations of 50, 100, 200, 400, and 800mg/L were performed for each of the 80 larvae tested, assigned in quadruplicate.

Evaluation of larvicidal activity based on the essential oil composition
Mosquito larvicidal assays were performed based on the methodology of the World Health Organization Standard Procedures with slight modifications 6 .Twenty-eight days after the treatment application, early second instar larvae of Cx. quinquefasciatus were observed to measure the influence of the different essential oil concentrations on the developmental stages (second, third, and fourth instars, pupae and adults).For each treatment, groups of 20 larvae were selected and placed into a plastic beaker with 99mL of distilled water and 1 mL of the essential oil concentration to be evaluated.The mortality induced by the essential oil was evaluated using two criteria: I) when inside the plastic beaker, larvae did not show movement that was similar to that exhibited by larvae from the control group, then the larvae was considered to be dead; and II) when larvae was disturbed with a brush to the siphon in its cervical region and it did not show any reaction, it was considered dead.Each treatment was replicated four times.Dead and live larvae and pupae were counted and removed daily from each developmental stage, as well as the number of emergent adults measured 22 .Measurements of larval and pupal mortality, and adult emergence were recorded to calculate the relative growth index (RGI) using the following formula: The control group (no treatment application), containing 80 larvae, were conditioned with 400mL of distilled water.

Statistical analysis
Bioassays were established under a completely randomized design.The Minitab v17.0 system analysis of variance procedure was used and significant differences to p < 0.01 were calculated.The average larval mortality (LM) (second, third, and fourth instars) up to the calculation of RGI and total mortality (TM) (second, third, and fourth instars, pupae and adult) at the end of the experimental period were subjected to probit analysis to calculate the lethal concentration a half (LC50) and 90% (LC90) of mosquito population and chi-square values.In all tests, no mortality was detected unless essential oil had been provided as a stimulus; therefore, no correction was required based on the Abbott formula.

Larvicidal activity
The essential oil extracted from C. macrostemum had affectivity against larvae of the mosquito Cx. quinquefasciatus.Table 2 shows that the effect of the essential oil was high when early second instar larvae developed into the fourth instar stage, which indicates that the essential oil exerted pressure on insect development.The essential oil at a concentration of 50mg/L ensured a LM of 63.8% (in the fourth larval stage); however, when the dead pupae percentage was added at the end of the experimental period, the TM was increased to 72.5%.The mortality provoked by a 400mg/L essential oil concentration increased from 86.3% (LM) to 90.0% (TM).The highest concentration of essential oil (800mg/L) did not record any variation between LM and TM owing to the fact that the remaining 10% formed into pupae (i.e., were alive).The essential oil had a significant toxic effect on mosquito larvae of Cx. quinquefasciatus.Table 3 shows the lethal concentrations of LC50 and LC90 as being 22.49 and 833.35mg/L, respectively, when measuring the total number of dead larvae (second, third, and fourth instars), and LC50 and LC90 of 6.62 and 693.35mg/L, respectively, at the end of the experimental period, when the number of dead pupae were added to the total number of dead larvae.Additionally, the essential oil inhibited the growth and development of the mosquito larvae at a rate of 32% (RGI = 0.68) in the lowest concentration (50mg/L) and up to 47% (RGI = 0.53) in the highest concentration (800mg/L), compared to the control group that had a RGI of 1.04.The emulsifier agent (Tween-20 = 0.01% of polysorbate) was tested and did not show any biological activity against the mosquito developmental stages, which was the same as the data obtained for the control group (no treatment application).This showed that the inert suspension did not interfere with the development of the mosquito larvae.

DISCUSSION
The importance of public health care should be considered from the control of arbovirus vectors from their larval stage to stop global expansion of diseases.It is important to develop environmentally friendly biocontrol mechanisms that are not resistant to adaptations of mosquito species such as those developed by synthetic pesticides in species of the genus Culex.The present study is the first to report on the bioactivity of essential oil from C. macrostemum for the control of Cx. quinquefasciatus mosquito larvae.An early evaluation using aqueous extract from similar species did not show any activity against this mosquito 6 .The extraction method used in the present study was useful for evaluating the insecticidal properties of C. macrostemum.This is because the microwave-assisted extraction method is a more efficient method for this purpose because it enables the enhancement of several of the extracted essential oil properties, including toxicity, antimicrobial and larvicidal activities, in comparison to that obtained by conventional techniques 10,[22][23][24] .The major compounds identified in the present study and larvicidal activity against the mosquito species have been found in other Lamiaceae species and therefore represent a defense and attack system for Cx.quinquefasciatus.Our results showed a mortality effectiveness of up to 80% with an essential oil concentration of 400mg/L, with similar results reported by Ansari et al. 25 for the larvicidal activity of Mentha piperita L. essential oil against Cx.quinquefasciatus, in which were present several of the major compounds identified in the present study.In this respect, several monoterpenes such  Considering the presence of caryophyllene (6.3%) in the essential oil identified from C. macrostemum, it is possible to compare it with other species of genus Clinopodium, as reported by Chen et al. 7 , who documented a strong larvicidal activity of essential oil from C. gracile aerial parts against the fourth instar larvae of Ae. albopictus.This previous study reported a higher content of sesquiterpenoids (70.5%) than monoterpenoids (12.2%) and one of the major compounds was caryophyllene (5.2%), which was similar to the present study.
Another important compound identified in the present study was linalool, which exhibited the highest percentage (55.4%)and has been identified in the essential oils of Lavandula gibsoni Graham and Plectranthus mollis (Aiton) Spreng.Both plants have activity against fourth instar larvae of mosquitos such as Aedes (Stegomyia) aegypti (Linnaeus, 1762), Anopheles stephensi Liston, and Cx.quinquefasciatus 30 .Among the major components identified in these essential oils were linalool for L. gibsoni (the more abundant compound in the present study) and piperitone for P. mollis.It was reported by Borges de Castro et al. 31 that the synergic effect of all compounds in essential oils could enhance their larvicidal effectiveness, which is shown by the direct relationship between concentration and mortality effect.
We demonstrated for the first time the effect of the essential oil of C. macrostemum on Cx. quinquefasciatus mosquito larvae.The extraction method employed in the present study (MAH) could be responsible for the larvicidal effectiveness of the essential oil obtained.The results show the potential of this essential oil as a means of biological control for mosquitoes, which are vectors of diseases with high incidence in the tropics and constitute a serious public health problem.The essential oil evaluated here shows a high effectiveness against Cx.quinquefasciatus, since it provoked a TM of this mosquito species as high as 72.5% in a concentration as low as 50mg/L.The results could be useful in the search for newer, safer, and more effective natural larvicidals and inhibitors of growth agents against Cx.quinquefasciatus.

TABLE 1 :
Chemical composition of the essential oil obtained from Clinopodium macrostemum dried leaves by microwave-assisted hydrodistillation.

TABLE 2 :
Mortality (%) from the developmental stages of the second instar larvae of Culex quinquefasciatus treated with Clinopodium macrostemum essential oil obtained by microwave-assisted hydrodistillation.Data represent means of the experiments performed in triplicates (n = 3).Means followed by different letters within the same developmental stage are significantly different at P < 0.01 compared to the control group.
II: second instar larvae; III: third instar; IV: fourth instar; C: concentration; LM: larval mortality; DP: dead pupae; TM: total mortality at the end of the experiment.RGI: relative growth index; SD: standard deviation.