Larvicide and repellent activity of Hypenia irregularis (Benth.) Harley in the alternative control of mosquito Aedes aegypti

Larvicide and repellent activity of Hypenia irregularis (Benth.) Harley in the alternative control of mosquito Aedes aegypti Richard Dias Possel, Talita Pereira de Souza Ferreira*, Douglas Martins de Oliveira Bessa, Marcela Oliveira Ferreira, Diego Patrick Nusrala Dias, Gleys Kellen Aquino Moraes, Luana Fernandes Ferraz, Taciano Peres Ferreira, Ana Claudia Fernandes, Julcemar Didonet, Gil Rodrigues dos Santos, Bruna Letícia Dias, and Raimundo Wagner de Souza Aguiar


INTRODUCTION
Dengue is an emerging epidemic disease and it is among those transmitted by the Aedes aegypti, it is the one with the highest number of cases with death (Brasil, 2016).
The mosquito A. aegypti is the main transmission vector of dengue fever, yellow fever, zika virus and chikungunya (Jansen and Beebe, 2010;Brasil, 2016).
The large majority of the world's population lives in countries where the incidence of dengue fever is high, and between 50 and 100 million infections are estimated each year (Brady et al. (2012), in Brazil in 2016, according to the Brasil (2016), 1,438,624 probable cases of dengue were registered until the Epidemiological Week (SE), being these all the reported cases, except those already discarded. The mosquitoes vectors of this disease, the A. aegypti and A. albopictus, are highly adapted to the social dynamics and the environment of the cities, which makes dengue an illness typical of urban areas with specific characteristics (Johansen et al., 2014).
Currently, the most common form of combat to the mosquito is the use of synthetic insecticides, composed of organophosphates and pyrethroids or even other compositions that could be highly toxic. The frequent and intensive use and the increasing doses of these products show the main problems regarding the use of insecticides, creating mosquito populations that are resistant to these products, altering the chronological cycle of the environment and allowing the direct reach of the species that corroborate with the environmental balance, leading to adverse effects (David et al., 2018).
Essential oils are volatile compounds present in various plant organs. They are considered oils due to their lipophilic composition, even though they are chemically different from vegetable oils and fats. And they are called essential because of the association with the defense mechanisms and attraction of pollinators in the plants, functions considered essential for plant survival. Resin, leaf, flower, fruit and seed are organs that accumulate these substances (Siani et al., 2000).
The exploitation of natural resources in the production of insecticides prioritizes several beneficial factors, not only the vector restraint, but also for biodegradable reasons and to be fully derived from renewable resources. This would interpolate the non-resistance of insects, since their composition added to several active principles occurs in a prolonged way. Besides, plants just like insects are species that coevolve, provoking in mosquitoes diverse effects like repellency, inhibition of oviposition and feeding, developmental disorders, deformations, infertility and mortality (Dequech et al., 2009) Figure 1.
H. irregularis is a species regionally known in Jalapão as Cerrado's Rosemary and belongs to the family Lamiaceae, subfamily Nepetoideae, tribe Ocimeae and sub tribe Hyptidinae. Hyptidinae is widely confined in the Cerrado's central-brazilian region, and is endemic in the central and northern South America's savannas. Despite the great diversity of species living in these areas, the composition of the essential oil is only known from flowers' samples (Wannes et al., 2010). Therefore, in the search for renewable and biodegradable natural products to control the transmission of dengue that can be used as an active ingredient in insecticidal and repellent formulations, the essential oil extracted from leaves of H. irregularis were tested against larvae and adult mosquitoes of A. aegypti.

Collection of plant material
H. irregularis species, regionally known as Cerrado's Rosemary, was collected in Jalapão-Tocantins, (Latitude: 09º57'46" S and longitude: 47º40'38" W) located in the northern region of Brazil. Branches containing leaves and flowers of H. irregularis were collected for taxonomic identification. The assortment was carried out in the months of January and February of 2018.

Extraction and analysis of the essential oil
The extraction was performed by hydrodistillation in a modified Clevenger type apparatus, following the methodology of Guimarães et al. (2008). The H. irregularis's leaves were collected and dried in the shade and after that period were cut into small fragments using a pair of scissors. Subsequently, 1000 ml of distilled water and approximately 300 g of the plant were added to a round bottom flask.

Breeding of larvae and mosquitoes
The breeding of A. aegypti was established in the integrated pest management laboratory at the Federal University of Tocantins (Campus-Gurupi), according to the methodology of Aguiar et al. (2015). Mosquitoes of A. aegypti were originally collected in Gurupi-Tocantins, Brazil (Latitude: 11°43'45" S and longitude: 49°04'07" W), male vectors were fed with a 10% sucrose solution and female with rodent blood from alive Wistar (Rattus norvegicus albinus). Thus, no insecticide or derivative was used to control the mosquitoes. The larvae grew in plastic containers (35 cm × 5 cm) where they were fed with a sterilized diet (80/20 chow chick/yeast mixture). All bioassays were performed in photo period 12 h light / dark at 27 ± 1°C, 65.0 ± 6% RH.

Larvae bioassay
The larvicidal test was performed according to the methodology described by Cheng et al. (2003), with some modifications. Solutions containing water, 1.7% DMSO (Dimethyl sulfoxide) and essential oil were prepared in order to reach the following concentrations: 0.007 to 0.13 μl ml -1 . 30 ml of distilled water and twenty five larvae of A. aegypti in the 3rd instar (which was appropriate for the research) were added in disposable cups with a capacity of up to 100 ml. For higher average precision and the

Response time
For the creation of concentration-related response curves, each developmental stage and vector mortality were considered. The test consisted of the same methodology previously used, since the lethal time for 50% of the deaths was considered from the LC 50 and consequently 95% from the LC 95 . Mortality was cataloged in the first tests at 15 minutes and then after 60 minutes of exposure to the essential oil concentrations of H. irregularis, the results being later described as LT 50 and LT 95 , in minutes.

Test of repellency activity
The repellent activity of H. irregularis essential oil was tested according to the methodology described by Nério et al. (2010) and WHO (2015) with some modifications. Three acrylic boxes (24×24×24 cm 3 ) were prepared for the bioassay, using a total of one hundred and fifty female A. aegypti with four to seven days of age. Solutions were also prepared with the essential oils, which were dissolved in 99.8% ethanol, with the final concentrations being from 0.0033 to 0.167 μl.cm -2 . For the validation of the tests, eight volunteers were requested, and for each product tested fifty females were used per test in five replications.
Before each test the forearms of the volunteers were sanitized with 70% ethanol. After drying, an area of 300 cm 2 was measured and the remaining area was covered by latex gloves, because the sweat particles contain lactic acid, making it attractive to females. After these steps, the volunteers instead their forearms into the acrylic boxes for 3 min ( Figure 2). The test was performed at intervals of 30 min and continued until the first bite was recorded or, consequently, 135 min, being it the end of the planned time. Afterwards, the number of bites was counted to calculate the results.

Oviposition test
The effect of the essential oil on the A. aegypti's egg depositing was determined following the methodology described previously. Twenty-five females fed with mouse blood (1 to 3 days) and fifty males were inserted containing different concentrations and incubated at 28±2°C. The cages contained up to six replicates in plastic cups (100 ml) at appropriate concentrations (0.0833 to 0.2 μl.ml -1 ) in 30 ml of ethanolic solution with 1.7 % of the essential oil of H. irregularis. The test was repeated four times. The numbers of eggs were counted daily during the addition of the cups and summed at the end of the seven days.
The percentage of viability for both ovicide and oviposition tests was calculated by the following equation: %V = (T-I) / T×100, where % V is the viability percentage of the egg, T is the number of viable eggs in the treatment of control without application of essential oil and E is the number of viable eggs after the treatment with essential oil.

Gas chromatography coupled to mass spectrometer
The chemical composition of the oil was determined in the Analytical Center of the Institute of Chemistry of the University of São Paulo (IQUSP) using the Gas Chromatography/mass spectrometer (CG / MS) technique. The analysis was performed on Shimadzu GC-2010 equipment, equipped with a selective mass detector, model QP2010Plus.
In Gas Chromatography (GC) the compounds were subjected to analysis using Shimadzu GC-2010 instrument, equipped with RTX-5MS fused silica capillary column (30 m × 0.25 mm × 0.25 μm film thickness); with the following temperature planning in the column: 60 to 240°C (3°C/min); Injector temperature: 220°C; gas carrier helium; injection with a split rate (1: 100) with an injected volume of 1 μl of a 1: 1000 hexane solution. For the mass spectrometer (MS), the following conditions were applied: impact energy of 70 eV; temperature of the ion source and the interface: 200 °C.

Statistical analyzes
The method adopted for the analysis of the results was the use of statistical and non-parametric techniques, that is, mathematical models in which the larvae possessed exponential phases, where, depending on the concentration, they obtained the fastest death, and consequently in greater numbers.
Mortality concentration curves were estimated using the PROBIT procedure using POLO PLUS statistical software (LeOra Software Berkeley, CA, USA). Residual Activity Charts were plotted using the SIGMA PLOT 11.0 software (Systat Software, Inc. San Jose, USA). The results of the oviposition despersuasion and repulsion activity were submitted to variance analysis (ANOVA) followed by the Tukey's test performed in Graph Pad Prism software v.5.03 (San Diego, California, USA). Differences were considered significant when P < 0.05.

RESULTS AND DISCUSSION
The H. irregularis essential oil showed a high toxicity against 3rd instar A. aegypti larvae ( Table 1). The tests were performed with concentrations ranging from 0.007 to 0.13 μl.ml -1 for the oil studied, by comparing it with other oils studied before. To quantitatively and qualitatively analyze the present compounds of the oil in question, the gas chromatography coupled to the mass spectrometer was performed (Table 1).
The most widely used methods for quantitatively and qualitatively analyzing essential compounds according to European Pharmacopeia (2002), is the gas chromatography coupled to mass spectrometry because it is more precise and efficient.
The 2,5-dimethoxy-ρ-cymene constituent, or only ρcymene, is one of the main responsible for the antimicrobial activity already detected in studies with the use of essential oils tested in Staphylococcus aureus, for example. In addition, it is one of the main constituents of the Alpinia zerumbet essential oil (false cardamom) in according with the woks of Castro et al. (2016).
As already observed by Carvalho et al. (2003), which evaluated the larvicidal property of Lippia sidoides essential oil, thymol was also considered as one of the main toxic active ingredients against A. aegypti larvae, being able to induce 100% mortality in 90 minutes in the concentration of 0.017 μl.ml -1 . Brito et al. (2015) also performed studies evaluating these properties, finding thymol (84.95%) as the major constituent of the essential oil of this same species, followed by ρ-cymene (5.33%) and methyl carvacrol ether (3.01%).
According to Silva (2012), comparing the analyzed parameters values obtained in the essential oil tested with those in the literature, we can observe the presence of similarities. The small differences in the values found can be attributed to factors such as collecting period, storage time, genetic factors, edaphoclimatic factors and even different soil types, and according to Barros et al (2009), enzymatic actions can also be influenced by climatic conditions, and may lead to changes in certain secondary metabolites.
According to Cheng et al. (2003), LC 50 properties of less than 0.1 µl.ml -1 , based on their effectiveness, are defined as possible larvicidal agents.  Therefore, considering the LC 50 0.037 μl.ml -1 , the H. irregularis essential oil's presented results lower than this value, thus reinforcing the viability of its use as a larvicidal for A. Aegypti's larvae, especially considering that the essential oil has a low cost of production, easy acquisition, cultivation and high yield, considering that in the extraction process, with 300 g of H. irregularis' dried leaves, 1.25 ml of oil were extracted, yielding 0.35%. Complications are noticed when performing the comparative analysis with H. irregularis oil due to being a recently examined plant, for this purpose, making the literature associated with its studies inaccurate, however, in the tests highlighted, some related studies that also demonstrated good results in relation to lethal concentrations were referenced.
In the larvicidal test, it was possible to verify that the concentrations were, respectively, 0.037 and 0.122 μl.ml -1 for determination of LC 50 and CL 95 of H. irregularis essential oil (Table 2). In the literature, Assunção (2013) calculated that the LC 50 of the Citrus sinensis essential oil against Ae. aegypti was 99.014 μl.ml -1 , although it has a larvicidal effect, their study shows a concentration needed of almost 3000 times greater than the H. irregularis oil to reach the LC 50 effect. Senthilkumar and Venkatesalu (2012) tested the A. calamus' rhizome's essential oil for its larvicidal effect in Culex quinquefasciatus larvae and found the LC 50 value of 63.43 μl.ml -1 and CL 90 of 145.95 μl.ml -1 .
In the studies by Ríos et al. (2017), eleven essential oils were tested against A. aegypti and all of them reached LC 50 values lower than 115 μl ml -1 , the lowest LC 50 value was attributed to the Thymus vulgaris plant with 45.73 μl.ml -1 of oil and the highest LC 50 was attributed to the Cymbopogon martinii plant with 114.65 μl.ml -1 . In addition, the main components of oils with higher larvicidal activity were thymol (42 %) and ρcymene (26.4%), compounds similar to those found in the present study.
According to Pavela (2015), LC 50 values lower than 0.05 μl.ml -1 are considered effective in the larvicidal effect. The H. irregularis oil reached a value close to this, 0.037 μl.ml -1 , so it is within the standard of efficiency demonstrated in studies that tested more than 122 species of plants' essential oil as larvicide and repellent against different types of mosquitoes.
Cole (2008), tested the larvicidal activity of the extracts of the fruits of Schinus terebinthifolius against A. aegypti, and obtained an LC 50 result of 117.34 μl.ml -1 . In another case, Nunes (2017) determined the values of CL 50 and CL 95 regarding the action of the extract of L. sidoides and H. crenata for larvae of the 3rd instar of A. aegypti, resulting in L. sidoides CL 50 of 123 μl.ml -1 and CL 95 of 434.379 μl.ml -1 , and from H. crenata oil the values obtained were LC 50 of 0.035μl.ml -1 and CL 95 of 0.113μl.ml -1 . Silva et al. (2014) evaluated the Croton linearifolius species obtaining an LC 50 of 13.3 μl.ml -1 , Costa et al. (2005) presented an CL 50 of 0.0083 μl.ml -1 using the Piper marginatum oil, and Oliveira (2012), using Piper aduncum, that had its oil extracted using organic solvents, obtained an LC 50 of 0.29 μl.ml -1 . Thus, H. irregularis oil becomes viable in the face of studies already published, being an economical possibility to formulate repellents and other insect control strategies in general.
To determine the lethal times (LT 50 and LT 95 ) in A. aegypti 3rd instar larvae using H. irregularis essential oil, pre-defined concentrations correlated within 24 hours of total test duration were used. Therefore, in view of the obtained results, it can be inferred that the amount of essential oil related to the concentration can influence the result in the larval and / or adult phases or with differences in the metabolic detoxification.
In addition, there are no studies that demonstrate data on the characteristics and effects of H. irregularis essential oil in a complete way, which makes it an interesting alternative for future studies and the development of biological control products for insect vectors for tropical diseases and even microorganisms of agricultural and medical interest.
When evaluating the response times of H. irregularis oil (Table 3), it was possible to conclude that the LT 50 is reached in 20.79 min and the LT 95 in 27.41 min. Considering that the value of p (0.093) < x² (0.934), there is a significant difference between LT 50 and LT 95 . Regarding the literature, Aguiar et al. (2015) analyzed the essential oil of Siparuna guianensis, where the response time was also obtained using the concentrations of LC 95 determined for the third stage of A. aegypti, and concluded that the time required to reach 50 % was 21 minutes and 95 % mortality (LT 95 ) was 29 min. When comparing it with the studies of H. irregularis the former shoed superior results, considering that the LT 50 was 20.791 and LT 95 was 27.412 min.
In addition to toxicity in larvae, H. irregularis oil was shown to be very promising as a repellent (Table 3). The concentrations used above 0.167 μL cm -2 /skin were the ones that had the best protection time during the 135 min in 100% (Figure 4). In addition, repellent activity from essential oil concentrations was greater than that of the commercial product generally sold in Brazil as an insect repellent (with 15% N, N-diethyl-m-toluamide (DEET) in its active formulation), which was used as a positive control, and had a maximum protection of 69 min ( Figure  3).
According to the literature, Aguiar et al. (2015), 0.00045 μL cm -2 obtained 100% of repellency for A. aegypti. From these results, H. irregularis essential oil has the potential to be good candidate for a possible mosquito repellent formulation.
According to Pacheco (2013), Melaleuca alternifolia oil has a repellent potential of up to 98% in only 15 min of testing, however, such oil needs to be better formulated to be a commercial repellent candidate, with better fixation efficacy in the aromatic compounds of the skin and increased performance and duration of the repellent effect. In contrast, H. irregularis were able to demonstrate 100% repellency in the 135 min tested. Nevertheless, studies on the improvement of its fixation should be performed, taking into account all the formulations necessary to maintain the tested efficiency and increase its time of action in the skin.
It has been noted that the essential oil can also be dissuasive to oviposition. The percentage of oviposition of the essential oil of H. irregularis was calculated ( Figure  4), where the vertical axis corresponds to the percentage of eggs deposited and the vertical axis refers to concentrations tested with the oil. Thus, it is observed that the higher the concentration of essential oil used (0.2 μl ml -1 ) the less the deposited eggs (20%) when compared to the untreated tests. At the lowest concentration tested (0.08 μl ml -1 ), the percentage of eggs deposited reaches almost 74%.
At first, the oil showed high inhibition of the mosquito ovipositor, this can be explained due to its major components and also due to its concentrations compared with the control solution. It is also noticeable that in the  course of the days the essential oil volatization occurred, a variation in the values of the tests when compared to the control, however this factor was not limiting, because even then, a satisfactory inhibitory activity was still noted until the fourth day. Silva (2012) tested the species Etlingera elatioros and concluded that the major components of the essential oil represented a retarding action that would soon be analogous to the essential oil at a concentration of 0.05 μl ml -1 where there was a significant amount of death of more than 70% of the eggs deposited in the containers. It is important to note that A. aegypti females were not attracted only by clean water sources, indicating that the insect has some adaptability in the acceptance of laying substrates that vary in quality (Beserra et al., 2010). For Santos et al. (2017), the use of the essential oil of Syagrus coronata was also efficient regarding the deterrent effect in the pregnant females of A. aegypti. Their results indicated that the detergent activity may be linked to the presence of octanoic acids in the composition of the essential oil tested.
Thus, in addition to the larvicidal and repellent effect, the H. irregularis oil is able to stop the proliferation of insects, by destroying their eggs or impeding their oviposition and occlusion, including A. aegypti in several stages of its development, facilitating its control and, consequently, the control of diseases to which the insect is a vector.

Conclusion
The H. irregularis essential oil was shown to be effective in the larvicidal effect with an LC 50 of 0.037 μl ml -1 and an LC 95 of 0.122 μl ml -1 , for the response time LT 50 of 20.791 minutes and LT 95 of 27.412 minutes. In the oviposition bioassay it was observed that the higher the concentration of essential oil used (0.2 μl ml -1 ), the lower the number of eggs deposited, when compared with the untreated tests. The repellency test was 100% approved at a concentration of 0.167 μlcm -2 /skin and was more efficient than the commercial DEET repellent used as a positive control in 135 minutes of exposure to A. aegypti. Therefore, the results observed in the present study contribute strongly to the basis of possible new formulations.