Impact of Cymbopogon Flexuosus (Poaceae) Essential Oil and Primary Components on the Eclosion and Larval Development of Aedes Aegypti


 The current study describes the effects of sub-lethal concentrations and constituent compounds (citral and geranyl acetate) of Cymbopogon flexuosus essential oil (EO) on the development of Aedes aegypti. We treated eggs with 6, 18, and 30 mg.L-1 and larvae with 3 and 6 mg.L-1 EO concentrations. Citral and geranyl acetate were evaluated at 18, 30, and 42 mg.L-1 and compared to the commercial growth inhibitors (diflubenzuron and methoprene). We measured larval head diameter, siphon length, and body length. Finally, we examined concentrations of moult hormone (MH) and juvenile hormone III (JH III) using high-performance liquid chromatography coupled to mass spectrometry. The EO decreased egg hatching at all concentrations and altered molting among larval instars and between larvae and pupae, with an increase in the length (3 mg.L-1: 6 ± 0.0 mm; 6 mg.L-1: 6 ± 0.7 mm) and head width (3 mg.L-1: 0.8 ± 0 mm; 6 mg.L-1: 0.8 ± 0.0 mm) compared with the control group. We did not detect chromatographic signals of MH and JH III in larvae treated with C. flexuosus EO or their major compounds. The sub-lethal concentrations C. flexuosus EO caused a similar effect to diflubenzuron, decreasing hormone concentration, extending the larval period, and death.


Introduction
Annually, more than 2.5 billion people are a risk to get different arboviruses like Dengue, Zika, and Chikungunya virus in urban and peri-urban areas due to the vector Aedes aegypti 1,2 . Given that there is no effective vaccine for any of these three diseases 3,4 , the principal strategy to mosquitoes control consists in the population decrease using chemical insecticides on juvenile stages (temephos) and adults (deltamethrin, lambda-cyhalothrin, cy uthrin, and malathion) 4,5 . However, continued application of these products leads to resistant populations as well as unwanted environmental effects 6,7,8,9,10 .
In the search for new mosquito control alternatives, the juvenile stages are underexplored as an ideal target of control because these stages have developed exclusively in aquatic environments. Any interferences or alterations in these environments caused by an external source of hormonal or growth regulation can interrupt the normal life cycle and cause abnormal development in adult stages 11 . and larvicidal activity was reported in plants of genera Cymbopogon against Ae. aegypti 27, 28 , Soonwera and Phasomkusolsil (2016) 29 reported morphological abnormalities over juvenile stages of Ae. aegypti and Anopheles dirus larvae and deformed pupae, as well as incomplete hatching and mortality due to the action of Cymbopogon citratus EO.
Previously, studies evaluated the activity of Cymbopogon exuosus (Poaceae) EO against Ae. aegypti in terms of larvicidal activity, repellence, or dissuasive e cacy using a lethal concentration (LC) (CL 50 = 17.2 mg.L -1 ; CL 95 = 49.9 mg.L -1 ) 21,22 . However, these studies did not mention the effect of this EO on development alterations. Therefore, the objectives of this study were to describe the effect on Ae. aegypti eggs and larval development when treated with of sub-lethal concentrations of Cymbopogon exuosus EO and its major compounds: citral (consist of a mixture of two isomers geranial and neral) and geranyl acetate. Additionally, considering that the major compounds of this plant have structural similarity ( Figure 1) with the commercial growth regulators and developmental inhibitors, we used highperformance liquid chromatography (HPLC) coupled with mass spectrometry to examine the potential variations of the concentrations of moult hormone (MH) and juvenile hormone III (JH III) caused by C. exuosus EO and its major compounds on Ae. aegypti larvae.

Materials And Methods
Essential oil extraction and isolation of major compounds C. exuosus EO was provided and characterised by the 'Centro de Investigación en biomoléculas (CIBIMOL) and Centro Nacional de Investigación para la Agroindustrialización de Plantas Aromáticas y Medicinales Tropicales (CENIVAM), of the Universidad Industrial de Santander (Colombia). The oil extraction methodology, as well as its chemical characterization, was carried out following the methodology described by Stashenko et al. (2004) 30 . The essential oil was obtained by hydrodistillation (HD) and microwave-assisted hydrodistillation (MWHD). The components were identi ed by comparing their relative retention times and mass spectrometry with the standard compounds 30 . The description of EO chemical characterisation was reported by Vera et al. (2014) 21 : citral (geranial 37.5% and neral 28.2%), geranyl acetate (10.0%), geraniol (9.0%), and β-Bourboneno trans-β-cario lene (2.0%). The major compounds (citral and geranyl acetate) were commercially acquired from Sigma-Aldrich (St. Louis, MI).

Biological material
The experiments were performed with a colony of Ae. aegypti, Rockefeller strain, maintained in an insectary at 25 ± 5 °C, with humidity of 70 ± 5% and photoperiod (12:12 concentrations previously establish (6, 18, and 30 mg.L -1 ), a cup as a control treatment (49 mL of water + 1 mL of 0.5% DMSO), and a cup as a positive control (di ubenzuron at 6, 18, and 30 mg.L -1 ). To collect and count the eggs, each cup was coated inside with half of a Whatman ® # 1 lter paper, folded in a cone shape. The oviposition lasted for eight consecutive days, changing the lter paper daily. Nine replicates were used for each concentration by substance (EO and Di ubenzuron), distributed on three different days. When the females oviposited, between 50 and 100 embryonated eggs (recollected after 72 hours) per cup were taken at random. The eggs were transferred to individual containers, according to the evaluated concentration, and they were counted and examined under a stereoscope to verify integrity. The hatching percentage of the eggs was evaluated up to 120 h after oviposition by submerging the eggs obtained in mineral water. The emerged rst instar larvae were counted under a microscope, and eggs that did not hatch after seven days were considered not viable. The hatching value was estimated as the percentage of eggs that went on to the larval stage.

Larvicidal activity
For those experiments, we establish two sublethal concentrations of 3 and 6 mg.L -1 of C. exuosus EO.
To evaluate the major compounds citral and geranyl acetate, we also investigated the 3 and 6 mg.L -1 concentrations. However, we did not observe any effect on the larvae. For this reason, we increased the concentrations until establishing three sublethal concentrations of 18, 30, and 42 mg.L -1 . Methoprene PESTANAL ® (Sigma-Aldrich) and di ubenzuron PESTANAL ® (Sigma-Aldrich) at 3 and 6 mg.L -1 were used as a comparison factor, and dimethylsulfoxide (DMSO 0.5%) as a negative control.
To determine the effect on larvae development, we used the methodology by Leyva et al. (2013) 31 with some modi cations. Brie y, ten larvae in stage L3 were selected and transferred by Pasteur pipettes to 200 mL plastic cups with 99.5 mL of chlorine-free water and 0.5 mL of each treatment (negative control, C. exuosus EO, citral major component, geranyl acetate major component, and methoprene and di ubenzuron as positive controls). All larvae treatments were supplied with 2% sh feed in chlorine-free water to ensure their survival. After 24 hours of treatment, the dead larvae were removed, and the survivors remained in the water until they pupated. At 48 hours, ve larvae were taken for each treatment and were used to measure morphological parameters (in mm): larval length, cephalic diameter, and siphon length. Nine replicates were used for each concentration by substance (EO and Di ubenzuron), distributed on three different days. Octadecylsiloxane-ODS column Zorbax XDB-C18 (4.6 mm x 10 mm, 2.0μm) was used.
Water (0.1% formic acid) plus acetonitrile (0.1% formic acid) and water (0.1% formic acid) plus methanol (0.1% formic acid) were used as the mobile phase, applying a binary gradient at a ow of 0.30 mL/min, with an injection volume of 10 μL. The total running time of the equipment was 15 min. The retention time corresponding to the signal of each hormone was obtained, and the concentration levels of the hormones in each treatment were determined.

Statistical analyses
All data were subjected to normality tests. The xed factors in each experiment consisted of the substances (Cymbopogon exuosus EO, Citral, geranyl acetate, di ubenzuron, and Methoprene) and the concentrations evaluated. The random factors in each experiment were the different effects of the substances against Ae. aegypti mosquito (% of hatching eggs, morphological parameters (in mm) of larvae, % of individuals in each development stage). When the data presented a normal distribution, we used an ANOVA and subsequently, Tukey's test. If the distribution was not normal, non-parametric tests were applied and subsequently the Kruskal-Wallis test. Statistical signi cance with values of p ≤ 0.05. The results were analysed with Statistica Software V11.

Results
Ovicidal activity We obtained a 65% decrease in hatching of eggs treated with C. exuosus EO at concentrations of 6, 18,  Figure 9). With the major concentration (42 mg.L -1 ), we obtained 100% mortality in the larvae at the rst day of treatment (Figure 7).
Juvenile hormone and moult hormone levels in larvae treated with EO We measured juvenile hormone III (JH III) and moult hormone (MH) from larvae (L4) by HPLC coupled with mass spectrometry using methanol as a mobile phase. The MH was followed at 6.77 min retention time ( Supplementary Fig. 1a) that corresponds to the molecular ion (mass/H + of 481.1 g.mol -1 ). The JH III was followed at 9.86 min retention time ( Supplementary Fig. 2a) that corresponds to 266.6 g.mol -1 from molecular ion mass.
To analyse the chromatograms of Ae. aegypti larvae (L4) treated with C. exuosus EO and its major compounds (citral and geranyl acetate), we compared the values of areas under the curve observed for MH and JH III in the positive controls (methoprene and di ubenzuron) with the values observed with C.

Discussion
The action mechanism of most synthetic pesticides explains the interaction or union of a synthetic molecule with a speci c target from a protein or biomolecule. This union or interaction triggers a series of biochemical events that modify the physiological functions or cause death 33 . Although the synthetic pesticide has four or six targets, the majority act directly on neuronal receptors or ions channels. The action on these targets makes them highly speci c, contributing to the generation of resistance mechanisms 33,34 .
The search of new molecules that counter the resistance levels has prompted the screening of substances of natural origin like essential oils and major compounds 33,35 . The importance of these natural substances resides in a wide range of molecular targets on proteins (enzymes, receptors, ions channels, structural proteins), nucleic acids, biomembranes, and interfered on different metabolic pathways 36 .
Ovicidal activity One of the major problems in the Ae. aegypti control resides in that its eggs are highly resistant to desiccation periods, remaining in latency near to the end of embryonic development 37  Ae. aegypti eggs are characterised by two layers, the external (exo-chorion) and internal (endo-chorion).
On the inner side of the endo-chorion is a serosal cuticle, which is formed during the embryogenesis process and protects the embryo from external factors like desiccation or the presence of bacteria or insecticides 37 . However, this serosal cuticle can be disrupted by lipophilic substances 39 like terpenes citral and geranyl acetate, major compounds of C. exuosus EO. The hatching inhibition by these terpenes is due to an interruption of embryo development caused by physiological alterations in water and gas exchanges, enzymatic modi cations, and hormonal changes. These physiological alterations cause the embryo to not fully develop, decreasing the percentage of hatching 39,40 , as observed in the results of the present study ( Figure 2).

Activity on larvae and the life cycle
We found that the sub-lethal concentrations of C. exuosus EO affect the normal development of the juvenile phases of Ae. aegypti. The EO caused development alterations and morphological changes in larvae, represented in the lack of change in stage, an increase in the size of the larva, the width of the head, and length of the siphon (Figure 3 and 4). These results agree with the report by Soonwera and Phasomkusolsil (2016) 29 , who found morphological abnormalities (in Ae. aegypti larvae, pupae, and adults), and high percentages of larvae mortality that do not reach the pupae stage under concentrations of 1, 5, and 10% of C. citratus EO.
We observed similar morphological changes in larvae treated with C. exuosus EO and methoprene, with an increase in the width of the head and the size of the larva (Figure 3 and 4). Because methoprene is a juvenile hormone analogue, it acts directly on the moult, permitting the increase of larvae size but preventing the change to pupae stage 42 . This inhibition effect on the juvenile stage change was also observed in larvae treatment with C. exuosus EO, with a maximum duration period in the larval stage of 12 days. On the other hand, the measures of the morphological parameters with di ubenzuron treatment were lower than measures in the presence of C. exuosus EO (Figure 6). Di ubenzuron inhibits chitin synthesis, causing alterations in the cuticle layers during the moult 11,43 . These alterations also alter the normal duration of development stages. In our case, they caused larval development inhibition, which was a duration greater than 12 days of observation.
To verify if the effect observed by C. exuosus EO is due to the action of the major compounds (citral and geranyl acetate), we found that the increase of the size of the larva was the unique parameter with a similar change to the effect caused by EO. Related to larval stage duration, we did not observe a notable inhibition at concentrations of 18 and 30 mg.L -1 . However, at the higher concentration (42 mg.L -1 ), both compounds caused larval mortality (Figure 8 and 9).
The major compound in C. exuosus EO is citral. It is a monoterpene with an acyclic aldehyde functional group, responsible from the aroma in species from the genus Cymbopogon, with different biological properties. The mortality per cent obtained in the present study with citral is similar as reported by other studies, with a mortality per cent of 100% in Ae. aegypti larvae (Figure 8) 27,44 . Concerning size modi cations in larvae, the mechanism of action involved may be related to the study by Chaimovitsh et al. (2010) 45 . These authors reported that the principal target of citral is the microtubules, which directly interact with tubulin and cause damage in the cell membrane in both animal and vegetable cells.
Additionally, Orhan et al. (2008) 46 found that citral is a reversible competitive inhibitor of acetylcholinesterase (AChE), affecting nerve impulse transmission. Finally, Matsuura et al. (2006) 47 found that citral has an inhibitory activity against tyrosinase, an enzyme responsible for different biological process, among those found the exoskeleton consolidation in arthropods during the molt process 48 .
Concerning the bioactivity observed under geranyl acetate treatment (an acyclic monoterpene), the results of the present study are similar to those reported by Michaelakis et al. (2014) 49 who evaluated the repellent and larvicide activity on Ae. aegypti of different compounds and derivatives. They argue that although the larvicidal activity of geranyl acetate is minor to that of citral, geranyl acetate presented higher repellent and larvicide activity related to his functionality and degree of saturation. Additionally, Cheng et al. (2009) 50 reported a larvicide activity of 100% with geranyl acetate against Ae. albopictus larvae. Although the exact mechanism of action involved is unknown, many authors mentioned that the monoterpenes structure and their functional modi cations play a fundamental role in the activity against mosquitoes, potentialised in acetylated forms 51 .
Considering these results, we can infer that C. exuosus EO in sublethal doses cause a similar effect as the juvenile hormone analogue, probably causing alterations in the homeostasis of hormones involved in the moulting process, which leads to abnormal larval development and growth 52, 53 . Rattan (2010) 36 mentioned that the EO and its constituents affect biochemical processes of insects, especially endocrine balance, with alterations in the morphogenesis process. However, this effect is not caused exclusively by the major compounds of the oil, citral, and geranyl acetate. This may be due to the synergic effect of their components, generating a greater biological response 55 .

Measuring of juvenile hormone and moult hormone levels
With the HPLC technique coupled with mass spectrometry, we identi ed the signals corresponding to molt hormone (MH) and juvenile hormone III (JH III) in untreated individuals. With di ubenzuron and methoprene standards treatments, we did not detected a peak corresponding to JH III. Considering that the treatments were performed in larvae L3, it is possible that the absence of JH III is due to natural diminution of the concentration of this hormone, which is higher during the rst larval stages and decreases in the L4 stage to permit the pupation and metamorphosis process 55 When we examined the chromatogram corresponding to larvae (L4) treated with C. exuosus EO and its major compounds (citral and geranyl Acetate), there was no clear peak corresponding to MH or JH III, or any of their fractions. Considering the diminution of the JH III in L4 and the possible effect of the EO on larval development, the concentration of this hormone could have been below the limit of detection based on the calibration curves performed for the JH or MH. In relation to the MH, the alteration in the process of ecdysis and chitin formation could be altered due to the action of the majority citral component, causing changes in the concentration of this hormone.

Conclusions
The EO from C. exuosus used at sublethal concentrations has bioactivity in eggs and larvae of the Ae. aegypti mosquito. In the egg stage, the EO can alter the development of the embryo, decreasing the number of hatched individuals when penetrating through the chorion. In early larval stages (L1, L2, and L3), it causes an effect similar to a juvenile hormone analogue, lengthening the larval period and making pupation impossible. Additionally, the majority component citral may cause alterations in the normal process of ecdysis, a process directly related to the moulting hormone. Using HPLC technique coupled with mass spectrometry, it was possible to identify the signal corresponding to JH III and MH in untreated individuals. However, in larvae treated with C. exuosus EO and its major compounds (citral and geranyl acetate), there was no clear peak. This result demonstrates an alteration in the concentration of JH III and MH hormones due to the action of EO, an effect that cannot be attributed exclusively to the major components of EO.