Introduction

Aedes aegypti transmits dengue, for which there are no competent vaccines available. Controlling vectors or avoiding their bites is the only way to prevent disease transmission, as daytime feeder Ae. aegypti can be best avoided by repelling them. Generally, the use of synthetic repellents is in common practice. However, the frequent and injudicious use of synthetic repellents has already made the exposed mosquito populations resistant and thereby rendered these chemicals gradually ineffective for repelling mosquito at the recommended doses. Besides, the repeated uses of synthetic repellents resulted in numerous health issues like neurological problems, allergy and skin irritation, etc., in human being along with associated environmental hazards. DEET, which is considered as the gold standard repellent, has been reported to initiate seizures, bradycardia, nausea, vomiting, encephalopathy and anaphylaxis, especially in pregnant women and children after dermal application [1].

These negative effects of synthetic repellents have ignited the researchers to think for eco-friendly alternatives of which botanicals are getting much importance. The past few decades have witnessed extensive research on the exploration of plant products for implications in pest control [2,3,4]. As a result, plant-derived repellents, neem oil, etc., are getting attention [5].

Continuing the search for effective natural repellents, in the proposed study, we selected essential oils (EO) derived from Citrus species. The genus Citrus of the family Rutaceae deserves importance as the source of a most commonly used fruit. The essential oils and compounds derived from Citrus are known for their medicinal, insecticidal, antibacterial, antifungal, antimicrobial properties and for their good demand as flavoring agents in food processing [6]. Essential oil is aromatic, volatile liquids derived by steam or hydro-distillation from plant material. EO comprises 20–60 compounds of which the major constituent compounds usually determine the bioactivity [7]. The essential oils and compounds find widespread application in medicine [8, 9], pest control [10], and it has also been implicated in allelopathy, the phenomenon in which one plant uses chemical inhibitors to interfere with the growth and development of another plant [11]. As Citrus is an edible fruit, there would be less chances of harmful effect of Citrus-derived essential oil on user and on environment as well. To be used by the common people the repellent product should be commercially viable or locally available. In this respect, Citrus plants are widely distributed and commercially grown mainly for obtaining fruits. Notably the North Eastern part of India is hub of Citrus sp. where mosquito species and mosquito-transmitted diseases are considerable.

Constituent compounds and biological activity vary in the same species in different environmental conditions [12]. So, to evaluate if the Citrus species present in our locality (North-East India) possess mosquito repellent property, we considered the EO of six commonly available Citrus species that are reported to have medicinal and insecticidal activities [10, 13] to assess repellent activity against Ae. aegypti. Citral and limonene are the major constituent compounds of Citrus aurantifolia as recorded in our earlier works [13]. Therefore, the repellency of these two constituent compounds was also assessed, along with a reference repellent compound DEET against the target mosquito.

Repellents mainly mediate their action via the olfactory sensory neurons of insects. The odorant-binding proteins (OBP) connect to the sensory neurons and play a pivotal role in the uptake of volatiles. OBPs transport the hydrophobic odorants through the lymph of the sensilla to their receptors. In Ae. aegypti, OBP1 and OBP22 have been reported as the mediator of olfaction [14]. Acetylcholinesterase (AChE), a central nervous system-associated enzyme plays an inevitable role in the normal physiology of the insects, was reported as the target of DEET and EOs for their contact toxicity [15]. So, molecular docking analysis of the citral, limonene and DEET was performed toward OBP1, OBP22 and AChE to examine their binding affinity.

Materials and Methods

Mosquitoes

The colony of Ae. aegypti was maintained in the Laboratory of Entomology, Gauhati University at 27 ± 2˚C and 70 ± 5% RH and 12:12 light/dark cycle. The culture was maintained similar to our earlier works [13]. Initially, eggs were collected from the ICMR-RMRC Dibrugarh. Furthermore, the mosquito was confirmed as Ae. aegypti using the identification key provided by the Indian Council of Medical Research. Museum specimen was submitted to the Biodiversity Museum Gauhati University with voucher specimen number A-10/ARI-31.

Plant Materials

Six locally available Citrus species, namely C. aurantifolia, C. maxima, C. aurantium, C. limon, C. medica, C. paradise, were collected and identified in the Department of Botany, Gauhati University.

Extraction of EO and Collection of the Constituent Compounds

EOs were extracted from the fresh peel (200 g) by hydrodistillation using Clevenger’s apparatus. The peel of citrus fruits was carefully removed and chopped into small pieces. Those pieces were put into the bowel of the apparatus (Volume 1L) with 500 ml water. The apparatus was set in a heating mantle, and tap water was allowed to run continuously through the condenser for six hours. After the peel is heated fully, the volatile compounds evaporate, cool at the condenser region, and descend down and decant at the surface of the water near the oil delivery cock. The oil was then collected in a glass vial. Moisture was removed using crystals of anhydrous sodium sulfate and stored at 4 °C [16].

Selected terpene compounds, i.e., limonene and citral and synthetic repellent DEET were purchased from Sigma-Aldrich having 97–98% purity.

Repellent Bioassay

Repellent bioassay was performed by the arm-in-cage method using human volunteers [17]. Hands were washed with tap water, dried with a towel, and the prepared concentrations (0.1, 0.2, 0.25, 0.5, 1, 2, 3, 4, 5 & 6 mg/cm2) were applied (one concentration at a time) on 25 cm2 area of the dorsal side of the arm. As a solvent and negative control, ethanol was used, whereas DEET was used as a positive control. The treated surface was exposed to 100 non-blood-fed female mosquitoes (5–7 days old). Tests were conducted from 09:00 to 16:00 h in the natural lightroom and in the laboratory temperature (28–32 °C). Each concentration of EOs and compounds was evaluated in triplicate on the same human subject. The number of bites was counted for 1 min after every five minutes up to 4 h. The percentage of repellency was calculated using the following formula

$$ P \, = \, \left( {Nc \, - \, Nt} \right)/Nc \, \times 100 $$

where P is the percentage of repellency/protection, Nc is the total number of mosquitoes landing and/or biting at the control area, and Nt is the total number of mosquitoes landing and/or biting at a treated area.

Statistical Analysis

The EC50 dose of selected oils and compounds against the adult stage of Ae. aegypti was calculated by probit analysis [18] using the SPSS software (version 20) and Minitab software.

Retrieval/Modeling of 3D Structures of Proteins and Molecular Docking

3D Structures of OBP1 and OBP22 and AChE

The 3D structures of the OBP1 and OBP22 were found accessible from the RCSB protein data bank (http://www.rcsb.org) with PDB id 3k1e (for OBP1) and 6p2e (for OBP22). Those structures were downloaded in PDB format and used for docking analysis. However, AChE was not found to be available in the RCSB protein data bank, so a model of AChE was built using modeler software.

Homology Modeling for AChE

The AChE enzyme of Ae. aegypti was modeled using Modeller 9.21 software [19]. Fasta format of AChE (Accession: XP_021699617.1) was selected from NCBI and blast was performed for the selection of templates. Six templates having more than 93% query coverage were selected from blast results. The PDB format of the selected templates (4fng, 5ivk, 5tym, 5ch3, 5c8v, 4fmn) was downloaded from the RCSB protein data bank. Following the instructions of basic modeling, suitable files were created to generate the required model. Alignment between the target sequence and templates was made in the modeler command prompt with a specified path. The created models were validated using the procheck online portal [20] that generate Ramachandran plots. Based on the Ramachandran plot, the best model was selected for docking analysis.

Ligand Preparation

The three-dimensional structures of citral, limonene, and DEET were then downloaded in SDF format from PubChem and converted to PDB using Open Babel software (OpenEye Scientific under GPL, GNU Public License).

Protein–Ligand Docking Studies

Docking analysis was performed in AutoDock Vina [21]. Blind docking was performed with grid box coordinates set for OBP1, OBP22, and AChE covering the whole protein with a spacing of 1°A. A configuration file was prepared to mention the coordinates of the search area, receptor, ligand, output filename, and the exhaustiveness of the search. After running the autodock Vina through the command prompt, output files were obtained with ten predictive docking conformations. The most negative binding energy with RMSD value zero was considered the best docking pose, and this docked conformation is presented in a 2-dimensional format using LigPlot and the crystal structures are presented using UCSF Chimera.

Results and Discussion

Repellency of EOs of Selected Citrus Species and Major Constituent Compounds Against Ae. aegypti

Among the tested six EOs, the oil of C. aurantifolia was found to possess the highest repellency having an EC50 value of 0.46 mg/cm2 and 100% protection up to 3 h at 1 mg/cm2 dose (Fig. 1). The present finding was in conformity with the findings of Soonwera [4]. C. aurantifolia was followed by the EO of C. maxima (0.67 mg/cm2), C. aurantium (0.79 mg/cm2), C. limon (2.41 mg/cm2), C. paradisi (2.63 mg/cm2) and C. medica (2.88 mg/cm2), respectively (Table S1). The findings are also in conformity with the findings of Amer and Mehlhorn [22], where they presented the repellency of 41 essential oils against Ae aegypti, Anopheles stephensi, and Culex quinquefasciatus mosquitoes. Deletre et al. [23] reported the repellent properties of 20 essential oils against malaria vector Anopheles gambiae.

Fig. 1
figure 1

Percent repellency of selected EO, compounds and DEET at 4 h exposure period at a concentration of 1 mg/cm2 (% repellency ± SE)

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The results of repellency of the three tested pure compounds showed the highest efficacy for DEET (EC50 0.14 mg/cm2) followed by limonene (0.35 mg/cm2) and citral (0.38 mg/cm2). As predicted, our study revealed the protection time of citral and limonene was higher than the parent oil C. aurantifolia (Table S1). This finding is consistent with the findings of Giatropoulos et al. [24] where they found higher repellency of citral than its parent oil against Aedes albopictus. Although DEET showed the highest repellency in the study, the negative implications of DEET related to health and environment demand safe plant-based alternatives as to the pressing priority of the modern time, although having moderate efficacy.

The observed repellent activity of both crude essential oil and selected compounds could be due to the effect of the repellents on the olfactory sensory neurons of the insect. This system is highly specific. These odorant-binding proteins connect to the sensory neurons. These proteins are highly abundant and play a pivotal role in the uptake of volatiles [25]. DEET exerts its effects in a similar fashion [26]. Odorant-binding proteins and odorant receptors are central to insect olfaction [27].

Protein 3D Structure of OBP 1 and OBP22 and Model of AChE

The 3D structure of the OBP1 and OBP22 was downloaded from RCSB PDB and was used for docking with the selected ligands (Fig. 2). Earlier the modes of repellent action of DEET on the sensory neurons have been established [26]. It is assumed that when the repellent compound binds tightly to the OBP it can transport it and elicit a better response.

Fig. 2
figure 2

Odorant-binding proteins (OBP1 AND OBP22) of Ae. aegypti and their interaction with citral limonene and DEET. Here, the repellent compounds are shown as spheres. Citral is shown in yellow, limonene as magenta and DEET red. Interaction of a citral with OBP1 b Limonene with OBP1 c DEET with OBP1 d citral with OBP22 e Limonene WITH OBP22 f DEET with OBP 22 using UCSF chimera (Color figure online)

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Among the ten models produced for AChE using modeler software, the best model (Figure S1A) was selected based on Ramachandran plot generated through Procheck online portal. Ramachandran plot (Fig S1E) showed the presence of 90.1% amino acid residues in the most favored regions, 8.5% amino acid residues in the additional allowed regions, 1% in generously allowed regions and 0.4% in the disallowed regions.

Docking Analysis of Citral, Limonene and DEET with OBP1 & OBP22 and AChE

Citral, limonene and DEET were found to perform good docking with the OBPs of Ae. aegypti (Figs. 2 and 3). From table (Table S2) of the binding energy values, it is seen that the three compounds had the binding efficiency almost at par. It signified that all the selected compounds exerted their mode of action after binding with these OBPs. Previously, Tsitsanou et al. [28] demonstrated that the repellent activity of DEET was due to its binding with OBP1 of Anopheles gambiae (AgamOBP1). While docking citral, limonene and DEET with OBP1 the binding energy was found to be − 6.4, − 6.5 and − 6.9 kcal/mol, respectively, and that for OBP22 it was found to be − 6.3, − 6.7, − 6.7 kcal/mol, respectively (Table S2). From the ligplot and docking poses it can be observed that these compounds get properly fitted within the 3D structure of the OBPs (Fig. 2 and Table S2) with hydrophobic interactions (Fig. 3). Citral, limonene and DEET bound to OBPs utilizing some of the common amino acid residues (Phe15, Ile125, Phe123, Trp114, and Met84) of OBP1 and (Leu11, Ile116 and Phe37) of OBP22 (Fig. 3 and Table S2). This indicated that DEET and the plant-based repellent compounds bind to OBPs with common amino acid residues at their active sites and also with almost equal binding affinity and similar conformation, evoking similar behavioral responses. The OBPs transport the repellent compound to the odorant receptors which reside in contact with the dendrites of sensory neurons. The information is then relayed to the central nervous system. A study conducted by Syed and Leal [29] showed that Culex quinquefasciatus mosquitoes are endowed with DEET detecting olfactory receptor neurons, that mediates behavioral response on detecting DEET in the environment. These neurons transport the odor of the repellent compound and elicit behavioral changes. Similar physiology is expected to occur in Ae. aegypti.

Fig. 3
figure 3

Presentation of the interaction of citral, limonene and DEET with OBP1 and OBP22 in two-dimensional format generated through ligplus software. Interaction of a Citral with OBP1 b Limonene with OBP1 c DEET with OBP1 d Citral with OBP22 e Limonene with OBP22 f DEET with OBP 22

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Taking AChE as the receptor it was observed that DEET possesses more affinity (− 6.6 kcal/mol) to the AChE enzyme of Ae. aegypti than limonene (− 6.1 kcal/mol) and citral (− 6.0 kcal/mol) (Fig. 4, Table S2 and Figure S1). It was observed that the ligand that binds more exhaustively, for instance, DEET, to the receptor is more effective in repelling mosquitoes (Table S1). Our earlier study also established the affinity of different terpene compounds toward AChE of Ae. aegypti [30].

Fig. 4
figure 4

Presentation of the interaction of citral, limonene and DEET with modeled AChE of Ae. aegypti in two-dimensional diagram generated through ligplus software. a DEET with AChE. b Citral with AChE. c Limonene with AChE. d Depiction of different bonds involved in the interaction of ligand and protein

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Conclusion

The study found that all the essential oils have the potential to repel Ae. aegypti. It showed 50% repellency up to 4 h at 1 mg/cm2 area for three citrus species, namely C. aurantifolia, C. maxima, and C. aurantium. EO of C. aurantifolia exhibited the highest repellent efficacy. The binding affinity of both DEET, citral and limonene was found to be almost similar and with the same amino acid residues. This could mean that DEET and the plant-based repellent compounds bind to OBPs utilizing common amino acid residues at their active sites and also with almost equal binding affinity and similar conformation, eliciting similar behavioral responses. Considering ones’ health and the environment, the authors recommend the locals to produce their own mosquito repellents from the byproducts of Citrus used in the households and use in place of DEET against Ae. aegypti.