Acaricidal and Insect Antifeedant Effects of Essential Oils From Selected Aromatic Plants and Their Main Components

This work has demonstrated the ixodicidal and insect antifeedant effects of essential oils from 14 experimentally cultivated aromatic plants. The strong ixodicidal and antifeedant oils corresponded to Thymus zygis, Thymus vulgaris, Satureja montana, Oreganum virens, and Mentha suaveolens. The moderately active oils were from Lavandula angustifolia, Mentha piperita, Mentha spicata, Artemisa herba-alba, and Rosmarinus officinalis. The most effective larvicidal and antifeedant compounds were piperitenone oxide, carvacrol, piperitenone, and thymol, explaining the effects of the most active essential oils. The rest of the tested compounds were not ixodicidal or antifeedant. Therefore, the activity of moderately active oils cannot be explained by their main components (linalyl acetate, linalool, menthone, menthol, limonene, camphor, 1,8-cineole, p-cymene, α-pìnene, and carvone), suggesting synergistic effects. Considering the ixodicidal and antifeedant effects of these extracts, the plants have been ranked in relation to Thymus vulgare, a commercial biopesticide ingredient, for their potential as botanical pesticides. T. zygis, S. montana, and M. suaveolens ranked over T. vulgaris as ixodicidal agents and S. montana as insecticidal. Therefore, we propose the plant populations of S. montana, T. zygis, and M. suaveolens tested here for further development as biopesticide ingredients.


INTRODUCTION
Food safety and environmental concerns related to the use of pesticides have resulted in more restricted regulatory frameworks worldwide, reducing the number of commercial products available for crop protection and other pest management sectors including the control of vectors of human and livestock diseases. Therefore, new safer and effective insecticides are needed. Botanical pesticides are emerging as a solution to meet part of the demand (Isman, 2020a). Essential oils (EOs) that are composed of volatile secondary metabolites, mostly terpenes (Bakkali et al., 2008), are among the most important extracts acting as botanical insecticides (Regnault-Roger et al., 2012;Pavela and Benelli, 2016), and some are being commercialized as commercial pesticide ingredients (Isman, 2020b).
Arthropods, including economically important disease vectors and insect pests, are an important target of the biological effects of EOs (Ntalli et al., 2019;Isman, 2020a,b). Tick-borne diseases are a serious health and economic problem, responsible for over 100,000 cases of human diseases worldwide (de la Fuente et al., 2008) and billions of dollars in losses to the livestock industry (Lotfi and Karima, 2020). Additionally, ticks are in expansion due to climate change (Abbas et al., 2018). For example, Hyalomma ticks, vectors of the Crimean-Congo hemorrhagic fever virus, have spread from their original distribution (African and Mediterranean environments) to other European countries, becoming an increasing public health concern (Chitimia-Dobler et al., Hansford et al., 2019;Buczek et al., 2020;Grandi et al., 2020). For many years, tick control has been carried out with synthetic acaricides, leading to the appearance of resistance (reviewed by Abbas et al., 2014) and being harmful to the environment. Therefore, new effective and safer tick control agents are needed. In this context, EOs have been reported as being toxic and/or repellent to ticks (Benelli et al., , 2017aBenelli and Pavela, 2018;Salman et al., 2020).
Crop yield damages caused by pest infestations and pesticide use are significant (Oerke, 2006;Gregory et al., 2009) and increasing with global warming. Adaptation measures to increased pest damage related to global warming may involve greater use of pesticides with detrimental effects on health, environmental damage, and increased pesticide resistance (Deutsch et al., 2018). Some important crop pests include the Egyptian cotton leafworm, Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), a highly polyphagous insect labeled as an A2 quarantine pest by the OEPP/EPPO (2015) due to its host range (Alford, 2007) and distribution (Centre for Agricultural Bioscience International, 2020a). The green peach aphid, Myzus persicae Sulzer (Hemiptera: Aphididae), is the most economically important aphid crop pest worldwide (van Emden and Harrington, 2017) due to its distribution (Centre for Agricultural Bioscience International, 2020b), host range (Blackman and Eastop, 2000), mechanisms of plant damage, life cycle, and its ability to evolve resistance to insecticides (Bass et al., 2014). The bird cherry-oat aphid, Rhopalosiphum padi L., is a global pest of cereals (van Emden and Harrington, 2017) and a vector of yellow dwarf viruses that cause significant crop losses in cereals (Finlay and Luck, 2011). Many EOs are good insecticidal candidates because of their direct effects, biodegradability, and their low level of toxicity to mammals (Isman, 2020a,b).
The commercial production of a botanical insecticide depends on the sustainable production of plant biomass for extraction. Therefore, the domestication and cultivation of aromatic and medicinal plants (AMPs) for the production of EOs contributes to species conservation and provides sustainability of the production and lower variations in active ingredients. For example, a selected chemotype of wormwood, Artemisia absinthium (Asteraceae), that lacks the toxic terpene β-thujone but produces other novel terpenoids that are toxic and antifeedant to a range of pest insects has been domesticated for cultivation and registered as a new plant variety . As part of an ongoing project on the domestication and valorization of selected AMPs, plant species belonging to the genera Artemisia, Hyssopus, Lavandula, Mentha, Origanum, Rosmarinus, Satureja, Tanacetum, and Thymus have been experimentally cultivated at a small scale. These genera include species traditionally used in medicinal, food, and flavor applications due to their contents in bioactive EOs (Fathiazad and Hamedeyazdan, 2011;Chishti et al., 2013;Kumar and Tyagi, 2013;Tepe and Cilkiz, 2016;Aprotosoaie et al., 2017;Singh and Pandey, 2018;Borges et al., 2019;Li et al., 2019;Isman, 2020a,b).
In this work, essential oils from selected species of aromatic and medicinal plants cultivated experimentally (Table 1) have been evaluated against arthropods of importance in public health and animal and crop production: the tick (Hyalomma lusitanicum) and three insect pests (S. littoralis, M. persicae, and R. padi). Thymus vulgaris has been included in this study as a reference to compare the rest of the selected species because it is one of the most important aromatic plants grown worldwide (Southern and Central Europe, Southeast Asia, North America, and Africa), and it is an ingredient of botanical insecticides because of its thymol content . Additionally, the composition of the most active EOs has been analyzed and the ixodicidal and insecticidal activities of their main components (Figure 1) tested.

Plant Material
Fourteen plant species belonging to the families Asteraceae and Lamiaceae (Table 1) were selected for the study. The plants come from Spanish native flora and have been experimentally cultivated in several locations in Aragon (Spain) as described (Burillo, 2003;Burillo et al., 2017;Navarro-Rocha et al., 2020).
Aerial parts of these plants were collected at the flowering stage. EOs were obtained in the laboratory by Clevenger hydrodistillation (European Pharmacopoeia, 1975).

Essential Oil Analysis
The essential oils were analyzed by gas chromatographymass spectrometry (GC-MS) using a Shimadzu GC-2010 gas chromatograph coupled to a Shimadzu GCMS-QP2010 Ultra mass detector (electron ionization, 70 eV) and equipped with a 30-m × 0.25-mm i.d. capillary column (0.25 µm film thickness) Teknokroma TRB-5 (95%) dimethyl-(5%) diphenylpolisiloxane. The working conditions were as follows: split ratio, 20:1; injector temperature, 300 • C; temperature of the transfer line connected to the mass spectrometer, 250 • C; initial column temperature, 70 • C; then heated to 290 • C at 6 • C/min. The relative amounts of the individual components were calculated based on the peak area without using a correction factor. Electron ionization mass spectra, retention data, and the calculated linear retention indices (LRIs) were used to assess the identity of the compounds by comparing them with those of standards or those found in the Wiley 229 Mass Spectral Database.

Ixodicidal Activity
Hyalomma lusitanicum engorged females were collected from red deer in Ciudad Real (Central Spain) and maintained under laboratory conditions [22-24 • C and 80% relative humidity (RH)] until oviposition and egg hatching.
Tick bioassays were performed according to Ruiz-Vásquez et al. (2017). Briefly, 50 µl of the test solution was added to 25 mg of powdered cellulose at different concentrations (initial concentration of 40 or 20 µg/mg for EOs or pure compounds, respectively) and the solvent was evaporated. The ticks and cellulose were then placed in laboratory glass tubes and carefully mixed by rotating the glass several times to ensure full tickcellulose contact. After mixing, the tubes were kept under laboratory conditions for 24 h. For each test, three replicates with 20 active older than 6 weeks larvae. To validate the tests, three replicates of negative (cellulose, 25 mg) and positive (thymol, 20 µg/mg) controls were also used.
Ticks were considered dead when they could not move from one place to another. Dead ticks were counted after 24 h of contact with the treated cellulose at the laboratory conditions described using a binocular magnifying glass. The larvicidal activity data are presented as percent mortality corrected according to Schneider-Orelli's formula (Püntener, 1981). Effective lethal doses (LC 50 and LC 90 ) were calculated by Probit analysis (1:2 serial dilutions to cover a range of activities between 100 and <50% mortality with a minimum of three doses) (STATGRAPHICS Centurion XVI, version 16.1.02).

Insect Antifeedant Activity
Spodoptera littoralis, M. persicae, and R. padi colonies are maintained at ICA-CSIC, reared on artificial diet, bell pepper (Capsicum annuum) and barley (Hordeum vulgare) plants, respectively, and kept at 22 ± 1 • C and >70% RH, with a photoperiod of 16:8 h (L/D) in a custom-made walk-in growth chamber.
The bioassays were conducted as described (Navarro-Rocha et al., 2018). The upper surfaces of the C. annuum and H. vulgare leaf disks or fragments (1.0 cm 2 ) were treated with 10 µl of the test substance. The EOs and products were tested at an initial dose of 10 or 5 µg/µl (100 or 50 µg/cm 2 ), respectively. Five to seven Petri dishes or 20 ventilated plastic boxes (2 × 2 cm) with two sixth-instar S. littoralis larvae (>24 h after molting) or 10 apterous aphid adults (24-48 h old) each were allowed to feed in a growth chamber (until 75% larval consumption of the control disks or 24 h for aphids, environmental conditions as above). Each experiment was repeated twice. Feeding inhibition or aphid settling was calculated by measuring the disk surface consumption (digitalized with https://imagej.nih. gov/ij/) (Rueden et al., 2017) or by counting the number of aphids on each leaf fragment. Feeding/settling inhibition (%FI or %SI) was calculated as %FI/SI = [1 -(T/C) × 100], where a Values (in percent) are the means of three replicates corrected according to Schneider-Orelli's formula (Püntener, 1981). b Lethal doses (upper-lower 95% confidence limits) calculated to give 50% (LD 50 ) or 90% (LD 90 ) mortality by Probit analysis.
T and C represent feeding/settling on the treated and control leaf disks, respectively. The antifeedant effects (%FI/SI) were analyzed for significance by the non-parametric Wilcoxon paired signed-rank test comparing the consumption/settling between the treatment and control leaf disks. Extracts and compounds with an SI >70% were further tested in a dose-response experiment (1:2 serial dilutions to cover a range of activities between 100 and <50% feeding inhibition with a minimum of three doses) to calculate their relative potency (EC 50 , the effective dose to give a 50% settling reduction) from the linear regression analysis (%FI/SI on Log-dose, STATGRAPHICS Centurion XVI, version 16.1.02).

Plant Species Ranking
Considering the ixodicidal and antifeedant effects of the tested EOs, the plants have been ranked in relation to T. vulgaris ( Table 4) for their potential as botanical pesticide ingredients. The ranking index has been established as [T. vulgaris EC 50 value/ranked species EC 50 value] for each test with significant effects (see Tables 2, 3).

DISCUSSION
This work has demonstrated the ixodicidal and insect antifeedant effects of EOs from experimentally cultivated AMPs. Furthermore, more EOs were ixodicidals than insect antifeedants, probably because of their different feeding ecologies (blood sucking vs. herbivores). Ticks are obligate hematophagous ectoparasites (Basu and Charles, 2017) and therefore have not evolved adaptations to plant secondary metabolites. On the other hand, insect herbivores have coevolved with plants and their chemical defenses/secondary metabolites (Maron et al., 2019). These differences in feeding adaptations could explain the selective toxicity of EOs toward the ticks observed here.
The EOs grouped as strong ixodicidal agents corresponded to T. zygis, T. vulgaris, S. montana, M. suaveolens, and Origanum virens. Similarly, the EOs grouped as strong antifeedants corresponded to S. montana, T. zygis, and T. vulgaris, followed by O. virens and M. suaveolens.
Thymus vulgaris EO, an ingredient of botanical pesticides , has been included in this work as a reference for further species selection. In this work, the EO from T. vulgaris (thymol/p-cymene) was the third most ixodicidal and the second most antifeedant against the insect species tested. The most common T. vulgaris chemotypes are thymol/carvacrol (György et al., 2020), which have reported ixodicidal effects including repellency against nymphs of Ixodes ricinus and adults of
has already been included in an agronomic development program for the production of biopesticides (Navarro-Rocha et al., 2020). However, this is the first report on the ixodicidal activity of this EO. The M. suaveolens population selected for this work was rich in piperitenone oxide/piperitenone. This EO was the second most effective ixodicidal extract tested here (more effective than T. vulgaris), along with S. montana, and showed stronger antifeedant effects against M. persicae than T. vulgaris. M. suaveolens is native of Africa, temperate Asia, and Europe (Abbaszadeh et al., 2009). There are three chemotypes described for M. suaveolens: pulegone, piperitenone oxide, and piperitenone oxide/piperitone oxide (Oumzil et al., 2002;BoŽović et al., 2015). Previously, M. suaveolens EOs (pulegone and menthone) showed ovicidal and larvicidal effects against the tick Hyalomma aegyptium (Laghzaoui et al., 2019). This species' EOs also have reported insecticidal replicates. c EC 50 (95% lower-upper confidence limits), concentration needed to produce 50% feeding/setting inhibition.
effects against stored-product pests such as Sitophilus oryzae (piperitenone oxide and piperitenone oxide/piperitenone chemotypes) (Zekri et al., 2013), Rizopertha dominica (piperitenone/pulegone/piperitone) (Benayad et al., 2012), and Triboleum castaneum (menthone/pulegone) (Kasrati et al., 2015) and larvicidal activity against C. quinquefasciatus (piperitenone oxide) . Antifeedant effects against L. decemlineata and M. persicae have been reported for a piperitenone oxide/piperitone epoxide Mentha chemotype (Kimbaris et al., 2017). However, this is the first report on the ixodicidal effects of a piperitenone oxide/piperitenone M. suaveolens chemotype. The ixodicidal and antifeedant effects (stronger than those of T. vulgaris) of the M. suaveolens EO tested in this work support further agronomic development of this species for the production of biopesticides. The chemotype of O. vulgare subsp. virens (p-cymene, carvacrol, and linalool) tested here showed ixodicidal effects similar to T. vulgaris, but was less antifeedant, affecting only the aphid M. persicae. O. vulgare have reported toxic or repellent activities against I. ricinus (Soutar et al., 2019) and acute and fumigant toxicity against aphids including M. persicae (Ikbal and Pavela, 2019). However, this is the first report on the ixodicidal and aphid antifeedant effects of O. vulgare subsp. virens EO. O. vulgare is a widespread species native to the Mediterranean, the Euro-Siberian, and the Irano-Turanian regions and is one of the most traded and consumed spice plants (Lukas et al., 2015). O. vulgare subsp. virens is a heterogeneous subspecies characterized by essential oils rich in acyclic and/or cymyl compounds (Lukas et al., 2015). Since the effects of the EO from O. vulgare subsp. virens tested here were similar to these of T. vulgaris, its further development for the production of biopesticides against arthropods is not supported. However, we suggest the valorization of its essential oil production residues (biomass: hydrolate) as a potential source of biopesticidal ingredients.
The EO from L. angustifolia tested here (linalyl acetate/linalool) showed moderate ixodicidal effects against H. luxitanicum larvae, lower than the effects of T. vulgaris, without significant insect antifeedant effects. Previous reports have shown interference with the host-seeking behaviors of H. marginatum and D. reticulatus for this species' EO (Mkolo and Magano, 2007;Štefanidesová et al., 2017) and toxicity to Rhipicephalus (Boophilus) annulatus (Pirali-Kheirabadi and Teixeira da Silva, 2010) for this species' EO. Additionally, a similar EO from the hybrid Lavandula × intermedia (rich in linalyl acetate and linalool) was also toxic to H. lusitanicum larvae and moderately antifeedant to S. littoralis (Ortiz de Elguea-Culebras et al., 2018). L. angustifolia, distributed in the sub-Mediterranean region, has a great economic importance in perfumery, cosmetics, food, pharmaceutical industries, and aromatherapy (Demasi et al., 2018). However, our results do not support its agronomic production as a biopesticide when compared to T. vulgaris, but suggest the valorization of its essential oil production residues (biomass: hydrolate) as a source of biopesticidal ingredients.
Rosmarinus officinalis (camphor/1,8-cineole/α-pinene) showed moderate ixodicidal effects in this work. Previous reports have shown a moderate post-ingestive toxicity to S. littoralis for a similar R. officinalis EO (Santana et al., 2014). R. officinalis EOs rich in 1,8-cineole were toxic to larvae of Hyalomma scupense (Djebir et al., 2019) and I. ricinus nymphs (Elmhalli et al., 2019), while an EO rich in α-pinene showed low-moderate toxicity against larvae of R. (B.) microplus (Martinez-Velazquez et al., 2011). This plant is cultivated worldwide as a food flavoring and preservative due to its antioxidant and antimicrobial potential (Borges et al., 2019). Our results showed lower effects for R. officinalis than T. vulgaris. However, being a commercial plant available worldwide, we suggest the valorization of its essential oil production residues (biomass: hydrolate) as a source of biopesticidal ingredients.
The chemotype of A. herba-alba (camphor/1,8-cineole/pcymene) tested in this work showed low-moderate larvicidal effects against H. lusitanicum. A. herba-alba is a medicinal and aromatic shrub that grows wild in arid areas of the Mediterranean Basin, being abundant in the Iberian Peninsula (Mohamed et al., 2010), showing chemical diversity (Salido et al., 2004). An A. herba-alba EO rich in piperitone showed repellency against I. ricinus nymphs (El-Seedi et al., 2017), and a thujone/camphor chemotype was antifeedant and moderately toxic to S. littoralis (Santana et al., 2014). Our results showed lower effects for a camphor/1,8-cineole A. herba-alba chemotype than those reported or T. vulgaris. Given the chemical diversity of A. herba-alba wild populations, we suggest further research on chemotype-bioactivity correlations for this plant species prior to its selection for agronomical development.
Considering the plant species' rank based on the ixodicidal and antifeedant effects of their EOs, we propose the plant populations of S. montana, T. zygis, and M. suaveolens tested here for further agronomical development as biopesticide ingredients for the control of ticks and insects. These EOs (S. montana, T. zygis, and M. suaveolens) have additional biopesticidal effects such as strong nematicidal action against root-knot nematodes (Meloidogyne javanica), with S. montana being the most effective (LC 50 = 0.041 µg/µl) (Andrés et al., 2012).
To further understand the effects of the active EOs, their main components (Figure 1) were also tested against the selected targets. The most effective larvicidal and antifeedant compounds were piperitenone oxide, carvacrol, piperitenone, and thymol.
The activity of piperitenone oxide and piperitenone explained the effects of M. suaveolens EO. Thymol explained the effects of the EOs from T. zygis and T. vulgaris, while carvacrol was responsible for the effects of S. montana and O. vulgare subsp. virens.

CONCLUSION
This work has demonstrated the ixodicidal and insect antifeedant effects of EOs from experimentally cultivated AMPs. The EOs grouped as strong ixodicidal agents corresponded to T. zygis, T. vulgaris, S. montana, M. suaveolens, and O. vulgare subsp. virens. Similarly, the EOs grouped as strong antifeedants corresponded to S. montana, T. zygis, and T. vulgaris, followed by O. vulgare subsp. virens and M. suaveolens. The moderate ixodicidal EOs were from L. angustifolia, M. piperita, M. spicata, A. herba-alba, and R. officinalis.
T. zygis, S. montana, and M. suaveolens were better ixodicidals and S. montana was a better antifeedant than T. vulgaris. Therefore, we propose the plant populations of S. montana, T. zygis, and M. suaveolens tested here for further development as biopesticide ingredients for the control of ticks and insect pests.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AUTHOR CONTRIBUTIONS
AG-C conceptualized the study. AG-C, AO, MG, JN-R, and FV curated the data. MG, FV, and MA did the formal analysis. AG-C, AO, and FV helped with funding acquisition and resources. MG, JN-R, FV, AO, MA, and AG-C did the investigation. AO, FV, AG-C, and MA helped with the methodology. FV and AG-C wrote the original draft. AG-C, AO, MA, FV, and JN-R did the writing-review and editing. All authors have read and agreed to the published version of the manuscript.