essential oils and formamidines as insecticides/acaricides: what are the molecular targets? Wolfgang Blenau, Eva Rademacher, Arnd Baumann

To cite this version:

Wolfgang Blenau, Eva Rademacher, Arnd Baumann. Plant essential oils and formamidines as in- secticides/acaricides: what are the molecular targets?. Apidologie, Springer Verlag, 2012, 43 (3), pp.334-347. ￿10.1007/s13592-011-0108-7￿. ￿hal-01003531￿

HAL Id: hal-01003531 https://hal.archives-ouvertes.fr/hal-01003531 Submitted on 1 Jan 2012

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Apidologie (2012) 43:334–347 Review article * INRA, DIB and Springer-Verlag, France, 2011 DOI: 10.1007/s13592-011-0108-7

Plant essential oils and formamidines as insecticides/ acaricides: what are the molecular targets?

1 2 3 Wolfgang BLENAU , Eva RADEMACHER , Arnd BAUMANN

1Institut für Bienenkunde (Polytechnische Gesellschaft), Goethe-Universität Frankfurt am Main, Karl-von-Frisch-Weg 2, 61440 Oberursel, Germany 2Institute of Biology, Freie Universität Berlin, 14195 Berlin, Germany 3Institute of Complex Systems—Cellular Biophysics-(ICS-4), Forschungszentrum Jülich, 52425 Jülich, Germany

Received 16 May 2011 – Revised 29 August 2011 – Accepted 21 October 2011

Abstract – The parasitic mite Varroa destructor is the main cause of the severe reduction in beekeeping during the last few decades. Therefore, efforts have been made to develop chemical treatments against the parasite. In the past, synthetic products were preferentially used to combat Varroa mites. Nowadays, mainly plant essential oils and organic acids are applied because they are safer and impose less unfavorable effects on the environment. Essential oils contain mixtures of mostly volatile and odorous terpenoid constituents. The molecular targets of these substances are tyramine and/or octopamine receptors that control and modulate vital functions ranging from metabolism to behavior. Disturbing the native function of these receptors in the mite results in deleterious effects in this parasite. This overview considers not only tyramine and octopamine receptors but also other potential targets of essential oils including ionotropic GABAA receptors, TRP type ion channels, and acetylcholinesterase.

GABA / G protein-coupled receptor / octopamine / / tyramine

1. PLANT ESSENTIAL OILS— as acetylcholinesterase (AChE) or that manipulate AN INTRODUCTION the overall metabolic status of the parasite (for a review, see Isman 2006). Substances The availability of environmentally safe and such as synthetic pyrethroids inhibit voltage- efficient chemicals against arthropod pests is an gated sodium channels that are important for important aspect for veterinary and crop the firing of neuronal action potentials. Phenyl- industries. At present, chemical compounds pyrazolines or polychloroalkanes (e.g., lindane) that rapidly deliver their insecticidal effect block the activity of GABAA channels, important are preferentially used to achieve a timely members of the ligand-gated ion channel family reduction of pests and ectoparasites on their involved in inhibitory signal generation in respective hosts. This condition is fulfilled by neurons. Organophosphates inhibit the enzymatic compounds that modify the activity of voltage- activity of AChE, an enzyme controlling the gated and/or ligand-gated ion channels in the concentration of the excitatory neurotransmitter central nervous system (CNS) and by compounds acetylcholine (ACh) in the synaptic cleft. Iso- that impair the activity of neuronal enzymes such flavonoids such as rotenone poison mitochondria and thus cause the breakdown of cellular energy Corresponding author: W. Blenau, production. Metabotropic (G protein-coupled) [email protected] octopamine receptors are the target of formami- Manuscript editor: Bernd Grünewald dine pesticides such as amitraz which manipulate Molecular targets of plant essential oils 335 cellular signal transduction processes. Despite the Similar to their heterogeneous composi- efficient and wide use of these substances, they tion, plant essential oils display a broad seem to harbor certain disadvantages. (1) Parasites spectrum of biological activity. They can be can develop resistance to the chemicals, probably toxic for insects and microbes, but can also because the generation times of the arthropod act as insect antifeedants and repellents pests are short. (2) Cross-reactivity of such (Anthony et al. 2005;Isman2006; Nerio et substances might also be significant in beneficial al. 2010;Vigan2010).Thetoxiceffectof insects such as the honeybee. In addition, these essential oils is most likely mediated by substances might cause undesired allergic or toxic neurological mechanisms. Indeed, plant essen- effects in humans. For such reasons, there is a tial oils have been suggested to exert their continual demand for compounds that, on the one bioactivity by interacting with various molec- hand, efficiently reduce parasites or pests and, on ular targets including tyramine and octop- the other hand, are safe for the host, the amine receptors (Enan 2001, 2005a, b;Price environment, and non-target species, including and Berry 2006), ionotropic GABA receptors man, that come into contact with such insecti- (Priestley et al. 2003; Tong and Coats 2010), cides/acaricides. Over the years, plant essential and AChE (Grundy and Still 1985; Ryan and oils have been considered as an attractive alterna- Byrne 1988; Keane and Ryan 1999). As tive that might fulfill these criteria. previously mentioned, octopamine receptors Plant essential oils are complex mixtures of have also been identified as a target site of the odorous substances obtained from botanical raw formamidine class of insecticides (Evans and materials by water vapor extraction, dry distil- Gee 1980;Goleetal.1983;Downeretal. lation, or mechanical treatment without heating 1985;Dudaietal.1987;Chenetal.2007). (Isman 2006; Vigan 2010). Because of their In mammalian tissues, p-tyramine, β- odorous character, plant essential oils are widely phenylethylamine, tryptamine, and octopamine used as fragrances and flavorings in perfume are present at very low (nanomolar) concen- and food industries. To identify and to further trations and are therefore referred to as “trace characterize the individual components of es- amines.” Trace amine-associated receptors sential oils, either separation by gas chromatog- (TAARs) have recently been discovered in raphy or HPLC has to be performed. These mammals (for a review, see Zucchi et al. analyses have identified monoterpenes, sesqui- 2006). It is worth emphasizing, however, that terpenes, and related aromatic compounds as the thus far, only two members of the TAAR family main constituents of plant essential oils. Terpe- have been shown to be responsive to trace noids are derived from five-carbon isoprene amines. In addition, mammalian TAARs are units. Coupling of two isoprene units leads to not closely related to arthropod tyramine and ten-carbon structures known as monoterpenoids, octopamine receptors. Thus, it appears that in whereas coupling of three isoprene units leads mammals, TAARs evolved independently to sesquiterpenoids (Table I). Terpenoids can be from the arthropod tyramine and octopamine linear (acyclic) or contain cyclic structures receptors and acquired the ability to interact (Table I). Further biochemical modifications by with different amines, including the decar- endogenous plant enzymes can cause oxidation, boxylated thyroid hormone derivatives known rearrangement, or additional cyclization (Table I) as thyronamines, several volatile amines, and that increases the variability of essential oil possibly other as yet unidentified endogenous constituents (Chappell 1995; Holstein and Hohl compounds (for a review, see Zucchi et al. 2004). At approximately 90%, the mono- and 2006). Therefore, arthropod tyramine and sesquiterpenoids outnumber the other constitu- octopamine receptors might emerge as prom- ents of plant essential oils. Nevertheless, some ising targets for insecticides/acaricides (e.g., aromatic compounds are worth mentioning plant essential oil components) with no or low (Table I). toxicity in vertebrates. 336 W. Blenau et al.

Table I. Naturally occurring essential oil components and synthetic substances mentioned in the text.

Compound Molecular Substance class Sources (examples) formula

Linalool [licareol=(R)-(−)- C10H18O Monoterpenoid alcohol, Coriandrum sativum ; coriandrol=(S)- linear (coriander) (+)-linalool] Ocimum basilicum () Citrus × sinensis (sweet orange)

Citral [geranial=citral A; C10H16O Monoterpenoid aldehyde, Backhousia citriodora neral=citral B] linear (lemon myrtle) Cymbopogon spp. (lemongrass) Leptospermum liversidgei (lemon tea tree)

Menthol C10H20O Monoterpenoid alcohol, × piperita monocyclic, unsaturated (peppermint)

α-Terpineol C10H18O Monoterpenoid alcohol, Melaleuca leucadendra monocyclic, unsaturated (cajuput oil) Pinus spp. (pine oil) Citrus × aurantium (petitgrain oil)

Carveol C10H16O Monoterpenoid alcohol, cis-(−)-Carveol: monocyclic, unsaturated Mentha spicata (spearmint)

Pulegone C10H16O Monoterpenoid ketone, Nepeta cataria (catnip) monocyclic Mentha × piperita (peppermint) Mentha pulegium (pennyroyal)

R-(−)-Carvone, S-(+)- C10H14O Monoterpenoid ketone, S-(+)-carvone carvone monocyclic, unsaturated Carum carvi (caraway) Anethum graveolens (dill) R-(−)-carvone Mentha spicata (spearmint)

α-Terpinene, g-terpinene C10H16 Monoterpenoid, monocyclic, Melaleuca alternifolia unsaturated (tea tree oil) Citrus × limon (bergamot oil) p-Cymene C10H14 Monoterpenoid, monocyclic, Cuminum cyminum () aromatic Chenopodium ambrosioides (epazote) Peumus boldus (boldo)

Carvacrol C10H14O Monoterpenoid phenol Origanum vulgare () Thymus spp. () Lepidium spp. (pepperwort)

Thymol C10H14O Monoterpenoid phenolic Thymus spp. (thyme) derivative of cymene Trachyspermum copticum (ajwain) Origanum vulgare (oregano)

Anethole (para- C10H12O Monoterpenoid ether, Pimpinella anisum (anise) methoxyphenylpropene, aromatic, unsaturated Foeniculum vulgare (fennel) Molecular targets of plant essential oils 337

Table I continued. Compound Molecular Substance class Sources (examples) formula

p-propenylanisole, Syzygium anisatum isoestragole) (anise myrtle)

Eugenol C10H12O2 Monoterpenoid, methoxy-phenol, Syzygium aromaticum unsaturated ( oil) Pimenta dioica (allspice) Myristica fragrans (nutmeg)

(+)-, (−)-camphor C10H16O Monoterpenoid ketone, (+)-Camphor bicyclic (camphor laurel) Rosmarinus officinalis () (−)-Camphor Tanacetum parthenium (feverfew) Matricaria recutita (German chamomile)

α-Pinene; β-pinene C10H16 Monoterpenoid, bicyclic, Pinus spp. (pine resin) unsaturated Rosmarinus officinalis (rosemary)

(+)-Borneol C10H18O Monoterpenoid ketone, Cinnamomum camphora bicyclic (camphor oil) Coriandrum sativum (coriander) Tanacetum vulgare (tansy)

Bornyl acetate C12H20O2 Acetic acid ester of borneol Abies spp. (fir) Pinus spp. (pine) Picea spp. (spruce)

Eucalyptol (1,8-cineol, 1, C10H18O Monoterpenoid, spp. 8-cineole, cineol, cineole) oxa-bicyclic (eucalyptus oil) Cinnamomum camphora (camphor laurel) Laurus nobilis (bay leaf)

Farnesol C15H26O Sesquiterpene, linear Cymbopogon spp. (lemongrass, citronella oil) Citrus × aurantium (bitter orange, neroli oil) Polianthes tuberosa (tuberose)

Cinnamic alcohol C9H10O Aromatic, unsaturated Liquidambar orientalis alcohol (storax) Myroxylon balsamum (balsam Peru) Cinnamomum spp. ()

Cinnamaldehyde C9H8O Aromatic, unsaturated aldehyde Cinnamomum spp. (cinnamon)

Capsaicin C18H27NO3 Capsaicinoid, 8-methyl- Capsicum spp. () 338 W. Blenau et al.

Table I continued. Compound Molecular Substance class Sources (examples) formula

N-vanilyl-6-nonenamide, aromatic Chlordimeform C10H13ClN2 Amidine, organochloride Synthetic

Demethylchlordimeform C9H11ClN2 Amidine, organochloride, Synthetic aromatic Amitraz C19H23N3 Amidine, triazapentadiene, Synthetic aromatic

For each substance, the molecular formula, characteristic structural properties, and a selection of natural sources synthesizing the compound are given

2. USE OF PLANT ESSENTIAL OILS co-infected with a greater number of pathogens FOR VARROA TREATMENT compared with control populations, suggesting OF HONEYBEE COLONIES an interaction between pathogens and other stress factors in CCD and a possible legacy effect of The honeybee, Apis mellifera, pollinates Varroa parasitism (Vanengelsdorp et al. 2009). about 80% of crop and wild . This makes Numerous efforts have been made to develop it the fourth most important farm animal in chemical treatments against V. destructor Germany (after cattle, pigs, and poultry). Hence, (Mutinelli and Rademacher 2003; Rosenkranz it is alarming that the honeybee stocking density et al. 2010). Nowadays, products based on has rapidly declined over the last few decades. organic acids (e.g., formic acid and oxalic acid; One reason for this dramatic reduction in Rademacher and Imdorf 2004; Rademacher beekeeping is the parasitic mite Varroa destructor, and Harz 2006; Calderone 2010), essential oils which was introduced into Germany in the (e.g., thymol; Imdorf et al. 1995, 1999; 1970s. V. destructor can only reproduce in a Rademacher and Radtke 2001;Florisetal. honeybee colony, precisely in sealed brood cells. 2004), pyrethroids (e.g., fluvalinate and flu- It attaches to the body of the larva, pupa, or adult methrin), organophosphates (e.g., coumaphos; bee and weakens its host by sucking hemolymph Milani and Lob 1998), and formamidines (e.g., (for reviews, see Rademacher 1990; Sammataro amitraz; Floris et al. 2001) are used to treat et al. 2000; Rosenkranz et al. 2010). The mite infected colonies. Essential oils (and formami- acts as a vector and spreads RNA viruses such as dines) most probably exert their therapeutic deformed wing virus to the bee (for reviews, see effects by binding to tyramine and/or octopamine Genersch and Aubert 2010; Le Conte et al. receptors. Amongst others, thymol (e.g., 2010). A significant mite infestation will lead to Apiguard and Thymovar) and combinations the death of the honeybee colony, usually during of thymol and other essential oils such as the summer or winter months. V. destructor is the , camphor, and (e.g., Api parasite with the most pronounced economic LifeVAR)arealsousedforVarroa treatment impact on the beekeeping industry. It is a major (Imdorf et al. 1999) and have been approved as contributing factor to colony collapse disorder veterinary drugs in many European countries. (CCD) and to non-CCD winter losses, which Calderone and Spivak (1995) found that a constitute severe problems in beekeeping world- blend of natural products similar to Api Life wide (Vanengelsdorp et al. 2009; Guzman- VAR provided more than 98.5% control of V. Novoa et al. 2010; for a review, see Le Conte destructor. In a recent study, Ghasemi et al. et al. 2010). Honeybees in CCD colonies are (2011) determined the fumigant toxicity of characterized by higher pathogen loads and are essential oils taken from Thymus kotschyanus, Molecular targets of plant essential oils 339

Ferula assa-foetida,andEucalyptus camaldu- Verlinden et al. 2010a; Blenau and Thamm lensis against V. destructor and A. mellifera. 2011). In the honeybee, three dopamine receptors Interestingly, whereas the essential oil of T. (AmDOP1: Blenau et al. 1998; et al. kotschyanus is the most potent fumigant for V. 2003; AmDOP2: Humphries et al. 2003; destructor, it has the lowest insecticidal activity Mustard et al. 2003;AmDOP3:Beggsetal. against A. mellifera (Ghasemi et al. 2011). 2005), one tyramine receptor (AmTYR1: Blenau Noteworthy is that menthol is used to treat hives et al. 2000; Mustard et al. 2005), one octopamine infested with the tracheal mite, Acarapis woodi, receptor (AmOA1: Grohmann et al. 2003), and which is an internal parasite of honeybees. On two serotonin receptors (Am5-HT1A:Thammet the one hand, essential oils have not yet led to al. 2010; Am5-HT7: Schlenstedt et al. 2006) resistant mite populations, but on the other hand, have been characterized. Aminergic receptors they are not consistently highly active in reduc- have also been characterized in locusts (Locusta ing mite populations in all situations (Le Conte et migratoria: Vanden Broeck et al. 1995; Schisto- al. 2010). In addition, treatments using thymol as cerca gregaria: Verlinden et al. 2010b) and the their active substance are suspected to have American cockroach (Periplaneta americana; adverse effects on honeybee colonies. Interest- Bischof and Enan 2004; Rotte et al. 2009; ingly, honeybee responses to Apiguard treatment Troppmann et al. 2010). These hemimetabo- change with honeybee age (Mondet et al. 2011). lous insects not only are pest insects but also While 2-day-old bees respond neutrally to serve as model organisms for basic research in Apiguard, foragers appear to be repelled by neurobiology and pharmacology. The successful Apiguard, but can become habituated to the completion of various insect genome sequencing treatment (Mondet et al. 2011). projects has led to the annotation of additional receptor genes by bioinformatics (Drosophila 3. OCTOPAMINE AND TYRAMINE melanogaster: Brody and Cravchik 2000; RECEPTORS AS MOLECULAR Anopheles gambiae:Hilletal.2002; A. melli- TARGETS OF PLANT ESSENTIAL fera:Hauseretal.2006; Tribolium castaneum: OILS AND FORMAMIDINE Hauser et al. 2008). Unfortunately, knowledge of PESTICIDES aminergic receptors in mites remains limited. So far, only one tyramine receptor (Baxter and Biogenic amines are small organic com- Barker 1999) and one serotonin receptor (Chen pounds that act as neurotransmitters, neuromodu- et al. 2004) of the cattle tick, Boophilus micro- lators, and/or neurohormones in vertebrates and in plus, and two dopamine receptors (Meyer et al. invertebrates. They represent an important group 2011) of the blacklegged tick, Ixodes scapularis, of messenger substances and mediate their diverse have been molecularly identified. To date, no effects by binding to membrane receptors that molecular data on aminergic receptors are belong to the large gene family coding for G available for V. destructor. protein-coupled receptors. In arthropods, the The neurotoxic activity of essential oils and/ group of biogenic amine messengers consists of or their purified constituents (, α- five members: dopamine, tyramine, octopamine, terpineol, cinnamic alcohol) against various serotonin, and histamine. Receptor activation insect species is probably attributable to binding leads to changes in the concentration of to tyramine and octopamine receptors (Enan intracellular second messengers (e.g., cAMP 2001). Octopamine and essential oil constituents 2+ or InsP3/Ca ). For each amine, multiple (10 nM) have been found to cause a significant receptor subtypes exist that couple to various increase in intracellular cAMP concentration intracellular signaling pathways in a receptor- ([cAMP]i) in the cotton bollworm, Helicoverpa specific manner (for reviews, see Blenau and armigera (Kostyukovsky et al. 2002). The Baumann 2001, 2003; Evans and Maqueira octopamine receptor antagonist phentolamine 2005;Scheineretal.2006;Hauseretal.2006; effectively inhibits the increase in [cAMP]i 340 W. Blenau et al. induced by essential oil treatment. These results of formamidines interacting with octopamine support the hypothesis that native octopamine receptors. Interestingly, some formamidines receptors are targets of essential oil constituents show an even higher affinity for octopamine (Kostyukovsky et al. 2002). receptors in the CNS of insects than the natural This hypothesis has subsequently been ligand octopamine itself (Hiripi et al. 1994; supported by evidence from cloned and Roeder 1995). The interaction of formamidines heterologously expressed receptors. Enan with octopamine receptors results in hyperexcit- (2005a) has shown that the toxicity rank order ability, abnormal behavior, paralysis, and death of p-cymene, thymol, , α-terpineol, and in insects (Dudai et al. 1987), and also in the R-(−)-carvone for D. melanogaster correlates detachment and mortality of parasitic acarines with their binding affinity to a heterologously (Stone et al. 1974). Chlordimeform HCl (K-79) expressed tyramine receptor (TyrR). The main has been tested for the treatment of varroosis and route by which this receptor acts causes a has been shown to be highly toxic for V. reduction in [cAMP]i. When heterologously destructor, but only slightly toxic for bees expressed in D. melanogaster S2 cells, the (Rademacher 1981; Wachendörfer et al. 1981). activation of the tyramine receptor also causes In veterinary medicine, amitraz is used to control an increase in the intracellular Ca2+ concentration ectoparasitic mites and insects (Hollingworth (Enan 2005a). Interestingly, two compounds, i.e., 1976). In some European countries, amitraz is thymol and carvacrol, are not toxic for a applied in the form of special plastic strips that tyramine receptor mutant (TyrRneo30;Enan slowly release the agent to control V. de st ru ct or 2005a). In this mutant, the insertion of a P (Floris et al. 2001). In other countries, amitraz is element abolishes the expression of a functional applied as an aerosol or a spray. In this form, receptor protein (Cooley et al. 1988). Together, amitraz is highly unstable and becomes rapidly these results suggest that thymol and carvacrol degraded into various partly poisonous metabo- mediate their toxic effects via tyramine receptors. lites. For this reason, the compound is not Notably, the specific binding of essential oil licensed for the treatment of honeybee colonies components (eugenol, cinnamic alcohol, trans- in Germany. Another side effect of amitraz anethole) to an octopamine receptor from D. treatment is that mites can become resistant to melanogaster and its ortholog from the cock- the chemical (Elzen et al. 2000;forareview,see roach, P. americana (Pa oa1), has also been Jonsson and Hope 2007). Notably, amino acid reported (Enan 2005b). substitutions have been identified in the receptor As early as the middle of the 1980s, the sequence coding for a putative octopamine/ formamidines demethylchlordimeform (DMCD), tyramine receptor in the cattle tick, B. microplus, BTS-27271, and amitraz were shown to mimic the in two amitraz-resistant strains (Chen et al. 2007). action of octopamine in elevating adenylyl cy- These substitutions are absent in all amitraz- clase activity in the nervous tissue of P. americana susceptible strains. The discovery of such muta- (Gole et al. 1983;Downeretal.1985). In D. tions in amitraz-resistant ticks provides evidence melanogaster, however, DMCD and chlordime- for these amino acid residues being part of the form inhibit octopamine-stimulated adenylyl pesticide-binding site of the wild-type receptor. cyclase activity, whereas amitraz activates the enzyme (Dudai et al. 1987). Both octopamine and 4. GABAA RECEPTOR ION CHANNELS chlordimeform potentiate contractions of the AS MOLECULAR TARGETS locust slow extensor tibiae muscle (Evans and OF PLANT ESSENTIAL OILS Gee 1980). Furthermore, chlordimeform and DMCD elicit light production in the firefly Fast neuronal signaling by the neurotransmitter (Photinus pyralis) lantern, which is controlled g-aminobutyric acid (GABA) is mediated by by octopaminergic neurons (Hollingworth and ionotropic GABAA receptors. These are pen- Murdock 1980). All these results argue in favor tameric complexes with an integral GABA-gated Molecular targets of plant essential oils 341 anion channel (for a review, see Buckingham et centrally by mimicking or facilitating the action al. 2005). Insect GABAA receptors are targets for of GABA (Waliwitiya et al. 2010). several insecticides, such as dieldrin, lindane, A closer look at the above data reveals that BIDN, and fipronil, all of which act as antago- micromolar concentrations are needed for all nists (for a review, see Bloomquist 1996). The described effects of essentials oil components monoterpenoid thymol, applied in concentrations on GABAA receptor channels. This makes the between 1 and 100 μM, potentiates the action of hypothesis of GABAA receptors being respon- GABA at recombinant human GABAA receptors sible for the insecticidal activity of essential oils with various subunit compositions and also at an rather questionable. insect (D. melanogaster) ionotropic GABA receptor (Priestley et al. 2003). The enhanced 5. ION CHANNELS OF THE TRP response to GABA is likely to be the result of a FAMILY ARE INHIBITED/ positive allosteric action of thymol (Priestley et ACTIVATED al. 2003). The direct agonistic action of thymol BY MONOTERPENOIDS has been observed at both mammalian and insect recombinant receptors between 100 and 500 μM Transient receptor potential (TRP) channels are (Priestley et al. 2003). Thymol does not appear to essential components of signaling cascades in compete with other GABAergic ligands. Thus, sensory cells specialized to detect painful stimuli, the effect of thymol is possibly mediated by a such as heat, cold, and pressure, and chemicals, binding site that is not yet characterized and that such as acids or irritants. The seven subfamilies of could represent a new avenue in insecticide TRP channels are clustered into two groups, with research (Priestley et al. 2003). Recently, Tong five members in group 1, i.e., TRPC (“C” for and Coats (2010) evaluated the pharmacological canonical), TRPV (“V” for vanilloid), TRPM action of five monoterpenoids (α-terpineol, (“M” for melastatin), TRPN, and TRPA. Two carvacrol, linalool, pulegone, and thymol) on subfamilies constitute group 2, i.e., TRPP (“P” for native insect GABAA receptors from houseflies polycystic) and TRPML (“ML” for mucolipin; for and American cockroaches using radiotracer a review, see Venkatachalam and Montell 2007). methods. The binding of [3H]t-butylbicycloor- The D. melanogaster TRPC channels, TRP and thobenzoate ([3H]-TBOB) to membrane prepara- TRP-like (TRPL), are the founding members of tions of housefly heads was enhanced by the TRP superfamily. In D. melanogaster,TRP carvacrol, pulegone, and thymol with EC50 channels are specifically expressed in photore- values of 48 μM, 432 μM, and 6 mM, respec- ceptor cells of the complex eye. Upon illumina- tively. Moreover, these three monoterpenoids at tion, TRP channels are activated and cause the concentrations of 500 μM and 1 mM also depolarization of the photoreceptor cell (for a − significantly increased the GABA-induced 36Cl review, see Raghu and Hardie 2009). The uptake in membrane microsacs prepared from thermoTRPs, which include TRPV1-4, TRPM8, American cockroach ventral nerve cords. The and TRPA1, constitute a subgroup of TRP authors concluded that carvacrol, pulegone, and channels that are activated by either hot or cold thymol are all positive allosteric modulators at temperatures and also by natural compounds insect GABAA receptors (Tong and Coats 2010). evoking “hot” (e.g., ) or “cold” (e.g., In contrast, α-terpineol and linalool showed little menthol) sensations, respectively (Macpherson et or no effect on both [3H]-TBOB binding and al. 2006;Xuetal.2006; Vogt-Eisele et al. 2007; − 36Cl uptake assays (Tong and Coats 2010). In Lee et al. 2008). the blowfly, Phaenicia sericata, thymol reduced Recently, Parnas et al. (2009) have demon- the frequency of flight muscle impulses and, strated, by patch clamp whole cell recording consequently, inhibited wing beat frequency from D. melanogaster S2 and D. melanogaster (Waliwitiya et al. 2010). The similarity of thymol photoreceptor cells, that carvacrol (500 μM), a and GABA actions suggests that thymol acts known activator of the mammalian ther- 342 W. Blenau et al.

moTRPs, TRPV3, and TRPA1, acts as an The Ki values were: eucalyptol, 220 nM; inhibitor of native TRP channels and of both pulegone, 32 μM; linalool, 240 μM; citral, native and heterologously expressed TRPL 330 μM; (−)-bornyl acetate, 510 μM (Keane channels. Furthermore, thymol (1 mM), eugenol and Ryan 1999). In addition, eucalyptol inhibits (3 mM), (IC50=1.6 mM), men- AChE activity in homogenates obtained from thol (IC50=1.81 mM), and carveol (IC50= head louse with an IC50 of 77 mM (Picollo et al. 4.22 mM), which are activators of mammalian 2008). Leaf oils of E. camaldulensis and their TRPV3, have also been found to inhibit the constituents (e.g., eucalyptol, p-cymene, g- TRPL channel (Parnas et al. 2009). Interestingly, terpinene) act as both fumigants and contact high concentrations of borneol (5 mM) and toxicants on termites, Coptotermes formosanus; camphor (10 mM) activate the TRPL channel this is most likely attributable to the inhibition of (Parnas et al. 2009). Whether the interaction of AChE activity (Siramon et al. 2009). β-Pinene millimolar concentrations of plant essential oil (Ki=2.8 μM) and menthol (Ki=48 μM) are components with ion channels of the TRP family potent inhibitors of AChE from the rice weevil, correlates with their insecticidal/acaricidal prop- Sitophilus oryzae, one of the main stored grain erties needs further validation. pests (Lee et al. 2001). In conclusion, many terpenoids might act as 6. INHIBITION AChE inhibitors, both in arthropods and in OF ACETYLCHOLINESTERASE vertebrates (for a review, see Houghton et al. ACTIVITY BY MONOTERPENOIDS 2006), but at relatively high concentrations (see above; Kostyukovsky et al. 2002). Whether ACh is an important neurotransmitter in both terpenoid toxicity is correlated with their ability the CNS and the peripheral nervous system of to inhibit AChE activity needs further experi- many organisms including arthropods. Once mental evidence (Grundy and Still 1985; Lee et released, ACh has a short half-life because of al. 2001). the enzymatic activity of AChE, which hydro- lyzes ACh to choline and acetate in the synaptic 7. PLANT ESSENTIAL OILS cleft. Thus, AChE inhibitors (e.g., organophos- AS INSECTICIDES/ACARICIDES— phates or carbamates such as physostigmine or AN OUTLOOK neostigmine) can be used to increase the ACh concentration and to prolong its functional Monoterpenoids and related aromatic com- activity in the nervous system. pounds show a broad spectrum of actions in As early as 1985, however, Grundy and Still insects and mites, with tyramine and octop- demonstrated that (+)-pulegone and related amine receptors being likely candidates for monoterpenoids inhibit both housefly and their molecular targets. In addition to affect- Madagascar roach (Gromphadorhina porten- ing tyramine and/or octopamine receptors, tosa) AChE activity in vitro (Grundy and Still some of the compounds show other modes 1985). In a pioneering study on “plant–insect of action. Thymol, for example, affects insect co-evolution and inhibition of acetylcholines- GABAA receptors (Priestley et al. 2003; Tong terase,” monoterpenoids (i.e., eucalyptol, pule- and Coats 2010)andD. melanogaster TRP and gone, linalool, citral, and (−)-bornyl acetate) TRPL channels (Parnas et al. 2009). were convincingly shown to inhibit AChE So far, nothing is known about the aminergic purified from the electric eel (Ryan and Byrne receptors of V. destructor. This is surprising as 1988). Similar to the results obtained for AChE substances belonging to two different chemical from electric eel, the five monoterpenoids were classes used for Varroa treatment exert their found to be reversible competitive inhibitors of actions by binding to aminergic receptors. To AChE purified from the brain of the waxmoth, understand the mode of action of essential oils Galleria mellonella (Keane and Ryan 1999). and formamidines, the molecular signature(s) of Molecular targets of plant essential oils 343 tyramine and octopamine receptors expressed in Huang 2008). Similar receptor models should V. destructor and other arthropod pests therefore be established for the tyramine and octopamine need to be unraveled. receptors of arthropod parasites (including V. A major concern about compounds currently destructor) and pests. These models can then be used for Varroa treatment is their (potential) used to design new pharmaceutical leads for toxicity for the bees. Hoppe (1990) examined use as potent and relatively specific agonists/ 55 essential oils for mite and bee toxicity. After antagonists for tyramine and octopamine recep- 72 h, 24 essential oils produced a mite mortality tors, thereby acting as potential pest control agents. of more than 90%. However, of these 24 oils, only nine resulted in bee mortality below 10%! ACKNOWLEDGMENTS Consequently, when applying these compounds, special care has to be taken to use concen- We wish to thank Prof. B. Grünewald (Oberursel) trations that are toxic for mites but that have no for the invitation to submit this review article. The or only minor toxicity for bees. For example, work of the authors was supported by the German concentrations of 5–15 μg thymol, 50–150 μg Science Foundation (BL 469/7). camphor, or 20–60 μg menthol per liter of air killed nearly 100% of the mites without a Huiles essentielles de plantes et formamidines, comme noticeable loss of bees (Imdorf et al. 1999). insecticides/acaricides: quelles sont leurs cibles By likewise testing compounds that are moléculaires? active on tyramine and/or octopamine receptors GABA / récepteur couplé à une protéine G / of V. destructor and orthologous receptors of the octopamine / thymol / tyramine bee, dose–response curves for receptors expressed in both species can be determined. Ätherische Öle und Formamidine als Insektizide/ In this way, compounds or combinations of Akarizide. 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