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Effects of essential oils on native and recombinant acetylcholinesterases of Rhipicephalus microplus

Efeito de óleos essenciais sobre acetilcolinesterases nativa e recombinante de Rhipicephalus microplus

Abstract

This study reports the action of essential oils (EO) from five plants on the activity of native and recombinant acetylcholinesterases (AChE) from Rhipicephalus microplus. Enzyme activity of native susceptible AChE extract (S.AChE), native resistant AChE extract (R.AChE), and recombinant enzyme (rBmAChE1) was determined. An acetylcholinesterase inhibition test was used to verify the effect of the EO on enzyme activity. EO from Eucalyptus globulus, Citrus aurantifolia, Citrus aurantium var.dulcis inhibited the activity of S.AChE and R.AChE. Oils from the two Citrus species inhibited S.AChE and R.AChE in a similar way while showing greater inhibition on R.AChE. The oil from E. globulus inhibited native AChE, but no difference was observed between the S.AChE and R.AChE; however, 71% inhibition for the rBmAChE1 was recorded. Mentha piperita oil also inhibited S.AChE and R.AChE, but there was significant inhibition at the highest concentration tested. Cymbopogon winterianus oil did not inhibit AChE. Further studies are warranted with the oils from the two Citrus species that inhibited R.AChE because of the problem with R. microplus resistant to organophosphates, which target AChE. C. winterianus oil can be used against R. microplus populations that are resistant to organophosphates because its acaricidal properties act by mechanism(s) other than AChE inhibition.

Keywords:
Cattle tick; Rhipicephalus microplus; acetylcholinesterase inhibition; acaricide resistance; essential oils

Resumo

Este estudo relata a ação de óleos essenciais de cinco plantas na atividade de acetilcolinesterases (AChE) nativas e recombinantes de Rhipicephalus microplus. A atividade enzimática do extrato de acetilcolinesterase nativa suscetível (S.AChE) e resistente (R.AChE) e da enzima recombinante (rBmAChE1) foi determinada. Um teste de inibição da AChE foi utilizado, para verificar o efeito dos óleos essenciais sobre a atividade enzimática. Óleos essenciais de Eucalyptus globulus, Citrus aurantifolia, Citrus aurantium var. dulcis inibiram a atividade de S.AChE e R.AChE. Os óleos das duas espécies de Citrus inibiram S.AChE e R.AChE de maneira semelhante, mas mostraram maior inibição sobre R.AChE. O óleo de E. globulus inibiu a AChE nativa, mas sem diferença entre a S.AChE e a R.AChE; no entanto, 71% de inibição para rBmAChE1 foi observada. O óleo de Mentha piperita também inibiu S.AChE e R.AChE, mas houve inibição significativa apenas nas concentrações mais altas testadas. O óleo de Cymbopogon winterianus não inibiu a AChE. Estudos adicionais são necessários com os óleos das duas espécies de Citrus que inibiram a R.AchE, devido ao problema de R. microplus resistente aos organofosforados ter como alvo AChE. O óleo de C. winterianus pode ser usado contra populações de R. microplus, que são resistentes a organofosforados, porque suas propriedades acaricidas agem por mecanismos diferentes.

Palavras-chave:
Carrapato bovino; Rhipicephalus microplus; acetilcolinesterase; resistência; óleos essenciais

Introduction

The tick Rhipicephalus (Boophilus) microplus (Canestrini, 1888) (Acari, Ixodidae) is an economically important ectoparasite of cattle that impairs livestock production systems in tropical and subtropical parts of the world (Pérez de León et al., 2020Pérez de León AA, Mitchell RD 3rd, Watson DW. Ectoparasites of cattle. Vet Clin North Am Food Anim Pract 2020; 36(1): 173-185. http://dx.doi.org/10.1016/j.cvfa.2019.12.004. PMid:32029183.
http://dx.doi.org/10.1016/j.cvfa.2019.12...
). The cattle tick R. microplus causes direct host damage through its obligate blood feeding habit and is a vector of pathogens, including species of Babesia and Anaplasma that cause bovine babesiosis and anaplasmosis, respectively (Roy et al., 2018Roy BC, Krucken J, Ahmed JS, Majumder S, Baumann MP, Clausen PH, et al. Molecular identification of tick-borne pathogens infecting cattle in Mymensingh district of Bangladesh reveals emerging species of Anaplasma and Babesia. Transbound Emerg Dis 2018; 65(2): e231-e242. http://dx.doi.org/10.1111/tbed.12745. PMid:29119682.
http://dx.doi.org/10.1111/tbed.12745...
). Synthetic chemicals with acaricidal properties are used to treat livestock infestation with R. microplus, which is associated with high expenses to farmers (Reginato et al., 2017Reginato CZ, Cadore GC, Menezes FRD, Sangioni LA, Vogel FSF. Efficacy of commercial synthetic pyrethroids and organophosphates associations used to control Rhipicephalus (Boophilus) microplus in Southern Brazil. Rev Bras Parasitol Vet 2017; 26(4): 500-504. http://dx.doi.org/10.1590/s1984-29612017054. PMid:29091122.
http://dx.doi.org/10.1590/s1984-29612017...
; Ferreira et al., 2018Ferreira FM, Delmonte CC, Novato TLP, Monteiro CMO, Daemon E, Vilela FMP, et al. Acaricidal activity of essential oil of Syzygium aromaticum, hydrolate and eugenol formulated or free on larvae and engorged females of Rhipicephalus microplus. Med Vet Entomol 2018; 32(1): 41-47. http://dx.doi.org/10.1111/mve.12259. PMid:28833280.
http://dx.doi.org/10.1111/mve.12259...
). In Brazil, the annual economic losses of R. microplus is at least $3.2 billion (Grisi et al., 2014Grisi L, Leite RC, Martins JRDS, Barros ATMD, Andreotti R, Cançado PHD, et al. Reassessment of the potential economic impact of cattle parasites in Brazil. Rev Bras Parasitol Vet 2014; 23(2): 150-156. http://dx.doi.org/10.1590/S1984-29612014042. PMid:25054492.
http://dx.doi.org/10.1590/S1984-29612014...
).

The indiscriminate use of acaricides has made several classes of these chemical agents ineffective due to the development and selection of resistant R. microplus populations (Reck et al., 2014Reck J, Klafke GM, Webster A, Dall’agnol B, Scheffer R, Souza UA, et al. First report of fluazuron resistance in Rhipicephalus microplus: a field tick population resistant to six classes of acaricides. Vet Parasitol 2014; 201(1-2): 128-136. http://dx.doi.org/10.1016/j.vetpar.2014.01.012. PMid:24560364.
http://dx.doi.org/10.1016/j.vetpar.2014....
; Rodriguez-Vivas et al., 2018Rodriguez-Vivas RI, Jonsson NN, Bhushan C. Strategies for the control of Rhipicephalus microplus ticks in a world of conventional acaricide and macrocyclic lactone resistance. Parasitol Res 2018; 117(1): 3-29. http://dx.doi.org/10.1007/s00436-017-5677-6. PMid:29152691.
http://dx.doi.org/10.1007/s00436-017-567...
). Commercially available classes of acaricides that are used extensively include the organophosphates (OP) and carbamates (CB). Synthetic chemicals in these classes of acaricides exert their inhibitory action on acetylcholinesterase (AChE) (Anderson & Coats, 2012Anderson JA, Coats JR. Acetylcholinesterase inhibition by nootkatone and carvacrol in arthropods. Pestic Biochem Physiol 2012; 102(2): 124-128. http://dx.doi.org/10.1016/j.pestbp.2011.12.002.
http://dx.doi.org/10.1016/j.pestbp.2011....
), which is a hydrolase enzyme that plays a vital role in cholinergic neurotransmission. Inhibition of AChE activity results in hyperexcitability of neurons that leads to seizures, nervous system collapse, and death of the organism (Sharifi et al., 2017Sharifi M, Ghadamyari M, Gholivand K, Valmoozi AAE, Sajedi RH. Characterization of acetylcholinesterase from elm left beetle, Xanthogaleruca luteola and QSAR of temephos derivatives against its activity. Pestic Biochem Physiol 2017; 136: 12-22. http://dx.doi.org/10.1016/j.pestbp.2016.08.010. PMid:28187825.
http://dx.doi.org/10.1016/j.pestbp.2016....
; Temeyer, 2018Temeyer KB. Molecular biology of tick acetylcholinesterases. Front Biosci 2018; 23(4): 1320-1337. http://dx.doi.org/10.2741/4646. PMid:28930602.
http://dx.doi.org/10.2741/4646...
).

Invertebrates have different AChE isoforms (Baxter & Barker, 2002Baxter GD, Barker SC. Analysis of the sequence and expression of a second putative acetylcholinesterase cDNA from organophosphate-susceptible and organophosphate-resistant cattle ticks. Insect Biochem Mol Biol 2002; 32(7): 815-820. http://dx.doi.org/10.1016/S0965-1748(01)00168-0. PMid:12044498.
http://dx.doi.org/10.1016/S0965-1748(01)...
). In R. microplus three paralogous genes encoding AChEs (rBmAChE1, rBmAChE2 and rBmAChE3) were confirmed and expressed in neural and non-neural tissues (Temeyer et al., 2010Temeyer KB, Pruett JH, Olafson PU. Baculovirus expression, biochemical characterization and organophosphate sensitivity of rBmAChE1, rBmAChE2, and rBmAChE3 of Rhipicephalus (Boophilus) microplus. Vet Parasitol 2010; 172(1-2): 114-121. http://dx.doi.org/10.1016/j.vetpar.2010.04.016. PMid:20451328.
http://dx.doi.org/10.1016/j.vetpar.2010....
). The different biochemical properties of these isoforms and the variation in enzymatic activity between tissues indicates the physiological plasticity of AChE in R. microplus (Temeyer et al., 2020Temeyer KB, Schlechte KG, Olafson PU, Drolet BS, Tidwell JP, Osbrink WLA, et al. Association of salivary cholinesterase with arthropod vectors of disease. J Med Entomol 2020; 57(6): 1679-1685. http://dx.doi.org/10.1093/jme/tjaa096. PMid:32459332.
http://dx.doi.org/10.1093/jme/tjaa096...
). For example, AChE1 is expressed in the salivary glands, ovaries and synganglion whereas AChE2 is only expressed in the synganglion (Baxter & Barker, 2002Baxter GD, Barker SC. Analysis of the sequence and expression of a second putative acetylcholinesterase cDNA from organophosphate-susceptible and organophosphate-resistant cattle ticks. Insect Biochem Mol Biol 2002; 32(7): 815-820. http://dx.doi.org/10.1016/S0965-1748(01)00168-0. PMid:12044498.
http://dx.doi.org/10.1016/S0965-1748(01)...
; Temeyer et al., 2013Temeyer KB, Tuckow AP, Brake DK, Li AY, Pérez De León AA. Acetylcholinesterases of blood-feeding flies and ticks. Chem Biol Interact 2013; 203(1): 319-322. http://dx.doi.org/10.1016/j.cbi.2012.09.010. PMid:23036311.
http://dx.doi.org/10.1016/j.cbi.2012.09....
). However, it is known that AChE insensitivity is related to resistance to OPs and CBs, and that in R. microplus this is a primary mechanism of resistance to compounds belonging to those classes of acaricides (Temeyer et al., 2010Temeyer KB, Pruett JH, Olafson PU. Baculovirus expression, biochemical characterization and organophosphate sensitivity of rBmAChE1, rBmAChE2, and rBmAChE3 of Rhipicephalus (Boophilus) microplus. Vet Parasitol 2010; 172(1-2): 114-121. http://dx.doi.org/10.1016/j.vetpar.2010.04.016. PMid:20451328.
http://dx.doi.org/10.1016/j.vetpar.2010....
).

Essential oils are among the repertoire of natural products that can be used as alternative treatment against tick infestations because they offer advantages over synthetic acaricides (Hüe et al., 2015Hüe T, Cauquil L, Fokou JB, Dongmo PM, Bakarnga-Via I, Menut C. Acaricidal activity of five essential oils of Ocimum species on Rhipicephalus (Boophilus) microplus larvae. Parasitol Res 2015; 114(1): 91-99. http://dx.doi.org/10.1007/s00436-014-4164-6. PMid:25300420.
http://dx.doi.org/10.1007/s00436-014-416...
; Valente et al., 2017Valente PP, Moreira GHFA, Serafini MF, Facury-Filho EJ, Carvalho AU, Faraco AAG, et al. In vivo efficacy of a biotherapic and eugenol formulation against Rhipicephalus microplus. Parasitol Res 2017; 116(3): 929-938. http://dx.doi.org/10.1007/s00436-016-5366-x. PMid:28058537.
http://dx.doi.org/10.1007/s00436-016-536...
). These include the slow development of resistance by pests, low toxicity to mammals, low environmental impact and reduction of residues in products of animal origin (Abdelgaleil et al., 2009Abdelgaleil SA, Mohamed MI, Badawy ME, El-Arami SA. Fumigant and contact toxicities of monoterpenes to Sitophilus oryzae (L.) and Tribolium castaneum (Herbst) and their inhibitory effects on acetylcholinesterase activity. J Chem Ecol 2009; 35(5): 518-525. http://dx.doi.org/10.1007/s10886-009-9635-3. PMid:19412756.
http://dx.doi.org/10.1007/s10886-009-963...
; Salman et al., 2020Salman M, Abbas RZ, Israr M, Abbas A, Mehmood K, Khan MK, et al. Repellent and acaricidal activity of essential oils and their components against Rhipicephalus ticks in cattle. Vet Parasitol 2020; 283: 109178. http://dx.doi.org/10.1016/j.vetpar.2020.109178. PMid:32652458.
http://dx.doi.org/10.1016/j.vetpar.2020....
). As compared to conventional synthetic acaricides, essential oils that are efficacious can enhance the safety of treatment for livestock infested with R. microplus (Gross et al., 2017Gross AD, Temeyer KB, Day TA, Pérez De León AA, Kimber MJ, Coats JR. Interaction of plant essential oil terpenoids with the southern cattle tick tyramine receptor: A potential biopesticide target. Chem Biol Interact 2017; 263: 1-6. http://dx.doi.org/10.1016/j.cbi.2016.12.009. PMid:27986436.
http://dx.doi.org/10.1016/j.cbi.2016.12....
; Wang et al. 2019Wang HH, Teel PD, Grant WE, Soltero F, Urdaz J, Pérez Ramírez AE, et al. Simulation tools for assessment of tick suppression treatments of Rhipicephalus (Boophilus) microplus on non-lactating dairy cattle in Puerto Rico. Parasit Vectors 2019; 12(1): 185. http://dx.doi.org/10.1186/s13071-019-3443-6. PMid:31029149.
http://dx.doi.org/10.1186/s13071-019-344...
). The composition of essential oils includes volatile secondary metabolites known for their significant role in plant defense mechanisms (Silva Lima et al., 2018Silva Lima A, Milhomem MN, Santos Monteiro O, Arruda ACP, Castro JAM, Fernandes YML, et al. Seasonal analysis and acaricidal activity of the thymol-type essential oil of Ocimum gratissimum and its major constituents against Rhipicephalus microplus (Acari: ixodidae). Parasitol Res 2018; 117(1): 59-65. http://dx.doi.org/10.1007/s00436-017-5662-0. PMid:29152690.
http://dx.doi.org/10.1007/s00436-017-566...
). Essential oils are a complex mixture of substances from various chemical families, however the most common compound found are terpenes (mono and sesquiterpenes) and phenylpropanoids (Dhifi et al., 2016Dhifi W, Bellili S, Jazi S, Bahloul N, Mnif W. Essential oils chemical characterization and investigation of some biological activities: a critical review. Medicines 2016; 3(4): 25. http://dx.doi.org/10.3390/medicines3040025. PMid:28930135.
http://dx.doi.org/10.3390/medicines30400...
; Salman et al., 2020Salman M, Abbas RZ, Israr M, Abbas A, Mehmood K, Khan MK, et al. Repellent and acaricidal activity of essential oils and their components against Rhipicephalus ticks in cattle. Vet Parasitol 2020; 283: 109178. http://dx.doi.org/10.1016/j.vetpar.2020.109178. PMid:32652458.
http://dx.doi.org/10.1016/j.vetpar.2020....
).

Essential oils are known to have pesticidal and repellent properties (Pinto et al., 2015Pinto ZT, Sánchez FF, Santos AR, Amaral AC, Ferreira JL, Escalona-Arranz JC, et al. Chemical composition and insecticidal activity of Cymbopogon citratus essential oil from Cuba and Brazil against housefly. Rev Bras Parasitol Vet 2015; 24(1): 36-44. http://dx.doi.org/10.1590/S1984-29612015006. PMid:25909251.
http://dx.doi.org/10.1590/S1984-29612015...
; Soares et al., 2016Soares AMS, Penha TA, Araújo SAD, Cruz EMO, Blank AF, Costa-Junior LM. Assessment of different Lippia sidoides genotypes regarding their acaricidal activity against Rhipicephalus (Boophilus) microplus. Rev Bras Parasitol Vet 2016; 25(4): 401-406. http://dx.doi.org/10.1590/s1984-29612016087. PMid:27982301.
http://dx.doi.org/10.1590/s1984-29612016...
; Carroll et al., 2017Carroll JF, Demirci B, Kramer M, Bernier UR, Agramonte NM, Baser KHC, et al. Repellency of the Origanum onites L. essential oil and constituents to the lone star tick and yellow fever mosquito. Nat Prod Res 2017; 31(18): 2192-2197. http://dx.doi.org/10.1080/14786419.2017.1280485. PMid:28278656.
http://dx.doi.org/10.1080/14786419.2017....
) and some essential oils have also been investigated for their inhibition capacity of AChE (Salleh & Khamis, 2020Salleh W, Khamis S. Chemical composition and anticholinesterase inhibitory activity of Pavetta graciliflora Wall. ex Ridl. essential oil. Z Naturforsch 2020; 75(11-12): 467-471. http://dx.doi.org/10.1515/znc-2020-0075. PMid:32469335.
http://dx.doi.org/10.1515/znc-2020-0075...
). As example, essential oil of Origanum syriacum inhibited AChE of Culex quinquefasciatus (López et al., 2019López V, Pavela R, Gómez-Rincon C, Les F, Bartolucci F, Galiffa V, et al. Efficacy of Origanum syriacum Essential Oil against the Mosquito Vector Culex quinquefasciatus and the Gastrointestinal Parasite Anisakis simplex, with Insights on Acetylcholinesterase Inhibition. Molecules 2019; 24(14): 2563. http://dx.doi.org/10.3390/molecules24142563. PMid:31311079.
http://dx.doi.org/10.3390/molecules24142...
), and Eucalyptus globulus essential oil inhibited AChE of Rhipicephalus annulatus (Arafa et al., 2020Arafa WM, Aboelhadid SM, Moawad A, Shokeir KM, Ahmed O. Toxicity, repellency and anti-cholinesterase activities of thymol-eucalyptus combinations against phenotypically resistant Rhipicephalus annulatus ticks. Exp Appl Acarol 2020; 81(2): 265-277. http://dx.doi.org/10.1007/s10493-020-00506-1. PMid:32472469.
http://dx.doi.org/10.1007/s10493-020-005...
). However, there are no scientific reports on the ability of essential oils from Eucalyptus globulus, Citrus aurantifolia, Citrus aurantium var. dulcis, Mentha piperita, and Cymbopogon winterianus to inhibit AChE from R. microplus. In this study we investigated the action of essential oils from those plants on AChE activity in larvae of acaricide susceptible and resistant strains of R. microplus, and on rBmAChE1.

Materials and Methods

Tick populations

Ticks from two populations of R. microplus, a susceptible (Porto Alegre strain) and other resistant (resistant to organophosphate, synthetic pyrethroids, phenylpyrazole, amidinic, macrocyclic lactone, and benzoylphenyl urea derivatives - Jaguar strain) (Reck et al., 2014Reck J, Klafke GM, Webster A, Dall’agnol B, Scheffer R, Souza UA, et al. First report of fluazuron resistance in Rhipicephalus microplus: a field tick population resistant to six classes of acaricides. Vet Parasitol 2014; 201(1-2): 128-136. http://dx.doi.org/10.1016/j.vetpar.2014.01.012. PMid:24560364.
http://dx.doi.org/10.1016/j.vetpar.2014....
) were obtained by artificial infestations of cattle, which were not recently exposed to acaricide. The experimental procedures were approved by the animal research ethics committee of the Federal University of Maranhão (UFMA) under protocol number 23115.008186/2017-18.

Fully engorged females were naturally detached, and then were collected, washed with distilled water, dried on filter paper, weighed and separated into groups containing ten specimens each (maximum weight difference was ± 0.5 g). The ticks were incubated (27 °C and relative humidity ≥ 80%), for 14-21 days, for oviposition (Silva Lima et al., 2018Silva Lima A, Milhomem MN, Santos Monteiro O, Arruda ACP, Castro JAM, Fernandes YML, et al. Seasonal analysis and acaricidal activity of the thymol-type essential oil of Ocimum gratissimum and its major constituents against Rhipicephalus microplus (Acari: ixodidae). Parasitol Res 2018; 117(1): 59-65. http://dx.doi.org/10.1007/s00436-017-5662-0. PMid:29152690.
http://dx.doi.org/10.1007/s00436-017-566...
) and subsequent hatching to produce larvae used in preparation of crude larval extracts.

Obtaining the native and recombinant AChEs

In order to extract the multiple AChEs present in the larval extracts, R. microplus larvae were macerated using a mortar and pestle for 5 min in 100 mM sodium phosphate buffer, pH 7.0, containing 5 mM EDTA, 0.5% (v/v) Triton X-100, and 5 μL.mL-1 protease Inhibitor mix (Sigma-Aldrich, St. Louis, MO, USA), at a 1:25 ratio (larva weight/buffer volume). The extract was left standing for 25 min at 4 °C and centrifuged at 4 °C for 30 min at 15.000 x g. The supernatant was recovered, stored at 4 °C, and used as a source of AChEs of susceptible and resistant strain named respectively, native susceptible AChE extract (S.AChE) and native resistant AChE extract (R.AChE).

The recombinant enzyme (rBmAChE1) was obtained according to Temeyer et al. (2010)Temeyer KB, Pruett JH, Olafson PU. Baculovirus expression, biochemical characterization and organophosphate sensitivity of rBmAChE1, rBmAChE2, and rBmAChE3 of Rhipicephalus (Boophilus) microplus. Vet Parasitol 2010; 172(1-2): 114-121. http://dx.doi.org/10.1016/j.vetpar.2010.04.016. PMid:20451328.
http://dx.doi.org/10.1016/j.vetpar.2010....
. Briefly, total RNA was isolated from pooled larvae of susceptible strain. Gene-specific primers were utilized to direct synthesis of first-strand cDNA from RNA template using Reverse Transcriptase, and complete BmAChE1 coding regions were amplified by high fidelity PCR from cDNA, sequenced, and expressed in baculovirus vectors. Recombinant expression clones were assembled, sequenced, and expressed in baculovirus infected Sf21 cell cultures.

Protein concentration was determined using bovine serum albumin (BSA) as standard (Bradford, 1976Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72(1-2): 248-254. http://dx.doi.org/10.1016/0003-2697(76)90527-3. PMid:942051.
http://dx.doi.org/10.1016/0003-2697(76)9...
). Results were expressed in milligrams of proteins per milliliter (mg.mL-1).

Determination of AChE activity

The S.AChE, R.AChE and rBmAChE1 activity was determined according to Ellman et al. (1961)Ellman GL, Courtney KD, Andres V Jr, Featherstone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961; 7(2): 88-95. http://dx.doi.org/10.1016/0006-2952(61)90145-9. PMid:13726518.
http://dx.doi.org/10.1016/0006-2952(61)9...
, modified as described by Li et al. (2005)Li AY, Pruett JH, Davey RB, George JE. Toxicological and biochemical characterization of coumaphos resistance in the San Roman strain of Boophilus microplus (Acari: ixodidae). Pestic Biochem Physiol 2005; 81(3): 145-153. http://dx.doi.org/10.1016/j.pestbp.2004.12.002.
http://dx.doi.org/10.1016/j.pestbp.2004....
. The reaction mixture consisted of 10 μL of the S.AChE or R.AChE (1.5 mg protein mL-1 final concentration) or rBmAChE1 diluted 30 X with buffer (50mM sodium phosphate, pH 7.5), 100 μL 50 mM sodium phosphate buffer, pH 7.5, and 100 μL of the reaction solution. The reaction solution contained 0.24 mM acetylthiocholine iodide (Sigma-Aldrich) and 0.64 mM 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) (Sigma-Aldrich) prepared in the above sodium phosphate buffer. In the blank sample, the AChE aliquot was replaced by the buffer. Reaction was conducted in a 96 well microplate for 30 min and monitored every 5 min by recording the absorbance (Abs) at 405 nm (Microplate Reader, Biochrom). Activity was calculated using the equation: Activity (abs/mL/min) = [(T30-T0)/30] x 100, where T0 and T30 = sample absorbance - blank absorbance measured at zero and 30 min reaction. Only linear reactions throughout the monitoring period were considered.

Inhibition of AChE activity

Essential oils from E. globulus (chemical composition - 83% 1.8-cineol, 9% limonene, 4% alfa-pinene, 3% p-cimene), C. aurantifolia (chemical composition - 57% limomene, 14% γ-terpinene, 12% β-pinene, 2% α-pinene, 1.5% mircene, 1.5% geranial), C. aurantium var. dulcis (chemical composition - 96% limonene, 1.8% mircene, 0.5% α-pinene, 0.3% sabinene), M. piperita (35% menthol, 26% menthone, 6.0% 1.8-cineol, 5.0% isomenthone, 5.0% methyl acetate, 4.0% neomenthol) and C. winterianus (chemical analysis was not performed) were commercially purchased (Ferquima). The chemical analysis were performed by company and informed to the authors. The essential oils were individually diluted in ethanol to 20 mg.mL-1 (stock solution). From the stock solution, an essential oil solution at 2 mg.mL-1 was prepared in 50 mM sodium phosphate buffer, pH 7.5. The final essential oil concentrations tested were 1.00, 0.67, 0.44, 0.30, 0.20, 0.13, 0.088, 0.058, 0.039, 0.026, 0.017 and 0.012 mg.mL−1. These concentrations were selected by a preliminary pilot test performed.

The AChE inhibitory activity was evaluated by mixing 10 μL of the S.AChE, R.AChE or rBmAChE1 with 100 μL of the essential oil and 100 μL of the reaction solution described above. Propoxur (Sigma-Aldrich) was used as a positive control. It was similarly prepared as the essential oil, but at 0.25, 0.05, 0.025, 0.005, 0.0025 and 0.0005 mM final concentrations (Prado-Ochoa et al., 2014Prado-Ochoa MG, Ramírez-Noguera P, Díaz-Torres R, Garrido-Fariña GI, Vázquez-Valadez VH, Velázquez-Sánchez AM, et al. The action of two ethyl carbamates on acetylcholinesterase and reproductive organs of Rhipicephalus microplus. Vet Parasitol 2014; 199(3-4): 215-224. http://dx.doi.org/10.1016/j.vetpar.2013.10.028. PMid:24315691.
http://dx.doi.org/10.1016/j.vetpar.2013....
). In the negative control, essential oils were replaced by the phosphate buffer and ethanol. Reactions were conducted in a 96 well microplate for 30 min and monitored every 5 min by recording the absorbance (Abs) at 405 nm (Microplate Reader, Biochrom).The percentage of enzyme inhibition was calculated by comparison with the negative control as follows: AChE inhibition (%) = 100 - [(As / Ac) x 100], where: As = AChE activity for each concentration; Ac = negative control (AChE activity without essential oil). Only linear regression reactions throughout the monitoring period were considered.

Statistical analyses

The data were obtained from the triplicate inhibition assays of two independent experiments for each essential oil on the S.AChE and R.AChE and rBmAChE1. The data were initially transformed to log (X) and normalized; subsequently, nonlinear regression was performed to obtain the IC50 (50% inhibition concentration) and the F test was used by pair to compare the curves. All analysis were performed using the GraphPad Prism 7.0 software (GraphPad Inc., San Diego, CA, USA).

Results and Discussion

Acaricidal and repellent activities, and egg hatch inhibition are among the biological properties against ticks reported for essential oils of plant species in the genus Citrus (Pazinato et al., 2016Pazinato R, Volpato A, Baldissera MD, Santos RC, Baretta D, Vaucher RA, et al. In vitro effect of seven essential oils on the reproduction of the cattle tick Rhipicephalus microplus. J Adv Res 2016; 7(6): 1029-1034. http://dx.doi.org/10.1016/j.jare.2016.05.003. PMid:27857849.
http://dx.doi.org/10.1016/j.jare.2016.05...
; Stefanidesova et al., 2017; Vinturelle et al., 2017Vinturelle R, Mattos C, Meloni J, Nogueira J, Nunes MJ, Vaz IS Jr, et al. In Vitro Evaluation of essential oils derived from Piper nigrum (Piperaceae) and Citrus limonum (Rutaceae) against the tick Rhipicephalus (Boophilus) microplus (Acari: ixodidae). Biochem Res Int 2017; 2017: 5342947. http://dx.doi.org/10.1155/2017/5342947. PMid:29123924.
http://dx.doi.org/10.1155/2017/5342947...
). In our experiments C. aurantium var. dulcis and C. aurantifolia inhibited S.AChE and R.AChE in varying degrees. Stronger inhibition against R.AChE was exhibited by C. aurantifolia oil (64.0 ± 13.1%) at 0.44 mg.mL-1 and C. aurantium var. dulcis (49.8 ± 8.5%) at 0.67 mg.mL-1. However, the inhibition decreased at highest concentrations (Figures 1A and 1B). There was statistically significant difference among IC50 of S.AChE, R.AChE and rBmAChE1 after treatment with C. aurantium var. dulcis, and C. aurantifolia.

Figure 1
Effect of different concentrations of essential oils (mg.mL-1) on the acetylcholinesterase activity of R. microplus expressed as percentage of inhibition (%) and its . The essential oils used (A) Citrus aurantifolia; (B) Citrus aurantium var. dulcis; (C) Eucalyptus globulus; (D) Mentha piperita, and (E) Cymbopogon winterianus. S.AChE: Native susceptible AChE extract; R.AChE: Native resistant AChE extract; rBmAChE1: Recombinant enzyme; IC50: 50 percent inhibitory concentration. Each point represents the mean of the values obtained from two independent experiments performed in triplicate.

The Citrus oil inhibition profiles for S.AChE and R.AChE reported here suggest that structural difference between the AChE of susceptible and resistant tick larvae result in different sensitivities to Citrus oils, which is consistent with the known mechanism of OP resistance in R. microplus associated with AChE insensitivity (Temeyer, 2018Temeyer KB. Molecular biology of tick acetylcholinesterases. Front Biosci 2018; 23(4): 1320-1337. http://dx.doi.org/10.2741/4646. PMid:28930602.
http://dx.doi.org/10.2741/4646...
). Bioassays are warranted to determine if our in vitro results translate into acaricidal and/or repellent activity against R. microplus by the oils of C. aurantifolia and C. aurantium var. dulcis. By comparison, monoterpenes in oils of other Citrus plants have been shown to be active against R. microplus and Dermacentor reticulatus (Pazinato et al., 2016Pazinato R, Volpato A, Baldissera MD, Santos RC, Baretta D, Vaucher RA, et al. In vitro effect of seven essential oils on the reproduction of the cattle tick Rhipicephalus microplus. J Adv Res 2016; 7(6): 1029-1034. http://dx.doi.org/10.1016/j.jare.2016.05.003. PMid:27857849.
http://dx.doi.org/10.1016/j.jare.2016.05...
; Stefanidesova et al., 2017). In this regard, the monoterpene limonene has been shown to be acaricidal against R. microplus (Ferrarini et al., 2008Ferrarini SR, Duarte MO, da Rosa RG, Rolim V, Eifler-Lima VL, von Poser G, et al. Acaricidal activity of limonene, limonene oxide and beta-amino alcohol derivatives on Rhipicephalus (Boophilus) microplus. Vet Parasitol 2008; 157(1-2): 149-153. http://dx.doi.org/10.1016/j.vetpar.2008.07.006. PMid:18755549.
http://dx.doi.org/10.1016/j.vetpar.2008....
; Vinturelle et al., 2017Vinturelle R, Mattos C, Meloni J, Nogueira J, Nunes MJ, Vaz IS Jr, et al. In Vitro Evaluation of essential oils derived from Piper nigrum (Piperaceae) and Citrus limonum (Rutaceae) against the tick Rhipicephalus (Boophilus) microplus (Acari: ixodidae). Biochem Res Int 2017; 2017: 5342947. http://dx.doi.org/10.1155/2017/5342947. PMid:29123924.
http://dx.doi.org/10.1155/2017/5342947...
).

Eucalyptus globulus oil inhibited 37.5 ± 7.5 and 45.5 ± 8.3% of S.AChE and R.AChE at 1 mg.mL-1, respectively. Significant difference in inhibitory activity was observed between the S.AChE (IC50 = 0.29 mg.mL-1) and R.AChE (IC50 = 0.27 mg.mL-1). However, the oil from E. globulus strongest inhibited the rBmAChE1 with IC50 of 0.10 mg.mL-1 (Figure 1C). Because the other oils tested did not inhibit rBmAChE1, we hypothesize that differences between their components afford them various levels of inhibitory activity against the AChE isoforms considering that R. microplus has at least three functional AChEs (Temeyer et al., 2010Temeyer KB, Pruett JH, Olafson PU. Baculovirus expression, biochemical characterization and organophosphate sensitivity of rBmAChE1, rBmAChE2, and rBmAChE3 of Rhipicephalus (Boophilus) microplus. Vet Parasitol 2010; 172(1-2): 114-121. http://dx.doi.org/10.1016/j.vetpar.2010.04.016. PMid:20451328.
http://dx.doi.org/10.1016/j.vetpar.2010....
), and that the larval extracts obtained in this study likely contained the three native AChEs. The pesticidal activity of this oil was associated with a high content of 1.8-cineole, also known as eucalyptol (Miresmailli et al., 2006Miresmailli S, Bradbury R, Isman MB. Comparative toxicity of Rosmarinus officinalis L. essential oil and blends of its major constituents against Tetranychus urticae Koch (Acari: Tetranychidae) on two different host plants. Pest Manag Sci 2006; 62(4): 366-371. http://dx.doi.org/10.1002/ps.1157. PMid:16470541.
http://dx.doi.org/10.1002/ps.1157...
; George et al., 2009George DR, Masic D, Sparagano OA, Guy JH. Variation in chemical composition and acaricidal activity against Dermanyssus gallinae of four eucalyptus essential oils. Exp Appl Acarol 2009; 48(1-2): 43-50. http://dx.doi.org/10.1007/s10493-008-9225-z. PMid:19089590.
http://dx.doi.org/10.1007/s10493-008-922...
). In a previous study with R. microplus, eucalyptol showed greater AChE inhibition against the resistant strain (IC50 0.36 mg.mL-1) than the susceptible strain (IC50 3.41 mg.mL-1) (Cardoso et al., 2020Cardoso AS, Santos EGG, Lima ADS, Temeyer KB, Pérez De León AA, Costa LMJ, et al. Terpenes on Rhipicephalus (Boophilus) microplus: acaricidal activity and acetylcholinesterase inhibition. Vet Parasitol 2020; 280: 109090. http://dx.doi.org/10.1016/j.vetpar.2020.109090. PMid:32208306.
http://dx.doi.org/10.1016/j.vetpar.2020....
). Furthermore, 0.01 M of eucalyptol inhibited AChE 64.9% in larvae of the beetle Tribolium castaneum (Abdelgaleil et al., 2009Abdelgaleil SA, Mohamed MI, Badawy ME, El-Arami SA. Fumigant and contact toxicities of monoterpenes to Sitophilus oryzae (L.) and Tribolium castaneum (Herbst) and their inhibitory effects on acetylcholinesterase activity. J Chem Ecol 2009; 35(5): 518-525. http://dx.doi.org/10.1007/s10886-009-9635-3. PMid:19412756.
http://dx.doi.org/10.1007/s10886-009-963...
). The observed differences in rBmAChE1 inhibition by essential oils are strongly suggestive as to their role in the total activity of AChE present in tick larvae, indicating that rBmAChE1 is only partially responsible for the total AChE pool activity, since C. aurantifolia and C. aurantium var. dulcis oils predominantly target other tick AChEs, in contrast, E. globulus oil which inhibited rBmAChE1.

Mentha piperita (popularly known as peppermint) essential oil also showed AChE inhibition. The inhibition rates recorded at the highest concentration (1 mg.mL-1) tested were 31.4 ± 5.7%, 19.4 ± 2.2%, and 11.2 ± 3.6% for the R.AChE (IC50 = 0.58 mg.mL-1), S.AChE (IC50 = 0.66 mg.mL-1), and rBmAChE1 (IC50 = 0.18 mg.mL-1), respectively (Figure 1D). These results suggest that AChE inhibition was caused by a relatively minor component of the peppermint oil or the majors component had low activity. Peppermint oil is acaricidal, repellent, and known to contain menthol and menthone as major compounds (Chagas et al., 2016Chagas AC, Oliveira MC, Giglioti R, Santana RC, Bizzo HR, Gama PE, et al. Efficacy of 11 Brazilian essential oils on lethality of the cattle tick Rhipicephalus (Boophilus) microplus. Ticks Tick Borne Dis 2016; 7(3): 427-432. http://dx.doi.org/10.1016/j.ttbdis.2016.01.001. PMid:26867819.
http://dx.doi.org/10.1016/j.ttbdis.2016....
). M. piperita oil also has fumigating action and this activity is promoted by the rapid volatilization of 1.8-cineole (Mkolo et al., 2011Mkolo NM, Olowoyo JO, Sako KB, Mdakane STR, Mitonga MMA, Magano SR. Repellency and toxicity of essential oils of Mentha piperita and Mentha spicata on larvae and adult of Amblyomma hebraeum (Acari: ixodidae). Sci J Microbiol 2011; 1(1): 1-7.).

Cymbopogon winterianus (popularly known as Citronella) essential oil is widely used and commercialized for its repellent and acaricidal activity. These properties of citronella oil are attributed to the presence of volatile substances such as citronellal, eugenol, geraniol, which are major components that act synergistically (Olivo et al., 2008Olivo CJ, Carvalho NM, Silva JHS, Vogel FF, Massariol P, Meinerz G, et al. Óleo de citronela no controle do carrapato de bovinos. Cienc Rural 2008; 38(2): 406-410. http://dx.doi.org/10.1590/S0103-84782008000200018.
http://dx.doi.org/10.1590/S0103-84782008...
; Singh et al., 2014aSingh NK, Jyoti, Vemu B, Nandi A, Singh H, Kumar R, et al. Acaricidal activity of Cymbopogon winterianus, Vitex negundo and Withania somnifera against synthetic pyrethroid resistant Rhipicephalus (Boophilus) microplus. Parasitol Res 2014a; 113(1): 341-350. http://dx.doi.org/10.1007/s00436-013-3660-4. PMid:24178747.
http://dx.doi.org/10.1007/s00436-013-366...
, bSingh NK, Jyoti, Vemu B, Nandi A, Singh H, Kumar R, et al. Laboratory assessment of acaricidal activity of Cymbopogon winterianus, Vitex negundo and Withania somnifera extracts against deltamethrin resistant Hyalomma anatolicum. Exp Appl Acarol 2014b; 63(3): 423-430. http://dx.doi.org/10.1007/s10493-014-9791-1. PMid:24647800.
http://dx.doi.org/10.1007/s10493-014-979...
). Citronella oil did not inhibit the R. microplus AChEs in our experiments (Figure 1E) and the IC50 were not obtained. Thus, C. winterianus oil can be used against R. microplus populations that are resistant to carbamates and organophosphates because its acaricidal properties act by mechanism(s) other than AChE inhibition.

Although the oils tested in this study had shown to be active against ticks of different species including R. microplus (George et al., 2009George DR, Masic D, Sparagano OA, Guy JH. Variation in chemical composition and acaricidal activity against Dermanyssus gallinae of four eucalyptus essential oils. Exp Appl Acarol 2009; 48(1-2): 43-50. http://dx.doi.org/10.1007/s10493-008-9225-z. PMid:19089590.
http://dx.doi.org/10.1007/s10493-008-922...
; Singh et al., 2014bSingh NK, Jyoti, Vemu B, Nandi A, Singh H, Kumar R, et al. Laboratory assessment of acaricidal activity of Cymbopogon winterianus, Vitex negundo and Withania somnifera extracts against deltamethrin resistant Hyalomma anatolicum. Exp Appl Acarol 2014b; 63(3): 423-430. http://dx.doi.org/10.1007/s10493-014-9791-1. PMid:24647800.
http://dx.doi.org/10.1007/s10493-014-979...
; Chagas et al., 2016Chagas AC, Oliveira MC, Giglioti R, Santana RC, Bizzo HR, Gama PE, et al. Efficacy of 11 Brazilian essential oils on lethality of the cattle tick Rhipicephalus (Boophilus) microplus. Ticks Tick Borne Dis 2016; 7(3): 427-432. http://dx.doi.org/10.1016/j.ttbdis.2016.01.001. PMid:26867819.
http://dx.doi.org/10.1016/j.ttbdis.2016....
), questions remained on their mode of action. The pesticidal effect of essential oils results from the synergistic interactions between their bioactive components (Isman, 2015Isman MB. A renaissance for botanical insecticides? Pest Manag Sci 2015; 71(12): 1587-1590. http://dx.doi.org/10.1002/ps.4088. PMid:26251334.
http://dx.doi.org/10.1002/ps.4088...
). Essential oil components can act simultaneously on different molecular targets (Politi et al., 2019Politi FAS, Fantatto RR, da Silva AA, Moro IJ, Sampieri BR, Camargo-Mathias MI, et al. Evaluation of Tagetes patula (Asteraceae) as an ecological alternative in the search for natural control of the cattle tick Rhipicephalus (Boophilus) microplus (Acari: ixodidae). Exp Appl Acarol 2019; 77(4): 601-618. http://dx.doi.org/10.1007/s10493-019-00368-2. PMid:31076974.
http://dx.doi.org/10.1007/s10493-019-003...
). Based on our experience (Costa-Júnior et al., 2016Costa-Júnior LM, Miller RJ, Alves PB, Blank AF, Li AY, Pérez de León AA. Acaricidal efficacies of Lippia gracilis essential oil and its phytochemical against organophosphate-resistant and susceptible strains of Rhipicephalus (Boophilus) microplus. Vet Parasitol 2016; 228: 60-64. http://dx.doi.org/10.1016/j.vetpar.2016.05.028. PMid:27692332.
http://dx.doi.org/10.1016/j.vetpar.2016....
; Cardoso et al., 2020Cardoso AS, Santos EGG, Lima ADS, Temeyer KB, Pérez De León AA, Costa LMJ, et al. Terpenes on Rhipicephalus (Boophilus) microplus: acaricidal activity and acetylcholinesterase inhibition. Vet Parasitol 2020; 280: 109090. http://dx.doi.org/10.1016/j.vetpar.2020.109090. PMid:32208306.
http://dx.doi.org/10.1016/j.vetpar.2020....
), the in vitro assays reported here focused on the inhibition of AChE in R. microplus to investigate the mode of action of essential oils from the five plants selected for this study.

Conclusion

The oils of E. globulus, C. aurantifolia, C. aurantium var. dulcis and M. piperita showed various degrees of inhibition on S.AChE and R.AChE, but only E. globulus oil inhibited rBmAChE1. The profiles of AChE inhibition for the five essential oils tested provided useful information to understand their mode of acaricidal activity, corroborating for further studies on the use of essential oils as candidates for new acaricides. Further studies are needed to determine the utility of these essential oils under field conditions to manage populations of R. microplus that are resistant to commercially available synthetic acaricidal chemicals.

Acknowledgements

The U.S. Department of Agriculture is an equal opportunity employer and provider. We thank the FAPEMA (Maranhão State Research Foundation) for financial support under process UNIVERSAL-00739/17 and for awarding a fellowship to Soares, A.M.S., under process BEPP-02011/18); Santos, E.G.G and Bezerra, W.A.S. We thank CNPq (Brazilian National Council for Scientific and Technological Development) for awarding a fellowship to L.M. Costa-Junior. We also thank the FINEP (Funding Authority for Studies and Projects) and FAPEMA for supporting the IECT (Science and Technology Institute of Maranhão) Biotechnology.

  • How to cite: Santos EGG, Bezerra WAS, Temeyer KB, Pérez de León AA, Costa-Junior LM, Soares AMS. Effects of essential oils on native and recombinant acetylcholinesterases of Rhipicephalus microplus. Braz J Vet Parasitol 2021; 30(2): e002221. https://doi.org/10.1590/S1984-29612021024

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Publication Dates

  • Publication in this collection
    28 May 2021
  • Date of issue
    2021

History

  • Received
    04 Feb 2021
  • Accepted
    18 Feb 2021
Colégio Brasileiro de Parasitologia Veterinária FCAV/UNESP - Departamento de Patologia Veterinária, Via de acesso Prof. Paulo Donato Castellane s/n, Zona Rural, , 14884-900 Jaboticabal - SP, Brasil, Fone: (16) 3209-7100 RAMAL 7934 - Jaboticabal - SP - Brazil
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