Effects of Piper aduncum (Piperales: Piperaceae) Essential Oil and Its Main Component Dillapiole on Detoxifying Enzymes and Acetylcholinesterase Activity of Amblyomma sculptum (Acari: Ixodidae)

Amblyomma sculptum is a species of tick in the family Ixodidae, with equids and capybaras among its preferred hosts. In this study, the acaricidal activity of the essential oil (EO) from Piper aduncum and its main component, Dillapiole, were evaluated against larvae of A. sculptum to establish lethal concentration values and assess the effects of these compounds on tick enzymes. Dillapiole exhibited slightly greater activity (LC50 = 3.38 mg/mL; 95% CI = 3.24 to 3.54) than P. aduncum EO (LC50 = 3.49 mg/mL; 95% CI = 3.36 to 3.62) against ticks. The activities of α-esterase (α-EST), β-esterase (β-EST), and glutathione-S-transferase (GST) enzymes in A. sculptum larvae treated with Dillapiole showed a significant increase compared to the control at all concentrations (LC5, LC25, LC50 and LC75), similar results were obtained with P. aduncum EO, except for α-EST, which did not differ from the control at the highest concentration (LC75). The results of the acetylcholinesterase (AChE) activity show an increase in enzyme activity at the two lower concentrations (LC5 and LC25) and a reduction in activity at the two higher, lethal concentrations (LC50 and LC75) compared to the control. These results suggest potential mechanisms of action for these natural acaricides and can provide guidance for the future development of potential plant-derived formulations.


Introduction
Amblyomma sculptum Berlese, 1888 is a species of tick commonly found in the most populated states of Brazil, as well as Paraguay and northern Argentina [1].Its preferred hosts are equids, but it can also parasitize cattle, other domestic animals, and wildlife [2,3].The species is part of a specific complex called Amblyomma cajennense sensu lato, with a wide distribution in the Americas [4].
Of great importance is its ability to transmit pathogens during the blood-feeding process on vertebrate hosts.In the scientific field, it is well-established that A. sculptum serves as a vector for the bacterium Rickettsia rickettsii Lima, 1916, which can be transmitted to humans when an infected tick feeds on human blood, leading to Brazilian Spotted Fever (BSF) [5][6][7][8].Recent years have seen reported cases of BSF in Brazil, highlighting the urgent need for alternative tick control methods [9,10].
In the search for alternatives for acaricidal activity on A. sculptum, essential oils (EOs) and isolated compounds from EOs can represent excellent sources of biologically active natural products [11,12].Notably, their toxicity is considerably lower, making them less harmful to the environment when compared to synthetic acaricides available on the market [13].Additionally, they exhibit more environmentally friendly behavior, i.e., they are biodegradable and potentially reduce ecological impact compared to synthetic alternatives [14][15][16].
Piper aduncum L. is an example of a plant whose EO has potential acaricidal activity [17][18][19], which makes this species a promising candidate for tests against A. sculptum.This plant is a shrubby plant belonging to the Piperaceae family that grows 6 to 7 m tall with lance-shaped leaves, native to southern Mexico, the Caribbean, and abundant in South and Central America [20,21].
The ability to detoxify compounds with insecticidal/acaricidal activities is associated with an increase in the expression or activity of specific enzyme families, such as esterases (EST) and glutathione-S-transferases (GST), and these are the possible resistance mechanisms in arthropods [22][23][24][25].Consequently, monitoring the levels of these enzymes is of utmost importance for evaluating the potential use of new substances derived from plants with acaricidal activity.
Another important enzyme to be evaluated in the proposal of new acaricidal substances is acetylcholinesterase (AChE), whose action is crucial in the propagation of nerve impulses.Inhibition of AChE is reported as one of the main activities of organophosphates and carbamates [26,27].However, mutations can produce ticks that are resistant to this class of insecticides, altering the enzyme's structure in such a way that it is no longer effectively inhibited by the insecticides [28].There are data in the literature demonstrating that natural compounds found in EOs, such as thymol and carvacrol, can increase the activity of enzymes related to the detoxification process in the cattle tick, Rhipicephalus microplus Canestrini, 1887 [29].As part of an effort to contribute to the sustainable, economical, and ecological reduction in tick populations, the study of plants with acaricidal activity has been gaining prominence in research.Therefore, the present study aims mainly to evaluate the acaricidal activity of the EO of P. aduncum and its main compound, Dillapiole, on larvae of A. sculptum, establish lethal concentration values, and assess the effects of these compounds on some tick enzymes.

Oil Composition Analysis
The EO of P. aduncum was analyzed by GC-MS to determine its main constituents.Identification of these constituents was based on library search, retention index (RI), and, when available, the use of standard compounds.The relative percentages of each constituent are presented in Table 1.A total of thirty-six compounds were identified in the oil, which were classified as hydrocarbon monoterpenes, hydrocarbon sesquiterpenes, oxygenated monoterpenes, oxygenated sesquiterpenes, and phenylpropanoids.The major compound in the EO, determined through GC-MS analysis, was the phenylpropanoid Dillapiole (81.9% relative concentration), which was selected for use in the tests (Figure 1).

Effect of Piper aduncum EO and Dillapiole on Larval Mortality
The effect of P. aduncum EO and its major constituent, Dillapiole, against A. sculptum larvae was evaluated after 24 h of treatment, following the conditions described in Section 4.3.Dillapiole exhibited greater activity (LC50 = 3.38 mg/mL; 95% CI = 3.24 to 3.54) compared to P. aduncum EO (LC50 = 3.49 mg/mL; 95% CI = 3.36 to 3.62); however, there was no significant difference between the EO and the main compound due to overlapping confidence intervals.The lethal concentrations (LC5, LC25, LC50, and LC75) of the compounds for the larvae are listed in Table 2.

Effect of Piper aduncum EO and Dillapiole on Detoxifying Enzymes of Larvae
The effects of P. aduncum EO and of its major constituent, Dillapiole, on the detoxifying enzymes of A. sculptum larvae were evaluated under the conditions described in Sections 4.4.2 and 4.4.3.The activities of α-esterase (α-EST), β-esterase (β-EST), and glutathione-S-transferase (GST) enzymes in the A. sculptum larvae treated with P. aduncum EO and Dillapiole showed a significant increase compared to the control group at all concentrations of P. aduncum EO and Dillapiole tested, with the exception of α-EST treated with the LC75 of P. aduncum EO (Figure 2), which did not significantly differ from the control.

Effect of Piper aduncum EO and Dillapiole on Larval Mortality
The effect of P. aduncum EO and its major constituent, Dillapiole, against A. sculptum larvae was evaluated after 24 h of treatment, following the conditions described in Section 4.3.Dillapiole exhibited greater activity (LC 50 = 3.38 mg/mL; 95% CI = 3.24 to 3.54) compared to P. aduncum EO (LC 50 = 3.49 mg/mL; 95% CI = 3.36 to 3.62); however, there was no significant difference between the EO and the main compound due to overlapping confidence intervals.The lethal concentrations (LC 5 , LC 25 , LC 50 , and LC 75 ) of the compounds for the larvae are listed in Table 2.

Effect of Piper aduncum EO and Dillapiole on Detoxifying Enzymes of Larvae
The effects of P. aduncum EO and of its major constituent, Dillapiole, on the detoxifying enzymes of A. sculptum larvae were evaluated under the conditions described in Sections 4.4.2 and 4.4.3.The activities of α-esterase (α-EST), β-esterase (β-EST), and glutathione-S-transferase (GST) enzymes in the A. sculptum larvae treated with P. aduncum EO and Dillapiole showed a significant increase compared to the control group at all concentrations of P. aduncum EO and Dillapiole tested, with the exception of α-EST treated with the LC 75 of P. aduncum EO (Figure 2), which did not significantly differ from the control.

Effect of Piper aduncum EO and Dillapiole on Acetylcholinesterase (AChE) Activity of Larvae
The effects of P. aduncum EO and its major constituent, Dillapiole, on the AChE activity of A. sculptum larvae were evaluated under the conditions described in Section 4.4.4.In our experiments, both P. aduncum EO and Dillapiole treatment caused an initial increase in enzyme activity at lower concentrations (LC5 and LC25) in contrast with a reduction in AChE activity at higher concentrations (LC50 and LC75), compared to controls, with the exception of the AChE activity of the larvae treated with P. aduncum EO at LC50 (3.49 mg/mL; AChE: 1.6 ± 0.08 Abs/min/ng protein) which did not differ significantly from the control group (AChE: 1.5 ± 0.15 Abs/min/ng protein) (Figure 3).

Effect of Piper aduncum EO and Dillapiole on Acetylcholinesterase (AChE) Activity of Larvae
The effects of P. aduncum EO and its major constituent, Dillapiole, on the AChE activity of A. sculptum larvae were evaluated under the conditions described in Section 4.4.4.In our experiments, both P. aduncum EO and Dillapiole treatment caused an initial increase in enzyme activity at lower concentrations (LC 5 and LC 25 ) in contrast with a reduction in AChE activity at higher concentrations (LC 50 and LC 75 ), compared to controls, with the exception of the AChE activity of the larvae treated with P. aduncum EO at LC 50 (3.49mg/mL; AChE: 1.6 ± 0.08 Abs/min/ng protein) which did not differ significantly from the control group (AChE: 1.5 ± 0.15 Abs/min/ng protein) (Figure 3).

Scanning Electron Microscopy
Ten images were obtained from the larvae in each treatment group, with five ventral and five dorsal views under the conditions described in Section 4.5.Only one image from each group (dorsal and ventral) was selected for data presentation (Figure 4).The scanning electron microscopy did not reveal cuticle alterations in A. sculptum larvae after in vitro exposure to P. aduncum EO and Dilapiolle (Figure 4).The cuticles remained intact, like what was observed in the vehicle control group.

Scanning Electron Microscopy
Ten images were obtained from the larvae in each treatment group, with five ventral and five dorsal views under the conditions described in Section 4.5.Only one image from each group (dorsal and ventral) was selected for data presentation (Figure 4).The scanning electron microscopy did not reveal cuticle alterations in A. sculptum larvae after in vitro exposure to P. aduncum EO and Dilapiolle (Figure 4).The cuticles remained intact, like what was observed in the vehicle control group.

Discussion
While there are few confirmed records of resistance to the acaricides used for the controlling of the Amblyomma complex until now [30][31][32], it is important to note that resistance to insecticides and acaricides is a phenomenon that can develop over time due to the selective pressure exerted by these chemicals [26,33].Thus, the search for acaricides derived from EOs and isolated compounds has led to an increase in the number of studies that have caught the attention of the scientific community [13].The present study showed that the EO of P. aduncum and its main component have an acaricidal effect on A. sculptum and interfere with the activity of detoxifying enzymes and AChE.These results reinforce what has been shown in the literature regarding the acaricidal activity of this Piper species [17].This also is the first time the effect of P. aduncum EO and of the phenylpropanoid Dillapiole have been shown on important detoxifying enzymes (GST, α-Est, β-Est) and on AChE in the tick A. sculptum.
The EO of P aduncum contains more than 30 compounds, most of which are terpenes that are present in very low concentrations and together represent less than 20% of the essential oil content.The major component in the P. aduncum EO, the phenylpropanoid Dillapiole (81.9% of EO content), was selected for use in our tests.Dillapiole on its own exhibited greater activity (LC 50 = 3.38 mg/mL; 95% CI = 3.24 to 3.54) than P. aduncum EO (LC 50 = 3.49 mg/mL; 95% CI = 3.36 to 3.62); however, there was no significant difference between the EO and the major component.The acaricidal potential of P. aduncum EO has already been demonstrated in other tick species such as R. microplus, where it resulted in 100% mortality in larvae at a concentration of 0.1 mg/mL [17].Regarding Dillapiole, to our knowledge, there is currently no data on acaricidal activity in any species.However, it has been reported that phenylpropanoids such as (E)-cinnamaldehyde [34] and Eugenol [12] have acaricidal activity on larvae of A. sculptum.This may indicate the possible susceptibility of A. sculptum larvae to compounds of the phenylpropanoid class.However, testing with other compounds of this class on A. sculptum larvae is necessary to corroborate this hypothesis.
The need to perform biological activity tests with the major compound(s) found in the chromatographic analysis of an EO is necessary due to two main reasons: the first is that EOs can exhibit variations in their compound composition due to varying environmental conditions, soil composition, growth stages, biotic factors, and genetic diversity within the plant [35][36][37][38].The second reason is that EOs are complex mixtures of monoterpenes, sesquiterpenes, and phenylpropanoids, and the primary components can interact synergistically or antagonistically with minor constituents, thereby producing results that differ from what might be expected for individual compounds [39].Another advantage of compounds found EOs is their standardization and the ease of obtaining the active ingredient on a large scale for developing commercial formulations [13].Based on the data obtained from the Acaricidal Effect X Chromatographic Analysis, we conclude that there seems to be no significant interaction (antagonistic or synergistic) of the major molecule, Dillapiole, with the other minor components present in the P. aduncum EO.
An increase in the activity of detoxifying enzymes (GST, α-EST, β-EST) was observed for A. sculptum larvae treated with P. aduncum EO and pure Dillapiole, reaching its peak at the sublethal concentration (LC 50 ).This elevation in the activity of detoxifying en-zymes from arthropods is, in general, a result of their defense mechanisms against certain substances [40,41].Similar to the results obtained in this study, a significant increase in GST activity was found in a susceptible strain of R. microplus (Porto Alegre) when treated with sublethal concentrations (LC 1 , LC 25 , and LC 50 ) of two monoterpenes (carvacrol and thymol) [29].In arthropods, GSTs are a set of versatile enzymes engaged in oxidative protection that confer resistance via direct metabolism or sequestration of chemicals, but also indirectly by providing protection against oxidative stress induced by insecticide exposure [42,43].A notable reduction in the activity of the analyzed detoxifying enzymes, GST, α-EST, and β-EST, was evident in the A. sculptum larvae after exposure to the LC 75 of both P. aduncum EO and Dillapiole, when compared to the larvae treated with the LC 50 , and in one case (α-EST activity after treatment with LC 75 of both P. aduncum EO and Dillapiole) showing a lack of increase in enzyme activity compared to the control.This reduction in enzyme activity after treatment can be attributed to the higher exposure concentration, which likely facilitates the entrance of a greater quantity of toxic substances into the tissues, causing increased oxidative stress and, consequently, reducing the levels of antioxidant enzyme activity [29].
In our experiments, both P. aduncum EO and Dillapiole progressively inhibited the activity of AChE in the larvae of A. sculptum when applied at higher concentrations, with increased inhibition upon exposure to the LC 75 .This suggests that in addition to affecting the activity of detoxification enzymes, both P. aduncum EO and Dillapiole may also interfere with the tick's neurotransmission.In the literature, EOs from the leaves and stems of Piper austrosinense, Piper puberulum, Piper flaviflorum, Piper betle, and Piper hispidimervium have shown robust AChE inhibitory activity in vitro, with IC 50 values ranging from 1.51 to 13.9 mg/mL [44].In other arthropods such as Aedes aegypti, A. albopictus, and Culex quinquefasciatus, phenylpropanoids Asaricin 1 and Isoasarone 2 displayed potent inhibition on acetylcholinesterase, with relative IC 50 values of 0.73 to 1.87 µg/mL, respectively [45].
Three hypotheses can explain the potential mechanisms by which P. aduncum EO and Dillapiole disrupt certain enzymes in the present study: 1-P.aduncum EO and its major component may inhibit intracellular signaling pathways that regulate the production of detoxifying enzymes, for example, modulating intracellular signaling cascades; 2-P.aduncum EO and Dillapiole may influence the expression of genes that encode detoxifying enzymes; 3-P.aduncum EO and Dillapiole may directly bind to the active site of the enzymes under study, thereby blocking their enzymatic activity.In this way, future studies are necessary to test these hypotheses and to bring clarity to the ideas regarding the relationship between new compounds and specific enzyme families (Figure 5).
At the enzymatic level, after data analysis, the EO of P. aduncum and Dilapiolle have the potential to alter the enzymatic activity of important detoxifying enzymes and of neurotransmission in A. sculptum larvae.At the morphological level, the investigation sought to determine whether the EO of P. aduncum and its major compound could affect the cuticle of A. sculptum, using scanning electron microscopy.In the current literature, it is already known that P. aduncum EO has the ability to destroy the cuticle of arthropods, as evidenced by the fact that in A. aegypti, the EO of this species was able to cause a strong rupture throughout the extent of the cuticle [46].Based on this idea, we sought to verify whether P. aduncum EO and its major compound could affect the cuticle of A. sculptum.
No morphological alterations at the cuticle level were observed in both groups (treated vs. control).The EO of P. aduncum and its major compound did not cause changes at the cuticle level, although damage may have occurred in the internal tissues and organs, as there was a reduction in the activity of antioxidant enzymes, as discussed above.Therefore, future studies focusing on the internal tissues of the tick are necessary for a better understanding and verification of these compounds in the action on tick tissues.component may inhibit intracellular signaling pathways that regulate the production of detoxifying enzymes, for example, modulating intracellular signaling cascades; 2-P.aduncum EO and Dillapiole may influence the expression of genes that encode detoxifying enzymes; 3-P.aduncum EO and Dillapiole may directly bind to the active site of the enzymes under study, thereby blocking their enzymatic activity.In this way, future studies are necessary to test these hypotheses and to bring clarity to the ideas regarding the relationship between new compounds and specific enzyme families (Figure 5).At the enzymatic level, after data analysis, the EO of P. aduncum and Dilapiolle have the potential to alter the enzymatic activity of important detoxifying enzymes and of neurotransmission in A. sculptum larvae.At the morphological level, the investigation sought to determine whether the EO of P. aduncum and its major compound could affect the cuticle of A. sculptum, using scanning electron microscopy.In the current literature, it is

Plant Material, Identification, and Extraction of Essential Oils
The leaves of Piper aduncum were collected in the period of January to June 2020 in the Institute of Chemistry-USP (46 • 43 ′ 32 ′′ W; 23 • 33 ′ 54 ′′ S).A specimen of the plant was deposited at the Herbário do Jardim Botânico do Rio de Janeiro under registration (K-0057), and the identification was made by Dr. Elsie Franklin Magalhães.The specimen collection was made under protocol number #59161-1 from SISBIO (Sistema de Autorização e Informação em Biodiversidade).
The EO was extracted from fresh leaves and submitted to hydrodistillation in a Clevenger-type apparatus for 4 h, using 300-500 g of fresh leaves and 500 mL of distilled water [47,48].The EO was collected and dried with anhydrous sodium sulfate and stored in amber bottles in a refrigerator at 4 • C until the experiments and analysis by GC-MS were performed according to the method described elsewhere [46].
The main compound from P. aduncum EO characterized by GC-MS and used in this study was Dillapiole.Pure Dillapiole was obtained by fractionation using an Isolera Flash Chromatography system (Biotage INC) according to the method described in [46].

Tick Collection
Engorged A. sculptum females were obtained from naturally infested horses at the Veterinary School of UFMG farm, located in the municipality of Pedro Leopoldo, MG, Brazil.They were maintained in a biochemical oxygen demand (BOD) incubator under a controlled temperature of 28 ± 2 • C and 90 ± 5% humidity until oviposition.Eggs laid in the lab were collected and placed in 15 mL conical-bottom tubes closed with cotton to allow air and moisture flow.Subsequently, they were incubated under the same temperature and humidity conditions described above.This population has been maintained in the laboratory for over 10 years until the present day and has been characterized as susceptible to synthetic acaricides of the pyrethroid class according to the FAO guidelines [49].The colony is maintained at the Laboratory of Hematophagous Arthropods-LAH, following the recommendations of the Ethics Committee (CEUA-UFMG) at the Federal University of Minas Gerais, Brazil, under protocol number 60/2020.

Larval Immersion Test
The larval immersion test (LIT) was performed following the method described by Sabatini et al., 2001 [50].Piper aduncum EO and Dillapiole were tested at concentrations of 0.6; 0.84; 1.2; 1.7; 2.4; 3.5; 5; and 6 mg/mL on A. sculptum.All dilutions were prepared in a solution containing 1% acetone and 0.02% Triton X-100.The LIT was performed in duplicate (two technical replicates) at each concentration, including for the control group, and was repeated five times on different days (five biological replicates per treatment group).The control group was treated with a solvent solution of 1% acetone and 0.02% Triton X-100.Approximately 100 larvae were immersed in each concentration for 10 min.The tubes were immediately sealed, shaken for 5 s, and allowed to rest for 10 min.Afterwards, the larvae were placed on filter paper to dry and then transferred to a dry filter paper package (8.5 cm × 7.5 cm) and sealed with plastic clips.The packages were placed in a BOD incubator at a temperature of 27 ± 1 • C and relative humidity (RH) ≥ 80% for 24 h.After incubation, dead and live larvae were counted using a vacuum suction pump.Tick larvae that showed no movement after CO 2 stimulation were considered dead.2) for larval treatment.The tubes were immediately closed, shaken for 5 s, and then left to rest for 10 min.After this period, the larvae were dried on filter paper, transferred to filter paper packages (10 cm × 6 cm), and closed with clips.The packages were placed in a B.O.D. incubator at 27 • C and RH ≥ 80% for 24 h, and larvae that survived the treatments were collected for further processing.
Surviving larvae were immediately macerated in PBS buffer (NaCl 136.8 mM; KCl 2.7 mM; Na 2 HPO 4 4.76 mM; KH 2 PO 4 1.76 mM pH 7.4) with 0.1% Triton X-100 and centrifuged (12,000× g, 5 min at 4 • C).The supernatant was removed and transferred to another microcentrifuge tube, where a mixture containing protease inhibitors at final concentrations of 1 mM Pepstatin A, 2 mM PMSF, 1.5 mM EDTA, and 10 µM E64 was added.The tubes were then kept at −80 • C until the measurement of enzymatic activity.The total protein content of all extracts was quantified using the Bradford method [51].
A negative control was established using a diluent (1% acetone and 0.02% Triton X-100) to prepare samples from larvae not exposed to EO or Dillapiole.Blanks were prepared by replacing the 10 µL of sample with 10 µL of PBS with 0.1% Triton X-100.All test sample absorbances were subtracted from the average of the blank absorbance for enzyme activity assays.All enzyme activity assays below were performed in duplicate (two technical replicates) at each concentration, including for the control group, and were repeated five times (five biological replicates per treatment group) on different days.

Glutathione-S-Transferase Activity Assay
The glutathione S-transferase (GST) activity of the larval protein extracts was determined according to the method described by Habig, 1974 [52].The assay intended for the measurement of total GST activity is based upon the GST-catalyzed reaction between the substrates glutathione (GSH) and CDNB (1-chloro-2,4-dinitrobenzene).
Ten µL of protein extract from treated larvae was transferred to a plate (BIOFLOAT™ 96-Well Plate).Then, 190 µL of the mixture (100 µL of 100 mM potassium phosphate buffer, pH 6.5, 78 µL of distilled water, 2 µL of CDNB 50mM in methanol, and 10 µL GSH 50 mM) was added to the wells and the plate was kinetically read at 340 nm for 60 min at 30 s intervals at 30 • C using a Versamax ELISA plate reader (Molecular Devices, Versamax, Battle Creek, MI, USA).The initial rate of absorbance increase was registered and the product concentration was calculated using the molar extinction coefficient of 9.6 mM/cm for S-(2,4-dinitrophenyl glutathione).One unit (U) of enzymatic activity is defined as the amount of enzyme that produces 1 µmol of product per minute.The activity was expressed as µU/µg protein./4.4.3.Esterase Activity Assay Esterase activity was evaluated with two different substrates: α-naphthyl acetate (for α-Esterase) and β-naphthyl acetate (for β-Esterase), according to the method described by van Asperen (1962) [53].Ten µL of protein extract from treated larvae was transferred to a plate (Biofloat™ 96-Well Plate).Then, 190 µL mixture (100 µL of 40 mM sodium phosphate buffer, pH 7.2, 80 µL of distilled water, and 10 µL of 30 mM α -naphthyl or β -naphthyl acetate substrate in acetone, depending on the enzyme being studied, α or β-Esterase) was added.
The plate was incubated for 30 min at 30 • C.After incubation, 50 µL of staining reagent (3.4% SDS and 0.3% Fast Blue) was added to each well of the plate and the plate was incubated for 5 min at 30 • C. The absorbance of the samples was measured at 570 nm using a Versamax ELISA plate reader (Molecular Devices, Versamax, Biloxi, MS, USA).The activity was expressed as µU/µg protein.

Determination of Acetylcholinesterase Activity
Acetylcholinesterase activity (AChE) of the larval protein extracts was determined according to Li et al. (2005) [54], with some modifications.The reaction mixture consisted of 10 µL of 20 mM acetylthiocholine iodide, 100 µL of 100 mM sodium phosphate buffer, pH 7.8, and 80 µL of distilled water.A total of 190 µL of the mixture was transferred to a plate (BIOFLOAT™ 96-Well Plate), and 10 µL of protein extract from treated larvae was added to each well and incubated for 60 min at 30 • C.After incubation, 50 µL of staining reagent containing 2% SDS, 6 mM 5,5 ′ -Dithiobis (2-nitrobenzoic acid) (DTNB) and 10 mM sodium phosphate buffer, pH 7,8, was added to each well and incubated for 5 min at 30 • C. The absorbance of the samples was measured at 410 nm using a Versamax ELISA plate reader (Molecular Devices, Versamax, Biloxi, MS, USA).The activity was expressed as Abs/min/ng protein.

Scanning Electron Microscopy (SEM)
Larvae of A. sculptum were sorted into the following treatment groups for a period of 24 h: Vehicle Control Group (1% acetone and 0.02% Triton X-100); LC 50 -P.aduncum Group; LC 75 -P.aduncum Group; LC 50-Dillapiole Group; LC 75 -Dillapiole Group.Ten larvae from each group were subsequently fixed in a solution of 2% paraformaldehyde + 2% glutaraldehyde for 21 days.After fifteen days, the fixative was changed and left for an additional 5 days to complete the 21 days.Subsequently, the fixative solution was completely removed, followed by four washes in PBS buffer (15 min each), and placed in 70% alcohol for 24 h.The samples were dehydrated in a graded series of alcohol (80, 90, 95, and 100%) for 30 min each.In the final bath, the samples were transferred to adapted Eppendorf 1.5 mL tubes and placed inside a 15 mL Falcon tube with 100% alcohol, which was then sent to the Multi-User High-Resolution Microscopy Laboratory at the Federal University of Goias (LABMIC/UFG) for drying and image capture.
After dehydration, hexamethyldisilazane (HMDS) was applied for 5 min.Subsequently, excess HMDS were removed, and the Eppendorf tube was left open at room temperature until complete removal of the reagent.After drying, the samples were mounted on cylindrical "Stub" sample holders on double-sided copper conductive tape.Subsequently, the samples were coated with a conductive material, gold, using the Desk V Gold Film Deposition System, Denton Vacuum LLC, Moorestown, NJ, USA.The samples were then analyzed using a Scanning Electron Microscope (SEM) (JSM-6610, Jeol, Tokyo, Japan) equipped with EDS (Thermo Scientific NSS Spectral Imaging) at an acceleration voltage of 8 kV.

Statistical Analysis
The data were organized in spreadsheets using Microsoft Excel (Office 2007) software.To calculate the LCs, the data were initially transformed to log(X), normalized, and then the nonlinear regression was calculated to obtain LC 50 (50% lethal concentration) using the GraphPad Prism 7.0 software (GraphPad Inc., San Diego, CA, USA).The LC 5 , LC 25 , and LC 75 were estimated by using ECanything from LC 50 (https://www.graphpad.com/quickcalcs/Ecanything1.cfm) access date 12 April 2023, Quick Calcs-GraphPad; EC (effective concentration); and entering the LC 50 HillSlope of each compound (EO P aduncum or Dillapiole).Data distribution and normality were tested using the Kolmogorov-Smirnov test (p > 0.05).Comparisons between groups were carried out using a one-way ANOVA followed by Tukey's Multiple Comparison post hoc test (p < 0.05).

Conclusions
The EO from P. aduncum and its major phenylpropanoid Dillapiole showed acaricidal activity in larvae of the tick A. sculptum.The potential increase in enzymatic activity (α-EST, β-EST, and GST) may serve as a response to minimize the damage caused by P. aduncum EO and Dillapiole.Conversely, examination of AChE after treatment with both P. aduncum EO and Dillapiole revealed a reduction in the activity of this enzyme at higher concentrations, suggesting the potential of these two products as substitute for synthetic pesticides such as organophosphates and carbamates.However, the P. aduncum EO and Dillapiole caused no damage to the tick cuticle.Additionally, P. aduncum EO and Dillapiole had a simultaneous effect on different detoxifying enzymes and on neurotransmission, which suggests that they trigger a complex response in A. sculptum and could potentially delay the development of resistance against these products.Further studies are needed to help clarify the exact mechanisms by which these compounds function and to strengthen the evidence supporting their potential to control ticks.

Figure 1 .
Figure 1.The GC-MS chromatograms of the essential oil (EO) of Piper aduncum show that Compound 33 corresponds to the major compound Dillapiole.The remaining compounds are identified in Table1.

Figure 2 .
Figure 2. Effect of the essential oil (EO) of Piper aduncum (A-C) and of Dillapiole (D-F) on the enzymatic activity of α-esterase (α-EST), β-esterase (β-EST), and glutathione-S-transferase (GST) of Amblyomma sculptum larvae.Means with the different letters are significantly different from each other (p < 0.05) in an ANOVA followed by Tukey post hoc test.Bars represent the mean ± standard deviation of 5 biological replicates.

Figure 2 .
Figure 2. Effect of the essential oil (EO) of Piper aduncum (A-C) and of Dillapiole (D-F) on the enzymatic activity of α-esterase (α-EST), β-esterase (β-EST), and glutathione-S-transferase (GST) of Amblyomma sculptum larvae.Means with the different letters are significantly different from each other (p < 0.05) in an ANOVA followed by Tukey post hoc test.Bars represent the mean ± standard deviation of 5 biological replicates.

Figure 3 .
Figure 3.Effect of the essential oil (EO) of Piper aduncum (A) and of Dillapiole (B) on acetylcholinesterase activity of Amblyomma sculptum larvae.Means with the different letters are significantly different from each other (p < 0.05) in an ANOVA followed by Tukey's post hoc test.Bars represent the mean ± standard deviation of 5 biological replicates.

Figure 3 .
Figure 3.Effect of the essential oil (EO) of Piper aduncum (A) and of Dillapiole (B) on acetylcholinesterase activity of Amblyomma sculptum larvae.Means with the different letters are significantly different from each other (p < 0.05) in an ANOVA followed by Tukey's post hoc test.Bars represent the mean ± standard deviation of 5 biological replicates.Int.J. Mol.Sci.2024, 25, x FOR PEER REVIEW 7 of 16

Figure 5 .
Figure 5. Overview of three hypotheses that can explain the potential mechanisms by which P. aduncum EO and Dillapiole disrupt certain enzymes in the present study.The chemical structure of dillapiole was obtained from the ChemSpider website.Some figure templates used in this overview were obtained from the BioRender software©2024.

Figure 5 .
Figure 5. Overview of three hypotheses that can explain the potential mechanisms by which P. aduncum EO and Dillapiole disrupt certain enzymes in the present study.The chemical structure of dillapiole was obtained from the ChemSpider website.Some figure templates used in this overview were obtained from the BioRender software©2024.

4. 4 .
Determination of Enzymatic Activity in Larvae 4.4.1.Treatment and Processing of Larvae Larvae of A. sculptum were treated with LC 5 , LC 25 , LC 50 , and LC 75 of EO P. aduncum or Dillapiole.Approximately 1000 larvae were added to microcentrifuge tubes containing the lethal concentrations of EO P. aduncum or Dillapiole (shown in Table

Table 1 .
Chemical composition of the essential oil of Piper aduncum.
a -retention index calculated against C8-C40 n-alkanes using an HP-5ms column.RI b -retention index values from the literature (Adams 2007).S Compound identity confirmed with an authentic standard.The remaining compounds were identified by comparing the RI and mass spectra with the Adams and Wiley databases (see text for details).* Unidentified compound.Int.J. Mol.Sci.2024, 25, x FOR PEER REVIEW 4 of 16