Different plant essential oils have been studied due to their properties as efficient alternatives for tick control. The toxicity of these compounds has been demonstrated by immersion test (Drummond test) or contact tests with impregnated surfaces, as well as vapor exposure to essential oils. Some experiments have determined through microscopy techniques the effect of synthetic substances and plant extracts on different R. sanguineus tissues, important alterations on the morphology of the tissues exposed to the assessed substances have been found. ( Roma et al., 2010; Araujo., 2012; Roma et al., 2012; Roma et al., 2013a; Roma et al., 2013b; Remedio et al., 2015; Pereira et al., 2017; Matos et al., 2018). TMEO has shown acaricidal activity against different tick species, being a potential opportunity for the development of new acaricides, (García et al., 2012; Da Silva et al., 2016; França et al., 2017).
The results found in the CG / MS analysis showed some variations in the TMEO samples analyzed; a total of 21 compounds were identified, of which Eucaliptol, (Z)-β-ocimene, o-cimene, (E)- tagetona, (Z)-tagetona, (Z)-cariofileno, Verbenona, Espatulenol were present in the TMEO samples extracted from leaves and flowers PD3, PD6, PD7, PD8 and PD2 respectively. Regardless of the type of plant material, place, and date of collection, the major compounds found were always (Z)-β-ocimeno, ocimeno, Z)-tagetona and Verbenona. Andreotti et al., (2013) found as TMEO major compounds dyhidrotagetone (54.10%), Limonene (6.68%), tagetona (6.73%) and (e)-β-ocimene (5.11%) which presented significant acaricide activity against larvae, nymphs and adults of R. sanguineus. García et al., (2012) described the presence of limonene (6.96%), β-Ocimene (5.11%), dihydrotagetone (54.21%), and Tagetona (6.73%) as major compounds, the same authors found that the TMEO is highly effective against several tick species including R. sanguineus. Kaul et al. (2005) assessed the TMEO composition finding a total of 24 compounds which represent 93.7% of the analyzed EO, the major compounds were limonene y beta-felandreno (4.7%), (Z)-β-ocimeno (36.8%), dihidrotagetona, (E)-β-ocimeno (15.5%), (Z)-tagetona (17.1%), (Z)-tagetenona (3.0%) and (E)-tagetenona (7.5%) the authors identified compounds not reported here in both TMEO flowers and leaves. The chemical composition variability of the same species EO could be the result of different chemotypes related to biological variations linked to factors like soil, temperature, climate conditions, illuminance, etc (Gakuubi et al., 2016). Therefore, there is a clear variation of the chemical profile on the TMEO when compared with the results found in the present study. Different factors may have influenced these findings, some variations in the EO composition can be due to the vegetable material used in the extraction either leaves or flowers, also the local and collection date could influence the composition of the different samples, Morais (2009) suggests the effect of abiotic factors like temperature, light intensity, seasonal effects among others in the EO composition. There was a mild difference in the concentration of the major and minor compounds related to the geographical location, that sort of variation was described by Salehi et al., (2018), meanwhile Gobbo-Neto & Lopes (2007) suggest that factors like time of collection, seasonality and development phase, temperature and ultraviolet radiation could affect the EO composition.
The compounds found in TMEO are classified as terpenes, whose structure is formed of isoprene units, which are hydrocarbons of 5 carbons. Monoterpenoides are those compounds formed by 2 isoprene units and a molecular structure of 10 carbon atoms (Perveen, 2018), as in compounds like (Z)-β-ocimene, (e)-tagetone, β-pinene, eucalyptol, (E)-β-ocimene, o-cimene, (Z)-tagetone, eugenol, (L)-α-terpineol, that correspond to most of the found compounds. Terpenes cover a wide variety of substances of vegetable origin and their ecological importance as defensives is well studied (Viegas, 2003).
The component β-cariophylene present in Piper aduncun EO and also present in TMEO when evaluated individually against Tetranycgys urticae presented important acaricidal activity (Araujo et al., 2012), could have influenced the tissue alterations in the present study. Ayoub et al., (2018) evaluated the gender Thymus species EO which caused high mortality on Sitophilus oryzae those EO present some constituents also found in TMEO such as β-pinene, o-cymene, eucalyptol, α-humulene, e-β-ocezen, Germacreno D, cariophilene oxide. Artemisia campestris EO which chemical profile is similar to TMEO include β-pinene, α-pinene, mircene, germacreno D, (Z)-β-ocemine showed toxicity against Culex quinquasciatus and Musca domestica. Similarly, Arabic pulicaria and Saccocalyx satureioides EO which contains compounds like epi-α-Cadinol, Δ-Cadinene, α-Cadinol and Germacrene D-4-OL and Timol, α-terpineol, Borneol and P-Cimen respectively, some of them also present in TMEO were active against the Spodoptera littoralis agricultural moth (Ammar et al., 2020). Huang et al., (2002) mentioned the contact toxic effect of Eugenol, isoeugenol, and methyleugenol against Sitophilus zeamais. Furthermore, a recent study found that eugenol in association with thymol and carvacrol had a synergistic acaricidal effect in Amblyomma sculptum, therefore suggesting that different compounds show a synergistic effect when acting simultaneously (Vale et al., 2021). The proven toxicity of Eugenol in insects and tick may have contributed to the toxic effect reported in this study.
Murraya microphylla EO which chemical profil have similarities to the found in TMEO, showed insecticide activity and repellent effect against tobacco beetle Lasioderma serricorne, that suggest a possible synergistic effect of the components of the TMEO (You et al., 2015). The insecticidal effect of (Z)-β-ocimene has also been analyzed individually agaisnt Anopheles spp, Aedes spp, Culex spp (Govindarajan & Benelli., 2016). Hüe et al., (2014) assessed the (Z)-β-ocimene acaricidal effect at 2% concentration on R. microplus without remarkable effects. Though the EO compounds toxicity have been recognized, their mechanism of action has not been totally understood, the evidence indicates that most of the compounds differs in their mode of action (Gross et al., 2017; Viega, 2003; Quadros et al., 2020). Monoterpenoids compounds mode of action may not be represented by a single mechanism (Grodnitzky & Coats, 2002). Cardoso et al., (2020) noted that the inhibition of ACHE may be the main mechanism associated with the effect of some terpenoids on R. microplus. Meanwhile, Ali et al., (2014) reported the cytotoxic effect of TMEO on MCF-7 line mammary tumor cells, showing moderate cytotoxic activity, the authors raise a possible synergistic effect between the different compounds. According to Gross et al., (2014), the mixture of the different components present in an EO could competitively be coupled to octopamine and tyramine receptors associated with G protein. Another proposed mode of action is the inhibition of the monoxidase enzyme (MAO) causing a neurotoxic effect associated with the increase of cyclic monophosphate adenosin (cAMP); others could be the possible TMEO mechanism of action, as in the case of Timol and Carvacrol which interact by inhibiting ACHE in R. (B.) microplus (Cardoso et al., 2020). The hydrophobic nature of EO could simultaneously exert a mechanical effect on the parasite well obstructing the area of spiracles entailed to death by water stress or suffocation as suggested by Burgess (2009) could be considered another mechanism of action involved.
The histological findings suggest a toxic causal effect over the tissues exposed to the different TMEO concentrations. R. sanguineus ticks when exposed to Tween 20 at 2% showed no considerable alterations in the synganglion morphology. These findings correspond to the R. sanguineus synganglion normal structure described by Roma et al. (2012) as well as by other authors who confirmed similar findings (Pereira et al., 2017, Matos et al., 2018). However, the ticks exposed to amitraz at 12.5% (50% sublethal doses) and the TMEO different concentrations, the synganglion morphology showed gradual alterations, mainly in the cortex and neuropile, these alterations were related to the EO concentration. The ticks exposed to amitraz presented alterations almost all over the synganglion. Although the neurilemma was intact, the perineurium presented vacuoles and cells with cytoplasm alterations, as well as significant signs of nuclear alteration like condensation, marginalization, and fragmentation of the chromatin were present, such alterations at the nuclear level are suggestive of irreversible cell injury (Damjanov, 2009). The cortex cells showed some morphological alterations like swelling, shape loss, and vacuolization, there were also vacuolization at the extracellular matrix; similar alterations were reported in R. sanguineus synganglion exposed to permethrin (Roma et al., 2013). It is considered that vacuolization related to autophagy is a cell mechanism to eliminate possible toxic substances or damaged structures, if the injuring effect is higher than the mechanisms of detoxification and repair, then the toxic substances will lead to irreversible lesions and the consequent cell death (Roma et al., 2013a; Elmore et al., 2016; Aki et al., 2012). Occasionally, the vacuolation process in response to injury is reversible, while in other circumstances, continuous exposure to levels of a certain substance, entails cell death, in a likely scenario the vacuole will end tearing up, causing the rupture of the cell membrane (Henics & Wheatley, 1999).
There are similar studies where ticks were exposed to amitraz, showing cell injuries similar than the observed in the present study. Kanapadinchareveethyl et al., (2019) suggest that Amitraz could cause vacuolization in the R. annulatus oocytes phase II cytoplasm; Likewise, Mangia et al., 2018 found that cell cultures of Ixodes ricinus exposed to different doses of Amitraz, Permethrin, and Fipronil, showed signs of cellular injury like increase in extracellular waste and loss of cell shape, which are evidence of death in addition to a reduction in the total count of cells; the authors suggested that toxic effects in vitro are due to oxidative stress which is a trigger of the apoptosis process. Some signs of structural disorganization in the neuropile are evident, presumably due to the amitraz effect in the cortex cells whose nerve fibers are projected into the neuropile; these alterations are more evident in the region underlying the subperineurium that shows widespread vacuolization. Roma et al., (2013) described a similar pattern in the neuropile in ticks exposed to permethrin. Although the amitraz mechanism of action is not fully understood, it is known that the effects of this substance in insects at a molecular level are related to an increase in the tyramine and octopamine levels; the amitraz is generally classified as an agonist receptor of octopamine and enzyme Oxidase inhibitor (Evans & Gee, 1980, Davenport et al., 1985 & Kita et al., 2016). Formamidines, such as the Clordimeform (CDM) and Amitraz, are considered prodrugs that are metabolically converted into agonists of octopamine receptors (OAR), DimethylClordimeform (DCDM) and N2- (2,4-dimethylphenyl) -n1-methyformamidine (DPMF) respectively; It has been shown that DPMF increases the intracellular levels of CA2 + in the Silkworm Bombyx mori although both amitraz and DPMF potently act in the octopamine receptors BMOAR1 and BMAAR2 of R (B.) microplus, increasing CA2 + levels and cAMP respectively (Kita et al., 2016).
The histological alterations observed in synganglion of R. sanguineus exposed to Amitraz are compatible with the type of cellular injury associated with acute cell death (Elmore et al., 2016). The agonistic effect of amitraz on octopamine receptors increases the calcium levels (Ca2+) and cAMP Cytosolic, in turn the receptors coupled to G protein are activated, like the case of the octopaminergic receptor (Roeder, 1999), Besides that, this will lead to the production of inositol triphosphate (IP3), which releases CA2 + from the endoplasmic reticulum (Blenau et al., 2012). The increase in CA2 + affects the cell through the predominant role of this element in the cell signaling, the cell injuries can also be a product of the direct effect of the CA2 + which induces the activation of hydrolases, the alterations at the cellular level induced by CA2 + are generally acute, rapid and in some circumstances irreversible; in the CA2 + case, this mechanism of cell injury would likely lead to cell death (Orrenius et al., 2011, Dong et al., 2006). Secondly, it could be possible that the continued increase of cAMP entails depletion of the ATP cytosolic reserves, which could trigger the cell death process. It has been evidenced that calcium ionophores induce apoptosis under ATP supply conditions, but in conditions of exhaustion of ATP, this can trigger cell death by necrosis (Eguchi et al., 1997). Thus amitraz could act at cellular level through these two mechanisms, more studies are required to elucidate the action of amitraz in cell components of R. sanguineus.
The histological slides of the ticks exposed to TMEO concentrations at 1.25% and 2.5% showed similar patterns of alterations compared with the control and the amitraz treatment, as well as in other studies where the synganglion of tick exposed to different substances was analyzed. The neurilemma did not show evident alterations but at the perineurium were vacuolization signs not being possible to identify any cellular structure, similar finds were described by Roma et al., (2013) in ticks treated with permethrin. Remedio et al., (2015) described similar tissue injuries in synganglion of tick exposed to Nim Azaridacta indica. Besides the cortex cells showed signs of volume increase and shape loss, condensation, and chromatin marginalization, suggesting a progressive cellular injury, those signs would correspond to a reversible cell lesion (Miller & Zachary, 2017). The persistence of the damaging agent can cause an irreversible injury that would result in cells death; additionally, an irregular distribution of the extracellular matrix is evident in the cortex region with the presence of vacuolization, similar findingss were already reported (Roma et al., 2013; Pereira et al., 2017, Matos et al., 2018). The increase in cytoplasmic and vacuoles volume may suggest that the cell membrane function was altered due to the effects of the TMEO constituents. Some cells of the cortex were in advanced state of degradation, which suggests that the mechanisms of detoxification (vacuolization) cannot reverse the effects of toxic compounds or eliminating them.
Ticks exposed to TMEO at 5% showed alterations similar to those described at the 1.25% and 2.5% concentrations, but was possible to observe more drastic effects. The neurilemma is intact but at the perineurium level there is an evident detachment of this structure next to the cortex, with a space between both structures, even in some areas the perineurium is no longer visible or shows structural alterations, suggesting that TMEO components affect the permeability of the neurilemma which would allow the contact of the TMEO with the interior of the synganglion. The perineurium composed mostly by glial cells showed degradation signs such as vacuolization and nuclear alterations. Once in contact with outer substances the cells would put into function detoxifying mechanisms, if fails, the cell would enter into an irreversible process, the empty areas that were found here probably corresponded to cells degrades. Remedio et al., (2015) described similar alteration in R. sanguineus synganglion exposed to Azadirachta indica EO, due that perineurium cells make first contact with exogenous substances, these cells show effects similar than the observed with the TMEO small concentrations, there is a remarkable alteration of a large number of cortex cells when compared to other treatments, with different degree of cell injury, some with an increase in volume or hydropic degeneration suggesting a reversible cell lesion. Other type of biochemical alterations associated with an eventual depletion of ATP and increase in CA + + intracellular levels may be involved in this case triggering irreversible alterations at the nuclear level such as picnosis, cariorrexis and karyolysis which are irreversible cell damage signs, (Miller & Zachary, 2017) then according to these findings is possible to suggest that TMEO components could affect the cell homeostasis mechanisms.
Similarly, to the ticks exposed to Amitraz a loss of continuity between the cortex and the sub-perineurium was observed at the neuropil, there is a considerable structural disorganization with signs of vacuolization, however, not as intense as observed in the Amitraz group or the reported in synganglion exposed to permethrin by Pereira et al., (2017). The structural alteration observed in neuropil can be linked to the injuries observed at the level of the cortex, like mentioned above the neuropil corresponds to nerve fibers from the cells located in the Cortex region.