The immersion tests performed on semi-engorged R. sanguineus s. l. females treated with the dichloromethane A. oleracea extract indicate high efficiency at concentrations higher than 10 mg/mL, considering decreased egg number and viability, reduced egg mass and reduced fertility and hatchability, among other biological parameters. The best results, of 95% efficacy, were obtained at 25 mg/mL. The 12.5 and 50 mg/mL extracts also provided excellent results, with control rates of 67% and 85%, respectively. Chungsamarnyart et al. (1991) and Chungsamarnyart and Jansawan (1993), when studying R. microplus larvae, obtained different results for aqueous and ethanolic A. oleracea extracts. The aqueous extract presented no acaricidal action at any concentration (Chungsamarnyart and Jansawan, 1993), while the ethanolic extract presented moderate action at 100 mg/mL (36% mortality after 48h) (Chungsamarnyart et al., 1991). Castro et al. (2014) reported positive results for engorged R. microplus females treated with a hexane A. oleracea extract, albeit at concentrations higher than 100 mg/mL (efficacy of 59%). Finally, Cruz et al. (2016) reported positive results for engorged R. microplus females treated with a methanolic A. oleracea extract at 50 mg/mL (efficacy of 85.2%).
Concerning the cytotoxic tests, the ovaries of individuals belonging to the control groups I and II presented typical morphohistological characteristics, i.e., ovaries containing oocytes in five developmental stages (I-V) (Denardi et al., 2004; Oliveira et al., 2005). Individuals belonging to both groups showed no significant differences compared to each other. However, the immersion tests revealed control rates of 21.55% in control group II. The functional meaning of this data is not clear, considering that this characteristic was not confirmed by histological images, in which oocytes presented a completely normal aspect. No other oocyte alterations were observed, and this characteristic probably did not affect oocyte development in the studied ticks. Thus, it is concluded that the use of 50% ethanol and 1% DMSO did not interfere in the obtained results, only aiding in the extract dilution process.
Regarding the treatment groups, the assessed A. oleracea extract resulted in disorganized and vacuolated areas in oocytes in initial development stages (oocytes I, II and III). The damage was progressive, i.e., the higher the concentration, the heavier the damage, leading to more intense autophagic processes, including vacuolation, where cytoplasm portions become damage and required degradadation (Roma et al., 2011; Denardi et al., 2010). At higher concentrations, oocyte cytoplasm presented extensive vacuolated and disorganized areas occupying more than 50% of the cell, which can lead to death, probably through autophagy, a type of programmed cell death. Similar results were reported by Oliveira et al. (2008, 2009) and Roma et al. (2010, 2011) for R. sanguineus exposed to fipronil and permethrin, respectively.
The plasma membrane of oocytes in initial development stages presented numerous small to large pleats, resulting in slight to significant deformity, respectively. These alterations were also reported by Vendramini et al. (2012) for R. sanguineus treated with andiroba oil and Denardi et al. (2010) for ticks treated with neem aqueous extracts. This may have occurred due to the absence of the chorion, a resistant protective membrane (Denardi et al., 2004; Oliveira et al., 2005) which is fully deposited in oocytes in later development stages, as the plasma membrane alone was not capable to prevent the numerous damages caused by the A. oleracea extracts.
Another relevant alteration was observed in the oocyte nuclei in initial development stages, mainly at 12.5 mg/mL and 25 mg/mL, where pleated membranes and nuclear vacuoles were observed. As discussed previously, these structures indicate degenerative processes. Similar findings were reported by Oliveira et al. (2016) for R. microplus exposed to a hexane A. oleracea extract and Roma et al. (2010, 2011) for R. sanguineus exposed to permethrin.
Furthermore, the nucleoli were also fragmented. Other nucleoli alterations have been reported by other authors. For example, Denardi et al. (2010), Roma et al. (2010, 2011) and Vendramini et al. (2012) demonstrated nucleoli emergence as a ring-shaped compact mass after exposing R. sanguineus to neem, permethrin, andiroba oil and neem oil extracts, respectively. Such nuclear alterations indicate genetic germ cell material damage. This is a serious effect that can impair ectoparasite survival and which may be lethal or even transmitted to the next parasite generation, impairing the following cycle, as the hatched individuals (in case they are successful in hatching) will probably be less resistant or less adapted.
The present study also demonstrated that the investigated extract affected oocytes in initial development stages, reducing the number and size of yolk granules. The difference in staining regarding the yolk granules of treated individuals seem to indicate that their chemical composition itself was altered. These data are justified by extract action on the cell cytoplasm, resulting in damage and affecting the yolk granules, which are essential for embryo nutrition (Denardi et al., 2004; Oliveira et al., 2005). Consequently, the oocytes cannot store sufficient yolk to develop, i.e., would not be capable of reaching further development stages, not complete vitellogenesis or form the embryo, leading to impaired development of new individuals. Similar data have been reported by Oliveira et al. (2008, 2009) for R. sanguineus exposed to fipronil.
The assessed A. oleracea extract resulted in disorganized and vacuolated regions in mature oocytes (oocytes IV and V). Oocytes IV presented extensively disorganized areas, while oocytes V exhibited fewer damage. These findings allow us to infer that the chorion, a protective membrane in mature oocytes formed within stages IV and V (Denardi et al., 2004; Oliveira et al., 2005), was not capable of preventing A. oleracea extract compounds from penetrating oocytes IV, since degenerative processes were observed at this developmental stage. On the other hand, the chorion is fully deposited in stage V and is capable of preventing significant damage, since few and concentrated alterations were observed in the peripheral region of the investigated cells. These data are in contrast to those reported in the literature regarding ticks treated with other natural compounds, such as aqueous neem extracts (Denardi et al., 2010) and andiroba oil (Vendramini et al., 2012), as those compounds caused minimum damage to mature germ cells.
Considering the data obtained for oocytes in initial development stages and the fact that an immersion test was performed, it can be inferred that the extract compounds crossed the integument and the hemolymph until reaching the ovary, where they penetrated the oocytes via the plasma membrane, damaging intracellular components. On the other hand, oocytes IV displayed significant alterations, mainly in near the pedicel, which suggests that the extract compounds established another entrance path when the chorion began its deposition, i.e., via the pedicel. This is a very efficient entrance path, since a significant reduction in the number and size of the yolk granules in the cytoplasm, disorganized areas and extensive vacuolation throughout the cell were observed. According to Oliveira et al. (2009) and Vendramini et al. (2012), extract compounds are able to cross the oocyte wall in ticks treated with fipronil and andiroba oil. However, Roma et al. (2010, 2011) and Denardi et al. (2010) exposed ticks to permethrin and an aqueous neem extract and reported that extract compounds are transferred to the oocytes via pedicel cells.
In the present study, the investigated A. oleracea extract caused damage to the midgut of R. sanguineus s. l. females. The first significant damage was regarding the shape of the generative cells (Stem cells), which became irregular and presented several plasma membrane deformities (ridges and scallops), which was sometimes ruptured, releasing cell content..
Another important alteration was generative cell (stem cells) disorganization and vacuolation. Round-shaped areas (probably lipid drops) were no longer observed due to weak or complete absence of staining. These results are justified by the action of the extract compounds, which damaged the plasma membrane, causing internal damage, including chemical cytoplasm composition alterations. Additionally, heavy vacuolation was observed in the cytoplasmic region in contact with the digestive cell. These structures confirm that these cell components were damaged and undergoing degradation processes, i.e., are evidence of a cell degeneration process (Scudeler et al., 2016). As generative cells are responsible for replacing all the dead cells throughout the digestive processes (Harrison and Foelix, 1999), the observed damage was severe and would certainly impair cell proliferation and differentiation, i.e., the damaged cells would produce faulty cells, or even be incapable of producing cells at all. Therefore, blood nutrient absorption would be prevented, leading to undernourished ectoparasites.
Another relevant alteration was associated with endosome number, size, location and color, digestive vacuoles and digestive residues found in the cytoplasm of digestive cells. Sessile digestive cells and residual sessile digestive cells presented significant decreases in endosome number and size, as well as digestive vacuoles and digestive residues, which turned a greenish color. This damage indicates that A. oleracea extract compounds affect digestive cells (destroying the plasma membrane, for example) resulting in impaired functions, including blood collection and intake (Agyei and Runham ,1995), blood cell lysis in digestive vacuoles (Sonenshine and Roe, 2014; Harrison and Foelix, 1999) and the formation of digestive residues (Sonenshine and Roe, 2014;; Agyei and Runham, 1995; Harrison and Foelix, 1999). Other authors have reported alterations in digestive cells after treatment with natural and/or synthetic chemicals, such as Oliveira et al. (2014), who investigated R. sanguineus treated with fluazuron and Scudeler et al. (2016), who exposed insects to neem oil (Azadirachta indica).
Kathrina and Antonio (2004) reported that the secondary metabolites displaying pesticide effects are capable of inhibiting parasite feeding processes, as well as growth and development, reproduction or behavior. Maciel et al. (2010) and Menezes (2005) described three different modes of action for botanical substances on pests, namely toxic action (neurotoxic), action on organs or target molecules or action by ingestion. In the present study, the immersion and cytotoxic test results suggest that the A. oleracea extract compounds may act via the nervous system (neurotoxic action) or crossing the integument through the hemolymph, reaching internal R. sanguineus s. l. female organs, such as the ovary and midgut, affecting them indirectly and directly, respectively. The extract action via the nervous system was confirmed, as abnormal, repetitive and disordered movements were observed at some extract concentrations in the bioassays. The present study also suggests that A. oleracea extract compounds do not necessarily result in tick death, but instead, cause alterations which impair their biological success. As the extract used herein was obtained according to Barbosa et al. (2016b), through a liquid/liquid extraction method using a separatory funnel, organic solvents and fraction isolation, approximately 84–100% spilanthol contents are obtained, allowing for the inference that spilanthol caused the damages reported herein.