Chemical acaricides to control R. microplus have become limited in cattle farming over time, and due to the high selective pressure, resistant strains have been seen in several dairy farms (Higa et al. 2015; Klafke et al. 2017). Therefore, a more sustainable, less harmful, and more effective alternative approach is needed to control R. microplus. One of the most promising strategies is the identification and characterization of plants from which to obtain extracts with acaricidal properties, especially when dealing with resistant tick strains (Borges et al. 2011; Ghosh et al. 2015). Along with the variety of actions of herbal medicines on parasites when compared to synthetic products, herbal medicines are less toxic to mammals and the environment and are less likely to induce the selection of resistant strains (Wanzala 2017). This information suggests the need for the discovery of new botanical specimens that are potentially useful for the control of R. microplus.
In the present study, we showed that tick populations resistant to cypermethrin and trichlorfon, two common active ingredients used in acaricides against R. microplus, are a common finding in dairy farms of southeast Brazil. In addition, the effect of extracts or the latex of eight plants commonly found in this region were tested against a cypermethrin-resistant population, and four of them showed acaricide efficiency with a potential to be used as alternative controls.
As expected, the sensitivity and resistance level of the Porto Alegre and Jaguar reference strains were classified as sensitive and resistance level IV, respectively. The other seven tick populations from MG dairy farms were classified as resistance levels I to IV (Klafke et al. 2017), confirming previous work that acaricide-resistant populations are a common finding in MG (Domingues et al. 2012; Faza et al. 2013) and other states of Brazil (Higa et al. 2015, 2016). Resistance levels from III on indicate that there is a low effect of the acaricide, and that it should not be used against the tick population (Higa et al. 2015). Therefore, cypermethrin and trichlorfon are no longer indicated for use for tick control on 71.4 and 42.9%, respectively, of the farms in the present work.
The Boa Vitoria field population was selected for further experiments using plant products, as it was the one with the highest level of acaricide resistance. It is of note that Boa Vitoria ticks were collected from Marchigiana (Bos taurus taurus) cattle on a farm that used endectocide drug treatments for many years without technical supervision, which is a drug that is based on doramectin salt and other formulations containing pyrethroids and organophosphates. At that farm, doramectin-resistant H. contortus were also identified, and along with other ectoparasites, incriminated as causing the death of adult animals (non-published data). The parasitic control used at the farm suggests the presence of multi-resistant ticks.
Extracts derived from the species C. brasiliense, S. brasiliensis, X. americana, and C. procera and the latex of C. procera showed the highest efficiency against R. microplus larvae, with mortality rates varying between 25 and 62% at 100 mg/mL. The native species with the greatest effectiveness was C. brasiliense, while C. procera was the best among the exotic ones tested. They showed greater acaricidal potential against the strains that were resistant to pyrethroids and organophosphates from the Boa Vitoria farm. The extracts of C. procera, S. brasliensis, and X. americana plants reduced the ELC and H rate of R. microplus females. It is of note that the acaricidal efficiency of C. procera EA at a concentration of 25 mg/mL showed an efficacy of 96.8%, which was significantly higher than the cypermethrin efficiency.
Calotropis procera is an exotic plant with several known medicinal proprieties and activities against arthropods (Wadhwani et al. 2021). Several studies were carried out with ticks. In a previous study, the EE of C. procera induced a 63.2% mortality of R. microplus larvae at 100 mg/mL (Shyma et al. 2014), which is a percentage similar to the one found here. On the other hand, Khan et al. (2019) used 21.15 mg/mL and described a lethal effect of 90%, which is a considerably higher acaricidal activity in relation to the present work. Notably, these authors used whole parts of the C. procera plant, which may have likely increased the acaricidal activity against larvae. In addition, ticks used in the previous studies were not characterized as resistant to acaricides.
It is interesting to note that the lower concentration of the C. procera AE (25 mg/mL) was the one with highest efficiency (Table 6). This finding is in line with previous studies that observed that the highest concentration is not necessarily the one that induced the highest activity against fully engorged females. Lazaro et al. (2012) carried out AITs to evaluate the effect of C. procera EA on the reproductive parameters of R. microplus and observed an acaricidal efficiency of 98.3, 97, and 96% at the concentrations of 5, 25, and 100%, respectively. The effects of C. procera against R. microplus may reflect the presence of different active compounds in different combinations. The exact components present in the extracts and the latex used in the present work were not assessed, but previous work has shown the presence of cardenolides, flavonoids, sterols, oxypregnanes, triterpenoids, glycosides, among others, with a prevalence of flavonoids and glycosids in the leaves and steroids and cardenolides in the latex (Wadhwani et al. 2021). Indeed, Bhaskar et al. (2021) found that the AE of C. procera contained 3-Isopropoxy-1,1,1,7,7,7-hex as a major compound, and the AE showed a tick-killing effect against adult R. microplus of up to 93.33% at concentrations up to 360 mg/mL.
Extracts from the plants S. brasiliensis, X. americana, and C. brasiliense also had interesting acaricidal activities on R. microplus larvae at 100 mg/mL, which has not been described in the literature so far. In another study, extracts of X. americana and S. brasiliensis significantly reduced the ELC and H of the tick D. nitens, showing an acaricide efficiency above 75% at 100 mg/mL (Vasconcelos et al. 2018). In addition to the acaricidal activity, the extracts have also been demonstrated to have antimicrobial, insecticide, and anthelminthic activity (Maikai et al. 2009; Santos et al. 2014; Morais-Costa et al. 2015; Morais et al. 2020; Linhares et al. 2022). The composition of the EE of S. brasiliensis and X. americana used in the present work was characterized in a previous work (Vasconcelos et al. 2018). The extracts contained a predominance of polyphenols with a major presence of tannins in S. brasiliensis and tannins and flavonoids in X. americana. Leaf EE of C. brasiliense was also previously characterized, with flavonoids as the major compound (Morais et al. 2020), but the plants were collected in a different site and the real composition of the EE used here remains to be characterized. Nonetheless, tannins and flavonoids have been reported to have acaricidal activities (Fernández-Salas et al. 2011; Madzimure et al. 2011) and are believed to be the main component inducing the mortality of larvae.
Although M. indica had low acaricidal activity in the present work, previous studies have shown that its leaf AE can induce up to a 54% mortality on R. microplus larvae and up to a 100% mortality on other tick species and mosquito larvae (Rajakumar et al. 2015). A similar effect occurred for P. viridiflora, whose EE was previously shown to contain tannins and showed an efficiency of up to 98.7% against D. nitens females (Vasconcelos et al. 2018) and an anthelminthic action against H. contortus larvae (Morais-Costa et al. 2015); however, it is important to note that variations of the activity of similar plant species is a common finding. Such differences may be due to the location, collection method, extraction, and processing of the plants, which may influence the concentration of a given bioactive compound (Gobbo-Neto and Lopes 2007).
The differences in the plant acaricide efficiency between the present work and previous literature may also be due to the resistant tick strains used in the experiments. Acaricide-resistant ticks may have genetic and physiological differences in cuticle production and detoxification enzymes, among others (De Rouck et al. 2023), that could influence the cuticle penetration and detoxification of the products. A previous work has reported that acaricide-resistant R. microplus have a lower reproductive performance (Davey et al. 2006) and a higher susceptibility to carvacrol (Costa-Júnior et al. 2016), which is a monoterpene commonly found in the essential oils of plants with acaricide effects against ticks (Quadros et al. 2020).
The findings of the present work, in addition to those available in previous literature, encourages future investigations of the effect of plants from the Brazilian savanna against ticks, and also suggest that further evaluation on the effect of C. procera is needed. The identification of the bioactive molecules responsible for the tick-killing effects of C. procera could be useful for the generation of a formulation that optimizes the effects of these molecules. Furthermore, it is essential to determine whether the properties of C. procera are restricted to ticks and if they are potentially harmful to other animals, including humans.