Amblyomma sculptum and R. sanguineus s.l. are among the most important vectors of diseases for animals and humans worldwide (de la Fuente et al. 2008), including the lethal BSF caused by R. rickettsii. Although all evolutionary stages of A. sculptum and R. sanguineus s.l. can parasitize man, the nymph stage is considered the most important due to its greater parasitic success, increasing the chances of BSF transmission (Szabó et al. 2013; Labruna et l. 2017; Luz et al. 2019). One of the main ways of prevention is the use of synthetic repellents, which can cause numerous adverse effects on animals, humans and the environment. The search for alternative compounds (e.g., EOs and its terpenes) against these ixodids is mandatory. Furthermore, EOs and terpenes are highly volatile reducing their repellent effect time, and the microencapsulation should be an alternative to decrease the volatility and increase the effect.
The encapsulation yield of carvacrol and thymol in yeast cell walls in the present study was 74.5% and 85.7%, respectively, showing the encapsulation efficiency of these compound. Furthermore, the encapsulation process did not degrade the yeast cell walls, remaining thick and without deformities after containing the terpenes. Similar efficacy was observed in the encapsulation of extract of curcumin (Paramera et al. 2011) and carvacrol (Lima et al. 2017; 2019).
Overall, carvacrol and thymol exhibited repellent effect against A. sculptum and R. sanguineus s.l. nymphs. This suggests these monoterpenes as alternative compounds in prevention and control of these ixodids and its tick borne disease. Carvacrol and thymol are known for their acaricidal activity, especially to Amblyomma, Rhipicephalus e Ixodes (Novelino et al. 2007; Daemon et al. 2009; Oliviera Monteiro et al. 2010; Silva Mendes et al. 2011; Carroll et al. 2017; Tabari et al. 2017; Lima et al. 2019; Vale et al. 2021). Furthermore, carvacrol and thymol are considered promising compounds against other arthropods such as mosquitoes, flies and mites (Lindberg et al. 2000; Park et al. 2012; Tababari et al. 2015).
Although both carvacrol and thymol had repellent activity against A. sculptum nymphs, nonencapsulated carvacrol was most effective. This result was unexpected, since both compounds are highly volatile and encapsulation with S. cerevisiae cell walls tends to increase their repellent effect over time (Bakkali et al. 2008; Shi et al. 2008; Lima et al. 2019). One hypothesis would be methodological, where the time from 1 to 15 minutes was not enough to observe the increase in repellent activity, requiring a longer time for a better release of the compound from the encapsulated and, consequently, having a greater activity. In addition, the species and stage of the tick may have influenced, since studies of the repellent activity of encapsulated monoterpenes (e.g., carvacrol) were carried out on Rhipicephalus larvae for up to 6h (Lima et al. 2019). Therefore, larvae would be more sensitive than nymphs for their morphophysiological characteristics (e.g., size, little chitin, and skin respiration). Thus, further studies on the repellent activity of encapsulated and nonencapsuled thymol and carvacrol against A. sculptum nymphs are needed (e.g., larvae and adults). Interestingly, carvacrol and thymol have high acaricidal activities against A. sculputm immatures, reaching a mortality rate > 90% (Vale et al. 2021), indicating the potential of these compound to control these ixodids.
Rhipicephalus sanguineus s.l. was more sensitive to encapsulated carvacrol when compared to the other compounds (P < 0.05), including the positive control (DEET). These results agree with those reported by Lima et al. (2019) for R. (B.) microplus larvae, in which encapsulated carvacrol was also more effective. Nonencapsulated carvacrol also showed high repellent activity against R. sanguineus s.l., but with repellent effect 22.5, 9.8, 9.3 and 5.5 less than encapsulated carvacrol. Lima et al. (2019) also reported that the repellent activity of encapsulated carvacrol was 3 to 5 times more potent than nonencapsulated carvacrol against R. (B.) microplus, concluding that the encapsulation technique protected and increased the repellent activity of carvacrol. However, thymol (nonencapsulated and encapsulated) also showed a high repellent activity against R. sanguineus s.l., in a greater similarity between DEET and encapsulated thymol. As observed for A. sculputm, thymol encapsulated and nonencapsulated had a varied repellent effect against R. sanguineus s.l.. Therefore, it is possible that thymol encapsulation with S. cerevisiae cell walls does not change its repellent activity over time as seen for encapsulated carvacrol in the present study and by Lima et al. (2019). Thus, the time from 1 to 15 minutes may have been a limiting factor, requiring further investigation. Importantly, thymol is toxic to a variety of ticks and mites of public and animal health importance, including Rhipicephalus spp. (Monteiro et al. 2012; Barimani et al. 2016; Tabari et al. 2017).
The tick R. sanguineus s.l. was more sensitive to the repellent effects of most compounds than A. sculptum (P < 0.05), except for nonencapsulated carvacrol (5, 10 and 15 minutes) and DEET (10 and 15 minutes) (P < 0.05) (Table 1). These results confirm the toxic effect of carvacrol and thymol against ticks of the genus Rhipicephalus (Araújo et al. 2016; Tabari et al. 2017; Lima et al. 2019), and the need of further investigation of these monoterpenes against ticks of the genus Amblyomma. Senra et al. (2013) using a concentration of 2.5 mg/mL of carvacrol and thymol, recorded a lethality of 100% for R. sanguineus s.l. nymphs, and of 45.0–62.7% for A. sculptum nymphs. It is possible that Rhipicephalus spp. tends to suffer more the effects of these monoterpenes than Ambyomma spp., indicating a possible natural resistance from this last group to the monterpenes carvacrol and thymol. In addition, A. sculptum parasitizes a variety of wild and domestic hosts, and during the non-parasitic phase they are found on the forest vegetation and dirty fields (Barros-Battesti et al. 2010; Guglielmone et al. 2014). Already R. sanguineus s.l. parasite the domestic dog, never found in forest areas or fields. Thus, it is possible that the constant contact of A. sculptum with botanical compound at the environments has evolved some type of resistance of this species against the toxicity of different plant compounds.
Due to their acaricide and repellent effects against ticks, low toxicity to humans, animals and the environment, EOs and it’s terpenes have become promising compounds in the control of these ectoparasites, and in the prevention of transmitted diseases (e.g., Lyme Disease, Tick-Borne Encephalitis and BSF). Although with distinct repellent activities, the results in the present study show a significant repellent efficacy of carvacrol and thymol against nymphs of R. sanguineus s.l. and A. sculptum. Also, the effectiveness of the yeast wall in encapsulating these monoterpenes confirming that the yeast wall is a promising structure in encapsulating molecules with high volatility (e.g., carvacrol and thymol), acting as a physical barrier against temperature and humidity variations.