Efficacy of resin and ethanol extract from leaves of Protium spruceanum (Bent) Engl. combined with Cypermethrin to control Rhipicephalus (Boophilus) microplus (Canestrini, 1887)

Rhipicephalus microplus is one of the most important ectoparasites in cattle because it affects animal production and is a vector of bovine babesiosis. Although conventional control of the tick depends mainly on the use of chemical acaricides, the intensive use of these drugs favors the selection of resistant populations. Thus, the objective of this study was to evaluate the in vitro acaricide activity of ethanolic extract (EE) of leaves and crude resin of Protium spruceanum (Benth) in association with a synthetic acaricidal product in two strains of R. microplus using larval package test (LPT) and biocarrapaticidogram test. The qualitative characterization was performed by colorimetric analyses. EE and resin combined with cypermethrin showed efficacy above 90% on R. microplus larvae at concentrations of 50.96 mg ml −1 + 75 μg ml −1 (EE + cypermethrin) and 5.08 mg ml −1 + 15 μg ml −1 (resin + cypermethrin) for both as strains used. High efficacy was not observed in females when the combination was used. However, the values were significantly higher than the efficacy for cypermethrin. The results of phytochemical analysis showed the presence of secondary metabolites of the following classes: phenolic compounds, tannins, alkaloids, terpenes, flavonoids and saponins for EE of P. spruceanum leaves. Only tannins, alkaloids and flavonoids were detected for the crude resin. The association between EE, crude resin and cypermethrin has implications for the control of immature and female stages engendered with R. microplus, which can reduce concentration and cost. 5,08 ml 15 (resina + cipermetrina) para las cepas utilizadas. No se observó una alta eficacia en las hembras hinchadas cuando se utilizó la combinación. Sin embargo, los valores fueron significativamente más altos que la eficacia de la cipermetrina. Los resultados del análisis fitoquímico mostraron la presencia de metabolitos secundarios de las siguientes clases: compuestos fenólicos, taninos, alcaloides, terpenos, flavonoides y saponinas para for the control of immature stages and engorged females of R. microplus . The results of phytochemical analysis showed the presence of phenolic compounds, tannins, alkaloids, terpenes, flavonoids and saponins in the ethanol extract of P. spruceanum leaves and for raw resin only tannins, alkaloids and flavonoids were detected. Finally, it is suggested these association tested in non-target organisms, to have more reliability in their use and advance to in vivo experiments.


Introduction
The tick Rhipicephalus microplus causes economic losses in several continents, and infests cattle in tropical and subtropical countries (Khan et al., 2019). It is not only a vector of microbial pathogens responsible for serious diseases such as babesiosis and anaplasmosis, but also directly promotes weight loss, significant decrease in milk and meat production, and affects the quality of leather (Feder et al., 2019). Control of R. microplus is often accomplished using synthetic acaricides.
However, indiscriminate use of these drugs can select resistant tick populations, cause poisoning of animals and humans, and promote environmental contamination (Lazcano Díaz et al., 2019). Multidrug resistance in ticks has been reported in several continents (Fular et al., 2018;Bisht et al., 2021). Santo et al. (2021) studied nine acaricides of different brands and observed multidrug resistance to cypermethrin and amitraz in tick strains in southern Bahia.
Studies have shown that the combination of compounds of plant origin can enhance the activity of these compounds and can be synergistic, additive, or antagonistic (Berthoud, 2013;Geary, 2013;Lederer et al., 2019). The synergism of these compounds helps improves efficiency, reduces the accumulation of waste in the environment, avoids poisoning of animals, and reduces the concentrations of chemical compounds used, which consequently reduce the cost of acaricide application (Gallardo et al., 2012;Vale et al., 2021). The use of these combinations also reduces the selection of ticks resistant to synthetic products (Greco et al., 1996;Ma & Motsinger-Reif, 2019).
Protium spruceanum (Benth) Engler is locally known as breu branco, almecega, almescla, almecegueira, or almecegavermelha. (Vieira et al., 2010). It is traditionally used as an expectorant and anti-inflammatory agent in topical treatments in folk medicine. (Amparo et al., 2019). In addition, ethanol extract (EE) and ethyl acetate (EA) from leaves of P. spruceanum showed acaricidal activity against females and larvae of R. microplus in vitro (Figuereido et al., 2019). The aim of this study was to demonstrate the efficacy of the combination of EE from leaves and resin of P. spruceanum with cypermethrin.

Plant collection and identification
Plant material was collected from the Água Doce River Basin, Bonito de Minas municipality, Minas Gerais, Brazil The leaves of adult plants were washed under running water and inspected, and those with lesions and damage were sorted out. They were then dehydrated in an oven with forced air circulation (TE 394/4, Equipamentos Técnicos Científicos, Tecnal, Piracicaba, SP, Brazil) at 40 °C for 72 h, ground in an industrial blender to obtain the dry powder, and stored in paper bags under light protection until use. The resin was collected by randomly selecting adult individuals after cutting the bark of the stems using a stainless steel knife. The crude material was stored in an airtight plastic bottle and cooled to approximately 4 °C in an isothermal box.

Extract preparation
Ethanolic extract (EE) was prepared by adding 100 g of dry leaf powder to 1000 mL of absolute ethyl alcohol PA 99.5° GL. The material was then stored in an amber jar at room temperature for 10 days. Subsequently, it was filtered in a glass funnel with gauze and cotton and dehydrated in a forced-air oven at 40 ± 5 °C. The dry extract was then separated and stored in dark bottles. The dry matter of three subsamples containing 2 g each were determined by the AOAC (Association of Official Analytical Chemists) technique for standardization and calculation of the tested concentrations.

Assessed tick populations
The first population evaluated was from a farm in the municipality of São João da Ponte, Minas Gerais. Infested females were collected from crossbred cattle (Gyr × Holstein) that were naturally infested and had no contact with acaricides for at least 60 days. After collection, the ticks were placed in plastic containers and transported to the laboratory, where they were incubated in an oven (BOD) at 28 °C and relative humidity of approximately 80% until use.
The other strain studied, Santa Rita, was kindly provided by Prof. Lívio Martins Costa Júnior of the Federal University of Maranhão (Universidade Federal do Maranhão (UFMA)). This strain is resistant to amidines and synthetic pyrethroids and sensitive to organophosphates. Larvae were maintained in artificially infested cattle at the experimental farm of the Institute of Agrarian Sciences of the Federal University of Minas Gerais (Instituto de Ciências Agrárias da Universidade Federal de Minas Gerais (ICA/UFMG)), Montes Claros, Minas Gerais, Brazil. The engorged females were morphologically selected and used for immersion tests of engorged females and kept in BOD ovens at 28 °C and relative humidity of approximately 80% for 15 days until hatching to obtain larvae. Both strains were approved for use by the Ethics Committee for Research, Society and Development, v. 11, n. 12, e75111234252, 2022 (CC BY 4.

Effects of association between EE and P. spruceanum resin and cypermethrin on R. microplus larvae Larval Packet Test (LPT)
EE LC90 concentrations were estimated at 101.92 mg ml −1 and resin at 50.18 mg ml −1 for the calculations in the formulations (Figueiredo et al., 2019). Larvae aged between 15-28 days post-hatch were used. The tests were carried out according to the methodology proposed by Stone and Haydock (1962) with modifications. Approximately 100 larvae were inserted into filter paper packages (Whatmann No 1) measuring 6 × 6 cm. Subsequently, the filter paper packets were sealed with staplers and separately impregnated with 300 µL of the binary combination ( Table 1). The entire experiment consisted of four replicates per treatment. Source: Authors.
The filter paper packets containing larvae were kept in petri dishes and placed in an oven (BOD) at 27 °C and 80% humidity for 24 h. The filter paper packets were then placed in the oven. Subsequently, the filter paper packets were opened on a white surface and the live and dead larvae were counted. The relative number of dead larvae compared to the total number of larvae was used for analysis of variance, and the means were compared using the Scott-Knott test at the 5% significance level in a completely randomized design. Lethal concentrations killing 90% of infectious larvae (LC90) were estimated using probit regression analysis of the SAEG 9.1 statistical package (SAEG 2007).

Immersion test in engorged females
Efficacy of the combination of P. spruceanum resin or EE with cypermethrin was evaluated at the concentrations shown in Table 1. The efficacy of the formulations was evaluated using the acaricide resistance test as described by Research, Society and Development, v. 11, n. 12, e75111234252, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i12.34252 5 Drummond et al. (1973). Engorged adult females 4-6 mm in size were selected, washed in distilled water, and dried on paper towels. They were then divided into homogeneous groups of five (05) adult females according to size and weight and immersed in 5 ml of the combination solution for 5 min. The excess solution was removed with a paper towel, placed in a Petri dish, and maintained at 28 °C and 70% relative humidity in a BOD incubator. All procedures were repeated five times (Leite, 1995).
After 15 days of incubation, egg masses from each group of adult females were weighed on an analytical balance and then transferred to 3 ml disposable syringes, sealed with hydrophilic cotton, and stored at 28 °C and 70% relative humidity (Drummond et al., 1973). Thirty days after the onset of larval hatching, the contents of the syringe were transferred to petri dishes containing 3 ml of a solution of water and detergent (50:50). Then, three 200 μl aliquots of this suspension were placed on glass slides to count the eggs, larval eggs, and larvae under an optical microscope with a 10x objective and to determine the hatching rate of each group. All procedures related to hatching tests were triplicated (Vasconcelos et al., 2018).
A modified version of the formula described by Bennett (1974) was used to determine egg laying capacity (ELC): ELC = (egg mass weight/initial female weight) × 100 Treatment efficacy (product efficacy) was estimated using the method of Drummond et al. (1973): RE (reproductive efficiency) = (egg mass weight × % incubation × 20,000)/initial female weight,

PE (product efficiency) = ((RE control group − RE treated group)/RE control group)) × 100
Tests were repeated five times, data were transformed and submitted to analysis of variance, and means for each group were compared using the Scott-Knott test (α = 0.05). The concentration of EE sufficient to inhibit 90% of hatching (IC90) was estimated by probit analysis using the SAEG 9.1 statistical package.

Phytochemical analysis
The protocols presented by Royo et al. (2015) were applied for phytochemical analysis. Qualitative characterization was performed with colorimetric and precipitation analyses of the classes of phenolic compounds, tannins, saponins, flavonoids, and terpenes. The reagents used for the photochemical tests were ferric chloride, 10% sodium hydroxide, Bertrand reagent, Dragendorff reagent, Mayer reagent, Lieberman-Burchard reagent, aluminum chloride, and distilled water. The intensity of the reaction was classified as (+) weak positive, (++) moderate positive and (+++) strong positive, and (−) negative.

Effects on larval mortality
The combinations containing EE or resin combined with cypermethrin showed efficacy of over 90% against R. microplus larvae (Tables 2 and 3).  The combination of crude resin with cypermethrin used against SJP and Santa Rita strains showed efficacy above 93% at all concentrations tested, which is similar to the results observed for cypermethrin in the control with cypermethrin (p < 0.05). To the best of our knowledge, to date, no study has evaluated the effect of the combination of a plant extract with an acaricidal compound on the control of R. microplus. However, essential oil combinations have already been analyzed. The combination of the monoterpenes thymol and carvacrol with the phenylpropanoid eugenol was synergistic and effective in controlling larvae of R. microplus and R. sanguinius (Araujo et al., 2016). A moderate synergistic effect was observed when controlling larvae of Amblyomma sculptum with the combinations carvacrol + thymol and carvacrol + (E)-cinnamaldehyde (Novato et al., 2015).

Effects on reproductive parameters of females of Rhipicephalus microplus
High efficacy was not observed when the association of EE or resin with cypermethrin was applied to engorged females of R. microplus at any of the concentrations evaluated (Tables 4 and 5). However, a higher efficacy of the combination of EE or resin with cypermethrin was observed when comparing the efficacy of cypermethrin alone on engorged females (p < 0.05).  In this study, combinations of plant extract and synthetic acaricide at low concentrations retained acaricidal activity in R. microplus larvae; this is in contrast to what was observed in engorged females, although the associations increased efficacy levels compared to cypermethrin alone. Larvae are the first stage of contact with the ruminant and initiate the parasitic phase.
Thus, the use of formulations in ruminants that eliminate larvae greatly reduces tick infestation and consequent damage caused by these ectoparasites.
The differences between the results obtained for larvae and mature females of R. microplus obtained in this study could be attributed to the morphological and physiological characteristics of the larvae. The larval body surface area is small, thereby making it easier for the products to penetrate the larval cuticle (Sonenshine and Roe, 2014;Vasconcelos et al, 2014).

Photochemical characterization of Protium spruceanum extract and resin
The results of phytochemical analysis showed the presence of secondary metabolites of the following classes: phenolic compounds, tannins, alkaloids, terpenes, flavonoids, and saponins in the EE from leaves (Table 6). However, only tannins, alkaloids, and flavonoids were detected in the crude resin.  (Barata et al., 2020). Additionally, Ruedigera et al. (2007) found substances such as terpenes and flavonoids in the essential oil extracted from the leaves and resin of P. spruceanum (Ruedigera et al., 2007). In this study, acaricidal compounds were found both in the EE and crude resin from P. spruceanum leaves. The efficacy of these bioproducts was observed in larvae and adult females of R. microplus when combinations of these compounds were used. In this way, substances of plant origin can act synergistically with drugs or chemicals already used in tick control, thereby allowing the reduction of doses required for the desired effect.

Conclusion
The association between ethanol extract, crude resin from P. spruceanum leaves and cypermethrin has implications for the control of immature stages and engorged females of R. microplus. The results of phytochemical analysis showed the presence of phenolic compounds, tannins, alkaloids, terpenes, flavonoids and saponins in the ethanol extract of P. spruceanum leaves and for raw resin only tannins, alkaloids and flavonoids were detected.
Finally, it is suggested that these association be tested in non-target organisms, to have more reliability in their use and advance to in vivo experiments.