Evaluation of the performance of synthetic acaricides and the essential oil of Plectranthus amboinicus (Lour.) Spreng (Lamiaceae) for the control of Rhipicephalus (Boophilus) microplus

[Avaliação de acaricidas sintéticos e do oléo essencial de Plectranthus amboinicus (Lour.) Spreng (Lamiaceae) para o controle de Rhipicephalus (Boophilus) microplus]

ABSTRACT

Chemical acaricides are used as the primary method for controlling Rhipicephalus (Boophilus) microplus. However, the incorrect use of these compounds over the years has led to the emergence of resistant tick populations. This study aimed to evaluate the effectiveness of different combinations of synthetic acaricides and the essential oil of Plectranthus amboinicus on engorged females of R. (B.) microplus. Engorged females were obtained from infested bovines in dairy farms in the Agreste mesoregion of Pernambuco, Brazil. Five different combinations of commercial synthetic acaricides: I) Chlorpyrifos 30.0g + Cypermethrin 15.0g + Fenthion 15.0g; II) Chlorpyrifos 8.5g + Cypermethrin 15.0g + Ethion 16.0g; III) Deltamethrin 50.0g; IV) Amitraz 12.5g; V) Chlorpyrifos 15.0g + Cypermethrin 25.0g + Piperonyl butoxide 50.0g, and the 1% essential oil of P. amboinicus were used for the Adult Immersion Test. Only the combinations I and V of synthetic acaricides and essential oil showed efficacy over 95%. The findings of this study demonstrate that ticks’ resistant populations are present in the study area, and the essential oil of P. amboinicus is a promising compound for developing products used for tick control. Finally, it is paramount to implement educational activities to improve farmers' awareness of the rational use of acaricides.

Keywords:
commercial acaricides; resistance; Phytotherapeutic

RESUMO

Os acaricidas químicos são utilizados como o principal método para controlar Rhipicephalus (Boophilus) microplus. No entanto, o uso incorreto desses compostos ao longo dos anos levou ao surgimento de populações de carrapatos resistentes. Este estudo teve como objetivo avaliar in vitro a eficácia de diferentes combinações de acaricidas sintéticos e do óleo essencial de Plectranthus amboinicus em fêmeas ingurgitadas de R. (B.) microplus. Fêmeas ingurgitadas foram obtidas de bovinos infestados, criados em fazendas leiteiras localizadas na mesorregião do Agreste do estado de Pernambuco, Brasil. Foram utilizadas cinco combinações diferentes de acaricidas sintéticos comerciais: I) Clorpirifós 30,0g + Cipermetrina 15,0g + Fention 15,0g; II) Clorpirifós 8,5g + Cipermetrina 15,0g + Etion 16,0g; III) Deltametrina 50,0g; IV) Amitraz 12,5g; V) Clorpirifós 15,0g + Cipermetrina 25,0g + Butóxido de piperonila 50,0g, e o óleo essencial de P. amboinicus a 1% para o teste de imersão de adultos. Apenas as combinações I e V de acaricidas sintéticos, juntamente com o óleo essencial, mostraram eficácia superior a 95%. Os resultados deste estudo demonstram que populações de carrapatos resistentes estão presentes na área de estudo, e o óleo essencial de P. amboinicus é uma substância promissora para o desenvolvimento de produtos utilizados no controle de carrapatos. Por fim, é fundamental a implementação de atividades educacionais para melhorar a conscientização dos produtores quanto ao uso racional de acaricidas.

Palavras-chave:
acaricidas comerciais; resistência; fitoterápico

INTRODUCTION

Rhipicephalus (Boophilus) microplus (Acari: Ixodidade) is the most important ectoparasite of cattle in tropical and subtropical areas worldwide. This tick species is responsible for economic losses in livestock animals due to reduced meat and milk production and transmission of pathogens (e.g., Babesia spp. and Anaplasma spp.) that may induce anemia and mortality (Jabbar et al., 2015). In Brazil, it is estimated that an annual cost of around three billion dollars spent for the control of this ixodid (Grisi et al., 2014; Hurtado and Giraldo-Ríos, 2018; Rodriguez-Vivas et al., 2018).

The control of R. (B.) microplus is based on chemical acaricides belonging to different drug classes, including organophosphates, pyrethroids, amidines, macrocyclic lactones, phenylpyrazoles, and benzoylphenylureas (Koller et al., 2019). In recent years, only a few new products have been introduced, and many existing market options have become ineffective due to the emergence of resistance resulting from their incorrect and prolonged usage (Mendes et al., 2022). The resistance of R. (B.) microplus to synthetic acaricides is developed through the increase of gene expression and enzymatic activity or due to mutations in sodium channels of neuroreceptors, which reduces and impedes the penetration of chemical compounds (Ffrench et al., 2004; Dzemo et al., 2022).

Reports of multi-resistant tick populations are frequent in areas infested by R. (B.) microplus. Additionally, concerns have arisen among producers regarding environmental contamination and the presence of residues of these acaricides in meat and milk. In their haphazard attempts at control using conventional acaricides, producers inadvertently exacerbate the resistance problem (Mendes et al., 2022; Agwunobi et al., 2021). In Brazil, resistance of this tick to various chemical formulations has previously been reported in the northern, northeastern, central-western, southeastern, and southern regions (Santos and Vogel, 2012; Santana et al., 2016; Cruz, 2017; Silva et al., 2020; Valsoni et al., 2020).

Due to the necessity of finding alternative methods for controlling these ectoparasites, research involving plant derivatives has been encouraged and shows promising results. Remarkably, using phytomedicines rich in bioactive compounds with action against different life stages of ticks has gained attention (Banumathi et al., 2017; Vinturelle et al., 2021). For example, Plectranthus amboinicus (Lour.) Spreng (Lamiaceae), Cuban oregano, is a pantropically cultivated plant rich in bioactives such as carvacrol and thymol. It exhibits insecticidal, larvicidal, antioxidant, anti-inflammatory, antibacterial, and antifungal properties and acaricidal potential (Pinheiro et al., 2015; Arumugam et al., 2016; Leesombun et al., 2022).

This study evaluated the in vitro effectiveness of different combinations of synthetic acaricides and the essential oil of P. amboinicus on engorged females of R. (B.) microplus.

MATERIAL AND METHODS

Engorged females were obtained from animals reared in a dairy cattle farm in Bom Conselho (9° 10’ 6” S, 36° 41’ 8” W), Agreste region, state of Pernambuco, Brazil. The area experiences an average annual temperature of 21°C, with a mean precipitation ranging from 700 to 1150 mm, influenced by the region’s varied topography. This municipality stands as one of the major milk producers in the state, with its economy predominantly centered around agriculture and livestock. The prevalent bovine breed in this area is the Holstein, which exhibits low resistance to ectoparasites despite its high production capabilities (Hurtado and Giraldo-Ríos, 2018; Efetivo, 2021).

The study received approval from the Ethics Committee on Animal Use (CEUA) of the Federal Rural University of Pernambuco under protocol number 8717300420/ID 192/2020.

R. (B.) microplus engorged females were manually collected from infested cattle without ectoparasiticide treatment in the last 30 days. Specimens were stored in plastic vials and transported to the laboratory for further processing. They were morphologically identified, quantified, and weighted for the formation of groups.

The essential oil was obtained from P. amboinicus leaves using the steam distillation technique with helium gas, following Craveiro et al. (1976). This method involves suspending the botanical material in water, boiling it, and collecting and cooling the vapor. This process separates the condensed material into two phases: an aqueous phase and an organic phase, which corresponds to the essential oil. Subsequently, the chemical composition of the oil was evaluated using the Gas Chromatography-Mass Spectrometry (GC-MS) technique.

Seven homogeneous groups containing ten females (~ 2 g) each were formed and separated as follows: I) Chlorpyrifos 30.0g + Cypermethrin 15.0g + Fenthion 15.0g; II) Chlorpyrifos 8.5g + Cypermethrin 15.0g + Ethion 16.0g; III) Deltamethrin 50.0g; IV) Amitraz 12.5g; V) Chlorpyrifos 15.0g + Cypermethrin 25.0g + Piperonyl butoxide 50.0g; 1% P. amboinicus essential oil, and a control group (distilled water). All treatments were conducted in triplicate, and the result was obtained by calculating the averages of the treatments. Synthetic acaricides were used according to the manufacturer's instructions.

Engorged females were subjected to the Adult Immersion Test (AIT) for in vitro detection of acaricide effectiveness (Drummond et al., 1973). After immersion for five minutes, specimens were placed in an incubator for oviposition under controlled conditions (26 ± 1°C, 80 ± 5% relative humidity) for 15 days. Subsequently, the egg mass was weighed, stored in sealed plastic vials, and incubated under controlled conditions (see above) for 25 days. Upon egg hatching, the hatching percentage was visually determined, and the product's efficacy was calculated according to Drummond et al. (1973). The product was considered adequate if the efficacy value was ≥ 95%.

RESULTS

The chemical characterization of the P. amboinicus essential oil is presented in Table 1. The compound Carvacrol (54.81%) predominated, followed by various terpenes.

Table 1
Proportion of compounds present in the essential oil of Plectranthus amboinicus (Lour.) Spreng (Lamiaceae)

Compound	Relative Area Quantification (%)
1,2,3,4,5-Pentamethyl-cyclopentane	0.50
Bicyclo [3.1.0] hex-2-ene, 2-methyl-5-(1-methylethyl)	0.27
β-Myrcene	0.99
ο-Cymene	0.19
3-Carene	8.92
γ-Terpinene	0.40
5-Isopropyl-2-methylbicyclo [3.1.0] hexan-2-ol	10.66
Bicyclo[3.1.0]hexan-2-ol, 2-methyl-5-(1-methylethyl)-, (1α.,2β.,5α.)	1.08
Terpinen-4-ol	0.31
Bicyclo[2.2.1]heptan-2-one, 1,7,7-trimethyl-, (1S)	0.60
Thymol	0.39
Carvacrol	54.81
Bicyclo[7.2.0]undec-4-ene, 4,11,11-trimethyl-8-methylene	12.51
α-Caryophyllene	3.15
Caryophyllene oxide	3.63
Ledol	1.03
Deconexent	0.56

Groups I, V, and P. amboinicus essential oil were effectiveness against R. (B.) microplus (Table 2).

Table 2
Efficacy of synthetic acaricides and P. amboinicus oil against R. (B.) microplus

Treatment	Egg Weight (g)	Hatch Rate (%)	Reproductive Efficiency (%)	Treatment Efficacy (%)
Control	0.90	100.0	100.0	-
I	0.27	10.0	3.0	97.0
II	0.40	24.0	10.67	89.33
III	0.90	92.70	92.70	7.30
IV	0.87	95.0	91.83	8.70
V	0.20	5.33	1.18	98.86
Essential oil	0.13	7.66	1.11	98.89

Among the synthetic products, the treatments that exhibited efficacy involved combinations of different active ingredients or compound synergies, such as treatments I and V, with respective efficacies of 97.0% and 98.86%. Notably, there was a reduction in egg laying of 70.0% in treatment I and 77.78% in treatment V when compared to the control group, accompanied by a decrease in the egg laying rate of 90.0% and 94.67%, respectively. All females of all groups remained alive during the whole study period, but the egg production and hatchability were altered.

DISCUSSION

This study demonstrates that tick-resistant populations are present in the study area, and the essential oil of P. amboinicus was effective against R. (B.) microplus. Despite no mortality of engorged females, synthetic compounds used for the treatment I and V inhibited egg laying and hatchability. Likely, higher concentrations of organophosphates with pyrethroids in treatment I and the potentiation of the insecticidal effect of organophosphates by piperonyl butoxide synergism in treatment V were responsible for the efficacy of these treatments (Koller et al., 2019). None of the synthetic compounds or the essential oil achieved a complete efficacy (100%) as previously reported in the study area (Santana et al., 2013). Actually, resistant populations of R. (B.) microplus to combinations of cypermethrin with chlorpyrifos and cypermethrin, chlorpyrifos, and citronellol were previously reported in the Garanhuns microregion, Pernambuco (Santana et al., 2013).

The low efficacy of deltamethrin and amitraz (treatments III and IV) underscores the exacerbation of the tick resistance to two different chemical groups simultaneously observed in the same region almost a decade ago (Santana et al., 2016). Resistance by ticks to more than one group of compounds simultaneously has been reported in various regions of Brazil by different studies (Santos and Vogel, 2012; Santana et al., 2016; Cruz, 2017; Silva et al., 2020; Valsoni et al., 2020).

The worsening of parasitic resistance arises from the need to implement combinations of different classes of compounds within the same product, as seen in treatments I, II, and V, replacing acaricides that lack such class associations (treatments III and IV). However, these non-associated acaricides lose their efficacy more rapidly due to intrinsic and extrinsic factors related to ticks, such as mutations and the artificial selection pressure imposed by improper management of commercial acaricides (Koller et al., 2019; Dzemo et al., 2022). Nevertheless, even acaricide combinations lose efficacy over time, as observed in treatment II. Therefore, it is necessary to introduce compounds that synergize with other products, like piperonyl butoxide used in treatment V, which, while not lethal to ticks exposed to acaricides, hindered their reproduction due to its cytotoxicity (Banumathi et al., 2017; Rodriguez-Vivas et al., 2018).

The tick resistance to synthetic acaricides is exacerbated by the incorrect use of these chemical compounds, which are sold everywhere without any control. In Brazil, most veterinary use acaricides are sold without needing a prescription as long as registered and approved by the Ministry of Agriculture, Livestock, and Supply. The artificial selection pressure on these parasites generates resistant populations (Koller et al., 2019). This insensitivity of a portion of the R. (B.) microplus population to treatments contributes to the difficulty in establishing effective control alternatives worldwide (Agwunobi et al., 2021).

As a result, research on plant extracts and essential oils with insecticidal and acaricidal bioactive compounds has intensified in recent decades. Also, due to the reduced efficacy of synthetic acaricides and the lower environmental and product residue left by these phytotherapies compared to synthetic acaricides (Hurtado and Giraldo-Ríos, 2018). Particularly the use of phytotherapy based on the exploitation of bioactive present in plants, especially those with known insecticidal action, such as carvacrol, which is the main component of P. amboinicus essential oil (Araujo et al., 2016; Leesombun et al., 2022).

The higher efficacy of the essential oil herein observed can be attributed to carvacrol and thymol. The combination of these terpenes demonstrates toxicity against larvae of R. (B.) microplus and R. sanguineus, enhancing the larvicidal effect through the synergism of these bioactives (Araujo et al., 2016). Furthermore, carvacrol has a cytotoxic effect on semi-engorged R. sanguineus females, leading to morphological alterations in oocytes and germinal vesicles and cytoplasmic vacuolization. Although it may not be lethal to all ticks, the survivors have their reproduction altered, resulting in effective long-term control (Souza et al., 2019).

A wide range of bioactives derived from the secondary metabolism of P. amboinicus, especially terpenes, serve as defense compounds for the plant. However, they also promise arthropod control by enhancing, through synergy, the effect of the primary compounds in the essential oil, such as carvacrol (Camilo et al., 2017). The use of terpenes against arthropod control has been reported as an alternative measure of control to be used integrated with chemical compounds.

CONCLUSION

The resistance of R. (B.) microplus to synthetic acaricide formulations and their combinations has led to the increased use of products with higher concentrations by rural producers. The lack of efficacy in treatments II, III, and IV follows the same pattern observed in other regions of Brazil, emphasizing the need for educational efforts to promote the rational use of acaricides. Additionally, phytotherapies, such as P. amboinicus essential oil, represent an alternative tick control source. Further in vitro and in vivo studies with different concentrations are required to better characterize this natural compound's role in the control of ectoparasites.

REFERENCES

• AGWUNOBI, D.O.; YU, Z.; LIU, J. A retrospective review on ixodid tick resistance against synthetic acaricides: implications and perspectives for future resistance prevention and mitigation. Pest. Biochem. Physiol., v.173, p.104776, 2021.
• ARAUJO, L.X.; NOVATO, T.P.L.; ZERINGOTA, V. et al. Synergism of thymol, carvacrol and eugenol in larvae of the cattle tick, Rhipicephalus microplus, and brown dog tick, Rhipicephalus sanguineus. Med. Vet. Entomol., v.30, p.377-382, 2016.
• ARUMUGAM, G.; SWAMY, M.K.; SINNIAH, U.R. Plectranthus amboinicus (Lour.) Spreng: botanical, phytochemical, pharmacological and nutritional significance. Molecules, v.21, p.369, 2016.
• BANUMATHI, B.; VASEEHARAN, B.; RAJASEKAR, P. et al. Exploitation of chemical, herbal and nanoformulated acaricides to control the cattle tick, Rhipicephalus (Boophilus) microplus - a review. Vet. Parasitol., v.244, p.102-110, 2017.
• CAMILO, C.J.; NONATO, C.F.A.; GALVÃO-RODRIGUES, F.F. et al. Acaricidal activity of essential oils: a review. Trends Phytochem. Res., v.1, p.183-198, 2017.
• CRAVEIRO, A.A.; MATOS, F.J.A.; ALENCAR, J.W. A simple and inexpensive steam generator for essential oils extraction. J. Chem. Educ., v.53, p.652, 1976.
• CRUZ, B.C. Aspectos ecológicos, biológicos e de resistência de Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) na região de Jaboticabal, São Paulo, Brasil. 2017. Tese (Doutorado) - Universidade Estadual Paulista Faculdade de Ciências Agrárias e Veterinárias, Jaboticabal, SP.
• DRUMMOND, R.O.; ERNST, S.E.; TREVINO, J.L. et al. Boophilus annulatus and B. microplus: laboratory tests of insecticides. J. Econ. Entomol., v.66, p.130-133, 1973.
• DZEMO, W.D.; THEKISOE, O.; VUDRIKO, P. Development of acaricide resistance in tick populations of cattle: a systematic review and meta-analysis. Heliyon, v.8, p.e08718, 2022.
• EFETIVO de rebanhos. Pesquisa da pecuária municipal. Rio de Janeiro: IBGE, 2021.
• FFRENCH, R.H.; DABORN, P.J.; LE GOFF, G. The genetics and genomics of insecticide resistance. Trends Genet., v.20, p.163-170, 2004.
• GRISI, L.; LEITE, R.C.; MARTINS, J.R.S. et al. Reassessment of the potential economic impact of cattle parasites in Brazil. Rev. Bras. Parasitol. Vet., v.23, p.150-156, 2014.
• HURTADO, O.J.B.; GIRALDO-RÍOS, C. Economic and health impact of the ticks in production animals. Ticks Tick Borne Pathogens, v.9, p.1-9, 2018.
• JABBAR, A.; ABBAS, T.; SANDHU, Z.D. et al. Tick-borne diseases of bovines in Pakistan: major scope for future research and improved control. Parasit Vectors, v.8:283, 2015.
• KOLLER, W.W.; HIGA, L.O.S.; ZIMMERMANN, N.M. et al. Resistência dos carrapatos aos acaricidas. Carrapatos na cadeia produtiva de bovinos. Brasília, DF: Embrapa, 2019. 240p.
• LEESOMBUN, A.; SUNGPRADIT, S.; BOONMASAWAI, S. et al. Insecticidal activity of Plectranthus amboinicus essential oil against the stable Fly Stomoxys calcitrans (Diptera: Muscidae) and the horse fly Tabanus megalops (Diptera: Tabanidae). Insects, v.13, p.255, 2022.
• MENDES, M.C.; PEREIRA, J.R.; PRADO, A.P. Sensitivity of Boophilus microplus (Acari: Ixodidae) to pyrethroids and organophosphate in farms in the Vale do Paraíba region, São Paulo, Brazil. Arq. Inst. Biol., v.74, p.81-85, 2022.
• PINHEIRO, P.F.; COSTA, A.V.; ALVES, T.A. et al. Phytotoxicity and cytotoxicity of essential oil from leaves of Plectranthus amboinicus, carvacrol, and thymol in plant bioassays. J. Agric. Food Chem., v.63, p.8981-8990, 2015.
• RODRIGUEZ-VIVAS, R.I.; JONSSON, N.N.; BHUSHAN, C. Strategies for the control of Rhipicephalus microplus ticks in a world of conventional acaricide and macrocyclic lactone resistance. Parasitol. Res., v.117, p.3-29, 2018.
• SANTANA, B.B.; RAMOS, R.A.N.; FAUSTINO, M.A.G. et al. Evaluation of the efficacy of cypermethrin and amitraz against Rhipicephalus (Boophilus) microplus, in the State of Pernambuco, Brazil. Arq. Inst. Biol., v.82, p.1-4, 2016.
• SANTANA, B.B.; RAMOS, R.A.N.; SANTANA, M.A. et al. Susceptibility of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) to pyrethroids and their associations in Pernambuco, Brazil. Rev. Bras. Parasitol. Vet., v.22, p.276-280, 2013.
• SANTOS, F.CC.; VOGEL, F.S.F. Resistência do carrapato Rhipicephalus (Boophilus) microplus frente ao amitraz e cipermetrina em rebanhos bovinos no Rio Grande do Sul de 2005 a 2011. Rev. Port. Ciênc. Vet., v.107, p.121-124, 2012.
• SILVA, R.; SANTOS, M.P.A.; FIGUEIREDO, M.A.P. Avaliação comparativa in vitro da sensibilidade de Rhipicephalus (Boophilus) microplus a acaricidas comercializados no município de Rolim de Moura, Rondônia, Brasil. Ars Vet., v.36, p.163-168, 2020.
• SOUZA, J.R.L.; OLIVEIRA, P.R.; ANHOLETO, L.A. et al. Effects of carvacrol on oocyte development in semi-engorged Rhipicephalus sanguineus sensu lato females ticks (Acari: Ixodidae). Micron, v.116, p.66-72, 2019.
• VALSONI, L.M.; FREITAS, M.G.; ECHEVERRIA, J.T. et al. Resistance to all chemical groups of acaricides in a single isolate of Rhipicephalus microplus in Mato Grosso do Sul, Brazil. Int. J. Acarol., v.46, p.276-280, 2020.
• VINTURELLE, R.; MATTOS, C.; MELONI, J. et al. Evaluation of essential oils as an ecological alternative in the search for control Rhipicephalus microplus (Acari: Ixodidae). Vet. Parasitol. Reg. Stud. Rep., v.23, p.100523, 2021.

Publication Dates

• Publication in this collection
  03 May 2024
• Date of issue
  May-Jun 2024

History

• Received
  03 Oct 2023
• Accepted
  22 Nov 2023