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Activity of essential oils from leaves, flower buds and stems of Tetradenia riparia on Rhipicephalus (Boophilus) microplus larvae

Atividade dos óleos essenciais das folhas, botões florais e caules de Tetradenia riparia sobre as larvas de Rhipicephalus (Boophilus) microplus

Abstract

Around the world, the main problems of livestock are caused by ectoparasites, however, commercial acaracide are toxic to the environment and detrimental to One Health. Therefore, research has increasingly focused on development of natural products as alternatives for tick control. The purpose of this study was to evaluate the larvicidal effect on Rhipicephalus (Boophilus) microplus, through use of essential oils (EOs) extracted from the leaves, flower buds and stems of Tetradenia riparia. The chemical composition of these EOs was determined through gas chromatography coupled to mass spectrometry (GC-MS). They were tested on larvae at concentrations of 100.000 to 40 µg/mL, using the larval packet test and under semi-natural conditions. The main class of compounds in the chemical composition was sesquiterpenes (both oxygenates and hydrocarbons), whereas the predominant compounds in the leaves, flower buds and stems were 14-hydroxy-9-epi-caryophyllene, T-cadinol and 6-7-dehydroroyleanone, respectively. The leaves proved to be the most effective, with highest larvicidal activity (LC99.9 = 83.53 µg/mL). When tested under semi-natural conditions, the oils obtained efficiency above 98% in all compound tests. The results indicated that these EOs were effective against R. (B.) microplus larvae in vitro and ex-situ, proving that this plant has bioactive molecules with significant larvicidal activity.

Keywords:
Larvicide; essential oils; ticks; Tetradenia riparia; Rhipicephalus microplus

Resumo

Os principais problemas para a pecuária estão relacionados às ectoparasitoses, e ao fato dos carrapaticidas apresentarem elevada toxicidade ao meio ambiente e à saúde única. Surgem, então, demandas na busca por inovações e desenvolvimento de produtos naturais, como alternativas para o controle dos carrapatos. O objetivo deste trabalho foi avaliar o potencial da atividade larvicida sobre Rhipicephalus (Boophilus) microplus a partir dos óleos essenciais de Tetradenia riparia (TrOEs) extraídos das folhas, botões florais e caules. A composição química foi determinada por cromatografia gasosa acoplada à espectrometria de massa (GC/MS). Os TrOEs foram testados sobre larvas nas concentrações de 100.000 a 40 µg/mL pelo teste de pacote de larvas e em condições seminaturais. Na composição química, a classe majoritária foi os sesquiterpenos (oxigenados e hidrocarbonetos); já os compostos em destaques foram 14-hidroxy-9-epi-caryophyllene, T-cadinol e 6-7-dehidroroyleanone para folhas, botões florais e caules, respectivamente. As folhas demonstraram ser mais eficientes e com maior poder larvicida (CL99.9 = 83.53 µg/mL). Quando testado em condições seminaturais os óleos obtiveram eficiência acima de 98% em todos os compostos testados. Os resultados indicaram que os TrOEs, foram eficazes sobre as larvas de R. (B.) microplus in vitro e ex-situ, evidenciando que esta planta possui moléculas bioativas com ação larvicidas significativas.

Palavras-chave:
Larvicida; óleo essencial; carrapatos; Tetradenia riparia; Rhipicephalus microplus

Introduction

Brazil has the largest commercial herd of cattle in the world, with approximately 214.8 million head. It is currently the fourth largest producer of milk, producing about 25.4 billion liters/year. The livestock sector accounts for 8.5% of Brazil’s gross domestic product (GDP) (IBGE, 2020Instituto Brasileiro de Geografia e Estatística – IBGE. Agricultura, pecuária e outros [online]. Rio de Janeiro: IBGE; 2020 [cited 2021 Jan 12]. Available from: https://www.ibge.gov.br/estatisticas/economicas/agricultura-e-pecuaria.html
https://www.ibge.gov.br/estatisticas/eco...
), which shows the strength of this sector in the Brazilian economy. However, farmers suffer productivity losses relating to ectoparasitosis, especially due to infestations of Rhipicephalus (Boophilus) microplus Canestrini, 1887 (Acari: Ixodidae). This ectoparasite is responsible for causing losses in Brazil of the order of US$ 3.2 billion/year (Grisi et al., 2014Grisi L, Leite RC, Martins JRS, Barros ATM, Andreotti R, Cançado PHD, et al. Reassessment of the potential economic impact of cattle parasites in Brazil. Rev Bras Parasitol Vet 2014; 23(2): 150-156. http://dx.doi.org/10.1590/S1984-29612014042. PMid:25054492.
http://dx.doi.org/10.1590/S1984-29612014...
). Worldwide, ticks are among the main pests of livestock. Experts estimate that losses due to tick infestations could reach US$ 30 billion/year (Lew-Tabor & Valle, 2016Lew-Tabor AE, Valle MR. A review of reverse vaccinology approaches for the development of vaccines against ticks and tick borne diseases. Ticks Tick Borne Dis 2016; 7(4): 573-585. http://dx.doi.org/10.1016/j.ttbdis.2015.12.012. PMid:26723274.
http://dx.doi.org/10.1016/j.ttbdis.2015....
).

Most of the negative economic impacts arise through direct expenditure on treating the damage that ticks cause to the animals’ health, such as blood loss, dermatitis, immune suppression, food inappetence and stress. These are due mainly to a complex of diseases known as cattle tick fever, which are transmitted by these ticks (Araújo et al., 2015Araújo LX, Novato TPL, Zeringota V, Matos RS, Senra TOS, Maturano R, et al. Acaricidal activity of thymol against larvae of Rhipicephalus microplus (Acari: Ixodidae) under semi-natural conditions. Parasitol Res 2015; 114(9): 3271-3276. http://dx.doi.org/10.1007/s00436-015-4547-3. PMid:26040843.
http://dx.doi.org/10.1007/s00436-015-454...
; Garcia et al., 2019Garcia MV, Rodrigues VS, Koller WW, Andreotti R. Biologia e importância do carrapato Rhipicephalus (Boophilus) microplus. In: Andreotti R, Garcia MV, Koller WW, editors. Carrapatos na cadeia produtiva de bovinos. Brasília: Embrapa; 2019. p. 17-28.). The consequences of cattle tick fever include weight loss, reduced milk production, falling birth rates, leather depreciation and increased mortality (Andreotti et al., 2019Andreotti R, Garcia MV, Koller WW. Controle estratégico dos carrapatos nos bovinos. In: Andreotti R, Garcia MV, Koller WW, editors. Carrapatos na cadeia produtiva de bovinos. Brasília: Embrapa; 2019. p. 125-136.). Moreover, indirect expenditure is incurred with regard to the cost of labor, acquisition of acaricide products and investments in infrastructure, equipment and logistics that are used exclusively in combating ticks.

Although there are a multitude of methods for controlling these ectoparasites, farmers always use methods that are easy to apply, with lower costs. These characteristics are commonly found among manufactured chemicals (Higa et al., 2019Higa LOS, Garcia MV, Barros JC, Bonatte P Jr. Controle do carrapato-do-boi por meio de acaricidas. In: Andreotti R, Garcia MV, Koller WW, editors. Carrapatos na cadeia produtiva de bovinos. Brasília: Embrapa; 2019. p. 137-147.), especially pyrethroids and organophosphates (Mendes et al., 2013Mendes MC, Duarte FC, Martins JR, Klafke GM, Fiorini LC, Barros AT. Characterization of the pyrethroid resistance profile of Rhipicephalus (Boophilus) microplus populations from the states of Rio Grande do Sul and Mato Grosso do Sul, Brazil. Rev Bras Parasitol Vet 2013; 22(3): 379-384. http://dx.doi.org/10.1590/S1984-29612013000300010. PMid:24142169.
http://dx.doi.org/10.1590/S1984-29612013...
).

Given the magnitude of the losses, the fact that commercial acaricides present high toxicity to the environment, animals and humans and the high standards demanded within international markets, there is a need for research relating to innovation and development of natural products. The aim is to search for economically viable alternatives that promote strategic tick control, combined with sustainable development throughout the production chain (Chagas et al., 2002Chagas ACS, Passos WM, Prates HT, Leite RC, Furlong J, Fortes ICP. Efeito acaricida de óleos essenciais e concentrados emulsionáveis de Eucalyptus spp em Boophilus microplus. Braz J Vet Res Anim Sci 2002; 39(5): 247-253. http://dx.doi.org/10.1590/S1413-95962002000500006.
http://dx.doi.org/10.1590/S1413-95962002...
; Medeiros et al., 2019Medeiros JP, Bortollucci WC, Silva ES, Oliveira HLM, Campos CFAA, Gonçalves JE, et al. Biocidal potential of Eugenia pyriformis essential oil in the control of Rhipicephalus (Boophilus) microplus in the free-living cycle. Pesqui Vet Bras 2019; 39(11): 879-888. http://dx.doi.org/10.1590/1678-5150-pvb-6434.
http://dx.doi.org/10.1590/1678-5150-pvb-...
).

Among the various species with potential for the development of bioactive products, Tetradenia riparia stands out. This is a shrub plant belonging to the family Lamiaceae that is native to the African continent but which has been introduced and duly adapted to all Brazilian biomes. It is used as an ornamental and aromatic plant, and is popularly known as false myrrh, lavandula or mist plum (Gazim et al., 2011Gazim ZC, Demarchi IZ, Lonardoni MVC, Amorim ACL, Hovell AMC, Rezende CM, et al. Acaricidal activity of the essential oil from Tetradenia riparia (Lamiaceae) on the cattle tick Rhipicephalus (Boophilus) microplus (Acari; Ixodidae). Exp Parasitol 2011; 129(2): 175-178. http://dx.doi.org/10.1016/j.exppara.2011.06.011. PMid:21762693.
http://dx.doi.org/10.1016/j.exppara.2011...
). Essential oils (EOs) with different types of biological activity such as antimalarial, antibacterial, fungicidal, anthelmintic and acaricide activity can be extracted from this plant (Campbell et al., 1997Campbell WE, Gammon DW, Smith P, Abrahams M, Purves TD. Composition and antimalarial activity in vitro of the essential oil of Tetradenia riparia. Planta Med 1997; 63(3): 270-272. http://dx.doi.org/10.1055/s-2006-957672. PMid:9225614.
http://dx.doi.org/10.1055/s-2006-957672...
; Gazim et al., 2011Gazim ZC, Demarchi IZ, Lonardoni MVC, Amorim ACL, Hovell AMC, Rezende CM, et al. Acaricidal activity of the essential oil from Tetradenia riparia (Lamiaceae) on the cattle tick Rhipicephalus (Boophilus) microplus (Acari; Ixodidae). Exp Parasitol 2011; 129(2): 175-178. http://dx.doi.org/10.1016/j.exppara.2011.06.011. PMid:21762693.
http://dx.doi.org/10.1016/j.exppara.2011...
; Kakande et al., 2019Kakande T, Batunge Y, Eilu E, Shabohurira A, Abimana J, Akinola SA, et al. Prevalence of dermatophytosis and antifungal activity of ethanolic crude leaf extract of Tetradenia riparia against dermatophytes isolated from patients attending Kampala International University Teaching Hospital, Uganda. Dermatol Res Pract 2019; 2019: 9328621. http://dx.doi.org/10.1155/2019/9328621. PMid:31379937.
http://dx.doi.org/10.1155/2019/9328621...
; Van Puyvelde et al., 2018Van Puyvelde L, Liu M, Veryser C, Borggraeve WMD, Mungarulire J, Mukazayire J, et al. Active principles of Tetradenia riparia. IV. Anthelmintic activity of 8(14),15-sandaracopimaradiene-7α,18-diol. J Ethnopharmacol 2018; 216: 229-232. http://dx.doi.org/10.1016/j.jep.2018.01.024. PMid:29366765.
http://dx.doi.org/10.1016/j.jep.2018.01....
).

The aim of the present study was to evaluate the potential for larvicidal activity on R. (B.) microplus larvae, through use of T. riparia essential oils (EOs) extracted from the following botanical structures: leaves, flower buds and stems.

Materials and Methods

Plant material

Botanical structures (leaves, flower buds and stems) of T. riparia were collected in the winter season (May-July) of 2020. The plants had been cultivated in the Medicinal Garden of Universidade Paranaense (UNIPAR), located in the city of Umuarama, northwestern region of the state of Parana, Brazil (latitude 23° 46' 22” S, longitude 53° 16' 73” W; altitude 391 m). The plant was identified by Professor Ezilda Jacomasi of the Department of Pharmacy of Paranaense Univeresity (UNIPAR), Paraná. A voucher specimen was deposited at the UNIPAR Herbarium (code number 2502). This species is registered in the National System for Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) under the registration number AFFE469.

Obtaining essential oils

Tetradenia riparia essential oils (EOs) were obtained separately from different botanical structures (leaves, flower buds and stems). All the plant material was dried at room temperature in a covered and ventilated place and was then ground up in a knife mill. The extraction process consisted of hydrodistillation for three hours in a modified Clevenger apparatus (Gazim et al., 2010Gazim ZC, Amorim ACL, Hovell AMC, Rezende CM, Nascimento IA, Ferreira GA, et al. Seasonal variation, chemical composition, and analgesic and antimicrobial activities of the essential oil from leaves of Tetradenia riparia (Hochst.) Codd in southern Brazil. Molecules 2010; 15(8): 5509-5524. http://dx.doi.org/10.3390/molecules15085509. PMid:20714310.
http://dx.doi.org/10.3390/molecules15085...
). After extraction and separation, the essential oils (EOs) were stored in amber flasks and kept under refrigeration at 4 °C until the time of the experiment. The essential oil yield was determined through calculating the ratio of the mass of dry leaves, fresh flower buds and stems (g) divided by the mass of essential oil (g) (%).

Chemical identification of essential oils

Chemical identification of the EOs was performed by means of gas chromatography-mass spectrometry (GC-MS) using a gas chromatograph (Agilent 7890B) coupled to a mass spectrometer, (Agilent 5977A MSD) equipped with capillary column HP5-MS UI 5% (30 × 250 µm × 0.25 µm; Agilent Technologies) and with automatic injector (CTC PAL Control). In order to perform a proper separation of the analytes in the GC-MS system, 2 μL of EO were injected into the column using Split injection mode in a 1:30 ratio, with injector temperature of 260 oC, flow of carrier gas He of 1 mL min-1 and under the following oven conditions: initial temperature of 80 °C, then ramp from 4 °C min-1 to 260 °C and lastly ramp from 40 °C min-1 to 300 °C. The transfer line was kept at 280 °C and the ionization source and quadrupole at 230 °C and 150 °C, respectively. The detection system was EM (Gain 1.5), in scan mode, in the mass-to-charge ratio range of 40 to 550 m/z, with a “solvent delay” of 3 min. The volatile compounds were identified by comparisons with mass spectra found with the mass spectra from the NIST Library version 11.0 and with retention indices (RI) that the been obtained from a homologous series of n-alkane (C7-C40) standards (Adams, 2017Adams RP. Identification of essential oil components by gas chromatography/mass spectrometry. Carol Stream: Allured Publishing; 2017 [cited 2022 Jun 10]. Available from: https://vdoc.pub/documents/identification-of-essential-oil-components-by-gas-chromatography-mass-spectrometry-1sa7rroj71d8
https://vdoc.pub/documents/identificatio...
).

Physicochemical indexes of essential oils from T. riparia.

The refractive index was determined in an ABBE refractometer, model RL3, brand PZO Warszawa at 20 °C (Korolkovas et al., 1988Korolkovas A, Colombo AJ, Cunha BCA, Melhem D, Bacchi EM, Ferreira EI, et al. Farmacopéia brasileira. São Paulo: Atheneu; 1988.).

The specific optical rotation was determined using an ACA TEC automated polarimeter (Korolkovas et al., 1988Korolkovas A, Colombo AJ, Cunha BCA, Melhem D, Bacchi EM, Ferreira EI, et al. Farmacopéia brasileira. São Paulo: Atheneu; 1988.). The solvent used for dilution of the EOs was absolute alcohol. The concentration of the EOs from leaves and stems used in this test was 0.156% and the concentration from flower buds was 0.039%, at a temperature of 20 °C, using a 10 cm tube.

The absolute density (g/mL) was determined in a 5 µL graduated capillary tube and was calculated as the ratio of the mass (g) of EOs divided by the volume (mL) that this oil occupied in the capillary tube, both at 20 °C, in accordance with the technique previously described (Korolkovas et al., 1988Korolkovas A, Colombo AJ, Cunha BCA, Melhem D, Bacchi EM, Ferreira EI, et al. Farmacopéia brasileira. São Paulo: Atheneu; 1988.).

Principal Component Analysis (PCA)

Multivariate exploratory evaluation in the form of principal component analysis (PCA) was conducted. This enabled joint evaluation of the major chemical compounds and the chemical classes of all compounds present in the essential oils of the leaves, flower buds and stems. The results from the analysis were presented in graphic form (Biplot), which helped to characterize the groups of variables analyzed (Moita & Moita, 1998Moita JM No, Moita GC. Uma introdução à análise exploratória de dados multivariados. Quím Nova 1998; 21(4): 467-469. http://dx.doi.org/10.1590/S0100-40421998000400016.
http://dx.doi.org/10.1590/S0100-40421998...
). For each sample of EOs obtained from the leaves, flower buds and stems, the major chemical compounds identified, their respective chemical classes and the amounts (area % on the plots) were transformed into orthogonal latent variables called principal components, which were linear combinations of the original variables created, with the eigenvalues of the data covariance matrix. The Kaiser criterion was used to select the principal components. Their eigenvalues preserved relevant information when they were greater than unity (Camacho et al., 2010Camacho J, Picó J, Ferrer A. Data understanding with PCA: structural and variance information plots. Chemom Intell Lab Syst 2010; 100(1): 48-56. http://dx.doi.org/10.1016/j.chemolab.2009.10.005.
http://dx.doi.org/10.1016/j.chemolab.200...
; Ferré, 1995Ferré L. Selection of components in principal component analysis: a comparison of methods. Comput Stat Data Anal 1995; 19(6): 669-682. http://dx.doi.org/10.1016/0167-9473(94)00020-J.
http://dx.doi.org/10.1016/0167-9473(94)0...
). This analysis was performed in two ways: the first contained only the data referring to the major compounds identified in the EOs of the leaves, flower buds and stems. The second contained the data referring to the chemical classes to which these compounds belonged. Both analyses were performed using the Statistica 13 software (StatSoft Inc.).

Collection of engorged females

Engorged females of R. (B) microplus were randomly collected (n ≅ 300) from naturally infested dairy cattle belonging to a farm in the municipality of São Tomé, northwestern region of the state of Parana, Brazil (latitude 23° 31' 22” S; longitude 52° 35' 08” W). On this farm, the cattle were only receiving homeopathic treatments and, at the time of tick collection, they had gone approximately 240 days without receiving any treatment.

The ticks collected were transported in a container with adequate aeration to the UNIPAR Parasitology Laboratory. In the laboratory, only the engorged females were selected, on the basis of normal appearance and motility, intact body and maximum engorgement with homogeneous weight (Medeiros et al., 2019Medeiros JP, Bortollucci WC, Silva ES, Oliveira HLM, Campos CFAA, Gonçalves JE, et al. Biocidal potential of Eugenia pyriformis essential oil in the control of Rhipicephalus (Boophilus) microplus in the free-living cycle. Pesqui Vet Bras 2019; 39(11): 879-888. http://dx.doi.org/10.1590/1678-5150-pvb-6434.
http://dx.doi.org/10.1590/1678-5150-pvb-...
). These were placed, without treatment, in a chamber with temperature-controlled environmental conditions that resembled the tick’s natural environment: approximately 28 °C; relative humidity (RH) ≥ 80%; and light/dark photoperiods of 12 h each. The aim was to produce larvae for the experimental tests. The average age of larvae used in the experiments was 21 days after hatching.

In vitro larvicidal activity of essential oils from T. riparia

For the R. (B.) microplus larvae to undergo the larval packet test (LPT), they were placed in sealed filter paper envelopes (Stone & Haydock, 1962Stone BF, Haydock KP. A method for measuring the acaricide-susceptibility of the cattle tick Boophilus microplus (Can.). Bull Entomol Res 1962; 53(3): 563-578. http://dx.doi.org/10.1017/S000748530004832X.
http://dx.doi.org/10.1017/S0007485300048...
), as adapted by Monteiro et al. (2012)Monteiro CM, Maturano R, Daemon E, Catunda-Junior FEA, Calmon F, Senra TS, et al. Acaricidal activity of eugenol on Rhipicephalus microplus (Acari: Ixodidae) and Dermacentor nitens (Acari: Ixodidae) larvae. Parasitol Res 2012; 111(3): 1295-1300. http://dx.doi.org/10.1007/s00436-012-2964-0. PMid:22622689.
http://dx.doi.org/10.1007/s00436-012-296...
. The EOs were applied homogeneously at concentrations of (100,000; 50,000; 25,000; 12,500; 6,250; 3,120; 1,560; 780; 390; 190; 95 and 40 µg/mL) (mass/volume) using 2.00% polysorbate 80 (v/v) as the emulsifier and purified water as the solvent (Chagas et al., 2012Chagas ACS, Barros LD, Cotinguiba F, Furlan M, Giglioti R, Oliveira MCS, et al. In vitro efficacy of plant extracts and synthesized substances on Rhipicephalus (Boophilus) Microplus (Acari: ixodidae). Parasitol Res 2012; 110(1): 295-303. http://dx.doi.org/10.1007/s00436-011-2488-z. PMid:21695568.
http://dx.doi.org/10.1007/s00436-011-248...
). An aqueous solution of 2.00% polysorbate 80 (v/v) was used as a negative control (NC). A commercial solution at 0.125% (v/v) containing 15.00% cypermethrin, 25.00% chlorpyrifos and 1.00% citronellal was used as a positive control (PC). Larvae that were found to be immobile upon being touched, 24 hours after application of the solution, were considered dead. The percentage larval mortality was calculated from this (Equation 1).

M o r t a l i t y % = d e a d l a r v a e x 100 / t o t a l l a r v a e (1)

The lethal concentrations (LCs) that killed 99.9% (LC99.9) of the tick larvae population, with their respective confidence intervals (CI), were calculated by means of Probit analysis (ED 50 Plus, version 1.0). All tests were performed in triplicate. The EO concentrations that gave rise to LC99.9, obtained from the LPT, were used in a subsequent test for ex situ control of ectoparasites in plastic pots.

Larvicidal activity of essential oils from T. riparia under semi-natural conditions

Plastic pots (n = 15) of dimensions 25 cm in height and 25 cm in diameter were each filled with 2.20 kg of soil and seedlings of the grass species Brachiaria decumbens were planted. The plants were kept in a greenhouse for three months with irrigation. At the time of the tests, the leaves of B. decumbens were cut 40 cm from the soil surface, and adhesive tape was placed around the rim of each pots as a physical barrier to contain the larvae of R. (B.) microplus (40 mg) which were deposited on the soil surface of each plant pot. After 24 h, it was observed that the larvae had migrated to the apex of the grass leaves (Araújo et al., 2015Araújo LX, Novato TPL, Zeringota V, Matos RS, Senra TOS, Maturano R, et al. Acaricidal activity of thymol against larvae of Rhipicephalus microplus (Acari: Ixodidae) under semi-natural conditions. Parasitol Res 2015; 114(9): 3271-3276. http://dx.doi.org/10.1007/s00436-015-4547-3. PMid:26040843.
http://dx.doi.org/10.1007/s00436-015-454...
). Each group of three pots constituted one treatment. The LC99.9 obtained through the LPT for the EOs from the leaves, flower buds and stems were used for the treated group, plus 20.00% mineral oil, 2.00% polysorbate 80 (v/v) and distilled water qsp (sufficient quantity), up to a volume of 15.00 mL.

A commercial acaricide solution was used as the positive control and an aqueous solution of 2.00% polysorbate 80 (mass/volume) plus 20.0% mineral oil was used as the negative control. For each treatment, 5.00 mL of each solution was sprayed on separately, in the respective pots, starting at the top of the plant and going down to the base only once. This simulated application of commercial acaricides to pastures. After 24 h, the leaves of B. decumbens were cut and, with the aid of a stereomicroscope, the larvae were counted. Larvae that did not show motility upon being touched were considered dead. Next, the mean numbers of live larvae in the NC group and in the group of larvae treated with EOs were determined. From these data, the percentage efficiency of the EOs was calculated, as shown in Equation 2, as described by Bittencourt et al. (2003)Bittencourt VREP, Bahiense TC, Fernandes EK, Souza EJ. Avaliação da ação in vivo de Metarhizium anisopliae (Metschnikoff, 1879) Sorokin, 1883 aplicado sobre Brachiaria decumbens infestada com larvas de Boophilus microplus (Canestrini, 1887) (Acari: ixodidae). Rev Bras Parasitol Vet 2003; 12(1): 38-42..

E f f i c i e n c y o f T r E O s % = A B / A x 100 (2)

Where A = average number of live larvae in the negative control group and B = average number of live larvae in the groups treated with TrEOs.

Statistical analysis

The experimental design was completely randomized (DIC). Data were subjected to analysis of variance (ANOVA) and differences between arithmetic means and standard deviation were determined using Tukey's test, with a significance level of 5%. The lethal concentrations (LCs) that killed 50.0% (LC50.0) and 99.9% (LC99.9) of R. (B.) microplus larvae and their respective CIs were calculated by means of Probit analysis (ED 50 Plus version 1.0). All tests were performed in triplicate.

Results

The essential oils extracted from T. riparia (EOs) have a characteristic smell of incense, and their color ranges in intensity from lighter orange (stems) to darker orange in the leaves and reddish orange in the flower buds. The absolute density (g/mL), specific optical rotation, refractive index and yield (%) are shown in Table 1. The chemical composition of the EOs obtained through GC-MS indicated the presence of 61 compounds in the leaves, 49 in the flower buds and 55 in the stems. Compounds in the class of sesquiterpenes (both oxygenates and hydrocarbons) were most abundant, accounting for 61.12%, 60.68% and 67.01% of the compounds found in leaves, flower buds and stems, respectively (Table 2).

Table 1
Physicochemical indices for absolute density (g/mL), refractive index, specific optical rotation and yield (%), of the essential oils from the leaves, flower buds and stems of Tetradenia riparia.
Table 2
Chemical composition of the essential oils from the leaves, flower buds and stems of Tetradenia riparia.

Class projections from principal component analysis (PCA) indicated that factor 1 accounted for 97.02% of the variability of the EOs found in the different botanical structures. EOs from leaves and stems exhibited a vector with a smaller angle, thus revealing that these structures were positively correlated with the class of oxygenated sesquiterpenes (38.74% and 39.13%, respectively). Moreover, the EOs of the flower buds and stems showed a positive correlation with the class of hydrocarbon sesquiterpenes (29.76% and 27.88%, respectively) (Figure 1 and Table 2).

Figure 1
Biplot representing the projection of chemical classes of essential oils from the leaves, flower buds and stems of Tetradenia riparia, obtained by means of gas chromatography coupled to mass spectrometry (GC/MS).

The projections from PCA on compound groupings indicated that factor 1 accounted for 67.28% of the variability of the TrEOs found in the different botanical structures. The EOs of the leaves and stems did not show angle separation and indicated that they were absolutely correlated with α-cadinol compounds (7.56% and 4.85%, respectively), fenchone (7.09% and 4.51%) and 6-7-dehydroroyleanone (7.27% and 9.58%). Although the EOs of the flower buds exhibited a vector with a large angle in relation to the other structures, there were positive correlations with the compounds 14-hydroxy-9-epi-caryophyllene (10.16%, 9.61% and 6.26%), caryophyllene (5.84%, 6.37% and 6.35%) and 9β,13β-epoxy-7-abietene (6.47%, 6.86% and 5.40%) for the leaves, flower buds and stems, respectively (Figure 2 and Table 2).

Figure 2
Biplot representing the projection of major compounds in the essential oils from the leaves, flower buds and stems of Tetradenia riparia, obtained by means of gas chromatography coupled to mass spectrometry (GC/MS).

The results regarding the larvicidal potential of R. (B.) microplus exposed to different concentrations of EOs through the LPT test indicated that the EOs from the botanical structures of the leaves, flower buds and stems yielded high mortality rates at concentrations of 100,000 and 50,000 µg/mL, without significant differences in relation to the PC, which killed 100% of the larvae.

At the concentration of 25,000 µg/mL, the leaves and stems presented mortality rates that were considered high (80.54% and 91.63%, respectively), without any significant difference between these structures. However, the floral buds showed worse performance (66.36%). It is important to point out that the negative control showed zero percentage mortality with regard to EOs from all three botanical structures, and that this result differed significantly from the other results (Table 3).

Table 3
Mortality rate (%) among larvae of Rhipicephalus (Boophilus) microplus subjected to treatments with essential oils from the leaves, flower buds and stems of Tetradenia riparia, at different concentrations.

The lethal concentrations (LC) were determined by means of Probit analysis. The results showed that the TrEOs from the leaves were most effective, requiring the lowest concentration (LC99.9 = 83.53 µg/mL), in comparison with the other botanical structures: floral buds and stems showed (LC99.9 = 91.91 and 85.42 µg/mL), respectively. In all treatments, LC99.9 were significantly different for positive control (Table 4).

Table 4
Lethal concentrations (LC50.0 and LC99.9) in µg/mL, as the arithmetic mean with standard deviation and confidence interval (CI), of the essential oils from leaves, flower buds and stems of Tetradenia riparia on larvae of Rhipicephalus (Boophilus) microplus by Probit analysis.

After all LC99.9 values had been determined (Table 4), further assays were performed under semi-natural conditions (ex situ) to determine the product efficiency (PE) of the TrEOs used in each treatment. The effectiveness of the TrEOs from all the botanical structures (leaves, flower buds and stems) were found to be 98.25%, 98.48% and 98.14%, respectively. These values were not significantly different from the positive control, which showed efficacy of 100%. The negative control showed zero percent PE, which was significantly different from all the treatments (Table 5).

Table 5
Mean lethal concentration (LC99.9 µg/mL), average number of live larvae (ALL) in pot tests (ex situ) and product efficiency (PE%) of the essential oils from the leaves, flower buds and stems of Tetradenia riparia, acting against the larvae of Rhipicephalus (Boophilus) microplus, determined through Probit analysis.

Discussion

The EOs presented yields of between 0.13% and 0.86%, and the OE of the leaves was the one that presented the highest yield (0.86%) (Table 1). Comparatively, in similar studies on EOs extracted from T. riparia leaves, yields of between 0.26% and 0.29% were found (Cardoso et al., 2015Cardoso BM, Mello TFP, Lopes SN, Demarchi IG, Lera DSL, Pedroso RB, et al. Antileishmanial activity of the essential oil from Tetradenia riparia obtained in different seasons. Mem Inst Oswaldo Cruz 2015; 110(8): 1024-1034. http://dx.doi.org/10.1590/0074-02760150290. PMid:26602873.
http://dx.doi.org/10.1590/0074-027601502...
; Gazim et al., 2010Gazim ZC, Amorim ACL, Hovell AMC, Rezende CM, Nascimento IA, Ferreira GA, et al. Seasonal variation, chemical composition, and analgesic and antimicrobial activities of the essential oil from leaves of Tetradenia riparia (Hochst.) Codd in southern Brazil. Molecules 2010; 15(8): 5509-5524. http://dx.doi.org/10.3390/molecules15085509. PMid:20714310.
http://dx.doi.org/10.3390/molecules15085...
; Zardeto-Sabec et al., 2020Zardeto-Sabec G, Jesus RA, Oliveira HLM, Campo CFAA, Jacomassi E, Gonçalves JE, et al. Tetradenia riparia (Lamiaceae) essential oil: an alternative to Rhipicephalus sanguineus. Aust J Crop Sci 2020; 14(10): 1608-1615. http://dx.doi.org/10.21475/ajcs.20.14.10.p2389.
http://dx.doi.org/10.21475/ajcs.20.14.10...
). These previous studies all used leaf structures collected in winter, which was also the seasonal period of the present study.

Araújo (2014)Araújo LLN. Características morfofisiológicas, produção e composição de óleo essencial em folhas de Tetradenia riparia (Hochst) Codd-Lamiaceae cultivada em diferentes níveis de sombreamento [dissertation]. Goiânia: Universidade Federal de Goiás; 2014. evaluated the effect of luminosity on T. riparia and found that the maximum yield of EOs was 0.26% in situations of 30% shading, thus proving the effect of light incidence on the yield of EOs. The availability of solar radiation interferes with the leaf morphophysiology of plants and is strongly related to abiotic factors (Araújo et al., 2019Araújo LLN, Melo HC, Castiglioni GL, Gonçalves LA. Intensidade de radiação influenciando características morfofisiológicas em folhas de Tetradenia riparia (Hochst.) Codd. Iheringia Sér Bot 2019; 74: e2019001. http://dx.doi.org/10.21826/2446-82312019v74e2019001.
http://dx.doi.org/10.21826/2446-82312019...
). Consequently, it influences the yield of EOs from many plant species. This would explain the different rates of TrEO yields found by different authors.

The physicochemical parameters of the EOs from all botanical structures were also analyzed, with the following results: absolute density (AD) between 0.83 and 0.90 (g/mL); refractive index (RI) between 1.4971 and 1.5588; and specific optical rotation (SOR) between -12.01° and -26.33°. The ranges of these parameters were equivalent to those found in the literature for EOs extracted from T. riparia leaves, regarding AD 0.88 to 0.95 (g/mL) and RI (1.2916 to 1.4685), but the range of SOR found here was significantly different to what had previously been reported in the literature (Campbell et al., 1997Campbell WE, Gammon DW, Smith P, Abrahams M, Purves TD. Composition and antimalarial activity in vitro of the essential oil of Tetradenia riparia. Planta Med 1997; 63(3): 270-272. http://dx.doi.org/10.1055/s-2006-957672. PMid:9225614.
http://dx.doi.org/10.1055/s-2006-957672...
; Gazim et al., 2010Gazim ZC, Amorim ACL, Hovell AMC, Rezende CM, Nascimento IA, Ferreira GA, et al. Seasonal variation, chemical composition, and analgesic and antimicrobial activities of the essential oil from leaves of Tetradenia riparia (Hochst.) Codd in southern Brazil. Molecules 2010; 15(8): 5509-5524. http://dx.doi.org/10.3390/molecules15085509. PMid:20714310.
http://dx.doi.org/10.3390/molecules15085...
). Hypothetically, these variations in SOR can be explained in terms of differences between geographical regions, seasonal periods and climatic and edaphic conditions, in relation to those of the present study (Table 1). Nevertheless, these indices need to be taken into account in industrial-scale production, in order to determine the final quality of the product, in view of the instability of EOs in relation to light, humidity, heat and oxygen levels (Medeiros et al., 2019Medeiros JP, Bortollucci WC, Silva ES, Oliveira HLM, Campos CFAA, Gonçalves JE, et al. Biocidal potential of Eugenia pyriformis essential oil in the control of Rhipicephalus (Boophilus) microplus in the free-living cycle. Pesqui Vet Bras 2019; 39(11): 879-888. http://dx.doi.org/10.1590/1678-5150-pvb-6434.
http://dx.doi.org/10.1590/1678-5150-pvb-...
).

The EOs from the leaves, flower buds and stems showed a complex mixture of chemical compounds. The analyses performed by means of GC/MS showed that the largest class of compounds identified were sesquiterpenes (both hydrocarbons and oxygenates), which accounted for 61.12%, 60.68% and 67.01% of the EOs from the leaves, flower buds and stems, respectively (Table 2). In the multivariate exploratory analysis to determine the major classes through PCA, it was seen that the EOs from the leaves and stems, and those from the flower buds and stems, were positively correlated with the classes of oxygenated and hydrocarbon sesquiterpenes, respectively (Figure 1).

Similar fractions of these classes of compounds were found by Zardeto-Sabec et al. (2020)Zardeto-Sabec G, Jesus RA, Oliveira HLM, Campo CFAA, Jacomassi E, Gonçalves JE, et al. Tetradenia riparia (Lamiaceae) essential oil: an alternative to Rhipicephalus sanguineus. Aust J Crop Sci 2020; 14(10): 1608-1615. http://dx.doi.org/10.21475/ajcs.20.14.10.p2389.
http://dx.doi.org/10.21475/ajcs.20.14.10...
, in leaves (61.41%) and flower buds (65.25%) of T. riparia, with positive correlations of sesquiterpenes (both oxygenates and hydrocarbons) for EOs from both botanical structures. In a study conducted by Campbell et al. (1997)Campbell WE, Gammon DW, Smith P, Abrahams M, Purves TD. Composition and antimalarial activity in vitro of the essential oil of Tetradenia riparia. Planta Med 1997; 63(3): 270-272. http://dx.doi.org/10.1055/s-2006-957672. PMid:9225614.
http://dx.doi.org/10.1055/s-2006-957672...
on the African continent, the EOs diverged regarding the class of compounds, such that monoterpenes (both hydrocarbons and oxygenates) comprised the majority (69.00%), thus confirming that these plants produce their secondary metabolites in accordance with their physiological needs and the conditions of the environment.

Evaluation of larvicidal activity showed that all the EOs from the three botanical structures analyzed significantly promoted mortality at concentrations of 100,000 and 50,000 µg/mL. This larvicidal action was already foreseeable, given the different substances with proven biological activity previously found in the EOs from this plant, with insecticidal (Fernandez et al., 2014Fernandez CMM, Barba EL, Fernandez ACM, Cardoso BK, Borges IB, Takemura OS, et al. Larvicidal activity of essential oil from Tetradenia riparia to control of Aedes aegypti larvae in function of season variation. J Essent Oil-Bear Plants 2014; 17(5): 813-823. http://dx.doi.org/10.1080/0972060X.2014.892841.
http://dx.doi.org/10.1080/0972060X.2014....
; Oliveira et al., 2022Oliveira AC, Simões RC, Tavares CPS, Lima CAP, Sá ISC, Silva FMA, et al. Toxicity of the essential oil from Tetradenia riparia (Hochstetter.) Codd (Lamiaceae) and its principal constituent against malaria and dengue vectors and non-target animals. Pestic Biochem Physiol 2022; 188: 105265. http://dx.doi.org/10.1016/j.pestbp.2022.105265. PMid:36464370.
http://dx.doi.org/10.1016/j.pestbp.2022....
). The results from the present study confirm the existence of acaricidal activity caused by EOs, which was demonstrated for the first time by Gazim et al. (2010)Gazim ZC, Amorim ACL, Hovell AMC, Rezende CM, Nascimento IA, Ferreira GA, et al. Seasonal variation, chemical composition, and analgesic and antimicrobial activities of the essential oil from leaves of Tetradenia riparia (Hochst.) Codd in southern Brazil. Molecules 2010; 15(8): 5509-5524. http://dx.doi.org/10.3390/molecules15085509. PMid:20714310.
http://dx.doi.org/10.3390/molecules15085...
.

Studies have correlated monoterpenes and sesquiterpenes with the biocidal action of EOs from different plants. Although the mechanism of action is not fully understood, it is already described in the literature, several monoterpenes are neurotoxic because they inhibit the enzyme acetylcholinesterase, and have the ability to interfere with the metabolic functions of arthropods (Jankowska et al., 2018Jankowska M, Rogalska J, Wyszkowska J, Stankiewicz M. Molecular targets for components of essential oils in the insect nervous system - a review. Molecules 2018; 23(1): 34. http://dx.doi.org/10.3390/molecules23010034. PMid:29295521.
http://dx.doi.org/10.3390/molecules23010...
; Medeiros et al., 2019Medeiros JP, Bortollucci WC, Silva ES, Oliveira HLM, Campos CFAA, Gonçalves JE, et al. Biocidal potential of Eugenia pyriformis essential oil in the control of Rhipicephalus (Boophilus) microplus in the free-living cycle. Pesqui Vet Bras 2019; 39(11): 879-888. http://dx.doi.org/10.1590/1678-5150-pvb-6434.
http://dx.doi.org/10.1590/1678-5150-pvb-...
; Yang et al., 2021Yang P, Jia M, Zhu L. Acaricidal activity of the essential oil from Senecio cannabifolius and its constituents eucalyptol and camphor on engorged females and larvae of Rhipicephalus microplus (Acari: ixodidae). Exp Appl Acarol 2021; 83(3): 411-426. http://dx.doi.org/10.1007/s10493-021-00590-x. PMid:33590356.
http://dx.doi.org/10.1007/s10493-021-005...
). For example, 1,8-cineol (eucalyptol) extracted from Lippia alba has remarkable insecticidal activity (Lima et al., 2021Lima TA, Baptista NMQ, Oliveira APS, Silva PA, Gusmão NB, Correia MTS, et al. Insecticidal activity of a chemotype VI essential oil from Lippia alba leaves collected at Caatinga and the major compound (1,8-cineole) against Nasutitermes corniger and Sitophilus zeamais. Pestic Biochem Physiol 2021; 177: 104901. http://dx.doi.org/10.1016/j.pestbp.2021.104901. PMid:34301362.
http://dx.doi.org/10.1016/j.pestbp.2021....
). Moreover, eucalyptol extracted from the EOs of Senecio cannabifolius (Yang et al., 2021Yang P, Jia M, Zhu L. Acaricidal activity of the essential oil from Senecio cannabifolius and its constituents eucalyptol and camphor on engorged females and larvae of Rhipicephalus microplus (Acari: ixodidae). Exp Appl Acarol 2021; 83(3): 411-426. http://dx.doi.org/10.1007/s10493-021-00590-x. PMid:33590356.
http://dx.doi.org/10.1007/s10493-021-005...
), Melinis minutiflora (Prates et al., 1998Prates HT, Leite RC, Craveiro AA, Oliveira AB. Identification of some chemical componentes of the essential oil from Molasses Grass (Melinis minutiflora Beauv.) and their activity against Cattle-Tick (Boophilus microplus). J Braz Chem Soc 1998; 9(2): 193-197. http://dx.doi.org/10.1590/S0103-50531998000200013.
http://dx.doi.org/10.1590/S0103-50531998...
) and Eucalyptus globulus (Chagas et al., 2002Chagas ACS, Passos WM, Prates HT, Leite RC, Furlong J, Fortes ICP. Efeito acaricida de óleos essenciais e concentrados emulsionáveis de Eucalyptus spp em Boophilus microplus. Braz J Vet Res Anim Sci 2002; 39(5): 247-253. http://dx.doi.org/10.1590/S1413-95962002000500006.
http://dx.doi.org/10.1590/S1413-95962002...
) was found to be able to kill 100% of R. (B.) microplus larvae in the respective studies. Limonene extracted from the EOs of Pilocarpus spicatus was found to have intense repellent activity against R. (B.) microplus (Nogueira et al., 2020Nogueira JAP, Figueiredo A, Duarte JL, Almeida FB, Santos MG, Nascimento LM, et al. Repellency effect of Pilocarpus spicatus A. St.-Hil essential oil and nanoemulsion against Rhipicephalus microplus larvae. Exp Parasitol 2020; 215: 107919. http://dx.doi.org/10.1016/j.exppara.2020.107919. PMid:32442440.
http://dx.doi.org/10.1016/j.exppara.2020...
). In a study by Gazim et al. (2011)Gazim ZC, Demarchi IZ, Lonardoni MVC, Amorim ACL, Hovell AMC, Rezende CM, et al. Acaricidal activity of the essential oil from Tetradenia riparia (Lamiaceae) on the cattle tick Rhipicephalus (Boophilus) microplus (Acari; Ixodidae). Exp Parasitol 2011; 129(2): 175-178. http://dx.doi.org/10.1016/j.exppara.2011.06.011. PMid:21762693.
http://dx.doi.org/10.1016/j.exppara.2011...
on TrEOs, the compounds fenchone and limonene were found to be responsible for intense biocidal activity in different life stages of R. (B.) microplus.

High synergism with high larval mortality rates for R. (B.) microplus has been demonstrated in relation to combinations of carvacrol, eugenol and thymol extracted from the EOs of Syzygium aromaticum, Cinnamomum zeylanicum and Cymbopogon citratus (Jyoti et al., 2019Jyoti, Singh NK, Singh H, Mehta N, Rath SS. In vitro assessment of synergistic combinations of essential oils against Rhipicephalus (Boophilus) microplus (Acari: ixodidae). Exp Parasitol 2019; 201: 42-48. http://dx.doi.org/10.1016/j.exppara.2019.04.007. PMid:31034814.
http://dx.doi.org/10.1016/j.exppara.2019...
). Similar studies conducted on other species of mites and even on insects have shown that thymol is 100% effective in its larvicidal activity against the species Rhipicephalus annulatus, at a concentration of 2.50% (Arafa et al., 2020Arafa WM, Aboelhadid SM, Moawad A, Shokeir K, Ahmed O. Toxicity, repellency and anti-cholinesterase activities of thymol-eucalyptus combinations against phenotypically resistant Rhipicephalus annulatus ticks. Exp Appl Acarol 2020; 81(2): 265-277. http://dx.doi.org/10.1007/s10493-020-00506-1. PMid:32472469.
http://dx.doi.org/10.1007/s10493-020-005...
). The high proportion of thymol in the EOs of Lantana camara has also been correlated with intense insecticidal activity against Sitophilus granarius, thus confirming that the larvicidal action of monoterpenes is efficiently expanded even to arthropods of other classes.

With regard to sesquiterpenes, Sadgrove et al. (2020)Sadgrove NJ, Senbill H, Van Wyk BE, Greatrex BW. New labdanes with antimicrobial and acaricidal activity: terpenes of Callitris and Widdringtonia (Cupressaceae). Antibiotics 2020; 9(4): 173. http://dx.doi.org/10.3390/antibiotics9040173. PMid:32290471.
http://dx.doi.org/10.3390/antibiotics904...
demonstrated that the compounds spathulenol and guaiol had antimicrobial and acaricidal activity. The chemical components aromadendrene, valencene and caryophyllene oxide, extracted from the leaves of Eugenia langsdorffii, proved to be potent acaricides that led to mortality rates above 80%, as observed using the residual contact method (Moraes et al., 2012Moraes MM, Camara CAG, Santos ML, Fagg CW. Essential oil composition of Eugenia langsdorffii O. Berg.: relationships between some terpenoids and toxicity against Tetranychus urticae. J Braz Chem Soc 2012; 23(9): 1647-1656. http://dx.doi.org/10.1590/S0103-50532012005000029.
http://dx.doi.org/10.1590/S0103-50532012...
). Another sesquiterpene, T-cadinol, isolated from the EOs of Casearia sylvestris, was found to induce mitochondrial impairment of Trypanosoma cruzi, and thus demonstrated protozoicidal effects of importance with regard to formulating future drugs for combating Chagas disease (Santos et al., 2021Santos AL, Amaral M, Hasegawa FR, Lago JHG, Tempone AG, Sartorelli P. (-)-T- Cadinol-a Sesquiterpene isolated from Casearia sylvestris (Salicaceae)-displayed in vitro activity and causes hyperpolarization of the membrane potential of Trypanosoma cruzi. Front Pharmacol 2021; 12: 734127. http://dx.doi.org/10.3389/fphar.2021.734127. PMid:34803682.
http://dx.doi.org/10.3389/fphar.2021.734...
). Because of this high biocidal power seen in relation to several groups of parasites, as confirmed through scientific evidence in the literature, it can be presumed that the larvicidal action seen in the present study was strongly associated with the large amounts of monoterpenes (both hydrocarbons and oxygenates) (17.42%, 17.90% and 12.30%) and sesquiterpenes (both hydrocarbons and oxygenates) (61.12%, 60.68% and 67.01%) that were present in the EOs of the leaves, flower buds and stems of T. riparia, respectively (Table 2).

Based on the larval mortality rate results presented in Table 3, progress was made in conducting other laboratory tests (in vitro and ex situ) to determine the LC50.0 and LC99.9, along with the product efficiency (PE) in relation to the larvae of R. (B.) microplus (Tables 4-5). The LCs were determined through Probit analyses (Table 4), and the botanical structure that proved to be the source of EOs of highest efficiency and greatest larvicidal power was the leaves, from which the concentration of EOs required was the lowest (LC99.9 = 83.53 µg/mL). In a study conducted by Gazim et al. (2011)Gazim ZC, Demarchi IZ, Lonardoni MVC, Amorim ACL, Hovell AMC, Rezende CM, et al. Acaricidal activity of the essential oil from Tetradenia riparia (Lamiaceae) on the cattle tick Rhipicephalus (Boophilus) microplus (Acari; Ixodidae). Exp Parasitol 2011; 129(2): 175-178. http://dx.doi.org/10.1016/j.exppara.2011.06.011. PMid:21762693.
http://dx.doi.org/10.1016/j.exppara.2011...
using EOs obtained from leaves, but harvested in summer, the LC99.9 was 11.38 (g/mL), a LC higher than that of the present study, thus demonstrating lower biocidal effect.

These disparate results can be explained in terms of interference from environmental variations, given that synthesis of secondary metabolites in plants is quantitatively and qualitatively affected by differences in climatic conditions (Hartmann, 1996Hartmann T. Diversity and variability of plant secondary metabolism: a mechanistic view. Entomol Exp Appl 1996; 80(1): 177-188. http://dx.doi.org/10.1111/j.1570-7458.1996.tb00914.x.
http://dx.doi.org/10.1111/j.1570-7458.19...
; Khakdan et al., 2021Khakdan F, Govahi M, Mohebi Z, Ranjbar M. Water deficit stress responses of monoterpenes and sesquiterpenes in different Iranian cultivars of basil. Physiol Plant 2021; 173(3): 896-910. http://dx.doi.org/10.1111/ppl.13485. PMid:34161632.
http://dx.doi.org/10.1111/ppl.13485...
), biotic and abiotic factors (Gobbo-Neto & Lopes, 2007Gobbo-Neto L, Lopes NP. Plantas medicinais: fatores de influência no conteúdo de metabólitos secundários. Quím Nova 2007; 30(2): 374-381. http://dx.doi.org/10.1590/S0100-40422007000200026.
http://dx.doi.org/10.1590/S0100-40422007...
), geographical locations and edaphic conditions (Swamy et al., 2016Swamy MK, Akhtar MS, Sinniah UR. Antimicrobial properties of plant essential oils against human pathogens and their mode of action: an updated review. Evid Based Complement Alternat Med 2016; 2016: 3012462. http://dx.doi.org/10.1155/2016/3012462. PMid:28090211.
http://dx.doi.org/10.1155/2016/3012462...
) and EO extraction and storage methods (Nea et al., 2020Nea F, Kambiré DA, Genva M, Tanoh EA, Wognin EL, Martin H, et al. Composition, seasonal variation, and biological activities of Lantana camara essential oils from Côte d’Ivoire. Molecules 2020; 25(10): 2400. http://dx.doi.org/10.3390/molecules25102400. PMid:32455772.
http://dx.doi.org/10.3390/molecules25102...
). According to Gazim et al. (2010)Gazim ZC, Amorim ACL, Hovell AMC, Rezende CM, Nascimento IA, Ferreira GA, et al. Seasonal variation, chemical composition, and analgesic and antimicrobial activities of the essential oil from leaves of Tetradenia riparia (Hochst.) Codd in southern Brazil. Molecules 2010; 15(8): 5509-5524. http://dx.doi.org/10.3390/molecules15085509. PMid:20714310.
http://dx.doi.org/10.3390/molecules15085...
, the species T. riparia showed high chemical variability of its EOs between the different seasons of the year. This was confirmed by Cardoso et al. (2015)Cardoso BM, Mello TFP, Lopes SN, Demarchi IG, Lera DSL, Pedroso RB, et al. Antileishmanial activity of the essential oil from Tetradenia riparia obtained in different seasons. Mem Inst Oswaldo Cruz 2015; 110(8): 1024-1034. http://dx.doi.org/10.1590/0074-02760150290. PMid:26602873.
http://dx.doi.org/10.1590/0074-027601502...
, who observed seasonality effects relating to the concentrations of TrEO compounds.

After defining LC99.9, the larvicidal activity was tested under semi-natural conditions (ex situ), to verify the chemical stability of the formula when applied in the field under uncontrolled conditions. Twenty-four hours after application of EOs from the leaves (LC99.9 = 83.53 µg/mL), flower buds (LC99.9 = 91.91 µg/mL) and stems (LC99.9 = 85.42 µg/mL), more than 95% of the larvae had died. This demonstrated that the TrEOs has a significant larvicidal effect, and that there were no significant differences between the treatments (Table 5). These results also fell within the guidelines of the World Association for the Advancement of Veterinary Parasitology (WAAVP), which recommend that effectiveness > 95% is required, for commercial acaricides to be described as good quality (Holdsworth et al., 2006Holdsworth PA, Kemp D, Green P, Peter RJ, Bruin CD, Jonsson NN, et al. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guidelines for evaluating the efficacy of acaricides against ticks (Ixodidae) on ruminants. Vet Parasitol 2006; 136(1): 29-43. http://dx.doi.org/10.1016/j.vetpar.2005.11.011. PMid:16377090.
http://dx.doi.org/10.1016/j.vetpar.2005....
).

The high mortality rate presented in this study indicates that the TrEOs from the three botanical structures (leaves, flower buds and stems) showed chemical stability with regard to the formulation components (emulsifiers and fixing agents). No oxidation or hydrolysis reactions occurred in these biomolecules due to abiotic factors (light, humidity, O2 and temperature).

The PE results were extremely satisfactory. All the TrEOs tested showed excellent efficacy: PE = 98.25%, 98.48% and 98.14% for EOs from the leaves, flower buds and stems, respectively. The commercial product used as the PC presented PE of 100%, without any significant differences between the treatments, which proved that the TrEOs were reaching their maximum efficiency of larvicidal activity against the species R. (B.) microplus under semi-natural laboratory conditions (Table 5). The PE of the TrEOs was higher than 95%, which is the minimum percentage effectiveness required by the Brazilian Ministry of Agriculture, Livestock and Supply (MAPA), for new registrations of acaricides in Brazil (Brasil, 1997Brasil. Ministério da Agricultura, Pecuária e Abastecimento. Portaria n. 48 de 12 de maio de 1997. Regulamento técnico para licenciamento e/ou renovação de licença de produtos antiparasitários de uso veterinário. Diário Oficial da República Federativa do Brasil, Brasília, may 1997 [cited 2021 Sep 20]. Available from: https://sistemasweb.agricultura.gov.br/sislegis/action/detalhaAto.do?method=visualizarAtoPortalMapa&chave=72818869#:~:text=REGULAMENTO%20T%C3%89CNICO%20PARA%20LICENCIAMENTO%20E,o%20disposto%20na%20legisla%C3%A7%C3%A3o%20vigente.
https://sistemasweb.agricultura.gov.br/s...
). Thus, the TrEOs of the present study can form excellent prospection sources for synthesis of biomolecules for new chemical formulations of acaricides.

Comparison of the present results with data from similar studies in the literature that used other EOs to combat R. (B.) microplus larvae showed that the TrEOs tested in the present study had better PE, with potential for biocidal activity. Medeiros et al. (2019)Medeiros JP, Bortollucci WC, Silva ES, Oliveira HLM, Campos CFAA, Gonçalves JE, et al. Biocidal potential of Eugenia pyriformis essential oil in the control of Rhipicephalus (Boophilus) microplus in the free-living cycle. Pesqui Vet Bras 2019; 39(11): 879-888. http://dx.doi.org/10.1590/1678-5150-pvb-6434.
http://dx.doi.org/10.1590/1678-5150-pvb-...
studied the EOs of Eugenia pyriformis and found one EO with PE = 72.20% and LC99.9 = 24.60 mg/mL. Their results were considered low because of the high LC. Araújo et al. (2015)Araújo LX, Novato TPL, Zeringota V, Matos RS, Senra TOS, Maturano R, et al. Acaricidal activity of thymol against larvae of Rhipicephalus microplus (Acari: Ixodidae) under semi-natural conditions. Parasitol Res 2015; 114(9): 3271-3276. http://dx.doi.org/10.1007/s00436-015-4547-3. PMid:26040843.
http://dx.doi.org/10.1007/s00436-015-454...
studied thymol, a monoterpene present in the EOs of several plant species, and obtained very similar results, with EP = 99.87% but at a concentration of 20.0 mg/mL, i.e., with a LC that was much higher than that of the present study.

Approximately 95% of the ticks on any farm are found in the pastures in the free-living form (Garcia et al., 2019Garcia MV, Rodrigues VS, Koller WW, Andreotti R. Biologia e importância do carrapato Rhipicephalus (Boophilus) microplus. In: Andreotti R, Garcia MV, Koller WW, editors. Carrapatos na cadeia produtiva de bovinos. Brasília: Embrapa; 2019. p. 17-28.). Moreover, all treatments in the present study showed active high performance, such that they acted efficiently in the initial stages of the parasite cycle, both under laboratory conditions (in vitro) and under semi-natural conditions (ex situ). In addition, the life stage of ticks is an important factor that needs to be considered in order to achieve success in strategic control (Silva et al., 2020Silva LC, Perinotto WMS, Sá FA, Souza MAA, Bitencourt ROB, Sanavria A, et al. In vitro acaricidal activity of Cymbopogon citratus, Cymbopogon nardus and Mentha arvensis against Rhipicephalus microplus (Acari: ixodidae). Exp Parasitol 2020; 216: 107937. http://dx.doi.org/10.1016/j.exppara.2020.107937. PMid:32535114.
http://dx.doi.org/10.1016/j.exppara.2020...
). In this light, it can be asserted that TrEOs constitute alternatives that in the future may help in combating cattle ticks with minimal environmental impact, thereby improving on the existing protocols for treatments at this stage of the cycle of this ectoparasite.

Conclusion

From the results presented in the tests (in vitro and ex situ), it was possible to conclude that the T. riparia essential oils EOs were effective against the larvae of R. (B.) microplus, thus showing that this plant has bioactive molecules with significant larvicidal action. Further studies are needed in order to demonstrate any potential toxicity of EOs towards non-target mammals and other organisms, so as to validate the safety of use of these EOs with regard to the environment and health. Subsequently, the biocidal molecules found in TrEOs could possibly be incorporated into economically viable commercial products for strategic control of cattle ticks.

Acknowledgements

The authors thank Universidade Paranaense, Universidade do Estado do Amazonas, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação Araucária for the support and the fellowship.

  • How to cite: Cella W, Rahal IL, Silva GCC, Jacomassi E, Piau Junior R, Gonçalves JE, et al. Activity of essential oils from leaves, flower buds and stems of Tetradenia riparia on Rhipicephalus (Boophilus) microplus larvae. Braz J Vet Parasitol 2023; 32(1): e013522. https://doi.org/10.1590/S1984-29612023011

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Publication Dates

  • Publication in this collection
    20 Feb 2023
  • Date of issue
    2023

History

  • Received
    07 Oct 2022
  • Accepted
    25 Jan 2023
Colégio Brasileiro de Parasitologia Veterinária FCAV/UNESP - Departamento de Patologia Veterinária, Via de acesso Prof. Paulo Donato Castellane s/n, Zona Rural, , 14884-900 Jaboticabal - SP, Brasil, Fone: (16) 3209-7100 RAMAL 7934 - Jaboticabal - SP - Brazil
E-mail: cbpv_rbpv.fcav@unesp.br