Chemical characterization and evaluation of acaricidal potential of rosemary essential oil and its main compound α-pinene on the two-spotted spider mite, Tetranychus urticae

Abstract Two-spotted spider mite causes significant damage to various crops, often requiring synthetic acaricides for their control. However, the frequent use of these products causes several environmental problems. Thus, this work aimed to evaluate the acaricidal action, using the application by fumigation on female adults of Tetranychus urticae of essential oil from the leaves of Rosmarinus officinalis L., and its major compound, α-pinene. In addition, the essential oil was characterized by gas chromatography and mass spectrometry. α-pinene was the compound with the highest relative area in the oil (29.2%). In fumigation tests, α-pinene showed more significant toxicity than rosemary essential oil on two-spotted spider mite females with LC50 and LC90 values of 1.58 and 49.61 µL/L air, showing the impact of the chemical composition of the essential oil on the biological activity. Graphical Abstract


Introduction
Tetranychus urticae (Koch, 1836), known as the two-spotted spider mite, belongs to the family Tetranychidae (Cazaux et al. 2014). Each female lays an average of 50-60 eggs. The biological cycle (egg to adult) varies from 10 to 12 days at temperatures close to 25 C. The longevity of the adult two-spotted spider mite is around 20-25 days, depending on the conditions of food availability (Ritzinger and Souza 2000;Fadini et al. 2004;Balbino 2006;Moro et al. 2012;Bernardi et al. 2015). With these specifications, the two-spotted spider mite can cause significant damage to several cultures, so its control is essential for maintaining production.
The primary method of controlling the two-spotted spider mite is synthetic acaricides. However, due to the continuous use of these substances, many producers have encountered severe problems with the development of resistance by mites, in addition to this type of product being persistent and ending up leaving residues in food and in the environment, which results in severe problems of contamination (Potenza et al. 2006;Sato et al. 2009;Grbi c et al. 2011;Agut et al. 2018).
The acaricides of natural origin can be an alternative for their safety aspects and the conservation of the agroecosystem balance. However, the option that presents a great advantage for developing safer and more effective bioacaricides are essential oils because they have low toxicity for what is not the target of control and degrade faster (Miguel et al. 2013;Fernandez et al. 2020;Garay et al. 2020;Pereira Junior et al. 2020;Cimmino et al. 2021;El-Mustapha et al. 2021;Gonc¸alves et al. 2021). There is a wide variety of plants with essential oils with numerous properties. We have Rosmarinus officinalis L., commonly known as rosemary, a shrub belonging to the Lamiaceae family, native to the Mediterranean basin (Ban et al. 2016;Oualdi et al. 2022). Rosemary essential oil contains antioxidant, antimicrobial, anti-inflammatory, anticarcinogenic, antidiabetic, antinociceptive, antithrombotic, antiulcerogenic, diuretic, and hepatoprotective properties (Dias et al. 2000;Haloui et al. 2000;Yamamoto et al. 2005;Ra skovi c et al. 2014;Hamidpour et al. 2017).
According to the above information, the objective of this work was to extract and determine the chemical compounds present in the essential oil of R. officinalis leaves and to evaluate the acaricidal effect of rosemary essential oil and its main compound on T. urticae by the route of application by fumigation.

Chemical characterization
The results are represented in Table S1. After analyzing the yield of the essential oil of rosemary, it is possible to observe that the hydrodistillation method is very efficient. The time for the beginning of the condensation was, on average, 40 min, being possible in this time to perceive the formation of the essential oil. At the end of the extraction after 4 h, an average essential oil yield of 1.13% m/m was obtained.
The chromatographic analysis of rosemary essential oil (R. officinalis) made it possible to identify 14 chemical compounds with a relative area greater than 1% (Table  S1). It can be observed that the component with the largest area of the essential oil was a-pinene (29.2%).
In a work by Capatina et al. (2020) with the essential oil of R. officinalis, eucalyptol was found as the main compound, whereas in work by Neves et al. (2019), the main compound was camphor. Based on the results in Table S1 and the literature, we can see that some compounds (a-pinene, camphor, and eucalyptol) are always constant in R. officinalis oil. We see the change of a-pinene compounds, camphor, and eucalyptol as the protagonist of the oil, with the variation of the plant in the different works that approached its essential oil.
The results of the chemical characterization obtained in this work demonstrated that the major compounds identified are similar to the data described in the literature. However, some variations in chemical composition can be attributed to several factors such as different plant origins, location, or age (Miresmailli et al. 2006).

Acaricidal activity of rosemary essential oil and its main compound a-pinene by fumigation
The estimated lethal concentration values (LC 50 and LC 90 ) calculated for the essential oil of R. officinalis, and its main compound, a-pinene, are described in Table S2.
The results obtained show that rosemary essential oil and a-pinene are toxic to T. urticae (Table S2). Higher lethality was observed for a-pinene when compared to essential oil (Figures S1 and S2).
In a work by Miresmailli et al. (2006) with the essential oil of R. officinalis against T. urticae, all the oil's main constituents were tested, the main one being camphor eucalyptol and a-pinene. Miresmailli et al. (2006) found that a-pinene showed less significant toxicity than the other constituents of the essential oil against T. urticae. Miresmailli et al. (2006) also found that rosemary essential oil is more toxic than the isolated compound a-pinene. Contrary to the findings in our experiments (Table S2), a-pinene is more toxic than essential oil.
The indication for a-pinene to present more significant toxicity than the essential oil from R. officinalis, in our experiments (Table S2) may be due to its composition, which in this work has verbenone (25.7%) as the second constituent with the most significant area (Table S1). Contrary to the work of Miresmailli et al. (2006), eucalyptol occupies the second position. In this case, the difference between the proportions of the constituents between the two essential oils could be responsible for the a-pinene being more toxic than the rosemary essential oil in our investigations.
Everything indicates that verbenone would not play the same action and toxicity that eucalyptol together with a-pinene as reported in the work of Miresmailli et al. (2006). Future studies with verbenone on T. urticae may provide information about its toxicity and participation in the toxicity of rosemary oil.
Even though the present study on the essential oil of R. officinalis and its main compound, a-pinene, showed promising results and confirmed its toxicity on T. urticae under laboratory conditions, further studies are needed and carried out in greenhouses, seeking greater effectiveness in controlling the two-spotted spider mite. Studies are also needed to evaluate the toxicity of these oils compared with synthetic acaricides, aiming at more effective control alternatives and less aggressive to the environment, consumer, and producer.

Conclusion
Through the chromatographic analysis of rosemary essential oil, it was possible to identify the presence of 14 compounds. a-pinene was shown to be the main compound with 29.2% relative area. In comparison with the oil, the a-pinene presented more significant toxicity. LC 50 and LC 90 estimated for the essential oil of R. officinalis of 3.03; 64.00 mL/L air, and para and a-pinene were 1.58; 49.61 mL/L air. These results show the impact of the chemical composition of the essential oil on the biological activity since the rosemary oil studied here presents the chemical composition and biological activity different than that studied by Miresmailli et al. (2006).

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The author(s) reported there is no funding associated with the work featured in this article.