ANTI-TERMITE ACTIVITY OF ESSENTIAL OILS FROM ROSMARINUS OFFICINALIS AND CHENOPODIUM AMBROSIOIDES AGAINST TERMITES NASUTITERMES CORNIGER

Objective: This study evaluates the antitermitic activity of Rosmarinus officinalis and Chenopodium ambrosioides essential oils against Nasutitermes corniger termites. Theoretical Framework: Termite control is done using pesticides, but many are toxic to the environment and human health. Among termite species, Nasutitermes corniger is considered a pest in urban areas, due to its high destructive power in construction wood and plants. Method: Essential oils were obtained by hydrodistillation and analyzed by GC-MS. Antitermitic activity was evaluated by bioassay without food choice, at concentrations of 12.5; 25.0; 50.0 and 100 mg/mL, with five repetitions. Results and Discussion: In the composition of R. officinalis essential oils, 51 compounds were identified representing 99.43% and 99.52% (dry and fresh leaves, respectively), with camphor being the majority. For C. ambrosioides , 29 compounds were identified, representing 93.76% and 95.06% of the compositions (dried and fresh leaves, respectively), and the main constituent of both oils was α-terpinene (63.29% and 57.50%). In the antithermic test, the concentration of 100 mg/mL of R. officinalis showed 100% mortality, while the essential oil of C. ambrosioides showed strong antithermic activity with high mortality from the concentration of 25 mg/mL. Research Implications: The research presents the antitermitic potential of the essential oils of Rosmarinus officinalis and Chenopodium ambrosioides against Nasutitermes corniger termites. Originality/Value: The search for new antitermitic products with high biodegradability and low toxicity has become necessary, with essential oils being promising due to their variety of biological properties.


INTRODUCTION
Termites are responsible for the degradation of wood, since they use cellulose as a source of food.Among the termite species, the species Nasutitermes corniger (Isoptera: Termitidae: Nasutitermitinae) is considered a pest in urban areas due to its high destructive power in construction wood and plants, having great proliferation in buildings (Batista et al¹., 2020).The successful transition of this species from rural to urban environments is related to its ability to adapt to the most varied conditions in different environments (Costa et al., 2020).
Termite control is a major challenge around the world.Several pesticides are used to control the pest, but many of its active ingredients are synthetic compounds that are toxic to the environment and human health and still exhibit a high level of environmental persistence (Akbar et al., 2021).Thus, essential oils become an important option for evaluating their antithermic activity, because of their vast potential for biological activities.
This study aimed to evaluate the antithermitic activity of the essential oils of Rosmarinus officinalis and Chenopodium ambrosioides L. against termites of theChenopodium ambrosioides L. The present study contributes to the identification of plants with potential to fight termites, since natural products can be an alternative to the synthetic antithermitytic insecticides currently used.4

THEORETICAL FRAME
The search for new antithermic products with high biodegradability and low toxicity to the environment and to man has become necessary and urgent.In this context, essential oils extracted from aromatic and medicinal plants are promising, as they have a variety of biological properties, including insecticidal activity against different species, among them termites (Clerici et al., 2018;Yang et al., 2023).These properties are attributed to their volatile constituents, mainly terpenes, when acting alone or synergistically (Ferreira et al., 2023).The condition of volatility also allows their rapid elimination from the environment (Pandey et al., 2012).
Among the plants that produce essential oils and that show biological activity, the species Rosmarinus officinalis and Chenopodium ambrosioides were chosen for the evaluation of their antithermitic activities for the species Nasutitermes corniger.Pandey et al. (2012) found a strong anti-thermitic activity of Rosmarinus officinalis essential oil against Odontotermes assamensis Holmgren termites (Isoptera: Termitidae) in Northeast India.Yang et al. (2023) tested the antithermitic activity of nine commercial essential oils of the Lamiaceae family, including the species R. officinalis, but the study was carried out with termites of the speciesReticulitermes dabieshanensis.
The species R. officinalis is a plant in the family Labiatae (Lamiaceae) of the Mediterranean region of Europe.It is also popularly known as rosemary, garden rosemary and rosemary (Lorenzi e Matos, 2006) and its essential oil has a vast biological activity.Among these are the insecticidal activity against the mosquito Aedes aegypti (Duarte, 2015), the Pristiphora abietina (Göktürk, 2022), besides the larvicicidal and adulticidal activities against the mosquito Culex pipiens¹ (Diptera: Culicidae) (Bosly, 2022) and theDroila melanogaster (Pedroso, 2023).Among the main components of its essential oil to which its different biological activities have been attributed are 1,8-cineole, camphor and α-pinene (Borges et al., 2019).
Chenopodium L. (Chenopodiaceae) is a genus comprising about 150 species that grow in arid or semi-arid regions.Essential oils, obtained mainly from C. ambrosioides L. have antifungal and antimicrobial activities (Yilmaz and Kolak, 2024), including insecticide and repellent activities against various insects found in stored products (Colorado et al., 2012).It is also used as an anthelmintic commonly attributed to the high concentration of ascaridol present in the chemical composition of its essential oil (Falcão e Moraes, 2020).Its aqueous extract applied to soil presented high potential (99.3%) in reducing Meloidogyne incognita infection in tomato plant (Maia et al., 2023).

EXTRACTION OF ESSENTIAL OIL
The extractions of the essential oils were carried out by the conventional method of hydrodistillation, using the Clevenger apparatus.The fresh leaves (200g) were ground shortly after harvest and the oils immediately extracted, while another portion of leaves was put to dry in the shade for eight days.The dried leaves (200g) were ground and extracted.The extraction period of the essential oil was 2h, both of the dry leaves and of the fresh leaves.The oils obtained were then treated with anhydrous sodium sulfate, packaged in an amber bottle and kept under refrigeration.

GAS CHROMATOGRAPHY ANALYZES COUPLED TO THE MASS SPECTROMETER (CG-MS)
Essential oil analyzes were performed using a Agilent Technologies (Palo Alto, CA, USA) 5975C simple quadrupole CG-MS equipped with a scientific J&WS nonpolar fused capillary column of dimensions (30m x 0.25mm internal diameter, with film thickness of 0.25mm).The oven was initially kept at 60°C for 3 min, then increased from 2.5°C/min to 240°C and kept at this temperature for 10 min.The drag gas was helium supplied at a constant pressure of 100 kPa, and the split/splitless injector was kept at 250 °C.The ionization potential applied was 70eV, the scanning range was 40 to 350 m/z and the scanning rate was 0.5 per second.Samples (1µL containing 2mg/mL of essential oils in hexane) were introduced into the column with the injector in splitless mode.
The linear retention indices (LRI) were determined for the individual components of essential oils by co-injection of a sample with a mixture of linear hydrocarbons C8-C30 under the conditions described above and subsequent application of the Van de Dool and Kratz equation (1963).The constituents were identified by comparing the LRI values with those published in the literature and confirmed by MS correspondence acquired with those in the CG-MS system library (NIST, Gaitherburg, MD, USA and Wiley, New Jersey, NJ, USA) and with other published data (Adams, 2007).

ANTITHERMAL BIOASSAY
Antithermitic activity was evaluated by a bioassay without a choice of feeding based on the method described by Kang et al. (1990), with some adaptations (Santana et al., 2010).Each experimental unit consisted of a Petri dish (90 X 15 mm) and a 4 cm diameter filter paper disk soaked with 200 µl of essential oil at concentrations of 12.5, 25.0, 50.0 and 100 mg/mL; acetone was used for negative control.A total of 20 termites (16 workers and 4 soldiers) were transferred to each plate.The bioassay was performed in five replicates for each concentration.
The evaluation of termites' survival was done daily for seven days.Survival rates were obtained for each treatment and expressed as mean.The Student t-test was used to compare the means.The differences were considered statistically significant at P <0.05.All statistical analyzes were performed with Origin 7.0 (Microcal, USA).

IDENTIFICATION OF THE CONSTITUENTS OF ESSENTIAL OIL
The chemical identification of essential oils of Rosmarinus officinalis and Chenopodium ambrosioides has been determined and the results shown in Tables 1 and 2.
A total of 51 compounds were identified, representing 99.43% and 99.52% of the composition of the essential oils of the dry and fresh leaves of R. officinalis, respectively (Table 1).Of the total, seven compounds were present only in the fresh leaves and three were detected only in the dry leaves, however, all of these compounds showed very low levels.The fraction of monoterpenes is 98.56% and 98.23% of dry and fresh leaf oils, while the fraction of sesquiterpenes is 0.87% and 1.29%, respectively.The drying of the leaves reduced the content of the major constituents α-pinene (16.44% to 14.39%), myrcene (13.39% to 10.47%) and 1.8cineol (16.95% to 15.69%), while the camphor content increased (22.62% to 31.00%).
Interestingly, while verbenone is a minor constituent, camphor is the most abundant compound which, according to Carreiro et al. (2020) a Constituents listed in order of elution of a nonpolar DB-5 capillary column; bIndexes of linear retention in the literature; cIndexes of linear retention calculated from the retention times relative to those of a series of n-alkanes separated in a nonpolar DB-5 capillary column; dContent (in% of total oil)eNot detected.
In the essential oils obtained from the dry and fresh leaves of Chenopodium ambrosioides, a total of 29 compounds were identified, basically monoterpenes, representing 93.76% and 95.06%, respectively, of their chemical compositions (Table 2).The main constituent of both oils was α-terpinene (63.29% and 57.50%).Other major constituents were o-cymene (17.36% and 21.39%) and ascaridol (6.88% and 12.04%).However, these results differ from those found by Degenhardt et al. (2016), which identified two main compounds pcymene (42.32%) and ascaridol (49.77%) that accounted for 92.09% of the composition of C. ambrosioides essential oil.This is probably because the chemical composition of the essential oil varies greatly depending on the location and time of harvest of the plant material (Sá et al, 2015).
When comparing the chemical constituents of the essential oil of fresh and dried leaves, it was observed that the drying of the leaves reduced the content of ascaridol (12.04% to 6.88%).
On the contrary, the oil from fresh leaves has a lower number of chemical constituents than the oil sample from dry leaves, however, all of these compounds show very low levels.These results are in accordance with the findings of Yadegari et al. (2013) and are important parameters for assessing oil quality.aConstituents listed in order of elution of a nonpolar DB-5 capillary column; bLiterature linear retention indices; cLinear retention indices calculated from retention times relative to those of a series of n-alkanes separated in a nonpolar DB-5 capillary column; dContent (in% of total oil) andNot detected.These results can be explained by the similarity in the chemical composition of the oils.
The essential oil of the dry leaves of Chenopodium ambrosioides showed strong antithermitic activity against Nasutitermes corniger at all concentrations tested, 100% mortality since the first day of bioassay (Figure 2a).The same was observed in the fresh leaves.However, exceptions were shown for the 12.5 mg/mL concentration, which showed 90% survival from the second day.
The oil from the dried leaves of C. ambrosioides showed a greater number of chemical constituents when compared to the fresh leaves of the oil, but both presented the-cymene, αterpinene and ascaridol as main constituents.Nagawa et al. (2015) showed that p-cymene and γ-terpinene have high antithermitic activity against Macrotermes bellicosus.
The essential oil of Alpinia galanga exhibited anti-feeding activity, repellent and toxic effect against the termites Coptotermes gestroi andCoptotermes curvignathus.The activities were attributed to 1,8-cineol, the main constituent of the oil (Abdullah et al., 2015).In the present study, 1,8-cineol was found only in dry leaves, albeit in small quantities.The anti- Larger and more mature groups stay alive longer because they resist stress (Chouvenc, et al.,2014;Patel, et al., 2020).It can be observed that there was mortality of termites in the negative control of the experiments R. officinalis x N. corniger,but, when compared with the concentrations tested, it can be observed that the constituents of the oils showed antithermitic activity.

CONCLUSION
In this study, the antithermitic activity of essential oils of fresh and dried leaves of R.
officinalis and C. ambrosioides was determined against termitesN.corniger.Antithermitic activity was assessed by a bioassay without choice, and the 100 mg/mL concentration of R.
officinalis showed mortality of 100%, with camphor, 1,8-cineol, myrcene and α-pinene oils being their main chemical constituents and recognized as having insecticide activity.The essential oil of C. ambrosioides also showed strong antithermitic activity with high mortality (100%) at the concentrations tested, since the first day, with the exception of the concentration of 12.5 mg/mL, with the main chemical constituents being α-terpinene and-oocymene, compounds proven to be antithermitic.The results suggest that the essential oils of these plants can be considered strong candidates for the development of new natural products for termite control.
___________________________________________________________________________ Rev. Gest.Soc.Ambient.| Miami | v.18.n.8 | p.1-16 | e08525 | 2024.10 4.2 ANTI-THERMITIC ACTIVITYThe antithermitic activity of the essential oil of the dried leaves of Rosmarinus officinalis showed similarity at the concentration of 12.5 and 25 mg/mL, a survival of approximately 50% from the second day of bioassay (Figure1 -A).The 50 mg/mL concentration showed approximately 10% survival on the second day of bioassay.The concentration of 100 mg/mL showed mortality of 100% on the second day of the bioassay.In fresh leaves of Rosmarinus officinalis the concentration of 12.5 mg/mL showed 65% survival on the second day.The concentrations of 25, 50 and 100 mg/mL showed similarity in mortality of approximately 100% on the second day (Figure1 -B).

Figure 1
Figure 1Antithermitic effect of the essential oil of dried leaves (A) and fresh leaves (B) of Rosmarinus officinalis in Nasutitermes corniger the oil may have been enhanced by the synergism of the main constituents, which present, on their own, insecticide activity.Two termite nests were used to perform the antithermitic activity bioassay.One nest for the bioassay of R. officinalis x N. corniger and the other nest for the bioassay of █C.It is believed that because different nests have been used, the size of each individual for each nest is differentiated, interfering with termite behavior.The size of the nest influences its activities.
Yang et al. (2023)l of R. officinalis can be characterized as a chemotype of camphoraceous odor.Already Capella et al. (2022)identified 16 compoundsin the essential oil of Rosmarinus officinalis, with cineole (47.91%) being the major component, while camphor had a content of 17.92%.Yang et al. (2023)found, for the essential oil of R. officinais commercial, values of 11.33% and 45.56%, respectively, for camphor and 1,8-cineol, while other components, present in the chemical composition of this study, were not found in the composition of this oil, such as myrcene, phelandrene, limonene, β-cymene, linalool, terpine-4-ol, citronelol and pulegone.Constituents of the essential oil of the dried and fresh leaves of Rosmarinus officinalis characterized by Gas Chromatography-Mass Spectrometry

Table 2
Constituents of the essential oil of fresh dry leaves of Chenopodium ambrosioides characterized by Gas Chromatography-Mass Spectrometry Roszaini et al. (2013)attributed the insecticide activity of the essential oil of Cinnamomum camphora to the presence of at least two of the main constituents, camphor and linalool, while the antithermitic activity of the essential oil of Alpinia galanga was ascribed to Anti-Termite Activity of Essential Oils From Rosmarinus Officinalis and Chenopodium Ambrosioides Against Termites Nasutitermes Corniger ___________________________________________________________________________ Rev. Gest.Soc.Ambient.| Miami | v.18.n.8 | p.1-16 | e08525 | 2024.