Chemical composition and larvicidal activity of the essential oil of Tagetes minuta Linnaeus from the southern Brazilian highlandsAbstract
Tagetes minuta L. is a naturalized species that is not endemic to the high-altitude fields of Santa Catarina in southern Brazil. In this study, we investigated the composition of the essential oil (EO) from the flowers of the plant and its larvicidal activity against Aedes aegypti. The EO was obtained by hydrodistillation using a Clevenger apparatus. The chemical composition was determined using gas chromatography coupled with mass spectrometry (GC-MS). The EO extraction yield was 4.9%, and the major compounds (Z)-tagetone, (Z)-β-ocimene, and dihydrotagetone were identified. The 24 h larvicidal activity was verified against that of L3 larvae of the Rockefeller strain of Ae. Aegypti. The EO showed larvicidal activity against Ae. aegypti, with LC50 = 17.28 μg/mL-1. The results indicate that the bioinsecticidal potential of T. minuta EO is promising for the development of research in the field of natural products.
Keywords:
Tagetes minuta; Essential oil; Larvicidal; Brazilian highlands; Tagetone
INTRODUCTION
Aedes aegypti Linnaeus (Diptera, Culicidae) is the main vector for arboviruses that cause dengue, Zika, chikungunya, and yellow fever (Pierson, Diamond, 2020), which are diseases that have a great impact on global health.
Control strategies are based on vector control, by employing synthetic and biological chemicals integrated with environmental management programs that mainly use chemical insecticides (Silva et al., 2019). However, frequent use of chemical insecticides favors the development of resistant insect populations (Dusfour et al., 2019; Vivekanandhan et al., 2021). In the search for inputs of natural origin as alternatives for the control of the Ae. aegypti mosquito, essential oil (EO)-based bioinsecticides have been created (Valli et al., 2021; Vivekanandhan et al., 2018; Vivekanandhan et al., 2020; Vivekanandhan et al., 2021; Vivekanandhan et al., 2024). Promising bioinsecticide molecules can overcome the majority of the challenges associated with synthetic insecticides, and they are biodegradable and have moderate selectivity, minimizing the emergence of resistant insects ( Isman, 2020).
Tagetes minuta L (family Asteraceae) is a herbaceous plant native to Central and South America that is found in grasslands and elevated regions of Argentina, Peru, Bolivia, Chile, and Paraguay (McVaugh, 1943). With the Spanish colonization of the Americas, T. minuta was introduced into different countries (Soulé, 1993) and is currently widely spread in Europe, Africa, Asia, and Oceania (Salehi et al., 2018). It is commonly known as wild marigold, wild clove, clove, chinchilho, rocket stick, or rocket butt. Is an aromatic, small-sized plant with small yellow flowers, hence the species name minuta. It is considered a source of economic interest mainly due to the broad spectrum of uses of its EO in folk medicine (Martinez et al., 2020). This plant is commercially cultivated in several countries to extract EO from leaves and flowers (Singh et al., 2015; Walia, Kumar, 2020).
T. minuta has biological properties of economic importance in the pharmaceutical, cosmetic and food industries, particularly in its EO (Salehi et al., 2018; Walia, Kumar, 2020). Studies have described the EO as a biocidal, herbicidal, nematicidal, larvicidal, anti-inflammatory, antibacterial, and antiviral agent (Singh et al., 2015; Wanzala et al., 2016; Salehi et al., 2018; Kumar, Pandey, Varshney, 2019). Its chemical composition is rich in secondary metabolites including monoterpenes, sesquiterpenes, flavonoids, and thiophenes (Walia et al., 2020).
T. minuta L. is an annual plant and is considered a weed. In Santa Catarina, it occurs spontaneously in fallow land or along roads and paths. The aim of this study was to investigate and characterize the composition of the EO produced by this species from the high-altitude fields in southern Brazil and evaluate its larvicidal activity against Ae. aegypti.
MATERIAL AND METHODS
Plant Material
Flowers of T. minuta L. were collected from the southern Brazilian highlands (28°24’57.3”S and 49°44’29.8”W) in the autumn (June), at an altitude of 1230 m above sea level, with temperature of 10 °C. The present study was authorized by the Genetic Heritage Management Council (CGEN), a legislative and deliberative body under the Ministry of the Environment of Brazil, under number 02001.001165 / 2013-47.
Extraction of EO
T. minuta L. was dried at room temperature for 20 days. Subsequently, the sample was pulverized using a cutting mill. The EO was extracted from 100 g of the flower by hydrodistillation for 6 h using a Clevenger apparatus. The EO was then stored in a sealed amber jar glass at -8 °C until subsequent analysis.
Gas chromatography-mass spectrometry (GC-MS) analysis
The EO from T. minuta was analyzed by gas chromatography-mass spectrometry using a Shimadzu GC-MS-QP 2010 Plus analyzer equipped with a capillary column Rtx -5MS (30 m × 0.25 mm × 0.25 μm.) with a splitless injector mode at 250 °C, and an ion source and interface at 300 °C. The mass window was analyzed from m/z 40 and m/z 350, using helium as the carrier gas. Ramp injection for analysis had the injector temperature set at 250 °C, pressure of 20 psi column, starting at 50 °C for 5 min and increasing to 200 °C at a rate of 5 °C/ min. Identification of the oil components was undertaken by comparing their Kovats indices and mass spectra with the NIST library as well as comparing them with those reported in the literature (Adams, 2007).
Larvicidal activity against Ae. aegypti
The methodology applied was adapted from the WHO (2005) and Betim et al. (2019). The eggs of Ae. aegypti (eggs from the Rockefeller strain made available by Oswaldo Cruz Foundation - Fiocruz) were placed in dechlorinated water and incubated in a B.O.D incubator at a controlled temperature of 27 °C and relative humidity of 80%. The larval diet consisted of fish feed (Aldon basic, MEP 200 complex), from hatching until they reach the third developmental larval stage.
The EO was diluted in 0.5% of polysorbate 80 (obtained from Sigma Aldrich), and then dissolved in dechlorinated water to obtain the desired concentration. An aqueous solution of 0.5% of polysorbate 80 was used as a negative control and the insecticide temephos was used as a positive control. The positive control was used at a concentration of 6 μg/mL (twice the lethal concentration that causes 99% mortality of susceptible strains) according to the protocol recommended.
After hatching, 10 larvae in the 3rd stage were placed in contact with the negative and positive control and essential oil at concentrations (1000, 100 and 10 μg/mL) for 24h and then live and dead larvae were counted. Four repetitions were used for each treatment giving a total of 40 larvae to each sample dose.
Statistical analysis
The probit method (Finney, 1971) was used to determine the lethal concentration (LC50 and LC90) values, as well as the corresponding 95% confidence intervals and chi square values for the assays with Ae. aegypti, using the SPSS Statistical Software Package version 23.0.
RESULTS AND DISCUSSION
Essential Oil Yield and Composition
Hydrodistillation of the flowers of T. minuta produced a characteristic yellow, fruit scented EO with a yield of 4.9%, with the highest volume obtained in the first 30 min.
T. minuta produces the highest EO yield among all cultivated species of the genus Tagetes. Oliveira et al. (2018) obtained a 2.09% EO yield in a sample from Brazil, while an extraction South Africa retrieved 0.33% (Igwaran et al., 2017), and Indian EO from the Himalayan region returned 0.37 to 0.79% (Walia et al., 2020). According to Kumar et al., (2014), EO yield can vary according to environmental factors, geographical location, collection time, cultivation method, and climatic conditions.
The present study was conducted in southeastern Santa Catarina, where the Atlantic Forest biome in southern Brazil has grassland formations called Campos de Altitude do Planalto das Araucárias and Campos de Cima da Serra. These areas predominate at higher altitudes, with elevations greater than 1000 m above sea level (Boldrini, 2009).
The climate is characterized, according to the Köppen-Geiger classification, as humid mesothermal (Cfb), with mild summers, cold winters, well-distributed rainfall during the year, and an average temperature of 14.4 ºC . The city is considered one of the coldest in Brazil, with records of frost and snowfall in the winter months. The maximum and minimum annual average temperatures are 26.0 ºC and - 9.2 ºC , respectively (Potter et al., 2004).
The high EO yield obtained in our study compared to those listed above may be related to post-flowering collection phase and the particular climatic and altitude conditions of the geographical region, corroborating the results of studies conducted in India (Kumar et al., 2014). In addition, the altitude difference appears to have affected the chemical composition of the EO (Rathore, Walia, Kumar, 2018).
GC/MS analysis detected and identified 11 terpenederived compounds (mono- and sesquiterpenes) corresponding to 97.72% of the components present in the EO of T. minuta (Table I).
Of the 11 compounds, 10 (99.8%) were monoterpenes and 1 (0.2%) was a sesquiterpene. The predominance of monoterpenes is in agreement with the results obtained in most studies on Tagetes species (Salehi et al., 2018).
The main compounds found were monoterpenes (Z)-tagetone (62.69%), (Z)-β-ocimene (21.34%) and dihydrotagetone (5.61%).
Z-tagetone, dihydrotagetone, and ocimene are associated with antimicrobial, insecticidal, herbicidal, and nematicidal activity. Ocimene is used in the perfume and fragrance industries (Salehi et al., 2018). In addition, tagetone and ocimene derivatives have been reported to be chemical constituents of EOs with potential cytotoxic effects (Ali et al., 2014; Shirazi et al., 2014). The result obtained is consistent with the characterization of EOs from previous research; however, a quantitative difference was observed.
The genus Tagetes is known to contain species rich in aromatic compounds, which are generally high in monoterpene hydrocarbons (ocimenes, limonene, terpinene, and myrcene), and acyclic monoterpene ketones (tagetone, dihydrotagetone, and tagetenone), which are the primary odorants. Lower amounts of sesquiterpene hydrocarbons and oxygenated compounds are also present (Ibrahim et al., 2016). T. lucida and T. filifolia are the only species with marked differences in EO composition, dominated by phenylpropanoids such as methyleugenol, methylchavicol, and anethole (Salehi et al., 2018). According to Singh et al. (2015), the EOs from T. minuta can be grouped into two chemotypes: those dominated by (Z)-β-ocimene and monoterpenes, and those with mainly tagetone and dihydrotagetone derivatives.
The EOs of T. minuta from Argentina showed different chemical compositions, as dihydrotagetone (42.9%) was the main constituent present in leaves, whereas β-ocimene (45.4%) was found in flowers (Chamorro et al., 2008). In another study, there was a predominance of limonene (66.3%) (Gillij, Gleiser, Zygadlo, 2008), whereas in our research, we only identified traces of this compound (0.80%).
Verbenone was the most abundant compound obtained in Uganda (Kyarimpa et al., 2014) and Turkey (Bahadirli, 2020), with 15% and 32.68%, respectively. Verbenone was not identified in the present study.
In Brazil, we observed variations in the chemical composition of EO across different geographical regions (Table II).
Garcia et al. (2012) found dihydrotagetone as the main compound (54.2%) in the EO from the central-western region, whereas the EO from the northeastern region was dominated by piperitone (86.3%) (Macedo et al., 2013). On the coast of the southern region near sea level, there was also a predominance of dihydrotagetone 67.64% (Chaaban et al., 2019).
Comparing our results with the data in Table II, it is clear that the composition of the EO of T. minuta grown in Brazil differs with respect to geographical region. The EO obtained from the central region showed higher tagetone content and traces of terpenes, while that from the northeastern region was dominated by piperitone (86.27%) and limonene (13.73%).
In the pioneer study by (Craveiro et al., 1988), the EO of T. minuta from the northeastern region of Brazil, Bahia, and Pernambuco, was observed to be rich in dihydrotagetone (reaching 99%), while in our study, the cold-climate plants from the altitude region of Santa Catarina produced an essential oil rich in (Z)-tagetone and β-ocimene, similar to the results obtained by Oliveira et al. (2018). The sample from Pernambuco contained thymol (7.4%), a compound that was not identified in our study.
Larvicidal activity against Aedes aegypti
The EO of T. minuta showed potential larvicidal activity for third-stage larvae of Ae. aegypti, with an LC50 of 17.28 μg.mL-1 (concentration required to inhibit 50% of treated larvae), as shown in Table III.
The EO of T. minuta has insecticidal and repellent properties against a number of pests and insects (Walia, Kumar, 2020), including Pediculus humanus capitis (Cestari et al., 2004), Brevicoryne brassicae, and Rhipicephalus appendiculatus (Wanzala et al., 2020). The EO extracted from T. minuta seeds showed insecticidal activity against pests found in grain storage: Tribolium castaneum, Rhyzopertha dominica, and Callosobruchus maculatus (Jayaram et al., 2022).
Studies have been conducted to evaluate the repellent and insecticidal effects of EO on different mosquito species, such as Anopheles arabiensis, A. stephensi, and Culex quinquefasciatus (Walia, Kumar 2020).
The repellent activity of EO against Ae. aegypti has been described in Argentina (Gillij, Gleiser, Zygadlo, 2008) and Kenya (Wanzala, Ogoma, 2013), with limonene being the major compound in both cases. In Peru, T. filifolia obtained a higher larvicidal activity (LC50=47.7 ppm) for A. aegypti when compared to that of T. minuta (LC50=52.3 ppm), probably due to its high concentration of trans-anethol (88.2%) (Ruiz et al., 2011).
The result obtained in our work is similar to the study by Kyarimpa et al. (2014), in which an LC50 of 1.49 mg/L was obtained against Anopheles gambiae, with an EO mainly composed of terpenic derivatives. Although that study also noted the presence of verbenone, which is a compound known to be nematicidal. In contrast, Furtado et al. (2005) obtained an LC50 of 72.85 mg/mL, which is lower than our results.
The EO insecticidal activity of T. minuta against Ae. aegypti can be attributed to the presence of the main constituents, limonene, E-ocimenone, dihydrotagetone, β-ocimene, and (Z)-tagetone (Bakshi, Ghosh 2022).
Green et al. (1991) reported the larvicidal activity of the EO of three species of Tagetes grown under controlled laboratory conditions. They reported that the EO of the flowers of T. minuta had the greatest biocidal effect on the larvae of Ae. aegypti. However, the components of the EO responsible for this activity were not characterized. Notably, the plant materials were obtained through controlled cultivation, which can directly interfere with the chemical composition of the EO. Lima et al. (2009) evaluated the larvicidal activity of the EO (of an unknown composition) obtained from a commercially acquired T. minuta of Indian origin. The authors reported a LC50 of 0.24 mL·L-1, and suggested that the compounds derived from thiophene and ocimenone were responsible for the toxicity in Ae. aegypti.
In this study, T. minuta was collected in situ from different soils and climatic conditions to characterize the chemotype of the EOs and elucidate their primary composition. β-ocimene, (E)-ocimenone, dihydrotagetone, and (Z)-tagetone were the major compounds identified, and possibly the ones responsible for the larvicidal activity, however, EOs are complex mixtures of substances, and synergism may occur with the minority compounds and their combinations (Jayaram et al., 2022). These compounds are believed to act on the insect nervous system by altering GABAergic functions (Bloomquist et al., 2008) and inhibiting acetylcholinesterase (Abdelgaleil et al., 2015), causing toxicity.
CONCLUSION
The major compounds found in the EO of the flowers of T. minuta from the altitude fields of Santa Catarina, southern Brazil, were the monoterpenes (Z)-tagetone (62.69%), (Z)-β-ocimene (21.34%) dihydrotagetone (5.61%), and (E)-ocimenone (3.64%). The EO yield obtained by vapor drag hydrodistillation was 4.9%, favoring its use in the pharmaceutical, cosmetic (perfumes and fragrances), and food industries. The high yield obtained can be correlated to the edaphoclimatic characteristics of the Campos de Altitude in the Serra Catarinense. Planting of the altitude cultivar should be encouraged, favoring the production chain of the EO and generating a new source of income for the cultivation region. The EO showed larvicidal activity against Ae. aegypti, with an LC50 = 17.28 μg.mL-1, which is promising for the generation of bioinsecticidal products for mosquito control. The results of this study demonstrate the need for further research with the Brazilian high-altitude chemotype of T. minuta EO, particularly in terms of the structural elucidation of its components, as well as its bioactivity and toxicity.
ACKNOWLEDGMENTS
The authors would like to thank Federal University of Paraná, the Araucaria Foundation, the Paraná State Secretariat of Science, Technology and Higher Education (SETI-PR) and NAPI-Genomics Consortium.
REFERENCES
- Abdelgaleil SA, Mohamed MI, Shawir MS, Abou-Taleb HK. Chemical composition, insecticidal and biochemical effects of essential oils of different plant species from Northern Egypt on the rice weevil, Sitophilus oryzae L. J Pest Sci. 2015;89:219-229.
- Adams, RP. Identification of essential oil components by gas chromatography / mass spectrometry. 4 ed. Carol Stream IL: Allured Publishing; 2007.804 p.
- Ali NAA, Sharopov FS, Al-kaf AG, Hill GM, Arnold N, Al-Sokari SS, et al. Composition of essential oil from Tagetes minuta and its cytotoxic, antioxidant and antimicrobial Activities: Nat Prod Commun. 2014;9(2):265-68.
- Bahadirli NP. Essential oil content and compositions of naturalized Tagetes minuta L. (wild marigold). Nat Volatiles Essent Oils. 2020;7(2):17-21.
- Bakshi L, Ghosh R. Marigold biopesticide as an alternative to conventional chemical pesticides. J Adv Sci Res. 2022;13(05):26-33.
- Betim FCM, Oliveira CF, Souza AM, Szabo EM, Zanin SMW, Miguel OG, et al. Ocotea nutans (Nees) Mez (Lauraceae): chemical composition, antioxidant capacity and biological properties of essential oil. Braz J Pharm Sci. 2019;55:1-10.
- Bloomquist JR, Boina DR, Chow E, Carlier PR, Reina M, Gonzalez-Coloma A. Mode of action of the plant-derived silphinenes on insect and mammalian GABAA receptor/chloride channel complex. Pestic Biochem Physiol. 2008;91(1):17-23.
- Boldrini II, organizator. Biodiversidade dos campos do planalto das araucárias. Brasília: Ministério do Meio Ambiente; 2009. 240 p. Portuguese.
- Cestari IM, Sarti SJ, Waib CM, Branco AC. Evaluation of the potential insecticide activity of Tagetes minuta (Asteraceae) essential oil against the head lice Pediculus humanus capitis (Phthiraptera: Pediculidae). Neotrop Entomol. 2004;33(6):805-807.
- Chaaban A, Santos VMCS, Martins CEN, Brum JS, Bertoldi FC, Molento MB. Tissue damage and cytotoxic effects of Tagetes minuta essential oil against Lucilia cuprina. Exp Parasitol. 2019;198:46-52.
- Chamorro ER, Ballerini G, Sequeira AF, Velasco GA, Zalazar MF. Chemical composition of essential oil from Tagetes minuta L. leaves and flowers. J Argent Chem Soc. 2008;96(1-2):80-86.
- Craveiro AA, Matos FJA, Machado MIL, Alencar JW. Essential oils of Tagetes minuta from Brazil. Flavour Fragr J. 1988;13(5):35-36.
- Dusfour I, Vontas J, David JP, Weetman D, Fonseca DM, Corbel V, et al. Management of insecticide resistance in the major Aedes vectors of arboviruses: Advances and challenges. PLOS Negl Trop Dis. 2019;13(10):e0007615.
- Finney DJ. Probit Analysis. 3th ed. Cambridge: Cambridge University Press; 1971.
- Furtado RF, Lima MG, Neto MA, Bezerra JN, Silva MGDV. Atividade larvicida de óleos essenciais contra Aedes aegypti L. (Diptera: Culicidae). Neotrop Entomol . 2005;34:843-847. Portuguese.
- Garcia MV, Matias J, Barros JC, Lima DP, Lopes RS, Andreotti R. Caracterização química e efeito acaricida do óleo essencial de Tagetes minuta Linnaeus (Asteraceae) em carrapatos. Rev Bras de Parasitol Vet. 2012;21(4):405-411. Portuguese.
- Gillij YG, Gleiser RM, Zygadlo JA. Mosquito repellent activity of essential oils of aromatic plants growing in Argentina. Bioresour Technol. 2008;99(7):2507-2515.
- Green MM, Singer JM, Sutherland DJ, Hibben CR. Larvicidal activity of Tagetes minuta (marigold) toward Aedes aegypti. JAMCA. 1991;7(2):282-286.
- Ibrahim SRM, Abdallah HM, El-Halawany AM, Mohamed GA. Naturally occurring thiophenes: isolation, purification, structural elucidation, and evaluation of bioactivities. Phytochem Rev. 2016;15(2):197-220.
- Igwaran A, Iweriebor BC, Okoh SO, Nwodo UU, Obi LC, Okoh AI. Chemical constituents, antibacterial and antioxidant properties of the essential oil flower of Tagetes minuta grown in Cala community Eastern Cape, South Africa. BMC Complement. Med Ther. 2017;17(1):1-10.
- Isman MB. Botanical Insecticides in the twenty-first century-fulfilling their promise? Annu Rev Entomol. 2020;65:233-249.
- Jayaram CS, Chauhan N, Dolma SK, Reddy SGE. Chemical Composition and insecticidal activities of essential oils against the Pulse Beetle. Molecules. 2022;27(2):568.
- Kumar R, Pandey A, Varshney VK. 2019. Antifungal activity of the essential oil of Tagetes minuta against some fungi of forestry importance. J Biol Act Prod Nat. 2019;9(1):67-72.
- Kumar R, Sharma S, Ramesh K, Pathania V, Prasad R. Irradiance stress and plant spacing effect on growth, biomass and quality of wild marigold (Tagetes minuta L.) - an industrial crop in western Himalaya. J Essent Oil Res. 2014;26(5):348-358.
- Kyarimpa CM, Böhmdorfer S, Wasswa J, Kiremire BT, Ndiege IO, Kabasa JD. Essential oil and composition of Tagetes minuta from Uganda. Larvicidal activity on Anopheles gambiae. Ind Crops Prod. 2014;62:400-404.
- Lima WP, Chiaravalloti Neto F, Macoris MLG, Zuccari DAPC, Dibo MR. Estabelecimento de metodologia para alimentação de Aedes aegypti (Diptera-Culicidae) em camundongos swiss e avaliação da toxicidade e do efeito residual do óleo essencial de Tagetes minuta L (Asteraceae) em populações de Aedes aegypti. Rev Soc Bras Med Trop. 2009;42:638-641. Portuguese.
- Macedo ITF, Oliveira LMB, Camurça-Vasconcelos ALF, Ribeiro WLC, Santos JML, Morais SM, et al. In vitro effects of Coriandrum sativum, Tagetes minuta, Alpinia zerumbet and Lantana camara essential oils on Haemonchus contortus. Rev Bras Parasitol Vet. 2013;22(4):463-469.
- Martinez F, Massuh Y, Aguilar JJ, Ojeda MS, Contigiani MS, Núñez SC, Konigheim BS. Cultivars of Tagetes minuta L. (Asteraceae) as a source of potential natural products with antiviral activity. J Herb Med. 2020;24:100397
- McVaugh R. Botanical collections of the La Plata Expedition of 1853-1855. Brittonia. 1943;5(1):64-79.
- Oliveira DH, Abib PB, Giacomini RX, Lenardão EJ, Schiedeck G, Wilhelm EA, et al. Antioxidant and antifungal activities of the flowers’essential oil of Tagetes minuta, (Z)-tagetone and thiotagetone. J Essent Oil Res . 2018;31(2):160-169.
- Pierson TC, Diamond MS. The continued threat of emerging flaviviruses. Nat Microbiol. 2020;5(6):796-812.
- Potter RO, Carvalho AP, Flores CA, Bognola I. Solos do Estado de Santa Catarina. Rio de Janeiro: Embrapa Solos. Boletim de Pesquisa e Desenvolvimento; 2004. 745 p. Portuguese.
- Rathore S, Walia S, Kumar R. 2018. Biomass and essential oil of Tagetes minuta influenced by pinching and harvesting stage under high precipitation conditions in the western Himalayas. J Essent Oil Res . 2018;30(5):360-368.
- Ruiz C, Cachay M, Domínguez M, Velásquez C, Espinoza G, Ventosilla P, et al. Chemical composition, antioxidant and mosquito larvicidal activities of essential oils from Tagetes filifolia, Tagetes minuta and Tagetes elliptica from Perú. Planta Med. 2011;77:PE30.
- Salehi B, Valussi M, Morais-Braga MFB, Carneiro JNP, Leal ALAB, Coutinho HDM et al. Tagetes spp. Essential oils and other extracts: chemical characterization and biological activity. Molecules. 2018;23(11):2847.
- Shirazi MT, Gholami H, Kavoosi G, Rowshan V, Tafsiry A. Chemical composition, antioxidant, antimicrobial and cytotoxic activities of Tagetes minuta and Ocimum basilicum essential oils. Food Sci Nutr. 2014;2(2):146-155.
- Silva PT, Santos HS, Teixeira AMR, Bandeira PN, Holanda CL, Vale JPC, et al. Seasonal variation in the chemical composition and larvicidal activity against Aedes aegypti of essential oils from Vitex gardneriana Schauer. S Afr J Bot. 2019;124:329-332.
- Singh P, Krishna A, Kumar V, Krishna S, Singh K, Gupta M, et al. Chemistry and biology of industrial crop Tagetes Species: a review. J Essent Oil Res . 2015;28(1):1-14.
- Soulé JA. Tagetes minuta: A potential new herb from South America. New Crops. 1993;649-654.
- Valli M, Atanázio LCV, Monteiro GC, Coelho RR, Demarque DP, Andricopulo AD et al. The potential of biologically active brazilian plant species as a strategy to search for molecular models for mosquito control. Planta Med . 2021;87(1-2):6-23.
- Vivekanandhan P, Alahmadi TA, Ansari MJ, Subala SP. Biocontrol efficacy of cajeput oil against Anopheles stephensi L. mosquito and its effect on non-target species. Front Physiol. 2024;15:1357411.
- Vivekanandhan P, Thendralmanikandan A, Kweka EJ, Mahande AM. Resistance to temephos in Anopheles stephensi larvae is associated with increased cytochrome P450 and α-esterase genes overexpression. Int J Trop Insect Sci. 2021;41:2543-2548.
- Vivekanandhan P, Usha-Raja-Nanthini A, Valli G, Subramanian Shivakumar MS. Comparative efficacy of Eucalyptus globulus (Labill) hydrodistilled essential oil and temephos as mosquito larvicide. Nat Prod Res. 2020;34(18):2626-2629.
- Vivekanandhan P, Venkatesan R, Ramkumar G, Karthi S, Senthil-Nathan S, Shivakumar MS. Comparative analysis of major mosquito vectors response to seed-derived essential oil and seed pod-derived extract from Acacia nilotica. IJERPH. 2018;15(2):388.
- Walia S, Kumar R. Wild marigold (Tagetes minuta L.) an important industrial aromatic crop: liquid gold from the Himalaya. J Essent Oil Res . 2020;32(5):373-393.
- Walia S, Mukhia S, Bhatt V, Kumar R, Kumar R. Variability in chemical composition and antimicrobial activity of Tagetes minuta L. essential oil collected from different locations of Himalaya. Ind Crops Prod . 2020;150:112449.
- Wanzala W, Hassanali A, Mukabana WR, Takken W, Minyoso SI. The effect of essential oils of Tagetes minuta and Tithonia diversifolia on-host behaviour of the brown ear tick Rhipicephalus appendiculatus. AJMAP. 2020;6(2):55-71.
- Wanzala W, Ogoma SB. Chemical Composition and mosquito repellency of essential oil of Tagetes minuta from the Southern Slopes of Mount Elgon in Western Kenya. J Essent Oil-Bear Plants. 2013;16(2):216-232.
- World Health Organization (WHO). Guidelines for laboratory and field testing of mosquito larvicides. Geneve: World Health Organization. No. WHO/CDS/ WHOPES/GCDPP. 2005.
