Research Article | Volume: 8, Issue: 6, Nov-Dec, 2020
Efficacy of commercial botanical pure essential oils of garlic (Allium sativum) and anise (Pimpinella anisum) against larvae of the mosquito Aedes aegypti
1Department of Public Health and Health Promotion, College of Allied Health Sciences, Suan Sunandha Rajabhat University, Thailand.
2College of Allied Health Sciences, Suan Sunandha Rajabhat University, Thailand.
Open Access
Published: Nov 25, 2020
DOI: 10.7324/JABB.2020.80614Aedes aegypti, also called the dengue or yellow fever mosquito, is a significant vector species of several viruses especially the dengue virus. Reducing Ae. aegypti population can directly reduce dengue outbreaks in the community. The research aim is to assess the efficacy of commercial botanical pure essential oils of garlic (Allium sativum) and anise (Pimpinella anisum) against Ae. aegypti larvae, the mortality of larvae was monitored after 24- and 48-h of exposure. The larvicidal activity of garlic oil after 24-h exposure was 0.005 ppm for LC25, 0.006 ppm for LC50, and 0.012 ppm for LC90, and after 48 h exposure was 0.004 ppm for LC25, 0.006 ppm for LC50, and 0.014 ppm for LC90. Meanwhile, the larvicidal activity of anise oil after 24-h exposure was 0.016 ppm for LC25, 0.023 ppm for LC50, and 0.043 ppm for LC90, and after 48-h exposure was 0.014 ppm for LC25, 0.020 ppm for LC50, and 0.037 ppm for LC90, whereas, Aedes larval mortalities increased with an increase in the concentration of garlic and anise essential oils after both 24- and 48-h exposure. The results of this study were clear evidence for the efficiency of commercial plant oil for dengue vector larval control, especially garlic essential oils.
Laojun S, Damapong P, Damapong P, Wassanasompong W, Suwandittakul N, Kamoltham T, Chaiphongpachara T. Efficacy of commercial botanical pure essential oils of garlic (Allium sativum) and anise (Pimpinella anisum) against larvae of the mosquito Aedes aegypti. J App Biol Biotech. 2020;8(6):88-92. https://doi.org/10.7324/JABB.2020.80614
The organic essential oils, also called volatile liquids or ethereal oils, are natural volatile compounds obtained from parts of aromatic plants, for example, leaves, roots, stems, seeds, fruits, and flowers depending on the type of plant [1]. Botanical pure essential oils are easily available on the market and are widely used in many industries including in pharmaceutical, food applications, perfumery, sanitary, medicine, and cosmetic products [2]. Products developed from the essential oils of many plants have been recognized and popular for use in repelling adult mosquitoes including flowering plants in the Lamiaceae (also called Labiatae) family such as Ocimum basilicum (basil), Mentha spp. (mint), Hyptis suaveolens (hyptis), Lavandula spp. (lavender), Salvia spp. (sage), and Thymus spp. (thyme); plants in the Myrtaceae family such as Eucalyptus spp. (eucalyptus) and Melaleuca spp. (tea tree); and plants in the Poaceae family such as Cymbopogon spp. (citronella, lemongrass, and palmarosa) [3]. In addition, it was reported that the essential oils of various species of plants including camphor, thyme, lemon, cedarwood, frankincense, dill, myrtle, juniper, black pepper, verbena, helichrysum, sandalwood, cassia, cinnamon, East Indian lemongrass, bay, sweet basil, holy basil, and ginger can be used to control immature stages of many mosquito species that are vectors of dangerous infectious diseases [4,5].
The garlic plant (Allium sativum), belonging to the family Liliaceae, and anise plant (Pimpinella anisum), belonging to the family Apiaceae, are important components of many foods and contain important substances that are beneficial to human health [6,7]. The previous research has reported that the essential oils of both of these plants are very effective in controlling many insects including the mealworm beetle [8], the cotton leafworm [9], and Culex mosquitoes [10].
Mosquitoes are insect vectors and each mosquito species has specificity to different diseases [11]. Aedes aegypti, also called the yellow fever mosquito, is a significant vector species of several viruses, especially the dengue virus [11,12]. Reducing Ae. aegypti population can directly reduce dengue outbreaks in the community [13]. Although the use of synthetic chemical insecticides is a highly effective method to control insects, including mosquito vectors, it directly affects living organisms, which includes human and animal health [14]. In addition, these harmful substances can leave residue and accumulate for a long time in the environment [15]. Therefore, using natural insecticides, which are non-toxic to the environment, are a necessary community need [16,17]. Some essential oils have been observed to be potential candidates to aid in the control of Ae. aegypti mosquito populations.
Garlic and anise oils could possibly be implemented to control mosquito larvae, but there is no clear evidence for their effectiveness in mosquito control. Thus, the aim of the present study is to assess the effectiveness of commercial garlic (A. sativum) and anise (P. anisum) essential oils in killing Ae. aegypti larvae. The research results will confirm the efficacy of killing larvae of the dengue vector using garlic and anise and could become evidence for further use of essential oils to control mosquito vectors.
Commercial essential oils of A. sativum (garlic) and P. anisum (anise) from the Chemipan Corporation Company Limited in Thailand were used for testing to evaluate the efficacy of Ae. aegypti larvae removal in this research. Both plant oils are pure essential oils that have been extracted from seeds by a steam distillation method.
The second stage and early third stage Ae. aegypti larvae were obtained from the Department of Medical Sciences, Ministry of Public Health, Thailand. Obtained larvae of Ae. aegypti were placed in a tray of filtered water (length 14 × width 11 × depth 7 inches). The experiment was conducted in 2018 at the biological laboratory of the College of Allied Health Sciences, Suan Sunandha Rajabhat University, Thailand. The laboratory conditions included a 12 h light and 12 h dark cycle at 25–28°C and relative humidity levels at 70–80%. Ground dog kibble was used for larval food and given only once a day to prevent spoilage. After about 2 days, larvae developed into late third-stage larvae of Ae. aegypti, which were then used for larvicidal testing in the next step.
The garlic and anise essential oils were diluted with filtered water in 250 mL glass beakers. The serial dilutions of the tested essential oils for this testing were at concentrations of 0.005, 0.012, 0.025, 0.037, and 0.050 ppm. The concentration range of the larvicidal test was conducted in accordance with recommendations from the World Health Organization. After the essential oil solutions were prepared, 25 late third-stage larvae were put in each 250 ml beaker containing the diluted essential oil for each of the previously mentioned concentrations. Afterward, Aedes larvae were observed and counted. Larval mortality was calculated 24- and 48-h after treatment, where the death of larvae was noted by their inactivity. Experiments for each concentration were performed in four replicates and one control group (beakers filled with water that contained no essential oils).
Larval mortality data were used in log-probit analyses to obtain the 50% and 90% lethal concentrations (LC50 and LC90), and Chi-square values. The log-probit analysis and lethal concentration graphing in this research were conducted using the program LdP Line which can be downloaded at http://www.ehabsoft.com/ldpline/. For statistical comparisons of the mortality of the mosquito larvae among essential oils, the analysis of variance (ANOVA) was used followed by the Duncan’s multiple range test (DMR) in R statistical software package as a free program. p< 0.05 was accepted to be statistically significant in this study.
To examine the efficacy of commercial botanical pure essential oils of garlic (Allium sativum) and anise (Pimpinella anisum) against Ae. aegypti larvae, the mortality of larvae was monitored after 24- and 48-h of exposure. The study results of larval mortality after 24- and 48-h exposure in the five different concentrations (0.005, 0.012, 0.025, 0.037, and 0.050 ppm) of garlic and anise essential oils are presented as percentages in Table 1. Aedes larval mortalities increased with an increase in the concentration of garlic and anise essential oils after both 24- and 48-h exposure. In this experiment, no larvae died in the control groups.
Table 1: Mortality of mosquito larvae after 24- and 48-h exposure to garlic and anise oils in each concentration.
| Concentrations (ppm) | % mortality (means±SE) | |||
|---|---|---|---|---|
| Garlic essential oils | Anise essential oils | |||
| 24 h | 48 h | 24 h | 48 h | |
| 0.005 | 25.00±1.91 | 28.00±1.63 | 3.00±3.00 | 4.00±4.00 |
| 0.012 | 93.00±3.42 | 100.00±0 | 7.00±3.00 | 7.00±3.00 |
| 0.025 | 99.00±1.00 | 99.00±1.00 | 35.00±9.57 | 49.00±11.47 |
| 0.037 | 100.00±0 | 100.00±0 | 96.00±2.83 | 100.00±0 |
| 0.050 | 100.00±0 | 100.00±0 | 100.00±0 | 100.00±0 |
| Control | 00.00±0 | 00.00±0 | 00.00±0 | 00.00±0 |
ppm: parts per million, 10−6, %: Percentage, SE: Standard error, h: Hours
Table 2 shows the results of probit analysis which reveal larvicidal efficacy of garlic and anise oils against Ae. aegypti. The garlic essential oil was more effective than the anise oil. The larvicidal activity of garlic oil after 24-h exposure was 0.005 ppm for LC25, 0.006 ppm for LC50, and 0.012 ppm for LC90, and after 48 h exposure was 0.004 ppm for LC25, 0.006 ppm for LC50, and 0.014 ppm for LC90. Meanwhile, the larvicidal activity of anise oil after 24-h exposure was 0.016 ppm for LC25, 0.023 ppm for LC50, and 0.043 ppm for LC90, and after 48-h exposure was 0.014 ppm for LC25, 0.020 ppm for LC50, and 0.037 ppm for LC90.
Table 2: LC25, LC50 and LC90 (ppm) of garlic and anise oils on Aedes mosquito larvae.
| Essential oils | Time (hours) | LC25 (ppm) | LC50 (ppm) | LC90 (ppm) | Slope ± SE | χ2 |
|---|---|---|---|---|---|---|
| Garlic | 24 | 0.005 | 0.006 | 0.012 | 5.031±0.520 | 5.495 |
| 48 | 0.004 | 0.006 | 0.014 | 3.933±0.623 | 20.840 | |
| Anise | 24 | 0.016 | 0.023 | 0.043 | 4.698±0.294 | 63.351 |
| 48 | 0.014 | 0.020 | 0.037 | 4.857±0.367 | 134.592 |
LC25: Lethal concentration that kills 25% of exposed Aedes larvae, LC50: Lethal concentration that kills 50% of exposed Aedes larvae, LC90: Lethal concentration that kills 90% of exposed Aedes larvae, ppm: Parts per million, SE: Standard error, χ2: Chi-square
Statistical analyses showed that the larvicidal efficacy between the 24-and 48-h exposure periods of essential oils was not different except for the LC90 value for anise oil, as shown in Figures 1-3, respectively. In addition, analyses revealed the difference of larvicidal efficacy between garlic and anise oils against Ae. aegypti.
 | Figure 1: Relative toxicity based on LC25 values of pure garlic and anise essential oils on Ae. aegypti larvae after 24- and 48-h exposure. The different red letters that appear at the end of description words indicate statistically significant differences between groups (P < 0.05) (in the top left corner) [Click here to view] |
 | Figure 2: Relative toxicity based on LC50 values of pure garlic and anise essential oils on Ae. aegypti larvae after 24- and 48-h exposure. The different red letters that appear at the end of description words indicate statistically significant differences between groups (P < 0.05) (in the top left corner) [Click here to view] |
 | Figure 3: Relative toxicity based on LC90 values of pure garlic and anise essential oils on Ae. aegypti larvae after 24- and 48-h exposure. The different red letters that appear at the end of description words indicate statistically significant differences between groups (P < 0.05) (in the top left corner) [Click here to view] |
Natural products are an important alternative that is recognized by the community as being environmentally friendly and many studies recognize essential oils as a potential effective alternative [18, 19]. This research clearly demonstrated that commercial pure garlic (A. sativum) and anise (P. anisum) essential oils are highly effective against Ae. aegypti larvae (LC50: Garlic = 0.006 ppm and anise = 0.020 ppm at after 24-h exposure). Our results were compared with the criteria of Cheng et al.[20] which describes that if the LC50<50 ml/L (or ppm), means that it has a highly efficacy against the larvae of mosquitoes. This result was consistent with the previous biological research, which found that garlic and anise essential oils are toxic to mosquito species including Culex restuans (LC50: garlic = 2.7 ppm) and Cx. pipiens (LC50: Garlic = 7.5 ppm and anise = 28.7 ppm) [10,21]. According to Öz et al.’s study [10], anethole has been identified as a major component of anise essential oil (94.48%). Anethole is an organic compound found in several plants and is reportedly toxic to insects [22]. Regarding garlic essential oil, the previous research of Muturi et al. [21] found that a major component was diallyl disulfide (49.13%). Diallyl disulfide is an organosulfur compound found commonly in onions, garlic, and a few other plants in the genus Allium. This substance is reportedly toxicity to mosquito larvae [23].
During our evaluation of the efficacy of both garlic and anise essential oils in the killing larvae of Ae. aegypti, we found that garlic essential oil has a stronger effect than anise essential oil. This result is in line with the previous research [10,21]. Statistical analyses show that the efficiency of garlic and anise essential oils for Aedes larval removal between 24- and 48-h post-exposure was not different, except concerning anise essential oil (which had different value for LC9O). Both commercial garlic and anise essential oils have high larvicidal activity at 24- and 48-h after exposure. Commercial essential oils are often used as cosmetic components, which require pure oils without any dilution. This may account for the strength of these oils in larvicidal activity.
This study supports the use of natural products particularly botanical essential oils as alternative methods for controlling the larvae of Ae. aegypti, a dengue vector, in water containers around the houses. Nowadays, essential oils are natural products that are readily available and good quality because they are important components of common cosmeceutical products. The results of this study were clear evidence for the efficiency of commercial plant oil for dengue vector larval control, especially garlic essential oils. This research paves the way for further use of essential oils as natural mosquito vector controls.
This research has been supported by the Suan Sunandha Rajabhat University, Bangkok, Thailand.
The authors declare that they have no conflicts of interest.
None.
1. Aktar W, Sengupta D, Chowdhury A. Impact of pesticides use in agriculture: Their benefits and hazards. Interdiscip Toxicol 2009;2:1-12. [CrossRef]
2. Amer A, Mehlhorn H. Larvicidal effects of various essential oils against Aedes, Anopheles, and Culex larvae (Diptera, Culicidae). Parasitol Res 2006;99:466-72. [CrossRef]
3. Bekele D. Review on insecticidal and repellent activity of plant products for malaria mosquito control. Biomed Res Rev 2018;2:1-7. [CrossRef]
4. Bongiorno PB, Fratellone PM, LoGiudice P. Potential health benefits of garlic (Allium sativum): A narrative review. J Complement Integr Med 2008;5:1-24. [CrossRef]
5. Chaiphongpachara T, Sumchung K, Bumrungsuk A, Chansukh KK. Larvicidal and adult mosquito vector attractant activity of Tremella fuciformis berk mushroom extract on Aedes aegypti (L.) and Culex sitiens Wiedemann (Diptera: Culicidae). J Appl Pharm Sci 2018;8:7-10. [CrossRef]
6. Chaiphongpachara T, Sumchung K, Chansukh KK. Larvicidal and adult mosquito attractant activity of Auricularia auricula-judae mushroom extract on Aedes aegypti (L.) and Culex sitiens Wiedemann. J Appl Pharm Sci 2018;8:21-5. [CrossRef]
7. Chaiphongpachara T, Laojun S, Wassanasompong W. Screening seven commercial essential herb oils for larvicidal activity against the mosquito Aedes aegypti (Linnaeus), a vector of the dengue virus. J Appl Pharm Sci 2020;10:43-50.
8. Cheng SS, Chang HT, Chang ST, Tsai KH, Chen WJ. Bioactivity of selected plant essential oils against the yellow fever mosquito Aedes aegypti larvae. Bioresour Technol 2003;89:99-102. [CrossRef]
9. Dias CN, Moraes DF. Essential oils and their compounds as Aedes aegypti L. (Diptera: Culicidae) larvicides: Review. Parasitol Res 2014;113:565-92. [CrossRef]
10. Hamada HM, Awad M, El-Hefny M, Moustafa MA. Insecticidal activity of garlic (Allium sativum) and ginger (Zingiber officinale) oils on the cotton leafworm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae). Afr Entomol 2018;26:84-94. [CrossRef]
11. Kimbaris AC, Kioulos E, Koliopoulos G, Polissiou MG, Michaelakis A. Coactivity of sulfide ingredients: A new perspective of the larvicidal activity of garlic essential oil against mosquitoes. Pest Manag Sci 2009;65:249-54. [CrossRef]
12. Mossa AT, Mohafrash SM, Chandrasekaran N. Safety of natural insecticides: Toxic effects on experimental animals. Biomed Res Int 2018;16:4308054. [CrossRef]
13. Muturi EJ, Ramirez JL, Zilkowski B, Flor-Weiler LB, Rooney AP. Ovicidal and larvicidal effects of garlic and asafoetida essential oils against west nile virus vectors. J Insect Sci 2018;18:1-6. [CrossRef]
14. Nicolopoulou-Stamati P, Maipas S, Kotampasi C, Stamatis P, Hens L. Chemical pesticides and human health: The urgent need for a new concept in agriculture. Front Public Health 2016;4:1-8. [CrossRef]
15. Özz E, Koç S, Çinbilgel ?, Yan?ko?lu A, Çetin H. Chemical composition and larvicidal activity of essential oils from Nepeta cadmea boiss and Pimpinella anisum L. on the larvae of Culex pipiens L. Marmara Pharm J 2018;22:322-7. [CrossRef]
16. Palazzolo E, Laudicina VA, Germanà MA. Current and potential use of citrus essential oils. Curr Org Chem 2013;17:3042-9. [CrossRef]
17. Plata-Rueda A, MartÃnez LC, Dos Santos MH, Fernandes FL, Wilcken CF, Soares MA, et al. Insecticidal activity of garlic essential oil and their constituents against the mealworm beetle, Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae). Sci Rep 2017;7:46406. [CrossRef]
18. Powell JR, Gloria-Soria A, Kotsakiozi P. Recent history of Aedes aegypti: Vector genomics and epidemiology records. Bioscience 2018;68:854-60. [CrossRef]
19. Roiz D, Wilson AL, Scott TW, Fonseca DM, Jourdain F, Müller P, et al. Integrated Aedes management for the control of Aedes borne diseases. PLoS Negl Trop Dis 2018;12:e0006845. [CrossRef]
20. Sabahi Q, Hamiduzzaman MM, Barajas-Pérez JS, Tapia-Gonzalez JM, Guzman-Novoa E. Toxicity of anethole and the essential oils of lemongrass and sweet marigold to the parasitic mite Varroa destructor and their selectivity for honey bee (Apis mellifera) workers and larvae. Psyche 2018;2018:6196289. [CrossRef]
21. Service M. Medical Entomology for Students. 4th ed. England: Cambridge University Press; 2008.
22. Tongnuanchan P, Benjakul S. Essential oils: Extraction, bioactivities, and their uses for food preservation. J Food Sci 2014;79:1231-49. [CrossRef]
23. Vecchio MG, Gulati A, Minto C, Lorenzoni G. Pimpinella Anisum and Illicium Verum: The multifaceted role of anise plants. Open Agric J 2016;10:81-6. [CrossRef]
1. Aktar W, Sengupta D, Chowdhury A. Impact of pesticides use in agriculture: Their benefits and hazards. Interdiscip Toxicol 2009;2:1-12. https://doi.org/10.2478/v10102-009-0001-7 | |
2. Amer A, Mehlhorn H. Larvicidal effects of various essential oils against Aedes, Anopheles, and Culex larvae (Diptera, Culicidae). Parasitol Res 2006;99:466-72. https://doi.org/10.1007/s00436-006-0182-3 | |
3. Bekele D. Review on insecticidal and repellent activity of plant products for malaria mosquito control. Biomed Res Rev 2018;2:1-7. https://doi.org/10.15761/BRR.1000114 | |
4. Bongiorno PB, Fratellone PM, LoGiudice P. Potential health benefits of garlic (Allium sativum): A narrative review. J Complement Integr Med 2008;5:1-24. https://doi.org/10.2202/1553-3840.1084 | |
5. Chaiphongpachara T, Sumchung K, Bumrungsuk A, Chansukh KK. Larvicidal and adult mosquito vector attractant activity of Tremella fuciformis berk mushroom extract on Aedes aegypti (L.) and Culex sitiens Wiedemann (Diptera: Culicidae). J Appl Pharm Sci 2018;8:7-10. https://doi.org/10.7324/JAPS.2018.8902 | |
6. Chaiphongpachara T, Sumchung K, Chansukh KK. Larvicidal and adult mosquito attractant activity of Auricularia auricula-judae mushroom extract on Aedes aegypti (L.) and Culex sitiens Wiedemann. J Appl Pharm Sci 2018;8:21-5. https://doi.org/10.7324/JAPS.2018.8803 | |
7. Chaiphongpachara T, Laojun S, Wassanasompong W. Screening seven commercial essential herb oils for larvicidal activity against the mosquito Aedes aegypti (Linnaeus), a vector of the dengue virus. J Appl Pharm Sci 2020;10:43-50. | |
8. Cheng SS, Chang HT, Chang ST, Tsai KH, Chen WJ. Bioactivity of selected plant essential oils against the yellow fever mosquito Aedes aegypti larvae. Bioresour Technol 2003;89:99-102. https://doi.org/10.1016/S0960-8524(03)00008-7 | |
9. Dias CN, Moraes DF. Essential oils and their compounds as Aedes aegypti L. (Diptera: Culicidae) larvicides: Review. Parasitol Res 2014;113:565-92. https://doi.org/10.1007/s00436-013-3687-6 | |
10. Hamada HM, Awad M, El-Hefny M, Moustafa MA. Insecticidal activity of garlic (Allium sativum) and ginger (Zingiber officinale) oils on the cotton leafworm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae). Afr Entomol 2018;26:84-94. https://doi.org/10.4001/003.026.0084 | |
11. Kimbaris AC, Kioulos E, Koliopoulos G, Polissiou MG, Michaelakis A. Coactivity of sulfide ingredients: A new perspective of the larvicidal activity of garlic essential oil against mosquitoes. Pest Manag Sci 2009;65:249-54. https://doi.org/10.1002/ps.1678 | |
12. Mossa AT, Mohafrash SM, Chandrasekaran N. Safety of natural insecticides: Toxic effects on experimental animals. Biomed Res Int 2018;16:4308054. https://doi.org/10.1155/2018/4308054 | |
13. Muturi EJ, Ramirez JL, Zilkowski B, Flor-Weiler LB, Rooney AP. Ovicidal and larvicidal effects of garlic and asafoetida essential oils against west nile virus vectors. J Insect Sci 2018;18:1-6. https://doi.org/10.1093/jisesa/iey036 | |
14. Nicolopoulou-Stamati P, Maipas S, Kotampasi C, Stamatis P, Hens L. Chemical pesticides and human health: The urgent need for a new concept in agriculture. Front Public Health 2016;4:1-8. https://doi.org/10.3389/fpubh.2016.00148 | |
15. Öz E, Koç S, Çinbilgel ?, Yan?ko?lu A, Çetin H. Chemical composition and larvicidal activity of essential oils from Nepeta cadmea boiss and Pimpinella anisum L. on the larvae of Culex pipiens L. Marmara Pharm J 2018;22:322-7. https://doi.org/10.12991/mpj.2018.70 | |
16. Palazzolo E, Laudicina VA, Germanà MA. Current and potential use of citrus essential oils. Curr Org Chem 2013;17:3042-9. https://doi.org/10.2174/13852728113179990122 | |
17. Plata-Rueda A, Martínez LC, Dos Santos MH, Fernandes FL, Wilcken CF, Soares MA, et al. Insecticidal activity of garlic essential oil and their constituents against the mealworm beetle, Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae). Sci Rep 2017;7:46406. https://doi.org/10.1038/srep46406 | |
18. Powell JR, Gloria-Soria A, Kotsakiozi P. Recent history of Aedes aegypti: Vector genomics and epidemiology records. Bioscience 2018;68:854-60. https://doi.org/10.1093/biosci/biy119 | |
19. Roiz D, Wilson AL, Scott TW, Fonseca DM, Jourdain F, Müller P, et al. Integrated Aedes management for the control of Aedes borne diseases. PLoS Negl Trop Dis 2018;12:e0006845. https://doi.org/10.1371/journal.pntd.0006845 | |
20. Sabahi Q, Hamiduzzaman MM, Barajas-Pérez JS, Tapia- Gonzalez JM, Guzman-Novoa E. Toxicity of anethole and the essential oils of lemongrass and sweet marigold to the parasitic mite Varroa destructor and their selectivity for honey bee (Apis mellifera) workers and larvae. Psyche 2018;2018:6196289. https://doi.org/10.1155/2018/6196289 | |
21. Service M. Medical Entomology for Students. 4th ed. England: Cambridge University Press; 2008. | |
22. Tongnuanchan P, Benjakul S. Essential oils: Extraction, bioactivities, and their uses for food preservation. J Food Sci 2014;79:1231-49. https://doi.org/10.1111/1750-3841.12492 | |
23. Vecchio MG, Gulati A, Minto C, Lorenzoni G. Pimpinella Anisum and Illicium Verum: The multifaceted role of anise plants. Open Agric J 2016;10:81-6. https://doi.org/10.2174/1874331501610010084 | |
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