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Combinations of plant-derived insecticidal compounds may exhibit synergistic or antagonistic interactions against pests. Given the rapid spread of diseases carried by Aedes mosquitoes and the increasing resistance of Aedes mosquito populations to traditional insecticides, twenty-eight combinations of terpene compounds based on plant essential oils were formulated and tested against the larval and adult stages of Aedes aegypti. Five plant essential oils (EOs) were initially evaluated for their larvicidal and adult-use efficacy, and two major compounds were identified in each EO based on GC-MS results. The main identified compounds were purchased, namely diallyl disulfide, diallyl trisulfide, carvone, limonene, eugenol, methyl eugenol, eucalyptol, eudesmol and mosquito alpha-pinene. Binary combinations of these compounds were then prepared using sublethal doses and their synergistic and antagonistic effects were tested and determined. The best larvicidal compositions are obtained by mixing limonene with diallyl disulfide, and the best adulticidal compositions are obtained by mixing carvone with limonene. The commercially used synthetic larvicide Temphos and the adult drug Malathion were tested separately and in binary combinations with terpenoids. The results showed that the combination of temephos and diallyl disulfide and malathion and eudesmol was the most effective combination. These potent combinations hold potential for use against Aedes aegypti.
Plant essential oils (EOs) are secondary metabolites containing various bioactive compounds and are becoming increasingly important as an alternative to synthetic pesticides. Not only are they environmentally friendly and user-friendly, but they are also a mixture of different bioactive compounds, which also reduces the likelihood of developing drug resistance1. Using GC-MS technology, researchers examined the constituents of various plant essential oils and identified more than 3,000 compounds from 17,500 aromatic plants2, most of which were tested for insecticidal properties and are reported to have insecticidal effects3,4. Some studies highlight that the toxicity of the compound’s main component is the same as or greater than that of its crude ethylene oxide. But the use of individual compounds may again leave room for the development of resistance, as is the case with chemical insecticides5,6. Therefore, the current focus is on preparing mixtures of ethylene oxide-based compounds to improve insecticidal effectiveness and reduce the likelihood of resistance in target pest populations. Individual active compounds present in EOs may exhibit synergistic or antagonistic effects in combinations reflecting the overall activity of the EO, a fact that has been well emphasized in studies conducted by previous researchers7,8. The vector control program also includes EO and its components. The mosquitocidal activity of essential oils has been extensively studied on Culex and Anopheles mosquitoes. Several studies have attempted to develop effective pesticides by combining various plants with commercially used synthetic pesticides to increase overall toxicity and minimize side effects9. But studies of such compounds against Aedes aegypti remain rare. Advances in medical science and the development of drugs and vaccines have helped combat some vector-borne diseases. But the presence of different serotypes of the virus, transmitted by the Aedes aegypti mosquito, has led to the failure of vaccination programs. Therefore, when such diseases occur, vector control programs are the only option to prevent the spread of the disease. In the current scenario, control of Aedes aegypti is very important as it is a key vector of various viruses and their serotypes causing dengue fever, Zika, dengue hemorrhagic fever, yellow fever, etc. The most noteworthy thing is the fact that the number of cases of almost all vector-borne Aedes-borne diseases are increasing every year in Egypt and are increasing worldwide. Therefore, in this context, there is an urgent need to develop environmentally friendly and effective control measures for Aedes aegypti populations. Potential candidates in this regard are EOs, their constituent compounds, and their combinations. Therefore, this study attempted to identify effective synergistic combinations of key plant EO compounds from five plants with insecticidal properties (i.e., mint, holy basil, Eucalyptus spotted, Allium sulfur and melaleuca) against Aedes aegypti.
All selected EOs demonstrated potential larvicidal activity against Aedes aegypti with 24-h LC50 ranging from 0.42 to 163.65 ppm. The highest larvicidal activity was recorded for peppermint (Mp) EO with an LC50 value of 0.42 ppm at 24 h, followed by garlic (As) with an LC50 value of 16.19 ppm at 24 h (Table 1).
With the exception of Ocimum Sainttum, Os EO, all other four screened EOs showed obvious allercidal effects, with LC50 values ranging from 23.37 to 120.16 ppm over the 24-hour exposure period. Thymophilus striata (Cl) EO was most effective in killing adults with an LC50 value of 23.37 ppm within 24 hours of exposure, followed by Eucalyptus maculata (Em) which had an LC50 value of 101.91 ppm (Table 1). On the other hand, the LC50 value for Os has not yet been determined as the highest mortality rate of 53% was recorded at the highest dose (Supplementary Figure 3).
The two major constituent compounds in each EO were identified and selected based on NIST library database results, GC chromatogram area percentage, and MS spectra results (Table 2). For EO As, the main compounds identified were diallyl disulfide and diallyl trisulfide; for EO Mp the main compounds identified were carvone and limonene, for EO Em the main compounds identified were eudesmol and eucalyptol; For EO Os, the main compounds identified were eugenol and methyl eugenol, and for EO Cl, the main compounds identified were eugenol and α-pinene (Figure 1, Supplementary Figures 5–8, Supplementary Table 1–5).
Results of mass spectrometry of the main terpenoids of selected essential oils (A-diallyl disulfide; B-diallyl trisulfide; C-eugenol; D-methyl eugenol; E-limonene; F-aromatic ceperone; G-α-pinene; H-cineole; R-eudamol).
A total of nine compounds (diallyl disulfide, diallyl trisulfide, eugenol, methyl eugenol, carvone, limonene, eucalyptol, eudesmol, α-pinene) were identified as effective compounds that are the main components of EO and were individually bioassayed against Aedes aegypti at larval stages. . The compound eudesmol had the highest larvicidal activity with an LC50 value of 2.25 ppm after 24 hours of exposure. The compounds diallyl disulfide and diallyl trisulfide have also been found to have potential larvicidal effects, with mean sublethal doses in the range of 10–20 ppm. Moderate larvicidal activity was again observed for the compounds eugenol, limonene and eucalyptol with LC50 values of 63.35 ppm, 139.29 ppm. and 181.33 ppm after 24 hours, respectively (Table 3). However, no significant larvicidal potential of methyl eugenol and carvone was found even at the highest doses, so LC50 values were not calculated (Table 3). The synthetic larvicide Temephos had a mean lethal concentration of 0.43 ppm against Aedes aegypti over 24 hours of exposure (Table 3, Supplementary Table 6).
Seven compounds (diallyl disulfide, diallyl trisulfide, eucalyptol, α-pinene, eudesmol, limonene and carvone) were identified as the main compounds of effective EO and were tested individually against adult Egyptian Aedes mosquitoes. According to Probit regression analysis, Eudesmol was found to have the highest potential with an LC50 value of 1.82 ppm, followed by Eucalyptol with an LC50 value of 17.60 ppm at 24-hour exposure time. The remaining five compounds tested were moderately harmful to adults with LC50s ranging from 140.79 to 737.01 ppm (Table 3). The synthetic organophosphorus malathion was less potent than eudesmol and higher than the other six compounds, with an LC50 value of 5.44 ppm over the 24-hour exposure period (Table 3, Supplementary Table 6).
Seven potent lead compounds and the organophosphorus tamephosate were selected to formulate binary combinations of their LC50 doses in a 1:1 ratio. A total of 28 binary combinations were prepared and tested for their larvicidal efficacy against Aedes aegypti. Nine combinations were found to be synergistic, 14 combinations were antagonistic, and five combinations were not larvicidal. Among the synergistic combinations, the combination of diallyl disulfide and temofol was the most effective, with 100% mortality observed after 24 hours (Table 4). Similarly, mixtures of limonene with diallyl disulfide and eugenol with thymetphos showed good potential with an observed larval mortality of 98.3% (Table 5). The remaining 4 combinations, namely eudesmol plus eucalyptol, eudesmol plus limonene, eucalyptol plus alpha-pinene, alpha-pinene plus temephos, also showed significant larvicidal efficacy, with observed mortality rates exceeding 90%. The expected mortality rate is close to 60-75%. (Table 4). However, the combination of limonene with α-pinene or eucalyptus showed antagonistic reactions. Likewise, mixtures of Temephos with eugenol or eucalyptus or eudesmol or diallyl trisulfide have been found to have antagonistic effects. Likewise, the combination of diallyl disulfide and diallyl trisulfide and the combination of either of these compounds with eudesmol or eugenol are antagonistic in their larvicidal action. Antagonism has also been reported with the combination of eudesmol with eugenol or α-pinene.
Of all 28 binary mixtures tested for adult acidic activity, 7 combinations were synergistic, 6 had no effect, and 15 were antagonistic. Mixtures of eudesmol with eucalyptus and limonene with carvone were found to be more effective than other synergistic combinations, with mortality rates at 24 hours of 76% and 100%, respectively (Table 5). Malathion has been observed to exhibit a synergistic effect with all combinations of compounds except limonene and diallyl trisulfide. On the other hand, antagonism has been found between diallyl disulfide and diallyl trisulfide and the combination of either of them with eucalyptus, or eucalyptol, or carvone, or limonene. Similarly, combinations of α-pinene with eudesmol or limonene, eucalyptol with carvone or limonene, and limonene with eudesmol or malathion showed antagonistic larvicidal effects. For the remaining six combinations, there was no significant difference between expected and observed mortality (Table 5).
Based on synergistic effects and sublethal doses, their larvicidal toxicity against a large number of Aedes aegypti mosquitoes was ultimately selected and further tested. The results showed that the observed larval mortality using the binary combinations eugenol-limonene, diallyl disulfide-limonene and diallyl disulfide-timephos was 100%, while the expected larval mortality was 76.48%, 72.16% and 63.4%, respectively (Table 6 ). . The combination of limonene and eudesmol was relatively less effective, with 88% larval mortality observed over the 24-hour exposure period (Table 6). In summary, the four selected binary combinations also demonstrated synergistic larvicidal effects against Aedes aegypti when applied on a large scale (Table 6).
Three synergistic combinations were selected for the adultocidal bioassay to control large populations of adult Aedes aegypti. To select combinations to test on large insect colonies, we first focused on the two best synergistic terpene combinations, namely carvone plus limonene and eucalyptol plus eudesmol. Secondly, the best synergistic combination was selected from the combination of synthetic organophosphate malathion and terpenoids. We believe that the combination of malathion and eudesmol is the best combination for testing on large insect colonies due to the highest observed mortality and very low LC50 values of the candidate ingredients. Malathion exhibits synergism in combination with α-pinene, diallyl disulfide, eucalyptus, carvone and eudesmol. But if we look at the LC50 values, Eudesmol has the lowest value (2.25 ppm). The calculated LC50 values of malathion, α-pinene, diallyl disulfide, eucalyptol and carvone were 5.4, 716.55, 166.02, 17.6 and 140.79 ppm. respectively. These values indicate that the combination of malathion and eudesmol is the optimal combination in terms of dosage. The results showed that the combinations of carvone plus limonene and eudesmol plus malathion had 100% observed mortality compared with an expected mortality of 61% to 65%. Another combination, eudesmol plus eucalyptol, showed a mortality rate of 78.66% after 24 hours of exposure, compared to an expected mortality rate of 60%. All three selected combinations demonstrated synergistic effects even when applied on a large scale against adult Aedes aegypti (Table 6).
In this study, selected plant EOs such as Mp, As, Os, Em and Cl showed promising lethal effects on the larval and adult stages of Aedes aegypti. Mp EO had the highest larvicidal activity with an LC50 value of 0.42 ppm, followed by As, Os and Em EOs with an LC50 value of less than 50 ppm after 24 h. These results are consistent with previous studies of mosquitoes and other dipterous flies10,11,12,13,14. Although the larvicidal potency of Cl is lower than other essential oils, with an LC50 value of 163.65 ppm after 24 hours, its adult potential is the highest with an LC50 value of 23.37 ppm after 24 hours. Mp, As and Em EOs also showed good allercidal potential with LC50 values in the range of 100–120 ppm at 24 h of exposure, but were relatively lower than their larvicidal efficacy. On the other hand, EO Os demonstrated a negligible allercidal effect even at the highest therapeutic dose. Thus, the results indicate that the toxicity of ethylene oxide to plants may vary depending on the developmental stage of mosquitoes15. It also depends on the rate of penetration of EOs into the insect’s body, their interaction with specific target enzymes, and the detoxification capacity of the mosquito at each developmental stage16. A large number of studies have shown that the main component compound is an important factor in the biological activity of ethylene oxide, since it accounts for the majority of the total compounds3,12,17,18. Therefore, we considered two main compounds in each EO. Based on the GC-MS results, diallyl disulfide and diallyl trisulfide were identified as the major compounds of EO As, which is consistent with previous reports19,20,21. Although previous reports indicated that menthol was one of its main compounds, carvone and limonene were again identified as the main compounds of Mp EO22,23. The composition profile of Os EO showed that eugenol and methyl eugenol are the main compounds, which is similar to the findings of earlier researchers16,24. Eucalyptol and eucalyptol have been reported as the main compounds present in Em leaf oil, which is consistent with the findings of some researchers25,26 but contrary to the findings of Olalade et al.27. The dominance of cineole and α-pinene was observed in melaleuca essential oil, which is similar to previous studies28,29. Intraspecific differences in the composition and concentration of essential oils extracted from the same plant species in different locations have been reported and were also observed in this study, which are influenced by geographic plant growth conditions, harvest time, developmental stage, or plant age. appearance of chemotypes, etc.22,30,31,32. The key identified compounds were then purchased and tested for their larvicidal effects and effects on adult Aedes aegypti mosquitoes. The results showed that the larvicidal activity of diallyl disulfide was comparable to that of crude EO As. But the activity of diallyl trisulfide is higher than EO As. These results are similar to those obtained by Kimbaris et al. 33 on Culex philippines. However, these two compounds did not show good autocidal activity against the target mosquitoes, which is consistent with the results of Plata-Rueda et al 34 on Tenebrio molitor. Os EO is effective against the larval stage of Aedes aegypti, but not against the adult stage. It has been established that the larvicidal activity of the main individual compounds is lower than that of crude Os EO. This implies a role for other compounds and their interactions in crude ethylene oxide. Methyl eugenol alone has negligible activity, whereas eugenol alone has moderate larvicidal activity. This conclusion confirms, on the one hand,35,36, and on the other hand, contradicts the conclusions of earlier researchers37,38. Differences in the functional groups of eugenol and methyleugenol may result in different toxicities to the same target insect39. Limonene was found to have moderate larvicidal activity, while the effect of carvone was insignificant. Similarly, the relatively low toxicity of limonene to adult insects and the high toxicity of carvone support the results of some previous studies40 but contradict others41. The presence of double bonds at both intracyclic and exocyclic positions may increase the benefits of these compounds as larvicides3,41, while carvone, which is a ketone with unsaturated alpha and beta carbons, may exhibit a higher potential for toxicity in adults42. However, the individual characteristics of limonene and carvone are much lower than the total EO Mp (Table 1, Table 3). Among the terpenoids tested, eudesmol was found to have the greatest larvicidal and adult activity with an LC50 value below 2.5 ppm, making it a promising compound for the control of Aedes mosquitoes. Its performance is better than that of the entire EO Em, although this is not consistent with the findings of Cheng et al.40. Eudesmol is a sesquiterpene with two isoprene units that is less volatile than oxygenated monoterpenes such as eucalyptus and therefore has greater potential as a pesticide. Eucalyptol itself has greater adult than larvicidal activity, and results from earlier studies both support and refute this37,43,44. The activity alone is almost comparable to that of the entire EO Cl. Another bicyclic monoterpene, α-pinene, has less of an adult effect on Aedes aegypti than a larvicidal effect, which is the opposite of the effect of full EO Cl. The overall insecticidal activity of terpenoids is influenced by their lipophilicity, volatility, carbon branching, projection area, surface area, functional groups and their positions45,46. These compounds may act by destroying cell accumulations, blocking respiratory activity, interrupting the transmission of nerve impulses, etc. 47 The synthetic organophosphate Temephos was found to have the highest larvicidal activity with an LC50 value of 0.43 ppm, which is consistent with Lek’s data -Utala48. Adult activity of the synthetic organophosphorus malathion was reported at 5.44 ppm. Although these two organophosphates have shown favorable responses against laboratory strains of Aedes aegypti, mosquito resistance to these compounds has been reported in different parts of the world49. However, no similar reports of the development of resistance to herbal medicines have been found50. Thus, botanicals are considered as potential alternatives to chemical pesticides in vector control programs.
The larvicidal effect was tested on 28 binary combinations (1:1) prepared from potent terpenoids and terpenoids with thymetphos, and 9 combinations were found to be synergistic, 14 antagonistic and 5 antagonistic. No effect. On the other hand, in the adult potency bioassay, 7 combinations were found to be synergistic, 15 combinations were antagonistic, and 6 combinations were reported to have no effect. The reason why certain combinations produce a synergistic effect may be due to the candidate compounds interacting simultaneously in different important pathways, or to the sequential inhibition of different key enzymes of a particular biological pathway51. The combination of limonene with diallyl disulfide, eucalyptus or eugenol was found to be synergistic in both small and large scale applications (Table 6), while its combination with eucalyptus or α-pinene was found to have antagonistic effects on larvae. On average, limonene appears to be a good synergist, possibly due to the presence of methyl groups, good penetration into the stratum corneum, and a different mechanism of action52,53. It has previously been reported that limonene may cause toxic effects by penetrating insect cuticles (contact toxicity), affecting the digestive system (antifeedant), or affecting the respiratory system (fumigation activity), 54 while phenylpropanoids such as eugenol may affect metabolic enzymes 55. Therefore, combinations of compounds with different mechanisms of action may increase the overall lethal effect of the mixture. Eucalyptol was found to be synergistic with diallyl disulfide, eucalyptus or α-pinene, but other combinations with other compounds were either non-larvicidal or antagonistic. Early studies showed that eucalyptol has inhibitory activity on acetylcholinesterase (AChE), as well as octaamine and GABA receptors56. Since cyclic monoterpenes, eucalyptol, eugenol, etc. may have the same mechanism of action as their neurotoxic activity, 57 thereby minimizing their combined effects through mutual inhibition. Likewise, the combination of Temephos with diallyl disulfide, α-pinene and limonene was found to be synergistic, supporting previous reports of a synergistic effect between herbal products and synthetic organophosphates58.
The combination of eudesmol and eucalyptol was found to have a synergistic effect on the larval and adult stages of Aedes aegypti, possibly due to their different modes of action due to their different chemical structures. Eudesmol (a sesquiterpene) may affect the respiratory system 59 and eucalyptol (a monoterpene) may affect acetylcholinesterase 60 . Co-exposure of the ingredients to two or more target sites may enhance the overall lethal effect of the combination. In adult substance bioassays, malathion was found to be synergistic with carvone or eucalyptol or eucalyptol or diallyl disulfide or α-pinene, indicating that it is synergistic with the addition of limonene and di. Good synergistic allercide candidates for the entire portfolio of terpene compounds, with the exception of allyl trisulfide. Thangam and Kathiresan61 also reported similar results of the synergistic effect of malathion with herbal extracts. This synergistic response may be due to the combined toxic effects of malathion and phytochemicals on insect detoxifying enzymes. Organophosphates such as malathion generally act by inhibiting cytochrome P450 esterases and monooxygenases62,63,64. Therefore, combining malathion with these mechanisms of action and terpenes with different mechanisms of action may enhance the overall lethal effect on mosquitoes.
On the other hand, antagonism indicates that the selected compounds are less active in combination than each compound alone. The reason for antagonism in some combinations may be that one compound modifies the behavior of the other compound by changing the rate of absorption, distribution, metabolism, or excretion. Early researchers considered this to be the cause of antagonism in drug combinations. Molecules Possible mechanism 65. Similarly, possible causes of antagonism may be related to similar mechanisms of action, competition of constituent compounds for the same receptor or target site. In some cases, non-competitive inhibition of the target protein may also occur. In this study, two organosulfur compounds, diallyl disulfide and diallyl trisulfide, showed antagonistic effects, possibly due to competition for the same target site. Likewise, these two sulfur compounds showed antagonistic effects and had no effect when combined with eudesmol and α-pinene. Eudesmol and alpha-pinene are cyclic in nature, whereas diallyl disulfide and diallyl trisulfide are aliphatic in nature. Based on the chemical structure, the combination of these compounds should increase the overall lethal activity since their target sites are usually different34,47, but experimentally we found antagonism, which may be due to the role of these compounds in some unknown organisms in vivo. systems as a result of interaction. Similarly, the combination of cineole and α-pinene produced antagonistic responses, although researchers previously reported that the two compounds have different targets of action47,60. Since both compounds are cyclic monoterpenes, there may be some common target sites that may compete for binding and influence the overall toxicity of the combinatorial pairs studied.
Based on LC50 values and observed mortality, the two best synergistic terpene combinations were selected, namely the pairs of carvone + limonene and eucalyptol + eudesmol, as well as the synthetic organophosphorus malathion with terpenes. The optimal synergistic combination of malathion + Eudesmol compounds was tested in an adult insecticide bioassay. Target large insect colonies to confirm whether these effective combinations can work against large numbers of individuals over relatively large exposure spaces. All of these combinations demonstrate a synergistic effect against large swarms of insects. Similar results were obtained for an optimal synergistic larvicidal combination tested against large populations of Aedes aegypti larvae. Thus, it can be said that the effective synergistic larvicidal and adulticidal combination of plant EO compounds is a strong candidate against existing synthetic chemicals and can be further used to control Aedes aegypti populations. Likewise, effective combinations of synthetic larvicides or adulticides with terpenes can also be used to reduce the doses of thymetphos or malathion administered to mosquitoes. These potent synergistic combinations may provide solutions for future studies on the evolution of drug resistance in Aedes mosquitoes.
Eggs of Aedes aegypti were collected from the Regional Medical Research Centre, Dibrugarh, Indian Council of Medical Research and kept under controlled temperature (28 ± 1 °C) and humidity (85 ± 5%) in the Department of Zoology, Gauhati University under the following conditions: Arivoli were described et al. After hatching, larvae were fed larval food (dog biscuit powder and yeast in a 3:1 ratio) and adults were fed a 10% glucose solution. Beginning on the 3rd day after emergence, adult female mosquitoes were allowed to suck the blood of albino rats. Soak filter paper in water in a glass and place it in the egg-laying cage.
Selected plant samples namely eucalyptus leaves (Myrtaceae), holy basil (Lamiaceae), mint (Lamiaceae), melaleuca (Myrtaceae) and allium bulbs (Amaryllidaceae). Collected from Guwahati and identified by the Department of Botany, Gauhati University. The collected plant samples (500 g) were subjected to hydrodistillation using a Clevenger apparatus for 6 hours. The extracted EO was collected in clean glass vials and stored at 4°C for further study.
Larvicidal toxicity was studied using slightly modified standard World Health Organization procedures 67 . Use DMSO as an emulsifier. Each EO concentration was initially tested at 100 and 1000 ppm, exposing 20 larvae in each replicate. Based on the results, a concentration range was applied and mortality was recorded from 1 hour to 6 hours (at 1 hour intervals), and at 24 hours, 48 hours and 72 hours after treatment. Sublethal concentrations (LC50) were determined after 24, 48 and 72 hours of exposure. Each concentration was assayed in triplicate along with one negative control (water only) and one positive control (DMSO-treated water). If pupation occurs and more than 10% of the larvae of the control group die, the experiment is repeated. If the mortality rate in the control group is between 5-10%, use the Abbott correction formula 68.
The method described by Ramar et al. 69 was used for an adult bioassay against Aedes aegypti using acetone as a solvent. Each EO was initially tested against adult Aedes aegypti mosquitoes at concentrations of 100 and 1000 ppm. Apply 2 ml of each prepared solution to the Whatman number. 1 piece of filter paper (size 12 x 15 cm2) and let the acetone evaporate for 10 minutes. Filter paper treated with only 2 ml of acetone was used as a control. After the acetone has evaporated, the treated filter paper and control filter paper are placed in a cylindrical tube (10 cm deep). Ten 3- to 4-day-old non-blood feeding mosquitoes were transferred to triplicates of each concentration. Based on the results of preliminary tests, various concentrations of selected oils were tested. Mortality was recorded at 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 24 hours, 48 hours and 72 hours after mosquito release. Calculate LC50 values for exposure times of 24 hours, 48 hours and 72 hours. If the mortality rate of the control lot exceeds 20%, repeat the entire test. Likewise, if the mortality rate in the control group is greater than 5%, adjust the results for the treated samples using Abbott’s formula68.
Gas chromatography (Agilent 7890A) and mass spectrometry (Accu TOF GCv, Jeol) were performed to analyze the constituent compounds of the selected essential oils. The GC was equipped with an FID detector and a capillary column (HP5-MS). The carrier gas was helium, the flow rate was 1 ml/min. The GC program sets Allium sativum to 10:80-1M-8-220-5M-8-270-9M and Ocimum Sainttum to 10:80-3M-8-200-3M-10-275-1M-5 – 280, for mint 10:80-1M-8-200-5M-8-275-1M-5-280, for eucalyptus 20.60-1M-10-200-3M-30-280, and for red For a thousand layers they are them 10: 60-1M-8-220-5M-8-270-3M.
The major compounds of each EO were identified based on the area percentage calculated from the GC chromatogram and mass spectrometry results (referenced to the NIST 70 standards database).
The two major compounds in each EO were selected based on GC-MS results and purchased from Sigma-Aldrich at 98–99% purity for further bioassays. The compounds were tested for larvicidal and adult efficacy against Aedes aegypti as described above. The most commonly used synthetic larvicides tamephosate (Sigma Aldrich) and the adult drug malathion (Sigma Aldrich) were analyzed to compare their effectiveness with selected EO compounds, following the same procedure.
Binary mixtures of selected terpene compounds and terpene compounds plus commercial organophosphates (tilephos and malathion) were prepared by mixing the LC50 dose of each candidate compound in a 1:1 ratio. The prepared combinations were tested on larval and adult stages of Aedes aegypti as described above. Each bioassay was performed in triplicate for each combination and in triplicate for the individual compounds present in each combination. Death of target insects was recorded after 24 hours. Calculate the expected mortality rate for a binary mixture using the following formula.
where E = expected mortality rate of Aedes aegypti mosquitoes in response to a binary combination, i.e. connection (A + B).
The effect of each binary mixture was labeled as synergistic, antagonistic, or no effect based on the χ2 value calculated by the method described by Pavla52. Calculate the χ2 value for each combination using the following formula.
The effect of a combination was defined as synergistic when the calculated χ2 value was greater than the table value for the corresponding degrees of freedom (95% confidence interval) and if the observed mortality was found to exceed the expected mortality. Similarly, if the calculated χ2 value for any combination exceeds the table value with some degrees of freedom, but the observed mortality is lower than the expected mortality, the treatment is considered antagonistic. And if in any combination the calculated value of χ2 is less than the table value in the corresponding degrees of freedom, the combination is considered to have no effect.
Three to four potentially synergistic combinations (100 larvae and 50 larvicidal and adult insect activity) were selected for testing against a large number of insects. Adults) proceed as above. Along with the mixtures, individual compounds present in the selected mixtures were also tested on equal numbers of Aedes aegypti larvae and adults. The combination ratio is one part LC50 dose of one candidate compound and part LC50 dose of the other constituent compound. In the adult activity bioassay, selected compounds were dissolved in the solvent acetone and applied to filter paper wrapped in a 1300 cm3 cylindrical plastic container. The acetone was evaporated for 10 minutes and the adults were released. Similarly, in the larvicidal bioassay, doses of LC50 candidate compounds were first dissolved in equal volumes of DMSO and then mixed with 1 liter of water stored in 1300 cc plastic containers, and the larvae were released.
Probabilistic analysis of 71 recorded mortality data was performed using SPSS (version 16) and Minitab software to calculate LC50 values.
Post time: Jul-01-2024