In a previous project testing local food processing plants for mosquitoes in Thailand, the essential oils (EOs) of Cyperus rotundus, galangal and cinnamon were found to have good anti-mosquito activity against Aedes aegypti. In an attempt to reduce the use of traditional insecticides and improve control of resistant mosquito populations, this study aimed to identify the potential synergism between the adulticidal effects of ethylene oxide and the toxicity of permethrin to Aedes mosquitoes. aegypti, including pyrethroid-resistant and sensitive strains.
To evaluate the chemical composition and killing activity of EO extracted from rhizomes of C. rotundus and A. galanga and bark of C. verum against the susceptible strain Muang Chiang Mai (MCM-S) and the resistant strain Pang Mai Dang (PMD-R). ) Adult active Ae. Aedes aegypti. An adult bioassay of the EO-permethrin mixture was also performed on these Aedes mosquitoes to understand its synergistic activity. aegypti strains.
Chemical characterization using GC-MS analytical method showed that 48 compounds were identified from the EOs of C. rotundus, A. galanga and C. verum, accounting for 80.22%, 86.75% and 97.24% of the total components, respectively. Cyperene (14.04%), β-bisabolene (18.27%), and cinnamaldehyde (64.66%) are the main components of cyperus oil, galangal oil, and balsamic oil, respectively. In biological adult killing assays, C. rotundus, A. galanga and C. verum EVs were effective in killing Ae. aegypti, MCM-S and PMD-R LD50 values were 10.05 and 9.57 μg/mg female, 7.97 and 7.94 μg/mg female, and 3.30 and 3.22 μg/mg female, respectively. Efficiency of MCM-S and PMD-R Ae in killing adults. aegypti in these EOs was close to piperonyl butoxide (PBO values, LD50 = 6.30 and 4.79 μg/mg female, respectively), but not as pronounced as permethrin (LD50 values = 0.44 and 3.70 ng/mg female respectively). However, combination bioassays found synergy between EO and permethrin. Significant synergism with permethrin against two strains of Aedes mosquitoes. Aedes aegypti was noted in the EM of C. rotundus and A. galanga. The addition of C. rotundus and A. galanga oils significantly reduced the LD50 values of permethrin on MCM-S from 0.44 to 0.07 ng/mg and 0.11 ng/mg in females, respectively, with synergy ratio (SR) values of 6.28 and 4.00 respectively. In addition, C. rotundus and A. galanga EOs also significantly reduced the LD50 values of permethrin on PMD-R from 3.70 to 0.42 ng/mg and 0.003 ng/mg in females, respectively, with SR values of 8.81 and 1233.33, respectively. .
Synergistic effect of an EO-permethrin combination to enhance adult toxicity against two strains of Aedes mosquitoes. Aedes aegypti demonstrates a promising role for ethylene oxide as a synergist in enhancing anti-mosquito efficacy, especially where traditional compounds are ineffective or inappropriate.
The Aedes aegypti mosquito (Diptera: Culicidae) is the main vector of dengue fever and other infectious viral diseases such as yellow fever, chikungunya and Zika virus, posing a huge and persistent threat to humans[1, 2]. . Dengue virus is the most serious pathogenic hemorrhagic fever affecting humans, with an estimated 5–100 million cases occurring annually and more than 2.5 billion people worldwide at risk [3]. Outbreaks of this infectious disease place a huge burden on the populations, health systems and economies of most tropical countries [1]. According to the Thai Ministry of Health, there were 142,925 cases of dengue fever and 141 deaths reported nationwide in 2015, more than three times the number of cases and deaths in 2014 [4]. Despite historical evidence, dengue fever has been eradicated or greatly reduced by the Aedes mosquito. Following control of Aedes aegypti [5], infection rates increased dramatically and the disease spread throughout the world, due in part to decades of global warming. Elimination and control of Ae. Aedes aegypti is relatively difficult because it is a domestic mosquito vector that mates, feeds, rests and lays eggs in and around human habitation during the day. In addition, this mosquito has the ability to adapt to environmental changes or disturbances caused by natural events (such as drought) or human control measures, and can return to its original numbers [6, 7]. Because vaccines against dengue fever have only recently been approved and there is no specific treatment for dengue fever, preventing and reducing the risk of dengue transmission depends entirely on controlling the mosquito vectors and eliminating human contact with the vectors.
In particular, the use of chemicals for mosquito control now plays an important role in public health as an important component of comprehensive integrated vector management. The most popular chemical methods include the use of low-toxic insecticides that act against mosquito larvae (larvicides) and adult mosquitoes (adidocides). Larval control through source reduction and regular use of chemical larvicides such as organophosphates and insect growth regulators is considered important. However, the adverse environmental impacts associated with synthetic pesticides and their labor-intensive and complex maintenance remain a major concern [8, 9]. Traditional active vector control, such as adult control, remains the most effective means of control during viral outbreaks because it can eradicate infectious disease vectors quickly and on a large scale, as well as reduce the lifespan and longevity of local vector populations [3]. , 10]. Four classes of chemical insecticides: organochlorines (referred to only as DDT), organophosphates, carbamates, and pyrethroids form the basis of vector control programs, with pyrethroids considered the most successful class. They are highly effective against various arthropods and have low effectiveness. toxicity to mammals. Currently, synthetic pyrethroids constitute the majority of commercial pesticides, accounting for about 25% of the global pesticide market [11, 12]. Permethrin and deltamethrin are broad-spectrum pyrethroid insecticides that have been used worldwide for decades to control a variety of pests of agricultural and medical importance [13, 14]. In the 1950s, DDT was selected as the chemical of choice for Thailand’s national public health mosquito control program. Following the widespread use of DDT in malaria-endemic areas, Thailand gradually phased out the use of DDT between 1995 and 2000 and replaced it with two pyrethroids: permethrin and deltamethrin [15, 16]. These pyrethroid insecticides were introduced in the early 1990s to control malaria and dengue fever, primarily through bed net treatments and the use of thermal fogs and ultra-low toxicity sprays [14, 17]. However, they have lost effectiveness due to strong mosquito resistance and lack of public compliance due to concerns about public health and the environmental impact of synthetic chemicals. This poses significant challenges to the success of threat vector control programs [14, 18, 19]. To make the strategy more effective, timely and appropriate countermeasures are necessary. Recommended management procedures include substitution of natural substances, rotation of chemicals of different classes, addition of synergists, and mixing of chemicals or simultaneous application of chemicals of different classes [14, 20, 21]. Therefore, there is an urgent need to find and develop an eco-friendly, convenient and effective alternative and synergist and this study aims to address this need.
Naturally derived insecticides, especially those based on plant components, have shown potential in the evaluation of current and future mosquito control alternatives [22, 23, 24]. Several studies have shown that it is possible to control important mosquito vectors by using plant products, especially essential oils (EOs), as adult killers. Adulticidal properties against some important mosquito species have been found in many vegetable oils such as celery, cumin, zedoaria, anise, pipe pepper, thyme, Schinus terebinthifolia, Cymbopogon citratus, Cymbopogon schoenanthus, Cymbopogon giganteus, Chenopodium ambrosioides, Cochlospermum planchonii, Eucalyptus ter eticornis . , Eucalyptus citriodora, Cananga odorata and Petroselinum Criscum [25,26,27,28,29,30]. Ethylene oxide is now used not only on its own, but also in combination with extracted plant substances or existing synthetic pesticides, producing varying degrees of toxicity. Combinations of traditional insecticides such as organophosphates, carbamates and pyrethroids with ethylene oxide/plant extracts act synergistically or antagonistically in their toxic effects and have been shown to be effective against disease vectors and pests [31,32,33,34,35]. However, most studies on the synergistic toxic effects of combinations of phytochemicals with or without synthetic chemicals have been conducted on agricultural insect vectors and pests rather than on medically important mosquitoes. Moreover, most of the work on the synergistic effects of plant-synthetic insecticide combinations against mosquito vectors has focused on the larvicidal effect.
In a previous study conducted by the authors as part of an ongoing research project screening intimicides from indigenous food plants in Thailand, ethylene oxides from Cyperus rotundus, galangal and cinnamon were found to have potential activity against adult Aedes. Egypt [36]. Therefore, this study aimed to evaluate the effectiveness of EOs isolated from these medicinal plants against Aedes mosquitoes. aegypti, including pyrethroid-resistant and sensitive strains. The synergistic effect of binary mixtures of ethylene oxide and synthetic pyrethroids with good efficacy in adults has also been analyzed to reduce the use of traditional insecticides and increase resistance to mosquito vectors, especially against Aedes. Aedes aegypti. This article reports the chemical characterization of effective essential oils and their potential to enhance the toxicity of synthetic permethrin against Aedes mosquitoes. aegypti in pyrethroid-sensitive strains (MCM-S) and resistant strains (PMD-R).
Rhizomes of C. rotundus and A. galanga and bark of C. verum (Fig. 1) used for essential oil extraction were purchased from herbal medicine suppliers in Chiang Mai Province, Thailand. The scientific identification of these plants was achieved through consultation with Mr. James Franklin Maxwell, Herbarium Botanist, Department of Biology, College of Science, Chiang Mai University (CMU), Chiang Mai Province, Thailand, and scientist Wannari Charoensap; in the Department of Pharmacy, College of Pharmacy, Carnegie Mellon University, Ms. Voucher specimens of each plant are stored in the Department of Parasitology at Carnegie Mellon University School of Medicine for future use.
Plant samples were shade-dried individually for 3–5 days in an open space with active ventilation and an ambient temperature of approximately 30 ± 5 °C to remove moisture content before extraction of natural essential oils (EOs). A total of 250 g of each dry plant material was mechanically ground into a coarse powder and used to isolate essential oils (EOs) by steam distillation. The distillation apparatus consisted of an electric heating mantle, a 3000 mL round-bottom flask, an extraction column, a condenser, and a Cool ace device (Eyela Cool Ace CA-1112 CE, Tokyo Rikakikai Co. Ltd., Tokyo, Japan). Add 1600 ml distilled water and 10-15 glass beads to the flask and then heat it to approximately 100°C using an electric heater for at least 3 hours until distillation is complete and no more EO is produced. The EO layer was separated from the aqueous phase using a separatory funnel, dried over anhydrous sodium sulfate (Na2SO4) and stored in a sealed brown bottle at 4°C until chemical composition and adult activity were examined.
The chemical composition of essential oils was carried out simultaneously with the bioassay for the adult substance. Qualitative analysis was performed using a GC-MS system consisting of a Hewlett-Packard (Wilmington, CA, USA) 7890A gas chromatograph equipped with a single quadrupole mass selective detector (Agilent Technologies, Wilmington, CA, USA) and an MSD 5975C (EI). (Agilent Technologies).
Chromatographic column – DB-5MS (30 m × ID 0.25 mm × film thickness 0.25 µm). The total GC-MS run time was 20 minutes. The analysis conditions are that the injector and transfer line temperatures are 250 and 280 °C, respectively; the furnace temperature is set to increase from 50°C to 250°C at a rate of 10°C/min, the carrier gas is helium; flow rate 1.0 ml/min; injection volume is 0.2 µL (1/10% by volume in CH2Cl2, split ratio 100:1); An electron ionization system with an ionization energy of 70 eV is used for GC-MS detection. The acquisition range is 50–550 atomic mass units (amu) and the scanning speed is 2.91 scans per second. Relative percentages of components are expressed as percentages normalized by peak area. Identification of EO ingredients is based on their retention index (RI). RI was calculated using the equation of Van den Dool and Kratz [37] for the n-alkanes series (C8-C40) and compared with retention indices from the literature [38] and library databases (NIST 2008 and Wiley 8NO8). The identity of the compounds shown, such as structure and molecular formula, was confirmed by comparison with available authentic samples.
Analytical standards for synthetic permethrin and piperonyl butoxide (PBO, positive control in synergy studies) were purchased from Sigma-Aldrich (St. Louis, MO, USA). World Health Organization (WHO) adult testing kits and diagnostic doses of permethrin-impregnated paper (0.75%) were commercially purchased from the WHO Vector Control Center in Penang, Malaysia. All other chemicals and reagents used were of analytical grade and were purchased from local institutions in Chiang Mai Province, Thailand.
The mosquitoes used as test organisms in the adult bioassay were freely mating laboratory Aedes mosquitoes. aegypti, including the susceptible Muang Chiang Mai strain (MCM-S) and the resistant Pang Mai Dang strain (PMD-R). Strain MCM-S was obtained from local samples collected in the Muang Chiang Mai area, Chiang Mai Province, Thailand, and has been maintained in the entomology room of the Department of Parasitology, CMU School of Medicine, since 1995 [39]. The PMD-R strain, which was found to be resistant to permethrin, was isolated from field mosquitoes originally collected from Ban Pang Mai Dang, Mae Tang District, Chiang Mai Province, Thailand, and has been maintained at the same institute since 1997 [40]. PMD-R strains were grown under selective pressure to maintain resistance levels by intermittent exposure to 0.75% permethrin using the WHO detection kit with some modifications [41]. Each strain of Ae. Aedes aegypti was colonized individually in a pathogen-free laboratory at 25 ± 2 °C and 80 ± 10% relative humidity and a 14:10 h light/dark photoperiod. Approximately 200 larvae were kept in plastic trays (33 cm long, 28 cm wide and 9 cm high) filled with tap water at a density of 150–200 larvae per tray and fed twice daily with sterilized dog biscuits. Adult worms were kept in moist cages and continuously fed with a 10% aqueous sucrose solution and a 10% multivitamin syrup solution. Female mosquitoes regularly suck blood to lay eggs. Females two to five days old that have not been blood-fed can be used continuously in experimental adult biological assays.
A dose-mortality response bioassay of EO was performed on adult female Aedes mosquitoes. aegypti, MCM-S and PMD-R using a topical method modified according to the WHO standard protocol for susceptibility testing [42]. EO from each plant was serially diluted with a suitable solvent (e.g. ethanol or acetone) to obtain a graduated series of 4-6 concentrations. After anesthesia with carbon dioxide (CO2), mosquitoes were weighed individually. The anesthetized mosquitoes were then kept motionless on dry filter paper on a custom cold plate under a stereomicroscope to prevent reactivation during the procedure. For each treatment, 0.1 μl of EO solution was applied to the female’s upper pronotum using a Hamilton handheld microdispenser (700 Series Microliter™, Hamilton Company, Reno, NV, USA). Twenty-five females were treated with each concentration, with mortality ranging from 10% to 95% for at least 4 different concentrations. Mosquitoes treated with solvent served as control. To prevent contamination of test samples, replace the filter paper with new filter paper for each EO tested. Doses used in these bioassays are expressed in micrograms of EO per milligram of living female body weight. Adult PBO activity was also assessed in a similar manner to EO, with PBO used as a positive control in synergistic experiments. Treated mosquitoes in all groups were placed in plastic cups and given 10% sucrose plus 10% multivitamin syrup. All bioassays were performed at 25 ± 2 °C and 80 ± 10% relative humidity and repeated four times with controls. Mortality during the 24-hour rearing period was checked and confirmed by the mosquito’s lack of response to mechanical stimulation and then recorded based on the average of four replicates. Experimental treatments were repeated four times for each test sample using different batches of mosquitoes. The results were summarized and used to calculate the percentage mortality rate, which was used to determine the 24-hour lethal dose by probit analysis.
The synergistic anticidal effect of EO and permethrin was assessed using a local toxicity assay procedure [42] as previously described. Use acetone or ethanol as a solvent to prepare permethrin at the desired concentration, as well as a binary mixture of EO and permethrin (EO-permethrin: permethrin mixed with EO at LD25 concentration). Test kits (permethrin and EO-permethrin) were evaluated against MCM-S and PMD-R strains of Ae. Aedes aegypti. Each of 25 female mosquitoes was given four doses of permethrin to test its effectiveness in killing adults, with each treatment repeated four times. To identify candidate EO synergists, 4 to 6 doses of EO-permethrin were administered to each of 25 female mosquitoes, with each application repeated four times. PBO-permethrin treatment (permethrin mixed with LD25 concentration of PBO) also served as a positive control. The doses used in these bioassays are expressed in nanograms of test sample per milligram of live female body weight. Four experimental evaluations for each mosquito strain were conducted on individually reared batches, and mortality data were pooled and analyzed using Probit to determine a 24-hour lethal dose.
The mortality rate was adjusted using the Abbott formula [43]. The adjusted data were analyzed by Probit regression analysis using the computer statistics program SPSS (version 19.0). Lethal values of 25%, 50%, 90%, 95% and 99% (LD25, LD50, LD90, LD95 and LD99, respectively) were calculated using the corresponding 95% confidence intervals (95% CI). Measurements of significance and differences between test samples were assessed using the chi-square test or Mann-Whitney U test within each biological assay. Results were considered statistically significant at P < 0.05. The resistance coefficient (RR) is estimated at the LD50 level using the following formula [12]:
RR > 1 indicates resistance, and RR ≤ 1 indicates sensitivity. The synergy ratio (SR) value of each synergist candidate is calculated as follows [34, 35, 44]:
This factor divides the results into three categories: an SR value of 1±0.05 is considered to have no apparent effect, an SR value of >1.05 is considered to have a synergistic effect, and an SR value of A light yellow liquid oil can be obtained by steam distillation of the rhizomes of C. rotundus and A. galanga and the bark of C. verum. Yields calculated on dry weight were 0.15%, 0.27% (w/w), and 0.54% (v/v). w) respectively (Table 1). GC-MS study of the chemical composition of oils of C. rotundus, A. galanga and C. verum showed the presence of 19, 17 and 21 compounds, which accounted for 80.22, 86.75 and 97.24% of all components, respectively (Table 2 ). C. lucidum rhizome oil compounds mainly consist of cyperonene (14.04%), followed by carralene (9.57%), α-capsellan (7.97%), and α-capsellan (7.53%). The main chemical component of galangal rhizome oil is β-bisabolene (18.27%), followed by α-bergamotene (16.28%), 1,8-cineole (10.17%) and piperonol (10.09%). While cinnamaldehyde (64.66%) was identified as the main component of C. verum bark oil, cinnamic acetate (6.61%), α-copaene (5.83%) and 3-phenylpropionaldehyde (4.09% ) were considered minor ingredients. The chemical structures of cyperne, β-bisabolene and cinnamaldehyde are the main compounds of C. rotundus, A. galanga and C. verum, respectively, as shown in Figure 2.
Results from three OOs assessed adult activity against Aedes mosquitoes. aegypti mosquitoes are shown in Table 3. All EOs were found to have lethal effects on MCM-S Aedes mosquitoes at different types and doses. Aedes aegypti. The most effective EO is C. verum, followed by A. galanga and C. rotundus with LD50 values of 3.30, 7.97 and 10.05 μg/mg MCM-S females respectively, slightly higher than 3.22 (U = 1 ), Z = -0.775, P = 0.667), 7.94 (U = 2, Z = 0, P = 1) and 9.57 (U = 0, Z = -1.549, P = 0.333) μg/mg PMD -R in women. This corresponds to PBO having a slightly higher adult effect on PMD-R than the MSM-S strain, with LD50 values of 4.79 and 6.30 μg/mg females, respectively (U = 0, Z = -2.021, P = 0.057 ). ). It can be calculated that the LD50 values of C. verum, A. galanga, C. rotundus and PBO against PMD-R are approximately 0.98, 0.99, 0.95 and 0.76 times lower than those against MCM-S, respectively. Thus, this indicates that the susceptibility to PBO and EO is relatively similar between the two Aedes strains. Although PMD-R was more susceptible than MCM-S, the sensitivity of Aedes aegypti was not significant. In contrast, the two Aedes strains differed greatly in their sensitivity to permethrin. aegypti (Table 4). PMD-R demonstrated significant resistance to permethrin (LD50 value = 0.44 ng/mg in women) with a higher LD50 value of 3.70 compared to MCM-S (LD50 value = 0.44 ng/mg in women ) ng/mg in women (U = 0, Z = -2.309, P = 0.029). Although PMD-R is much less sensitive to permethrin than MCM-S, its sensitivity to PBO and C. verum, A. galanga, and C. rotundus oils is slightly higher than MCM-S.
As observed in the adult population bioassay of the EO-permethrin combination, binary mixtures of permethrin and EO (LD25) showed either synergy (SR value > 1.05) or no effect (SR value = 1 ± 0.05). Complex adult effects of an EO-permethrin mixture on experimental albino mosquitoes. Aedes aegypti strains MCM-S and PMD-R are shown in Table 4 and Figure 3. Addition of C. verum oil was found to slightly reduce the LD50 of permethrin against MCM-S and slightly increase the LD50 against PMD-R to 0.44–0 .42 ng/mg in women and from 3.70 to 3.85 ng/mg in women, respectively. In contrast, addition of C. rotundus and A. galanga oils significantly reduced the LD50 of permethrin on MCM-S from 0.44 to 0.07 (U = 0, Z = -2.309, P = 0.029) and to 0.11 (U = 0 ). , Z) = -2.309, P = 0.029) ng/mg women. Based on the LD50 values of MCM-S, the SR values of the EO-permethrin mixture after addition of C. rotundus and A. galanga oils were 6.28 and 4.00, respectively. Accordingly, the LD50 of permethrin against PMD-R decreased significantly from 3.70 to 0.42 (U = 0, Z = -2.309, P = 0.029) and to 0.003 with the addition of C. rotundus and A. galanga oils (U = 0 ). , Z = -2.337, P = 0.029) ng/mg female. The SR value of permethrin combined with C. rotundus against PMD-R was 8.81, whereas the SR value of galangal-permethrin mixture was 1233.33. Relative to MCM-S, the LD50 value of the positive control PBO decreased from 0.44 to 0.26 ng/mg (females) and from 3.70 ng/mg (females) to 0.65 ng/mg (U = 0, Z = -2.309, P = 0.029) and PMD-R (U = 0, Z = -2.309, P = 0.029). The SR values of the PBO-permethrin mixture for strains MCM-S and PMD-R were 1.69 and 5.69, respectively. These results indicate that C. rotundus and A. galanga oils and PBO enhance permethrin toxicity to a greater extent than C. verum oil for strains MCM-S and PMD-R.
Adult activity (LD50) of EO, PBO, permethrin (PE) and their combinations against pyrethroid-sensitive (MCM-S) and resistant (PMD-R) strains of Aedes mosquitoes. Aedes aegypti
[45]. Synthetic pyrethroids are used worldwide to control almost all arthropods of agricultural and medical importance. However, due to the harmful consequences of the use of synthetic insecticides, especially in terms of the development and widespread resistance of mosquitoes, as well as the impact on long-term health and the environment, there is now an urgent need to reduce the use of traditional synthetic insecticides and develop alternatives [35, 46, 47]. In addition to protecting the environment and human health, the advantages of botanical insecticides include high selectivity, global availability, and ease of production and use, making them more attractive for mosquito control [32,48, 49]. This study, in addition to elucidating the chemical characteristics of effective essential oils through GC-MS analysis, also assessed the potency of adult essential oils and their ability to enhance the toxicity of synthetic permethrin. aegypti in pyrethroid-sensitive strains (MCM-S) and resistant strains (PMD-R).
GC-MS characterization showed that cypern (14.04%), β-bisabolene (18.27%) and cinnamaldehyde (64.66%) were the main components of C. rotundus, A. galanga and C. verum oils, respectively. These chemicals have demonstrated diverse biological activities. Ahn et al. [50] reported that 6-acetoxycyperene, isolated from the rhizome of C. rotundus, acts as an antitumor compound and can induce caspase-dependent apoptosis in ovarian cancer cells. β-Bisabolene, extracted from the essential oil of myrrh tree, exhibits specific cytotoxicity against human and mouse mammary tumor cells both in vitro and in vivo [51]. Cinnamaldehyde, obtained from natural extracts or synthesized in the laboratory, has been reported to have insecticidal, antibacterial, antifungal, anti-inflammatory, immunomodulatory, anticancer, and antiangiogenic activities [52].
The results of the dose-dependent adult activity bioassay showed good potential of the tested EOs and showed that the Aedes mosquito strains MCM-S and PMD-R had similar susceptibility to EO and PBO. Aedes aegypti. A comparison of the effectiveness of EO and permethrin showed that the latter has a stronger allercidal effect: LD50 values are 0.44 and 3.70 ng/mg in females for strains MCM-S and PMD-R, respectively. These findings are supported by many studies showing that naturally occurring pesticides, especially plant-derived products, are generally less effective than synthetic substances [31, 34, 35, 53, 54]. This may be because the former is a complex combination of active or inactive ingredients, while the latter is a purified single active compound. However, the diversity and complexity of natural active ingredients with different mechanisms of action may enhance biological activity or hinder the development of resistance in host populations [55, 56, 57]. Many researchers have reported the anti-mosquito potential of C. verum, A. galanga and C. rotundus and their components such as β-bisabolene, cinnamaldehyde and 1,8-cineole [22, 36, 58, 59, 60,61, 62,63 ,64]. However, a review of the literature revealed that there have been no previous reports of its synergistic effect with permethrin or other synthetic insecticides against Aedes mosquitoes. Aedes aegypti.
In this study, significant differences in permethrin susceptibility were observed between the two Aedes strains. Aedes aegypti. MCM-S is sensitive to permethrin, whereas PMD-R is much less sensitive to it, with a resistance rate of 8.41. Compared to the sensitivity of MCM-S, PMD-R is less sensitive to permethrin but more sensitive to EO, providing a basis for further studies aimed at increasing the effectiveness of permethrin by combining it with EO. A synergistic combination-based bioassay for adult effects showed that binary mixtures of EO and permethrin reduced or increased mortality of adult Aedes. Aedes aegypti. Addition of C. verum oil slightly decreased the LD50 of permethrin against MCM-S but slightly increased the LD50 against PMD-R with SR values of 1.05 and 0.96, respectively. This indicates that C. verum oil does not have a synergistic or antagonistic effect on permethrin when tested on MCM-S and PMD-R. In contrast, C. rotundus and A. galanga oils showed a significant synergistic effect by significantly reducing the LD50 values of permethrin on MCM-S or PMD-R. When permethrin was combined with EO of C. rotundus and A. galanga, the SR values of the EO-permethrin mixture for MCM-S were 6.28 and 4.00, respectively. Additionally, when permethrin was evaluated against PMD-R in combination with C. rotundus (SR = 8.81) or A. galanga (SR = 1233.33), SR values increased significantly. It is worth noting that both C. rotundus and A. galanga enhanced the toxicity of permethrin against PMD-R Ae. aegypti significantly. Similarly, PBO was found to increase the toxicity of permethrin with SR values of 1.69 and 5.69 for strains MCM-S and PMD-R, respectively. Since C. rotundus and A. galanga had the highest SR values, they were considered to be the best synergists in enhancing permethrin toxicity on MCM-S and PMD-R, respectively.
Several previous studies have reported the synergistic effect of combinations of synthetic insecticides and plant extracts against various mosquito species. A larvicidal bioassay against Anopheles Stephensi studied by Kalayanasundaram and Das [65] showed that fenthion, a broad-spectrum organophosphate, was associated with Cleodendron inerme, Pedalium murax and Parthenium hysterophorus. Significant synergy was observed between the extracts with a synergistic effect (SF) of 1.31. , 1.38, 1.40, 1.48, 1.61 and 2.23, respectively. In a larvicidal screening of 15 mangrove species, petroleum ether extract of mangrove stilted roots was found to be most effective against Culex quinquefasciatus with an LC50 value of 25.7 mg/L [66]. The synergistic effect of this extract and the botanical insecticide pyrethrum was also reported to reduce the LC50 of pyrethrum against C. quinquefasciatus larvae from 0.132 mg/L to 0.107 mg/L, in addition, an SF calculation of 1.23 was used in this study. 34,35,44]. The combined effectiveness of Solanum citron root extract and several synthetic insecticides (eg, fenthion, cypermethrin (a synthetic pyrethroid) and timethphos (an organophosphorus larvicide)) against Anopheles mosquitoes was evaluated. Stephensi [54] and C. quinquefasciatus [34]. The combined use of cypermethrin and yellow fruit petroleum ether extract showed a synergistic effect on cypermethrin in all ratios. The most effective ratio was the 1:1 binary combination with LC50 and SF values of 0.0054 ppm and 6.83, respectively, relative to An. Stephen West[54]. While a 1:1 binary mixture of S. xanthocarpum and temephos was antagonistic (SF = 0.6406), the S. xanthocarpum-fenthion combination (1:1) exhibited synergistic activity against C. quinquefasciatus with an SF of 1.3125 [ 34]]. Tong and Blomquist [35] studied the effects of plant ethylene oxide on the toxicity of carbaryl (a broad-spectrum carbamate) and permethrin to Aedes mosquitoes. Aedes aegypti. The results showed that ethylene oxide from agar, black pepper, juniper, helichrysum, sandalwood and sesame increased the toxicity of carbaryl to Aedes mosquitoes. aegypti larvae SR values vary from 1.0 to 7.0. In contrast, none of the EOs were toxic to adult Aedes mosquitoes. At this stage, no synergistic effects have been reported for the combination of Aedes aegypti and EO-carbaryl. PBO was used as a positive control to enhance the toxicity of carbaryl against Aedes mosquitoes. The SR values of Aedes aegypti larvae and adults are 4.9-9.5 and 2.3, respectively. Only binary mixtures of permethrin and EO or PBO were tested for larvicidal activity. The EO-permethrin mixture had an antagonistic effect, while the PBO-permethrin mixture had a synergistic effect against Aedes mosquitoes. Larvae of Aedes aegypti. However, dose response experiments and SR evaluation for PBO-permethrin mixtures have not yet been performed. Although few results have been achieved regarding the synergistic effects of phytosynthetic combinations against mosquito vectors, these data support the existing results, which open the prospect of adding synergists not only to reduce the applied dose, but also to increase the killing effect. Efficiency of insects. Additionally, the results of this study demonstrated for the first time that C. rotundus and A. galanga oils synergistically exert significantly higher efficacy against pyrethroid-susceptible and pyrethroid-resistant strains of Aedes mosquitoes compared to PBO when combined with permethrin toxicity. Aedes aegypti. However, unexpected results from the synergistic analysis showed that C. verum oil had the greatest anti-adult activity against both Aedes strains. Surprisingly, the toxic effect of permethrin on Aedes aegypti was unsatisfactory. Variations in toxic effects and synergistic effects may be due in part to exposure to different types and levels of bioactive components in these oils.
Despite efforts to understand how to improve efficiency, the synergistic mechanisms remain unclear. Possible reasons for the different efficacy and synergistic potential may include differences in the chemical composition of the products tested and differences in mosquito susceptibility associated with resistance status and development. There are differences between the major and minor ethylene oxide components tested in this study, and some of these compounds have been shown to have repellent and toxic effects against a variety of pests and disease vectors [61,62,64,67,68]. However, the main compounds characterized in C. rotundus, A. galanga and C. verum oils, such as cypern, β-bisabolene and cinnamaldehyde, were not tested in this paper for their anti-adult and synergistic activities against Ae, respectively. Aedes aegypti. Therefore, future studies are needed to isolate the active ingredients present in each essential oil and elucidate their insecticidal efficacy and synergistic interactions against this mosquito vector. In general, insecticidal activity depends on the action and reaction between poisons and insect tissues, which can be simplified and divided into three stages: penetration into the insect body skin and target organ membranes, activation (= interaction with the target) and detoxification. toxic substances [57, 69]. Therefore, insecticide synergism resulting in increased effectiveness of toxicant combinations requires at least one of these categories, such as increased penetration, greater activation of accumulated compounds, or less reduced detoxification of the pesticide active ingredient. For example, energy tolerance delays cuticle penetration through a thickened cuticle and biochemical resistance, such as enhanced insecticide metabolism observed in some resistant insect strains [70, 71]. The significant effectiveness of EOs in increasing the toxicity of permethrin, especially against PMD-R, may indicate a solution to the problem of insecticide resistance by interacting with resistance mechanisms [57, 69, 70, 71]. Tong and Blomquist [35] supported the results of this study by demonstrating a synergistic interaction between EOs and synthetic pesticides. aegypti, there is evidence of inhibitory activity against detoxifying enzymes, including cytochrome P450 monooxygenases and carboxylesterases, which are closely associated with the development of resistance to traditional pesticides. PBO is not only said to be a metabolic inhibitor of cytochrome P450 monooxygenase but also improves the penetration of insecticides, as demonstrated by its use as a positive control in synergistic studies [35, 72]. Interestingly, 1,8-cineole, one of the important components found in galangal oil, is known for its toxic effects on insect species [22, 63, 73] and has been reported to have synergistic effects in several areas of biological activity research [74]. . ,75,76,77]. In addition, 1,8-cineole in combination with various drugs including curcumin [78], 5-fluorouracil [79], mefenamic acid [80] and zidovudine [81] also has a permeation-promoting effect. in vitro. Thus, the possible role of 1,8-cineole in synergistic insecticidal action is not only as an active ingredient but also as a penetration enhancer. Due to greater synergism with permethrin, especially against PMD-R, the synergistic effects of galangal oil and trichosanthes oil observed in this study may result from interactions with resistance mechanisms, i.e. increased permeability to chlorine. Pyrethroids increase the activation of accumulated compounds and inhibit detoxifying enzymes such as cytochrome P450 monooxygenases and carboxylesterases. However, these aspects require further study to elucidate the specific role of EO and its isolated compounds (alone or in combination) in synergistic mechanisms.
In 1977, increasing levels of permethrin resistance were reported in major vector populations in Thailand, and over the following decades, the use of permethrin was largely replaced by other pyrethroid chemicals, especially those replaced by deltamethrin [82]. However, vector resistance to deltamethrin and other classes of insecticides is extremely common throughout the country due to excessive and persistent use [14, 17, 83, 84, 85, 86]. To combat this problem, it is recommended to rotate or reuse discarded pesticides that were previously effective and less toxic to mammals, such as permethrin. Currently, although the use of permethrin has been reduced in recent national government mosquito control programs, permethrin resistance can still be found in mosquito populations. This may be due to exposure of mosquitoes to commercial household pest control products, which mainly consist of permethrin and other pyrethroids [14, 17]. Thus, successful repurposing of permethrin requires the development and implementation of strategies to reduce vector resistance. Although none of the essential oils tested individually in this study were as effective as permethrin, working together with permethrin resulted in impressive synergistic effects. This is a promising indication that the interaction of EO with resistance mechanisms results in the combination of permethrin with EO being more effective than the insecticide or EO alone, particularly against PMD-R Ae. Aedes aegypti. The benefits of synergistic mixtures in increasing efficacy, despite the use of lower doses for vector control, may lead to improved resistance management and reduced costs [33, 87]. From these results, it is pleasing to note that A. galanga and C. rotundus EOs were significantly more effective than PBO in synergizing permethrin toxicity in both MCM-S and PMD-R strains and are a potential alternative to traditional ergogenic aids.
The selected EOs had significant synergistic effects in enhancing adult toxicity against PMD-R Ae. aegypti, especially galangal oil, has an SR value of up to 1233.33, indicating that EO has broad promise as a synergist in enhancing the effectiveness of permethrin. This may stimulate the use of a new active natural product, which together could increase the use of highly effective mosquito control products. It also reveals the potential of ethylene oxide as an alternative synergist to effectively improve upon older or traditional insecticides to address existing resistance problems in mosquito populations. Using readily available plants in mosquito control programs not only reduces dependence on imported and expensive materials, but also stimulates local efforts to strengthen public health systems.
These results clearly show the significant synergistic effect produced by the combination of ethylene oxide and permethrin. The results highlight the potential of ethylene oxide as a plant synergist in mosquito control, increasing the effectiveness of permethrin against mosquitoes, especially in resistant populations. Future developments and research will require synergistic bioanalysis of galangal and alpinia oils and their isolated compounds, combinations of insecticides of natural or synthetic origin against multiple species and stages of mosquitoes, and toxicity testing against non-target organisms. Practical use of ethylene oxide as a viable alternative synergist.
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