Several factors affected the extraction yield of plant essential oil, such as agricultural practice, cultivation area, and extraction method26. The observed essential oil yields from the five plants in this study were checked and confirmed against the values reported in the literature, and mostly, they agreed very well. Namely, the essential oil yield of C. citratus observed in this study was 1.15% v/w, while papers by Soonwera and Sittichok24, Aungtikun et al.25, and Verma et al.26 reported the yield of C. citratus as 0.50–1.5% v/w; the observed C. verum EO yield was 1.10% v/w, well-matched with 1.01–1.14% v/w reported by Aungtikun and Soonwera27 and Li et al.31; the observed E. globulus EO yield was 1.21% v/w (water distillation extraction method), well-matched with 1.2–3.0% w/w (water distillation) reported by Sebei et al32; and 1.10% v/w (steam distillation) reported by Joshi et al.28 and Soonwera and Phasomkusolsil29. The observed I. verum EO yield was 3.23% v/w, slightly lower than the 4.0–4.5% v/w, (water distillation) reported by Gholivand et al.33 and the 4.0% v/w (water distillation) reported by Aungtikun et al.25, and the observed yield of Z. limonella EO was 9.27% v/w, slightly lower than the 9.63% v/w reported by Charoensup et al.30. Moreover, although local species of C. citratus in Thailand and in India have the same number of constituents, the percentages of the constituents in their chemical profile are different34. Therefore, those yield percentages were determined and checked against the values in recent literature. Many factors influence the percentage of a major constituent in an EO’s chemical profile, such as plant maturity, harvesting time, and good harvesting practice25. The observed GC-MS chemical profiles of every EO were well-matched with those reported in a previous study. Namely, the percentage of geranial, the major chemical constituent of C. citratus EO observed in this study, was 46.33%, while papers by Soonwera and Sittichok24, Aungtikun et al.25, and Chauhan et al.34 reported the percentage geranial at 42.40–49.69% of the EO chemical profile; in addition, the observed major constituent of C. verum EO was cinnamaldehyde at 75.23%, well matched with 73.21% of the chemical profile reported by Aungtikun and Soonwera 27 and with 74.49% reported by Li et al.31, and lower than 90.17% reported by Chansang et al.35; the observed major constituent of E. globulus EO was 1,8-cineole, at 45.82%, well-matched with 42.60–44.54% of the chemical profile reported by Soonwera and Sittichok24 and Cotchakaew and Soonwera36; the observed major constituent of I. verum EO was trans-anethole, at 92.24%, well-matched with 88.32–94.0% of the chemical profile reported by Aungtikun et al.25 and Junior et al.37; and the observed major constituent of Z. limonella EO was limonene, at 28.13%, slightly higher than 18.62% reported by Imphat and Woottisin38, and lower than 43.63% reported by Charoensup et al.30, and 57.94% reported by Wongkattiya et al.39.
Nevertheless, there were some slight discrepancies, such as the yield and percentage in the chemical profile of the major constituents of I. verum and Z. limonella EOs. These discrepancies were likely due to the usage of different plant parts for EO extraction, a difference in the extraction procedures, and variations in genotypic, phenotypic, and agroecological factors (e.g., plant age, growth phase in plant development, amount of sunlight and moisture, as well as the temperature at the farm’s geographical location40.
High toxicity of plant EO in soybean oil against insect pests has been reported in recent literature. A study on EO toxicity against head louse eggs by Soonwera et al.41 concluded that Zingiberaceae plant EOs in soybean oil exhibited high toxicity to the eggs of head louse (Pediculus humanus capitis; Pediculidae: Phthiraptera), with a full 100% inhibition rate. In this study, 10% I. verum EO and the combined EO formulation in soybean oil showed high egg inhibition rates of 96.9% and 99.3%, respectively, against P. americana. This result was expected before the experiment started because the outcomes of many studies on I. verum EO against eggs of insect pests other than P. americana have been reported in the literature. Specifically, Sinthusiri and Soonwera42 reported that 10% I. verum EO exhibited markedly high oviposition deterrent and ovicidal activities against female houseflies (Musca domestica; Muscidae: Diptera), while Matos et al.43 reported that I. verum EO exhibited a high inhibition rate against the eggs of cowpea weevil, Callosobruchus maculatus (Bruchidae: Coleoptera). In addition to a high repellent activity against the adults, 10% C. verum EO and the combined formulation in soybean oil also exhibited a high repellent activity against P. americana adults—100% and 83.4% and 100% and 89.5% repellent activity at 1 and 48 h after exposure, respectively. Many papers have reported a high repellent activity of C. verum EO against several insect pests such as Plodia interpunctella (Pyralidae: Lepidoptera), Bemisia tabaci (Aleyrodidae: Homoptera), Ae. aegypti (Culicidae: Diptera), Sitophilus zeamais (Curculionidae: Coleoptera)44, Sitophilus oryzae (Curculionidae: Coleoptera)45, and ant46.
Unlike a large number of papers on individual EOs against P. americana mentioned above, papers on combined EO formulations against P. americana are limited. An example is a paper by Zibaee and Khorram16 reporting that a combined formulation of E. globulus + R. officinalis EOs exhibited a stronger insecticidal effect against adult P. americana than that of each individual EO component. Aungtikun et al.25 reported that a combined formulation of 0.5% I. verum EO + 0.5% geranial showed a higher insecticidal effect against adult house flies (M. domestica) than each individual EO.
Most notably, all EO formulations in soybean oil exhibited higher ovicidal and repellent activities against P. americana eggs and adults than all EO formulations in ethyl alcohol, in full agreement with findings from Sittichok and Soonwera47 that eight herbal EOs in soybean oil showed a higher inhibition rate against P. americana eggs than the same EOs in ethyl alcohol. Findings on repellent activity against adult mosquitoes in studies by Soonwera and Phasomkusosil48 and Phasomkusosil and Soonwera49 showed the same trend: 10% EOs from C. citratus, C. nardus, Cananda odorata, Ocimum basilicum, and Z. cassumunar in soybean oil weremore repellent and feeding deterrent against adult females of Anopheles minimus, Ae. aegypti, and Culex quinquefasciatus than the same EOs in ethyl alcohol. In the same vein, in this study, C. verum EO and the combined EO formulation exerted their potency better in soybean oil than in ethyl alcohol.
The soybean oil solvent in this study was pure soybean oil, but the ethyl alcohol solvent was 70% (v/v) ethyl alcohol in water. Therefore, the reason that a soybean oil solution of an EO is more repellent than the same EO in ethyl alcohol solution is that soybean oil is more lipophilic than ethyl alcohol, and a lipophilic solvent retards evaporation of EOs49. In this study, C. verum EO and the combined EO formulation in soybean oil provided a longer, more persistent repellent activity against P. americana than the same EO or combined EOs in ethyl alcohol, and the higher activity was attributed to this reason. On the other hand, an EO in a hydrophilic solvent evaporated faster and hence shorter, less persistent repellent activity. This claim is supported by a paper by Phasomkusosil and Soonwera49: 10% Citrus sinensis EO in soybean oil was more repellent (30–60 min) against adult females of Cx. quinquefasciatus, Ae. aegypti, and An. minimus than the EO in ethyl alcohol (less than 1.0 min). Another paper by Phasomkusosil and Soonwera49 and Phukerd and Soowera50 reported that 10% Z. cassumunar EO in soybean oil was more repellent (70 min) against adult females of Ae. aegypti than the EO in ethyl alcohol (30.0 min).
On the other hand, even though the reason that I. verum EO and the combined EO formulation were more potent in ovicidal activity in soybean oil than in ethyl alcohol is also the higher lipophilicity of soybean oil, but the mechanisms of action were not the same. The mechanism responsible for the higher ovicidal activity is that a lipophilic solvent can carry an EO through insect cuticles more readily because insect cuticles are mainly lipophilic41, hence more EO passes into the insect, providing more ovicidal activity from a higher amount of EO in its body41,51,52. This claim is supported by a conclusion from Rajashekar and Shivanandappa53 and Jankowska et al.54 that embryo and egg mortality were strongly affected by EO’s high cuticle permeability. The outcomes of this experiment confirm the expectation that every EO in soybean oil was more effective than the same EO in ethyl alcohol.
Regarding the high ovicidal activity (96.9–99.3%) against P. americana eggs of 10% I. verum EO among all tested individual EO formulations, this high activity is likely to be provided by its main constituent, trans-anethole25. Strong potency of trans-anethole against mosquitoes and houseflies was reported by Aungtikun et al.25 and Pavela55. Its mechanism of action was reported in Jankowska et al.54 and Bosch-Serra et al.56: namely, trans-anethole inhibited the development of embryo by blocking cytochrome P450 detoxification enzymes, retarding cell growth and juvenile hormone production and weakening the immune system, leading to eventual embryo death.
In the same way, the high repellent activity (100% at 1 h) against P. americana adults of 10% C. verum EO is likely to be provided by its main constituent, cinnamaldehyde47. Cinnamaldehyde has been reported to strongly repel P. americana adults (75.23%)47. The repellent activity of cinnamaldehyde is strong because it causes serious damage to their respiratory system47, so they always try to avoid it. This conclusion is supported by a claim by Devi and Devi57 that the mechanisms of action of cinnamaldehyde against insect pests were inhibition of its respiratory system by enzyme inhibition, cell membrane alteration, and reduced cell membrane integrity, as well as reduced cell respiration. Because of these reasons, C. verum EO showing the lowest ovicidal efficacy (in Table 2) makes sense because it does not have a constituent that strongly inhibited the eggs of P. americana like trans-anethole that I. verum EO has, hence it provided a low inhibition rate against the eggs of P. americana and house fly, as reported by previous works of Sittichok and Soonwera47 and Sinthusiri and Soonwera42. In the same vein, the graph of repellent activity of C. citratrus EO in Fig. 3 looks similar to that of C. verum EO but not that of I. verum EO because of them have geranial and cinnamaldehyde as their major constituent, while I. verum EO do not.
The overall findings from this study indicate that the combined formulation of 5% C. verum EO + 5% I. verum EO would be a good current alternative to cypermethrin because, first, it is equally or more potent than cypermethrin in ovicidal and repellent activities and, second, they should be less harmful to humans and non-target organisms and not be as persistent as cypermethrin in the environment. Findings supporting the first reason were as follows. First, against P. americana adults, C. verum EO and the combined EO formulation were 1.71–1.97 times more potent than cypermethrin in repellent activity, while the ovicidal activity of cypermethrin was only slightly more potent than those of I. verum EO and the combined EO formulation. Moreover, these findings are supported well by findings from a previous study by Sinthusiri and Soonwera42 that 10% cypermethrin showed a 100% inhibition rate against house fly eggs, while 10% I. verum EO showed a 97.33% rate. Second, findings by Ichikawa58 and Sharma et al.59 support the safety reason for replacing cypermethrin with a plant essential oil: cypermethrin is seriously toxic to the nervous and immune systems of humans, especially pregnant women and children, while findings by Aungtikun and Soonwera27 and Patra et al.60 support the claim that EOs are quickly degraded in the environment. Moreover, its safety to humans has long been established in Southeast Asia because it has been widely used as folk medicine since ancient times, and today, it is an irreplaceable food ingredient on some Thai menus as well as an active ingredient in modern, scientifically tested medicine (Tamiflu, an anti-influenza drug).
To conclude, the findings in this study indicate that 10% EO of I. verum provided the highest ovicidal activity against P. americana eggs among the five tested EOs, while 10% C. verum provided the highest repellent activity against P. americana adults. Moreover, they suggest that a formulation of their combination (5% I. verum EO+ 5% C. verum EO) in soybean oil can replace cypermethrin as an equally potent but much safer alternative agent for controlling P. americana populations.