INTRODUCTION

Synthetic insecticides are immensely important in combatting mosquito populations throughout the world (Gramiccia and Beales 1988; Roberts et al. 2000; Lengeler 2004; Mabaso et al. 2004; WHO 2006, 2013). In particular, pyrethroid, organophosphate, and organochlorine insecticides have been the primary tools by which wild mosquito populations have been controlled (Nauen 2007, Pocquet et al. 2013). By promoting the continued influx of sodium ions across the membrane of neurons in insects, pyrethroid insecticides cause hyper-excitability of the insect nervous system, contributing to spastic paralysis and death (Narahashi 1962). Because pyrethroids readily migrate across the insect cuticle and diffuse throughout the insect, they exert toxic effects minutes after the target insect comes into contact with the compounds (Narahashi 1972). One important symptom of pyrethroid intoxication is rapid immobilization (knockdown). Unlike some slow-acting insecticides, pyrethroids are ideal for public health vector control because this knockdown effect immediately prevents mosquitoes from feeding on future hosts (Norris et al. 2015, Gross et al. 2017a). Moreover, it likely contributes to the mortality of the intoxicated insect through various scenarios. As suggested in a previous study, this knockdown may lead to increased desiccation, susceptibility to predation, and the inhibition of grooming (which mitigates the accumulation of fungal spores on the insect), all of which may lead to higher levels of mortality in field applications. As such, this knockdown effect is a crucial consideration in the development of future insecticidal formulations.

One of the obstacles facing the identification of new insecticidal formulations for the control of mosquito populations is ensuring that these new mixtures will be fast-acting and cause high mortality to the target species while simultaneously being safe for application in residential areas. Although some potent synthetic insecticides exist that cause rapid intoxication by interfering with the normal physiology of the insect nervous system, their high toxicity to other nontarget organisms and relatively long half-lives in the environment can prevent them from being viable avenues to follow in the search for new mosquito adulticides. Moreover, registration of new insecticides for the control of adult mosquitoes is prohibitively costly (Rose 2001, Ridley et al. 2017). It is paramount to identify candidate insecticides that have low mammalian toxicity, degrade rapidly, and still quickly immobilize and kill mosquitoes. Some plant essential oil compounds manifest these prerequisite characteristics due to their low cost, ready availability, rapid degradation in the environment, and safety to mammals (Isman 2011). Because of the rapid development of resistance to many of the currently available insecticides on the market (Rose 2001, Oxborough 2016), these plant essential oil components may be viable alternatives in future insecticidal formulations.

The bioactivity of plant essential oils is rooted in diverse molecular mechanisms of action that lead to rapid knockdown and/or mortality of exposed insects. These active sites include, but are not limited to, octopamine receptors, tyramine receptors, acetylcholinesterase, nicotinic acetylcholine receptors, and gamma-aminobutyric acid-gated chloride channels (Essam 2001, Anderson and Coats 2012, Tong et al. 2012, Gross et al. 2017b). Numerous other studies suggest that other sites of toxic action may be involved. Because some compounds making up the most common plant essential oils are neuroactive in various insect models, their utility as insecticides and their various modes of insecticidal action need to be further characterized. Due to the unique mechanisms of action of essential oils compared with other currently utilized synthetic insecticides, cross-resistance with synthetic insecticides on the market today is unlikely (Norris et al. 2015, Gross et al. 2017a). Plant essential oils also contain multiple terpenoids, which all may possess their own mechanisms of insecticidal action (Isman 2000). The development of resistance to these plant oil insecticides may be delayed due to the multiple mechanisms of action represented by different terpenoids within the formulation (Georghiou 1994). Instead, plant essential oils, and particularly the terpenoids they are made up of, may represent novel approaches in the field of vector control.

Our lab has identified numerous plant essential oils that cause rapid knockdown when applied topically to adult female Aedes aegypti L. alone or in combination with synthetic insecticides (Norris et al. 2015, Gross et al. 2017a). Moreover, these plant essential oils cause significant knockdown in combination with other synthetic insecticides. This effect is described as the immobilization or the inability of exposed mosquitoes to orient in the upright posture. We postulate that by more completely characterizing this effect, it will be possible to further explore the potential of natural products in the control of public health pests. For this study, we screened numerous plant essential oils with the goal of identifying which compounds cause knockdown at 1 h after exposure at low concentrations. Furthermore, our goal was to compare this knockdown effect to the 24-h mortality from the same plant oils to determine whether this knockdown effect occurred at comparable exposure thresholds as the toxicity of these oils. Finally, to evaluate whether knockdown may cause some level of delayed lethality in the laboratory, we monitored the 24-h survival of mosquitoes that were immobilized immediately postexposure.

MATERIALS AND METHODS

Topical application

Similar methods as described in Norris et al. (2015) were used for the topical application of various doses of 32 plant essential oils (Table 1) to the pronotum of adult, female Ae. aegypti with some minor exceptions. This study differed in the doses of plant essential oils used and the duration of monitoring. Concentrations used caused between 5% and 95% knockdown at 1 h. The dose response of knockdown effect was then compared with the concentrations that caused mortality at 24 h.

Table 1. The KD₅₀ after topical exposure to multiple plant essential oils compared to the calculated LD₅₀ (mortality recorded at 24 h).

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Survival curves for the 2 most successful oils (clove leaf and patchouli) were compared with each other within each experiment to see if delayed mortality was caused by significant knockdown (i.e., statistically significant from the control at 0.2 μl of acetone). Experiments consisted of applying either an effective dose (ED₀₅ or ED₇₅) as compared to a control treatment for the 2 most successful plant essential oils to 30 individual Ae. aegypti females. The ED₀₅ was used as an estimation of the no adverse effects level of the plant essential oil. This dosage was compared with the effective concentration (EC₇₅) of each oil to identify whether 1-h KD would also produce delayed lethality.

RESULTS

The 5 most successful plant essential oils that provided knockdown (measured as KD₅₀) values of less than 3,000 ppm (μg/g mosquito) in Ae. aegypti were patchouli, clove leaf, cinnamon leaf, clove bud, and cassia oil, with KD₅₀ values of 210, 2,000, 2,100, 2,100, and 2,300 μg/g, respectively (Table 1). Of all the oils screened, the most potent (patchouli) produced a KD₅₀ value of 210 ppm, whereas the least potent (black pepper) produced a KD₅₀ value of 34,000 ppm in Ae. aegypti. Comparing the knockdown effectiveness of both oils at this level we found that patchouli was more than 161-fold more toxic than black pepper.

Overall, plant essential oils produced lower KD₅₀ values than LD₅₀ values, with some exceptions. Among all plant essential oils screened, 16 caused KD₅₀ values at concentrations that were dissimilar to concentrations that caused mortality. An example of the differences between the 1-h KD and 24-h mortality dose response is patchouli oil (Fig. 1). It is evident that significant 1-h KD occurs at lower concentrations than those that lead to mortality at 24 h, as evidenced by the lack of 95% confidence interval overlap between the 2 oils (Table 1). Among all the plant essential oils screened, 13 caused 1-h KD at concentrations that were lower than those that cause mortality (patchouli, clove leaf, cinnamon leaf, clove bud, thyme, guaiacwood, catnip, basil [Egyptian], celery seed, sassafras, nutmeg [East Indies], rosemary, and wintergreen oil) (Table 1). Alternatively, cedarwood (Moroccan), orange, and sesame oil caused 1-h KD at concentrations that were higher than those that caused mortality.

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That said, we found that a Pearson correlation coefficient of 0.723 was observed (Fig. 2), indicating that there is a strong positive, direct relationship between the KD₅₀ value and the LD₅₀ value of each oil. The P value of less than 0.001 suggests that the model accurately describes the correlation of these 2 datasets.

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Adult mosquitoes exposed to clove leaf or patchouli oil (both being the 2 most capable oils causing sublethal knockdown at 1 h) when applied at either a KD₀₅ or KD₇₅ resulted in delayed mortality (Fig. 3) compared to controls. Approximately 87% (26 out of 30) of acetone (control) -exposed Ae. aegypti survived at the end of the 9-day experimental interval (Fig. 3A, 3B). This was in stark contrast to 30% (9 out of 30) and 40% (12 out of 30) of surviving mosquitoes that were exposed to the KD₀₅ value for clove leaf oil and patchouli oil, respectively. Curves of these 2 oils were statistically significant from the control at that level but were equivalent to each other. Similar to the ED₀₅ level, mosquitoes treated with an ED₇₅ of patchouli oil resulted in 23% survival (7 out of 30) after 9 days. As for the ED₇₅ exposure of clove leaf, 37% of mosquitoes (11 out of 30) survived until the 9th experimental day. For the KD₀₅ and KD₇₅ of both oils, survival significantly differed from the control at day 9. Survival curves for mosquitoes exposed to clove leaf and patchouli were significantly different compared to the control group (P < 0.05).

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DISCUSSION

Based on our bioassay screening data, numerous plant essential oils caused significant knockdown at 1 h after individual mosquitoes were exposed to them. This knockdown is unique to mortality as it occurs at distinct concentrations from those that cause mortality. The most potent plant essential oils that cause knockdown in Ae. aegypti were patchouli, clove leaf, cinnamon leaf, clove bud, and cassia (Table 1). The KD₅₀ values of these oils ranged from 210 to 2,300 ppm compared with the range in LD₅₀ values of 1,500–4,200 ppm. The lower dosages required to cause knockdown in these oils may be an indicator for utility in mosquito control programs. Even at low doses, these plant essential oils rapidly immobilize adult mosquitoes; it is likely that this would prevent mosquitoes from blood feeding and thereby potentially transmitting pathogens. However, rapid immobilization of adult mosquitoes may allow for their use in future insecticidal formulations. By rapidly immobilizing exposed mosquitoes, plant oils may prevent insects from transmitting pathogens. Rapidly preventing mosquitoes from feeding on vertebrate hosts is an important characteristic of all successful public health pest insecticides. Furthermore, this immobilization may lead to death due to other environmental processes. These might include increased levels of predation, increased toxicity from entomopathogenic fungi due to decreased grooming, and desiccation from the inability to seek water. This phenotype is best illustrated when mosquitoes are exposed to patchouli oil (Fig. 1). Here it is evident that the concentrations required to cause this knockdown are much lower than those required to cause mortality at 24 h. Moreover, the concentrations that induce knockdown can be as low as 79 ppm, which corresponded to a 1-h KD value of approximately 30 ± 14% (for patchouli oil). These concentrations that cause bioactivity are comparable to concentrations at which synthetic insecticides are active (Pridgeon et al. 2008).

Out of the 32 oils screened in this study, 13 caused knockdown at concentrations that were significantly lower than the lethal concentrations. The mode or modes of action that underpin this knockdown is not known. It is possible that these plant essential oils and their constituents are active at various nervous system targets and are readily detoxified after this initial bioactivity. This may lead to inactivation and excretion of these components from the insect, which would necessitate higher doses in order to lead to mortality. Waliwitiya et al. (2009) illustrated that the toxicity of terpenoids, the primary constituents of a majority of plant essential oils, were more toxic after a preapplication of piperonyl butoxide. This may suggest that the inhibition of various detoxification enzymes, such as cytochromes P-450, may allow terpenoids within these plant oils to remain bioactive for longer periods within the exposed organism, thereby causing higher percentage toxicity.

Our current study also highlighted a correlation between plant essential oil KD₅₀ and LD₅₀ values. With a Pearson correlation coefficient of 0.723, we showed that the capacity of a plant essential oil to cause knockdown is strongly correlated with mortality at a later time. However, some outliers were noted in our datasets. The most significant outliers in this analysis were sassafras, nutmeg (East Indies), rosemary, and wintergreen oil. All these oils have significantly lower KD₅₀ values compared with their corresponding LD₅₀ values. It may be that the terpenoids that cause rapid immobilization in these oils are more readily degraded and thus cause low levels of mortality at 24 h. These data could be valuable in the formulation of new insecticidal formulations that rely on plant essential oils or the terpenoids that are in them, as more insecticidal oils are more likely to cause rapid knockdown. This information could be utilized in the development of future blends of plant oils that cause rapid knockdown.

We also demonstrated that delayed toxicity was caused by some plant essential oils screened in this study. For this exploration, we chose to examine the latent mortality caused by the 2 oils that possessed the lowest KD₅₀ values. At both an ED₀₅ level (Fig. 3A) and an ED₇₅ level (Fig. 3B) of plant essential oil applied, significant mortality was observed out to 9 days compared to the control treatment. This may highlight the potential of these botanical products in the control of wild mosquito populations in the future. Interestingly, in both the ED₀₅ and ED₇₅ exposure levels, significant delayed mortality was observed for both clove leaf and patchouli oils.

The delayed mortality caused by some of the plant essential oils screened in this study may be due to oxidative stress brought about by the detoxification of the natural insecticides at sublethal levels (at 24 h). It is well known that CYP450-dependent detoxification can lead to the increase of reactive oxygen species within cells (Hrycay and Bandiera 2015). Based on the disparity between KD and mortality according to dose, we postulate that the mortality effect is not correlated to initial immobilization of the insect by these knockdown agents in the laboratory. Very little knockdown was observed in the ED₀₅-exposure experiment compared with the ED₇₅-exposure experiment; however, delayed percentage mortality was similar in both experiments. This indicates that this delayed mortality is caused by an alternative mechanism, which warrants further investigation. Promisingly, treatments of plant essential oils at concentrations that caused little to no mortality at 24 h postexposure caused significant delayed mortality as observed out to 9 days postexposure in the Kaplan-Meier survival analysis.

In summary, this study highlights the potential of plant essential oils and natural products in the control of adult mosquitoes. The use of only 24 h mortality as a metric of efficacy (Oxborough et al. 2015) may exclude potential bioactive compounds from subsequent testing regimens and development as future insecticidal products. Here we demonstrate the utility of plant essential oils as immobilizing agents for mosquitoes, measured by the metric of percentage knockdown at 1 h. Moreover, the delayed mortality caused by a number of the most potent plant essential oils also reveals interesting new lines of research for potential novel modes of action for natural products and plant essential oils as mosquito control strategies. As insecticide resistance causes the failure of many insecticidal chemistries that have been extremely valuable in the control of mosquito populations, new agents must be explored as additives in future insecticidal formulations. Plant essential oils may represent a viable source of novel insecticidal additives.