Essential Oils from Cameroonian Aromatic Plants as Effective Insecticides against Mosquitoes, Houseflies, and Moths

Recently, spices have attracted the attention of scientists and agrochemical companies for their potential as insecticidal and acaricidal agents, and even as repellents to replace synthetic compounds that are labeled with detrimental impacts on environment and human and animal health. In this framework, the aim of this study was to evaluate the insecticidal potential of the essential oils (EOs) obtained from three Cameroonian aromatic plants, namely Monodora myristica (Gaertn.) Dunal, Xylopia aethiopica (Dunal) A. Rich., and Aframomum citratum (J. Pereira) K. Schum. They were produced by hydrodistillation, with yields of 3.84, 4.89, and 0.85%, respectively. The chemical composition was evaluated by GC-MS analysis. The EOs and their major constituents (i.e., geraniol, sabinene, α-pinene, p-cymene, α-phellandrene, and β-pinene) were tested against the polyphagous moth pest, i.e., Spodoptera littoralis (Boisd.), the common housefly, Musca domestica L., and the filariasis and arbovirus mosquito vector, Culex quinquefasciatus Say. Our results showed that M. myristica and X. aethiopica EOs were the most effective against M. domestica adults, being effective on both males (22.1 µg adult−1) and females (LD50: 29.1 µg adult−1). The M. myristica EO and geraniol showed the highest toxicity on S. littoralis, with LD50(90) values of 29.3 (123.5) and 25.3 (83.2) µg larva−1, respectively. Last, the EOs from M. myristica and X. aethiopica, as well as the major constituents p-cymene and α-phellandrene, were the most toxic against C. quinquefasciatus larvae. The selected EOs may potentially lead to the production of cheap and effective botanical insecticides for African smallholders, although the development of effective formulations, a safety evaluation, and an in-depth study of their efficacy on different insect species are needed.


Introduction
Spices are aromatic plants or their specific parts, including bark, flowers, fruits, and seeds, owning a peculiar aroma given by flavoring-odorous compounds such as volatile terpenes and phenylpropanoids. Aromatic plants have attracted the attention of explorers since ancient civilizations because of their numerous culinary and medical applications [1]. Most of the plants classified as 'spices' come from aromatic plants growing in tropical regions, notably Asia and Africa, from where they have been brought to Europe since

Insecticidal Activity
The effects of M. myristica, X. aethiopica, and A. citratum EOs and their major compounds on M. domestica adult mortality are presented in Table 2. All our tested EOs were toxic to the insect species included in this study. However, significant differences were observed between the EOs. EOs from M. myristica and X. aethiopica showed the highest efficacy, with an LD 50(90) of 29.1(137.6) and 30.7(164.2) µg adult −1 on females, respectively, and 22.1(127.6) and 31.5(178.5) µg adult −1 , respectively, on males ( Table 2).
The efficacy of M. myristica EO against houseflies was roughly equivalent to that of the major compound p-cymene, with the LD 50 estimated as 28.4 µg adult −1 for females and 32.6 µg adult −1 for males. The other major compounds showed better efficacy compared to EOs, with special reference to the LD 50 values estimated on males, which were more sensitive; nevertheless, LD 90 values were approximately on the same level as those estimated for the EOs.
The tested EOs showed promising efficacy also against S. littoralis larvae (Table 3). Again, the highest efficacy was provided by the M. myristica EO with LD 50(90) estimated as 29.3(123.5) µg larva −1 . However, given the overlapping confidence intervals (CI 95 ), it is impossible to determine with certainty whether the efficacy was significantly better. Among individual major compounds, a significantly better efficacy was shown by geraniol, which achieved a significantly lower LD 50 , if compared to other tested substances (Table 3).

Essential Oil Chemical Composition
The three EOs were analyzed using GC-MS, and the relative content of each component was determined by comparing the ratio of the peak area of each detected compound with the total area of all detected compounds. It resulted in the identification and quantification of both the major and minor compounds that are responsible for the insecticidal activity, acting synergistically. Regarding M. myristica harvested in the locality of Dschang in west Cameroon, Massodi et al. [21] found that the most abundant compounds in the EO were α-phellandrene (52.2%), followed by α-pinene (6.3%), myrcene (4.4%), limonene (3.7%), and α-thujene (2.9%). Similarly, the most represented class reported in this latter study was that of monoterpene hydrocarbons, representing 69.5% of the total composition. Meffo Dongmo et al. [26] identified 20 compounds in M. myristica EO from Bafoussam locality (west Cameroon) with the predominant compound as in the case of Massodi et al. [21] being α-phellandrene (61.5%) followed by germacradienol (7.9%) and δ-cadinene (4.2%). The analysis of this EO from the forest of Lobaye in Central African Republic by Koudou et al. [36] showed that α-phellandrene (34.4%) and p-cymene (22.2%) were the most abundant among 30 identified compounds. While Owokotomo and Ekundayo [37] have demonstrated that the EO of M. myristica seed harvested in Iwaro-Oka, Nigeria, was rich in germacrene-D-4-ol (25.48%), tricyclo [5.2.1(1,5)] dec-2-ene (13.35%), δ-cadinene (11.09%), and linalool (15.10%) on 22 identified compounds.
The results obtained by Meffo Dongmo et al. [26] about the composition of A. citratum corroborate with what we found to the extent that the major compound present in both studies was geraniol (96.8%).
The differences in chemical composition and percentage of compounds of an EO from a different location can be justified by the fact that many factors such as genetics as well as environmental ones (maturity of fruits, geographical conditions, and climate and seasonal changes) can influence the yield and quantitative-qualitative composition of EOs [40,41].

Insecticidal Activity
Botanical-based pesticides represent a concrete and eco-friendly opportunity for small farmers worldwide, being able to exert their action through multiple mechanisms, therefore limiting resistance development. In this scenario, Africa can play a key role in botanical pesticide discovery, development, and commercialization [42,43]. After a careful analysis of the literature, it has been noted that our knowledge on the insecticidal activity of the A. citratum, M. myristica, and X. ethiopica EOs is patchy. Indeed, the available studies on EOs, only tested the X. ethiopica EO against several stored product insect pests [27][28][29][30][31][32]44]. Only a study has been done on M. myristica EO, still on stored product beetles [45], while the insecticidal activity of A. citratum EO has never been investigated. Further research has been conducted on extracts from the three plants. For example, M. myristica ethanolic extracts have been tested against dermestid beetles attacking stored fish [46], while M. myristica and A. citratum extracts exerted toxic effects on bruchid beetle species developing on stored pulses [44], and acted as antifeedants against S. littoralis larvae [47].
Our results pointed out that M. myristica and X. aethiopica EOs were effective against M. domestica adults (♀LD 50 29.1; ♂LD 50 22.1 µg adult −1 ). Bioactivity rates of both EOs are significant, even if still lower if compared to other promising plant EOs, such as the Carlina acaulis L. one, recently tested against houseflies [48]. On the other hand, EOs from these plants are much more available [49,50] and at the same time are characterized by a high extraction yield, which ranges between 4-5%.
The insecticidal effectiveness of EOs depends on many factors, such as the content of major constituents and their synergistic or antagonistic relationships, post-application conditions, and/or the modes of action of the major compounds [51,52]. Also in our case, it was found that the effectiveness of EOs was in line with the effectiveness of most substances.
Against houseflies, the EO from M. myristica and its major compound p-cymene showed comparable effectiveness, while other major compounds performed better in terms of LD 50 while LD 90 values were close to those calculated for EOs. In addition, on S. littoralis larvae, the M. myristica EO was the most effective one, with the major constituent geraniol, showing higher toxicity if compared to the other tested molecules. Last, the EOs from M. myristica and X. aethiopica, as well as the major constituents p-cymene and α-phellandrene, were the most toxic against C. quinquefasciatus larvae. Although the relatively good insecticidal effectiveness of the EOs tested by us on the three insect species was found, it will be important to study other options to make the effectiveness of EOs more efficient, so that the lowest possible amount of active substance is applied in practice. An increase in biological effectiveness can be achieved, for example, by using suitable formulation methods, which include encapsulation or nanoemulsion [8,53,54]. These methods can both extend the persistence time and significantly increase the insecticidal efficacy of EOs themselves. However, it will be equally important to study the effect of sublethal doses or concentrations on target and non-target organisms [55]. It was found that even a sublethal concentration or a short period of exposure to EOs can subsequently significantly reduce the vitality of insects [53,56,57]. Thanks to this phenomenon, not only the high mortality of treated larvae or adults can occur, but also the fertility of the next generation can be significantly reduced, and this can lead to a significant reduction in the number of pests or vectors.
Although EOs are generally considered environmentally safe [6,[58][59][60][61], our further research will be directed to testing the effect of EOs on selected characteristics of non-target organisms, with the aim of confirming the environmental safety of botanical insecticide applications based on our selected EOs. It will also be important to study the possibility of increasing the content of EOs in plants, on the one hand by using suitable elicitation methods and on the other hand by developing more profitable cultivation technologies, and technique of extractions [62][63][64] that will lead to a higher yield of EOs, like the case of other aromatic plants.
Overall, there is still a long way ahead of us, which will lead from the basic screening we performed to the development and production of botanical insecticides based on EOs from M. myristica and X. aethiopica. However, this does not change the fact that we have managed to select EOs that may potentially lead to the development and production of effective botanical insecticides for African smallholders [43].

Plant Material and Essential Oil Extraction
The plant material was bought in a local market of Yaounde (3 • 52 00" N, 11 • 31 00" E, 726 m a.s.l.), Cameroon, in December 2019. The seeds from the three aromatic plants were dried away from the sun at room temperature. Plant specimens were identified by one of us (N. Tsabang). The dried seeds (500 g for M. myristica, 584 g for X. aethiopica, and 550 g for A. citratum) were ground to reduce them into smaller pieces, then inserted in a 10 L Pyrex flask, which was filled with 6 L of distilled water. EOs were obtained by hydrodistillation using a Clevenger-type apparatus for 4 h. The calculation of the oil yields was based on a dry weight (w/w) matter.

GC-MS Analysis
The M. myristica, X. aethiopica, and A. citratum EOs were prepared by a 1:100 dilution with hexane and analyzed with an Agilent 6890N-5973N GC-MS system operating in the EI mode at 70 eV, using a HP-5MS (5% phenylmethylpolysiloxane, length 30 m, internal diameter 0.25 mm, film thickness 0.1 µm; J & W Scientific, Folsom, CA, USA) capillary column. The total duration of the run was around 66 min with the following temperature program: 60 • C for 5 min, afterward up to 220 • C at 4 • C min −1 , then up to 280 • C at 11 • C min −1 and maintained for 15 min. The carrier gas used in this analysis was helium at a flow rate of 1 mL min −1 . The injection volume was 2 µL and the split ratio 1:50. The range of acquisition was 29-400 m z −1 . The combination of linear retention indices (RIs) and mass spectra (MS) with those appearing in libraries such as Adams (2007), FFNSC2 (2012), and NIST17 (2017) was the method used for the peak identification unless no analytical standard (purchased from Merck, Milan, Italy) was available. The analytical standards of the major EO components, namely geraniol, sabinene, α-pinene, p-cymene, α-phellandrene, and β-pinene, were purchased from Merck (Milan, Italy). Relative peak area percentage for each identified compound was extracted from the total area in the chromatogram without using correction factors.

Insects
Insects used for the tests were obtained from established laboratory colonies, reared under controlled conditions for >20 generations. Uniform larvae of S. littoralis (third instar, mean larval weight 12 ± 3 mg), C. quinquefasciatus larvae (third instar), and adults of M. domestica (males and females, 3-5 days old) were selected for the experiments. The rearing methods of the three species mentioned above were recently described by Benelli et al. [65]). All the species were maintained at 25 ± 1 • C, 70 ± 3% R.H., and 16:8 h (L:D). All below described experiments were carried out under the same conditions.
To calculate the lethal doses/concentrations for M. myristica, X. aethiopica, and A. citratum EOs and their main compounds on each insect target, we used a minimal series of at least five different doses/concentrations that resulted in mortality rates in the range of 10-90%. The experiment was replicated four times in total (20 insects per replication). Insect mortality was assessed 24 h after treatment. In insecticidal experiments, mortality was corrected, where needed, through the Abbott's formula [66], LC 50 and LC 90 and the associated 95% confidence limits were estimated by probit analysis [67].

Conclusions
In this work, three EOs extracted from the Cameroonian plants M. myristica, X. aethiopica, and A. citratum were analyzed, and their insecticidal activity was evaluated. Their efficacy against M. domestica, S. littoralis, and C. quinquefasciatus proved to be strongly influenced by the synergistic and antagonistic interactions of the major constituents. In particular, M. myristica and X. aethiopica and their major components p-cymene and α-phellandrene resulted the most active against both houseflies and C. quinquefasciatus, while S. littoralis larvae showed sensitivity to M. myristica EO. Due to their activity and wide distribution in the African continent, these EOs have a great potential to develop insecticide products on a large scale. Future steps in this direction will be the encapsulation of the EOs in nanoformulations and the evaluation of the effect of sublethal doses or concentrations on target and non-target organisms.

Data Availability Statement:
The data presented in this study are available on request from the corresponding authors.