The Laboratory and Semi-Field Larvicidal Effects of Essential Oil Extracted from Feronia limonia against Anopheles arabiensis Patton

This study intended to evaluate the larvicidal activity of Feronia limonia leaf essential oil against the wild population of Anopheles arabiensis Patton larvae in laboratory and semi-field environments. Larvae mortality was observed after 12, 24, 48, and 72 hours of exposure. In laboratory condition, the essential oil showed good larvicidal activity against An. arabiensis (LC50 = 85.61 and LC95 = 138.03 ppm (after 12 hours); LC50 = 65.53 and LC95 = 117.95 ppm (after 24 hours); LC50 = 32.18 and LC95 = 84.59 ppm (after 48 hours); LC50 = 8.03 and LC95 = 60.45 ppm (after 72 hours), while in semi-field experiments, larvicidal activity was (LC50 = 91.89 and LC95 = 134.93 ppm (after 12 hours); LC50 = 83.34 and LC95 = 109.81 ppm (after 24 hours); LC50 = 66.78 and LC95 = 109.81 (after 28 hours); LC50 = 47.64 and 90.67 ppm (after 72 hours). These results give an insight on the future use of F. limonia essential oils for mosquitoes control.


Introduction
Mosquito control in recent past has been challenged by the wide spread of insecticide resistance among the vector populations [1,2]. The decline of efficacy of currently used vector control tools (long-lasting insecticidal nets and indoor residual spray) have threatened the achieved efforts across Sub-Saharan Africa [1,2]. The search for alternative insecticides with different mode of action is of paramount importance. The targeting of breeding habitats has shown a great impact on larvae and subsequently reduction of adult mosquitoes and disease incidences [3]. The search of the natural products to overcome the resistance by targeting the aquatic stages of mosquitoes has shown an effect in laboratory and semi-field evaluation [3]. The use of botanical extracts for pests control has shown to have no side effect on environment and non-targeted organisms [4,5]. Previous studies have shown that the essential oil from plants displayed a great impact on larvicidal, adulticidal, and repellent effects [6]. To date, the plant extracts resistance by vectors has not been reported [6]. The aim of this study was to assess the larvicidal activities of Feronia limonia leaf essential oil against An. arabiensis.

Material and Methods
2.1. Plant Collection and Essential Oil Extraction. The leaves of F. limonia were collected from Keerapalayam [11°26 ′ ;03 N, 079°39 ′ ;02 E], Cuddalore District, Tamil Nadu, India, and the voucher specimen (AUBOT# 209) is deposited at the Herbarium, Department of Botany, AU. The fresh leaves were subjected to hydro-distillation using Clevenger-type apparatus for 4 hours. The essential oil was dried over anhydrous sodium sulphate, and the purified essential oil was stored at +4°C for mosquito larvicidal activity.
The essential oil volatile constituents were determined by Gas chromatography-mass spectrometry (GC-MS) by using Varian 3800 Gas Chromatography equipped with Varian 1200 L single quadrupole mass spectrometer. The mass spectrometer was operated in the electron impact (IE) mode at 70 eV. Ion source and transfer line temperature were kept at 250°C. The mass spectra were obtained by centroid scan of the mass range from 40 to 1000 amu. The compounds were identified based on the comparison of their retention indices (RI), retention time (RT), and data bank mass spectra of Wiley library (Table 1).

Mosquito Larvae
Rearing. The An. arabiensis Patton eggs were obtained from wild gravid mosquitoes collected from cowsheds in Lower Moshi near rice irrigation schemes. The wild population of Anopheles gambiae s.l. in this area has been confirmed to be composed of 100% An. arabiensis [7]. The larvae room was maintained at the temperature of 27 ± 2°C and relative humidity of 78 ± 2%. The larvae were fed with 0.003 g of Tetra mine per larva as shown in previous study [8]. The An. arabiensis larvae were reared until when they were stage three instars and used for screening as per WHO guidelines.

Mosquito Larvicidal
Assay. The F. limonia essential oil was dissolved in 1 ml of acetone. Then, serial dilution was made from this stoke solution into six concentrations 3.125, 6.25, 12.5, 25, 50, and 100 ppm with distilled water as per WHO guidelines. Each concentration had six replicates, each with twenty-third instar larvae of An. arabiensis. There were two controls: one control (C1) contained distilled water, while the other (C2) contained 1 ml aqueous solution of acetone. During these assays, food was provided to the larvae after 24 hours. The mortality of larvae was monitored after 12, 24, 48, and 72 hours of exposure period in both treatments and controls. The dead and moribund larvae were both considered dead. Experiments were set in both laboratory and semi-field conditions as described elsewhere [9].

Data
Analysis. Data were entered into excel sheet and transferred into IBM SPSS Version 26 (IMB Corp., Armonk, NY, USA) for analysis. The lethal concentrations LC 50 and LC 95 and their 95% confidence limit of upper and lower confidence levels were calculated by probit analysis [10]. The comparison of larvae mortality among treatments, between treatments and control, and between laboratory and semi-field environment were conducted using Chi-square test.

Chemical Composition.
The GC-MS chemical analysis of the F. limonia leaf essential oil revealed 51 chemical compounds with estragole as the highest abundant compound with 34.69%, while cis-dihydro-β-terpineol and ρ-Cymene were revealed to have the least amount of 0.03% each. The other 48 compounds occurred in different percentage compositions (Table 1).

Mortality
Effect of the Essential Oils. The mortality of the larvae of An. arabiensis from 12 to 72 hours of observation in both laboratory and semi-field environment was found to be dosage dependent (Figure 1), and the proportion of larvae that died between laboratory and semi-field environments was found to have statistically significant difference ( Table 2). The observed mortality rate was higher in the laboratory environment as compared to semi-field environment, it ranged between 20.83% in 3.125 ppm and 91.88% in 100 ppm (Table 2). Also, the mortality effect was found to be exposure time dependent, with percentage mortality significant different revealed in some time intervals (Table 3).

Lethal Dose.
The lethal dose enough to kill 50% (LC 50 ) and 95% (LC 95 ) of the larvae exposed varied with exposure time in both laboratory and semi-field environments ( Table 4). In both LC 50 and LC 95 , the lowest values were found in laboratory than in the semi-field conditions ( Table 4). The proportions of lethal dose comparison were found to differ significantly between laboratory and semifield experiments (Table 4).

Discussion
The findings of this study have highlighted the impact of F. limonia essential oil against An. arabiensis larvae. The mortality of the larvae in both laboratory and semi-field was found to be dosage dependent with higher mortality observed in laboratory. These findings are similar to the previous study conducted using larvae of Aedes aegypti, Anopheles stephensi, Culex quinquefasciatus, and An. gambiae s.s. having more mortality in laboratory than semi-field [11]. This mortality decreases in semi-field compared to the laboratory and it might have been attributed to the exposure of the essential oil to the sunlight, which might cause compound degradation into secondary metabolites with low toxicity [12]. To maintain the effectiveness of the botanical larvicides, repeated application is needed frequently [13]. The modification of the plant-based biolarvicides is of paramount importance to extend its longevity in environment by making it in slow-release technology. This technology has been practical to Bacillus thuringiensis israelensis (Bti), which in application lasts for 5-7 days [14,15], but with improved slow-release technology, it has lasted active against larvae for 6 months with no effects to non-targeted organisms [3,16].
In recent years, the non-particle technologies have enhanced natural products to be more stable and effective. The recent nanoparticles incorporated in natural products have seen to be effective for public health and agriculture pests [17].
The larval source management currently is mostly done with frequent application of organophosphate [15] and insect growth regulators [18,19]. So, taking up plant-and fungal-based natural product in improved synthesis in 2 Journal of Parasitology Research small-scale trials can be paving a way to manage insecticide resistance and reduce vector abundance. The findings of this study have shown that the essential oils of F. limonia have impact on mosquito mortality, but in semi-field environment has to be modified to maintain the laboratory observed mortality results.
The larvicidal mortality seen in this study might have been caused by the occurrence of β-pinene and estragole (methyl chavicol). Estragole is a common chemical constituent in plants' essential oils [20]. Essential oils with methyl chavicol from different have shown high mortality against mosquito larvae. Also, this compound has shown to exhibit fumigant and contact toxicity against Ceratitis capitata, Bactrocera dorsalis, Bactrocera cucurbitae [21], and other storage post-harvest pests [22,23]. In F. Limonia, β-pinene occurrence was higher and past studies have shown high mortality effect of larvae [24]. The β-pinene-rich in essential oil has shown to impact mortality of Aedes aegypti fourth instar Ae. aegypti larvae. Also, there might be an impact of elements considered to be minor components of the essential oils in larvicidal activity of essential oils [25]. In Ocimum suave essential oils, the linalool among the least occurring ingredient was found to be a major source of mortality [26]. In this case, the natural products from plants have shown to be the major alternative of synthetic pesticides if well moderated and composed.

Conclusion
The findings of this study have shown that the essential oils of F. limonia have impact on mosquito mortality, but in semi-field environment has to be modified to maintain the laboratory observed mortality results.

Data Availability
All data generated in this study have been provided in the manuscript.

Ethical Approval
This study did not require ethical clearance.

Conflicts of Interest
The authors declare that they have no conflicts of interest.