Synthetic pesticides are the cornerstone of vector-borne disease control, but alternatives are urgently needed to tackle the growing problem of insecticide resistance and concerns over environmental safety. Leptospermum scoparium J.R. Forst and G. Forst (manuka) essential oil and its four fractions were analyzed for chemical composition and toxicity against Aedes aegypti larvae. The use of bio-based amylose-N-1-hexadecylammonium chloride inclusion complexes (Hex-Am) as an emulsifier for L. scoparium essential oil was also investigated. Fraction 1 was inactive, fractions 2 (LC50 = 12.24 ppm) and 3 (LC50 = 20.58 ppm) were more toxic than the whole essential oil (LC50 = 47.97 ppm), and fraction 4 (LC50 = 35.87 ppm) had similar toxicity as the whole essential oil. Twenty-one chemical constituents were detected in L. scoparium essential oil compared to 16, 5, 19 and 25 chemical constituents in fractions, 1, 2, 3 and 4 respectively. The two most dominant chemical constituents were calamenene (17.78%) and leptospermone (11.86%) for L. scoparium essential oil, calamenene (37.73%) and ledene (10.37%) for fraction 1, leptospermone (56.6%) and isoleptospermone (19.73) for fraction 2, cubenol (24.30%) and caryophyllene oxide (12.38%) for fraction 3, and γ-gurjunene (21.62%) and isoleptospermone (7.88%) for fraction 4. Alpha-pinene, ledene, and aromandendrene were 2–7 times less toxic than the whole essential suggesting that the toxicity of L. scoparium essential oil was either due to other chemical constituents that were not tested or due synergist interactions among chemical constituents. Leptospermum scoparium essential oil-Hex-Am emulsion (LC50 = 29.62) was more toxic than the whole essential oil. These findings suggest that L. scoparium essential oil is a promising source of mosquito larvicide and that Hex-Am is an excellent emulsifier for L. scoparium essential oil for use as a larvicide.
Citation: Muturi EJ, Selling GW, Doll KM, Hay WT, Ramirez JL (2020) Leptospermum scoparium essential oil is a promising source of mosquito larvicide and its toxicity is enhanced by a biobased emulsifier. PLoS ONE 15(2):
Editor: Ahmed Ibrahim Hasaballah, Al-Azhar University, EGYPT
Received: December 13, 2019; Accepted: January 28, 2020; Published: February 20, 2020
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability: All relevant data are within the manuscript.
Funding: This research was supported by the U.S. Department of Agriculture, Agricultural Research Service. All authors are employed by the USDA. The specific roles of each author are articulated in the ‘author contributions’ section. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Mosquito-borne diseases remain one of the most pressing public health challenges facing mankind today. Vector control is one of the primary strategies that have effectively been used to break the transmission cycle and has historically relied on the use of synthetic insecticides. Unfortunately, the widespread use of synthetic insecticides has resulted in alarming levels of insecticide resistance among the target mosquito species, raising concerns that current gains in the fight against mosquito-borne diseases could be lost. Moreover, the use of some synthetic insecticides in vector control has been discontinued or restricted due to their potential to disrupt ecological processes and cause harm to non-target organisms. These challenges have reinforced the urgent need for effective and environmentally-friendly vector control strategies.
Some plants produce essential oils that contain a spectrum of chemical compounds which provide protective role against herbivores and pathogens. Some of these essential oils have insecticidal and repellent activity against mosquitoes [1–6] and possess other traits that make them suitable alternatives to synthetic insecticides. These include low mammalian toxicity, rapid degradation in the environment, limited non-target effects, multiple modes of action that may inhibit development of insecticide resistance, and commercial availability at an affordable cost [7, 8]. As a result, significant research effort has been devoted towards the discovery and development of essential oil-based insecticides for pest and vector management.
Manuka (Myrtaceae: Leptospermum scoparium J.R. Forst and G. Forst) also known as “tea tree”, is one of the most abundant and widespread indigenous shrub species in New Zealand. Early records report the traditional use of the bark, leaves, sap, and seed capsules from manuka for treatment of various diseases and ailments including fever, cough, mouth and throat sores, running nose, dysentery, diarrhea, colic pain, breast inflammation, back stiffness, eye problems, and scald and burn injuries [9, 10]. Essential oil derived from L. scoparium is also used as a strong antimicrobial and antifungal agent in creams, soaps, toothpastes and other preparations [11, 12]. During World War II, L. scoparium essential oil was provided in the first aid kits of serving Australian soldiers for use as a general antimicrobial agent and insect repellent . Other studies have demonstrated the toxicity of L. scoparium essential oil and some of its fractions against arthropods of economic and medical significance including the spotted wing drosophila Drosophila suzukii Matsumura, itch mite, Sarcoptes scabiei Linnaeus, poultry red mite, Dermanyssus gallinae De Geer, stored food mite, Tyrophagus putrescentiae Schrank, and house dust mites, Dermatophagoides farinae Hughes and D. pteronyssinus Troussart [13–16]. Leptospermum scoparium essential oil has also been shown to be an attractive bait for the redbay ambrosia beetle, Xyleborus glabratus Eichhoff . A recent study by our research group also demonstrated that L. scoparium essential oil is toxic to Aedes aegypti Linnaeus larvae (LC50 = 53.0 ppm) and interacted synergistically with oregano essential oil and antagonistically with clove bud essential oil . In general, however, knowledge regarding the insecticidal activity of L. scoparium essential oil against arthropods of medical and economic significance is limited. Additionally, most studies on insecticidal properties of essential oils focus on the whole essential oil, yet some studies have shown that bioassay-guided fractionation of some essential oils may yield fractions that are more toxic than the oil itself [3, 18–20]. The use of essential oils as mosquito larvicides also remains a challenge due to their chemical instability, high volatility, and poor solubility in water. Thus, technologies that improve the solubility and environmental stability of essential oils when used as biopesticides are urgently needed.
Oil-in-water emulsions are considered to be efficient delivery systems for hydrophobic compounds by dispersing the lipid phase as a colloidal dispersion . Here, two immiscible liquids are stabilized through the addition of a surfactant (emulsifier) which prevents droplet coalescence by lowering the interfacial tension . Amylose from starch has attracted significant interest as a low-cost material for the synthesis of emulsifying agents. When amylose is combined with suitable ligands such as fatty amine salts, the hydrophobic portion of the ligand associates with the hydrophobic internal cavity of the amylose helix to form water-soluble amylose-inclusion complexes [23, 24]. These complexes have been shown to be surface active polymers with superior emulsion activity compared to commercial modified starch emulsifiers  and have been used to reduce volatility, increase stability, enhance bioactivity and extend the shelf life of the target bioactive compounds [25, 26]. Additionally, amylose inclusion complexes are biodegradable and non-toxic, making them appealing emulsifiers for the development of ecofriendly biopesticides .
In this study we analyzed the chemical composition of L. scoparium essential oil and its fractions and evaluated their toxicity against larvae of the yellow fever mosquito, Aedes aegypti. We also evaluated the use of amylose-N-1-hexadecylammonium chloride inclusion complexes (Hex-Am) as emulsifiers for L. scoparium essential oil for use as mosquito larvicides. Our overall goal was to develop a better understanding of the insecticidal activity of L. scoparium essential oil against disease vectors and to generate new knowledge that may guide the development of effective biopesticides based on essential oils.
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Materials and methods
Separation of L. scoparium essential oil fractions was performed using preparative flash chromatography (Cheetah MP200, Bonna-Agela Technologies Inc., Newcastle, DE). The column (Supel Dlash Catridge, 80g, 40–60 μm silica) was equilibrated with hexane for 10 min at a flow rate of 60 mL per min. The oil sample (5 mL) was injected into the column using a 10-mL syringe and the column was developed with hexane-ethyl ether gradient method over 27 minutes as follows: 100% hexane for 2 min, 0–100% ethyl ether for 20 min, and 100% ethyl ether for 5 min. The effluent was monitored at 254 nm and fractions were collected by volume (60 mL). Fractions containing each absorbance peak were pooled and placed in the fume hood for evaporation of organic solvent. The procedure was repeated until adequate amounts of different fractions were obtained. All fractions were labeled and stored in amber-colored glass bottles until use.
Gas Chromatography-Mass Spectrophotometry (GC-MS) analysis
Identification of the chemical constituents of L. scoparium essential oil and its fractions was accomplished as previously described . Briefly, two different Agilent 7890 (Santa Clara, CA) gas chromatographs, each using Agilent’s Mass Hunter software were used to acquire and process the data. A 5975 mass spectrometry detector using NIST05 library (National Institute of Standards and Technology, Gaithersburg, MD) was employed for product identification, whereas flame ionization detection (FID) was used for quantitation. Samples of ~10 μL were diluted in 1 mL of heptane and 1 μL was injected by autosampler using a 50:1 split ratio and analyzed on Agilent/J&W DB35-MS column (30m × 320 mm, 0.25 mm film thickness). Helium flow in the column was maintained at 1.37 mL per minute. Oven temperature was programmed at 40°C for 3 min, 10°C min-1 to 190°C for 5 min, and 25°C min-1 to 340°C. Commercial compounds were purchased when available, diluted in heptane, and used for comparison of retention time. To determine the relative retention time, a GC sample of alkanes, ranging in size from decane to tetracosane (10 to 24 carbon atoms) was made and ran on the GC and GC-MS under identical conditions to the sample analysis. The retention times of each of the alkanes was determined, and then all of the relative retention times were calculated according to the formula 7 in ASTM D6730-19 as follows:
Preparation of amylose-complexes and oil emulsions
Amylose-complexes were produced following the procedure outlined previously [28–30]. A dispersion of high-amylose starch (100.0 g of starch) and deionized water (1800 mL) was passed through a Penick & Ford laboratory model continuous steam jet cooker (Penford Corp., Cedar Rapids, IA) operating under the following conditions: hydroheater temperature 140°C, steam back pressure 380 kPa, steam line pressure 550kPa and pumping rate of 1 L min−1. Cooked dispersions were collected in a Dewar flask to prevent rapid temperature loss. A solution of N-1-hexadecylammonium chloride was prepared as previously described  and added to the hot starch dispersion immediately after jet-cooking. The mixture was rapidly stirred for 1 min, and then cooled to 25°C in an ice bath. Spray drying of amylose-N-1-hexadecylammonium chloride inclusion complexes (Hex-Am) was performed using a Niro atomizer spray dryer (Niro, Columbia, MD, USA) as previously described . Materials were collected and stored at room temperature until use. Spray dried amylose-complexes were used as emulsifiers to prepare oil-in-water (O/W) emulsions. Emulsions were prepared by mixing water, L. scoparium essential oil, and spray-dried Hex-Am at 92.5: 5: 2.5 ratio, respectively. A mixture totaling 10 g, was placed in a 30 mL glass beaker and homogenized for 180 seconds at 20,000 rpm using a Power Gen 35 handheld micro homogenizer (Fisher Scientific, Pittsburgh, PA).
Dynamic light scattering
Dynamic light scattering (DLS) analysis to determine the particle size and distribution was conducted using a Horiba LB-550 Dynamic Light Scattering Particle-Size Analyzer (HORIBA Instruments Incorporated, Irvine, CA). The analysis was conducted at 25°C using a 1 cm path-length cell having a volume of 1.25 mL. Aqueous emulsions (minimum three samples tested) of L. scoparium essential oil and Hex-Am were diluted ~1000x to obtain spectra. Horiba software was used to analyze and process the hydrodynamic diameter distribution data to determine the median hydrodynamic diameter. Intensity % for each diameter was calculated by dividing its value by the total area for the spectral curve multiplied by 100.
Aedes aegypti (Rockefeller strain) larvae were reared on yeast: lactose albumin (1:1) diet in batches of ~200 larvae at 26°C, 70% relative humidity (RH) and 10:14 h (light: dark cycle). Larvae from all rearing containers were pooled before the bioassays. With exception of the water volume and the starting number of larvae per container, the toxicity bioassays followed the standard World Health Organization guidelines . Twenty late third instar larvae of Ae. aegypti were added into 120 mL of DI water held in 400 mL tripour beakers. Treatments included L. scoparium essential oil purchased from Sigma-Aldrich and its four fractions obtained via flash chromatography. The oil and its fractions were diluted in absolute ethanol to create stock solutions of similar concentrations to oil emulsions (50,000 ppm). The treatments were tested at varying concentrations depending on their degree of toxicity. Leptospermum scoparium essential oil, fraction 1 and fraction 4 were tested at 7 concentrations ranging from 20–80 ppm. Fraction 2, fraction 3 and L. scoparium essential oil-Hex-Am emulsion were more toxic and were tested at lower concentrations. Fractions 2 and 3 were tested at 7 concentrations ranging from 5–35 ppm for fraction 2 and 16–34 ppm for fraction 3. Leptospermum scoparium essential oil-Hex-Am emulsion was tested at 6 concentrations ranging from 20–45 ppm. A control group was treated with absolute ethanol without oil/fraction/emulsion treatment. Each treatment was replicated 3 times, and 3 separate trials with different batches of mosquitoes were conducted. The containers were held at room temperature and the total number of larvae surviving 24 hours post-treatment were counted and recorded. Probit analysis conducted using “ecotox” package in R version 3.3.2 was used to calculate the LC50 and LC90 values for each oil/fraction/emulsion. To determine the contribution of some individual constituents to the toxicity of L. scoparium essential oil, three L. scoparium essential oil chemical constituents that were commercially available at affordable prices were purchased from Millipore Sigma (Saint Louis, MO) and tested at a concentration of L. scoparium essential oil (70 ppm) expected to kill 90% of the test larvae. The chemical constituents were alpha-pinene, ledene, and aromandendrene and were tested using the experimental procedures described above.
A total of 29.5 g of L. scoparium essential oil was processed yielding 13.06 (44.27%), 7.05 (23.90%), 1.43 (4.85%) and 0.96 g (3.25%) of fractions 1, 2, 3, and 4 respectively. GC-MS analysis revealed qualitative and quantitative differences in the chemical composition of L. scoparium essential oil and its fractions (Table 1). Twenty-one chemical constituents were detected in L. scoparium essential oil compared to 16, 5, 19 and 25 chemical constituents in fractions, 1, 2, 3 and 4 respectively (Table 1). Calamenene (17.78%), leptospermone (11.86%), α-selinene (7.17%) and α-cadinene (6.40%) were the four most abundant chemical constituents in L. scoparium essential oil. The four most abundant chemical constituents in fraction 1 were calamenene (37.73%), ledene (10.37%), α-selinene (9.20%), and α-copaene (7.96%). Fraction 2 was predominantly leptospermone (56.6%), isoleptospermone (19.73%), flavesone (16.82%), and γ-muurolene (5.42%). For fraction 3, the dominant constituents were cubenol (24.30%), caryophyllene oxide (12.38%), leptospermone (10.89%), and flavesone (6.78%). The dominant constituents in fraction 4 were γ-gurjunene (21.62%), isoletospermone (7.88%), eudesma-4(14),11 diene (6.61%), and unidentified compound (6.00%). Venn diagrams were used to summarize the chemical constituents that were present/absent in L. scoparium essential oil and its fractions (Fig 1). All 16 constituents detected in fraction 1 were present in the whole essential oil, but 5 constituents present in the whole essential oil were not detected in fraction 1. These were α-pinene, isoleptospermone, leptospermone, cubenol, and γ-muurolene. Similarly, all 5 chemical constituents detected in fraction 2 were present in the whole essential oil. With exception of α-cubebene, these compounds were more abundant in fraction 2 than in the whole essential oil. Seven compounds were shared between L. scoparium essential oil and fraction 3, 14 were only detected in L. scoparium essential oil and 12 were only detected in fraction 3. Five constituents were shared between L. scoparium essential oil and fraction 4, with 16 constituents only detected in L. scoparium essential oil and 20 constituents only detected in fraction 4. Overall, 11, 0, 4, and 12 compounds were unique to L. scoparium essential oil, fraction 2, fraction 3 and fraction 4, respectively and only 2 constituents were shared among the four treatments. When only the four fractions were considered, 10, 0, 4, and 12 constituents were unique to fractions 1, 2, 3 and 4 respectively and no compounds were shared among all four fractions.
Table 1. Chemical composition of L. scoparium essential oil and its fractions.
LP, L. scoparium essential oil, F1-F4, fractions 1–4. Also included are retention time from the GC-FID with relative retention index (RRI), and the major fragmentations ions observed by GC-MS listed in order of relative abundance. Dash (-) indicates that the compound was not detected.
The larvicidal activity of L. scoparium essential oil fractions and emulsions against Ae. aegypti was evaluated relative to the whole essential oil. Fraction 1 was inactive, fractions 2 (LC50 = 12.24 ppm) and 3 (LC50 = 20.58 ppm) were 4 and 2 times more toxic than the whole essential oil (LC50 = 47.97 ppm), and fraction 4 (LC50 = 35.87 ppm) had similar activity as the whole essential oil (Table 2). The three chemical constituents of L. scoparium essential oil tested (α-pinene, ledene, and aromandendrene) were 2–7 times less toxic than the whole essential oil (Fig 2). Leptospermum scoparium essential oil-Hex-Am emulsion (LC50 = 29.62 ppm) was more toxic to Ae. aegypti larvae than the whole essential oil. Dynamic light scattering analysis of L. scoparium essential oil-Hex-Am emulsion revealed that the complexes had a median (± SD) hydrodynamic diameter of 1.96 ± 0.74 microns.
Fig 2. Toxicity of three chemical constituents of manuka (L. scoparium) essential oil against Aedes aegypti larvae relative to whole essential oil.
Error bars represent the standard error of the mean.
Table 2. LC50 and LC90 values for Leptospermum scoparium essential oil and its fractions and emulsions produced with hexadecyl ammonium chloride amylose inclusion complexes.
ND, not determined because it was outside the range of concentrations tested. LP, Leptospermum scoparium.
Leptospermum scoparium essential oil is known for its many medicinal applications, but its insecticidal properties remain poorly understood. Here, we show that L. scoparium essential oil can serve as an important source of larvicides for mosquito control. Fractions 2 and 3, respectively were 4 and 2 times more toxic than the whole essential oil, while fraction 4 had similar toxicity as the whole essential oil. The World Health Organization (WHO) has not established a standard criterion for determining the larvicidal activity of natural products but several scientists have developed their own criteria. Komalamisra et al.  considered products showing LC50 <50 mg/L active, 50 mg/L50<100 mg/L moderately active, 100 mg/L50<750 mg/L effective, and LC50>750 mg/L inactive.
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