Development of cellulose nanocrystal-stabilized Pickering emulsions of massoia and nutmeg essential oils for the control of Aedes albopictus

We investigated the larvicidal potential of 10 plant essential oils (EOs) against the Asian tiger mosquito Aedes albopictus. Among the EOs, larvicidal activity against Ae. albopictus was strongest in those derived from massoia (Massoia aromatica) and nutmeg (Myristica fragrans). Larvicidal activities of massoia and nutmeg EOs against Ae. albopictus were 95.0% and 85.0% at 50 μg/mL, respectively. A total of 4 and 14 compounds were identified from massoia and nutmeg, respectively, and two massoia lactones, C10 and C12, were isolated from massoia EO. Among the identified compounds, benzyl salicylate, terpinolene, C12 massoia lactone, sabinene, benzyl benzoate, methyl eugenol, and C10 massoia lactone exhibited the strong larvicidal activity. Cellulose nanocrystal (CNC)-stabilized Pickering emulsions of massoia and nutmeg EOs were developed to overcome the insolubility of EOs in water. CNC/massoia and CNC/nutmeg emulsions were stable for at least 10 days, and larvicidal activities of CNC/massoia PE and CNC/nutmeg were higher than those of crude massoia and nutmeg EOs. This study presents a CNC-stabilized PE, a suitable formulation for EOs, as a potential larvicide against Ae. albopictus.

Identification of C10 and C12 massoia lactones. C10 and C12 massoia lactones were identified and obtained using bioassay-guided isolation because two compounds were commercially unavailable. The chemical structures of the isolated compounds were confirmed by 1 H, 13 C nuclear magnetic resonance (NMR) and mass spectrometer data. The purities of isolated C10 massoia lactone and C12 massoia lactone were 95% and 95%, respectively.
Larvicidal activity of major constituents identified in massoia and nutmeg Eos. The larvicidal activities against Ae. albopictus of the constituents derived from massoia and nutmeg EOs are shown in Table 3. Among the identified compounds, benzyl salicylate, C12 massoia lactone, terpinolene, C10 massoia lactone, sabinene, benzyl benzoate, and methyl eugenol showed > 80% larvicidal activity at 25 μg/mL. Larvicidal activi- Physical properties of CNC-based PEs. The stability of PEs can be evaluated by observing the phase separation of the emulsion solution and the change in emulsion fraction for long-term storage at different CNC contents (Fig. 1). Phase separation of the emulsion solution was observed at 135 and 180 mg of CNC/mL massoia PEs. It resulted from insufficient CNC content for emulsifying the oils. In contrast, 270 mg of CNC/mL massoia or more formed a stable emulsion state without phase separation for 10 days (Fig. 1A). The fraction of emulsion decreased after 10 days to approximately 20% for 135 and 180 mg CNC/mL massoia PEs, but no big change was shown for 270 mg CNC/mL massoia and more content (Fig. 1B). Microscopic images showed a shell structure of PEs due to the densely packed CNCs at the surface ( Fig. 2A). Typically, PE size decreased as CNC content increased. The PEs ranged in size from 3 to 15 μm and the size distribution of PEs prepared at 135 and 180 mg CNC/mL massoia was wider than that of the other PEs ( Fig. 2B-D). Colloidal stability of nutmeg EO PEs was achieved at a lower CNC content compared with massoia PEs (Fig. 3A). Nutmeg EO PEs were stable at 180 and 225 mg of CNC/mL nutmeg after storage for 10 days, while the emulsion fraction decreased to approximately 20% after storage for 1 day due to the phase separation of the emulsion at 45, 90, and 135 mg of CNC/mL nutmeg (Fig. 3B). Microscopic images revealed a CNC shell structure at the surface of the PEs (Fig. 4A). The PEs ranged in size from 4 to 10 μm and the size decreased as the CNC content increased (Fig. 4B-D). The massoia and nutmeg EOs encapsulated in PEs was imaged by confocal microscopy (Fig. 5). Blue fluorescence represented CNCs stained with Calcofluor white, and red fluorescence represented EOs stained with Nile red. Both massoia and nutmeg EOs were encapsulated in the CNC PEs (Fig. 5A,B). The CNCs formed a shell structure at the surface of the PEs and the EOs were contained inside the shells. The densely packed CNC shells formed a rigid structure, reducing the aggregation of particles and maintaining colloidal stability for an extended period.
92.5 ± 4.8ab 85.0 ± 6.5ab 55.0 ± 6.5b 25.0 ± 6.5b -- www.nature.com/scientificreports/ albopictus were 55.65, 28.61, and 35.98 mg/L, respectively. However, the values for ( +)-α-pinene, (-)-α-pinene, myrcene, and ( +)-terpinen-4-ol were > 100 μg/mL in this study. The larvicidal activities of ( +)-limonene, α-terpinene, α-phellendrene, and γ-terpinene reported in previous study 40 were slightly stronger than those in this study, but the larvicidal activity of terpinolene was slightly weaker than that of this study. bin Jantan et al. 37 reported that the LC 50 values of benzayl salicylate, benzyl benzoate, and safrole were 5.5, 6.5, and 28.0 μg/mL, respectively. Larvicidal activity of benzyl salicylate was weaker than that of this study, but larvicidal activities of benzyl benzoate and safrole were slightly stronger. A previous research effort 37 and this study discovered different larvicidal activities associated with eugenol and methyleugenol. Larvicidal activity of eugenol was stronger than that of methyleugenol in a previous study 37 but the relationship was reversed in this study. In a report by Seo et al. 41 the insecticidal activities of myristicin and elemicin were similar to those of this study. The differences in larvicidal activities of EOs constituents against Ae. albopictus may be attributable to the methodological differences, as bin Janten et al. 37 used fourth instar larvae for larvicidal tests and ethanol as a solvent for EOs, unlike the method of this study. C10 and C12 massoia lactones are reportedly the main constituents of massoia EO 23,33 , and they possess an α-β-unsaturated δ-lactone moiety. Massoia lactone groups have been reported to show biological activities, including antimicrobial, cytotoxic, anti-inflammatory and phytotoxic activities 22,24,43,44 . However, larvicidal activity against Ae. albopictus has not been reported for C10 and C12 massoia lactones.
Differences in the chemical structure of C10 and C12 massoia lactones include the aliphatic chain length at the C6 position, and this may be responsible for the difference in larvicidal activity against Ae. albopictus. Previous studies indicated that the chain length of compounds with similar chemical structures can play an important role in insecticidal and nematicidal activity 45,46 . Hammond and Kubo 45 evaluated the larvicidal activity of alkanols with C 1 -C 20 chain lengths against the mosquito Culiseta incidens. They found that larvicidal activities of dodecanol, tridecanol, and undecanol, with chain lengths of 12, 13, and 11, respectively, were stronger than those of alkanols with other chain lengths. Seo et al. 46 also reported that an optimal chain length of aliphatic compounds was necessary for nematicidal activity against the pine wood nematode Bursaphelenchus xylophilu. Nematicidal activities of aliphatic compounds with a C 9 -C 11 chain length were stronger than those of other aliphatic compounds with other chain lengths. Larvicidal activities of eugenol and its derivatives, such as methyleugenol, isoeugenol, and methylisoeugenol, showed that the methoxy group and a double-bond position affected activity. Larvicidal activities of eugenol and methyl eugenol with an allyl group were stronger than those of isoeugenol and methylisoeugenol with a propenyl group, and larvicidal activities of methyl eugenol and methylisoeugenol with two methoxy groups at a benzene ring were stronger than those of eugenol and isoeugenol with one methoxy www.nature.com/scientificreports/ group. Bhardwaj et al. 47 reported that larvicidal activity of methyl eugenol was stronger than that of eugenol against the tobacco armyworm, Spodopter litura. Methyleugenol exhibited strong contact toxicity against the cigarette beetle, L. serricorne compared with methylisoeugenol 32 . The insolubility of plant EOs in water requires emulsion-based formula for the practical use of plant EOs as larvicides against mosquitoes. Emulsions generally comprise small spherical droplets of two liquids stabilized with surfactants or surface-active polymers 48 , and are used widely in the pesticide industry 20 . However, conventional emulsions made with synthetic emulsifiers such as alkylphenol ethoxylates and organic-phase emulsifiers such as toluene and xylene have many drawbacks 20 , requiring the development of environmentally friendly emulsions, and PEs stabilized by solid particles are considered alternatives for conventional emulsions [49][50][51][52] . CNCs are appropriate solid particles for manufacturing PEs due to their high aspect ratio of crystalline fibrils and amphiphilicity 48,53 . In this study, CNC-stabilized PEs of massoia and nutmeg EOs were made and their physiological properties and larvicidal activity were investigated. The PEs exhibited visual differences depending on CNC content after storage for 24 h (Figs. 1A, 3A). This study shows that a critical concentration of CNCs is required to cover EOs completely and ensure the long-term stabilization of CNC-based PEs. The critical concentrations of CNC/mL EOs for massoia and nutmeg EOs were approximately 270 mg and 180 mg, respectively. Shin et al. 19  The results of this study demonstrate that CNC-based PEs of massoia and nutmeg EOs as promising larvicides which can be applied in field, since the larvicidal activies of CNC/massoia and CNC/nutmeg PEs were stronger than those of crude massoia and nutmeg EOs. Also, CNC-based PEs have showed stability for at least 10 days after the production, with improved solubility of massoia and nutmeg EOs in water. Another advantage of CNC-based PEs may be controlled release of EOs, as EOs and their constituents easily evaporate when treated in water 15,41 . However, further studies about proper dilution of the formulation, safety to non-target organisms, radiation and temperature effect after application and other factors such as costs are required for practical application of CNC-based PEs of massoia and nutmeg EOs.

Methods
Plant essential oils. The information about EOs used in this experiment are shown in Table 1. Blue cypress, Hinoki, Texas cedar, Japanese cedar, Golden-lotus, Cubeb and massoia EOs were purchased from Oshadhi Ltd. (Cambridge, UK). Fir needle, Spice, spruce and Nutmeg EOs were purchased from Jin Aromatics (Anyang, Republic of Korea).

Insects. Cultures of Ae. albopictus were supplied from Korean Disease Control and Prevention Agency
(Cheongju, Republic of Korea). They were reared in an insectary at 26 ± 1 °C and a relative humidity of 60 ± 5% under a 16:8 h light/dark cycle. We supplied the larva with a sterilized diet composed of 40-mesh chick chow powder and yeast (4:1). All procedures involving the use of animals were performed in compliance with the ARRIVE guidelines for animal studies. A live mouse was provided as a blood-meal source, using a method approved by the Institutional Animal Care and Use Committee (approval no. SNU-190418-1-2; title: Providing rodents for blood-feeding mosquitoes to assess the effectiveness of insecticides against mosquitoes). www.nature.com/scientificreports/ Larvicidal activity test. Each EO or compound (50 mg) was dissolved in 5 mL of acetone (10,000 μg/mL) to make a stock solution. For each EO or compound solution, up to 6 more concentrations (5,000, 2,500, 1,250, 625, 312.5 and 156.25 μg/mL) were prepared by serial dilution. Likewise, 0.5 g of CNC-stabilized PEs of massoia and nutmeg EOs (100 mg/g) were dissolved in 5 mL of double distilled water (10,000 μg/mL) to make a stock solution. For each CNC-stabilized PEs solution, up to 3 more concentrations (5,000, 2,500 and 1,250 μg/mL) were prepared by serial dilution. Each test solution (1 mL) was suspended in 99 mL of water in 6.5-oz paper cups. 10 early-third-instar larvae of Ae. albopictus were used for each treatment 55 . 1 mL of acetone and CNC-stabilized PE without EOs was used for negative control, and temephos was used as a positive control. Treated and control larvae were kept at the same condition for maintenance. Larval mortality was recorded 48 h post-treatment.  Preparation of sulfated-CNC. Sulfated CNCs were prepared from 10 g of cotton pulp (Whatman, Grade 2, Kent, UK) and 100 mL of 60% (w/w) sulfuric acid solution (Junsei Chemical Co. Ltd., 95% w/w purity, Tokyo, Japan) 19,57 . Finely cut filter paper was mixed with a sulfuric acid solution and stirred at 45 °C for 60 min. The hydrolyzed CNC solution was centrifuged with de-ionized water at 6,000 rpm for 15 min. The well-dispersed CNC solution was dialyzed for 7 days using a cellulose dialysis membrane (MWCO 12-14 kDa, Spectra/Por, Breda, the Netherlands).
Preparation of CNC-stabilized massoia and nutmeg PEs. Massoia and nutmeg EO PEs were mixed with the CNC solution at a 10% ratio and tip-sonicated at a 60% amplitude for 1 min using an ultrasonic processor (VCX 130, Sonics & Materials Inc., Newtown, CT, USA). The CNC contents were fixed at 135, 180, 270, 360 and 450 mg per 1 mL of massoia EO and 45, 90, 135, 180 and 225 mg per 1 mL of nutmeg EO. Each PE solution was stored for 10 days at room temperature to allow for observation of phase separation of the solution, and the height of phase separation was measured using ImageJ software (1.52a, National Institutes of Health, Bethesda, MA, USA). The fraction of each emulsion was calculated by dividing the height of the creaming layer by the height of the total solution. The droplet shape and the size of the PEs were observed by a polarized light microscope (LV100, Nikon, Tokyo, Japan) in dark-field mode. The first droplet size was measured after diluting 20 times, and the second droplet size was measured after diluting the solution diluted 20 times again 2 times (40 times) 58 . Size distribution of the PE droplets was characterized using ImageJ. The CNCs were stained with Calcofluor white dye (Sigma-Aldrich, St. Louis, USA) while massoia and nutmeg EOs were stained with Nile red (sigma-Aldrich, St. Louis, USA) for confocal laser scanning microscopy (SP8 X, Leica, Wetzlar, Germany).

Statistical analysis.
Mortality data were transformed to arcsine square root values for one-way analysis of variance (ANOVA). Treatment means were compared and separated by Scheffe's test. All statistical analysis was carried out in IBM SPSS Statistics 26.0 (2019).

Conclusion
Our results showed that CNC-stabilized PEs of massoia and nutmeg EOs were stable and dispersed easily in water. In addition, larvicidal activities of the CNC-stabilized PEs of massoia and nutmeg EOs were stronger than those of crude EOs. This indicates that a CNC-stabilized PE is a suitable formulation for EOs as a larvicide against mosquitoes in field applications.