Liquid Marbles in Nature: Craft of Aphids for Survival | Langmuir

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Liquid marbles in nature: Craft of aphids for survival Moe Kasahara, Shin-ichi Akimoto, Takahiko Hariyama, Yasuharu Takaku, Shinichi Yusa, Shun Okada, Ken Nakajima, Tomoyasu Hirai, Hiroyuki Mayama, Satoshi Okada, Shigeru Deguchi, Yoshinobu Nakamura, and Syuji Fujii Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b00771 • Publication Date (Web): 16 Apr 2019 Downloaded from http://pubs.acs.org on April 19, 2019

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Liquid marbles in nature: Craft of aphids for survival Moe Kasahara1, Shin-ichi Akimoto2, Takahiko Hariyama3, Yasuharu Takaku3 Shin-ichi Yusa4, Shun Okada1, Ken Nakajima5, Tomoyasu Hirai6,7 Hiroyuki Mayama8, Satoshi Okada9, Shigeru Deguchi9, Yoshinobu Nakamura6,7 and Syuji Fujii*6,7 1

Division of Applied Chemistry, Graduate School of Engineering Osaka Institute of Technology, 5-16-1, Omiya, Asahi-ku, Osaka 535-8585, Japan 2 Department of Ecology and Systematics, Graduate School of Agriculture Hokkaido University, Kita 8, Nishi 5, Kita-ku, Sapporo, Japan 3 Preeminent Medical Photonics Education & Research Center, Institute for NanoSuit Research, Hamamatsu University School of Medicine Higashi-ku, Hamamatsu 431-3192, Japan 4 Department of Applied Chemistry, University of Hyogo 2167 Shosha, Himeji, Hyogo, Japan 5 Department of Chemical Science and Engineering School of Materials and Chemical Technology, Tokyo Institute of Technology 2-12-1, O-okayama, Meguro, Tokyo 152-8552 Japan 6 Department of Applied Chemistry, Faculty of Engineering Osaka Institute of Technology, 5-16-1 Omiya, Asahi-ku Osaka 535-8585, Japan. 7 Nanomaterials Microdevices Research Center Osaka Institute of Technology, 5-16-1 Omiya, Asahi-ku Osaka 535-8585, Japan. 8 Department of Chemistry, Asahikawa Medical University 2-1-1-1 Midorigaoka-Higashi, Asahikawa 078-8510, Japan. 9 R&D Center for Marine Biosciences Japan Agency for Marine-Earth Science and Technology (JAMSTEC) 2-15, Natsushima-cho, Yokosuka, Kanagawa 237-0061, Japan.

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Abstract Some aphids that live in the leaf galls of the host plant are known to fabricate liquid marbles consisting of honeydew and wax particles as an inner liquid and a stabilizer, respectively. In this study, the liquid marbles fabricated by the galling aphids, Eriosoma moriokense, were extensively characterized with respect to size and size distribution, shape, nanomorphology, liquid/solid weight ratio, and chemical compositions. The stereo microscopy studies confirmed that the liquid marbles have a near-spherical morphology and that the number-average diameter was 368±152 m, which is one order of magnitude smaller than the capillary length of the honeydew. The field emission scanning electron microscopy studies indicated that micrometer-sized wax particles with fiber- and dumpling-like shapes coated the honeydew droplets, which rendered the liquid marbles hydrophobic and nonwetting. Furthermore, the highly magnified scanning electron microscopy images confirmed that the wax particles were formed with assemblage of submicrometer-sized daughter fibers. The contact angle measurements indicated that the wax was intrinsically hydrophobic and that the liquid marbles were stabilized by the wax particles in the Cassie-Baxter model. The weight ratio of the honeydew and the wax particles was determined to be 96/4, and the honeydew consisted of 19 wt% nonvolatile components and 81 wt% water. The 1H nuclear magnetic resonance, Fourier transform infrared spectroscopy and mass spectroscopy studies confirmed that the wax mainly consisted of triglycerides and that the honeydew mainly consisted of saccharides (glucose and fructose) and ribitol. The atomic force microscopy studies confirmed that honeydew is sticky in nature.

Key words: liquid marble, aphid, wax, adsorption *Author to whom correspondence should be addressed: [email protected]


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1. Introduction Liquid marbles (LMs) are liquid droplets stabilized by solid particles adsorbed at the gas-liquid interface [1-4]. LMs can be artificially fabricated by coating the liquid droplets with hydrophobic solid particles and can move easily on various solid surfaces because of their nonwetting nature. Studies have been conducted on the effects of solid particulate stabilizers (hydrophilic-hydrophobic balance of surface [5, 6] and shape [7]) and inner liquid droplet phase (polarity) [8] on the structure and stability of LMs. Recently, LMs have gained interest in material science in view of their potential applications in areas including sensors [9-11], microfluidics [12], miniature reactors [13-17], cosmetics [18], pressure-sensitive adhesives [19] and delivery carrier materials [20, 21]. In nature, some gall-forming aphids, small sap-sucking insects, have been known to fabricate honeydew LMs and treat the liquid as nonwetting materials [22-25]. Accumulation of honeydew in the galls brings potentially fatal problems [26]: (a) the aphids drown in the honeydew [27] and (b) pathogens thrive on the carbohydrate-rich honeydew [28]. To avoid these problems, the aphids wrap the honeydew using wax particles, which are secreted from their bodies, to form LMs and remove them out of the galls [29, 30]. Removal of the honeydew from the galls also permits the aphids to utilize its limited space more efficiently and to utilize plant sap more efficaciously [31]. Pike et al. reported that fibrous wax particles coat honeydew droplets to form LMs and studied LM size and the dynamics of LM movement [24]. However, there have been few studies on LMs fabricated in nature from the perspective of interface chemistry, and no investigations have focused on the nanostructure of LMs. In this study, the interface chemistry of LMs fabricated by the eriosomatine aphid, Eriosoma moriokense (E. moriokense), was extensively characterized using stereo and 3 ACS Paragon Plus Environment

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scanning electron microscopy, microbalance, 1H nuclear magnetic resonance, Fourier transform infrared spectroscopy and mass spectroscopy with respect to size and size distribution,

shape,

nanomorphology,

liquid/solid

weight

ratio

and

chemical

compositions. The hydrophilic-hydrophobic balance of the wax was studied by measuring the contact angle of water. The surface tension and tackiness of the honeydew were characterized by a glass capillary method and atomic force microscopy, respectively. The motivation of this study is understanding/learning the structure and interface chemistry of LMs in nature, which could help us guide the design and fabrication of artificial functional LMs based on biomimetics.

2. Experimental 2.1. Materials Unless otherwise stated, all materials were guaranteed reagent grade. n-Hexane (dehydrated) and acid fuchsin were obtained from Kanto Chemical Co., Inc. (Tokyo, Japan) and were used without further purification. Deuterium oxide (D2O, 99.9 atom % D) and benzene-d6 (99.6 atom % D) from Sigma-Aldrich Co. LLC (St. Louis, USA) were also used without further purification. Deionized water (< 0.06 S cm-1) was prepared with a purifier (Advantec®, MFS RFD240NA: GA25A-0715, Toyo Roshi Kaisha, Co., Ltd., Tokyo, Japan) and was used for contact angle measurements. Glass capillaries (FPT-100, Fujiston) with inner and outer diameters of 0.60 mm and 1.0 mm, respectively, were obtained from Fujirika Co., Ltd., Osaka, Japan.

2.2 Aphids


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E. moriokense [32] is a gall-forming eriosomatine aphid that alternates host plants seasonally between the Japanese elm, Ulmus davidiana var. japonica, and herbaceous species such as Sedum spp. The first-instar foundress, after hatching from an overwintered egg, induces a leaf-roll gall on U. davidiana in mid-May. During gall formation, a young leaf is deformed such that one edge of the leaf blade is rolled inwards, and the rolled part inflates slightly with the color turning yellowish [32]. The gall is not completely closed, and predators sometimes invade it through a slit-like opening. The foundress parthenogenetically produces some hundreds of offspring, with some developing into wingless adults and others into winged adults. The second-generation wingless adults also produce offspring, all of which develop into winged adults and migrate to the secondary host plants. The gall generation lasts from mid-May to mid-July, with the maximum number of aphids in a gall being approximately 500. Approximately 30 galls were collected on and near the campus of Hokkaido University (43°04'11"N, 141°20'27"E) for analyses in June and July.

2.3. Characterization of LMs Size analysis of LMs The size of the LMs was determined using stereomicroscopy images of the LMs taken using an SZX12 Olympus camera (Floyd Light, Wraymer Inc., Osaka, Japan). The mean LM diameter was taken as the number-average mean diameter (Dn), which is mathematically expressed as Dn =  DiNi /  Ni, where Di is the diameter of individual LMs and Ni is the number of LMs corresponding to a specific diameter.

Contact angle measurements The static contact angles of water droplets (15 L) at 25 °C were determined 15 min after deposition onto the samples using an SImage02V apparatus (Excimer Inc., Atsugi, Japan) with 5 ACS Paragon Plus Environment

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V01-VI Version 1.004 software. At least three separate droplets were investigated at different locations.

Anus diameter measurements One gall of E. moriokense (gall no. 74) was collected from a U. davidiana tree at the Botanical Garden, Hokkaido University, Sapporo City, and was preserved in a mixture of ethanol and deionized water (80% ethanol) on June 28, 2017. The gall sample was kept in a solution of ethanol and water for 1 week. The samples for measurement were randomly selected from the aphids in the gall, including adults and larvae, and approximately 200 aphids were mounted on glass slides. Permanent slides were prepared following Blackman and Eastop’s method [33], in which the sample aphids were mounted on glass slides using Canada balsam after staining with acid fuchsin. Images of the specimens were captured on a computer using a microscope eyepiece camera (Dino-Eye, AnMo Electronics Corporation, New Taipei City, Taiwan) installed on a microscope (Axiophot, Carl Zeiss, Oberkochen, Germany). The voucher specimens are preserved in Systematic Entomology, Graduate School of Agriculture, Hokkaido University. The diameters of the anal pores were measured based on the images using ImageJ software [34]. An anal pore is oblong and present between the cauda and anal plate [35], and its rim is thickened and conspicuous. For the measurements, 188 specimens were used.

Scanning electron microscopy (SEM) Field emission scanning electron microscopy (FE-SEM) studies were carried out with a JEM-7100F (JEOL. Ltd. Tokyo, Japan) operating at an acceleration voltage of 1.0 kV. The vacuum level of the observation chamber was 10-3 - 10-6 Pa. The signals of secondary electrons were obtained from the lower detector. Other details are as follows: working distance: 8 mm, aperture size: φ 100 µm, scan speed of each beam: 10-15 frames/sec. The FE-SEM studies were 6 ACS Paragon Plus Environment

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performed as previously described [36,37]. Briefly, without any conventional pretreatments, such as chemical fixation, dehydration and ultrathin coating of electrically conducting materials, the specimens were directly introduced into the SEM where a thin membrane, NanoSuit®, formed following irradiation by the electron beam.

Surface tension measurement The LMs freshly harvested from the gall were centrifuged at 2400 g for 1 min at room temperature to separate the honeydew (bottom layer) from the wax particles (top layer) using an MX-107 system (Tomy Seiko Co. Ltd., Tokyo, Japan). A glass capillary was plugged into the honeydew liquid, and the height (h) to which the honeydew rose was measured. The surface tension of the honeydew () was determined from the h value using Jurin’s law (Eq. 1) [38]

𝜸=

𝝆𝒈𝑹𝒄𝒂𝒑𝒉 𝟐

(Eq. 𝟏) 　　　　　　　　　　　　　　　　　　　　　　　

where ρ is the density of the honeydew, 𝒈 is the gravitational acceleration, and 𝑹𝒄𝒂𝒑 is the inner radius of the glass capillary. The accuracy of the measurements was confirmed using pure water (Tables S1 and S2). The density of the honeydew was estimated gravimetrically with an SE2 Ultra microbalance (Sartorius AG, Göttingen, Germany) using a glass capillary with a known inner diameter with and without the honeydew inside.

Fourier transform infrared (FT-IR) spectroscopy The chemical compositions of the wax and honeydew samples dispersed on BF2 substrate were studied using FT-IR (FTIR-6600, JASCO Corp., Tokyo, Japan) spectroscopy. The measurements were performed at 128 scans per spectrum with 4 cm-1 resolution. 7 ACS Paragon Plus Environment

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Direct analysis in a real-time mass spectrometer (DART-MS) The DART-MS studies were carried out at an independent laboratory (Shimadzu Techno-Research, Inc., Japan). A DART-MS system consisting of a DART ion source (IonSense, Saugus, MA, USA) and a single quadrupole liquid chromatograph-mass spectrometer (LCMS-2020, Shimadzu Scientific Instruments, Kyoto, Japan) was used. The operating conditions of the DART ion source were as follows: positive ion mode; helium flow: 5.0 L min−1; heater temperature: 350 °C; block heater temperature: 400 °C; desolvation line temperature: 250 °C; and desolvation line voltage: 0 V. The conditions of the MS were as follows: monitored mass range: m/z 100–1000 and acquisition time: 0.2 s/scan.

1H

nuclear magnetic resonance (NMR) measurements

The diffusion coefficient (𝐷) was calculated from the best fit to the two-dimensional diffusion-ordered NMR spectroscopy (2D DOSY) data. The 2D DOSY data were obtained on a Bruker DRX-500 NMR using the pulse program stebpgp1s (Bruker TopSpin 1.3, MA, USA). The hydrodynamic radius (𝑅ℎ) was estimated from DOSY and is given by the Stokes–Einstein equation.

𝑹𝒉 =

𝒌𝑩𝑻 𝟔𝝅𝜼𝑫

(Eq. 𝟐) 　　　　　　　　　　　　　　　　　　　　　　　　　　　　

where kB is the Boltzmann constant, T is the absolute temperature, and η is the solvent viscosity.

Atomic force microscopy (AFM) 8 ACS Paragon Plus Environment

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The honeydew (approximately 65 mg) was dried on a Ni-coated metal substrate (diameter, 20 mm; K-Y10200167, Hitachi High-Tech Science Corp., Tokyo, Japan) in air for 3 days. Force curves were measured using an AFM (AFM5100N, Hitachi High-Tech Science Corp., Tokyo, Japan) in contact mode using a cantilever made from Si3N4 (SN-AF01-S-NT, spring constant: 0.11 N/m, the nominal value of curvature radius at tip: 15 nm, the back is coated with Au, Hitachi High-Tech Science Corp., Tokyo, Japan). The relationship between the stress applied to the probe tip and the deformation of the specimen surface was characterized [39]. Because the stress applied to the probe tip is on the order of nN, the nanoscale stress-deformation curve of the honeydew could be obtained. The Youngʹs modulus and the adhesive energy were calculated from the force curves using the Johnson-Kendall-Roberts (JKR) two-point method [40], which has been applied to viscoelastic materials (pressure-sensitive adhesive tapes) [41]. The measurements were conducted at 22.0 °C and 65%RH.


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3. Results and discussion LMs in galls E. moriokense induces a gall by rolling up an elm leaf including the midrib, forming a ball-like or pouch-like structure (Figure 1a): the upper side of the leaf, which faces sunlight, forms outside of the gall. The typical gall size was approximately 1 cm and 3 cm in the lengthwise and crosswise directions, respectively (Figure 1a inset). Many kinds of aphids secrete wax, which may help them to obtain water-proofing abilities, to avoid honeydew contamination, to hide or protect from parasites, predators, dehydration, frost and/or entomopathogens and to have an antimolestation function [42]. The trick of coating honeydew with wax particles is widespread in aphids and has been known for over 100 years [43]. Active removal of the honeydew in the form of LMs is known for some species of aphids [29,44-46]: Pemphigidae and Hormaphididae push the LMs out of the galls with their heads. Interestingly, in the case of E. moriokense, the LMs selectively came out of the gall by knocking, and the aphids did not tend to come out (Supporting Information Movie 1), which means that the

Figure 1. (a) Digital photograph of a gall fabricated by Eriosoma moriokense on Japanese elm, Ulmus davidiana var. japonica. The inset is a magnified image of the gall. (b) Stereo microscopy image of the inside of the gall. The inset shows an aphid carrying waxes on its body. 10 ACS Paragon Plus Environment

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Figure 2. (a) Stereo microscopy image of liquid marbles fabricated by Eriosoma moriokense. The liquid marbles were placed on a glass substrate. (b) Size distribution of the liquid marbles. The number of liquid marbles obtained in the same gall is shown in the same color. vibrations generated by wind, rain and/or animals can lead to the removal of the honeydew LMs from the gall. Another noteworthy observation is that the water droplets, which were partially coated with wax particles, came out of the gall, and no/few aphids came out if the pure water droplets were added from an opening of the gall (Supporting Information Movie 2). This result indicates that there is no damage to the aphids even if the rain droplets enter the gall through the opening. When the gall was opened using tweezers, the existence of aphids and LMs was 11 ACS Paragon Plus Environment

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confirmed by stereomicroscopy (Figure 1b and Supporting Information Movie 3). Generally, two generations of aphids (2nd and 3rd generations) were observed in the gall, reaching up to 500 individuals in total. Their dorsum was densely covered with a white-colored wax, which attached to the caudal part of the body (near their anus) (Figure 1b inset), and the wax detached from the body covered the inner surface of the gall. Some psyllids possess wax gland pores on their circumanal ring to efficiently cover their honeydew with wax and minimize their contamination with the honeydew [47]. Of note, some aphids were carrying honeydew droplets covered with wax particles, which attached to their anus. The LMs were stable enough against disruption for the aphids to walk on them. The wax particle coating on the honeydew droplets offers an air-wax gap between the LMs and can also avoid the coalescence of the droplets. The formation mechanism of the LM is under investigation, but the wax attached near the anus is expected to be simultaneously adsorbed onto the honeydew droplet when secreted. Another possibility is that after the wax is detached from the aphid body, it remains on the inner gall surface and is adsorbed to the honeydew droplet surface. Interestingly, the LMs and the aphids on the inner wall of the gall were not washed away, even if pure water droplets were placed on them (Supporting Information Movie 4). If the water droplets contacted the LMs and the aphids, the excess wax particles attached on the LMs and the wax particles on the dorsum of the aphids were transferred onto the water droplet surfaces and worked as sacrificial protecting material for the aphids and the LMs. The LMs remained intact even after transfer from the gall onto solid substrates, such as glass or plastic (see Figure 2a), as well as after transfer onto a planar air-water surface. The LMs remained stable on the planar air-water interface, which was prepared in a petri dish, for approximately 20 s. This stability indicates that the liquid phase inside the LMs was shielded by the vapor gap formed by the wax particles between the inner honeydew and the planar water 12 ACS Paragon Plus Environment

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surface formed in the petri dish. The average number of LMs per gall was determined to be 165±165 (number of measured galls, 14), and the maximum and minimum numbers were 678 and 7, respectively (Figure S1). The shape of the LMs was nearly spherical, and their sizes were polydisperse. The number-average diameter of the LMs (Dn) was measured to be 368±152 m (number of measured LMs, 2315) (Figure 2b), which is on the same order of magnitude as those measured for the LMs fabricated by other aphids (200 m for Pemphigus spyrothecae [24] and 400 m for C. clematis [48]); the maximum and minimum diameters were 1089 m and 94 m, respectively. There was no apparent relationship between the number of LMs per gall and their size (Figure S1). The application of stress on the LM between glass substrates led

Figure 3. (a) Optical microscopy images of the anus of an Eriosoma moriokense. (b) Diameter distribution of the anuses. 13 ACS Paragon Plus Environment

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to disruption and leakage of the inner honeydew, which indicates that the wax particle coated the honeydew droplet (Figure S2).

Physical chemistry of the LMs The surface tension of the honeydew () was determined by the glass capillary method to be 65.0±4.9 mN/m, which was slightly lower than that of pure water (71.6±1.2 mN/m). This result indicates that some surface active compounds are included in the honeydew. (Note that there is a possibility that wax components with lower surface tension than water were adsorbed to the air-honeydew interface.) The density of the honeydew () was measured to be 1079±6 kg/m3. With these  and  values, the capillary length (𝜿 ―𝟏) was calculated to be 2.48 mm for the honeydew using Eq. 3.

𝜿 ―𝟏 =

𝜸 (Eq. 3) 　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　 𝝆𝒈

The number-average radii of the LMs were one order of magnitude smaller than the capillary length, which indicates that gravity is dominated by capillaries, and the LM shape was nearly spherical, consistent with the stereo microscopy observations. The number-average diameter of the aphid anus (𝐷𝑎) was measured to be 56±13 m (Figure 3) (number of measured anuses, 188), which agrees relatively well with the results obtained from the SEM studies (Figure S3). The calculated honeydew drop diameter (𝐷ℎ) was estimated to be 1.27 mm using Eq. 4 by balancing between the gravitational and capillary forces at the aphid anus.

𝑫 𝒉 = 𝟐𝟑

𝟑𝜸𝑫𝒂 𝟒𝝆𝒈

(𝐄𝐪. 𝟒) 　　　　　　　　　　　　　　　　　　　　　　　　　 14 ACS Paragon Plus Environment

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The estimated drop diameter was two orders of magnitude larger than the measured diameter. The honeydew droplet expulsion is actively controlled by the opening and closing of the anus, and the drop size could be smaller than 1.27 mm. There is another possible reason for the smaller LM diameter: the coverage of the honeydew droplets with the wax particles and the shear stress applied to the droplet attached to the anus during the aphid’s movement in the gall. Note that honeydew production rates are 40-150 g per aphid per hour [49], which should be slow enough for the wax particles to coat the honeydew droplet. The wide distribution of the LM diameter should arise from several causes, which include the distribution of aphid anus diameters, the coalescence of incompletely wax-coated honeydew droplets and the evaporation of water in honeydew [23,31].

Structure and chemistry of the LMs The stereo micrograph of the LMs confirmed that the LMs were coated with wax particles with fibrous and atypical dumpling-shaped morphologies, and the inner liquid phase could be observed via an opening among the wax particles (Figure 2a). There are two types of wax producing-structures on the aphid body, namely, wax pores and small wax pits, which result in the production of wax particles with various morphologies for the wooly oak aphid Stegophylla brevirostris Quednau [50]. The layer consisted of fibrous wax particles and air formed on the LM surface was thick in comparison with that formed by the spherical particles, and the honeydew was protected by the thick layer from leakage and wetting to the substrate that contacted the LMs. The fibrous particles could also offer higher stability against physical stress to the LMs. Recently, the effects of particulate LM stabilizer shape on the dynamic behavior of LMs were investigated, and the results clarified that LMs coated with rod-shaped particles 15 ACS Paragon Plus Environment

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showed a higher stiffness than that of LMs stabilized with spherical particles. The rod-shaped particles form a three-dimensional network structure, and therefore the LMs stabilized with the particles show a high resistance to deformation [7]. The fibrous wax particles on the LM surfaces could be transferred to the glass substrate after rolling on it (Figure S4), and the number-average length was measured to be 71±71 m (number of measured waxes, 263): the maximum and minimum lengths were 518 m and 5 m, respectively. (Note that the original fibrous wax length should be longer because the wax fiber could be cut during rolling on the glass substrate.) The FE-SEM studies using the NanoSuit® method revealed the morphology of the wax particles in detail (Figure 4). The NanoSuit® method was developed for in situ observation of living things using SEM; hence, imaging in a hydrous/wet state closely approximating the natural condition is possible [36,37].


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Figure 4. (a) FE-SEM images of the LM fabricated by Eriosoma moriokense and (b-d) waxes adsorbed to the liquid marble surface. Figs. (c) and (d) are magnified FE-SEM images of the fiber-shaped and dumpling-shaped waxes, respectively. Fibrous and dumpling-shaped wax particles covered the LM surfaces, and the fibers


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Figure 5. FE-SEM images of liquid marbles after application of shear stress: Liquid marbles (a, b) after and (c, d) before removal of water by evaporation. Figs. (b) and (d) are magnified images of Figs (a) and (c), respectively. protruded out of the LM surfaces (Figure 4a). Interestingly, regular helix-like undulation was observed in some fibrous wax particles (Figure 4b). The number-average diameter of the fibrous wax particles was 2.7±1.3 m (number of measured fibrous wax particles, 250), and the wax particles consisted of a bundle of daughter wax fibers with a diameter of 204±44 nm (number of measured daughter wax fibers, 20) (Figure 4c and S5). On the other hand, the diameter of the dumpling-shaped wax particles was 5.7±1.6 m (number of measured dumpling-shaped wax particles, 206), and the wax particles consisted of a network of daughter wax fibers with a diameter of 129±41 nm (number of measured daughter wax fibers, 20) (Figure 4d and S5). The reason and mechanism for the generation of these two kinds of wax morphologies is under investigation, but the differences in aphid generation and the secretion part of the aphid body (secretion hole size and shape) possibly play some roles [50]. 18 ACS Paragon Plus Environment

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Figure 6. (a) FT-IR and (b) 1H NMR spectra obtained for waxes and honeydew. When the LMs were left on the glass substrate at room temperature, the LM surface buckled due to the evaporation of the inner water, which is a volatile component of the honeydew (Figure S6). The wax particles are irreversibly adsorbed at the honeydew droplet surface with high adsorption energy, and the surface area of the LM remains almost constant. Thus, the LM adjusts its surface-to-volume ratio by buckling [51]. The LM is covered by wax particles, and the area of the bare air–honeydew interface, where water can evaporate, is smaller than that of the bare spherical honeydew droplet. Therefore, the evaporation speed of the inner water could be expected to be low in comparison with that of the pure honeydew droplets without wax coating at the initial stage [52]. The amount of water loaded in the LMs was gravimetrically determined to be 78 wt% after drying under silica gels. Cross-sectional FE-SEM observation of the dried LM after breaking using a tweezer indicated that the wax particles exist only on the LM surface and do not intrude into the honeydew phase (Figure 5a, b and S7). The amounts of loaded wax particles and the nonvolatile water-soluble component of the honeydew in the LM were gravimetrically determined to be 4 wt% and 18 wt%, respectively, by measuring the weights of dried LMs before and after extraction of the wax particles using n-hexane. 19 ACS Paragon Plus Environment

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The chemical compositions of the wax particles and nonvolatile water-soluble component in the honeydew were characterized by FT-IR, 1H NMR and DART-MS after purification by manual removal of any contaminating aphids and their cast-off skins. The FT-IR spectrum of the wax particles indicated that long wax esters should be the major components (Figure 6a) [50]. In the spectrum of the wax particles, the characteristic peaks at 2916 and 2848 cm-1 could correspond to the CH2 antisymmetric and symmetric stretching vibrations, respectively. The positions of these peaks are believed to be strongly related to the degree of conformational order. When the methylene chain formed a trans-zigzag conformation, peaks were observed near 2920 and 2850 cm-1. In contrast, the methylene peaks in the gauche conformation could be observed near 2926 and 2856 cm-1 [53]. One might conclude that the alkyl chain in the wax particles forms a trans-zigzag conformation, leading to a highly ordered structure. Moreover, the peaks corresponding to the C=O stretching mode could be observed at 1730 and 1701 cm-1, implying that the wax particles contain ester groups and/or carboxyl groups. The broad peak centered at approximately 3340 cm-1 could be assigned to the O-H vibration, which should indicate the presence of fatty acids [50]. The FT-IR spectrum of the nonvolatile water-soluble component in the honeydew shows a peak at 2930 cm-1 (Figure 6a). In general, in the IR spectra of sugars, namely, fructose, sucrose and glucose, a peak at 2930-2950 cm-1 is also observed and is assigned to methylene antisymmetric vibration [54]. Additionally, the broad absorption centered at approximately 3400 cm-1 should be assigned to the OH groups in the sugars. The 1H NMR studies on the wax particles confirmed many peaks between 0.7 ppm and 2.0 ppm, which indicates that the wax particles are a mixture of aliphatic compounds and mono-substituted aliphatic compounds (Figure 6b). Additionally, small peaks at approximately 3-4 ppm were observed, suggesting that there may be ether compounds. The 2D DOSY data also confirmed that the wax particles consisted of several large compounds with low diffusion 20 ACS Paragon Plus Environment

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constants in benzene-d6 compared to the water molecules. In the 1H NMR spectrum of the nonvolatile compounds in honeydew, many peaks were observed between 3 ppm and 4 ppm and 5.2 ppm, which should indicate the mixture of sugar compounds: the observed peaks correspond well with those measured for fructose, sucrose and glucose [55-57]. Fructose, sucrose and glucose have been reported to be contained in the honeydew of aphids [58]. The DOSY spectrum also indicates that the honeydew consisted of a mixture of various compounds with low diffusion constants and thus high molecular weights compared with the water molecule. A chemical with hydrodynamic radii (Rh) of up to 2.4 nm was measured in the wax (Rh value of water: ~0.15 nm). In the positive DART-MS spectrum of the wax particles, peaks at m/z 723, 807, 611 and 667 were detected, which can be attributed to the molecular ions of triglycerides, namely, trimyristin, tripalmitin, 2-hexano-1,3-dimyristin and 2-hexano-1,3-dipalmitin, respectively. In addition to these molecular ions, fragments of triglycerides were observed at m/z 388 and 411, assigned to 2-hexano-1,3-dimyristin and 2-hexano-1,3-dipalmitin, respectively, and at m/z 551, assigned to 2-hexano-1,3-dipalmitin [59]. In the negative DART-MS spectrum, a peak at m/z 255 was detected, which can be assigned to palmitic acid. Of note, m/z 667, 681, 695, 709, 723, 737, 751, 765 and 779 with a difference of multiples of m/z 14 were detected, which indicates that hydrocarbon units with different numbers of CH2 exist in the wax. In this work, only solid wax components were studied, although volatile substances, which can work as an alarm odor, have been reported to be contained [60]. In the positive spectrum of the honeydew, a strong peak at m/z 180 was detected, which should be assigned to monosaccharides (glucose and fructose) [49,61]. A peak at m/z 152 and other peaks at m/z > 400 (e.g., 401, 462, 686, 698 and 758) could be attributable to ribitol and oligosaccharides. Due to these sugars, the honeydew tastes sweet to us. 21 ACS Paragon Plus Environment

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The wettability of the wax was studied by measuring the apparent contact angles of water droplets on the wax (Figure S8). The wax particles entirely coated the inner wall of the gall, and the contact angles measured on it should be the values for the rough surface made from the wax particles. The contact angle on the dried wax particles was measured to be 130±14°, which is in relatively good accordance with the values of approximately 160° and 150° measured for the wax particles excreted by two other aphids, Pemphigus spyrothecae [24] and C. clematis [48], respectively; this contact angle indicates the hydrophobic character of the inner wall of the gall. The surface roughness formed by the wax particles allows air to be trapped under the water droplet, and the air-wax dual surface is responsible for the high contact angle. The water contact angle on the wax particles after exposure to water vapor and that followed by drying under silica gels were 129±16° and 123±14°, respectively. These results indicated that water was not condensed on the wax particle surface from the vapor phase and that the Cassie-Baxter wetting mode was attained on the intrinsically hydrophobic wax surface. The water contact angle on the smooth wax surface was determined to be 97±3°, which is close to that on a hydrophobic candle wax surface (110°) [24]. These results are different from those obtained for the lotus leaf surface [62]: the contact angle decreased below 90° and varied roughly between 40° and 60° from approximately 160° after exposure to water vapor, which indicates a change in the wetting mode from the meta-stable Cassie-Baxter state to the Wenzel state due to water condensation on the wax surface with intrinsically hydrophilic nature. The contact angle on the air-solid composite rough surface (𝜃𝑅) can be expressed using Eq. 5:

cos 𝜃𝑅 = 𝑓(1 + cos 𝜃𝑒𝑞) ―1

(Eq. 5)

where f is the fraction of the planar area of water in contact with wax particles and 𝜃𝑒𝑞 is the 22 ACS Paragon Plus Environment

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equilibrium contact angle of water on the smooth wax surface. When f approaches zero and the substrate is hydrophobic (𝜃𝑒𝑞 > 90°), the contact angle increases toward 180°. Using the 𝜃𝑅 and 𝜃𝑒𝑞 values of 130° and 97°, the f value could be estimated to be 0.41.

Nature of honeydew in the LMs


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Figure 7. Force-deformation curve obtained for honeydew fabricated by Eriosoma moriokense. The honeydew is a sticky liquid and has thread-forming properties. The FE-SEM studies indicated that the threads were formed after stretching the LM before water evaporation (Figures 5c, d). The force curve measured using the AFM for the honeydew is shown in Figure 7. The original force curve shows the relationship between the force detected by the cantilever and the piezo scanner displacement during the approaching process of the cantilever to the sample and the withdrawing process. The force curve was converted to a force (F)-deformation (𝛿) curve in accordance with the procedure performed by Nakajima et al. [39]. In the approaching process, a sample deformation of 320 nm was observed at a force of 5 nN. During the withdrawing process, the large sample deformation over -1450 nm with a force of -21 nN was measured. This force curve is similar to those measured for pressure-sensitive adhesives [63], which should indicate that honeydew is a sticky material. The force curve was analyzed by the JKR two-point method according to the method proposed by Sun et al. [40] To calculate the Youngʹs modulus (E) and the adhesive energy (W), the balance point (A) and the maximum adhesion point (B) were set as (δ0, 0) and (δ1, F1), respectively, as shown in Figure 7. The E and 24 ACS Paragon Plus Environment

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W were calculated using Eqs. 6 and 7:

3(1 ― 𝜈2) 1 + 3 16 E= 4 3

(

2𝐹1

W= ― 3𝜋𝑅

)

3 2

― 𝐹1 𝑅(𝛿0 ― 𝛿1)3

(F 1 < 0)

(F 1 < 0)

　　　　

(Eq. 6)

(Eq. 7)

where ν is the Poisson ratio and R is the radius of curvature at the tip of the cantilever. In this study, ν is assumed to be 0.5, which is a typical value for rubber, and R is 15 nm, which is the nominal value of the cantilever. The Youngʹs modulus was calculated to be 0.1 MPa, and the adhesive energy was calculated to be 0.30 J/m2.

4. Conclusions In summary, the LMs fabricated by the aphids were successfully characterized based on interface chemistry. The stereo microscopy studies confirmed that the LMs have a near-spherical morphology, and their diameters are several hundreds of micrometers, which are one order of magnitude smaller than the capillary length of the honeydew. The FE-SEM studies indicated that the honeydew droplets were coated with fiber- and dumpling-shaped micrometer-sized hydrophobic wax particles, which render the LMs hydrophobic and nonwetting. Furthermore, the FE-SEM images confirmed that the wax particles were formed with the assemblage of submicrometer-sized daughter fibers. The contact angle measurements indicated that the wax is intrinsically hydrophobic and repels water droplets even after exposure to a high-humidity atmosphere. The weight ratio of the honeydew and the wax particles was determined to be 96/4, and the honeydew consisted of 19 wt% nonvolatile components and 81 wt% water. The wax mainly consisted of triglycerides, and the honeydew mainly consisted of saccharides 25 ACS Paragon Plus Environment

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(glucose and fructose) and ribitol, as confirmed by the 1H NMR, FT-IR, and DART-MS spectroscopy studies. The AFM studies indicated the sticky nature of the honeydew. The structure and interface chemistry of the LMs in nature, which have been clarified in this study, should contribute to the construction of design guidelines for artificial functional LMs based on biomimetics [19].

Supporting Information. Experimental details on cryo-SEM. Characteristics of the LMs, wax and gall/leaf. Movies capturing the inside of the gall and illustrating the water-repellent behavior of the gall.

Acknowledgments The authors thank Dr. Izumi Yano (Hokkaido Univ.) for assistance with the electric balance. SF and SA thank Prof. Masatsugu Shimomura (Chitose Institute of Science and Technology) for providing the opportunity to work together. The authors also thank Keyence Co. for contributing to the stereo microscopy studies. This work was supported by a Grant-in-Aid for Scientific Research (B) (JSPS KAKENHI Grant Number JP16H04207)

and

Scientific

Research

on

Innovative

Areas

“Engineering

Neo-Biomimetics (JSPS KAKENHI Grant Number JP15H01602)”, “New Polymeric Materials Based on Element-Blocks (JSPS KAKENHI Grant Number JP15H00767)”, “Molecular Robotics (JSPS KAKENHI Grant Number JP15H00791)” and “Molecular Soft Interface Science (JSPS KAKENHI Grant Number JP23106720)”.

Supplementary Data Available Supplementary data are available free of charge via the Internet.


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(33)Blackman, R. L.; Eastop, V. F. Aphids on the World's Crops. An Identification and Information Guide, 2nd ed., John Wiley & Sons, Chichester, 2000. (34) Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2012. (35)Miyazaki, M. Morphology of aphids. Minks, A. K.; Harrewijn, P. (eds): Aphids: Their Biology, Natural Enemies and Control, 1-25, Elsevier, Amsterdam, 1987. (36)Takaku, Y.; Suzuki, H.; Ohta, I.; Ishii, D.; Muranaka, Y.; Shimomura, M.; Hariyama, T. A thin polymer membrane, nano-suit, enhancing survival across the continuum between air and high vacuum. PNAS 2013, 110, 7631-7635. (37)Takehara, S.; Takaku, Y.; Suzuki, H.; Ohta, I.; Shimomura, M.; Hariyama, T. A ‘NanoSuit’ successfully protects petals of cherry blossoms in high vacuum: Examination of living plants in an FE-SEM. Sci. Reports 2018, 8, 1685. (38)Quéré, D.; Brochard-Wyart, F.; De Gennes, P.-G. Capillarity and Wetting Phenomena - Drops, Bubbles, Pearls, Waves, Springer, New York, NY, 2004. (39)Nakajima, K.; Ito, M.; Wang, D.; Liu, H.; Nguyen, H. K.; Liang, X.; Kumagai, A.; Fujinami, S. Nano-palpation AFM and its quantitative mechanical property mapping. Microscopy 2014, 63, 193-207. (40)Sun, Y.; Akhremitchev, B.; Walker, G. C. Using the adhesive interaction between atomic force microscopy tips and polymer surfaces to measure the elastic modulus of compliant samples. Langmuir 2004, 20, 5837-5845. (41)Canetta, E.; Adya, A. K. Atomic force microscopic investigation of commercial pressure sensitive adhesives for forensic analysis. Forensic Sci. Int. 2011, 210, 16-25. (42)Pope, R. D. Some aphid waxes, their form and function (Homoptera: Aphididae). J. Nat. Hist. 1983, 17, 489-506. (43)Buckton, G. B. Monograph of the British aphides. London: Ray Society, 1876.


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(53)Sato, T.; Ozaki, Y.; Iriyama, K. Molecular aggregation and photoisomerization of langmuir-blodgett films of azobenzene-containing long-chain fatty acids and their salts studied by ultraviolet-visible and infrared spectroscopies. Langmuir 1994, 10, 2363-2369. (54)Max, J.; Chapados, C.; Glucose and fructose hydrates in aqueous solution by IR spectroscopy. J. Phys. Chem. A 2007, 111, 2679-2689. (55)Cazor, A.; Deborde, C.; Moing, A.; Rolin, D.; This, H. Sucrose, glucose, and fructose extraction in aqueous carrot root extracts prepared at different temperatures by means of direct NMR measurements. J. Agr. Food Chem. 2006, 54, 4681-4686. (56)Hohmann, M.; Koospal, V.; Bauer-Christoph, C.; Christoph, N.; Wachter, H.; Diehl, B.; Holzgrabe, U. Quantitative 1H NMR analysis of egg yolk, alcohol, and total sugar content in egg liqueurs. J. Agr. Food Chem. 2015, 63, 4112-4119. (57)Weberskirch, L.; Luna, A.; Skoglund, S.; This, H. Comparison of two liquid-state NMR methods for the determination of saccharides in carrot (Daucus carota L.) roots. Anal. Bioanal. Chem. 2011, 399, 483-487. (58)Klingauf, F. A. Feeding, adaptation and excretion. Minks, A. K.; Harrewijn, P. (eds): Aphids; Their Biology, Natural Enemies and Control, 225-253, Elsevier, Amsterdam, 1987. (59)Callow, R. K.; Greenway, A. R.; Griffiths, D. C. Chemistry of the secretion from the cornicles of various species of aphids. J. Insect Physiol. 1973, 19, 737-748. (60)Kislow, C. J.; Edwards, L. J. Repellent odour in aphids. Nature 1972, 235, 108-109. (61)Wool, D.; Hendrix, D. L.; Shukry, O. Seasonal variation in honeydew sugar content of galling aphids (Aphidoidea: Pemphigidae: Fordinae) feeding on Pistacia: Host ecology and aphid physiology. Basic. Appl. Ecol. 2006, 7, 141-151. (62)Cheng, Y.-T.; Rodak, D. E. Is the lotus leaf superhydrophobic? Appl. Phys. Lett. 2005, 86, 144101.


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(63)Paiva, A.; Sheller, N.; Foster, M. D.; Crosby, A. J.; Shull, K. R. Study of the surface adhesion of pressure-sensitive adhesives by atomic force microscopy and spherical indenter tests. Macromolecules 2000, 33, 1878–1881.


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For use in the Table of Contents only

“Liquid marbles in nature: Craft of aphids for survival”

Moe Kasahara, Shin-ichi Akimoto, Takahiko Hariyama, Yasuharu Takaku, Shin-ichi Yusa Shun Okada, Ken Nakajima, Tomoyasu Hirai, Hiroyuki Mayama, Satoshi Okada, Shigeru Deguchi Yoshinobu Nakamura and Syuji Fujii*


Liquid Marbles in Nature: Craft of Aphids for Survival | Langmuir

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