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Wing wettability of Odonata species as a function of quantity of epicuticular waxes
Vibrational Spectroscopy 75 (2014) 173–177
Contents lists available at ScienceDirect
Vibrational Spectroscopy journal homepage: www.elsevier.com/locate/vibspec
Wing wettability of Odonata species as a function of quantity of epicuticular waxes夽 Song Ha Nguyen a , Hayden K. Webb a , Jafar Hasan a , Mark J. Tobin b , David E. Mainwaring a , Peter J. Mahon a , Richard Marchant c , Russell J. Crawford a , Elena P. Ivanova a,∗ a
Faculty of Science, Engineering, and Technology, Swinburne University of Technology, PO Box 218, Hawthorn, VIC 3122, Australia Australian Synchrotron, 800 Blackburn Rd., Clayton, VIC 3168, Australia c Melbourne Museum, 11 Nicholson St., Carlton, VIC 3053, Australia b
a r t i c l e
i n f o
Article history: Available online 1 August 2014 Keywords: Insect wings Long-chain aliphatic hydrocarbons Surface topography Wettability
a b s t r a c t Dragonflies have gained much attention due to their sophisticated wing surface structure, and their associated superhydrophobic, self-cleaning and bactericidal properties. In this work, we compared and contrasted the chemical composition and surface morphology of the wing membranes of four species of dragonfly and damselfly from the Odonata family collected in 1970s (Diplacodes melanopsis and Xanthagrion erythroneurum) and 2011 (Diplacodes bipunctata, and Ischnura heterosticta). Diplacodes species are dragonflies, whilst Xanthagrion and Ischnura are damselflies. Fourier-transform infrared spectroscopy data obtained from the Australian Synchrotron were used to classify the fundamental components of all four of the insect species’ wings. The spectra of all species were dominated by C H stretching, amide I and amide II and O H stretch absorbance indicating the presence of a similar membrane composition of chitin, protein and wax in all four species. Although the samples were collected 40 years apart, there was no evidence of degradation having taken place during this time. Despite the overall similarities in spectral profile, species-specific differences were observed, most notably in the intensity of the CH2 peaks, which in part reflected the amount of waxes present on the wings, which appeared to be different between individual species. The surface topography also contained minor differences in the diameter and the spacial distribution of its nanopillars. It is postulated that the differences in surface wettability of the wings could be attributed to these minor differences in surface chemistry and surface topography. For example, X. erythroneurum presented the highest water contact angle (WCA) of 160◦ whilst the D. melanopsis wings exhibited the lowest WCA (138◦ ), and the wettability of their wings was found to directly correlate with the intensity of hydrocarbon peaks found in their respective IR specta. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Insects first evolved the ability to fly at least 400 million years ago and are one of the largest classes, representing half of all living organisms on Earth [1]. In order to adapt to ever-changing environments, they have developed strategies to cope with different stresses [2]. Investigation into the mechanisms that they have adopted to enable them to cope with these environmental stresses has provided the scientific community with great insights for many
夽 Paper presented at the 7th International Workshop on Infrared Microscopy and Spectroscopy with Accelerator-Based Sources (WIRMS), Melbourne, Australia, 10–13th November 2014. ∗ Corresponding author. Tel.: +61 3 9214 5137. E-mail address: [email protected] (E.P. Ivanova). http://dx.doi.org/10.1016/j.vibspec.2014.07.006 0924-2031/© 2014 Elsevier B.V. All rights reserved.
useful applications [3–8]. For example, the mechanism by which some beetles collect water from fog-laden wind on their back can be applied in water-trapping systems, such as water condensers and engines [9]. The anti-reflective properties of the eyes of some moth species have been used as an inspiration for various optical applications [10]. Colourful butterfly wings have been studied extensively as a template for the fabrication of smart materials [11]. Superhydrophobicity and self-cleaning properties are also two important characteristics possessed by many insects. In order to maintain their high levels of functionality, their surfaces have evolved to possess highly specific structures and surface chemistries [12–19]. Dragonfly and damselfly species belong to the order Odonata, and their wing surfaces are known for their superhydrophobicity and self-cleaning properties. A surface that exhibits a WCA greater than 150◦ is considered to be superhydrophobic. When water droplets remain in a spherical shape and readily roll off a
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S.H. Nguyen et al. / Vibrational Spectroscopy 75 (2014) 173–177
surface with a tilted angle of less than 10◦ , this surface is considered to be both superhydrophobic and self-cleaning [20–22]. It is well-documented that superhydrophobic surfaces, including insect wings, commonly adhere to the Cassie–Baxter regime [21,23–25], which describes the effect of a composite interface on its wettability. Previously published work has revealed that the fundamental components of the cuticular layer of dragonfly wings consists of peptide linked structural protein, chitin components that comprise the bulk of the wing membrane [26], and waxy components including long chain aliphatic hydrocarbons (C14 –C30 ) together with some carboxylic acids (palmitic acid and stearic acid). These components make up the outermost layer of the insect wing epicuticle [27]. These components contribute to not only the superhydrophobicity of the wing, but also to the hierarchical structure of the wing, which has been found to impart bactericidal properties to some wing surfaces [28]. In light of the discovery of these anti-bacterial properties, this paper explores the diversities of natural nanostructures and their corresponding surface chemistry. This has been achieved by comparing four different wings, including aged and fresh samples, belonging to Diplacodes melanopsis, Xanthagrion erythroneurum, Diplacodes bipunctata, and Ischnura heterosticta. Recent work has shown heterogeneity in the wax distribution on the surface of the wing membrane of the cicada Psaltoda claripennis, which may relate to the superhydrophobic and self cleaning qualities of these membranes [29]. For this reason high spatial resolution maps were acquired from each sample using the synchrotron beamline, to reveal any chemical heterogeneity in the species studied here. 2. Experimental 2.1. Materials Fresh dragonfly D. bipunctata and damselfly I. heterosticta wing samples were obtained from the suburban regions of South Australia in early to mid-2011 and preserved in sterile Petri dishes at room temperature (ca. 22 ◦ C). Aged dragonfly D. melanopsis and damselfly X. erythroneurum wing samples that had been preserved at the Melbourne Museum in the 1970s were also used in this study. When required, the wings were aseptically removed from the insect body and stored at room temperature in sterile plastic containers. 2.2. Synchrotron radiation Fourier-transform infrared spectrometry (SR-FTIR) The distribution of the organic functional groups present across the wing membrane of the dragonfly species was obtained using Synchrotron Radiation-Fourier Transform Infrared (SR-FTIR) microspectroscopy at the Australian Synchrotron. The samples were scanned in transmission mode over several areas typically of 100 m × 100 m using a Bruker Hyperion 2000 FTIR microscope (Bruker Optic GmbH, Ettlingen, Germany) using a 36× objective and condenser (numerical aperture 0.5), an aperture of 5 m × 5 m and step size of 3 m in x and y directions. 8 spectra were co-added at a resolution of 8 cm−1 per measurement position. OPUS software version 6.5 was used to operate the microscope and spectrometer and OPUS versions 6.5 and 7.2 were used for subsequent analysis of the data. 2.3. Scanning electron microscopy (SEM) The surface morphology of each wing surface was obtained using a field emission scanning electron microscope (FeSEM – SUPRA 40VP, Carl Zeiss GmbH, Jena, Germany) at a fixed voltage of 3 kV. The samples were attached to metallic substrata using conductive double-sided adhesive tape. Samples were sputter coated
Fig. 1. IR absorbance spectra of four insect wings. The intensity of the CH2 stretching bands provides an indication of the amount of aliphatic hydrocarbons present on the wing surface.
with gold using a JEOL NeoCoater (model MP-19020NCTR) prior to imaging. 2.4. Wettability of the wing surfaces The contact angles of Milli-Q water on the wing surfaces were measured using the sessile drop method [30–33]. The contact angle measurements were carried out in air using an FTA1000 (First Ten Ångstroms, Inc., Portsmouth, VA, USA) instrument. At least ten measurements were performed to obtain an average contact angle for each of the samples, in duplicate. Evaluation of the resulting contact angles was performed by recording 50 images in 2 s with a Pelcomodel PCHM 575-4 camera using FTA Windows Mode 4 software. To eliminate the effect of the vein structure on the wing surface, the contact angle measurement was performed on regions of the wing that were sufficiently large so that the entire droplet footprint could be accommodated. All measurements were taken under the ambient conditions of 21 ◦ C and relative humidity of 60–70%. 3. Results and discussion 3.1. Surface chemistry The FTIR microspectroscopy beam line at the Australian Synchrotron was used to obtain infrared spectral maps of each wing sample. The spectra from each sample were broadly similar, containing three major bands or band groups at 3480–3230 cm−1 , 3000–2800 cm−1 and 1750–1480 cm−1 which correspond to hydroxyl, alkyl hydrocarbons, ester carbonyl and amide groups, respectively (Fig. 1). The spectra of all four wings were dominated by amide I and amide II absorption bands, due to C O bond stretching coupled to N H bending (1610–1695 cm−1 ) and C N stretching coupled to N H bending (1480–1575 cm−1 ), respectively [34–37]. The presence of amide groups can be attributed to the chitin and protein components of the wings, as they represent the major structural components that make up the bulk of insect cuticle [38–42]. The 2D spectral maps of the insect wings showed spatial variations in the intensities of the symmetric and antisymmetric CH2 peaks and the amide I and amide II peaks. Fig. 2a shows distribution of the integrated area of the as CH2 peak of the damselfly Ischnura heterosticta, integrated between 2913 cm−1 and 2931 cm−1 . Subtraction of a spectrum from areas of low as CH2 (average of three points) from a spectrum from high as CH2 (average of three
S.H. Nguyen et al. / Vibrational Spectroscopy 75 (2014) 173–177
175
Fig. 2. Heterogeneous distribution of wax across the wing membrane. (A) Map of integrated area of as CH2 across a wing area of 100 m × 100 m of the damselfly Ischnura heterosticta. Scale bar = 20 m. (B) The spectral difference in the CH spectral region resulting from subtraction of spectra from low CH regions of the map shown in 2a from spectra from high CH areas of the map.
points) resulted in a spectrum containing just as CH2 and s CH2 peaks at 2924 cm−1 and 2854 cm−1 respectively (Fig. 2b). This is suggesting that the variation in CH2 observed in Fig. 2a arises from heterogeneous distribution of wax across the wing membrane. Amide I variations which were also observed in FTIR maps, are likely due to variations in the thickness or density of the wing membrane. A small ester carbonyl stretching band was present at approximately 1735 cm−1 in all of the recorded spectra. The C H stretching region (2840–3000 cm−1 ) represents the symmetric (s ) and anti-symmetric (as ) stretching vibrations of CH2 and CH3 functional groups. The presence of C H stretching bands with a
prevalence of methylene bands is an indication of the presence of long-chain aliphatic hydrocarbons. This is consistent with previous reports that highlighted the presence of cuticular waxes on the surface of dragonfly wings [26,27]. It can therefore be concluded that the key components of the Odonata wings are similar for all wing samples tested, highlighting that these have not essentially changed over the past 40 years. This result suggests that the wings possess remarkable stability. Notably, however, the intensity of the C H stretching bands, which provides an indication of the amount of waxes present on the wings, was seen to vary between species. The I. heteosticta and X. erythroneurum wings contained the highest
Fig. 3. Scanning electron micrographs showing the surface morphologies of Odonata wing surfaces and their corresponding 3D SEM. (A) Diplacodes melanopsis, (B) Diplacodes bipunctata, (C) Xanthagrion erythroneurum, and (D) Ischnura heterosticta. Scale bar = 1 m.
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Fig. 4. Variation in wettability of the wings of Odonata species. Average static water contact angles are given in the graph, with sample water droplet contact angles are given in the figures above. Results were an average of at least 10 independent measurements of WCA. Error bars indicates standard deviation, n = 10.
absorption intensity, whereas the Diplacodes species contained the lowest wax levels. 3.2. Surface morphology on the wings of Odonata species High-resolution scanning electron micrographs of the wing samples revealed the presence of a layer of nanopillar-like structures that covered the wing surfaces (Fig. 3). Surface-view micrographs revealed that these pillars were randomly distributed, with differences in pillar diameter and spatial distribution between species being evident. These nanopillars were present on all samples, suggesting that long-term storage of the wings did not significantly affect their surface structure, however, some minor differences between species were observed. The pillars on the surface of the D. melanopsis and I. heterosticta wings appeared to form clusters. The D. bipunctata and X. erythroneurum, however, appeared to possess distinct pillars, but their spatial distribution was much smaller than that of the other two species. Despite their ages being 40 years apart, the D. melanopsis and X. erythroneurum wing chemical composition and nano-structure appeared consistent over time, highlighting the robustness of the wings structure. 3.3. Variation in wettability between dragonflies and damselflies The freshly collected Odonata wing samples, D. bipunctata and I. heterosticta have been reported to possess superhydrophobic properties, displaying WCA greater than 150◦ (152◦ and 158◦ respectively) [28,43]. The older wing samples, however, exhibited a wider variation in WCA; D. melanopsis and X. erythroneurum displayed WCAs of 138◦ and 160◦ , respectively. The D. Melanopsis wing would therefore not be considered superhydrophobic (Fig. 4). The two species of damselfly exhibited similar degrees of wettability, despite their difference in age. The dragonfly species, on the other hand, exhibited a difference in WCA. As previously noted, the wettability of a surface depends on both the chemistry and the morphology of the surface. The X. erythroneurum and I. heterosticta wings presented the highest CH2 stretching band intensity in the IR spectra (Fig. 1); these two species also exhibited the high-
est WCA among the four species tested. Therefore, a correlation exists between the amount of cuticular waxes present on the surface of the wing and their surface wettability. This is consistent with known theories of wettability, as chemical composition is one of the main two factors that affect the wettability of surfaces. The way by which surface morphology contributes to surface wettability has been well documented [44–46]. Nosonovsky and Bhushan calculated the requirement for optimal superhydrophobic surfaces found in natural surfaces with hierarchical structures [47,48]. Even though the surface chemical composition is also one of the two major contributing factors to surface wettability, no work had addressed the extend of this influence. Nguyen et al. evaluated the influence of both surface chemistry and surface morphology at the same time [26]. In particular, it was found out that surface composition contributed not only to surface wettability but also to the surface structure of dragonfly wing, Hemicordulia tau.
4. Conclusion In this study, the natural diversity in the properties of the wings of Odonata species were determined. The compositions of the major components of the insect wings were found to be qualitatively similar according to SR-FTIR analyses; all samples contained protein/chitin components, with waxy compounds also being present. It was also shown that surface chemistry is an important contributing factor in determining the wettability of these natural surfaces. The damselfly wings, X. erythroneurum and I. heterosticta, exhibited the highest WCA among the four species investigated, and these also contained the highest amount of wax compounds on the surface of each of the wings tested. The data obtained from this work also highlighted the durability and robustness of these insect wings. The surface morphology, surface chemistry, and wettability of the wings appeared to have been maintained over an archive period of 40 years. Further investigation of the properties of these insect wings may lead to a greater understanding of how the environments in which these insects live have influenced the pattern of evolution of these insects.
S.H. Nguyen et al. / Vibrational Spectroscopy 75 (2014) 173–177
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Contents lists available at ScienceDirect
Vibrational Spectroscopy journal homepage: www.elsevier.com/locate/vibspec
Wing wettability of Odonata species as a function of quantity of epicuticular waxes夽 Song Ha Nguyen a , Hayden K. Webb a , Jafar Hasan a , Mark J. Tobin b , David E. Mainwaring a , Peter J. Mahon a , Richard Marchant c , Russell J. Crawford a , Elena P. Ivanova a,∗ a
Faculty of Science, Engineering, and Technology, Swinburne University of Technology, PO Box 218, Hawthorn, VIC 3122, Australia Australian Synchrotron, 800 Blackburn Rd., Clayton, VIC 3168, Australia c Melbourne Museum, 11 Nicholson St., Carlton, VIC 3053, Australia b
a r t i c l e
i n f o
Article history: Available online 1 August 2014 Keywords: Insect wings Long-chain aliphatic hydrocarbons Surface topography Wettability
a b s t r a c t Dragonflies have gained much attention due to their sophisticated wing surface structure, and their associated superhydrophobic, self-cleaning and bactericidal properties. In this work, we compared and contrasted the chemical composition and surface morphology of the wing membranes of four species of dragonfly and damselfly from the Odonata family collected in 1970s (Diplacodes melanopsis and Xanthagrion erythroneurum) and 2011 (Diplacodes bipunctata, and Ischnura heterosticta). Diplacodes species are dragonflies, whilst Xanthagrion and Ischnura are damselflies. Fourier-transform infrared spectroscopy data obtained from the Australian Synchrotron were used to classify the fundamental components of all four of the insect species’ wings. The spectra of all species were dominated by C H stretching, amide I and amide II and O H stretch absorbance indicating the presence of a similar membrane composition of chitin, protein and wax in all four species. Although the samples were collected 40 years apart, there was no evidence of degradation having taken place during this time. Despite the overall similarities in spectral profile, species-specific differences were observed, most notably in the intensity of the CH2 peaks, which in part reflected the amount of waxes present on the wings, which appeared to be different between individual species. The surface topography also contained minor differences in the diameter and the spacial distribution of its nanopillars. It is postulated that the differences in surface wettability of the wings could be attributed to these minor differences in surface chemistry and surface topography. For example, X. erythroneurum presented the highest water contact angle (WCA) of 160◦ whilst the D. melanopsis wings exhibited the lowest WCA (138◦ ), and the wettability of their wings was found to directly correlate with the intensity of hydrocarbon peaks found in their respective IR specta. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Insects first evolved the ability to fly at least 400 million years ago and are one of the largest classes, representing half of all living organisms on Earth [1]. In order to adapt to ever-changing environments, they have developed strategies to cope with different stresses [2]. Investigation into the mechanisms that they have adopted to enable them to cope with these environmental stresses has provided the scientific community with great insights for many
夽 Paper presented at the 7th International Workshop on Infrared Microscopy and Spectroscopy with Accelerator-Based Sources (WIRMS), Melbourne, Australia, 10–13th November 2014. ∗ Corresponding author. Tel.: +61 3 9214 5137. E-mail address: [email protected] (E.P. Ivanova). http://dx.doi.org/10.1016/j.vibspec.2014.07.006 0924-2031/© 2014 Elsevier B.V. All rights reserved.
useful applications [3–8]. For example, the mechanism by which some beetles collect water from fog-laden wind on their back can be applied in water-trapping systems, such as water condensers and engines [9]. The anti-reflective properties of the eyes of some moth species have been used as an inspiration for various optical applications [10]. Colourful butterfly wings have been studied extensively as a template for the fabrication of smart materials [11]. Superhydrophobicity and self-cleaning properties are also two important characteristics possessed by many insects. In order to maintain their high levels of functionality, their surfaces have evolved to possess highly specific structures and surface chemistries [12–19]. Dragonfly and damselfly species belong to the order Odonata, and their wing surfaces are known for their superhydrophobicity and self-cleaning properties. A surface that exhibits a WCA greater than 150◦ is considered to be superhydrophobic. When water droplets remain in a spherical shape and readily roll off a
174
S.H. Nguyen et al. / Vibrational Spectroscopy 75 (2014) 173–177
surface with a tilted angle of less than 10◦ , this surface is considered to be both superhydrophobic and self-cleaning [20–22]. It is well-documented that superhydrophobic surfaces, including insect wings, commonly adhere to the Cassie–Baxter regime [21,23–25], which describes the effect of a composite interface on its wettability. Previously published work has revealed that the fundamental components of the cuticular layer of dragonfly wings consists of peptide linked structural protein, chitin components that comprise the bulk of the wing membrane [26], and waxy components including long chain aliphatic hydrocarbons (C14 –C30 ) together with some carboxylic acids (palmitic acid and stearic acid). These components make up the outermost layer of the insect wing epicuticle [27]. These components contribute to not only the superhydrophobicity of the wing, but also to the hierarchical structure of the wing, which has been found to impart bactericidal properties to some wing surfaces [28]. In light of the discovery of these anti-bacterial properties, this paper explores the diversities of natural nanostructures and their corresponding surface chemistry. This has been achieved by comparing four different wings, including aged and fresh samples, belonging to Diplacodes melanopsis, Xanthagrion erythroneurum, Diplacodes bipunctata, and Ischnura heterosticta. Recent work has shown heterogeneity in the wax distribution on the surface of the wing membrane of the cicada Psaltoda claripennis, which may relate to the superhydrophobic and self cleaning qualities of these membranes [29]. For this reason high spatial resolution maps were acquired from each sample using the synchrotron beamline, to reveal any chemical heterogeneity in the species studied here. 2. Experimental 2.1. Materials Fresh dragonfly D. bipunctata and damselfly I. heterosticta wing samples were obtained from the suburban regions of South Australia in early to mid-2011 and preserved in sterile Petri dishes at room temperature (ca. 22 ◦ C). Aged dragonfly D. melanopsis and damselfly X. erythroneurum wing samples that had been preserved at the Melbourne Museum in the 1970s were also used in this study. When required, the wings were aseptically removed from the insect body and stored at room temperature in sterile plastic containers. 2.2. Synchrotron radiation Fourier-transform infrared spectrometry (SR-FTIR) The distribution of the organic functional groups present across the wing membrane of the dragonfly species was obtained using Synchrotron Radiation-Fourier Transform Infrared (SR-FTIR) microspectroscopy at the Australian Synchrotron. The samples were scanned in transmission mode over several areas typically of 100 m × 100 m using a Bruker Hyperion 2000 FTIR microscope (Bruker Optic GmbH, Ettlingen, Germany) using a 36× objective and condenser (numerical aperture 0.5), an aperture of 5 m × 5 m and step size of 3 m in x and y directions. 8 spectra were co-added at a resolution of 8 cm−1 per measurement position. OPUS software version 6.5 was used to operate the microscope and spectrometer and OPUS versions 6.5 and 7.2 were used for subsequent analysis of the data. 2.3. Scanning electron microscopy (SEM) The surface morphology of each wing surface was obtained using a field emission scanning electron microscope (FeSEM – SUPRA 40VP, Carl Zeiss GmbH, Jena, Germany) at a fixed voltage of 3 kV. The samples were attached to metallic substrata using conductive double-sided adhesive tape. Samples were sputter coated
Fig. 1. IR absorbance spectra of four insect wings. The intensity of the CH2 stretching bands provides an indication of the amount of aliphatic hydrocarbons present on the wing surface.
with gold using a JEOL NeoCoater (model MP-19020NCTR) prior to imaging. 2.4. Wettability of the wing surfaces The contact angles of Milli-Q water on the wing surfaces were measured using the sessile drop method [30–33]. The contact angle measurements were carried out in air using an FTA1000 (First Ten Ångstroms, Inc., Portsmouth, VA, USA) instrument. At least ten measurements were performed to obtain an average contact angle for each of the samples, in duplicate. Evaluation of the resulting contact angles was performed by recording 50 images in 2 s with a Pelcomodel PCHM 575-4 camera using FTA Windows Mode 4 software. To eliminate the effect of the vein structure on the wing surface, the contact angle measurement was performed on regions of the wing that were sufficiently large so that the entire droplet footprint could be accommodated. All measurements were taken under the ambient conditions of 21 ◦ C and relative humidity of 60–70%. 3. Results and discussion 3.1. Surface chemistry The FTIR microspectroscopy beam line at the Australian Synchrotron was used to obtain infrared spectral maps of each wing sample. The spectra from each sample were broadly similar, containing three major bands or band groups at 3480–3230 cm−1 , 3000–2800 cm−1 and 1750–1480 cm−1 which correspond to hydroxyl, alkyl hydrocarbons, ester carbonyl and amide groups, respectively (Fig. 1). The spectra of all four wings were dominated by amide I and amide II absorption bands, due to C O bond stretching coupled to N H bending (1610–1695 cm−1 ) and C N stretching coupled to N H bending (1480–1575 cm−1 ), respectively [34–37]. The presence of amide groups can be attributed to the chitin and protein components of the wings, as they represent the major structural components that make up the bulk of insect cuticle [38–42]. The 2D spectral maps of the insect wings showed spatial variations in the intensities of the symmetric and antisymmetric CH2 peaks and the amide I and amide II peaks. Fig. 2a shows distribution of the integrated area of the as CH2 peak of the damselfly Ischnura heterosticta, integrated between 2913 cm−1 and 2931 cm−1 . Subtraction of a spectrum from areas of low as CH2 (average of three points) from a spectrum from high as CH2 (average of three
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Fig. 2. Heterogeneous distribution of wax across the wing membrane. (A) Map of integrated area of as CH2 across a wing area of 100 m × 100 m of the damselfly Ischnura heterosticta. Scale bar = 20 m. (B) The spectral difference in the CH spectral region resulting from subtraction of spectra from low CH regions of the map shown in 2a from spectra from high CH areas of the map.
points) resulted in a spectrum containing just as CH2 and s CH2 peaks at 2924 cm−1 and 2854 cm−1 respectively (Fig. 2b). This is suggesting that the variation in CH2 observed in Fig. 2a arises from heterogeneous distribution of wax across the wing membrane. Amide I variations which were also observed in FTIR maps, are likely due to variations in the thickness or density of the wing membrane. A small ester carbonyl stretching band was present at approximately 1735 cm−1 in all of the recorded spectra. The C H stretching region (2840–3000 cm−1 ) represents the symmetric (s ) and anti-symmetric (as ) stretching vibrations of CH2 and CH3 functional groups. The presence of C H stretching bands with a
prevalence of methylene bands is an indication of the presence of long-chain aliphatic hydrocarbons. This is consistent with previous reports that highlighted the presence of cuticular waxes on the surface of dragonfly wings [26,27]. It can therefore be concluded that the key components of the Odonata wings are similar for all wing samples tested, highlighting that these have not essentially changed over the past 40 years. This result suggests that the wings possess remarkable stability. Notably, however, the intensity of the C H stretching bands, which provides an indication of the amount of waxes present on the wings, was seen to vary between species. The I. heteosticta and X. erythroneurum wings contained the highest
Fig. 3. Scanning electron micrographs showing the surface morphologies of Odonata wing surfaces and their corresponding 3D SEM. (A) Diplacodes melanopsis, (B) Diplacodes bipunctata, (C) Xanthagrion erythroneurum, and (D) Ischnura heterosticta. Scale bar = 1 m.
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Fig. 4. Variation in wettability of the wings of Odonata species. Average static water contact angles are given in the graph, with sample water droplet contact angles are given in the figures above. Results were an average of at least 10 independent measurements of WCA. Error bars indicates standard deviation, n = 10.
absorption intensity, whereas the Diplacodes species contained the lowest wax levels. 3.2. Surface morphology on the wings of Odonata species High-resolution scanning electron micrographs of the wing samples revealed the presence of a layer of nanopillar-like structures that covered the wing surfaces (Fig. 3). Surface-view micrographs revealed that these pillars were randomly distributed, with differences in pillar diameter and spatial distribution between species being evident. These nanopillars were present on all samples, suggesting that long-term storage of the wings did not significantly affect their surface structure, however, some minor differences between species were observed. The pillars on the surface of the D. melanopsis and I. heterosticta wings appeared to form clusters. The D. bipunctata and X. erythroneurum, however, appeared to possess distinct pillars, but their spatial distribution was much smaller than that of the other two species. Despite their ages being 40 years apart, the D. melanopsis and X. erythroneurum wing chemical composition and nano-structure appeared consistent over time, highlighting the robustness of the wings structure. 3.3. Variation in wettability between dragonflies and damselflies The freshly collected Odonata wing samples, D. bipunctata and I. heterosticta have been reported to possess superhydrophobic properties, displaying WCA greater than 150◦ (152◦ and 158◦ respectively) [28,43]. The older wing samples, however, exhibited a wider variation in WCA; D. melanopsis and X. erythroneurum displayed WCAs of 138◦ and 160◦ , respectively. The D. Melanopsis wing would therefore not be considered superhydrophobic (Fig. 4). The two species of damselfly exhibited similar degrees of wettability, despite their difference in age. The dragonfly species, on the other hand, exhibited a difference in WCA. As previously noted, the wettability of a surface depends on both the chemistry and the morphology of the surface. The X. erythroneurum and I. heterosticta wings presented the highest CH2 stretching band intensity in the IR spectra (Fig. 1); these two species also exhibited the high-
est WCA among the four species tested. Therefore, a correlation exists between the amount of cuticular waxes present on the surface of the wing and their surface wettability. This is consistent with known theories of wettability, as chemical composition is one of the main two factors that affect the wettability of surfaces. The way by which surface morphology contributes to surface wettability has been well documented [44–46]. Nosonovsky and Bhushan calculated the requirement for optimal superhydrophobic surfaces found in natural surfaces with hierarchical structures [47,48]. Even though the surface chemical composition is also one of the two major contributing factors to surface wettability, no work had addressed the extend of this influence. Nguyen et al. evaluated the influence of both surface chemistry and surface morphology at the same time [26]. In particular, it was found out that surface composition contributed not only to surface wettability but also to the surface structure of dragonfly wing, Hemicordulia tau.
4. Conclusion In this study, the natural diversity in the properties of the wings of Odonata species were determined. The compositions of the major components of the insect wings were found to be qualitatively similar according to SR-FTIR analyses; all samples contained protein/chitin components, with waxy compounds also being present. It was also shown that surface chemistry is an important contributing factor in determining the wettability of these natural surfaces. The damselfly wings, X. erythroneurum and I. heterosticta, exhibited the highest WCA among the four species investigated, and these also contained the highest amount of wax compounds on the surface of each of the wings tested. The data obtained from this work also highlighted the durability and robustness of these insect wings. The surface morphology, surface chemistry, and wettability of the wings appeared to have been maintained over an archive period of 40 years. Further investigation of the properties of these insect wings may lead to a greater understanding of how the environments in which these insects live have influenced the pattern of evolution of these insects.
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