J Biosci Vol. 43, No. 4, September 2018, pp. 673–684 Ó Indian Academy of Sciences DOI: 10.1007/s12038-018-9793-y Fine nanostructural variation in the wing pattern of a moth Chiasmia eleonora Cramer (1780) 1 2 SHAUNAK GHOSH and MONALISA MISHRA * 1Department of Biotechnology, Heritage Institute of Technology, Chowbaga Road, Anandapur, P.O. East Kolkata Township, Kolkata, West Bengal 700107, India 2Neural Developmental Biology Lab, Department of Life Science, NIT Rourkela, Rourkela, Odisha 769008, India *Corresponding author (Email, [email protected]) MS received 23 February 2018; accepted 11 June 2018; published online 16 August 2018 Butterflies and moths possess diverse patterns on their wings. Butterflies employ miscellaneous colour in the wings whereas moths use a combination of dull colours like white, grey, brown and black for the patterning of their wings. The exception is some of the toxic diurnal moths which possess bright wing colouration. Moths possess an obscure pattern in the dorsal part of the wings which may be a line, zigzag or swirl. Such patterns help in camouflage during resting period. Thus, the dorsal wing pattern of the moth is used for both intra- as well as inter-specific signal communication. Chiasmia eleonora is a nocturnal moth of greyish black colouration. The dorsal hindwing possesses yellow and black colour patches. A white- coloured oblique line crosses both left and right fore- and hindwings to form a V-shaped pattern across the dorsal wing. This V-shaped pattern possesses a UV signal. Closer to the body, the colour appears darker, which fades towards the margin. The fine nanostructural variation is observed throughout the wings. This study elucidates the wing pattern of the geometrid moth C. eleonora using high-resolution microscopy techniques that has not been described in previous studies. Keywords. Chiasmia eleonora; moth wing pattern; scanning electron microscope; structural and pigmentary colouration 1. Introduction background matching is poor (Merilaita and Tullberg 2005). In this context, prey with disruptive countershading with The family Lepidoptera includes members of butterflies and close likeness to the surroundings survived better compared moths. The members of this family possess unique charac- to a few cryptic prey that accurately matched the environ- teristic pattern in its wings (Nijhout 2001; Monteiro et al. ment (Merilaita and Lind 2005). Disruptive colouration 2006). The wing colouration is produced due to diverse function is independent of background thus gives better types of scales present throughout the body. The wing pat- survivability to the insect than cryptic ones (Sherratt et al. tern arises to provide maximum advantage to the insect in 2005). Crypsis and disruptive colouration increases the that environment (Hazel 2002; Monteiro et al. 2006). The efficacy of the insect to survive in different environments scales present on the body help in insulation, thermoregu- (Schaefer and Stobbe 2006). Most of the geometrid moths lation, hormone production (in case of a male) (Hill et al. possess such types of patterns on their wings. 2002) and supporting smooth flight. Besides this, the most In moths, the wing pattern shares similarity with the important function of the scale is to send signals for survival resting substratum. The moths’ behaviour is comprehen- as well as propagation. Thus, the scales help in camouflage, sively studied for its preferences to choose the background which is essential to safeguard the insect and find a potential substrate. Most moths consider two different parameters mate (Papke et al. 2007; Kolle et al. 2010). Moths are well while selecting its resting site. (1) Selection of a matching known for their camouflage mechanism. background colour (Stuart-Fox et al. 2004) and (2) appro- Camouflage mechanisms are of two types: (1) cryptic and priate resting orientation. Landing behaviour of a geometrid (2) disruptive colouration (Cuthill et al. 2005). In the cryptic moth was studied by various authors (Webster et al. 2009; mechanism, the contrast between prey and background is Kang et al. 2012; Wang and Schaefer 2012). According to reduced (Endler 1978). However, crypsis is specific to a this study, the moth cannot recognize the fine pattern of the particular background and it is not an effective camouflage trunk or wing or boundaries formed by shadow. Most of the (Lev-Yadun et al. 2004; Ruxton et al. 2004) when the geometrid moths possess a typical pattern on the dorsal side http://www.ias.ac.in/jbiosci 673 674 Shaunak Ghosh and Monalisa Mishra of the wing (Sihvonen et al. 2011; Kang et al. 2012). colourations of various regions were imaged and transferred However, a structural study from the wing pattern of a to ImageJ. geometrid moth is missing from the literature. Chiasmia eleonora Cramer (1780), a moth belonging to the Geometridae family and found in parts of south-west 2.4 Field-emission scanning electron microscopy Asia countries like India. We analysed the wing pattern for (FESEM) the following reasons. (1) The wing pattern of the geometrid moth is not described at high resolution in earlier studies. (2) Various coloured regions as described for optical micro- What contributes for the colour of the wing? Is it the scopy were mounted on a stub. Each stub has double structure or pigment is also not known. (3) Moreover, moth adhesive carbon tape on it. Various wing regions were patterning is less studied in comparison to butterfly. Thus, a coated with platinum with a sputter coater (JEOL- study on wing patterning of C. eleonora will be beneficial JFC1600 auto find coater) for 15 mins. Samples were for physicists, biologists as well as for material scientists. analysed under a FESEM (FEI, NOVA NANOSEM 450) at an operating voltage of 12 kV. Images were captured at various magnifications and processed and analysed using 2. Materials and methods ImageJ. Measurements of various morphological param- eters like the distance between both the ribs, length of the 2.1 Collection of moths lamellae, microrib, crossrib, trabeculae and area of win- dows were conducted using the SEM images. Species of C. eleonora were collected from the National For morphological measurements, 10 moth wings were Institute of Technology, Rourkela campus, Odisha, India imaged using SEM. From each moth 20 measurements were (N22°1405700, E84°5205800) during June–July 2017 in live taken for each parameter. All the values were presented as condition. Moths were collected in the morning perched on mean ± standard deviation. green leaves of Jasminum grandiflorum and Ixora coccinea in a sitting posture. During early afternoon, moths were collected from damped walls of the corridor. Thirty moths 3. Results were collected and kept in a zipper bag at 4 °C. Later, fore- and hindwings were dissected out using a sharp blade. The C. eleonora possess body length of 1.8–2 cm and a dissected wings were kept in a glass petri dish in desiccators wingspan of 4–4.5 cm. The length of the forewing is at room temperature. Twenty wings were imaged with a ruler 1.21 ± 0.06 cm and the hindwing is 1.19 ± 0.02 cm. The and various measurements of the wings were taken using wings are brown in colour with white shades. The brown ImageJ. colour appears like a gradient in both dorsal and ventral regions (figure 1A and B). Closer to the body, the wing colour appears darker which fades towards the margin. 2.2 Analysis of UV signals The dark and light regions are separated by means of a white oblique line in both fore- and hindwings. A very The intact moth was placed inside a Gel Doc (Bio-Rad, thin black border is present on the side of the white band USA), and the images were captured under UV light. The of the hindwing, which gradually vanishes from the images were taken from both dorsal as well as ventral sides. forewing. The white band passes to the maximum extent Images were transferred to ImageJ for further analysis. of wings and covers both left and right half of the body to make a V-shaped structure over the wing (figure 1Aand B). In the hindwing, a rectangular-shaped black patch is 2.3 Optical microscopy analysis found. Next to the black patch another mixed yellow and black colour patch was found. The marginal region of the Various regions of the wings having distinct coloured pat- wing has white shedding and thorax is golden brown in terns were chosen for analysis. From the forewing region, (1) colour. brown (closer to the body), (2) white part and (3) greyish- Analysis of the wing under UV light: Under UV light, white part were chosen. Similarly, from the hindwing, (1) the V-shaped white pattern and the marginal region of the dark brown (closer to the body), (2) white, (3) yellow and (4) fore- and hindwings possess UV signals (figure 1C and D). black regions were selected for analysis. Appropriate regions The same pattern of the UV signal is observed for both were cut and placed on a glass slide. Slides were observed dorsal and ventral wings. under the optical-light microscope in reflection mode (Carl Light and optical microscopy (scale stacking): Various Zeiss, Inverted Optical light microscope, reflection mode) at regions of the dorsal and ventral fore- and hindwings were 409 in a 90° inverted position. In the reflection mode, the analysed under an optical microscope. Nanostructural variation in wing of C.elenora 675 Figure 1. An individual of C. eleonora:(A) dorsal and (B) ventral view. Note the colour variation between the dorsal and ventral side. The dorsal hindwing has black patch surrounded by yellow colour which is absent from the ventral hindwing. Moth under UV illumination: (C) dorsal and (D) ventral view. 3.1 Dorsal forewing scales are found in the marginal region which appears dull blue under the optical microscope (figure 2F).