Arthropod Structure & Development 53 (2019) 100887 Contents lists available at ScienceDirect Arthropod Structure & Development journal homepage: www.elsevier.com/locate/asd High accuracy of color-generating nanoarchitectures is kept in lowland and mountainous populations of Polyommatus dorylas (Lepidoptera: Lycaenidae: Polyommatinae) * Zsolt Balint a, , Gergely Peter Katona a, Zsolt Endre Horvath b, Krisztian Kertesz b, Gabor Piszter b,Laszl oP eter Biro b a Hungarian Natural History Museum, Baross utca 13, Budapest, H-1088, Hungary b Institute of Technical Physics and Materials Science, Centre for Energy Research, P.O. Box 49, Budapest, H-1525, Hungary article info abstract Article history: It is known that the size of the scales covering the surface of the Lepidoptera wings is in correlation with Received 25 July 2019 body size: larger species possess larger scales. However, butterfly individuals representing the various Accepted 8 October 2019 generations of the same species but differing in body size were not investigated in this respect. Similarly, Available online xxx the question whether different scale size may influence structural color generation based on nano- architectures in the scale lumen was never addressed. Populations of lowland (environment of Budapest, Keywords: Hungary) and upland (Carpathian Mountains, Romania) Polyommatus dorylas were compared in terms of Wing scale size voltinism, wing and scale size, and the structural origin of blue coloration. Data analysis showed that the Polyommatina Sexual signal univoltine upland population exhibits a larger wing and scale size. On the other hand, the nano- Spectral properties morphology of the blue color-generating scales was identical when compared between univoltine and Voltinism bivoltine populations. Coloration was also identical when measured with a spectrophotometer under Nanoarchitecture ultraviolet and visible light. This high accuracy present in the male structural coloration suggests that it is controlled genetically. Body size alteration for enhanced thermal fitness has no influence on the fine structure of the nanoarchitecture present in the scale lumen. © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction Paulus, 1970; Tilley and Eliot, 2002; Wilts et al., 2009; Biro and Vigneron, 2010). Due to the role this color generation mechanism The Blue butterflies, the tribe Polyommatini of the Lycaenidae, is plays in the mating strategy, the physical color of the lycaenids is one of the most typical day flying lepidopteran tribes in the subjected to strict selection (Piszter et al., 2016), and exhibits a northern hemisphere (Talavera et al., 2013; Vila et al., 2011). They stress stability which by far exceeds the stability of the pigment- are important members of habitats characterized by open land- based pattern found on the ventral side of the wings (Kertesz et scapes in the Carpathian Basin (Szabo, 1956). In the wind-swept al., 2017). Moreover, as our previous experiments revealed, the meadows, polyommatine lycaenid females spend most of their sexual signaling color of Polyommatus icarus (Rottemburg, 1775) time feeding and basking at ground level. They find their mate(s) exhibits an amazing spectral stability, both in space and in time because of the special prezygotic strategy the tribe employs: fe- (Kertesz et al., 2019). males detect species-specific optical signals present on the dorsal An important strategy in the adaptation to the environment is wing surfaces of males. Females prefer brightly colored males the regulation of generation numbers. For example, the widely (Imafuku and Kitamura, 2018). The male sexual signaling colors are distributed typical grassland species, P. icarus, may have a single species-specific and precisely tuned spectrally (Balint et al., 2012). brood in northern latitudes (Henriksen and Kreutzer, 1982) but The physical background of this signal is provided by photonic produces up to four broods in regions close to the Mediterranean nanoarchitectures located in the lumen of the scales (Schmidt and Sea (Tshikolovtes, 2011). Therefore, the voltinism of this species is highly influenced by various local conditions. When the environ- ment turns less favorable, some of the populations are forced to * Corresponding author. reduce their brood number in order to adopt a more economical E-mail address: [email protected] (Z. Balint). way to use the available recourses for breeding. Because of this, the https://doi.org/10.1016/j.asd.2019.100887 1467-8039/© 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 2 Z. Balint et al. / Arthropod Structure & Development 53 (2019) 100887 size of the imagines in populations with different number of gen- all specimens on a millimeter-scaled graph paper background. erations can also differ in body weight and wing surface size, as Images were acquired with a Camedia C 7070 (Olympus, Tokyo, previously documented for P. icarus (Nygren et al., 2008). Japan) digital camera. On the other hand, according to recent investigations in Lepi- Using CorelDRAW X6 (Corel, Ottawa, Ontario, Canada) software, doptera, there is a strong correlation between the wing surface three measurements of the left forewing were taken: (1) the length area, location and the size of the scales (Simonsen and Kristensen, of the forewing costa measured from the wing base (vein erection) 2003; Dhungel and Otaki, 2014). Here, one has to observe that in to the apex (vein R2 terminus); (2) the length of the outer margin the case of photonic nanoarchitectures, a very close relationship measured from the apex (vein R2 terminus) to the tornus (anal vein exists between the geometric size of the wing scale nano- terminus) and (3) the length of the anal margin measured from the architectures at the nanometer level and the spectral properties, wing base (vein erection) to the tornus (anal vein terminus) which provide the species-specific signals for polyommatine (Fig. 1B). Using these three measurements we calculated the area lycaenids (Balint et al., 2012). Therefore, it is very useful to inves- between the white lines on Fig. 1A. tigate species in which the switching from two generations to a Then, using optical microscope images of the right forewing single generation is associated with an easily measurable change in (taken with a 2.5 Â objective), we marked an area of 1.96 mm2 body and wing size (Balint, 1987; Nygren et al., 2008), and the between the vein M3 and the Cubitus. We determined the number consequences of the change in wing size, both in terms of the of blue cover scales in this area on wings of five randomly selected associated scale size and the eventual spectral modifications. male exemplars of Magna, Dorylas I, and Dorylas II. In this paper, we studied one polyommatine species that is typically found in calciferous open landscapes in the Western 2.4. Optical microscopy Palearctic region: Polyommatus dorylas (Denis and Schiffermüller, 1775). In general, this species is bivoltine across most of its range Optical imaging of the wing scales was carried out using an Axio (Tolman and Levington, 1997; Tshikolovtes, 2011), but there are Imager A1 microscope (Carl Zeiss AG, Jena, Germany) using re- records of univoltine populations, some of which have been flected light. The wing scales usually stand at an angle of approxi- discriminated taxonomically (Balint,1985, 1987). It appears that the mately 15 relative to the wing membrane, so for better visibility, univoltine populations of this species produce imagines with we used focus stacking (Adobe Photoshop, Adobe systems Inc., CA, conspicuously larger wing surface areas as compared to bivoltine USA) to compensate for the narrow depth of field of the high- populations. We aimed to compare the wing surface and the color- resolution microscope objectives. generating scale sizes of populations with different voltinisms. We Single scales were removed using a sharp needle, and placed on also investigated their spectral properties and, in particular, a microscope glass slide. The scales were inspected in reflected attempted to determine whether there is any correlation between light, and we measured their maximum width and length using the size of the scales and the number of generations and whether CorelDRAW X6. the spectral properties of the populations with different voltinisms are common or different. 2.5. Scanning and transmission electron microscopy (SEM and TEM) 2. Materials and methods SEM images were taken using a LEO 1540 XB electron micro- 2.1. Species investigated scope (Carl Zeiss AG, Jena, Germany). Wing pieces were cut and mounted on a sample holder with double-sided conductive tape; P. dorylas is distributed westwards from the Dnieper Plain single scales were also placed on conductive tape. To ensure that throughout Central Europe to the Pyrenees, and southwards to the the original structure of the wing scales was preserved, no other Balkans and Anatolia, and the Caucasus region (Tshikolovtes, 2011). treatment was applied. For TEM, a standard sample preparation For Central Europe in general, this species is documented as being procedure was performed: after fixing and dehydration, a few bivoltine (Szabo, 1956; Slamka, 2004). In mountainous areas, to- millimeters of wing pieces were embedded in Spurr's resin (SPI wards the edge of its range, populations