Abstract The Saharan silver ant Cataglyphis bombycina is one of the terrestrial living organisms best adapted to tolerate high temperatures. It has recently been shown that the hairs covering the ant’s dorsal body part are responsible for its silvery appearance. The hairs have a triangular cross-section with two corrugated surfaces allowing a high optical reflection in the visible and near-infrared (NIR) range of the spectrum while maximizing heat emissivity in the mid-infrared (MIR). Those two effects account for remarkable thermoregulatory properties, enabling the ant to maintain a lower thermal steady state and to cope with the high temperature of its natural habitat. In this paper, we further investigate how geometrical optical and high reflection properties account for the bright silver color of C. bombycina. Using optical ray-tracing models and attenuated total reflection (ATR) experiments, we show that, for a large range of incidence angles, total internal reflection (TIR) conditions are satisfied on the basal face of each hair for light entering and exiting through its upper faces. The reflection properties of the hairs are further enhanced by the presence of the corrugated surface, giving them an almost total specular reflectance for most incidence angles. We also show that hairs provide an almost 10-fold increase in light reflection, and we confirm experimentally that they are responsible for a lower internal body temperature under incident sunlight. Overall, this study improves our understanding of the optical mechanisms responsible for the silver color of C. bombycina and the remarkable thermoregulatory properties of the hair coat covering the ant’s body.

Citation: Willot Q, Simonis P, Vigneron J-P, Aron S (2016) Total Internal Reflection Accounts for the Bright Color of the Saharan Silver Ant. PLoS ONE 11(4): e0152325. https://doi.org/10.1371/journal.pone.0152325 Editor: Matthew Shawkey, University of Akron, UNITED STATES Received: November 20, 2015; Accepted: March 11, 2016; Published: April 13, 2016 Copyright: © 2016 Willot et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper. Funding: This work was supported by the Wallonia-Brussels Federation (PS), the Université de Namur (grant ARC2010-2015♯033) (JPV), the Université Libre de Bruxelles (ARC2010-2015 ♯5) (SA), the Belgian FRIA (QW), the FRS-FNRS (PS, SA), and the Belgian Fonds National de la recherche Scientifique (FRFC 2.4516.11) (SA). The authors acknowledge using resources from the Interuniversity Scientific Computing Facility located at the University of Namur (Belgium), which is supported by the FRS-FNRS under convention 2.4617.07. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Cataglyphis bombycina, the”silver ant” of the Sahara, the Sinai and the deserts of the Arabian Peninsula, is famous for its ability to withstand extremely high temperatures [1,2]. Workers come out from the nest during the hottest midday period, when temperatures exceed 50°C, to scavenge corpses of heat-stricken animals. By restricting foraging activity to the hottest period of the day, the ants minimize the chances of encountering their most frequent predator—a lizard that ceases all activities when the temperature becomes unbearable. This, however, requires that the ants themselves are effectively protected against excessive temperatures. Workers strictly limit the time of exposure to the light from the high sun and the heat radiated by the hot sand [2]. They are equipped with legs much longer than other ants relative to their body shape, allowing them to keep their body at a greater distance from the hot surface, and to run much faster reducing the duration of their sorties while maximizing cooling by convection [1,3,4]. In contrast to other organisms, workers produce heat-shock proteins before emerging from the nest, thereby avoiding a slow adaptation to the sudden heat exposure [5,6]. Recently, it has been shown that silver ants have evolved remarkable thermoregulatory solutions to cope with the thermally stressful conditions of their natural environment [7]. The dorsal side of the workers’ head, thorax and abdomen is covered with a dense array of triangularly shaped hairs that are responsible for the ants silvery appearance and produce strong thermoregulatory effects (Fig 1). By measuring reflectance values in the visible and near-infrared (NIR) spectra, Shi et al [7] have shown that the presence of triangular hairs greatly enhances light reflection. Sunlight is presumably deflected by a combination of scattering within the triangular hairs (Mie scattering) and total internal reflection (TIR) on their bottom face. Based on radiative heat transfer experiments, these authors showed with thermal camera images that hairs allow the workers to maintain lower body temperatures. The latter are achieved by minimizing energy absorption in the visible and NIR ranges of the spectrum, and by maximizing heat emissivity in mid-infrared (MIR) where the blackbody radiation spectrum of the ant’s body is at its maximum [7]. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. The silver ant Cataglyphis bombycina. Colonies contain a caste of spindly workers and a caste of soldiers with large heads and saber-shaped mandibles. (a) In full sunlight, workers and soldiers show a metallic sheen that justifies the vernacular name of the species. Photo copyright: P. Landmann. (b,c) The hairs covering the dorsal side of the workers’ head, thorax (b) and abdomen (c) follow the cuticle’s curvatures. https://doi.org/10.1371/journal.pone.0152325.g001 In this paper, we further investigate the geometrical optics underlying the high reflectivity of C. bombycina hairs. Using theoretical and empirical approaches, we demonstrate that the bright silver color of the ant C. bombycina stems from the TIR associated with the hairs that densely cover the ant body. First, we examined hair morphology using scanning and transmission electron microscopy. Second, using ray-tracing optical models, as well as bi-directional reflectance distribution function (BRDF) and attenuated total reflection (ATR) experiments, we provide evidence that the triangular shape of hairs allows TIR for a large range of incident angles. We also show that hairs are corrugated on their upper faces and that this enhances reflection properties, giving hairs an almost total specular reflectance beyond the critical angle at which TIR occurs, thus the silver color. Finally, we bring additional evidence on the effect of hairs as an adaptation to heat tolerance, by comparing reflectance and internal body heating rate between hairy workers and ‘shaved’ workers whose hairs were removed.

Materials and Methods Samples of C. bombycina were collected in the sand dunes of Erg Chebbi, Merzouga, in South-eastern Morocco (3108’45”N, 0359’55”W). The dunes are public areas and the silver ant is not an endangered or protected species; no permission was required to collect the samples. To compare the reflectance and heating rate between hairy, untreated workers and ‘shaved’ workers whose hairs were removed, ants were anesthetized by CO 2 exposure and hairs covering the dorsal side of the abdomen were completely removed using a sharp scalpel blade Electron microscopy analyses of hairs morphology Samples for scanning electron microscopy (SEM) were prepared by first removing the legs from workers, and then gluing the head, thorax and abdomen with silver paint on a metallic sample holder. The whole mount was coated in metal (20 nm of gold) in order to ease charge elimination, and placed in a JEOL InTouchScope JSM-6010LV microscope. For transmission electron microscopy (TEM), samples were prepared by embedding a piece of cuticle with hairs in an epoxy resin infiltrating the structure at a constant temperature of 35°C for 48 hours, and hardened at 60°C for 72 hours. Then, 90 nm-slices were cut perpendicular to the hairs’ axis and examined with a FEI Tecnai transmission electron microscope. Experimentally testing total internal reflection To experimentally test the TIR model, we first measured the direction and spectral radiance of 2 mm2 of the abdomen of a C. bombycina worker using a viewing angle measurement system (Eldim EZContrast L80) that gives the full spectral bi-directional reflectance distribution function (BRDF). The detector gives accurate data for emissions ranging from 400 nm to 700 nm. This method allowed us to obtain a full BRDF image for each wavelength measured. In order to obtain reflectance values, spectral radiance was normalized with radiance of incident light. Reflectance of hairy workers was compared with that of shaved workers. Data were obtained for 3 individuals in each group; no significant intra-group difference was observed. Second, we experimentally tested attenuated total reflection (ATR) by placing hairs on a selectively absorbing surface, thereby greatly reducing TIR. Under ATR conditions, the light is transmitted rather than internally reflected in the hair [8]. Hairs were deposited flat face down on a copper grid (support for TEM samples) and examined with an optical microscope (Olympus BX61) in bright-field epi-illumination mode. The objective (x60) had a numerical aperture (0.85) large enough to illuminate a single hair under angles > 34.9°, which corresponds to conditions for observing TIR (see Results). Ant heating rate A 0.075mm diameter thermocouple (K-type Thermocouple chromel–alumel, RS Components Ltd) connected to a digital thermometer (Digital Thermometer 206–3738, RS Components Ltd) was inserted through a small incision in the rectum of a freshly killed ant. The same position under the first tergite was always reached. We did not witness any bleeding or liquid loss that might have been a source of evaporative cooling. We let the abdomens reach room temperature (21°C) prior to measurements. The dorsal surface of each abdomen was exposed perpendicular to the light emitted by a solar simulator (ORIEL Corporation USA, No. 81172), away from any possible interfering surfaces. The device uses a 1000 W xenon arc lamp (power supply: ORIEL No. 68820). Wavelengths above 1100 nm were removed with a water filter to limit blackbody radiation measuring only the effect of visible and NIR radiation (ORIEL No. 61945). Temperature elevation of the abdomen was recorded every 5 sec for 90 sec to approximate temperature equilibrium. Measures were repeated 3 times per abdomen, on 7 hairy and 7 shaved individuals; only ants of same size were used. All experiments were performed under constant temperature in an air-conditioned room. Initial rate of heat gain K was estimated following [9], as K = (T ex *0.5)/t 1/2 ), with T ex being the excess temperature (i.e., the difference between ambient and sample temperature) and t 1/2 corresponding to the heating half-time. Comparisons of mean temperatures between hairy and shaved C. bombycina were performed using unpaired t-tests, using Graphad Prism6 software.

Discussion Our results show that total internal reflection in the hairs of the Saharan ant C. bombycina is responsible for its bright silver sheen. Each hair has the shape of a prism with a triangular cross-section within which the conditions for TIR are met for a wide range of incidence angles. In addition, our simulations indicate that corrugations on the entrance and exit faces of the hairs further increase reflectance across the whole visible wavelength range. For incidence angles ranging from 34.9° to 90°, the reflectance is very close to 100% for all azimuthal angles. For smaller incidence angles, light can leak through a hair. However, the light transmitted will be reflected by other hairs underneath. Together, these physical properties account for a strong mirror effect in the whole visible range, hence, the silver color of the ant. Consistent with this, ATR experiments show that the proximity of a metallic surface attenuates TIR, suppressing the silver color. Our data show that the hairs covering the ant dorsal face reflect up to 10 times more light than shaved cuticles over the visible spectrum (425–700 nm). The values of reflectance obtained in the present study are similar to those reported by Shi et al. [7]. These authors measured reflectance values with a Fourier-transform based spectrometer allowing full visible and NIR spectral range, while our measures of radiance were performed by BRDF in the visible range only. The energy of direct sunlight is composed approximately of 40% visible and 50% NIR radiation (UV below 400 nm and IR beyond 1500 nm account for 2–4% and 6–8% of this energy, respectively;[18]). NIR radiation is therefore a major source of incoming energy that may greatly affect the thermal steady state of the ants. It was shown [7] that hairs also largely enhance NIR reflectance. The thermoregulatory properties of the hairs coating stem from at least two effects [7]: (i) a reduction of solar energy intake by the mirror effect in the visible and NIR range of the spectra, which is further confirmed by our own results, and (ii) enhanced heat dissipation by radiation in the mid-IR where the black body radiation spectrum of the ant is at its maximum. By exposing individuals to a high-power xenon lamp to simulate the solar spectral distribution at the desert sand surface, these authors conducted an elegant set of thermodynamic experiments both in vacuum (to examine the effect of thermal radiation) and in air (to study the interplay of thermal radiation and convection). Thermal camera images gave evidence that hairy workers maintain significantly lower body temperatures than those with the hairs removed. Our heating rate experiments, based on direct measures of temperature in the body of ants, corroborate these results. They show that hairy workers exposed to solar simulated light experience a reduction in excess internal temperature up to 2°C compared with shaved workers. Even a small difference in body temperature may prove highly ecologically relevant for C. bombycina, as it can make the difference between life and death for such a small insect foraging so close to its upper critical thermal limit. If such a hair coating is an efficient way to prevent overheating, why hasn’t it evolved in more desert insects? Activity period of ectotherms is limited both by their lower and upper temperature tolerance. During the cooler Saharan season, dark-colored diurnal ectotherms will be at advantage by absorbing more direct solar energy, thereby increasing their daily activity. Outside this period, they typically hide during the hottest hours of the day [19]. In contrast, foraging activity in C. bombycina is restricted to a narrow thermal window (48–51°C) delimited by heat stress (upper limit) and predatory pressure by their main lizard predator (Acanthodactylus dumerili) (lower limit) [2]. Thus, their lower limit of activity is far above physiological demands, whereas their upper limit depends on their ability to withstand heat. Consequently, unlike other desert insects, C. bombycina would gain in reflecting incident sunlight all year round. So far, TIR has been reported as limiting light extraction in fireflies [20], and as a side effect in superhydrophobic leaves immersed in water [21]. Nature often uses other (more complicated) methods to produce a bright metallic coloration [14,22]. Bragg mirrors, for instance, have been described in other Hymenoptera. This kind of mirror uses light-wave interference in a one-dimensional layered structure to produce a high specular reflection, but often over a limited range of wavelengths. A reflectance covering a broader range of wavelengths can be obtained with a stack of layers with graded-thickness, such as those encountered in some Scarabaeidae [23]. In beetles of the subfamily Rutelinae, the alternation of refractive index values in the multilayer is caused by the helical distribution of anisotropic chitin bars, in the form of a discrete Bouligand structure [24–26]. The large number of layers allows a highly efficient broadband reflectance that avoids iridescence. This structure is obviously much more complex than that found in the ant C. bombycina. The triangular form of the hairs in the Saharan ant contrasts with the typical cylindrical shape [27,28] or flattened plate-like shape [29] of bristles documented in most arthropods. Depending on the species and their body position, the morphology of hairs has evolved to serve several functions including defense, locomotion, prey capture, pheromone dispersal, or temperature sensing [28,30,31]. The triangular shape that evolved in C. bombycina optimizes the backscattering of light and is responsible for the silver sheen of the ant. While reflection allows insects to gain less heat under direct sunlight [7,32], it could also serve other purposes such as camouflage [33] or communication [34]. Current studies indeed show that triangularly shaped hairs also occur in ground-dwelling ants from the rainforest, which are exposed to low insulation conditions (Simonis, unpubl. data). Therefore, several mechanisms might have been jointly implicated in positive selection for the reflective coating in the silver ant.

Acknowledgments This work was supported by the Belgian FRIA (QW), the FRS-FNRS (PS, SA), and the Université Libre de Bruxelles (ARC 2010–2015 #5; SA). The authors acknowledge using resources from the Interuniversity Scientific Computing Facility located at the University of Namur (Belgium), which is supported by the FRS-FNRS under convention 2.4617.07. We thank M. Oualim, Y. Bahou and M. Harmouchi for collecting samples, G. Ullens de Schooten for sample preparation, H. Darras for drawing the figures, D. Bolsée and the Belgian Spatial Aeronomy Institute (IASB) for their help with the solar simulator. S. Berthier, J.L. Deneubourg, B. Gassner, S. Massar, and J.M. Pasteels made helpful comments on a first draft of the manuscript. We also thank two anonymous reviewers for their comments and suggestions, which have substantially improved the manuscript.

Author Contributions Conceived and designed the experiments: QW PS JPV SA. Performed the experiments: QW PS. Analyzed the data: QW PS SA. Contributed reagents/materials/analysis tools: PS SA. Wrote the paper: QW PS SA.