© 2016. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2016) 219, 2837-2843 doi:10.1242/jeb.137885 RESEARCH ARTICLE Differences in spectral selectivity between stages of visually guided mating approaches in a buprestid beetle Michael J. Domingue1,*, Jonathan P. Lelito2, Andrew J. Myrick1,György Csóka3, Levente Szöcs3, Zoltan Imrei4 and Thomas C. Baker1 ABSTRACT Domingue et al., 2014), making them promising candidates for the Spectral mating preferences were examined in male Agrilus exploration of the mechanisms of color selection in beetles. The angustulus (Buprestidae: Coleoptera), a member of a taxon known Buprestidae is a highly diverse family of 15,000 species (Bellamy, for its high species diversity and striking metallic coloration. The 2008), whose members are known for their dazzling arrays of spectral emission profile of a typical A. angustulus female displays metallic coloration patterns. Here, we examined more closely the low chroma, broadly overlapping that of the green oak leaves they role of color in mate-finding behaviors of an arboreal buprestid feed and rest upon, while also including longer wavelengths. To species, Agrilus angustulus (Illiger 1803). In addition to pinpoint behaviorally significant spectral regions for A. angustulus determining whether color preferences exist in their stereotypical males during mate selection, we observed their field approaches to mate-finding flights, we also explored the questions of whether such females of five Agrilus planipennis color morphs that have greater preferences might change during these brief flights, and what chroma than the normal conspecific female targets. Agrilus ecological factors might provide the selective pressures shaping angustulus males would initially fly equally frequently toward any of these behaviors. the three longest wavelength morphs (green, copper and red) whose The stereotypical mate-location behavior in arboreal Agrilus spectral emission profiles all overlap that of typical A. angustulus buprestids involves a visual response of flying males toward females females. However, they usually only completed approaches toward resting on the exposed upper surfaces of sunlit leaves. This behavior the two longest wavelength morphs, but not the green morphs. Thus, is characterized by the male rapidly descending from up to a meter ‘ ’ spectral preference influenced mate selection by A. angustulus above the female to pounce directly onto her. The repeatability of males, and their discrimination of suitable targets became greater the visually guided mating behavior was first documented in detail as these targets were approached. This increasing spectral for Agrilus planipennis Fairmaire 1888 males approaching dead discrimination when approaching targets may have evolved to allow pinned specimens (Lelito et al., 2007, 2009). Similar behavior was female emissions to remain somewhat cryptic, while also being later determined to exist for other arboreal Agrilus species visible to conspecifics as distinct from the background vegetation and (Domingue et al., 2011; Lelito et al., 2011). Many such arboreal heterospecific competitors. buprestids cryptically reflect light at wavelengths similar to the leaves they rest upon, while also reflecting some light at longer or KEY WORDS: Buprestidae, Color vision, Crypsis, Flight, Species shorter wavelengths (Muskovits and Hegyessy, 2002; Bellamy, recognition 2008). It was further noted in studies involving the mating behaviors of multiple Agrilus species that despite coloration and size INTRODUCTION differences, male pouncing behavior could also often occur For arthropods, there have been extensive studies regarding the role toward heterospecific specimens (Domingue et al., 2011, 2013, of color preferences with respect to flower selection (Johnson and 2014; Lelito et al., 2011). Midgley, 2001; Lunau et al., 2006; Yaku et al., 2007; Martinez- Here, further insight into color discrimination and preference in Harms et al., 2012; McCall et al., 2013), prey location (Taylor et al., Agrilus was obtained by directly observing male mating approaches 2014) and the efficacy of traps for monitoring and detection of pests to females with varied spectral emission profiles. Five female color (Francese et al., 2010; Gadi and Reddy, 2014). However, morphs of A. planipennis, differing from each other in their peak documentation of color preferences in mate selection has been spectral reflectance, were pinned to the leaves of European oaks in less commonly described (Silberglied and Taylor, 1978; Kemp and the field to assess the preferences of local A. angustulus males. The Rutowski, 2011; Sánchez-Guillén et al., 2013). Male buprestid A. planipennis color morphs have greater chroma than a typical beetles (Coleoptera) perform visually guided mating flights toward A. angustulus female target, whose spectral emission profile spans females (Gwynne and Rentz, 1983; Lelito et al., 2007, 2011; that of three of these morphs. This experiment thus allowed us to determine which particular portions of the spectrum emitted by A. angustulus females are most attractive in eliciting male mating 1The Pennsylvania State University, Department of Entomology, University Park, PA 16802, USA. 2United States Department of Agriculture, Animal and Plant Health responses, both at the initiation and at the completion of such Inspection Service, Plant Protection and Quarantine, Brighton, MI 48116, USA. stereotypical mating flights. 3NARIC Forest Research Institute, Department of Forest Protection, Mátrafüred 3232, Hungary. 4Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest 1022, Hungary. MATERIALS AND METHODS Agrilus planipennis color morphs *Author for correspondence ([email protected]) All A. planipennis female color morphs were obtained from a large- M.J.D., 0000-0002-3055-3533 scale rearing facility in Brighton, MI, USA, run by the USDA Animal and Plant Health Inspection Service. The facility Received 22 January 2016; Accepted 29 June 2016 continuously rears A. planipennis using natural host plant Journal of Experimental Biology 2837 RESEARCH ARTICLE Journal of Experimental Biology (2016) 219, 2837-2843 doi:10.1242/jeb.137885 material. Most individuals of the species are observed to be green, were not intended to be characteristic of the spectral reflectance but rarely phenotypes emitting longer (red) or shorter (blue) patterns of the leaves used in the experiment, but rather to provide wavelengths have been observed (<1% of all beetles). Pairs of the reader with a visualization of where in the spectrum peak similar rare phenotypes were mated to each other in an attempt to reflectance due to chlorophyll pigmentation typically occurs in produce true-breeding lines of these differently colored beetles. comparison to the beetles. Measurements were performed using a Lines were obtained in which at least half the offspring had mutant Lambda 950 photospectrometer (PerkinElmer, Bridgeville, PA, phenotypes, but a detailed genetic analysis of these lines will not be USA) with a 150 mm integrating sphere equipped with a presented here. Within the short-wavelength lines, two visibly microfocus lens and mechanical iris to establish a beam size of distinct phenotypes, blue and violet, were noticed (Fig. 1A). 2 mm, which had an angle of incidence of 0 deg. All sample spectra Likewise, within the long-wavelength lines there were often two were referenced to a Spectralon™ (Labsphere, Inc., North Sutton, other discrete phenotypes, copper and red (Fig. 1A). Sample sets of NH, USA) reflectance standard. Specimens were prepared using females of each color morph were set aside for behavioral elytra from dead specimens that were less than 1 year old and experiments. The females were killed by freezing overnight maintained at room temperature in closed containers to prevent and pinned through the anterior portion of the pronotum. The degradation that might influence coloration. The beam illuminating mean (±s.e.m.) lengths of the beetles in these samples were not the specimens was variably larger than the elytra of the A. angustulus substantially different across color morphs (violet: 11.60±0.19 mm, specimens. Thus, accurate comparative measurements of total blue: 11.73±0.37 mm, green: 12.14±0.09 mm, copper: 11.55± reflectance of this species were not possible. The wavelength of 0.05 mm, red: 11.96±0.19 mm). Similarly, the mean (±s.e.m.) peak reflectance from each trace was assessed after smoothing using pronotum widths deviated little among morphs (violet: 2.90± cubic spline interpolation. The smoothed traces were compared with 0.04 mm, blue: 2.83±0.06 mm, green: 2.91±0.03 mm, copper: the original data to ensure that the shape and position of the peaks 2.85±0.05 mm, red: 2.87±0.05 mm). A single specimen of each did not shift. Although this procedure was used to provide the most color morph was also set aside for spectral reflectance analysis. accurate estimate of peak reflectance, unaltered spectral traces are Because such analysis required a destructive preparation, and the used in all displays of the data. number of color morphs obtained was limited, we did not perform measurements from all specimens from all lines. Destroying the Field site samples to perform such measurements would preclude the Observations and collections were performed in a mixed oak forest possibility of future behavioral experiments involving these rare site near Mátrafüred, Hungary (47°50′17″N, 19°59′50″E). Within morphs and