Northeastern Naturalist B147 S.T. Deyrup, et al. 2017 Vol. 24, Special Issue 7 Escape into Winter: Does a Phenological Shift by Ellychnia corrusca (Winter Firefly) Shield It from a Specialist Predator (Photuris)? Stephen T. Deyrup1, Riley G. Risteen2, Kathareeya K. Tonyai2, Madalyn A. Farrar2, Bailey E. D’Antonio2, Zenab B. Ahmed1, Brian T. Christofel1, Nicole R. Howells1, and Scott R. Smedley2,* Abstract - Ellychnia corrusca (Winter Firefly) is one of few winter-active insects. Exposed throughout the season, this beetle appears vulnerable to insectivorous predators, but was recently shown to possess lucibufagins (LBGs), potent chemical defenses. The Winter Firefly is closely related to summer-active fireflies. To provide an adaptive explanation for this apparent phenological shift, we hypothesized that winter activity may protect the Winter Firefly from summer-active fireflies in the genus Photuris, predators that specialize on LBG-containing prey. To test this hypothesis, we studied the Winter Firefly and Photuris that occur sympatrically, but asynchronously as adults, at our Connecticut field sites. Through 2 experiments, we demonstrated that Photuris selectively consumes the Winter Firefly and sequesters its LBGs. Our findings are consistent with a hypothesis that winter activity by the Winter Firefly, by enabling early spring reproduction, provides phenological escape from a specialist predator. Introduction Typically ectothermic, insects face challenges in the winter environments of temperate and boreal biomes of northeastern North America and beyond (Heinrich 2003, Marchand 2013), where winter is commonly a season of diapause (Tauber et al. 1986). Complex life cycles afford insects with multiple stages in which to potentially overwinter. Many species diapause in the egg or pupa stage, both of which effectively protect the organism from the environment (Danks 1978, Leather et al. 1993). There are, however, exceptional insects and related arthropods that overwinter as adults that are active during warmer winter days. At the proximate level of analysis (MacDougall-Shackleton 2011, Tinbergen 1963), the mechanisms of cold tolerance underlying winter insect-activity have received a fair amount of attention (Block 1990, Danks 1978, Denlinger and Lee 2010, Sinclair et al. 2003, Teets and Denlinger 2013). However, at the ultimate level, an understanding of the adaptive value of these shifts to wintertime activity by insects is less developed, often speculative, and frequently focused on greater capacity for dispersal and reduced predation (Hågvar 2010). 1Department of Chemistry and Biochemistry, Siena College, Loudonville, NY 12211. 2Department of Biology, Trinity College, Hartford, CT 06106. *Corresponding author - scott. smedley@trincoll.edu. Manuscript Editor: Sara Lewis Winter Ecology: Insights from Biology and History 2017 Northeastern Naturalist 24(Special Issue 7):B147–B166 Northeastern Naturalist S.T. Deyrup, et al. 2017 B148 Vol. 24, Special Issue 7 Given the phyletic dominance of beetles, with over 350,000 described species (Bouchard et al. 2009, McHugh and Leibherr 2009), there are disproportionately few reports of winter-active coleopterans, and those appear largely restricted to species that overwinter in the relatively mild subnivean zone (Aitchison 1979, Jaskuła and Soszyńska-Maj 2011). Among North American fireflies (Coleoptera: Lampyridae), Ellychnia corrusca L. (Winter Firefly) is unique because it overwinters as an adult, becoming active on warm winter days. Ambiguity surrounds the taxonomy of this species and a number of researchers consider it a species complex (Fender 1970, Lewis 2016, Lloyd 2003). The Winter Firefly has an extensive geographic range, extending from the Canadian Maritimes to the southeastern US, and westward into the Great Plains (Lloyd 1993, Luk et al. 2011, Majka 2012). Recent phylogenetic analyses (Martin et al. 2017, Stanger-Hall and Lloyd 2015) suggest Ellychnia as the sister group of Photinus, a wide-spread genus of summer-active fireflies. Aspects of the winter ecology of the Winter Firefly are well-documented (Faust 2012, Rooney and Lewis 2000, Williams 1917), particularly its reproductive phenology. In northeastern Massachusetts, Winter Firefly adults appear from late-summer through autumn on tree bark (Fig. 1A), where they overwinter (Rooney and Lewis 2000). The insects remain exposed in this microhabitat throughout the winter and mate diurnally in the early spring, before dispersing. That generation of adults dies by late spring or early summer. In the fall, the recently emerged adults have substantial fat reserves, but no internal evidence of sexual maturation (Rooney and Lewis 2000). Adult Winter Fireflies in eastern Tennessee typically appear on tree trunks from October to February, depending on conditions during the previous larval growing season, with mating and dispersal starting in mid-March (Faust 2012, 2017). Considering that the Winter Firefly’s diurnal mating behavior deviates from the typical nocturnal courtship and mating of fireflies, it is not surprising that adults lack a light organ (Stanger-Hall et al. 2007) and apparently rely on pheromonal signaling (Ming and Lewis 2010, South et al. 2008), as do other diurnal lampyrids (Lloyd 1972). Figure 1 (following page). Adult Winter Firefly, adult Photuris, and their experimental interaction. (A) Winter Firefly (indicated by arrow) in typical resting pose in furrow of Quercus rubra L. (Red Oak) bark, 4 October 2016, Andover, CT. (B) Winter Firefly active during mating period on surface of Qurecus alba L. (White Oak), 8 May 2015, Andover, CT. (C) Winter Firefly reflex bleeding during predatory attack simulated by gentle pinch with forceps. (D) Photuris reflex bleeding (exposed hemolymph indicated with arrows) along margins of elytra. Hemolymph was analyzed for lucibufagin content in Experiment I. (E) Photuris left elytron (anterior to panel’s left) showing 4 pores along dorsal surface of outer margin (scanning electron micrograph). These pores have a thin cuticular seal that ruptures during reflex bleeding releasing underlying hemolymph. Seal is slightly elevated by escaping hemolymph in F. (G–I) Sequence during attack and consumption of a Winter Firefly by Photuris. (G) Soon after initiation of attack, ventral surface of the Winter Firefly facing upward beneath embracing Photuris. (H) Partial consumption (27 min following G). (I) Near-complete consumption of softer portions, with disarticulated wings, legs, pronotum, and head remaining (57 min following H), rating a consumption score of 3. Scale bars: B = 5 mm, D = 2 mm, E = 100 μm, and F = 10 μm. Northeastern Naturalist B149 S.T. Deyrup, et al. 2017 Vol. 24, Special Issue 7 Several lines of evidence suggested that the Winter Firefly is likely chemically protected from predators. It is exposed on tree trunks (Fig. 1A, B) to insectivorous predators for extended winter periods, when other insect prey are of limited Figure 1. [Caption on preceeding page.] Northeastern Naturalist S.T. Deyrup, et al. 2017 B150 Vol. 24, Special Issue 7 availability. It is visually aposematic (Fig. 1A–C). When disturbed, the Winter Firefly reflex-bleeds (Fig. 1C), which is a common response of insects with hemolymphborne chemical defenses (Eisner 2003, Eisner et al. 2005). Salticid spiders find Winter Fireflies unpalatable (Long et al. 2012). Finally, close relatives of the Winter Firefly in the genus Photinus are known to possess steroidal chemical defenses known as lucibufagins, hereafter LBGs (Eisner et al. 1978; Goetz et al. 1979, 1981, Meinwald et al. 1979). Our recent investigation of the Winter Firefly’s chemistry revealed a mixture of 8 LBGs (Fig. 2), 4 of which were previously unknown compounds (Smedley et al. 2017). We also demonstrated the highest LBG content for a firefly known to date, and that reflex bleeding by the Winter Firefly does externalize these compounds, previously shown to have potent antifeedant activity against both vertebrate (Eisner et al. 1978) and invertebrate generalist predators (Eisner et al. 1997, González et al. 1999). Members of the firefly genus Photuris occur sympatrically with the Winter Firefly at our Connecticut study sites and throughout much of its geographic range. Unlike the Winter Firefly, Photuris spp. occur as summer-active adults that do not appear to possess endogenous LBGs (Eisner et al. 1997). However, certain Photuris spp. acquire them otherwise. In a classic example of aggressive mimicry, females of particular Photuris species mimic the reply of a female Photinus given in response to the courtship flash of her conspecific male. The duped male Photinus is then eaten by the larger female Photuris (Lloyd 1965, 1975), which proceeds to sequester his LBGs for her own defense (Eisner et al. 1997) and that of her eggs (González et al. 1999). Beyond aggressive mimicry, other behaviors of these specialist predators seem likely, but unexamined, avenues of Figure 2. Structures of lucibufagins found in Winter Firefly. Molecules 1–4 are previously known from Photinus, while 5–8 represent recently reported novel structures (Smedley et al. 2017). Northeastern Naturalist B151 S.T. Deyrup, et al. 2017 Vol. 24, Special Issue 7 LBG acquisition: hawking and kleptoparasitism. In hawking, the female Photuris locates a male Photinus, by detecting his courtship flash signal, and then attacks him in flight (Lloyd and Wing 1983). In kleptoparastism (Faust et al. 2012), the female steals captured Photinus from spider webs, likely responding to the aposematic flash signal produced by the prey. Merging this understanding of the predatory ecology of Photuris with our newly aquired knowledge of the chemical defenses of the Winter Firefly, we developed a hypothesis to attempt to explain the adaptive significance of the winter activity of adult Winter Fireflies as a prelude to spring reproduction and adult senescence. Additionally, the Winter Firefly’s diurnal activity, while not unique (Stanger-Hall and Lloyd 2015), is not the norm for lampyrids. Could these phenological and diel shifts by the Winter Firefly have evolved to afford protection from summer-active, nocturnal Photuris, by eliminating or greatly minimizing temporal overlap between potential prey and the LBG-seeking predator? Demonstration that Photuris spp. consume Winter Fireflies, potentially acquiring their LBGs, and thus imposing selective pressure as a predator, would be necessary support for this hypothesis. To address this question, we performed 2 laboratory experiments that brought together these naturally asynchronous species. Based on these encounters, we here report that, given the opportunity, female Photuris, will indeed consume Winter Fireflies and sequester their LBGs. Field-site Description All field sites were located in the rural, eastern Connecticut towns of Andover and Bolton. Winter Firefly and Photuris were common and sympatric at these sites. However, phenological overlap of their adult stages was nearly non-existent. Locally, the period of adult activity for Winter Firefly was mid-September to late May/ early June (with very rare encounters after late May), while that of Photuris was early June to early August. The adult Winter Fireflies that we used in this study were readily encountered in the early spring on tree trunks as they concluded their overwintering and initiated mating. Our 2 source-sites for these beetles were Camp Johnson, Bolton, CT (41°46'39.83''N, 72°25' 26.22''W), and a woodland in Andover, CT (41°43'44.62''N, 72°21'56.45''W). General habitat was secondary mixed deciduous forest. Tree species with more deeply furrowed bark (e.g., Fraxinus spp. [ash] and Quercus spp. [oaks]) were preferred substrates. We collected adult female Photuris in early June 2015 in a wooded cattle pasture and adjacent hayfield (bordered by forest) on the Siismets Farm, Andover, CT (41°43'58.15''N, 72°23'2.51''W). Taxonomically, Photuris is an extremely challenging genus (Faust 2017, Lewis 2016). A series of vouchers of male Photuris collected at the same time as the above females consisted of specimens potentially representing multiple, morphologically indistinguishable species of the versicolor complex (J. Lloyd, University of Florida, Gainesville, FL, pers. comm.). Consequently, throughout this paper, we refer to them generically as Photuris. During the early June 2015 collection period, several species of Photinus were flying with Northeastern Naturalist S.T. Deyrup, et al. 2017 B152 Vol. 24, Special Issue 7 the Photuris at the study site. Although we did not encounter Winter Fireflies then, we did find them earlier in mid-May 2015 on the pasture’s trees and in flight, and again on those trees in October. The hayfield also served as a source of larval Photuris in fall 2015, as did lawns of 2 nearby yards (41°44'27.13''N, 72°23'28.46''W and 41°44'9.16''N, 72°23'25.43''W), both within 1.1 km of the h ayfield. We deposited voucher specimens from our study within the Florida State Collection of Arthropods, Florida Department of Agriculture, Gainesville, FL: Winter Firefly adults (n = 26, “SmedBC#01” to “SmedBC#26”); Photuris male adults collected on 10 June 2015 with female Photuris used in Experiment I (n = 6, “SmedBC#30” to “SmedBC#35”); and Photuris adults reared from a series of field-collected larvae that also produced the female Photuris used in Experiment II (n = 4, “SmedBC#36” to “SmedBC#39”). Methods To determine whether adult Photuris females can consume adult Winter Fireflies, and if so, whether they acquire LBGs from these prey, we conducted 2 experiments that brought together these sympatric fireflies that do not overlap temporally as adults in the field. Experiment I: Field-collected Photuris vs. previously frozen Winter Fireflies Collection and storage of adult Winter Firefly. We collected adult Winter Fireflies at Camp Johnson on 4 May 2015, near the end of their active season. We were careful to prevent disturbance and resultant reflex bleeding. We placed beetles singly into separate 18-ml (5-dram) plastic vials, froze, and stored them at -20 °C until female Photuris were available for experimentation. Collection of adult female Photuris. We collected adult female Photuris on the Siismets farm, with 1 exception: an individual collected nearby (1.1 km to the NW). We made our collections during 10–17 June 2015, the earliest part of the local Photuris flight season, to minimize the opportunity for these females to have acquired LBG as femme fatales or via related behavior. Females were typically spotted at night as they emitted their flash signal while perched on vegetation. They were individually housed in plastic vials. General experimental procedure. In the laboratory the morning following collection, we confirmed the sex of each specimen through stereomicroscopic examination of the sexually dimorphic light organ of Photuris (Lewis 2016, Williams 1916). We then transferred each beetle to its own 60 mm x 15 mm plastic Petri dish supplied with a 1.5 cm x 1.0 cm (length x diameter) piece of braided dental cotton saturated with 10% honey water (v/v %) and randomly assigned each one to an experimental group. Treatment (n = 16) and control (n = 16) Photuris received either frozen Winter Firefly (unsexed) or frozen adult Tenebrio molitor L. (Mealworm Beetle), respectively, as a potential meal according to the schedule in Table 1A. We selected Mealworm Beetle as a negative control because our analysis (1H NMR, described below) of a sample of 30 adults, showed that this species lacked LBGs (S.T. Deyrup et al., unpubl. data). Approximately Northeastern Naturalist B153 S.T. Deyrup, et al. 2017 Vol. 24, Special Issue 7 24 h after the presentation of each meal, we collected any remnants of the potential prey item for later scoring of the degree of consumption by the Photuris. We maintained the Photuris at 22 °C under humidified conditions with a 14-h light/10-h dark cycle (hereafter referred to as standard rearing conditions). Sampling of reflex bleeding. Due to the field origin of these Photuris, with unknown behavior prior to capture, we sought to establish the baseline LBG content of each individual at the onset of the experiment. Thus, we collected a sample of hemolymph, released via reflex bleeding, on Day 2 (Table 1A), which allowed >24 h for the organisms to incorporate LBG from any meal obtained in the field prior to collection. To elicit reflex bleeding, we gently squeezed the beetle across the thorax/abdomen with fine-tipped forceps. We collected the resulting droplets of hemolymph, released along the elytral margins (Fig. 1D–F) and coxal joints, with a 5-μl glass microcapillary tube. We determined the mass of the hemolymph to the nearest 0.01 mg, then broke off and froze (-20 °C) the portion of the microcapillary tube containing the sample until chemical analysis. Using the same procedure, we obtained a second hemolymph sample on Day 6, at the close of the individual’s experimental tenure, at which time we determined each individual’s overall mass and froze each specimen as a whole-body sample for LBG analysis. Scoring of meal consumption. Stereomicroscopic inspection allowed the condition of the remnants of each meal (see above) to be categorized on a 4-grade scale: 0 = no apparent damage; 1 = minor puncture damage, apparently resulting from mandibular insertion without tearing of the cuticle; 2 = major cuticular tearing/ breakage, but no dismemberment of body; and 3 = dismemberment (Fig. 3). For each Photuris, we calculated a cumulative consumption score (0–9) by summing the scores of each of its 3 meals (provided on Day 2–Day 4; Table 1A). Table 1. Photuris femmes fatales vs. Winter Firefly experiments. Schedules for key activities. Experiment I (A): field-collected Photuris presented previously frozen Winter Firefly and Mealworm Beetle; Experiment II (B): reared Photuris presented live Winter Firefly and Mealworm Beetle. Day Activity (A) Experiment I 0 Field capture 1 Set-up 2 Hemolymph sample; meal 1 3 Change honey water; meal 2 4 Meal 3 5 • 6 Hemolymph sample; obtain whole body sample (B) Experiment II ≤0 Adult emergence in lab; potential refrigeration 1 Set-up; meal 1 2 Meal 2 3 Change honey water; meal 3 4 • 5 Obtain whole-body sample Northeastern Naturalist S.T. Deyrup, et al. 2017 B154 Vol. 24, Special Issue 7 Experiment II: Reared Photuris vs. live Winter Firefly By rearing field-collected Photuris larvae to adulthood in the laboratory, we were able to shift their natural phenology to cause them to mature early and thus overlap as adults with naturally occurring, live adult Winter Fireflies. Unlike in Experiment I, we were certain that those adults had not acquired LBG as femme fatales or via related behavior prior to experimental use. Thus, we were able to conduct a similar experiment, but without the necessity of establishing baseline LBG-levels via hemolymph sampling. Collection and storage of adult Winter Firefly. We collected Winter Firefly adults from Camp Johnson on 3 March 2016 and the Andover woodland on 24 March 2016, following the previously described procedure, and stored them alive at 4 °C until presentation to the female Photuris. Collection and rearing of larval Photuris. We collected larval Photuris nocturnally on 7 evenings from 30 September to 13 October 2015 at the sites indicated above. The larvae were conspicuous, due to their bioluminescence, and were easily found on the ground and low vegetation. The next day, we transferred 10 larvae per dish to 15.5 cm x 2.5 cm plastic rearing dishes lined with 2 sheets of 15-cm diameter (P8 qualitative grade) filter paper that had been moistened with deionized water. We placed these dishes in humidified plastic boxes, which were stored at 10 °C in a dark incubator, following a rearing procedure modified from McClean et al. (1972). On 22 October 2015, we placed the following items in each dish: 2 live Eisenia fetida (Savigny) (Red Wiggler Earthworm), 3 chopped Red Wiggler Earthworms, and 5 pieces of dry cat chow (Stop and Shop Companion Brand®, Blended Formula), which had been soaked in warm tap water. We then placed the dishes under standard rearing conditions for ~24 h, then transferred the larvae to new dishes Figure 3. Adult Winter Firefly (ventral view) representing the 4 scores (0–3) for the degree of meal consumption. (A) uninjured. (B) arrow indicates minor puncture wound (< 0.5 mm diameter) in abdominal sternite. (C) arrow indicates a substantial gash (2 mm wide) on thoracic venter. (D) dismembered body parts following major consumption. Scale bar in A = 5 mm, with identical magnification throughout. Northeastern Naturalist B155 S.T. Deyrup, et al. 2017 Vol. 24, Special Issue 7 without food, and returned the dishes to the 10 °C/dark conditions. We repeated this feeding regimen every 21 days. Following the feeding on 14 January 2016, we transferred each larva to its own pupation container, a 59.2 ml (2-oz) plastic soufflé cup (Solo® product B200) with a cap. Photuris larvae naturally pupate in a subterranean chamber; thus, we lined the cup with 25 ml of soil from the organic layer at the main larval collection site (dried, then sifted through a #18 sieve) and moistened with 13 ml of deionized water. We partially buried a 3-cm piece of braided cotton that served as a wick to augment moisture levels. The cups were collectively stored in humidified boxes under standard rearing conditions, with regular provisioning of a fresh piece of soaked cat chow. We inspected the cups daily, and adult Photuris began to emerge in late February. Upon emergence, beetles were either immediately employed in Experiment II or stored (4 °C) until a sufficient number were available. General experimental procedure. We maintained and scored meal consumption of female Photuris as in Experiment I; however, the beetles were organized into triads (n = 8) based on emergence date, and no hemolymph sampling took place. We randomly assigned members of a triad to either of 2 treatments—presentation of a live male or female Winter Firefly, respectively—or a live Mealworm Beetle control. The Winter Firefly has a more pliable cuticle than the Mealworm Beetle; thus, we excised the control’s right elytron to make it potentially more vulnerable to attack by Photuris. Experiment II was conducted according to the schedule in Table 1B. At the culmination of the experiment (Day 6) for a triad, we collected a whole-body sample for each of its members. Analytical chemistry: Lucibufagin detection To establish whether individual Photuris contained LBGs, we employed proton nuclear magnetic resonance (1H NMR) and ultra-high–pressure liquid chromatography– high-resolution mass spectrometry (UHPLC-HRMS) to analyze whole-body and hemolymph samples, respectively. Each sample was presented to the chemists in a blind manner, without disclosing an individual beetle’s treatment group. NMR analysis. Each Photuris whole-body sample was placed in a glass scintillation vial, pulverized with a glass stir-rod, extracted with 1 ml CDCl3, and filtered through cotton into an NMR tube. To acquire a 1H NMR spectra, analysts employed a 500-MHz spectrometer (Bruker, Ascend®500, Bruker Corporation, Coventry, UK) at room temperature using standard parameters (pulse program = zg30; TD = 65,536; acquisition time = 3.277 sec; D1 = 1.0 sec) and 256 scans. NMR spectra were processed using Mnova 9.0 (MestreLab Research S.L., A Coruña, Spain), and the residual solvent peak was used for calibration (CHCl3, d = 7.260 ppm). We considered LBGs to be present if NMR signals indicative of an α-pyrone moiety occurred in the spectrum (d = 7.68 ppm, dd, J = 9.8 Hz, 2.6 Hz; d = 7.39 ppm, dd, J = 2.6 Hz, 0.8 Hz; d = 6.29 ppm, dd, J = 9.8 Hz, 0.8 Hz) at a signal-to-noise ratio of 3:1 or greater. UHPLC-HRMS analysis. We pulverized the microcapillary tube segments containing hemolymph samples with a glass rod in separate 3.6-ml (1-dram) glass vials, rinsed the resulting fragments 3 times with 0.2 ml HPLC-grade acetonitrile, Northeastern Naturalist S.T. Deyrup, et al. 2017 B156 Vol. 24, Special Issue 7 and transferred the samples into 1.5-ml Agilent HPLC vials (Agilent Technologies, Santa Clara, CA) for analysis. UHPLC separations were performed on an Agilent 1290 Infinity® Binary LC system using an Agilent Zorbax Eclipse Plus® C18 column (2.1 mm x 50 mm, 1.8 m) with an injection volume of 1 ml. The solvent system was 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B), with the method consisting of 80% solvent A for 1 min followed by a linear gradient to 100% solvent B over 8 min and a 4-min hold at 100% solvent B. The flow rate was maintained at 260 ml/min throughout the 12-min protocol. We acquired HRMS data on a Bruker Impact HD® qTOF spectrometer operating in positive electrospray ionization mode. The observed range was 25–1000 m/z, and the acquisition rate was 3 spectra/s. Masses were calibrated using a sodium acetate solution as an internal standard. HRMS parameters were as follows: gas temperature 200 °C, gas flow 9.0 L/min, nebulizer gas pressure 1.0 Bar, capillary voltage 4500 V. We produced for each sample extracted-ion chromatograms (XIC) for 491 m/z and analyzed LBG content by measuring the intensity of the 491 m/z peak at retention time = 2.8–3.0 min. We ran blank injections after each sample to minimize possible carry-over between samples. We considered LBGs to be present if the intensity of the 491 m/z peak exceeded 1950 units, a value at least twice as high as that of background levels (Fig. 4B, E, F). Statistical analysis Except where noted, we performed all statistical analyses in GraphPad Prism 7.0a for Mac OS X. The D’Agostino-Pearson omnibus test of normality suggested that certain data series were non-Gaussian, and since there was no variance in the data for other treatment groups, we employed non-parametric tests. Fifty percent of the cells in the 3 x 2 contingency table for LBG presence/absence in Experiment II (Table 2B) had expected values of less than 5; thus, we performed the Freeman-Halton extension of the Fisher exact probability test for contingency (Freeman and Halton 1951) instead of the chi-square test in VassarStats (Lowery 2016). Results Consumption of Winter Firefly by female Photuris Female Photuris readily consumed Winter Firefly (Fig. 1G–I), but they did not eat Mealworm Beetle, as evidenced by comparison of the summation of each individual’s consumption scores (Fig. 5). Video of a female Photuris nearing completion of a meal of Winter Firefly in a laboratory encounter is offered as Supplemental File 1 (available online at http://www.eaglehill.us/NENAonline/suppl-files/n24-sp7- N1468x-Smedley-s1, and, for BioOne subscribers, at http://dx.doi.org/10.1656/ N1468x.s1). Photuris consumed more dead (previously frozen, unsexed) Winter Fireflies than Mealworm Beetles (Experiment I; Fig. 5A). Photuris consumed more live male Winter Firefly than live Mealworm Beetle, while consumption of live female Winter Fireflies was at an intermediate level, not differing from either their male counterparts or the other coleopteran (Experiment II; Fig. 5B). Northeastern Naturalist B157 S.T. Deyrup, et al. 2017 Vol. 24, Special Issue 7 Acquisition of Winter Firefly LBGs by female Photuris Whole-body analyses. NMR analysis of whole-body samples of female Photuris revealed major variation in their LBG content (Fig. 6). The presence of LBG in female Photuris was strongly associated with their treatment group in both Experiments I (Table 2A) and II (Table 2B): females with access to Winter Fireflies had a much greater frequency of detectable LBG than those with access to Mealworm Beetles. Photuris whole-body mass did not differ between treatment groups in either experiment (Experiment I: Mann-Whitney U = 87, P = 0.13; Experiment II: Kruskal-Wallis statistic = 1.04, P = 0.59). Hemolymph analyses. Analysis (UHPLC-HRMS) of Photuris female hemolymph samples showed variation in their LBG content (Fig. 4). For a given individual in Experiment I, we compared the intensity (I ) of the LBG peak (at 491 m/z) for the hemolymph samples obtained prior to treatment and at the close of the Figure 4. Representative UHPLC-HRMS chromatograms used to assign presence or absence of lucibufagins in hemolymph of female Photuris. Each panel represents an extracted- ion chromatogram (XIC) at m/z 491. (A, D) LBG sample purified from Winter Fireflies. (B) hemolymph from a Photuris prior to consuming a Winter Firefly. (C) hemolymph of same individual in B, but after consuming a Winter Firefly. (E) hemolymph from a Photuris prior to exposure to Mealworm Beetle. (F) hemolymph from same individual as in E, but after exposure to Mealworm Beetle. Northeastern Naturalist S.T. Deyrup, et al. 2017 B158 Vol. 24, Special Issue 7 experiment (i.e., ΔI = IDay6 - IDay2). Within the treatment group of females receiving Winter Firefly (n = 14 for which both hemolymph samples were obtained), there was a substantial increase in hemolymph LBG content (Wilcoxon signed-rank W = 105, P < 0.0001; median ΔI = 30,616 units). However, within the control group of females receiving Mealworm Beetles (n = 15 for which both samples were Figure 5. Female Photuris consumption of E. corrusca (Winter Firefly) or T. molitor (Mealworm Beetle): frequency of Photuris achieving each of the 10 possible cumulative consumption scores. See Methods section for scoring procedure. Experiment I (A): field-collected Photuris (n = 16/treatment) presented previously frozen Winter Firefly and Mealworm Beetle; Experiment II (B): reared Photuris (n = 8/treatment) presented live Winter Firefly and Mealworm Beetle. In all cases, arrow signifies median value. In Experiment I, Winter Firefly was consumed at a higher level than Mealworm Beetle controls (Mann- Whitney U = 8, P < 0.0001). In Experiment II, consumption level varied with the potential prey offered (Kruskal-Wallis statistic = 10.59, P = 0.005). Multiple comparison (Dunn’s test) results are presented with red lower-case letters. Medians, representing the respective frequency distributions, sharing a letter did not vary at an experiment-wide a = 0.05. Northeastern Naturalist B159 S.T. Deyrup, et al. 2017 Vol. 24, Special Issue 7 obtained), there was no change in hemolymph LBG content (W = 61, P = 0.09; median ΔI = 57 units). Furthermore, while IDay2 did not vary between the 2 groups (Mann-Whitney U = 76, P = 0.051), at the experiment’s close, IDay6 was greater for the treatment group receiving Winter Fireflies than for the control group receiving Mealworm Beetles (Mann-Whitney U = 15, P < 0.0001). Two female Photuris assigned to the Winter Firefly treatment and 2 assigned to the Mealworm Beetle control showed elevated hemolymph LBG levels prior to experimen tal use (IDay2 > Table 2. Female Photuris whole-body lucibufagin content (determined by 1H NMR) as a function of dietary access to Winter Firefly or Mealworm Beetle: contingency-table analyses. Experiment I (A): field-collected Photuris presented previously frozen Winter Firefly and Mealworm Beetle; Experiment II (B): reared Photuris presented live Winter Firefly and Mealworm Beetle. In Experiment I, Photuris with access to Winter Firefly were more likely to have detectable LBG levels than those with access to Mealworm Beetle controls (χ2 Yates Correction = 18.66, df = 1, P < 0.0001). In Experiment II, a higher frequency of individuals with detectable LBG levels was associated with the 2 Winter Firefly treatments compared to the Mealworm Beetle control (Fisher exac t test: P < 0.0001). (A) Experiment I Treatment LBG detected LBG not detected Winter Firefly 16 0 Mealworm Beetle 3 13 (B) Experiment II Treatment LBG detected LBG not detected Winter Firefly, male 8 0 Winter Firefly, female 8 0 Mealworm Beetle 1 7 Figure 6. Selected portions of the 1H NMR spectra of female Photuris: (A) an individual that did not contain lucibufagins, and (B) an individual that contained LBGs, following consumption of Winter Firefly. Boxes highlight the regions of the spectra that were used to determine presence or absence of LBGs. Residual solvent peaks are labeled above their signals. Northeastern Naturalist S.T. Deyrup, et al. 2017 B160 Vol. 24, Special Issue 7 1980 units, in all 4 cases). The mass of the paired Photuris hemolymph samples was not different between the Winter Firefly treatment group (W = -5, P = 0.89) and the Mealworm Beetle control group (W = 19, P = 0.54). There was also no significant difference in the mass of the hemolymph samples between the treatment and control groups overall (Kruskal-Wallis statistic = 0.39, P = 0.96). Discussion The Winter Firefly and members of the genus Photuris occur sympatrically in ecological communities throughout eastern North America. Considering that the Winter Firefly is closely related to the summer-active genus Photinus (Martin et al. 2017, Stanger-Hall and Lloyd 2015) and that western North American Ellychnia are active as adults during warmer months (Fender 1970), the wintertime activity of adult Winter Fireflies is likely a derived feature. By overwintering as an adult and reproducing in spring (Faust 2012, Rooney and Lewis 2000), the LBG-possessing Winter Firefly (Smedley et al. 2017) greatly minimizes temporal overlap with summer- active Photuris, LBG-seeking specialist predators of fireflies. To support this ultimate-level hypothesis explaining the Winter Firefly’s phenological shift, it is necessary to determine that it is vulnerable to predatory Photuris. A scenario under which the Winter Firefly would be immune to Photuris would be if it maintained LBGs as a defense against generalists, but had evolved another line of chemical defense effective against these specialists. Both theoretical (Brockhurst et al. 2014) and empirical (Becerra et al. 2009, Berenbaum and Feeny 1981) coevolutionary studies have suggested that organisms may diversify their chemical defenses as an escalatory response to specialist consumers that have overcome chemical armature targeted towards generalists. Given the constraints of present-day firefly seasonality and diel activity patterns, to determine the potential vulnerability of the Winter Firefly vis-à-vis Photuris, it proved necessary to bring these sympatric, but asynchronous, species together in a laboratory setting. In both Experiments I (field-collected Photuris vs. previously frozen Winter Firefly) and II (lab-reared Photuris vs. live Winter Firefly), Photuris consumed Winter Fireflies preferentially to the Mealworm Beetle control; in fact, none of the latter were eaten (Fig. 5), including those rendered more vulne rable due to elytron removal (Experiment II). This result is clearly consistent with Photuris responding to an LBG-positive Winter Firefly (Smedley et al. 2017). LBGs are demonstrated phagostimulants for Photuris (Gronquist et al. 2006): LBG-free items, such as Mealworm Beetle, fail to elicit ingestion by Photuris; however, treatment of such items with LBG leads to their consumption (Gronquist et al. 2006). Consumption of Winter Fireflies was slightly higher (Mann-Whitney U = 73, P = 0.0319) in Experiment I than that in Experiment II (male and female Winter Firefly combined), perhaps due to the differences in the beetles between the 2 experiments. In Experiment I, the Winter Fireflies were dead, while in Experiment II they were alive and therefore more capable of evading attack. In Experiment I, the Photuris were field-collected, and, thus, potentially more vigorous than their lab-reared counterparts in Experiment II. The arenas in which experimental encounters of Photuris Northeastern Naturalist B161 S.T. Deyrup, et al. 2017 Vol. 24, Special Issue 7 and Winter Fireflies took place were clearly much less structurally complex than the substrates upon which Photuris predator–prey interactions naturally occur, and, thus, may have influenced the frequency of prey capture in Experiment II (see Lewis et al. 2012). However, the laboratory conditions we employed seemed an appropriate setting for an initial assessment of whether or not Photuris is capable of consuming the Winter Firefly and potentially acquiring its LBGs. Whole-body analysis of female Photuris at the close of both experiments showed that those with access to Winter Firefly prey had elevated LBG content compared to those that received the Mealworm Beetle controls (Table 2). This finding, coupled to the observed preferential consumption of the Winter Firefly, suggests that its ingestion by Photuris leads to the predator’s sequestration of LBG, as has been demonstrated for other LBG-possessing prey, Photinus (Eisner et al. 1997) and Lucidota atra (G. Oliver) (Black Firefly; Gronquist et al. 2006). The selective pressure favoring such predation by female Photuris is apparent: acquisition of exogenous LBG protects both them and their eggs from predation (Eisner et al. 1997, González et al. 1999). In all instances in Experiment II, where female Photuris inflicted no visible damage (n = 6, with consumption score (Σ) = 0; Fig. 5B) upon their potential Winter Firefly prey, those females nonetheless showed detectable LBG levels. During Experiment II, disturbance by Photuris was observed to result in reflex bleeding by the Winter Firefly, so LBG sequestration by these individuals may well have stemmed from imbibing the LBG-laden hemolymph (Smedley et al. 2017) that was released defensively. The increase in hemolymph LBG levels over the course of Experiment I for female Photuris receiving Winter Firefly, but not for those receiving Mealworm Beetle further supports that access to the Winter Firefly results in LBG uptake by Photuris. Interestingly, the random assignment of the female Photuris (field-collected as adults) in Experiment I resulted in 2 members of the treatment group and 2 members of the control group that displayed elevated LBG levels prior to involvement (i.e., on Day 2). A likely explanation for this result is that these individuals were successful femmes fatales or otherwise obtained LBG prior to capture. When provided with an opportunity during our laboratory experiments, Photuris females ate Winter Fireflies and sequestered their LBGs, which suggests that if the 2 species were to co-occur as adults in the field, the Winter Firefly would be vulnerable to this LBG-seeking predator. Thus, one could envision that selective pressure by this specialist predator may have favored a phenological shift to wintertime activity by the Winter Firefly. In Photinus, another genus with LBGs, females prefer more-conspicuous male courtship flashes (Branham and Greenfield 1996, Cratsley and Lewis 2003), but also subject those males to higher predation risk by Photuris femmes fatales (Woods et al. 2007). As previously mentioned, the adult activity patterns of the Winter Firefly appear to represent 2 temporal shifts from what is typical for fireflies: 1 phenological (to winter, coupled with spring reproduction) and the other diel (to the daytime). Diurnal activity would seem prerequisite for the phenological shift because the cool conditions of early spring nights would preclude reproductive behavior in Northeastern Naturalist S.T. Deyrup, et al. 2017 B162 Vol. 24, Special Issue 7 ectothermic fireflies; thus, diurnal activity (the genus Ellychnia is entirely diurnal [Fender 1970]) may have served as an exaptation for the unique phenology of the Winter Firefly. Assuming that ancestral Ellychnia also contained LBGs and occurred with Photuris, avoidance of this nocturnal predator could be 1 possible explanation for their diurnal activity, but any selective force leading to daytime activity would have predisposed the Winter Firefly to undergo this phenological shift. Within the LBG-possessing genus Photinus, P. indictus (LeConte) and P. cooki Green, which as adults are synchronous with Photuris adults, have independently lost their light organs (Stanger-Hall and Lloyd 2015) and have become diurnal. Lloyd (1990) has speculated that P. indictus became day-active in response to Photuris predation. When experimentally paired with Photuris, members of the diurnal, LBG-containing genus Lucidota (Gronquist et al. 2006), are eaten. This result suggets that their daytime activity could minimize such predation in nature (Gronquist et al. 2006, Lewis et al. 2012). Surveying additional diurnal taxa for the presence of LBGs and susceptibility to Photuris predation is clearly essential to test the prediction that such fireflies indeed contain LBGs and are vulnerable to the pred ator. By shifting its adult activity to a different season than that of Photuris, the Winter Firefly might have gained further protection than that afforded simply from a diel shift; under the latter scenario adults of the 2 species are nonetheless present in the same community at the same time, just less likely to encounter one another. Such protection might be particularly important to the Winter Firefly, given its extreme levels of LBGs (Smedley et al. 2017). Although its phenological shift is not as pronounced as that of the Winter Firefly, Pyractomena borealis (Randall) employs a combination of diurnal and nocturnal adult behavior (Faust 2012) and is reproductively active largely before the appearance of Photuris. We have recently detected LBGs in this species (S.T. Deyrup et al., unpubl. data), which is known to be vulnerable to Photuris femme fatales (Lloyd 1973). It will be interesting to see if P. borealis has relatively high LBG levels, thus suggesting a potential trend between elevated levels and a phenological shift. Phenological escape by prey animals to avoid predation has received limited attention (Evans 1990, Wissinger et al. 2006), although more focus has been placed on plants’ use of this strategy to avoid herbivores and granivores (Agrawal 2000, Aide 1992, Evans et al. 1989, Janzen 1969, Kinsman and Platt 1984, Parker et al. 2010). The Winter Firefly appears to have undergone a dramatic phenological shift to inhabit a temporal environment free of a specialist predator. Not only is it diurnal, as are some other fireflies, but it is exceptional among North American lampyrids with its wintertime adult activity. In doing so, however, it has subjected itself to other potential predation pressures. Insectivorous birds regularly traverse the tree trunks upon which the Winter Firefly overwinters, yet mark–recapture analysis suggests that this beetle experiences minimal winter mortality (Rooney and Lewis 2000). Avian predators are known to reject LBG-containing fireflies (Eisner et al. 1978, Gronquist et al. 2006) and normally palatable food treated with isolated LBG (Eisner et al. 1978). Winter Fireflies, with their LBG content of 625 ± 65 mg/beetle (mean ± SE) have the highest quantity of these compounds known for lampyrids, over 4 times that of Northeastern Naturalist B163 S.T. Deyrup, et al. 2017 Vol. 24, Special Issue 7 Photinus (Smedley et al. 2017). One wonders whether such high levels arose after the shift to winter activity, given these compounds’ anti-predatory activity against birds, and the fact that they would have conceivably made the Winter Firefly even more attractive in an environment with foraging Photuris. Finally, it should be noted that there may be multiple, non-mutually exclusive, ultimate-level hypotheses to explain the phenological shift to winter activity by adult Winter Fireflies. The Winter Firefly is a frequent host of a phorid parasitioid, Apocephalus antennatus Malloch, a specialist on lampyrids (Brown 1994, Faust 2012, Lloyd 1973). J. Lloyd ( pers. comm.) has proposed that the wintertime exposure of the Winter Firefly on tree trunks subjects them to extreme cold and freeze–thaw cycling that may prove detrimental to the parasitoid and therefore beneficial to the host. Evidence of the timing of infection of Winter Fireflies by A. antennatus is thus critical to evaluate this hypothesis. We are currently examining whether Winter Fireflies harbor this phorid while overwintering or whether the parasitoid strikes in the spring. Regardless of this outcome, the evidence obtained to date is consistent with the Winter Firefly’s unusual phenology functioning, at least in part, as a means of temporal escape from a specialist predator, Photuris. Acknowledgments This research was supported by Trinity College and Siena College, including a Trinity Faculty Research Committee stipend to Riley Risteen. The electron microscopy was funded in part by the National Science Foundation (MRI-1039588). Field-site access was kindly granted by Camp Johnson, Bolton, CT; the Town of Andover, CT; and the Sissmets, Palmer, and Smedley families. Thanks to Yunming Hu of the Trinity Electron Microscopy Facility for assistance and to Dr. Kris Kolonko of the SAInT Center at Siena College for aiding in acquisition of UHPLC-HRMS. We thank David Posner and Faizan Rahim for extraction and NMR data acquisition of Mealworm Beetle samples. We are grateful for field assistance provided by Glory Kim, various Trinity students, and Dr. Fred Oliver. 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