The origin of photic behavior and the evolution of sexual communication in fireflies (Coleoptera: Lampyridae)

Through a phylogenetic analysis using adult morphological characters, we show that the origin of bioluminescence in cantharoid beetles appears to predate the origin of the family Lampyridae. The ability to produce and emit photic signals was first gained by larvae and appears to function as an aposematic warning display; it was subsequently gained in adults and is used as a sexual signal. Our analysis also suggests that while pheromonal sexual signals are used basally in the family, they are used in conjunction with and then subsequently replaced by photic signals in some lampyrid lineages. Both photic signals and the photic organs used to produce them have become greatly elaborated in the fireflies that no longer employ pheromonal sexual signals. In addition, the ability to produce a flashed sexual signal appears to have arisen at least three times in the family Lampyridae. Convergent evolution is also evident in a number of adult male photic organ morphologies. Further, we recommend that individual signal system components be compared rather than overall signal system complexity. The use of this strategy may allow one to recognize and better interpret adaptive correlations despite convergence or loss. We demonstrate that phylogenetic analysis is a powerful tool even for rapidly evolving traits.     

Flash signals, nuptial gifts and female preference in photinus fireflies

The evolution of male courtship signals such as the bioluminescentflashes of fireflies may be shaped, at least in part, by femalepreference for particular characteristics of the male signal.These female preferences for male courtship signals may ariseas a result of the benefits of choosing males with particulartraits. One possible benefit of mate choice occurs if femalescan use male courtship signals as an honest indicator of malenutritional contributions at mating, nuptial gifts. This paperreviews female preference for male flash characteristics inPhotinus fireflies (Coleoptera: Lampyridae), and the potentialfor females to use male flash characteristics to predict nuptialgift quality. In Photinus firefly species with single pulseflashes females preferentially respond to flashes of greaterintensity and duration. Male Photinus provide a nuptial giftto females at mating in the form of a spermatophore and flashduration serves as a good predictor of spermatophore mass formales collected early in the season. However, Photinus firefliesdo not feed as adults, so spermatophore mass decreases withsubsequent matings. In response, nutrient-limited females maystop preferentially responding to longer duration flashes, increasingtheir overall responsiveness later in the mating season as theyforage for spermatophores. Therefore, the evolution of malecourtship signals in Photinus fireflies is the product not onlyof female preference for male flash characteristics, but alsothe costs and benefits of female choice that shape these preferences.     

Firefly Courtship: Behavioral and Morphological Predictors of Male Mating Success in Photinus greeni

Differences in male mating success can generate selection on male morphological traits and courtship behaviors involved in male–male competition or female mate choice. In Photinus fireflies (Coleoptera: Lampyridae), courtship is based on bioluminescent flash signals produced by both sexes. We conducted field observations of Photinus greeni fireflies engaged in competitive courtships, in which females are able to simultaneously assess several males, to identify male morphological traits and courtship behaviors that might predict male mating success. Male morphological traits did not differ between males that successfully mated compared with unsuccessful males (dialoging males that did not mate). However, courtship behavioral interactions differed: successful males tended to have higher flash pattern rates (number of flash patterns per minute), and their courtship flashes were more likely to be answered by females. We also examined how the risk of predation by Photuris fireflies altered courtship behavior of their Photinus prey. When predatory Photuris fireflies were present, P. greeni females were less likely to mate, and showed decreased flash responses to most males. However, P. greeni males that did successfully mate in spite of Photuris presence were males that maintained high flash pattern rates that elicited female responses. These results suggest that both female mate choice and Photuris predation exert strong selective pressures on the evolution of courtship signals in Photinus fireflies.     

Correlated evolution of female neoteny and flightlessness with male spermatophore production in fireflies (Coleoptera: Lampyridae)

The beetle family Lampyridae (fireflies) encompasses ～100 genera worldwide with considerable diversity in life histories and signaling modes. Some lampyrid males use reproductive accessory glands to produce spermatophores, which have been shown to increase female lifetime fecundity. Sexual dimorphism in the form of neotenic and flightless females is also common in this family. A major goal of this study was to test a hypothesized link between female flight ability and male spermatophore production. We examined macroevolutionary patterns to test for correlated evolution among different levels of female neoteny (and associated loss of flight ability), male accessory gland number (and associated spermatophore production), and sexual signaling mode. Trait reconstruction on a molecular phylogeny indicated that flying females and spermatophores were ancestral traits and that female neoteny increased monotonically and led to flightlessness within multiple lineages. In addition, male spermatophore production was lost multiple times. Our evolutionary trait analysis revealed significant correlations between increased female neoteny and male accessory gland number, as well as between flightlessness and spermatophore loss. In addition, female flightlessness was positively correlated with the use of glows as female sexual signal. Transition probability analysis supported an evolutionary sequence of female flightlessness evolving first, followed by loss of male spermatophores. These results contribute to understanding how spermatophores have evolved and how this important class of seminal nuptial gifts is linked to other traits, providing new insights into sexual selection and life-history evolution.     

Fireflies with or without prespermatophores: Evolutionary origins and life-history consequences

During mating, some male North American fireflies produce spermatophores from prespermatophores in their paired reproductive accessory glands. Other species of fireflies have neither prespermatophores nor spermatophores. To establish a pattern of spermatophore occurrence across firefly species, we examined the male internal reproductive system in 20 Japanese species belonging to 10 genera for the presence or absence of prespermatophores. Twelve species from seven genera produced prespermatophores, while eight species from three genera did not. Superimposed on a molecular phylogeny of Japanese fireflies based on mitochondrial 16S ribosomal DNA sequences, the basal group was prespermatophore producers. Prespermatophores appear to have been lost in two different lineages. Species without prespermatophores are characterized by degeneration of both the forewings and hindwings, and by body gigantism in females.     

Flash signal evolution in Photinus fireflies: Character displacement and signal exploitation in a visual communication system

Animal communication is an intriguing topic in evolutionary biology. In this comprehensive study of visual signal evolution, we used a phylogenetic approach to study the evolution of the flash communication system of North American fireflies. The North American firefly genus Photinus contains 35 described species with simple ON–OFF visual signals, and information on habitat types, sympatric congeners, and predators. This makes them an ideal study system to test hypotheses on the evolution of male and female visual signal traits. Our analysis of 34 Photinus species suggests two temporal pattern generators: one for flash duration and one for flash intervals. Reproductive character displacement was a main factor for signal divergence in male flash duration among sympatric Photinus species. Male flash pattern intervals (i.e., the duration of the dark periods between signals) were positively correlated with the number of sympatric Photuris fireflies, which include predators of Photinus. Females of different Photinus species differ in their response preferences to male traits. As in other communication systems, firefly male sexual signals seem to be a compromise between optimizing mating success (sexual selection) and minimizing predation risk (natural selection). An integrative model for Photinus signal evolution is proposed.     

The evolution of adult light emission color in North American fireflies

Firefly species (Lampyridae) vary in the color of their adult bioluminescence. It has been hypothesized that color is selected to enhance detection by conspecifics. One mechanism to improve visibility of the signal is to increase contrast against ambient light. High contrast implies that fireflies active early in the evening will emit yellower luminescence to contrast against ambient light reflected from green vegetation, especially in habitats with high vegetation cover. Another mechanism to improve visibility is to use reflection off the background to enhance the light signal. Reflectance predicts that sedentary females will produce greener light to maximize reflection off the green vegetation on which they signal. To test these predictions, we recorded over 7500 light emission spectra and determined peak emission wavelength for 675 males, representing 24 species, at 57 field sites across the Eastern United States. We found support for both hypotheses: males active early in more vegetated habitats produced yellower flashes in comparison to later‐active males with greener flashes. Further, in two of the eight species with female data, female light emissions were significantly greener as compared to males.     

Spontaneous flash communication of females in an Asian firefly

Fireflies are well known for the use of bioluminescence for sexual communication. In species using flash signals for pair formation, species and sexual identity are conferred by flash timing parameters such as flash duration, flash interval, flash number, and response delay. In dialog fireflies in North America, the male is the advertiser and the female is the responder. In these species, the male flash signal parameter varies depending on species, but the female flash signal parameter is limited only to response delay. However, in fireflies other than dialog fireflies, sexual flash communication is not well studied. Although many female-advertisement-like fireflies are reported, we have no confirmed case of sexual communication in a female-advertisement species. Here, we report the sexual flash communication of an Asian firefly, Luciola (Hotaria) parvula, in which the female flashes spontaneously. By using an electronic firefly, we confirm experimentally that males are specifically attracted to flashes with a female-specific flash duration. This is the first experimental report of sexual communication of a female advertiser in firefly communication. In this species, females call males usually with spontaneous flashes unlike dialog fireflies.     

Energy and predation costs of firefly courtship signals

Animal courtship signals include many highly conspicuous traits and behaviors, and it is generally assumed that such signals must balance the benefits of attracting mates against some fitness costs. However, few studies have assessed the multiple costs potentially incurred by any one courtship signal, so we have limited understanding of the relative importance of different costs. This study provides the first comprehensive assessment of signal costs for Photinus fireflies (Coleoptera: Lampyridae), using controlled experiments to measure both the energy and predation costs associated with their bioluminescent courtship signals. We measured energy required to generate bioluminescent flashes, using differential open-flow respirometry, and found that flash signaling results in only a nominal increase in energy expenditure above resting levels. These results suggest that the energy required to generate bioluminescent flashes represents a minor component of the total cost of firefly courtship. However, controlled field experiments revealed that visually oriented predators imposed major costs on firefly courtship signals, with higher signaling rates significantly increasing the likelihood of predation. Together with previous results demonstrating that female fireflies prefer more conspicuous courtship signals, these results support the importance of multiple-receiver communication networks in driving signal evolution.     

Examining the Role of Cuticular Hydrocarbons in Firefly Species Recognition

During animal courtship, multiple signals transmitted in different sensory modalities may be used to recognize potential mates. In fireflies (Coleoptera: Lampyridae), nocturnally active species rely on long-range bioluminescent signals for species, sex, and mate recognition, while several diurnally active species rely on pheromonal signals. Although in many insects non-volatile cuticular hydrocarbons (CHC) also function in species and sex discrimination, little is known about the potential role of CHC in fireflies. Here, we used gas chromatography to characterize species and sex differences in the CHC profiles of several North American fireflies, including three nocturnal and two diurnal species. Additionally, we conducted behavioral bioassays to determine whether firefly males (the searching sex) were differentially attracted to extracts from conspecific vs. heterospecific females. Gas chromatography revealed that nocturnal Photinus fireflies had low or undetectable CHC levels in both sexes, while diurnal fireflies showed higher CHC levels. No major sex differences in CHC profiles were observed for any firefly species. Behavioral bioassays demonstrated that males of the diurnal firefly Ellychnia corrusca were preferentially attracted to chemical extracts from conspecific vs. heterospecific females, while males of the remaining species showed no discrimination. These results suggest that while CHC may function as species recognition signals for some diurnal fireflies, these compounds are unlikely to be important contact signals in nocturnal Photinus fireflies.     

Lampyridae (Coleoptera): A plethora of mollicute associations

Beetles (Coleoptera) harbor many species ofAcholeplasma andSpiroplasma (division Tenericutes, class Mollicutes). Mollicutes were isolated from guts and/or hemocoels of firefly beetles (Lampyridae) from the United States (Maryland and West Virginia), Ecuador, and Tobago. Firefly beetles were frequent hosts for the group XIV spiroplasma, isolated from Ellychnia corrusca, and the group XIX spiroplasma, isolated fromPhoturis spp. The most unusual feature of the firefly-mollicute association is the carriage of four Mycoplasma species. Recent phylogenetic studies indicate that these species are members of a clade that includes a vertebrate pathogen,Mycoplasma mycoides. The high rate of occurrence ofMycoplasma species (which are, otherwise, infrequent in insects) in lampyrid beetles suggests that the association is significant. The unusual light-producing physiology of lampyrids (which is dependent on large pools of energy) and the production of large amounts of cardenolides from cholesterol (a critical growth factor for many mollicutes) may favor colonization by mollicutes. Offprint requests to: K. J. Hackett.     

Pyrazine emission by a tropical firefly: An example of chemical aposematism?

Although famous for photic courtship displays, fireflies (Coleoptera: Lampyridae) are also notable for emitting strong odors when molested. The identity of volatile emissions and their possible role, along with photic signals, as aposematic warnings of unpalatability have been little explored, especially in tropical species. Pursuant to the observation that the widespread Neotropical fireflies, Photuris trivittata and Bicellonycha amoena, emit pungent odors, glows, and flashes when handled, we investigated their cuticular and headspace chemistry. Gas chromatography–mass spectrometry analyses revealed that both fireflies have species‐specific cuticular hydrocarbon profiles. Photuris trivittata headspace was dominated by 2‐methoxy‐3‐(1‐methylpropyl) pyrazine (hereafter, pyrazine), on the order of 1.59 ng/individual and a suite of sesquiterpenes, while B. amoena emitted 3‐methoxy‐2‐butenoic acid methyl ester and a few ketones. This is the first report of such compounds in fireflies. We investigated the role of pyrazine in P. trivittata's interactions with potential predators: sympatric ants, toads, and bats. Solvent‐washed P. trivittata painted with pyrazine incurred lower ant predation than did their solvent‐washed counterparts. Pyrazine significantly repelled ants at baits in concentrations as low as 9.8 × 10^?4 ng/μl. The toad, Rhinella marina, readily accepted intact fireflies, pyrazine‐coated and uncoated mealworms. Both Myotis nigricans and Molossus molossus bats rejected fireflies, but accepted both pyrazine‐coated and uncoated mealworms. While pyrazine repels ants, its role as an aposematic signal warning other potential predators of firefly distastefulness requires further investigation. Our results underscore the idea that multiple enemies exert conflicting selection on firefly defenses.     

Flash communication between the sexes of the firefly, Photuris lucicrescens

ABSTRACT. The courtship signal of the male firefly, Photuris lucicrescens Barber (Coleoptera, Lampyridae), is a brilliant crescendo flash which grows in intensity, reaches a peak and abruptly terminates. It was found to be triggered by a long neural burst from the brain. Males and females produce weak, twinkling flashes which induce male crescendo flashes. Female responses were triggered by a slowly rising intensity, and female response latency is therefore variable. Male and female P.lucicrescens fireflies produce two different types of flashes and both these flashes play an integral part in their courtship communication.     

Genome size of 14 species of fireflies (Insecta,Coleoptera, Lampyridae)

Eukaryotic genome size data are important both as the basis for comparative research into genome evolution and as estimators of the cost and difficulty of genome sequencing programs for non-model organisms.In this study,the genome size of 14 species of fireflies (Lampyridae) (two genera in Lampyrinae,three genera in Luciolinae,and one genus in subfamily incertae sedis) were estimated by propidium iodide (PI)-based flow cytometry.The haploid genome sizes of Lampyridae ranged from 0.42 to 1.31 pg,a 3.1-fold span.Genome sizes of the fireflies varied within the tested subfamilies and genera.Lamprigera and Pyrocoelia species had large and small genome sizes,respectively.No correlation was found between genome size and morphological traits such as body length,body width,eye width,and antennal length.Our data provide additional information on genome size estimation of the firefly family Lampyridae.Furthermore,this study will help clarify the cost and difficulty of genome sequencing programs for non-model organisms and will help promote studies on firefly genome evolution.     

The production and transfer of spermatophores in three Asian species of Luciola fireflies

During mating, many male insects transfer sperm packaged within a spermatophore that is produced by reproductive accessory glands. While spermatophores have been documented in some North American fireflies (Coleoptera: Lampyridae), little is known concerning either production or transfer of spermatophores in the aquatic Luciola fireflies widespread throughout Asia. We investigated this process in Japanese Luciola lateralis and L. cruciata by feeding males rhodamine B, a fluorescent dye known to stain spermatophore precursors. We then mated males with virgin females, and dissected pairs at various timepoints after mating. In both of these Luciola species, spermatophores were produced by three pairs of male accessory glands and were transferred to females during the second stage of copulation. Male spermatophores were highly fluorescent, and were covered by a thin outer sheath; a narrow tube leading from an internal sperm-containing sac fit precisely into the female spermathecal duct, presumably for sperm delivery. Both L. lateralis and L. cruciata females have a spherical spermatheca as well as a highly extensible gland where spermatophore breakdown commences by 24h post-mating. Similar reproductive anatomy was observed for both sexes in Luciola ficta from Taiwan. These results suggest that nuptial gifts may play an important role in many firefly-mating systems.     

Determinants of reproductive success across sequential episodes of sexual selection in a firefly

Because females often mate with multiple males, it is critical to expand our view of sexual selection to encompass pre-, peri- and post-copulatory episodes to understand how selection drives trait evolution. In Photinus fireflies, females preferentially respond to males based on their bioluminescent courtship signals, but previous work has shown that male paternity success is negatively correlated with flash attractiveness. Here, we experimentally manipulated both the attractiveness of the courtship signal visible to female Photinus greeni fireflies before mating and male nuptial gift size to determine how these traits might each influence mate acceptance and paternity share. We also measured pericopulatory behaviours to examine their influence on male reproductive success. Firefly males with larger spermatophores experienced dual benefits in terms of both higher mate acceptance and increased paternity share. We found no effect of courtship signal attractiveness or pericopulatory behaviour on male reproductive success. Taken together with previous results, this suggests a possible trade-off for males between producing an attractive courtship signal and investing in nuptial gifts. By integrating multiple episodes of sexual selection, this study extends our understanding of sexual selection in Photinus fireflies and provides insight into the evolution of male traits in other polyandrous species.     

The evolutionary process of bioluminescence and aposematism in cantharoid beetles (Coleoptera: Elateroidea) inferred by the analysis of 18S ribosomal DNA

Cantharoid beetles are distinctive for their leathery soft elytra and conspicuous color or bioluminescence, and many of the members are equipped with chemical defenses. Thus, the vivid coloration of Cantharidae and Lycidae and the bioluminescence in Lampyridae and Phengodidae appear to be aposematic signals. However, the evolutionary aspect of their aposematism is not well understood, because the classification of the families remains controversial. In this study, we performed molecular phylogenetic analyses of species from cantharoid families, based on nucleotide sequence comparisons of nuclear 18S ribosomal DNA. The results shows that the luminous species Rhagophthalmus ohbai, which had sometimes been classified in Lampyridae, is excluded from a lampyrid clade and associates with the taxa of Phengodidae. The molecular data also suggests that four major subfamilies of Cantharidae (Cantharinae, Chauliognathinae, Malthininae, and Silinae) form a clade. The six subfamilies of Lampyridae are grouped and classified into two sublineages: Amydetinae + Lampyrinae + Photurinae and Cyphonocerinae + Luciolinae +Ototretinae. Genera Drilaster and Stenocladius are the members of Ototretinae in Lampyridae. These results conform to traditional taxonomy but disagree with more recent cladistic analyses. Based on these findings, we propose an evolutionary process of bioluminescence and aposematism in cantharoids: the clades of Cantharidae, Lampyridae, Lycidae, and Phengodidae have evolved aposematic coloration; subsequently Lampyridae and Phengodidae acquired bioluminescence; and these four major cantharoid families achieved their current adaptive diversities.     

Firefly luciferase genes from the subfamilies Psilocladinae and Ototretinae (Lampyridae, Coleoptera)

Firefly luciferase genes have been isolated from approximately 20 species of Lampyrinae, Luciolinae, and Photurinae. These are mostly nocturnal luminescent species that use light signals for sexual communication. In this study, we isolated three cDNAs for firefly luciferase from Psilocladinae (Cyphonocerus ruficollis) and Ototretinae (Drilaster axillaris and Stenocladius azumai), which are diurnal non-luminescent or weakly luminescent species that may use pheromones for communication. The amino acid sequences deduced from the three cDNAs showed 81-89% identities to each other and 60-81% identities with known firefly luciferases. The three purified recombinant proteins showed luminescence and fatty acyl-CoA synthetic activities, as observed in other firefly luciferases. The emission maxima by the three firefly luciferases (λmax, 545-546 nm) were shorter than those by known luciferases from the nocturnal fireflies (λmax, 550-568 nm). These results suggest that the primary structures and enzymatic properties of luciferases are conserved in Lampyridae, but the luminescence colors were red-shifted in nocturnal species compared to diurnal species.     

Flash communication systems of Japanese fireflies

Japanese fireflies range from nocturnal luminescent species to diurnal non-luminescent species. Their communication systems are classified into 6 types based on the following criteria: 1) Female responds to male's flashes after a fixed delay; 2) Male is directly attracted by female's light signal, the male perches on a leaf near the female, then the male changes his flashes with twinkling, and copulation behavior is released. However, the female may not respond to the male; 3) Male seeks female calling signal during the male's flying and synchronous flashing, then the male approaches the female, emitting flashes with various patterns, displaying walking-luminescing, sedentary signaling, chasing, and copulating; 4) Male is attracted by continuous luminescent signals of the female, and male perches near the female, then the male distinguishes the female's light organs shape. Thereafter, the male copulation behavior is released by her sex pheromone; 5) Male and female flight occurs in the daytime; when the male approaches the female, copulation is released by the female's pheromone; weak luminescent signals may be fulfilling the function of supplementary communication signals; 6) Luminescent signals have nothing to do with communication between male and female, and copulation is released by a sex pheromone.