Illuminating Firefly Diversity: Trends, Threats and Conservation Strategies.

Fireflies are a diverse group of bioluminescent beetles belonging to the family Lampyridae. Recent research on their diversity, evolution, behavior and conservation has greatly advanced our scientific understanding of these charismatic insects. In this review, we first summarize new discoveries about their taxonomic and ecological diversity, then focus on recent endeavors to identify and protect threatened fireflies around the world. We outline the main threats linked to recent population declines (habitat loss and degradation, light pollution, pesticide overuse, climate change and tourism) and describe relevant risk factors that predict which species will be particularly vulnerable to these threats. Although global coordination of firefly conservation efforts has begun only recently, considerable progress has already been made. We describe work by the IUCN SSC Firefly Specialist Group to identify species currently facing elevated extinction risks and to devise conservation strategies to protect them. To date, IUCN Red List assessments have been completed for 150 firefly taxa, about 20% of which face heightened extinction risks. The conservation status for many species has yet to be determined due to insufficient information, although targeted surveys and community science projects have contributed valuable new data. Finally, we highlight some examples of successful firefly habitat protection and restoration efforts, and we use the framework of the IUCN SSC Species Conservation Cycle to point out high-priority actions for future firefly conservation efforts.

Artificial light impacts the mate success of female fireflies.

Anthropogenic light pollution is a novel environmental disruption that affects the movement, foraging and mating behaviour of nocturnal animals. Most of these effects are sublethal, and their net impact on reproductive fitness and population persistence is often extrapolated from behavioural data. Without dedicated tracking of wild individuals, however, it is impossible to predict whether populations in light-polluted habitats will decline or, instead, move to shaded refuges. To disentangle these conflicting possibilities, we investigated how artificial light affects mating and movement in North American Photinus, a genus of bioluminescent fireflies known to experience courtship failure under artificial light. The degree to which artificial light reduced mate success depended on the intensity of the light treatment, its environmental context, and the temporal niche of the species in question. In the laboratory, direct exposure to artificial light completely prevented mating in semi-nocturnal Photinus obscurellus. In the field, artificial light had little impact on the movement or mate success of local Photinus pyralis and Photinus marginellus but strongly influenced mate location in Photinus greeni; all three species are relatively crepuscular. Our nuanced results suggest greater appreciation of behavioural diversity will help insect conservationists and dark sky advocates better target efforts to protect at-risk species.

Costs and benefits of "insect friendly" artificial lights are taxon specific.

The expansion of human activity into natural habitats often results in the introduction of artificial light at night, which can disrupt local ecosystems. Recent advances in LED technology have enabled spectral tuning of artificial light sources, which could in theory limit their impact on vulnerable taxa. To date, however, experimental comparisons of ecologically friendly candidate colors have often considered only one type of behavioral impact, sometimes on only single species. Resulting recommendations cannot be broadly implemented if their consequences for other local taxa are unknown. Working at a popular firefly ecotourism site, we exposed the insect community to artificial illumination of three colors (blue, broad-spectrum amber, red) and measured flight-to-light behavior as well as the courtship flash behavior of male Photinus carolinus fireflies. Firefly courtship activity was greatest under blue and red lights, while the most flying insects were attracted to blue and broad-spectrum amber lights. Thus, while impacts of spectrally tuned artificial light varied across taxa, our results suggest that red light, rather than amber light, is least disruptive to insects overall, and therefore more generally insect friendly.

Effects of artificial light on growth, development, and dispersal of two North American fireflies (Coleoptera: Lampyridae).

Holometabolous insects exhibit complex life cycles in which both morphology and ecological niche change dramatically during development. In the larval stage, many insects have soft, slow-moving bodies and poor vision, limiting their ability to respond to environmental threats. Artificial light at night (ALAN) is an environmental perturbation known to severely impact the fitness of adult insects by disrupting both temporal and spatial orientation. The impact of ALAN on earlier life stages, however, is largely unknown. We conducted a series of laboratory experiments to investigate how two distinct forms of ALAN affect the development and movement of immature Photuris sp. and Photinus obscurellus fireflies. Although long-term exposure to dim light at night (dLAN), akin to urban skyglow, did not impact overall survivorship or duration of egg, larval, and pupal stages in either species, it did accelerate weight gain in early-instar Photuris larvae. Late-instar Photuris exposed to point sources of ALAN at the start of their nightly foraging period were also significantly more likely to burrow beneath the soil surface, rather than disperse across it. ALAN may therefore impede dispersal of firefly larvae away from illuminated areas, which could have downstream consequences for the reproductive fitness of adults.

Differences in signal contrast and camouflage among different colour variations of a stomatopod crustacean, Neogonodactylus oerstedii.

Animal colouration is often a trade-off between background matching for camouflage from predators, and conspicuousness for communication with con- or heterospecifics. Stomatopods are marine crustaceans known to use colour signals during courtship and contests, while their overall body colouration may provide camouflage. However, we have little understanding of how stomatopods perceive these signals in their environment or whether overall body coloration does provide camouflage from predators. Neogonodactylus oerstedii assess meral spot colour during contests, and meral spot colour varies depending on local habitat. By calculating quantum catch for N. oerstedii's 12 photoreceptors associated with chromatic vision, we found that variation in meral spot total reflectance does not function to increase signal contrast in the local habitat. Neogonodactylus oerstedii also show between-habitat variation in dorsal body colouration. We used visual models to predict a trichromatic fish predator's perception of these colour variations. Our results suggest that sandy and green stomatopods are camouflaged from a typical fish predator in rubble fields and seagrass beds, respectively. To our knowledge, this is the first study to investigate signal contrast and camouflage in a stomatopod. These results provide new insight into the function and evolution of colouration in a species with a complex visual system.

The impact of artificial light at night on nocturnal insects: A review and synthesis.

In recent decades, advances in lighting technology have precipitated exponential increases in night sky brightness worldwide, raising concerns in the scientific community about the impact of artificial light at night (ALAN) on crepuscular and nocturnal biodiversity. Long-term records show that insect abundance has declined significantly over this time, with worrying implications for terrestrial ecosystems. The majority of investigations into the vulnerability of nocturnal insects to artificial light have focused on the flight-to-light behavior exhibited by select insect families. However, ALAN can affect insects in other ways as well. This review proposes five categories of ALAN impact on nocturnal insects, highlighting past research and identifying key knowledge gaps. We conclude with a summary of relevant literature on bioluminescent fireflies, which emphasizes the unique vulnerability of terrestrial light-based communication systems to artificial illumination. Comprehensive understanding of the ecological impacts of ALAN on diverse nocturnal insect taxa will enable researchers to seek out methods whereby fireflies, moths, and other essential members of the nocturnal ecosystem can coexist with humans on an increasingly urbanized planet.

Firefly genomes illuminate parallel origins of bioluminescence in beetles.

Fireflies and their luminous courtships have inspired centuries of scientific study. Today firefly luciferase is widely used in biotechnology, but the evolutionary origin of bioluminescence within beetles remains unclear. To shed light on this long-standing question, we sequenced the genomes of two firefly species that diverged over 100 million-years-ago: the North American Photinus pyralis and Japanese Aquatica lateralis. To compare bioluminescent origins, we also sequenced the genome of a related click beetle, the Caribbean Ignelater luminosus, with bioluminescent biochemistry near-identical to fireflies, but anatomically unique light organs, suggesting the intriguing hypothesis of parallel gains of bioluminescence. Our analyses support independent gains of bioluminescence in fireflies and click beetles, and provide new insights into the genes, chemical defenses, and symbionts that evolved alongside their luminous lifestyle.

Molecular characterization of firefly nuptial gifts: a multi-omics approach sheds light on postcopulatory sexual selection.

Postcopulatory sexual selection is recognized as a key driver of reproductive trait evolution, including the machinery required to produce endogenous nuptial gifts. Despite the importance of such gifts, the molecular composition of the non-gametic components of male ejaculates and their interactions with female reproductive tracts remain poorly understood. During mating, male Photinus fireflies transfer to females a spermatophore gift manufactured by multiple reproductive glands. Here we combined transcriptomics of both male and female reproductive glands with proteomics and metabolomics to better understand the synthesis, composition and fate of the spermatophore in the common Eastern firefly, Photinus pyralis. Our transcriptome of male glands revealed up-regulation of proteases that may enhance male fertilization success and activate female immune response. Using bottom-up proteomics we identified 208 functionally annotated proteins that males transfer to the female in their spermatophore. Targeted metabolomic analysis also provided the first evidence that Photinus nuptial gifts contain lucibufagin, a firefly defensive toxin. The reproductive tracts of female fireflies showed increased gene expression for several proteases that may be involved in egg production. This study offers new insights into the molecular composition of male spermatophores, and extends our understanding of how nuptial gifts may mediate postcopulatory interactions between the sexes.

Multimodal signals: ultraviolet reflectance and chemical cues in stomatopod agonistic encounters.

Complex signals are commonly used during intraspecific contests over resources to assess an opponent's fighting ability and/or aggressive state. Stomatopod crustaceans may use complex signals when competing aggressively for refuges. Before physical attacks, stomatopods assess their opponents using chemical cues and perform threat displays showing a coloured patch, the meral spot. In some species, this spot reflects UV. However, despite their complex visual system with up to 20 photoreceptor classes, we do not know if stomatopods use chromatic or achromatic signals in contests. In a field study, we found that Neogonodactylus oerstedii meral spot luminance varies with sex, habitat and, more weakly, body length. Next, we conducted an experimental manipulation which demonstrated that both chemical cues and chromatic signals are used during contests. In the absence of chemical cues, stomatopods approached an occupied refuge more quickly and performed offensive behaviours at a lower rate. When UV reflectance was absent, stomatopods performed offensive behaviours more frequently and contest duration trended towards shorter fights. These results provide new evidence that UV reflectance and/or visible spectrum luminance is used to amplify threat displays. Our results are the first to demonstrate that chemical and chromatic cues comprise a multimodal signal in stomatopod contests.

Emerging issues in the evolution of animal nuptial gifts.

Uniquely positioned at the intersection of sexual selection, nutritional ecology and life-history theory, nuptial gifts are widespread and diverse. Despite extensive empirical study, we still have only a rudimentary understanding of gift evolution because we lack a unified conceptual framework for considering these traits. In this opinion piece, we tackle several issues that we believe have substantively hindered progress in this area. Here, we: (i) present a comprehensive definition and classification scheme for nuptial gifts (including those transferred by simultaneous hermaphrodites), (ii) outline evolutionary predictions for different gift types, and (iii) highlight some research directions to help facilitate progress in this field.

Using RNA sequencing to characterize female reproductive genes between Z and E Strains of European Corn Borer moth (Ostrinia nubilalis).

BACKGROUND: Reproductive proteins often evolve rapidly and are thought to be subject to strong sexual selection, and thus may play a key role in reproductive isolation and species divergence. However, our knowledge of reproductive proteins has been largely limited to males and model organisms with sequenced genomes. With advances in sequencing technology, Lepidoptera are emerging models for studies of sexual selection and speciation. By profiling the transcriptomes of the bursa copulatrix and bursal gland from females of two incipient species of moth, we characterize reproductive genes expressed in the primary reproductive tissues of female Lepidoptera and identify candidate genes contributing to a one-way gametic incompatibility between Z and E strains of the European corn borer (Ostrinia nubilalis). RESULTS: Using RNA sequencing we identified transcripts from ~37,000 and ~36,000 loci that were expressed in the bursa copulatrix or the bursal gland respectively. Of bursa copulatrix genes, 8% were significantly differentially expressed compared to the female thorax, and those that were up-regulated or specific to the bursa copulatrix showed functional biases toward muscle activity and/or organization. In the bursal gland, 9% of genes were differentially expressed compared to the thorax, with many showing reproduction or gamete production functions. Of up-regulated bursal gland genes, 46% contained a transmembrane region and 16% possessed secretion signal peptides. Divergently expressed genes in the bursa copulatrix were exclusively biased toward protease-like functions and 51 proteases or protease inhibitors were divergently expressed overall. CONCLUSIONS: This is the first comprehensive characterization of female reproductive genes in any lepidopteran system. The transcriptome of the bursa copulatrix supports its role as a muscular sac that is the primary site for disruption of the male ejaculate. We find that the bursal gland acts as a reproductive secretory body that might also interact with male ejaculate. In addition, differential expression of proteases between strains supports a potential role for these tissues in contributing to reproductive isolation. Our study provides new insight into how male ejaculate is processed by female Lepidoptera, and paves the way for future work on interactions between post-mating sexual selection and speciation.

Sexual dimorphism, mating systems, and nuptial gifts in two Asian fireflies (Coleoptera: Lampyridae).

Many insect taxa show conspicuous sexual dimorphism in which females lack functional wings and are therefore incapable of flight. In fireflies, this loss of female flight is correlated with male production of spermatophore nuptial gifts, with species with flightless females also lacking gift production. In this study we further examined the relationship between sexual dimorphism and nuptial gifts by examining the reproductive ecology of two Asian fireflies, the sexually monomorphic Aquatica ficta (Luciolinae) and dimorphic Pyrocoelia pectoralis (Lampyrinae). We describe the reproductive anatomy of P. pectoralis males and females, and the time course of A. ficta spermatophore transfer. Through a series of mating experiments, we determined the effect of the number of matings on female fecundity, egg hatching success and female lifespan. The spermatophore-producing A. ficta was found to be monandrous, and female lifespan doubled as a result of that single mating. P. pectoralis was found to lack spermatophores but females were polyandrous and fecundity and egg hatching success both increased with additional matings. These results contradict patterns known from North American firefly species and provide insight into the role of male-derived substances in mating systems.

The influence of male ejaculate quantity on female fitness: a meta-analysis.

Although the primary function of mating is gamete transfer, male ejaculates contain numerous other substances that are produced by accessory glands and transferred to females during mating. Studies with several model organisms have shown that these substances can exert diverse behavioural and physiological effects on females, including altered longevity and reproductive output, yet a comprehensive synthesis across taxa is lacking. Here we use a meta-analytic approach to synthesize quantitatively extensive experimental work examining how male ejaculate quantity affects different components of female fitness. We summarize effect sizes for female fecundity (partial and lifetime) and longevity from 84 studies conducted on 70 arthropod species that yielded a total of 130 comparisons of female fecundity and 61 comparisons of female longevity. In response to greater amounts of ejaculate, arthropod females demonstrate enhanced fecundity (both partial and lifetime) but reduced longevity, particularly for Diptera and Lepidoptera. Across taxa, multiply mated females show particularly large fecundity increases compared to singly mated females, indicating that single matings do not maximize female fitness. This fecundity increase is balanced by a slight negative effect on lifespan, with females that received more ejaculate through polyandrous matings showing greater reductions in lifespan compared with females that have mated repeatedly with the same male. We found no significant effect size differences for either female fecundity or longevity between taxa that transfer sperm packaged into spermatophores compared to taxa that transfer ejaculates containing free sperm. Furthermore, females that received relatively larger or more spermatophores demonstrated greater lifetime fecundity, indicating that these seminal nuptial gifts provide females with a net fitness benefit. These results contribute to our understanding of the evolutionary origin and maintenance of non-sperm ejaculate components, and provide insight into female mate choice and optimal mating patterns.

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.

Pheromone production by male Tribolium castaneum (Coleoptera: Tenebrionidae) is influenced by diet quality.

Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), the red flour beetle, is a common cosmopolitan pest exploiting a variety of stored products. We experimentally manipulated diet nutritional quality by using non-nutritive filler to examine how this influenced pheromone production and olfactory attractiveness of T. castaneum adult males. Volatiles released by individual males reared on high versus low nutrition diets were collected using solid phase microextraction, and gas chromatography coupled to mass spectrometry was used to identify and quantify the Tribolium aggregation pheromone 4, 8-dimethyldecanal (DMD). Males kept on high nutrition diet showed a three-fold increase in daily DMD production, which suggests the possibility that this pheromone could act as a condition-dependent mating signal. In pitfall trap assays, there was no significant difference in the mean response of virgin females to discs kept with low versus high nutrition males, although discs carrying male cues were significantly more attractive than blank discs. These results suggest that DMD production rates by T. castaneum males will depend on the nutritional quality of various stored products, but such differences may not alter males' ability to attract females.

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.

An integrative view of sexual selection in Tribolium flour beetles.

Sexual selection is a major force driving the evolution of diverse reproductive traits. This evolutionary process is based on individual reproductive advantages that arise either through intrasexual competition or through intersexual choice and conflict. While classical studies of sexual selection focused mainly on differences in male mating success, more recent work has focused on the differences in paternity share that may arise through sperm competition or cryptic female choice whenever females mate with multiple males. Thus, an integrative view of sexual selection needs to encompass processes that occur not only before copulation (pre-mating), but also during copulation (peri-mating), as well as after copulation (post-mating), all of which can generate differences in reproductive success. By encompassing mechanisms of sexual selection across all of these sequential reproductive stages this review takes an integrative approach to sexual selection in Tribolium flour beetles (Coleoptera: Tenebrionidae), a particularly well-studied and economically important model organism. Tribolium flour beetles colonize patchily distributed grain stores, and juvenile and adult stages share the same food resources. Adults are highly promiscuous and female reproduction is distributed across an adult lifespan lasting approximately 1 year. While Tribolium males produce an aggregation pheromone that attracts both sexes, there appears to be little pre-mating discrimination among potential mates by either sex. However, recent work has revealed several peri-mating and post-mating mechanisms that determine how offspring paternity is apportioned among a female's mates. During mating, Tribolium females reject spermatophore transfer and limit sperm numbers transferred by males with low phenotypic quality. Although there is some conflicting evidence, male copulatory leg-rubbing appears to be associated with overcoming female resistance to insemination and does not influence a male's subsequent paternity share. Evidence suggests that Tribolium beetles have several possible post-mating mechanisms that they may use to bias paternity. Male sperm precedence has been extensively studied in Tribolium spp. and the related Tenebrio molitor, and several factors influencing male paternity share among a female's progeny have been identified. These include oviposition time, inter-mating interval, male strain/genotype, the mating regimen of a male's mother, male starvation, and tapeworm infection. Females exert muscular control over sperm storage, although there is no evidence to date that females use this to differentiate among mates. Females could also influence offspring paternity by re-mating with additional males, and T. castaneum females more readily accept spermatophores when they are re-mating with more attractive males. Additional work is needed to examine the possible roles played by both male and female accessory gland products in determining male paternity share. Sexual selection during pre-mating episodes may be reinforced or counteracted by peri- and post-copulatory selection, and antagonistic coevolution between the sexes may be played out across reproductive stages. In Tribolium, males' olfactory attractiveness is positively correlated with both insemination success and paternity share, suggesting consistent selection across different reproductive stages. Similar studies across sequential reproductive stages are needed in other taxa to provide a more integrative view of sexual selection.

Flash signal evolution, mate choice, and predation in fireflies.

Many key advances in our understanding of firefly biology and signaling have been made over the past two decades. Here we review this recent research, which includes new phylogenetic results that shed light on the evolution of courtship signal diversity within the family Lampyridae, new insights into firefly flash control, and the discovery of firefly nuptial gifts. We present a comprehensive overview of sexual selection in lampyrids, including evidence from Photinus fireflies that females choose their mates on the basis of male flash signals, and discuss the importance of examining both precopulatory and postcopulatory sexual selection in this group. Finally, we review recent findings on firefly chemical defenses, and discuss their implications for flash signal evolution in response to generalist predators as well as specialist predatory fireflies. This review provides new insight into how firefly flash signals have been shaped by the dual evolutionary processes of sexual selection (mate choice) and natural selection (predation), and proposes several exciting directions for future research.

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.