Firefly Photinus Research Articles

The role of pigments in light color variation of the firefly Photinus pyralis.

Fireflies use bioluminescent signals to communicate with their mates. Luciferase has been thought to be the sole contributor to light color; however, populations of the Photinus pyralis firefly display variation in the color of their emitted signals yet have identical luciferase sequences. Here, we examined whether pigments could be present in the light organs of the twilight-active species P. pyralis and contribute to this variation. We detected patterns of expression that suggest ommochrome and pterin screening pigments are expressed in P. pyralis light organs and could filter light emitted by luciferase and play a role in signal tuning. There were no significant differences between the pigment gene expression of P. pyralis individuals with yellower and greener signals. Our study provides alternative mechanisms that could influence pigments in P. pyralis light organs that could also play a role in modifying signal color.

Improved security of medical images using DWT–SVD watermarking mechanisms based on firefly Photinus search algorithm

Security is the primary concern in the transmission of medical images, as it involves sensitive patient information. This study introduces an optimized watermarking approach, constructed using discrete wavelet transform and singular value decomposition. The Low- level frequency bands (LL3) sub-band singular values of the host image were embedded with the singular values of a binary watermark using multiple scaling factors. These MSFs were optimized using a recently proposed firefly Photinus algorithm to balance robustness and imperceptibility. The proposed method was applied to various images, including computed tomography images, where the visual quality of the signed and attacked images was evaluated by peak signal-to-noise ratio (PSNR) and normalized cross-correlation. The performance of the proposed algorithm demonstrates significant improvements in the embedding and extraction processes, showing an enhancement in the balance between robustness and imperceptibility, with a PSNR above 79.28 dB, compared to other related works.

Deceptive Seduction by Femme Fatale Fireflies and Its Avoidance by Males of a Synchronous Firefly Species (Coleoptera: Lampyridae).

Photuris female fireflies attract males of different firefly species by responding to their flashing signals; then, they try to capture and feed on them. This aggressive mimicry is considered a major selective pressure on the communication systems of the fireflies of the American continent. The intensity of this selective pressure is a function of its efficiency in prey capture. In this study, the rates of attraction and capture of males of the synchronous firefly Photinus palaciosi by the predatory females of Photuris lugubris are reported. Although the females attract numerous males, their hunting success is low. This result is consistent with the few previous measurements published. In agreement with the predicted coevolutionary race between predator and prey, behaviors consistent with predation avoidance in P. palaciosi and increasing prey encounters and prey deception by P. lugubris were observed.

Development of a Novel ATP Bioluminescence Assay Based on Engineered Probiotic Saccharomyces boulardii Expressing Firefly Luciferase.

Quantitative analysis of adenosine triphosphate (ATP) has been widely used as a diagnostic tool in the food and medical industries. Particularly, the pathogenesis of a few diseases including inflammatory bowel disease (IBD) is closely related to high ATP concentrations. A bioluminescent D-luciferin/luciferase system, which includes a luciferase (FLuc) from the firefly Photinus pyralis as a key component, is the most commonly used method for the detection and quantification of ATP. Here, instead of isolating FLuc produced in recombinant Escherichia coli, we aimed to develop a whole-cell biocatalyst system that does not require extraction and purification of FLuc. To this end, the gene coding for FLuc was introduced into the genome of probiotic Saccharomyces boulardii using the CRISPR/Cas9-based genome editing system. The linear relationship (r2 = 0.9561) between ATP levels and bioluminescence generated from the engineered S. boulardii expressing FLuc was observed in vitro. To explore the feasibility of using the engineered S. boulardii expressing FLuc as a whole-cell biosensor to detect inflammation biomarker (i.e., ATP) in the gut, a colitis mouse model was established using dextran sodium sulfate as a colitogenic compound. Our findings demonstrated that the whole-cell biosensor can detect elevated ATP levels during gut inflammation in mice. Therefore, the simple and powerful method developed herein could be applied for non-invasive IBD diagnosis.

Inferring the demographic history of the North American firefly Photinus pyralis.

The firefly Photinus pyralis inhabits a wide range of latitudinal and ecological niches, with populations living from temperate to tropical habitats. Despite its broad distribution, its demographic history is unknown. In this study, we modelled and inferred different demographic scenarios for North American populations of P. pyralis, which were collected from Texas to New Jersey. We used a combination of ABC techniques (for multi-population/colonization analyses) and likelihood inference (dadi, StairwayPlot2, PoMo) for single-population demographic inference, which proved useful with our RAD data. We uncovered that the most ancestral North American population lays in Texas, which further colonized the Central region of the US and more recently the North Eastern coast. Our study confidently rejects a demographic scenario where the North Eastern populations colonized more southern populations until reaching Texas. To estimate the age of divergence between of P. pyralis, which provides deeper insights into the history of the entire species, we assembled a multi-locus phylogenetic data covering the genus Photinus. We uncovered that the phylogenetic node leading to P. pyralis lies at the end of the Miocene. Importantly, modelling the demographic history of North American P. pyralis serves as a null model of nucleotide diversity patterns in a widespread native insect species, which will serve in future studies for the detection of adaptation events in this firefly species, as well as a comparison for future studies of other North American insect taxa.

On the inference of a southern origin of the North American firefly Photinus pyralis

On the inference of a southern origin of the North American firefly Photinus pyralis

Morphological changes in the tracheal system associated with light organs of the firefly Photinus pyralis (Coleoptera: Lampyridae) across life stages.

Oxygen is an important and often limiting reagent of a firefly's bioluminescent chemical reaction. Therefore, the development of the tracheal system and its subsequent modification to support the function of firefly light organs are key to understanding this process. We employ micro-CT scanning, 3D rendering, and confocal microscopy to assess the abdominal tracheal system in Photinus pyralis from the external spiracles to the light organ's internal tracheal brush, a feature named here for the first time. The abdominal spiracles in firefly larvae and pupae are of the biforous type, with a filter apparatus and appear to have an occlusor muscle to restrict airflow. The first abdominal spiracle in the adult firefly is enlarged and bears an occlusor muscle, and abdominal spiracles two through eight are small, with a small atrium and bilobed closing apparatus. Internal tracheal system features, including various branches, trunks, and viscerals, were homologized across life stages. In adults, the sexually dimorphic elaboration and increase in volume associated with tracheal features of luminous segments emphasizes the importance of gas exchange during the bioluminescent process.

Optimized Loop-Mediated Isothermal Amplification (LAMP) Allows Single Copy Detection Using Bioluminescent Assay in Real Time (BART).

The bioluminescent assay in real time (BART) is a molecular diagnostic tool for the detection of nucleic acid amplification by recording light output. The key component for BART is a thermostable luciferase derived from the firefly Photinus pyralis. Coupling BART detection with the isothermal amplification method loop-mediated isothermal amplification (LAMP) enables rapid molecular diagnostic results using simple equipment. LAMP-BART provides quantitative results from a closed tube and is appropriate to microliter standard tests and nanoliter microfluidic assays. In this chapter, we introduce a protocol to amplify and detect genetic markers using LAMP with BART. Furthermore, we provide advice to optimize LAMP assays for high sensitivity and specificity and to eliminate the incidence of "false positive" results which can occur from the components of the assay. The optimization of genetically modified (GM) maize by targeting the nopaline synthase terminator (NOSt) and 35S promoter (35Sp) sequences is described.

The size of signal detection and emission organs in a synchronous firefly: sexual dimorphism, allometry and assortative mating.

The size of the organs responsible for emitting and detecting sexual communication signals is a likely target for selection. Communication via bioluminescent signals in synchronous fireflies is a promising model to test hypotheses regarding differences between males and females in the effect of the size of signal emission and detection organs on fitness components. Synchronous firefly species congregate in large numbers during the mating season, displaying bioluminescent signals aimed at potential mates during relatively short nightly periods. Operational sex ratios are male-biased and, thus, the so-called typical sex roles (indiscriminate males and choosy females) are expected to evolve. We studied the synchronous firefly Photinus palaciosi, a species that during the mating season congregates in forests of central Mexico offering a magnificent natural show that attracts numerous tourists. P. palaciosi females have reduced wings (brachyptery) and cannot fly. Our field study tested the hypothesis that the male-biased operational sex ratio and the short daily mating period result in strong male-male competition that selects for males with larger lanterns and larger eyes, and against male mate choice, whereas female-female mate competition is absent and, thus, no selection on lantern or eye size is expected. Even though lantern, eye or body size do not predict the probability of being found in copula for either sex, sexual dimorphism in these features, along with allometric slopes of lantern size and assortative mating in terms of relative lantern size, support not only the hypothesis of intense sexual selection among males, but the possibility of subtle mechanisms of sexual selection among females. Trade-offs between investment in signaling (lanterns) versus detection (eyes) structures, or with pressures different from sexual selection such as those imposed by predators, are also likely to be important in shaping the evolution of sexual signaling in these fireflies.

Genetically engineered bio-nanoparticles with co-expressed enzyme reporter and recognition element for IgG immunoassay

A bioluminescence-based immunoassay, relying on genetically engineered multifunctional outer membrane vesicles (OMVs, ∼50 nm) with co-expressed enzyme reporter and IgG-binding domain, has been developed for detection of immunoglobulin G (IgG), an important biomacromolecule in humoral immunity. OMVs with co-expressed IgG-binding domain (Z domain) and ATP-independent NanoLuc luciferase (∼150-fold more active than either the firefly (Photinus pyralis) or Renilla reniformis luciferase) were produced through simple fermentation of genetically engineered nano-vesicle-forming E. coli. The size and concentration of the as-produced multifunctional OMVs were determined by Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis, respectively. To reduce the non-specific binding of OMVs during immunoassay, blocking agent test was systematically carried out before IgG detection. Sensitive detection of IgG was further demonstrated with the limit of detection of 5 ng/mL IgG, comparable to that of commercial IgG ELISA kit. The advantages of genetically engineered bio-nanoparticles (bio-NPs) containing both reporter and recognition element are cost-effective and scalable through simple cell cultures with high reproducibility and pre-determined biological functions, in comparison with the conventional costly enzyme-antibody conjugation used in immunoassay. This study opens a new pathway on the low-cost immunoassay by rational design of genetically engineered bio-NPs with integrated reporter/recognition function.

Mutagenesis and Structural Studies Reveal the Basis for the Activity and Stability Properties That Distinguish the Photinus Luciferases scintillans and pyralis.

The dazzling yellow-green light emission of the common North American firefly Photinus pyralis and other bioluminescent organisms has provided a wide variety of prominent research applications like reporter gene assays and in vivo imaging methods. While the P. pyralis enzyme has been extensively studied, only recently has a second Photinus luciferase been cloned from the species scintillans. Even though the enzymes share very high sequence identity (89.8%), the color of the light they emit, their specific activity and their stability to heat, pH, and chemical denaturation are quite different with the scintillans luciferase being generally more resistant. Through the construction and evaluation of the properties of chimeric domain swapped, single point, and various combined variants, we have determined that only six amino acid changes are necessary to confer all of the properties of the scintillans enzyme to wild-type P. pyralis luciferase. Altered stability properties were attributed to four of the amino acid changes (T214N/S276T/H332N/E354N), and single mutations each predominantly changed emission color (Y255F) and specific activity (A222C). Results of a crystallographic study of the P. pyralis enzyme containing the six changes (Pps6) provide some insight into the structural basis for some of the documented property differences.

Rational Side-Chain Amino Acid Substitution in Firefly Luciferase for Improved Thermostability

Improving the thermostability of the luciferase from firefly (Photinus pyralis) needs to be solved to broaden its industrial applications. In this paper, molecular dynamic (MD) simulations were used to identify 4 amino acid substitutions (P183V, E325K, Q338V, and E354K) which might have a significant influence on the thermostability of luciferase. Root-mean-square deviation values were calculated to further evaluate the effect of these mutations on thermostability of the enzyme and demonstrated that the thermostability of the corresponding protein variants was in the order E354K > E325K > WT > P183V > Q338V. Following the MD simulation, the enzyme variants were expressed in a recombinant host, and the results showed that the t1/2, T50, and Tm of mutant E354K were increased 2.32-fold, and 4.5 and 3.3°C more compared with the wild type, respectively. MD simulations, as well as circular dichroism and fluorescence spectroscopy were further applied to elucidate the conformational differences between the wild-type and E354K luciferases. The results indicated that a possible explanation for the improved thermostability of E354K enzyme lies in the formation of a salt bridge between Lys354 and Glu311 and alteration of protein conformation.

Fireflies thwart bat attack with multisensory warnings.

Many defended animals prevent attacks by displaying warning signals that are highly conspicuous to their predators. We hypothesized that bioluminescing fireflies, widely known for their vibrant courtship signals, also advertise their noxiousness to echolocating bats. To test this postulate, we pit naïve big brown bats (Eptesicus fuscus) against chemically defended fireflies (Photinus pyralis) to examine whether and how these beetles transmit salient warnings to bats. We demonstrate that these nocturnal predators learn to avoid noxious fireflies using either vision or echolocation and that bats learn faster when integrating information from both sensory streams-providing fundamental evidence that multisensory integration increases the efficacy of warning signals in a natural predator-prey system. Our findings add support for a warning signal origin of firefly bioluminescence and suggest that bat predation may have driven evolution of firefly bioluminescence.

Male Fireflies (Photinus pyralis (Linnaeus) and Photinus sabulosus Green) are at Special Risk of Capture by Spider Predators and are then Stolen by Kleptoparasitic Fireflies in the Genus Photuris Dejean (Coleoptera: Lampyridae)

Abstract Males of Photinus pyralis (Linnaeus) and Photinus sabulosus Green were the primary victims of spider attacks at a field site in northern Virginia from 1 July 2017 to 21 August 2017. Males of these species, as is true for other members of the genus, appear to be at far greater risk of predation than are females, a reflection of the mating system of fireflies in which males fly in search of females that are largely sedentary during the mating period. Once trapped, males are sometimes eaten by kleptoparasitic fireflies in the genus Photuris Dejean, which can usually steal wrapped prey from the spiders that have captured them, as has been demonstrated elsewhere as well, but occasionally fall prey to others of their genus that have been attracted to spider webs.

Firefly Luciferase Mutant with Enhanced Activity and Thermostability.

The luciferase isolated from the firefly Photinus pyralis (Ppy) catalyzes a two-step reaction that results in the oxidation of d-luciferin accompanied by emission of yellow–green light with a peak at 560 nm. Among many applications, Ppy luciferase has been used extensively as a reporter gene in living cells and organisms. However, some biological applications are limited by the low stability of the luciferase and limited intracellular luciferin concentration. To address these challenges, efforts to protein engineer Ppy luciferase have resulted in a number of mutants with improved properties such as thermostability, pH tolerance, and catalytic turn over. In this work, we combined amino acid mutations that were shown to enhance the enzyme’s thermostability (Mutant E) with those reported to enhance catalytic activity (LGR). The resulting mutant (YY5) contained eight amino acid changes from the wild-type luciferase and exhibited both improved thermostability and brighter luminescence at low luciferin concentrations. Therefore, YY5 may be useful for reporter gene applications.

Unnaturally aglow with a bright inner light.

A bioluminescent system enables imaging single cells deep inside small animals

Electrostatic Catalysis Induced by Luciferases in the Decomposition of the Firefly Dioxetanone and Its Analogue.

The variations of the barrier heights in the decomposition of the firefly dioxetanone and its analogues with the electrostatic field produced by the active site amino acid residues in the firefly luciferase are examined by a density functional theory study of the high-energy intermediates of the three luciferins. The positive electric field along the long-axis direction of the luciferins lowers the activation energy and acts as an electrostatic catalyst in the thermolysis process. The calculated barrier heights for the firefly dioxetanone and its analogues surrounded by the firefly Photinus pyralis luciferase show that the energy barrier of the firefly dioxetanone is lowered by the luciferase but is raised for the other analogue. Thus, the thermolysis rate is enhanced for the natural d-luciferin and reduced for the other analogue by the firefly luciferase, which elucidates why the luciferase acts as a catalyst for the natural d-luciferin but makes some luciferins emit weaker light signals.

ATP-bioluminescence technique for fruit quality assessments: a detailed methodology for analysis of adenylate nucleotides

The bioluminescence technique has been used during a long time for measurementof adenylate nucleotides (ATP, ADP, and AMP) in several investigation fields.Until the end of the seventies, the assay was complicated and the results uncertainbecause of the low purity reagents, which were initially, extracted directly fromdesiccated lanterns of firefly (Photinus pyralis), and poorly controlled reactionconditions at the laboratory. With the introduction of a new type of luciferinluciferasereagent, the ATP Monitoring Reagent (AMR), these disadvantages havebeen overcome, and therefore, the application of the assay has been extended.The new reagent results in the emission of light of an almost constant intensity,proportional to the ATP concentration. This is in contrast to the flash obtainedwith conventional reagents. This new technique facilitates the measurements andimproves the sensitivity of the assay. The reagent may also be used to monitorchanges in ATP concentration because the ratio between the ATP concentration andlight intensity remains constant. This work describes the principle and a detailedmethodology for determination of ATP, ADP, and AMP by the luciferin-luciferasereaction in fruits such as apples, pears and sweet cherries and some applications.

ATP-bioluminescence technique for fruit quality assessments: a detailed methodology for analysis of adenylate nucleotides

The bioluminescence technique has been used during a long time for measurementof adenylate nucleotides (ATP, ADP, and AMP) in several investigation fields.Until the end of the seventies, the assay was complicated and the results uncertainbecause of the low purity reagents, which were initially, extracted directly fromdesiccated lanterns of firefly (Photinus pyralis), and poorly controlled reactionconditions at the laboratory. With the introduction of a new type of luciferinluciferasereagent, the ATP Monitoring Reagent (AMR), these disadvantages havebeen overcome, and therefore, the application of the assay has been extended.The new reagent results in the emission of light of an almost constant intensity,proportional to the ATP concentration. This is in contrast to the flash obtainedwith conventional reagents. This new technique facilitates the measurements andimproves the sensitivity of the assay. The reagent may also be used to monitorchanges in ATP concentration because the ratio between the ATP concentration andlight intensity remains constant. This work describes the principle and a detailedmethodology for determination of ATP, ADP, and AMP by the luciferin-luciferasereaction in fruits such as apples, pears and sweet cherries and some applications.

Sulfoluciferin is Biosynthesized by a Specialized Luciferin Sulfotransferase in Fireflies.

Firefly luciferin is a specialized metabolite restricted to fireflies (family Lampyridae) and other select families of beetles (order Coleoptera). Firefly luciferin undergoes luciferase-catalyzed oxidation to produce light, thereby enabling the luminous mating signals essential for reproductive success in most bioluminescent beetles. Although firefly luciferin and luciferase have become widely used biotechnological tools, questions remain regarding the physiology and biochemistry of firefly bioluminescence. Here we report sulfoluciferin to be an in vivo derivative of firefly luciferin in fireflies and report the cloning of luciferin sulfotransferase (LST) from the North American firefly Photinus pyralis. LST catalyzes the production of sulfoluciferin from firefly luciferin and the sulfo-donor PAPS. Sulfoluciferin is abundant in several surveyed firefly genera as well as in the bioluminescent elaterid beetle Pyrophorus luminosus at a low level. We propose that sulfoluciferin could serve as a luciferin storage molecule in fireflies and that LST may find use as a new tool to modulate existing biotechnological applications of the firefly bioluminescent system.