Genome assembly of Genji firefly (Nipponoluciola cruciata) reveals novel luciferase-like luminescent proteins without peroxisome targeting signal.

The Genji firefly, Nipponoluciola cruciata, is an aquatic firefly endemic to Japan, inhabiting a wide area of the Japanese archipelago. The luminescence of fireflies is a scientifically interesting phenomenon, and many studies have evaluated this species in Japan. In this study, we sequenced the whole genome of male N. cruciata and constructed a high-quality genome assembly of 662 Mb with a BUSCO completeness of 99.1% in the genome mode. Using the detected set of 15,169 protein-coding genes, the genomic structures and genetic background of luminescence-related genes were also investigated. We found four new firefly luciferase-like genes in the genome. The highest bioluminescent activity was observed for LLa2, which originated from ancestral PDGY, a mitochondrial acyl-CoA synthetase. A thioesterase candidate, NcruACOT1, which is involved in d-luciferin biosynthesis, was expressed in the lantern. Two opsins were also detected and the absorption wavelength of the UV-type opsin candidate shifted from UV to blue. These findings provide an important resource for unravelling the adaptive evolution of fireflies in terms of luminescence and vision.

Discovery of Red-Shifting Mutations in Firefly Luciferase Using High-Throughput Biochemistry.

Photinus pyralis luciferase (FLuc) has proven a valuable tool for bioluminescence imaging, but much of the light emitted from the native enzyme is absorbed by endogenous biomolecules. Thus, luciferases displaying red-shifted emission enable higher resolution during deep-tissue imaging. A robust model of how protein structure determines emission color would greatly aid the engineering of red-shifted mutants, but no consensus has been reached to date. In this work, we applied deep mutational scanning to systematically assess 20 functionally important amino acid positions on FLuc for red-shifting mutations, predicting that an unbiased approach would enable novel contributions to this debate. We report dozens of red-shifting mutations as a result, a large majority of which have not been previously identified. Further characterization revealed that mutations N229T and T352M, in particular, bring about unimodal emission with the majority of photons being >600 nm. The red-shifting mutations identified by this high-throughput approach provide strong biochemical evidence for the multiple-emitter mechanism of color determination and point to the importance of a water network in the enzyme binding pocket for altering the emitter ratio. This work provides a broadly applicable mutational data set tying FLuc structure to emission color that contributes to our mechanistic understanding of emission color determination and should facilitate further engineering of improved probes for deep-tissue imaging.

Key homeobox transcription factors regulate the development of the firefly's adult light organ and bioluminescence.

Adult fireflies exhibit unique flashing courtship signals, emitted by specialized light organs, which develop mostly independently from larval light organs during the pupal stage. The mechanisms of adult light organ development have not been thoroughly studied until now. Here we show that key homeobox transcription factors AlABD-B and AlUNC-4 regulate the development of adult light organs and bioluminescence in the firefly Aquatica leii. Interference with the expression of AlAbd-B and AlUnc-4 genes results in undeveloped or non-luminescent adult light organs. AlABD-B regulates AlUnc-4, and they interact with each other. AlABD-B and AlUNC-4 activate the expression of the luciferase gene AlLuc1 and some peroxins. Four peroxins are involved in the import of AlLUC1 into peroxisomes. Our study provides key insights into the development of adult light organs and flash signal control in fireflies.

Cloning and characterization of luciferase from an Asian firefly Pygoluciola qingyu and its comparison with other beetle luciferases.

The bioluminescence system of luminescent beetles has extensive applications in biological imaging, protein labeling and drug screening. To explore wild luciferases with excellent catalytic activity and thermal stability, we cloned the luciferase of Pygoluciola qingyu, one species living in areas of high temperature and with strong bioluminescence, by combining transcriptomic sequencing and reverse transcription polymerase chain reaction (RT-PCR). The total length of luciferase gene is 1638 bp and the luciferase consists 544 amino acids. The recombinant P. qingyu luciferase was produced in vitro and its characteristics were compared with those of eight luciferases from China firefly species and two commercial luciferases. Compared with these luciferases, the P. qingyu luciferase shows the highest luminescence activity at room temperature (about 25-28 â„ƒ) with similar KM value for D-luciferin and ATP to the Photinus pyralis luciferase. The P. qingyu luciferase activity was highest at 35 â„ƒ and can keep high activity at 30-40 â„ƒ, which suggests the potential of P. qingyu luciferase for in vivo and cell application. Our results provide new insights into P. qingyu luciferase and give a new resource for the application of luciferases.

Luminescent characteristics and mitochondrial COI barcodes of nine cohabitated Taiwanese fireflies.

Background: Over 50 Taiwanese firefly species have been discovered, but scientists lack information regarding most of their genetics, bioluminescent features, and cohabitating phenomena. In this study, we focus on morphological species identification and phylogeny reconstructed by COI barcoding, as well as luminescent characteristics of cohabited Taiwanese firefly species to determine the key factors that influenced how distinct bioluminescent species evolved to coexist and proliferate within the same habitat. Methods: In this study, 366 specimens from nine species were collected in northern Taiwan from April to August, 2016-2019. First, the species and sex of the specimens were morphologically and genetically identified. Then, their luminescent spectra and intensities were recorded using a spectrometer and a power meter, respectively. The habitat temperature, relative humidity, and environmental light intensity were also measured. The cytochrome oxidase I (COI) gene sequence was used as a DNA barcode to reveal the phylogenetic relationships of cohabitated species. Results: Nine species-eight adult species (Abscondita chinensis, Abscondita cerata, Aquatica ficta, Luciola curtithorax, Luciola kagiana, Luciola filiformis, Curtos sauteri, and Curtos costipennis) and one larval Pyrocoelia praetexta-were morphologically identified. The nine species could be found in April-August. Six of the eight adult species shared an overlap occurrence period in May. Luminescent spectra analysis revealed that the λ max of studied species ranged from 552-572 nm (yellow-green to orange-yellow). The average luminescent intensity range of these species was about 1.2-14 lux (182.1-2,048 nW/cm2) for males and 0.8-5.8 lux (122.8-850 nW/cm2) for females, and the maximum luminescent intensity of males was 1.01-7.26-fold higher than that of females. Compared with previous studies, this study demonstrates that different λ max, species-specific flash patterns, microhabitat choices, nocturnal activity time, and/or an isolated mating season are key factors that may lead to the species-specific courtship of cohabitated fireflies. Moreover, we estimated that the fireflies start flashing or flying when the environmental light intensity decreased to 6.49-28.1 lux. Thus, based on a rough theoretical calculation, the sensing distance between male and female fireflies might be 1.8-2.7 m apart in the dark. In addition, the mitochondrial COI barcode identified species with high resolution and suggested that most of the studied species have been placed correctly with congeners in previous phylogenies. Several cryptic species were revealed by the COI barcode with 3.27%-12.3% variation. This study renews the idea that fireflies' luminescence color originated from the green color of a Lampyridae ancestor, then red-shifted to yellow-green in Luciolinae, and further changed to orange-yellow color in some derived species.

Biosensing firefly luciferin synthesis in bacteria reveals a cysteine-dependent quinone detoxification route in Coleoptera.

Luciferin biosynthetic origin and alternative biological functions during the evolution of beetles remain unknown. We have set up a bioluminescent sensing method for luciferin synthesis from cysteine and benzoquinone using E. coli and Pichia pastoris expressing the bright Amydetes vivianii firefly and P. termitilluminans click beetle luciferases. In the presence of D-cysteine and benzoquinone, intense bioluminescence is quickly produced, indicating the expected formation of D-luciferin. Starting with L-cysteine and benzoquinone, the bioluminescence is weaker and delayed, indicating that bacteria produce L-luciferin, and then racemize it to D-luciferin in the presence of endogenous esterases, CoA and luciferase. In bacteria the p-benzoquinone toxicity (IC50 ~ 25 µM) is considerably reduced in the presence of cysteine, maintaining cell viability at 3.6 mM p-benzoquinone concomitantly with the formation of luciferin. Transcriptional analysis showed the presence of gene products involved with the sclerotization/tanning in the photogenic tissues, suggesting a possible link between these pathways and bioluminescence. The lack of two enzymes involved with the last steps of these pathways, indicate the possible accumulation of toxic quinone intermediates in the lanterns. These results and the abundance of cysteine producing enzymes suggest that luciferin first appeared as a detoxification byproduct of cysteine reaction with accumulated toxic quinone intermediates during the evolution of sclerotization/tanning in Coleoptera.

Expanding luciferase reporter systems for cell-free protein expression.

Luciferases are often used as a sensitive, versatile reporter in cell-free transcription-translation (TXTL) systems, for research and practical applications such as engineering genetic parts, validating genetic circuits, and biosensor outputs. Currently, only two luciferases (Firefly and Renilla) are commonly used without substrate cross-talk. Here we demonstrate the expansion of the cell-free luciferase reporter system, with two orthogonal luciferase reporters: N. nambi luciferase (Luz) and LuxAB. These luciferases do not have cross-reactivity with the Firefly and Renilla substrates. We also demonstrate a substrate regeneration pathway for one of the new luciferases, enabling long-term time courses of protein expression monitoring in the cell-free system. Furthermore, we reduced the number of genes required in TXTL expression, by engineering a cell extract containing part of the luciferase enzymes. Our findings lead to an expanded platform with multiple orthogonal luminescence translation readouts for in vitro protein expression.

Bioluminescence Color-Tuning Firefly Luciferases: Engineering and Prospects for Real-Time Intracellular pH Imaging and Heavy Metal Biosensing.

Firefly luciferases catalyze the efficient production of yellow-green light under normal physiological conditions, having been extensively used for bioanalytical purposes for over 5 decades. Under acidic conditions, high temperatures and the presence of heavy metals, they produce red light, a property that is called pH-sensitivity or pH-dependency. Despite the demand for physiological intracellular biosensors for pH and heavy metals, firefly luciferase pH and metal sensitivities were considered drawbacks in analytical assays. We first demonstrated that firefly luciferases and their pH and metal sensitivities can be harnessed to estimate intracellular pH variations and toxic metal concentrations through ratiometric analysis. Using Macrolampis sp2 firefly luciferase, the intracellular pH could be ratiometrically estimated in bacteria and then in mammalian cells. The luciferases of Macrolampis sp2 and Cratomorphus distinctus fireflies were also harnessed to ratiometrically estimate zinc, mercury and other toxic metal concentrations in the micromolar range. The temperature was also ratiometrically estimated using firefly luciferases. The identification and engineering of metal-binding sites have allowed the development of novel luciferases that are more specific to certain metals. The luciferase of the Amydetes viviani firefly was selected for its special sensitivity to cadmium and mercury, and for its stability at higher temperatures. These color-tuning luciferases can potentially be used with smartphones for hands-on field analysis of water contamination and biochemistry teaching assays. Thus, firefly luciferases are novel color-tuning sensors for intracellular pH and toxic metals. Furthermore, a single luciferase gene is potentially useful as a dual bioluminescent reporter to simultaneously report intracellular ATP and/or luciferase concentrations luminometrically, and pH or metal concentrations ratiometrically, providing a useful tool for real-time imaging of intracellular dynamics and stress.

Cloning and molecular properties of a novel luciferase from the Brazilian Bicellonycha lividipennis (Lampyridae: Photurinae) firefly: comparison with other firefly luciferases.

Several firefly luciferases eliciting light emission in the yellow-green range of the spectrum and with distinct kinetic properties have been already cloned, sequenced, and characterized. Some of them are currently being applied as analytical reagents and reporter genes for bioimaging and biosensors, and more recently as potential color tuning indicators of intracellular pH and toxic metals. They were cloned from the subfamilies Lampyrinae (Photinini: Photinus pyralis, Macrolampis sp2; Cratomorphini: Cratomorphus distinctus), Photurinae (Photuris pennsylvanica), Luciolinae (Luciola cruciata, L. lateralis, L. mingrelica, L. italica, Hotaria parvula), and Amydetinae (Amydetes vivianii) occurring in different parts of the world. The largest number has been cloned from fireflies occurring in Brazilian biomes. Taking advantage of the large biodiversity of fireflies occurring in the Brazilian Atlantic rainforest, here we report the cloning and characterization of a novel luciferase cDNA from the Photurinae subfamily, Bicellonycha lividipennis, which is a very common firefly in marshlands in Brazil. As expected, multialignements and phylogenetic analysis show that this luciferase clusters with Photuris pennsylvanica adult isozyme, and with other adult lantern firefly luciferases, in reasonable agreement with traditional phylogenetic analysis. The luciferase elicits light emission in the yellow-green region, has kinetics properties similar to other adult lantern firefly luciferases, including pH- and metal sensitivities, but displays a lower sensitivity to nickel, which is suggested to be caused by the natural substitution of H310Y.

First complete mitochondrial genomes of Ototretinae (Coleoptera, Lampyridae) with evolutionary insights into the gene rearrangement.

The subfamily Ototretinae represents an important and unusual lineage of fireflies. Here, we sequenced and annotated three mitogenomes for this subfamily, with two Stenocladius species and one Drilaster species as representatives. The mitogenome of Stenocladius exhibits a rearranged gene order between trnC and trnW caused by transposition, which is a novel finding in Lampyridae. Meanwhile, a long intergenic space (241 to 376 bp) exists between the two rearranged genes, and some remnants (23 bp) of trnW are present within this non-coding region. Moreover, phylogenetic analyses did not recover the monophyly of Ototretinae, in which Drilaster is shown at a basal lineage in Lampyridae, but Stenocladius seems more related to Luciolinae. Therefore, the gene rearrangement in Stenocladius is presumed to result from independent evolutionary events, suggesting that this genus should be placed in a separate lineage. Nevertheless, more representative mitogenomes from different groups are required to verify the present results.

Firefly genomes illuminate the evolution of beetle bioluminescent systems.

Fireflies are one of the best-known bioluminescent organisms, and the reaction mechanism and ecological utility of bioluminescence have been well-studied. Genome assemblies of six species of bioluminescent beetles have recently been published. These studies have focused on the evolution of novelties; luciferase, and the biosynthesis of luciferin and defensive chemicals. For example, clustering of the luciferase gene with acyl-CoA synthetase genes on a chromosome in luminous beetle genomes suggests the involvement of tandem gene duplications and neofunctionalization during the evolution of beetle bioluminescence. Several candidate genes for critical roles in beetle bioluminescence have been identified, but their functional analyses are still ongoing. The establishment of a long-term mass-rearing system and strain will be the key for the post-genome research on bioluminescent beetles. Lastly, the application of contemporary chromosome-scale genome assembly techniques to luminous beetles will help resolve outstanding evolutionary questions, such as how many times bioluminescence evolved in this clade.

Molecular cloning, characterization, and evolution analysis of the luciferase genes from three sympatric sibling fireflies (Lampyridae: Lampyrinae, Diaphanes).

Firefly adult bioluminescence functions as signal communication between sexes. How sympatric sibling species with similar glow pattern recognize their conspecific mates remains largely unknown. To better understand the role of the luciferases of sympatric fireflies in recognizing mates, we cloned the luciferase genes of three sympatric forest dwelling fireflies (Diaphanes nubilus, Diaphanes pectinealis, and Diaphanes sp2) and evaluated their enzyme characteristics. Our data show that the amino acid (AA) sequences of all three luciferases are highly conserved, including the identities (D. nubilus vs D. pectinealis: 99%; D. nubilus vs Diaphanes sp2: 98.5%; D. pectinealis vs Diaphanes sp2: 99.4%) and the protein structures. Three recombinant luciferases produced in vitro all possess significant luminescence activity at pH 7.8, and similar maximum emission spectrum (D. nubilus: 562 nm; D. pectinealis and Diaphanes sp2: 564 nm). They show the highest activity at 10 °C (D. pectinealis, Diaphanes sp2) and 15 °C (D. nubilus), and completely inactivation at 45 °C. Their KM for D-luciferin and ATP were 2.7 µM and 92 µM (D. nubilus), 3.7 µM and 49 µM (D. pectinealis), 3.5 µM and 46 µM (Diaphanes sp2). Phylogenetic analyses support that D. nubilus is sister to D. pectinealis with Diaphanes sp2 at their base, which further cluster with Pyrocoelia. All combined data indicate that sympatric Diaphanes species have similar luciferase characteristics, suggesting that other strategies (e.g., pheromone, active time, etc.) may be adopted to recognize mates. Our data provide new insights into Diaphanes luciferases and their evolution.

Mutant firefly luciferase enzymes resistant to the inhibition by sodium chloride.

OBJECTIVES: Firefly luciferase, one of the most extensively studied enzymes, has numerous applications. However, luciferase activity is inhibited by sodium chloride. This study was aimed at obtaining mutant luciferase enzymes resistant to the sodium chloride inhibition. RESULTS: We first obtained two mutant luciferase enzymes whose inhibition were alleviated and determined the mutations to be Val288Ile and Glu488Val. Under medical dialysis condition (140 mM sodium chloride), the wild type was inhibited to 44% of its original activity level. In contrast, the single mutants, Val288Ile and Glu488Val, retained 67% and 79% of their original activity, respectively. Next, we introduced Val288Ile and Glu488Val mutations into wild-type luciferase to create a double mutant using site-directed mutagenesis. Notably, the double mutant retained its activity more than 95% of that in the absence of sodium chloride. CONCLUSIONS: The mutant luciferase, named luciferase CR, was found to retain its activity in various concentrations of sodium chloride. The luciferase CR may be extensively useful in any bioassay which includes firefly luciferase and is employed in the presence of sodium chloride.

The nearly complete mitogenome of the Southeast Asian firefly Pteroptyx tener (Coleoptera: Lampyridae).

The estuarine firefly, Pteroptyx tener, aggregates in the thousands in mangrove trees lining tidal rivers in Southeast Asia where they engage one another in a nocturnal, pre-mating ritual of synchronised courtship flashes. Unfortunately, populations of the species by virtue of being restricted to isolated estuarine rivers systems in the region, are at risk of genetic isolation. Because of this concern we undertook the task of sequencing and characterising the mitochondrial DNA genome of P. tener, as the first step towards helping us to characterise and better understand their genetic diversity. We sequenced and assembled the mitochondrial DNA genome of P. tener from two male and female specimens from the district of Kuala Selangor in Peninsular Malaysia and announce the molecules in this publication. We also reconstructed the phylogenetic trees of all available lampyrids mitogenomes and suggest the need to re-examine our current understanding of their classification which have largely been based on morphological data and the cox1 gene. Separately, our analysis of codon usage patterns among lampyrid mitogenomes showed that the codon usage in a majority of the protein-coding genes were non-neutral. Codon usage patterns between mitogenome sequences of P. tener were, however, largely neutral. Our findings demonstrate the usefulness of mitochondrial genes/mitogenomes for analysing both inter- and intra- specific variation in the Lampyridae to aid in species discovery in this highly variable genus; and elucidate the phylogenetic relationships of Pteroptyx spp. from the region.

Selection, drift, and constraint in cypridinid luciferases and the diversification of bioluminescent signals in sea fireflies.

Understanding the genetic causes of evolutionary diversification is challenging because differences across species are complex, often involving many genes. However, cases where single or few genetic loci affect a trait that varies dramatically across a radiation of species provide tractable opportunities to understand the genetics of diversification. Here, we begin to explore how diversification of bioluminescent signals across species of cypridinid ostracods ("sea fireflies") was influenced by evolution of a single gene, cypridinid-luciferase. In addition to emission spectra ("colour") of bioluminescence from 21 cypridinid species, we report 13 new c-luciferase genes from de novo transcriptomes, including in vitro assays to confirm function of four of those genes. Our comparative analyses suggest some amino acid sites in c-luciferase evolved under episodic diversifying selection and may be associated with changes in both enzyme kinetics and colour, two enzymatic functions that directly impact the phenotype of bioluminescent signals. The analyses also suggest multiple other amino acid positions in c-luciferase evolved neutrally or under purifying selection, and may have impacted the variation of colour of bioluminescent signals across genera. Previous mutagenesis studies at candidate sites show epistatic interactions, which could constrain the evolution of c-luciferase function. This work provides important steps toward understanding the genetic basis of diversification of behavioural signals across multiple species, suggesting different evolutionary processes act at different times during a radiation of species. These results set the stage for additional mutagenesis studies that could explicitly link selection, drift, and constraint to the evolution of phenotypic diversification.

Genomic and experimental data provide new insights into luciferin biosynthesis and bioluminescence evolution in fireflies.

Fireflies are among the most charismatic insects for their spectacular bioluminescence, but the origin and evolution of bioluminescence remain elusive. Especially, the genic basis of luciferin (D-luciferin) biosynthesis and light patterns is largely unknown. Here, we present the high-quality reference genomes of two fireflies Lamprigera yunnana (1053 Mb) and Abscondita terminalis (501 Mb) with great differences in both morphology and luminous behavior. We sequenced the transcriptomes and proteomes of luminous organs of two species. We created the CRISPR/Cas9-induced mutants of Abdominal B gene without luminous organs in the larvae of A. terminalis and sequenced the transcriptomes of mutants and wild-types. Combining gene expression analyses with comparative genomics, we propose a more complete luciferin synthesis pathway, and confirm the convergent evolution of bioluminescence in insects. Using experiments, the function of the firefly acyl-CoA thioesterase (ACOT1) to convert L-luciferin to D-luciferin was validated for the first time. Comparisons of three-dimension reconstruction of luminous organs and their differentially expressed genes among two species suggest that two positive genes in the calcium signaling pathway and structural difference of luminous organs may play an important role in the evolution of flash pattern. Altogether, our results provide important resources for further exploring bioluminescence in insects.

Comparative transcriptomic analysis of fireflies (Coleoptera: Lampyridae) to explore the molecular adaptations to fresh water.

Aquatic insects are well adapted to freshwater environments, but the molecular basis of these adaptations remains largely unknown. Most firefly species (Coleoptera: Lampyridae) are terrestrial, but the larvae of several species are aquatic. Here, larval and adult transcriptomes from Aquatica leii (freshwater) and Lychnuris praetexta (terrestrial) were generated to test whether the genes associated with metabolic efficiency and morphology have undergone adaptive evolution to fresh water. The aquatic fireflies had a significantly lower ratio of nonsynonymous to synonymous substitutions than the terrestrial insects, indicating a genomewide evolutionary constraint in the aquatic fireflies. We identified 341 fast-evolving genes and 116 positively selected genes in the aquatic fireflies. Of these, 76 genes exhibiting both fast evolution and positive selection were primarily involved in ATP production, energy metabolism and the hypoxia response. We identified 7,271 differentially expressed genes (DEGs) in A. leii (adults versus larvae) and 8,309 DEGs in L. praetexta (adults versus larvae). DEGs specific to the aquatic firefly (n = 1,445) were screened via interspecific comparisons (A. leii versus L. praetexta) and were significantly enriched for genes involved in metabolic efficiency (e.g., ATP production, hypoxia, and immune responses) and certain aspects of morphology (e.g., cuticle chitin, tracheal and compound eye morphology). These results indicate that sequence and expression-level changes in genes associated with both metabolic efficiency and morphological attributes related to the freshwater lifestyle contributed to freshwater adaptation in fireflies. This study provides new insights into the molecular mechanisms of aquatic adaptation in insects.

Comparative Transcriptomics Reveals Gene Families Associated with Predatory Behavior in Photuris femme fatale Fireflies.

Identifying the basis of phenotypic variation is a key objective of genetics. This work has been mostly limited to model systems with a plethora of genetic manipulation and functional characterization tools. With the development of high-throughput sequencing and new computational tools, it is possible to identify candidate genes related to phenotypic variation in non-model organisms. Fireflies are excellent for studying phenotypic variation because of their diverse and well-characterized behaviors. Most adult fireflies emit a single mating flash pattern and do not eat. In contrast, adult females of many species in the genus Photuris employ multiple flash patterns and prey upon mate-seeking males of other firefly species. To investigate the genetic basis for this variation, we used comparative transcriptomics to identify positively selected genes between a predatory firefly, Photuris sp., and a non-predatory relative, Photuris frontalis, controlling for genes generally under selection in fireflies by comparing to a Photinus firefly. Nine gene families were identified under positive selection in the predatory versus non-predatory Photuris comparison, including genes involved in digestion, detoxification, vision, reproduction, and neural processes. These results generate intriguing hypotheses about the genetic basis for insect behavior and highlight the utility of comparative transcriptomic tools to investigate complex behaviors in non-model systems.

Evaluation of the population structure and phylogeography of the Japanese Genji firefly, Luciola cruciata, at the nuclear DNA level using RAD-Seq analysis.

The Genji firefly, Luciola cruciata, is widely distributed throughout the major Japanese islands (Honshu, Shikoku, and Kyushu) and distinguished into two ecological types on the basis of the flash interval of the mate-seeking males (4-sec slow-flash or 2-sec fast-flash intervals). The boundary of the ecological types corresponds to the Fossa Magna, a great rupture zone that separates eastern and western Japan. Although the degree of genetic differentiation of the two types has been evaluated using allozyme and mitochondrial DNA sequence data, it has not been evaluated using genome-wide data. Based on the genome-wide data obtained using single-end restriction-site-associated DNA (RAD-Seq), principal component, gene-level phylogenetic tree, admixture, and Wright's fixation index analyses, we identified three phylogenetic groups in L. cruciata: East-Honshu, West-Honshu, and Kyushu. This grouping corresponds to the ecological types: East-Honshu to the slow-flash type and West-Honshu and Kyushu to the fast-flash type. Although introgression was exceptionally observed around adjacent or artificially transplanted areas, gene flow among the groups was almost absent in the natural populations. The phylogenetic tree under the coalescent model also evaluated differentiation among the East-Honshu, West-Honshu and Kyushu groups. Furthermore, because the distribution patterns of the three groups are consistent with the geological history of Japanese islands, a vicariant speciation scenario of L. cruciata is concluded. In addition, we identified genetic markers that can be used to distinguish the three genetic groups for genetic management of firefly transplantation in nature conservation and regeneration.

Identification of potential candidate genes involved in the sex determination cascade in an aquatic firefly, Sclerotia aquatilis (Coleoptera, Lampyridae).

Sexual differentiation, dimorphism, and courtship behavior are the downstream developmental programs of the sex determination cascade. The sex determination cascade in arthropods often involves key genes, transformer (tra), doublesex (dsx), transformer-2 (tra2), and fruitless (fru). These genes are conserved among insect taxa; however, they have never been reported in fireflies. In this study, the candidate genes for these key genes were identified for the first time in an aquatic firefly, Sclerotia aquatilis using transcriptome analysis. A comparative protein-protein interaction (PPI) network of sex determination cascade was reconstructed for S. aquatilis based on a network of a model insect, Drosophila melanogaster. Subsequently, a sex determination cascade in S. aquatilis was proposed based on the amino acid sequence structures and expression profiles of these candidates. This study describes the first efforts toward understanding the molecular control of sex determination cascade in fireflies.