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PubMed1992

Expression of luciferase genes from different origins in Bacillus subtilis.

A group of vectors for luciferase expression in Bacillus subtilis was constructed. So far, only bacterial luciferases have been expressed in Bacillus, but in this study we wanted also to express genes encoding eukaryotic luciferases to perform direct comparisons of the light levels produced by the two different systems in B. subtilis. The vectors constructed can replicate both in Escherichia coli and B. subtilis, and the luciferase expression is strictly regulated due to the dual plasmid system used. Nearly a 100-fold increase in light production compared to previous results was achieved when genes encoding bacterial luciferase were inserted into the constructs and transformed into B. subtilis. An additional tenfold increase in light production was obtained when luciferase genes from the North American firefly (Photinus pyralis) or a click beetle (Pyrophorus plagiophtalamus) were introduced in a similar fashion into B. subtilis. Measurement of the light emission was performed without disruption of bacterial cells in a real-time manner, which is a common feature when working with all of these constructions. Structures of the shuttle vector constructs and results from light emission measurements are presented.

Amino Acid Sequence↗
PubMed1988

ATP formation onset lag and post-illumination phosphorylation initiated with single-turnover flashes. I. An assay using luciferin-luciferase luminescence.

The great sensitivity of the luciferin-luciferase ATP detection system allows direct observation of ATP formation derived from single-turnover flashes in a thylakoid reaction mixture. The method can measure the energization threshold--the number of flashes required for the initiation of ATP formation--as well as detect post-illumination ATP formation after the last flash of a flash sequence. We describe the characteristics of this post-illumination phosphorylation which can be observed after a series of phosphorylating flashes (PIP+) or when the assay for ATP formation was performed in a "traditional" manner where the ADP and Pi were added after the flash-energization period (PIP-). Comparing PIP+ yields and kinetics of the PIP+ decay under various treatments can give information about membrane energization events only if it is clearly established that different PIP+ yields and decay rates are not due to limitations of the luciferase-catalyzed reaction. Experiments showing that the PIP+ ATP yield and kinetics were due to membrane-limited deenergization events (proton efflux) rather than luciferase limitations include: (1) An uncoupler, nigericin, added after the last flash reduced the PIP+ yield, but had no effect on the luciferase reaction. (2) The kinetics of the luminescence after adding standard ATP were much faster than the PIP+ kinetics. (3) Valinomycin and K+ stimulated the PIP+ yield but had no influence on the luciferase reaction. (4) Lowering the pH from 8 to 7 increased both the PIP- (an assay independent of luciferase kinetics) and the PIP+ ATP yields, an expected result owing to the greater endogenous buffering power encountered by the proton gradient when the external pH is 7. In spite of the last-mentioned point, the threshold flash number for ATP formation onset was the same for pH 7 and 8 (valinomycin, K+ present) at slow flash frequencies. This is consistent with a membrane-localized rather than a delocalized gradient. The accompanying reports (W. A. Beard, G. Chiang and R. A. Dilley, and W. A. Beard and R. A. Dilley, J. Bioenerg. Biomembr.) show that different conditions can lead to observing either localized or delocalized proton gradient coupling in the PIP+ event and the ATP onset threshold flash number.

Adenosine Triphosphate↗
PubMed1994

Luciferase activity as a marker of tumor burden and as an indicator of tumor response to antineoplastic therapy in vivo.

The gene encoding firefly luciferase has been used as a reporter gene for the study of gene function. The luciferase catalyzes its substrate and subsequently produces luminescence. In addition, it is not present in mammalian cells. We have therefore explored its use in monitoring the growth of tumors in vivo. The luciferase gene was transfected into two murine tumor lines, i.e. c162 melanoma and M109 lung carcinoma, and the luciferase activity associated with the cells was determined by a rapid chemiluminescent reaction. Luciferase activity was well-correlated with the number of tumor cells in vitro. Luciferase activity also correlated with the tumor burden in vivo, as well as with the effect of an adoptive T cell transfer therapy in the syngeneic C3H/HeN mice experimental tumor model. This assay offers the advantage of being quantitative, rapid, and reliable for the detection of tumor burden and for the evaluation of the effect of antineoplastic therapy.

Animals↗
PubMed2001

Enhanced luciferin entry causes rapid wound-induced light emission in plants expressing high levels of luciferase.

In-vivo imaging of transgenic tobacco plants (Nicotiana tobacum L.) expressing firefly luciferase under the control of the Arabidopsis phenylalanine ammonialyase 1 (PAL1)-promoter showed that luciferase-catalyzed light emission began immediately after the substrate luciferin was sprayed onto the leaves and reached a plateau phase after approximately 60 min. This luminescence could easily be detected for up to 24 h after luciferin application although the light intensity declined continuously during this period. A strong and rapid increase in light emission was observed within the first minutes after wounding of luciferin-sprayed leaves. However, these data did not correlate with luciferase activity analysed by an in-vitro enzyme assay. In addition, Arabidopsis plants expressing luciferase under the control of the constitutive 35S-promoter showed similar wound-induced light emission. In experiments in which only parts of the leaves were sprayed with luciferin solutions, it was shown that increased uptake of luciferin at the wound site and its transport through vascular tissue were the main reasons for the rapid burst of light produced by preformed luciferase activity. These data demonstrate that there are barriers that restrict luciferin entry into adult plants, and that luciferin availability can be a limiting factor in non-invasive luciferase assays.

Arabidopsis↗
PubMed2005

Bioluminescence spectra of native and mutant firefly luciferases as a function of pH.

Bioluminescence spectra of the wild-type recombinant Luciola mingrelica firefly luciferase and its mutant form with the His433Tyr point mutation were obtained within the pH 5.6-10.2 interval. The spectra are shown to be a superposition of the spectra of the three forms of the electronically excited reaction product oxyluciferin: ketone (lambdamax = 618 nm), enol (lambdamax = 587 nm), and enolate-ion (lambdamax = 556 nm). The shift in lambdamax by 40 nm to the red region in the mutant luciferase bioluminescence at the pH optimum of enzyme activity (pH 7.8) is explained by the change in the relative content of different oxyluciferin forms due to the shift in the ketone <--> enol <--> enolate equilibria. A computer model of the luciferase-oxyluciferin-AMP complex was constructed and the structure of amino acid residues participating in the equilibrium is proposed. Computer models of the protein region near the His433 residue for the wild type and mutant luciferases are also proposed. Comparison of the models shows that the His433Tyr mutation increases flexibility of the polypeptide loop that binds the N and C domains of luciferase. As a result, the flexibility of the C domain amino acid residues in the emitter microenvironment increases, and this increase may be the reason for the observed differences in the bioluminescence spectra of the native and mutant luciferases.

Adenosine Monophosphate↗
PubMed1988

Delineation of bacterial luciferase aldehyde site by bifunctional labeling reagents.

Previously we have established that a highly reactive cysteinyl group on the alpha subunit is at the aldehyde site of the (alpha beta) dimeric Vibrio harveyi luciferase. Three isomeric bifunctional reagents have been synthesized and used to further delineate the luciferase aldehyde site. These probes differ in their relative positions of and distances between the two functional groups active in chemical and photochemical labelings, respectively. Each of the probes can effectively and reversibly inactivate luciferase by forming a disulfide linkage primarily to the reactive cysteinyl residue. Upon subsequent photolysis, a diazoacetate arm in each probe was activated for photochemical labeling of amino acid residues within reach. After reductive regeneration of the reactive cysteinyl residue, 0.35-0.40 probe per dimeric luciferase was found to have been photochemically incorporated, correlating well with the degree of irreversible enzyme inactivation. Low but significant amounts of the three isomeric probes initially attached to the alpha reactive cysteine through a disulfide have been found to photochemically tag certain residues on beta. The latter residues are estimated to be no more than 8-11 A away from the alpha reactive cysteine. Thus the reactive cysteinyl residue, and hence the aldehyde site, must be at or near the alpha beta subunit interface. Furthermore, the structural integrity of the microenvironment surrounding this reactive cysteinyl residue is crucial to luciferase activity. An HPLC method for the isolation of luciferase alpha and beta subunits has also been developed.

Affinity Labels↗
PubMed1992

Fatty acid-enhanced binding of flavin mononucleotide to bacterial luciferase measured by steady-state fluorescence.

Bacterial luciferase catalyzes the oxidation of reduced flavin mononucleotide and tetradecanal resulting in the emission of light. We have investigated the interactions of a recombinant luciferase from a terrestrial bacterium Xenorhabdus luminescens with the reaction products, FMN and myristic acid, using steady-state fluorescence spectroscopy. Quenching of the intrinsic fluorescence and FMN fluorescence on binding of FMN to luciferase was found to be greatly stimulated in the presence of myristic acid, corresponding to a reduction of more than 30-fold in the FMN dissociation constant of the enzyme. In addition, the FMN-luciferase complex exhibits distinct fatty acid-dependent fluorescence properties. These results indicate that luciferase forms a ternary complex with FMN and myristic acid with a significantly different conformation from that of the binary FMN-luciferase complex.

Enterobacteriaceae↗
PubMed1993

Luciferase from the east European firefly Luciola mingrelica: cloning and nucleotide sequence of the cDNA, overexpression in Escherichia coli and purification of the enzyme.

We have cloned cDNA encoding luciferase in Luciola mingrelica, fireflies living near the Black Sea in southern Russia, and obtained high level expression of the cloned sequences in Escherichia coli. The nucleotide sequences of two isolated clones were determined; five single base differences were observed, but none resulted in a change in the encoded amino acid residue. The cDNA encoded a protein of 548 amino acid residues. The overall amino acid sequence identity with the luciferase from Photinus pyralis, the North American firefly, was 67%, while comparison of the L. mingrelica luciferase with L. cruciata and L. lateralis, both indigenous to Japan, showed about 80% of the residues were strictly conserved. A novel overexpression system which employs the regulatory genes of the luminous bacterium Vibrio fischeri allowed growth of cultures to high cell density and high luciferase content, facilitating purification of the enzyme. Luciferase was purified to homogeneity in good yield from lysates of recombinant E. coli by ammonium sulfate fractionation and chromatography on columns of DEAE Sephadex and Blue Sepharose. The physicochemical properties of the luciferases from the available recombinant sources are significantly different and should allow detailed investigations into the mechanism of the bioluminescence reaction and the physical basis of the differences in the color of light emitted from the various enzymes.

Amino Acid Sequence↗
PubMed1996

Engineering the C-terminus of firefly luciferase as an indicator of covalent modification of proteins.

Protein kinase recognition sequences and proteinase sites were engineered into the cDNA encoding firefly luciferase from Photinus pyralis in order to establish whether these modified proteins could be developed as bioluminescent indicators of covalent modification of proteins. Two key domains of the luciferase were modified in order to identify regions of the protein in which peptide sequences may be engineered whilst retaining bioluminescent activity; one between amino acids 209 and 227 and the other at the C-terminus, between amino acids 537 and 550. Mutation of amino acids between residues 209 and 227 reduced bioluminescent activity to less than 1% of wild-type recombinant. In contrast engineering peptide sequences at the C-terminus resulted in specific activities ranging from 0.06-120% of the wild-type recombinant. Addition of cyclic AMP dependent protein kinase catalytic subunit, to a variant luciferase incorporating the kinase recognition sequence, LRRASLG, with a serine at amino-acid position 543 resulted in a 30% reduction in activity. Alkaline phosphatase treatment restored activity. The bioluminescent activity of a variant luciferase containing a thrombin recognition sequence, LVPRES, with the cleavage site positioned between amino acid 542 and 543, decreased by 50% when incubated in the presence of thrombin. The results indicate regions within luciferase where peptide sequences may be engineered while retaining bioluminescent activity and have shown changes in bioluminescent activity when these sites are subjected to covalent modification. Changes in secondary structure, charge and length at the C-terminus of luciferase disrupt the microenvironment of the active site, leading to alterations in light emission. This has important implications both in understanding the evolution of beetle bioluminescence and also in development of bioluminescent indicators of the covalent modification of proteins.

Amino Acid Sequence↗
PubMed1989

Cloning and sequence analysis of cDNA for luciferase of a Japanese firefly, Luciola cruciata.

Luciferases of Japanese and North American fireflies act on a common substrate (luciferin) but the resulting lights emitted are of different colors. As a step toward an understanding of the molecular mechanism of the luciferase reaction, a cDNA clone (pGLf1) was isolated from a cDNA library of lantern poly(A)+RNA of the Japanese firefly, Luciola cruciata ('Genji-botaru' in Japanese), using a cDNA of North American firefly luciferase. The isolated 2-kb cDNA sequence was able to direct the synthesis of active luciferase in Escherichia coli under the control of the lac promoter. The primary structure of Genji firefly luciferase deduced from the nucleotide sequence was shown to consist of 548 amino acids (aa) with an Mr of 60,024. Homology between the amino acid sequences of the Genji and North American firefly luciferases was 67%, but a number of amino acid changes were found in the first 200 aa from the N terminus.

Amino Acid Sequence↗
PubMed2006

Thermostable red and green light-producing firefly luciferase mutants for bioluminescent reporter applications.

Light emission from the North American firefly Photinus pyralis, which emits yellow-green (557-nm) light, is widely believed to be the most efficient bioluminescence system known, making this luciferase an excellent tool for monitoring gene expression. We present studies on the production of a set of thermostable red- and green-emitting luciferase mutants with bioluminescent properties suitable for dual-color reporter assays, biosensor measurements with internal controls, and imaging techniques. Starting with the luciferase variant Ser284Thr, we introduced the mutations Thr214Ala, Ala215Leu, Ile232Ala, Phe295Leu, and Glu354Lys to produce a new red-emitting enzyme with a bioluminescence maximum of 610 nm, narrow emission bandwidth, favorable kinetic properties, and excellent thermostability at 37 degrees C. By adding the same five changes to luciferase mutant Val241Ile/Gly246Ala/Phe250Ser, we produced a protein with an emission maximum of 546 nm, providing a set of thermostable enzymes whose bioluminescence maxima were separated by 64 nm. Model studies established that the luciferases could be detected at the attomole level and six orders of magnitude higher. In microplate luminometer format, mixtures containing 1.0 fmol total luciferase were quantified from measurements of simultaneously emitted red and green light. The results presented here provide evidence that it is feasible to monitor two distinct activities at 37 degrees C with these novel thermostable proteins.

Animals↗
PubMed2005

Bacterial luciferase is naturally destabilized in Mycobacterium tuberculosis and can be used to monitor changes in gene expression.

Reporter systems efficient at monitoring temporal gene expression in slow-growing mycobacteria would significantly aid the characterization of gene expression in specific environments. Bacterial luciferase is a reporter that has not been widely used to study gene expression in mycobacteria. This report describes the determination of the degradation of bacterial luciferase in Mycobacterium tuberculosis H37Ra and its utility as a reporter of temporal gene expression in this slow-growing mycobacterium. The inducible/repressible alanine dehydrogenase promoter of M. tuberculosis H37Rv was used to track the decay kinetics of Vibrio harveyi luciferase in both mid-log phase and stationary phase grown M. tuberculosis H37Ra, which proved to be highly similar during both phases of growth. The luciferase reporter was then used to detect changes in expression from the heat-shock promoter, phsp60, of M. bovis BCG during M. tuberculosis H37Ra growth in culture. Quantitative real-time PCR analysis of groEL2, the hsp60 homologue in M. tuberculosis, displayed a similar pattern of expression to phsp60-driven luciferase. These results strongly suggest that the luciferase reporter can be used to monitor temporal changes in gene expression in M. tuberculosis and may serve as a novel system to examine gene expression under specific conditions.

Alanine Dehydrogenase↗
PubMed2004

Characterization of CG6178 gene product with high sequence similarity to firefly luciferase in Drosophila melanogaster.

This is the first identification of a long-chain fatty acyl-CoA synthetase in Drosophila by enzymatic characterization. The gene product of CG6178 (CG6178) in Drosophila melanogaster genome, which has a high sequence similarity to firefly luciferase, has been expressed and characterized. CG6178 showed long-chain fatty acyl-CoA synthetic activity in the presence of ATP, CoA and Mg(2+), suggesting a fatty acyl adenylate is an intermediate. Recently, it was revealed that firefly luciferase has two catalytic functions, monooxygenase (luciferase) and AMP-mediated CoA ligase (fatty acyl-CoA synthetase). However, unlike firefly luciferase, CG6178 did not show luminescence activity in the presence of firefly luciferin, ATP, CoA and Mg(2+). The enzymatic properties of CG6178 including substrate specificity, pH dependency and optimal temperature were close to those of firefly luciferase and rat fatty acyl-CoA synthetase. Further, phylogenic analyses strongly suggest that the firefly luciferase gene may have evolved from a fatty acyl-CoA synthetase gene as a common ancestral gene.

Adenosine Monophosphate↗
PubMed2004

A new additional reporter enzyme, dinoflagellate luciferase, for monitoring of gene expression in mammalian cells.

Dinoflagellate luciferase (DL) catalyses the oxidation of dinoflagellate luciferin by molecular oxygen, resulting in an electronically excited species that emits blue light (lambda(max)=474 nm). Luciferase has three catalytic domains in its single polypeptide chain (M(r)=ca. 140 kDa), and each domain (about 40 kDa) is enzymatically active when expressed individually in recombinant fusion proteins in E. coli. Thus, DL should be useful as a reporter enzyme in studies of gene expression in mammalian cells. Expression plasmids consisting of one domain of luciferase (dDL) cDNA linked to different several promoters were introduced into a series of mammalian cell lines. Following transfection, dDL activities in cell extracts were determined by a rapid light emission assay of luciferase activity. For dual and multiple reporter assays, it is possible to exchange dDL for the firefly or renilla luciferases, and use the new luciferase for control or target reporter genes. Thus, the triple-reporter assay can identify three transcriptional activities of different genes at the same time. This work establishes the DL gene as a new efficient marker of gene expression in mammalian cells.

Animals↗
PubMed1998

Is there a specific receptor for anesthetics? Contrary effects of alcohols and fatty acids on phase transition and bioluminescence of firefly luciferase.

Firefly luciferase emits a burst of light when mixed with ATP and luciferin (L) in the presence of oxygen. This study compared the effects of long-chain n-alcohols (1-decanol to 1-octadecanol) and fatty acids (decanoic to octadecanoic acids) on firefly luciferase. Fatty acids were stronger inhibitors of firefly luciferase than n-alcohols. Myristyl alcohol inhibited the light intensity by 50% (IC50) at 13.6 microM, whereas the IC50 of myristic acid was 0.68 microM. According to the Meyer-Overton rule, fatty acids are approximately 12,000-fold stronger inhibitors than corresponding alcohols. The Lineweaver-Burk plot showed that myristic acid inhibited firefly luciferase in competition with luciferin, whereas myristyl alcohol inhibited it noncompetitively. The differential scanning calorimetry (DSC) showed that an irreversible thermal transition occurred at approximately 39 degrees C with a transition DeltaHcal of 1.57 cal g-1. The ligand effects on the transition were evaluated by the temperature where the irreversible change is half completed. Alcohols decreased whereas fatty acids increased the thermal transition temperature of firefly luciferase. Koshland's transition-state theory (Science. 1963. 142:1533-1541) states that ligands that bind to the substrate-recognition sites induce the enzyme at a transition state, which is more stabilized than the native state against thermal perturbation. The long-chain fatty acids bound to the luciferin recognition site and stabilized the protein conformation at the transition state, which resisted thermal denaturation. Eyring's unfolding theory (Science. 1966. 154:1609-1613) postulates that anesthetics and alcohols bind nonspecifically to interfacial areas of proteins and reversibly unfold the conformation. The present results showed that alcohols do not compete with luciferin and inhibit firefly luciferase nonspecifically by unfolding the protein. Fatty acids are receptor binders and stabilize the protein conformation at the transition state.

Alcohols↗
PubMed2000

Use of firefly luciferase in ATP-related assays of biomass, enzymes, and metabolites.

The kinetics of ATP reagents not affected by product inhibition or other forms of inactivation of luciferase during the measurement time has been clarified. Under these conditions the decay rate of the light emission expressed as percentage per minute is a measure of luciferase activity and can be given as the rate constant k (min-1), directly reflecting the degradation of ATP in the luciferase reaction. Three types of reagents with different analytical characteristics and different application possibilities have been identified. Stable light-emitting reagents are suitable for measurements of ATP down to 1000 amol. This is the only type of reagent suitable for monitoring ATP-converting reactions, i.e., assays of enzymes or metabolites, assays of oxidative phosphorylation, photophosphorylation, and so on. A higher luciferase activity resulting in a slow decay of the light emission by approximately 10% per minute (k = 0.1 min-1) gives a reagent suitable for measurements down to 10-100 amol. The slow decay of light emission allows use of manual luminometers without reagent dispensers. A further increase of the luciferase activity resulting in a decay rate of approximately 235% per min (k = 2.35 min-1) and only 10% of the light emission remaining after 1 min is suitable for measurements down to 1 amol corresponding to half a bacterial cell. With this type of flash reagent the total light emission can be calculated from two measurements of the light intensity on the decay part of the light emission curve. This new measure is not affected by moderate variations in luciferase activity, but only by changes in quantum yield and self-absorption of the light in the sample. Flash-type reagents require the use of reagent dispensers. The stringent requirements for ATP-free cuvettes, pipette tips, and contamination-free laboratory techniques make it unlikely that flash reagents would be useful in nonlaboratory surroundings. A potential application for this type of reagent is sterility testing. In general, it is concluded that one should select the ideal ATP reagent carefully for each application. Obviously the reagents used in a particular application do not have to match the decay rates given earlier exactly. However, various applications of the ATP technology and the properties of manual and automatic luminometers fall quite nicely into categories corresponding to the properties of the three reagents described. The rapidly growing interest in ATP technology has already resulted in the development of a greater variety of luminometers, from hand-held instruments to high-throughput systems. The continuation of efforts in both reagent and instrument development will undoubtedly result in many new applications.

Adenosine Triphosphate↗
PubMedDate not supplied

Sensitivity of virally-driven luciferase reporter plasmids to members of the steroid/thyroid/retinoid family of nuclear receptors.

During a series of transfection experiments, the pRSV-luc plasmid used as an internal control was found to be sensitive to cotransfection with expression vectors for several members of the steroid/thyroid/retinoid superfamily of nuclear receptors. Therefore, a survey of the effect of these expression vectors on the activity of four reporter plasmids was conducted. In CV-1 cells, the activity of pRSV-luc, which contains the P. pyralis luciferase gene, was repressed by co-transfection of PPARalpha and ARP-1 and was activated by COUP-TFI. Expression of pSV40-luc, containing the same luciferase gene, was repressed by PPARalpha and HNF-4 and activated by both COUP-TFI and ARP-1. All four of these expression vectors reduced the expression of the pRL-TK plasmid, which contains the luciferase gene from Renilla reniformis. RXR expression vectors had no effect on luciferase activity in CV-1 cells but induced luciferase activity in H4IIEC3 hepatoma cells. This activation was blocked by the addition of ligand, 9-cis retinoic acid. pSV2-CAT, which contains the chloramphenicol acetyltransferase gene, was insensitive to all receptor expression vectors tested. Both the P. pyralis and R. reniformis luciferase genes appear to contain sequences that render them responsive to steroid/thyroid/retinoid nuclear receptors.

Animals↗
PubMed2003

Genomic structure of the luciferase gene and phylogenetic analysis in the Hotaria-group fireflies.

The luminescent fireflies have species specific flash patterns, being recognized as sexual communication. The luciferase gene is the sole enzyme responsible for bioluminescence. We describe here the complete nucleotide sequence and the exon-intron structure of the luciferase gene of the Hotaria-group fireflies, H. unmunsana, H. papariensis and H. tsushimana. The luciferase gene of the Hotaria-group firefly including the known H. parvula spans 1950 bp and consisted of six introns and seven exons coding for 548 amino acid residues, suggesting highly conserved structure among the Hotaria-group fireflies. Although only one luciferase gene was cloned from H. papariensis, each of the two sequences of the gene was found in H. unmunsana (U1 and Uc) and H. tsushimana (T1 and T2). The amino acid sequence divergence among H. unmunsana, H. papariensis, and H. tsushimana only ranged from zero to three amino acid residues, but H. parvula differed by 10-11 amino acid residues from the other Hotaria-group fireflies, suggesting a divergent relationship of this species. Phylogenetic analysis using the deduced amino acid sequences of the luciferase gene resulted in a monophyletic group in the Hotaria excluding H. parvula, suggesting a close relationship among H. unmunsana, H. papariensis and H. tsushimana. Additionally, we also analyzed the mitochondrial cytochrome oxidase I (COI) gene of the Hotaria-group fireflies. The deduced amino acid sequence of the COI gene of H. unmunsana was identical to that of H. papariensis and H. tsushimana, but different by three positions from H. parvula. In terms of nucleotide sequences of the COI gene, intraspecific sequence divergence was sometimes larger than interspecies level, and phylogenetic analysis placed the three species into monophyletic groups unresolved among them, but excluded H. parvula. In conclusion, our results suggest that H. unmunsana, H. papariensis and H. tsushimana are very closely related or might be an identical species, at least based on the luciferase and COI genes.

Amino Acid Sequence↗