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Beetle luciferases catalyze the bioluminescent oxidation of D-luciferin, producing bioluminescence colors ranging from green to red, using two catalytic steps: adenylation of D-luciferin to produce D-luciferyl-adenylate and PPi, and oxidation of D-luciferyl-adenylate, yielding AMP, CO2, and excited oxyluciferin, the emitter. Luciferases and CoA-ligases display a similar fold, with a large N-terminal domain, and a small C-terminal domain which undergoes rotation, closing the active site and promoting both adenylation and oxidative reactions. The effect of C-terminal domain deletion was already investigated for Photinus pyralis firefly luciferase, resulting in a red-emitting mutant with severely impacted luminescence activity. However, the contribution of C-terminal in the bioluminescence activities and colors of other beetle luciferases and related ancestral luciferases were not investigated yet. Here we compared the effects of the C-terminal domain deletion on green-emitting luciferases of Pyrearinus termitilluminans (Pte) click beetle and Phrixothrix vivianii railroadworm, and on the red-emitting luciferase of Phrixothrix hirtus railroadworm and luciferase-like enzyme of Zophobas morio. In all cases, the domain deletion severely impacted the overall bioluminescence activities and, slightly less, the oxidative activities, and usually red-shifted the bioluminescence colors. The results support the involvement of the C-terminal in shielding the active site from the solvent during the light emitting step. However, in Pte luciferase, the deletion caused only a 10 nm red-shift, indicating a distinctive active site which remains more shielded, independently of the C′-terminal. Altogether, the results confirm the main contribution of the C-terminal for the catalysis of the adenylation reaction and for active site shielding during the light emitting step.

Firefly luciferases usually emit green-yellow bioluminescence at physiological pH values. However, under acidic conditions, in the presence of heavy metals and, at high temperatures they emit red bioluminescence. To understand the structural origin of bioluminescence colors and pH-sensitivity, about 20 firefly luciferases have been cloned, sequenced and investigated. The proton and metal-binding site responsible for pH- and metal sensitivity in firefly luciferases was shown to involve the residues H310, E311 and E354 in firefly luciferases. However, it is still unclear how and why pH-sensitivity arose and evolved in firefly luciferases. Here, we cloned and characterized two novel luciferase cDNAs from the fat body and lanterns of the Brazilian firefly Aspisoma lineatum. The larval fat body isozyme (AL2) has 545 residues, and displays very slow luminescence kinetics and a pH-insensitive spectrum. The adult lantern isozyme (AL1) has 548 residues, displays flash-like kinetics and pH and metal sensitive bioluminescence spectra, and is at least 10 times catalytically more efficient than AL2. Thermostability and CD studies showed that AL2 is much more stable and rigid than the AL1 isozyme. Multialignment and modelling studies show that the E310Q substitution (E310 in AL2 and Q310 in AL1) may have been critical for the origin of pH-sensitivity in firefly luciferases. The results indicate that the lantern efficient flash-emitting pH-sensitive luciferases arose from less efficient glow-type pH-insensitive luciferases found in the fat body of ancestral larval fireflies by enzyme structure flexibilization and substitution at position 310.

Larvae of O. fultoni (Keroplatidae: Keroplatinae), which occur along river banks in the Appalachian Mountains in Eastern United States, produce the bluest bioluminescence among insects from translucent areas associated to black bodies, which are located mainly in the anterior and posterior parts of the body. Although closely related to Arachnocampa spp (Keroplatidae: Arachnocampininae), O.fultoni has a morphologically and biochemically distinct bioluminescent system which evolved independently, requiring a luciferase enzyme, a luciferin, a substrate binding fraction (SBF) that releases luciferin in the presence of mild reducing agents, molecular oxygen, and no additional cofactors. Similarly, the closely related Neoceroplatus spp, shares the same kind of luciferin-luciferase system of Orfelia fultoni. However, the molecular properties, identities and functions of luciferases, SBF and luciferin of Orfelia fultoni and other luminescent members of the Keroplatinae subfamily still remain to be fully elucidated. Using O. fultoni as a source of luciferase, and the recently discovered non-luminescent cave worm Neoditomiya sp as the main source of luciferin and SBF, we isolated and initially characterized these compounds. The luciferase of O. fultoni is a stable enzyme active as an apparent trimer (220 kDa) composed of ~70 kDa monomers, with an optimum pH of 7.8. The SBF, which is found in the black bodies in Orfelia fultoni and in smaller dark granules in Neoditomiya sp, consists of a high molecular weight complex of luciferin and proteins, apparently associated to mitochondria. The luciferin, partially purified from hot extracts by a combination of anion exchange chromatography and TLC, is a very polar and weakly fluorescent compound, whereas its oxidized product displays blue fluorescence with an emission spectrum matching the bioluminescence spectrum (~460 nm), indicating that it is oxyluciferin. The widespread occurrence of luciferin and SBF in both luminescent and non-luminescent Keroplatinae larvae indicate an additional important biological function for the substrate, and therefore the name keroplatin.

The Neotropical region is the richest in bioluminescent Coleoptera species, however, its bioluminescence megadiversity is still underexplored in terms of genomic organization and evolution, mainly within the Phengodidae family. The railroad worm Phrixothrix hirtus is an important biological model and symbolic species due to its bicolor bioluminescence, being the only organism that produces true red light among bioluminescent terrestrial species. Here, we performed the partial genome assembly of P. hirtus, combining short and long reads generated with Illumina sequencing, providing an important source of genomic information and a framework for comparative genomic analyses for the evaluation of the bioluminescent system in Elateroidea. The estimated genome size has ∼3.4Gb, 32% of GC content, and 67% of repetitive elements, being the largest genome described in the Elateroidea superfamily. Several events of gene family expansions associated with anatomical development and morphogenesis, as well as distinct odorant-binding receptors and retrotransposable elements were found in this genome. Similar molecular functions and biological processes are shared with other studied species of Elateriformia. Common genes putatively associated with bioluminescence production and control, including two luciferase genes that displayed 7 exons and 6 introns, and genes that could be involved in luciferin biosynthesis were found, indicating that there are no clear differences about the presence or absence of gene families associated with bioluminescence in Elateroidea. In P. hirtus the conversion of L- to D-luciferin seems to involve additional steps using a Palmitoyl-CoA thioesterase instead of an Acyl-CoA synthetase, which was found in Lampyridae species.HighlightsFirst draft genome assembly of Phengodidae, the largest one described in Coleoptera;Gene family expansions associated with anatomical development and morphogenesis;Bioluminescent control and luciferin biosynthesis genes are common within Elateroidea;Despite similar bioluminescent system, metabolic routes may have evolved independently

Beetle luciferases produce bioluminescence (BL) colors ranging from green to red, having been extensively used for many bioanalytical purposes, including bioimaging of pathogen infections and metastasis proliferation in living animal models and cell culture. For bioimaging purposes in mammalian tissues, red bioluminescence is preferred, due to the lower self-absorption of light at longer wavelengths by hemoglobin, myoglobin and melanin. Red bioluminescence is naturally produced only by Phrixothrix hirtus railroad worm luciferase (PxRE), and by some engineered beetle luciferases. However, Far-Red (FR) and Near-Infrared (NIR) bioluminescence is best suited for bioimaging in mammalian tissues due to its higher penetrability. Although some FR and NIR emitting luciferin analogs have been already developed, they usually emit much lower bioluminescence activity when compared to the original luciferin-luciferases. Using site-directed mutagenesis of PxRE luciferase in combination with 6&prime, modified amino-luciferin analogs, we finally selected novel FR combinations displaying BL ranging from 636&ndash, 655 nm. Among them, the combination of PxRE-R215K mutant with 6&prime, (1-pyrrolidinyl)luciferin proved to be the best combination, displaying the highest BL activity with a catalytic efficiency ~2.5 times higher than the combination with native firefly luciferin, producing the second most FR-shifted bioluminescence (650 nm), being several orders of magnitude brighter than commercial AkaLumine with firefly luciferase. Such combination also showed higher thermostability, slower BL decay time and better penetrability across bacterial cell membranes, resulting in ~3 times higher in vivo BL activity in bacterial cells than with firefly luciferin. Overall, this is the brightest FR emitting combination ever reported, and is very promising for bioimaging purposes in mammalian tissues.

Bioluminescence spectra of firefly luciferases are affected by pH, heavy metals and high temperatures. Previously, we compared the effect of pH and heavy metals on the bioluminescence spectra of different firefly luciferases and showed that such spectral sensitivity can be harnessed to ratiometrically estimate the pH inside cells and metal concentration. Here, we compared the effect of temperature on the spectral sensitivity of four firefly luciferases (Amydetes vivianii: 539 nm; Cratomorphus distinctus: 548 nm; Photinus pyralis: 558 nm and Macrolampis sp2: 594 nm) and investigated whether a ratiometric curve could be used to estimate temperature. The ratio of intensities of bioluminescence at two wavelengths (green and red) at different temperatures (5–35 °C) was determined. The results confirm that, in the case of pH-sensitive luciferases, the more blue-shifted the bioluminescence spectrum, the more thermostable the enzyme and the less sensitive the emission spectrum to temperature. An almost linear relationship between temperature and the ratio of bioluminescence intensities in the green and red region of the spectrum was found for the four luciferases: the more blue-shifted and less sensitive luciferases exhibit a smaller slope and the more red-shifted luciferases exhibit a steeper slope in the following order: Amy < Crt < Ppy < Mac. This relationship offers the possibility of using firefly luciferases as ratiometric indicators of temperature and may allow the compensation of the effect of temperature in the ratiometric analysis of intracellular pH and heavy metal concentration for each enzyme.

Blue shining fungus gnats (Diptera) had been long reported in the Waitomo caves of New Zealand (Arachnocampa luminosa Skuse), in stream banks of the American Appalachian Mountains (Orfelia fultoni Fisher) in 1939 and in true spore eating Eurasiatic Keroplatus Bosc species. This current report observes that similar blue light emitting gnat larvae also occur nearby the Betary river in the buffer zone of High Ribeira River State Park (PETAR) in the Atlantic Forest of Brazil, where the larvae were found when on fallen branches or trunks enveloped in their own secreted silk. The new species is named Neoceroplatus betaryiensis nov. sp. (Diptera: Keroplatidae: Keroplatinae: Keroplatini) based on a morphological analysis. Neoceroplatus betaryiensis nov. sp. larvae emit blue bioluminescence that can be seen from their last abdominal segment and from two photophores located laterally on the first thoracic segment. When touched, the larvae can actively stop its luminescence, which returns when it is no longer being agitated. The in vitro bioluminescence spectrum of N. betaryiensis nov. sp. peaks at 472 nm, and cross-reactivity of hot and cold extracts with the luciferin-luciferase from Orfelia fultoni indicate significant similarity in both enzyme and substrate of the two species, and that the bioluminescence system in the subfamily Keroplatinae is conserved.

Firefly luciferases have been widely used for bioanalytical purposes during the last 5 decades. They usually emit yellow-green bioluminescence and are pH-sensitive, displaying a color change to red at acidic pH and higher temperature and in the presence of heavy metals. Besides the usual applications as bioanalytical reagents and as reporter genes, firefly luciferases' pH- and metal-sensitivities have been recently harnessed for intracellular metal and pH biosensing. Previously we cloned the luciferase of the Brazilian Amydetes vivianii firefly which displays the most blue-shifted color among known firefly luciferases. Here we purified it, characterized and investigated the kinetic properties and the pH, metal and thermal sensitivities of this firefly luciferase. This luciferase displays the lowest reported KM for ATP, the highest catalytic efficiencies, and the highest thermostability among the studied recombinant beetle luciferases, making this enzyme and its cDNA an ideal reagent for sensitive ATP assays and reporter gene. The blue-shifted spectrum, higher thermostability, lower pH- and thermal-sensitivities and protein fluorescence studies indicate a more rigid active site during light emission. This enzyme displays an unmatched selective spectral sensitivity for cadmium and mercury, making it a promising ratiometric indicator of such toxic metals. Finally, the weaker thermal-sensitivity compared to other firefly luciferases makes this enzyme a better ratiometric pH indicator at temperatures above 30 °C, suitable for mammalian cell assays.

Bioluminescence, the emission of visible light in a living organism, is an intriguing phenomenon observed in different species and environments. In terrestrial organisms, the bioluminescence is observed mainly in beetles of the Elateroidea superfamily (Coleoptera). Several phylogenetic studies have been used different strategies to propose a scenario for the origin and evolution of the bioluminescence within this group, however some of them showed incongruences, mainly about the relationship of the bioluminescent families. In order to increase the number of molecular markers available for Elateroidea species and to propose a more accurate phylogeny, with high supported topology, we employed the Next-Generation Sequencing (NGS) methodology to perform the RNA-Seq analysis of luminescent (Elateridae, Phengodidae, Rhagophthalmidae, and Lampyridae) and non-luminescent (Cantharidae) species of Neotropical beetles. We used the RNA-Seq data to construct a calibrated phylogeny of Elateroidea superfamily using a large number of nuclear molecular markers. The results indicate Lampyridae and Phengodidae/Rhagophthalmidae as sister-groups, suggesting that the bioluminescence evolved later in Elateridae than other families (Lampyridae, Phengodidae, and Rhagophthalmidae), and indicating the Upper Cretaceous as the period for the main diversification of Elateroidea bioluminescent species.

Bioluminescence is widely used in biosensors. Firefly luciferase-based bioluminescent sensors are among the most popular ones. Firefly luciferases are pH-sensitive, displaying a large red shift at acidic pH, a property that has been considered undesirable for most applications. Currently, biosensors that can detect intracellular pH are in demand, and some fluorescent biosensors are available. However, pH sensors using bioluminescence have not been used yet. Thus, we decided to harness a firefly luciferase to measure the intracellular pH in mammalian cells. For this purpose, we engineered the luciferase derived from Macrolampis sp2 firefly to localize it on the cytosol or nucleus, in order to observe pH variation in these compartments during biological activities. We first calibrated the emission ratios (R = Igreen/Ired) at different pH values. As expected, we observed a red shift of light emission under acidic conditions when the cells were subjected to different pH conditions in the presence of the K+/H+ ionophore, nigericin. Based on these results, we concluded that this firefly luciferase can be used as a diagnostic tool for measuring the intracellular pH variation in pathogenic cells or in cells during apoptosis. This is the first example of real time-monitoring of pH change using color tuning luciferase.

Firefly luciferases display a typical change in bioluminescence color to red at acidic pH, high temperatures and in the presence of heavy metals. Recently, the proton and metal sensing site responsible for the pH-sensitivity of firefly luciferases, which involves the salt bridges between E311-R337 and H310-E354, was identified. However, it is unclear what other residues contribute to the distinct degrees of pH-sensitivity observed in other firefly luciferases. A multialignment of primary structures of a large set of pH-sensitive and pH-insensitive beetle luciferases showed that the conserved E270 among adult firefly luciferases is substituted by Gly (railroad worms)/Gln (click-beetles) in pH-insensitive ones. Site-directed mutagenesis studies using Macrolampis sp2 and Amydetes vivianii firefly luciferases indeed showed that E270 is important for the pH-dependent activity and spectral profiles: the substitution E270A/G drastically decreases the spectral pH-sensitivity, and extends the activity profile above pH 9.0. These mutations also decrease the sensitivity to metals such as zinc, mercury and cadmium. Modelling studies showed that the residue E270 is located in a three-glutamate motif (269EEE271) at the N-terminal of α-helix-10. The results suggest that at acidic pH, the protonation of E270 carboxylate may extend a turn of the helix at the N-terminal, misaligning the pH-sensor and luciferin phenolate binding site residues: S286, I288 and E311. In contrast, the substitution of E270A/G may unwind a turn of the α-helix-10, indirectly increasing the interaction of the pH-sensor and other residues at the bottom of the luciferin binding site, stabilizing the green light emitting conformation.

Beetle luciferases catalyze the bioluminescent oxidation of D-luciferin, producing bioluminescence colors ranging from green to red, using two catalytic steps: adenylation of D-luciferin to produce D-luciferyl-adenylate and PPi, and oxidation of D-luciferyl-adenylate, yielding AMP, CO

Bioluminescence in the superfamily Elateroidea is produced by specific light organs distributed in different parts of the body depending on the life stage and family. Whereas the luciferin-luciferase system is well known, the molecular control of bioluminescence, the biosynthetic route of luciferin, and the molecular differentiation of photogenic tissue in Elateridae remain enigmatic. With the aim to improve the knowledge about these questions, we performed the RNA-Seq analysis of lanterns and fat body of the Brazilian click beetle larvae: Pyrearinus fragilis (luminescent) and Conoderus sp. (non-luminescent) and compared the gene products contents and their abundance among these tissues. For the first time, we showed that the fat body of luminescent click beetle displays luminescence. We also analyzed some gene products expression patterns in lantern and fat body of Elateridae. Several of them, which are potentially involved with bioluminescence and its control are also found in the transcriptional profile of the firefly Aspisoma lineatum (Lampyridae) and the railroad worm Phrixothrix hirtus (Phengodidae). These gene products were observed in both lanterns and fat body of elaterid species, however with low abundance in the fat body. The results indicate that in Elateridae, the bioluminescent tissue also originated from the fat body. In P. fragilis, we observed two luciferase/luciferase-like enzymes with high abundance, one in the lantern and a different one in the fat body, indicating the possibility of two distinct isoenzymes involved in the bioluminescence in the lantern and fat body of Elateridae species, such as observed in fireflies.

The Pyrearinus pumilus species group from South America includes luminescent click beetles (Agrypninae: Pyrophorini) associated with the phenomena of “luminescent termite mounds” and “luminous canga caves”. The latter was recently reported in the state of Pará, Brazil. This group includes six species based on the morphology of adults, of which two have immature stages already described. In this work we present the morphology and biological aspects of mature larva and pupa of Pyrearinus pumilus (Candèze, 1863), from the canga caves. Moreover, we provide a key and illustrations for identification of male adults and the known larvae. Our study shows that: (1) morphological characters of immatures support the close relationship of P. pumilus with their allies in the P. pumilus species group; (2) the traits of the known larvae of the P. pumilus species group are reliable for species identification.

In fireflies, the bioluminescent system involves a benzothiazolic luciferin, ATP, a luciferase, and complex lanterns consisting of different tissues including a photogenic one. Whereas the biochemical reaction and the structure of lanterns are quite well known, the evolutionary and developmental origins of photogenic tissue as well as the biosynthetic origin of luciferin remain enigmatic. One approach to better understand the origin of the photogenic tissue and biosynthetic origin of luciferin is to investigate the molecular differentiation among photogenic and non-photogenic tissues. Thus, here we compared - for the first time - the transcriptomes of the photogenic tissue of lanterns (specialized tissue) and the fat body (non-specialized tissue) of larvae, adult lanterns, and eggs stages of the common Southeastern Brazilian firefly Aspisoma lineatum . The similarities between the photogenic tissue of lanterns and the fat body, with the occurrence of several luciferase isozymes, luciferin-regenerating enzyme, L-DOPA metabolism related enzymes, and nitric oxide synthase, support the hypothesis that in fireflies, the photogenic tissue evolved from the fat body.

Bioluminescence in beetles is found in the Elateroidea superfamily, which includes Elateridae, Lampyridae, Rhagophthalmidae, and Phengodidae families. Despite sharing the same biochemical system, it is unclear whether the lanterns and the bioluminescent system share a common origin or not. To better understand the molecular differentiation of photogenic from non-photogenic tissues and the molecular physiology of these tissues, we compared the transcriptional profiles of the lateral lanterns and fat body in Phrixothrix hirtus railroad worm (Phengodidae) and the abdomen of the non-luminescent Chauliognathus flavipes (Cantharidae). We observed gene products involved in pheromone biosynthesis in all tissues and four distinct opsin gene products in C. flavipes, more than expected when compared to the transcriptional analysis of lampyrid eyes. We also observed several gene products potentially associated with bioluminescence in P. hirtus lantern and in C. flavipes body, whereas in the fat body of P. hirtus we found only a single copy of luciferase homolog enzyme. Altogether these results suggest that the photogenic tissue of phengodid lanterns has distinct origin from fireflies lanterns, and also the existence of a latent bioluminescent potential in the fat body tissue of some Elateroidea families.

How the unique luciferase of Phrixothrix hirtus (PxRE) railroad worm catalyzes the emission of red bioluminescence using the same luciferin of fireflies, remains a mystery. Although PxRE luciferase is a very attractive tool for bioanalysis and bioimaging in hemoglobin rich tissues, it displays lower quantum yield (15%) when compared to green emitting luciferases (>40%). To identify which parts of PxRE luciferin binding site (LBS) determine bioluminescence color, and to develop brighter and more red-shifted emitting luciferases, we compared the effects of site-directed mutagenesis and of larger 6′-substituted aminoluciferin analogues (6′-morpholino- and 6′-pyrrolidinyl-LH) on the bioluminescence properties of PxRE and green-yellow emitting beetle luciferases. The effects of mutations in the benzothiazolyl and thiazolyl parts of PxRE LBS on the KM and catalytic efficiencies, indicated their importance for luciferin binding and catalysis. However, the absence of effects on the bioluminescence spectrum indicated a less interactive LBS in PxRE during light emission. Mutations at the bottom of LBS of PxRE blue-shifted the spectra and increased catalytic efficiency, suggesting that lack of interactions of this part of LBS with excited oxyluciferin phenolate underlie red light emission. The much higher bioluminescence activity and red-shifted spectra of PxRE luciferase with 6′-morpholino- (634 nm) and 6′-pyrrolidinyl-luciferins (644 nm), when compared to other beetle luciferases, revealed a larger luciferin phenolate binding pocket. The size and orientation of the side-chains of L/I/H348 are critical for amino-analogues accommodation and modulate bioluminescence color, affecting the interactions and mobility of excited oxyluciferin phenolate. The PxRE luciferase and 6′-aminoluciferins provide potential far-red combinations for bioimaging applications.

Bioluminescence in Diptera is found in the Keroplatidae family, within Arachnocampininae and Keroplatinae subfamilies, with reported occurrences in Oceania, Eurasia, and Americas. Larvae of Orfelia fultoni, which inhabit stream banks in the Appalachian Mountains, emit the bluest bioluminescence among insects, using it for prey attraction, similarly to Arachnocampa spp. Although bioluminescence has a similar prey attraction function, the systems of Arachonocampininae and Keroplatinae subfamilies are morphologically/biochemically distinct, indicating different evolutionary origins. To identify the possible coding genes associated with physiological control, ecological adaptations, and origin/evolution of bioluminescence in the Keroplatinae subfamily, we performed the RNA-Seq analysis of O. fultoni larvae during day and night and compared it with the transcriptomes of Arachnocampa luminosa, and reanalyzed the previously published proteomic data of O. fultoni against the RNA-Seq dataset. The abundance of chaperones/heat-shock and hexamerin gene products at night and in luciferase enriched fractions supports their possible association and participation in bioluminescence. The low diversity of copies/families of opsins indicate a simpler visual system in O. fultoni. Noteworthy, gene products associated with silk protein biosynthesis in Orfelia were more similar to Lepidoptera than to the Arachnocampa, indicating that, similarly to the bioluminescent systems, at some point, the biochemical apparatus for web construction may have evolved independently in Orfelia and Arachnocampa.

Beetle luciferases, the enzymes responsible for bioluminescence, are special cases of CoA-ligases which have acquired a novel oxygenase activity, offering elegant models to investigate the structural origin of novel catalytic functions in enzymes. What the original function of their ancestors was, and how the new oxygenase function emerged leading to bioluminescence remains unclear. To address these questions, we solved the crystal structure of a recently cloned Malpighian luciferase-like enzyme of unknown function from Zophobas morio mealworms, which displays weak luminescence with ATP and the xenobiotic firefly d-luciferin. The three dimensional structure of the N-terminal domain showed the expected general fold of CoA-ligases, with a unique carboxylic substrate binding pocket, permitting the binding and CoA-thioesterification activity with a broad range of carboxylic substrates, including short-, medium-chain and aromatic acids, indicating a generalist function consistent with a xenobiotic-ligase. The thioesterification activity with l-luciferin, but not with the d-enantiomer, confirms that the oxygenase activity emerged from a stereoselective impediment of the thioesterification reaction with the latter, favoring the alternative chemiluminescence oxidative reaction. The structure and site-directed mutagenesis support the involvement of the main-chain amide carbonyl of the invariant glycine G323 as the catalytic base for luciferin C4 proton abstraction during the oxygenase activity in this enzyme and in beetle luciferases (G343).

The Malpighian tubules in insects play an essential role in osmoregulation, through the transport of ions during excretion, whereas the fat body is usually associated with the intermediary metabolism. The tubules also are involved in excretion of organic solutes and xenobiotics. However, with the exception of a preliminary transcriptional survey of the Zophobas morio (Tenebrionidae) larval tubules, there are no detailed transcriptional analysis of this organ in Coleoptera. A luciferase-like enzyme that displays weak luminescence activity in the presence of firefly D-luciferin and ATP was cloned from the tubules of Z. morio larvae. In order to better understand the molecular physiology of Malpighian tubules and fat body in Coleoptera larvae, and to investigate the occurrence and functions of AMP-CoA ligases in these tissues, we performed a comparative transcriptional analysis of these tissues using Z. morio giant-mealworms. As expected, the tubules displayed organic and inorganic transporters, xenobiotic metabolism enzymes, V-ATPases, channels, and pumps. The fat body showed proteins that are synthesized in this tissue and secreted to the hemolymph, as well as enzymes involved in lipid and carbohydrate metabolism. These tissues are also involved in common pathways, such as nitrogen metabolism to degradation/excretion, eye pigments biosynthesis, immunity, and detoxification. The presence of coumarate-CoA ligase-like enzymes in these tissues suggest their involvement in the degradation of coumaric acid derivatives obtained from the diet, or alternatively, in the biosynthesis of compounds structurally related to coumaric acids such as eye pigments. Our results confirm to the physiological versatility of tubules and fat body in larval Coleoptera.

Bioluminescence in Diptera is found in the family Keroplatidae, in the glowworms of the genera Arachnocampa, Orfelia and Keroplatus. Despite belonging to the same family, Arachnocampa spp. and Orfelia fultoni display morphologically and biochemically distinct bioluminescence systems: Arachnocampa spp. produce light by the terminal ends of Malpighian tubules using ATP, a luciferin and a luciferase, whereas Orfelia fultoni produces light by translucent areas associated with rows of black bodies in the anterior and posterior parts of the body, using a 140 kDa luciferase and a luciferin which do not cross-react with the Arachnocampa luciferin–luciferase system, and a substrate binding fraction (SBF) which apparently releases luciferin in the presence of reductants. While several other keroplatids are not luminescent, we recently discovered a non-luminescent web-constructing keroplatid larva living in the roofs of caves in the Atlantic rainforest in Brazil, which noteworthily has a compound with Orfelia luciferin-like activity and its associated binding protein (SBF). Both the Neoditomyia luciferin-like compound and SBF cross-react with purified Orfelia luciferase to produce light in the same blue region of the Orfelia luciferin–luciferase system (479 nm). We also checked for the presence of Orfelia-type luciferin in Arachnocampa luminosa and Aedes aegytpi larval bodies, but no traces were found. Molecular studies indicate that Neoditomyia sp. is phylogenetically closer to Keroplatus and Orfelia than to Arachnocampa species. The presence of luciferin and its associated binding protein in this non-bioluminescent keroplatid larva indicates that luciferin may display another important biochemical function in keroplatid larvae and suggests that bioluminescence could be a recently evolved trait in Keroplatidae.

Firefly luciferases produce yellow-green light under physiological and alkaline conditions, however at acidic pH, higher temperatures or in the presence of heavy metals the color changes to red, a property called pH-sensitivity. Despite many decades of studies, the proton and metal binding sites responsible for pH-sensitivity remain enigmatic. Previously we suggested that the salt bridge E311/R337 keeps a closed conformation of the luciferin phenolate binding site. Here we further investigated the effect of this salt bridge and mutations of the neighbor residues H310 and E/N354, on metal and pH-sensitivity of firefly luciferases emitting distinct bioluminescence colors (Cratomorphus distinctus: 548 nm; Macrolampis sp2: 569 nm). The substitutions of H310 and E/N354 modulate metal sensitivity, whereas the carboxylate of E311 may work as the catalytic base essential for green bioluminescence and pH-sensitivity. Modeling studies showed that H310, E311 and E354 side-chains coordinate Zinc, constituting the metal binding site and the pH-sensor. Electrostatic potential and pKa calculations suggest that the external couple H310/E354 is affected by pH, whereas E311/R337 make a stabilized internal pair which retains excited oxyluciferin ejected proton near its phenolate group into a high energy state, promoting yellow-green bioluminescence. Protonation or metal binding weaken these electrostatic gates and their ability to retain the excited oxyluciferin released proton near its phenolate, promoting red light emission.

Summary Bioluminescence, the creation and emission of light by organisms, affords insight into the lives of organisms doing it. Luminous living things are widespread and access diverse mechanisms to generate and control luminescence [1–5]. Among the least studied bioluminescent organisms are phylogenetically rare fungi—only 71 species, all within the ∼9,000 fungi of the temperate and tropical Agaricales order—are reported from among ∼100,000 described fungal species [6, 7]. All require oxygen [8] and energy (NADH or NADPH) for bioluminescence and are reported to emit green light (λ max 530 nm) continuously, implying a metabolic function for bioluminescence, perhaps as a byproduct of oxidative metabolism in lignin degradation. Here, however, we report that bioluminescence from the mycelium of Neonothopanus gardneri is controlled by a temperature-compensated circadian clock, the result of cycles in content/activity of the luciferase, reductase, and luciferin that comprise the luminescent system. Because regulation implies an adaptive function for bioluminescence, a controversial question for more than two millennia [8–15], we examined interactions between luminescent fungi and insects [16]. Prosthetic acrylic resin "mushrooms," internally illuminated by a green LED emitting light similar to the bioluminescence, attract staphilinid rove beetles (coleopterans), as well as hemipterans (true bugs), dipterans (flies), and hymenopterans (wasps and ants), at numbers far greater than dark control traps. Thus, circadian control may optimize energy use for when bioluminescence is most visible, attracting insects that can in turn help in spore dispersal, thereby benefitting fungi growing under the forest canopy, where wind flow is greatly reduced.

Fungus-gnats of the genus Arachnocampa are unique among bioluminescent insects for displaying blue–green bioluminescence, and are responsible for one of the most beautiful bioluminescence spectacles on the roofs of the Waitomo Caves. Despite morphological studies showing that Arachnocampa larval lanterns involve specialization of the Malpighian tubules, the biochemical origin of their bioluminescence remains enigmatic. Using a cDNA library previously constructed from lanterns of the New Zealand glowworm A. luminosa, we carried out the first transcriptional analysis of ~ 500 expressed sequence tags (ESTs) to identify putative candidate proteins for light production, and to better understand the molecular physiology of the lanterns and their relationship with Malpighian tubule physiology. The analysis showed an abundance of hexamerin-like proteins, as well as luciferase-like enzymes, indicating a possible critical role for these proteins in bioluminescence. These findings were corroborated by proteomic analysis of lantern extracts, which showed the presence of hexamerins and luciferase-like enzymes. Other gene products typical of Malpighian tubules, such as detoxifying enzymes, were also found. The results support the existence of an evolutionary link between Malpighian tubule detoxification and the origin of bioluminescence in these Diptera. Copyright © 2015 John Wiley & Sons, Ltd.

Artificial night lighting is gaining attention as a new type of pollution; however, studies of its impacts are scarce. Fireflies provide good models to investigate its effects on nocturnal wildlife, since they depend on their bioluminescence for reproduction. This study investigated the impact of artificial illumination on firefly activity at the new campus of the Federal University of S&atildeo Carlos (Sorocaba, Brazil). The flashing activity of different firefly species, especially Photinus sp1 (82% of all occurrences), was investigated during 3 years, before and after the installation of multi metal vapor spotlights. Quantitative and qualitative analysis, performed in transects at different distances from the artificial light sources, showed significant negative effects on Photinus sp1 occurrence. This study proposes fireflies as potential flagship species and bioindicators for artificial night lighting and for the first time quantifies its effects, providing subsidies for future conservationist legislations regarding photopollution.

Background Luciferases, enzymes that catalyze bioluminescent reactions in different organisms, have been extensively used for bioanalytical purposes. The most well studied bioluminescent system is that of firefly and other beetles, which depends on a luciferase, a benzothiazolic luciferin and ATP, and it is being widely used as a bioanalytical reagent to quantify ATP. Protein kinases are proteins that modify other proteins by transferring phosphate groups from a nucleoside triphosphate, usually ATP. Methods Here, we used a red-light emitting luciferase from Phrixotrix hirtus railroad worm to determine the activity of kinases in a coupled assay, based on luminescence that is generated when luciferase is in the presence of its substrate, the luciferin, and ATP. Results In this work we used, after several optimization reactions, creatine kinase isoforms as well as NEK7 protein kinase in the absence or presence of ATP analogous inhibitors to validate this new luminescence method. Conclusion With this new approach we validated a luminescence method to quantify kinase activity, with different substrates and inhibition screening tests, using a novel red-light emitting luciferase as a reporter enzyme. Electronic supplementary material The online version of this article (doi:10.1186/s12858-017-0087-z) contains supplementary material, which is available to authorized users.

Firefly luciferases are widely used as bioluminescent reporter genes for bioimaging and biosensors. Aiming at simultaneous analyses of different gene expression and cellular events, luciferases and GFPs that exhibit distinct bioluminescence and fluorescence colors have been coupled with each promoter, making dual and multicolor reporter systems. Despite their wide use, firefly luciferase bioluminescence spectra are pH-sensitive, resulting in a typical large red shift at acidic pH, a side-effect that may affect some bioanalytical purposes. Although some intracellular pH-indicators employ dual color and fluorescent dyes, none has been considered to benefit from the characteristic spectral pH-sensitivity of firefly luciferases to monitor intracellular pH-associated stress, an important indicator of cell homeostasis. Here we demonstrate a linear relationship between the ratio of intensities in the green and red regions of the bioluminescence spectra and pH using firefly luciferases cloned in our laboratory (Macrolampis sp2 and Cratomorphus distinctus), allowing estimation of E. coli intracellular pH, thus providing a new analytical method for ratiometric intracellular pH-sensing. This is the first dual reporter system that employs a single luciferase gene to simultaneously monitor intracellular pH using spectral changes, and gene expression and/or ATP concentration using the bioluminescence intensity, showing great potential for real time bioanalysis of intracellular processes associated with metabolic changes such as apoptosis, cell death, inflammation and tissue acidification, among the other physiological changes.

Bioluminescence is widely used in biosensors. For water toxicity analysis, the naturally bioluminescent bacteria Vibrio fischeri have been used extensively. We investigated the suitability of two new beetle luciferases for Escherichia coli light off biosensors: Macrolampis firefly and Pyrearinus termitilluminans click beetle luciferases. The bioluminescence detection assay using this system is very sensitive, being comparable or superior to V. fischeri. The luciferase of P. termitilluminans produces a strong and sustained bioluminescence that is useful for less sensitive and inexpensive assays that require integration of the emission, whereas Macrolampis luciferase displays a flash-like luminescence that is useful for fast and more sensitive assays. The effect of heavy metals and sanitizing agents was analyzed. Zinc, copper, 1-propanol, and iodide had inhibitory effects on bioluminescence and growth assays; however, in these cases the bioluminescence was not a very reliable indicator of cell growth and metabolic activity because these agents also inhibited the luciferase. On the other hand, mercury and silver strongly affected cell bioluminescence and growth but not the luciferase activity, indicating that bioluminescence was a reliable indicator of cell growth and metabolic activity in this case. Finally, bioluminescent E. coli immobilized in agarose matrix gave a more stable format for environmental assays.

Bioluminescence in beetles is found mainly in the Elateroidea superfamily (Elateridae, Lampyridae and Phengodidae). The Neotropical region accounts for the richest diversity of bioluminescent species in the world with about 500 described species, most occurring in the Amazon, Atlantic rainforest and Cerrado (savanna) ecosystems in Brazil. The origin and evolution of bioluminescence, as well as the taxonomic status of several Neotropical taxa in these families remains unclear. In order to contribute to a better understanding of the phylogeny and evolution of bioluminescent Elateroidea we sequenced and analyzed sequences of mitochondrial NADH2 and the nuclear 28S genes and of the cloned luciferase sequences of Brazilian species belonging to the following genera: (Lampyridae) Macrolampis, Photuris, Amydetes, Bicellonycha, Aspisoma, Lucidota, Cratomorphus; (Elateridae) Conoderus, Pyrophorus, Hapsodrilus, Pyrearinus, Fulgeochlizus; and (Phengodidae) Pseudophengodes, Phrixothrix, Euryopa and Brasilocerus. Our study supports a closer phylogenetic relationship between Elateridae and Phengodidae as other molecular studies, in contrast with previous morphologic and molecular studies that clustered Lampyridae/Phengodidae. Molecular data also supported division of the Phengodinae subfamily into the tribes Phengodini and Mastinocerini. The position of the genus Amydetes supports the status of the Amydetinae as a subfamily. The genus Euryopa is included in the Mastinocerini tribe within the Phengodinae/Phengodidae. Copyright © 2013 John Wiley & Sons, Ltd.

The origin of luciferases and of bioluminescence is enigmatic. In beetles, luciferases seem to have evolved from AMP-CoA-ligases. How the new oxygenase luminogenic function originated from AMP-ligases leading to luciferases is one of the most challenging mysteries of bioluminescence. Comparison of the cloned luciferase-like enzyme from the nonluminescent Zophobas morio mealworm and beetle luciferases showed that the oxygenase activity may have emerged as a stereoselective oxidative drift with d-luciferin, a substrate that cannot be easily thioesterified to CoA as in the case of the l-isomer. While the overall kcat displayed by beetle luciferases is orders of magnitude greater than that of the luciferase-like enzyme, the respective oxidation rates and quantum yields of bioluminescence are roughly similar, suggesting that the rate constant of the AMP-ligase activity exerted on the new d-luciferin substrate in beetle protoluciferases was the main enzymatic property that suffered optimization during the evolution of luciferases. The luciferase-like enzyme and luciferases boost the rate of luciferyl-adenylate chemiluminescent oxidation by factors of 10(6) and 10(7), respectively, as compared to the substrate spontaneous oxidation in buffer. A similar enhancement of luciferyl-adenylate chemiluminescence is provided by nucleophilic aprotic solvents, implying that the peptide bonds in the luciferin binding site of beetle luciferase could provide a similar catalytically favorable environment. These data suggest that the luciferase-like enzyme and other similar AMP-ligases are potential alternative oxygenases. Site-directed mutagenesis studies of the luciferase-like enzyme and the red light-producing luciferase of Phrixotrix hirtus railroadworm confirm here a critical role for T/S345 in luciferase function. Mutations such as I327T/S in the luciferase-like enzyme, which simultaneously increases luciferase activity and promotes blue shifts in the emission spectrum, could have been critical for evolving functional bioluminescence from red-emitting protoluciferases. Through the combination of I327T/S mutations and N-terminal fusion, the luminescence activity of this enzyme was increased to visible levels, with the development of a totally new orange-emitting luciferase. These results open the possibility of engineering luciferase activity in a set of AMP-CoA-ligases.

The biochemistry of firefly bioluminescence is well understood; however, the molecular physiology of the lanterns is still poorly studied, especially the biosynthesis and origin of beetle luciferin which are almost unknown. Using a cDNA library previously constructed from Macrolampis sp2 lanterns, we randomly selected and sequenced 572 cDNAs in order to have a first transcriptional profile of the most represented messages found in the lanterns and therefore to better understand their molecular physiology. As expected, high percentage of the gene products (~ 22%) displayed high similarity with Coleoptera genome products. About 7% represented mitochondrial genes, including several copies of cytochrome oxidase, which are also expected for this tissue. Luciferase genes were especially abundant, representing ca 2% of the products. Gene products involved with cysteine and sulfur metabolism such as the cystathionine β-lyase and the S-adenosylmethionine synthetase were abundant. Noteworthy, an abundance of proteins involved with hormone metabolism was found, suggesting a possible link between bioluminescence and hormone metabolism.

The use of agrochemicals has increased considerably in recent years, and consequently, there has been increased exposure of ecosystems and human populations to these highly toxic compounds. The study and development of methodologies to detect these substances with greater sensitivity has become extremely relevant. This article describes, for the first time, the use of atomic force spectroscopy (AFS) in the detection of enzyme-inhibiting herbicides. A nanobiosensor based on an atomic force microscopy (AFM) tip functionalised with the acetolactate synthase (ALS) enzyme was developed and characterised. The herbicide metsulfuron-methyl, an ALS inhibitor, was successfully detected through the acquisition of force curves using this biosensor. The adhesion force values were considerably higher when the biosensor was used. An increase of ∼250% was achieved relative to the adhesion force using an unfunctionalised AFM tip. This considerable increase was the result of a specific interaction between the enzyme and the herbicide, which was primarily responsible for the efficiency of the nanobiosensor. These results indicate that this methodology is promising for the detection of herbicides, pesticides, and other environmental contaminants.

Firefly luciferases have been used extensively as bioanalytical reagents and their cDNAs as reporter genes for biosensors and bioimaging, but they are in general unstable at temperatures above 30°C. In the past few years, efforts have been made to stabilize some firefly luciferases for better application as analytical reagents. Novel luciferases from different beetle families, displaying distinct bioluminescence colours and kinetics, may offer desirable alternatives to extend the range of applications. In the past years, our group has cloned the largest variety of luciferases from the three main families of bioluminescent beetles (Elateridae: P. termitilluminans, F. bruchi, P. angustus; Phengodidae: P. hirtus, P. vivianii; and Lampyridae: A. vivianii, C. distinctus and Macrolampis sp2) occurring in Brazilian biomes. We compared the thermostability of these recombinant luciferases and investigated their relationships with bioluminescence spectra and kinetics. The most thermostable luciferases were those of Pyrearinus termitilluminans larval click beetle (534 nm), Amydetes vivianii firefly (539 nm) and Phrixotrix vivianii railroad worm (546 nm), which are the most blue-shifted examples in each family, confirming the trend that the most blue-shifted emitting luciferases are also the most thermostable. Comparatively, commercial P. pyralis firefly luciferase was less thermostable than P. termitilluminans click beetle and A. vivianii firefly luciferases. The higher thermostability in these luciferases could be related to higher degree of hydrophobic packing and disulfide bond content (for firefly luciferases).

Bioluminescent click-beetles display a wide variation of bioluminescence colors ranging from green to orange, including an unusual intra-specific color variation in the Jamaican Pyrophorus plagiophthalamus. Recently, we collected individuals of the Pyrophorus angustus species from the Southern Amazon forest, in Brazil, which displays an orange light emitting abdominal lantern. This species was also previously described from Central America, but displaying a bioluminescence spectrum from 536 nm (dorsal) to 578 nm (ventral). The biogeographic variation of the bioluminescence color in this species could be an adaptation to environmental reflectance and inter/intraspecific sexual competition. Here, we cloned, sequenced, characterized and performed site-direct mutagenesis of this new orange emitting luciferase. The in vitro luciferase spectrum displayed a peak at 594 nm, KM values for ATP and d-luciferin of 160 μM and 17 μM, respectively, and an optimum pH of approximately 8.5. Comparative multialignment and site-directed mutagenesis using different color emitting click-beetle luciferases from P. angustus, Fulgeochlizus bruchi and Pyrearinus termitilluminans luciferases cloned by our group showed an integral role of residue 247 in bioluminescence color modulation.

The control region (CR) or A + T-rich region in Coleoptera mt genome is poorly characterized, including the Elateroidea bioluminescent species. Here, we provided the first attempt to characterize and compare the structure and organization of the CR of different species within Elateridae. We also revisited some sequenced Coleoptera CR and observed consensus T-stretches, non-conserved sequences near the stem-loop and unusual inner tRNAs-like sequences. All these features are probably involved in the replication start of the mt genome. The phylogenetic relationships in Elateridae bioluminescent groups using partial sequence of CR showed the monophyly of Pyrearinus pumilus group and Pyrearinus as a polyphyletic genus, corroborating our previous results. The wider genetic variation obtained by CR analysis could separate two different lineages that occur within P. termitilluminans populations. In Elateridae, the CR exhibited high polymorphism within and between populations, which was also observed in other Coleoptera species, suggesting that the CR could be described as a suitable molecular marker to be applied in phylogenetic and phylogeographic studies.

Brazil hosts the richest biodiversity of bioluminescent beetles in the world. Several species are found in the Atlantic rain forest, one of the richest and most threatened tropical forests in the world. We have catalogued the biodiversity of bioluminescent species mainly of Elateroidea superfamily occurring in one of the last largest and most preserved remnants of Atlantic rain forest, located at the Biological Station of Boracéia of São Paulo University (Salesopolis, SP, Brazil). This site accounted with the largest diversity ever found for a single place in São Paulo State, with 39 species: Lampyridae (30), Phengodidae (5), Elateridae (3) and Staphylinidae (1). This fauna has unique species that were not found in any other places of Atlantic forest in São Paulo state, especially fireflies from the Lampyrinae tribes (Cratomorphini, Lamprocerini, Lucidotini), the subfamilies Amydetinae and Photurinae, and the phengodids Pseudophengodes and Brasilocerus sp.2. Most species are found in dense Ombrophyl forest or at their border, and a few ones are found dwelling in the few open fields around the forest. There is a predominance of glowing patterns in the green region among forest inhabiting species when compared with open field fireflies.

Bioluminescent click-beetles emit a wide range of bioluminescence colors (λ(Max) = 534-594 nm) from thoracic and abdominal lanterns, which are used for courtship. Only the luciferases from Pyrophorus and Pyrearinus species were cloned and sequenced. The Brazilian Fulgeochlizus bruchi click-beetle, which inhabits the Central-west Cerrado (Savannas), is noteworthy because, differently from other click-beetles, the adult stage displays only a functional abdominal lantern, which produces a bright green bioluminescence for sexual attraction purposes, and lacks functional thoracic lanterns. We cloned the cDNA for the abdominal lantern luciferase of this species. Notably, the primary sequence of this luciferase showed slightly higher identity with the green emitting dorsal lantern luciferases of the Pyrophorus genus instead of the abdominal lanterns luciferases. This luciferase displays a blue-shifted spectrum (λ(Max) = 540 nm), which is pH-insensitive from pH 7.5 to 9.5 and undergoes a slight red shift and broadening above this pH; the lowest K(M) for luciferin among studied click-beetle luciferases, and the highest optimum pH (9.0) ever reported for a beetle luciferase. At pH 9.0, the K(M) for luciferin increases, showing a decrease of affinity for this substrate, despite the higher activity. The slow luminescence decay rate of F. bruchi luciferase in vitro reaction could be an adaptation of this luciferase for the long and sustained in vivo luminescence display of the click-beetle during the courtship, and could be useful for in vivo intracellular imaging.

Aspisoma lineatum (Gyllenhal) is a common firefly in the Southeastern region of Brazil. Adults and larvae were collected in the municipality of Campinas, state of São Paulo, Brazil, and the immature stages were described and reared in the laboratory. Four generations were reproduced in the laboratory, and a method for its rearing was established. The life cycle usually lasts 6 months, but under optimal laboratory conditions, it lasted from 2 to 4 months. Larvae were fed with Bradybaena similaris and Bulimulus tenuissimus snails since the beginning of the larval stage. This species was found to be easily adapted to environments under anthropic influence, such as urban areas and farms.

The structural origin and evolution of bioluminescent activity of beetle luciferases from AMP/CoA ligases remains a mystery. Previously we cloned the luciferase-like enzyme from Zophobas morio mealworm, a reasonable protoluciferase model that could shine light on this mystery. Kinetic characterization and studies with D- and L-luciferin and their adenylates showed that stereoselectivity constitutes a critical feature for the origin of luciferase activity in AMP/CoA ligases. Comparison of the primary structures and modeling studies of this protoluciferase and the three main families of beetle luciferases showed that the carboxylic acid substrate binding site of this enzyme is smaller and more hydrophobic than the luciferin binding site of beetle luciferases, showing several substitutions of otherwise conserved residues. Thus, here we performed a site-directed mutagenesis survey of the carboxylic binding site motifs of the protoluciferase by replacing their residues by the respective conserved ones found in beetle luciferases in order to identify the structural determinants of luciferase/oxygenase activity. Although most of the substitutions had negative impact on the luminescence activity of the protoluciferase, only the substitution I327T improved the luminescence activity, resulting in a broad and 15 nm blue-shifted luminescence spectrum. Such substitution indicates the importance of the loop motif 322YGMSEI327 (341YGLTETT347 in Photinus pyralis luciferase) for luciferase activity, and indicates a possible route for the evolution of bioluminescence function of beetle luciferases.