Role of Histidine 310 in Amydetes vivianii firefly luciferase pH and metal sensitivities and improvement of its color tuning properties.

Firefly luciferases emit yellow-green light and are pH-sensitive, changing the bioluminescence color to red in the presence of heavy metals, acidic pH and high temperatures. These pH and metal-sensitivities have been recently harnessed for intracellular pH indication and toxic metal biosensing. However, whereas the structure of the pH sensor and the metal binding site, which consists mainly of two salt bridges that close the active site (E311/R337 and H310/E354), has been identified, the specific role of residue H310 in pH and metal sensing is still under debate. The Amydetes vivianii firefly luciferase has one of the lowest pH sensitivities among the group of pH-sensitive firefly luciferases, displaying high bioluminescent activity and special spectral selectivity for cadmium and mercury, which makes it a promising analytical reagent. Using site-directed mutagenesis, we have investigated in detail the role of residue H310 on pH and metal sensitivity in this luciferase. Negatively charged residues at position 310 increase the pH sensitivity and metal sensitivity; H310G considerably increases the size of the cavity, severely impacting the activity, H310R closes the cavity, and H310F considerably decreases both pH and metal sensitivities. However, no substitution completely abolished pH and metal sensitivities. The results indicate that the presence of negatively charged and basic side chains at position 310 is important for pH sensitivity and metals coordination, but not essential, indicating that the remaining side chains of E311 and E354 may still coordinate some metals in this site. Furthermore, a metal binding site search predicted that H310 mutations decrease the affinity mainly for Zn, Ni and Hg but less for Cd, and revealed the possible existence of additional binding sites for Zn, Ni and Hg.

Biophosphorescence in fluorescent millipedes (Diplopoda: Xystodesmidae) and its relationships with bioluminescence.

Three types of luminescence have been reported in living organisms: bioluminescence (BL), ultraweak chemiluminescence and biofluorescence (FL). In millipedes, both BL and FL have been reported in Motyxia sequoiae and related Xystodesmidae species. Noteworthy, when walking at night with a UV lantern at the Biological Station of Highlands, I found three blue-fluorescent millipedes (Deltotaria brimleii, Deltotoria sp and Euryus orestes) that also displayed phosphorescence after turning off the UV source. The phosphorescence of the cuticle was in the green region (λmax = 525 nm). The phosphorescence remained associated with cuticle and pellets, but frozen fluorescent supernatants, also displayed phosphorescence. The fluorescent compounds extracted from the cuticles in water and methanol and separated by TLC, displayed fluorescence spectra similar to that of 6-pteridine carboxylic acid. In contrast to Motyxia sequoiae cuticle extracts, no bioluminescence was found in Deltatoria and Euryus extracts  in the presence of MgATP, but weak green chemiluminescence was detected with H2O2 and superoxide. The spectral overlapping of phosphorescence of these millipedes with the bioluminescence of Motyxia (~ 507 nm) and the intimate association of both types of luminescence with the cuticles, raises the possibility that bioluminescence in Motyxia may arise from chemiluminescence reactions preferentially generating triplet excited states instead of singlet states.

Effect of pH on the secondary structure and thermostability of beetle luciferases: structural origin of pH-insensitivity.

Beetle luciferases were classified into three functional groups: (1) pH-sensitive yellow-green-emitting (fireflies) which change the bioluminescence color to red at acidic pH, high temperatures and presence of heavy metals; (2) the pH-insensitive green-yellow-emitting (click beetles, railroad worms and firefly isozymes) which are not affected by these factors, and (3) pH-insensitive red-emitting. Although the pH-sensing site in firefly luciferases was recently identified, it is unclear why some luciferases are pH-insensitive despite the presence of some conserved pH-sensing residues. Through circular dichroism, we compared the secondary structural changes and unfolding temperature of luciferases of representatives of these three groups: (1) pH-sensitive green-yellow-emitting Macrolampis sp2 (Mac) and Amydetes vivianii (Amy) firefly luciferases; (2) the pH-insensitive green-emitting Pyrearinus termitilluminans larval click beetle (Pte) and Aspisoma lineatum (Al2) larval firefly luciferases, and (3) the pH-insensitive red-emitting Phrixotrix hirtus railroadworm (PxRE) luciferase. The most blue-shifted luciferases, independently of pH sensitivity, are thermally more stable at different pHs than the red-shifted ones. The pH-sensitive luciferases undergo increases of α-helices and thermal stability above pH 6. The pH-insensitive Pte luciferase secondary structure remains stable between pH 6 and 8, whereas the Al2 luciferase displays an increase of the ß-sheet at pH 8. The PxRE luciferase also displays an increase of α-helices at pH 8. The results indicate that green-yellow emission in beetle luciferases can be attained by: (1) a structurally rigid scaffold which stabilizes a single closed active site conformation in the pH-insensitive luciferases, and (2) active site compaction above pH 7.0 in the more flexible pH-sensitive luciferases.

Genome analysis of Phrixothrix hirtus (Phengodidae) railroad worm shows the expansion of odorant-binding gene families and positive selection on morphogenesis and sex determination genes.

Among bioluminescent beetles of the Elateroidea superfamily, Phengodidae is the third largest family, with 244 bioluminescent species distributed only in the Americas, but is still the least studied from the phylogenetic and evolutionary points of view. The railroad worm Phrixothrix hirtus is an essential 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 partial genome assembly of P. hirtus, combining short and long reads generated with Illumina sequencing, providing the first source of genomic information and a framework for comparative analyses of the bioluminescent system in Elateroidea. This is the largest genome described in the Elateroidea superfamily, with an estimated size of ∼3.4 Gb, displaying 32 % GC content, and 67 % transposable elements. Comparative genomic analyses showed a positive selection of genes and gene family expansion events of growth and morphogenesis gene products, which could be associated with the atypical anatomical development and morphogenesis found in paedomorphic females and underdeveloped males. We also observed gene family expansion among distinct odorant-binding receptors, which could be associated with the pheromone communication system typical of these beetles, and retrotransposable elements. Common genes putatively regulating bioluminescence production and control, including two luciferase genes corresponding to lateral lanterns green-emitting and head lanterns red-emitting luciferases with 7 exons and 6 introns, and genes potentially 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.

Spectral tuning of bioluminescence and visual sensitivity in males of Brazilian firefly species inhabiting dim light environments (Coleoptera: Elateroidea: Lampyridae).

Bioluminescence in fireflies is essential for sexual communication, and each species has evolved a specific bioluminescence emission capable of being detected by its visual system. This spectral "tuning" between visual sensitivity and bioluminescent emission has been established in 14 species of North American fireflies inhabiting diverse photoecological niches. Here we extend that research to three Brazilian species. Macrolampis omissa inhabits the Cerrado (savannas), while Photinus sp1 and Pyrogaster moestus are often sympatric species inhabiting borders of mesophyll rain forests and secondary growth. P. moestus particularly favors humid areas of the forest. M. omissa and Photinus sp1 are twilight-active fireflies emitting yellow bioluminescence. P. moestus is a "twi-night" species emitting green bioluminescence. It initiates flashing at the end of twilight and continues activity into the night. The visual spectral sensitivity of dark-adapted compound eyes in these three species is similar, showing a maximum in the yellow-green wavelengths and a secondary peak in the near-UV, suggesting the presence of two receptors. The bioluminescence emission spectrum in each species is tuned to its yellow-green visual sensitivity peak. Green chromatic adaptation experiments on Photinus sp1 and P. moestus suggest the presence of a blue receptor. The presence of near-UV, blue, and long-wavelength receptors in the compound eyes would enable a trichromatic color vision in Brazilian firefly species active in dim illumination.

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.

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.

A Novel Brighter Bioluminescent Fusion Protein Based on ZZ Domain and Amydetes vivianii Firefly Luciferase for Immunoassays.

Immunoassays are widely used for detection of antibodies against specific antigens in diagnosis, as well as in electrophoretic techniques such as Western Blotting. They usually rely on colorimetric, fluorescent or chemiluminescent methods for detection. Whereas the chemiluminescence methods are more sensitive and widely used, they usually suffer of fast luminescence decay. Here we constructed a novel bioluminescent fusion protein based on the N-terminal ZZ portion of protein A and the brighter green-blue emitting Amydetes vivianii firefly luciferase. In the presence of D-luciferin/ATP assay solution, the new fusion protein, displays higher bioluminescence activity, is very thermostable and produces a sustained emission (t1/2 > 30 min). In dot blots, we could successfully detect rabbit IgG against firefly luciferases, Limpet Haemocyanin, and SARS-CoV-2 Nucleoprotein (1-250 ng), as well as the antigen bound antibodies using either CCD imaging, and even photography using smartphones. Using CCD imaging, we could detect up to 100 pg of SARS-CoV-2 Nucleoprotein. Using this system, we could also successfully detect firefly luciferase and SARS-CoV-2 nucleoprotein in Western Blots (5-250 ng). Comparatively, the new fusion protein displays slightly higher and more sustained luminescent signal when compared to commercial HRP-labeled secondary antibodies, constituting a novel promising alternative for Western Blotting and immunoassays.

Synthesis of bioluminescent gold nanoparticle-luciferase hybrid systems for technological applications.

Bioluminescent gold nanoparticles (AuNPs) were synthesized in situ using dithiol-terminated polyethylene glycol (PEG(SH)2) as reducer and stabilizing agents. Hybrid Au/F3O4 nanoparticles were also produced in a variation of synthesis, and both types of nanostructures had the polymer capping replaced by L-cysteine (Cys). The four types of nanoparticles, PEG(SH)2AuNPs, PEG(SH)2Au/F3O4NPs, CysAuNPs, and CysAu/F3O4NPs were associated with purified recombinant Pyrearinus termitilluminans green emitting click beetle luciferase (PyLuc) and Phrixotrix hirtus (RELuc) red-emitting railroad worm luciferase. Enzyme association with PEG(SH)2 was also investigated as a control. Luciferases were chosen because they catalyze bioluminescent reactions used in a wide range of bioanalytical applications, including ATP assays, gene reporting, high-throughput screening, bioluminescence imaging, biosensors and other bioluminescence-based assays. The immobilization of PyLuc and RELuc promoted partial suppression of the enzyme luminescence activity in a functionalization-dependent way. Association of PyLuc and RELuc with AuNPs increased the enzyme operational stability in relation to the free enzyme, as evidenced by the luminescence intensity from 0 to 7 h after substrate addition. The stability of the immobilized enzymes was also functionalization-dependent and the association with CysAuNPs was the condition that combined more sustained luminescent activity with a low degree of luminescence quenching. The higher enzymatic stability and sustained luminescence of luciferases associated with nanoparticles may improve the applicability of bioluminescence for bioimaging and biosensing purposes.

RNA-Seq analysis of the blue light-emitting Orfelia fultoni (Diptera: Keroplatidae) suggest photoecological adaptations at the molecular level.

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.

Influence of the C-terminal domain on the bioluminescence activity and color determination in green and red emitting beetle luciferases and luciferase-like enzyme.

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.

Selective inhibition of Zophobas morio (Coleoptera: Tenebrionidae) luciferase-like enzyme luminescence by diclofenac and potential suitability for light-off biosensing.

The accumulation of toxic carboxylic compounds may cause severe effects on the environment and living organisms. A luciferase-like enzyme, previously cloned from the Malpighian tubules of the non-luminescent Zophobas morio mealworm, displays thioesterification activity with a wide range of carboxylic substrates, and produces weak red luminescence in the presence of ATP and firefly d-luciferin, a xenobiotic for this organism. To better investigate the function of this enzyme in carboxylic xenobiotic detoxification, we analyzed the inhibitory effect of different xenobiotic carboxylic acids on the luminescence activity of this enzyme, including environmental pollutants and pharmaceutical compounds. Noteworthy, the anti-inflammatory drug diclofenac severely inhibited this luciferase-like enzyme luminescence activity, both in in vitro (IC50 20 µM) and in vivo in bacterial cells assays, when compared with other beetle luciferases. Similar results were obtained with its brighter I327S mutant. Kinetic analysis of diclofenac's effect on luminescence activity indicated mixed-type inhibition for both ATP and d-luciferin. Modelling studies showed five potential binding sites for diclofenac, including the coenzyme A binding site, which showed one of the highest binding constant. Taken together, these results raise the possibility of using this luciferase-like enzyme for the development of novel whole-cell luminescent biosensors for diclofenac and similar drugs.

A novel brighter bioluminescent fusion protein based on ZZ domain and amydetes vivianii firefly luciferase for immunoassays

Immunoassays are widely used for detection of antibodies against specific antigens in diagnosis, as well as in electrophoretic techniques such as Western Blotting. They usually rely on colorimetric, fluorescent or chemiluminescent methods for detection. Whereas the chemiluminescence methods are more sensitive and widely used, they usually suffer of fast luminescence decay. Here we constructed a novel bioluminescent fusion protein based on the N-terminal ZZ portion of protein A and the brighter green-blue emitting Amydetes vivianii firefly luciferase. In the presence of D-luciferin/ATP assay solution, the new fusion protein, displays higher bioluminescence activity, is very thermostable and produces a sustained emission (t1/2 > 30 min). In dot blots, we could successfully detect rabbit IgG against firefly luciferases, Limpet Haemocyanin, and SARS-CoV-2 Nucleoprotein (1–250 ng), as well as the antigen bound antibodies using either CCD imaging, and even photography using smartphones. Using CCD imaging, we could detect up to 100 pg of SARS-CoV-2 Nucleoprotein. Using this system, we could also successfully detect firefly luciferase and SARS-CoV-2 nucleoprotein in Western Blots (5–250 ng). Comparatively, the new fusion protein displays slightly higher and more sustained luminescent signal when compared to commercial HRP-labeled secondary antibodies, constituting a novel promising alternative for Western Blotting and immunoassays.

Morphology of immature stages of Pyrearinus pumilus (Candèze, 1863 (Coleoptera: Elateridae: Pyrophorini): the click beetle responsible for the luminous canga caves in the state of Pará, Brazil.

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.

A new brilliantly blue-emitting luciferin-luciferase system from Orfelia fultoni and Keroplatinae (Diptera).

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.

A Very Bright Far-Red Bioluminescence Emitting Combination Based on Engineered Railroad Worm Luciferase and 6'-Amino-Analogs for Bioimaging Purposes.

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'-modified amino-luciferin analogs, we finally selected novel FR combinations displaying BL ranging from 636-655 nm. Among them, the combination of PxRE-R215K mutant with 6'-(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.

Temperature effect on the bioluminescence spectra of firefly luciferases: potential applicability for ratiometric biosensing of temperature and pH.

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.