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1

Campello, Lucas, Stephanie Vaz, José R. M. Mermudes, André L. D. Ferreira, and Luiz F. L. Silveira. "Comparative morphology and key to Amydetinae genera, with description of three new firefly species (Coleoptera, Lampyridae)." ZooKeys 1114 (July 27, 2022): 131–66. http://dx.doi.org/10.3897/zookeys.1114.77692.

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Amydetinae is an exclusively Neotropical subfamily of fireflies, distributed among three genera: Amydetes Illiger, 1807, Magnoculus McDermott, 1964, and Memoan Silveira & Mermudes, 2013. Here, we describe three new species of Amydetinae: two belonging to Amydetes (A. alexisp. nov. and A. marolaesp. nov.) and one to the previously monotypic Memoan (Me. conanisp. nov.). All three species are known only from the Atlantic Forest in southeastern Brazil. Endoskeletal structures of Memoan and Magnoculus species are described and compared with those of Amydetes for the first time. After studying the type material, Photinus fruhstorferi Pic, 1942 is transferred to Memoan, generating Memoan fruhstorfericomb. nov., and Me. ciceroi Silveira & Mermudes, 2013 syn. nov. is placed as a junior synonym. We also redescribe Magnoculus obscurus Olivier, 1885 and compare it to other species of genus and to other amydetine taxa to identify potential new diagnostic traits for the Amydetinae and its constituting genera. We provide an updated diagnosis for Memoan, illustrations for all four species, and a distribution map for the three new species, as well as a key to adult males of the three amydetine genera, and an updated key to Amydetes species based on males.
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2

Campello, Lucas, Stephanie Vaz, José R. M. Mermudes, André L. D. Ferreira, and Luiz F. L. Silveira. "Comparative morphology and key to Amydetinae genera, with description of three new firefly species (Coleoptera, Lampyridae)." ZooKeys 1114 (July 27, 2022): 131–66. https://doi.org/10.3897/zookeys.1114.77692.

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Amydetinae is an exclusively Neotropical subfamily of fireflies, distributed among three genera: Amydetes Illiger, 1807, Magnoculus McDermott, 1964, and Memoan Silveira & Mermudes, 2013. Here, we describe three new species of Amydetinae: two belonging to Amydetes (A. alexi sp. nov. and A. marolae sp. nov.) and one to the previously monotypic Memoan (Me. conani sp. nov.). All three species are known only from the Atlantic Forest in southeastern Brazil. Endoskeletal structures of Memoan and Magnoculus species are described and compared with those of Amydetes for the first time. After studying the type material, Photinus fruhstorferi Pic, 1942 is transferred to Memoan, generating Memoan fruhstorferi comb. nov., and Me. ciceroi Silveira & Mermudes, 2013 syn. nov. is placed as a junior synonym. We also redescribe Magnoculus obscurus Olivier, 1885 and compare it to other species of genus and to other amydetine taxa to identify potential new diagnostic traits for the Amydetinae and its constituting genera. We provide an updated diagnosis for Memoan, illustrations for all four species, and a distribution map for the three new species, as well as a key to adult males of the three amydetine genera, and an updated key to Amydetes species based on males.
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3

Pelentir, G. F., V. R. Bevilaqua, and V. R. Viviani. "A highly efficient, thermostable and cadmium selective firefly luciferase suitable for ratiometric metal and pH biosensing and for sensitive ATP assays." Photochemical & Photobiological Sciences 18, no. 8 (2019): 2061–70. http://dx.doi.org/10.1039/c9pp00174c.

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4

Viviani, Vadim R., Danilo Amaral, Rogilene Prado, and Frederico G. C. Arnoldi. "Correction: A new blue-shifted luciferase from the Brazilian Amydetes fanestratus (Coleoptera: Lampyridae) firefly: molecular evolution and structural/functional properties." Photochemical & Photobiological Sciences 14, no. 11 (2015): 2128. http://dx.doi.org/10.1039/c5pp90033f.

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Correction for ‘A new blue-shifted luciferase from the Brazilian Amydetes fanestratus (Coleoptera: Lampyridae) firefly: molecular evolution and structural/functional properties’ by Vadim R. Viviani et al., Photochem. Photobiol. Sci., 2011, 10, 1879–1886.
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5

Silveira, Luiz Felipe Lima Da, and José Ricardo M. Mermudes. "Systematic review of the firefly genus Amydetes Illiger, 1807 (Coleoptera: Lampyridae), with description of 13 new species." Zootaxa 3765, no. 3 (2014): 201–48. https://doi.org/10.11646/zootaxa.3765.3.1.

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Silveira, Luiz Felipe Lima Da, Mermudes, José Ricardo M. (2014): Systematic review of the firefly genus Amydetes Illiger, 1807 (Coleoptera: Lampyridae), with description of 13 new species. Zootaxa 3765 (3): 201-248, DOI: 10.11646/zootaxa.3765.3.1
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6

Serain, Alessandra Freitas, Daniel Rangel de de Souza, and Vadim R. Viviani. "Fast and Sensitive Swab-Based Bioluminescent Detection Method for Meat and Chicken Microbiological Contamination Using Amydetes vivianii Firefly Luciferase." Chemosensors 13, no. 2 (2025): 27. https://doi.org/10.3390/chemosensors13020027.

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New faster and more sensitive detection methods are required for food microbiological contamination quality control throughout the entire handling process. The adenosine triphosphate (ATP) bioluminescence detection method based on the firefly luciferin–luciferase system has been a widely used approach for decades due to its practicality, efficiency, and rapidity. The luciferase of the Amydetes vivianii firefly, cloned and produced in our laboratory, displays a high bioluminescence and stability, showing desirable properties for ATP assays. Using this enzyme, in this study we developed and validated a swab-based ATP detection method for meat, chicken, and milk, allowing us to distinguish between contaminated and non-contaminated forms of these foods. This method demonstrated robust interday and intraday precision, an accuracy of 70–71% across different matrices, and a limit of detection of 1.0 × 104 cps for the dilutions and 2.7 × 103 cps for the swabs, fulfilling all validation criteria and ensuring reliability for routine applications. Except for milk, which has very low endogenous ATP levels due to pasteurization, thus requiring sample pre-processing, the method allowed us to luminometrically detect ATP on the surface of meat and chicken in less than an hour using an assay solution. The method showed higher sensitivity compared to an available commercial kit due to its intense signal, with a remarkable ~22-fold increase in luminescence intensity when comparing the highest ATP concentration of Amydetes luciferase with a commercially available luciferase, allowing for detecting microbiological contamination at lower levels or using less sensitive luminometers.
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7

Carrasco-López, César, Juliana C. Ferreira, Nathan M. Lui, et al. "Beetle luciferases with naturally red- and blue-shifted emission." Life Science Alliance 1, no. 4 (2018): e201800072. http://dx.doi.org/10.26508/lsa.201800072.

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The different colors of light emitted by bioluminescent beetles that use an identical substrate and chemiexcitation reaction sequence to generate light remain a challenging and controversial mechanistic conundrum. The crystal structures of two beetle luciferases with red- and blue-shifted light relative to the green yellow light of the common firefly species provide direct insight into the molecular origin of the bioluminescence color. The structure of a blue-shifted green-emitting luciferase from the firefly Amydetes vivianii is monomeric with a structural fold similar to the previously reported firefly luciferases. The only known naturally red-emitting luciferase from the glow-worm Phrixothrix hirtus exists as tetramers and octamers. Structural and computational analyses reveal varying aperture between the two domains enclosing the active site. Mutagenesis analysis identified two conserved loops that contribute to the color of the emitted light. These results are expected to advance comparative computational studies into the conformational landscape of the luciferase reaction sequence.
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8

Bevilaqua, Vanessa Rezende, Jaqueline Rodrigues da Silva, and Vadim Ravara Viviani. "Demonstração bioluminescente de ATP com luciferase recombinante de vagalume (Amydetes vivianii Silveira & Mermudes, 2014) em aulas práticas de bioenergética." Revista de Ensino de Bioquímica 20, no. 2 (2022): 209–28. http://dx.doi.org/10.16923/reb.v20i2.1006.

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A vida na Terra depende de energia. O ATP é a molécula universal que armazena e transporta energia nas células. Entre as reações que requerem a energia inicial do ATP, está a bioluminescência do vagalume. Nesta reação catalisada pela enzima luciferase, um composto chamado de luciferina é oxidado produzindo luz. Devido à dependência da reação bioluminescente pelo ATP, a luciferina e a luciferase têm sido utilizadas para de quantificação analítica desta molécula. Nesta aula prática, utilizamos uma luciferase recombinante do vagalume Amydetes vivianii para detectar e quantificar visualmente e fotograficamente a presença do ATP em amostras biológicas, por meio da bioluminescência. Ao misturar luciferina e luciferase às amostras de bactérias lisadas e corpo gorduroso da larva de Zophobas morio, a bioluminescência produzida pode ser facilmente visualizada em ambientes escuros e fotografada com câmeras fotográficas de telefones celulares, propiciando ao estudante um registro fotográfico dos resultados para análise. Na presença de padrões de concentração conhecida de ATP, o estudante pode estimar a concentração deste composto na amostra analisada. Ciências Biológicas e Engenharia Florestal, podendo ser utilizada também em aulas de Biofísica e Microbiologia.
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9

Viviani, Vadim R., Gabriel Felder Pelentir, Vanessa Rezende Bevilaqua, Jaqueline Rodrigues da Silva, and Atílio Tomazini Júnior. "Aula Prática: Efeito de cádmio e pH na função de uma enzima: a luciferase de vagalume." Revista de Ensino de Bioquímica 21, no. 2 (2023): 164–78. http://dx.doi.org/10.16923/reb.v21i2.1048.

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A desnaturação de proteínas pode ser causada por pHs extremos e metais pesados, como chumbo e mercúrio. Pequenas variações de pH ou baixas concentrações de metais pesados também afetam a estrutura e função de proteínas como enzimas, tendo efeitos tóxicos no metabolismo, entretanto, tais efeitos não são facilmente visualizados. A enzima luciferase, que catalisa a reação de oxidação da luciferina em vagalumes, produz luz verde-amarela em pH fisiológico. Em pH ácido ou na presença de certos metais pesados a cor da luz emitida muda para o laranja-avermelhado. Estas enzimas ao ligar os metais pesados ou prótons, mudam de conformação, afetando as interações eletrostáticas entre os aminoácidos do sítio-ativo e a molécula-produto (oxiluciferina), mudando a cor da luz emitida. Nesta aula prática a luciferase clonada do vagalume Amydetes vivianii, que é especialmente sensível ao cádmio, é utilizada para indicar variações de concentração deste metal e, também, para mudanças de pH, por meio da alteração da cor da luz do verde-amarelo-laranja. A bioluminescência produzida é, facilmente, visualizada em ambiente escuro, podendo ser fotografada com câmera de celular convencional.
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10

Viviani, Vadim R., Danilo Amaral, Rogilene Prado, and Frederico G. C. Arnoldi. "A new blue-shifted luciferase from the Brazilian Amydetes fanestratus (Coleoptera: Lampyridae) firefly: molecular evolution and structural/functional properties." Photochem. Photobiol. Sci. 10, no. 12 (2011): 1879–86. http://dx.doi.org/10.1039/c1pp05210a.

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11

DA SILVEIRA, LUIZ FELIPE LIMA, and JOSÉ RICARDO M. MERMUDES. "Systematic review of the firefly genus Amydetes Illiger, 1807 (Coleoptera: Lampyridae), with description of 13 new species." Zootaxa 3765, no. 3 (2014): 201. http://dx.doi.org/10.11646/zootaxa.3765.3.1.

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12

Viviani, Vadim R., Murilo S. Teixeira, and Gabriel F. Pelentir. "CadmiLume: A Novel Smartphone-Based Bioluminescence Color-Tuning Assay and Biosensor for Cadmium and Heavy Metal Detection in Water Samples." Methods and Protocols 8, no. 2 (2025): 33. https://doi.org/10.3390/mps8020033.

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Heavy metal contamination of soil and water is a growing environmental concern, especially mercury, lead, and cadmium. Therefore, fast and reliable methodologies to assess contamination in the field are in demand. However, many methodologies require laborious, expensive, and cumbersome equipment that is not convenient for rapid field analysis. Mobile phone technology coupled with bioluminescent assays provides accessible hands-on alternatives that has already been shown to be feasible. Previously, we demonstrated that firefly luciferases can be harnessed as luminescence color-tuning sensors for toxic metals. An assay based on such a principle was already successfully applied for teaching biochemistry laboratory lessons, which demonstrates the effect of cadmium on enzyme function based on bioluminescence color change. For analytical detection of cadmium in water, here, we developed a novel bioluminescence assay using the cadmium-sensitive Amydetes vivianii firefly luciferase coupled with a cell phone provided with a program to quantify cadmium concentration based on luminescence color discrimination. The application has proven to be efficient with high precision between 0.10 and 2 mM of cadmium, being appliable to diluted water samples (0.1–2 µM) upon concentration and relying on reference cadmium standards values. The light emitted by the reference standards and samples in a dark box is captured by the smartphone’s camera, which, using computer vision, automatically quantifies cadmium according to the RGB color. CadmiLume is a simple and easy luminescent enzymatic biosensor for cadmium contamination in water samples, which instantaneously can provide results with the convenience of a smartphone in the palm of one’s hands.
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13

Viviani, Vadim R., Gabriel F. Pelentir, and Vanessa R. Bevilaqua. "Bioluminescence Color-Tuning Firefly Luciferases: Engineering and Prospects for Real-Time Intracellular pH Imaging and Heavy Metal Biosensing." Biosensors 12, no. 6 (2022): 400. http://dx.doi.org/10.3390/bios12060400.

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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.
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14

Bevilaqua, Vanessa R., Gabriel F. Pelentir, Moema A. Hausen, Eliana A. R. Duek, and Vadim R. Viviani. "Selection and Engineering of Novel Brighter Bioluminescent Reporter Gene and Color- Tuning Luciferase for pH-Sensing in Mammalian Cells." Biosensors 15, no. 1 (2025): 18. https://doi.org/10.3390/bios15010018.

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Firefly luciferases have been extensively used for bioanalytical applications, including their use as bioluminescent reporters, biosensors, and for bioimaging biological and pathological processes. Due to their intrinsic pH- sensitivity, in recent years we have demonstrated that firefly luciferases can also be harnessed as color- tuning sensors of intracellular pH. However, it is known that mammalian cells require temperatures higher than 36 °C, which red-shift the bioluminescence spectra of most firefly luciferases, decreasing their activities and the resolution of ratiometric pH analysis. Therefore, we prospected and engineered novel pH-sensitive firefly luciferases for mammalian cells. We humanized the luciferases of Amydetes vivianii (Amy-Luc) and Cratomorphus distinctus (Crt-Luc) fireflies, inserted them into the pCDNA3 vector, and compared their bioluminescence and pH-sensing properties with those of Macrolampis firefly luciferase (Mac-Luc) inside fibroblasts. The transfected COS-1 with Mac-Luc and Crt-Luc displayed lower bioluminescence activity and considerably red-shifted spectra (611 and 564 nm, respectively) at 37 °C, whereas Amy-Luc displayed the highest bioluminescence activity and spectral stability at 37 °C inside cells, displaying the most blue-shifted spectrum at such temperatures (548 nm) and the best spectral resolution at different pH values, making it possible to ratiometrically estimate the pH from 6.0 to 8.0. These results show that Amy-Luc is a novel brighter reporter gene and suitable pH- indicator for mammalian cells. Furthermore, whereas at pH 8.0 the spectrum was thermally stable, at pH 6.0 Amy-Luc showed higher temperature sensitivity, raising the possibility of using this luciferase as an intracellular temperature sensor. Thus, the improved bioluminescence properties as compared to existing luciferases could offer advantages for in vivo imaging and pH- sensing for the study of mammalian cellular physiology.
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15

Ferreira, Vinicius S., Oliver Keller, Felipe Francisco Barbosa, and Michael A. Ivie. "Integrative systematics of Cheguevaria Kazantsev, 2007 (Coleoptera, Lampyridae, Cheguevariinae) identifies genetic stability in color-polymorphic individuals and a disjoint relationship with Amydetinae." Insect Systematics and Diversity 8, no. 6 (2024). http://dx.doi.org/10.1093/isd/ixae033.

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Abstract The genus Cheguevaria (Kazantsev 2007) is currently comprised of three species which are endemic to the Greater Antilles: Cheguevaria angusta Kazantsev, 2007 (Hispaniola), Cheguevaria taino Kazantsev, 2007 and Cheguevaria montana Kazantsev, 2008 (both from Puerto Rico). Cheguevaria is the sole member of Cheguevariinae, and a previous morphology-based phylogenetic hypotheses suggested a close relationship with the subfamily Amydetinae. Recent collecting trips to Puerto Rico and the survey of other scientific collections allowed us to identify and locate more specimens of the genus, including several color morphs of specimens tentatively identified as C. montana. We used this material to revise the genus, and to describe Cheguevaria cubensisnew species, the first record of the genus from Cuba. We also produced DNA barcodes based on partial cytochrome c oxidase subunit 1 (COI), which were used to investigate the species limits within color morph species of C. montana. Our results showed a high level of genetic stability in those color morphs, indicating that all specimens belong to the same species. We present a discussion to explain possible scenarios leading to such variation in color patterns. We also generated amplicons for two specimens of Amydetinae, represented by two Amydetes Illiger, 1807 species, and we tested their relationship with Cheguevaria in a phylogenetic context based on a four gene dataset. Our analyzes indicated a disjoint relationship between Cheguevaria and Amydetes, corroborating previously published morphology-based phylogenies, and we maintained the status of Cheguevaria as an independent lineage within Lampyridae.
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16

Viviani, Vadim R., Jaqueline Rodrigues Silva, and Paulo Lee Ho. "A Novel Brighter Bioluminescent Fusion Protein Based on ZZ Domain and Amydetes vivianii Firefly Luciferase for Immunoassays." Frontiers in Bioengineering and Biotechnology 9 (October 18, 2021). http://dx.doi.org/10.3389/fbioe.2021.755045.

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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.
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17

Pelentir, Gabriel F., Atílio Tomazini, Vanessa R. Bevilaqua, and Vadim R. Viviani. "Role of Histidine 310 in Amydetes vivianii firefly luciferase pH and metal sensitivities and improvement of its color tuning properties." Photochemical & Photobiological Sciences, May 1, 2024. http://dx.doi.org/10.1007/s43630-024-00570-1.

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18

de Souza, Daniel Rangel, Jaqueline Rodrigues Silva, Ariele Moreira, and Vadim R. Viviani. "Biosensing firefly luciferin synthesis in bacteria reveals a cysteine-dependent quinone detoxification route in Coleoptera." Scientific Reports 12, no. 1 (2022). http://dx.doi.org/10.1038/s41598-022-17205-z.

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AbstractLuciferin 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.
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19

Santos, Raphael M., Marcelo Nivert Schlindwein, and Vadim R. Viviani. "Survey of Bioluminescent Coleoptera in the Atlantic Rain Forest of Serra da Paranapiacaba in São Paulo State (Brazil)." Biota Neotropica 16, no. 1 (2016). http://dx.doi.org/10.1590/1676-0611-bn-2015-0045.

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Brazil is the country with the largest number of bioluminescent beetle species in the world. However, estimates suggest that this number could much be higher, since many species remain to be discovered. In this work we made a survey of the species of bioluminescent beetles in Serra de Paranapiacaba - the largest remnant of Atlantic Forest in São Paulo State. The survey was done at Intervales State Park, Carlos Botelho State Park and municipality of Tapiraí and the following species were collected: Aspisoma lineatum, Aspisoma physonotum, Aspisoma fenestrata, Cratomorphus besckey, Cratomorphus distinctus, Photinus penai, Photinus sp1, Photinus sp9, Ethra aff. malledicta or axilaris, Ethra aff. adicta , Lucidotini incertae sedis, Cladodes flabellicornis, Cladodes demoulini, Amydetes lucernuta, Bicellonycha sp8, Bicellonycha ornaticollis, Pyrogaster lunifer, Pyrogaster moestus, Pyrogaster sp2, Pyrogaster sp5, Pyrogaster sp6, Photuris lugubris, Photuris sp1, Photuris sp7, Stenophrixotrix sp1, Brasilocerus sp1, Pseudophengodes sp1, Hapsodrilus pyrotis, Hypsiophtalmus sp1, Ptesimopsia luculenta, Pyroptesis cincticollis, Pyrearinus brevicolis, Pyrearinus candelarius, Pyrearinus micatus, Pyrophorus divergens. Our data show that Serra de Paranapiacaba is the second richest area in São Paulo state, especially in elaterids, with unique species typical of this area and species common to other investigated sites such as the Biological Station of Boracéia (in Salesópolis county) and the urbanized areas in the between Campinas - Sorocaba- São Paulo, originally covered to the Atlantic Rainforest.
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