Glowing wonders: exploring the diversity and ecological significance of bioluminescent organisms in Brazil.

Bioluminescence, the emission of light by living organisms, is a captivating and widespread phenomenon with diverse ecological functions. This comprehensive review explores the biodiversity, mechanisms, ecological roles, and conservation challenges of bioluminescent organisms in Brazil, a country known for its vast and diverse ecosystems. From the enchanting glow of fireflies and glow-in-the-dark mushrooms to the mesmerizing displays of marine dinoflagellates and cnidarians, Brazil showcases a remarkable array of bioluminescent species. Understanding the biochemical mechanisms and enzymes involved in bioluminescence enhances our knowledge of their evolutionary adaptations and ecological functions. However, habitat loss, climate change, and photopollution pose significant threats to these bioluminescent organisms. Conservation measures, interdisciplinary collaborations, and responsible lighting practices are crucial for their survival. Future research should focus on identifying endemic species, studying environmental factors influencing bioluminescence, and developing effective conservation strategies. Through interdisciplinary collaborations, advanced technologies, and increased funding, Brazil can unravel the mysteries of its bioluminescent biodiversity, drive scientific advancements, and ensure the long-term preservation of these captivating organisms.

Conjugated Dienoic Acid Peroxides as Substrates in Chaetopterus Bioluminescence System.

Biochemistry of bioluminescence of the marine parchment tubeworm Chaetopterus has been in research focus for over a century; however, the results obtained by various groups contradict each other. Here, we report the isolation and structural elucidation of three compounds from Chaetomorpha linum algae, which demonstrate bioluminescence activity with Chaetopterus luciferase in the presence of Fe2+ ions. These compounds are derivatives of polyunsaturated fatty acid peroxides. We have also obtained their structural analogues and demonstrated their activity in the bioluminescence reaction, thus confirming the broad substrate specificity of the luciferase.

Bioluminescent Dinoflagellates as a Bioassay for Toxicity Assessment.

Dinoflagellates bioluminescence mechanism depends upon a luciferin-luciferase reaction that promotes blue light emission (480 nm) in specialized luminogenic organelles called scintillons. The scintillons contain luciferin, luciferase and, in some cases, a luciferin-binding protein (LBP), which prevents luciferin from non-enzymatic oxidation in vivo. Even though dinoflagellate bioluminescence has been studied since the 1950s, there is still a lack of mechanistic understanding on whether the light emission process involves a peroxidic intermediate or not. Still, bioassays employing luminous dinoflagellates, usually from Gonyaulax or Pyrocystis genus, can be used to assess the toxicity of metals or organic compounds. In these dinoflagellates, the response to toxicity is observed as a change in luminescence, which is linked to cellular respiration. As a result, these changes can be used to calculate a percentage of light inhibition that correlates directly with toxicity. This current approach, which lies in between fast bacterial assays and more complex toxicity tests involving vertebrates and invertebrates, can provide a valuable tool for detecting certain pollutants, e.g., metals, in marine sediment and seawater. Thus, the present review focuses on how the dinoflagellates bioluminescence can be applied to evaluate the risks caused by contaminants in the marine environment.

Fungal bioassays for environmental monitoring.

Environmental pollutants are today a major concern and an intensely discussed topic on the global agenda for sustainable development. They include a wide range of organic compounds, such as pharmaceutical waste, pesticides, plastics, and volatile organic compounds that can be found in air, soil, water bodies, sewage, and industrial wastewater. In addition to impacting fauna, flora, and fungi, skin absorption, inhalation, and ingestion of some pollutants can also negatively affect human health. Fungi play a crucial role in the decomposition and cycle of natural and synthetic substances. They exhibit a variety of growth, metabolic, morphological, and reproductive strategies and can be found in association with animals, plants, algae, and cyanobacteria. There are fungal strains that occur naturally in soil, sediment, and water that have inherent abilities to survive with contaminants, making the organism important for bioassay applications. In this context, we reviewed the applications of fungal-based bioassays as a versatile tool for environmental monitoring.

A putative chordate luciferase from a cosmopolitan tunicate indicates convergent bioluminescence evolution across phyla.

Pyrosomes are tunicates in the phylum Chordata, which also contains vertebrates. Their gigantic blooms play important ecological and biogeochemical roles in oceans. Pyrosoma, meaning "fire-body", derives from their brilliant bioluminescence. The biochemistry of this light production is unknown, but has been hypothesized to be bacterial in origin. We found that mixing coelenterazine-a eukaryote-specific luciferin-with Pyrosoma atlanticum homogenate produced light. To identify the bioluminescent machinery, we sequenced P. atlanticum transcriptomes and found a sequence match to a cnidarian luciferase (RLuc). We expressed this novel luciferase (PyroLuc) and, combined with coelenterazine, it produced light. A similar gene was recently predicted from a bioluminescent brittle star, indicating that RLuc-like luciferases may have evolved convergently from homologous dehalogenases across phyla (Cnidaria, Echinodermata, and Chordata). This report indicates that a widespread gene may be able to functionally converge, resulting in bioluminescence across animal phyla, and describes and characterizes the first putative chordate luciferase.

Evaluation of Phenolic Compound Toxicity Using a Bioluminescent Assay with the Fungus Gerronema viridilucens.

Basidiomycetes (phylum Basidiomycota) are filamentous fungi characterized by the exogenous formation of spores on a club-shaped cell called a basidium that are often formed on complex fruiting bodies (mushrooms). Many basidiomycetes serve an important role in recycling lignocellulosic material to higher trophic levels, and some show symbiotic relationships with plants. All known bioluminescent fungi are mushroom-forming basidiomycetes in the order Agaricales. Hence, the disruption of the basidiomycete community can entirely compromise the carbon cycle in nature from fungi to higher trophic levels. The fungus Gerronema viridilucens was used in the present study to investigate the toxicity of a phenolic compound series based on the inhibition of its bioluminescence. The median effect concentration (EC50) obtained from curves of bioluminescence inhibition versus log [phenolic compound] showed that 2,4,6-trichlorophenol was the most toxic compound in the series. The log EC50 values of all phenolic compounds were then used for the prediction of their toxicity. The univariate correlation of log EC50 values obtained from 6 different phenolic compounds was stronger with the dissociation constant (pKa ) than with 1-octanol/water partition coefficient (KOW ). Nevertheless, the toxicity can be better predicted by using both parameters, suggesting that the phenol-driven uncoupling of fungus mitochondrial adenosine triphosphate synthesis is the origin of phenolic compound toxicity to the test fungus. Environ Toxicol Chem 2020;39:1558-1565. © 2020 SETAC.

Chaetopterus variopedatus Bioluminescence: A Review of Light Emission within a Species Complex.

Chaetopterus variopedatus has been studied for over a century in terms of its physiology, ecology and life history. One focus of research is on its intrinsic bioluminescent emissions, which can be observed as a blue light emitted from the extremities of individual body segments, or as a secreted mucus. Even though research shows that C. variopedatus is a species complex miscategorized as a single species, all of the variants of this polychaete produce light, which has been investigated in terms of both physiology and biochemistry. Despite decades of study, there are still many questions about the luminescence reaction, and, as of yet, no clear function for light emission exists. This review summarizes the current knowledge on C. variopedatus luminescence in addition to briefly describing its morphology, life cycle and ecology. Possible functions for luminescence were discussed using observations of specimens found in Brazil, along with a comparison of previous studies of other luminescent organisms. Further study will provide a better understanding of how and why C. variopedatus produces luminescence, and purifying the protein and luciferin involved could lead to new bioanalytical applications, as this reaction is unique among all known luminescent systems.

Neoceroplatus betaryiensis nov. sp. (Diptera: Keroplatidae) is the first record of a bioluminescent fungus-gnat in South America.

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.

Characterizing the Bioluminescence of the Humboldt Squid, Dosidicus gigas (d'Orbigny, 1835): One of the Largest Luminescent Animals in the World.

Bioluminescence is found in a number of cephalopods, such as Watasenia scintillans and Sthenoteuthis oualaniensis; however, many species remain poorly studied, including the Humboldt squid, Dosidicus gigas. This is the largest member of the Ommastrephidae family and grows to 2 m in length, making it one of the largest luminescent animals ever observed. Humboldt squid have small photophores all over their body that emit a brilliant blue luminescence. Using lyophilized photophores from squid caught off the coast of Chile, experiments were conducted to isolate the luciferin and protein involved in its bioluminescence. Methanolic extracts of the photophores were shown to contain dehydrocoelenterazine, and a membrane-bound photoprotein was shown to be involved. This photoprotein was purified using ion exchange chromatography, and SDS-PAGE showed a clean band of approximately 60 kDa. The excised band was analyzed by LC/MS, and the obtained data were compared against the transcriptome data of D. gigas, allowing us to find two gene products which displayed high coverage (>80%), the enzymes symplectin and vanin-2, which potentially associate with light emission process in this organism. Finally, the purified photoprotein was shown to emit a blue light (470 nm) in the presence of dehydrocoelenterazine.

Genetically encodable bioluminescent system from fungi.

Bioluminescence is found across the entire tree of life, conferring a spectacular set of visually oriented functions from attracting mates to scaring off predators. Half a dozen different luciferins, molecules that emit light when enzymatically oxidized, are known. However, just one biochemical pathway for luciferin biosynthesis has been described in full, which is found only in bacteria. Here, we report identification of the fungal luciferase and three other key enzymes that together form the biosynthetic cycle of the fungal luciferin from caffeic acid, a simple and widespread metabolite. Introduction of the identified genes into the genome of the yeast Pichia pastoris along with caffeic acid biosynthesis genes resulted in a strain that is autoluminescent in standard media. We analyzed evolution of the enzymes of the luciferin biosynthesis cycle and found that fungal bioluminescence emerged through a series of events that included two independent gene duplications. The retention of the duplicated enzymes of the luciferin pathway in nonluminescent fungi shows that the gene duplication was followed by functional sequence divergence of enzymes of at least one gene in the biosynthetic pathway and suggests that the evolution of fungal bioluminescence proceeded through several closely related stepping stone nonluminescent biochemical reactions with adaptive roles. The availability of a complete eukaryotic luciferin biosynthesis pathway provides several applications in biomedicine and bioengineering.

Identification of hispidin as a bioluminescent active compound and its recycling biosynthesis in the luminous fungal fruiting body.

We previously showed that luminous fungi share a common mechanism in bioluminescence, and identified hispidin as a luciferin precursor in Neonothopanus nambi mycelium. Here we showed the presence of hispidin as a bioluminescent active compound at 25-1000 pmol g-1 in the fruiting bodies of Mycena chlorophos, Omphalotus japonicus, and Neonothopanus gardneri. These results suggest that luminous mushrooms contain hispidin as a luciferin precursor. We also found that non-luminous "young" fruiting bodies exhibited luminescence by hispidin treatment. Furthermore, we observed a gradual luminescence enhancement of the cell-free fruiting body extract by the addition of hispidin biosynthetic components, namely caffeic acid, ATP and malonyl-CoA. These findings suggest that continuous weak glow of luminous mushrooms is regulated by slow recycling biosynthesis of hispidin.

Mechanism and color modulation of fungal bioluminescence.

Bioluminescent fungi are spread throughout the globe, but details on their mechanism of light emission are still scarce. Usually, the process involves three key components: an oxidizable luciferin substrate, a luciferase enzyme, and a light emitter, typically oxidized luciferin, and called oxyluciferin. We report the structure of fungal oxyluciferin, investigate the mechanism of fungal bioluminescence, and describe the use of simple synthetic α-pyrones as luciferins to produce multicolor enzymatic chemiluminescence. A high-energy endoperoxide is proposed as an intermediate of the oxidation of the native luciferin to the oxyluciferin, which is a pyruvic acid adduct of caffeic acid. Luciferase promiscuity allows the use of simple α-pyrones as chemiluminescent substrates.

Selected Least Studied but not Forgotten Bioluminescent Systems.

Bioluminescence is a form of chemiluminescence generated by luminous organisms. Luminous taxa have currently been reported from about 800 genera and probably over 10 000 species in the world. On the other hand, their bioluminescent systems, including chemical structures of luciferins/chromophores and the genes encoding luciferases/photoproteins, have been elucidated from only a few taxonomic groups, for example beetles, bacteria, dinoflagellates, ostracods and some cnidarians. Research efforts to understand unknown bioluminescence systems are being conducted around the world, and recently, for example, novel luciferin structures of luminous enchytraeid potworms and fungi were identified by the authors. In this study, we review the current status and perspectives, in the context of postgenomic era, of most likely novel but less-revealed bioluminescence systems of ten selected organisms: earthworm, parchment tubeworm, fireworm, scaleworm, limpet, millipede, brittle star, acorn worms, tunicate and shark, which indeed are the next focus of our international collaboration.

Circadian control sheds light on fungal bioluminescence.

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 (λmax 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.

Current status of research on fungal bioluminescence: biochemistry and prospects for ecotoxicological application.

Over the last half decade the study of fungal bioluminescence has regained momentum since the involvement of enzymes has been confirmed after over 40 years of controversy. Since then our laboratory has worked mainly on further characterizing the substances involved in fungal bioluminescence and its mechanism, as well as the development of an ecotoxicological bioluminescent assay with fungi. Previously, we proved the involvement of a NAD(P)H-dependent reductase and a membrane-bound luciferase in a two-step reaction triggered by addition of NAD(P)H and molecular oxygen to generate green light. The fungal luminescent system is also likely shared across all lineages of bioluminescent fungi based on cross-reaction studies. Moreover, fungal bioluminescence is inhibited by the mycelium exposure to toxicants. The change in light emission under optimal and controlled conditions has been used as endpoint in the development of toxicological bioassays. These bioassays are useful to better understand the interactions and effects of hazardous compounds to terrestrial species and to assist the assessment of soil contaminations by biotic or abiotic sources. In this work, we present an overview of the current state of the study of fungal luminescence and the application of bioluminescent fungi as versatile tool in ecotoxicology.

Evidence that a single bioluminescent system is shared by all known bioluminescent fungal lineages.

Since the early 20th century, many researchers have attempted to determine how fungi are able to emit light. The first successful experiment was obtained using the classical luciferin-luciferase test that consists of mixing under controlled conditions hot (substrate/luciferin) and cold (enzyme/luciferase) water extracts prepared from bioluminescent fungi. Failures by other researchers to reproduce those experiments using different species of fungi lead to the hypothesis of a non-enzymatic luminescent pathway. Only recently, the involvement of a luciferase in this system was proven, thus confirming its enzymatic nature. Of the 100,000 described species in Kingdom Fungi, only 71 species are known to be luminescent and they are distributed unevenly amongst four distantly related lineages. The question we address is whether the mechanism of bioluminescence is the same in all four evolutionary lineages suggesting a single origin of luminescence in the Fungi, or whether each lineage has a unique mechanism for light emission implying independent origins. We prepared hot and cold extracts of numerous species representing the four bioluminescent fungal lineages and performed cross-reactions (luciferin × luciferase) in all possible combinations using closely related non-luminescent species as controls. All cross-reactions with extracts from luminescent species yielded positive results, independent of lineage, whereas no light was emitted in cross-reactions with extracts from non-luminescent species. These results support the hypothesis that all four lineages of luminescent fungi share the same type of luciferin and luciferase, that there is a single luminescent mechanism in the Fungi, and that fungal luciferin is not a ubiquitous molecule in fungal metabolism.

The enzymatic nature of fungal bioluminescence.

The uncertainty about the possible involvement of a luciferase in fungal bioluminescence has not only hindered the understanding of its biochemistry but also delayed the characterization of its constituents. The present report describes how in vitro light emission can be obtained enzymatically from the cold and hot extracts assay using different species of fungi, which also indicates a common mechanism for all these organisms. Kinetic data suggest a consecutive two-step enzymatic mechanism and corroborate the enzymatic proposal of Airth and Foerster. Finally, overlapping of light emission spectra from the fungal bioluminescence and the in vitro assay confirm that this reaction is the same one that occurs in live fungi.

Fungi bioluminescence revisited.

A review of the research conducted during the past 30 years on the distribution, taxonomy, phylogeny, ecology, physiology and bioluminescence mechanisms of luminescent fungi is presented. We recognize 64 species of bioluminescent fungi belonging to at least three distinct evolutionary lineages, termed Omphalotus, Armillaria and mycenoid. An accounting of their currently accepted names, distributions, citations reporting luminescence and whether their mycelium and/or basidiomes emit light are provided. We address the physiological and ecological aspects of fungal bioluminescence and provide data on the mechanisms responsible for bioluminescence in the fungi.