Analyses of the bioluminescence mechanism in the land snail, Quantula weinkauffiana.

The mechanism of bioluminescence in the luminous land snails remains largely unknown. Here, we analyzed basic biochemical properties of the luminous land snail, Quantula weinkauffiana. The luminescence activity was extracted from the light organ located near the mouth using a neutral buffer containing detergent. The reaction of the crude buffer extract was triggered by the addition of only hydrogen peroxide (H2O2). These results are inconsistent with the single precedent report on the bioluminescence in the Quantula striata by Shimomura and Haneda in 1986, in which the luminescence of the buffer extract (without detergent) from the light organ was induced by the coaddition of three indispensable components H2O2, ferrous ion, and 2-mercaptoethanol. Based on the present findings, we suggested that an insoluble photoprotein is involved in the bioluminescence of the luminous land snails and the luminescence reaction is simply triggered by H2O2.

14-3-3 proteins are luciferases candidate proteins from lanternfish Diaphus watasei.

The lanternfish is a deep-sea fish with ventral-lateral and head photophores. It uses its ventral-lateral photophores to camouflage its ventral silhouette, a strategy called counterillumination. The bioluminescent reaction of lanternfish involves coelenterazine as a substrate luciferin but the enzyme catalyzing the bioluminescent reaction has not been identified. We report a candidate enzyme of luciferase from lanternfish Diaphus watasei. We purified the luciferase and performed SDS-PAGE analysis resulted in two bands corresponding to the activity, and following mass spectrometry analysis detected three 14-3-3 proteins of which functions is known to exhibit protein-protein interactions. The molecular weights and isoelectric points of the 14-3-3 proteins were almost consistent with the luciferase properties. The addition of two 14-3-3 binding compounds, R18 peptide and fusicoccin, resulted in the inhibition of the luciferase activity. However, the two 14-3-3 recombinant proteins showed very slight luminescence activity. These results suggested that the 14-3-3 proteins are candidate luciferases of D. watasei.

A new species of the genus Aleurodiscus sensu lato (Russulales, Basidiomycota) from Hachijo Island, Japan.

Aleurodiscus sagittisporus sp. nov. is described and illustrated. This species is characterized by producing basidiomata with a monomitic hyphal system, clampless-septate hyphae, arrowhead-shaped, amyloid, finely verrucose basidiospores, gloeocystidia, dendrohyphidium-like branched paraphysoid hyphae, and variously shaped swelling cells in the hymenium. Phylogenetic analyses based on nuclear rDNA LSU and ITS sequences revealed that the species is distinct from the lineage of Aleurodiscus s. str. and related genera in the Aleurodiscus s. lat. clade. Basidiomata of A. sagittisporus have been collected only from dead petioles attached to living trees of Livistona chinensis var. subglobosa on Hachijo Island, Japan.

Bioluminescence chemistry of fireworm Odontosyllis.

Marine polychaetes Odontosyllis undecimdonta, commonly known as fireworms, emit bright blue-green bioluminescence. Until the recent identification of the Odontosyllis luciferase enzyme, little progress had been made toward characterizing the key components of this bioluminescence system. Here we present the biomolecular mechanisms of enzymatic (leading to light emission) and nonenzymatic (dark) oxidation pathways of newly described O. undecimdonta luciferin. Spectral studies, including 1D and 2D NMR spectroscopy, mass spectrometry, and X-ray diffraction, of isolated substances allowed us to characterize the luciferin as an unusual tricyclic sulfur-containing heterocycle. Odontosyllis luciferin does not share structural similarity with any other known luciferins. The structures of the Odontosyllis bioluminescent system's low molecular weight components have enabled us to propose chemical transformation pathways for the enzymatic and nonspecific oxidation of luciferin.

Etmopterus lantern sharks use coelenterazine as the substrate for their luciferin-luciferase bioluminescence system.

The lantern shark genus Etmopterus contains approximately 40 species of deep-sea bioluminescent cartilaginous fishes. They emit blue light mainly from the ventral body surface. The biological functions of this bioluminescence have been discussed based on the luminescence patterns, but the bioluminescence mechanism remains uncertain. In this study, we detected both coelenterazine and coelenterazine-dependent luciferase activity in the ventral photophore tissue of Etmopterus molleri. The results suggested that bioluminescence in lantern sharks is produced using coelenterazine as the substrate for the luciferin-luciferase reaction, as some luminous bony fishes.

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.

Reflector of the body photophore in lanternfish is mechanistically tuned to project the biochemical emission in photocytes for counterillumination.

Lanternfish, a family Myctophidae, use ventro-lateral body photophores for camouflage of the ventral silhouette, a strategy called counterillumination. While other deep-sea fishes possess pigmented filters and silver reflectors to match sunlight filtering down through the depths, myctophids developed a blue-green reflector for this purpose. In this study, we showed in a lanternfish Diaphus watasei that the reflector comprised monolayered iridophores containing multilayered guanine crystals which enable high reflection with light interference colouration. Platelets shape in body photophores is an unique near-regular hexagonal, probably to allow the homogeneity of reflection angle of the luminescence from photocytes. Focus point of the parabola-like reflector is positioned on the photocytes that ensures the light produced from the photocytes is redirected to the ventral direction. In vitro luminescence reaction using purified luciferase and the substrate coelenterazine showed the light emission at λmax 454 nm, while reflection spectra of the iridophores exhibit peaks at longer wavelength, which accomplish to alter the luminescence emitted from photocytes to longer wavelength to fit the mesopelagic light environment. Taken together, we revealed multiple mechanistic elaborations in myctophid body photophores to achieve effective control of biochemical luminescence for counterillumination.

Bioluminescence properties of Thelepus japonicus (Annelida: Terebelliformia).

Terebelliformia is a benthic group of marine annelid worms. The bioluminescence of several species has been reported in taxonomical and histological literature, but very little information is known about the biochemical aspects of this phenomenon. In this study, we examined the basic properties of the luminescence system using an extract of the Japanese terebelliform worm, Thelepus japonicus. The bioluminescence extract was soluble in water, and emitted blue-green light at λmax 508 nm following the addition of divalent cations. This triggering action was highly specific to Fe2+ and addition of ATP, H2 O2 or coelenterazine did not enhance activity. The bioluminescence was inactivated by heat treatment and organic solvents, indicating the involvement of a protein component. These results suggested that Thelepus worm produces light using a novel system that differs from that in other known luminescent annelids.

Luciferase of the Japanese syllid polychaete Odontosyllis undecimdonta.

Odontosyllis undecimdonta is a marine syllid polychaete that produces bright internal and exuded bioluminescence. Despite over fifty years of biochemical investigation into Odontosyllis bioluminescence, the light-emitting small molecule substrate and catalyzing luciferase protein have remained a mystery. Here we describe the discovery of a bioluminescent protein fraction from O. undecimdonta, the identification of the luciferase using peptide and RNA sequencing, and the in vitro reconstruction of the bioluminescence reaction using highly purified O. undecimdonta luciferin and recombinant luciferase. Lastly, we found no identifiably homologous proteins in publicly available datasets. This suggests that the syllid polychaetes contain an evolutionarily unique luciferase among all characterized luminous taxa.

2-S-cysteinylhydroquinone is an intermediate for the firefly luciferin biosynthesis that occurs in the pupal stage of the Japanese firefly, Luciola lateralis.

Firefly luciferin is a natural product that is well-known to function as the substrate of the bioluminescence reaction in luminous beetles. However, the details of the biosynthetic system are still unclear. In this study, we showed by LC-MS/MS analysis that stable isotope-labeled 2-S-cysteinylhydroquinone was incorporated into firefly luciferin in living firefly specimens. Comparison of the incorporation efficiency among the developmental stages suggested that firefly luciferin is biosynthesized predominantly in the pupal stage. We also accomplished the in vitro biosynthesis of firefly luciferin using 2-S-cysteinylhydroquinone and the crude buffer extract of firefly pupae, suggesting the presence of a biosynthetic enzyme in the pupal extract.

Biochemical characteristics and gene expression profiles of two paralogous luciferases from the Japanese firefly Pyrocoelia atripennis (Coleoptera, Lampyridae, Lampyrinae): insight into the evolution of firefly luciferase genes.

Two paralogous genes of firefly luciferase, Luc1 and Luc2, have been isolated from the species in two subfamilies, Luciolinae and Photurinae, of the family Lampyridae. The gene expression profiles have previously been examined only in the species of Luciolinae. Here we isolated Luc1 and Luc2 genes from the Japanese firefly Pyrocoelia atripennis. This is the first report of the presence of both Luc1 and Luc2 genes in the species of the subfamily Lampyrinae and of the exon-intron structure of Luc2 in the family Lampyridae. The luminescence of both gene products peaked at 547 nm under neutral buffer conditions, and the spectrum of Luc1, but not Luc2, was red-shifted under acidic conditions, as observed for Luc2 in the Luciolinae species. The semi-quantitative reverse transcription-polymerase chain reaction suggested that Luc1 was expressed in lanterns of all the stages except eggs, while Luc2 was expressed in the non-lantern bodies of eggs, prepupae, pupae, and female adults. These expression profiles are consistent with those in the Luciolinae species. Considering the distant phylogenetic relationship between Lampyrinae and Luciolinae in Lampyridae, we propose that fireflies generally possess two different luciferase genes and the biochemical properties and gene expression profiles for each paralog are conserved among lampyrid species.

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.

Identification and characterization of the Luc2-type luciferase in the Japanese firefly, Luciola parvula, involved in a dim luminescence in immobile stages.

Nocturnal Japanese fireflies, Luciola parvula, emit from their lanterns a yellow light, one of the most red-shifted colors found among fireflies. Previously, we isolated and characterized two different types of luciferase gene, Luc1 and Luc2, from the fireflies Luciola cruciata and Luciola lateralis; Luc1 is responsible for the green-yellow luminescence of larval and adult lanterns, whereas Luc2 is responsible for the dim greenish glow of eggs and pupal bodies. The biological role of firefly lanterns in adults is related to sexual communication, but why the eggs and pupae glow remains uncertain. In this study, we isolated the gene Luc2 from L. parvula, and compared its expression profiles and enzymatic characteristics with those of Luc1. A semi-quantitative reverse transcription polymerase chain reaction showed that Luc1 was predominantly expressed in larvae, prepupae, pupae and adults, whereas Luc2 was expressed in eggs, prepupae, pupae and adult females. Enzymatic analyses showed that the luminescent color of Luc1 matches the visual sensitivity of L. parvula eyes, whereas that of Luc2 is very different from it. These results suggest that the biological role of Luc2 expressed in immobile stages is not intraspecific communication.

The role of doublesex in the evolution of exaggerated horns in the Japanese rhinoceros beetle.

Male-specific exaggerated horns are an evolutionary novelty and have diverged rapidly via intrasexual selection. Here, we investigated the function of the conserved sex-determination gene doublesex (dsx) in the Japanese rhinoceros beetle (Trypoxylus dichotomus) using RNA interference (RNAi). Our results show that the sex-specific T. dichotomus dsx isoforms have an antagonistic function for head horn formation and only the male isoform has a role for thoracic horn formation. These results indicate that the novel sex-specific regulation of dsx during horn morphogenesis might have been the key evolutionary developmental event at the transition from sexually monomorphic to sexually dimorphic horns.

The Chemical Basis of Fungal Bioluminescence.

Many species of fungi naturally produce light, a phenomenon known as bioluminescence, however, the fungal substrates used in the chemical reactions that produce light have not been reported. We identified the fungal compound luciferin 3-hydroxyhispidin, which is biosynthesized by oxidation of the precursor hispidin, a known fungal and plant secondary metabolite. The fungal luciferin does not share structural similarity with the other eight known luciferins. Furthermore, it was shown that 3-hydroxyhispidin leads to bioluminescence in extracts from four diverse genera of luminous fungi, thus suggesting a common biochemical mechanism for fungal bioluminescence.

Global species delimitation of the cosmopolitan marine littoral earthworm Pontodrilus litoralis (Grube, 1855).

The marine littoral earthworm Pontodrilus litoralis (Grube, 1855) is widely distributed and is reported as a single species. This study utilized an integrative taxonomic approach based upon morphological examination, phylogenetic reconstruction, and molecular species delimitation, to test whether the taxon is a single species or a species complex. For this, a total of 114 P. litoralis specimens collected from North America, Africa, Australia and Oceania, Europe and Asia were used. The phylogenetic analyses revealed deeply divergent mitochondrial lineages and a high level of genetic diversity among P. litoralis populations. Both single and multi-locus species delimitation analyses yielded several molecular operational taxonomic units. Therefore, due to the homogeneity of morphological characteristics, it is likely that the morphospecies P. litoralis is a complex of four or more cryptic species, suggesting that more sampling is required and that the population structure genetic data and gene flow need to be investigated.

Bioluminescence of a firefly pupa: involvement of a luciferase isotype in the dim glow of pupae and eggs in the Japanese firefly, Luciola lateralis.

We isolated cDNA for a luciferase isotype, LlLuc2, from the ovary of the Japanese firefly, Luciola lateralis. The gene product LlLuc2 showed 59% amino acid identity with LlLuc1, which had been isolated from the adult L. lateralis lantern. Molecular phylogenetic analysis indicated that LlLuc2 is an orthologue of LcLuc2 from Luciola cruciata. The spectral maxima of the luminescence by recombinant LlLuc1 and LlLuc2 were 550 and 539 nm, respectively. Quantitative PCR analysis revealed that LlLuc1 was expressed predominantly in larvae and adults, and LlLuc2 was expressed in eggs and pupae, which glow dimly, and we found that the in vivo luminescence spectra of the egg and pupa in L. lateralis were in good agreement with the in vitro luminescence spectrum by LlLuc2. These results suggest that, in L. lateralis, LlLuc1 is responsible for the yellowish luminescence of larval and adult lanterns, and LlLuc2 is responsible for the dim, greenish glow of eggs and whole pupae. Similar results were obtained in L. cruciata.

The Luminous Fungi of Japan.

Luminous fungi have long attracted public attention in Japan, from old folklore and fiction to current tourism, children's toys, games, and picture books. At present, 25 species of luminous fungi have been discovered in Japan, which correspond to approximately one-fourth of the globally recognized species. This species richness is arguably due to the abundant presence of mycophiles looking to find new mushroom species and a tradition of night-time activities, such as firefly watching, in Japan. Bioluminescence, a field of bioscience focused on luminous organisms, has long been studied by many Japanese researchers, including the biochemistry and chemistry of luminous fungi. A Japanese Nobel Prize winner, Osamu Shimomura (1928-2018), primarily focused on the bioluminescence system of luminous fungi in the latter part of his life, and total elucidation of the mechanism was finally accomplished by an international research team with representatives from Russia, Brazil, and Japan in 2018. In this review, we focused on multiple aspects related to luminous fungi of Japan, including myth, taxonomy, and modern sciences.

Flickering flash signals and mate recognition in the Asian firefly, Aquatica lateralis.

Nocturnal fireflies sometimes use intricate bioluminescent signal systems for sexual communication. In this study, we examined flash signals and mate recognition in the Asian firefly, Aquatica lateralis, under natural field conditions. We found that the flash pattern of females changes after copulation, from simple short flashes to flashes with longer duration and flickering. To understand the functions of flickering, we video-recorded and analyzed the flashes of sedentary males, receptive females, and mated females. The results showed that the flashes of these three adult phases can be discriminated from each other by two parameters, flash duration and flicker intensity, with little overlap. Male attraction experiments using an artificial LED device termed 'e-firefly' confirmed that flying and sedentary males are attracted to flashes with shorter durations and lower flicker intensities. The range of attraction success was much wider for flying males and narrower for sedentary males, and the latter was close to the range of receptive female's flashes. These findings suggest that in addition to flash duration, flicker intensity is a flash signal parameter of mate recognition in A. lateralis males.

A new discovery of the bioluminescent terrestrial snail genus Phuphania (Gastropoda: Dyakiidae).

The mysterious world of the bioluminescent molluscs in terrestrial ecosystems is mesmerizing, but Quantula striata was previously the only terrestrial mollusc known to be luminescent. Here, we document the new discovery of bioluminescence in four land snails, namely Phuphania crossei, P. globosa, P. carinata, and P. costata. Our observations establish clearly that these four species of Phuphania produce a continuous greenish light from the light-emitting cells located within the mantle and the foot, and that its bright luminescence is intracellular and is not due to any luminous secretion. Although both Quantula and Phuphania can produce a green light, the luminescence patterns are different. The luminescence displayed by Quantula is rhythmical blinking or flashing, while Phuphania glows continuously. In addition, the bioluminescence in Q. weinkauffiana is confirmed, which is similar to that in the related species, Q. striata.