Luminous Beetle Research Articles

Multiple Origins of Bioluminescence in Beetles and Evolution of Luciferase Function.

Bioluminescence in beetles has long fascinated biologists, with diverse applications in biotechnology. To date, however, our understanding of its evolutionary origin and functional variation mechanisms remains poor. To address these questions, we obtained high-quality reference genomes of luminous and nonluminous beetles in 6 Elateroidea families. We then reconstructed a robust phylogenetic relationship for all luminous families and related nonluminous families. Comparative genomic analyses and biochemical functional experiments suggested that gene evolution within Elateroidea played a crucial role in the origin of bioluminescence, with multiple parallel origins observed in the luminous beetle families. While most luciferase-like proteins exhibited a conserved nonluminous amino acid pattern (TLA346 to 348) in the luciferin-binding sites, luciferases in the different luminous beetle families showed divergent luminous patterns at these sites (TSA/CCA/CSA/LVA). Comparisons of the structural and enzymatic properties of ancestral, extant, and site-directed mutant luciferases further reinforced the important role of these sites in the trade-off between acyl-CoA synthetase and luciferase activities. Furthermore, the evolution of bioluminescent color demonstrated a tendency toward hypsochromic shifts and variations among the luminous families. Taken together, our results revealed multiple parallel origins of bioluminescence and functional divergence within the beetle bioluminescent system.

Firefly genomes illuminate the evolution of beetle bioluminescent systems.

Fireflies are one of the best-known bioluminescent organisms, and the reaction mechanism and ecological utility of bioluminescence have been well-studied. Genome assemblies of six species of bioluminescent beetles have recently been published. These studies have focused on the evolution of novelties; luciferase, and the biosynthesis of luciferin and defensive chemicals. For example, clustering of the luciferase gene with acyl-CoA synthetase genes on a chromosome in luminous beetle genomes suggests the involvement of tandem gene duplications and neofunctionalization during the evolution of beetle bioluminescence. Several candidate genes for critical roles in beetle bioluminescence have been identified, but their functional analyses are still ongoing. The establishment of a long-term mass-rearing system and strain will be the key for the post-genome research on bioluminescent beetles. Lastly, the application of contemporary chromosome-scale genome assembly techniques to luminous beetles will help resolve outstanding evolutionary questions, such as how many times bioluminescence evolved in this clade.

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.

One-pot non-enzymatic formation of firefly luciferin in a neutral buffer from p-benzoquinone and cysteine.

Firefly luciferin, the substrate for the bioluminescence reaction of luminous beetles, possesses a benzothiazole ring, which is rare in nature. Here, we demonstrate a novel one-pot reaction to give firefly luciferin in a neutral buffer from p-benzoquinone and cysteine without any synthetic reagents or enzymes. The formation of firefly luciferin was low in yield in various neutral buffers, whereas it was inhibited or completely prevented in acidic or basic buffers, in organic solvents, or under a nitrogen atmosphere. Labelling analysis of the firefly luciferin using stable isotopic cysteines showed that the benzothiazole ring was formed via the decarboxylation and carbon-sulfur bond rearrangement of cysteine. These findings imply that the biosynthesis of firefly luciferin can be developed/evolved from the non-enzymatic production of firefly luciferin using common primary biosynthetic units, p-benzoquinone and cysteine.

Quantum Yields and Kinetics of the Firefly Bioluminescence Reaction of Beetle Luciferases

Quantum yields of firefly bioluminescence reactions were determined for beetle luciferases from the three main families of luminous beetles emitting different bioluminescence colors. Quantum yield (QY) was significantly correlated with luminescence spectrum. The green light-emitting luciferase of the Brazilian click beetle, Pyrearinus termitilluminans, whose luminescence spectrum had the shortest peak wavelength of all the luciferases investigated, had the highest QY (0.61). Mutant analyses of active site-substituted Pyrocoelia miyako luciferases showed that, although k(cat) was decreased by the mutations, the QY was not significantly affected.

A Gigantic Bioluminescent Starworm (Coleoptera: Rhagophthalmidae) From Northwest Yunnan, China

“Starworm” is the common name for larvae and larviform females of the rhagophthalmid genus Diplocladon (Haneda, 1950; 1985). Such a name results from their three longitudinal rows of lights, one dorso-central and two laterals, produced by three spot-like light organs located on contiguous body segments (Harvey, 1952; Braham and Wenzel, 2001; Ohba, 2004). Although Diplocladon was erected by Gorham in 1883, only two species (D. hasseltii Gorham, 1883, and D. haplocladon indicum Gorham, 1903) have been reported from India and Indonesia (Wittmer, 1944). Diplocladon hasseltii is known from both sexes (Haneda, 1950), but D. haplocladon indicum Gorham known only from the male. In addition, Harvey (1952) thought that luminous larvae or larviform females of a few unidentified species of Diplocladon (some up to 5 cm long) lived in Hangzhou (Zhejiang Province, China, Waterhouse, 1889) and Malaya (Gahan, 1924). Ridley (1934) also dealt with some Diplocladon larva from Singapore and pointed out the large (also circa 5 cm long) luminous insects of Clark (1865) from India belonged in this genus. Because Diplocladon males are nonluminous, they are less commonly collected and correctly associated with the conspecific females. Females and larvae are usually edaphic and relatively cryptic, making them also difficult to collect. Perhaps, the scarcity of specimens has made the systematics of Diplocladon problematic. For instance, Diplocadon has been placed in the Drilidae (Gorham, 1883; Olivier, 1910; Wittmer, 1944; Harvey, 1952), Phengodidae (Crowson, 1972), or Rhagophthalmidae (Lawrence et al., 2000; Branham, 2000). Herein, we follow Lawrence et al. (2000). Having been woken up by his barking dog at midnight, Mr. Guojian Liao, a coal worker at the Tongda Coal Company, found a luminous object on a slope about 50 meters from his home [Baiguhe Village, Tongda Township, Huaping County, Lijiang Prefecture, Yunnan Province, China (N26.75541o, E101.10284o; 2234 m)] on June 6, 2006. The object turned out to be a gigantic (circa 9 cm long) luminous worm. At first sight, its body shape and coloration are very similar to those of larvae and larviform females of the luminous beetle genus Rhagophthalmus (Rhagophthalmidae). Rhagophthalmus larvae are predatory but larviform females rarely feed (Ohba, 1997). The luminous worm was kept in a bottle

Mitochondrial genomes of two luminous beetles, Rhagophthalmus lufengensis and R. ohbai (Arthropoda, Insecta, Coleoptera)

We determined the mitochondrial DNA (mtDNA) sequences of two luminous beetles (Arthropoda, Insecta, Coleoptera), Rhagophthalmus lufengensis from Yunnan, China and Rhagophthalmus ohbai from Yaeyama Island, Japan. We identified all the 37 mtDNA genes of R. lufengensis (15,982 bp) and the 34 genes of R. ohbai (15,704 bp). R. lufengensis and R. ohbai genomes have higher A + T contents than other coleopteran genomes although the gene arrangements are similar. Interestingly, in a study of the evolutionary relationship among R. lufengensis, R. ohbai and the firefly Pyrocoelia rufa, the phylogenetic tree inferred from lrRNA genes from mitochondrial genomes indicates a biogeographic relationship among the bioluminescent insects in East Asia and the phylogenetic tree inferred from luciferase-related genes from nuclear genomes shows an appropriate relationship among coleopterans, reflecting the evolutionary origin of bioluminescence. Thus, the mtDNAs of luminescent beetles can provide an insight into their evolutionary origin and biogeography.

Phylogenetic relationships of click beetles (Coleoptera: Elateridae) inferred from 28S ribosomal DNA: Insights into the evolution of bioluminescence in Elateridae

Although the taxonomy of click beetles (family Elateridae) has been studied extensively, inconsistencies remain. We examine here the relationships between species of Elateridae based on partial sequences of nuclear 28S ribosomal DNA. Specimens were collected primarily from Japan, while luminous click beetles were also sampled from Central and South America to investigate the origins of bioluminescence in Elateridae. Neighbor-joining, maximum-parsimony, and maximum-likelihood analyses produced a consistent basal topology with high statistical support that is partially congruent with the results of previous investigations based on the morphological characteristics of larvae and adults. The most parsimonious reconstruction of the “luminous” and “nonluminous” states, based on the present molecular phylogeny, indicates that the ancestral state of Elateridae was nonluminous. This suggests that the bioluminescence in click beetle evolved independent of that of other luminous beetles, such as Lampyridae, despite their common mechanisms of bioluminescence.

Bioluminescent signals and the role of reflectors

Organisms in a well lit environment use optical signals derived from the selective reflection of ambient light. In a dim or dark environment it is very difficult (because of low photon numbers) to detect the contrast between light reflected from the organism and that from the background, and many organisms use bioluminescent signals instead. The use of such signals on land is largely restricted to sexual signalling by the luminous beetles, but in the deep ocean their use is widespread, involving both many different organisms and a range of uses which parallel those of reflective signals on land. Some bioluminescent signals rely almost entirely on an optically unmodified light source (e.g. a secretion) but others depend upon complex optical structures, particularly reflectors, in the light-emitting organs. Reflectors in the light organs of many shrimp, squid and fish are based on constructive interference systems but employ different biological materials. They and other structures modify the angular, spectral and intensity distributions of bioluminescent signals. The ready availability of highly efficient biological reflectors has been a formative influence in the evolution of bioluminescent signalling in the sea.

THE CHEMICAL MECHANISM and EVOLUTIONARY DEVELOPMENT OF BEETLE BIOLUMINESCENCE

Abstract— Bioluminescence, as a phenotype, has many evolutionary origins, and thus is an example of natural reinvention many times over. Although peculiar, it arises from the same biochemical principles and evolutionary mechanisms as other biochemical reactions. Of these many different bioluminescent systems, that of the luminous beetles is one of the best understood, having been extensively studied for over 50 years. The luminescence ensues from oxidation of a molecule unique to luminous beetles, beetle luciferin, through a catalytic mechanism evolved from ancestral coenzyme A synthetases. Thus, the character of this bioluminescent reaction is in part a consequence of that evolutionary history. Beetle bioluminescence is furthermore unusual in having a range of luminescent colors found among different beetle species and sometimes even within individual beetles. Structural features of the luciferases are responsible for these color differences, although the underlying mechanism is not yet clear.

Luminous Beetles of Florida

Luminous Beetles of Florida