Adult Lantern Research Articles

Cloning and molecular properties of a novel luciferase from the Brazilian Bicellonycha lividipennis (Lampyridae: Photurinae) firefly: comparison with other firefly luciferases.

Several firefly luciferases eliciting light emission in the yellow-green range of the spectrum and with distinct kinetic properties have been already cloned, sequenced, and characterized. Some of them are currently being applied as analytical reagents and reporter genes for bioimaging and biosensors, and more recently as potential color tuning indicators of intracellular pH and toxic metals. They were cloned from the subfamilies Lampyrinae (Photinini: Photinus pyralis, Macrolampis sp2; Cratomorphini: Cratomorphus distinctus), Photurinae (Photuris pennsylvanica), Luciolinae (Luciola cruciata, L. lateralis, L. mingrelica, L. italica, Hotaria parvula), and Amydetinae (Amydetes vivianii) occurring in different parts of the world. The largest number has been cloned from fireflies occurring in Brazilian biomes. Taking advantage of the large biodiversity of fireflies occurring in the Brazilian Atlantic rainforest, here we report the cloning and characterization of a novel luciferase cDNA from the Photurinae subfamily, Bicellonycha lividipennis, which is a very common firefly in marshlands in Brazil. As expected, multialignements and phylogenetic analysis show that this luciferase clusters with Photuris pennsylvanica adult isozyme, and with other adult lantern firefly luciferases, in reasonable agreement with traditional phylogenetic analysis. The luciferase elicits light emission in the yellow-green region, has kinetics properties similar to other adult lantern firefly luciferases, including pH- and metal sensitivities, but displays a lower sensitivity to nickel, which is suggested to be caused by the natural substitution of H310Y.

Transcriptomes from the photogenic and non-photogenetic tissues and life stages of the Aspisoma lineatum firefly (Coleoptera: Lampyridae): Implications for the evolutionary origins of bioluminescence and its associated light organs

In fireflies, the bioluminescent system involves a benzothiazolic luciferin, ATP, a luciferase, and complex lanterns consisting of different tissues including a photogenic one. Whereas the biochemical reaction and the structure of lanterns are quite well known, the evolutionary and developmental origins of photogenic tissue as well as the biosynthetic origin of luciferin remain enigmatic. One approach to better understand the origin of the photogenic tissue and biosynthetic origin of luciferin is to investigate the molecular differentiation among photogenic and non-photogenic tissues. Thus, here we compared - for the first time - the transcriptomes of the photogenic tissue of lanterns (specialized tissue) and the fat body (non-specialized tissue) of larvae, adult lanterns, and eggs stages of the common Southeastern Brazilian firefly Aspisoma lineatum. The similarities between the photogenic tissue of lanterns and the fat body, with the occurrence of several luciferase isozymes, luciferin-regenerating enzyme, L-DOPA metabolism related enzymes, and nitric oxide synthase, support the hypothesis that in fireflies, the photogenic tissue evolved from the fat body.

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 function of Hox and appendage-patterning genes in the development of an evolutionary novelty, the Photuris firefly lantern.

Uncovering the mechanisms underlying the evolution of novel traits is a central challenge in biology. The lanterns of fireflies are complex traits that lack even remote homology to structures outside luminescent beetle families. Representing unambiguous novelties by the strictest definition, their developmental underpinnings may provide clues to their origin and offer insights into the mechanisms of innovation in developmental evolution. Lanterns develop within the context of abdominal Hox expression domains, and we hypothesized that lantern formation may be instructed in part by these highly conserved transcription factors. We show that transcript depletion of Abdominal-B in Photuris fireflies results in extensive disruption of the adult lantern, suggesting that the evolution of adult lanterns involved the acquisition of a novel regulatory role for this Hox gene. Using the same approach, we show that the Hox gene abdominal-A may control important secondary aspects of lantern development. Lastly, we hypothesized that lantern evolution may have involved the recruitment of dormant abdominal appendage-patterning domains; however, transcript depletion of two genes, Distal-less and dachshund, suggests that they do not contribute to lantern development. Our results suggest that complex novelties can arise within the confines of ancestral regulatory landscapes through acquisition of novel targets without compromising ancestral functions.

Correction: Biosynthesis of Firefly Luciferin in Adult Lantern: Decarboxylation of L-Cysteine is a Key Step for Benzothiazole Ring Formation in Firefly Luciferin Synthesis

Correction: Biosynthesis of Firefly Luciferin in Adult Lantern: Decarboxylation of L-Cysteine is a Key Step for Benzothiazole Ring Formation in Firefly Luciferin Synthesis

Biosynthesis of Firefly Luciferin in Adult Lantern: Decarboxylation of ʟ-Cysteine is a Key Step for Benzothiazole Ring Formation in Firefly Luciferin Synthesis

BackgroundBioluminescence in fireflies and click beetles is produced by a luciferase-luciferin reaction. The luminescence property and protein structure of firefly luciferase have been investigated, and its cDNA has been used for various assay systems. The chemical structure of firefly luciferin was identified as the ᴅ-form in 1963 and studies on the biosynthesis of firefly luciferin began early in the 1970’s. Incorporation experiments using 14C-labeled compounds were performed, and cysteine and benzoquinone/hydroquinone were proposed to be biosynthetic component for firefly luciferin. However, there have been no clear conclusions regarding the biosynthetic components of firefly luciferin over 30 years.Methodology/Principal FindingsIncorporation studies were performed by injecting stable isotope-labeled compounds, including ʟ-[U-13C3]-cysteine, ʟ-[1-13C]-cysteine, ʟ-[3-13C]-cysteine, 1,4-[D6]-hydroquinone, and p-[2,3,5,6-D]-benzoquinone, into the adult lantern of the living Japanese firefly Luciola lateralis. After extracting firefly luciferin from the lantern, the incorporation of stable isotope-labeled compounds into firefly luciferin was identified by LC/ESI-TOF-MS. The positions of the stable isotope atoms in firefly luciferin were determined by the mass fragmentation of firefly luciferin.ConclusionsWe demonstrated for the first time that ᴅ- and ʟ-firefly luciferins are biosynthesized in the lantern of the adult firefly from two ʟ-cysteine molecules with p-benzoquinone/1,4-hydroquinone, accompanied by the decarboxylation of ʟ-cysteine.

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.

Bioluminescent Fat Body of Larval Aspisoma lineatum (Coleoptera: Lampyridae) Firefly: Ontogenic Precursor of Lantern's Photogenic Tissue

Abstract Previously, we found that the fat body of Aspisoma lineatum Gyll (Coleoptera: Lampyridae) firefly larvae is weakly bioluminescent. This tissue is very different from that of other insect larvae. It is macroscopically distinguished by its color (pinkish and whitish), morphology, and the absence of oenocytes. It is composed of trophocytes that are arranged in groups of globular units covered by a layer of basal lamina. The cytochemistry indicated that the trophocytes have glycoproteins, which are produced by a well-developed rough endoplasm reticulum (RER). Expanded RER cisterns indicated intense protein synthesis by the trophocytes. Lipid droplets are also present in the trophocytes. Charge-coupled device imaging showed that the fat body produces a continuous bioluminescence whose intensity is 2–3 orders of magnitude lower than that of the lanterns, a result that is explained by the lower contents of luciferin and luciferase in the fat body compared with the lanterns. Expression of different luciferase isozymes in the fat body and lanterns is confirmed by bioluminescence spectral and kinetic analyses. Trophocytes were identified as the emitting cells, suggesting that the larval and adult lantern's photocytes may have evolved from fat body trophocytes.

Sea Urchin FGFR Muscle-Specific Expression: Posttranscriptional Regulation in Embryos and Adults

We have shown previously byin situhybridization that a gene encoding a fibroblast growth factor receptor (SpFGFR) is transcribed in many cell types during the initial phases of sea urchin embryogenesis (Strongylocentrotus purpuratus) (McCoonet al., J. Biol. Chem. 271,20119–20195, 1996). Here we demonstrate by immunostaining with affinity-purified antibody that SpFGFR protein is detectable only in muscle cells of the embryo and appears at a time suggesting that its function is not in commitment to a muscle fate, but instead may be required to support the proliferation, migration, and/or differentiation of myoblasts. Surprisingly, we find thatSpFGFRtranscripts are enriched in embryo nuclei, suggesting that lack of processing and/or cytoplasmic transport in nonmuscle cells is at least part of the posttranscriptional regulatory mechanism. Western blots show that SpFGFR is also specifically expressed in adult lantern muscle, but is not detectable in other smooth muscle-containing tissues, including tube foot and intestine, or in coelomocytes, despite the presence ofSpFGFRtranscripts at similar concentrations in all these tissues. We conclude that in both embryos and adults, muscle-specific SpFGF receptor synthesis is controlled primarily at a posttranscriptional level. We show by RNase protection assays that transcripts encoding the IgS variant of the ligand binding domain of the receptor, previously shown to be enriched in embryo endomesoderm fractions, are the predominant, if not exclusive,SpFGFRtranscripts in lantern muscle. Together, these results suggest that only a minority ofSpFGFRtranscripts are processed, exported, and translated in both adult and embryonic muscle cells and these contain predominantly, if not exclusively, IgS ligand binding domain sequences.

Release of Octopamine From the Photomotor Neurones of the Larval Firefly Lanterns

ABSTRACT The biogenic amine, octopamine, has been proposed as the transmitter in the firefly light organ (Evans, 1980; Robertson, 1981). Whereas initial studies implicated adrenaline (Smalley, 1965; Carlson, 1968a), octopamine was found to be more potent in inducing luminescence than any amine other than synephrine (Carlson, 1968b). Octopamine, but not synephrine, was found in adult lantern segments (Robertson & Carlson, 1976; Copeland & Robertson, 1982) and in larval lantern tissue (Christensen, Sherman, McCaman & Carlson, 1983). Evidence that cyclic nucleotides mediate the effect of octopamine was obtained by Oertel & Case (1976). An octopamine-sensitive adenylate cyclase has been found in the larval lantern and application of exogenous octopamine strongly stimulates cyclic AMP production in adult (Nathanson, 1979) and larval (Nathanson & Hunnicutt, 1979) light organs.

Synthesis of active firefly luciferase by in vitro translation of RNA obtained from adult lanterns

Poly (A) + RNA was isolated from the lanterns of adult fireflies, Photinus pyralis . The Poly (A) + RNA was translated in a cell-free translation mixture from rabbit reticulocytes and the synthesis of enzymatically active firefly luciferase was demonstrated. The translation products were immunoprecipitated with anti-luciferase and then subjected to SDS gel electrophoresis. It was shown that a newly synthesized polypeptide exhibited an identical electrophoretic mobility as the purified enzyme.

Biochemical and morphological changes accompanying light organ development in the firefly, Photuris pennsylvanica

The larval light organs of the firefly, Photuris pennsylvanica, regress and are replaced by the adult lantern during metamorphosis. Larval and adult light organs are present and capable of periodic light emission during the latter stages of pupation and the early adult. The whole pupa emits a continuous, low level, glow throughout pupation. During pupation levels of luciferase and luciferin, the enzyme and substrate required in the light reaction, were found to remain constant in the posterior half of the pupa and to show an initial increase followed by a decrease in the anterior half. Levels of luciferase and luciferin in anterior halves were not affected by ablation of the larval light organs. The ratio of luciferase to luciferin concentrations changed from less than 1, in larval and pupal stages, to greater than 1, in the adult. Changes in the concentration and the localization of luciferase and luciferin were correlated with observed light organ development.