Relative isotopic peak intensities (%) of the parent and its fragment mass from firefly luciferin in the lantern extracts after injecting L-Cys[1-¹³C] or L-Cys[3-¹³C] with 1,4-hydroquinone into the adult of <i>L. lateralis.</i>

<p>^a (<b><i>a</i></b>) represents the parent mass of firefly luciferin with MH⁺281 (+0, 100%). (<b><i>b</i></b>) and (<b><i>c</i></b>) represent the fragment ion mass from firefly luciferin with MH⁺235 (+0, 100%) and MH⁺177 (+0, 100%), respectively, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084023#pone-0084023-g002" target="_blank">Fig. 2</a>. The numbers in bold are the significant mass peaks containing the incorporated stable isotope atoms.</p

Identification of arbutin in <i>L. lateralis</i> by HPLC analysis.

<p>A. HPLC analysis of the extracts from an adult <i>L. lateralis</i> by using a fluorescence detector. (a) authentic arbutin, (b) the extracts of <i>L. lateralis</i> lantern. The arbutin fraction between the vertical dashed lines is used for hydrolysis as in Fig. 10C. B. HPLC analysis of the hydrolyzed arbutin fraction in Fig. 10A–b. (a) authentic 1,4-hydroquinone (labeled peak 1) containing benzoquinone (labeled peak 2), (b) the hydrolyzed products of the peak fraction between the dashed lines in Fig. 10A–b. Asterisk indicates 1,4-hydroquinone from arbutin. C. A scheme of acid hydrolysis of arbutin to 1,4-hydroquinone by acid treatment with HCl.</p

Maximum <i>p</i>-distance for genus vs. the number of species analyzed.

<p>No strong correlation was found between the two variables (Spearman’s rank correlation coefficient <i>r_s</i> = 0.356).</p

Strategy to study on the biosynthetic pathway of firefly luciferin in an adult lantern of a living firefly by injecting the stable isotope-labeled compounds, and the bioluminescence reaction catalyzed by firefly/beetle luciferase.

<p>A. Proposed biosynthetic pathway of firefly luciferin (<b>I</b>) from <i>p</i>-benzoquinone and two L-cysteines in an adult lantern, and the luminescence reaction of luciferin with firefly luciferase, followed by the formation of 2-cyano-6-hydroxybenzothiazole (<b>III</b>) from oxyluciferin (<b>II</b>). B. Stable isotope-labeled L-cysteines used in the experiments. Asterisk indicates the position of a ¹³C atom. C. Preparation of <i>p</i>-[D₄]-benzoquinone from 1,4-[D₆]-hydroquinone by the oxidation reaction using silver oxide with H₂O₂.</p

Incorporation of <i>p</i>-[D₄]-benzoquinone or 1,4-[D₆]-hydroquinone with L-cysteines into firefly luciferin in an adult lantern of <i>L. lateralis</i>.

<p>A. Predicted firefly luciferins incorporated from <i>p</i>-[D₄]-benzoquinone and L-cysteine. B. Predicted firefly luciferins incorporated from 1,4-[D₆]-hydroquinone and L-cysteine. C. Predicted firefly luciferins incorporated from 1,4-[D₆]-hydroquinone and L-Cys[3-¹³C₃]. The number in parenthesis on the right indicates the number of the stable isotope atoms incorporated into firefly luciferin. Asterisk indicates the position of a ¹³C-atom.</p

Map of Japan with sampling localities (red circles), created using Google Maps.

<p>Taxonomic lists of the Japanese elaterids have been published by some authors [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116612#pone.0116612.ref011" target="_blank">11</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116612#pone.0116612.ref013" target="_blank">13</a>], and the number of species listed has steadily increased (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116612#pone.0116612.t001" target="_blank">Table 1</a>). At present, approximately 130 elaterid genera and 770 species have been recorded in Japan (reviewed in Ôhira, 2013 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116612#pone.0116612.ref003" target="_blank">3</a>]), although the most updated species list was not published with this review. This number of elaterid species in Japan is comparable with that of the whole Nearctic region (965 species) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116612#pone.0116612.ref002" target="_blank">2</a>] and much larger than that of the British Islands (73 species) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116612#pone.0116612.ref014" target="_blank">14</a>], which are as large as the Japanese Islands. These numbers highlight the high elaterid biodiversity in Japan. Adult elaterids are relatively common in Japan. They are often collected in various traps during environmental impact statement research and thus appear as significant members in the prefectural lists of wild insects. For example, Elateridae constitutes 5.1% and 4.9% of the beetle species collected in Tochigi [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116612#pone.0116612.ref015" target="_blank">15</a>] and Okayama [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116612#pone.0116612.ref016" target="_blank">16</a>] prefectures, respectively.</p

DNA Barcoding of Japanese Click Beetles (Coleoptera, Elateridae)

<div><p>Click beetles (Coleoptera: Elateridae) represent one of the largest groups of beetle insects. Some click beetles in larval form, known as wireworms, are destructive agricultural pests. Morphological identification of click beetles is generally difficult and requires taxonomic expertise. This study reports on the DNA barcoding of Japanese click beetles to enable their rapid and accurate identification. We collected and assembled 762 cytochrome oxidase subunit I barcode sequences from 275 species, which cover approximately 75% of the common species found on the Japanese main island, Honshu. This barcode library also contains 20 out of the 21 potential pest species recorded in Japan. Our analysis shows that most morphologically identified species form distinct phylogenetic clusters separated from each other by large molecular distances. This supports the general usefulness of the DNA barcoding approach for quick and reliable identification of Japanese elaterid species for environmental impact assessment, agricultural pest control, and biodiversity analysis. On the other hand, the taxonomic boundary in dozens of species did not agree with the boundary of barcode index numbers (a criterion for sequence-based species delimitation). These findings urge taxonomic reinvestigation of these mismatched taxa.</p></div

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

<div><p>Background</p><p>Bioluminescence 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 D-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.</p><p>Methodology/Principal Findings</p><p>Incorporation studies were performed by injecting stable isotope-labeled compounds, including L-[U-¹³C₃]-cysteine, L-[1-¹³C]-cysteine, L-[3-¹³C]-cysteine, 1,4-[D₆]-hydroquinone, and <i>p</i>-[2,3,5,6-D]-benzoquinone, into the adult lantern of the living Japanese firefly <i>Luciola lateralis</i>. 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.</p><p>Conclusions</p><p>We demonstrated for the first time that D- and L-firefly luciferins are biosynthesized in the lantern of the adult firefly from two L-cysteine molecules with <i>p</i>-benzoquinone/1,4-hydroquinone, accompanied by the decarboxylation of L-cysteine.</p></div

Distributions of the maximum intraspecific <i>p</i>-distances (red) and the nearest neighbor distance for each species (green).

<p>Sequences below 500 bp were eliminated. (A) Japanese click beetles (project name, JEBP). (B) Combination of plots from eight public DNA barcoding datasets (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116612#pone.0116612.s004" target="_blank">S4 Fig.</a> for plots of each of the eight datasets) that were used to describe the BIN system by Ratnasingham and Hebert [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116612#pone.0116612.ref032" target="_blank">32</a>]. The public data were acquired from the BOLD system on 7 April 2014.</p

Relative isotopic peak intensities (%) of the parent and its fragment mass from firefly luciferin in the lantern extracts after injecting L-Cys[U-¹³C₃] with 1,4-hydroquinone or <i>p</i>-benzoquinone into the adult of <i>L. lateralis.</i>

<p>^a (<b><i>a</i></b>) represents the parent mass of firefly luciferin with MH⁺281 (+0, 100%). (<b><i>b</i></b>) and (<b><i>c</i></b>) represent the fragment ion mass from firefly luciferin with MH⁺235 (+0, 100%) and MH⁺177 (+0, 100%), respectively, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084023#pone-0084023-g002" target="_blank">Fig. 2</a>. The numbers in bold are the significant mass peaks containing the incorporated stable isotope atoms.</p