Determination of the Luciferin Contents in Luminous

and Non-Luminous Beetles

Yuichi OBA,1;y Takeru SHINTANI,1 Takanori NAKAMURA,1

Makoto OJIKA,1 and Satoshi INOUYE2

1Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan2Yokohama Research Center, Chisso Co., 5-1 Okawa, Kanazawa, Yokohama 236-8605, Japan

Received January 9, 2008; Accepted January 12, 2008; Online Publication, May 7, 2008

[doi:10.1271/bbb.80017]

The contents of firefly luciferin in luminous and non-

luminous beetles were determined by the methods of

HPLC with fluorescence detection and the luminescence

reaction of luciferin and firefly luciferase. Luminous

cantharoids and elaterids contained various amounts

of luciferin in the range of pmol to hundreds of nmol,

but no luciferin was detected in the non-luminous

cantharoids and elaterids.

Key words: bioluminescence; Cantharidae; Elateridae;

Lampyridae; Lycidae

A remarkable feature of beetles (Coleoptera) isbioluminescence that is found in many cantharoidbeetles and some click beetles (Elateridae). The cantha-roid beetles, formerly the superfamily Cantharoidea,1)

consist of Lampyridae (firefly), Phengodidae (railroadworm), Lycidae (net-winged beetle), Cantharidae (sol-dier beetle), and six other small families.2) All knownspecies of Lampyridae and Phengodidae are luminous,3)

but the members of the other cantharoid families arenon-luminous.1) The family Elateridae consists ofluminous and non-luminous species: all luminouselaterids belong to the subfamily of Pyrophorinae.4)

The fireflies, railroad worms, and luminous clickbeetles use the same luciferin (firefly luciferin) in theirluciferin-luciferase reaction (L-L reaction).5) The luci-ferase genes were cloned from various species ofLampyridae, Phengodidae and Elateridae,6) and theprimary sequences show high similarities (>48%) amongthem.7) Thus, the bioluminescence systems of luminousbeetles are essentially the same (Eqs. 1 and 2).5,7)

Luciferinþ ATP � Luciferyl-AMPþ PPi ð1Þ

Luciferyl-AMPþ O2

! Oxyluciferinþ CO2 þ Light ð2Þ

The amount of luciferin has been measured in severalspecies of Lampyridae by the different methods.

Approximately 9.0mg of luciferin was crystallized from15,000 adult lanterns of the North American firefly,Photinus pyralis (2.1 nmol/lantern).8) Thereafter, ap-proximately 5.5mg of luciferin was crystallized from12,000 adult abdomens of the Japanese firefly, Luciolacruciata (1.6 nmol/abdomen).9) Strause et al.10) deter-mined the amount of luciferin in Photuris pennsylvanicaby the L-L reaction and reported that a specimen of larvaand adult contains 90 nmol and 100 nmol of luciferin,respectively. The amount of luciferin in a larva of theIranian firefly, Lampyris turkestanicus, was measured byHPLC with a UV detector,11) and was approximately0.5 nmol/mg of dried lantern. The amount of luciferinin L. lateralis was recently determined by HPLC witha fluorescence detector, and the result showed that alarva and an adult have approximately 0.1 and 1 nmolof luciferin, respectively.12)

The aim of this study is to determine the amountof luciferin in luminous and non-luminous beetles. Weapplied two different methods to analyze the luciferincontent in a specimen: the L-L reaction and an HPLCanalysis with fluorescence detection. The analyticalmethods have an advantage and a disadvantage indetermining the luciferin contents in beetles. Themethod using the L-L reaction is highly sensitive, butthe amount of luciferin might be underestimated due tothe presence of inhibitory factors for the L-L reaction,such as long-chain fatty acids.13) On the other hand, themethod using HPLC with a fluorescence detector is alsohighly sensitive, but the result might be overestimatedwhen the signal of luciferin overlaps with the peak ofother components. Thus, it would be preferable to applytwo independent methods for accurately estimating theamount of luciferin.The mealworm beetle, Tenebrio molitor, was pur-

chased from Jupiter Co. (Tochigi, Japan). The larvae ofL. cruciata and L. lateralis were purchased from ChikyuCo. (Hyogo, Japan). A frozen adult of Pyrophorus sp.collected at Santa-Fe in Panama was kindly supplied by

y To whom correspondence should be addressed. Tel/Fax: +81-52-789-4280; E-mail: oba@agr.nagoya-u.ac.jp

Abbreviation: L-L reaction, luciferin-luciferase reaction

Biosci. Biotechnol. Biochem., 72 (5), 1384–1387, 2008

Note

Tama Zoological Gardens (Tokyo, Japan). All the otherbeetles were collected in Aichi, Gifu, and Nagano,Japan. The specimens were frozen by liquid nitrogen,lyophilized with an FDU-506 freeze dryer (TokyoRikakikai, Tokyo, Japan), and then homogenized withten times the weight per volume of hot water at 95 �C.After centrifugation, the supernatant was washed withan equal volume of n-hexane, and the aqueous layer wassubjected to centrifugal filtration with Ultrafree-MC(PVDF, 0.45 mm; Millipore, Bedford, MA, USA). Toestimate the efficiency of luciferin recovery, the adult ofT. molitor or A. binodulus was extracted in the presenceof 100 pmol of authentic firefly luciferin (potassium salt;Wako Pure Chemicals, Osaka, Japan). The recovery ofluciferin was calculated to be 80–90%. For the L-Lreaction assay, the reaction mixture (50 ml) in a 96-wellplate contained the insect extract (38 ml, but 16 ml forthe small species of Plateros coracinus), 5mM ATP,0.25mM CoA and 5mM MgCl2 in 500mM Tris–HCl(pH 7.8). The L-L reaction was initiated by adding 50 mlof recombinant P. pyralis luciferase (1.49 mg in 500mM

Tris–HCl; Promega, Madison, WI, USA). After tenseconds, the light intensity was measured for 60 s byusing a JNR AB-2100 luminometer (Atto, Tokyo,Japan). After the measurement, the pH value of thereaction solution was confirmed to be unchanged. Forthe HPLC analyses, an extract (22–33 ml) was subjectedto reversed-phase HPLC (Jasco, Tokyo, Japan), using aDevelosil ODS UG-5 column (4:6� 250mm; NomuraChemical, Seto, Japan) eluted with a linear gradientof 10–70% MeOH in 80mM Tris–HCl (pH 9.0; thefluorescence intensity of firefly luciferin under the basiccondition is higher than that under the acidic condition)5)

for 30min at a flow rate of 0.6ml/min. Fluorescencewas detected (excitation at 383 nm; emission at 528 nm)with an FP-1520 fluorescence detector (Jasco). Theretention time and peak area were analysed by Bowinsoftware (Jasco), using authentic firefly luciferin. Underthe foregoing conditions for HPLC analysis, luciferinwas eluted at 21min. When a peak was detected at asimilar retention time to that of luciferin, authenticluciferin was mixed with the extract and reanalyzed todistinguish true luciferin from false.

The calibration curve and detection limit of luciferinin the L-L reaction (Fig. 1A) and HPLC (n ¼ 5,Fig. 1B) analyses were determined by using authenticfirefly luciferin. The concentration of authentic fireflyluciferin was determined spectrophotometrically, usingan absorption coefficient of 18,450 M�1 cm�1 at 328 nmin water.14) Quantitative linearity (correlation efficientR2 > 0:999) was observed from 10 fmol to 20 pmol perwell by the L-L reaction (Fig. 1A) and from 15 fmol to20 pmol/injection by HPLC (Fig. 1B). The detectionlimit of luciferin (the mean value� SE is significantlydifferent (Student’s t-test, P < 0:05) from that withoutluciferin) was 5 fmol/well by the L-L reaction. Thedetection limit by the HPLC (signal to noise ratio, S/N>3) was 15 fmol/injection.

The results showed that adult fireflies (Lampyridae) ofL. cruciata, L. lateralis, and L. parvula contain approx-imately 3.5, 0.5, and 1.5 nmol of luciferin per specimen,respectively (Table 1). These amounts of luciferin areapproximately equal to the values previously reported:L. lateralis by HPLC with a fluorescence detector;12)

P. pyralis and L. cruciata by purification.8,9) Theamount of luciferin in an adult of L. parvula isapproximately three times higher than that in L. later-alis, although they have roughly same body size. Theamount of luciferin in larval L. cruciata determined bythe L-L reaction was higher than that by HPLC(Student’s t-test, P < 0:05). The luciferin in larvalL. lateralis was approximately 20 pmol/insect, whichis nearly the same as the result by Niwa et al.12)

L-Luciferin is not a substrate for the bioluminescencereaction,15) and Niwa et al.12) suggested that the larvaof L. lateralis possesses approximately 1:1 mixture ofD- and L-luciferin by an HPLC analysis using a chiralcolumn. In our analysis, the content of luciferin in thelarva of L. lateralis measured by HPLC analysis waslarger than that by the L-L reaction analysis (althoughstatistically not significant; Table 1). This may beexplained by the presence of L-luciferin as suggestedby Niwa et al.12) or by the presence of compounds thatinhibit the L-L reaction in the extract. The adult ofLucidina biplagiata is a diurnal and seems to commu-nicate between male and female by pheromones.16) Theadult of L. biplagiata is usually considered to be non-

Rel

ativ

e lig

ht u

nits

106

108

104

102

A

R2=0.9994

Luciferin (fmol)10-1 101 103 1050

//

B

102

104

106R2=0.9998

Pea

k ar

ea0 -1 101 103 105

Luciferin (fmol)

//10

Fig. 1. Calibration Curves of Luciferin Determined by the Luciferin-

Luciferase Reaction (A) and by HPLC Analysis with a Fluorescence

Detector (B).

Luciferin Contents in Luminous and Non-Luminous Beetles 1385

luminous or almost non-luminous: the bioluminescenceis undetectable by the human eye, although very weakluminescence was detected by a photomultiplier.16) Inthis study, the amount of luciferin in an adult ofL. biplagiata was determined to be approximately3 pmol/insect (Table 1). Our result suggests that thevery weak luminescence of L. biplagiata is caused bythe L-L reaction, as same as the other luminous fireflies.

The beetles in Cantharidae and Lycidae are diurnaland non-luminous,1,3) and the families of Cantharidaeand Lycidae are supposed to be closely related toLampyridae.3,17) Our results indicated that the species ofCantharidae have no luciferin (Table 1). In Lycidae(Lycostomus modestus and P. coracinus), luciferin couldnot be detected by the L-L reaction, but a small peakcorresponding to luciferin on an HPLC chromatogramwas detected in the extract. If this peak was luciferin, thecontent of luciferin was approximately 50 fmol/insectwhich can be readily detected by the L-L reaction. Thus,the peak detected in the extract of lycids by the HPLCanalysis would not be luciferin.

Click beetles (Elateridae) are widespread throughoutthe world, but the luminous species are only found intropical and subtropical America and Melanesia.4) Theluciferin in luminous click beetles was determined to beidentical to firefly luciferin,5) but the amount of luciferinhas not been estimated. In the present study, the amountof luciferin in an adult of the luminous elaterid,Pyrophorus sp., was determined to be approximately250 nmol, which is the highest value in our present

analysis (Table 1). It is known that the luminescenceintensity of luminous elaterids, especially of the genusPyrophorus, is much brighter than that in lampyrids.18)

No luciferin in four non-luminous elaterid species wasdetected, suggesting that non-luminous elaterids haveno luciferin (Table 1). Among the four species, A. bi-nodulus and Cryptalaus berus belong to the subfamilyPyrophorinae, the same as luminous Pyrophorus.19) Wehave recently cloned and characterized the orthologousgene of the beetle luciferase of A. binodulus (AbLL).20)

The gene product of AbLL is fatty acyl-CoA synthetase,and not luciferase. Thus, the non-luminous click beetleA. binodulus lacks both luciferin and the luminescenceactivity of the beetle luciferase-like gene, in spite of itsclose relationship to luminous click beetles.The mealworm beetle T. molitor (Tenebrionidae) is

an outgroup taxa of cantharoids and elaterids.17,21) Ourresults showed that luciferin was not detected in larvaand adult of T. molitor (Table 1). We also found that thehomologous genes of beetle luciferase in T. molitorhave fatty acyl-CoA synthetic activity but not luciferaseactivity with luciferin.21) Thus, T. molitor lacks both aluciferin and a luciferase.On the basis of the morphological analysis, Crowson1)

suggested that the luminosity of Pyrophorus (clickbeetle) and of various cantharoids (firefly and railroadworm) derived from a common ancestor is unlikely. Ourmolecular phylogenetic analysis also suggested that theancestral state of the Elateridae was non-luminous.19) Itmay be hypothesized that the larvae of luminous click

Table 1. Luciferin Contents in Luminous and Non-Luminous Beetles

FAMILYStagea

Body length Dry weight Luciferin (pmol/mg dry weight) Luciferin (pmol/insect)

Species (average, mm) (average, mg) L-L reactionb HPLC L-L reactionb HPLC

LAMPYRIDAE

Luciola cruciata Adult (1) �3 16.0 25.9 140� 42:1 145� 50:6 3;560� 1;260 3;670� 1;550

Larva (1) �3 18.7 101.8 0:527� 0:159 0:175� 0:074 53:7� 16:3 17:8� 7:50

Luciola lateralis Adult (1) �3 7.3 3.6 124� 29:1 160� 60:4 439� 102 548� 81:8

Larva (1) �3 9.7 6.7 3:65� 4:16 5:76� 2:96 19:1� 13:4 34:5� 6:73

Luciola parvula Adult (1) �3 8.0 3.4 369� 67:0 525� 163 1260� 489 1810� 880

Lucidina biplagiata Adult (2) �3 8.2 2.6 1:09� 0:65 1:17� 0:59 2:63� 1:20 2:84� 1:02

CANTHARIDAE

Lycocerus suturellus Adult (4) �1 16.8 40.3 ND�� ND� — —

Lycocerus vitellinus Adult (13) �1 8.9 7.8 ND�� ND� — —

LYCIDAE

Lycostomus modestus Adult (8) �1 8.9 3.7 ND�� (0.0198)c — (0.0732)c

Plateros coracinus Adult (15) �1 6.8 2.0 ND� (0.0194)c — (0.0427)c

ELATERIDAE

Pyrophorus sp. Adult (1) �1 32.0 309.1 948 753 293,000 233,000

Agrypnus binodulus Adult (2) �1 14.0 43.5 ND�� ND� — —

Cryptalaus berus Adult (1) �1 32.0 502.2 ND�� ND�� — —

Hemicrepidius secessus Adult (2) �1 11.6 10.9 ND�� ND� — —

Melanotus legatus Adult (2) �1 17.0 46.5 ND�� ND� — —

TENEBRIONIDAE

Tenebrio molitor Adult (2) �1 13.5 36.7 ND�� ND� — —

Larva (3) �1 19.0 43.4 ND�� ND� — —

Luminous species are indicated in bold. ND, not detected (undetectable level). �< 5 fmol; ��< 2 fmol.aParentheses indicate the numbers of specimens used for sample preparation. Lampyrid specimens were measured at n ¼ 3. Other specimens were measured at n ¼ 1.bL-L represents the analysis by bioluminescence using recombinant P. pyralis luciferase.cParentheses indicate the value based on the assumption that the HPLC peak is luciferin.

1386 Y. OBA et al.

beetles, which are carnivorous in the larval stage, alsoobtain luciferin from luminous cantharoids such asLampyridae and Phengodidae sympatrically occurring inCentral and South America. However, this possibility isinvalid, because adults of Pyrophorus were luminouseven when the specimens were reared from egg to adultby feeding only T. molitor as bait (K. Ogura, personalcommunication). These results suggested that the lumi-nous click beetle and luminous cantharoids independ-ently developed the ability of luciferin biosynthesis.

Acknowledgments

The authors thank Kanjirou Ogura (Tama ZoologicalGardens, Tokyo) for information about the laboratoryrearing of luminous click beetles, Dr. Hitoo Ohira forthe specimen identification, and Tooru Ojika forsupplying specimens.

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Luciferin Contents in Luminous and Non-Luminous Beetles 1387