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

0022-I

910 7’) 0401-0399

w.no

0

BIOCHEMICAL AND MORPHOLOGICAL CHANGES ACCOMPANYING LIGHT ORGAN DEVELOPMENT IN THE FIREFLY, PHOTURIS PENNSYLVANZCA L. G. STRAUSE,M. Department

of Biology,

University

DELUCA* and J. F. CASE

of California,

Santa

Barbara.

CA 93106. U.S.A

(Received I3 Seprember 1978; revised 24 Ocroher 1978) larval light organs of the firefly, Photurispennsy/vanica. 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 Icvel. glow throughout pupation. Durmg 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 I, in larval and pupal stages, to greater than I. in the adult. Changes in the concentration and the localization of luciferase and luciferin were correlated with observed light organ development.

Abstract-The

MATERIALS

INTRODUCTION Dr KIM; metamorphosis radical changes. Larval slow, neurally controlled

firefly light organs undergo light organs, capable only of glowing (OERTELand CASE,

!976), are replaced by an adult lantern that produces complex flash displays (BUCKand CASE,1961; BUCKet al., 1963; BUCKet al., 1965; CARLSON,1961; CAKEand BUCK, 1963). These events are accompanied by neural reorganization involving at least the terminal ventral nerve cord ganglia innervating the light organs. Although many aspects of the control of luminescence have been studied in both larvae and adults, and the biochemistry of adult firefly luminescence is well known (MCELROYand DELUCA, 1973; DELUCA and MCELROY, 1974; DELUCA, 1976), little is known regarding morphological, physiological and biochemical events and their coordination during metamorphosis. Here we describe concentrations and localization of luciferase and luciferin during development, and correlate changes in these with known morphological (SMITH. 1963; OFRTELer of., 1975; CASEand LINBERG, in preparation) and physiological (BUCK and CASE, 1961; BUCKet al., 1963; CASEand BUCK, 1963; OERTEL and CASE, 1976) differences in larval and adult light organs and luminescence behaviour patterns. We also show that the generally distributed ‘whole body’ luminescence characteristic of the pupa (WILLIAMS, 1916) is correlated with widespread increasing levels of luciferin and luciferase. These increments are not a consequence of release during larval light organ degeneration, but result from synthesis throughout the pupa. At maturity these compounds are localized in the region of the adult lantern.

_.___

___~_

* Dcp.lrtmcnt of (‘hcmistry. Lnl\crslt) Diego, La Jolla. CA 92093, U.S.A.

_ 01‘ California.

San

AND METHODS

Photuris pennsylvanica larvae were collected in Autumn and Spring in the vicinity of Columbia, Maryland, stored at 10°C in closed plastic containers lined with moist filter paper and maintained on a 12L: 12D cycle. Twice a month they were fed beef liver, cleaned and provided with fresh filter paper (MCLEAN et al., 1972). In addition, lyophilized Photinus pyalis adults, provided by M. DeLuca, were used in some biochemical assays. Adults of both species gave similar results which are, therefore. tabulated together. Gross morphological

changes

and light emission

Pupation was induced by placing 20-30 mature larvae in a closed, transparent plastic container, 17.5 x 12.5 x 6 cm, containing a depth of 1-3 cm of moistened loam soil, and maintaining them at 25°C on a 16L:8D cycle. The soil was sufficiently deep to conceal the buried larvae (MCLEAN et al.. 1972) yet allowed observation of the pupal chamber from below, providing an easy means of locating metamorphosing pupae without disturbance. High humidity was maintained by frequent spraying of the soil with water. Developing pupae were isolated in 4 cm dia. petri dishes lined with moist sponge. Spontaneous luminescence was recorded from individual pupating insects by a photomultiplier positioned below the petri dish and registering on a chart recorder. To ascertain the relative contributions to luminescence of larva! and adult light organs during the period when both arC present, the larval light organs of a number of larvae were excised prior to pupation. Minima! bleeding occurred and blood was washed away with saline (OERTELand CASE, 1976) before returning the larvae to their pupating containers. Prior to pupation the experimental larvae were divided into four groups: Spring-collected, Autumncollected, Spring-collected with ablated light organs, and Autumn-collected with ablated light organs. The

339

340

L. G.

STRAUSE.

M. DELUCAAND J. F.

data from both groups of Spring- and Autumncollected larvae were similar. Consequently our results compare development of normal larvae to the development of insects from larvae with ablated light organs, without respect to collecting season. Biochemical analysis

Total luciferin and luciferase concentrations were determined for critical development stages from mature larva through pupal stages to the adult, both on normal insects and on insects developing from larvae with ablated larval light organs. Insects were quickly frozen in a dry-ice and acetone bath, bisected between the thorax and first abdominal segment, lyophilized for 24 hr, and stored with a desiccant at 4°C until needed. Half insects were weighed and ground in 1 ml of 0.1 M phosphate buffer, pH 7.8, at 0°C. Crystalline firefly luciferase was purified by the method of GREENand MCELROY (1956). Luciferase concentration was determined by measuring absorbance at 278 nm, at which 1 mg/ml of luciferase has an absorbance of 0.75 (GREENand MCELROY,1956). All luciferin assays used a 200-fold dilution of purified luciferase, a concentration of 0.63 nmole/ml. o-Luciferin was synthesized according to the method of SETO et al. (1963). A 10m3 M solution of purified luciferin was used in all luciferase assays. Lx&erase assay. Luciferase was assayed as the magnitude of the initial flash of light emitted upon injection of excess (0.1 ml of 0.02 M) ATP into a mixture containing 0.0215 M glycyl-glycine buffer, pH 7.8,5.3 mM magnesium-sulfate, 0.08 mM purified luciferin and 0.01 ml of the ground extract, final vol 0.51 ml. Light intensity was measured with an Aminco Chem-glow photometer and Aminco recorder. All assays were carried out in triplicate. Luciferin assay. The luciferin assay system contained 0.024 M glycyl-glycine buffer, pH 7.8, 6 mM magnesium-sulfate, 0.01 ml of 0.033 mg/ml purified luciferase, and 0.1 ml of 0.02 M ATP to a final vol of 0.51 ml. The light reaction was initiated by injection of 0.1 ml ground extract into the assay mixture. When the light reaction proceeds in the presence of excess luciferase, ATP and MgSO,, luciferin is rate-limiting, resulting in a flash height proportional to its concentration in the extract. Light intensity was measured as described above and all assays were carried out in triplicate. Standards. All measurements were standardized relative to dry wt of the insect. Flash heights were converted to pmole/ml according to standard curves prepared for each set of assays for both purified luciferase and luciferin. The concentration of the purified luciferase was 12.6 mg/ml based on a mol. wt of 100,000 (DELUCA, 1976). The relationship between peak light intensity and luciferase concentration, in the range of 2000-fold to lOO-fold dilutions, was used in construction of standard luciferase activity curves. Standard luciferin activity curves, flash height vs were determined for luciferin con~mole/ml, centrations in the range of 10m3 to low6 M. Final concentrations of luciferase and luciferin were expressed in terms of pmole/mg dry wt. Differences in luciferase and luciferin concentrations were compared using Student’s r-test.

CASE

RESULTS Gross morphological changes and light emission Correlation of biochemical changes with the extensive changes occurring during metamorphosis required subdivision of the developmental period into identifiable stages. Four such characteristic stages were defined (Fig. 1). Stage one (SI). The period between the last larval moult and the beginning of deteetable eye pigmentation in the intact specimen (Fig. 1b). During this period, which lasts 3-5 days at 25°C all external adult structures are visible. The whole pupa is colourless except for late appearing reddish, speckled pigmentation at the periphery of the eyes. A small number of unpigmented bristles are present on the head and abdominal segments. Pupae are capable of turning but normally orient with ventral surface upwards. The paired larval light organs, located ventrally in the 8th abdominal segment, periodically glow at this stage (Fig. 2a). Both larval light organs were always observed to glow simultaneously. implying continued CNS control. Stage two (SII). -The 4-day period between completion of eye pigmentation and the beginning of antenna1 pigmentation (Fig. lc). The eyes protrude and are darkly pigmented. Antennae, mouth parts. abdominal segments, thorax and legs are lightly pigmented. Bristles in the head region have darkened. Rotation of the body still occurs. The larval light organs are still capable of spontaneous light emission. Stage three (SIII). The period of 4-6 days between completion of antenna1 pigmentation and the beginnmg of wing pigmentation (Fig. Id). Eyes, mouth parts and antennae are darkly pigmented, and wings begin to darken. Only the abdominal segments and legs remain unpigmented. All abdominal bristles have darkened. The pupa is usually found ventral side up and abdominal movement is reduced. During this stage, the adult light organs appear as uniformely white tissue located in the 6th and 7th abdominal segments. Both larval and adult light organs glow periodically (Fig. 2b), not necessarily in synchrony. Stagefour (SW). The 3-5 days before emergence of the adult firefly (Fig. le). Eyes, mouth parts, antennae. thorax, wings and legs are completely pigmented. The top of the head (vertex) is dark while the remainder of the head and the abdominal segments remain light. The insect lies ventral side up until eclosion is completed (Fig. If). The newly emerged adult, after shedding the epicuticle of the last instar, is soft and colourless. A period of 10-12 hr is required for darkening and hardening of the cuticle. The larval light organs are still present (Fig. lg,h) and capable of spontaneous and simultaneous luminescence (Fig. 2c) in 75% of the newly emerged adults. Within 2-24 hr after eclosion the larval light organs are no longer observed to luminesce. Development in the absence of larval light organs.

Larval light organs were excised, prior to pupation, to permit independent study of pupal luminescence and functional states of the differentiating adult lantern. SI pupae with ablated larval light organs still exhibited continuous luminescence originating throughout the pupa (Fig. 3a). In an SI pupa, immediately after bisection between the thorax and 1st abdominal

Fig. 1. Developmental stages of Photufis. (a) Dorsal view of a mature larva. (b). (c), (d) and (e) Ventral view of pupal stages 1,2,3 and 4, respectively. (f) Ventral view of an adult at eclosion. (g) and (h) Time exposures oflight emission from larval(L.L.O.)and adult (A.L.O.)light organsin an adult. (i)Ventralviewofa mature adult female.

342

Fig. 3. Pupal glow from a Stage 2 pupa. (a) Intact pupa. (b) Pupa bisected between the thorax abdominal segment. Photographs were taken from a video-image intensifier system.

and 1st

Biochemical

al

and morphological

I

i

changes

in Phoruris pmns~k~nica

343

b

d

Fig. 7. Spontaneous light emission recorded from intact specimens. (a) Stage 1pupa with larval light organs only. (b) Stage 3 pupa. both larval and adult light organs present. (c) Newly emerged adult, both larval and adult light organs present. (d) Stage 3 pupa with larval light organs excised, only adult light organs present. Scale = I2 set segment, luminescence is apparent in both the anterior and posterior regions, confirming that the pupal glow is not due to reflection from a localized luminescence source (Fig. 3b). The intensity of the pupal glow, as recorded by a high gain photomultiplier, is constant until the appearance of the adult light organ in SIII, whereupon periodic glows emitted by the early adult light organs are superimposed upon the pupal glow (Fig. 2d). High sensitivity recordings of luminescence from mature larvae, with the body cavity opened and the larval light organs removed, and from anterior halves of mature adults with the body cavity opened, revealed no diffuse glow.

Biochemical analysis Luci$erase assay. Figure 4 shows the relationship between developmental stages and the concentration of luciferase in the posterior half containing the light organ and the anterior half of the insect. When posterior halves of mature larvae were assayed, the flash heights recorded corresponded to an average of 1.1 x 1O- ? pmole luciferase/mg dry wt. Assays of the corresponding anterior halves resulted in peak light intensities which were not within the linear range of enzyme activity, representing less than 0.01 nmole/ml. The concentration of luciferase in pupal posterior halves remains reIatively constant during pupation

Fig. 4. Luciferase concentrations (mean k S.D.) from anterior (0) and posterior ( x ) halves of normally developing insects. * The lower error bar was omitted, for graphical clarity, when the mean minus the SD. approached zero. In Figs 4-8, SI-SIV refer to pupal stages as described in text. (A = anterior. P = posterior).

344

L. G. STRAUSE. M. DELUCA

AND

J. F. CAKE

I-

-5 LOW3

SI

I

I

SII

Sill

A SE

Adult

Stage Fig.

5. Luciferin

concentrations developing

(mean + S.D.) from anterior (0) and posterior ( x ) halves of normally insects. * As in Fig. 4. (A = anterior. P = posterior).

and approximates the concentration found in the mature larvae. Assays of the corresponding anterior halves show an increase in enzyme concentration, with a maximum at SII and a mimimum at SIII and SIV (Fig. 4). Mature adults of Photuris and Photinw contain more luciferase than both anterior and posterior halves of their larval and pupal stages. The concentration of luciferase in the posterior, light organ-containing half (0.1 pmole luciferaseimg dry wt), is always greater than that in the anterior half (4 x lo- 3 pmole luciferase/mg dry wt) (Fig. 4). Luciferin assay. The posterior halves of mature larvae contain an average of 4.5 x 10A3 pmole luciferinimg dry wt. In all pupal stages, the concentration of luciferin in the posterior halves remains relatively constant and equal to the concentration found in the mature larval stage (Fig. 5). During pupation, the concentration of luciferin in the anterior halves shows an initial increase, as seen for luciferase, but does not show a significant decrease

until the adult stage. In anterior halves. of both larval and adult stages, the peak light intensity, produced by the rapid injection of ground extract into the normal assay mixture, did not fall within the linear range of luciferin activity, and therefore contained less than 10m4pmole/ml luciferin. However, the posterior, light organ-containing half, of mature adults shows a dramatic increase in luciferin concentration, to an average of 1.5 pmole luciferinimg dry wt (Fig. 5). Development

in the absence of larval light organs.

Assays on insects developed from larvae with excised larval light organs confirmed that an absolute increase in the levels of both luciferase and luciferin occurs in both anterior and posterior halves, throughout pupation, rather than a possible re-distribution by release from degenerating larval light organs. The concentration of luciferase in the posterior halves of pupae without larval light organs shows a continual increase during pupation. A significant but lower concentration of luciferase was measured in the

x P

/

-5

SI

SII Stage,

SIII

SIP

Adult

(no LLO)

Fig. 6. Luciferaae concentrations (mean & S.D.) from anterior (0) and pobtcrior ( x ) hul\c\ insects v.ith larval light organs cxiscd. (A = antcwx. P = pohtcrior)

ofdc\clopins

Biochemical

and morphological

_orvoi

su

Sl

Stage, Fig. 7. Luciferin

in Phoruris penns_vlvunica

changes

SItI

SIX

345

Adult

(no LLO)

concentrations (mean rf: S.D.) from anterior (0) and posterior ( x ) halves of developing insects with larval light organs excised. (A = anterior. P = posterior).

anterior halves (Fig. 6). A maximum luciferase concentration in the anterior half was reached at an earlier stage of development, (SI), in insects reared without larval light organs than in normal developing insects, (SII). This corresponds with the appearance of the adult light organ in intact insects without larval light organs as early as SII, as compared to SIII in normal developing insects. The concentration of luciferin in pupae developed from larvae with ablated light organs also shows a significant increase in both anterior and posterior halves. The average concentration of luciferin was approximately equal in both halves, 0.36 x 10e3 pmole/mg dry wt. and remained constant throughout pupation (Fig. 7). The average concentration in the anterior halves of insects reared without larval light organs was comparable to that found for normally developing insects. Total luciferase and lucifirin concentrations. The relationship between luciferase and luciferin concentrations was expressed as a luciferin/luciferase ratio. During larval and pupal stages the ratio was consistently less than 1, an average of0.33 in the larval

corresponding

IO

stage and between 0.28 and 0.52 in the pupal stages. Luciferin is the rate-limiting compound in the light reaction during these stages. In the mature adult, however, the ratio of luciferin to luciferase was much greater than 1, averaging 9.9 (Fig. 8). Thus luciferin is in excess in the mature adult and luciferase is ratelimiting in the light reaction. There is a significant difference in the luciferin and luciferase concentrations, in the anterior as compared to posterior halves, of all developmental stages (Student’s f-test, P~0.01) (Table 1). The concentrations of luciferin and luciferase in the posterior portions of mature larvae are also shown to be significantly different from the corresponding concentrations in the mature adult (Student’s t-test, P
r i

7

0' LOWI

Fig. R. Changes

in luciferiniluciferase

T

SI

I

-x-x Stl

SIU

Stage ratios of whole insects during

SIP

normal

I Adult

development.

L. G.

346

STRAUSE,M.

DELUCAAND J. F. CAKE

Table 1. Changes in luciferin and luciferase concentrations

during light organ development in the firefly Photuris

pennsylvanica

Average dry Stage

,m”t

Larval ( 1O)t Anterior Posterior

20

SI (8) Anterior Posterior

23

SII (8) Anterior Posterior

24

SIII (7) Anterior Posterior

19

SIV (7) Anterior Posterior

20

Adult (9) Anterior Posterior

10

Luciferin* LH, +_ S.D. 0 4.6 2 0.002

0 11.3 f 0.006

0.2 f 0.0025 3.6 + 0.002

0.8 + 0.001 I I .9 * 0.007

0.25 k 0.0002 4.3 + 0.002

I.0 + 0.002 11.8 + 0.005

0.14 * 0.0001 9.1 + 0.007

0.28 k 0.0004 13.9 + 0.009

0.2 + 0.0002 4.7 + 0.003

0.32 + 0.0002 10.7 + 0.006

0 1500 f 100

P

Luciferase* Lase + S.D.

Lase:

LHZ

Lase

0.09

0.2

0.001

0.001

0.09

0.3

0.001

0.001

0.1

0.3

0.001

0.001

0.18

0.27

0.01

0.01

0. I

0.22

0.002

0.001

0. I

0.22

0.002

0.001

4.3 + 0.003 90 * 30

*pmole/mg dry wt x 10m3. t No. animals tested. t pmole/insect. Sl-SIV designates pupal stages as described in text. especially the nervous system, which, together with the simultaneous regression of the larval light organs and assembly of the adult light organs, presents an initially obscure picture. Fortunately one conspicuous uniformity is seen through development at the biochemical level where the light-producing reactions remain essentially the same. We have, therefore, started investigation of the developmental physiology of the luminescent system of the firefly with a correlation of changes in the levels of luciferin and luciferase with gross developmental changes. The lantern of the newly emerged adult contains 15 pmole of luciferin and 1 pmole of luciferase, based on an average adult dry wt of 10 mg (Table 1). Assuming a quantum efficiency for luciferin of 0.88, this is enough for 10,000 flashes [lOI photons/flash (SELIGER, personal communication)]. An adult male P. pennsylvanica can be estimated to deliver about 10,000 flashes in its lifetime (assuming 14 day life, 3 hr of patrolling per night). Thus there appears to be no necessity for synthesis of luciferin during the adult stage, assuming that it is not utilized except for bioluminescent displays. Direct biochemical evidence on the matter of biosynthesis of luciferin in the adult stage is not available, although OKADA et al. (1974) postulate the possible in vivo conversion of oxyluciferin to luciferin in adult fireflies and MCCAPRA and RAZAVI (1976) report incorporation of DL-cystine into luciferin in the luminescent click beetle, Pyrophorus

pellucens.

In contrast, the larva has a very small reserve of luciferin and luciferase. Posterior halves of larvae weighing an average of 20 mg dry wt, contains only 9 x 10e2

pmole of luciferin and 0.2 pmole of luciferase. There must, therefore, be extensive synthesis of luciferin and luciferase during metamorphosis. During this period of active synthesis, spontaneous glows are emitted by both larval and early adult light organs against a background of continuous, low level glowing from the whole pupa. Since the pupal glow persists after excision of the larval light organs it cannot be caused by dispersal of the contents of the larval light organ and implies widespread synthesis of luciferin and luciferase throughout the pupa. To test for developmental interactions between the light organs, the larval light organs were removed prior to pupation. Our results show that the adult light organs develop without the larval light organs, as originally reported by HARVEY and HALL (1929), but suggest that the larval organs may exert an inhibitory influence on the development of the adult light organs, as implied by the earlier luciferase peak and the earlier appearance of the adult light organ in insects developed without larval light organs (Fig. 6). Obvious adult light organ functional capacity is seen 7-10 days after pupal ecdysis as evidenced by changes in the luminescence pattern and intensity in pupae developed from larvae with excised light organs (Fig. 2d). Concomitantly, luciferin and luciferase begin to decrease in the anterior half of the insect. This may possibly be a result of either a localization of differentiating luminous cells, in the region of the 6th and 7th abdominal segments, or termination of synthesis in cells not destined to become photocytes. The results, in either case, would be a concentration of luciferin and luciferase in the region of the adult

Biochemical

and morphological

lantern. The source of photocytes has not been the subject of a modern study. HESS (1922) suggests they are derived from the fat body. The longer latency, (600-800 msec) and duration (6-10 set) of the larval (OERTEL and CASE, 1976) and early adult (STRAUSE and CASE, in preparation) light organ luminescence, ascompared to the adult flash (60 msec latency and 100 msec duration; BUCK et al., 1963). may be accounted for by the kinetics of the photogenic reaction. Stop-flow studies (DELUCA and MCELROY, 1974) show that a 60-msec latency to peak emission is observed when oxygen is added to the already anaerobically formed active intermediate’ luciferyl adenylate. However, a latency of 300-500 msec is observed when luciferin, luciferase and the required cofactors are combined simultaneously. Light emission is inhibited once luciferase is bound by the product. oxyluciferin (DELUCA and MCELROY, 1974; DELUCA, 1976). The adult light organ may not be capable of emitting an adult type flash, until all the luciferase is in a bound state and consequently inhibited by the enzyme-product complex. Once this ‘saturated state’ is reached, neural control might involve disinhibition of the product complex, resulting in a flash (CASE and STRAUSE, 1978). Our findings that the ratio of the concentration of luciferin to the concentration of luciferase changes from much less than one in the larval and pupal stages to much greater than one in the mature adult provides a biochemical basis for this hypothesis (Fig. 8). Only in the adult would it be possible to completely saturate all the enzyme with the substrate. Morphological changes in light organ structure and innervation parallel these changes in the biochemical state. The concept of an altered biochemical state. responsible for the transition from the glow seen in larval and pupal stages to the adult flash, is correlated in the larva with lack of a tracheal end-organ and of synapses directly on the photocyte membrane (OERTEL er al., 1975). In contrast the adults, with a highly organized tracheal system (SMITH, 1963), have synapses on the tracheolar cell (CASE and LINBERG, in preparation). Thus, in the larva direct excitation of the photocytes may lead to the activation of luciferin, luciferase. and cofactors producing the long latency and duration of the larval and pupal glow. A peripheral excitation-sequence involving: (1) the trigdering of nerve terminals within the tracheal endorgan; (2) transmission to the photocyte membrane (by a mechanism still unknown); and (3) activation of the luciferyl adenylate intermediate would result in the rapid adult flash. This mechanism would appear to require more precise regulation of oxygen admission to the reaction-site, since luciferyl adenylate requires only oxygen to emit light (DELUCA, 1976), which might be the physiological explanation of the elaborate tracheal end-organ structure and indirect innervation seen in the adult.

changes

in Photuris pennsylvanica

347

firefly larvae. Supported

by NSF Grant BNS 76-80246. the UCSB Quantum Institute and Faculty Research Funds of the University of California.

REFERENCES

BUCKJ. and CAKEJ. F. (1961) Control of flashing in tirellies-em I. The lantern as a neuroeffector organ. Eiol Bull mar biol Lab.. Woods Hole 121, 234-256. BUCK J.. CASE J. F. and HANSON F. E. 41963) Control of flashing in fireflies--III. Peripheral excitation. Viol Bull mar. biol. Lab Woods Hole 125, 251-269. BUCK J.. CA.SE J. F. and HANSON F. E. (1965) Control of flashing in fireflies. Proc X1111/1 Inrt,rn (‘oryr Entomolog_v p. 226. CARLSON A. D. (1961 j Effects of neural activity on the fire& pseudoflash. Biol Bull. mar. hiol. Lob Wk~ds Hole 12i. 265-276. CASE J. F. and BUCK K. (1963) Control of flashing in fireflies-II. Role of central nervous system. Bwl Bull mar. biol. Lab Wbods Hole 125, 234-250. CASE J. F. and STRAUSE L. G. (1978) Neurally controlled luminescent systems. In Bioluminescence in At,tion (Ed. by HERRING P.). Chap. 10. Academic Press. London. (in press). DELUCA M. ( 1976) Firefly luciferase. .4(/l, En:w Rel Areas Molec Biol 44, 37-68. DELUCA M. and MCELROY W. D. (1974) Kinetics of the firefly luciferase catalyzed reactions. Biochemisrn, 13, 921-925. GREEN A. and MCELROY W. D. (1956) Crystalline tirefly luciferase. Biochim. biophys. Acra 20, 17&l 76. HARVEY E. N. M and HALL R. T. (1929) Will the adult firefly luminesce if its larval organs are entirely removed? Scienw Wash LXIX ,- ‘53-254. HESS W. N. (1922) Origin and development of the light organs of Phoruris penns_vlwmica De Geer. .J. Morphol. 36, 245-263. MCCAPRA F. and RAZAVI J. (1976) Biosynthesis of luciferin in Pyrophorus pellucens J them. Sot Chem Comnw~ 5, I531 54. MCELROY W. D. and DELUCA M. (1973) Chemical and enzymatic mechanisms of firefly lummescencr. In Chemiluminescence and Bioluminescenc~e (Ed. .by CORMIER M. J.. HERCULES D. M. and LEE J.). pp. 285-309. Plenum Press. New York. MCLEAU M.. BUCK J. and HANSON F. E. (1972) Culture and larval behaviour of photurid fireflles. Am. Midland Narurulist 87, 133-145. OERTEL D. and CASE J. F. (1976) Neural excitation of the larval firefly photocyte: slow depolarization possibly mediated by a cyclic nucleotide. J. e.rp Biol. 65, 2 I 3-227. OERTEL D.. LINBERG K. A. and CASE J. F. (1975) Ultrastructure of the larval firefIy light organ as related to control of light emission. Cell Tiss. Res 164, 3744. OKADA K.. IJO H.. KUBUTA I. and Gore T. (1974) Frretly bioluminescence-III. Conversion of oxylucifertn to luciferin in firefly. Tetrahedron Levr 32, 2771-2774. SETOS.. OGURA K. and NISHIYAMA Y. (1963) A convenient synthetic method of 2-carbamoyl-6-methoxybenthlazole. dne of the intermediates for -the synthesis of firefly luciferin. Bull Chem !k Jao. 36, 331-333. SMITH D. (1963) Organizatidn and innervation of the luminescent organ in a firefly, Phoruris penns~~lvunica J Cell Biol 16, 323-359. ,4~~~no~~~/t,d~e,?lml_We are grateful to Dr. FRANK WILLIAMS F. X. (I 9 16) Photogenic organs and embryology of lampyrids. J Morph 28. 145-207. HA\\FOU and CAROLYN CFARL.EYfor assistance in collecting