Photochemisrw nnd Photohioloyy, 1976, Vol. 24, pp. 279-285. Pergamon Prcss Printed In Great Britain

BIOLUMINESCENCE OF THE AUSTRALIAN

HARRISON GLO W-W ORM, ARAC H N OCAMPA R I C H A R DSAE

JOHN LEE Bioluminescence Laboratory, Department of Biochemistry, University of Georgia,

Athens, GA 30602, U.S.A.

(Received 13 January 1976; acceptvd 19 March 1976)

Abstract--Stable dry powders of the light organ of the glow-worm, Aruchnocampa richardsae (Diptera) have been prepared and although the amounts of material are extremely limited, a semi-quantitative description of the bioluminescence reaction can be given. The in viuo and in v i m bioluminescence spectra are the same, maximum 488nm (corrected) and the shape of the in vitro emission spectrum is not influenced by pH (5.9-8.5). Light is produced by addition of buffer (optimum pH 7) to the powder, but is stimulated several fold if the buffer contains adenosine 5’-triphosphate (ATP), about 0.3 mM being required for half maximum stimulation. A number of other high-energy phosphates do not stimulate. Ethylenediaminctetraacetate quenches the ATP stimulation implicating a Mg2 + re- quirement, but not ethylene-bis(oxyethy1ene-nitri10)-tetraacetate which chelates C d 2 + but not Mg2 +. Inorganic pyrophosphate (2 mM) also quenches if added with the ATP but in contrast to the firefly (Coleoptera) reaction, does not stimulate the light emission if added post-ATP. The decay of the ATP-stimulated glow-worm bioluminescence is first-order unlike the strongly product inhibited firefly kinetics. Oxygen is required for both in uiuo and in vitro bioluminescence. A glow-worm can emit more than 1015 photons in its lifetime but at any one time, appears to possess only about 10% of this total capacity

INTRODUCTION

Among the insects the only orders that have been established to have bioluminescent members are the Coleoptera (beetles), Diptera (flies), Collembola and Hemiptera (Harvey, 1952; McElroy et al., 1974). The bioluminescence system of the firefly (Coleoptera) has received detailed biochemical study, greatly aided by the ready availability of material. Besides the Coleop- tera, there are no biochemical descriptions of the bio- luminescence systems of the other orders of insects, except for a short report on the New Zealand glow- worm Arachnocarnpu luminosa (Diptera) by Shimo- mura et al. (1966).

The Dipteran glow-worm is the larva of a fungus gnat (Mycetophilidae) and occurs in fair abundance only in Australia and New Zealand. A number of biological studies of this insect have been reported (Harvey, 1952; CSIRO, 1970) and the more recent ones include studies of anatomy, behavior and tax- onomy (Richards, 1960; Gatenby, 1960; Harrison, 1966). There is a popular description by Richards (1964). Glow-worms inhabit wet caves and often infest abandoned railway or mining tunnels. They are also to be found in rock crevices which are damp and well-shaded but collection of large numbers of speci- mens from such areas is impractical. The light is emitted from a whitish area associated with the excre- tory organ, occurring at the end of the darkly pig- mented larva. The size of specimens ranges 3-40mm long and it sits in the middle of a snare it constructs, using the bioluminescence to lure small insects into

the trap (Richards, 1960). Several species within thc genus Aruchnocampa have now been established, among them A. luminosu which is found only in New Zealand, A . tasmaniensis in Tasmania, Australia and A. richardsae from further north in New South Wales. Australia (Richards, 1960; Gatenby, 1960; Harrison, 1966).

Since bioluminescence is a specific and easily measured reaction, studies of quite crude material can yield useful information about the biochemical mechanism. Thus using powdered whole insect mater- ial from several thousand specimens of A. Ittminosa, Shimomura et al. (1966) showed that, among a number of possible cofactors tested, only adenosine 5’-triphosphate (ATP) and adenosine 5’-diphosphate (ADP) were effective in stimulating the luminescence obtained on adding buffer to the powder. The reac- tion was also found to be inhibited by pyrophosphate and these observations are strongly reminiscent of the properties of the firefly reaction (McElroy and Seliger, 1961).

In view of the analogy between the biochemical mechanisms of the classes of coelenterates and the strong similarity in chemical reaction mechanism of many bioluminescence systems (Cormier et ul., 1975), it would not be surprising if the bioluminescence mechanisms in insects possessed common features. The purpose of this work is to confirm and extend the observations of Shimomura et al. (1966) to another species of glow-worm and to compare and contrast the properties of the glow-worm and firefly bioluminescence.

219

280 JOHN LEE

MATERIALS AND METHODS

Specimens were collected from an abandoned railway tunnel near Newnes, New South Wales, Australia. About 200 could be collected in one day and a total of 600 were used for the present experiments. Individuals were picked out of their snare and placed in a 2dram glass vial stop- pered with moist cotton and maintained at 5-10°C until returned to the laboratory. Within about 6 h of collection they were rapidly frozen in liquid N,, then freeze-dried for 2 days. The light organ was then cut off from the remainder of the body, coarsely ground (IOpg) and stored desiccated at -5°C. The bioluminescence activity of these preparations remains stable for several months and even survives intercontinental mailing. Fragility of the dried in- sect prcvented clean separation of the light organ so that the powder was contaminated about 50% by the body material. The total dry weight of material was about 150 mg.

Total bioluminescence emission was measured by pho- tometers constructed here and calibrated for absolute pho- ton response with the luminol chemiluminescence standard (Lee c't ul., 1966). This standard is traceable to the National Bureau of Standards standard lamp (Lee and Seliger, 1965).

Emission spectra were taken in uiuo on site with a Fastic -Ebert 1/4 meter, j73 spectrometer and in uitro with either a Bausch & Lomb 1/2m, ,f/4 computer controlled monochromator (Wampler and DeSa, 1970) or a Bausch & Lomb 1/4 m high intensity monochromator. All spectra are corrected by reference to the National Bureau of Standards standard of spectral radiance.

All chemicals used were the best commcrcial grades mostly from Sigma Chemical Co., St. Louis, MO and Cal- biochem, La Jolla, CA.

RESULTS

The population at the collecting site was counted several times in the period 1971-76, and varied in the range 10,00&20,000. Samples of 1000-2000/yr would therefore not he expected to disturb this popu- lation. Within the snare at least 95% were emitting light estimated in the range 10'o-lO1l quanta s - ' . Loud noise o r bright light did not affect any change in emission rate. The New Zealand species are also insensitive to noise and light except for the popula- tion in the Waitomo Caves (Richards, 1960, 1964). On returning the collection to the laboratory only 10% would be still emitting visibly (> 10'' quanta s '), the rest having turned down to about lo7 quanta s- ' . They did not restore their light even several weeks after having rebuilt their snare in the collection vial, which they commence doing as soon as they are removed from their snare, and which takes only a few hours.

Light emission in vivo. Three individuals that were emitting at a rate over 10" quanta s- ' were selected for long-term study. Their sizes were 3, 10 and 15 mm representative of the range in this population. No diurnal variation in light level was evident, not too surprising for a species living in continuous darkness. The level varied daily, however, from below lo3 quanta s- ' (undetectable) to 1OI2 quanta s-'. There was no quantitative effect of external light on the level but a slight vibration outside the photometer could often elicit a rapid increase, falling back to a

Figure 1. Bioluminescence response of a glow-worm, Arachnorampu richurdsur, to exposure at time zero to a

vapor of ether (20°C).

lower level over a 10-min period. This could be repeated several times until exhaustion. The insect would recover this response again after several days. A dramatic increase also occurred if they were allowed to warm (35"C), but they also expired after several hours.

Evacuation of air above the specimen dimmed the light after several min and readmission of air caused a more intense glow which then recovered to the ori- ginal level. An oxygen requirement can also be demonstrated in the in uitro bioluminescence (vide in-

All viable (moving) specimens, regardless of whether they were giving light or not, responded to a vapor of ether in the manner shown in Fig. 1. A lag of several min is followed by a rapid biolumines- cence increase often to multiple maxima, then an ap- proximately logarithmic decay of half-life about 20 s. Less total light was stimulated by chloroform and C 0 2 but methanol and ethyl acetate were ineffective. The latter causes the firefly to glow continuously.

If an ATP solution is added to a freshly excised crushed light organ or to the lyophilized material, an "in uitro" bioluminescence is produced. Table 1

f r 4 .

Table 1. Total bioluminescence emission per individual glow-worm*

Average Number of total light Range

Procedure specimens (10'' hv) (10" hv)

I n uivot 3 400 270-500 Warm to 35°C 5 30 2-70 Ether 25 17 0.7-140 Light organ in uitro$ 10 13 0.2-22 Lyophilized powderf 200 3 __

*Measured at room temperature. ?Measured over 3 weeks. $With ATP (10 mM) in phosphate buffer (0.05 M, PH 7).

Bioluminescence of the glow-worm 281

I I

600

WAVELENGTH (nm)

Figiirc 2. Bioluminesccnce spectra (corrected) from the glow-worm i r i vioo (-) and in uitro (----) from the lyo- pliilixd light organ with ATP (10 mM, pH 7). Slit widths

5 nm.

compares the average total light obtained on a per specimen basis, for each of five procedures-con- tinuous in uiuo, warming it to 35°C until it expires, ether stimulation, ATP addition to crushed light organ or to the lyophilized powder. The total in vim bioluminescence did not depend on the size of the specimen. If it can be considered that each glow- worm should be capable of producing at least twice as much light as actually measured over the 3-week period, it is clear that this is in considerable excess over that which can be elicited at any one time by o n e of the other techniques.

The lyophilization procedure successfully preserves a t least 20% of the activity as measured for instance by ether stimulation. Less than 1% of this light organ activity is Found in a powder of the remainder of the larva.

Biolurninescmce spectra. Figure 2 shows the in uivo (full line) and in uitro (dash line) bioluminescence spectra. The in uivo spectra were taken from unstimu- lated specimens at the collection site and were unaf- fected by change in temperature from ambient (8OC) to 30°C. The in vitro spectra were taken from the lyophilized powder to which was added ATP (10 mM) in glycylglycine (25 mM, pH 7) (much of the firefly study has been done in glycylglycine but usually a t

- 1

I ATP

I Li lb ;@ 3b 40 50 60

TIME ( 5 )

Figure 3. I n umo bioluminescence from lyophilized powder on adding buffer (glycylglycine 25 mM, pH 7) followed by

ATP (10 mM in buffer) (23°C).

Table 2. Cofactor requirement for glow-worm bioluminescence

Specific light Addition* yield (lo* hvlpg)

Buffer Adenosine triphosphate Adenosine triphosphatet Adenosine diphosphate Cytidine triphosphate Uridine triphosphate Guanosine triphosphate Creatine phosphate? Arginine phosphate

12 140 150 20 25 47 30 24 12

* 10 mM in buffer a t 23"C, pH 7. All in glycylgly- cine (25 mM) except +phosphate (50 mM).

pH 7.7). Pigmentation from contaminating body parts requires a correction for self-absorption to be applied to the short wavelength end of the in vitro spectrum and this is difficult to do precisely.' Therefore the slight discrepancy between the two spectra in this region is not regarded as significant.

Shimomura et al. (1966) reported 487nm for the maximum of emission from A. lurninosa but their spectra are uncorrected, and so it is uncertain whether this value differs from the present case.

There is no change in the in uitro spectrum on changing the pH to 5.9, 7.9 or 8.5.

The light reaction. The addition of buffer alone to the lyophilized powder produces a flash of light. Figure 3 shows that if ATP is added subsequent to the buffer the light is increased several-fold and this will be referred to as 'bioluminescence enhancement'. The specific light yield (total quanta per pg) is about ten times more with ATP in the initial buffer than without (Table 2). No bioluminescence enhancement occurs if the ATP addition is made after the buffer induced emission has ceased. There are no soluble inhibitors in the spent solution since it may be added to a fresh powder to produce the same light yield. No additional light is obtained on adding firefly D-luciferin to the spent reaction, not does it stimulate when added during the bioluminescence. In contrast to the firefly reaction, the post-addition of inorganic pyrophosphate or CoA causes no bioluminescence en- hancement (McElroy, 1951 : Airth et d., 1958).

The light yield is strongly reduced if the initiating buffer contains 0.5 M ammonium sulfate or ethylene- diaminetetraacetate (EDTA). A soluble luciferin is not stabilized by these solutions since an aliquot diluted 100 times into ATP produces no light. No luciferin could be extracted by methanol or dimethylsulfoxide. There was no evidence for a luciferin-luciferase reac- tion by hot water-cold water extracts. Shimomura et al. (1966) also failed to solubilize luciferin in the New Zealand species. Sodium dodecyl sulfate (1%). manni- to1 (2 M ) and 2-mercaptoethanol (0.1 M ) were without effect on the light reaction.

282 JOHN LEE

- I

I

(ATP C O N C E N T R A T I O N ~ ' ( ~ M Y '

0 5

Figure 4. Reciprocal plots for ATP concentration against activity as assayed by specific light yield (ATP in initiating buffer) or bioluminescence enhancement (buffer, then ATP when [I/(, = 0.1). is the intensity with ATP. Buffer

50 mM phosphatc. pH 7 , 23°C.

Loss of activity accompanies fine grinding of the powdcr as also observed by Shimomura rt al. (1966) but a buffer volume dependence of the light yield reported by them is not confirmed in this case. All these reactions were made with around 0.1 ml of solu- tion added to a 5&250 pg weighed portion of lyophi- lized powder. The specific light yields (per microgram) showed considerable scatter resulting in a coefficient of variation of 50% (4-8 determinations). The preci- sion was not improved by taking larger samples. Nevertheless these data allow conclusions of a semi- quantitative nature to be made.

Table 2 compares the specific light yields when the initiating buffer contains a 10 mM concentration of a number of nucleotides and high energy phos- phagens. Besides ATP, only uridine triphosphate shows stimulation significantly above the buffer alone. In a crude firefly extract, myokinases are known to be responsible for the slight activity of ADP and othcr nucleotides and this would be a likely explana- tion hcrc too. Also the lack of effect of creatine and arginine phosphate along with other cofactors like riboflavin 5'-phosphate, nicotine adenine dinucleotide and H 2 0 , tested by Shimomura et al. (1966) makes it likely that ATP itself is the specific cofactor for the glow-worm bioluminescence. Phosphate ion at 50 mM concentration shows no quenching of the light over glycylglycine, whereas a three-fold quenching is seen in the firefly reaction (Green and McElroy, 1956).

In Fig. 4 a reciprocal plot is shown of ATP concen- tration against bioluminescence activity, either by a specific light yield measurement (filled circles) or bio- luminescence enhancement (open circles). The latter is determined by adding ATP when the light intensity I has fallen to about 10% of its maximum level lo with buffer alone (Fig. 3). Though scattered both sets of data fall on the same line yielding a half-saturation value for ATP of 0.3mM. This would be an upper limit of the Michaelis-Menten constant for ATP if it is involved in the light reaction. It is about five times the half-saturation for ATP in the crude firefly system (McElroy and Strehler, 1949). At 10 mM ATP

both the bioluminescence enhancement and the speci- fic light yield are approximately doubled in the pres- ence of Mg2+ (10 mM). Higher concentration of ATP inhibits the light slightly as is also observed for the firefly system (McElroy ef ul., 1953).

Supporting evidence for a metal cofactor require- ment is the quenching of bioluminescence enhance- ment by EDTA (Fig. 5). To the buffer initiated reac- tion is added ATP (10 mM) in a solution containing various concentrations of EDTA. The biolumines- cence enhancement in the absence of EDTA. E = IATl,/I and in its presence, E, I)TA = I,,.,, (EDTA)/I are seen to be related by a regular quenching equation, E / E , , , , = I + KCEDTA] (Fig. 5 , open circles). The filled circle is with ethylene-bis(oxyethylenenitri1o)- tetraacetate (EGTA) which chelates Mg'+ weakly but binds Ca2+ as strongly as does EDTA (Portzehl et a/., 1964). The single point is enough to demonstrate that EGTA has little effect and may not be different from its general salt effect, which would be more prominent at higher concentration.

The specific light yield has an optimum around p H 7 about 0.5 units more acid than firefly (Green and McElroy, 1956). Heat inactivates the lyophilized powder completely in less than 1 min at 80°C and by 50% in about 1Omin at 50°C.

Pyrophosphute quenching. Figure 6 shows the reduc- tion in specific light yield (open and filled circles) if inorganic pyrophosphate is included in the initiating buffer along with ATP. For ATP 10 mM hall quench- ing occurs at 2.5 mM pyrophosphate but this is ATP concentration dependent, requiring 5 mM pyrophos- phate when ATP is 1 mM. There is no significant effect of pyrophosphate on the bioluminescence en- hancement (triangles) where ATP (10 mM) and pyro- phosphate are not added until after the buffer. Shimo- mura et al. (1966) also noted a strong effect of pyro- phosphate at 1 mM. For the firefly reaction, 20pM pyrophosphate added prior to the ATP reduces the light yield by one-half (McElroy et ul., 1953).

CONCENTRATION OF EDTA (rn/M)

Figure 5. Quenching of bioluminescence enhancement by EDTA (0) or EGTA (0). E / E , ,,, , is the enhancement ratio

without to with chelator.

Bioluminescence of the glow-worm 283

\ 46 m

2 4 6 8 1 0

PYROPHOSPHATE CONCENTRATION (mM)

Figure 6. Effect of inorganic pyrophosphate on specific light yield for ATP lOmM (0); or 1 mM (0). There is no effect of pyrophosphate on bioluminescence enhance-

ment (ATP lOmM (A)).

Kinetics o j the light reaction. Figure 7 shows that on addition of ATP (10 p M ) to the lyophilized pow- dered light organ, the bioluminescence intensity rapidly rises to a maximum value and then falls in a manner which can be described as the sum of two first-order rate processes, k l and k2, At 23°C and pH 7 (glycylglycine, 25 mM) these rates are t, = 8 s, k , = 0.08 s - ', k 2 = 0.02 s - ' (coefficient of variation 50%) and are not affected by the concentration of ATP. There is a slight effect of quenchers in slowing all rates (phosphate 200mM, EDTA 20mM and pyrophosphate 4 mM). There is a pH effect increasing k , on the basic side and increasing the contribution of k? on the acid side.

.~

TIME (sl

Figure 7. Addition of ATP (10mM) in glycylglycine (25 mM, 23"C, pH 7) to lyophilized powder of glow-worm

light organ. Dark current has been subtracted.

17 m

- 1

320 340 360 (TEMPERATURE )F' 1 0 ~ 1 ~ (K-?

Figure 8. Arrhenius plots of the glow-worm biolumines- cence kinetics; t,, rise-time (0); k l , k, , decay rate constants

(0, 0); initial intensity, I,, (A).

Lowering the temperature slows all rates and in- creases the contribution of the slower decay modc so that.it contains 25% of the total decay pathway at 0°C. Arrhenius plots of the rates and of 1,) are shown in Fig. 8. All rates have the same activation energy of 46kJ mol-', but for I,, it is 71 kJ mol-' due to the larger contribution of the slow decay mode at low temperature. A comparable activation energy

i AT 0 2

5 0 I00 300

TIME ( 5 )

Figure 9. Glow-worm bioluminescence in uitro, left-hand portion, on addition of buffer (phosphate SO mM, pH 7, 23°C) at low oxygen concentration, followed at 300s by

air-saturated ATP (10 mM).

284 JOHN LEE

of 77 kJ mol- is observed for I,, in the firefly bio- luminescence (McElroy and Strehler, 1949). The speci- fic light yield is unaffected by temperature up to 30"C, but decreases above this.

Oxygen requirement. The specific light yield is strongly depressed in the absence of oxygen. Figure 9 shows a typical reaction where the buffer is first deoxygenatcd by bubbling nitrogen for 15 min, then added to the lyophilized powder under nitrogen. Some light is emitted but its yield is down about ten times from that obtained with air-saturated buffer. The rise-time is also slowed. If ATP (10 mM) is added within 5 min another flash of light occurs with about the same total light yield as the first. The rate of rise is much faster than for the normal air-saturated assay and the decay is more complex usually, with the slower component making a more substantial contribution. After about 5 min under nitrogen the light yield on addition of ATP diminishes rapidly.

DISCUSSlON

The firefly reaction is known to proceed by acti- vation of firefly luciferin [LH, ; D( -)-2-(6'-hydroxy- 2'-benzothiazolyl)-A'-thiazoline-4-carboxylic acid] on firefly luciferase ( E ) by ATP and Mg2+, followed by oxidation to give light (McElroy and Seliger, 1961; McElroy and-DeLu& 1973; McElroy et

Mg2+ E + LH2 + ATP-E-LHZAMP

I O2

E-LO + AMP 4 C02 +

al., 1974)

+ PP

hv

Inorganic pyrophosphate reverses the equilibrium with the luciferyl adenylate (E-LH,AMP) and there- fore inhibits if added prior to the ATP. Reaction with molecular oxygen decarboxylates this compound to oxyluciferin (LO) and light is emitted in the process. When ATP is added to thc firefly system the bio- luminescence rises rapidly (0.2s) and then falls in a doubly exponential (under some conditions) manner ( k , = 3 s-', k , = 0.05 s-'), in which the slower decay is by far the major pathway. This is explained by inhibition of the luciferase by dehydroluciferin, a by-product of firefly luciferin oxidation, and can be relieved by post addition of pyrophosphate or CoA which produce a bioluminescence enhancement by displacing the inhibitor and making more free enzyme available for catalysis. Stimulation by increase in pyrophosphate concentration has been suggested as the mechanism by which the firefly controls its flash (McElroy and Hastings, 1955).

An ATP requirement for the glow-worm biolumi- nescence seems likely based on the present results and those of Shimomura et al. (1966). There is probably ATP in the crude powder itself, sufficient to initiate

the bioluminescence on addition of buffer alone. However some caution must be added before con- cluding that ATP is involved in the same way as in the firefly case. Crude extracts of the bioluminescent coelenterate, Renilla renijormis, produced light on addition of ATP (Cormier, 1959) due both to a con- tamination by the genuine cofactor adenosine 3',5'-diphosphate and to a coupling reaction which also produced it. Although this cofactor has bccn found ineffective for the glow-worm reaction (Shimo- mura et al., 1966) the possibility of other contami- nants or of coupling reactions must not bc cxcluded. In conjunction with the ATP requirement, EDTA quenches the reaction by removing Mgz+. but not EGTA which only removes Ca2+. The I<, for ATP in the glow-worm reaction is only about five-fold greater than for the firefly and would probably de- crease to a similar value on purification of the bio- luminescence system.

The firefly flash in uitro is inhibited by a factor of about two by addition of 20pM pyrophosphate prior to the ATP (McElroy et a!., 1953) but the glow- worm requires about 2 m M (Fig. 6). If prior incuba- tion were possible in the latter perhaps inhibition would be more effective. Interpretation of pyrophos- phate quenching is complicated by its ATP concen- tration dependence (Fig. 6). Taken as a whole the results are consistent with an ATP activation in glow- worm bioluminescence like that in the firefly.

Oxygen is clearly a requirement for both in uii:o and in uitro glow-worm bioluminescence and the oxygen reaction appears to be rate-limiting. In the absence of oxygen the activity is stabilized in solution suggesting that glow-worm luciferin is subject to rapid competitive autooxidation in free solution. If its adenylate were not bound as tightly to glow-worm luciferase as for the firefly case, higher concentrations of pyrophosphate would be required for quenching, as is observed. Separation of luciferin from luciferase in this case will require vacuum manipulation tech- niques.

There are important points of difference in the con- trol of bioluminescence and the emission spectra between the firefly and glow-worm bioluminescence systems. The glow-worm glows continuously and does not flash like the firefly. It does control its biolumi- nescence, since it can change the level of intensity dramatically over several min, and under ether anaes- thetization discharges its bioluminescence completely. But in uitro the product inhibition-like kinetics are not exhibited and there is no pyrophosphate stimu- lation. It would not need such a rapid control mechanism.

The second difference is in the emission wavelength maximum of 488nm, far to the blue of the shortest observed firefly emission, 547 nm (McElroy at al., 1974). Fireflies have a species-dependent maximum ranging from 547-594 nm and since the same luciferin can be utilized, this shift is attributed to a perturba- tion of thc cxcitcd statc cncrgy lcvcl by thc environ-

Bioluminescence of the glow-worm 285

ment of the active site o n the luciferase. A shift to 488nm would be a rather large perturbation to be produced by an environmental effect, though not out of the question. More likely, and since firefly luciferin does not cross-react, glow-worm luciferin has a differ- ent chemical structure, but the change would not have to be very great. For instance if the ionization of the 6-hydroxyl is prevented firefly luciferin fluoresces in the blue (McElroy et a/., 1974).

The final point is the lack of pH or temperature effect on the glow-worm bioluminescence spectrum. At higher temperature or acid pH, the firefly produces a red emission at 617 nm, interpreted as an acid--base tautomeric shift of the oxyluciferin excited state. No such effect is to be seen in the glow-worm case which again suggests a different chemical structure for its oxyluciferin.

In conclusion these preliminary results show that the bioluminescence systems of the firefly and the

glow-worm differ in many respects. Perhaps the differ- ences will not turn out to be major-for instance the differences in the properties of the bioluminescence spectra could arise from only a slight change in sub- stitution in the luciferin chemical structure. The glow- worm bioluminescence also appears to be ATP-acti- vated, but until some partial purification of the sys- tem can be achieved, this is not proven.

Acknowledgements-I thank W. Kilkeary for assistance col- lecting glow-worms, the Lands Department of New South Wales for permission to collect, H. H. Seliger for the loan of a portable spectrometer, A. M. Richards and M. DeLuca for their comments on the manuscript. and J. H. Green and members of the staff of Macquarie University, Sydney, for their hospitality during the course of these experiments. This work was supported by grants from the National In- stitutes of Health (GM 19163) and National Science Foun- dation (BMS 74-19890).

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