E. Newton Harvey

“ Interest in the light of animals from a physical standpoint has centred around questions of quality, efficiency and intensity, but in only one group of luminous animals, the beetles, have accurate measurements of these characteristics been made. This is due in part to the abundance of these forms and their appeal to human interest and in part because they are among the brightest of luminous organisms. Weak lights are not only difficult to measure but, when dispersed to form spectra, give bands so faint that their limits are very difficult to see and more so to photograph. ”

“ It should be clearly borne in mind that if we place luminous organisms, say bacteria or fungi, in an atmosphere devoid of oxygen and find that no light is produced, this may merely mean that certain functions of the cell are interfered with, including light production, but does not necessarily indicate that oxygen is actually used up in the photogenic process. ”

“ We have seen that Bioluminescence is an oxylumines [Pg 111] cence, that the light is probably due to the oxidation of a compound, luciferin, in presence of air and water and that the oxidation is accelerated by an enzyme-like substance, luciferase. We also saw in Chapter 2 that light production is of fairly common occurrence during the oxidation of many organic compounds, provided the oxidation is carried out in the proper way. Many of these organic compounds must be oxidized by relatively strong alkali or such strong oxidizing agents as would have a very deleterious action on living cells. ”

“ Luciferase solution must always contain the oxidation product of luciferin, oxyluciferin. In presence of nascent H this is reduced to luciferin, and since the reaction of the medium is alkaline and luciferase is present this is oxidized with light production, when, by shaking, air is dissolved. The light can never become very bright except at the surface because of the deficiency of oxygen in the solution. It would seem, then, that the action of bacteria, yeast, muscle cells, etc., on oxyluciferin must be due not entirely to their acid reaction but to their reducing power as well. ”

“ Perhaps the best known case among aquatic animals is Sapphirina, a marine copepod living at the surface of the sea, and especially likely to be collected with other luminous forms. Its cuticle is so ruled with fine lines as to diffract the light and flash on moving much as a fire opal. Needless to say no trace of light is given off from this animal in a totally dark room.
It has often been supposed that the eye of a cat or of other animals is luminous. The eyes of a moth, also, [Pg 15] can be seen to glow like beads of fire when it is flying about a flame. ”

“ I can state from my own experiments that luminous bacteria will live in normal human urine, but not well. In albuminous urines it is very likely that they would live better, and it is possible that the luminous urines reported are the results of luminous bacterial infection. On the other hand, the light may be purely chemical, due to the oxidation of some compound, an abnormal incompletely oxidized product of metabolism, which oxidizes spontaneously in the air. ”

“ Expecting this animal to be of great interest, I examined it further only to find that the frog had just finished a hearty meal of fireflies, whose light was shining through the belly with considerable intensity.
Infection with luminous bacteria is especially liable to occur in any dead marine animal. The flesh is an excellent culture medium. I have seen non-luminous species of squid, recently killed, covered with minute growing colonies, quite evenly spaced, so as to closely resemble luminous species whose light is restricted to scattered light organs over the surface of the body. ”

“ We have just seen that the effect of cooling a Cypridina extract containing luciferin and luciferase and luminescing with a bluish light, is to reduce the intensity and change the shade toward the yellow. Velocity of oxidation must be lowered and with the same concentration of luciferase lowered velocity means more light of the longer wave-lengths. A very instructive experiment on color of the light can be carried out with animals having different colored lights and so closely related that their luciferins and luciferases will interact with each other. ”

“ Some of the earlier workers on fireflies and Noctiluca obtained light even after placing these organisms in absence of oxygen, but they did not realize how low is the amount of oxygen necessary to produce light. It is difficult to remove traces of oxygen from the water, traces which are nevertheless sufficient to cause luminescence. If the organisms are numerous, as in an emulsion of luminous bacteria, they will themselves use up all the oxygen and the liquid soon ceases to glow except at the surface in contact with air. ”

“ The firefly turns on its light by stimulation through nerves of the luminous organ. Noctiluca flashes on stimulation of any kind, even the slightest agitation causing a brilliant emission of light. If the stimulation process means merely the admission of oxygen to the photogenic cells we have a mechanism in the cell itself for automatically producing the light. The admission of oxygen results in aërobic conditions and luciferin in presence of luciferase can then oxidize to oxyluciferin with luminescence. ”

“ The metre-kerze is a unit of illumination, not of intensity, and is incorrectly used by Steche.
Luminous bacteria probably glow with less intensity than any other organism. The light from a single organism cannot be seen but that from a colony is visible to the dark-adapted eye. Even so we must remember that the eye is an exceedingly delicate instrument which can detect very small energy changes. ”

“ Visible radiation × visual sensibility is spoken of as luminosity; (3) by the amount of light (expressed in candles) produced in relation to a given expenditure of energy or in relation to the cost of the energy expended. Thus, of the radiation emitted from an incandescent electric lamp only a small per cent. is light, the rest being heat and actinic rays. It is therefore very far from being 100 per cent. efficient. If there were no infra-red or ultra-violet in the radiation from an incandescent lamp its efficiency would be 100 per cent. if we disregarded visual sensibility. ”

“ We may say, therefore, that the firefly has carried the striving for efficiency too far to be acceptable to human use; it has produced the most efficient light known, as far as amount of light for expenditure of energy is concerned, but has produced it at the (inevitable) expense of range of color. The most efficient light for human use, taking into account both color and energy-light relationships, would be a light similar to the firefly light containing no radiation beyond the visible spectrum, but differing from it by being white. ”

“ Because of difficulties in measuring low intensities of light which are constantly changing, no figures on light intensities can be given, but it is easy to establish the following facts: The greater the concentration of luciferin or luciferase the more intense the luminescence. The greater the concentration of luciferin the longer the duration of luminescence and the greater the concentration of luciferase, the shorter the luminescence lasts. Thus, if we mix concentrated luciferin and weak luciferase we get a bright light which lasts for a half hour or more, gradually growing more dim. ”

“ Another method of testing CO2 production is to measure the amount of heat produced during luminescence. Substances burned during respiration give off considerable heat, one gram of glucose to CO2 and H2O, as much as 4000 calories. We have seen in Chapter III that no infra-red radiation is produced in the light of the firefly. This does not mean, however, that no heat is produced by the reaction which produces the luminescence. A temperature change of a few thousandths or hundredths of a degree would evolve no measurable radiation. ”

“ Many of the luminous marine animals are to be found in the plankton, while the littoral luminous forms are in the minority. Some members of all the above groups are found at one or another of our marine labora [Pg 11] tories with the possible exception of Pholas, Phyllirrhoë and squid. Although earthworms and myriapods which produce light are found in the United States, they are rather rare and seldom observed forms.
Not only adult forms but the embryos and even the eggs of some animals are luminous. The egg of Lampyris emits light within the ovary and freshly laid eggs are quite luminous. ”

“ If the ratio of luciferin to luciferase in a single animal is 100:1, it would mean that luciferase could oxidize 10,000 times its weight of luciferin. A large excess of luciferin but not an indefinite quantity can be oxidized by luciferase, and I believe this is sufficient justification for considering luciferase an enzyme, although it is not an ideal example of an organic catalyzer. Quite a number of enzymes are known to be diminished during the course of the reaction they accelerate or to be poisoned by their reaction products. ”

“ If the radiant energy is light (visible or ultra-violet) we speak of photoluminescence, if cathode rays we have cathodoluminescence, if anode rays, anodoluminescence, and if X-rays (Röntgen rays) we have radioluminescence. Inasmuch as the α, β, and γ rays of radium correspond to the anode, cathode, and X-rays, respectively, radium radiation also produces luminescence in many kinds of material. If the material gives off the light only during the time it is radiated we speak of fluorescence; if the light persists we speak of phosphorescence. ”

“ I have been unable to oxidize with light production various mixtures of amino-acids (from tryptic digestion of beef and casein, or the acid hydrolysis products of luciferin itself) by means of luciferase, and consequently am led to believe that Cypridina luciferin is either a new [Pg 123] natural proteose, soluble in absolute alcohol and not digested by trypsin or that it belongs to some other group than the proteins. The absence of a biuret reaction would point in that direction and the question must await further study. ”

“ As we raise the temperature, chemiluminescence becomes more intense but we soon reach a temperature for maximum luminescence and above this the intensity diminishes. This is especially well seen in the action of various oxidizers on pyrogallol and H2O2 recorded in Table 10. At 100° C. practically no light is produced by many [Pg 146] oxidizers which are themselves unaffected at 100°. If we are to connect reaction velocity with intensity of luminescence we must conclude that the evolution of light is dependent rather on an optimum than a maximum reaction velocity. ”

“ Before considering luciferin and luciferase, however, let us study a few well-known chemiluminescent oxidations with special reference to concentration of reacting substances and temperature. [Pg 145]
The effect of temperature on luminescence is of special interest because it gives us a means of analysis for determining if the luminescence depends on reaction velocity. We know that photochemical reactions are very little affected by temperature because the reaction is dependent on the absorption of light, a physical process, and this increases only a small per cent. ”

“ They really left the question open as to whether the effect of Pyrophorus light on their bolometer was due to the visible band of rays or to this plus another band in the infra-red. "The firefly's actual efficiency as a light source is dependent to a large degree on the radiation being confined to the visible region. If there should be found infra-red of quantity comparable to the visible, the firefly, while still a very efficient source would not be, as usually supposed, the example of an ideally efficient light produced by nature." ”

“ We see then, that air (oxygen) , water, and some photogenic substance are necessary for the light production. Spallanzani's experiment, which has been confirmed for a great many luminous forms, shows also that animal luminescence is not a vital process, in the same sense that the conduction of a nerve impulse is a vital process. A nerve loses its characteristic property of conduction on drying or maceration while luminous cells still possess the power to luminesce after drying or maceration. ”

“ It was only in 1830 that Michaelis suspected the light of dead fish to be the result of some living thing and in 1854 Heller gave the name Sarcina noctiluca to the suspected organism. In 1875 Pflüger showed that nutrient media could be inoculated with small amounts of luminous fish and that these would increase in size, like bacterial [Pg 2] colonies, and we now know that the light of all dead fish and flesh is due to luminous bacteria.
In the early part of the nineteenth century it was surmised that the light of damp wood was connected with fungus growth because of a similarity in smell. ”

“ In the presence of the perhydridase the oxygen of water oxidizes the aldehyde and the hydrogen set free reduces any easily reducible substance which may be present. There is a perhydridase in fresh milk, spoken of as Schardinger's enzyme, which is destroyed by boiling. If some aldehyde is added, fresh milk will reduce methylene blue to its leuco-base or nitrates to nitrites, upon standing a short time. If shaken with air the blue color returns. There is no reduction unless an aldehyde is added or unless some boiled extract of a tissue such as liver is added. ”

“ If we add some Al powder and dilute NaOH to an oxyluciferin solution, H is given off and luciferin is formed. As oxyluciferin cannot be formed by the addition of alkali alone we must have in this experiment a reduction of oxyluciferin in alkaline medium by the nascent H produced. Luciferin can also be formed by merely adding Al or Zn or Mg dust to an oxyluciferin solution. Methylene blue can also be readily reduced to its leuco-base by Zn dust or Al + NaOH.
Indeed, if one adds some Al or Zn or Mg powder to a solution of luciferase, light will appear whenever the solution is shaken. ”

“ Bacteria, fungi, and a few fish produce light continuously and independently of stimulation. Its intensity varies only over long periods of time and is dependent on the nature of the nutrient medium or general physiological condition of the organism. All other forms give off no light until they are stimulated. Stimulation may of course come from the inside (nerves) or outside. Only under unfavorable conditions, such as will eventually lead to the destruction of the luminous cells, do these forms give off a continuous light. ”

“ However, the reaction which occurs during phosphorescence cannot be stated.
It is a fact that light will cause the reduction of oxyluciferin. A tube of oxyluciferin exposed to sunlight for six hours, or the mercury arc for two hours, will be partially converted into luciferin. It will luminesce when luciferase is added, while a control tube kept in darkness shows no trace of luciferin. The action is more marked with the ultra-violet as a solution of oxyluciferin in a quartz tube showed more reduction than one in a glass tube when exposed for the same length of time to the quartz mercury arc. ”

“ Phipson's description of the properties of noctilucin are too crude and inaccurate to be considered. Dubois did not study the chemical properties of luciferin and luciferase from Pyrophorus, the first form with which he worked, except to point out that Pyrophorus luciferase was destroyed on heating and was precipitated by alcohol while the Pyrophorus luciferin was not so affected. Luciferin was found only in the luminous organ of Pyrophorus, not in the blood; luciferase probably exists throughout the animal. ”

“ We know that luminous bacteria occasionally lose the power of lighting and that on certain culture media they develop as non-luminous forms. Luminescence is not indispensable to them. The same is true of some of the [Pg 83] fungi but Noctiluca and other animals are not known in a non-luminous condition, although we can see no definite value to the organism of this power of luminescence. ”

“ If we are to produce light artificially in the same way that animals do, the luciferins must be synthesized. The luciferin of Pholas will luminesce with KMnO4 and other oxidizing agents, and, although I have not yet succeeded in oxidizing Cypridina luciferin with oxidizing agents, I have no doubt but that some inorganic catalyzer will be found to take the place of luciferase and accelerate oxidation of Cypridina luciferin with light production. ”

“ However, addition of a fresh emulsion of yeast cells to a glowing Cypridina secretion is sufficient to rapidly extinguish the light, because the yeast is capable of utilizing the last trace of oxygen in the mixture. Light only appears when, by agitation, we cause more air to dissolve. The minimal concentration of oxygen for luminescence of Cypridina lies somewhere between 3.04 mm. and the amount which living yeast fails to extract from solution, a concentration approaching zero. ”

“ A substance, fluorescent in solution, has been found in a few luminous animals, notably in several species of fireflies and also in a non-luminous beetle. It is called pyrophorine or luciferesceine. Dubois (1886) has ascribed to pyrophorine the power of absorbing invisible rays and transforming them into visible ones, thus increasing the animal's light. That this is not the case has been shown by the work of Coblentz (1909) . He photographed the spectrum of the firefly's light and the fluorescent spectrum of luciferesceine. ”