Some forms only produce light at certain seasons of the year. According to Giesbrecht (1895) this is true of the copepods, which only light in summer and autumn, and according to Greene (1899) in the toad-fish; Porichthys, which can only be stimulated to luminesce during the spawning season in spring and early summer.

Some animals possess a periodicity of luminescence. They only luminesce at night and fail to respond to stimulation or are difficult to stimulate during the day. Bright light has an inhibiting effect. Perhaps correlated with this is the fact that most luminous forms are strongly negatively heliotropic. Fireflies lie hidden in the day, to appear about dusk and the ostracod crustacean, Cypridina, is difficult to obtain on moonlight nights.

The Ctenophores were the first forms in which the inhibiting effect of light was noticed. This was described by Allman (1862) and has been confirmed by a number of observers, especially Peters (1905). Massart found that Noctiluca was difficult to stimulate during the day and Ceratium, according to both Zacharias (1905) and Moore (1908), only luminesces at night, or if kept in darkness, for some little time. Crozier[4] finds a persistent day-night rhythm of light production when Ptychodera, a balanoglossid, is maintained for eight days in continued darkness. The animal is difficult to stimulate during the period which corresponds to day and luminesces brilliantly and at the slightest touch during the period which corresponds to night.

On the other hand, a great many forms are able to luminesce quite independently of previous illumination. According to Crozier[4] Chætopterus luminescence is not affected by an exposure to 3000 metre-candles for six hours.

In the case of animals with extracellular luminescence we may speak of luminous secretions and true luminous glands. A large number of forms possess luminous glands or gland cells, including some of the medusæ, the hydroids (probably), the pennatulids (?), the molluscs (Pholas and Phyllirhoë) (probably), some cephalopods (Heteroteuthis and Sepietta), most annelids, ostracods, copepods, some schizopods (Gnathophausia) and decapod (Heterocarpus and Aristeus) crustaceans, all myriapods, and the balanoglossids. The remaining organisms burn their material within the cell. These include the bacteria, fungi, protozoa, some medusæ (?), ctenophores (probably), most cephalopods, a few annelids (Tomopterus (?)), ophiuroids (?), some schizopod (Nyctiphanes, Euphasia, Nematocelis, Stylochiron) and decapod (Sergestes) crustacea, all(?) insects, Pyrosoma, and fishes (selachians and teleosts). It is among this latter type that the most complicated luminous organs have been developed. While a description of all the types of luminous organs and luminous structures cannot be attempted here (excellent descriptions have been given by Dahlgren and Mangold) it is necessary to understand the structural conditions in a few of the forms whose physiology has attracted most attention.

Luminous bacteria are so small that the light from a single individual cannot be seen. It is almost impossible to make out structural differences within the cell and we cannot definitely state in just what special region, if any, the luminescence is produced. We do know that the light is intracellular and that filtration of the bacteria from their culture medium gives a dark sterile filtrate absolutely free from any luminous secretion.

Among protozoa, in certain forms at least, it is easy to observe that luminescence is connected with globules or granules which were considered by the earlier observers to be oil droplets. Thus, in Noctiluca (Figs. 17 and 18), when the animal is violently stimulated or in the presence of reagents which slowly kill it, the whole interior appears a mass of starry points of light which can be traced to minute granules along the strands of protoplasm (Quatrefages, 1850).

Turning to the multicellular forms, we find the simplest development of luminosity in those animals which possess gland cells producing a luminous secretion. These cells may be scattered over the surface of the animal as in Chætopterus (Fig. 19) or Cavernularia, or restricted to certain areas [Pholas, (Fig. 19),] or more definitely localized to form an isolated group of gland cells as in Cypridina. True multicellular glands also occur. In every case, however, we find that the luminosity of these uni- or multicellular glands is connected with the presence of granules. They are often spoken of as luciferine granules, although it is not certain whether they are made up of luciferin or luciferase (see Chapter IV) or both. They are most similar to the zymogen granules found so abundantly in gland cells and thought to be the precursors of various enzymes. According to Dahlgren (1915), the luciferine granules stain blue-black by iron hæmatoxylon after fixation at the boiling point, and photogenic cells can be detected by this method of selective staining. Dubois (1914, book), who regards them as examples of bioprotein, comparable to the chondriosomes and handed on from one generation to another, gives them the name of vacuolides or macrozymases. In some forms he has described their transformation into crystals and believed at one time that animal light was a crystalloluminescence. His figures of the crystal transformation are not very convincing. Pierantoni (1915) has considered the granules to be symbiotic luminous bacteria, but this is certainly not the case.

The light of Chætopterus comes from a material mixed with a mucous secretion formed over almost the whole body surfaces of the animal. A section of the epithelium shows large mucous-producing cells and smaller granule-containing light cells (Fig. 20). These appear to be under nervous control, as a strong stimulation in one part of the body causes luminescence which spreads over the whole surface of the worm. The animal becomes fatigued rather readily, however. In the pennatulids, such as Cavernularia, we have also the formation of a luminous secretion over the whole surface of the body and the individual animals in this colonial form are also connected with nerves. A stimulation in any local region, as Panceri (1872) first showed (Fig. 21), will cause a wave of luminosity to spread from this point until it extends over the whole surface of the colony. In Pennatula the rate of this luminous wave is about 5 cm. per second.

Pholas dactylus possesses similar light cells to those of Chætopterus, but they are restricted to narrow bands on the siphon and mantle and a pair of triangular spots near the retractor muscles. Nerves pass to the luminous regions.

In many luminous animals the light secretion formed over the surface of the body is small in amount and adheres to the animal because it is embedded in the mucous skin secretions. In those forms which possess a true localized light gland the luminous secretion when expelled into the sea water (if the animal be a marine form) may persist as a luminous streak for some time and exhibit diffusion and convection movements. The most beautiful examples of luminous secretions are found among the ostracod crustacea.

In Cypridina hilgendorfii the luminous gland is situated on the upper lip near the mouth. It is made up of elongate (some 0.7 mm. in length), spindle-shaped cells, each one of which opens by a separate pore with a kind of valve. The openings are arranged on five protuberances. Muscle fibres pass between the gland cells in such a way that by contracting the secretion can be forced out. In the sea water the secretion luminesces brilliantly and the Japanese call these forms umi hotaru, or marine fireflies. Fig. 22 is a diagram showing the structure. Watanabe (1897), who first studied this form, and also Yatsu (1917) have described two kinds of granule-containing cells, one with large yellow globules, 4-10µ in diameter (Fig. 23), the other with small colorless granules 0.5, in diameter. I have observed in the living form these two types and also large colorless globules of the same size as the yellow globules. All dissolve when extruded into the sea water. Dahlgren[5] has described from sections four types of cells containing (1) large globules, (2) small granules, (3) a fat-like material, (4) a mucous material. Just what the significance and nature of these types of substance is cannot be stated at present. At least one, probably two, are concerned in light production. The others may possibly form digestive fluids which act on the food of the animal.

Turning now to the animals possessing light cells with intracellular luminescence we find in general that such light cells are localized to form definite light organs and that these may be single, as in the common fireflies, paired, as the prothoracic light organs of Pyrophorus, or scattered over the surface of the body, as in so many shrimps, cephalopods and fishes, when they are often called photophores. The light cells proper are often associated with reflectors, lenses, opaque screens and color screens.

The insects possess the simplest types of intracellular light organs, a mass of photogenic cells, which, in the common firefly (a lampyrid beetle) of Eastern North America, has probably been developed from the fat body, while in the New Zealand glowworm, the larva of a tipulid fly (Bolitophila luminosa), part of the Malpighian tubule cells have acquired photogenic power (Wheeler and Williams, 1915). This is illustrated in Fig. 24.

The photogenic organ of the firefly is made up of two kinds of cells, a dorsal mass of small cells several layers deep, the reflector layer, and a ventral mass of large cells with indistinct boundaries, the photogenic layer (Fig. 25). The photogenic cells contain a mass of granules, spherical in the male and short rods in the female. The photogenic cells are divided into groups by large tracheal trunks which pass into the light organ and branch to form tracheoles connected with tracheal end cells. The exact distribution varies in different species, but in all the arrangement is such as to give a very abundant oxygen supply. Each group of photogenic cells is surrounded by a clear ectoplasm containing no granules. The tracheoles pass through this and either end openly within the photogenic cells or anastomose with tracheoles from neighboring tracheæ. Nerves, but no blood-vessels—which are absent in insects—enter the organ. It is difficult to determine if the nerves supply the tracheal end cells or the photogenic cells.

The dorsal reflecting layer is made up of cells containing numerous minute crystals of some purin base, either xanthin or urates, or both. They have a white milky appearance and while they are certainly not good reflectors in the optical sense, they do act as a white background, scatter incident light, and partially prevent its penetration to the internal organs of the firefly. Although a few crystals similar to those of the reflector layer are found in the photogenic cells and in other cells of the body, it is known that the photogenic cells are not transformed into the reflector cells. The two layers are distinct and permanent from an early stage in development.

Curiously enough, the light organ of the larva of the firefly (glowworm) is quite distinct from that of the adult. Like so many other structures in insects, the adult organ is developed anew from potential photogenic cells during the pupal period. Even the egg of the firefly is luminous and glows with a steady light, and during the pupal period light may sometimes be seen coming from the thoracic region.

In the firefly there is no true lens, the light merely shining through the cuticle which is transparent over the light organ, whereas over the rest of the body it is dark and pigmented. In the deep sea shrimp, Acanthephyra debelis, with light organs scattered over the surface of the body, the cuticle covering the light organ forms a concavo-convex lens, behind which are the photogenic cells (Kemp, 1910). As may be seen from Fig. 26, the lens is made up of three layers which suggests that it may be corrected for chromatic aberration—a veritable "achromatic triplet." In an allied form, Sergestes (Fig. 27), the lens is of two layers and double convex. Optical studies of these lanterns have been made by Trojan (1907). The course of the light rays is shown in Fig. 28. The lens of these organs is also bluish in color which suggests that they may serve also as color filters. Behind the photogenic cells is a mass of connective tissues through which enters the nerve, for the light of these organs is under the control of the animal and may be flashed "at will."

All gradations in complexity of light organs may be found from the condition in the shrimp just described to that found among the squid and fish. Figs. 29 and 30 are sections of two of the more complicated types found in squid. The explanation given to the various structures is that of Chun (1903) to whom we are indebted for a careful histological investigation of these forms. It will be noted that in addition to photogenic and lens tissues there are various types of reflector cells and a line of pigment about the whole inner surface of the organ to effectively screen the animal's tissues from the light. In one form (Fig. 30) chromatophores are found about the region where the light is emitted and these no doubt serve as color filters. There are also an abundant blood supply and nerves passing to the organ. Figs. 30 and 31 are sections through light organs of fishes.

We thus see that light organs may be very simple and also very complicated. The latter must have evolved from the former, although it is not always possible to point out the intermediate stages. It is not within the scope of this book to discuss bioluminescence in its evolutionary aspects. It may be worth while, however, to point out briefly what is known concerning the use of the light to the animal. There are four possibilities.

(1) The light may be of no use whatever, purely fortuitous, an accompaniment of some necessary or even unnecessary chemical reaction.

This appears to be the case in the luminous bacteria and fungi and perhaps the great majority of forms which make up the marine plankton, Noctiluca, dinoflagellates, jelly-fish, ctenophores and even the sessile sea pens.

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 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.

In the case of sea pens, however, we might suppose that the light acts as an attraction to small organisms on which the sea pen feeds, although these creatures only luminesce when stimulated in some way, which rather detracts from the above suggestion.

(2) The light may act as a warning to scare away predacious animals which would otherwise feed on the luminous organism. Perhaps this is the case in the sea pens, although these forms possess nematocysts which should serve as adequate protection. The marine worm, Chætopterus, is brightly luminous and lives its whole life in an opaque parchment tube. If this tube were torn open by a predacious form we might conceive that the attacking animal would be alarmed by the light and refrain from destroying the worm. The Chætopterus, however, could not rebuild another tube and its light would only protect it in the night time. These cases will suffice to indicate the difficulties and perplexities of the problem. Perhaps we may add one more guess and suppose that the light of certain fishes is actually for blinding or distracting their enemies or blinding the forms on which they feed. Until this use of luminous organs has actually been observed, we can give little credence to it.

(3) The light may serve as a means of recognition or a sex signal to bring the sexes together for mating. It would seem from the work of Mast and of McDermott that this is the case in the common fireflies and it may be the case in the toad-fish, Poricthys, which is only luminous in the spawning season and in the worm, Odontosyllis, of Bermuda, which is brilliantly luminous while swarming when the eggs and sperm are shed. It is non-luminous at other times (Galloway and Welch, 1911.)

(4) Finally, it is possible that animals with complex luminous organs, such as squid, fish and shrimp, actually use these as lanterns. It is significant that most of them are deep sea forms, living in a region of perpetual darkness, and it is perfectly logical to suppose that they make use of their light organs for illuminating purposes.

The whole problem of the use and purpose of luminous organs is an exceedingly complex and difficult one. We have, perhaps, said enough to indicate this and may add that in most cases, so far as opinion is based on actual evidence and observation, that of the layman is of as great value as that of the scientist.

CHAPTER V
THE CHEMISTRY OF LIGHT PRODUCTION, PART I

"Exp. I.: Having procured a Piece of shining Wood, about the bigness of a groat or less, that gave a vivid Light, (for rotten Wood) we put it into a middle sized Receiver, so as it was kept from touching the Cement; and the Pump being set a-work, we observed not, during the 5 or 6 first Exsuctions of the Air, that the splendor of the included Wood was manifestly lessened (though it was never at all increased;) but about the 7th Suck, it seemed to glow a little more dim, and afterwards answered our Expectation, by losing of its Light more and more, as the Air was still farther pumped out; till at length about the 10th Exsuction, (though by the removal of the Candles out of the Room, and by black Cloaths and Hats we made the place as dark as we could, yet) we could not perceive any light at all to proceed from the Wood.

"Exp. II.: Wherefore we let in the outward Air by Degrees and had the pleasure to see the seemingly extinguished Light revive so fast and perfectly, that it looked to us almost like a little Flash of Lightning, and the Splendor of the Wood seemed rather greater than at all less, than before it was put into the Receiver."

Boyle proved that light from the wood was able to pass a vacuum and later showed that "shining fish" behaved as the "shining wood," but that a piece of white hot iron would not regain its light on readmitting air to the exhausted receiver and that the iron lost its glow under the air-pump merely because it cooled off. A piece of glowing coal, however, did lose its light in the absence of air and regained it on again admitting air, provided the air had not been removed for too long. Boyle was apparently impressed with the similarity of the light giving process in glowing coal and shining wood as he draws a comparison between the two which brings out the fundamental similarity of combustion processes.

"Resemblances:

VII. The Things wherein I observed a Piece of shining Wood and a burning Coal to agree or resemble each other are principally these five:

Differences:

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. If we find, however, that extracts of luminous cells or luminous secretions devoid of cells cease to light when the oxygen is removed and again luminesce when it is returned, we may be quite certain that the photogenic process itself requires free oxygen. As luminous extracts of fireflies, pennatulids, ostracods, Pholas and others give off no light when the oxygen is removed, we may safely conclude that for these luminescences, oxygen is necessary. Bacteria, fungi, and Noctiluca, whose light also disappears in absence of oxygen, although they are whole cells, we may by analogy also assume to require oxygen in the photogenic process.

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. We may gain an idea of the amount of oxygen necessary for luminescence from an experiment of Beijerinck (1902). He mixed luminous bacteria with an emulsion of clover leaves containing chloroplasts and kept the two in the dark until all the oxygen was used up and the bacteria ceased to glow. If now a match was struck for a fraction of a second, sufficient oxygen was formed by photosynthesis to cause the bacteria to luminesce for a short time.

Exact figures on the minimal concentration of oxygen for luminescence cannot be given. The luminescent secretion of Cypridina hilgendorfii will still give off much light if hydrogen containing only 0.4 per cent. of oxygen is bubbled through it, i.e., a partial oxygen pressure of 1/250 atmosphere (3.04 mm.Hg). 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. It is probably nearer the latter figure.

As the oxygen pressure is increased from 0 to about 7 mm., the intensity of the Cypridina luminescence increases and at the latter figure the light is just as bright as if the solution were saturated with air (152 mm.O₂). Thus, the luminescence requires only a low pressure of oxygen and the similarity to the saturation of hæmoglobin with oxygen is obvious. Just as hæmoglobin is nearly saturated with oxygen at low pressures and becomes bright red in color, so the luminous material becomes saturated with oxygen at low pressures and glows intensely.

Boyle also made many experiments to show that air was necessary for the life of animals and the germination of seeds and showed that repeatedly respired air was unfit for further breathing. About the same time R. Hooke discovered the true meaning of respiratory movements and by forcing a blast of air continuously through the lungs with bellows, was able to keep animals alive. He concludes "that as the bare Motion of the Lungs, without fresh air, contributes nothing to the life of the Animal, he being found to survive as well as when they were not moved as when they were; so it was not the Subsiding or Movelessness of the Lungs that was the immediate cause of death, or the stopping of the circulation of the Blood through the Lungs, but the Want of a sufficient Supply of fresh Air." The cause of death on collapse of the lungs could not be better stated to-day. Thus combustion, respiration and luminescence of flesh or wood were early recognized as related phenomena.

Although the "gas sylvestre" (CO₂) of burning charcoal and fermentation of wine was known to van Helmont (1577-1644) and Mayow (1646-1679) in 1674 showed that "spiritus nitroærens" (oxygen) was responsible for the life of animals and for combustion, a century elapsed before the true significance of these gases became known. In the meantime the phlogiston theory of combustion had been developed, Black (1728-1799) in 1755 had rediscovered carbon dioxide ("fixed air") in the expired air and Priestley (1733-1804) and Scheele (1742-1786) had both rediscovered oxygen ("dephlogisticated air") in 1774. About the same time Lavoisier overthrew the phlogiston doctrine and showed that in the combustion of organic substances water and CO₂ are formed.

Later it was realized that this slow combustion did not take place in the lungs, or in the blood, but in the tissues cells themselves and respiration in the chemical sense has come to mean this universal slow combustion in the cells of the body rather than the breathing movements of the lungs themselves. In anaerobic respiration, CO₂ is given off, but no oxygen absorbed. In aerobic respiration, oxygen is absorbed and CO₂ given off. In addition we know of many substances which oxidize by taking up oxygen without giving off CO₂. We have seen that oxygen must be absorbed for luminescence of animals and we may now inquire whether CO₂ is given off and the relation between respiration and light production.

To determine if CO₂ is given off during luminescence it is necessary to work with fairly pure luminous materials, obtained from luminous organisms. It is impossible to use the living organisms themselves as the CO₂ continually respired becomes a very disturbing factor. From Cypridina, a small crustacean, two materials soluble in water may be prepared (luciferin and luciferase), which will give a brilliant luminescence on mixing. It is possible to determine the H-ion concentration of the two solutions separately and of the mixture of the two after the luminescence has occurred.

If CO₂ is produced during luminescence the H-ion concentration of the luminous solution should increase. Measurements made electrometrically with the hydrogen electrode have failed to demonstrate any increase in acidity. The Ph of both solutions and of a mixture of the two is 9.04. This would indicate that CO₂ is not produced. As both luminous solutions contain proteins and the luminous substances themselves are probably proteins, which have a high buffer value, a method of this kind is none too sensitive. However, we can definitely state that not enough CO₂ is produced to be detected and that this may be due to the buffer action of the luminous substances themselves. After all, unless luminescence is connected with respiration, we should hardly expect CO₂ to be produced.

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. Coblentz (1912) first studied the problem of heat production in the firefly, using a thermocouple as the measuring instrument. He came to the conclusion that the temperature of the insect was slightly lower than the temperature of the air and that the luminous segments were slightly hotter than the non-luminous segments, whereas a dead firefly is of the same temperature as its surroundings. No definite increase or decrease in temperature could be established during the flash of the firefly. However, further work on the firefly is much to be desired.

The use of a living animal for such measurements introduces a possible source of error in that any contraction of the muscles of the animal will produce heat which may add to an increase or mask a decrease of temperature during luminescence. Utilization of extracts of luminous animals containing the luciferin and luciferase mentioned above avoids the complications due to muscular contraction. By bringing the solutions of luciferin and luciferase to the same temperature and then mixing them one can measure any increase or decrease of temperature which occurs during the luminescence which results from mixing. We can thus gain some idea of the heat of oxidation of luciferin.

As a determination of heat production is of considerable interest the method will be given in some detail. Although the experiment sounds very simple, it is actually somewhat difficult to carry out. The attainment of temperature equilibrium between two solutions is very slow when one wishes to obtain them to within 0.001° C. of the same temperature. After many attempts, the following arrangement of apparatus (Fig. 33) was found most satisfactory. About 10 c.c. luciferin solution was placed in the inner tube (D) of a special non-silvered thermos bottle (A). About 1 c.c. of luciferase solution was placed in a very thin-walled glass tube (E) which was immersed in the luciferin solution and connected with a small motor so that it could be slowly but constantly rotated, thus stirring the solutions. Thermocouples (L and M) of advance (.008 in)—copper (No. 30, B and S, enamel insulated) wire were paraffined and placed in each tube and the copper wires connected through a copper double throw switch (C) with a Leeds and Northrup d'Arsonval wall galvanometer (No. 34637, silver strip suspension) of 35 ohms resistance and 310 megohms sensitivity. The constant temperature junctions (N) were placed in a large Dewar flask (B) filled with water at approximately the same temperature as the luciferin solution. One mm. galvanometer scale division represented 0.003° C. and the division readings could be estimated to tenths. By means of a glass rod (F) placed in the tube containing luciferase solution, this tube could be broken and the luciferase and luciferin solution mixed.