mentation liquors were a rich source of a material (to which they gave the somewhat inappropriate name "copro- gen") which was very active in promot- ing the growth of Pilobolus. Third, Light and the Asexual Neilands (4) discovered a family of iron-containing compounds, the fer- richromes, which were also very active Reproduction of Pilobolus in promoting the growth of Pilobolus. Now, hemin, coprogen, and the fer- Responses to light play an role in the richromes have one property in com- important mon: they all contain iron in chelated development and reproduction of this fungus. form. It appears, therefore, that one distinctive nutritional requirement of Pilobolus is that it be supplied with Robert M. Page iron in specially chelated form. Al- though the identity of the material in dung remains unknown, it seems proba- ble, from the work of Lyr (5), that Pilobolus, a fungus assigned to the mals. For example, I have isolated bacteria in the dung are a major source order Mucorales, differs from all other strains- from such exotic sources as of this growth factor. It is possible, members of this order of terrestrial caribou dung collected near Point therefore, that this factor is similar to Phycomycetes in that it discharges its Barrow, Alaska, and wallaby dung col- coprogen, or that the two are identical. sporangia explosively. Because of this lected at Wilson's Promontory at the In addition to their requirement for spectacular method of spore dispersal southernmost tip of Australia (1). specially chelated iron, some species Pilobolus has attracted the attention of One question which arises immedi- of Pilobolus have a near-requirement biologists for almost three centuries, ately is, Why does Pilobolus grow on for fatty acids. Growth is very poor in but the genus is of current interest not dung? To be sure, one species, P. media containing carbohydrates as a only because of the dramatic way in oedipus, has been reported to grow on source of carbon, but growth is vigorous which it is adapted to its environment decomposing algae along river and canal in media containing appropriate concen- but also because it has attributes which banks, but all of the other species have trations of sodium acetate. All of the make it of interest in the study of been found only on dung. Is the genus species of Pilobolus tested have grown fundamental biological problems. restricted to this habitat only by the on synthetic media containing hemin, All of the species of Pilobolus except mechanics of its spore dispersal, or are acetate, a nitrogen source, and in- one occur on the dung of herbivorous nutritional factors involved as well? A organic salts (2). By far the most animals. The mycelium grows beneath partial answer to this question may be active nitrogen source is ammonia the surface of the substratum, and found in the results of nutritional (ammonium ion). Both vegetative after a few days it produces a crop of studies. growth and reproduction are greatly sporangiophores. When each sporan- stimulated by ammonia in certain con- gium reaches maturity it is shot off centrations (6). forcibly, and if, at the end of its tra- Nutritional Requirements It appears from the results of nutri- jectory, it strikes a blade of grass, the tional studies that Pilobolus is bound sporangium adheres to it. If a grazing Early attempts to grow Pilobolus on to its habitat as much by its nutritional animal eats a blade of grass with ad- synthetic media of known chemical requirements as by its method of spore herent sporangia, the thousands of composition were unsuccessful. The dispersal, and that its nutrition is as spores contained within each sporan- fungus grew well only on dung or on specialized as its morphology. More- gium pass unharmed through its di- aqueous extracts of dung. Within the over, from a practical point of view gestive tract and emerge with the dung, past decade, however, investigations in the results of nutritional studies have in which they germinate to repeat this several laboratories have provided some been useful because they have made it asexual reproductive cycle. Pilobolus information on the nutritional require- possible to grow the fungus under much also reproduces sexually in a manner ments of some members of the genus. more rigidly controlled conditions than similar to that of other Mucorales. First, through fractionation of aqueous is possible on a dung medium. Also, the Pilobolus is widely distributed, and extracts of cow dung, it was found that growth and development of the fungus although it is not often observed in the material in dung which was re- can be observed more conveniently on the field because its sporangiophores quired by Pilobolus was heat-stable synthetic media than on natural sub- are ephemeral, it appears frequently and soluble in water but not in fat strata. on samples of dung brought to the solvents. It was not adsorbed by anionic laboratory and kept under moist con- or cationic exchange resins, but it was ditions for a few days (Fig. 1). The strongly absorbed by charcoal and cer- Growth and Development genus has been reported from many tain other nonspecific adsorbants. The parts of the world, and it occurs on the growth-promoting activity of this factor On transparent synthetic agar media dung of many kinds of herbivorous ani- can be imitated by hemin, but relatively it is possible to follow the growth of high concentrations (10 mg/ml) are the mycelium and the development of The author is associate professor in the de- required (2). Second, workers at the the asexual structures of partment of biological sciences, Stanford Uni- reproductive versity, Stanford, Calif. Lederle Laboratories (3) found that fer- Pilobolus in detail (Fig. 2). The my- 1238 SCIENCE, VOL. 138 sporangium, which is later separated from the rest of the sporangiophore by a crosswall. Sooner or later the upper part of the sporangiophore swells to form a conspicuous subsporangial swelling or vesicle, and the wall of the sporangium becomes jet black. In the meantime the contents of the sporan- gium cleave to form a large number of multinucleate spores. As the sporan- gium reaches maturity its wall separates at the base, so that the gelatinous ma- terial surrounding the spores is ex- posed. Finally, the wall of the sub- Fig. 1. Sporangiophores of Pilobolus sp. on cow dung. (About X 5) sporangial vesicle ruptures suddenly just below the sporangium. The sporan- gium is shot off, propelled by the liquid celium, which grows beneath the sur- conditions, the mycelium begins to in the sporangiophore and the empty face of the medium, may be some- form trophocysts, which are first recog- sporangiophore is thrown flat on the what restricted, as in Pilobolus crystal- nizable as swollen regions along certain medium by the recoil. linus Fries and its relatives, or it may hyphae. These swellings enlarge as The force with which the sporangia spread rapidly to fill the medium in a protoplasm flows into them, and finally are discharged is impressive. Sporangia petri dish in a few days, as is the case each trophocyst is completed by the which strike the lid of a petri dish with P. kleinii van Tiegh or P. sphaero- formation of crosswalls which isolate it make a clearly audible "plink," and sporus Palla. The mycelium is coeno- from the parent hypha. Later, a stout those whose progress is not interrupted cytic; the hyphae are devoid of cross- hypha, the sporangiophore, grows from are thrown for remarkable distances. walls except for those which delimit each trophocyst, increases in length, All records for altitude and distance are reproductive structures or wall off small emerges from the medium, and con- held by sporangia reported by Buller branch hyphae from which the pro- tinues to elongate in the air. After a (7); he found that large sporangia toplasm has been withdrawn. Sooner or time the sporangiophore stops elongat- of Pilobolus kleinii and P. longipes are later, depending on the species and ing and its tip swells to form the shot to a height of 6 feet 0.5 inch. .... : - . 0 :. :-, C) 0 Fig. 2. Schematic drawing (based on P. crystallinus) showing the development of asexual reproductive structures of Pilobolus. Trophocysts and sporangia are represented as developing in clockwise sequence on hyphae emanating from a sporangium. 14 DECEMBER 1962 1239 When sporangiophores were aimed at form sporangia in the dark, P. sphaero- a 45-degree angle above the horizontal, sporus is more suitable for studies on the maximum range was 8 feet 0.5 inch periodicity, and it has been shown to for P. kleinii and 8 feet 7.5 inches for have an endogenous rhythm which is P. longipes. susceptible of entrainment (11). From the maximum height to which The phototropic responses of Pilobo- sporangia are shot, Buller calculated lus have been studied far more inten- that the muzzle velocity would be 19.6 sively than its morphogenic responses feet per second if air resistance is to light (12). It is well known that neglected. Pringsheim and Czurda (8) the fungus discharges its sporangia attempted to measure the muzzle veloc- toward a source of light, and the ity directly by allowing sporangia to accuracy with which the sporangio- pass through holes in a rotating disk phores direct the sporangia at an illu- and strike a second disk rotating on the minated target has been demonstrated same shaft. From the results, they cal- by many workers. To illustrate this culated that the muzzle velocity was Fig. 3. Sporangia of Pilobolus kleinii ad- accuracy, a target of translucent paper approximately 14 meters per second. hering to a paper target of which the was attached to the underside of the In order to avoid some of the errors center was illuminated (see text).