BRAZILIAN CHYTRIDS. VI. RHOPALOPHLYCTIS AND CHYTRIOMYCES, TWO NEW CHITINOPHYLLIC OPERCULATE GENERA

FRESH-WATER chytrids which inhabit the chitinous exuviae of insects are fairly common in nature, but until recently they have not received much attention from mycologists. The first study devoted exclusively to these saprophytes was made in 1903 by Petersen in Denmark, and it was not until 1937 that they were again given serious consideration. In the latter year Sparrow (1937) recorded the occurrence of Petersen's Danish species in the New World and described several new American species from the exuviae of caddisflies, midges, and of other insects, the larval stages of which develop in fresh water. Since that time, several additional species have been reported by the author (1944) from the Amazon Valley in Brazil, which indicates that chitinophyllic chytrids are widespread in nature and more abundant than has been generally realized. Such chytrids grow well on the chitinous skeletons of insects and appear to derive most of their energy from chitin. These unusual growth requirements suggested to the author, particularly after Mliss Anne Hanson, a graduate student at Columbia University, had found that some species can be grown successfully on thin strips of shrimp shell, that insect-inhabiting chytrids might be grown on artificial chitinous media. Although such chytrids may be readily trapped and isolated on shrimp shells, this type of bait may also become heavily infected with other aquatic fungi and protozoa and is not wholly satisfactory. However, if all but the pure chitin is removed, better results may be obtained. In purifying the chitin, the procedures of Benecke (1905), Folpmers (1921), Johnson (1932), Benton (1934), Zobell and Rittenberg (1938), and Hock (1940) were used. Shrimp, crab, and lobster shells were scrubbed clean of dirt and decalcified in cold one per cent hydrochloric acid which was changed several times in the course of a week. The material was then washed in water and soaked in two per cent potassium hydroxide for ten days to remove the protein and other organic matter except chitin. Following this treatment, the limp leathery shells were boiled in several chatiges of ethyl alcohol for tlhree days or until no solid matter was obtained on evaporation of a portion of the extract, after which the shells were dried and stored for future use. The treatment with alkali and alcohol removes all or most of the pigment, leaving the chitin greyishwhite or semi-transparent. The ventral scales of the slhrimp's tail are usually quite thin and almost transparent, and when treated in the manner described above and cut into thin strips, make a satisfactory substratum for trapping, growing, and studying mi1 Received for publication March 1, 1945. This work has been greatly facilitated by a Grant-inAid of Research from the Graduate Faculties of Columbia University. croscopically certain chitinophyllic chytrids. Up to the present time species of Rhizidium, Obelidium, Asterophlyctis, Siphonaria, Rhopalophlyctis, Chytriomyces, Rhizoclosmatium, and Polychytrium have been isolated from fresh-water cultures in Brazil and the U.S.A. on bits of pure chitin. Processed shrimp, lobster, and crab shells may be ground in a powder mill and added directly to nutrient agar, but this does not make a very satisfactory medium. Chitin is difficult to pulverize in a mill, and the fragments usually obtained are so large that they sink to the bottom of an agar plate and are not directly available to monocentric chytrids sown on the surface. In the case of polycentric species like Polychytrium aggregatum (Ajello, 1942), however, good results may be obtained because the rhizomycelium grows downward in the agar and soon spreads from one bit of chitin to another. A more satisfactory and highly dispersed chitin for inclusion in agar media may be obtained by dissolving chitin in a 1:1 solution of sulfuric acid overnight in the refrigerator and then precipitating with a twenty per cent potassium hydroxide solution. The milky-white precipitate is then filtered and washed with distilled water on a Buehner funnel until the extract shows neutral with litmus. The flocculated chitin may be added in varying amounts to plain or enriched agar as desired, autoclaved, and poured. The flask, however, should be shaken carefully to disperse the chitin evenly before pouring. Chitinagar media prepared in this fashion are usually milky-white in appearance. Chitinophyllic chvtrids planted on such media digest the chitin as the rhizoids and rhizomycelium spread, and as a result a clear zone develops around each thallus or colony within a few days. The present contribution concerns the structure and life cycles of two new chitinophyllic operculate genera which were isolated on chitin and grown in the manner described above. These fungi were first found on unidentified insects in fresh water in Brazil and later collected in abundance on the exuviae of mayflies in Connecticut, New York, and Virginia, U.S.A. The first of these genera is characterized by predominantly clavate, septate or continuous, extramatrical sporangia, an intramatrical bushy tuft of thread-like rhizoids, and by posteriorly uniflagellate zoospores which swarm in a vesicle outside of the sporangium. Because of the characteristic shape of its sporangia and its occurrence on exuviae of insects, the name Rhopalophlyctis sarcoptoides is proposed for this fungus. The second genus includes two species and is distinguished by extramatrical sporangia, extensive intramatrical rhizoids which arise from the base of the sporangium or apophysis, posteriorly uniflagellate zoospores which swarm in a vesicle outside