INFECTION POTENTIAL AND GROWTH OF LAGENIDIUM CHTHAMALOPHILUM

JOHNSON, T. W., JR. (Duke U., Durham, N. C.) Infection potential and growth of Lagenidium chthamalophilum. Amer. Jour. Bot. 47(5): 383-385. Illus. 1960.-Temperature and salinity influence infection of Chthamalus fragilis and Balanus amphitrite denticulata ova by Lagenidium chthamalophilum. Optimum conditions for infection are 20-220C. in raw (as opposed to aged) sea water of 34 0/00 salinity. With increases in incubation temperature, the percentage of infection occurring in egg lamellae submerged in hypersaline waters also increases. Nutrition has essentially the same effect on salinity tolerance as water temperature: only on enriched agar media will L. chthamalophiliam grow vegetatively at salinities well above that of normal sea water. Ova of B. amphitrite denticulata become infected by L. chthamalophilum only at a temperature of 150C. in hypersaline water, or if the Balanus ova are subjected to below-freezing temperatures before exposure to the fungus. Lagenidiium chthamalophilum Johnson (1958) is a destructive pathogen in unfertilized ova and embryonic stages (not nauplii) of the barnacle Chthamalus fragilis Darwin.2 Two characteristics of L. chthamalophilum bear significantlv on pathogenicity and epidemiology. First, development of the inoculating units (planonts) from migration of undifferentiated protoplasm into the discharge vesicle to spore discharge (Johnson, 1958, p. 210, and fig. 13-17) occurs in about 20 min. in raw, unsterilized sea water. Many inoculating units may, therefore, be produced by sporulating hyphae in a relatively short time. Second, the time lapse between inoculation (contact of the planont with the ovum membrane) and infection (germ tube penetration and establishment of a hyphal rudiment within the egg) seldom exceeds 25 min. (Johnson, 1958, p. 209, and fig. 18a-c). Within 2 davs after a peripheral ovum in a lamella is invaded, all the remaining ova may be penetrated and the embryos killed by the fungus. Lagenidiumn chthamalophilum neither invades moribund Chthamalus ova, nor, apparently, infects eggs of the associated barnacle Balanus amphitrite denticulata, under natural conditions. Although Balanus ova may not be immune to attack by Lagenidium, the eggs are certainly highly resistant in nature (Johnson, 1958). The soundness of this premise has been investigated further. Three environmental factors are probably involved in epidemiology: (1) salinity-rapid and profuse planont development in raw sea water of about 34 0/00; (2) temperature-maximum mycelial development and accelerated sporulation at water temperatures of 25-27?C; (3) fouled water-retardation or inhibition of sporulation in water par1 Received for publication September 16, 1959. Financial support by the National Science Foundation, through Grant G-2324 and G-7178 is gratefully acknowledged. I am especially indebted to Drs. C. G. Bookhout, F. K. Sparrow and F. A. Wolf for their review and criticisms of the manuscript. 2 The host, Chthamalus fragilis denticulata, was misnamed when the fungus was first described. The varietal name, denticulata, applies to Balanus amphitrite. tially depleted of oxvgen (or high in carbon dioxide). What are the temperature and salinity tolerance limits of L. chthamalophilum? What influence does environment have on infection? These and other questions bear on epidemiology. The pathogen can be cultivated on agar in pure culture. and hence may be investigated autecologically, as was done by Mrs. Vishniac (1958) with Haliphthoros milfordensis, occurring in ova and embryos of Urosalpinx cinerea Say, the oyster drill. Mrs. Vishniac's study is one successful approach to epidemiology. A second approach, favored by the writer, deals with infection under more nearly natural conditions without employing pure cultures of either the pathogen or suscept. In the summers of 1957 and 1958, a general investigation was made of environmental effects on infection of Cht hamalus ova by Lagenidium chthamalophilum. The results of this exploratory ecological study are presented. MATERIALS AND METHODS.-Since the investigation dealt primarily with infection, a basic technique for insuring inoculation was necessary. Egg lamellae were dissected from Chthamalus, and placed in a few drops of sterile, raw sea water in Petri dishes. The lamellae subsequently were examined with a dissecting microscope to separate parasitized and unparasitized ones. Uninvaded ova masses were placed in drops of sterile, raw sea water and incubated for 24 hr. at 250C. These lamellae were examined, and if not visihly infected were subsequently used in the infection experiments. Sea water in all inoculation-infection experiments was obtained at 1-meter depth and filtered through cheesecloth. The water was used immediately (raw), or diluted with distilled water and then used. Partial evaporation of sea water in enamel pans provided hypersaline water. Fresh water came from a local pond. For inoculation, 2 lamellae (one infected, one uninfected) were placed in a Petri dish. Ten ml. of water (at a given salinity) were then added, the lamellae thus being submerged. Infection was re-