FUNGOUS DISEASES OF TflE HONEYBEE 15 



and separate the spores. The dry spores were then shaken only 

 lightly over the brood. If too many spores were shaken into the cells, 

 the worker bees set to work at once to clean them out, and sometimes 

 removed the brood before any infection could be determined. If the 

 inoculated brood is not removed by the workers within 24 hours, it is 

 usually allowed to remain in the comb unless death from infection 

 occurs. 



A somewhat surer method was used with fungi that do not produce 

 abundant spores, and when the evidence obtained with dry spores was 

 not considered conclusive. Water suspensions of spores were pre- 

 pared and small drops added to the larval food with a pipette drawn 

 to a slender tapering point. Larvae were rarely removed from the 

 cells following this treatment except after infection occurred and 

 death had resulted. Close observation, however, was always necessary 

 to determine the exact fate of the inoculated brood. In strong colo- 

 nies the workers often remove the brood before infection can result if 

 the inoculation is heavy, or they may remove them from the cells, 

 often piecemeal, before symptoms of the disease appear. This method 

 offers quick and easy means, however, of testing the pathogenicity 

 of a large number of organisms for brood of bees. 



While examining brood inoculated by the methods just described 

 it was occasionally noticed that tufts of mycelium appeared on the 

 brood before other symptoms of diseases had developed. (PI. 1, D.) 

 Microscopical examination of such larvae showed that these tufts had 

 developed from mycelium beneath the skin of the larvae. This 

 mycelium was much more abundant in the tissues beneath the tufts 

 than elsewhere. If infection 'occurred within the alimentary canal 

 one would expect to find mycelium more extensively developed there, 

 at least immediately following infection, than in the tissues just be- 

 neath the larval skin. It appeared that the mycelium observed on a 

 few larvae soon after inoculation originated from infection through 

 the skin rather than from within the alimentary canal. 



To find whether germ tubes from spores germinating upon the 

 moist skin of larvae can penetrate the skin to the tissues beneath, 

 masses of spores, each about the size of a pinhead, were smeared on 

 a number of larvae in brood combs. The position of these within the 

 comb was marked by removing the brood from the two surrounding 

 rows of cells. The infected larvae were examined twice daily during 

 the three days following. In another method that was used larvae 

 were removed from the comb and kept in watch crystals on a 70 per 

 cent solution of sugar sirup or honey during the experiment. Masses 

 of spores were placed on the larvae in the same manner as if they had 

 been left in the comb. The watch crystals with the larvae were kept 

 between observations in an incubator at 36° C. This method was 

 finally used to the exclusion of the f ormerj for when the larvae were 

 left in the hive, spores placed upon the skins were often removed by 

 the workers or mixed with the food in the cells as the result of move- 

 ments of the inoculated larvae. This frequently resulted in infection 

 through the alimentary canal. If care was used in placing the moist 

 mass of spores on the skin of the larvae in watch crystals, spores 

 rarely reached the alimentary canal. Larvae move very little when 

 on sirup ; consequently the spores remained in the position in which 

 they were placed. 



