266 Journal oj Agricultural Research ^'ol. 45, No. 5 



In the first series of inoculations the number of spores fed each 

 larva ranged from approximately 5,000 down to 1. None of the 

 larvae inoculated developed disease. (Table 2.) Later a second 

 series of inoculations was made. The same colonies were used be- 

 cause of the limited number available^ but the larvae inoculated were 

 in a different comb in each colony and a different color was used to 

 mark the cells. In these inoculations the number of spores fed 

 ranged from 5,000,000 down to 1,000 per larva. No disease devel- 

 oped from this set of inoculations. 



It vv-as thought possible that the nurse bees might be removing 

 most, if not all, of the inoculated sugar sirup before the larvae had 

 had time to ingest a sufficient number of spores to bring about infection. 

 Therefore, in a third series of experiments each inoculated comb was 

 placed in a screen-wire queen-nucleus introducing cage, and this cage 

 was put back in the colony for periods ranging from one-half to one 

 hour before the unprotected comb was replaced in the colony, thus 

 theoretically giving the larvae time to ingest some of the sugar sirup 

 before the nurse bees had access to the inoculated cells. In these 

 tests the larvae were kept from the bees so long that many of them, 

 becoming hungry, were starting to crawl from the cells. The number 

 of spores fed ranged from 50,000,000 down to 500,000 per larva. 

 Twenty-four hours after the larvae were fed it was found that all re- 

 ceiving 50,000,000 and 25,000,000 spores had been removed from the 

 cells, while those receiving a smaller number of spores were either 

 partly removed or remained in the cells, according to the strength of 

 the dilution and the length of time that the larvae were kept away 

 from the nurse bees. (Table 2.) 



Two days later another set of larvae was inoculated with the same 

 dilutions as were previously used for these colonies but on the other 

 ■side of the same combs. In this series the combs were kept away 

 from_ the bees for periods ranging from 5 minutes for the heaviest 

 dilution to 30 minutes for the weakest. Again all the larvae receiving 

 the 50,000,000 and 25,000,000 spores were removed, while those 

 receiving the 5,000,000, which were kept from the bees for half an 

 hour, were partly removed, and those receiving 7,500,000 or 10,000,000 

 were not removed. Apparently there are two factors concerned in 

 the removal of the larvae — the length of time they are kept away 

 from the bees and the amount of foreign matter in the sirup, as indi- 

 cated by the spore content, that is given to the larvae. 



The results of the last two series of inoculations showed that in the 

 colonies in wliicli the larvae were not removed, or were not entirely 

 removed, several larvae in the colony receiving 10,000,000 spores per 

 larva developed disease, while those in the colonies receiving a smaller 

 number remained healthy. (Table 2.) This work should be repeated 

 with a different colony for each set of inoculations, although appar- 

 ently the disease did not spread in the colonies used. Only one colony 

 of the entire number developed disease, xilthough a certain degree 

 of success was obtained, these results seem to bear out Toumanoff's 

 {29) conclusion that the artificial infection of individual larvae is not 

 brought about so easily as one had been in the habit of believing. 

 Apparently, also, a considerable number of spores are necessary to 

 establish an infection under these conditions. 



