152 Journal of Agricultural Besearch voi. xxviii, No. 2 



be found that most of the glycogen has disappeared, although the iodin solu- 

 tion gives a light yellowish brown color. The presence of a trace of reducing 

 sugar also occasionally can be demonstrated with Benedict's solution in dis- 

 eased material of this type where vegetative organisms are stiU actively present. 

 In material which has decomposed completely, has reached the dark brown 

 ropy stage (fig. 9), and contains only spores of Bacillus larvae, glycogen is found 

 to be completelj' absent, nor can any reducing sugar be demonstrated, the 

 sugars having been completely destroyed. 



This type of material stained with Sudan III or osmic acid {S^, p. 78) shows 

 fat globules in practically the same condition and amount as in healthy larvae, 

 so that fat is apparently not acted upon by Bacillus larvae even after drying down 

 to the scale stage. 



Glycogen of the fat body of the healthy larva is hydrolyzed to dextrose to be 

 used in metamorphosis, by the action of enzyms during the histolytic processes 

 subsequent to sealing and prior to metamorphosis. This enzym action is demon- 

 strated by the following e.xperiments: 



EXPERIMENTAL PBOCEDURE 



Several series of 50 healthy prepupae each that had reached the period of 

 quiescence were macerated in 25 cubic centimeters of 50 per cent alcohol and 

 incubated at 37° C. for from 3 to 24 hours. The extract was then filtered and 

 diluted with an equal amount of water. A series .of test tubes were prepared, 

 using for each tube 5 cubic centimeters of this extract and 5 cubic centimeters of 

 0.4 per cent glycogen in water, and also another series using 5 cubic centimeters 

 each of a 0.1 per cent soluble starch. Both glycogen and starch were used, since 

 it has been shown by Bradley and KeUersberger (S), as well as bj* experiments 

 by the writer using commercial Taka-diastase, that diastase acts similarly on 

 both glycogen and starch. These tubes were incubated for various periods and 

 then tested with iodin solution for the presence of glycogen and starch (Table 

 VI) . Hydrolysis of both glycogen and starch seems to be complete after incuba- 

 tion for about five hours, and positively complete after incubation overnight, 

 demonstrating the presence of diastase in the prepupae. 



In another experiment 50 prepupae were macerated in 50 cc. of water and in- 

 cubated at 37° C. for 24 hours. Then sufiicient 95 per cent alcohol was added to 

 precipitate any glycogen present, and the solution was filtered and tested with 

 both the qualitative and the quantitative Benedict's solutions. In both cases 

 definite traces of reducing sugar could be demonstrated, none having been present 

 in the original solution before incubation, again demonstrating enzym activity 

 of the larval tissues. This may have been due to action by bacterial contamina- 

 tion, but if such had been the case the sugar would probably have been fermented 

 and could not have been demonstrated. 



In a similar manner extracts with 50 per cent alcohol were made of ropy dis- 

 eased material, enzym activity being demonstrated in the same manner as above. 

 This, however, does not indicate whether the organism causing the disease has 

 any diastatic power or whether the reaction was due to enzyms remaining in 

 the decomposed tissues. Further extracts were made with 25 per cent and 50 

 per cent alcohol of several 48-hour vegetative cultures of Bacillus larvae grown on 

 egg-yolk suspension medium. These extracts showed definite enzym activity 

 with glycogen after a few hours' incubation, and more positive activity after 

 incubation overnight (Table VI), while with starch marked hydrolysis was shown 

 'after only a few hours' incubation. 



