FUNGOUS DISEASES OP THE HONEYBEE 23 



In contrast to the findings of Eduard Buchner {J^), in his work on 

 the extraction of zymase from yeast, the enzymes of several species 

 of Aspergillus and Mucor were found to exist in much greater quan- 

 tities in the medium in which cultures have grown than within the 

 mycelium. Evidence in support of the view that the effect of patho- 

 genic fungi on bees is chiefly enzymatic and toxic was obtained from 

 the following experiments : 



Enzymes from the liquid medium in which pure cultures of Asper- 

 gillus flmnts, A. efwsm, and A. fwmgahDs had matured were ex- 

 tracted by precipitation with alcohol. After thorough drying the 

 precipitate was dissolved in distilled water. The digestive tract and 

 muscle tissues of bees that had been recently killed, entire bees, and 

 each of the three divisions of the body were submerged in this solu- 

 tion and kept at 37° C. while the action was observed. Tissues di- 

 rectly exposed to the action of enzymes were soon discolored and in 

 from 5 to 10 hours offered no noticeable resistance to pressure. Tis- 

 sues of thoraces, abdomens, and heads of bees with the exoskeleton 

 of each of these body divisions attached were affected similarly but 

 somewhat more slowly. The chitinous exoskeleton appeared to be 

 unaffected after 24 hours, whereas no evidence of enzymatic action 

 on the internal tissues of entire bees was noticeable after 30 hours. 

 It appears that the exoskeleton is not extensively attacked by any 

 of the enzymes produced by these fungi, although conidiophores 

 and mycelium penetrate the body wall of the bee when excessive 

 moisture is present. Ordinarily the only charmel of infection is 

 through the alimentary canal. 



The experiments described above were repeated with the filtered 

 medium from cultures, with sterile medium that had not been used 

 for culturing, and with distilled water. With the medium from 

 cultures the action was similar but somewhat slower than with ex- 

 tracted enzymes. With sterile, freshly prepared medium and with 

 water as checks, no enzymatic action upon tissues was noticeable after 

 48 hours. The medium, which was of a high sugar content (25 per 

 cent), appeared to act as a preservative, since bacterial decay of the 

 tissue occurred later than in distilled water. 



Dbmonstkation of a Toxic Substance Peoducbd by Aspekgikltts flavtjs Aobc 



It is a familiar fact that many of the diseases of animals and 

 plants are due to microorganisms which elaborate toxic substances 

 of some form or other. The exact nature of these substances, which 

 are at times partially responsible for the symptoms of the various 

 diseases, is not completely known. The inability of chemists to 

 determine their constitution is probably due to the impurity com- 

 plexity, and unstable nature of the molecule. It is possible by various 

 procedures to extract from cultures of pathogenic organisms sub- 

 stances that are more or less toxic. When toxins are properly ad- 

 ministered in the absence of the pathogenic organisms they may pro- 

 duce the symptoms of the disease. Severe poisoning may also be 

 produced by toxic products of nonpathogenic organisms. The po- 

 tency of these toxins, which may be many times as effective as strych- 

 nine, IS well known. Toxic substances are not uncommon among the 



