22 TECHNICAL BULLETIN 149, U. S. DEPT. OF AGHICULTTJEE 



It appears that one must look for other causes responsible for disease 

 and death in the bees infected by fungi. 



To determine these causes of death, a histological and chemical 

 study was made of infected tissues from diseased and dead bees. 

 The chemical action of the fungus was determined by observing the 

 infected tissues, and -by testing the action of certain metabolic prod- 

 ucts produced by the fungus, when applied to the tissues in the 

 absence of the fungus. 



It was determined by examination of sections of the ventriculus 

 that hyphae penetrate the tissues soon after the spores germinate. 

 The wall is usually attacked first and is soon permeated with 

 mycelium. Other tissues of the abdomen and thorax are also 

 attacked by the fungus. 



That a mechanical effect is produced in the tissues by the advanc- 

 ing mycelium is shown by the forcing apart of the muscle fibers when 

 the hyphae grow among them. Later the mycelium becomes densely 

 interwoven among all of the softer tissues, and the mechanical effect 

 of this is probably sufficient to cause death. 



In view of the rapid softening of the tissues and the appearance of 

 weakness, with the consequent crawling condition, among infected 

 bees, it appears that enzymatic and toxic substances contribute 

 materially to the symptoms. The tissues of the bee are attacked by 

 the fungous enzymes in advance of the growing mycelium, but this 

 digestion is not completed until the mycelium is well developed. 

 The resultant softening of the muscle tissues of the thorax is followed 

 by a loss of the brownish tinge characteristic of healthy tissues and 

 the appearance of a dull white color. A sarcolemma is absent 

 about the wing-muscle fibers of the honeybee, and no support other 

 than the sarcoplasm is known to exist. The semifluid sarcoplasm 

 surrounding the sarcostyles is softened, permitting the muscle fibers 

 to separate readily. This probably accounts for the softening and 

 collapse of muscle tissue and the ready separation of the threads of 

 muscle fibers under pressure. 



Softening of the sarcoplasm surrounding the muscle fibers is 

 followed by the death of the sarcoplasm of the sarcotyles and the 

 disappearance of their membranous walls. In advanced stages of 

 digestion, i. e., after death, the sarcostyles break up into segments, 

 frequently separating at the telophragmata. After complete diges- 

 tion only formless granules remain. 



The muscles of the thorax are most easily studied, but the wall of 

 the ventriculus may be used successfully since it is attacked soon after 

 infection occurs. The digestion of sarcoplasm of the tissues of the 

 bee is probably largely proteolytic in nature, for protein is the chief 

 constituent of muscle tissue. It appeared, however, from some com- 

 parative measurements made by the writer that pathogenicity of the 

 organisms studied is in no way related to the quantitative production 

 of protease. It is possible that the sarcoplasm is killed by fungous 

 toxins before enzymatic action begins. 



The observations described above on infected tissues indicate that 

 the changes which occur after the death of the tissues are apparently 

 due to enzymatic action. If this is true, muscle tissue should be sim- 

 ilarly affected in fungus-free culture media or in solutions of ex- 

 tracted enzymes. 



