16 B. GRASSI AND A. SANDIAS. 



The experiment was therefore repeated several times with 

 increased care in the selection of specimens, and with the em- 

 ployment of wood which had been occupied by Termes (many 

 parasitic Protozoa of which are not conspecific with those of 

 Calotermes). 



In this way it was possible successfully to establish several 

 nests which remained free from Protozoa for over a month. 

 Nevertheless only a few examples from among all those of 

 suitable age became substitute forms. 



It is thereby demonstrated that the mere ab- 

 sence of Protozoa is insufficient by itself to stimu- 

 late the maturation of the genital organs. But it 

 is certain that the first indications which point 

 to the development of a substitute form date from 

 their disappearance. However, this disappearance 

 also takes place in examples approaching an ecdysis, 

 and by the use of tube-nests it can be made out with 

 certainty that the incipient substitute royal forms 

 (larvae) undergo a moult a few days after the eyes 

 begin to darken. The ecdysis may therefore be re- 

 sponsible by itself for the disappearance (death) of 

 the Protozoa, which would then be indicated as a 

 secondary phenomenon. 



11. Nests of examples free from Protozoa are difficult to 

 keep alive. This raises the question whether the cause of 

 death is due to the absence of the parasites, or simply that the 

 inmates are in process of moulting, or are just entering or 

 quitting a moult. Recently moulted examples have an espe- 

 cially tender cuticle, and undoubtedly require a greater amount 

 of moisture in the air and food ; they are therefore more deli- 

 cate, and this alone may sufficiently explain the difficulty in 

 question. But I shall return to this point later. 



13. Examples in process of becoming royal substitutes 

 reached maturity in the tubes in very few cases (after three or 

 four months ; the tubes in question were packed in a box and 

 taken to North Italy and Germany). As a rule, the nest dies 

 away before they are fully developed. 



