CAUSE OF CUTICULA COLORS. 139 



sterile test tubes and diluted with 1 cc. of distilled water. After twelve 

 hours, into each of these tubes was placed the pronotum of the corresponding 

 species, which had been taken from the pupa: before the enzyme had de- 

 veloped in the hypodermis below, and had been freed from all hypodermal 

 cells. After thirty-six hours the tubes were examined, and each pronotum 

 had developed a normal dark-brown color over its entire surface, but without 

 any tendency to form spots. 



Experiment II. A series of tubes were prepared as in Experiment I, and 

 were placed at temperatures of o ,5 ,io ,i5 ,20 ,30 ,35,40 , 42 , and45 

 C, and at the expiration of forty-eight hours the cultures were examined. In 

 the tubes at o, 5 , and io C. no action had taken place. In those from 15 

 to 30 C. results like those in Experiment I were found. In tubes kept at 35 

 to 40 C. the development of color was accelerated with the increase of tem- 

 perature, but at 42 C. development was practically wanting, and at 45 C. 

 no color whatever appeared. At the expiration of seventy-two hours, in the 

 tubes at low temperatures, color was found to be developing in those at io 

 C, and at 5 C. a trace was found, but none at o C. At the end of ninety- 

 six hours color development had ceased in all tubes above 15 C, the deepest 

 colors being produced in tubes kept at 35 to 40 C. At the end of ten days 

 the color development had entirely ceased in all tubes, with the result that at 

 O C. no color developed, at 5 to io C. dark color appeared, from 15 to 

 30 C. color as in Experiment I, and at higher temperatures as stated above. 



Experiment III. To tubes of extract prepared as in Experiment I above, 

 the pronota of species not proper to the extract were added ; i e., in extract 

 from signaticollis pronota of decemlineata were placed, and vice versa. These 

 were kept at laboratory temperature (17 to 22 C). At the end of three 

 days the tubes were examined and a trace of color was found ; at the end of 

 six days this had deepened somewhat, but rather irregularly, and all activity 

 had ceased at the end of ten days. 



From the preceding experiments and study of the development of these 

 colors in Leptinotarsa, it follows that cuticula colors deevlop as the direct 

 result of an enzyme action upon the primary cuticula, which produces azo 

 colors and at the same time a hardening of the cuticula. It further appears 

 that color development in experiment is stopped at o C. and at 40 C, that 

 it is most developed at about io C. and at 35 to 40 C, and that there are 

 average conditions between these two. Color appears in spots under natural 

 conditions, and over the entire surface in experiment, which indicates that 

 the color-producing enzymes are produced only in certain areas and not over 

 the entire surface. It seems probable that a group of closely related enzymes 

 are at work, the most general one being chitase, which produces the harden- 

 ing of the cuticula. 



