138 COLORATION IN LEPTINOTARSA. 



of decemlineata, undecimlineata, signaHcollis, nmltitceniata, and dilecta were 

 taken just as the development of pigment began, and the specimens of each 

 species were divided into three lots. Of these one lot was killed in a vapor of 

 HCN, and then placed in an atmosphere saturated with chloroform vapor; 

 another was put alive into the same conditions, and the third was kept for 

 control. Of those put into the atmosphere of chloroform vapor, all, both 

 living and dead, showed color development, and more than half in a norm.al 

 manner. 



I have shown in a former paper that if the pupae are put into chloroform 

 vapor before the pigment is ready to develop, color formation is permanently 

 inhibited; and this I find to be true with several species of Leptinotarsa. 

 These experiments point to the conclusion I had before reached that the 

 development of cuticula color is not due to external agencies, nor to secre- 

 tions, but to the action of an enzyme or katalytic agent upon the cuticula itself. 



During the last two years I have investigated the development of these 

 enzymes, especially in Leptinotarsa, and I find my later results wholly con- 

 firm those given in my former paper, but I have not been able to learn more 

 of the nature of the chemical processes involved nor of the compounds 

 produced. 



The existence of enzymes in the hypodermal cells can be shown in two 

 ways — by microscopical examination, or by extraction and partial isolation. 

 Their action has been demonstrated by microscopical study and experiment 

 with the extracted enzymes. 



The microscopical proof of the existence of enzymes and their action is 

 demonstrated by the study of the developing cuticula color in sections after 

 proper killing and staining. I have studied these substances in L. decem- 

 lineata, multitceniata, signaticollis, and dilecta with the same results in each 

 species, so that their development is probably the same in the entire genus. 



In the extraction of the chitases of Leptinotarsas I have made use of the 

 same methods given in a former paper (1903). Most of the extractions were 

 made in 35 per cent alcohol and 2 per cent glacial acetic acid, or in 50 per 

 cent alcohol and 10 per cent glycerin. Both proved satisfactory solvents. 



With extracts of decemlineata, midtitccniata, and signaticollis experiments 

 were tried that confirm and extend the observations given in my 1903 paper. 

 With the extract made from the pronotum of decemlineata experiments were 

 performed as follows : 



The experiments were always performed under as nearly aseptic conditions 

 as possible, in sterilized dishes, with distilled sterile water, and always in the 

 presence of chloroform vapor, thus insuring as nearly as may be freedom 

 from error. 



Experiment I. — One cubic centimeter of the alcoholic extract of the en- 

 zymes from the pronotum of decemlineata, and a similar amount from the 

 pronotum of signaticollis, plus o.i per cent glacial acetic acid, were placed in 



