H. A. SCHNEIDERMAN 53 



ingly low respiratory quolienls (R.Q.) are commonly recorded, especially al 

 low temperatures. Indeed, in our own experience, continuous measurements of 

 oxygen uptake and carbon dioxide output of Cecropia pupae at io°C over as 

 long as 4 days often yielded R.Q.'s as low as o.i. These low R.Q.'s have formed 

 the basis of various theories of diapause, most recently the theory of Agrell (i), 

 which held that diapause was due to a defect in the enzyme systems responsible 

 for decarboxylation, more particularly a deficiency of thiamine necessary for 

 cocarboxylase. Under this view, PGH repaired the defect in the decarboxylase 

 system. 



In our efforts to understand these low R.Q's we soon became aware of their 

 remarkable origin. While the pupa consumes oxygen continuously, it stores 

 most of its metabolic carbon dioxide and releases it in brief 'bursts' which in 

 Cecropia at io°C occur every 3 to 7 days (25, 28, ^^, 37). When this discontinu- 

 ous release of carbon dioxide is taken into account in respiratory measurements, 

 the R.Q. of the pupa regains normal proportions, namely 0.78. 



Since the over-all pattern of gas exchange of the diapausing insect reveals no 

 qualitative peculiarities, let us examine the pathways of intermediary metab- 

 olism. It had been observed more than 20 years ago by Bodine and Boell (6, 7, 

 26) that in the eggs of the grasshopper Melanoplus dijfereiitialis pronounced 

 alterations in the cytochrome system occurred in synchrony with the termina- 

 tion of diapause. For this reason attention focuses on the role of terminal oxi- 

 dases in relation to the action of PGH (cf. 47, 51). 



The principal terminal oxidases which have been demonstrated in animals 

 are cytochrome c oxidase, cytochromes of the b type, flavoproteins and tyro- 

 sinase. An effective method of detecting the participation of the various ter- 

 minal oxidases in respiration and in physiological processes like growth is the 

 use of metabolic inhibitors, especially cyanide, phenylthiourea and carbon 

 monoxide. Cyanide inhibits cytochrome oxidase, catalase, peroxidase and 

 tyrosinase, while phenylthiourea inhibits only tyrosinase. In insects where 

 hemoglobins are absent, carbon monoxide inhibits both cytochrome oxidase 

 and tyrosinase but apparently fails to inhibit any other enzymes or substrates. 

 Carbon monoxide's inhibition of cytochrome oxidase is reversed by light, its 

 inhibition of tyrosinase is not, and it therefore affords a remarkably specific 

 tool for tracking the participation of the cytochrome oxidase system in bio- 

 logical reactions. 



The effects of carbon monoxide on cytochrome oxidase are reversed by oxy- 

 gen, that is, inhibition depends on the CO/O2 ratio. A ratio of 16:1 inhibits 

 about 70 per cent, while 25: i inhibits 85 per cent. To achieve such ratios with- 

 out lowering the oxygen pressure to a point where it limits respiration, respira- 

 tory measurements were performed at positive pressures. As Paul Bert (5) long 

 ago showed, gaseous pressure per se is without conspicuous effects on insects 

 unless it is exceedingly high. Animals were enclosed in a specially designed high 



