1921] Muttkowski: Respiration of Insects 151 
II. OxyGEN AND RESPIRATORY PROTEINS IN INSECT BLOoD. 
To determine the oxygen content of insect blood directly is a matter 
of some difficulty, primarily because such reagents as are available are 
suitable only for considerable quantities of material, and are not adapt- 
able for use with the microscopic quantities dealt with in the present 
experiments. For instance, an excellent reagent is pyrogallic acid, 
which in presence of an alkali absorbs oxygen with avidity. When 
testing for oxygen in the few drops of blood obtained from an insect, 
the difficulty lay in occluding atmospheric oxygen. Yet several experi- 
ments indicated the presence of oxygen in the blood; for the reagent 
darkened much mcre rapidly when applied to insect blood (pyrogallic 
acid turns brown to black with absorbed oxygen) than in the blank 
ontrol tests. Yet at best this was unsatisfactory, as at least scme 
portion of the reagent was in contact with atmospheric oxygen and 
tended to obscure the blood reaction. Nor could this be obviated with 
the use of cell slides and vaselined cover slips. 
However, the same reagent was applied to Dytiscus larvae by another 
method which was more effective. Freshly prepared pyrogallic acid 
solution was injected into the body cavity of the larvze, followed by 
weak hydroxide. Death ensued quickly, in periods ranging from tén 
seconds to three minutes. Even before dissection the semi-transparent 
larvee showed brown stains inside. The dissected larvee showed brown 
blotches in the haemocoel and among the tissues, indicating that 
oxygen had been absorbed by the reagent. In the vicinity of the 
tracheze the reagent was deeper brown, while the tracheze themselves 
were almost black. Here, too, once the animal was cut open, atmos- 
pheric air had access to the reagent, which then blackened rapidly, 
obscured the tissues, and prevented more detailed study. 
No doubt some of this reaction was due to oxygen dissolved in the 
blood serum, although the serum has no power to combine with oxygen 
like a respiratory protein. This oxygen, in mammalian serum, amounts 
to 94% of the amount soluble in water and the serum loses its supply 
with increased temperature. 
These experiments, which were repeated a number of times with 
Dytiscus, Aeshna, Chironomus, and other larve, are significant from 
at least the qualitative standpoint. They do not indicate whether 
an active agent was present which combined loosely with the oxygen, 
or if the gas was in physical solution as in the water. The topic was, 
therefcre broached from another angle—namely, that of the presence 
or absence of respiratory proteins. The obvious postulate was: If a 
respiratory pigment can be demonstrated in the blood of insects. its 
purpose must be to fix and transport oxygen. In that case, the blood 
of insects has the additional function of aiding in the respiration. 
Such a respiratory protein need not necessarily be visible to the 
naked eye. For, in the first place, among the insects we are dealing 
with minute quantities of blood. Secondly, such a pigment wculd play 
only a subsidiary role, as the primary supply of oxygen is received by 
way of the trachee. The quantities of gas in soluticn in the blood 
