154 Annals Entomological Society of America [Vol. XIV, 
and less, until it is zero at boiling point of the water. Furthermore, 
as the temperature of water rises above zero, decomposition and oxida- 
tion of wastes increase, so that the available supply of dissolved oxygen 
is used up in inverse ratio to its absorption by the water. 
If respiration in aquatic insects, specifically in water-breathers, 
proceeded on the basis of a physical gas equilibrium on two sides of a 
membrane, the insect would soon show a deficiency of oxygen, and 
that at a time when it is most active and its metabolism demands a 
high rate of oxygenation. Yet this is precisely what we do not find. 
Regardless of the impoverished oxygen in warm pond or swamp water, 
the insect blood contains a plentiful supply of oxygen, and metabolism 
proceeds at its normal rate. Thus, in Aeshna, Dytiscus, and Chiro- 
nomid larvee, purposely kept in covered jars filled with decaying plant 
and animal matter, I found normal activity and the blood reacted 
copiously with injections of Pyrogallic acid. It is evident, therefore, 
that the blood possesses a protein which is capable of binding oxygen in 
excess of the amounts dissolved in the water. The incinerations showed 
that this protein is a copper compound. 
From this standpoint, the various respiratory structures of insects, 
especially the gill filaments and gill pouches of Trichoptera larve and 
aquatic caterpillars, the caudal blood gills of Chironomus larve, of 
Culicid larvze, Simulium larve, etc., acquire a real significance. These 
structures are purely blood gills, consisting of a thin cuticle and epi- 
dermis and their lumina are continuous with the haemocoel, so that 
the blood courses freely through them. With this type of gill, a 
respiratory protein to fix oxygen is very effective. On the other hand, 
unless such a protein is present, these structures loose their significance, 
in fact, appear useless as organs and inefficient physiologically. 
Ill. CArBoN D1o0xIDE In INSEcT BLOOD. 
The presence of carbon dioxide in insect blood has been reported 
frequently. During the summer of 1920 I was able to verify this fact 
in several series of experiments. The insects used were the same species 
utilized in the incinerations listed in the preceding section. The 
reagents employed were Potassium and Barium hydroxides, Lead 
acetate, Rosolic Acid, and halogen acids, the latter for carbonates. Of 
these Potassium hydroxide was perhaps the least satisfactory, as it 1s 
difficult to find the proper concentration of this reagent to give a definite 
reaction. Barium hydroxide reacts well with insect blood, yielding an 
amorphous precipitate. Lead acetate when applied directly will 
precipitate some of the blood proteins in addition to the carbon dioxide. 
When used with a micro-still, however, it is more effective and con- 
clusive. I have used it as follows: A few drops of insect blood are 
caught on glass wool in a small glass or porcelain crucible, over which is 
placed a slide with a hanging drop of Lead acetate. Gentle heating 
releases the carbon dioxide, which forms Lead carbonate with the 
reagent. 
