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The composition of insect blood, or hemolymph; has been extensively investigated, a recent review on the subject being that of Buck (1953). Most of this work has been concerned with the chemical differences between phylogenetic groups or with the changes in major groups of metabolites during metamorphosis. Only one study of which we axe aware (Levenbook, 1950 c) had as its object the formulation of a physiological solution for use in studies on an insect's tissues. The majority of "insect Ringer solutions" in use (see Buck, 1953, p. 161) bear no relation to the composition of insect body fluids, and the lack of a balanced physiological solution hindered experiments by one of us (S. S. W.) on culturing tissues of the silkworm, Bombyx mori L. To assist in formulation of such a solution, it was decided to obtain more detailed information on the composition of silkworm hemolymph, making use of some recently developed techniques, notably chromatography on filter paper. Our principal object was the estimation of individual compounds, such as free amino acids and sugars, on which there is little published :information. We also measured hydrogen ion concentration and nitrogen and phosphorus distribution. To give the results more physiological significance, we examined hemolymph from a series of developmental stages of the Silkworm, and also single samples from each of two other insect species. Although it was necessary to conclude the study while it was very incomplete, the results were of value in the formulation of a tissue culture medium, as described in the preceding paper (S. S. Wyatt, 1956), The results also demonstrate that a substantial proportion of the organic components of insect hemolymph is yet unidentified, and suggest that further work on the composition and physiology of this fluid would be rewarding.

I953 2. After injection of 82p into young rats the specific activity of the free glycerylphosphorylethanolamine after short periods was always greater than that of the phosphatidylethanolamine. This suggests that glycerylphosphorylethanolamine is not an in vivo breakdown product of phosphatidylethanolamine, although other considerations indicate that it does not lie on the main synthetic pathway. 3. The specific activity of a combined form of glycerylphosphorylethanolamine labile to 10% trichloroacetic acid at 40 was always identical with that of phosphatidylethanolamine. The specific activity of the ethanolamine-containing acetal-, phospholipid was also similar. 4. The synthesis of radioactive glycerylphosphorylethanolamine by minced rat brain has been accomplished. No invitro synthesis ofphosphatidylethanolarnine could be demonstrated.

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A curious feature of insect blood, shown by many analyses, is the apparent absence of all but minute amounts of sugar (for reviews, see Beutler, 1939;Babers, 1941;Buck, 1953). Reducing substances are abundant, but (except in the case of a few species) the greater part of these are not fermentable by yeast and are therefore presumably not sugar. Typical are data on the silkworm, Bombyx mori, in which the reported levels of blood sugar range from zero up to about 30 rag. per 100 ml. (Florkin, 1937;Kuwana, 1937; and others) --remarkably small amounts for a metabolically active animal with a high dietary intake of sugars. Twenty years ago, however, Kuwana (1937) made the significant discovery that acid hydrolysis of Bombyx hemolymph caused release of much reducing sugar from a substance which was not glycogen. Even earlier, Ronzoni and Bishop (1929) had reported an unidentified non-reducing "polysaccharide below glycogen" in the blood of honeybee larvae. These reports received little attention. Recently, unaware of them, we made essentially the same finding. The blood of Bombyx mori and of two other insect species was found to contain large amounts of anthrone-reactive material which was neither a reducing sugar, sucrose, nor glycogen (Wyatt, Loughheed, and Wyatt, 1956).As already reported in preliminary form (Wyatt and Kalf, 1956), we have now identified as a major sugar of insect plasma the non-reducing disaccharide a,a-trehalose. In the present paper, we give details of the isolation and characterization of this sugar together with some quantitative analyses of trehalose and certain other carbohydrates in insect plasma. Their distribution and significance will be considered.