146 Journal of Agricultural ResearcJi voi. xxvm, No. 2 



well as Straus (Table IV), also give weights for larvse of known age, but 

 in order to eliminate the danger of variations due to the eflfect of different 

 seasonal and environmental conditions, the average age of the larvse analyzed 

 from various groups of 25 was determined by comparison with a series of weigh- 

 ings of larvse of known age that "were made during this same period (35) . The 

 various series of weights, with the corresponding determinations of reducing 

 sugar, were arranged in age groups, 24 hours apart, as shown ip Table V. In 

 some cases, such as the small two-day larvae, or the quiescent prepupffi, where 

 the amount of unassimilated sugar is small, 50 larvae were taken for analysis, 

 but usually 25 proved satisfactory. 



PREPAEATION OF MATERIAL FOE ANALYSIS 



Several difficulties were encountered in the preparation of material for sugar 

 determination. At first, attempts to extract the sugar were made by macerating 

 the larvae with distilled water and filtering through filter paper. This produced 

 a cloudy opalescent liquid, indicating the presence of colloidal material, and this 

 solution did not give the characteristic reaction with the Benedict reagent. 

 Various clarification methods were tried. Precipitation with both neutral and 

 basic lead acetate (10, p. 276) solutions proved unsatisfactory, something stiU 

 remaining to interfere with the reaction. Mercuric nitrate solution, which is 

 sometimes used to clarify liquids of animal origin such as blood, urine, and milk, 

 was tried {10, p. 447). This method occasionally gave good results, mainly 

 with the younger larvae, but often with older larvae and prepupae the colloidlike 

 material still remained in the filtrate, interfering with the reaction. Furthermore, 

 because of the numerous filtrations necessary to remove successive precipitates, 

 it was feared that more or less sugar is lost by adsorption to those precipitates, 

 even with careful washing. An attempt was made to clarify by filtration with 

 suction through a celloidin membrane, and this gave a clear solution which reacted 

 well with the test solution, but the method required too great time. The method 

 finally adopted was by extraction with 50 per cent alcohol, similar to the method 

 used in the extraction of sugars from grains and similar products (It). This 

 method proved successful, since the alcohol causes precipitation of all solid 

 matter, giving a clear filtrate which reacted properly with the Benedict's reagent. 

 Since glycogen in water solution is colloidal in nature, and thereby difficult to 

 remove by filtration from such a solution, it is doubtless the glycogen present in 

 the larva which prevented clarification and interfered with the reaction. It is 

 possible for this reason that Straus (43) failed to demonstrate reducing sugars. 

 To determine this point, a small amount of glycogen was added to a known solu- 

 tion of dextrose and tested with the copper sulphate solution, and the known 

 reducing sugars could not now be demonstrated quantitatively. Since glycogen 

 is insoluble in alcohol {10, p. 44S) the 50 per cent alcohol precipitates the glycogen 

 and thereby removes materials interfering with the reaction in the filtrate. 

 Even though there may stiU be a small loss of reducing sugar by adsorption or by 

 some other means, the results obtained are of value for purposes of comparison. 

 If any reducing sugar is lost by the method adopted, the amount is exceedingly 

 small and may therefore be disregarded, since repeated washings failed to demon- 

 strate its presence. 



TECHNIC ADOPTED 



After weighing, the larvae are renioved to a small porcelain mortar and mace- 

 rated in 30 CO. of 50 per cent alcohol. This material is then washed carefully 

 into a small flask and allowed to stand from two to three hours before filtering. 

 The precipitate is washed with 60 per cent alcohol. The filtrate is then made 

 up to 50 cc. with distilled water, and run into a burette. Five cc. of the stand. 



