Methods in Anopheles Research - MR4

Chapter 3 : Specific Anopheles Techniques3.6 Determination of Lipid, Glycogen, and Sugars in MosquitoesPage 1 of 43.6 Determination of Lipid, Glycogen and Sugars in MosquitoesAdapted from (Van Handel 1985a; Van Handel 1985b; Van Handel and Day 1988; Kaufmann andBrown 2008)Introduction by C. KaufmannThis method determines the lipid, glycogen, and sugar content of a single insect. It has the advantagethat reasonable results can be achieved with only a few numbers insects, which may be of greatsignificance in the field.The separation of lipid, glycogen and sugar is rather important because vast amounts of ingested sugar,as given via a feeding solution in the laboratory or sugar sources like nectar and honeydew that areingested in nature, can interfere with the lipid analysis (blue/violet coloring of the vanillin test). Also, thehot anthrone test does not differentiate between the ingested sugar within the crop and the carbohydratethat is already transferred to its storage form glycogen.The homogenization with sodium sulfate solution will help to co-precipitate the glycogen after the additionof the chloroform-methanol solution, in which the lipids and sugar dissolve; the addition of water allowsthe separation of the sugar and lipid (sugar in the upper phase and lipid in the lower phase). Be carefulwhen handling the chloroform and acid.Note that for larger insects, you should use different values of chloroform/methanol (2.8 ml instead of 1.6ml) and water (2 ml instead of 0.6 ml); the rest is the same.Materials1. Glass centrifuge tube2. Glass grinding pestle or stir rod3. Heating block at 90-110 o C4. Erlenmeyer flask5. CentrifugeReagents1. Sulfuric acid (95-98%)2. Anthrone3. Anhydrous glucose4. Sodium sulfate5. Methanol6. Commercially available vegetable oil (e.g. soybean oil)7. Vanillin8. Phosphoric acid (85%)9. ChloroformSolutions1. 2% sodium sulfate (NaSO 4 ) solution2. chloroform/methanol mixed 1:1 (v/v)3. Vanillin-phosphoric acid reagenta. Dissolve 600 mg vanillin in 100 mL DI hot water.b. Add 400 mL 85% phosphoric acid.c. Store in the dark. Stable for several months but discard if it darkens.4. Anthrone reagenta. Add 385 mL sulfuric acid (95-98%) to 150 mL DIb. Dissolve 750 mg anthrone