2. Measurement of glucose and trehalose in honey bee haemolymph
This protocol describes a sensitive enzymatic method for quantifying glucose and trehalose in honey bee haemolymph. Haemolymph sugar levels do not only need to be regulated, they also provide important information on the physiological state of carbohydrate metabolism, which is associated with gustatory sucrose responsiveness (Amdam et al., 2006; Wang et al., 2012). In general, the glucose titre in honey bee haemolymph is below 20 µg/µl and the trehalose titer is below 30 µg/µl (Wang et al., 2012).
The method allows measuring of glucose concentrations from 0.5 to 100 µg/µl, and trehalose from 0.4 to 94 µg/µl. Such sensitivity makes the method appropriate for estimation of glucose and trehalose in most insect body fluids, without prior concentration or extraction. Furthermore, these methods are specific, reproducible, sensitive, high throughput and do not require extensive sample preparation. Measuring other carbohydrates of the honey bee colony, such as sugar concentration in honey or the crop of foragers, does not require such a sensitive method and can be easily done by means of a refractometer.
The glucose method described herein is based on the reaction between glucose and adenosine triphosphate (ATP). Glucose can be phosphorylated by adenosine triphosphate (ATP) to form glucose- 6-phosphate (G6P) in a reaction catalysed by hexokinase. G6P is then oxidized to 6-phosphogluconate in the presence of oxidized nicotinamide adenine dinucleotide (NAD+) in a reaction catalysed by glucose-6-phosphate dehydrogenase (G6PDH). During this oxidation, an equimolar amount of NAD+ is reduced to NADH, which consequently can be spectophotometrically detected as an increase in absorbance at 340 nm (Peterson and Young, 1968; Bondar et al., 1974). The amount of NAD+ consumed in the reaction is directly proportional to the amount of glucose present in the sample. For quantifying trehalose, this disaccharide must first be hydrolysed to two molecules of D-glucose in a reaction catalysed by trehalase (Broughton et al., 2005; Flatt and Kawecki, 2007).