Carbohydrate accumulation and utilization by oocytes of Rhodnius prolixus.

The processes of accumulation and mobilization of carbohydrate stores in eggs of Rhodnius prolixus were analyzed. During oogenesis, the total amounts of glycogen, glucose, and trehalose increased with an accumulation of proteins, especially when oocytes grew from 1.0 to 1.5 mm in length. At 2.0 mm length, when oocytes were ready for oviposition, nutrient reserves did not increase appreciably and trehalose content decreased. Mating did not affect the final content of carbohydrates or proteins in oocytes of mated and virgin females. A trehalase activity was detected in follicles containing vitellogenic oocytes, 1.0 and 1.5 mm length, in both mated and virgin females. This activity was extremely low in chorionated, 2.0-mm oocytes. After oviposition, glycogen content decreased in fertilized eggs, but not in unfertilized ones, and some was present in newly hatched nymphs. Glucose content remained constant in unfertilized eggs, but increased in fertilized ones, while total protein amount was constant in both groups after egg laying.

Absorption of toxic beta-glucosides produced by plants and their effect on tissue trehalases from insects.

Trehalases present in body wall, Malpighian tubules, fat body, midgut and haemolymph from Tenebrio molitor (Coleoptera), Musca domestica (Diptera), Spodoptera frugiperda and Diatraea saccharalis (Lepidoptera) were assayed in the presence and absence of toxic beta-glucosides produced by plants or their aglycones. The glucosides used were phlorizin, amygdalin, prunasin and the aglycone mandelonitrile. In addition, T. molitor and S. frugiperda trehalases were assayed with and without esculin. More than 60% of total trehalase activity was found in the midgut of these insects. As a rule, trehalases present in each insect were inhibited by at least two of the glucosides. Prunasin was the best inhibitor in tissues with highest trehalase activity. S. frugiperda beta-glucosidases were not able to hydrolyze esculin. Nevertheless, their larval midguts absorb the intact glucoside that is recovered from the fat body, Malpighian tubules and mainly from haemolymph. Mature larvae fed on a diet containing 3 mM (0.1%) esculin have 0.2 mM esculin in their haemolymph, and weigh 60% of control larvae. In vitro, haemolymph trehalase activity is abolished by 0.5 mM esculin. This inhibition may play a role in the decrease of body weight and in animal survival. S. frugiperda larvae reared in 0.1% amygdalin-containing diet present higher trehalase activity in tissues than the larvae reared in 0.1% esculin-containing diet. Higher trehalase activity should be the reason why the S. frugiperda development is not impaired by 1% dietary amygdalin, in contrast to what is observed when insects are reared in 0.1% esculin. The data suggest that many plant beta-glucosides are toxic because they inhibit trehalase, a key enzyme controlling glucose availability in insects.

Determination of trehalose in biological samples by a simple and stable trehalase preparation.

A three step purification procedure for trehalase from Saccharomyces cerevisiae with a recovery of 76% of the original activity is presented. The enzyme was activated by a heat shock treatment prior to homogenization of the cells. A mutant strain deleted in SUC genes was used to avoid contamination by invertase. The lyophylized enzyme was stable for, at least, 5 months and could be used to determine trehalose in the range 25 to 500 nmol. The preparation was free of inspecific phosphatases allowing for trehalose determinations in yeast cell free extracts and in insect hemolymph.