Gene identification and enzymatic properties of a membrane-bound trehalase from the ovary of Rhodnius prolixus.

Trehalose represents the main hemolymph sugar in most insects and its metabolic availability is regulated by trehalase. In this study, trehalase activity associated with the reproductive system was investigated in the insect Rhodnius prolixus, a hematophagous hemipteran vector of Chagas' disease. A single-copy gene that encodes a membrane-bound trehalase (RpTre-2) was identified in the genome of R. prolixus. RpTre-2 deduced amino acid sequence is closely related to other insect membrane-bound trehalases. The expression of this gene was detected in all analyzed organs, including ovary, where total trehalase enzymatic activity was determined, and was highest at day 7 after blood meal. Ovary membranes showed a major trehalase specific activity, which confirmed the presence of a membrane-bound trehalase in this insect. This trehalase activity seemed not to be regulated at transcriptional level, as the expression of RpTre-2 gene in the ovary did not change over the days after feeding. Similarly, ovarian follicles at different developmental stages did not show any variation in the transcription level of this gene. The RpTre-2 kinetic parameters were also investigated. Activity was highest at pH 5.5 and followed Michaelis-Menten kinetics, with an apparent K(m) = 1.42 ± 0.36 mM and Vmax = 167.90 ± 12.91 nmol/mg protein/h. These data reveal the presence of a membrane-bound trehalase in R. prolixus that is active in ovary and probably takes part in the insect carbohydrate metabolism associated with the reproductive process.

Lipid accumulation and utilization by oocytes and eggs of Rhodnius prolixus.

Insect eggs must contain the necessary nutrients for embryonic growth. In this article, we investigated the accumulation of triacylglycerol (TAG) in growing oocytes and its utilization during embryonic development. TAG makes up about 60% of the neutral lipids in oocytes and accumulates as oocytes grow, from 2.2 ± 0.1 µg in follicles containing 1.0 mm length oocytes to 10.2 ± 0.8 µg in 2.0 mm length oocytes. Lipophorin (Lp), the hemolymphatic lipoprotein, radioactively labeled in free fatty acid (FFA) or diacylglycerol (DAG), was used to follow the transport of these lipids to the ovary. Radioactivity from both lipid classes accumulated in the oocytes, which was abolished at 4°C. The capacity of the ovary to receive FFA or DAG from Lp varied according to time after a blood meal and reached a maximum around the second day. (3) H-DAG supplied by Lp to the ovaries was used in the synthesis of TAG as, 48 hr after injection, most of the radioactivity was found in TAG (85.7% of labeling in neutral lipids). During embryogenesis, lipid stores were mobilized, and the TAG content decreased from 16.4 ± 2.1 µg/egg on the first day to 10.0 ± 1.3 µg on day 15, just before hatching. Of these, 7.4 ± 0.9 µg were found in the newly emerged nymphs. In unfertilized eggs, the TAG content did not change. Although the TAG content decreased during embryogenesis, the relative lipid composition of the egg did not change. The amount of TAG in the nymph slowly decreased during the days after hatching.

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.