An aspartic proteinase precursor, herein named BYC (Boophilus Yolk pro-Cathepsin) was isolated from eggs of the hard tick, Boophilus microplus. As judged by electrophoresis on sodium dodecyl sulfate polyacrylamide slab gel (SDS-PAGE), purified BYC presented 2 bands of 54 and 49 kDa, bearing the same NH2-terminal amino acid sequence. By Western blot analysis, BYC was also found in the haemolymph, indicating an extraovarian site of synthesis. Several organs were incubated in culture medium with [35S]methionine, and only the gut and fat body showed synthesis of BYC polypeptides. Protein sequencing of both the NH2-terminal and an internal sequence obtained after cyanogen bromide (CNBr) cleavage of BYC revealed homology with several aspartic proteinase precursors. Incubation at pH 3.5 resulted in autoproteolysis of BYC, which produced the mature form of the enzyme, that displayed pepstatin-sensitive hydrolytic activity against haemoglobin. Western blot analysis using anti-BYC monoclonal antibodies showed proteolytic processing of BYC during embryogenesis and suggested activation of the enzyme during development. A role of BYC in degradation of vitellin, the major yolk protein of tick eggs, is discussed.

BackgroundThe mosquito A. aegypti is vector of dengue and other viruses. New methods of vector control are needed and can be achieved by a better understanding of the life cycle of this insect. Embryogenesis is a part of A. aegypty life cycle that is poorly understood. In insects in general and in mosquitoes in particular energetic metabolism is well studied during oogenesis, when the oocyte exhibits fast growth, accumulating carbohydrates, lipids and proteins that will meet the regulatory and metabolic needs of the developing embryo. On the other hand, events related with energetic metabolism during A. aegypti embryogenesis are unknown.ResultsGlucose metabolism was investigated throughout Aedes aegypti (Diptera) embryonic development. Both cellular blastoderm formation (CBf, 5 h after egg laying - HAE) and germ band retraction (GBr, 24 HAE) may be considered landmarks regarding glucose 6-phosphate (G6P) destination. We observed high levels of glucose 6-phosphate dehydrogenase (G6PDH) activity at the very beginning of embryogenesis, which nevertheless decreased up to 5 HAE. This activity is correlated with the need for nucleotide precursors generated by the pentose phosphate pathway (PPP), of which G6PDH is the key enzyme. We suggest the synchronism of egg metabolism with carbohydrate distribution based on the decreasing levels of phosphoenolpyruvate carboxykinase (PEPCK) activity and on the elevation observed in protein content up to 24 HAE. Concomitantly, increasing levels of hexokinase (HK) and pyruvate kinase (PK) activity were observed, and PEPCK reached a peak around 48 HAE. Glycogen synthase kinase (GSK3) activity was also monitored and shown to be inversely correlated with glycogen distribution during embryogenesis.ConclusionsThe results herein support the hypothesis that glucose metabolic fate changes according to developmental embryonic stages. Germ band retraction is a moment that was characterized as a landmark in glucose metabolism during Aedes aegypti embryogenesis. Furthermore, the results also suggest a role for GSK3 in glycogen balance/distribution during morphological modifications.

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The AKT/GSK3 axis is conserved in tick in a way that integrates glycogen metabolism and cell survival, and exhibits phylogenic differences that could be important for the development of novel control methods.

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