Germ band retraction as a landmark in glucose metabolism during Aedes aegypti embryogenesis

Abstract

Background: The 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.

Results: Glucose 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.

Conclusions: The 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.

Figure 1

Aedes aegypti embryogenesis at 28°C. (A) 0 h after egg laying (HAE) embryo, detached from the endochorion. (B) 3-HAE embryo at the syncytial blastoderm stage. Insert shows the pole cells outside the blastoderm. (C) 5-HAE embryo, at the cellular blastoderm stage. Insert shows ventral-posterior region of the blastoderm detached from the endochorion and cell boundaries. (D) 10-HAE embryo in the middle of germ band extension. (E) 15-HAE embryo at the beginning of germ band retraction. (F) 24-HAE embryo at the germ band retraction stage. (G) 31-HAE embryo, at dorsal closure stage. Focal plane is located inside the embryo at the embryo-yolk junction region. (H) 48-HAE embryo at late organogenesis stage. Larvae segmentation is partially evident. (I) 62-HAE embryo at the end of embryogenesis showing the head, three fused thoracic segments, eight abdominal segments and the respiratory siphon and associated structures. In B-G, dorsal side is up. Scale bar = 100 μm. Arrow: cephalic region segmented. Arrowhead: serosal cuticle limits, detached from the endochorion. y: yolk. A-C, F, H, I: DIC microscopy. C insert: Bright field. D, E, G: Stereoscope.

Figure 2

Representative scheme for pathways of glucose metabolism. The scheme is based on enzyme activities and metabolites quantification evaluated during Aedes aegypti embryogenesis. HK - Hexokinase, G6PDH - Glucose 6-phosphate Dehydrogenase, PK - Pyruvate Kinase, PEPCK - Phosphoenolpyruvate Carboxykinase, PEP - Phosphoenolpyruvate and PPP - Penthose-Phosphate Pathway.

Figure 3

HK, PK and G6PDH activities during A. aegypti embryogenesis. The HK (A), PK (B) and G6PDH (C) activities were measured in egg homogenates on different hours of embryo development. Each experiment was replicated three times and error bars represent standard deviation of sample means. (*p < 0.05; ***p < 0.001, ANOVA). The dashed line indicates the germ band retraction stage 24 HAE.

Figure 4

Glucose and glycogen levels during A. aegypti embryogenesis. The glucose (open lozenge) and glycogen (black up-pointing triangle) concentration were measured in egg homogenates on different hours of embryo development. Each experiment was replicated six times and error bars represent standard deviation of sample means. The dashed line indicates the germ band retraction stage 24 HAE.

Figure 5

Glycogen Synthase Kinase-3 (GSK3) activity and relative expression during A. aegypti embryo development. The GSK3 activity (open lozenge) and GSK3 transcripts levels (black down-pointing triangle) (normalized by rp49 cDNA) were measured on different hours of embryo development. Each experiment was replicated three times and error bars represent standard deviation of sample means. Insert shows GSK3 transcripts levels in ovaries of A. aegypti females dissected in different hours after blood meal (**p < 0.01; ***p < 0.001, ANOVA). The dashed line indicates the germ band retraction stage 24 HAE.

Figure 6

Gluconeogenesis pathway during A. aegypti embryogenesis. (A) The protein concentration was measured in egg homogenates. (B) The PEPCK activity was measured in egg homogenates. Each experiment was replicated three times and error bars represent standard deviation of sample means. (**p < 0.01; ***p < 0.001, ANOVA). The dashed line indicates the germ band retraction stage 24 HAE.

Figure 7

Proposed scheme for glucose metabolism during the A. aegypti embryo development. The components represented herein were determined as described in Materials and Methods. HK: Hexokinase, G6PDH: Glucose 6-phosphate Dehydrogenase, PEP: Phosphoenolpyruvate, PEPCK: Phosphoenolpyruvate Carboxykinase, PK: Pyruvate Kinase, PPP: Pentose-Phosphate Pathway and GSK3: Glycogen Synthase Kinase 3.