BACKGROUND
the mosquito aedes aegypti is vector of urban yellow fever and also the main dengue vector  <cit> .
one of the major problems involving dengue transmission is that a. aegypti embryos enter dormancy at the end of embryogenesis, surviving and remaining viable for several months inside the egg  <cit> .
this extended viability is possible due to the acquisition of embryonic desiccation resistance, a biological mechanism that is believed to involve the formation and maturation of serosal cuticle, a layer covering the embryo  <cit> .
despite its importance as a vector, little attention is given to a. aegypti embryonic development.
taking into account the fact that mosquito populations are becoming resistant to the insecticides currently available for vector control  <cit> , it is imperative to establish new vector control methods.
these methods can be developed from a better comprehension of the biology of these insects, since some parts of their life cycle, such as embryogenesis, are still poorly understood.
as a rule, oviparous animals face embryogenesis in the absence of exogenous nutrient supply.
in this case, maternal nutrients are packaged into the female gamete  during oogenesis  <cit> .
in insect oogenesis the oocytes exhibit fast growth, accumulating carbohydrates, lipids and proteins that will meet the regulatory and metabolic needs of the developing embryo  <cit> .
in mosquitoes, the majority of yolk components are synthesized at extraovarian sites, primarily in the female fat body  <cit> .
subsequently, these yolk components are transported via haemolymph and incorporated into the oocytes  <cit> .
the sequential deposition of yolk components was evaluated during oogenesis in a. aegypti.
synchronous protein and lipid incorporation into the oocytes occurs in the first  <dig> h, while rapid glycogen incorporation happens between  <dig> and  <dig> h of oogenesis  <cit> .
the current literature provides ample information regarding metabolic events during larval and adult phases of a. aegypti  <cit> .
nevertheless, in a. aegypti embryogenesis, aspects concerning energy metabolism such as the activity of central metabolic pathways  or the determination of energy reserves to be used have been neglected.
in the fruit fly drosophila melanogaster an increase in glycogen content strongly correlated with protein levels in follicles and young embryos has been described  <cit> .
histochemical studies reveal that glycogen is the predominant form of carbohydrate storage in d. melanogaster eggs  <cit> .
additionally, the amount of carbohydrates was shown to decrease from late oocyte stages until after  <dig> h of embryogenesis, and increases up to the blastoderm stage, during later development  <cit> .
furthermore, changes in protein content occur in an opposite direction to that determined for the carbohydrate content  <cit> .
moreover, in d. melanogaster glycogen is abundantly stored in the midgut compartment during late stages of embryogenesis  <cit> .
insulin is a key regulator of energetic metabolism in many organisms.
it increases glucose transport, glycogen synthesis, diminishes gluconeogenesis, inhibits glycogenolysis, and regulates the expression of various genes  <cit> .
components of insulin signaling pathway have been discovered to be extremely conserved in organisms as distantly related as humans, d. melanogaster and caenorhabditis elegans  <cit> .
in a. aegypti, upstream components of insulin signaling pathway, as phosphatidylinositol 3-kinase   <cit>  and protein kinase b   <cit> , have been identified and correlated with glucose metabolism.
glycogen synthase kinase- <dig> , is a serine-threonine kinase present as two highly homologous forms, gsk3a and gsk3b, was first identified based on its action towards glycogen synthase , and is also considered a downstream component of insulin signaling cascade  <cit> .
gsk <dig> is now recognized as a key component of a surprisingly large number of cellular processes.
a previous study on the cattle tick rhipicephalus microplus conducted by our research group revealed that gsk <dig> activity was correlated with diminished glycogen content in eggs during embryogenesis  <cit> .
in this paper we correlated different biochemical parameters of glucose metabolism with morphological changes that take place during a. aegypti embryo development.
it was also demonstrated that glucose and glycogen levels are closely correlated with activity and transcription levels of gsk <dig> during embryogenesis.
it suggests a highly conserved participation of gsk <dig> in glycogen metabolism in arthropod embryogenesis.
to the best of our knowledge there is no work describing either function or activity of gsk <dig> in mosquitoes, or gsk <dig> involvement in glycogen metabolism.
RESULTS
embryonic development
in order to determine the timing of major morphological landmarks during a. aegypti embryogenesis at 28°c, embryos in distinct stages were clarified  <cit>  and observed .
zero hours after egg laying , eggs have just been fertilized and the embryos are detached from the surrounding endochorion .
three hae embryos are at the syncytial blastoderm stage, with the pole cells positioned outside the blastoderm   <cit> , while  <dig> hae embryos are right before or at the cellular blastoderm stage   <cit> .
ten and  <dig> hae embryos are in the middle of germ band extension and at the beginning of germ band retraction, respectively   <cit> .
twenty-four hae embryos are in the middle of germ band retraction   <cit> .
thirty-one hae, embryos are at the dorsal closure stage , while  <dig> hae embryos are at late organogenesis stage  <cit> , with evident larvae segmentation .
the embryonic development at 28°c is completed  <dig>  hae  <cit> .
accordingly,  <dig> hae embryos show all the features of larvae ready to hatch   <cit> .
glycolytic pathway increases after germ band formation in a. aegypti embryos
the glycolytic pathway transforms glucose into pyruvate obtaining atp during this process .
in a. aegypti embryogenesis the glycolytic pathway was evaluated determining the enzymatic activity of hexokinase  and pyruvate kinase  .
hk and pk are respectively the initial and final steps of glycolysis and both are regulatory enzymes that control the flux of glycolysis.
the pk and hk patterns of activity are positively correlated throughout embryogenesis.
this demonstrates that the activity of these enzymes occurs concomitantly, exerting a connected action at this phase of mosquito embryo development.
hk and pk activities are low during the first  <dig> h of embryogenesis .
glucose 6-phosphate is mainly destined to pentose-phosphate pathway before the germ band formation in a. aegypti embryos
the pentose-phosphate pathway, which produces nadph and ribose 5-phosphate , was investigated during embryogenesis by determining the activity profile of glucose 6-phosphate dehydrogenase , the rate limiting step of this pathway  <cit> .
the very beginning of mosquito embryogenesis is marked by high levels of g6pdh activity  <dig> hae, followed by a drastic decrease in the first  <dig> h of embryo development .
g6pdh activity was no longer detected  <dig> hae .
a. aegypti embryo gsk <dig> sequence analysis
rt-pcr from cdna obtained from a. aegypti embryos between  <dig> and  <dig> hae  using degenerated primers for gsk <dig> generated an approximately 600-bp product .
the cloned fragment was sequenced and showed a 100% identity with an a. aegypti gsk <dig> sequence deposited in genbank  and a. aegypti gene index .
then, the full-length mosquito gsk <dig> sequence was compiled from the data obtained by sequencing the cloned fragment and database sequences analysis.
the deduced amino acid sequence of the a. aegypti gsk <dig> was compared with orthologs from a number of other species .
the partial aegsk <dig> cdna is  <dig>  bp long and had an open reading frame of  <dig>  bp, beginning with the first atg codon at position  <dig> bp and with the stop codon at position  <dig>  bp.
the aegsk <dig> sequence was analyzed with blast and scanprosite tools.
the protein kinase atp-binding region  and serine/threonine protein kinase active-site signatures , which are important for determining its biological proprieties, were found .
the control of glycogen metabolism in a. aegypti embryos
glycogen metabolism in a. aegypti embryogenesis was investigated by determining glycogen and glucose content , and glycogen synthase kinase  activity and transcription .
total glycogen and glucose amount in eggs increased between  <dig> and  <dig> hae.
after that, glycogen and glucose content greatly decreased until the end of embryogenesis .
the level of gsk <dig> activity decreases from  <dig> hae to  <dig> hae , being inversely correlated with glycogen distribution from  <dig> to  <dig> hae.
from  <dig> hae until the end of embryogenesis gsk <dig> activity remains at low levels.
to identify the profile of gsk <dig> mrna transcription, cdnas obtained from eggs collected at different embryogenesis stages and from blood-fed and not blood-fed females ovaries were analyzed by qpcr.
figure  <dig> shows the relative amount of gsk <dig> mrna, normalized by rp <dig> mrna .
embryos at different stages of development were evaluated over the course of embryogenesis between  <dig> hae   and  <dig> hae.
the gsk <dig> gene expression decreased sharply between  <dig> and  <dig> hae, from 100% to 16% of initial relative amount of gsk <dig> mrna .
after this moment  the relative amount of gsk <dig> mrna remained stable, around 15% of its initial value, until the end of embryogenesis.
gsk <dig> mrna transcripts are present in ovaries from blood-fed and not blood-fed females .
in contrast to embryos, gsk <dig> relative expression increased significantly , when comparing ovaries of not blood-fed females  and ovaries of females  <dig> h after blood meal.
in ovaries of females  <dig> h after blood meal, gsk <dig> relative expression increased significantly by  <dig> times, when compared to not blood-fed females.
gluconeogenesis increases after germ band formation in a. aegypti embryos
the gluconeogenesis consists of the formation of glucose from noncarbohydrate precursors, such as the products of lipids and proteins breakdown .
the evaluation of gluconeogenesis during embryogenesis was carried out by correlating protein and glucose content in eggs , and the activity of phosphoenolpyruvate carboxykinase  , the key enzyme of this pathway.
glucose content increased until  <dig> hae, and then was gradually reduced towards embryogenesis completion .
total protein content in eggs increased until  <dig> hae, at the stage of cellular blastoderm formation.
from then until the middle of germ band retraction , protein content was maintained and then declined drastically between  <dig> and  <dig> hae, and remained at low levels until the end of embryogenesis .
pepck activity decreased continuously until  <dig> hae, being inversely correlated with glucose content up to that point .
pepck activity subsequently doubled between  <dig> and  <dig> hae, remaining at high levels until the end of embryogenesis, concomitantly with a significant decrease in protein content .
discussion
in a previous work with the cattle tick r. microplus embryos our group demonstrated a correlation between the kinetics of egg energetic components mobilization and the morphological changes occurring during early embryogenesis  <cit> .
in this work, stages of a. aegypti embryogenesis were visualized in clarified embryos  and compared with the metabolic modifications related to glucose metabolism throughout embryogenesis.
farnesi et al.
<cit>  demonstrated that a. aegypti embryonic development is completed around  <dig> hae when the specimens are reared at 28°c.
according to our analysis, a. aegypti exhibits the same pattern of embryo formation as described for d. melanogaster  <cit>  and for another mosquito, anopheles albitarsis  <cit> .
acquisition of drug resistance by insects vectors of diseases is a major global health problem, due to a rapid selection of insects resistant to compounds used as conventional insecticides  <cit> .
in fact, insecticide resistance is the fastest case of evolutive adaptation ever observed and it is extensively documented  <cit> .
the discovery of new drugs aimed at working as insecticides demand a high investment in research and these drugs might have a short lifetime efficacy due to the acquisition of resistance by insects.
therefore, the discovery of new drugs that can be used as new insecticides must be a constant process.
a strategy for development of new control methods can rely on the characterization of the existing metabolic pathways in insects, in order to identify targets present in the insect but not it the host.
this approach was successfully employed regarding chitin metabolism and the moulting process, with juvenile hormone analogues  <cit> .
so, further studies are needed to confirm the viability of this strategy to new metabolic pathways and distinct life stages, such as the embryo.
hexokinase and pyruvate kinase catalytic activities were detected throughout embryogenesis, and presented increase from  <dig> hae on which suggest that glycolysis is intensified after germ band retraction stage  remaining elevated until the end of embryogenesis.
at the very beginning of embryogenesis, however, the glucose 6-phosphate  produced by hexokinase would, most likely, be driven to the pentose-phosphate pathway , due to the high activity of g6pdh,  <dig> hae.
after, g6pdh catalytic activity declined abruptly between  <dig> and  <dig> hae, and was nearly null  <dig> hae.
this result suggests an intense participation of ppp in the initial part of a. aegypti embryogenesis.
our observations support an abrupt shift in glucose fate at the moment of cellular blastoderm formation , which occurs  <dig> hae.
before cbf, a. aegypti embryos must sustain a high synthesis of nucleic acids due to the intense nuclei division for the formation of the syncytial blastoderm occurring prior to cbf  <cit> .
at this moment the ppp would supply ribose 5-phosphate units for the synthesis of nucleotides.
the nadph produced by ppp would also be used for phospholipids necessary for blastoderm cellularization happening  <dig> hae.
a similar increase in g6pdh activity prior to cbf was observed during embryonic development of the hard tick r. microplus  <cit> .
total protein content in a. aegypti eggs was significantly increased between  <dig> and  <dig> hae.
elevations in protein levels were previously observed in d. melanogaster embryos, and an early elevation in protein content during embryogenesis can be explained by maternal mrna driven protein synthesis   <cit> .
our data suggest that such protein biosynthesis in a. aegypti embryos could be supported by concomitant generation of reducing potential  by the highly active ppp.
an increase in glucose and glycogen content was observed during the first hours of embryogenesis .
the peak of glucose, observed  <dig> hae, might be important for the synthesis of the chitinized serosal cuticle that happens at this very moment  <cit> .
this hypothesis is supported by evidences which indicate that the amino sugar pathway  is upregulated at this development stage .
therefore, chitin production , might be a sink for glucose consumption at this stage of development.
further experiments will confirm this hypothesis.
additionally, chitin production used for organogenesis and synthesis of the larval cuticle at late embryogenesis, starting from  <dig> hae  <cit> , might also be consuming glucose, although at this moment no peak of glucose is observed.
previous works in drosophila embryogenesis also identified an increase in glycogen and carbohydrates content, which nevertheless occurred at the earlier developmental stage of blastoderm formation  <cit> .
it has been previously observed that glycogen accumulates in embryos of the cattle tick r. microplus in parallel with an increase in pepck catalytic activity.
it was suggested that glycogen biosynthesis could be supported by concomitant gluconeogenesis  <cit> .
on the other hand, during a. aegypti embryogenesis, pepck activity declined between  <dig> and  <dig> hae , suggesting the availability of other carbohydrates source in insects, such as trehalose  <cit> , which should be mobilized and converted into glucose and/or glycogen.
after the progressive drop until  <dig> hae, a significant increase in pepck catalytic activity was observed between  <dig> and  <dig> hae, concomitant with a consistent reduction in total protein content, the main gluconeogenic substrate .
taken together, these results suggest a correlation between protein content, gluconeogenic pathway, and morphogenetic modifications that occur during germ band retraction , dorsal closure stage  and late organogenesis  .
in this scenario, glycogen would be produced using proteins as substrate, during the whole organogenesis process that takes place after germ band retraction  <cit> .
yamazaki and yanagawa  <cit>  also reported an oscillation in glycogen distribution during d. melanogaster embryogenesis.
conversely, after being almost totally consumed, glycogen content increased at the late stages of embryo formation  and accumulates in embryo midgut.
our previous work demonstrated that glycogen distribution throughout r. microplus embryogenesis was inversely associated with gsk <dig> activity  <cit> .
due to its role on glycogen synthesis regulation, both gsk <dig> activity and its relative expression were determined during a. aegypti embryogenesis .
we observed that gsk <dig> activity was directly related to gsk <dig> transcript levels in a. aegypti embryos.
both gsk <dig> activity and gene transcription dropped between  <dig> and  <dig> hae.
furthermore, gsk <dig> activity was inversely related to the glycogen content in the interval  <dig> to  <dig> hae  suggesting that glycogen accumulation in eggs can be regulated by gsk <dig> activity during cellular blastoderm formation and germ band extension .
gsk <dig> activity is classically described as negatively regulated by insulin signaling pathway  <cit> .
we suggest that such regulation may be conserved in invertebrate organisms, like mosquitoes.
in this perspective, a high insulin signal would be present during the first  <dig> h, leading to drop in gsk <dig> activity and increasing glycogen formation .
in fact, three out of the eight genes encoding insulin-like peptides in a. aegypti were previously reported to be expressed in eggs  <cit> .
on the other hand, it has been already demonstrated that gsk <dig> is involved with embryo dorsoventral axis formation in d. melanogaster  <cit>  and xenopus  <cit> , rather as a component of wnt signaling pathway than as regulator of the metabolism of glycogen.
further studies will be necessary to describe whether gsk <dig> plays a role on the control of cell differentiation and embryo polarity patterning during mosquito embryogenesis.
additionally, we compared the aegsk <dig> cdna amino acid sequence  and it was revealed a high homology indexes for vertebrates and invertebrates species .
several authors have reported the effect of mosquito/insect blood meal on gene expression  <cit> .
gsk <dig> was upregulated in ovaries from bloodfed a. aegypti females, when compared to unfed females.
hence, one must consider a possible role for gsk <dig> during mosquito oogenesis.
in a. aegypti mosquito, an insulin-like peptide was shown to regulate oocyte maturation and metabolism  <cit> .
monosaccharides were previously measured in mature oocytes well after follicular growth, and glycogen appeared during or even after oocyte chorionation  <cit> .
it has been suggested that chorionated oocytes must retain enzymes able to synthesize glycogen long after the end of oogenesis, presumably due to activation of glycogen synthase enzyme .
additionally, briegel et al.
<cit>  postulate that late accumulation of glycogen in a. aegypti developing oocytes could be due to gs activity inhibition.
the increase in gsk <dig> transcript levels observed during a. aegypti oogenesis suggests its activation as a mechanism to inhibit gs activity and regulate glycogen incorporation kinetics in developing oocytes.
therefore, from late oogenesis up to the initial developmental stages gsk <dig> activity and expression are high and glycogen content is relatively low.
from  <dig> hae on, glycogen metabolism appears to switch in order to reduce its biosynthesis and accumulation .
coincidentally, this same period of development is marked by a strong reduction in both gsk <dig> activity and expression .
CONCLUSIONS
the results presented here demonstrated that glucose metabolism is closely correlated to a. aegypti developmental embryonic stages.
furthermore, germ band retraction is a landmark regarding both glucose and glycogen metabolism.
it is important to stress that the shift in glucose metabolism is related with the cellular processes that are taking place before and after germ band retraction.
we intend to study these processes in detail in the future.
the results observed in the present study are schematically represented together in figure  <dig>  nevertheless, further elucidation of these phenomena would lead to a better understanding of the regulatory mechanisms in glucose metabolism during a. aegypti embryogenesis.

 the mosquito aedes aegypti is vector of urban yellow fever and also the main dengue vector. one of the major problems involving dengue transmission is that a. aegypti embryos enter dormancy at the end of embryogenesis, surviving and remaining viable for several months inside the egg. this extended viability is possible due to the acquisition of embryonic desiccation resistance, a biological mechanism that is believed to involve the formation and maturation of serosal cuticle, a layer covering the embryo. despite its importance as a vector, little attention is given to a. aegypti embryonic development. taking into account the fact that mosquito populations are becoming resistant to the insecticides currently available for vector control, it is imperative to establish new vector control methods. these methods can be developed from a better comprehension of the biology of these insects, since some parts of their life cycle, such as embryogenesis, are still poorly understood. as a rule, oviparous animals face embryogenesis in the absence of exogenous nutrient supply. in this case, maternal nutrients are packaged into the female gamete during oogenesis. in insect oogenesis the oocytes exhibit fast growth, accumulating carbohydrates, lipids and proteins that will meet the regulatory and metabolic needs of the developing embryo. in mosquitoes, the majority of yolk components are synthesized at extraovarian sites, primarily in the female fat body. subsequently, these yolk components are transported via haemolymph and incorporated into the oocytes. the sequential deposition of yolk components was evaluated during oogenesis in a. aegypti. synchronous protein and lipid incorporation into the oocytes occurs in the first h, while rapid glycogen incorporation happens between and h of oogenesis. the current literature provides ample information regarding metabolic events during larval and adult phases of a. aegypti. nevertheless, in a. aegypti embryogenesis, aspects concerning energy metabolism such as the activity of central metabolic pathways or the determination of energy reserves to be used have been neglected. in the fruit fly drosophila melanogaster an increase in glycogen content strongly correlated with protein levels in follicles and young embryos has been described. histochemical studies reveal that glycogen is the predominant form of carbohydrate storage in d. melanogaster eggs. additionally, the amount of carbohydrates was shown to decrease from late oocyte stages until after h of embryogenesis, and increases up to the blastoderm stage, during later development. furthermore, changes in protein content occur in an opposite direction to that determined for the carbohydrate content. moreover, in d. melanogaster glycogen is abundantly stored in the midgut compartment during late stages of embryogenesis. insulin is a key regulator of energetic metabolism in many organisms. it increases glucose transport, glycogen synthesis, diminishes gluconeogenesis, inhibits glycogenolysis, and regulates the expression of various genes. components of insulin signaling pathway have been discovered to be extremely conserved in organisms as distantly related as humans, d. melanogaster and caenorhabditis elegans. in a. aegypti, upstream components of insulin signaling pathway, as phosphatidylinositol 3-kinase and protein kinase b, have been identified and correlated with glucose metabolism. glycogen synthase kinase-, is a serine-threonine kinase present as two highly homologous forms, gsk3a and gsk3b, was first identified based on its action towards glycogen synthase, and is also considered a downstream component of insulin signaling cascade. gsk is now recognized as a key component of a surprisingly large number of cellular processes. a previous study on the cattle tick rhipicephalus microplus conducted by our research group revealed that gsk activity was correlated with diminished glycogen content in eggs during embryogenesis. in this paper we correlated different biochemical parameters of glucose metabolism with morphological changes that take place during a. aegypti embryo development. it was also demonstrated that glucose and glycogen levels are closely correlated with activity and transcription levels of gsk during embryogenesis. it suggests a highly conserved participation of gsk in glycogen metabolism in arthropod embryogenesis. to the best of our knowledge there is no work describing either function or activity of gsk in mosquitoes, or gsk involvement in glycogen metabolism.  embryonic development in order to determine the timing of major morphological landmarks during a. aegypti embryogenesis at 28°c, embryos in distinct stages were clarified and observed. zero hours after egg laying, eggs have just been fertilized and the embryos are detached from the surrounding endochorion. three hae embryos are at the syncytial blastoderm stage, with the pole cells positioned outside the blastoderm, while hae embryos are right before or at the cellular blastoderm stage. ten and hae embryos are in the middle of germ band extension and at the beginning of germ band retraction, respectively. twenty-four hae embryos are in the middle of germ band retraction. thirty-one hae, embryos are at the dorsal closure stage, while hae embryos are at late organogenesis stage, with evident larvae segmentation. the embryonic development at 28°c is completed hae. accordingly, hae embryos show all the features of larvae ready to hatch. glycolytic pathway increases after germ band formation in a. aegypti embryos the glycolytic pathway transforms glucose into pyruvate obtaining atp during this process. in a. aegypti embryogenesis the glycolytic pathway was evaluated determining the enzymatic activity of hexokinase and pyruvate kinase. hk and pk are respectively the initial and final steps of glycolysis and both are regulatory enzymes that control the flux of glycolysis. the pk and hk patterns of activity are positively correlated throughout embryogenesis. this demonstrates that the activity of these enzymes occurs concomitantly, exerting a connected action at this phase of mosquito embryo development. hk and pk activities are low during the first h of embryogenesis. glucose 6-phosphate is mainly destined to pentose-phosphate pathway before the germ band formation in a. aegypti embryos the pentose-phosphate pathway, which produces nadph and ribose 5-phosphate, was investigated during embryogenesis by determining the activity profile of glucose 6-phosphate dehydrogenase, the rate limiting step of this pathway. the very beginning of mosquito embryogenesis is marked by high levels of g6pdh activity hae, followed by a drastic decrease in the first h of embryo development. g6pdh activity was no longer detected hae. a. aegypti embryo gsk sequence analysis rt-pcr from cdna obtained from a. aegypti embryos between and hae using degenerated primers for gsk generated an approximately 600-bp product. the cloned fragment was sequenced and showed a 100% identity with an a. aegypti gsk sequence deposited in genbank and a. aegypti gene index. then, the full-length mosquito gsk sequence was compiled from the data obtained by sequencing the cloned fragment and database sequences analysis. the deduced amino acid sequence of the a. aegypti gsk was compared with orthologs from a number of other species. the partial aegsk cdna is bp long and had an open reading frame of bp, beginning with the first atg codon at position bp and with the stop codon at position bp. the aegsk sequence was analyzed with blast and scanprosite tools. the protein kinase atp-binding region and serine/threonine protein kinase active-site signatures, which are important for determining its biological proprieties, were found. the control of glycogen metabolism in a. aegypti embryos glycogen metabolism in a. aegypti embryogenesis was investigated by determining glycogen and glucose content, and glycogen synthase kinase activity and transcription. total glycogen and glucose amount in eggs increased between and hae. after that, glycogen and glucose content greatly decreased until the end of embryogenesis. the level of gsk activity decreases from hae to hae, being inversely correlated with glycogen distribution from to hae. from hae until the end of embryogenesis gsk activity remains at low levels. to identify the profile of gsk mrna transcription, cdnas obtained from eggs collected at different embryogenesis stages and from blood-fed and not blood-fed females ovaries were analyzed by qpcr. figure shows the relative amount of gsk mrna, normalized by rp mrna. embryos at different stages of development were evaluated over the course of embryogenesis between hae and hae. the gsk gene expression decreased sharply between and hae, from 100% to 16% of initial relative amount of gsk mrna. after this moment the relative amount of gsk mrna remained stable, around 15% of its initial value, until the end of embryogenesis. gsk mrna transcripts are present in ovaries from blood-fed and not blood-fed females. in contrast to embryos, gsk relative expression increased significantly, when comparing ovaries of not blood-fed females and ovaries of females h after blood meal. in ovaries of females h after blood meal, gsk relative expression increased significantly by times, when compared to not blood-fed females. gluconeogenesis increases after germ band formation in a. aegypti embryos the gluconeogenesis consists of the formation of glucose from noncarbohydrate precursors, such as the products of lipids and proteins breakdown. the evaluation of gluconeogenesis during embryogenesis was carried out by correlating protein and glucose content in eggs, and the activity of phosphoenolpyruvate carboxykinase, the key enzyme of this pathway. glucose content increased until hae, and then was gradually reduced towards embryogenesis completion. total protein content in eggs increased until hae, at the stage of cellular blastoderm formation. from then until the middle of germ band retraction, protein content was maintained and then declined drastically between and hae, and remained at low levels until the end of embryogenesis. pepck activity decreased continuously until hae, being inversely correlated with glucose content up to that point. pepck activity subsequently doubled between and hae, remaining at high levels until the end of embryogenesis, concomitantly with a significant decrease in protein content. discussion in a previous work with the cattle tick r. microplus embryos our group demonstrated a correlation between the kinetics of egg energetic components mobilization and the morphological changes occurring during early embryogenesis. in this work, stages of a. aegypti embryogenesis were visualized in clarified embryos and compared with the metabolic modifications related to glucose metabolism throughout embryogenesis. farnesi et al. demonstrated that a. aegypti embryonic development is completed around hae when the specimens are reared at 28°c. according to our analysis, a. aegypti exhibits the same pattern of embryo formation as described for d. melanogaster and for another mosquito, anopheles albitarsis. acquisition of drug resistance by insects vectors of diseases is a major global health problem, due to a rapid selection of insects resistant to compounds used as conventional insecticides. in fact, insecticide resistance is the fastest case of evolutive adaptation ever observed and it is extensively documented. the discovery of new drugs aimed at working as insecticides demand a high investment in research and these drugs might have a short lifetime efficacy due to the acquisition of resistance by insects. therefore, the discovery of new drugs that can be used as new insecticides must be a constant process. a strategy for development of new control methods can rely on the characterization of the existing metabolic pathways in insects, in order to identify targets present in the insect but not it the host. this approach was successfully employed regarding chitin metabolism and the moulting process, with juvenile hormone analogues. so, further studies are needed to confirm the viability of this strategy to new metabolic pathways and distinct life stages, such as the embryo. hexokinase and pyruvate kinase catalytic activities were detected throughout embryogenesis, and presented increase from hae on which suggest that glycolysis is intensified after germ band retraction stage remaining elevated until the end of embryogenesis. at the very beginning of embryogenesis, however, the glucose 6-phosphate produced by hexokinase would, most likely, be driven to the pentose-phosphate pathway, due to the high activity of g6pdh, hae. after, g6pdh catalytic activity declined abruptly between and hae, and was nearly null hae. this result suggests an intense participation of ppp in the initial part of a. aegypti embryogenesis. our observations support an abrupt shift in glucose fate at the moment of cellular blastoderm formation, which occurs hae. before cbf, a. aegypti embryos must sustain a high synthesis of nucleic acids due to the intense nuclei division for the formation of the syncytial blastoderm occurring prior to cbf. at this moment the ppp would supply ribose 5-phosphate units for the synthesis of nucleotides. the nadph produced by ppp would also be used for phospholipids necessary for blastoderm cellularization happening hae. a similar increase in g6pdh activity prior to cbf was observed during embryonic development of the hard tick r. microplus. total protein content in a. aegypti eggs was significantly increased between and hae. elevations in protein levels were previously observed in d. melanogaster embryos, and an early elevation in protein content during embryogenesis can be explained by maternal mrna driven protein synthesis. our data suggest that such protein biosynthesis in a. aegypti embryos could be supported by concomitant generation of reducing potential by the highly active ppp. an increase in glucose and glycogen content was observed during the first hours of embryogenesis. the peak of glucose, observed hae, might be important for the synthesis of the chitinized serosal cuticle that happens at this very moment. this hypothesis is supported by evidences which indicate that the amino sugar pathway is upregulated at this development stage. therefore, chitin production, might be a sink for glucose consumption at this stage of development. further experiments will confirm this hypothesis. additionally, chitin production used for organogenesis and synthesis of the larval cuticle at late embryogenesis, starting from hae, might also be consuming glucose, although at this moment no peak of glucose is observed. previous works in drosophila embryogenesis also identified an increase in glycogen and carbohydrates content, which nevertheless occurred at the earlier developmental stage of blastoderm formation. it has been previously observed that glycogen accumulates in embryos of the cattle tick r. microplus in parallel with an increase in pepck catalytic activity. it was suggested that glycogen biosynthesis could be supported by concomitant gluconeogenesis. on the other hand, during a. aegypti embryogenesis, pepck activity declined between and hae, suggesting the availability of other carbohydrates source in insects, such as trehalose, which should be mobilized and converted into glucose and/or glycogen. after the progressive drop until hae, a significant increase in pepck catalytic activity was observed between and hae, concomitant with a consistent reduction in total protein content, the main gluconeogenic substrate. taken together, these results suggest a correlation between protein content, gluconeogenic pathway, and morphogenetic modifications that occur during germ band retraction, dorsal closure stage and late organogenesis. in this scenario, glycogen would be produced using proteins as substrate, during the whole organogenesis process that takes place after germ band retraction. yamazaki and yanagawa also reported an oscillation in glycogen distribution during d. melanogaster embryogenesis. conversely, after being almost totally consumed, glycogen content increased at the late stages of embryo formation and accumulates in embryo midgut. our previous work demonstrated that glycogen distribution throughout r. microplus embryogenesis was inversely associated with gsk activity. due to its role on glycogen synthesis regulation, both gsk activity and its relative expression were determined during a. aegypti embryogenesis. we observed that gsk activity was directly related to gsk transcript levels in a. aegypti embryos. both gsk activity and gene transcription dropped between and hae. furthermore, gsk activity was inversely related to the glycogen content in the interval to hae suggesting that glycogen accumulation in eggs can be regulated by gsk activity during cellular blastoderm formation and germ band extension. gsk activity is classically described as negatively regulated by insulin signaling pathway. we suggest that such regulation may be conserved in invertebrate organisms, like mosquitoes. in this perspective, a high insulin signal would be present during the first h, leading to drop in gsk activity and increasing glycogen formation. in fact, three out of the eight genes encoding insulin-like peptides in a. aegypti were previously reported to be expressed in eggs. on the other hand, it has been already demonstrated that gsk is involved with embryo dorsoventral axis formation in d. melanogaster and xenopus, rather as a component of wnt signaling pathway than as regulator of the metabolism of glycogen. further studies will be necessary to describe whether gsk plays a role on the control of cell differentiation and embryo polarity patterning during mosquito embryogenesis. additionally, we compared the aegsk cdna amino acid sequence and it was revealed a high homology indexes for vertebrates and invertebrates species. several authors have reported the effect of mosquito/insect blood meal on gene expression. gsk was upregulated in ovaries from bloodfed a. aegypti females, when compared to unfed females. hence, one must consider a possible role for gsk during mosquito oogenesis. in a. aegypti mosquito, an insulin-like peptide was shown to regulate oocyte maturation and metabolism. monosaccharides were previously measured in mature oocytes well after follicular growth, and glycogen appeared during or even after oocyte chorionation. it has been suggested that chorionated oocytes must retain enzymes able to synthesize glycogen long after the end of oogenesis, presumably due to activation of glycogen synthase enzyme. additionally, briegel et al. postulate that late accumulation of glycogen in a. aegypti developing oocytes could be due to gs activity inhibition. the increase in gsk transcript levels observed during a. aegypti oogenesis suggests its activation as a mechanism to inhibit gs activity and regulate glycogen incorporation kinetics in developing oocytes. therefore, from late oogenesis up to the initial developmental stages gsk activity and expression are high and glycogen content is relatively low. from hae on, glycogen metabolism appears to switch in order to reduce its biosynthesis and accumulation. coincidentally, this same period of development is marked by a strong reduction in both gsk activity and expression.  the results presented here demonstrated that glucose metabolism is closely correlated to a. aegypti developmental embryonic stages. furthermore, germ band retraction is a landmark regarding both glucose and glycogen metabolism. it is important to stress that the shift in glucose metabolism is related with the cellular processes that are taking place before and after germ band retraction. we intend to study these processes in detail in the future. the results observed in the present study are schematically represented together in figure nevertheless, further elucidation of these phenomena would lead to a better understanding of the regulatory mechanisms in glucose metabolism during a. aegypti embryogenesis. 
this activity is correlated with the need for nucleotide precursors generated by the pentose phosphate pathway, 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 activity and on the elevation observed in protein content up to hae. both cellular blastoderm formation and germ band retraction may be considered landmarks regarding glucose 6-phosphate destination. germ band retraction is a moment that was characterized as a landmark in glucose metabolism during aedes aegypti embryogenesis. the results herein support the hypothesis that glucose metabolic fate changes according to developmental embryonic stages. furthermore, the results also suggest a role for gsk in glycogen balance/distribution during morphological modifications. 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. we observed high levels of glucose 6-phosphate dehydrogenase activity at the very beginning of embryogenesis, which nevertheless decreased up to hae. glycogen synthase kinase activity was also monitored and shown to be inversely correlated with glycogen distribution during embryogenesis. 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. glucose metabolism was investigated throughout aedes aegypti embryonic development.
 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.  glucose metabolism was investigated throughout aedes aegypti embryonic development. both cellular blastoderm formation and germ band retraction may be considered landmarks regarding glucose 6-phosphate destination. we observed high levels of glucose 6-phosphate dehydrogenase activity at the very beginning of embryogenesis, which nevertheless decreased up to hae. this activity is correlated with the need for nucleotide precursors generated by the pentose phosphate pathway, 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 activity and on the elevation observed in protein content up to hae. concomitantly, increasing levels of hexokinase and pyruvate kinase activity were observed, and pepck reached a peak around hae. glycogen synthase kinase activity was also monitored and shown to be inversely correlated with glycogen distribution during embryogenesis.  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 gsk in glycogen balance/distribution during morphological modifications. 