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Glycogen and glucose metabolism are essential for early embryonic development of the red flour beetle Tribolium castaneum.

Fraga A, Ribeiro L, Lobato M, Santos V, Silva JR, Gomes H, da Cunha Moraes JL, de Souza Menezes J, de Oliveira CJ, Campos E, da Fonseca RN - PLoS ONE (2013)

Bottom Line: In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis.Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality.Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos.

View Article: PubMed Central - PubMed

Affiliation: Laboratório Integrado de Bioquímica Hatisaburo Masuda (LIBHM), Núcleo de Pesquisas Ecológicas e Sócioambientais de Macaé (NUPEM), Universidade Federal do Rio de Janeiro (UFRJCampus Macaé), Rio de Janeiro, Brazil.

ABSTRACT
Control of energy metabolism is an essential process for life. In insects, egg formation (oogenesis) and embryogenesis is dependent on stored molecules deposited by the mother or transcribed later by the zygote. In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis. Previous studies in a few vector species showed a strong correlation of key morphogenetic events and changes in glucose metabolism. Here, we investigate glycogen and glucose metabolism in the red flour beetle Tribolium castaneum, an insect amenable to functional genomic studies. To examine the role of the key enzymes on glycogen and glucose regulation we cloned and analyzed the function of glycogen synthase kinase 3 (GSK-3) and hexokinase (HexA) genes during T. castaneum embryogenesis. Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality. Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos. Importantly, glycogen levels are upregulated after Tc-GSK-3 RNAi and glucose levels are upregulated after Tc-HexA1 RNAi, indicating that both genes control metabolism during embryogenesis and oogenesis, respectively. Altogether our results show that T. castaneum embryogenesis depends on the proper control of glucose and glycogen.

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Tc-GSK-3 knockdown affects T. castaneum egg laying, larvae hatching and glycogen content.(A) Tc-GSK-3 expression decreases after Tc-GSK-3 dsRNA injection. (B) Number of laid eggs diminishes 50% after Tc-GSK-3 RNAi when compared to the control. (C) Number of hatching larvae decreases after Tc-GSK-3 RNAi to about 20% of the control. (D) Glycogen content increases in Tc-GSK-3 RNAi eggs when compared to the control (LacZ RNAi eggs). Asterisk indicates that the difference between the two groups is statistically significant (p<0,05).
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pone-0065125-g008: Tc-GSK-3 knockdown affects T. castaneum egg laying, larvae hatching and glycogen content.(A) Tc-GSK-3 expression decreases after Tc-GSK-3 dsRNA injection. (B) Number of laid eggs diminishes 50% after Tc-GSK-3 RNAi when compared to the control. (C) Number of hatching larvae decreases after Tc-GSK-3 RNAi to about 20% of the control. (D) Glycogen content increases in Tc-GSK-3 RNAi eggs when compared to the control (LacZ RNAi eggs). Asterisk indicates that the difference between the two groups is statistically significant (p<0,05).

Mentions: Recently, during the writing of this manuscript, Bucher and co-authors have published a throughout description of Tc-GSK-3/shaggy RNAi phenotype [44]. We have observed a similar range of phenotypes in our analysis. Injection of high amounts of GSK-3 RNAi (2 µg/µL) lead to female sterility or large descrease in egg laying (data not shown), while decreasing dsRNA concentration (up to 50 ng/µL) allowed the recovery of RNAi embryos. The decrease in dsRNA concentration might affect the level of Tc-GSK-3 knockdown in the embryos. Thus, we analyzed Tc-GSK-3 expression in control (LacZ RNAi) and Tc-GSK-3 RNAi eggs by RT-PCR (Figure 8A). Injection of Tc-GSK-3 dsRNA (50 ng/µL) decreased its expression to levels of 20% of the control (Figure 8A), confirming that Tc-GSK-3 transcription was affected. After injection of Tc-GSK-3 dsRNA (50 ng/µL) egg laying was also reduced when compared to the control (Figure 8B), as well as the number of hatched larvae, which is largely decreased after Tc-GSK-3 dsRNA injection (Figure 8C).


Glycogen and glucose metabolism are essential for early embryonic development of the red flour beetle Tribolium castaneum.

Fraga A, Ribeiro L, Lobato M, Santos V, Silva JR, Gomes H, da Cunha Moraes JL, de Souza Menezes J, de Oliveira CJ, Campos E, da Fonseca RN - PLoS ONE (2013)

Tc-GSK-3 knockdown affects T. castaneum egg laying, larvae hatching and glycogen content.(A) Tc-GSK-3 expression decreases after Tc-GSK-3 dsRNA injection. (B) Number of laid eggs diminishes 50% after Tc-GSK-3 RNAi when compared to the control. (C) Number of hatching larvae decreases after Tc-GSK-3 RNAi to about 20% of the control. (D) Glycogen content increases in Tc-GSK-3 RNAi eggs when compared to the control (LacZ RNAi eggs). Asterisk indicates that the difference between the two groups is statistically significant (p<0,05).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672164&req=5

pone-0065125-g008: Tc-GSK-3 knockdown affects T. castaneum egg laying, larvae hatching and glycogen content.(A) Tc-GSK-3 expression decreases after Tc-GSK-3 dsRNA injection. (B) Number of laid eggs diminishes 50% after Tc-GSK-3 RNAi when compared to the control. (C) Number of hatching larvae decreases after Tc-GSK-3 RNAi to about 20% of the control. (D) Glycogen content increases in Tc-GSK-3 RNAi eggs when compared to the control (LacZ RNAi eggs). Asterisk indicates that the difference between the two groups is statistically significant (p<0,05).
Mentions: Recently, during the writing of this manuscript, Bucher and co-authors have published a throughout description of Tc-GSK-3/shaggy RNAi phenotype [44]. We have observed a similar range of phenotypes in our analysis. Injection of high amounts of GSK-3 RNAi (2 µg/µL) lead to female sterility or large descrease in egg laying (data not shown), while decreasing dsRNA concentration (up to 50 ng/µL) allowed the recovery of RNAi embryos. The decrease in dsRNA concentration might affect the level of Tc-GSK-3 knockdown in the embryos. Thus, we analyzed Tc-GSK-3 expression in control (LacZ RNAi) and Tc-GSK-3 RNAi eggs by RT-PCR (Figure 8A). Injection of Tc-GSK-3 dsRNA (50 ng/µL) decreased its expression to levels of 20% of the control (Figure 8A), confirming that Tc-GSK-3 transcription was affected. After injection of Tc-GSK-3 dsRNA (50 ng/µL) egg laying was also reduced when compared to the control (Figure 8B), as well as the number of hatched larvae, which is largely decreased after Tc-GSK-3 dsRNA injection (Figure 8C).

Bottom Line: In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis.Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality.Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos.

View Article: PubMed Central - PubMed

Affiliation: Laboratório Integrado de Bioquímica Hatisaburo Masuda (LIBHM), Núcleo de Pesquisas Ecológicas e Sócioambientais de Macaé (NUPEM), Universidade Federal do Rio de Janeiro (UFRJCampus Macaé), Rio de Janeiro, Brazil.

ABSTRACT
Control of energy metabolism is an essential process for life. In insects, egg formation (oogenesis) and embryogenesis is dependent on stored molecules deposited by the mother or transcribed later by the zygote. In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis. Previous studies in a few vector species showed a strong correlation of key morphogenetic events and changes in glucose metabolism. Here, we investigate glycogen and glucose metabolism in the red flour beetle Tribolium castaneum, an insect amenable to functional genomic studies. To examine the role of the key enzymes on glycogen and glucose regulation we cloned and analyzed the function of glycogen synthase kinase 3 (GSK-3) and hexokinase (HexA) genes during T. castaneum embryogenesis. Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality. Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos. Importantly, glycogen levels are upregulated after Tc-GSK-3 RNAi and glucose levels are upregulated after Tc-HexA1 RNAi, indicating that both genes control metabolism during embryogenesis and oogenesis, respectively. Altogether our results show that T. castaneum embryogenesis depends on the proper control of glucose and glycogen.

Show MeSH