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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-HexA RNAi affects oogenesis, glucose content, and reduces egg lay.(A,B) Ovary morphology in (A) control ovaries (injected with LacZ dsRNA) and (B,B’) After Tc-HexA1 dsRNA injection. (B)Tc-HexA1 dsRNA ovarioles are less numerous and display many oocytes undergoing apparent degeneration (black arrows) when compared to the control ovaries. Mature oocytes can be eventually identified in Tc-HexA1 dsRNA ovaries (arrowhead). Nurse cells of the Tc-HexA1 dsRNA ovarioles also appear reduced when compared to the control, although the germarium in some ovarioles seem not to be affected like in B’. (B’) Arrowheads highlights the germarium in Tc-HexA1 dsRNA ovaries, which appears similar to the control in some ovarioles. (C) Tc-HexA1 dsRNA injection largely reduces oviposition when compared to the WT. (D) Analysis of larvae hatching after Tc-HexA1 RNAi when compared to the control. Less than 10% of the laid eggs hatch, indicating an essential role of Tc-HexA1 during embryonic development. (E) Analysis of glucose content in ovaries injected with Tc-HexA1 dsRNA and the control (LacZ dsRNA). Asterisk indicates that the difference between the two groups is statistically significant (p<0,05).
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pone-0065125-g005: Tc-HexA RNAi affects oogenesis, glucose content, and reduces egg lay.(A,B) Ovary morphology in (A) control ovaries (injected with LacZ dsRNA) and (B,B’) After Tc-HexA1 dsRNA injection. (B)Tc-HexA1 dsRNA ovarioles are less numerous and display many oocytes undergoing apparent degeneration (black arrows) when compared to the control ovaries. Mature oocytes can be eventually identified in Tc-HexA1 dsRNA ovaries (arrowhead). Nurse cells of the Tc-HexA1 dsRNA ovarioles also appear reduced when compared to the control, although the germarium in some ovarioles seem not to be affected like in B’. (B’) Arrowheads highlights the germarium in Tc-HexA1 dsRNA ovaries, which appears similar to the control in some ovarioles. (C) Tc-HexA1 dsRNA injection largely reduces oviposition when compared to the WT. (D) Analysis of larvae hatching after Tc-HexA1 RNAi when compared to the control. Less than 10% of the laid eggs hatch, indicating an essential role of Tc-HexA1 during embryonic development. (E) Analysis of glucose content in ovaries injected with Tc-HexA1 dsRNA and the control (LacZ dsRNA). Asterisk indicates that the difference between the two groups is statistically significant (p<0,05).

Mentions: In all experiments, we injected the unrelated dsRNA LacZ as a negative control in a separate batch of females. These LacZ dsRNA females laid the normal amount of eggs, which hatched as larvae, indicating that injection of unrelated dsRNA had no effect on T. castaneum development. We then analyzed several parameters related to fecundity in the females injected with Tc-HexA1 dsRNA. First, egg laying of the Tc-HexA1 dsRNA injected females was drastically reduced to 10% of the control (Figure 5C). Second, among the few Tc-HexA1 RNAi laid eggs, only 5% of them hatched as larvae, indicating a strong requirement of this gene for embryonic development (Figure 5D). This extreme reduction in egg laying prevented the analysis of Hex activity or glucose content in RNAi embryos. We tried to circumvent this problem by analyzing glucose content in ovaries of Tc-HexA1 dsRNA and LacZ dsRNA (control) injected females. Interestingly, glucose content was higher in Tc-HexA1 dsRNA ovaries when compared to the control (Figure 5E). This reduction in egg laying stimulated us to compare ovary morphology in control and Tc-HexA1 dsRNA injected females. Morphological analysis via nuclear DAPI stainings of the ovarioles of control and Tc-HexA1 dsRNA injected females showed clear differences (Figure 5A,B). T. castaneum control ovaries display several tube-like projections, the ovarioles e.g.[37], which contains oocytes in different stages of maturation. In control ovaries, larger eggs are present in the distal part of the ovariole. Tc-Hex RNAi ovarioles showed distinct features. First, the ovariole number is reduced (Figure 5B and data not shown). Second, degenerated oocytes can be observed at the distal part (Figure 5B - black arrows), although some mature oocytes surrounded by follicle cells can also be observed (Figure 5B - arrowhead). Third, the germarium appears to be diminished in some Tc-HexA1 RNAi ovarioles (Figure 5B), while others appear similar to control ovaries (Figure 5B’-arrows). This abnormal ovary morphology is probably related to the large reduction in oviposition when compared to the control (Figure 5C). Finally, the few eggs observed after Tc-HexA1 RNAi stopped embryonic development before cellularization (8 hours of development - data not shown). To sum up, the analysis of Tc-HexA1 role during oogenesis and embryogenesis supports an essential role of Tc-HexA1 and glucose metabolism during these processes. Since glucose can be generated via glycogen degradation we sought to investigate how glycogen is regulated during T. castaneum embryogenesis.


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-HexA RNAi affects oogenesis, glucose content, and reduces egg lay.(A,B) Ovary morphology in (A) control ovaries (injected with LacZ dsRNA) and (B,B’) After Tc-HexA1 dsRNA injection. (B)Tc-HexA1 dsRNA ovarioles are less numerous and display many oocytes undergoing apparent degeneration (black arrows) when compared to the control ovaries. Mature oocytes can be eventually identified in Tc-HexA1 dsRNA ovaries (arrowhead). Nurse cells of the Tc-HexA1 dsRNA ovarioles also appear reduced when compared to the control, although the germarium in some ovarioles seem not to be affected like in B’. (B’) Arrowheads highlights the germarium in Tc-HexA1 dsRNA ovaries, which appears similar to the control in some ovarioles. (C) Tc-HexA1 dsRNA injection largely reduces oviposition when compared to the WT. (D) Analysis of larvae hatching after Tc-HexA1 RNAi when compared to the control. Less than 10% of the laid eggs hatch, indicating an essential role of Tc-HexA1 during embryonic development. (E) Analysis of glucose content in ovaries injected with Tc-HexA1 dsRNA and the control (LacZ dsRNA). Asterisk indicates that the difference between the two groups is statistically significant (p<0,05).
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Related In: Results  -  Collection

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pone-0065125-g005: Tc-HexA RNAi affects oogenesis, glucose content, and reduces egg lay.(A,B) Ovary morphology in (A) control ovaries (injected with LacZ dsRNA) and (B,B’) After Tc-HexA1 dsRNA injection. (B)Tc-HexA1 dsRNA ovarioles are less numerous and display many oocytes undergoing apparent degeneration (black arrows) when compared to the control ovaries. Mature oocytes can be eventually identified in Tc-HexA1 dsRNA ovaries (arrowhead). Nurse cells of the Tc-HexA1 dsRNA ovarioles also appear reduced when compared to the control, although the germarium in some ovarioles seem not to be affected like in B’. (B’) Arrowheads highlights the germarium in Tc-HexA1 dsRNA ovaries, which appears similar to the control in some ovarioles. (C) Tc-HexA1 dsRNA injection largely reduces oviposition when compared to the WT. (D) Analysis of larvae hatching after Tc-HexA1 RNAi when compared to the control. Less than 10% of the laid eggs hatch, indicating an essential role of Tc-HexA1 during embryonic development. (E) Analysis of glucose content in ovaries injected with Tc-HexA1 dsRNA and the control (LacZ dsRNA). Asterisk indicates that the difference between the two groups is statistically significant (p<0,05).
Mentions: In all experiments, we injected the unrelated dsRNA LacZ as a negative control in a separate batch of females. These LacZ dsRNA females laid the normal amount of eggs, which hatched as larvae, indicating that injection of unrelated dsRNA had no effect on T. castaneum development. We then analyzed several parameters related to fecundity in the females injected with Tc-HexA1 dsRNA. First, egg laying of the Tc-HexA1 dsRNA injected females was drastically reduced to 10% of the control (Figure 5C). Second, among the few Tc-HexA1 RNAi laid eggs, only 5% of them hatched as larvae, indicating a strong requirement of this gene for embryonic development (Figure 5D). This extreme reduction in egg laying prevented the analysis of Hex activity or glucose content in RNAi embryos. We tried to circumvent this problem by analyzing glucose content in ovaries of Tc-HexA1 dsRNA and LacZ dsRNA (control) injected females. Interestingly, glucose content was higher in Tc-HexA1 dsRNA ovaries when compared to the control (Figure 5E). This reduction in egg laying stimulated us to compare ovary morphology in control and Tc-HexA1 dsRNA injected females. Morphological analysis via nuclear DAPI stainings of the ovarioles of control and Tc-HexA1 dsRNA injected females showed clear differences (Figure 5A,B). T. castaneum control ovaries display several tube-like projections, the ovarioles e.g.[37], which contains oocytes in different stages of maturation. In control ovaries, larger eggs are present in the distal part of the ovariole. Tc-Hex RNAi ovarioles showed distinct features. First, the ovariole number is reduced (Figure 5B and data not shown). Second, degenerated oocytes can be observed at the distal part (Figure 5B - black arrows), although some mature oocytes surrounded by follicle cells can also be observed (Figure 5B - arrowhead). Third, the germarium appears to be diminished in some Tc-HexA1 RNAi ovarioles (Figure 5B), while others appear similar to control ovaries (Figure 5B’-arrows). This abnormal ovary morphology is probably related to the large reduction in oviposition when compared to the control (Figure 5C). Finally, the few eggs observed after Tc-HexA1 RNAi stopped embryonic development before cellularization (8 hours of development - data not shown). To sum up, the analysis of Tc-HexA1 role during oogenesis and embryogenesis supports an essential role of Tc-HexA1 and glucose metabolism during these processes. Since glucose can be generated via glycogen degradation we sought to investigate how glycogen is regulated during T. castaneum embryogenesis.

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
Related in: MedlinePlus