Glycogen and Glucose Metabolism Are Essential for Early Embryonic Development of the Red Flour Beetle <i>Tribolium castaneum</i>

<div><p>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 <i>Tribolium castaneum</i>, 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 <i>glycogen synthase kinase 3 (GSK-3)</i> and <i>hexokinase (HexA)</i> genes during <i>T. castaneum</i> embryogenesis. Expression analysis via <i>in situ</i> 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 <i>Tc-GSK-3</i> and <i>Tc-HexA1</i> RNAi embryos. Importantly, glycogen levels are upregulated after <i>Tc-GSK-3</i> RNAi and glucose levels are upregulated after <i>Tc-HexA1</i> RNAi, indicating that both genes control metabolism during embryogenesis and oogenesis, respectively. Altogether our results show that <i>T. castaneum</i> embryogenesis depends on the proper control of glucose and glycogen.</p></div

Simplified view of the metabolic pathways investigated in this study.

<p>Glycogen Synthase Kinase-3 (GSK-3-blue) is not only involved in glycogen synthase (GS) regulation, but also acts as a downstream component of the Wnt signaling pathway (<i>e.g. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065125#pone.0065125-KaidanovichBeilin1" target="_blank">[60]</a>). Glycogen Phosphorylase (GP) breaks up glycogen into glucose subunits. Hexokinase (Hex-red) is involved in producing Glucose-6-P from glucose, acting as an important enzyme of the glycolytic pathway. Other Glucose-6-P possible roles in different metabolic pathways are omitted for simplicity.</p

<i>Tc-HexA</i> RNAi affects oogenesis, glucose content, and reduces egg lay.

<p>(A,B) Ovary morphology in (A) control ovaries (injected with LacZ dsRNA) and (B,B’) After <i>Tc-HexA1</i> dsRNA injection. (B)<i>Tc-HexA1</i> 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 <i>Tc-HexA1</i> dsRNA ovaries (arrowhead). Nurse cells of the <i>Tc-HexA1</i> 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 <i>Tc-HexA1</i> dsRNA ovaries, which appears similar to the control in some ovarioles. (C) <i>Tc-HexA1</i> dsRNA injection largely reduces oviposition when compared to the WT. (D) Analysis of larvae hatching after <i>Tc-HexA1</i> RNAi when compared to the control. Less than 10% of the laid eggs hatch, indicating an essential role of <i>Tc-HexA1</i> during embryonic development. (E) Analysis of glucose content in ovaries injected with <i>Tc-HexA1</i> dsRNA and the control (LacZ dsRNA). Asterisk indicates that the difference between the two groups is statistically significant (p<0,05).</p

<i>Tc-GSK-3</i> knockdown affects <i>T. castaneum</i> egg laying, larvae hatching and glycogen content.

<p>(A) <i>Tc-GSK-3</i> expression decreases after <i>Tc-GSK-3</i> dsRNA injection. (B) Number of laid eggs diminishes 50% after <i>Tc-GSK-3</i> RNAi when compared to the control. (C) Number of hatching larvae decreases after <i>Tc-GSK-3</i> RNAi to about 20% of the control. (D) Glycogen content increases in <i>Tc-GSK-3</i> RNAi eggs when compared to the control (<i>LacZ</i> RNAi eggs). Asterisk indicates that the difference between the two groups is statistically significant (p<0,05).</p

A simplified model for the regulation of glucose and glycogen during early <i>T. castaneum</i> embryonic development.

<p>Three distinct time points of <i>Tribolium castaneum</i> development are highlighted (0–8, 8–20 and 20–72 hours). During the first eight hours of development fast cleavages occur and glycogen content maternally provided is degraded. Hexokinase activity and <i>Tc-HexA1</i> mRNA expression is high. Between 8–16 hours of development the extra-embryonic membranes, amnion (purple) and serosa (yellow) are established and <i>Tc-HexA1</i> and <i>Tc-GSK-3</i> expression are restricted to the embryonic tissue (see text for details). During these stages (8–20 hours) glycogen and glucose levels remain largely stable. Between 20–72 hours glycogen is degraded and glucose levels increase, as well as Hex activity. At 20 hours AEL head (H) and the posterior growth-zone (gz) can be visualized. pp – posterior pit, sw – serosal window, ac- amniotic cavity. For a morphological description of the embryonic events see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065125#pone.0065125-Handel1" target="_blank">[49]</a>.</p

Analysis of glucose content during <i>Tribolium castaneum</i> embryogenesis.

<p>Glucose content is low during the first 20 hours of embryonic development, and increases from 20 hours on towards maximal levels until larvae hatching (about 96 hours after egg laying). Grey box highlights the region with high glucose content.</p

<i>In situ</i> hybridization of <i>Tc-HexA1</i> and Hexokinase activity during the first 24 hours of beetle embryogenesis.

<p>(A–E) <i>In situ</i> hybridization and respective nuclear DAPI stainings (A’–E’). In all panels head is to the left and dorsal side up. (F) Hex activity during the first 24 hours of embryonic development. (A,A’) Eggs during the first four hours after egg lay (AEL), when rapid cleavages occur display ubiquitous <i>Tc-HexA1</i> mRNA. (B,B’) Eggs between four and eight hours (4–8 hours) also show ubiquitous <i>Tc-HexA1</i> expression. (C,C’) During gastrulation between 8–12 hours <i>Tc-HexA1</i> expression largely decreases, remaining low between 12–16 hours in D,D’. (E,E’) During germ band elongation (16–20 hours) <i>Tc-HexA1</i> expression is upregulated and occurs only in the embryonic region (emb in D), being absent in the serosa (ser). (F) Specific Hexokinase activity (U/mg protein). High activity is detected in egg extracts from 0–4 hours and after 16 hours, which correlates to <i>Tc-HexA1</i> mRNA expression pattern. pp - posterior pit, emb - embryonic tissue, ser - serosa.</p

<i>Hexokinase (Hex)</i> locus structure in <i>Tribolium</i> and <i>Hex</i> gene evolution in insects.

<p>(A) Snapshot of the Beetlebase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065125#pone.0065125-Kim2" target="_blank">[42]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065125#pone.0065125-Wang1" target="_blank">[43]</a> showing relative expression of <i>Tc-HexA1</i> (Tc-Glean00319) and <i>Tc-HexA2</i> (Tc-Glean00318) at 6 hours, 14 hours and 30 hours of embryonic cDNA libraries. Note that <i>Tc-HexA1</i> is expressed at early stages while <i>Tc-HexA2</i> seems to be upregulated only at later stages. (B) Phylogenetic analysis using maximum likelihood method. Amino acid substitution model: WAG+G. In Drosophillids four <i>Hex</i> genes exist (<i>HexC, HexT1, HexT2 and HexA),</i> while in most other insects only one <i>Hex</i> gene exists. Bootstrap values (1,000 replicates) are indicated as percentages. <i>Aae - Aedes aegypti</i>; <i>Ad - Anopheles darling</i>; <i>Am - Apis mellifera; Cq - Culex quinquefasciatus; Dmel - Drosophila melanogaster; Dpse - Drosophila pseudoobscura; Nv - Nasonia vitripennis; Tc - Tribolium castaneum</i>. Accession numbers for the NCBI are available upon request.</p

<i>Tc-GSK-3</i> is expressed in the embryonic tissue throughout embryogenesis.

<p>(A–D) <i>Tc-GSK-3</i> expression and respective nuclear DAPI stainings (A’–D’). (A,A’) In the first four hours after egg laying (AEL), <i>Tc-GSK-3</i> is ubiquitously expressed. Note the few nuclei at the periphery. In the next four hours (4–8 hours) a similar ubiquitous expression pattern is observed (data not shown). (B–B’) Between 8–12 hours AEL <i>Tc-GSK-3</i> expression is mainly concentrated at the embryonic cells (emb) and not in the extraembryonic polyploid serosal (ser) cells. (C–C’) This pattern of strong expression of <i>Tc-GSK-3</i> in the embryonic cells remains between 12–16 hours AEL, when serosal cells surround the embryonic ones. (D–D’) Between 16–20 hours AEL <i>Tc-GSK-3</i> expression is still largely confined to the embryo (emb), which is undergoing germ band elongation. At 20–24 hours AEL a similar expression profile is observed (data not shown).</p

Glycogen content decreases in two phases during <i>T. castaneum</i> embryogenesis.

<p>High glycogen content is detected at the first four hours of embryogenesis and decreases between 4 and 8 hours of embryonic development (dashed box). Glycogen level is maintained or slightly increased between 8–12, 12–16, 16–20 and 20–24 hours. During the next 24 hours glycogen content largely decreases (grey box) and remains low until 72 hours.</p