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

et al. (2013) Glycogen and Glucose Metabolism Are Essential for Early Embryonic Development of the Red Flour Beetle Tribolium castaneum. PLoS ONE 8(6): e65125. doi:10.1371/journal.pone.0065125 Glycogen and Glucose Metabolism Are Essential for Early Embryonic Development of the Red Flour Beetle Tribolium castaneum Amanda Fraga 0 Lupis Ribeiro 0 Mariana Lobato 0 Vito ria Santos 0 Jose Roberto Silva 0 Helga Gomes 0 Jorge Luiz da Cunha Moraes 0 Jackson de Souza Menezes 0 Carlos Jorge Logullo de 0 Oliveira 0 Eldo Campos 0 Rodrigo Nunes da Fonseca 0 Jo Anne Powell-Coffman, Iowa State University, United States of America 0 1 Laborato rio Integrado de Bioqu mica Hatisaburo Masuda (LIBHM), Nu cleo de Pesquisas Ecolo gicas e So cioambientais de Macae (NUPEM), Universidade Federal do Rio de Janeiro (UFRJCampus Macae ) , Rio de Janeiro , Brazil , 2 Programa de Po sgraduac a o em Produtos Bioativos e Biociencias (PPGPRODBIO), Universidade Federal do Rio de Janeiro (UFRJCampus Macae ) , Rio de Janeiro , Brazil , 3 Laborato rio de Qu mica e Func a o de Prote nas e Pept deos and Unidade de Experimentac a o Animal, Universidade Estadual Norte Fluminense Darcy Ribeiro (UENF) , Campos dos Goytacazes, Rio de Janeiro , Brazil , 4 Instituto Nacional de Ciencia e Tecnologia em Entomologia Molecular , Rio de Janeiro , Brazil 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. - Funding: Brazil is funded by FAPERJ, (Fundacao de Amparo a` Pesquisa do Estado do Rio de Janeiro (Portuguese: Research Support Foundation of the State of Rio de Janeiro; Brazil)) CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (Brazil)) and INCT-Entomologia Molecular. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Energy homeostasis is an essential process for life [1]. Carbon source conversion in living tissues involves tight regulation of enzymes of the glycolytic pathway. During oogenesis, the insect mother deposits lipids, proteins, carbohydrates and mRNAs which are essential for posterior embryonic development. Particularly in oviparous species the insect egg must contain all nutrients required for embryonic development being a closed and isolated system from the environment [2]. Classical insect studies have investigated metabolic activity in several organs during adulthood and oogenesis [3,4,5,6,7], but only recently metabolic activity during embryogenesis was analyzed in a few arthropod species [8,9]. A simplified model of energy utilization of glycogen mobilization into the glycolytic pathway (Figure 1) shows that glycogen storage is mobilized into the glycolytic pathway due to the action of Glycogen Phosphorylase (GP), while its synthesis is due to Glycogen Synthase (GS), whose activity is regulated by glycogen synthase kinase-3 (GSK-3) (reviewed in [10]). GSK-3 is not only involved in metabolic reactions but also acts as an essential kinase of the Wnt pathway [11,12], which plays an essential role during embryogenesis. Recently, GSK-3 was also shown to be involved in the integration of the Wnt and BMP/Smad1 pathways [13,14,15]. Thus, GSK-3 is an important hub linking important signaling pathways during embryogenesis to metabolic reactions. Glycogen break down generates glucose, which enters in the glycolytic pathway being converted into pyruvate (Figure 1). This process leads to ATP generation. Important enzymes for this process such as Hexokinase (Hex or HK) have been characterized in several organisms [16,17,18,19,20]. Hexokinase (ATP: hexose6-phosphotransferase, E.C. 2.7.1.1; Hex) catalyzes the first step in the oxidative metabolism of hexoses via glycolysis. Four distinct hexokinase isozymes are reported for mammalian tissues and are named as types IIV (also called types 14 or AD). Structurally, Hex IIII are 100 kDa proteins thought to have evolved by duplication and fusion of a gene encoding an ancestral 50 kDa hexokinase [16,17,21]. In insects, classical studies have isolated four Hex isozymes from different tissues of the fruit fly Drosophila melanogaster [22,23]. After D. melanogaster genome sequencing four genes encoding Hex proteins were identified [24,25,26]. Only recently Hex from the shrimp Litopenaeus vannamei was cloned and shown to be regulated by hypoxia as its mammal homolog [27]. Previous studies have analyzed metabolic regulation during embryogenesis in blood sucking arthropod species like the tick Riphicephalus (Boophilus) microplus [8] and the mosquito Aedes aegypti [9]. These studies have revealed important morphogenetic events which are associated with changes in the embryonic metabolic regulation, e.g., germ band retraction is correlated with an increase in glycolysis during mosquito embryogenesis [9]. Here, we have investigated the metabolic regulation in the red flour beetle Tribolium castaneum, which has emerged in the past few years as an excellent model for studies of embryogenesis and evolution of signaling pathways (reviewed in [28,29]). This beetle had its genome sequenced [30], is amenable to functional studies like RNA interference (RNAi) [31,32], and mutant and enhancer trap lines have been developed [33]. T. castaneum feeds on whole grain flour during all its motile stages. This stands in g (...truncated)