Redox imbalance induces remodeling of glucose metabolism in Rhipicephalus microplus embryonic cell line.

Carbohydrate metabolism not only functions in supplying cellular energy but also has an important role in maintaining physiological homeostasis and in preventing oxidative damage caused by reactive oxygen species. Previously, we showed that arthropod embryonic cell lines have high tolerance to H2O2 exposure. Here, we describe that Rhipicephalus microplus tick embryonic cell line (BME26) employs an adaptive glucose metabolism mechanism that confers tolerance to hydrogen peroxide at concentrations too high for other organisms. This adaptive mechanism sustained by glucose metabolism remodeling promotes cell survival and redox balance in BME26 cell line after millimolar H2O2 exposure. The present work shows that this tick cell line could tolerate high H2O2 concentrations by initiating a carbohydrate-related adaptive response. We demonstrate that gluconeogenesis was induced as a compensation strategy that involved, among other molecules, the metabolic enzymes NADP-ICDH, G6PDH, and PEPCK. We also found that this phenomenon was coupled to glycogen accumulation and glucose uptake, supporting the pentose phosphate pathway to sustain NADPH production and leading to cell survival and proliferation. Our findings suggest that the described response is not atypical, being also observed in cancer cells, which highlights the importance of this model to all proliferative cells. We propose that these results will be useful in generating basic biological information to support the development of new strategies for disease treatment and parasite control.

Aedes fluviatilis cell lines as new tools to study metabolic and immune interactions in mosquito-Wolbachia symbiosis.

In the present work, we established two novel embryonic cell lines from the mosquito Aedes fluviatilis containing or not the naturally occurring symbiont bacteria Wolbachia, which were called wAflu1 and Aflu2, respectively. We also obtained wAflu1 without Wolbachia after tetracycline treatment, named wAflu1.tet. Morphofunctional characterization was performed to help elucidate the symbiont-host interaction in the context of energy metabolism regulation and molecular mechanisms of the immune responses involved. The presence of Wolbachia pipientis improves energy performance in A. fluviatilis cells; it affects the regulation of key energy sources such as lipids, proteins, and carbohydrates, making the distribution of actin more peripheral and with extensions that come into contact with neighboring cells. Additionally, innate immunity mechanisms were activated, showing that the wAflu1 and wAflu1.tet cells are responsive after the stimulus using Gram negative bacteria. Therefore, this work confirms the natural, mutually co-regulating symbiotic relationship between W. pipientis and A. fluviatilis, modulating the host metabolism and immune pathway activation. The results presented here add important resources to the current knowledge of Wolbachia-arthropod interactions.

TOR as a Regulatory Target in Rhipicephalus microplus Embryogenesis.

Embryogenesis is a metabolically intensive process carried out under tightly controlled conditions. The insulin signaling pathway regulates glucose homeostasis and is essential for reproduction in metazoan model species. Three key targets are part of this signaling pathway: protein kinase B (PKB, or AKT), glycogen synthase kinase 3 (GSK-3), and target of rapamycin (TOR). While the role of AKT and GSK-3 has been investigated during tick embryonic development, the role of TOR remains unknown. In this study, TOR and two other downstream effectors, namely S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), were investigated in in vitro studies using the tick embryonic cell line BME26. First, we show that exogenous insulin can stimulate TOR transcription. Second, TOR chemical inhibition led to a decrease in BME26 cell viability, loss of membrane integrity, and downregulation of S6K and 4E-BP1 transcription. Conversely, treating BME26 cells with chemical inhibitors of AKT or GSK-3 did not affect S6K and 4E-BP1 transcription, showing that TOR is specifically required to activate its downstream targets. To address the role of TOR in tick reproduction, in vivo studies were performed. Analysis of relative transcription during different stages of tick embryonic development showed different levels of transcription for TOR, and a maternal deposition of S6K and 4E-BP1 transcripts. Injection of TOR double-stranded RNA (dsRNA) into partially fed females led to a slight delay in oviposition, an atypical egg external morphology, decreased vitellin content in eggs, and decreased larval hatching. Taken together, our data show that the TOR signaling pathway is important for tick reproduction, that TOR acts as a regulatory target in Rhipicephalus microplus embryogenesis and represents a promising target for the development of compounds for tick control.

Carbohydrate Metabolic Compensation Coupled to High Tolerance to Oxidative Stress in Ticks.

Reactive oxygen species (ROS) are natural byproducts of metabolism that have toxic effects well documented in mammals. In hematophagous arthropods, however, these processes are not largely understood. Here, we describe that Rhipicephalus microplus ticks and embryonic cell line (BME26) employ an adaptive metabolic compensation mechanism that confers tolerance to hydrogen peroxide (H2O2) at concentrations too high for others organisms. Tick survival and reproduction are not affected by H2O2 exposure, while BME26 cells morphology was only mildly altered by the treatment. Furthermore, H2O2-tolerant BME26 cells maintained their proliferative capacity unchanged. We evaluated several genes involved in gluconeogenesis, glycolysis, and pentose phosphate pathway, major pathways for carbohydrate catabolism and anabolism, describing a metabolic mechanism that explains such tolerance. Genetic and catalytic control of the genes and enzymes associated with these pathways are modulated by glucose uptake and energy resource availability. Transient increase in ROS levels, oxygen consumption, and ROS-scavenger enzymes, as well as decreased mitochondrial superoxide levels, were indicative of cell adaptation to high H2O2 exposure, and suggested a tolerance strategy developed by BME26 cells to cope with oxidative stress. Moreover, NADPH levels increased upon H2O2 challenge, and this phenomenon was sustained mainly by G6PDH activity. Interestingly, G6PDH knockdown in BME26 cells did not impair H2O2 tolerance, but generated an increase in NADP-ICDH transcription. In agreement with the hypothesis of a compensatory NADPH production in these cells, NADP-ICDH knockdown increased G6PDH relative transcript level. The present study unveils the first metabolic evidence of an adaptive mechanism to cope with high H2O2 exposure and maintain redox balance in ticks.

Non-Invasive Delivery of dsRNA into De-Waxed Tick Eggs by Electroporation.

RNA interference-mediated gene silencing was shown to be an efficient tool for validation of targets that may become anti-tick vaccine components. Here, we demonstrate the application of this approach in the validation of components of molecular signaling cascades, such as the Protein Kinase B (AKT)/Glycogen Synthase Kinase (GSK) axis during tick embryogenesis. It was shown that heptane and hypochlorite treatment of tick eggs can remove wax, affecting corium integrity and but not embryo development. Evidence of AKT and GSK dsRNA delivery into de-waxed eggs of via electroporation is provided. Primers designed to amplify part of the dsRNA delivered into the electroporated eggs dsRNA confirmed its entry in eggs. In addition, it was shown that electroporation is able to deliver the fluorescent stain, 4',6-diamidino-2-phenylindole (DAPI). To confirm gene silencing, a second set of primers was designed outside the dsRNA sequence of target gene. In this assay, the suppression of AKT and GSK transcripts (approximately 50% reduction in both genes) was demonstrated in 7-day-old eggs. Interestingly, silencing of GSK in 7-day-old eggs caused 25% reduction in hatching. Additionally, the effect of silencing AKT and GSK on embryo energy metabolism was evaluated. As expected, knockdown of AKT, which down regulates GSK, the suppressor of glycogen synthesis, decreased glycogen content in electroporated eggs. These data demonstrate that electroporation of de-waxed R. microplus eggs could be used for gene silencing in tick embryos, and improve the knowledge about arthropod embryogenesis.

Non-classical gluconeogenesis-dependent glucose metabolism in Rhipicephalus microplus embryonic cell line BME26.

In this work we evaluated several genes involved in gluconeogenesis, glycolysis and glycogen metabolism, the major pathways for carbohydrate catabolism and anabolism, in the BME26 Rhipicephalus microplus embryonic cell line. Genetic and catalytic control of the genes and enzymes associated with these pathways are modulated by alterations in energy resource availability (primarily glucose). BME26 cells in media were investigated using three different glucose concentrations, and changes in the transcription levels of target genes in response to carbohydrate utilization were assessed. The results indicate that several genes, such as glycogen synthase (GS), glycogen synthase kinase 3 (GSK3), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6 phosphatase (GP) displayed mutual regulation in response to glucose treatment. Surprisingly, the transcription of gluconeogenic enzymes was found to increase alongside that of glycolytic enzymes, especially pyruvate kinase, with high glucose treatment. In addition, RNAi data from this study revealed that the transcription of gluconeogenic genes in BME26 cells is controlled by GSK-3. Collectively, these results improve our understanding of how glucose metabolism is regulated at the genetic level in tick cells.

The conserved role of the AKT/GSK3 axis in cell survival and glycogen metabolism in Rhipicephalus (Boophilus) microplus embryo tick cell line BME26.

BACKGROUND: Tick embryogenesis is a metabolically intensive process developed under tightly controlled conditions and whose components are poorly understood. METHODS: In order to characterize the role of AKT (protein kinase B) in glycogen metabolism and cell viability, glycogen determination, identification and cloning of an AKT from Rhipicephalus microplus were carried out, in parallel with experiments using RNA interference (RNAi) and chemical inhibition. RESULTS: A decrease in glycogen content was observed when AKT was chemically inhibited by 10-DEBC treatment, while GSK3 inhibition by alsterpaullone had an opposing effect. RmAKT ORF is 1584-bp long and encodes a polypeptide chain of 60.1 kDa. Phylogenetic and sequence analyses showed significant differences between vertebrate and tick AKTs. Either AKT or GSK3 knocked down cells showed a 70% reduction in target transcript levels, but decrease in AKT also reduced glycogen content, cell viability and altered cell membrane permeability. However, the GSK3 reduction promoted an increase in glycogen content. Additionally, either GSK3 inhibition or gene silencing had a protective effect on BME26 viability after exposure to ultraviolet radiation. R. microplus AKT and GSK3 were widely expressed during embryo development. Taken together, our data support an antagonistic role for AKT and GSK3, and strongly suggest that such a signaling axis is conserved in tick embryos, with AKT located upstream of GSK3. GENERAL SIGNIFICANCE: The AKT/GSK3 axis is conserved in tick in a way that integrates glycogen metabolism and cell survival, and exhibits phylogenic differences that could be important for the development of novel control methods.

Exogenous insulin stimulates glycogen accumulation in Rhipicephalus (Boophilus) microplus embryo cell line BME26 via PI3K/AKT pathway.

Ticks are obligatory blood-feeding arthropods and important vectors of both human and animal disease agents. Besides its metabolic role, insulin signaling pathway (ISP) is widely described as crucial for vertebrate and invertebrate embryogenesis, development and cell survival. In such cascade, Phosphatidylinositol 3-OH Kinase (PI3K) is hierarchically located upstream Protein Kinase B (PKB). To study the insulin-triggered pathway and its possible roles during embryogenesis we used a culture of embryonic Rhipicephalus microplus cells (BME26). Exogenous insulin elevated cell glycogen content in the absence of fetal calf serum (FCS) when compared to cells without treatment. Moreover, in the presence of PI3K inhibitors (Wortmannin or LY294002) these effects were blocked. We observed an increase in the relative expression level of PI3K's regulatory subunit (p85), as determined by qRT-PCR. In the presence of PI3K inhibitors these effects on transcription were also reversed. Additionally, treatment with Wortmannin increased the expression level of the insulin-regulated downstream target glycogen synthase kinase 3 beta (GSK3beta). The p85 subunit showed elevated transcription levels in ovaries from fully engorged females, but was differentially expressed during tick embryogenesis. These results strongly suggest the presence of an insulin responsive machinery in BME26 cells, and its correlation with carbohydrate/glycogen metabolism also during embryogenesis.