Project description:The migratory locust, Locusta migratoria, is a serious agricultural pest and important insect model to study insect digestion and feeding behavior. The gut is one of the primary interfaces between the insect and its environment. Nevertheless, knowledge on the gut transcriptome of L. migratoria is still very limited. With the development of two EST databases from L. migratoria (whole body and central nervous system (CNS)) and one EST database from Schistocerca gregaria (CNS), an abundance of transcript data was made available for locusts. In addition, the genome of Locusta was also recently published in an effort to create a better understanding of swarm formation and flight behavior (Wang et al., 2014). While the transcript composition of nervous tissue was relatively well studied after the development of the specific CNS-derived EST-databases from both L. migratoria and S. gregaria, little transcript profiling information is available for the digestive system at the moment. Locusts are, however, widely used as physiological model organisms regarding the regulation and control of feeding and digestion, and improved knowledge on the gut transcriptome could contribute significantly to a better understanding of their gut physiology. Therefore, we aimed to use the available sequence data to specifically identify gut-expressed transcripts in 5th larval locusts. By means of two independent self-self microarray hybridizations for two distinct tissues, the gut and the brain, a selection could be made of those ESTs that are present in the gut and/or the brain. Here, sequences that were found to be expressed in gut but not brain were further functionally annotated to shed new light on the complex physiology of the locust digestive system. Since the gut is the single most important organ in digestion, and both tissues are assumed to be involved in the regulation thereof, the resulting subset of sequences can also be valuable for further in-depth studies on the regulation of digestion. In addition, the method allowed us to rank the signal intensities, using them as a rough indicator to compare relative transcript abundance in the gut. Therefore, the data complement previously published transcript and genomic data, and provide a clear overview of the expressed portion of the genome in the gut. Taken together, the present data provide significant insight into locust larval gut physiology, and will be valuable for future studies on the insect gut.

Project description:The migratory locust, Locusta migratoria, is a serious agricultural pest and important insect model to study insect digestion and feeding behavior. The gut is one of the primary interfaces between the insect and its environment. Nevertheless, knowledge on the gut transcriptome of L. migratoria is still very limited. With the development of two EST databases from L. migratoria (whole body and central nervous system (CNS)) and one EST database from Schistocerca gregaria (CNS), an abundance of transcript data was made available for locusts. In addition, the genome of Locusta was also recently published in an effort to create a better understanding of swarm formation and flight behavior (Wang et al., 2014). While the transcript composition of nervous tissue was relatively well studied after the development of the specific CNS-derived EST-databases from both L. migratoria and S. gregaria, little transcript profiling information is available for the digestive system at the moment. Locusts are, however, widely used as physiological model organisms regarding the regulation and control of feeding and digestion, and improved knowledge on the gut transcriptome could contribute significantly to a better understanding of their gut physiology. Therefore, we aimed to use the available sequence data to specifically identify gut-expressed transcripts in 5th larval locusts. By means of two independent self-self microarray hybridizations for two distinct tissues, the gut and the brain, a selection could be made of those ESTs that are present in the gut and/or the brain. Here, sequences that were found to be expressed in gut but not brain were further functionally annotated to shed new light on the complex physiology of the locust digestive system. Since the gut is the single most important organ in digestion, and both tissues are assumed to be involved in the regulation thereof, the resulting subset of sequences can also be valuable for further in-depth studies on the regulation of digestion. In addition, the method allowed us to rank the signal intensities, using them as a rough indicator to compare relative transcript abundance in the gut. Therefore, the data complement previously published transcript and genomic data, and provide a clear overview of the expressed portion of the genome in the gut. Taken together, the present data provide significant insight into locust larval gut physiology, and will be valuable for future studies on the insect gut. Self-self hybridisation of Cy5- and Cy3-labeled samples. One biological repeat per tissue type, i.e., brain and gut. Gut is a combination of foregut, midgut, gastric caeca and hindgut. Per tissue type, a pool was made from RNA from 3 pools of 5 locusts, and this for 3 different feeding conditions, resulting in samples derived from a total of 45 locust larvae. Feeding conditions were normally fed, fed with diet containing additional protease inhibitors (PIs), and starved locusts.

Project description:Plasmodium and Toxoplasma are parasites of major medical importance that belong to the Apicomplexa phylum of protozoa. These parasites transform into various stages during their life cycle and express a specific set of proteins at each stage. Although still little is known of how gene expression is controlled in Apicomplexa, histone modifications, particularly acetylation, are emerging as key regulators of parasite differentiation and stage conversion. Here, we investigated the anti-Apicomplexa effect of FR235222, a histone deacetylase (HDAC) inhibitor. We show that FR235222 is active against a variety of Apicomplexa genera, including Plasmodium and Toxoplasma, and is more potent than other HDACi such as TSA and the clinically relevant compound, pyrimethamine. We identify TgHDAC3 as the target of FR235222 in Toxoplasma tachyzoites and demonstrate the crucial role of the conserved and Apicomplexa HDAC-specific residue TgHDAC3 T99 in the inhibitory activity of the drug. We also show that FR235222 induces differentiation of the tachyzoite (replicative) into the bradyzoite (non replicative) stage. Additionally, via its anti-TgHDAC3 activity, FR235222 influences the expression of ~370 genes, a third of which are stage-specifically expressed. These results identify FR235222 as a potent HDAC inhibitor of Apicomplexa, and establish HDAC3 as a central regulator of gene expression and stage conversion in Toxoplasma and likely other Apicomplexa. Freshly released tachyzoites were needle-passed, and filtered using a 3-Âµm nucleopore membrane. Parasites were resuspended into fresh DMEM supplemented with 10% (v/v) FBS and 25 mM HEPES buffer pH7.2. Parasites were incubated in the presence of FR235222 (40 nM) or DMSO (0.1%) for 4 h at 37Â°C with 5% CO2. For ChIP-chip experiments freshly released tachyzoites (~5 x 109 at ~12 x 107 parasites/mL) were fixed for 15 min in 1% formaldehyde. Increase in AcH4 signals was verified by immunoblot to verify that FR235222 treatment was effective. To prepare chromatin samples, fixed parasites were lysed in MNase buffer (0.32 M Sucrose, 50 mM Tris-HCl pH7.8, 4 mM MgCl2, 3 mM CaCl2, 100 mM NaCl, 0.25% (v/v) NP40, 5% (v/v) glycerol, protease inhibitor EDTA-free cocktail (Roche)) and DNA was digested for 4 min at 37Â°C by MNase (2 units/mL). Digestion was stopped with 20 mM EDTA and chromatin was recovered in the soluble fraction after centrifugation at 10,000 g at 4Â°C; this constituted the S1 fractions. Pelleted materials were resuspended in dialysis buffer (1mM Tris-HCl pH7.8, 0.2 mM EDTA) containing 1 mM PMSF and protease inhibitor cocktail (RocheÂ®) and dialyzed overnight at 4Â°C against the same solution. Then dialyzed materials were centrifuged and supernatant were harvested; this constitutes the S2 fractions. For chromatin immunoprecipitations, fractions S1 and S2 were pooled and DNA quality was verified by electrophoresis on 2% agarose gels; oligonucleosome ladder of 100-1000 bp were obtained. The histone-DNA complexes were immunoprecipitated with anti-acetyl histone H4 (UpstateÂ®, catalog # 06-866) antibodies according to NimbleGenâs protocol (http://www.genomecenter.ucdavis.edu/expression_analysis/documents).

Dataset Information

Study of Gregarinidae (Apicomplexa) from Orthoptera

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