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Transcriptomic analysis of Aedes aegypti midgut epithelial cells upon heme exposure

ABSTRACT: The female mosquito Aedes aegypti requires amino acids and other nutrients like heme and iron from a blood meal to initiate vitellogenesis. Heme is a pro-oxidant molecule that acts as a nutrient, signaling molecule and in large quantities, as a toxin. Ae. aegypti has developed a few strategies to handle heme toxicity, as during a typical meal ~10mM is released into the midgut lumen. These strategies include heme aggregation to the peritrophic matrix and the degradation of heme by heme oxygenase in the cytosol of the midgut epithelium. However, despite the importance of heme as a nutrient and toxin, the mechanism of entry into the midgut epithelial cells is not currently known. As no heme transport proteins in have been identified in any dipteran, heme fluorescent analog studies were performed to visualize changes in expression caused by heme followed by global expression analyses performed in midgut tissues using NGS-based RNA sequencing with the end goal to identify the gene(s) that encode the membrane bound heme import proteins responsible for heme uptake during blood digestion. Examination of differential expression of mRNA transcripts at the gene level, found 65 significant DE genes at the adjusted p-value cut off of 0.0001, 38 of which are TM containing and only 2 of which showed high expression changes, AAEL019570 (-2.04 log2, ~0.243), unknown function, and AAEL000717 (3.91 log2, ~15.03), a protocadherin. This list was further reduced to 16 genes with potential heme import function and 7 genes with potential heme export function by examination of differential expression, number of TM domains and function relating to transport. As very few highly differentially expressed genes were found in the analysis, heme import may be controlled by a redundant system of multiple transport proteins instead of a single highly expressed one. Alternatively, heme transport in Ae. aegypti could be regulated post-translationally.

ORGANISM(S): Aedes aegypti

PROVIDER: GSE141651 | GEO | 2020/08/03

REPOSITORIES: GEO

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Project description:After a bloodmeal, mosquitoes import heme into the midgut epithelium. Heme acts as an essential signal for oogenesis in Aedes aegypti. However, the mechanisms behind heme import in Aedes aegypti are largely unexplored. In this study, RNA sequencing data from 4 different Aedes aegypti cell culture experiments where exposure to an overabundance or deficiency of heme was examined to identify heme-responsive genes. Zinc mesoporphyrin (ZnMP), a heme fluorescent analog, was used to measure changes in heme uptake prior to mRNA sequencing. A soft cluster analysis was performed to identify genes encoding potential membrane bound importers and exporters based on expression profiles across the samples for each experiment. Stronger candidates were obtained by comparing genes in each dataset to each other. When comparing all datasets to each other, 223 candidate genes with expression pattern changes consistent with importers were found to be heme-regulated in only 2 datasets, 46 were heme-regulated in 3 datasets and 2 was heme-regulated in all 4 datasets. In contrast, 114 candidate genes with patterns consistent with exporters were common to only 2 datasets, with just 11 present in 3 of the 4 datasets. A large number of genes containing transmembrane domains were isolated across the 4 datasets which showed downregulation or upregulation in response to heme overexposure conditions indicating the possibility that heme membrane bound import and export genes were identified respectively, with the reverse trend observed in the presence of heme deficiency. However, few transporter candidate genes were identified that showed high differential expression (above 4-fold or below 1/4th fold). Ultimately, as few strong candidates were identified by these studies, we consider the possibility that each cell line could have a redundant system of heme import, whereby multiple transport proteins each contribute to the total heme accumulation in the cell. Alternatively, it is possible that these cells lines do not regulate heme transport at the transcript level, but rather do so post-translationally.

Project description:Background: The piRNA pathway has been shown in model organisms to be involved in silencing of transposons thereby providing genome stability. In D. melanogaster the majority of piRNAs map to these sequences. The medically important mosquito species Aedes aegypti has a large genome size, a high transposon load which includes Miniature Inverted repeat Transposable Elements (MITES) and an expansion of the piRNA biogenesis genes. Studies of transgenic lines of Ae. aegypti have indicated that introduced transposons are poorly remobilized and we sought to explore the basis of this. We wished to analyze the piRNA profile of Ae. aegypti and thereby determine if it be responsible for transposon silencing in this mosquito. Results: Estimated piRNA sequence diversity was comparable between Ae. aegypti and D. melanogaster, but surprisingly only 19% of mosquito piRNAs mapped to transposons compared to 51% for D. melanogaster. Ae. aegypti piRNA clusters made up a larger percentage of the total genome than those of D. melanogaster but did not contain significantly higher percentages of transposon derived sequences than other regions of the genome. Ae. aegypti contains a number of protein coding genes that may be sources of piRNA biogenesis with two, traffic jam and maelstrom, implicated in this process in model organisms. Several genes of viral origin were also targeted by piRNAs. Examination of six mosquito libraries that had previously been transformed with transposon derived sequence revealed that new piRNA sequences had been generated to the transformed sequences, suggesting that they may have stimulated a transposon inactivation mechanism. Conclusions: Ae. aegypti has a large piRNA complement that maps to transposons but primarily gene sequences, including many viral-derived sequences. This, together the more uniform distribution of piRNA clusters throughout its genome suggest that some aspects of the piRNA system differ between Ae. aegypti and D. melanogaster.

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Transcriptomic analysis of Aedes aegypti midgut epithelial cells upon heme exposure

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