Transcriptomic analyses of Aedes aegypti cultured cells upon heme exposure

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. Overall design: 33 samples, 3 replicates of each type of sample. 4 total experiments were performed, 6, 9, 9, and 9 samples in each one. The untreated samples are the reference sample in all experiments