Transcriptomic analyses of Aedes aegypti cultured cells and ex vivo midguts in response to an excess or deficiency of heme: a quest for transcriptionally-regulated heme transporters

Abstract

Background: Aedes aegypti is the principle vector of many arboviruses, including dengue virus and Zika virus, which are transmitted when an infected female mosquito takes a blood meal in order to initiate vitellogenesis. During blood digestion,~10 mM heme-iron is ingested into the midgut lumen. While heme acts as both a nutrient and signaling molecule during blood digestion, it can also be highly toxic if left unchaperoned. Both signaling by, and degradation of, heme are intracellular processes, occurring in the nucleus and cytoplasm, respectively. However, the precise mechanism of heme uptake into the midgut epithelium is not currently known. Results: We used next generation RNA sequencing with the goal to identify genes that code for membrane bound heme import protein(s) responsible for heme uptake into the midgut epithelium. Heme deprivation increased uptake of a heme fluorescent analog in cultured cells, while treatment of midguts with an excess of heme decreased uptake, confirming physiological changes were occurring in these heme-sensitive cells/tissues prior to sequencing. A list of candidate genes was assembled for each of the experimental sample sets, which included Aag2 and A20 cultured cells as well as midgut tissue, based on the results of a differential expression analysis, soft cluster analysis and number of predicted transmembrane domains. Lastly, the functions related to heme transport were examined through RNAi knockdown. Conclusions: Despite a large number of transmembrane domain containing genes differentially expressed in response to heme, very few were highly differentially expressed in any of the datasets examined. RNAi knockdown of a subset of candidates resulted in subtle changes in heme uptake, but minimal overall disruption to blood digestion/egg production. These results could indicate that heme import in Ae. aegypti may be controlled by a redundant system of multiple distinct transport proteins. Alternatively, heme membrane bound transport in Ae. aegypti could be regulated post-translationally.

Identification and characterization of a heme exporter from the MRP family in Drosophila melanogaster

“…To study heme transport in D. melanogaster , a convenient screen system was established to detect heme transport in S2 cells. Zinc mesoporphyrin (ZnMP), a fluorescent heme analog, can be easily monitored by microscopy and flow cytometry [ 35 , 40 ]. With increasing concentrations and time, the fluorescence signals grew simultaneously, indicating that ZnMP absorption is dose- and time-dependent in S2 cells (Fig.…”

“…Despite that a transcriptional analysis has been performed in A. aegypti [ 35 , 44 , 45 ], there is no functional study of heme export in mosquito species. As both the model organism D. melanogaster and the mosquito species A. aegypti belong to the Diptera and are therefore related in evolutionary terms, we wondered whether the heme transporters identified in D. melanogaster are conserved in A. aegypti .…”

“…Previously, some putative heme importers or exporters have been identified in yellow fever mosquito through transcriptomic analyses. It is concluded that heme import may be controlled by a redundant mechanism [ 35 ]. Also, ABCB10 was identified in Rhipicephalus (Boophilus) microplus , which cannot self-synthesize heme, to function as the heme importer located in the hemosome of the midgut [ 36 , 37 ].…”

Aedes aegypti dyspepsia encodes a novel member of the SLC16 family of transporters and is critical for reproductive fitness

“…Among some of the candidate genes, we observed another Ae. aegypti cell line, A20, exhibited higher expression than Aag2 [41], and we used A20 cells for these genes for the downstream dual luciferase assay (Table 1). The original differential expression Gene silencing and luciferase reporter assay to verify iron transport by the candidate genes.…”

Mosquito metallomics reveal copper and iron as critical factors for Plasmodium infection

“…It is also a critical inducer of Plasmodium gametogenesis in the intestinal lumen of Anopheles stephensi mosquitoes [29]. Amongst the many unknowns in insect iron metabolism [30], recent efforts to identify a heme transporter [31] and unravel the function of multicopper oxidases [32,33] in iron trafficking have been unsuccessful (for related studies in D. melanogaster see [34][35][36]). Beyond iron, there has been little attention to other essential metals, such as copper, zinc, manganese, and molybdenum in mosquito biology.…”