Transcriptome assembly and differential gene expression of the invasive avian malaria parasite Plasmodium relictum in Hawai'i

Abstract

The malaria parasite Plasmodium relictum (lineage GRW4) was introduced less than a century ago to the native avifauna of Hawai'i, where it has since caused major declines of endemic bird populations. One of the native bird species that is frequently infected with GRW4 is the Hawai'i 'amakihi (Chlorodrepanis virens). To achieve a better understanding of the transcriptional activities of this virulent parasite, we performed a controlled challenge experiment of 15 'amakihi that were infected with GRW4. Blood samples containing malaria parasites were collected at two time points (intermediate and peak infection stages) from host individuals that were either experimentally infected by mosquitoes or inoculated with infected blood. We then used RNA sequencing to assemble a high-quality blood transcriptome of P. relictum GRW4, allowing us to quantify parasite expression levels inside individual birds. We found few significant differences (one to two transcripts) in GRW4 expression levels between host infection stages and between inoculation methods. However, 36 transcripts showed differential expression levels among all host individuals, indicating a potential presence of host-specific gene regulation across hosts. To reduce the extinction risk of the remaining native bird species in Hawai'i, genetic resources of the local Plasmodium lineage are needed to enable further molecular characterization of this parasite. Our newly built Hawaiian GRW4 transcriptome assembly, together with analyses of the parasite's transcriptional activities inside the blood of Hawai'i 'amakihi, can provide us with important knowledge on how to combat this deadly avian disease in the future.

Keywords: Chlorodrepanis; Hawaiʻi ʻamakihi; Plasmodium; gene expression; hemosporidia; transcriptomics.

FIGURE 1

(a) Image of Plasmodium relictum GRW4 on a Giemsa‐stained blood smear seen through a microscope. Red blood cells are pictured, each containing an elongated nucleus in dark purple color. The pink round shapes within some of the cells constitute the parasites. Image by Carter T. Atkinson. (b) The host species, Hawaiʻi ʻamakihi (Chlorodrepanis virens). Photograph by Loren Cassin‐Sackett

FIGURE 2

(a) Density curve of the Plasmodium relictum GRW4 transcriptome GC content (mean = 21.31%). (b) Proportion of GRW4 transcripts with the best blast match to species in the EBI TrEMBL protein database

FIGURE 3

No clustering of transcriptomes based on parasitemia intensity. Euclidian distance heatmap together with dendrogram show that Plasmodium relictum gene expression patterns do not cluster based on parasitemia levels (here denoted as low, intermediate, high). Right side of the graph lists host IDs in the same sample order as the parasitemia levels given at the bottom of the graph. Darker colors indicate greater distance between samples, and white boxes denote identical samples

FIGURE 4

Few transcriptional differences in Plasmodium relictum between intermediate and peak host infection stages. (a) Differential gene expression analyses identified two upregulated P. relictum transcripts during the peak stage (red points). (b) PCA of P. relictum transcriptomes show samples from the two infection stages largely overlapping in gene expression. Ellipses denote the 90% confidence intervals

FIGURE 5

Expression levels of 36 Plasmodium relictum transcripts that were significantly differentially expressed in one or several host individuals (likelihood ratio test, controlled for time and parasitemia). The y‐axis shows the transcripts' protein products and the x‐axis depicts host individual:infection stage. Warmer colors indicate higher expression levels (rlog‐transformed transcript expression values). Transcript IDs can be found in Table S4