Comparative transcriptomics of female and male gametocytes in Plasmodium berghei and the evolution of sex in alveolates

Abstract

Background: The clinical symptoms of malaria are caused by the asexual replication of Plasmodium parasites in the blood of the vertebrate host. To spread to new hosts, however, the malaria parasite must differentiate into sexual forms, termed gametocytes, which are ingested by a mosquito vector. Sexual differentiation produces either female or male gametocytes, and involves significant morphological and biochemical changes. These transformations prepare gametocytes for the rapid progression to gamete formation and fertilisation, which occur within 20 min of ingestion. Here we present the transcriptomes of asexual, female, and male gametocytes in P. berghei, and a comprehensive statistically-based differential-expression analysis of the transcriptional changes that underpin this sexual differentiation.

Results: RNA-seq analysis revealed numerous differences in the transcriptomes of female and male gametocytes compared to asexual stages. Overall, there is net downregulation of transcripts in gametocytes compared to asexual stages, with this trend more marked in female gametocytes. Our analysis identified transcriptional changes in previously-characterised gametocyte-specific pathways, which validated our approach. We also detected many previously-unreported female- and male-specific pathways and genes. Transcriptional biases in stage and gender were then used to investigate sex-specificity and sexual dimorphism of Plasmodium in an evolutionary context. Sex-related gene expression is well conserved between Plasmodium species, but relatively poorly conserved in related organisms outside this genus. This pattern of conservation is most evident in genes necessary for both male and female gametocyte formation. However, this trend is less pronounced for male-specific genes, which are more highly conserved outside the genus than genes specific to female development.

Conclusions: We characterised the transcriptional changes that are integral to the development of the female and male sexual forms of Plasmodium. These differential-expression patterns provide a vital insight into understanding the gender-specific characteristics of this essential stage that is the primary target for treatments that block parasite transmission. Our results also offer insight into the evolution of sex genes through Alveolata, and suggest that many Plasmodium sex genes evolved within the genus. We further hypothesise that male gametocytes co-opted pre-existing cellular machinery in their evolutionary history, whereas female gametocytes evolved more through the development of novel, parasite-specific pathways.

Keywords: Evolution; Gametocyte; Malaria; Next-generation sequencing; Plasmodium; Plasmodium berghei; RNA-seq; Sex; Transcriptome.

Fig. 1

Purification of different stages of P. berghei. a Life-cycle schematic highlighting female gametocytes (A), male gametocytes (B), and asexual stages (C). b Fluorescence-activated flow cytometry plot showing collected samples of RFP female gametocytes (A) and GFP male gametocytes (B)

Fig. 2

Comparative expression data for different stages. a Heatmap of expression, with genes on the vertical axis, and strains (male, ♂; female, ♀; asexual, A) on the horizontal axis. Replicates from each life-stage cluster independently (upper dendrogram). b Multi-dimensional scaling plot, showing clustering of sexual stages. c Venn diagram showing male and female genes that are differentially expressed compared to asexual genes, and the overlap between these groups. d Schematic depicting the asexual developmental precursor, and the differentiation into either female or male gametocytes. Thick arrows represent up- or downregulation, labelled with how many genes are affected, and arrow width proportional. Monochrome arrows represent shared up- or downregulation; red and green arrow pairs represent up- or downregulation specific to female or male gametocytes respectively. The green-red arrow pair on top represents genes identified by direct comparison of the two genders. e Fold-change of reads mapped to each chromosome compared to asexual samples. Each point represents a biological sample. The numbers of mapped reads were normalised to total reads mapped for that sample, then fold-change to the mean of the asexual counts for each chromosome plotted. Female and male samples with significant differences to asexual stages are marked by blue rectangles (p value < 0.05)

Fig. 3

Gene set enrichment analysis. The gametocyte-specific genes are to the left of the x-axis, and the asexual-specific genes are to the right. (See main text for a more extensive explanation). Female enrichment score is plotted with a red line, and male enrichment score with a green line. Statistically significant differences (p value < 0.05) are indicated by an asterisk (*). Lines in the “barcode” beneath each plot indicate the position of a gene from that gene set within the ranked list. a Enrichment analysis of two P. berghei datasets. b P. berghei genes were transformed into their P. falciparumsyntenic orthologues, then analysed for enrichment of P. falciparum datasets

Fig. 4

Phylogenetic comparisons of differentially-expressed transcripts. (a and b) Volcano plots of female- and male-specific genes. Each point represents a P. berghei gene from our analysis. The y-axis indicates confidence in log odds (B), and the x-axis shows magnitude of difference as log₂ (fold change ♀/♂), from most male-specific (♂) to most female-specific (♀). Diamond symbols within boxplots indicate the mean. All plots marked *** have p value < 0.001 a P. berghei genes with syntenic orthologues from P. falciparum are plotted in cyan; genes lacking syntenic orthologues are shown in pink. Inset boxplots show genes with syntenic orthologues are more likely to have been identified as differentially expressed between genders, based on log odds. Boxplots below the volcano plot demonstrate that genes with syntenic orthologues are more likely to have a lower magnitude of change in male-specific genes, with no difference in female-specific genes. b Genes concordant in both species, i.e. male-specific (MM) or female-specific (FF) in both, are marked in orange. Genes that are discordant, i.e. male-specific in P. berghei and female-specific in P. falciparum (MF) or the opposite (FM), are shown in purple. Inset boxplots show concordant genes are more likely to have been identified in our differential expression screen (higher log odds), and discordant gene are less likely. Boxplots below the volcano plot show that concordant genes have larger magnitude of differential expression, and discordant genes have a lower magnitude. c Phylogeny showing conservation of orthologous groups in different organisms. The central panel depicts the number of conserved general sex orthologous groups divided by total orthologous groups for each species. The right panel partitions these sex-related groups into shared sex genes (in both male and female gametocytes), male-specific genes, and female-specific genes. These are normalised for the total number of general-sex orthologous groups. N.B. these need not total 1.00, because a few orthologous groups contain genes from multiple sets. The vertical dashed lines reference the P. berghei data on the first row of the phylogeny, and are collinear with the category boundaries for this organism