Gene expression separates male and female single gametocytes into distinct populations. Gene expression separates male and female single gametocytes into distinct populations. (A) Principal component analysis (PCA) separates single gametocytes into male (M) and female (F) populations based on their gene expression. Furthermore, the female population is generally separated by stage, indicating differences in gene expression in mid- and late-stage female gametocytes. PC1, principal component 1. (B) The SINGuLAR Analysis Toolset 3.0 was used to perform unbiased hierarchical clustering on 89 mid- to late-stage gametocytes, 1 asexual cell, and 6 control samples, including positive and negative controls. Eighty-seven gametocyte-specific genes, two housekeeping genes, and two asexual markers were used in this analysis. Genes are clustered based on the Pearson method, while samples are clustered by the Euclidean method. Complete linkage was used to find similar clusters. The blue cluster denotes genes that are higher (enriched) in males, while the pink cluster denotes genes enriched in females. Katelyn A. Walzer et al. mSphere 2018; doi:10.1128/mSphere.00130-18