Fig. 4: Short-term DHA exposure results in a decrease of hemoglobin-derived peptides, and a temporary, reversible cell-cycle arrest in K13 mutant parasites, concomitant with distinct transcriptional responses between K13 mutant and WT parasites.

a Heat map of log₂ fold changes in DHA-treated Cam3.II^C580Y or Cam3.II^WT parasites vs. DMSO controls for the eight metabolites that showed a significant change in level after parasites were pulsed with either 70 or 350 nM DHA for 3 h. DHA treatment at rings and trophozoites resulted in a consistent down-regulation of host hemoglobin-derived peptides in both lines. For levels in untreated rings, see Supplementary Fig. 4f. b, c Transcriptome-based age estimation of the k13-edited R539T, C580Y, and WT in Dd2 or Cam3.II parasites after 6 h pulsed exposure to either 700 nM DHA (solid lines) or DMSO vehicle control (dashed lines). Parasites were sampled up to 48 h post treatment and developmental ages were determined using Spearman rank correlations. Results shown as means ± SEM (N = 3 for each Dd2 line and N = 1 for Cam3.II lines). ***P < 0.001, unpaired two-sided t-tests. P values in b are: 24 h time point—Dd2^WT DHA vs. DMSO P = 1.7E−5, Dd2^C580Y DHA vs. DMSO P = 1.7E−5, Dd2^R539T DHA vs. DMSO P = 4.7E−6; 32 h time point—Dd2^WT DHA vs. DMSO P = 2.5E−6, Dd2^C580Y DHA vs. DMSO P = 2.4E−6, Dd2^R539T DHA vs. DMSO P = 0.002; 48 h time point—Dd2^WT DHA vs. DMSO P = 3.7E−9, Dd2^R539T DHA vs. DMSO P = 3.8E−6. d Giemsa-stained parasite images showed that DHA-treated Cam3.II^WT parasites became pyknotic after 24 h, whereas Cam3.II^R539T parasites underwent a lag in progression through the IDC following DHA treatment, compared to DMSO mock-treated controls. These images are representative of the majority (>50%) of parasites observed at each time and condition per parasite line, with additional examples provided in Supplementary Fig. 6. Microscopy analyses were performed for at least 5000 cells counted across three independent experiments, which yielded similar results. e Heat map of log₂ ratios of DHA-treated/DMSO-treated mRNA expression levels for the 667 hierarchically clustered genes whose expression differed significantly in the Dd2^C580Y and/or Dd2^R539T mutants as compared with Dd2^WT parasites across 3–48 h post initiation of treatment (N = 3 independent experiments; two-factor ANOVA with adjusted P < 0.05; pathways listed in Supplementary Fig. 6 and Supplementary Data 7). A more divergent transcriptional response was observed with Dd2^R539T parasites that display high levels of ART resistance in vitro compared with Dd2^C580Y parasites¹⁶. f Functional enrichment analyses identified biological pathways significantly enriched (P < 0.05, hypergeometric tests) among the genes that were up- or down-regulated in the DHA-treated Dd2^R539T as compared with Dd2^WT parasites relative to DMSO controls in early rings after 3 and 6 h post DHA exposure. Up-regulated genes were relatively more abundant, reflecting increased transcript levels induced by DHA-treated Dd2^R539T parasites vs. Dd2^WT parasites as compared to their respective DMSO controls. Down-regulated genes reflected a reduction in transcription or faster turnover of these transcripts in DHA-treated Dd2^R539T parasites vs. Dd2^WT parasites, compared to their DMSO controls. Gene sets in bold or labeled as # were differentially expressed over the 48 h period of sampling or between DHA-treated Cam3.II^R539T parasites vs. isogenic Cam3.II^WT parasites respectively (see Supplementary Data 7 for pathways and genes).