Gametocyte production and transmission fitness of African and Asian Plasmodium falciparum isolates with differential susceptibility to artemisinins

Abstract

The emergence of Plasmodium falciparum parasites partially resistant to artemisinins (ART-R) poses a significant threat to recent gains in malaria control. ART-R has been associated with PfKelch13 (K13) mutations, which differ in fitness costs. This study investigates the gametocyte production and transmission fitness of African and Asian P. falciparum isolates with different K13 genotypes across multiple mosquito species. We tested three ART-sensitive (ART-S) isolates (NF54, NF135, and NF180) and three ART-R isolates (ARN1G, 3815, and PAT-023) for sexual conversion and transmission to Anopheles stephensi, An. gambiae, and An. coluzzii. ART-R levels were quantified in vitro using the Ring-stage Survival Assay (RSA), and the transmission-reducing effects of dihydroartemisinin (DHA) on mature gametocytes were assessed. Results showed that ART-S parasite lines consistently produced gametocytes and transmitted effectively in all three mosquito species. ART-R isolates showed variability: ARN1G maintained high transmission levels, whereas 3815 showed limited transmission potential despite higher sporozoite loads in An. coluzzii. The African ART-R isolate PAT-023 demonstrated low gametocyte commitment but was transmitted efficiently in both An. gambiae and An. coluzzii. DHA exposure reduced mosquito infectivity for all isolates, regardless of K13 genotype. These findings, based on a limited number of field isolates, suggest that ART-R parasites remain transmissible across different Anopheles species. However, ART-R does not appear to confer a direct transmission advantage. This study highlights the complexity of ART-R dynamics and underscores the need for further research to inform malaria control strategies in regions where ART-R parasites are circulating.

Keywords: K13 genotype; P. falciparum; artemisinin resistance; malaria; mosquito transmission.

Fig 1

Parasite survival after exposure to dihydroartemisinin (DHA) and gametocyte conversion rates of P. falciparum isolates with different K13 genotypes and genetic backgrounds. (A) Survival rate from the Ring-stage Survival Assay (RSA) of each parasite line following 6 h exposure to 700 nM DHA compared with DMSO controls in duplicate wells (4–6 replicates were performed for each parasite line). The dots represent biological replicate survival rates at 72 h post invasion from counts either by microscopy or flow cytometry, and the error bars represent the 95% confidence intervals. (B) Sexual conversion in a plate-based assay that quantifies conversion rates from a single asexual round using three different media types (shown in different color gradient of the bars). Conversion rates are calculated by dividing the final gametocytemia by the starting parasitemia in the same well. Dots represent a single well from independent plates; error bars represent 95% confidence intervals.

Fig 2

Infection of three mosquito species with the six different parasite lines. (A) Ookinete counts 20 h after blood meal. The ookinetes were stained with anti-Pfs25 488 conjugate and counted on a hemocytometer under a fluorescent microscope. Immature forms included rounded zygotes (or unfertilized females) and ookinetes with incomplete maturation (lighter shading bar), while mature forms were only the completely mature ookinetes (dark shaded bar). Dots represent a single count from an independent experiment, and the error bars represent 95% confidence intervals. (B) Average oocysts per midgut were dissected on day 7 post-bloodmeal. A total of 20 mosquitoes per group were dissected, and oocysts were counted by mercurochrome staining. Dots represent the counted oocysts from a single midgut; error bars represent 95% confidence intervals. (C) Salivary glands from individual mosquitoes were dissected, and sporozoites were quantified by qPCR. The dots represent the sporozoites per salivary gland from a single mosquito, and the error bars represent the confidence intervals.

Fig 3

Transmission reduction in the presence of DHA. (A) Oocyst densities observed upon exposing mature gametocytes to 700 and 7,000 nM DHA prior to being fed to mosquitoes in a blood meal. The dots represent the average oocysts per infected mosquito from a single cage. (B) Relative reductions in oocyst density compared with the no drug control. All error bars represent the confidence intervals.

Fig 4

Impact of resistance on transmission and commitment. Transmission (A) and conversion (B) plotted against survival rates from the RSA for each parasite line. The underlying detailed data on in vitro parasite resistance in the RSA and gametocyte commitment are presented in Fig. 1; data on transmission under DHA exposure are presented in Fig. 3.