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Lactic Acid Supplementation Increases Quantity and Quality of Gametocytes in Plasmodium falciparum Culture.

ABSTRACT: Malaria infection by Plasmodium falciparum continues to afflict millions of people worldwide, with transmission being dependent upon mosquito ingestion of the parasite gametocyte stage. These sexually committed stages develop from the asexual stages, yet the factors behind this transition are not completely understood. Here, we found that lactic acid increases gametocyte quantity and quality in P. falciparum culture. Low-passage-number NF54 parasites exposed to 8.2 mM lactic acid for various times were monitored using blood film gametocyte counts and RNA analysis throughout 2 weeks of gametocyte development in vitro for a total of 5 biological cohorts. We found that daily continuous medium exchange and 8.2 mM lactic acid supplementation increased gametocytemia approximately 2- to 6-fold relative to controls after 5 days. In membrane feeding mosquito infection experiments, we found that gametocytes continuously exposed to 8.2 mM lactic acid supplementations were more infectious to Anopheles stephensi mosquitoes, essentially doubling prevalence of infected midguts and oocyst density. Supplementation on days 9 to 16 did not increase the quantity of gametocytes but did increase quality, as measured by oocyst density, by 2.4-fold. Lactic acid did not impact asexual growth, as measured by blood film counts and luciferase quantification, as well as radioactive hypoxanthine incorporation assays. These data indicate a novel role for lactic acid in sexual development of the parasite.

SUBMITTER: West R

PROVIDER: S-EPMC7927926 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundUnderstanding the dynamics of gametocyte production in polyclonal Plasmodium falciparum infections requires a genotyping method that detects distinct gametocyte clones and estimates their relative frequencies. Here, a marker was identified and evaluated to genotype P. falciparum mature gametocytes using amplicon deep sequencing.MethodsA data set of polymorphic regions of the P. falciparum genome was mined to identify a gametocyte genotyping marker. To assess marker resolution, the number of unique haplotypes in the marker region was estimated from 95 Malawian P. falciparum whole genome sequences. Specificity of the marker for detection of mature gametocytes was evaluated using reverse transcription-polymerase chain reaction of RNA extracted from NF54 mature gametocytes and rings from a non-gametocyte-producing strain of P. falciparum. Amplicon deep sequencing was performed on experimental mixtures of mature gametocytes from two distinct parasite clones, as well as gametocyte-positive P. falciparum field isolates to evaluate the quantitative ability and determine the limit of detection of the genotyping approach.ResultsA 400 bp region of the pfs230 gene was identified as a gametocyte genotyping marker. A larger number of unique haplotypes was observed at the pfs230 marker (34) compared to the sera-2 (18) and ama-1 (14) markers in field isolates from Malawi. RNA and DNA genotyping accurately estimated gametocyte and total parasite clone frequencies when evaluating agreement between expected and observed haplotype frequencies in gametocyte mixtures, with concordance correlation coefficients of 0.97 [95% CI: 0.92-0.99] and 0.92 [95% CI: 0.83-0.97], respectively. The detection limit of the genotyping method for male gametocytes was 0.41 pfmget transcripts/µl [95% CI: 0.28-0.72] and for female gametocytes was 1.98 ccp4 transcripts/µl [95% CI: 1.35-3.68].ConclusionsA region of the pfs230 gene was identified as a marker to genotype P. falciparum gametocytes. Amplicon deep sequencing of this marker can be used to estimate the number and relative frequency of parasite clones among mature gametocytes within P. falciparum infections. This gametocyte genotyping marker will be an important tool for studies aimed at understanding dynamics of gametocyte production in polyclonal P. falciparum infections.

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Lactic Acid Supplementation Increases Quantity and Quality of Gametocytes in Plasmodium falciparum Culture.

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