Phenotypes

Phenotypes are given as log₂-fold change (log₂FC) in relative barcode abundances between developmental stages or on sequential days of blood infections. These values are normalised to control mutants (i.e., mutants that can be assumed not to have a phenotype) from the same experiment. Where screens were conducted in batches, the same control mutants were spiked into each batch, and comparability between batches is high. Variance is expressed as standard deviation (SD) between replicates.

To see how precisely phenotypes and variance were calculated in each screen, consult original publications provided here.

Individual phenotypes are visualised as follows:

• Blood stage growth: Relative asexual daily growth during days 4-7 after infection is plotted against a measure of confidence (Bushell et al., 2017).
• Blood stage growth 2: Unpublished data from 318 PlasmoGEM vectors with 3 kb homology arms that were made recently and not included in any of the published screens.
• Male gametocyte: Relative abundance of male gametocytes measured by Russell et al. (2023) as expression of a fluorescent reporter protein controlled by a male-specific promoter. Data from a screen that omitted most blood stage essential genes.
• Female gametocyte: Relative abundance of female gametocytes measured by Russell et al. (2023) as expression of a fluorescent reporter protein controlled by a female-specific promoter. Data from a screen that omitted most blood stage essential genes.
• Male fertility: Sayers et al. (2024) mutagenised parasites making only male gametocytes and transmitted them to mosquitoes by crossing with a female-only line. The phenotype relates to the relative ability of mutants to transmit to oocysts. Despite its late endpoint, the screen reports on male fertility because gene functions needed after fertilisation are largely complemented by the opposite sex. Both fertility screens focused on the same gene set as the gametocyte development screens.
• Female fertility: Sayers et al. (2024) mutagenised a female-only line and transmitted mutants to mosquitoes by crossing with a male-only line. The phenotype relates to the relative ability of mutants to transmit to oocysts. Despite its late endpoint, the screen reports on female fertility because gene functions needed after fertilisation are largely complemented by the genome of the opposite sex.
• Male egress: Sayers et al. (2024) conducted a subscreen of their male fertility hits for mutants depleted from a population of gametocytes that had undergone activation to form gametes, egress being the first sign of activation. Hits are genes required at any point prior to egress and include developmental mutants.
• Male motility: Sayers et al. (2024) also stratified their male fertility hits by selecting for mutants that could swim up from a cell pellet as a measure of flagellar motility.
• Oocyst: As part of an experiment to transmit all viable mutants through the life cycle, Stanway et al. (2019) measured the rate with which mutants converted from blood stage to oocyst when transmitted to Anopheles stephensi. This phenotype mostly relates to genes required for gamete fertility in both sexes. Genes whose first essential function is in the ookinete or oocyst were predictably not revealed in this screen since any disruption is heterozygous in the functionally diploid oocyst.
• Sporozoite: Stanway et al. (2019) also measured the ability of oocysts to convert to salivary gland sporozoites, but the ability to detect sporozoite essential genes was limited as expected, since even mutated sporozoites would inherit wild-type proteins from the oocyst.
• Liver: Stanway et al. (2019) measured the ability of pools of mutants from mosquito salivary glands to convert to blood stages when injected i.v. in large numbers. The analysis corrected for blood stage growth rates described by Bushell et al. (2017) to obtain a measure for how much a gene contributes to the parasite’s ability to pass through the liver. Some mutants appear to pass the liver more efficiently, but this probably results largely from an overcompensation for asexual growth rate.