Disruption of RAMA leads to defective rhoptry morphology and reduced rhoptry neck formation.
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Tightly synchronised mature schizonts from DMSO- or RAP-treated cultures of RAMAloxP clone 9C10 parasites were fixed and processed for TEM, then three sections from each sample taken and 20 schizonts examined per section. (A) Mean number of visible rhoptry profiles per schizont calculated from each set of 60 TEM images of DMSO-treated and RAP-treated schizonts. There was no statistically significant difference in the mean number of rhoptry profiles visible. Error bars, ±1 SD. p>0.05, non-significant (ns). (B) Representative TEM images from DMSO and RAP-treated RAMAloxP schizonts indicating that club-shaped and circular (i.e. likely spherical) rhoptry profiles were detected in both. Scale bar, 100 nm. (C) The proportion of club-shaped rhoptry profiles visible in DMSO-treated RAMAloxP schizonts was greater than that in RAP-treated RAMAloxP schizonts. Statistical significance was determined by unpaired t-test. Error bars depict ±1 SD. p≤0.01, **. (D) Plots of aspect ratio (longest diameter divided by shortest diameter) calculated from thin section electron micrographs for all rhoptries (both DMSO and RAP-treated) classified as non-circular. Non-circular rhoptries from DMSO-treated schizonts displayed a higher aspect ratio (i.e. were more elongated) than those from RAP-treated RAMAloxP schizonts. Statistical difference determined by two-tailed t-test. Error bars, ±1SD. p≤0.0001, ****. (E) Two dimensional cross section images and three-dimensional models derived from 200 nm thick section serial tomograms acquired from a DMSO-treated (upper two panels) and a RAP-treated RAMAloxP schizont (lower two panels). Rhoptries are depicted in various colours to easily distinguish individual structures. Whilst the rhoptries of the DMSO-treated schizonts appear predominantly club shaped, those of the RAP-treated parasites appear much more circular with stubby, non-conoid projections. Scale bars, 500 nm. All observations where zero rhoptry profiles were reported for a merozoite were deemed ‘failed’ observations for the purposes of this analysis and were not included in the analysis.