Virulence was measured by minimum red blood cell density (y-axis) in lines of P. chabaudi before (“ancestral lines;”black and gray symbols) and after serial passage through immunized (“I-lines;” red lines and triangles) or naïve (“N-lines,” green lines and circles) mice before (A) and after (B) mosquito transmission. Evolved and ancestral lines were compared in both naïve (solid lines) and immunized mice (broken lines). Filled symbols, before mosquito transmission; open symbols, after mosquito transmission. Lines were selected from an avirulent, “unadapted” clone (CW-0; left set of lines) and a virulent, “preadapted” ancestral population (CW-A; right): the latter was derived from the former by 12 serial passages in a previous experiment (). Each symbol (with ± 1 standard error based on the variance between subline means) represents the mean of mice infected with an ancestral line or a set of passaged lines (i.e., five sublines, two mice per subline). Prior to mosquito transmission (A), differences between the I-lines and N-lines were significant in three out of the four cases (p < 0.05 for lines from the unadapted line infecting naïve mice, p < 0.01 for unadapted infecting immunized, and p < 0.001 for preadapted infecting immunized): in the fourth case (p > 0.1 for preadapted infecting naïve), virulence of the ancestral line was already apparently near-maximal. After mosquito transmission (B), the differences between the I-lines and N-lines remained the same in naïve mice as before transmission (interaction between the mosquito transmission effect and the I-line-versus-N-line difference was p > 0.7 in both the unadapted and preadapted cases). However, these line differences were eliminated in immunized mice (interaction term: p = 0.02 for the unadapted case, p = 0.08 for the preadapted case). Mosquito transmission significantly reduced the virulence of the preadapted ancestral line in immunized mice (p = 0.03) but not in the other ancestral-line-by-immune-treatment combinations (p > 0.2 in these cases). In the selection lines, mosquito transmission significantly reduced the virulence in five out of the eight comparisons (p = 0.009 and p = 0.13 in the N-lines in naïve mice derived from CW-0 and CW-A, respectively, with values of p = 0.55 and p = 0.005 in N-lines in immunized mice, p = 0.022 and p = 0.26 in I-lines in naïve mice, and p = 0.006 and p < 0.0001 in I-lines in immunized mice). Ancestral pretransmission lines had similar levels of virulence in the separate pretransmission and posttransmission experiments, with the exception of the preadapted ancestral line in immunized mice, which had higher virulence in the latter than the former (p = 0.002). Similar results to the above were obtained when virulence was measured by maximum weight loss (unpublished data). No deaths occurred during the pretransmission experiments, but in addition to the one death that occurred early in the infection prior to the occurrence of any weight loss or anemia (excluded from analyses), five occurred in the posttransmission experiment, four of these in naïve mice (two in the N-lines, one in the I-lines, and one in the nontransmitted ancestral line, all derived from the preadapted line) and one in an immunized mouse (preadapted, nontransmitted ancestral line).

Each curve represents the mean asexual parasitemia (dark blue) and gametocytemia (light blue) over all parasite lines (ancestral and selected) in naïve (solid lines; n = 47) and immunized mice (broken lines; n = 50) during the pretransmission evaluation phase. Immunization reduced asexual parasitemia and gametocytemia throughout the infection (p < 0.001 based on the log₁₀ daily average taken over all days). The arrow indicates the day of transfer during the selection phase of the experiment.

Virulence, as measured by minimum red blood cell density, is plotted against maximum parasitemia in (A), and average daily gametocyte production (a measure of lifetime transmission potential) is plotted against virulence in (B). Data are all from pretransmission lines—ancestral and selected—measured in naïve (closed symbols; solid line) and immunized mice (open symbols; broken line). Regression analysis for both traits showed significant (p < 0.001) and similar (p > 0.05) slopes within both naïve and immunized mice, and significantly lower (p < 0.001) maximum parasitemia and gametocyte production in immunized than in naïve mice. When the two data points from naïve mice with values of above 3 × 10⁹ rbc/ml were excluded from the analyses, the slopes remained statistically similar (p > 0.05). Unselected ancestral populations, black squares; N-lines, green circles; I-lines, red triangles; avirulent unadapted ancestral population, small symbols; virulent preadapted ancestral population, large symbols.