Females stop laying eggs and expressing genes encoding egg protein when placed into tissue culture.

<p>Adult females from mixed sex infections were placed in culture with or without male parasites (Four female parasites per well of a 6 well plate). A) Total numbers of eggs per well produced during each 24 h period were counted. Results are pooled from 3 individual experiments. The rate of decline of egg production is significantly faster in un-paired females. Data were log transformed (Y = log(Y+1)) for analysis. Linear regression analysis revealed a highly significant (P<0.0001) difference in the slopes of egg production decline. A 2-way-ANOVA showed a significant interaction of pairing and time on egg production, and Bonferroni post-test determined significant effects of pairing on egg production at days 3 through 6. B) Eggs produced during each day of culture were photographed. C) Real-time RT-PCR analyses of p14 transcripts. Expression of S. mansoni alpha-tubulin was used as the endogenous control to calculate relative expression levels, and expression levels were normalized to ex vivo females. Data were log transformed for analysis. Expression of P14 declined significantly over time in culture in both paired and un-paired females. Linear regression analysis revealed a highly significant (P<0.0001) difference in the slopes of p14 expression decline between paired and un-paired females. Independent t-tests performed at each time point showed dramatic and highly significant differences in expression of p14 at days 7 and 11 between paired and un-paired females (P<0.0001). Females that were un-paired for 7 days in vitro, then re-paired for 4 days showed a trend of higher P14 expression at day 11 than females who were un-paired for the full 11 days. This suggests that re-pairing with the male may be sufficient to prevent further decline of eggshell protein expression. There were no significant changes in alpha-tubulin expression over the time period of the experiment. D) Real time RT-PCR analyis of p14 transcripts in virgin females from single sex infections following culture in vitro paired or un-paired with male parasites. Only worms that were actually paired for the 11day duration were considered paired. Expression of alpha-tubulin was used as the endogenous control to calculate relative expression levels, and expression levels were normalized to the ex vivo females. Expression of P14 increased significantly (P<0.01) in single sex females that were paired for 11 days in vitro. Furthermore, the already low expression of P14 in virgine females from single sex infections was further reduced upon culturing in the absence of males (P<0.05). Thus, the difference in P14 expression levels at day 11 in females that are paired vs un-paired is highly significant (P<0.0001). Data were log transformed for analysis. T-tests were done to compare expression between samples. E) Changes in cross sectional area of virgin females from single sex infections over time in culture with or without males. No significant differences in size over time with or without males were noted. Each data point represents a single female worm.