Fig 1.
Experimental set-up for each of the three D. magna clones.
1) Initially, 20 primiparous Daphnia (6 replicates per clone and treatment) were cultured without any plastic particles. 2) When the animals reached primiparity, they were transferred to a glass jar prepared 24h prior with fresh artificial medium, food and the corresponding plastic mixtures. 3) They were exposed to their respective treatments for over 48 hours. 4) After the exposure, 15 of the Daphnia of each replicate were preserved for (I) gene expression analyses (6 replicates per treatment). 5) From the remaining five animals, three were transferred individually into small glass jars (18 replicates per treatment). These were cultured until they produced the 5th brood for assessing (II) morphology and life-history parameters.
Fig 2.
Expression profile of D. magna clone BL2.2, K34J and Max4 after 48h exposure to plastic mix A and B. Reference genes used for normalization of gene expression were for BL2.2: SDH, TBP and UBC; for K34J: GAPDH and UBC and for Max4: STX16, UBC and GAPDH. Error bars indicate 95% confidence interval. Confidence intervals are not affected by the correction for multiple testing by the false discovery rate method. * p<0.05, ** p<0.01.
Fig 3.
Morphological parameters of adult D. magna.
Body length, body width and tail spine length of the clones Bl2.2, K34J and Max 4; measured 48h after primiparity and upon carrying the 3rd and 5th clutch. Statistical analyses of body width and tail spine length were performed using body length as a covariate in order to compensate for size-dependent differences. Likewise, for the figures body width and tail spine length were drawn as relative values of the body length in percent. Error bars indicate the 95% confidence intervals. Significance level against the control treatment is indicated by * p<0.05, ** p<0.01.
Fig 4.
Number of offspring from D. magna produced during the entire experimental period.
Error bars indicate the 95% confidence intervals.
Fig 5.
Morphological parameters of juvenile D. magna.
Body length, body width and tail spine length clones Bl2.2, K34J and Max 4. The statistical analyses of body width and tail spine length were performed using body length as a covariate in order to compensate for size-dependent differences and by nesting the five individuals of each replicate. Likewise for the figures body width and tail spine length were drawn as relative values of the body length in percent. The five individuals of each replicate were averaged to represent the nested individuals in the graphs. Error bars indicate the 95% confidence intervals. Significance level against the control treatment is indicated by * p<0.05, ** p<0.01.