A song for the unsung: The relevance of Plasmodium vinckei as a laboratory rodent malaria system

Rodent malaria parasites (RMPs) allow the study of malaria parasite biology across its entire life cycle through a vertebrate host and a mosquito vector under laboratory conditions. Among the four RMPs originally collected from wild thicket rats in sub-Saharan Central Africa and adapted to laboratory mice, Plasmodium vinckei has the largest geographical range and includes the largest number of sub-species, demonstrating its deep genetic diversity. Despite affording the same advantages as other RMP species and additionally displaying a large degree of phenotypic and genotypic diversity, P. vinckei has seen limited use in the laboratory. Here, we review the contribution of P. vinckei to our understanding of malaria and highlight the areas where it could offer an advantage over other RMP species in future studies

Effect of IL-10 and IL-6 on the regulation of hepcidin in (a) primary macrophages and (b) HepG2 cells. Data show fold-increase relative to mRNA in media control.

<p>Error bars indicate standard error of the mean. Bar plots show data from at least 3 independent experiments. ** <i>P</i><0.01, ***<i>P</i><0.001 (Mann-Whitney test, compared with Media control).</p

Proposed model of hepcidin in malaria infection.

<p>The regulation of hepcidin in response to infection may vary with cell type. A major response to infection occurs in hepatocytes in response to IL-6. However, our observations support the role of IL-10 in primary macrophages. Availability of iron to erythroid developing cells ultimately depends on macrophages and thus the high concentration of IL-10 may play a key regulatory role. Indeed, actively dividing cells like those found in the bone marrow are more susceptible to oxidative damage. In this context, both the direct anti-inflammatory effect of IL-10 and its indirect effect on iron restriction through the up-regulation of hepcidin may be beneficial.</p

Long untranslated regions and putative anti-sense non-coding RNAs in <i>Tg</i>VEG and <i>Nc</i>LIV.

<p>(A) Length distribution of 5’UTRs, 3’UTRs and CDS in <i>Toxoplasma gondii</i>, <i>Neospora caninum</i>, <i>Schizosaccharomyces pombe</i>, <i>Arabidopsis thaliana</i>, <i>Caenorhabditis elegans</i>, <i>Drosophila melanogaster</i> and <i>Homo sapiens</i>. 5’UTRs are found to be strikingly large in the parasites, almost 4 times higher than other eukaryotes. 3’UTRs are comparable to those in human and longer than other eukaryotes. (B) Sequence conservation across UTRs and their flanking intergenic regions. UTR regions are generally more conserved than their flanking intergenic regions. (C) Log abundance ratio of antisense non-coding RNA (ancRNA) and sense coding mRNA pair versus sense coding RNA. There is an inverse relation between abundances of ancRNA and their sense mRNA counterpart.</p

Summary of the manually curation of <i>Tg</i>VEG and <i>Nc</i>LIV (ToxoDb v8.0) genes.

<p>A gene model was “corrected” by adding/deleting exons or altering their exon-intron boundaries to conform to the transcript and peptide evidence. The corrected genes also include the models that were either “split” into two separate genes or “merged” into a single gene based on transcript splice-site evidence. “New” genes were annotated in open reading frames with clear expression evidence. Genes that lacked expression evidence and overlapped with an expressed gene model were considered spurious and “deleted”.</p

Rhythms in inflammatory cytokines follow rhythms in parasite development.

<p>Mean ± SEM (N = 4 per time point) for cytokines (A) IFN-gamma and (B) TNF-alpha for parasites matched and mismatched to the SCN rhythms of the host (matched: green, mismatched: orange). Sampling occurred every 3 hours on days 4–5 post infection. Matched parasites undergo schizogony around ZT 17, (indicated by green dashed line) and mismatched parasites undergo schizogony 6 hours later, around ZT 23 (indicated by orange dashed line). IFN-gamma peaks at ZT 21.29 in matched infections (green) and at ZT 0 in mismatched infections (orange). TNF-alpha peaks at ZT 19.26 in matched infections (green) and at ZT 1.29 in mismatched infections (orange). Light and dark bars indicate lights on and lights off (lights on: ZT 0, lights off: ZT 12).</p

Parasite rhythms in light and dark fed mice significantly diverge by day 5–6 post infection.

<p>The proportion of ring stage parasites across infections (light fed mice, red, and dark fed mice, blue) as a phase marker reveals that rhythms of parasites in light fed mice (red) and dark fed mice (blue) diverge. Mice were sampled at ZT 12 on days 2, 4 and 6 and at ZT 0 on days 3, 5 and 7 post infection (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006900#ppat.1006900.g003" target="_blank">Fig 3</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006900#ppat.1006900.s002" target="_blank">S2 Fig</a>). Consistent significant differences (**, <i>p</i> < 0.05; ***, <i>p</i> < 0.001) between feeding treatments begins on day 5. By days 6–7 post infection, rings in light fed mice are present at ZT12 while rings in dark fed mice are present at ZT 0, indicating that parasites in dark fed mice have rescheduled. Ring stages are presented as the phase marker because this is the most accurately quantified stage but other stages follow a similar pattern (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006900#ppat.1006900.s009" target="_blank">S3 Table</a>). Mean ± SEM is plotted and N = 10 for each treatment group.</p

Feeding mice in the day time affects blood glucose regulation.

<p>A) Mean ± SEM (N = 5 per group) for light fed mice (LF, white bars; allowed access to food from ZT 0-ZT 12) and dark fed mice (DF, grey bars; allowed access to food from ZT 12-ZT 0). Blood glucose concentration was measured every ~2 hours for 30 hours from ZT 0. Steep increases in blood glucose concentration occur as a result of the main bout of feeding in each group (i.e. just after lights on in LF mice and lights off in DF mice, illustrated by the regions with solid lines connecting before and after the main bout, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006900#ppat.1006900.s011" target="_blank">S5 Table</a>), and suggests glucose concentration is inverted during the night. Light and dark bars indicate lights on and lights off (lights on: ZT 0, lights off: ZT 12). B) as for A, but plotted as a polar graph with corresponding developmental stages for each treatment group (red, LF; blue DF) on the perimeter.</p

Parasite rhythms are inverted in hosts fed during the day compared to the night.

<p>(A) The asexual cycle of malaria parasites is characterised by five morphologically distinct developmental stages (ring, early trophozoite, mid trophozoite, late trophozoite) differentiated by parasite size within the red blood cell, the size and number of nuclei, and the appearance of haemozoin [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006900#ppat.1006900.ref031" target="_blank">31</a>]. (B) Mean ± SEM (N = 10 per group) proportion of observed parasites in the blood at ring stage in light fed mice (red; allowed access to food during the day, between ZT 0 and ZT 12) and dark fed mice (blue; allowed access to food during the night, between ZT 12 and ZT 24). The proportion of parasites at ring stage in the peripheral blood is highest at night (ZT 22) in dark fed mice but in the day (ZT 10) for light fed mice, illustrating the patterns observed for all other (rhythmic) stages (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006900#ppat.1006900.s002" target="_blank">S2 Fig</a>). (C) CoG (estimate of phase) in ZT (h) for each rhythmic parasite stage in the blood. Each violin illustrates the median ± IQR overlaid with probability density (N = 10 per group). The height of the violin illustrates the variation in the timing of the CoG between mice and the width illustrates the frequency of the CoGs at particular times within the distribution. Sampling occurred every 6 hours days 6–8 post infection. Light and dark bars indicate lights on and lights off (lights on: ZT 0, lights off: ZT 12).</p

Feeding nocturnal mice in the day time disrupts rhythms in body temperature and locomotor activity.

<p>(A) Hourly mean ± SEM body temperature and locomotor activity (number of transitions per hour is the average number of movements a mouse makes in an hour, between antennae on the Home Cage Analysis system, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006900#sec003" target="_blank">Materials and Methods</a>) and (B) interaction between time-of-day and treatment group on body temperature and locomotor activity (calculating the mean temperature/activity across the day, ZT 0–12, and night, ZT 12–24, ± SEM) averaged from 48 hours of monitoring mice before infection. N = 5 for each of the light fed (LF, red) and dark fed (DF, blue) groups. Light and dark bars indicate lights on and lights off (lights on: ZT 0/24, lights off: ZT 12).</p