Fig 1.
Preparation of borosilicate needles for egg disruption.
(A) Heat a borosilicate pipette on a Bunsen burner until the glass starts to melt, (B) twist the pipette once the glass starts to become viscous, (C) pull the ends of the pipet carefully until there remains only a hair-thin connection between the two ends, (D) remove the pipet from the flame, let it cool, and finally break the thin connection.
Fig 2.
Disruption of Taenia eggs using self-made borosilicate needles.
(A) Place a 4 μL droplet of RNAse-free PBS on a fresh cover slide and add two Taenia eggs, (B) place the needles in the droplet and gently move them towards the egg, (C) then push the needles against each other with mild force until you can visually confirm the disruption of the egg.
Fig 3.
Mapping of information on Taenia solium transmission versus size of population with an increased risk for taeniasis.
The color scheme indicates the size of the population exposed to T. solium, with dark red indicating a large population at risk for infection. The color code has been generated through a combination of epidemiology data from the WHO and the indices for living conditions provided by the World Bank. Each yellow dot represents a location mentioned in a publication about T. solium prevalence. Sources: Base map and data were obtained from OpenStreetMap and OpenStreetMap Foundation. The map contains information from OpenStreetMap and OpenStreetMap Foundation, which is made available under the Open Database License (https://www.openstreetmap.org/#map=2/20.5/22.0).
Fig 4.
Impact of pre-analytic storage and transport conditions on RNA quality.
(A) Our experimental storage and transport conditions comprised the following phases: collection at ambient temperature in Zambia, storage at 4°C for 5 days (without freezing!), subsequent transport on coolpacks, and final deep freezing at -80°C. (B) RNA-quality assessed on an Agilent 2100 Bioanalyzer after samples had been prepared using our workflow immediately after thawing before library preparation. Fluorescence Units (FU) as a value of RNA abundance are plotted against the size of the RNA molecules. The size of the RNA-fragments in [bp] is given on the X-axis. The high amount of long RNA molecules and the presence of 18S and 28S rRNA peaks indicates sufficiently good RNA integrity. (C) RNA-quality of visually intact eggs, that were kept for 2 days at 4°C after thawing from -80°C. The overall low FU-values and the high amount of very short RNA fragments indicates that RNA-quality rapidly deteriorates after thawing of the eggs. Therefore, we advise against several freeze-thaw cycles or longer storage after thawing.
Fig 5.
BWA MEM v0.7.12-alignment and per base coverage of the Taenia solium mitochondrial DNA (mtDNA).
(A) Screenshot of the ungapped alignment visualized with the IGV v2.3 viewer. Pink reads represent the forward, blue reads, the reverse sequence reads. (B) Annotation of the different genes on the T. solium mtDNA. All three panels A-C are drawn to the same scale and correspond to each other. (C) Per base coverage of the different transcripts from the mitochondrial genome. The coverage fluctuates between 4 orders of magnitude with highest coverages found for the mitochondrial ribosomal RNA and lowest for the ND5-subunit of mitochondrial complex I. Both samples #1 and #2 show the identical fluctuations across the mtDNA sequence.
Fig 6.
mRNA gene expression in Taenia solium eggs.
The bars depict the FPKM values (fragments per kilobase of exon per million reads mapped) of mitochondrial (mtRNA, pink) and nuclear (nRNA, green) encoded house-keeping genes in single T. solium eggs. The expression levels of worm mtDNA encoded genes are on average 10–100 fold higher than that of nuclear encoded house-keeping genes. The mtDNA encoded genes are sorted according to their coding function for subunits of respiratory chain complex I (CI), complex III (CIII), cytochrome C oxidase (CIV), ATPase (CV), the large and small fragments of the mitochondrial ribosomal RNA (rRNA), and the mitochondrial transfer-RNAs (m.tRNA). The bars depict the mean and SD of two biological replicates. acsl4, acyl-CoA synthetase long chain family member 4 (TsM_000719800); chrna3, cholinergic receptor nicotinic alpha 3 subunit (TsM_000436900); col4a6, collagen type IV alpha 6 chain (TsM_000925000); dmn1, dynamin 1 (TsM_000700600); golga2, golgin A2 (TsM_000292400); h2ac13, H2A clustered histone 13 (TsM_000346300); h2ax, H2A.X variant histone (TsM_000426400); hdac10, histone deacetylase 10 (TsM_000886700); hspa4l, heat shock protein family A (Hsp70) member 4 like (TsM_000256500); hspa8, Heat shock protein family A (Hsp70) member 8 (TsM_000375700); immt, Inner membrane mitochondrial protein (TsM_000168900); nup54, Nucleoporin 54 (TsM_000540600); pcdh10, Protocadherin 10 (TsM_000478100); polr1b, RNA polymerase I subunit B (TsM_000754600); polr3b, RNA polymerase III subunit B (TsM_000691900); timm21, Translocase of inner mitochondrial membrane 21 (TsM_001020500), the accession numbers in brackets refer to the transcripts from the PRJNA170813 BioProject [30]. Nota bene, due to the large expression differences we opted for a logarithmic rendition of FPKM values.
Fig 7.
BWA MEM v0.7.12-alignment of the Taenia solium mitochondrial RNA to the mtDNA of other Taenia species.
Alignment can only be found in areas of high conservation at the mtDNA genes encoding the subunit I of the cytochrome C oxidase (cox1) and the small and large mitochondrial rRNA fragments. (A) IGV visualization of the alignment to the T. saginata mtDNA, (B) annotation of the Taenia saginata mtDNA. (C) IGV visualization of the alignment to the Taenia asiatica mtDNA, (D) annotation of the Taenia asiatica mtDNA.