Paper Chromatographies

We assembled sequence data for our target taxa and three other congeners—Chinook salmon (O. tshawytscha), coho salmon (O. kisutch), and sockeye salmon (O. nerka)–to design qPCR assays in the NADH region of the mitogenome. We used NADH because it tends to show relatively high sequence divergence and is one of the most commonly archived mitochondrial sequences for salmonids. For YCT, we obtained sequences from a 3,483-bp region of the NADH subunits 1 and 2 for 17 individuals from across its range ([18 ]; GenBank accession numbers: EU186781.1 –EU186797.1; Table 1 and Fig 2). We obtained these same gene regions from whole mitogenomes for five RBT from Pacific North America (GenBank accession numbers: DQ288268.1 –DQ288271.1, L29771.1) and each of the Pacific salmon congeners listed above (GenBank accession numbers: AF392054.1 and NC_002980.1 for Chinook, EF126369.1 for coho, and EF055889.1 for sockeye). We used previously unpublished sequences from 96 WCT from across the species’ range (S1 Text). All tissue samples used in this study were stored in ethanol, lysis buffer, or dried on chromatography paper. All samples used in this study were provided by collaborators from previous studies conducted under appropriate scientific sampling permits or from collections under Montana Fish, Wildlife and Parks Scientific Collectors Permits 12–2001, 14–2001, and 19a-2009 issued to M. K. Young (Table 1, Fig 2). Sampling sites were accessed via public land and did not require special permission. Sampling of protected species was allowed under U.S. Fish and Wildlife Service Federal Fish and Wildlife Permit TE220826-0 issued to M. K. Young. Animals were captured via backpack electrofishing, a small fin tissue sample was collected, and then the animal was released at the place of capture in accordance with a protocol approved under these scientific sampling permits. DNA was extracted from these samples using QIAGEN DNeasy Blood and Tissue Kit following the manufacturer’s protocol.

The 2DE gel spots were excised and destained using 40% methanol 7% acetic acid. The gel plugs were dehydrated with acetonitrile, the acetonitrile was removed and 25mM DTT was allowed to absorb into the plug. The plug was heated to 100°C for 5 min, allowed to cool, and alkylated in the dark for 30 min with 75mM iodoacetamide. The gel plugs were washed using two repeats of acetonitrile dehydration and 20 mM ammonium bicarbonate rehydration. After a final dehydration, the gel plugs were dried in a speed vac. Peptide∶N-glycosidase F (PNGase F) was diluted with 20 mM ammonium bicarbonate pH 7 and allowed to adsorb into the gel plug. The gel plug was then covered with the same solution and allowed to incubate overnight at 37°C. The glycans were eluted from the gel plug by sonication in Milli-Q water three times; the elutant was pooled, dried down, and labeled with a 2AB dye (Ludger, Oxford, UK) according to the manufacturer's instructions. The glycans were then cleaned up using paper chromatography and filtered using a 0.22-µm syringe filter. Fluorescently labeled glycans were subsequently analyzed using the Waters Alliance high-performance liquid chromatography system with a normal phase column (TSK amide 80 columns) complemented with a Waters fluorescence detector and quantified using the Millennium Chromatography Manager (Waters Corporation, Milford, MA). The mobile phase consisted of solvent A (50 mM ammonium formate, pH 4.4) and solvent B (acetonitrile). The gradient used was as follows: linear gradient from 20% to 58% solvent A at 0.4 mL/minute for 152 min followed by a linear gradient from 58% to 100% solvent A for the next 3 min. The flow rate was increased to 1.0 mL/minute; the column was washed in 100% solvent A for 5 min. Following the wash step, the column was equilibrated in 20% solvent A for 22 min in preparation for the next sample. Glycan structures were identified by calculating the glucose uptake value and exoglycosidase digestion, as described previously [21] (link).

This was a cross-sectional health point prospective study. The study enrolled participants who before the start of the study were presented with malaria clinical characteristics and had resided in Kisii County for at least the last six months. However, the study excluded those who were reluctant to give consent to the study. In addition, participants with febrile clinical illnesses initiated by pathogens other than malaria were excluded from the study. Before collecting blood specimen, a complete medical examination and demographic information were obtained. All patients who had been suspected of having malaria infection by having a fever (≥ 38 °C) or having a history of fever in the past 24 h, were confirmed for the presence of P. falciparum using microscopy (as a confirmatory test). Briefly, thick and thin blood smears were stained with 2% Giemsa for 30 min. A smear was considered negative if no parasites were observed after examination under 100 high-powered fields. Thin smears were fixed in methanol before Giemsa-staining. Blood samples were collected by obtaining 1 ml of venous blood for the participants older than 2 years. 100 μL finger-pricked blood samples were collected in the case of children below 2 years of age. This procedure was repeated during the consequent follow-up visits. The blood spots were made on chromatography filter paper (ET31CHR; Whatman Limited, Kent, UK) and labelled with the participant identification number. Malaria-positive participants were followed up for a period of 28 days by evaluating clinical and parasitological parameters on days 1, 3, 7, 14, and 28, respectively, after AL treatment initiation. Finger pricks for follow-up were taken on days 1, 3, 7, 14 and 28 to check for the presence of P. falciparum [18 (link)].