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Open in new tab Download slide Collection sites of Paralithodes camtschaticus near the city of Juneau, Alaska over three years — Crabs were healthy or infected with the parasite Briarosaccus regalis.

Numbers of individuals are shown when more than one crab of the same sex and infection status were caught at the same site. Figure 1. Tissue samples were typically collected as soon as crabs were brought on board a vessel; however, inseven P.

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Tissue samples were taken from these crabs a few days prior to the collection. When feasible, crabs were collected and tissues were sampled from one infected and one healthy crab of the same sex, of similar size, and the same collection site. Crabs were considered infected if they had an externa or a scar from a lost externa; crabs with early infection stages, prior to emergence of the externa, were not sampled.

Samples were taken from the hepatopancreas, muscle, and hemolymph of P. Individuals without an externa or scar were dissected and examined with the naked eye to verify the absence of an interna before being considered healthy.

The interna of Briarosaccus is bright green in color Noever et al. The entire body bollner rs binary options was searched, with a focus on the hepatopancreas, where interna rootlets were always abundant in infected crabs. Infected L. Given the low prevalence of crabs with externae or scars during the collection years, it is unlikely that any crab with early internal stages of infection was misidentified as healthy; nevertheless, this possibility was taken into consideration during data analyses.

Within 30 min of pot recovery, tissue and hemolymph samples were collected using sterile, disposable sampling supplies and placed in sterile 2 ml cryogenic vials.

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The interna of Briarosaccus is interwoven within the tubules of the hepatopancreas. Separating these in a timely manner during field collection was not possible, so hepatopancreas samples from infected crabs likely included Briarosaccus tissue as well as crab tissue.

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The interna of Briarosaccus does not extend into the legs Noever et al. Sample extraction We extracted duplicate analytical replicates mg sub-samples from all field samples, following protocols described by Maity et al. While still frozen, sub-samples were first removed with sterilized stainless-steel instruments and weighed.

All methods utilized Agilent MassHunter software version B.

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A reference mass solution was infused by dual ESI spray needle. Reference masses During extraction of raw data, ions were identified based on their unique neutral mass, retention time, and abundance. Total ion chromatograms were viewed, and in rare cases 2 of samples one of the analytical replicates was removed from the sample set for either the polar or non-polar fractions due to large inconsistencies in chromatograms.

Minimum peak height was set at counts for the polar phase and 1, counts for the non-polar phase after examining background noise level. Charge state was set at 1, the minimum abundance was set at an absolute height of 5, counts, the lowest number of ions allowed in the isotopic distribution was set at 2, and the peak spacing tolerance was set at 0.

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In MPP, samples were grouped into four conditions healthy male, healthy female, infected male, infected female. Potential metabolites were aligned across all samples using a retention time window of 0. Indicator parabolc binary options bollner rs binary options approach to data mining can help reduce the number of false negative results Frank et al. The original data were re-mined using this algorithm, with a mass window of 15 ppm and a retention time 0.

All potential metabolites were checked manually to ensure proper alignment. Consistent clustering of analytical replicates in the PCA indicated minimal technical variation Supplementary material Fig.

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S1thus intensity values for analytical replicates were averaged to create a single record of potential metabolites for bollner rs binary options sample.

Blank filtering was used to remove contaminates.

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Statistical analysis and potential metabolites identification Statistical analyses were performed in Metaboanalyst 3. Peak intensity tables were uploaded and processed prior to statistical analyses.

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Missing data were replaced with one half the minimum detected value Xia et al. Data were bollner rs binary options normalized by the sum Ejigu et al.

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To view possible clustering of samples we used PCA, an unsupervised multivariate analysis technique. Initial tests were performed to determine whether differences could be detected between scarred crabs and those with an externa.

Molecular formulae were generated using an algorithm that combines carbon number of atoms allowed: 0—60hydrogen 0—nitrogen 0—30oxygen 0—30sulfur 0—3and chlorine 0—3.

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Only the compounds with the highest score i. For potential metabolites that had multiple possible identities with equally high scores, only the most plausible are reported, with synthetic compounds and rare metabolites excluded.

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After application of quality control filters to remove noise and blank filtering, and polar and non-polar potential metabolites remained for P.

There was no apparent clustering based on sex or infection state.

This article has been cited by other articles in PMC. Abstract Plant and algal prolyl 4-hydroxylases P4Hs are key enzymes in the synthesis of cell wall components.

Instead, the obvious clusters especially in the hemolymph resulted from collection year and location Fig. Figure 2.