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. 2018 Dec 3;9:5125. doi: 10.1038/s41467-018-07575-2

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — Transcripts and proteins in the Limnoria digestome and native hemocyanin extract. a Relative abundance of glycosyl hydrolase (GH) families detected in hindgut fluids based on molar percentage. Values represent sample mean, N = 3 (N consisting of material from 100 animals each). b Relative hemocyanin abundance in hindgut separated into tissue (including solid content) and fluids by comparison of relative molar percentage of hemocyanins. Circles represent sample values and bars sample mean ± SD, N = 3 (N consisting of material from 100 animals each), replicates were normalized intra-sample on the expectation of equal total protein amount between replicates when summed across all organs. c Top: differential relative gene expression of two members each of GH family 5, 7, and 9 cloned from Limnoria [GenBank accession numbers GU066826 (GH5A), GU066827 (GH5C), FJ940756 (GH7A), FJ940757 (GH7B), FJ940759 (GH9A), FJ940760 (GH9B)], measured by RT-qPCR: Body, remaining body part after removal of hindgut (Gut) and hepatopancreas (Hp). Values represent samples derived from pools of fifty organs each. Bottom: Spatial distribution of GH epitopes from families 5, 7, and 9 in hepatopancreas and hindgut, analyzed by western blots probed with anti-GH antibodies (ɑGH5, ɑGH7, ɑGH9). d Top: differential relative gene expression of four hemocyanin genes cloned from Limnoria [Hc1–4, GenBank accessions numbers GU166295 (Hc1), GU166296 (Hc2), GU166297 (Hc3), GU166298 (Hc4)] measured by RT-qPCR: Body, remaining body part after removal of hindgut and hepatopancreas (Hepatop.). Bar values represent sample mean ± SD, N = 3 (N consisting of five organs each; individual sample values are only shown as circles for hepatopancreas). Bottom: spatial distribution of hemocyanin epitopes in hepatopancreas and hindgut, analyzed by western probed with anti-hemocyanin antibodies (ɑHemocyanin). e Coomassie-stained SDS–PAGE gel of native Limnoria hemocyanin in pooled and concentrated fractions from gel filtration (50 µg of protein) compared with molecular size marker in kDa. Hc, band containing hemocyanin as identified by protein ID. f Uranyl acetate negative stained TEM images of native Limnoria hemocyanin extract showing hexamers and stacked multiples of these (scale bar, 50 nm)