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. 2020 Jan 21;10:787. doi: 10.1038/s41598-019-57409-4

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© The Author(s) 2020

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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Relative expression analysis of genes in the NLHS-VP modulated immune pathways. Expression level of genes in the prophenoloxidase system (PO1 and PO2), IMD pathway (IMD, IKKε, IKKβ, and Relish), Toll pathway (Toll1, Toll2, Toll3, MyD88, TRAF6, Pelle, Drosal, and Cactus), antimicrobial peptide (ALF AA-K, Penaedin 4a, and AMP type 2), and hemocyte homeostasis (TGase and Inhibitor of apoptosis protein) was determined by RT-qPCR in the hemocytes of VP_AHPND-challenged L. vannamei at 0 hpi (NH 0 hpi) and of NLHS-treated L. vannamei challenged with VP_AHPND at 0 hpi (H 0 hpi), 6 hpi (H 6 hpi), and 24 hpi (H 24 hpi). Relative expression ratios are calculated using EF-1α as the internal control. Relative expression level of each gene in hemocytes of NLHS-treated L. vannamei challenged with VP_AHPND at each time point after infection was normalized to that of NH 0 hpi to determine the effect of both NLHS and VP_AHPND challenge. The results were derived from triplicate experiments. Asterisks indicate significant differences at P < 0.05 from the NH 0 hpi. The expression profile of PO1 and PO2 under the NLHS condition was modified and re-presented from Fig. 4.