Table 1.
Transcript selection and primer design summary.
Size refers to qPCR amplicon; efficiency was calculated with a serial dilution of 4-fold.
Fig 1.
LC50 doses calculation at different time points in Litopenaeus vannamei after Vibrio parahaemolyticus IPNGS16 infection.
(A) LC50 standardization curve using a range of bacterial concentrations (from 350 to 3,000 CFU/mL), X-axis shows different time points where shrimp mortality was measured, Y- axis represents the percentage of cumulative shrimp mortality (n = 30). (B) The graph represents a Probit model, in the X- axis are the bacteria concentrations values [CFU/mL], in the Y- axis the Probit values for cumulative mortality, the dotted line represents the value of intersection to calculate the LC50. The LC50 value was determinate at [660.95 CFU/mL].
Fig 2.
Light micrographs of longitudinal sections of hepatopancreas (Hp) from Litopenaeus vannamei infected with Vibrio parahaemolyticus IPNGS16.
Sections were stained with Hematoxylin-eosin, five samples were analyzed for each different time point. Untreated sample at 0 h. Normal Hp has clear tubules (Tub,) (A’), a star-shaped lumen (Lum) (A’), and B-cells (HpB). B- F). Infected samples at different time point post-infection. B,-B’). Small spaces can be seen between epithelial cells indicating sloughing cells (Slo) at 12 hpi. C- C’) Necrosis (Nec), and hemocyte infiltration (Hem) is present in the tubular epithelium, at 24 hpi. D) Presence of hemocyte infiltration is accompanied by lumen (D’) and epithelium reduction at 24 and 48 hpi. E- E’) Atrophy in epithelial cells (Atr) and sloughing cells in tubules is seen at 72 hpi. F) 100× magnification to visualize granular (HemG) and hyaline (HemH) hemocyte infiltration. A’-F’) are magnified views of white dotted rectangle regions in panels (A- F) respectively. Scale bar: 100 μm (A–E) and 30 μm (F).
Table 2.
Summary of assembly and annotation.
Data from the hepatopancreas of Litopenaeus vannamei with AHPND.
Fig 3.
BLASTx statistics, transcript length distribution e- value probability and percentage of transcript identity.
RNAseq data was validated using the following parameters: A) Species distribution of the BLASTx using Swissprot and UniProtKB and filtered with the UniProtINV database which includes the invertebrate phyla: Arthropoda, Mollusca, Porifera, Cnidaria, Echinodermata, Platyhelminthes, Nematoda and Annelida. B) Transcript length distribution after trinity assembly. In the X- axis the total transcript set was grouped into different lengths (shown in base pairs, bp), the Y- axis shows the frequency of each transcript group. C) An e-value was calculated (X- axis) to determine the number of random hits in our BLASTx (Y- axis). D) Percentage of identity (in the X- axis) was also calculated after the BLASTx, shown in the Y- axis as number of BLASTx hits.
Fig 4.
Annotation of Litopenaeus vannamei transcripts.
Annotated transcripts were classified separately using three database resources: (A) Gene ontology (GO), transcripts were grouped in three different ontologies: biological process (BP) in blue, cellular component (CC) in red, and molecular function (MF) in green. The X- axis shows each GO ontology and all the biological categories that lie in each ontology. The Y- axis in the left hand side (LHS) shows the percentage of transcripts (%) for each individual category and the Y- axis in the right hand side (RHS) the number of transcripts in each category (B) The second functional classification was using the orthologous cluster group (COG). The X- axis represents the protein categories predicted which are listed in the RHS. (C) In the third classification, six categories were assigned using the Kyoto Encyclopedia of Genes and Genomes (KEGG) listed top of the chart: (a) Cellular processes, (b) Environmental Information Processing, (c) Genetic Information Processing, (d) Human Diseases, (e) Metabolism, and (f) Organismal Systems. In the X- axis are the gene function assignations for each category. On the Y- axis in the RHS are the number of transcripts found for each gene assignation.
Fig 5.
Differentially expressed transcripts.
Volcano plot shows the DETs from shrimp hepatopancreas challenged with Vibrio parahaemolyticus 16IPNIAV13 at 24 hpi. Log counts in the X- axis indicate transcript log-abundance. LogFC in the Y- axis indicates fold change; each dot represents one gene; red dots are either up or down-regulated genes and black dots are genes with no differential expression.
Fig 6.
DETs were grouped in three main GO categories: biological process (blue bars), molecular function (red bars), cellular component (green bars). A) Up-regulated transcripts. B) Down-regulated transcripts. The X-axis in both charts indicates the number of enriched terms (P<0.01).
Table 3.
Network 3 predicted Hub genes and their homologs.
Fig 7.
Thrombospondin gene interacting network.
A) Topology of the hub thrombospondin (yellow triangle) network with its first up and down -regulated gene neighbors. B) Circular interacting gene network, thrombospondin is in the unregulated group. In both networks, functional enriched up-regulated transcripts are in red triangles, down-regulated transcripts are in blue chevrons, the gray circle represents a set of non-immunological related genes predicted by a GeneMANIA.
Fig 8.
mRNA expression of immune system- related genes.
RT-qPCR results showing the relative mRNA expression of DETs. For all the charts, the X-axis shows different time point post-infection (shown as hpi), the Y-axis shows relative mRNA expression (target gene/LvRPL7); bars show the mean and error lines the standard error (n = 9). Dash line represents the normalized value and lower case letter(s) show statistically significant differences (p < 0.05). A) Cytosolic superoxide dismutase. B) Peroxiredoxin. C) Cottable Protein. D) Hemocyanin. E) Thrombospondin. F) Cellular apoptosis susceptibility protein.
Fig 9.
mRNA expression of metabolic process- related genes.
RT-qPCR results showing the relative mRNA expression of DETs. For all the charts, the X-axis shows different time point post-infection (shown as hpi), the Y-axis shows relative mRNA expression (target gene/LvRPL7); bars show the mean and error lines the standard error (n = 9). Dash line represents the normalized value and lower case letter(s) show statistically significant differences (p < 0.05). A) Phosphoenolpyruvate carboxykinase. B) Chymotrypsin. C) Hypoxia up-regulated protein. D) Cathepsin L protein.