Schematic alignment of transcript sequences of three antimicrobial peptides, <i>Cg</i>-<i>Defh</i>, <i>Cg</i>-<i>Defm</i> and <i>Cg</i>-<i>Prp</i> from H and L oyster lines.

<p>Transcript sequences were obtained by PCR from whole oyster body RNA, from L lines (L) and H line (H) oysters. Numbers between parentheses at the left of sequences indicate the number of identical sequences found. Black bars indicate polymorphic sites compared to the first sequence.</p

Alignment of deduced amino acid sequences of three antimicrobial peptides, <i>Cg</i>-Defh, <i>Cg</i>-Defm and <i>Cg</i>-Prp, from H and L oyster lines.

<p>Amino acid sequences were deduced from transcripts sequences obtained from L lines (L) and H line (H) oysters. Numbers between parentheses at the left of sequences indicate the number of identical sequences found. Black bars indicate substitution sites and dots show identical amino acids compared to the first sequence. Amino acids under positive selection are shown in gray (using the ratio of nonsynonymous to synonymous substitutions per codon).</p

Melting temperature from transcript amplicons of four antimicrobial peptides and protein and three reference genes in individual oysters from H and L lines.

<p>Graphs represent melting curves of qPCR amplicons of three antimicrobial peptides (<i>Cg-Defs</i>, <i>Cg-Defh</i>, and <i>Cg-Prp</i>), one antimicrobial protein (<i>Cg-BPI</i>), and three constitutively expressed genes (<i>Cg-EF1</i>, <i>Cg-RPL40</i> and <i>Cg-RPS6</i>) from two selected oyster lines (ten oysters per line). H oyster line is represented in grey and L oyster line in black. The three antimicrobial peptides (<i>Cg-Defs</i>, <i>Cg-Defh</i>, and <i>Cg-Prp</i>) display a high variation on their melting temperatures is significantly associated with the L line (Fisher test, <i>p</i><0.05) while other genes present same variations in each oyster lines (Fisher test, <i>p</i>>0.05).</p

Gene expression and sequence polymorphism of 42 immune related genes in two oyster lines.

<p>Selected genes belonging to nine functional categories are listed below the figure and represented as symbols in front of each gene name. <b>A</b>) Hierarchical clustering of the relative expression levels of 42 immune related genes in non-stimulated oysters of H and L line (three groups of ten oysters per line). Each cell in the matrix corresponds to the expression level of one gene in one sample. The intensity of the color from green to red indicates the magnitude of differential expression (see color scale at the bottom of the image). Relative expressions were calculated according the 2^−ΔΔCq method [21]. The dendrogram at the top of the figure indicate relationship among samples; while the dendrogram at the right of the figure indicate relationship among the relative expression levels of selected genes. Hierarchical clustering was constructed with Multiple ArrayViewer software using average linkage clustering with Spearman Rank Correlation as the default distance metric. Significant differences of relative expressions between oyster lines were determined by the Mann-Whitney U test and genes with significant variation are underlined (<i>p</i><0.05). (<b>B</b>) Hierarchical clustering of the melting temperatures of qPCR amplicons of 42 selected genes in non-stimulated oysters of H and L line (three groups of ten oysters per line). Melting temperature of each sample is represented as the deviation from the mean of melting temperatures of all samples for each gene. Each cell in the matrix corresponds to the deviation from the mean of melting temperature of one gene in one sample. The intensity of the color from green to red indicates the magnitude of the deviation of melting temperature from the mean of each gene (see color scale at the bottom of the image). The dendrogram at the top of the figure indicate relationship among samples; while the dendrogram at the right of the figure indicate relationship among variation of melting temperatures of selected genes. Hierarchical clustering was constructed with Multiple Array Viewer software using average linkage clustering with Pearson Correlation as the default distance metric. Genes who present variation equal or superior to 0.5°C between samples and/or a significant differences of melting temperature between oyster lines (Mann-Whitney U test, <i>p</i><0.05) are underlined. Asterisks (*) indicate genes who present a significant differential of expression together with a variation of melting temperature.</p

Correlation between basal gene expression and gene copy number for two antimicrobial peptides <i>Cg-Defs</i> and <i>Cg-Prp</i> from two oyster lines.

<p>Relative expression and gene copy number were estimated by qPCR (N=14 or 15) for (a) <i>Cg-Defs</i> and(b) <i>Cg-Prp</i>. Individuals from L oyster line are shown in black, individuals from H oyster line are shown in grey. Significant positive correlations between expression level and gene copy number (Spearman’s rank correlation coefficient) were detected for <i>Cg-Defs</i> (<i>p</i><0.05) and <i>Cg-Prp</i> (<i>p</i><0.1).</p

Spectrum of antimicrobial activities of recombinant ALFs and synthetic ALF β-hairpins.

<p>MIC (µM) values refer to the minimal inhibitory concentration required to achieve 100% growth inhibition. NT: not tested.</p

Differential LPS-binding properties of shrimp ALFs from Groups B and D.

<p>The ability of <i>Litsty</i> ALF-D1 (Group D, <i>open triangles</i>) to bind LPS was compared to that of <i>Penmon</i> ALF-B1 (Group B, <i>open squares</i>) in the <i>Limulus</i> amoebocyte lysate (LAL) assay. Polymixin B (PmB, <i>black circles</i>) was used as a positive control. Absorbance (405 nm) is indicative of LAL assay activation by LPS. LPS neutralization by LPS-binding peptides prevents LAL assay activation. It takes here 20-fold more <i>Litsty</i> ALF-D1 than <i>Penmon</i> ALF-B1 to neutralize an equivalent amount of LPS.</p

Nucleotide sequence of primers used in this study.

<p>Nucleotide sequence of primers used in this study.</p

Differential expression of ALF groups in response to an experimental infection.

<p>The transcript abundance of the four ALF Groups (<i>Litvan ALF-A</i>, <i>-B</i>, <i>-C</i>, and <i>-D</i>) was measured by RT-qPCR on circulating hemocytes of shrimp (three groups of three shrimps per condition) injected with the shrimp pathogen <i>Fusarium solani</i> (black boxes), sterile sea water (SSW, grey boxes) or non-injected (white boxes). Analyses were performed 24 and 48 hours after injection. Relative abundances of ALF transcripts are calculated according the 2^−ΔΔCq method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067937#pone.0067937-Livak1" target="_blank">[32]</a>, with the <i>L. vannamei</i> ribosomal protein L40 (<i>Litvan-rpl40</i>, GenBank: FE077602) used as reference gene. Results are expressed as mean values (central squares) ±SE (boxes) and ±SD (whiskers) of relative expressions. Significant differences between conditions for each ALF groups (Student’s t-test, <i>p</i><0.05) are indicated by different lowercase letters (a, b, and c). The use of a same letter indicates the absence of significant difference, while the use of different letters indicates significant difference.</p

Penaeid shrimp ALFs cluster into four groups.

<p>ALF sequences from penaeid shrimps and horseshoe crabs, and scygonadins from crabs (outgroup) were aligned with MAFFT alignment program prior to phylogenetic analysis. The tree was constructed using the Neighbour-Joining method (Pairwise deletion) in MEGA 4. Bootstrap sampling was reiterated 1,000 times. Sequences included in analyses were the following: (i) <u>Shrimp ALFs</u>: black tiger shrimp <i>Penaeus monodon</i> (<i>Penmon</i> ALF-A1 or ALF<i>Pm</i>2: ABP73291; <i>Penmon</i> ALF-A1 or ALF<i>Pm</i>1: ABP73290; <i>Penmon</i> ALF-B1 or ALF<i>Pm</i>3: ABP73289; <i>Penmon</i> ALF-B2 and -B3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067937#pone.0067937-Somboonwiwat3" target="_blank">[47]</a>; <i>Penmon</i> ALF-B4: ADC32520; <i>Penmon</i> ALF-C1 or ALF<i>Pm</i>6: ADM21460; <i>Penmon</i> ALF-C2 or ALF<i>Pm</i>6: AER45468), kuruma prawn (<i>Marjap</i> ALF-A1 or <i>Mj</i>ALF2: BAH22585; <i>Marjap</i> ALF-C1 or M-ALF: BAE92940), fleshy prawn <i>Fenneropenaeus chinensis</i> (<i>Fenchi</i> ALF-B1 or ALF<i>Fc</i>: AAX63831), Indian prawn <i>F. indicus</i> (<i>Fenind</i> ALF-B1: ADE27980; <i>Fenind</i> ALF-B2: ADK94454), pink shrimp <i>Farfantepenaeus paulensis</i> (<i>Farpau</i> ALF-B1 or ALF<i>Fpau</i>: ABQ96193), Atlantic white shrimp <i>Litopenaeus setiferus</i> (<i>Litset</i> ALF-D1: BE846661), Pacific white leg shrimp <i>L. vannamei</i> (<i>Litvan</i> ALF-A1 or <i>Lv</i>ALF1: EW713395; <i>Litvan</i> ALF-A2: FE087264; <i>Litvan</i> ALF-B1 or ALF<i>Lv</i>3: ABB22833; <i>Litvan</i> ALF-B2 or ALF<i>Lv</i>2: ABB22832; <i>Litvan</i> ALF-C1: FE153599; <i>Litvan</i> ALF-C2: FE176556; <i>Litvan</i> ALF-C3: FE058235; <i>Litvan</i> ALF-C4: FE079082; <i>Litvan</i> ALF-C5: FE088301; <i>Litvan</i> ALF-C6: FE078559; <i>Litvan</i> ALF-C7: FE079755; <i>Litvan</i> ALF-C8: FE105941; <i>Litvan</i> ALF-C9: FE090668; <i>Litvan</i> ALF-C10: FE052210; <i>Litvan</i> ALF-C11: FE088625; <i>Litvan</i> ALF-C12 or: <i>Lv</i>ALF2: EW713396; <i>Litvan</i> ALF-D1: FE152534; <i>Litvan</i> ALF-D2: FE151634; <i>Litvan</i> ALF-D3: FE110967; <i>Litvan</i> ALF-D4: FE115964), Southern white shrimp <i>L. schmitti</i> (<i>Litsch</i> ALF-B1 or ALF<i>Lsch</i>: ABJ90465) and blue shrimp <i>L. stylirostris</i> (<i>Litsty</i> ALF-B1: AGH32549; <i>Litsty</i> ALF-D1: AAY33769); (ii) <u>Horseshoe crab ALFs</u>: Chinese horseshoe crab <i>Tachypleus tridentatus</i> (TACTR_ALF: P07087; TACTR2_ALF: AAK00651), Atlantic horseshoe crab <i>Limulus polyphemus</i> (LIMPO_ALF: P07086) and Southeast Asian horseshoe crab <i>Carcinoscorpius rotundicauda</i> (CARRO_ALF: CK086627); (iii) <u>Scygonadins (outgroup):</u> giant mud crab <i>Scylla serrata</i> (<i>S serrata</i> scygonadin: AAW57403; <i>S serrata</i> scygonadin-2: ABI96918; <i>S serrata</i> SSAP: ABM05493) and green crab <i>Carcinus maenas</i> (<i>C. maenas</i> scygonadin: DY307310).</p