The expression profile of SpFLT-1 mRNA in hemocytes (Fig 4A) and gills (Fig 4B) post bacterial challenge using qPCR.

<p>The significant difference of SpFLT-1 transcripts between experimental group and control group is indicated with asterisks (*: <i>p</i> < 0.05). Bars indicate mean ±S.E. (n = 5). Immune-blotting analysis of expression of SpFLT-1 and <i>Vibrio</i> protein in hemocytes (Fig 4C) and gills (Fig 4D). N: normal group crabs; S: saline group crabs; B-3 h, B-6 h: crabs from 3 hpi or 6 hpi; V: <i>V</i>. <i>alginolyticus</i> alone. The figure is representative of results from three independent assays.</p

SpFLT-1 facilitates the endocytosis of <i>V</i>. <i>alginolyticus</i> in EPC cells.

<p>(A) & (B) & (C) Representative images of endocytosis of <i>V</i>. <i>alginolyticus</i> in 4°C treated EPC cells and cells transfected by pCMV-HA vector or pCMV-HA-FLT1 using confocal microscopy. DNA in the EPC cells was stained with DAPI and is shown in blue. FITC-labeled <i>V</i>. <i>alginolyticus</i> is green. Bar: 10 μm. (D) Immune-blotting analysis of expression of SpFLT-1 in EPC cells. GAPDH was used as a loading control. (E) Representative images of SpFLT-1 expression in pCMC-HA vector or pCMV-HA-FLT1 transfected cells using immunofifluorescence assay. SpFLT-1 protein detected with an antibody specific for HA-tag and anti-rabbit IgG FITC-conjugated secondary antibody is shown in green. DNA in the EPC cells was stained with DAPI and is shown in blue. Bar: 20 μm. (F) The endocytosis rates of the cells were determined using an iCys quantitative image cytometer. Data from three independent experiments were analyzed. Bars indicate mean ± S.E.</p

Expression of SpFLT-1 in different tissues and embryonic development stages from <i>S</i>. <i>paramamosain</i>.

<p>(A) Relative gene expression of SpFLT-1 in tissues. HC: hemocytes; OA: ovaries; MG: midgut gland; TG: thoracic ganglion mass; GI: gills; HT: heart; SP: spermathecae; MU: muscle; ST: stomach; EY: eyestalk; BR: brain; RT: reproductive tract; HP: hepatopancreas; SE: subcuticular epithelia. (B) Immune-blotting analysis of SpFLT-1 protein expression in selected tissues; (C & D) Immunofluorescence analysis of SpFLT-1 protein in HC and GI. SpFLT-1 protein was detected with an antibody specific for SpFLT-1 and anti-mouse Alexa Fuluor 647-conjugated secondary antibody and is shown in red. Pre-immune serum was used as a control. DNA in HC and GI were stained with DAPI and are shown in blue. Bar, HC: 10 μm, GI: 20 μm. (E) Relative gene expression of SpFLT-1 in maternal ovaries, zygote, early to late-staged embryos and the zoea 1 stage larva of <i>S</i>. <i>paramamosain</i>. M-OA: maternal ovaries; Zy: zygote; Em1, 2, 3, 4: embryo stages from early to late 1–4; Z1: zoea 1.</p

Silencing of SpFLT-1 inhibits the endocytosis rate of <i>V</i>. <i>alginolyticus</i> in the primary cultured hemocytes from <i>S</i>. <i>paramamosain</i>.

<p>(A) & (B) & (C) Representative images of endocytosis of <i>V</i>. <i>alginolyticus</i> in the primary cultured hemocytes treated by 4°C, GFP dsRNA or SpFLT-1 dsRNA using confocal microscopy. DNA in the hemocytes was stained with DAPI and is shown in blue. FITC-labeled <i>V</i>. <i>alginolyticus</i> is green. Bar: 10 μm. (D) & (E) SpFLT-1 expression was analyzed using semi-quantitative PCR (left) and immune-blotting (right) in the RNA interference assays. GAPDH was used as a loading control. (F) The endocytosis rates of the cells were determined using an iCys quantitative image cytometer. Data from three independent experiments were analyzed. Bars indicate mean ± S.E.</p

The expression profile of SpFLT-1 mRNA in hemocytes (Fig 4A) and gills (Fig 4B) post bacterial challenge using qPCR.

<p>The significant difference of SpFLT-1 transcripts between experimental group and control group is indicated with asterisks (*: <i>p</i> < 0.05). Bars indicate mean ±S.E. (n = 5). Immune-blotting analysis of expression of SpFLT-1 and <i>Vibrio</i> protein in hemocytes (Fig 4C) and gills (Fig 4D). N: normal group crabs; S: saline group crabs; B-3 h, B-6 h: crabs from 3 hpi or 6 hpi; V: <i>V</i>. <i>alginolyticus</i> alone. The figure is representative of results from three independent assays.</p

A New Membrane Lipid Raft Gene SpFLT-1 Facilitating the Endocytosis of <i>Vibrio alginolyticus</i> in the Crab <i>Scylla paramamosain</i>

<div><p>Pathogens can enter their host cells by way of endocytosis in which the membrane lipid raft gene flotillins are probably involved in the invasion process and this is an important way to cause infection. In this study, a new gene SpFLT-1 was identified in <i>Scylla paramamosain</i>, which shared high identity with the flotillin-1 of other species. The SpFLT-1 gene was widely distributed in tissues and showed the highest level of mRNA transcripts in the hemocytes. This gene might be a maternal gene based on the evident results that it was highly expressed in maternal ovaries and in the early developmental stages of the zygote and early embryo stage whereas it gradually decreased in zoea 1. SpFLT-1 positively responded to the challenge of <i>Vibrio alginolyticus</i> with a significantly increased level of mRNA expression in the hemocytes and gills at 3 hours post infection (hpi). The SpFLT-1 protein was detected densely in the same fraction layer where the <i>Vibrio</i> protein was most present in the hemocytes and gills at 3 hpi. Furthermore, it was found that the expression of SpFLT-1 decreased to the base level following disappearance of the <i>Vibrio</i> protein at 6 hpi in the gills. Silencing SpFLT-1 inhibited the endocytosis rate of <i>V</i>. <i>alginolyticus</i> but overexpression of the gene could facilitate bacterial entry into the epithelioma papulosum cyprinid cells. Our study indicated that SpFLT-1 may act as a key protein involved in the process of bacterial infection and this sheds light on clarifying the pathogenesis of pathogens infecting <i>S</i>. <i>paramamosain</i>.</p></div

A New Membrane Lipid Raft Gene SpFLT-1 Facilitating the Endocytosis of <i>Vibrio alginolyticus</i> in the Crab <i>Scylla paramamosain - Fig 1 </i>

<p>(A) Complementary DNA and predicted amino acid sequences of SpFLT-1 from <i>S</i>. <i>paramamosain</i>. The putative conserved domain of the flotillin/SPFH family is framed gray highlighted. The polyadenylation signal is boxed and the stop codon is indicated by “*”. (B) Phylogenetic analysis of deduced amino acid sequences from SpFLT-1 and the flotillin-1 of other species. SpFLT-1 is underlined. Numbers next to the branches indicate bootstrap value of each internal branch in the phylogenetic tree nodes from 1000 replicates. (C) Multiple alignment of amino acid sequences among SpFLT-1 and the flotillin-1 of other species. The pond sign and enclosed Pabove the alignment indicate the putative palmitoylation and phosphorylation sites. Secondary-structure predictions: six β strand (B1 to B6) and five α helices (H1 to H5) are accordingly shown in the solid and dashed line rounded rectangle. Both conserved N-terminal hydrophobic stretches are in framed boxes. EA-rich coiled-coil regions are designated in parentheses. Amino acid residues are numbered to the right of each sequence. Flotillin-1 amino acid sequences were obtained from GenBank as follows, Hs-FLT1: <i>H</i>. <i>sapiens</i> (human) (AC AAD40192); Mm-FLT1: <i>M</i>. <i>musculus</i> (house mouse) (AC NP_032053); Xl-FLT1: <i>X</i>. <i>laevis</i> (African clawed frog) (AC NP_001082374); Dr-FLT1: <i>D</i>. <i>rerio</i> (zebrafish) (AC NP_570988); Od-FLT1: <i>O</i>. <i>dancena</i> (brackish medaka) (AC ACN49164); Ss-FLT1: <i>S</i>. <i>salar</i> (Atlantic salmon) (AC ACN10783); Ci-FLT1: <i>C</i>. <i>intestinalis</i> (vase tunicate) (AC XP_002123705); Am-FLT1: <i>A</i>. <i>mellifera</i> (honey bee) (AC XP_623738); Dm-FLT1: <i>D</i>. <i>melanogaster</i> (fruit fly) (AC NP_477358); Sp-FLT1: <i>S</i>. <i>paramamosain</i> (AC FJ774690).</p