Figure 1.
Identification and characterization of Dscam in P. leniusculus.
A) Amino acid sequence of PlDscam; double underlining represents signal peptide, while the Immunoglobulin (Ig) and Fibronectin type III (FNIII) domains are indicated by light and dark gray shading, respectively. A conserved RGD motif between Ig6 and Ig7 is indicated by single underlining. In addition, the single transmembrane domain is shown in a box. B) Schematic diagram showing the domain organization of the PlDscam protein. C) 3′ UTR containing the polyadenylation signal (AATAA) is indicated by double underlining and a stop codon is bold and underlined.
Figure 2.
High diversity regions of Dscam in freshwater crayfish.
A) Diagram showing the locations of putative alternatively spliced exons of PlDscam. The variable regions are indicated at the N-terminal parts of Ig2 (red) and Ig3 (pink) and complete Ig7 (blue) and transmembrane (green) domains in crayfish. The number of alternative exons is presented by bars under the diagram. B–E) Multiple amino acid sequence alignment of PlDscam variable regions of Ig2(B), Ig3(C), Ig7(D) and transmembrane domain (E) aligned by ClustalW, Dark shading indicates the boundary of each domain.
Figure 3.
Tissue distribution and phylogenetic analysis.
A) The expression of PlDscam was studied in various tissues, such as HP = Hepatopancreas, ST = Stomach, IN = Intestine, G = Gill, TT = Testis, H = Heart, M = Muscle, B = Brain, HC = Hemocyte and N = abdominal nerve. A 40S ribosomal gene was used as an internal control. B) A phylogram based on multiple alignments between PlDscam and other Dscam proteins; Drosophila melanogaster (AAF71926), Apis mellifera (AAT96374), Homo sapiens (AF217525), Mus musculus (NP_112451), Danio rerio (AAT36313), Daphnia pulex (ACC65888), Xenopus tropicalis (NP_001136135), Gallus gallus (XP_416734), Bos taurus (XP_002693048), Canis familiaris (XP_546506) Rattus norvegicus (AAL57167), Macaca mulatta (XP_002803170), Callithrix jacchus (XP_002761477), Tribolium castaneum (NP_001107841), Nasonia vitripennis (XP_001599258), Acyrthosiphon pisum (XP_001949262), Equus caballus (XP_001491675), Aedes aegypti (EAT37388), Aplysia californica (ABS30432) and Litopenaeus vannamei (ACZ26466). A human junctional adhesion molecule A or JAMA was used as outgroup (NP_058642).
Figure 4.
Expression profiles of PlDscam in response to immune challenge.
PlDscam expression was significantly higher than the control from 6 h post-injection with LPS (A), E. coli (C) and S. aureus (D). In contrast, PG (B) and WSSV (E)-injection did not have any effect on the PlDscam expression level. The asterisk indicates that the expression levels are significantly different (*P<0.05, **P<0.01).
Figure 5.
Bacteria-induced PlDscam isoforms.
A) Different isoforms of PlDscam were amplified from hemocyte cDNA of normal crayfish, at 12 h post injection with E. coli or S. aureus. The PCR products were detected on 1.2% agarose gel. The PCR-products from normal crayfish (N) are shown in lanes 1–3, while PCR-products from E. coli (E) and S. aureus (S) injected animals are shown in lanes 4–6 and 7–9 respectively. B) Clustering of all PlDscam isoforms is based on the similarity of their amino acid sequence. The gray shaded boxes represent the chosen isoforms. C) Multiple amino acid sequence alignment of the isoforms E2, N6 and S9 aligned by ClustalW. Dark shading indicates the boundary of each domain.
Figure 6.
Bacteria-induced PlDscam isoforms have different binding ability to bacteria.
A) Different PlDscam isoforms were amplified from N6, E2 and S9 subclones and recombinant proteins were produced. The recombinant isoforms were subjected to 12% SDS and were confirmed by western blotting. Isoforms from non-injected animals in lanes 1 and 4; E. coli- induced isoform in lanes 2 and 5; and S. aureus-induced isoform in lanes 3 and 6. B) In vitro binding assays of rPlDscam isoforms of E. coli (left) and S. aureus (right), respectively. Binding of bacteria to different rPlDscam isoforms, consisting of N6 = isoforms from non-induced animal, E2 = E. coli induced isoforms, S9 = S. aureus induced isoforms or GST = control recombinant protein was determined using ELISA by measuring the absorbance of samples at 450 nm (OD450). C and D) The effect of bacteria induced isoforms on the clearance process in vivo was tested by pre-incubation of the bacteria E. coli (C) and S. aureus (D) with each recombinant isoform prior to injection into crayfish. The bacterial numbers in crayfish were determined as CFU/ml of crayfish in the hemolymph at 40 min (black) and 3 h (grey) after the bacterial challenge. This experiment was repeated three times and data represent means of these experiments. E) Phagocytosis and nodule formation of FITC-conjugated heat-killed bacteria by crayfish hemocytes. Black arrows show nodule formation under normal light microscopy. White arrows show the ingested FITC-conjugated heat-killed bacteria by crayfish hemocytes under UV light microscope. Red arrows show nodules that contain several ingested bacterial particles under UV light microscope. F) The percentage of crayfish hemocytes ingesting FITC-conjugated heat-killed E. coli (left) and S. aureus (right) that had been pre-incubated with each isoform of the recombinant proteins and the phagocytic hemocytes was determined at 1.5 h after challenge. Significant difference compared to control is marked by * = P<0.05 and ** = P<0.01.
Figure 7.
Effect of PlDscam silencing on viral replication of WSSV in vitro.
A) Silencing of PlDscam using dsRNA in cultured HPT cells was confirmed by RT-PCR before WSSV inoculation. B) WSSV VP28 expression in PlDscam silenced animals (lane 7–9) and the control (lane 4–6). UV-killed WSSV was served as control (lane 1–3), analyzed by semi-quantitative RT-PCR. C) Statistical analysis after comparing different band intensity of the results shown in (B). Significant differences are indicated by asterisks (P<0.05).