A mannose-specific C-type lectin from Fenneropenaeus merguiensis exhibited antimicrobial activity to mediate shrimp innate immunity.

Being one type of pattern recognition receptors (PRRs), lectins exhibit a crucial role in the defense mechanism of invertebrates which are deficient in an adaptive immune system. A new C-type lectin called FmLC3 was isolated from hepatopancreas of Fenneropenaeus merguiensis by cloning approaches, RT-PCR and 5' and 3' RACE (rapid amplification of cDNA ends). A full-length cDNA of FmLC3 contains 607 bp with one open reading frame of 480bp, encoding a 159-amino acids peptide. The predicted primary structure of FmLC3 is composed of a signal peptide, a carbohydrate recognition domain with an EPN motif and one Ca2+ binding site-2, including a double-loop region assisted by two conserved disulfide linkages. FmLC3 had a molecular mass of 17.96kDa and pI of 4.92. In normal or unchallenged shrimp, the mRNA expression of FmLC3 was detected only in hepatopancreas whilst its native proteins were found in hemolymph, heart, stomach and intestine but not in the expressed tissue, indicating that after being synthesized in hepatopancreas, FmLC3 would be secreted to other tissues. The significant up-regulation of FmLC3 was manifested in shrimp challenged with Vibrio harveyi or white spot syndrome virus. After knockdown with gene-specific double-stranded RNA and following by co-pathogenic inoculation, the FmLC3 expression was severely suppressed with coherence of increasing in cumulative mortality and reduction of the median lethal time. Recombinant FmLC3 (rFmLC3) had agglutinating activity towards diverse bacterial strains in a Ca2+-dependent manner. Its activity was inhibited by lipopolysaccharide and mannose, implying that FmLC3 was mannose-binding C-type lectin. Moreover, rFmLC3 could bind directly to various microbial strains with Ca2+-requirement. Otherwise, rFmLC3 exhibited the antimicrobial activity by inhibiting effectively the microbial growth in vitro. All these results signified that FmLC3 might act as PRR to recognize with a broad specificity for diverse pathogens, and contribute in shrimp immune response via the agglutination, binding and antimicrobial activity.

Dynamics of vitellogenin mRNA expression during vitellogenesis in the banana shrimp Penaeus (Fenneropenaeus) merguiensis using real-time PCR.

An open reading frame (ORF) of vitellogenin (Vg) cDNA was amplified from the ovaries of the banana shrimp, Penaeus merguiensis. An examination of Vg-deduced amino acid sequence revealed the presence of cleavage sites at a consensus motif for subtilisin-like endoproteases prior to the N-terminal sequences of purified vitellin (Vt) subunits. A comparison of the primary structures of Vg molecules in decapod crustacean species revealed the existence of a common characteristic structure, and phylogenetic analysis reflected the current taxonomic classifications of crustaceans. A PCR product of 1.1 kb encoding the 3'-end of Vg cDNA was cloned from the hepatopancreas. Although its sequence was almost identical to that of the same region of the ovarian Vg, with only 18 nucleotide differences, analysis suggests that they have been subjected to natural selection, indicating that there may be two different, tissue-specific Vg genes in P. merguiensis. This is consistent with the different expression patterns of Vg mRNA, as determined by real-time PCR. Vg mRNA levels were maintained at low levels during the previtellogenic stage and they increased as vitellogenesis progressed to reach a peak at the early vitellogenic stage in the ovary or at the vitellogenic stage in the hepatopancreas, and thereafter, levels decreased. Expression of Vg mRNA was much higher in the ovary compared to the hepatopancreas at all stages of ovarian development, implying that the ovary is mainly responsible for Vt synthesis. These indicate that penaeids constitute a unique model for vitellogenesis, showing intraovarian gene expression and synthesis of yolk protein.