Knockdown of a novel G-protein pathway suppressor 2 (GPS2) leads to shrimp mortality by exuvial entrapment during ecdysis.

A novel G-protein pathway suppressor 2 (GPS2) has been identified from hemocytes of the whiteleg shrimp Penaeus vannamei (Pv) and appears to play a role in ecdysis. The full-length of PvGPS2 cDNA consisted of a 1230-bp open reading frame, encoding 409 deduced amino acids with significant sequence homology to GPS2 sequences of crustaceans and insects. RT-PCR revealed that PvGPS2 was expressed in all P. vannamei tissues examined, but that expression was molt stage specific in eyestalk tissue. Relative expression was higher in the period before molting (i.e., intermolt and pre-molt stages) than in the post-molt stage. When double-stranded RNA (dsRNA)-mediated RNA interference was employed to inhibit PvGPS2 formation in shrimp, it led to significant mortality due to unsuccessful separation of new cuticle from old cuticle (exuvial entrapment) during ecdysis.

Knocking down a Taura syndrome virus (TSV) binding protein Lamr is lethal for the whiteleg shrimp Penaeus vannamei.

A cDNA encoding a laminin receptor protein (Lamr) has been isolated from hemocytes of the Pacific white shrimp Penaeus (Litopenaeus) vannamei (Pv), based on primers designed from a previously published Lamr sequence of a Taura syndrome virus (TSV) binding protein of the black tiger shrimp Penaeus monodon (Pm). The deduced amino acid sequence of PvLamr shares 97% identity with PmLamr and has significant homology to laminin receptors and ribosomal protein p40 from various organisms. Tissue distribution analysis by RT-PCR revealed that Lamr transcripts were widely expressed in all tested tissues of P. monodon and Penaeus vannamei. PmLamr was constructed and expressed in Escherichia coli, and the recombinant protein was purified and used to raise a polyclonal antibody. The antiserum reacted with purified recombinant PmLamr and crude muscle tissue proteins from both P. monodon and P. vannamei, but not with hemocyte-free shrimp hemolymph. Examination of protein localization by immunohistochemical analysis revealed the presence of Lamr positive cytoplasm in subcuticular epithelial cells, hematopoietic tissues, epithelial cells of the stomach, epithelial cells of the anterior midgut cecum, antennal gland epithelial cells, F cells of the hepatopancreas, cells in the ovarian zone of proliferation and spheroid cells in the lymphoid organ. RNA interference-mediated silencing of the messenger from Lamr in P. vannamei led to shrimp mortality and indicated an essential function of Lamr for shrimp viability. A negative consequence was that the effect of Lamr knockdown on shrimp infection by Taura syndrome virus could not be assessed.

Characterization of a shrimp serine protease homolog, a binding protein of yellow head virus.

A serine protease homolog (SPH) cDNA namely SPH516 was identified via a yeast two-hybrid screen between yellow head virus (YHV) proteins and hemocyte proteins of the black tiger shrimp Penaeus monodon. Initially, the C-terminal region of SPH516 (SPH516-C) was found to interact with a putative metal ion-binding domain (MIB) encoded by open-reading of frame ORF1b of the YHV genome. Subsequently, the full-length of SPH516 cDNA was obtained using 5' rapid amplification of cDNA ends (5' RACE) and it also bound specifically to the MIB domain only. Primers designed based on the SPH516 coding region amplified not only SPH516 but also an additional SPH named SPH509 from shrimp hemocytes using reverse transcriptase-polymerase chain reaction (RT-PCR). These new SPHs had high homology to MasSPH previously reported from P. monodon. All shared the same domain features including a putative signal peptide, glycine-rich repeat motifs, a clip domain, an HDG triad and a trypsin-like serine protease domain. It is interesting that these sequences were phylogenetically closer to a prophenoloxidase-activating factor (PPAF) from blue crab than to another SPH from the black tiger shrimp reported to be involved in cell adhesion. Our SPH transcripts were highly expressed in hemocytes and gills and were found to be down-regulated after YHV infection. Immunohistochemistry using a polyclonal antibody raised against shrimp protein SPH516-C heterologously expressed in Escherichia coli revealed that SPH516 was present almost exclusively in the shrimp hemolymph.

High expression of a novel leucine-rich repeat protein in hemocytes and the lymphoid organ of the black tiger shrimp Penaeus monodon.

A novel leucine-rich repeat (LRR) cDNA has been cloned from hemocytes of the black tiger shrimp Penaeus monodon by 5' rapid amplification of cDNA ends. The full-length of P. monodon LRR (PmLRR) consisted of 2604 bp with a 1686-bp open reading frame, encoding 561 amino acids. The deduced protein contained a high proportion of leucine residues (17%) and had significant homology to LRR-containing proteins from bacteria to humans. Sixteen tandem LRR motifs of 23-24 amino acids in length occurred in the primary sequence. The computed 3D structure revealed a horseshoe shape consisting of alternately repeated strand and helical domains. Such structures are generally considered to mediate protein - protein interactions and to our knowledge, this is the first report of an LRR protein from a crustacean. PmLRR expression was tissue-specific (i.e. highest in hemocytes, intestine and lymphoid organ) suggesting that it may play some roles in shrimp defense against pathogens. A preliminary test suggested that PmLRR may be down-regulated after viral injection.