Premruethai Supungul

Identification of immune-related genes in hemocytes of black tiger shrimp (Penaeus monodon).

An expressed sequence tag (EST) library was constructed from hemocytes of the black tiger shrimp (Penaeus monodon) to identify genes associated with immunity in this economically important species. The number of complementary DNA clones in the constructed library was approximately 4 × 105. Of these, 615 clones having inserts larger than 500 by were unidirectionally sequenced and analyzed by homology searches against data in GenBank. Significant homology to known genes was found in 314 (51%) of the 615 clones, but the remaining 301 sequences (49%) did not match any sequence in GenBank. Approximately 35% of the matched ESTs were significantly identified by the BLASTN and BLASTX programs, while 65% were recognized only by the BLASTX program. Of the 615 clones, 55 (8.9%) were identified as putative immune-related genes. The isolated genes were composed of those coding for enzymes and proteins in the clotting system and the prophenoloxidase-activating system, antioxidative enzymes, antimicrobial peptides, and serine proteinase inhibitors. Three full-length ESTs encoding antimicrobial peptides (antilipopolysaccharide and penaeidin homologues) and a heat shock protein (cpn10 homologue) are reported.

Discovery of immune molecules and their crucial functions in shrimp immunity

Several immune-related molecules in penaeid shrimps have been discovered, most of these via the analysis of expressed sequence tag libraries, microarray studies and proteomic approaches. These immune molecules include antimicrobial peptides, serine proteinases and inhibitors, phenoloxidases, oxidative enzymes, clottable protein, pattern recognition proteins, lectins, Toll receptors, and other humoral factors that might participate in the innate immune system of shrimps. These molecules have mainly been found in the hemolymph and hemocytes, which are the main sites where immune reactions take place, while some are found in other immune organs/tissues, such as the lymphoid organs, gills and intestines. Although the participation of some of these immune molecules in the shrimp innate immune defense against invading pathogens has been demonstrated, the functions of many molecules remain unclear. This review summarizes the current status of our knowledge concerning the discovery and functional characterization of the immune molecules in penaeid shrimps.

Cationic Antimicrobial Peptides in Penaeid Shrimp

Penaeid shrimp aquaculture has been consistently affected worldwide by devastating diseases that cause a severe loss in production. To fight a variety of harmful microbes in the surrounding environment, particularly at high densities (of which intensive farming represents an extreme example), shrimps have evolved and use a diverse array of antimicrobial peptides (AMPs) as part of an important first-line response of the host defense system. Cationic AMPs in penaeid shrimps composed of penaeidins, crustins, and anti-lipopolysaccharide factors are comprised of multiple classes or isoforms and possess antibacterial and antifungal activities against different strains of bacteria and fungi. Shrimp AMPs are primarily expressed in circulating hemocytes, which is the main site of the immune response, and hemocytes expressing AMPs probably migrate to infection sites to fight against pathogen invasion. Indeed, most AMPs are produced as early as the nauplii developmental stage to protect shrimp larvae from infections. In this review, we discuss the sequence diversity, expression, gene structure, and antimicrobial activities of cationic AMPs in penaeid shrimps. The information available on antimicrobial activities indicates that these shrimp AMPs have potential therapeutic applications in the control of disease problems in aquaculture.

Microsatellite polymorphism and the population structure of the black tiger shrimp (Penaeus monodon) in Thailand.

Abstract: Genetic variation and differentiation of Thai Penaeus monodon from five geographic locations (Chumphon, Trad, Phangnga, Satun, and Trang) were investigated using five microsatellite loci (CUPmo18, Di25, Di27, CSCUPmo1, and CSCUPmo2). The number of alleles across the five loci ranged from 19 to 30, and heterozygosities ranged from 0.49 to 0.95. The mean number of alleles and effective number of alleles per locus were 21.0 to 26.6 and 13.1 to 20.4, respectively. The average heterozygosity across all investigated samples was 0.78, indicating high genetic diversity in this species. Geographic heterogeneity analysis of the results from two of the loci, CUPmo18 and Di25, showed significant differences among the Gulf of Thailand (Trad and Chumphon) but not the Andaman samples. Comparison between regions revealed significant heterogeneity of the Andaman and Trad P. monodon (P < .001), whereas those from Chumphon and the Andaman were genetically similar (P > .05). Significant genetic differentiation was consistently observed between the Andaman-Trad samples (FST= 0.0101, P < .0001) and the Chumphon-Trad samples (FST= 0.0101, P < .0001). On the basis of our analyses, the investigated samples from five geographic locations were allocated to three distinct populations composed of the Andaman Sea (A), Chumphon (B), and Trad (C).

Genomic structure, expression pattern and functional characterization of crustinPm5, a unique isoform of crustin from Penaeus monodon.

A unique isoform of crustin, crustinPm5, was identified from a gill-epipodite cDNA library of the tiger shrimp, Penaeus monodon. The crustinPm5 cDNA contains an open reading frame (ORF) of 510 bp encoding a 169 amino acid protein. The deduced amino acid sequence of crustinPm5 showed 38% and 37% overall sequence identity with those of crustinPm1 and crustin-likePm, respectively, two crustin isoforms previously reported. The crustinPm5 gene contained four exons interrupted by three introns whilst the upstream sequence contains a putative promoter with two potential binding sites for NF-kappaB, one complete heat-shock regulatory element (HSE) and five putative GATA factor binding sites. The transcripts of crustinPm5 were primarily observed in the epipodite and eyestalk and not in hemocytes. Expression analysis revealed that the transcript levels of crustinPm5, crustinPm1 and crustin-likePm in epipodite were up-regulated upon heat treatment and hyperosmotic salinity stress. The recombinant crustinPm5 exhibited antimicrobial activity against some Gram-positive bacteria in vitro, but did not inhibit the growth of any Gram-negative bacteria tested. These results, together with the transcript expression pattern, indicate a diverse function of the proteins in the crustin family particularly crustinPm5 that might function as a stress mediator in addition to its antibacterial action.

Molecular characterization and expression analysis of a c-type and two novel muramidase-deficient i-type lysozymes from Penaeus monodon

Lysozyme is a widely distributed hydrolase possessing a hydrolytic activity against peptidoglycan in the bacterial cell wall and, hence, causing lysis of the bacteria. Two types of lysozymes; the c-type (PmLyzc) and the two catalytic residue ablated i-type lysozymes (PmLyzi1 and 2), were identified from the Penaeus monodon EST database (http://pmonodon.biotec.or.th). By RT-PCR, PmLyzc transcript was detected in all tissues: gill, antennal gland, epipodite, heart, hemocyte, hepatopancreas, eyestalk, lymphoid organ and intestine, and highly expressed in hemocyte. The expression of PmLyzi2 mRNA was highest in heart while undetected in gill, lymphoid organ and intestine. The PmLyzi1 transcript was expressed only in hepatopancreas. The up-regulation of mRNA transcription after bacterial challenge was observed only with PmLyzc. To investigate their biological activities, the three mature recombinant proteins were expressed in an Escherichia coli system. Although the turbidimetric assay revealed that only recombinant PmLyzc possessed the muramidase activity, all of them variably exhibited antimicrobial activity against both Gram-positive and -negative bacteria especially the shrimp pathogens, Vibrio species. The antimicrobial activities of recombinant PmLyzc was the most effective one. These results demonstrated that the ability of lysozyme to inhibit the growth of bacteria did not depend only on the muramidase activity. Differences in tissue expression pattern of these gene transcripts and their antimicrobial activities indicated the multifunction of lysozyme as immune defense and digestive enzymes in P. monodon.

A comparative study of antimicrobial properties of crustinPm1 and crustinPm7 from the black tiger shrimp Penaeus monodon

Several isoforms of crustin have been identified in the black tiger shrimp Penaeus monodon. These cationic cysteine-rich antimicrobial peptides contain a single whey acidic protein (WAP) domain at the C-terminus and exhibit antimicrobial activity against both Gram-positive and Gram-negative bacteria. In this paper, we investigate the binding properties and antimicrobial actions of crustinPm1 and crustinPm7, the two most abundant crustin isoforms found in the haemocyte of P. monodon. Previously, crustinPm1 showed strong inhibition against Gram-positive bacteria, whilst crustinPm7 acted against both Gram-positive and Gram-negative bacteria. A binding study showed that both crustins can bind to Gram-positive and Gram-negative bacterial cells. Enzyme-linked immunosorbent (ELISA) assay suggested that crustins bind to the cell wall components, lipoteichoic acid (LTA) and lipopolysaccharide (LPS) with positive cooperativity of Hill slope (H)>2. This indicates that at least two molecules of crustins interact with one LTA or LPS molecule. In addition, both crustins can induce bacterial agglutination and cause inner membrane permeabilization in Escherichia coli. Scanning Electron Microscopy (SEM) revealed the remarkable change on the cell surface of Staphylococcus aureus, Vibrio harveyi and E. coli after the bacteria were treated with the recombinant crustinPm7. Meanwhile, crustinPm1 can cause a visible change on the cell surface of S. aureus and E. coli only. This is in agreement with the fact that crustinPm1 has shown no antimicrobial activity against V. harveyi. It is likely that the antimicrobial activity of crustins mainly relies on their ability to agglutinate bacterial cells and to disrupt the physiochemical properties of bacterial surface.

Kazal-type serine proteinase inhibitors from the black tiger shrimp Penaeus monodon and the inhibitory activities of SPIPm4 and 5

Serine proteinase inhibitors (SPIs) play important roles in physiological and immunological processes involving proteinases in all multicellular organisms. In black tiger shrimp Penaeus monodon, nine different Kazal-type SPIs, namely SPIPm1-9, were identified from the cDNA libraries of hemocyte, hepatopancreas, hematopoietic tissue, ovary and lymphoid organ. They are multi-domain SPIs containing 2-7 and possibly more Kazal domains. Two interesting cDNA clones, SPIPm4 and SPIPm5 coding for two-domain Kazal-type SPIs, were identified from the heat-treated hemocyte cDNA libraries. The SPIPm4 and SPIPm5 consist of open reading frames of 387 and 399 bp coding for polypeptides of 128 and 132 amino acids with putative signal peptides of 21 and 19 amino acid residues and mature SPIs of 107 and 113 amino acid residues, respectively. Recombinant expression in an Escherichia coli expression system yielded recombinant proteins, rSPIPm4 and rSPIPm5, with molecular masses of 12.862 and 13.433 kDa, respectively. The inhibitory activities of SPIPm4 and SPIPm5 were tested against trypsin, chymotrypsin, subtilisin and elastase. The SPIPm4 exhibited potent inhibitory activity against subtilisin and weakly against chymotrypsin whereas the SPIPm5 strongly inhibited subtilisin and elastase. The inhibition was a competitive type with inhibition constants (K(i)) of 14.95 nM for SPIPm4 against subtilisin, 4.19 and 59.64 nM, respectively, for SPIPm5 against subtilisin and elastase. They had no bacteriostatic effect against Gram-positive bacteria: Bacillus subtilis, Bacillus megaterium, Staphylococcus aureus, and Gram-negative bacteria: Vibrio harveyi 639, E. coli JM109. Gene expression study revealed that the SPIPm5 gene was up-regulated in response to heat treatment suggesting the involvement of SPIs in stress responses.

HSP70 and HSP90 are involved in shrimp Penaeus vannamei tolerance to AHPND-causing strain of Vibrio parahaemolyticus after non-lethal heat shock

ABSTRACT Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus carrying toxin‐producing plasmid, has led to severe mortalities in farmed penaeid shrimp throughout Asia. Previous studies reported that a non‐lethal heat shock (NLHS) could enhance disease tolerance in aquatic animals. Here, we investigate whether the NLHS could enhance the survival of shrimp Penaeusvannamei upon challenge with an AHPND‐causing strain of V. Parahaemolyticus (VPAHPND). Two NLHS conditions, acute and chronic NLHSs, were used. The former abruptly exposed the juveniles shrimp from 28 °C to 38 °C for 30 min only once whereas the latter exposed the shrimp to 38 °C for 5 min every day for 7 days. The treated shrimp were, then, challenged with VPAHPND at day 3, day 7, and day 30 during the recovery time after the treatment. The results showed that the shrimp exposed to either acute or chronic NLHS had higher survival rate (>50%) than that of the non‐heated shrimp control (20%) when they were challenged with VPAHPND at day 3 recovery time. However, only those exposed to chronic NLHS showed the VPAHPND protection at day 7 and day 30 recovery times. Furthermore, the qRT‐PCR analysis revealed that the expression of heat shock proteins, LvHSP70, LvHSP90 as well as other immune‐related genes, LvproPO and LvCrustin, were induced upon exposure of shrimp to chronic NLHS. Interestingly, gene silencing of LvHSP70 and LvHSP90 eliminated the VPAHPND tolerance in the chronic NLHS shrimp and had decreasing PO activity suggesting that these LvHSPs played crucial roles in bacterial defense in shrimp. All together, we show for the first time that the NLHS enhance the shrimp tolerance to VPAHPND infection and this is likely mediated by the induction of LvHSP70, LvHSP90 and subsequent activation of the proPO system. HIGHLIGHTSNon‐lethal heat shock (NLHS) could induce shrimp tolerance to AHPND infection.LvHSP70, LvHSP90 as well as LvproPO and LvCrustin, were induced upon exposure of shrimp to chronic NLHS.LvHSP70 and LvHSP90 function to mediate the immunity of shrimp against VPAHPND.

Type I and type II crustins from Penaeus monodon, genetic variation and antimicrobial activity of the most abundant crustinPm4.

An antimicrobial protein, crustin, is involved in the innate immunity of crustacean by defending the host directly against the microbial pathogens. By data mining the Penaeus monodon EST database, two type I crustins, carcininPm1 and 2, and ten type II crustins, crustinPm1-10, were identified. The abundant crustins were crustinPm1, 4 and 7, each with variation in the length of Gly-rich repeat among its members. A few crustinPm1, 4 and 7 with deletion in the Cys-rich region were also observed. Furthermore, the crustinPm4 with the longest N-terminal Gly-rich region was characterized. The crustinPm4 allelic genes were expressed mainly from the hemocytes. Its expression was up-regulated readily by WSSV infection and gradually decreased to normal level afterwards. The recombinant crustinPm4-1 (rcrustinPm4-1) isoform was produced using the Escherichia coli expression system and tested for its antimicrobial activity. The rcrustinPm4-1 was able to inhibit the growth of a Gram-positive bacterium, Bacillus megaterium but not Bacillus subtilis, Micrococcus luteus and Staphylococcus aureus. It also inhibited the growth of two Gram-negative bacteria, E. coli 363 and Vibrio harveyi 639 at lower potency. The rcrustinPm4-1 affected the WSSV infection because the expression of an intermediate early gene ie1 in WSSV-infected hemocyte cell culture was reduced. It was shown further that the rcrustinPm4-1 could delay by about one and a half days the manifestation of disease by WSSV.