Molecular characterization of a short-chained pentraxin gene from kuruma shrimp Marsupenaeus japonicus hemocytes.

Pentraxins (PTXs) are a family of pattern recognition proteins (PRPs) that play a role in pathogen recognition during infection via pathogen-associated molecular patterns (PAMPs). Here, we characterized a short-chained pentraxin isolated from kuruma shrimp (Marsupenaeus japonicus) hemocytes (MjPTX). MjPTX contains the pentraxin signature HxCxS/TWxS (where x can be any amino acid), although the second conserved residue of this signature differed slightly (L instead of C). In the phylogenetic analysis, MjPTX clustered closely with predicted sequences from crustaceans (shrimp, lobster, and crayfish) displaying high sequence identities exceeding 52.67 %. In contrast, MjPTX showed minimal sequence identity when compared to functionally similar proteins in other animals, with sequence identities ranging from 20.42 % (mouse) to 28.14 % (horseshoe crab). MjPTX mRNA transcript levels increased significantly after artificial infection with Vibrio parahaemolyticus (48 h), White Spot Syndrome Virus (72 h) and Yellow Head Virus (24 and 48 h). Assays done in vitro revealed that recombinant MjPTX (rMjPTX) has an ability to agglutinate Gram-negative and Gram-positive bacteria and to bind microbial polysaccharides and bacterial suspensions in the presence of Ca2+. Taken together, our results suggest that MjPTX functions as a classical pattern recognition protein in the presence of calcium ions, that is capable of binding to specific moieties present on the surface of microorganisms and facilitating their clearance.

Molecular cloning, characterization and gene expression analysis of aminolevulinic acid synthase in Litopenaeus vannamei.

5-Aminolevulinic acid synthase (ALAS) is the rate-limiting enzyme in the biosynthesis of heme, a prosthetic group that is found in hemoproteins, including those involved in molting. To better understand the roles of ALAS in L. vannamei (LvALAS), we analyzed its sequence and tissue distribution, the effects of age and bacterial infection on its gene expression, and the effects of LvALAS gene silencing. We also examined the expressions of three hemoproteins, the cytochrome oxidase subunit I (COX I) and subunit IV (COX IV) and catalase. Three LvALAS splicing variants were found in the hepatopancreas, with the main splicing variant having an open reading frame that encodes 532 aa. LvALAS transcripts were found in each of the eleven tissues tested in this study, with the highest gene expression in the intestine. The transcript abundances of LvALAS, COX I and COX IV in the hepatopancreas and stomach tended to decrease with age. LvALAS and catalase gene expressions significantly increased in the stomach after V. parahaemolyticus infection. LvALAS gene expression in the hepatopancreas, stomach and intestine (12- and 24-hours post-injection) was relatively lower in dsALAS-injected shrimp than in PBS-injected shrimp. All the PBS-injected shrimp molted after 8-10 days while no molting activity was observed in the dsALAS-injected shrimp group within the 14 days post-injection period. Our results provide evidence that (1) only the housekeeping form of ALAS exists in L. vannamei; LvALAS gene expression (2) decreases with age and (3) increases after bacterial infection; and (4) an ALAS-dependent pathway is necessary for proper molting in L. vannamei.

Anti-PirA-like toxin immunoglobulin (IgY) in feeds passively immunizes shrimp against acute hepatopancreatic necrosis disease.

Acute hepatopancreatic necrosis disease (AHPND), caused by a toxin-producing Vibrio parahaemolyticus strain, has become a serious threat to shrimp aquaculture. The need to regulate antibiotic use prompted the development of alternative ways to treat infections in aquaculture including the use of chicken egg yolk immunoglobulin (IgY) for passive immunization. This study evaluated the protective effect of IgY against AHPND infection in Litopenaeus vannamei (Boone). IgY was isolated from eggs laid by hens immunized with recombinant PirA-like (rPirA) and PirB-like (rPirB) toxins. Whole-egg powders having IgY specific to rPirA (anti-PirA-IgY) and rPirB (anti-PirB-IgY) and IgY from non-immunized hen (control-IgY) were mixed with basal diets at 20% concentrations and used to prefeed shrimp 3 days before the bacterial challenge test. Survival rates of the challenged shrimp fed the anti-PirA-IgY, anti-PirB-IgY and control-IgY diets were 86%, 14% and 0%, respectively. Only the feed containing anti-PirA-IgY protected shrimp against AHPND. Increasing the concentration of rPirA antigen to immunize hens and lowering the amount of egg powder in feeds to 10% consistently showed higher survival rates in shrimp fed with anti-PirA-IgY (87%) compared with the control (12%). These results confirm that addition of anti-PirA-IgY in feeds could be an effective prophylactic method against AHPND infection in shrimp.

Effects of 5-Aminolevulinic Acid on Gene Expression, Immunity, and ATP Levels in Pacific White Shrimp, Litopenaeus vannamei.

With the emergence of several infectious diseases in shrimp aquaculture, there is a growing interest in the use of feed additives to enhance shrimp immunity. Recently, the use of 5-aminolevulinic acid (5-ALA), a non-protein amino acid that plays a rate-limiting role in heme biosynthesis, has received attention for its positive effect on immunity in livestock animals. To evaluate the effect of 5-ALA in the Pacific white shrimp, Litopenaeus vannamei, we conducted microarray analysis, a Vibrio parahaemolyticus immersion challenge test, an ATP level assay, and gene expression analysis of some hemoproteins and genes associated with heme synthesis and degradation. Out of 15,745 L. vannamei putative genes on the microarray, 101 genes were differentially expressed by more than fourfold (p < 0.05) between 5-ALA-supplemented and control shrimp hepatopancreas. 5-ALA upregulated 99 of the 101 genes, 41 of which were immune- and defense-related genes based on sequence homology. Compared to the control, the 5-ALA-supplemented group had a higher survival rate in the challenge test, higher transcript levels of porphobilinogen synthase, ferrochelatase, catalase, nuclear receptor E75, and heme oxygenase-1 and higher levels of ATP. These findings suggest that dietary 5-ALA enhanced the immune response of L. vannamei to V. parahaemolyticus, upregulated immune- and defense-related genes, and enhanced aerobic energy metabolism, respectively. Further studies are needed to elucidate the extent of 5-ALA use in shrimp culture.

Discrimination of juvenile yellowfin (Thunnus albacares) and bigeye (T. obesus) Tunas using mitochondrial DNA control region and liver morphology.

Yellowfin tuna, Thunnus albacares (Bonnaterre, 1788) and bigeye tuna, Thunnus obesus (Lowe, 1839) are two of the most economically important tuna species in the world. However, identification of their juveniles, especially at sizes less than 40 cm, is very difficult, often leading to misidentification and miscalculation of their catch estimates. Here, we applied the mitochondrial DNA control region D-loop, a recently validated genetic marker used for identifying tuna species (Genus Thunnus), to discriminate juvenile tunas caught by purse seine and ringnet sets around fish aggregating devices (FADs) off the Southern Iloilo Peninsula in Central Philippines. We checked individual identifications using the Neighbor-Joining Method and compared results with morphometric analyses and the liver phenotype. We tested 48 specimens ranging from 13 to 31 cm fork length. Morpho-meristic analyses suggested that 12 specimens (25%) were bigeye tuna and 36 specimens (75%) were yellowfin tuna. In contrast, the genetic and liver analyses both showed that 5 specimens (10%) were bigeye tuna and 43 (90%) yellowfin tuna. This suggests that misidentification can occur even with highly stringent morpho-meristic characters and that the mtDNA control region and liver phenotype are excellent markers to discriminate juveniles of yellowfin and bigeye tunas.