Molecular characterization and functional analysis of scavenger receptor class B from black tiger shrimp (Penaeus monodon)

BACKGROUND: Scavenger receptor class B (SRB) is a multifunctional protein in animals that participates in physiological processes, including recognition of a wide range of ligands. Astaxanthin is a major carotenoid found in shrimp. However, the molecular mechanism of astaxanthin and SRB protein binding has not been reported. RESULTS: In the present study, a member of the SRB subfamily, named PmSRB, was identified from the transcriptome of black tiger shrimp (Penaeus monodon). The open reading frame of PmSRB was 1557 bp in length and encoded 518 amino acids. The structure of PmSRB included a putative transmembrane structure at the N-terminal region and a CD36 domain. Multiple sequence alignment indicated that the CD36 domain were conserved. Phylogenetic analysis showed four separate branches (SRA, SRB, SRC, and croquemort) in the phylogenetic tree and that PmSRB was clustered with SRB of Eriocheir sinensis. Quantitative real-time polymerase chain reaction showed that the PmSRB gene was widely expressed in all tissues tested, with the highest expression level observed in the lymphoid organ and brain. Subcellular localization analysis revealed that PmSRB-GFP (green fluorescent protein) fusion proteins were predominantly localized in the cell membrane. The recombinant proteins of PmSRB showed binding activities against astaxanthin in vitro. CONCLUSIONS: PmSRB was identified and characterized in this study. It is firstly reported that PmSRB may take as an important mediator of astaxanthin uptake in shrimp.

Detecting Allergens From Black Tiger Shrimp Penaeus monodon That Can Bind and Cross-link IgE by ELISA, Western Blot, and a Humanized Rat Basophilic Leukemia Reporter Cell Line RS-ATL8

BACKGROUND: Black tiger shrimp Penaeus monodon is one of the common causes of shellfish allergy that is increasing worldwide. One of the important problems in the management of shellfish allergy is the lack of accurate diagnostic assay because the biological and immunological properties of allergens in black tiger shrimp have not been well characterized. This study aims to detect proteins with the ability to bind and cross-link immunoglobulin E (IgE) from black tiger shrimp by enzyme-linked immunosorbent assay (ELISA), Western blot, and a humanized rat basophilic leukemia reporter cell line RS-ATL8. METHODS: Sera from shrimp allergic subjects were subjected to ELISA and Western blots using raw or cooked shrimp extract as antigens. Pooled sera were used to sensitize the RS-ATL8 reporter cell line and cells were activated by shrimp extract. Eluted protein extracts separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were tested on the RS-ATL8 cell line and subjected to mass spectrometry to identify potential candidate allergens. RESULTS: Allergic sera reacted stronger to raw shrimp extract than cooked shrimp extract (P=0.009). Western blot demonstrated that major IgE reactivity protein bands were at 32–39 kDa and 91–230 kDa in both raw and cooked shrimp extracts. The eluted protein bands at the molecular weight of 38 and 115 kDa from raw shrimp extract induced IgE cross-linking as assayed by the RS-ATL8 cell line. These protein bands were subjected to mass spectrometry for analysis. Ubiquitin-activating enzyme and crustacyanin were identified as potential candidate novel shrimp allergens. CONCLUSIONS: The RS-ATL8 reporter cell line can be used to identify potential new shrimp allergens that can functionally cross-link IgE and induce mast cell degranulation.

Molecular variations in Vibrio alginolyticus and V. harveyi in shrimp-farming systems upon stress

A study was performed to investigate the genomic variations in the shrimp farm isolates of Vibrio alginolyticus and V. harveyi when the isolates were subjected to environmental stress. Samples of shrimps, water and sediment were collected from Southern Indian coastal shrimp farms. Vibrio isolates were biochemically identified and confirmed using 16S rDNA and gyrB gene specific PCR. The bacterial strains were genotyped by PCR fingerprinting using GTG(5) and IS (Insertion Sequence) primers. Seven strains each of V. alginolyticus and V. harveyi were subjected to 10 passages through trypticase soya broth (TSB), which contained different NaCl concentrations (3, 6 and 8%) and trypticase soya agar (TSA). V. alginolyticus was also passaged through TSB with a 12% NaCl concentration. PCR fingerprinting, which was performed on the strains that were passaged through different salt concentrations, confirmed that V. alginolyticus and V. harveyi could affect the genomic variations, depending on the environmental conditions of the culture. The study highlights the complex genotypic variations that occur in Vibrio strains of tropical aquatic environment because of varied environmental conditions, which result in genetic divergence and/or probable convergence. Such genetic divergence and/or convergence can lead to the organismal adaptive variation, which results in their ability to cause a productive infection in aquatic organisms or generation of new strains.

Impact of mineral deposition on shrimp, Penaeus monodon in a high alkaline water.

This study compares water quality parameters, shrimp growth and mortality rates, and biomass at harvest in two ponds of equal size, seeded with the same density (7 m-2) of White Spot Syndrome Virus (WSSV) and Monodon Baculo Virus (MBV) negative post-larvae (PL)-20 of shrimp, Penaeus monodon in the Vellar estuary of South India. The primary difference between the ponds was the water source; one was filled from the estuary and the second with water from bore wells with high alkalinity. Temperature in both ponds was similar and reached 32oC after 185 days of culture. Dissolved oxygen (DO) levels were within the acceptable range although levels in the alkaline pond were near the lower limit for the last 90 days before harvest. Salinity levels were similar in both ponds, above optimal levels, and increased over the 185 days. Alkalinity in the estuarine water was typically < 50 ppm and again 200-320 ppm in the alkaline pond. In the alkaline pond, beginning on the 75th day mineral deposits was observed covering all parts of the shrimp including the eye and the inner gill chambers, and by harvest, 42% of the shrimp showed this coating. Elemental analysis identified the major constituents as calcium, phosphorus and manganese. Survival rates in the estuarine-water-fed pond was 92% with a total pond biomass at harvest of 1.65 tons ha-1 compared to survival of 79% in the alkaline pond and a biomass at harvest of 1.020 tons ha-1. When well water must be used, its alkalinity should be monitored and diluted with water from other sources.

Isolation and characterization of genes functionally involved in ovarian development of the giant tiger shrimp Penaeus monodon by suppression subtractive hybridization (SSH)

Suppression subtractive hybridization (SSH) libraries between cDNA in stages I (previtellogenic) and III (cortical rod) ovaries of the giant tiger shrimp (Penaeus monodon) were established. In all, 452 ESTs were unidirectionally sequenced. Sequence assembly generated 28 contigs and 201 singletons, 109 of which (48.0 percent) corresponding to known sequences previously deposited in GenBank. Several reproduction-related transcripts were identified. The full-length cDNA of anaphase promoting complex subunit 11 (PmAPC11; 600 bp with an ORF of 255 bp corresponding to a polypeptide of 84 amino acids) and selenoprotein M precursor (PmSePM; 904 bp with an ORF of 396 bp corresponding to a polypeptide of 131 amino acids) were characterized and reported for the first time in penaeid shrimp. Semiquantitative RT-PCR revealed that the expression levels of PmSePM and keratinocyte-associated protein 2 significantly diminished throughout ovarian development, whereas Ser/Thr checkpoint kinase 1 (Chk1), DNA replication licensing factor mcm2 and egalitarian were down-regulated in mature ovaries of wild P. monodon (p < 0.05). Accordingly, the expression profiles of PmSePM and keratinocyte-associated protein 2 could be used as biomarkers for evaluating the degree of reproductive maturation in domesticated P. monodon.