Inhibitory effect of a reproductive-related serpin on sperm trypsin-like activity implicates its role in sperm maturation of Penaeus monodon.

In a number of marine animals, sperm serine proteases are important for fertilization. Penaeus monodon sperm require trypsin-like activity for a complete acrosome reaction, which exclusively occurs in sperm residing in the female thelycum. In this study, a complete cDNA sequence of reproductive tract-related Serine protease inhibitor (rrPmserpin) was identified. The longest open reading frame was composed of 1,366 nucleotides encoding 402 amino acids with a predicted pI of 6.86 and molecular mass of 44.88 kDa. The signal peptide cleavage site was identified as the 17th amino acid residue in the amino-terminus, and two potential N-glycosylation sites were predicted as post-translation modifications. A conserved reactive loop and fold similarities, identified through three-dimensional modeling, suggested that this gene is a member of the serpin family. The expression of rrPmserpin mRNA was prominent in the reproductive organs, including the testis, vas deferens, terminal ampoule containing the spermatophore, and the female thelycum. Inhibitory activity of recombinant rrPmSERPIN-6His was revealed from the negative correlation between the abundance of rrPmserpin mRNA and sperm trypsin-like activities, along with its inhibitory effects on chymotrypsin, trypsin, and thelycal proteases. Therefore, our results suggest that rrPmserpin participates in the regulation of the activity of a sperm protease and the decapacitation process.

Differential regulation of the lipoxygenase pathway in shrimp hepatopancreases and ovaries during ovarian development in the black tiger shrimp Penaeus monodon.

Dietary polyunsaturated fatty acids (PUFAs) are critical to the success of ovarian development in marine crustaceans, especially for domesticated species such as the black tiger shrimp Penaeus monodon. These fatty acids are stored in a midgut gland called the hepatopancreas and subsequently serve as an energy source or are incorporated in yolk during ovarian development. PUFAs are known precursors of hydroxy fatty acids, including hydroxyeicosatetraenoic acid and hydroxyeicosapentaenoic acid (HEPE), which are catalyzed by lipoxygenases (LOX). In previous studies, 8-HEPE has been shown to regulate female reproduction and adipogenesis in marine crustaceans. However, whether the biosynthesis of 8-HEPE in these species is the result of LOX activity has yet to be investigated. In this study, 8-HEPE was identified exclusively in P. monodon hepatopancreases using liquid chromatography-mass spectrometry. Treatment with nordihydroguaiaretic acid resulted in the reduction of 8-HEPE, suggesting the enzyme-dependent catalysis of 8-HEPE in hepatopancreases. Additionally, a full-length P. monodon LOX (PmLOX) was amplified from shrimp ovary cDNA. Sequence analysis revealed that the putative PmLOX contains domains and catalytic residues required for LOX catalytic function. Furthermore, PmLOX expression increased steadily as shrimp ovary maturation progressed, while PmLOX expression and the amount of 8-HEPE decreased in shrimp hepatopancreases. These findings not only suggest differential requirements for hydroxy fatty acid biosynthesis in shrimp ovaries and hepatopancreases during the P. monodon ovarian development, but also provide insights into the LOX pathway in marine crustaceans.

Enterocytozoon hepatopenaei sp. nov. (Microsporida: Enterocytozoonidae), a parasite of the black tiger shrimp Penaeus monodon (Decapoda: Penaeidae): Fine structure and phylogenetic relationships.

A new microsporidian species, Enterocytozoon hepatopenaei sp. nov., is described from the hepatopancreas of the black tiger shrimp Penaeus monodon (Crustacea: Decapoda). Different stages of the parasite are described, from early sporogonal plasmodia to mature spores in the cytoplasm of host-cells. The multinucleate sporogonal plasmodia existed in direct contact with the host-cell cytoplasm and contained numerous small blebs at the surface. Binary fission of the plasmodial nuclei occurred during early plasmodial development and numerous pre-sporoblasts were formed within the plasmodium. Electron-dense disks and precursors of the polar tubule developed in the cytoplasm of the plasmodium prior to budding of early sporoblasts from the plasmodial surface. Mature spores were oval, measuring 0.7x1.1microm and contained a single nucleus, 5-6 coils of the polar filament, a posterior vacuole, an anchoring disk attached to the polar filament, and a thick electron-dense wall. The wall was composed of a plasmalemma, an electron-lucent endospore (10nm) and an electron-dense exospore (2nm). DNA primers designed from microsporidian SSU rRNA were used to amplify an 848bp product from the parasite genome (GenBank FJ496356). The sequenced product had 84% identity to the matching region of SSU rRNA from Enterocytozoon bieneusi. Based upon ultrastructural features unique to the family Enterocytozoonidae, cytoplasmic location of the plasmodia and SSU rRNA sequence identity 16% different from E. bieneusi, the parasite was considered to be a new species, E. hepatopenaei, within the genus Enterocytozoon.

Using double-stranded RNA for the control of Laem-Singh Virus (LSNV) in Thai P. monodon.

Viral inhibition by double-stranded (ds)RNA is a potential therapeutic approach for controlling shrimp viral diseases. Here, we describe the successful oral application of dsRNA targeting Laem-Singh Virus (LSNV) to diminish monodon slow growth syndrome (MSGS) in Thai Penaeus monodon. Shrimp feed formulated with bacterially expressed LSNV-dsRNA was given to shrimp for 9 weeks. RT-PCR results revealed that all control shrimp were LSNV-positive at the end of experiment, while the shrimp that received dsRNA-feed exhibited 20-60% LSNV reduction. The average body weight of treated shrimp (number of shrimp=100) was significantly higher than that of the control group. Such increase is likely due to the elimination of MSGS caused by LSNV, as size variation of the treated group is much lower than that in the control group. This study demonstrates for the first time that feed with LSNV-specific dsRNA promotes the overall growth of P. monodon and relieves MSGS condition in LSNV-infected shrimp. The work reaffirms the potential of dsRNA application for controlling viral disease in shrimp farming.

Therapeutic effect of Artemia enriched with Escherichia coli expressing double-stranded RNA in the black tiger shrimp Penaeus monodon.

We exploited Artemia as a double-stranded (ds)RNA-delivery system to combat viral diseases in shrimp. First, the transformed Escherichia coli (E. coli) expressing red fluorescent protein (RFP) was tested in the Artemia enrichment process. RFP signals detectable in the gut of Artemia under confocal microscope were evident for the successful encapsulation. Second, the Artemia enrichment process was performed using E. coli producing Laem-Singh virus (LSNV)-specific dsRNA, which has been previously shown to inhibit the viral infection in the black tiger shrimp Penaeus monodon by intramuscular injection and oral administration. The enriched Artemia nauplii were confirmed to contain dsRNA-LSNV by RT-PCR, and were subjected to the feeding test with P. monodon postlarvae. Quantitative RT-PCR indicated that a number of LSNV copies in most of the treated shrimp were, at least, 1000-fold lower than the untreated controls. During 11-17weeks after feeding, average body weight of the treated group was markedly increased relative to the control group. A smaller differential growth rate of the treated group as compared to the control was also noticed. These results suggested that feeding shrimp with the dsRNA-enriched Artemia can eliminate LSNV infection, which is the cause of retarded growth in P. monodon. The present study reveals for the first time the therapeutic effect of dsRNA-enriched Artemia for shrimp disease control.