Mapping of essential residues in the predicted <i>P. monodon</i> PGES3 enzyme.

<p>Multiple sequence alignments of <i>P. monodon</i> PGES3 protein and their homologs reveal one conserved serine residue for the CKII phosphorylation site (black arrow), while the other phosphorylation site was not conserved (gray arrow). Genus and species used in this alignment are abbreviated as followed: <i>Penaeus</i> − <i>P. monodon</i>, Litopenaeus − <i>L. vannamei</i>, Danio − <i>Danio rerio</i>, <i>Pediculus</i> − <i>P. humanus corporis</i>, <i>Xenopus</i> − <i>Xenopus laevis</i>, <i>Caenorhabditis</i> − <i>Caenorhabditis elegans</i>, Drosophila − <i>Drosophila melanogaster</i>, Gallus − <i>Gallus gallus</i>, Bos − <i>B. taurus</i> and Homo − <i>H. sapiens</i>.</p

Insights into the Prostanoid Pathway in the Ovary Development of the Penaeid Shrimp <i>Penaeus monodon</i>

<div><p>The prostanoid pathway converts polyunsaturated fatty acids (PUFAs) into bioactive lipid mediators, including prostaglandins, thromboxanes and prostacyclins, all of which play vital roles in the immune and reproductive systems in most animal phyla. In crustaceans, PUFAs and prostaglandins have been detected and often associated with female reproductive maturation. However, the presence of prostanoid biosynthesis genes remained in question in these species. In this study, we outlined the prostanoid pathway in the black tiger shrimp <i>Penaeus monodon</i> based on the amplification of nine prostanoid biosynthesis genes: <i>cytosolic phospholipase A2, hematopoietic prostaglandin D synthase, glutathione-dependent prostaglandin D synthase, prostaglandin E synthase 1, prostaglandin E synthase 2, prostaglandin E synthase 3, prostaglandin F synthase</i>, <i>thromboxane A synthase</i> and <i>cyclooxygenase</i>. TBLASTX analysis confirmed the identities of these genes with 51-99% sequence identities to their closest homologs. In addition, prostaglandin F_2α (PGF_2α), which is a product of the prostaglandin F synthase enzyme, was detected for the first time in <i>P. monodon</i> ovaries along with the previously identified PUFAs and prostaglandin E₂ (PGE₂) using RP-HPLC and mass-spectrometry. The prostaglandin synthase activity was also observed in shrimp ovary homogenates using <i>in vitro</i> activity assay. When prostaglandin biosynthesis was examined in different stages of shrimp ovaries, we found that the amounts of <i>prostaglandin F synthase</i> gene transcripts and PGF_2α decreased as the ovaries matured. These findings not only indicate the presence of a functional prostanoid pathway in penaeid shrimp, but also suggest a possible role of the PGF_2α biosynthesis in shrimp ovarian development. </p> </div

Relative expression levels of <i>PmPGES</i> and <i>PmPGFS</i> genes in each ovary stage.

<p>Wild broodstock from the Andaman Sea (N=27) were captured and dissected to obtain ovary samples used in the real-time PCR analysis. Each graph represents the average copy number of prostaglandin biosynthesis gene transcripts normalized against <i>EF1α</i> in each ovary stage. (A) <i>PmPGES1</i>, (B) <i>PmPGES2</i>, (C) <i>PmPGES3</i> and (D) <i>PmPGFS</i>. Error bars show standard deviations and asterisks indicate significant changes between stages (p < 0.05).</p

Mapping of essential residues in the predicted <i>P. monodon</i> PGES1 enzyme.

<p>Multiple sequence alignments of <i>P. monodon</i> PGES1 and their homologs showed a conserved residue for the MAPEG family (white star), catalytic residues that interact with PGH₂ (white arrow head), essential residues for H-bonding to GSH (black arrows) and consensus sequence required for oxygenation product (underline). Genus and species used in this alignment are abbreviated as followed: <i>Penaeus</i> − <i>P. monodon</i>, Litopenaeus − <i>L. vannamei</i>, <i>Crassostrea</i> − <i>Crassostrea virginica</i>, Homarus − <i>H. americanus</i>, <i>Pediculus</i> − <i>Pediculus humanus corporis</i>, <i>Culex</i> − <i>Culex quinquefasciatus</i>, <i>Tribolium</i> − <i>Tribolium castaneum</i>, Equus − <i>Equus caballus</i>, Bos − <i>Bos taurus</i> and Homo − <i>Homo sapiens</i>.</p

Mapping of essential residues in the predicted <i>P. monodon</i> PGES2 enzyme.

<p>Multiple sequence alignments of <i>P. monodon</i> PGES2 protein and their homologs were performed, revealing conserved Cys residues at the active site (black arrows) and the N-terminal sequence of the mature enzyme (underline). Genus and species used in this alignment are abbreviated as followed: <i>Penaeus</i> − <i>P. monodon</i>, <i>Pediculus</i> − <i>P. humanus corporis</i>, Caligus − <i>Caligus rogercresseyi</i>, Mus − <i>Mus musculus</i> and Homo − <i>H. sapiens</i>.</p

The proposed prostanoid biosynthesis pathway in <i>P. monodon</i>.

<p>PUFAs, prostaglandins and full-length prostanoid biosynthesis genes identified in this study (black) were used to outline the <i>P. monodon</i> prostanoid pathway based on the previously published pathway in mammals. Prostanoids that have yet to be identified in <i>P. monodon</i> are shown in gray.</p

<i>In vitro</i> prostaglandin synthase activity assay using shrimp ovary homogenates.

<p>Shrimp ovary homogenates were incubated with 25 µM AA at 28 °C and collected at different time points (0, 30, 60, 120, 240 and 360 min). The homogenates were spun down and concentrations of PGE₂ (A) and PGF_2α (B) in the homogenate supernatant were estimated using EIA. The experiment was performed in triplicate and error bars indicate the standard deviation from the means. Asterisk indicates significant difference between the prostaglandin concentration at 0 h and the marked time point (<i>P</i><0.05).</p

Mapping of essential residues in the predicted <i>P. monodon</i> PGFS enzyme.

<p>Multiple sequence alignments of <i>P. monodon</i> PGFS protein and their homologs were performed, revealing residues that are important for substrate (black arrows) and the NADP⁺ cofactor (white arrow head) binding. Genus and species used in this alignment are abbreviated as followed: <i>Penaeus</i> − <i>P. monodon</i>, Litopenaeus − <i>L. vannamei</i>, Canis − <i>Canis lupus familiaris</i>, Ovis − <i>Ovis aries</i>, Bos − <i>B. taurus</i>, Equus − <i>Equus caballus</i> and Homo − <i>H. sapiens</i>.</p

Body length, body weight, spermatophore weight, and total sperm count of wild-caught and domesticated shrimp.

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Criteria for the identification of eicosanoids and PUFAs using retention time, precursor ion, proposed fragment ion, and m/z distribution.

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