RESUMO
The polychaete Perinereis nuntia is preferred over commercial feed pellets for boosting ovarian maturation of the female black tiger shrimp Penaeus monodon. High levels of prostaglandins in polychaetes are believed to enhance shrimp ovarian development. However, the impact of polychaete feeding on shrimp prostaglandin biosynthesis and fatty acid regulatory pathways have yet to be investigated. As polychaetes contain higher levels of arachidonic acid (ARA), eicosapentaenoic acid (EPA), prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) than feed pellets, we examined the effects of polychaete feeding alone and in combination with eyestalk ablation on shrimp hepatopancreases and ovaries. Shrimp fed with polychaetes contained higher levels of EPA, PGE2 and PGF2α in hepatopancreases than those of pellet-fed shrimp. Similarly, higher levels of ARA and higher transcription levels of cyclooxygenase (COX) and prostaglandin F synthase (PGFS) were detected in ovaries of polychaete-fed shrimp compared to those of pellet-fed shrimp. The combination of polychaete-feeding and eyestalk ablation, commonly practiced to induce ovarian development, increased levels of ARA and EPA and transcription levels of COX in hepatopancreases and ovaries of polychaete-fed shrimp compared to those of pellet-fed shrimp. In ovaries, prostaglandin biosynthesis gene transcripts were induced by polychaete feeding while transcriptional levels of fatty acid regulatory genes were regulated by shrimp feed and eyestalk ablation. Our findings not only elucidate the effects of polychaete consumption on shrimp prostaglandin biosynthesis and fatty acid regulatory pathways during larvae production, but also suggests that high levels of dietary ARA, EPA and prostaglandins are essential during P. monodon ovarian development.
Assuntos
Ração Animal/análise , Regulação da Expressão Gênica no Desenvolvimento , Larva/metabolismo , Ovário/metabolismo , Penaeidae/metabolismo , Poliquetos/fisiologia , Prostaglandinas/biossíntese , Animais , Feminino , Perfilação da Expressão Gênica , Larva/genética , Larva/crescimento & desenvolvimento , Lipogênese , Penaeidae/genética , Penaeidae/crescimento & desenvolvimentoRESUMO
The delay in ovarian maturation in farmed black tiger shrimp Penaeus monodon has resulted in the widespread practice of feeding broodstock with the polychaete Perinereis nuntia and their unilateral eyestalk ablation. Although this practice alters fatty acid content in shrimp ovaries and hepatopancreas, its effects on fatty acid regulatory genes are yet to be systematically examined. Here, microarray analysis was performed on hepatopancreas and ovary cDNA collected from P. monodon at different ovarian maturation stages, revealing that 72 and 58 genes in fatty acid regulatory pathways were differentially expressed in hepatopancreas and ovaries respectively. Quantitative real-time PCR analysis revealed that ovarian maturation was associated with higher expression levels of acetyl-CoA acetyltransferase, acyl-CoA dehydrogenase, acyl-CoA oxidase 3 and long-chain fatty acid transport protein 4 in hepatopancreas, whereas the expression levels of 15 fatty acid regulatory genes were increased in shrimp ovaries. To distinguish the effects of different treatments, transcriptional changes were examined in P. monodon with stage 1 ovaries before polychaete feeding, after 1 month of polychaete feeding and after eyestalk ablation. Polychaete feeding resulted in lower expression levels of enoyl-CoA hydratase and acyl-CoA synthetase medium-chain family member 4, while the expression level of phosphatidylinositide phosphatase SAC1 was higher in shrimp hepatopancreas and ovaries. Additionally, eyestalk ablation resulted in a higher expression level of long-chain fatty acid-CoA ligase 4 in both tissues. Together, our findings describe the dynamics of fatty acid regulatory pathways during crustacean ovarian development and provide potential target genes for alternatives to eyestalk ablation in the future.
Assuntos
Ácidos Graxos/metabolismo , Metabolismo dos Lipídeos/genética , Ovário/metabolismo , Penaeidae/genética , Técnicas de Ablação , Ração Animal , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Ovário/crescimento & desenvolvimento , Penaeidae/crescimento & desenvolvimento , Penaeidae/metabolismo , Poliquetos , Fatores de TempoRESUMO
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.
Assuntos
Hepatopâncreas/embriologia , Hepatopâncreas/enzimologia , Lipoxigenase/metabolismo , Ovário/embriologia , Ovário/enzimologia , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Masculino , Especificidade de Órgãos/fisiologia , Organogênese/fisiologia , Penaeidae/embriologia , Penaeidae/enzimologia , Transdução de Sinais/fisiologiaRESUMO
Eicosanoids, which are oxygenated derivatives of polyunsaturated fatty acids (PUFAs), serve as signaling molecules that regulate spermatogenesis in mammals. However, their roles in crustacean sperm development remain unknown. In this study, the testis and vas deferens of the black tiger shrimp Penaeus monodon were analyzed using ultra-high performance liquid chromatography coupled with Orbitrap high resolution mass spectrometry. This led to the identification of three PUFAs and ten eicosanoids, including 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and (±)15-hydroxyeicosapentaenoic acid ((±)15-HEPE), both of which have not previously been reported in crustaceans. The comparison between wild-caught and domesticated shrimp revealed that wild-caught shrimp had higher sperm counts, higher levels of (±)8-HEPE in testes, and higher levels of prostaglandin E2 (PGE2) and prostaglandin F2α in vas deferens than domesticated shrimp. In contrast, domesticated shrimp contained higher levels of (±)12-HEPE, (±)18-HEPE, and eicosapentaenoic acid (EPA) in testes and higher levels of 15d-PGJ2, (±)12-HEPE, EPA, arachidonic acid (ARA), and docosahexaenoic acid (DHA) in vas deferens than wild-caught shrimp. To improve total sperm counts in domesticated shrimp, these broodstocks were fed with polychaetes, which contained higher levels of PUFAs than commercial feed pellets. Polychaete-fed shrimp produced higher total sperm counts and higher levels of PGE2 in vas deferens than pellet-fed shrimp. In contrast, pellet-fed shrimp contained higher levels of (±)12-HEPE, (±)18-HEPE, and EPA in testes and higher levels of (±)12-HEPE in vas deferens than polychaete-fed shrimp. These data suggest a positive correlation between high levels of PGE2 in vas deferens and high total sperm counts as well as a negative correlation between (±)12-HEPE in both shrimp testis and vas deferens and total sperm counts. Our analysis not only confirms the presence of PUFAs and eicosanoids in crustacean male reproductive organs, but also suggests that the eicosanoid biosynthesis pathway may serve as a potential target to improve sperm production in shrimp.
Assuntos
Penaeidae , Animais , Ácido Araquidônico , Dinoprosta , Dinoprostona/metabolismo , Ácidos Docosa-Hexaenoicos , Eicosanoides , Ácido Eicosapentaenoico , Ácidos Graxos Insaturados , Masculino , Mamíferos/metabolismo , Prostaglandinas E , Sêmen/metabolismo , Contagem de Espermatozoides , Espermatozoides/metabolismoRESUMO
Cyclooxygenase (COX) is a two-step enzyme that converts arachidonic acid into prostaglandin H2, a labile intermediate used in the production of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α). In vertebrates and corals, COX must be N-glycosylated on at least two asparagine residues in the N-(X)-S/T motif to be catalytically active. Although COX glycosylation requirement is well-characterized in many species, whether crustacean COXs require N-glycosylation for their enzymatic function have not been investigated. In this study, a 1,842-base pair cox gene was obtained from ovarian cDNA of the black tiger shrimp Penaeus monodon. Sequence analysis revealed that essential catalytic residues and putative catalytic domains of P. monodon COX (PmCOX) were well-conserved in relation to other vertebrate and crustacean COXs. Expression of PmCOX in 293T cells increased levels of secreted PGE2 and PGF2α up to 60- and 77-fold, respectively, compared to control cells. Incubation of purified PmCOX with endoglycosidase H, which cleaves oligosaccharides from N-linked glycoproteins, reduced the molecular mass of PmCOX. Similarly, addition of tunicamycin, which inhibits N-linked glycosylation, in PmCOX-expressing cells resulted in PmCOX protein with lower molecular mass than those obtained from untreated cells, suggesting that PmCOX was N-glycosylated. Three potential glycosylation sites of PmCOX were identified at N79, N170 and N424. Mutational analysis revealed that although all three residues were glycosylated, only mutations at N170 and N424 completely abolished catalytic function. Inhibition of COX activity by ibuprofen treatment also decreased the levels of PGE2 in shrimp haemolymph. This study not only establishes the presence of the COX enzyme in penaeid shrimp, but also reveals that N-glycosylation sites are highly conserved and required for COX function in crustaceans.
Assuntos
Penaeidae/enzimologia , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Sequência de Bases , Inibidores de Ciclo-Oxigenase/farmacologia , Análise Mutacional de DNA/métodos , DNA Complementar/genética , Dinoprosta/metabolismo , Dinoprostona/metabolismo , Feminino , Glicosilação/efeitos dos fármacos , Células HEK293 , Hemolinfa/metabolismo , Humanos , Ibuprofeno/farmacologia , Peso Molecular , Ovário/metabolismo , Prostaglandina-Endoperóxido Sintases/química , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transfecção , Tunicamicina/farmacologiaRESUMO
Torrubiellones A-D (1-4), new pyridone and tetramic acid alkaloids, were isolated from the spider pathogenic fungus Torrubiella sp. BCC 2165. Torrubiellone A (1) exhibited antimalarial activity with an IC(50) value of 8.1 µM, while it showed very weak cytotoxic activity.
Assuntos
Alcaloides/isolamento & purificação , Alcaloides/farmacologia , Antimaláricos/isolamento & purificação , Antimaláricos/farmacologia , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Hypocreales/química , Piridonas/isolamento & purificação , Piridonas/farmacologia , Pirrolidinonas/isolamento & purificação , Pirrolidinonas/farmacologia , Aranhas/microbiologia , Alcaloides/química , Animais , Antimaláricos/química , Antineoplásicos/química , Concentração Inibidora 50 , Estrutura Molecular , Plasmodium falciparum/efeitos dos fármacos , Piridonas/química , Pirrolidinonas/químicaRESUMO
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 Penaeus monodon based on the amplification of nine prostanoid biosynthesis genes: 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, thromboxane A synthase and cyclooxygenase. TBLASTX analysis confirmed the identities of these genes with 51-99% sequence identities to their closest homologs. In addition, prostaglandin F2α (PGF2α), which is a product of the prostaglandin F synthase enzyme, was detected for the first time in P. monodon 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 in vitro activity assay. When prostaglandin biosynthesis was examined in different stages of shrimp ovaries, we found that the amounts of prostaglandin F synthase gene transcripts and PGF2α 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 PGF2α biosynthesis in shrimp ovarian development.
Assuntos
Proteínas de Artrópodes/genética , Vias Biossintéticas/genética , Enzimas/genética , Ovário/metabolismo , Penaeidae/metabolismo , Prostaglandinas/biossíntese , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Dinoprosta/metabolismo , Dinoprostona/metabolismo , Enzimas/classificação , Enzimas/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Oxirredutases Intramoleculares/classificação , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Ovário/crescimento & desenvolvimento , Penaeidae/crescimento & desenvolvimento , Filogenia , Prostaglandina-E Sintases , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
Torrubiellins A (1) and B (2), two new dimeric anthraquinones were isolated from the leafhopper pathogenic fungus Torrubiella sp. BCC 28517. The structures of the new compounds were elucidated by analyses of the NMR spectroscopic and mass spectrometry data. Torrubiellin B (2) exhibited broad range of biological activities.
Assuntos
Antraquinonas/isolamento & purificação , Antraquinonas/farmacologia , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Hemípteros/microbiologia , Hypocreales/química , Hypocreales/patogenicidade , Animais , Antraquinonas/química , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Antifúngicos/química , Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Antimaláricos/química , Antimaláricos/isolamento & purificação , Antimaláricos/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Dimerização , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura MolecularRESUMO
A new dimeric isocoumarin, bireticulol, was isolated from the terrestrial Streptomyces sp. and characterized as a 5-5' dimer of reticulol. In addition, reticulol and 8-hydroxy-6,7-dimethoxy-3-methyl isocoumarin, together with other known polyketides piericidin A, 2'-(2-hydroxyphenyl)-2,4'-bibenzoxazole-4-carboxylic acid methyl ester (UK-1) and 3-benzyl-4-hydroxy-5-methyldihydrofuran-2-one were also obtained. Bireticulol exhibited cytotoxic effects against KB (human epidermoid carcinoma, ATCC CCL-17) and NCI-H187 (human small cell lung cancer, ATCC CRL-5804) cell lines with IC(50) values of 24.4 and 8.31 µg ml(-1), respectively.