Ovaries Of Shrimp Research Articles

Killing of xenogenous and virally infected homogenous target cells by shrimp lymphocyte-like haemocytes

Haemocytes play a crucial role in the invertebrate's immune system. In our lab, five subpopulations of shrimp haemocytes were identified in the past: hyalinocytes, granulocytes, semi-granulocytes and two subpopulations of non-phagocytic cells. In the latter two subpopulations, their characteristics such as having small cytoplasmic rims and not adhering to plastic cell-culture plates are very similar to those of mammalian lymphocytes. Therefore, they were designated lymphocyte-like haemocytes. Although little is known about their function, we hypothesize, based on their morphology, that they may have a cytotoxic activity like natural killer cells, with the ability to recognize and kill target cells. In our study, K562 cells and Sf9 cells were used as xenogenous target cells to detect the cytotoxic activity of the shrimp non-adherent lymphocyte-like haemocytes. Non-adherent haemocytes were collected and mixed with K562 cells and Sf9 cells at a 5:1 ratio and the binding activity was examined under a microscope. The binding rate of non-adherent haemocytes to K562 cells and Sf9 cells reached 6.6 % and 2.4 % after 240 min of culture, respectively. Then, the killing activity of non-adherent haemocytes was detected by an EMA staining (fluorescence microscopy), which showed 3.75 % dead K562 cells and 1.025 % dead Sf9 cells, and by Sytox® blue staining (flow cytometry), which showed 4.97 % of dead K562 cells. Next, a killing assay was developed to visualize the killing activity of shrimp non-adherent haemocytes. Non-adherent haemocytes were pre-labeled in blue (CellTracker blue) and K562/Sf9 cells in green (CFSE); dead cells were differentially stained red with ethidium bromide. The cytotoxic activity increased and reached a level of 2.59 % in K562 cells and 0.925 % in Sf9 cells at 120 min after co-culture. Furthermore, in the co-cultures of non-adherent haemocytes with K562 cells and Sf9 cells, upregulation of the gene and protein expression of the cytotoxic molecules torso-like protein and granzyme B was observed by RT-qPCR at 240 min and western blotting at 180 min. Additionally, non-adherent haemocytes were co-cultured with WSSV-inoculated shrimp ovary and lymphoid organ cells to detect the cytotoxicity to homogenous target cells. The binding activity started at 60 min in both the ovary and lymphoid organ cultures and reached at 240 min 50.62 % and 40.7 %, respectively. The killing activity was detected by EMA staining and the percentage of dead ovary and lymphoid organ cells increased respectively from 10.84 % to 6.89 % at 0 min to 13.09 % and 8.37 % at 240 min. In conclusion, we demonstrated the existence of cytotoxic activity of shrimp lymphocyte-like haemocytes against xenogenous cells from mammals and insects and against WSSV-infected homogenous shrimp cells.

Potential receptors in Fenneropenaeus merguiensis ovary and role of saxophone, the bone morphogenetic protein receptor, in ovarian development

Receptors, which play an initial role in signaling pathways in several physiological processes, including reproduction, are among the several molecular factors that control ovarian development in organisms. This study aimed to identify and study receptors potentially involved in controlling the reproductive process of female banana shrimp, Fenneropenaeus merguiensis. Ovarian transcriptomes derived from 4 developmental stages were generated by RNA sequencing. A total of 53,763 transcripts were obtained from the de novo assembled transcriptome, and 663 genes were identified as receptors. Among them, 185 receptors were differentially expressed during ovarian development. Fifteen of these differentially expressed receptors showed distinct expression patterns that were validated by RT-qPCR. Bone morphogenetic protein receptors (BMPR) and their signaling genes were investigated for their roles in shrimp vitellogenesis. The expressions of F. merguiensis saxophone (FmSax), a BMP type I receptor, and BMP type II receptor (FmBMPRII) as well as FmMad, FmMed, and FmSMAD3 were significantly altered during ovarian development. RNA interference was used to investigate the role of FmSax in vitellogenesis. The result indicated that the expression of vitellogenin (Vg) was significantly reduced in both ovary and hepatopancreas of FmSax-knockdown shrimp compared to control shrimp. Furthermore, in FmSax-silencing shrimp, FmBMPRII, FmMad, and FmMed expressions were decreased as well as Vg expression. These findings suggest that FmSax positively regulates Vg synthesis via the BMP signaling pathway.

Microplastics and bisphenol A hamper gonadal development of whiteleg shrimp (Litopenaeus vannamei) by interfering with metabolism and disrupting hormone regulation

Gonadal development is a prerequisite for the reproductive success of an organism, and might be affected by environmental factors such as emergent pollutants. Although marine crustaceans are threatened by ubiquitous emergent pollutants such as microplastics (MPs) and bisphenol A (BPA) under realistic scenarios, studies about the impacts of these pollutants on the gonadal development of crustacean species are rare. In this study, the effects of MPs and BPA, alone or in combination, on gonadal development were investigated in whiteleg shrimp (Litopenaeus vannamei). The results obtained demonstrated that whiteleg shrimp exposed to MPs and BPA had significantly smaller gonad-somatic index (GSI) and an obvious delay in the gonad developmental stage. In addition, exposure of whiteleg shrimp to pollutants tested resulted in a reduction in the oxygen consumption rate, elevation in the ammonia excretion rate, decline in the O: N ratio, and downregulation in the expression of metabolism-related genes, indicating significant disruptions of shrimp metabolism by the pollutants. Furthermore, in addition to a few exceptions, both the in vivo contents of gonadal development-related hormones (GIH and MIH) and the expression of genes encoding regulatory hormones (GIH, MIH, and CHH) were upregulated by the exposure of whiteleg shrimp to the pollutants investigated, suggesting a significant obstruction of endocrine regulation. Moreover, MP-BPA coexposure was shown to be more toxic to whiteleg shrimp than the corresponding single exposures and significantly greater amount of BPA accumulated in the gonads (both testis and ovaries) of shrimp with the coexistence of MPs, which may be caused by the Trojan horse effect and summation of the toxic impacts on common targets. In general, the data obtained in this study demonstrated that MPs and BPA at environmentally realistic concentrations significantly inhibited the gonadal development of whiteleg shrimp probably by interfering with metabolism and disrupting endocrine regulation.

Retinoid X receptor modulates vitellogenin gene expression in black tiger shrimp, Penaeus monodon

Effective inducing of ovarian maturation in female shrimp broodstock is important for successful breeding programs. Vitellogenesis is a biochemical process during which a yolk protein precursor vitellogenin (Vg) is synthesized and thus, can be used to indicate ovarian maturation stage. In this study, transcriptional regulation of Vg synthesis in the black tiger shrimp, Penaeus monodon was investigated. Genome walking on 5′ upstream sequence of Vg gene revealed several putative binding sites of lipophilic retinoic acid response elements (RARE), and nuclear hormone responsive elements. Deletion of RARE significantly reduced the promoter activity to drive the expression of luciferase reporter gene in Sf-9 cells. To validate the trans-factor that potentially controls Vg expression through RARE, a cDNA encoding retinoid X receptor (PmRXR), one of the RARE-bound transcription factors was cloned from P. monodon's ovary. PmRXR expression was detected in various shrimp tissues, and was up-regulated during ovary development in a similar way to Vg expression. The DNA-binding domain of PmRXR protein showed specific binding to RARE-containing region on Vg 5′ upstream sequence as determined by Electrophoretic Mobility Shift Assay (EMSA). Furthermore, dsRNA-mediated PmRXR silencing in previtellogenic and vitellogenic shrimp revealed that suppression of PmRXR could reduce Vg transcript in both stages. Taken together, the results presented in this study indicate that RXR is possibly an activator protein that modulates Vg expression in shrimp ovary through the binding to RARE.

Deciphering the molecular regulatory mechanism orchestrating ovary development of the Pacific whiteleg shrimp Litopenaeus vannamei through integrated transcriptomic analysis of reproduction-related organs

Ovary development of decapod crustaceans is controlled by a complex neuro-endocrine regulatory network. In crustacean aquaculture, eyestalk ablation (ESA) technique has been widely used to induce ovary development and maturation. However, this technique can cause significant hormonal imbalance leading to inferior egg quality and weak immunity of the animals. Understanding the molecular regulatory mechanism of ovary development will help to develop alternative measures to expedite ovarian maturation. In this study, we performed a comprehensive transcriptomic analysis of five important organs related to ovary development, including the eyestalk ganglion, brain, thoracic ganglion, hepatopancreas, and ovary, at different ovary developmental stages of the Pacific white shrimp Litopenaeus vannamei. A total of 698,118,770 clean reads were obtained from the Illumina HiSeq™ 4000 sequencing platform, resulting in an assembly of 48,722 unique transcripts (or unigenes) with an N50 of 2463 bp and a mean length of 1244 bp. 18,661 unigenes can be matched to homologous sequences in public databases. Pairwise comparisons revealed that a wide variety of genes were differentially expressed and several gene expression trends were significantly enriched in different organs during ovary development. Through weighted gene co-expression network analysis, six tissue- or stage-specific modules of co-expressed genes were identified. GO and KEGG analysis further disclosed that a rich set of genes, including those associated with the CHH family hormones, ecdysone signaling, juvenile hormone signaling, Insulin-PI3K-Akt signaling, EGFR signaling, and TGFβ signaling pathways, were preferentially expressed in specific tissues, implying their crucial roles in the regulation of ovary development process. Taken together, the study reports a comprehensive analysis of transcriptomic modulation in major neuro-endocrine organs related to shrimp ovary development. The data obtained sheds light on the potential coordination between the nervous system and its target organs (i.e. ovary and hepatopancreas), and contributes to a deeper understanding of the regulatory network orchestrating ovary development of shrimp.

Polychaete consumption increased prostaglandin biosynthesis in female Penaeus monodon.

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.

Induction of vitellogenesis in female banana shrimp, Fenneropenaeus merguiensis by leucine-tyrosine-arginine motif-containing protein 5 (LYRM5)

Female ovarian development is crucial for the successful propagation of sexually reproducing organisms that involve several physiological events, including vitellogenin synthesis, oocyte proliferation, and metabolism. These processes are controlled by several genes. Among them, a leucine-tyrosine-arginine motif-containing protein 5 (LYRM5) was found to be differentially expressed between undeveloped and mature ovaries, in the transcriptome of Fenneropenaeus merguiensis's ovaries. This study aimed to investigate the expression analysis and the role of LYRM5 in vitellogenesis of female banana shrimp, F. merguiensis. The FmLYRM5 was phylogenetically analyzed to be a member of LYRM5 and showed high similarity to LYRM5 of other species. The FmLYRM5 was highly expressed in eyestalks, thoracic ganglia, and hepatopancreas, and less in brain, gills, and ovary. Its expression was significantly affected in the ovaries by the developmental stage and was decreased in the matured shrimp, but was unchanged in hepatopancreas. Also, it was up-regulated by 20-hydroxyecdysone (20-HE), methyl farnesoate (MF) and serotonin (5-HT) treatments in the primary ovarian cells. The FmLYRM5 localizes at the mitochondrial compartment in the CHO cells. The overexpression of FmLYRM5 in female shrimp, induced by the recombinant plasmid (pLYRM5His) injection, showed increased vitellogenin expression and oocyte proliferation. Furthermore, the total sugar level in hemolymph (1526.73 ± 392.05 μg/ml) and ovary (1.53 ± 0.3 μg/mg ovary) was induced in shrimp injected with pLYRM5His on day 14 compared with that in hemolymph (139.10 ± 17.5 μg/ml) and ovary (0.71 ± 0.3 μg/mg ovary) of control shrimp. The total lipid level was increased only in the hemolymph of FmLYRM5-overexpressing shrimp (54.19 ± 23.1 μg/ml) compared with that of control shrimp (5.52 ± 1.4 μg/ml) but was unchanged in their ovaries. Our study indicates one mitochondrial factor that plays roles in stimulating vitellogenesis and in metabolic regulation during ovarian development.

Dynamics of fatty acid regulatory genes during ovarian development in Penaeus monodon.

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.

Bioassay-Guided Fractionation of Emilia sonchifolia Extract on the Induction of Ovarian Maturation in Fenneropenaeus merguiensis

This study was carried out to identify the active compounds of Emilia sonchifolia on induction of ovarian maturation in Fenneropenaeus merguiensis. The crude extracts from dichloromethane and acetone were investigated in vitro. The crude extract from acetone induced up-regulation of shrimp ovarian peritrophin (SOP) and translationally controlled tumor protein (TCTP) genes, which were highly expressed during early phase of ovarian development higher than dichloromethane extract. Furthermore, fraction 14 (F14) from acetone extract up-regulated both of the SOP and the TCTP genes to the greatest extent. Leading to in vivo study, the effect of ribosomal protein L10a (RPL10a), which acts as shrimp ovarian stimulator plus with F14 on shrimp ovarian maturation was investigated by injection. The best result was observed in the group that received RPL10a plus with F14 at 0.8 μg/g body weight of shrimp. The RPL10a plus F14 enhanced the shrimp ovaries from undeveloped stage to 57% of early developing stage within 15 days. Meanwhile, the control group remained 100% of the undeveloped stage. Hence, F14 seems to play a positive role in the induction of shrimp ovarian maturation. The component of F14 was identified using mass spectroscopy and presented as 1,2-benzenedicarboxylic acid; 2,4-di-tert-butyl phenol; 2,4,5-trimethoybenzylidene;palmitic acid; 1-heneicosyl formate; 1-heptadecanol; ethyl-4-ethoxybenzoate and stearic acid.

Arachidonic acid in diets for early maturation stages enhances the final reproductive performances of Pacific white shrimp (Litopenaeus vannamei)

A 70-day feeding trial with Pacific white shrimp (Litopenaeus vannamei) broodstocks was conducted before eyestalk ablation to investigate the effects of arachidonic acid (ARA) in diets for early maturation stages on the final reproductive performances. Three isonitrogenous and isolipidic experimental diets were formulated to contain different ARA levels: the control diet without ARA supplementation (C, 0.87% ARA of total fatty acids (TFA)) and two diets with low (4.65% of TFA, ARA-L) or high ARA (12.39% of TFA, ARA-H) supplementation level. The diets were randomly assigned to 9 tanks of eight-month-old Pacific white shrimp broodstock (30 females and 30 males in each tank). Shrimp were reared in a flowing seawater system and fed to apparent satiation four times daily. At the end of the feeding trial, unilateral eyestalk of the females was ablated to stimulate the gonad maturation. Following the feeding trial (Period I) was a 28-day reproductive evaluation period (Period II), during which all shrimps were fed the same fresh invertebrate ingredients (a mixture of squid and polychaetes). Tissues samples of females were collected at the end of the feeding trial (Period I) and before spawning (ready to spawn) at the reproductive evaluation period (Period II) respectively to measure the gonadosomatic index (GI), hepatopancreas index (HI), estradiol level, and fatty acid composition. The spawning performance and quality of egg and larvae were assayed at Period II. The results showed that compared to the control group, ARA-L increased the GI at Period II while the ARA supplemented diets increased the estradiol synthesis at Period I. The HI was not influenced by dietary ARA level. Compared to the control group, ARA-L but not ARA-H significantly increased the spawning rate, multi-spawning rate, average spawning frequency and fecundity of female shrimp, diameter of fertilized egg, and crude metamorphosis rate of nauplii at 33h post spawning. The hatching rate significantly ranked as follows: C<ARA-H<ARA-L. The dietary ARA supplementation significantly improved the duration of nauplius and zoea larvae in the challenge test with low salinity. The experimental diets significantly affected the fatty acid compositions in hepatopancreas and ovaries of female shrimp, as well as those in fertilized eggs. In conclusion, these results suggested that the ARA supplementation in the diets for early maturation stages enhanced the final reproductive performances of Pacific white shrimp.

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.

Transcriptomic information from Pacific white shrimp (Litopenaeus vannamei) ovary and eyestalk, and expression patterns for genes putatively involved in the reproductive process

The increased use of massive sequencing technologies has enabled the identification of several genes known to be involved in different mechanisms associated with reproduction that so far have only been studied in vertebrates and other model invertebrate species. In order to further investigate the genes involved in Litopenaeus vannamei reproduction, cDNA and SSH libraries derived from female eyestalk and gonad were produced, allowing the identification of expressed sequences tags (ESTs) that potentially have a role in the regulation of gonadal maturation. In the present study, different transcripts involved in reproduction were identified and a number of them were characterized as full-length. These transcripts were evaluated in males and females in order to establish their tissue expression profiles during developmental stages (juvenile, subadult and adult), and in the case of females, their possible association with gonad maturation was assessed through expression analysis of vitellogenin. The results indicated that the expression of vitellogenin receptor (vtgr) and minichromosome maintenance (mcm) family members in the female gonad suggest an important role during previtellogenesis. Additionally, the expression profiles of genes such as famet, igfbp and gpcr in brain tissues suggest an interaction between the insulin/insulin-like growth factor signaling pathway (IIS) and methyl farnesoate (MF) biosynthesis for control of reproduction. Furthermore, the specific expression pattern of farnesoic acid O-methyltransferase suggests that final synthesis of MF is carried out in different target tissues, where it is regulated by esterase enzymes under a tissue-specific hormonal control. Finally, the presence of a vertebrate type steroid receptor in hepatopancreas and intestine besides being highly expressed in female gonads, suggest a role of that receptor during sexual maturation.

Molecular cloning and expression analysis of MAT1 gene in black tiger shrimp (Penaeus monodon).

MAT1 (ménage à trois 1), an assembly factor and targeting subunit of the CDK-dependent kinase (CAK), can regulate the cell cycle, transcription, and DNA repair. This study was intended to investigate the role of MAT1 in the reproductive maturation of black tiger shrimp (Penaeus monodon). In this study, the P. monodon MAT1 (PmMAT1) gene was identified and characterized. The full-length cDNA of PmMAT1 was 1490 bp in length with an open-reading frame of 993 bp corresponding to 330 amino acids. The temporal expression of PmMAT1 in various tissues was measured by quantitative real-time PCR with the highest expression observed in ovaries. In the ovaries, the PmMAT1 gene was continuously but differentially expressed during the maturation stages. Comparative analyses of MAT1, CDK7, and cyclin H in the CAK complex of P. monodon indicated that the expression of CDK7 and cyclin H coincided with that of MAT1 during the ovary maturation stages. Serotonin (5-HT) injection promoted the expression level of PmMAT1 in the ovaries of shrimp at 6-48 h post-injection. These results indicate that PmMat1 plays a prominent role in the process of ovarian maturation.

Cloning and expression analysis on a homolog of spermatogonial stem-cell renewal factor in Fenneropenaeus chinensis

The spermatogonial stem-cell renewal factor (SSRF) was named since its function in spermatogonial mitosis was reported in Japanese eel. Our previous study showed that a homolog of SSRF was highly expressed in the ovary of triploid shrimp, but not expressed in the ovary of diploid shrimp. To understand the function of SSRF in shrimp, the full-length cDNA of ssrf gene was cloned from Chinese shrimp Fenneropenaeus chinensis (Fcssrf) and its expression was analyzed. The full length of Fcssrf cDNA was 2588 bp and it contained an open reading frame encoding 450 amino acids. The predicted tertiary structure of FcSSRF was very similar to that of SSRF/eSRS34 from Anguilla japonica and TP/PD-ECGF from Homo sapiens. RT-PCR analysis showed that the Fcssrf was highly expressed in nerve, testis, hepatopancreas, gill, and stomach rather than in ovary. Expression of Fcssrf mRNA was not detected during embryonic stages and larval stages, from the nauplii to the post-larvae stage, in diploid, and triploid shrimp. However, it began to be expressed in juvenile stages (June–September) in diploid and triploid shrimp. Immunohistochemical analyses showed that FcSSRF was identified in both the diploid testis and triploid ovary. We inferred that the Fcssrf might be related to testis development.

Insights into the Prostanoid Pathway in the Ovary Development of the Penaeid Shrimp Penaeus monodon

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.

Expression profiles and localization of vitellogenin mRNA and protein during ovarian development of the giant tiger shrimp Penaeus monodon

Ovarian maturation of the giant tiger shrimp (Penaeus monodon) results from rapid synthesis and accumulation of a major yolk protein, vitellin which is derived from its precursor, vitellogenin. The expression profiles and localization of P. monodon vitellogenin 1 (PmVtg1) were examined. PmVtg1 mRNA was expressed only in ovaries and hepatopancreas but not in other tissues of female and testes of male broodstock. In wild intact broodstock, PmVtg1 mRNA was expressed at a low level in stage I ovaries and up-regulated in stages II and III being significantly reduced in stage IV ovaries (P<0.05). In eyestalk-ablated broodstock, the expression level of PmVtg1 mRNA was increased to nearly a peak level in vitellogenic (stage II) ovaries and peaked in cortical rod (stage III) ovaries (P<0.05). The level of PmVtg1 mRNA in hepatopancreas of intact shrimp with stages II and III ovaries was significantly greater than those with stages IV and V (post-spawning) ovaries. Interestingly, PmVtg1 mRNA in hepatopancreas was 25–40 times higher than that in ovaries of intact shrimp with stages I–III of ovarian development. In situ hybridization revealed that PmVtg1 mRNA was clearly localized in the cytoplasm of follicular cells surrounding late previtellogenic, vitellogenic and cortical rod oocytes. Quantitative real-time PCR against different periods of spawned eggs suggested that PmVtg1 mRNA was also synthesized in oocytes. Western blot analysis revealed a similar profile of ovarian P. monodon vitellin and PmVtg1 mRNA during ovarian development and also suggested that the Vtg protein in hepatopancreas and Pm-vitellin in ovaries should have encoded from different Vtg genes. Immunohistochemistry confirmed that Pm-vitellin was localized in developing, vitellogenic and mature oocytes but not in the follicular cells.

The roles of ribosomal protein S3a in ovarian development of Fenneropenaeus merguiensis (De Man)

Shrimp ovarian development is a pivotal step for aquaculture in order to maintain broodstocks. The molecular processes in the ovaries of Fenneropenaeus merguiensis De Man during vitellogenesis were investigated. A quantitative real time PCR (Q-PCR) assay showed a high expression of ribosomal protein S3a (RPS3a) in the early stage of ovarian development and a decline in the later stages. Serotonin (5-HT) increased the expression of RPS3a significantly at 4h after incubating with the ovarian explants. A full-length RPS3a was amplified by the RACE technique and expressed in Escherichia coli BL21 (DE3). Western blot analysis showed that the molecular weight of the recombinant histidine (His)-RPS3a was approximately 33kDa. Its function was tested by incubating undeveloped ovarian explants with His-RPS3a. In vitro assays indicated that the His-RPS3a stimulated the expression of shrimp ovarian peritrophin (SOP) and translational controlled tumor protein (TCTP) genes significantly when compared to the untreated ovarian explants. In addition, the SOP and TCTP genes had been previously reported to be highly expressed during the early stages of ovarian development. In addition, FITC-RPS3a was shown to be imported into the ovarian cells. Therefore, RPS3a may act as a stimulator for the development of shrimp ovaries.

A Homolog of the Cell Apoptosis Susceptibility Gene Involved in Ovary Development of Chinese Shrimp Fenneropenaeus chinensis1

The cell apoptosis susceptibility (CAS) gene is a homolog of the yeast chromosome segregation (CSE1) gene, which functions in cell proliferation and apoptosis. In the present study, a homolog of CAS was cloned from Chinese shrimp Fenneropenaeus chinensis (FcCAS). The full-length FcCAS cDNA is 3534 bp and contains an open reading frame encoding 968 amino acids. The predicted tertiary FcCAS structure is highly similar to that of CSE1 from the yeast Saccharomyces cerevisiae. RT-PCR analysis showed that the FcCAS gene is expressed mainly in testis, ovary, stomach, lymphoid organs, gills, and hemocytes. RNA in situ hybridization showed that FcCAS transcripts were distributed mainly in the cytoplasm of oocytes. Western blot analysis showed that FcCAS could be detected only in testis and ovary, and its expression levels differed at different developmental stages of ovaries. Immunohistochemical analysis showed that FcCAS existed in both the cytoplasm and the nucleus, which suggested that FcCAS might function as a nuclear protein. No transcript was detected in the abnormally developed ovaries of triploid shrimp. Therefore, we inferred that the FcCAS gene might be one of the key genes that is closely related to ovary development in shrimp.

Molecular mechanism of serotonin via methyl farnesoate in ovarian development of white shrimp: Fenneropenaeus merguiensis de Man

Ovarian maturation in shrimp broodstock is a complex process controlled by several factors. There is evidence that 5-HT (5-Hydroxytryptamine) can stimulate maturation. The mechanism of this effect was investigated by both in vitro and in vivo assays. Ovarian explants of Fenneropenaeus merguiensis de Man incubated with 5-HT had a significantly higher expression of the RPL10a (ribosomal protein L10a), SOP (shrimp ovarian peritrophin) and TCTP (translational controlled tumor protein) genes up to 4 h after being incubated in the presence of 1 μg/mL 5-HT, while there was no upregulation of the expression of these genes in 5-HT-treated muscle explants at any point of their incubation. The results of an in vivo assay indicated that 5-HT injection stimulated ovarian development to stage 3 (mature) of vitellogenesis. The transcripts of the RPL10a, SOP and TCTP in the ovaries at days 5 and 10 were significantly higher in the shrimp injected with 1 μg 5-HT/g B.W. compared to their controls. However, the RPL10a transcript was the only one that had a statistically significant increase from day 5 to day 10 ( P ≤ 0.05) after injection with 50 μg 5-HT/g B.W. 5-HT at 1 μg/g B.W. induced the presence of MF (methyl farnesoate) in the hemolymph and this increased to its highest level (1.1 ± 0.05 ng/mL) at day 5, but there was no significant increase observed in shrimp that received 50 μg/g B.W. of 5-HT at day 5. Although, ovarian development reached a mature stage of 29% after injection of both levels of 5-HT but the ovaries of shrimp injected with 50 μg 5-HT/g B.W. were observed to have 14% undeveloped ovaries. However, no undeveloped ovaries were detected in the group injected with 1 μg 5-HT/g B.W. Moreover, second stage ovaries were found at 29% and 14% in shrimp injected with 1 μg 5-HT/g B.W. and 50 μg 5-HT/g B.W., respectively. It was also shown that injection of 5-HT released MF into the hemolymph, and MF has been reported to be involved in the ovarian development in shrimp. There was a significant correlation observed between the ovarian development stimulated by 5-HT, the upregulation of the RPL10a, SOP and TCTP genes and the hemolymph MF level.

An improved method of cell culture system from eye stalk, hepatopancreas, muscle, ovary, and hemocytes of Penaeus vannamei

Improved methods of cell culture from eye stalk, hepatopancreas, muscle, ovary, and hemocytes of shrimp (Penaeus vannamei) were established using synthetic media and shrimp muscle extract (SME). For hemocytes and ovarian cell cultures, Grace's insect medium supplemented with 10% (v/v) fetal bovine serum and 10% SME (v/v) showed enhanced attachment and proliferation of the cells. The hemocyte and ovarian cell cultures could be maintained for 48 and 66 days, respectively, and have been sub-cultured four and six times, respectively. Both ovary and hemocyte cell cultures contained primarily epithelial-like cells. Cells derived from ovary tissue grew preferably between 26°C and 28°C with 5% CO(2). Although the temperature preference of hemocyte cells was the same as ovarian cells, CO(2) supplementation did not show any difference in the growth of hemocyte cells. When the shrimp were injected with lipopolysaccharide (8 μg/g of shrimp) and hemolymph was drawn 24 h post-injection, the in vitro multiplicity of hemocytes dramatically improved. The growth of eye stalk, hepatopancreas, and muscle-derived cells was much less compared to ovarian cells and hemocytes under the conditions described above. The optimal culture conditions for ovarian cells and hemocytes were also different from that for eye stalk, hepatopancreas, and muscle cell culture. The proliferation efficiencies of primary cultures of hepatopancreas, eyestalk, and muscle cells were about 30, 12, and <7 d, respectively. The improved culture conditions described here, particularly for hemocytes and ovary, will be very useful for in vitro studies involving viruses infecting shrimp and in shrimp genomic studies.