Prawn Macrobrachium Nipponense Research Articles

Nutrient Compositions and Properties of Antarctic Krill (Euphausia superba) Muscle and Processing By-Products

ABSTRACTThis study investigated the morphometrics, proximate chemical compositions, pH, total amino acid (TAA), fatty acid profile, and minerals of the processing by-products of Antarctic krill (Euphausia superba). The nutrient compositions and properties of the by-products were revealed by being compared to those of Antarctic krill muscle and the economically important species of freshwater prawns (Macrobrachium nipponense) and penaeid shrimps (Metapenaeus ensis). The by-products are worthy of utilization because of the high ratio to the total weight (65.7%). The crude protein contents in the muscle and by-products of krill are 17.4 and 11.7%, respectively. The krill proteins have higher contents of essential amino acids (EAAs). The EAAs constitute 42 and 37% of the TAAs in muscle and by-products, respectively. The krill processing by-products contain high levels of total lipid (3.3%), and polyunsaturated fatty acids constitute 34% of fatty acids with high levels of eicosapentaenoic acid (EPA; 19.08%) and docosahexaenoic acid (DHA; 10.02%). Krill meat provides considerable iron, zinc, calcium, selenium, and copper. It is imperative to lower the fluoride level (70.1 mg/kg, wet basis) in krill muscle. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) reveals that myosin heavy chain (MHC) and actin are the major proteins in muscle, and their contents vary between species.

Identification and characterization of a Macrobrachium nipponense ferritin subunit regulated by iron ion and pathogen challenge

Ferritin, a major iron storage protein in most living organisms, plays a crucial role in iron metabolism. In this study, the ferritin subunit MnFer was identified in the oriental river prawn (Macrobrachium nipponense) and functionally characterized. The full-length cDNA of MnFer is 999 bp in size with a 122-bp 5′-untranslated region (UTR), a 364-bp 3′-UTR and a 513-bp open reading frame that encodes a protein possessing 171 amino acids and a deduced molecular weight of 19.40 kDa. Prawn ferritin transcripts are expressed in muscle, heart, hepatopancreas, gill, hemocytes, ovary and testis. Quantitative real-time PCR revealed that the abundance of ferritin transcript was highest in the hepatopancreas followed by muscle. Ferritin transcript expression in muscle increased six-fold 3 h after the injection of iron. In the gill, a four-fold increase in ferritin transcript expression was detected 3 h post-injection; the expression remained elevated for 48 h. Heart ferritin mRNA expression increased up to seven-fold at 24 h post-injection. No significant difference was found in the hepatopancreas. The iron binding capacity of recombinant ferritin protein was also demonstrated in this study. In hemocyte experiments, the transcriptional expression of MnFer showed the strongest response to Aeromonas hydrophila. As a whole, our study suggested that the ferritin from M. nipponense may play critical roles in cellular and organismic iron homeostasis along with in innate immune defense.

A clip-domain serine proteinase homolog (SPH) in oriental river prawn, Macrobrachium nipponense provides insights into its role in innate immune response

In this study, a clip-domain serine proteinase homolog designated as MnSPH was cloned and characterized from a freshwater prawn Macrobrachium nipponense. The full-length cDNA of MnSPH was 1897 bp and contained a 1701 bp open reading frame (ORF) encoding a protein of 566 amino acids, a 103 bp 5′-untranslated region, and a 93 bp 3′-untranslated region. Sequence comparison showed that the deduced amino acids of MnSPH shared 30–59% identity with sequences reported in other animals. Tissue distribution analysis indicated that the MnSPH transcripts were present in all detected tissues with highest in the hepatopancreas and ovary. The MnSPH mRNA levels in the developing ovary were stable at the initial three developmental stages, then increased gradually from stage IV (later vitellogenesis), and reached a maximum at stage VI (paracmasis). Furthermore, the expression of MnSPH mRNA in hemocytes was significantly up-regulated at 1.5 h, 6 h, 12 h and 48 h post Aeromonas hydrophila injection. The increased phenoloxidase activity also demonstrated a clear time-dependent pattern after A. hydrophila challenge. These results suggest that MnSPH participates in resisting to pathogenic microorganisms and plays a pivotal role in host defense against microbe invasion in M. nipponense.

Molecular cloning and characterization of the lipopolysaccharide and β-1,3-glucan binding protein from oriental river prawn, Macrobrachium nipponense

The lipopolysaccharide and β-1,3-glucan binding protein (LGBP), one of the pattern recognition proteins, plays an important role in the innate immune response of invertebrates. A 1,506 bp full-length cDNA of a LGBP gene was cloned and characterized from the oriental river prawn Macrobrachium nipponense (named as MnLGBP). Analysis of the nucleotide sequence revealed that the cDNA clone has an open reading frame of 1,119 bp, encoding a protein of 372 amino acids including a 21-aa signal peptide. The calculated molecular mass of the mature protein (351 aa) was 39.9 kDa with an estimated pI of 4.63. The MnLGBP sequence contains: (1) two putative integrin-binding motifs, (2) a glucanase motif, (3) two putative N-glycosylation sites, (4) one protein kinase C phosphorylation site, and (5) a putative recognition motif for β-1,3-linkage of polysaccharides. Sequence comparison based on the deduced amino acid sequence of MnLGBP showed varied identity of 89, 76 and 74% with those of Macrobrachium rosenbergii LGBP, Marsupenaeus japonicus β-1,3-glucan binding proteins, and Fenneropenaeus chinensis LGBP, respectively. Quantitative RT-PCR results showed that MnLGBP was expressed in nerve, intestine, muscle, gill, heart, haemocytes and at the highest level in hepatopancreas. After challenge with the pathogen, Aeromonas hydrophila and Vibrio parahaemolyticus, the expression of MnLGBP mRNA was significantly upregulated in the hepatopancreas compared to the control group. At the same time, the mRNA level of MnproPO increased dramatically at 48 h after injection of bacteria. These data should be helpful to better understand the function of MnLGBP in the prawn immune system.

Calcium–calmodulin dependent protein kinase I from Macrobrachium nipponense: cDNA cloning and involvement in molting

Calcium–calmodulin dependent protein kinase I is a component of a calmodulin-dependent protein kinase cascade and involved in many physiological processes. The full-length cDNA of calcium–calmodulin dependent protein kinase I (MnCaMKI) was cloned from the freshwater prawn Macrobrachium nipponense and its expression pattern during the molt cycle and after eyestalk ablation is described. The full-length cDNA of MnCaMKI is 3262bp in length and has an open reading frame (ORF) of 1038bp, encoding a 345 amino acid protein. The expression of MnCaMKI in three examined tissues was upregulated in the premolt stage of the molt cycle. Its expression was induced after eyestalk ablation (ESA): the highest expression level was reached 1day after ESA in hepatopancreas, and 3days after ESA in muscle. By dsRNA-mediated RNA interference assay, expression of MnCaMKI and ecydone receptor gene (MnEcR) was significantly decreased in prawns treated by injection of dsMnCaMKI, while expression of these two genes was also significantly decreased in prawns treated by injection of dsMnEcR, demonstrating a close correlation between the expression of these two genes. These results suggest that CaMKI in M. nipponense is involved in molting.

Isolation and characterization of 27 novel polymorphic microsatellite markers in oriental river prawn Macrobrachium nipponense

We developed 27 polymorphic microsatellite loci by constructing a microsatellite-enriched genomic library. The number of alleles per locus ranged from two to seven, and observed and expected heterozygosities from 0.1905 to 0.8000 and from 0.1765 to 0.8017 respectively. Seven loci (X63, X195, X208, X348, X731, X795, X1214, and XB96) deviated from the Hardy–Weinberg equilibrium in the sampled population after BONFERRONI correction (adjusted P value = 0.003). These microsatellite markers will be useful for the study of population structure, genetic diversity and the construction of the genetic mapping of Macrobrachium nipponense.

Identification of spliced mRNA isoforms of retinoid X receptor (RXR) in the Oriental freshwater prawn Macrobrachium nipponense.

Retinoid X receptors (RXR) are members of the nuclear receptor family that are conserved from invertebrates to vertebrates, and they play an essential role in regulating reproductive maturation, molting, and embryo development. In this study, five RXR isoforms, named RXRL2 (L, long form), RXRL3, RXRS1 (S, short form), RXRS2, and RXRS3, containing six domains from A to F, were cloned from the prawn Macrobrachium nipponense using 5'- and 3'- rapid amplification of cDNA ends. Differences among their structures were observed not only in the D and E domains but also in the A/B domain, which were previously found in insects but not in crustaceans. This is the first report to show that differences occur in the A/B domain of RXR in crustaceans. RXR expressions were also examined in various tissues including the ovary, testis, muscle, hepatopancreas, heart, gill, stomach, intestine, and cuticle. Expression pattern investigations indicated that the five isoforms were differentially expressed. RXRS3 was only detected in the ovary, and the other RXRs were abundant in the ovary and testis. These data suggested that RXR mediates a series of processes related to reproduction.

Artificial infection of the native Korean freshwater prawn Macrobrachium nipponense (De Haan, 1849) (Decapoda, Palaemonidae) with white spot syndrome virus (WSSV)

White spot syndrome virus (WSSV), an important shrimp pathogen that can affect both salt- and freshwater shrimp, has a high mortality rate that can reach up to 100% in 3-10 days. Therefore, we assessed the susceptibility of Macrobrachium nipponense (De Haan, 1849), a freshwater prawn native to Korea, to artificial WSSV infection. Although rearing in water containing WSSV-infected carcasses did not result in infection, M. nipponense was infected with WSSV after being fed a WSSV-containing diet, per os infection, or injection with the purified virus. In the feeding test, the first mortality was observed on the 7th day and cumulative mortality reached 30% by 3 weeks post-infection. After per os infection, experimental animals started dying on the second day and within 3 weeks mortality had reached 90%. In the injection test, the mortality was found to be dose-dependent. Prawn injected with 2.5 × 104, 900 and 80 copies/g body weight exhibited the first mortality at 1, 2 and 4 days post inoculation, with 100, 100 and 50% final mortality at 2, 10 and 12 days post inoculation, respectively. Histological observation revealed inclusion bodies in the gills, antennal glands and other tissues of ectodermal origin. Real-time polymerase chain reaction revealed the increase of viral titers, indicating viral replication in the inoculated host. This forms the first report of the experimental susceptibility of M. nipponense to WSSV.

Oriental River Prawn of the Genus Macrobrachium in the Lower Reach of the Dniester River

Paper deals with findings of the oriental river prawn Macrobrachium nipponense De Haan in the Dniester liman, in the Dniester and Turunchuk rivers and in fish breeding ponds.

Molecular cloning, characterization and expression analysis of the fatty acid-binding protein (MnFABP), involved in dietary lipid sources response in oriental river prawn,Macrobrachium nipponense

A fatty acid-binding protein (FABP) gene designated as MnFABP10 was cloned and characterized from the freshwater prawn Macrobrachium nipponense. The full-length cDNA of MnFABP10 was 646 bp encoding a 130 amino acid. Real-time quantitative RT-PCR showed that the MnFABP10 gene was expressed in various tissues with the highest expression in the hepatopancreas. The MnFABP10 mRNA levels in the hepatopancreas and ovary of M. nipponense were dependent on the stages of ovarian development. Western blot results revealed a single immunoreactive band with an estimated molecular mass of approximate 14 kDa in the developmental ovary. Then, M. nipponense with an initial body weight of 0.090 ± 0.0010 g were fed with four isonitrogenous and isocaloric diets with different oils, that is, beef tallow (BT), soybean oil (SO), pollack fish oil (FO) and a mixture of fish oil and soybean oil (FO/SO 2 : 1 w/w) for 52 days. The mRNA levels of MnFABP10 in the hepatopancreas were influenced by different lipid sources, with a peak expression observed in prawns fed SO. This study suggests that MnFABP10 may have a putative function in ovary maturation, and its mRNA expression in the hepatopancreas can be regulated by the source of dietary lipids in M. nipponense.

Effect of nitrite exposure on metabolic response in the freshwater prawn Macrobrachium nipponense

Abstract The metabolic response of the freshwater prawn, Macrobrachium nipponense to nitrite toxicity was evaluated. The prawns were exposed to 0, 1, 2, 3 and 4 mg L−1 NO2-N concentrations for 48 h. The metabolic parameters in muscle were measured after 12, 24 and 48 h. Glucose level significantly increased after 24 h. Exposure to lower nitrite concentrations (1 and 2 mg L−1) resulted in significant increases in alanine aminotransferase (ALT) activities after 24 and 48 h. Aspartate aminotransferase (AST) activities treated with 2 and 3 mg L−1 nitrite-N at 48 h were significantly higher than those at 12 and 24 h. Intermediate sublethal nitrite concentrations produced significant elevations in lactate dehydrogenase (LDH) activities from 12 h up to 48 h. No significant changes were detected in any of the groups for triglycerides and creatine kinase (CK). To satisfy the increased energy demands caused by acute nitrite exposure, mobilization of lipids is not the main reason while utilization of amino acids seems to play a more important role. The results would be helpful for aquaculture farmers to prevent a potential depression of productivity caused by elevated nitrite levels.

Six chitinases from oriental river prawn Macrobrachium nipponense: cDNA characterization, classification and mRNA expression during post-embryonic development and moulting cycle

Chitinase plays crucial physiological roles in crustaceans, including the digestion of chitin-containing food, moulting and the defense of shrimp against viruses. However, in contrast to insect species, no genome-wide analysis has been carried out in crustacean species and cDNAs encoding chitinase and chitinase-like proteins have been characterized in relatively few species. In this study, we identified six chitinase genes in the oriental river prawn, Macrobrachium nipponense, according to the established expressed sequence tag (EST) information using Rapid Amplification of the cDNA Ends (RACE) technique and homology cloning. We assigned these genes to three different chitinase groupings, which were designated MnCht1A, 1B, 3A, 3B, 3C and 4. The domain organization analysis of the six MnCht proteins revealed that only MnCht3C and MnCht4 possessed full structure, while MnCht1A, 1B, 3A and 3B lacked the serine/threonine (S/T)-rich linker and chitin-binding domains (CBDs). Their expression in different tissues and different developmental stages suggested that all of them have a function in the digestion of chitinous foods, modification of gut peritrophic membrane and degradation of the chitin exoskeleton. Analysis of the stage-specific moulting cycle and different temperature stimulation provided further evidence that MnCht1A, 1B and 3B have pivotal roles in the moulting cycle, while MnCht 4 only assists in the moulting process. This study provides important information for further investigations on the functions of chitinase in M. nipponense and other crustaceans.

Ecdysone receptor gene from the freshwater prawn Macrobrachium nipponense: Identification of different splice variants and sexually dimorphic expression, fluctuation of expression in the molt cycle and effect of eyestalk ablation

The full-length cDNA of an ecdysone receptor gene (MnEcR) from Macrobrachium nipponense was cloned and the expression of the gene was investigated. MnEcR maintained a relatively low expression level in the early stages of embryos, but from nauplius stage, a steady increase in MnEcR expression was detected, it had the highest expression level in zoea stage. MnEcR was highly expressed in the hepatopancreas and gills among ten different tissues examined. MnEcR was rapidly upregulated in the premolt stage and rapidly downregulated in the postmolt stage. The expression of MnEcR was remarkably downregulated after eyestalk ablation in M. nipponense. An 18-amino-acid insertion/deletion and a 49-amino-acid substitution were found in the coding region of MnEcR, resulting in four splice variants: MnEcR-L1, -L2, -S1 and-S2. The expression of four splice variants of MnEcR in gonads was investigated using RT-PCR. Interestingly, the expression patterns of these splice variants differed between males and females. The dominant splice variants in testis were MnEcR-S1 and -S2, while in ovary they were MnEcR-L1 and -S2, indicating specific roles for these splice variants in male and female individuals.

Molecular cloning and expression analysis of two sex-lethal homolog genes during development in the oriental river prawn, Macrobrachium nipponense

In this study, two Sxl gene homologs, designated as Mnsxl1 and Mnsxl2, were cloned and characterized from the freshwater prawn Macrobrachium nipponense by rapid amplification of cDNA ends. The deduced amino acid sequences of Mnsxl1 and Mnsxl2 showed high sequence homology to the insect Sxl and contained conserved domains in two RNA-binding motifs. Real-time quantitative reverse transcription-polymerase chain reaction (RT-QPCR) showed that the Mnsxl1 and Mnsxl2 genes were expressed in all investigated tissues, with the highest level of expression in the intestine and liver. RT-QPCR also revealed that Mnsxl1 and Mnsxl2 mRNAs expressions were both significantly increased at 5 and 20 days post-larvae after metamorphosis. Thus, the results of the present study imply that Mnsxl1 and Mnsxl2 play complex and important roles in the sex differentiation of M. nipponense.

The retinoid-X receptor gene from the freshwater prawn Macrobrachium nipponense: cloning, expression pattern and different responses of two splice variants during the molting cycle

The retinoid-X receptor (RXR) is among the most conserved members of the nuclear receptor superfamily and is widely studied in vertebrate and invertebrate families. RXR plays an important role in regulation of molting and/or metamorphosis, development and reproduction. We cloned the full-length cDNA sequence of the RXR from the freshwater prawn Macrobrachium nipponense (De Haan, 1849) (MnRXR) and investigated the expression profile of MnRXR in different developmental stages of embryos, in different tissues and in the molting cycle. Two MnRXR splice variants were identified: One, MnRXR-L, the full length of which was 2472 bp, encoded a 449-amino-acid protein; the second, MnRXR-S, the full length of which was 1832 bp, encoded a 420-amino-acid protein, in which the first 29 amino-acid residues of MnRXR-L were absent. MnRXR was observed in all developmental stages of embryos and had the highest expression level in the embryonised-zoea stage, it was highly expressed in hepatopancreas, gill and intestine among the ten tissues examined. The expression of MnRXR was rapidly up-regulated in the premolt stage and rapidly down-regulated after molting. Moreover, of the two MnRXR splice variants, only MnRXR-S was induced during the molting cycle, suggesting that the two splice variants play different roles in the molting cycle.

Molecular characterization of cathepsin L cDNA and its expression during oogenesis and embryogenesis in the oriental river prawn Macrobrachium nipponense (Palaemonidae)

We identified the cDNA sequence of cathepsin L (CatL) in Macrobrachium nipponense, designated as MnCatL, for the first time. The MnCatL cDNA, isolated from the ovary, was 1710 bp in length, containing a 31-bp 5'-untranslated region, a 650-bp 3'-untranslated region, and an open reading frame of 1029 bp, encoding a 342-amino acid polypeptide with a predicted molecular mass of 37.7 kDa. The polypeptide is composed of an 18-amino acid signal peptide, a 106-amino acid propeptide and a 218-amino acid mature peptide. MnCatL mRNA was detected in all tissues that we examined, including the thoracic ganglia, heart, muscle, intestine, hemocytes, ovary, testis, gills, and hepatopancreas. MnCatL expression reached a maximum value in both hepatopancreas and ovaries at the later stages of vitellogenesis, suggesting that MnCatL is involved in ovarian maturation of the oriental river prawn. During embryogenesis, MnCatL expression decreased as the embryo developed. The expression of MnCatL in the ovary and embryo suggest that MnCatL plays an important role in the uptake of vitellogenin and yolk protein, which are deposited in the oocyte for ovary maturation and embryo development, during oogenesis and embryogenesis of M. nipponense.

Localization of germline marker vasa homolog RNA to a single blastomere at early cleavage stages in the oriental river prawn Macrobrachium nipponense: Evidence for germ cell specification by preformation

Germ cells are specified by the inheritance of maternal germline determinants (preformation mode) or inductive signals from somatic cells (epigenesis mode) during embryogenesis. However, the germline specification in decapod crustaceans is unclear so far. Using vasa homolog (MnVasa) as a germ cell marker, here we probed the early events of germline specification in the oriental river prawn Macrobrachium nipponense. Quantitative RT-PCR analysis of unfertilized eggs and embryos demonstrated that the prawn MnVasa mRNA is a maternal factor. Whole-mount in situ hybridization further indicated that MnVasa transcripts are maternally supplied to only one blastomere at the very early cleavage stages. As cleavage proceeds, the MnVasa-positive blastomere undergoes proliferation and increases in number. During gastrulation, the MnVasa-positive cells are found to be around a blastopore and could migrate into an embryo through the blastopore. At the zoea stage, clusters of the MnVasa-positive cells distribute not only in the gonad rudiment in the cephalothorax but also at an extragonadic site, dorsal to the posterior hindgut in the abdomen, suggesting that MnVasa-positive cells could migrate anteriorly to the genital rudiment through the hindgut. Based on the dynamic localization and number of MnVasa-positive cells during embryogenesis, we concluded that the MnVasa-positive cells are primordial germ cells (PGC) or founder cells of PGC that are separated from soma at the early cleavage stage. MnVasa mRNA might have a key function in the specification of the prawn germline cells as a maternal determinant. These results provide the first evidence that the germline specification in decapod crustaceans follows a preformation mode.

A novel effect of imidazole derivative KK-42 on increasing survival of Aeromonas hydrophila challenged prawn Macrobrachium nipponense

Imidazole derivative KK-42 is well known as the insect growth regulator. Here we find that KK-42 pretreatment could promote the survival of Macrobrachium nipponense infected with Aeromonas hydrophila, which is considered to be possibly related to the prophenoloxidase (proPO), a conserved copper-containing enzyme that plays an important role in defense against pathogens. In this study, a full-length of proPO gene from M. nipponense haemocytes, designated as MnproPO, was firstly cloned and characterized. The full-length cDNA contained 2428 bp with a 2013 bp open reading frame encoding a putative proPO protein of 671 amino acids with a predicted molecular mass of 76.5 kDa and pI of 7.31. It was predicted to possess all the expected features of proPO members, including two putative copper-binding sites with six histidine residues and a thiol ester-like motif. Sequence analysis showed that MnproPO exhibited the highest amino acid sequence similarity (93%) to a proPO of Macrobrachium rosenbergii. The gene was expressed highly in haemocytes and weakly in hepatopancreas. Real-time PCR analysis revealed that the MnproPO expression increased significantly at 3, 12 and 24 h after KK-42 treatment, the PO activity also importantly rose from 6 to 48 h in KK-42-treated prawns and reached the maximum at 24 h with a 2.3-fold higher than that in control group. Injection of A. hydrophila could stimulate the MnproPO transcription and PO activity whether or not the prawns were pretreated by KK-42, the mRNA level increased obviously only at 3 h and 6 h after the bacterium injection (challenged control), but increased constantly during the phase of experiment except at 6 h under the condition of KK-42 pretreatment (challenged treatment group). The change trend of PO activity was basically similar to that of MnproPO expression. Our present results demonstrate that the MnproPO expression as well as PO activity may be induced by KK-42, which is likely one of the molecular mechanisms of KK-42 acts for increasing survival of the prawn infected with A. hydrophila.

Prevention of Adhesion to Tank Wall above the Water Surface of Larvae of the Freshwater Prawn Macrobrachium nipponense Used Positive Phototaxis

Prevention of Adhesion to Tank Wall above the Water Surface of Larvae of the Freshwater Prawn Macrobrachium nipponense Used Positive Phototaxis

Current status and prospects of farming the giant river prawn (Macrobrachium rosenbergii) and the oriental river prawn (Macrobrachium nipponense) in China

The current status of the farming of Macrobrachium species in China is presented, including the scale of production and with an emphasis on its farming technology. The problems faced and research needed for the further development of freshwater prawn farming of China is also reviewed. Finally, this paper discusses the prospects and future expansion in this sector of national aquaculture.