Oriental River Prawn Research Articles

Accumulation of toxic elements in an invasive crayfish species (Procambarus clarkii) and its health risk assessment to humans

The accumulation and potential health risks of eight trace elements (Cu, Cd, Cr, Mn, Zn, Pb, Hg and As) were analyzed in the commercially important crayfish and oriental river prawn species from the Zhenjiang City, Jiangsu Province, China. The accumulation sequence of elements in different tissues were as gill > walking leg > muscle. Among the analyzed elements, majority of the elements were accumulated in the gill tissue, while Hg was concentrated in abdominal muscle and Zn in walking leg tissues. The elements Zn (95.9 μg g−1) and Cd (0.02 μg g−1) were the most and the least accumulated elements in the abdominal muscle tissue. The calculated estimated daily intake (EDI) and hazard indices (HI) of the analyzed elements in the tissue organs of Procambarus clarkii and Macrobrachium nipponense species were lower than the Chinese Food Health Criterion and international guideline values and results in no acute toxicities and thus, safe for human consumption. This study also suggests that Procambarus clarkii species can be used as an effective bio-indicator organism for examining the toxic heavy metals in the freshwater ecosystems.

Recombinant expression of an oriental river prawn anti-lipopolysaccharide factor gene in Pichia pastoris and its characteristic analysis

Anti-lipopolysaccharide factors (ALFs), as main host-defense molecules of crustaceans, form a unique family of antimicrobial peptides (AMPs). MnALF4 is one isoform of ALFs isolated from the freshwater prawn Macrobrachium nipponense. In the present study, MnALF4 gene was successfully expressed in the yeast Pichia pastoris and the recombinant MnALF4 protein exhibited efficient and broad-spectrum antimicrobial activities against both Gram-positive bacteria and Gram-negative bacteria in vitro. When prawns were injected with rMnALF4 before bacterial challenge with E. coli, the recombinant protein effectively promote the elimination of bacteria by the host. It manifested that rMnALF4 could effectively kill the invading bacteria in vivo. Treatment with rMnALF4 led to remarkable changes in bacterial morphology, such as spheroidization, oversized bacteria, and cell lysis. In addition, rMnALF4 showed weak hemolysis activity to the rabbit red blood cells. Our work suggests that MnALF4 plays an important role in Macrobrachium immunity and is worthy of further investigation as a potential antibacterial agent with high efficacy against bacterial infection and low toxicity to host cells.

Population structure and growth characteristics of oriental river prawn, Macrobrachium nipponense (De Haan, 1849) in Soon Lun Reservoir, Mandalay Region, Myanmar

Investigation on the population structure and growth characteristics of freshwater oriental river prawn, Macrobrachium nipponense was conducted in Soon Lun Reservoir of Myingyan Township, Mandalay Region, Myanmar from June 2009 to May 2010. This study was aimed to investigate size frequency distribution, length-weight relationship and condition factor from study area. A total of 2207 specimens including 955 (56.73%) males and 1252 (43.27%) females were collected. It was observed that the numbers of females were significantly different (p< 0.05) from males. The differences in size group between sexes indicated a sexual dimorphism in M. nipponense. The medium size class of male total length (TL) was 51-80 mm and female TL was 41-65 mm predominated throughout the study period. The correlation coefficient of male (r = 0.947), non berried female (r = 0.801) and berried female (r = 0.912) were strongly positive related (p<0.01). The value of coefficient (b) was 3.098 for male and 3.069 for non berried female indicate isometric growth and 3.424 for berried females show positive allometric growth. The mean monthly condition factor (K) ranged from 0.95 ± 0.11 – 1.49 ± 0.36 for males, from 0.99 ± 0.12 – 2.29 ± 0.27 for non berried females and 1.02 ± 0.11 – 1.81 ± 0.03 for berried females. The fluctuation was observed in the mean monthly condition factor (K) for both males and females and better condition factors of M. nipponense were found in cold season for both sexes.

Identification of neuropeptides from eyestalk transcriptome profiling analysis of female oriental river prawn (Macrobrachium nipponense) under hypoxia and reoxygenation conditions.

The oriental river prawn, Macrobrachium nipponense, is a commercial freshwater prawn species in China. It is highly sensitive to hypoxia, and this has posed a challenge to its intensive culturing. To date, the effects of hypoxia on reproduction in female prawns are not entirely clear, as are the underlying mechanisms of the effects of hypoxia. In this work, comparative transcriptome and gene expression analyses of the eyestalk were performed in M. nipponense females under hypoxia and reoxygenation conditions. Sequencing and de novo assembly of the combined reads yielded 43,583 unigenes with an average length of 1726bp. A total of 711 genes were found to be differentially expressed in the eyestalk under the hypoxia and reoxygenation conditions. With the help of functional and pathway enrichment analysis of the differentially expressed genes, a novel set of transcripts that were associated with several important functions, such as hormone biosynthesis and progesterone-mediated oocyte maturation, were identified. Additionally, ten neuropeptides were identified based on the differentially expressed transcripts, and they were validated by quantitative real-time polymerase chain reaction (qRT-PCR) and reverse transcription PCR (RT-PCR) analyses. Three neuropeptide genes were expressed in the neural tissue and ovary of the prawns; this indicates that they were involved in reproductive processes. In particular, RNA interference (RNAi) short neuropeptide F dramatically promoted ovary maturation, as indicated by the gonad somatic index. While the present findings do indicate that hypoxia affects reproductive function in M. nipponense females, in-depth functional analyses of the candidate neuropeptides should be conducted in the future to understand their role in hypoxia adaptation and the associated mechanisms that affect the reproductive capacity of this species.

Application of magnetic resonance imaging to study lipid deposition patterns in three commercial prawn species

The lipid deposition pattern in economical crustaceans determines their flavor and commercial value. In this study, magnetic resonance imaging (MRI) was used to compare the lipid deposition patterns of three prawn species: red swamp crawfish (Procambarus clarkii), oriental river prawn (Macrobrachium nipponense), and whiteleg shrimp (Litopenaeus vannamei). To our knowledge, this is the first report of the determination of the total volume of lipid tissues in these prawn species by using three-dimensional (3D) images. The MRI results showed that the lipids were deposited as only visceral adipose tissue; the lipids were specifically deposited in the hepatopancreas and seldom in the gill and muscle tissues. The volume of lipid tissues was consistent with that measured after traditional dissection. The chemical analysis indicated that the lipid content of the hepatopancreas, muscle, and whole body of the red swamp crayfish was significantly higher than that of the oriental river prawn and whiteleg shrimp. We found that gonadal analysis with MRI is a non-destructive method to evaluate the plumpness of the tissues in live female red swamp crawfish, and the results were consistent with those of the histological analysis. The male whiteleg shrimps were starved, and MRI of the mesenteric plumpness of the hepatopancreas and biochemical analysis successfully detected changes in lipid metabolism in response to starvation. Thus, MRI can be used to study the plumpness of the hepatopancreas and gonadal tissues and physiological state of crustaceans and help us to understand the lipid deposition patterns in intact and live crustaceans.

A 28.6-kD small heat shock protein (MnHSP28.6) protects Macrobrachium nipponense against heavy metal toxicity and oxidative stress by virtue of its anti-aggregation activity

Small heat shock proteins (sHSPs) are ATP-independent chaperones and involved into various physiological and stress processes. In the present study, a 28.6-kD sHSP coding gene, MnHSP28.6, was cloned and characterized from the oriental river prawn Macrobrachium nipponense. Tissue distribution analysis via qPCR and western blot revealed that MnHSP28.6 predominantly expressed in muscle. The temporal transcription of MnHSP28.6 in muscle after bacterial challenge, heavy metal exposure and doxorubicin (DOX) injection was investigated by qPCR. The results showed that the expression of MnHSP28.6 were strongly enhanced by both Cd2+ and Cu2+ exposure, as well as DOX injection, but not by bacterial infection. Aggregation assays showed that recombinant MnHSP28.6 could effectively prevent temperature-induced aggregation of citrate synthase, and reduction-induced aggregation of insulin in vitro. MnHSP28.6 also could protect muscle extracts from heat-induced protein denaturation and superoxide dismutase (SOD) inactivation. Expressing MnHSP28.6 in E. coli conferred host cell impressive protection against H2O2 compared to control. These results suggest a protective role of MnHSP28.6 in maintaining protein homeostasis, preventing aggregation, promoting resistance to heavy metal and keeping redox balance.

Dietary manganese requirement and its effects on antioxidant enzyme activities, intestinal morphology and microbiota in oriental river prawn Macrobrachium nipponense (De Haan)

Manganese (Mn) is an essential micronutrient for animals. An 8-week feeding experiment with oriental river prawn Macrobrachium nipponense was conducted to determine the effects of Mn supplementation on growth, antioxidative indices, intestinal morphology, and gut microbiota in juveniles of this species. Manganese sulfate monohydrate was added to a basal semi-purified diet at six levels: 5.4 (control), 13, 22, 34, 73 and 150 mg Mn kg−1 diet. Each diet was fed to juvenile prawns (mean weight 0.104 ± 0.003 g), twice daily to apparent satiation, in four replicates. Prawns fed the 22 mg Mn kg−1 diet achieved the highest weight gain. The Mn concentration in muscle significantly increased with increased dietary Mn supplementation, from 5.4 to 22 mg kg−1 diet, but leveled off in animals fed greater levels. Prawns fed the 34 mg Mn kg−1 diet had the lowest malondialdehyde content. Total superoxide dismutase activity in the hepatopancreas increased significantly with an increasing level of dietary Mn in the groups fed the 5.4 (control) to 73 mg kg−1 diets, but plateaued with higher levels of supplementation. Prawns fed the 13–150 mg Mn kg−1 diets had significantly higher Mn superoxide dismutase activity than those fed the 5.4 mg kg−1 diet. Epithelial cells of the intestine were closely arranged, with clear cell definition, in the groups fed the 5.4 (control) or 22 mg Mn kg−1 diet, whereas these cells were completely separated from the basement membrane in prawns fed the 150 mg Mn kg−1 diet. The estimator of community richness (Chao1) in the intestine was significantly higher for prawns fed the 22 mg Mn kg−1 diet as compared with animals fed the 5.4 (control) and 150 mg Mn kg−1 diets. The dominant phyla in the gut of M. nipponense were Proteobacteria, followed by Firmicutes and Bacteroidetes, regardless of the level of Mn supplementation. The intestinal microbiota phylotypes significantly differed among the groups fed the 5.4 (control), 22 and 150 mg Mn kg−1 diets. We conclude that Mn supplementation in formulated feeds for this species can improve growth performance and Mn deposition in muscle, whereas diets causing Mn deficiency or excess will induce oxidant stress, and very high levels of dietary Mn could damage the intestinal morphology. The intestinal core microbiota were not affected by the dietary Mn level, although the microbiota richness was somewhat affected. Based on weight gain, the optimal level of Mn supplementation for juvenile M. nipponense is 17.59 mg kg−1 diet.

Organophosphorus flame retardants in a typical freshwater food web: Bioaccumulation factors, tissue distribution, and trophic transfer

Water, sediment, and wild aquatic species were collected from an electronic waste (e-waste) polluted pond in South China. This study aimed to investigate the bioaccumulation, tissue distribution, and trophic transfer of organophosphorus flame retardants (PFRs) in these aquatic organisms. The concentrations of PFRs detected in the analyzed organisms were between 1.7 and 47 ng/g wet weight (ww). Oriental river prawn and snakehead exhibited the highest and lowest levels, respectively. Tri-n-butyl phosphate (TnBP), tris(2-chloroethyl) phosphate (TCEP), tris(2-chloroisopropyl) phosphate (TCPP) and triphenyl phosphate (TPhP) were dominant contaminants, accounting for approximately 86% of the total sum. The mean values of bioaccumulation factors (BCFs) and logarithmic biota-sediment accumulation factors (log BSAFs) for individual PFRs varied from 6.6 to 1109 and from −2.0 to 0.41, respectively. Both log BCFs and log BSAFs of PFRs were significantly and positively correlated with their octanol-water partitioning coefficient (log KOW). The concentrations of PFRs in tissues of large mud carp and snakehead were significantly and positively correlated with the lipid content (each p < 0.05) and the liver, kidney, and gill exhibited high PFR levels. When the concentration was expressed on a lipid basis, liver exhibited the lowest level, indicating the probable effects of metabolism. Significantly positive correlation was also found between lipid content and total PFR concentration in muscle of all aquatic organisms, given the strong correlation between lipid content and the concentration of TnBP. Trophic magnification factors (TMF) of TnBP and TPhP were lower than 1 (0.57 and 0.62), indicating that these PFRs undergo trophic dilution in this aquatic food web.

Identification of potentially novel functions of DNA polymerase zeta catalytic subunit in oriental river prawn, Macrobrachium nipoponense: cloning, qPCR, in situ hybridization and RNAi analysis.

The goal of this study was to analyze the functions of DNA polymerase zeta catalytic subunit (Rev3) in the oriental river prawn Macrobrachium nipponense (Mn-Rev3) with a focus on its potential roles in sex differentiation and development. The full length of Mn-Rev3 cDNA sequence was 6832 base pairs (bp) with an open reading frame of 6102bp encoding 2033 amino acids. Mn-Rev3 showed the closest evolutionary relationship with Penaeus vannamei. The highest expression level of Mn-Rev3 occurred in the hepatopancreas and strong signals were observed in hepatopancreas cells, suggesting that Mn-Rev3 played a role in the immune system. Expression levels of Mn-Rev3 also were relatively high in the androgenic gland and testis, suggesting its potential roles in male sexual differentiation and development. During development, expression of Mn-Rev3 was highest on larval day 15 and relatively high from post-larval day 1 (PL1) to PL15, indicating that it played essential roles in promoting metamorphosis and gonad differentiation and development in M. nipponense. Strong Mn-Rev3 signals were detected in spermatids, spermatocytes, and sperm in the testes, and Mn-Rev3 expression was higher in the testes during the reproductive season than in the non-reproductive season. This result indicated that Rev3 promoted whole testis development, and especially sperm development, in M. nipponense. The expression level of Mn-Rev3 was high from ovary V to ovary II stages, indicating that Rev3 may be involved in yolk deposition. The expression level of Mn-insulin-like androgenic gland hormone (Mn-IAG) and the content of testosterone showed the same expression pattern as that of Mn-Rev3 after injection of double-stranded RNA of Mn-Rev3, which indicated that Rev3 had positive effects on male sexual differentiation and development in M. nipponense. The results of this study advance our understanding of male sexual development in M. nipponense and provide the basis for further studies of male sexual differentiation and development in crustaceans.

Molecular insights into ovary degeneration induced by environmental factors in female oriental river prawns Macrobrachium nipponense

The oriental river prawn, Macrobrachium nipponense, is an important breeding species in China. The ovary development of this prawn is regulated by the genetic factors and external environmental factors and has obvious seasonal regularity. However, the molecular mechanism of regulating ovary degradation in M. nipponense remains unclear. To address this issue, we performed transcriptome sequencing and gene expression analyses of eyestalks, cerebral ganglia (CG) and thoracic ganglia (TG) of female M. nipponense between the full ovary stage and degenerate ovary stage. Differentially expressed genes enrichment analysis results identified several important pathways such as “phototransduction-fly,” “circadian rhythm-fly” and “steroid hormone biosynthesis secretion.” In the period of ovarian degeneration, the expressions of Tim, Per2 and red pigment concentration hormone (RPCH) were significantly decreased in the eyestalk, CG and TG. And expression of 7 genes in the steroid synthesis pathway, including steryl-sulfatase, cytochrome P450 family 1 subfamily A polypeptide 1, estradiol 17β-dehydrogenase 2, glucuronosyltransferase, 3-oxo-5-alpha-steroid 4-dehydrogenase 1, estradiol 17-dehydrogenase 1 and estrone sulfotransferase was significantly decreased in the CG. Food and light signals affect the expression of clock genes and thereby decrease the expression of RPCH and the estradiol synthesis-related genes in the nervous system, which may be the main cause of ovarian degeneration in M. nipponense. The results will contribute to a better understanding of the molecular mechanisms of ovarian development regulation in crustaceans.

Transcriptome analysis of oriental river Prawn(Macrobrachium nipponense)Hepatopancreas in response to ammonia exposure

The oriental river prawn, Macrobrachium nipponense, is an economically and nutritionally important species of the Palaemonidae family of decapod crustaceans. Ammonia is a major aquatic environmental pollutant that negatively affects the health of prawns and their associated commercial productivity. Here, we used high-throughput sequencing techniques for detecting the effects of ammonia stress (22.1 mg/L ammonia-N for 48 h) on gene expression in the hepatopancreas of M. nipponense. We generated 176,228,782 high-quality reads after eliminating adapter sequences and filtering out low-quality reads, which were assembled into 63453 unigenes. Comparative analysis of the expression profiles of the ammonia-treated and control groups identified 887 differentially expressed genes (P < 0.05), including 481 upregulated genes and 406 downregulated genes. Analyses of the GO and KEGG databases revealed significant differences between the two groups in 32 pathways. Immune-related pathways under ammonia stress included Complement and coagulation cascades, Platelet activation, B cell receptor signaling pathway, Antigen processing and presentation, Chemokine signaling pathway, NOD-like receptor signaling pathway, RIG-I-like receptor signaling pathway, T cell receptor signaling pathway and Toll-like receptor signaling pathway. Remarkably, ammonia stress altered the expression patterns of key immune genes (lectin3, syntenin, alpha-2-macroglobulin, cathepsin L, PIM3, serine protease inhibitor, suppressor of cytokine signaling-2 like protein), indicating that ammonia-stress induce immune response. These data provide new insights into the immune response of M. nipponense and pave a new way for fighting ammonia stress. The genes and pathways identified here represent valuable genetic resources for development of molecular markers and genetic breeding studies.

Types of carbohydrate in feed affect the growth performance, antioxidant capacity, immunity, and activity of digestive and carbohydrate metabolism enzymes in juvenile Macrobrachium nipponense

The present study evaluated the effect of different sources of dietary carbohydrate on the growth, immunity, antioxidant capacity, and digestive enzyme and carbohydrate metabolism enzyme activities in the juvenile oriental river prawn, Macrobrachium nipponense. The experimental diets, which comprised raw maize starch, pregelatinized maize starch, dextrin, maltose, glucose or cellulose, were fed to juvenile prawns for 56 days, respectively. We found that prawns fed with starch, dextrin or maltose achieved better growth. The highest digestive enzyme activities were noted in prawns fed with starch or dextrin. Furthermore, the prawns fed with starch, dextrin or maltose exhibited significantly higher levels of hepatopancreas hexokinase (HK) and phosphofructokinase (PFK) activities. Phosphoenolpyruvate carboxykinase (PEPCK) activity was higher in prawns fed with dextrin, maltose or cellulose, and higher glucose-6-phosphate dehydrogenase (G6PDH) activities were observed in prawns fed with starch or dextrin. We further evaluated the effect of different dietary carbohydrate sources on antioxidant capacity and immune competence. Prawns supplied with starch exhibited superior activities of superoxide dismutase (SOD), catalase (CAT), total antioxidant capacity (T-AOC), lysozyme and respiration burst, and had lower level of maleic dialdehyde (MDA). In summary, the dietary addition of starch is a promising option for juvenile prawn farming, given its positive effect on the growth, glycolysis, pentose phosphate pathway, antioxidant activity and immunity of prawns.

A toll receptor is involved in antibacterial defense in the oriental river prawn, Macrobrachium nipponense

Toll-like receptors (TLRs) play an important role in the activation of innate immune response in animals. In this study, we identified a TLR from the oriental river prawn, Macrobrachium nipponense (MnToll1) and investigated its functions in immunity. The MnToll1 protein shares similar structural characteristics with other known Toll family proteins. MnToll1 transcripts are broadly distributed in all of the examined tissues, and its expression level was significantly up-regulated by bacterial challenge. RNAi-mediated knockdown of MnToll1 significantly impaired the survivability of Vibrio-challenged prawns. RNAi experiments also revealed that the expression of several antimicrobial peptide genes were regulated by MnToll1. Moreover, we found the extracellular region of MnToll1 could directly bind to bacteria and bacterial glycoconjugates. These findings suggest that MnToll1 function as a pattern recognition receptor to recognize invading pathogen and initiate downstream gene expression, to participate in antibacterial defense of M. nipponense.

Effects of feed input and planting of submerged aquatic vegetation on methanotrophic communities in the surface sediments of aquaculture ponds

Methanotrophs represent the only biological sink for the greenhouse gas methane, and only few studies have focused on methanotrophic communities in aquaculture ponds. Illumina high-throughput sequencing and real-time quantitative PCR were employed to profile the methanotrophic bacterial community and investigate the pmoA gene abundance in surface sediments from crab, oriental river prawn, perch, and Wuchang fish aquaculture ponds. Results revealed that Gammaproteobacteria (mainly the environmental_samples_norank genera) and Alphaproteobacteria (mainly the Methylocystis genera) were the most and second most abundant methanotrophic bacterial classes, respectively. While the species cultivated in these ponds were significantly different, the planting of submerged vegetation and the feed input were the main factors found to shape the methanotrophic bacterial community. Submerged vegetation potentially influenced methanotrophs in two ways: by increasing the alpha diversity of the methanotrophic community or by increasing methanotrophic abundance through inducing anaerobic root zone conditions, thereby increasing methane oxidation potential. The amount of feed input also influenced the methanotrophic communities in the fishponds (especially Wuchang fishponds), possibly through deposition of nitrogen, phosphorous, and organic matter (especially organic matter). These results provide preliminary biological data for the regulation of methanotrophs in aquaculture ponds in the future.

Transcriptional responses to starvation stress in the hepatopancreas of oriental river prawn Macrobrachium nipponense

Various crustaceans are farmed using aquaculture, and food deprivation or fasting can occur due to changing of environmental or management strategies. However, the molecular mechanisms underlying responses to starvation in crustaceans remain unclear. To address this, 12 hepatopancreas transcriptomes were compared for oriental river prawn (Macrobrachium nipponense) from four fasting stages (0, 7, 14 and 21 d). Gene Ontology functional annotation and Kyoto Encyclopaedia of Genes and Genomes pathway enrichment analysis of differentially expressed genes were subsequently performed. During the early stages of starvation (0–7 d), drug metabolism via the cytochrome P450 pathway and metabolism of xenobiotics by the cytochrome P450 pathway were enriched, suggesting that they metabolised compounds generated under starvation stress. As starvation proceeded (7–14 d), the retinol (vitamin A) metabolism pathway was activated, based on three up-regulated genes (CYP3, ADH and UGT), along with the two p450 pathways. Meanwhile, vitamin A was gradually consumed. As acute starvation was reached (14–21 d), vitamin A deficiency decreased the mRNA expression levels of IGF-I that is involved in the mTOR signalling pathway, which ultimately affected the growth and development of M. nipponense. Our results implicate drug/xenobiotic metabolism by cytochrome P450s in adaptation to starvation stress. Furthermore, metabolic cascades (CYP and retinol pathways) and growth (mTOR signalling) pathways are clearly triggered in crustaceans during starvation. The findings expand our understanding of the genes associated with hepatopancreas functioning in M. nipponense, and the underlying molecular mechanisms that govern the responses of crustaceans to starvation stress.

2-Transmembrane C-type lectin from oriental river prawn Macrobrachium nipponense participates in antibacterial immune response

As a type of pattern-recognition proteins (PRRs), C-type lectins (CTLs) perform important functions in non-self recognition and clearance of pathogens in innate immunity. In this study, a unique 2-transmembrane CTL (designated as Mn-2TM-cLec) with a single carbohydrate recognition domain (CRD) was isolated from Macrobrachium nipponense. The full-length cDNA of Mn-2TM-cLec consisted of 3265 bp with an 837 bp open reading frame encoding a protein with 278 amino acids. Mn-2TM-cLec was ubiquitously distributed in various tissues of normal prawn, particularly in the hemocytes, hepatopancreas, and gills. The expression of Mn-2TM-cLec was significantly up-regulated in the gills and hepatopancreas after the prawns were challenged with Staphylococcus aureus and Vibrio parahaemolyticus. RNA interference knock-down of Mn-2TM-cLec gene decreased the transcription levels of three antimicrobial peptides (anti-lipopolysaccharide factor (ALF) 1, ALF2, and Crustin (Crus) 1) after V. parahaemolyticus infection. The recombinant CRD of Mn-2TM-cLec could bind lipopolysaccharide, peptidoglycans, and diverse bacterial strains and agglutinate S. aureus and V. parahaemolyticus in a Ca2+-dependent manner. In addition, the rCRD enhanced the clearance of V. parahaemolyticus injected in prawns. In summary, Mn-2TM-cLec might act as a PRR to participate in the prawn immune defense against pathogens through its antimicrobial activity.

Molecular and functional analysis of the insulin-like peptides gene in the oriental river prawn Macrobrachium nipponense

The insulin-like peptide (ILP) family is a group of evolutionarily conserved proteins that control body size and organ growth in metazoans. In the current study we describe, for the first time, the Mn-ILP gene in the oriental river prawn Macrobrachium nipponense. Full-length of the Mn-ILP cDNA was 1630 bp, encoding 174 amino acids. The deduced amino acid sequence of Mn-ILP had the typical features of ILP proteins, including two cleavage sites and six conserved cysteines. To define the function of Mn-ILP, the expression ofthe Mn-ILP gene in different growth stages of prawns of both sexes, in male prawns of different sizes, and in prawns at different stages of the molt cycle was analyzed by qRT-PCR. Mn-ILP expression was significantly higher 1) in the rapid growth stage than in the other stages of male prawns; 2) in the normal growth stage than in the gonad development stage of female prawns; 3) in big male prawns than in small male prawns; and 4) in the intermolt stage than in the other stages of the molt cycle in prawns of the same size. Further, silencing Mn-ILP expression by RNAi effectively slowed down the growth speed of M. nipponense. Thus, Mn-ILP appears to have an important role in the growth and development process of M. nipponense.

Effect of dietary vitamin E on growth, immunity and regulation of hepatopancreas nutrition in male oriental river prawn, Macrobrachium nipponense

We evaluated the effects of dietary vitamin E on growth, immunity and regulation of the hepatopancreas in male oriental river prawn, Macrobrachium nipponense. Shrimps were fed 0, 40, 80, 160, 320 or 640 mg vitamin E/kg for 60 days. The 80 mg/kg group had the highest weight gain rate, specific growth rate and lowest feed conversion rate while there were no significant differences in survival rate and hepatosomatic index. The highest crude fat and lowest crude protein content were observed in the 160 mg/kg group. In the hepatopancreas, lysozyme, acid phosphatase and alkaline phosphatase activities were highest in the 160 mg/kg group, while superoxide dismutase, catalase and glutathione peroxidase activities decreased with increasing vitamin E levels. Malondialdehyde content initially decreased then increased with vitamin E levels, whereas the reverse was seen with total antioxidant capacity. Linoleic acid, DPA, DHA, total n-3 polyunsaturated fatty acid and total polyunsaturated fatty acid first increased then decreased, while EPA and total saturated fatty acid rose with vitamin E levels. Total n-6 polyunsaturated fatty acid content declined while there were no significant differences in linolenic and total monounsaturated fatty acid content. Following a toxicity test with Aeromonas hydrophila, hepatopancreas ultrastructure revealed that appropriate vitamin E levels promote an increase in mitochondria, endoplasmic reticulum and Golgi bodies, but excess vitamin E can damage cell structure. These results provide evidence that 80–160 mg/kg dietary vitamin E has a positive impact on growth, immunity and regulation of the hepatopancreas in male shrimp.

Identification of multiple ferritin genes in Macrobrachium nipponense and their involvement in redox homeostasis and innate immunity

Based on the transcriptome database, we screened out four ferritin subunit genes (MnFer2-5) from the oriental river prawn Macrobrachium nipponense, which encode two non-secretory and two secretory peptides. MnFer2 and 4 possess a strictly conserved ferroxidase site, and MnFer3 has a non-typical ferroxidase site. MnFer5 seems to be a number of ferritin families, which has a distinct dinuclear metal binding motif, but lacks an iron ion channel, a ferroxidase site and a nucleation site. Diverse tissue-specific transcriptions of the four genes indicate their functional diversity in the prawn. Among them, MnFer2 is mainly expressed in hepatopancreas and intestines, MnFer3 and 4 are predominantly expressed in gills, and MnFer5 is widely expressed in various tissues with high presence in intestines, hepatopancreas and haemocytes. The transcription of all the four MnFer genes can be strongly induced by doxorubicin, indicating the involvement of these ferritin subunits in protection from oxidative stress. Upon Aeromonas hydrophila infection, only MnFer5 is persistently up-regulated, while other subunits including MnFer2-4 are down-regulated during the early stage, followed by recovery and even a slight increase at 48 h post bacterial challenge. Moreover, the iron binding capacity of recombinant MnFer2 is also demonstrated in vitro. The E. coli expressing MnFer2 displays increased resistance to hydrogen peroxidase cytotoxicity. These results suggest a protective role of ferritins from M. nipponense in iron homeostasis, redox biology and antibacterial immunity and shed light on the molecule evolution of crustacean ferritin subunits.

Histological analysis of an outbreak of red gill disease in cultured oriental river prawn Macrobrachium nipponense

An epidemic of red gill disease with a high mortality rate recently occurred in the cultured oriental river prawn Macrobrachium nipponense in China. The prawns had flaccid bodies and gills were red in color with numerous black spots. By wet smear microscopy and negative staining, numerous membrane-bound plasmodia or degenerated spores were observed. Histological sections indicated the infection was limited to the gills. The gill filaments were filled with masses of mononuclear parasites, multinucleate and multicellular plasmodia and became enormously hypertrophic. Host encapsulation response and inflammatory foci with phagocytized parasite spores were evident. Transmission electron microscopy (TEM) analysis confirmed the gross plasmodia almost filling the entire space of gill epithelial cell. The intracellular parasite was a multinucleate plasmodium enclosed in a vacuole within the cytoplasm of the host cell. Large numbers of trophonts were visible in each plasmodium. High degree of vesiculation of early sporonts was evident. Scanning electron microscopy (SEM) and TEM observations all revealed characteristic tubular extension from the surface of a late sporont. We recognized the risk of associating the presence of a parasite with a particular pathology in the absence of total evidence that demonstrates an unequivocal link between the parasite, the perceived pathology and a clear disease manifestation that would be demonstrated by e.g. experimental studies. However, given the widespread importance of the host in aquaculture, it is necessary to provide this short report to highlight to other researchers and to the industry the potential for a new and emerging disease. It is hoped that this will lead to additional sightings of the condition and additional research to confirm the link between parasite and the pathology seen. It is our intention to complete life-cycle studies with to satisfy Koch's postulate.