Detection and phylogenetic profiling of nodavirus associated with white tail disease in Malaysian <Emphasis Type="Italic">Macrobrachium rosenbergii</Emphasis> de Man

White tail disease (WTD) is a serious viral disease in the hatcheries and nursery ponds of Macrobrachium rosenbergii in many parts of the world. A new disease similar to WTD was observed in larvae and post larvae of M. rosenbergii cultured in Malaysia. In the present study, RT-PCR assay was used to detect the causative agents of WTD, M. rosenbergii nodavirus (MrNV) and extra small virus (XSV) using specific primers for MrNV RNA2 and XSV. The results showed the presence of MrNV in the samples with or without signs of WTD. However, XSV was only detected in some of the MrNV-positive samples. Phylogenetic analysis showed that the RNA2 of our Malaysian isolates were significantly different from the other isolates. Histopathological studies revealed myofiber degeneration of the tail muscles and liquefactive myopathy in the infected prawns. This was the first report on the occurrence of MrNV in the Malaysian freshwater prawn.     

Macrobrachium rosenbergii cathepsin L: Molecular characterization and gene expression in response to viral and bacterial infections

Cathepsin L (MrCathL) was identified from a constructed cDNA library of freshwater prawn Macrobrachium rosenbergii. MrCathL full-length cDNA is 1161 base pairs (bp) with an ORF of 1026 bp which encodes a polypeptide of 342 amino acid (aa) long. The eukaryotic cysteine proteases, histidine and asparagine active site residues were identified in the aa sequence of MrCathL at 143–154, 286–296 and 304–323, respectively. The pair wise clustalW analysis of MrCathL showed the highest similarity (97%) with the homologous cathepsin L from Macrobrachium nipponense and the lowest similarity (70%) from human. Phylogenetic analysis revealed two distinct clusters of the invertebrates and vertebrates cathepsin L in the phylogenetic tree. MrCathL and cathepsin L from M. nipponense were clustered together, formed a sister group to cathepsin L of Penaeus monodon, and finally clustered to Lepeophtheirus salmonis. High level of (P < 0.05) MrCathL gene expression was noticed in haemocyte and lowest in eyestalk. Furthermore, the MrCathL gene expression in M. rosenbergii was up-regulated in haemocyte by virus [M. rosenbergii nodovirus (MrNV) and white spot syndrome baculovirus (WSBV)] and bacteria (Vibrio harveyi and Aeromonas hydrophila). The recombinant MrCathL exhibited a wide range of activity in various pH between 3 and 10 and highest at pH 7.5. Cysteine proteinase (stefin A, stefin B and antipain) showed significant influence (100%) on recombinant MrCathL enzyme activity. The relative activity and residual activity of recombinant MrCathL against various metal ions or salts and detergent tested at different concentrations. These results indicated that the metal ions, salts and detergent had an influence on the proteinase activity of recombinant MrCathL. Conclusively, the results of this study imply that MrCathL has high pH stability and is fascinating object for further research on the function of cathepsin L in prawn innate immune system.     

Viral diseases of the giant fresh water prawn Macrobrachium rosenbergii: a review

The giant freshwater prawn Macrobrachium rosenbergii is cultivated essentially in Southern and South-eastern Asian countries such as continental China, India, Thailand and Taiwan. To date, only two viral agents have been reported from this prawn. The first (HPV-type virus) was observed by chance 25 years ago in hypertrophied nuclei of hepatopancreatic epithelial cells and is closely related to members of the Parvoviridae family. The second, a nodavirus named MrNV, is always associated with a non-autonomous satellite-like virus (XSV), and is the origin of so-called white tail disease (WTD) responsible for mass mortalities and important economic losses in hatcheries and farms for over a decade. After isolation and purification of these two particles, they were physico-chemically characterized and their genome sequenced. The MrNV genome is formed with two single linear ss-RNA molecules, 3202 and 1250 nucleotides long, respectively. Each RNA segment contains only one ORF, ORF1 coding for the RNA-dependant RNA polymerase located on the long segment and ORF2 coding for the structural protein CP-43 located on the small one. The XSV genome (linear ss-RNA), 796 nucleotides long, contains a single ORF coding for the XSV coat protein CP-17. The XSV does not contain any RdRp gene and consequently needs the MrNV polymerase to replicate.     

Experimental transmission and tissue tropism of Macrobrachium rosenbergii nodavirus (MrNV) and its associated extra small virus (XSV)

White tail disease (WTD) was found to be a serious problem in hatcheries and nursery ponds of Macrobrachium rosenbergii in India. The causative organisms have been identified as M. rosenbergii nodavirus (MrNV) and its associated extra small virus (XSV). Experimentally transmitted to healthy animals, they caused 100% mortality in post-larvae but failed to cause mortality in adult prawns. The RT-PCR assay revealed the presence of both viruses in moribund post-larvae and in gill tissue, head muscle, stomach, intestine, heart, hemolymph, pleopods, ovaries and tail muscle, but not in eyestalks or the hepatopancreas of experimentally infected adult prawns. The presence of these viruses in ovarian tissue indicates the possibility of vertical transmission. Pleopods have been found to be a suitable organ for detecting these viruses in brooders using the RT-PCR technique.     

Quantitative relationship of two viruses (MrNV and XSV) in white-tail disease of Macrobrachium rosenbergii

Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) were purified from diseased freshwater prawns M. rosenbergii and used to infect healthy post-larvae (PL) by an immersion method. Three groups of prawns were challenged with various combined doses of MrNV and XSV. Signs of white-tail disease (WTD) were observed in Groups 1 and 2, which had been challenged with combinations containing relatively high proportions of MrNV and low proportions of XSV. By contrast there was little sign of WTD in Group 3, which had been challenged with a higher proportion of XSV than MrNV. A 2-step Taqman real-time RT-PCR was developed and applied to quantify viral copy numbers in each challenged PL. Results showed that genomic copies of both viruses were much higher in Groups 1 and 2 than they were in Group 3, indicating that MrNV plays a key role in WTD of M. rosenbergii. The linear correlation between MrNV and XSV genome copies in infected prawns demonstrated that XSV is a satellite virus, dependent on MrNV, but its role in pathogenicity of WTD remains unclear.     

Dietary supplementation of green synthesized manganese-oxide nanoparticles and its effect on growth performance,muscle composition and digestive enzyme activities of the giant freshwater prawn Macrobrachium rosenbergii

The green synthesized Mn₃O₄ nanoparticles (manganese-oxide nanoparticles) using Ananas comosus (L.) peel extract was characterized by various techniques. HR-SEM photograph showed that manganese-oxide nanoparticles (Mn-oxide NPs) were spherical in shape, with an average size of 40–50 nm. The Zeta potential revealed the surface charge of Mn-oxide NPs to be negative. Further, the Mn-oxide NPs were dietary supplemented for freshwater prawn Macrobrachium rosenbergii. The experimental basal diets were supplemented with Mn-oxide NPs at the rates of 0 (control), 3.0, 6.0, 9.0, 12, 15 and 18 mg/kg dry feed weight. The as-supplemented Mn-oxide NPs were fed in M. rosenbergii for a period of 90 days. The experimental study demonstrated that prawns fed with diet supplemented with 3–18 mg Mn-oxide NPs/kg shows enhanced (P < 0.05) growth performance, including final weight and weight gain (WG). Significant differences (P < 0.05) in feed conversion ratio (FCR) were observed in prawn fed with different diets. Additionally, prawns fed with 3.0–18 mg/kg Mn-oxide NPs supplemented diets achieved significant (P < 0.05) improvement in growth performance, digestive enzyme activities and muscle biochemical compositions, while, the prawns fed with 16 mg/kg of Mn-oxide NPs showed enhanced performance. Prawns fed on diet supplemented with 16 mg/kg Mn-oxide NPs showed significantly (P < 0.05) higher total protein level. The antioxidants enzymatic activity (SOD and CAT) metabolic enzymes status in muscle and hepatopancreas showed no significant (P > 0.05) alterations in prawns fed with 3.0–18 mg/kg of Mn-oxide NPs supplemented diets. Consequently, the present work proposed that 16 mg/kg of Mn-oxide NPs could be supplemented for flexible enhanced survival, growth and production of M. rosenbergii. Therefore, the data of the present study recommend the addition of 16 mg/kg of Mn-oxide NPs diet to developed prawn growth and antioxidant defense system.     

Effect of infectious hypodermal and haematopoietic necrosis virus (IHHNV) infection on caspase 3c expression and activity in freshwater prawn Macrobrachium rosenbergii

Caspase 3c (MrCasp3c) was sequenced from the freshwater giant prawn Macrobrachium rosenbergii using Illumina Solexa Genome Analyzer Technique. MrCasp3c consisted of 2080 bp nucleotide encoded 521 polypeptide with an estimated molecular mass of 59 kDa. MrCasp3c sequence contains caspase family p20 domain profile and caspase family p10 domain profile at 236-367 and 378-468 respectively. The quantitative real time PCR analysis revealed a broad expression of MrCasp3c with the highest expression in haemocyte and the lowest in stomach. The expression of MrCasp3c after challenge with the infectious hypodermal and haematopoietic necrosis virus (IHHNV) was tested in haemocyte. In addition, MrCasp3c was expressed in Escherichia coli by prokaryotic expression plasmid pMAL-c2x. The enzyme activity of MrCasp3c was also found to be up-regulated by IHHNV in haemocyte and hepatopancreas tissues. This study suggested that MrCasp3c may be an effector caspase associated with the induction of apoptosis which is potentially involved in the immune defence of M. rosenbergii.     

Artemia as a possible vector for Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus transmission (XSV) to Macrobrachium rosenbergii post-larvae

Five developmental stages of Artemia were exposed to Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) by immersion and oral routes in order to investigate the possibility of Artemia acting as a reservoir or carrier of these viruses. The second objective was to determine if virus-exposed Artemia were capable of transmitting the disease to post-larvae (PL) of M. rosenbergii. There was no significant difference in percent mortality between Artemia control groups and groups challenged with these viruses. On the other hand, all the developmental stages of Artemia were positive for both viruses by nested RT-PCR, regardless of the challenge route. In horizontal transmission experiments, 100% mortality was observed in M. rosenbergii PL fed with Artemia nauplii exposed to MrNV and XSV by either challenge route. However, no mortality was observed in PL fed with virus-free Artemia. RT-PCR analysis of the M. rosenbergii PL confirmed the presence of MrNV and XSV in the challenge group and absence in the control group.     

Crustin,a WAP domain containing antimicrobial peptide from freshwater prawn Macrobrachium rosenbergii: Immune characterization

Crustin (MrCrs) was sequenced from a freshwater prawn Macrobrachium rosenbergii. The MrCrs protein contains a signal peptide region at N-terminus between 1 and 22 and a long whey acidic protein domain (WAP domain) at C-terminus between 57 and 110 along with a WAP-type ‘four-disulfide core’ motif. Phylogenetic results show that MrCrs is clustered together with other crustacean crustin groups. MrCrs showed high sequence similarity (77%) with crustin from Pacific white shrimp Litopenaeus vannamei and Japanese spiny lobster Panulirus japonicas. I-TASSER uses the best structure templates to predict the possible structures of MrCrs along with PDB IDs such as 2RELA and 1FLEI. The gene expressions of MrCrs in both healthy M. rosenbergii and those infected with virus including infectious hypodermal and hematopoietic necrosis virus (IHHNV) and white spot syndrome virus (WSSV) and bacteria Aeromonas hydrophila (Gram-negative) and Enterococcus faecium (Gram-positive) were examined using quantitative real time PCR. To understand its biological activity, the recombinant MrCrs gene was constructed and expressed in Escherichia coli BL21 (DE3). The recombinant MrCrs protein agglutinated with the bacteria considered for analysis at a concentration of 25 μg/ml, except Lactococcus lactis. The bactericidal results showed that the recombinant MrCrs protein destroyed all the bacteria after incubation, even less than 6 h. These results suggest that MrCrs is a potential antimicrobial peptide, which is involved in the defense system of M. rosenbergii against viral and bacterial infections.     

Effects of salinity on rates of protein synthesis and oxygen uptake in the post-larvae and juveniles of the tropical prawn Macrobrachium rosenbergii (de Man)

Protein synthesis is a major determinant of growth and yet little is known about the environmental factors that influence protein synthesis rates in farmed freshwater prawns. To this end, post-larvae and juveniles of Macrobrachium rosenbergii were exposed to various salinities (0, 14, 30‰) to determine whole-animal rates of fractional protein synthesis (k_s) and oxygen uptake. In the post-larvae that migrate upstream from brackish to freshwater areas, whole-animal k_s was unaffected by salinity, but rates of oxygen uptake were significantly lower at 14‰. In the freshwater juveniles, a different response was observed, as mean k_s was significantly higher at 14‰ compared with 0‰, but rates of oxygen uptake remained unchanged. Such differences are thought to be related to the energetic costs of osmoregulation and to the ability to maintain osmotic gradients in freshwater. In an additional experiment, acclimation temperature (20, 26, 30 °C) had a direct effect on k_s in juveniles held at 0‰. In all cases, changes in k_s resulted from alterations in RNA activity at constant RNA capacity. In juveniles at least, whole-animal rates of protein synthesis were highest at 14‰ and 30 °C which corresponds to the optimal salinity and temperature recommended for the growth and culture of M. rosenbergii.     

Effect of exposure to cadmium on the tropical freshwater prawn Macrobrachium rosenbergii

The effect of cadmium (Cd) on mortality, resistance and bioaccumulation in the tropical freshwater prawn Macrobrachium rosenbergii from Egypt were studied. Survival of prawns exposed to Cd doses over 60 μg l^?1 were significantly lower than that of prawns exposed to lower doses. After 96 h prawns exposed to >40 μg l^?1 of Cd had a greater reduction in total haemocyte count and phagocytic activity than those exposed to lower concentrations. Bioaccumulation of Cd in the gills, hepatopancreas and muscles was variable. Cadmium accumulated significantly in the gills and hepatopancreas, but Cd accumulation in the muscles increased only marginally. Macrobrachium rosenbergii manifested histopathological alterations in the gills, hepatopancreas and muscles when exposed to various concentrations of cadmium.     

Lead toxicity at different life stages of the giant prawn (Macrobrachium rosenbergii,de Man): considerations of osmoregulatory capacity and histological changes in adult gills

This study evaluated the acute toxicity of lead in different life stages of the freshwater prawn Macrobrachium rosenbergii, determined the effect of sublethal Pb concentrations on osmoregulatory capacity (OC), measured the Pb level in gills, and investigated the effect of Pb on the structure of the gills of adult prawns. The 24-, 48-, and 96-h LC₅₀ values for Pb to M. rosenbergii increased progressively with increasing life stage, from post-larvae (PL), juvenile to adult. The 24- and 48-h LC₅₀ values for post-larvae ranged from 0.01- to 0.09-mg Pb/L. The 24-, 48-, and 96-h LC₅₀ values for Pb were lower at 12 ppt than those at 0 ppt for either the juveniles or the adults. At 12 ppt, the 96-h LC₅₀ values in PL11, juvenile and adult were 0.47-, 0.58-, and 2.03-mg Pb/L, respectively. Meanwhile, at 12 ppt, the 96-h LC₅₀ values in PL11, juvenile and adult were 0.63-, 4.44-, and 7.98-mg Pb/L, respectively. In adults, the OC values of controls and prawns exposed to 2- and 4-mg Pb/L at 0 ppt were not significantly different. The OC of prawns exposed to and 2-mg Pb/L at 12 ppt increased by 72 and 109% from the OC of the control prawns. At media 12 ppt, the OC value of prawns exposed to 1-mg Pb/L was significantly different from that of prawns exposed to 2-mg Pb/L. The concentrations of Pb in gill tissues increased significantly in Pb exposed prawns both at 0 and 12 ppt. The level of Pb in gills of prawns exposed to 2-mg Pb/L at 12 ppt was not significantly different from those exposed at 0 ppt. The severe toxic actions of Pb were noted in gills of prawns exposed in media 12 ppt. Hyperplasia and necrosis were observed in gill lamellae, resulting in abnormal gill tips after Pb exposure at media 12 ppt. Since the effect of Pb is more pronounced in higher salinity (12 ppt) than in freshwater (0 ppt) it is clear that aquaculture of M. rosenbergii should be conducted in freshwater ponds.     

Microsatellite loci in the eastern form of the giant freshwater prawn (Macrobrachium rosenbergii)

Six microsatellite loci were identified and characterized in the eastern form of the widespread and commercially important giant freshwater prawn (Macrobrachium rosenbergii). The loci were detected by randomly screening for dinucleotide and trinucleotide repeat units within a partial genomic library developed for the species. In a sample of 29 prawns, number of alleles and heterozygosity per locus ranged from 12 to 18 and from 0.66 to 0.90, respectively. These markers provide powerful tools for the conservation and management of wild stocks, the improvement of cultured stocks of M. rosenbergii, and for investigating evolutionary processes underlying genetic divergence among populations.     

Differentially enhanced gene expression in hemocytes from Macrobrachium rosenbergii challenged in vivo with lipopolysaccharide

In order to better understand the immune response in prawns after treatment with the immunostimulant lipopolysaccharide (LPS), in this study, the differential gene expression of the hemocytes from LPS-injected versus non-injected prawns (Macrobrachium rosenbergii) were isolated and identified using suppression subtractive hybridization (SSH). The hemocytes were extracted after treatment for 1, 6, and 12 h. The upregulated genes (i.e., where gene expression was elevated) were identified and could be divided into four classes on the basis of physiological function: genes concerning defense-related molecules, genes involved in energy-production (respiration), genes related to protein synthesis and folding, and genes with unknown function. The time-course for gene expression indicated that, except for expression of the gene anti-microbial peptide (amp), which was increased at 12 h after LPS treatment, the expression of the other two immune-related genes was much earlier (at 1 h), including alpha-2-macroglobulin (α2-M) and Mas-like protein (mas). These results suggest that in the early phase of LPS stimulation some immune reactions regulated by α-2M and Mas may be induced, such as the activation of prophenoloxidase activating system, opsonization, and anti-microbial activity. In addition, six unigenes with unproven function were particularly interesting and worthy of further study because their expression in LPS-treated hemocytes was clearly enhanced.     

Induction of Female Breeding Characteristics by Ovarian Tissue Implants in Androgenic Gland Ablated Male Freshwater Prawns Macrobrachium rosenbergii (de Man) (Decapoda,Palaemonidae)

Summary

Implantation of ovarian tissue into androgenic gland ablated male prawns, Macrobrachium rosenbergii (de Man) induced the development of ovigerous and ovipositing setae and brood chambers, suggesting that in females, these secondary sexual characteristics are induced by the ovary. This model system has potential for the study of morphological changes associated with breeding in the Decapoda.     

Bromeliothrix metopoides,a boom and bust ciliate (Ciliophora,Colpodea) from tank bromeliads

We investigated the recently described colpodid ciliate Bromeliothrix metopoides in a series of laboratory experiments to reveal the environmental factors that constrain this species to its peculiar habitat, i.e. the tanks of bromeliads. Our results demonstrated that the various life stages of this ciliate (bacterivorous theronts and microstome trophonts, flagellate-feeding macrostomes) have specific demands in terms of food quality and quantity. Bromeliothrix required a high food threshold (>1.4 mg C L^?1) in order to thrive. Food quality also affected resting cyst formation of B. metopoides when the experimental containers dried out. Its maximum growth rates (μ_max = 4.71 d^?1, i.e. 6.8 doublings d^?1) belong to the highest ones recorded thus far for free-living ciliates. The pH niche of B. metopoides was relatively wide (pH ～4 to >9) under optimal food conditions. However, its high sensitivity to unfavourable environmental conditions let the population collapse within several hours. We conclude that B. metopoides is a boom and bust ciliate that is specifically adapted to its peculiar habitat but virtually unviable in other environments.