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.     

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.     

Novel polymorphic microsatellite DNA markers from Malaysian giant freshwater prawn, <Emphasis Type="Italic">Macrobrachium rosenbergii</Emphasis>

Seven single locus microsatellite markers were characterized in Malaysian giant freshwater prawn, Macrobrachium rosenbergii from an enriched genomic library Primer pairs were designed to flank the repeat sequences and the loci characterized for this species. The bands resulting from the PCR amplifications of these eight microsatellite loci were polymorphic with the number of alleles ranging from 8 to 26 alleles per locus, whereas the observed heterozygosity ranged from 0.0641 to 0.6564. These newly developed microsatellite markers should prove to be useful for population studies and in the management of genetic variations in broodstocks of freshwater prawn, M. rosenbergii.     

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.     

Hepatopancreas and ovary are sites of vitellogenin synthesis as determined from partial cDNA encoding of vitellogenin in the marine shrimp,Penaeus vannamei

Summary

The site of yolk protein synthesis in crustaceans has long been a subject of controversy. A portion of the vitellogenin gene structure was reported recently in a freshwater giant prawn (Macrobrachium rosenbergii) and black tiger shrimp (Penaeus monodori), in which the hepatopancreas was confirmed to be the extraovarian site of vitellogenin synthesis. The ovary is also frequently reported to be the site of yolk protein synthesis in penaeid shrimp. The same PCR product was obtained using cDNA from the hepatopancreas or the ovary as a template. The deduced amino acid sequence of Vg in P. vannamei showed high identities of 57% and 78% with those from M. rosenbergii and P. monodon, respectively. The same location of the intron in the sequenced region of genomic DNA was also found between these three species. We therefore concluded that the hepatopancreas and ovary are sites of vitellogenin synthesis in P. vannamei. The partial structure of the vitellogenin gene is further presented.     

Molecular epidemiological investigation of infectious hypodermal and hematopoietic necrosis virus and Taura syndrome virus in <Emphasis Type="Italic">Penaeus Vannamei</Emphasis> cultured in China

The Infectious hypodermal and hematopoietic necrosis virus (IHHNV) and Taura syndrome virus (TSV) are two important shrimp viruses in cultured shrimp in America. These two viruses were transmitted to China at the beginning of the 21^st century. In this study, 214 shrimp samples of Penaeus vannamei were collected from seven different areas of China and tested by PCR for IHHNV and TSV infection. The results showed that there were a high prevalence of IHHNV (65.42%) and low prevalence of TSV (3.27%) in the tested samples. Several samples were found to be co-infected with these two viruses. A 3 kb fragment of 7 positive IHHNV samples and a structure protein region (ORF2) of three TSV positive samples were amplified and sequenced. The sequence comparison indicated that both IHHNV and TSV sequenced in China have a low genetic variations compared with the prototype IHHNV and TSV from Hawaii. Phylogenetic analysis showed that TSV isolates were clustered into two groups, Asia and America group, which was genetically correlated to geographic distribution. Foundation item: This work was supported by 973 project (2006CB101801)     

Twelve polymorphic microsatellites in Oriental river prawn, Macrobrachium nipponense

Oriental river prawn, Macrobrachium nipponense, is a commercially important freshwater prawn species in China, Japan, Korea and Vietnam. Due to overfishing for food, the wild stocks M. nipponense are endangered. Twenty microsatellite loci were isolated from the M. nipponense. Twelve of these loci were polymorphic (seven to 16 alleles per locus), with expected heterozygosity ranging from 0.68 to 0.86 (n = 48). These polymorphic loci provide a valuable tool for assessing genetic diversity of wild and cultured populations.     

Historical demography and genetic differentiation of the giant freshwater prawn Macrobrachium rosenbergii in Bangladesh based on mitochondrial and ddRAD sequence variation

Macrobrachium rosenbergii, the giant freshwater prawn, is an important source of high quality protein and occurs naturally in rivers as well as commercial farms in South and South‐East Asia, including Bangladesh. This study investigated the genetic variation and population structure of M. rosenbergii sampled from four rivers in Bangladesh (sample size ranged from 19 to 20), assessing sequence variation, both in the mitochondrial cytochrome oxidase subunit 1 (CO1) gene and in 106 single nucleotide polymorphisms (SNPs) sampled randomly from the genome with double digest RAD sequencing (ddRADseq). The mitochondrial variation presented a shallow genealogy with high haplotype diversity (h = 0.95), reflecting an expansion in population size for the last ~82 kyr. Based on the CO1 variation the current effective population size (N_e) was 9.7 × 10⁶ (CI: 1.33 × 10⁶ – 35.84 × 10⁶) individuals. A significant population differentiation was observed with the mitochondrial CO1 sequence variation and based on the ddRADseq variation, which could be traced to the divergence of the population in the Naf River in the South‐East border with Myanmar from the other populations. A differentiation in mtDNA haplotype frequencies was also observed between the Biskhali River and the Karnaphuli Rivers in eastern Bangladesh. This study demonstrated the use of high‐throughput genotyping based on the ddRADseq method to reveal population structure at a small geographical scale for an important freshwater prawn. The information from this study can be utilized for management and conservation of this species in Bangladesh.     

Influence of lecithotrofic feeding on growth and development of larvae of freshwater prawn <Emphasis Type="Italic">Macrobrachium rosenbergii</Emphasis>

In the giant freshwater prawn Macrobrachium rosenbergii (De Man), lecithotrofic feeding was discovered at the zoea I stage, and facultative lecithotrofic feeding was found at the zoea II stage. Cases of the completion of the first two stages without feeding were detected. However, a delay in feeding at the zoea II stage caused the inhibition of the growth and development of larvae. In this connection, we recommend to introduce food to the aquaculture of the giant freshwater prawn on the end of the first day after hatching, when the first zoea II larvae emerge.     

Immunomodulatory effect of Withania somnifera supplementation diet in the giant freshwater prawn Macrobrachium rosenbergii (de Man) against Aeromonas hydrophila

The effect of Withania somnifera extract supplementation diets on innate immune response in giant freshwater prawn Macrobrachium rosenbergii (de Man) against Aeromonas hydrophila was investigated. The bacterial clearance efficiency significantly increased in prawn fed with 0.1% and 1.0% doses of W. somnifera supplementation diet against pathogen from weeks 1-4 as compared to the control. The innate immune parameters such as, phenoloxidase activity, superoxide anion level, superoxide dismutase activity, nitrate, and nitrite concentrations were significantly enhanced in prawn fed with 0.1% and 1.0% doses of W. somnifera supplementation diet from weeks 1-4 against pathogen. The total hemocyte counts (THC) significantly increased in prawn fed with 0.1% and 1.0% doses diet from weeks 1-4 against pathogen as compared to the control. These results strongly suggested that administration of W. somnifera through supplementation diet positively enhances the innate immune system and enhanced survival rate in M. rosenbergii against A. hydrophila infection.     

Size-selective predation by all-male prawns: implications for sustainable biocontrol of snail invasions

All-male populations of the freshwater prawn Macrobrachium rosenbergii were recently produced by a novel temporal RNA interference (RNAi)-based biotechnology for aquaculture purposes. This biotechnology opens the way to the wide use of all-male prawn populations as sustainable biocontrol agents against invading populations of freshwater snails, for which there is currently no environmentally friendly solution. Among the most damaging of the invasive freshwater snail species are the apple snails (Pomacea spp.), which inflict major damage on natural ecosystems and rice fields. The proposed use of all-male prawn populations as environmentally friendly biocontrol agents against invasive freshwater snails has several advantages: efficient predation by the prawns over a wide range of freshwater snails, the ready availability of the prawns, and the monosex non-reproductive nature of the biocontrol agents. Since the aquatic predators are strongly size selective, we quantified the predation rate as a function of body size of both predator and prey (M. rosenbergii and P. caniculata). Medium-sized and large prawns (~10–30 g) efficiently preyed small and medium-sized snails (up to 15 mm), while small prawns (up to 4 g) immediately and completely eradicated snail hatchlings. Medium-sized prawns (~22 g) exterminated a significant fraction of snail biomass within 24 h (up to 58% of their body mass) after being introduced into a tank of snails. A typical ‘climbing-to-the surface’ anti-predator behavior of the snails was recorded. The potential of all-male prawns as efficient biocontrol agents over hatchling and adult apple snails as part of an integrated pest management program is discussed. Our experiments set the stage for evaluating the ecological and economic implications of this generic solution for a wide variety of habitats.     

对虾白斑综合征病毒新株型WSSV-CN-Pc的毒力研究

对虾白斑综合征病毒(white spot syndrome virus,WSSV)是一种能够感染虾类并且造成其大面积死亡的环状双链DNA病毒。WSSV有多种分离株,其毒力有所差异。从克氏原螯虾(Procambarus clarkii)中分离得到1株WSSV新分离株WSSV-CN-Pc,其毒力尚不清楚。本研究采用肌肉注射和经口注射的方法,以WSSVTW型作为阳性对照,分别对克氏原螯虾(P.clarkii)和罗氏沼虾(Macrobrachium rosenbergii)进行活体实验。实验结果显示:肌肉注射WSSV-CN-Pc和WSSV-TW的克氏原螯虾均在第6天出现100%的死亡;罗氏沼虾在肌肉注射WSSV-TW后未出现死亡,但在注射WSSV-CN-Pc后的第9天死亡率达100%。经口注射WSSV-CN-Pc和WSSV-TW的克氏原螯虾均在第16天出现100%的死亡;罗氏沼虾经口注射WSSV-CN-Pc后的第19天死亡率为100%,但注射WSSV-TW的实验组并未出现死亡。结果表明,对于克氏原螯虾,WSSV-CN-Pc具有和WSSV-TW相似的毒力,而对罗氏沼虾存在明显的毒力差异。提示克氏原螯虾是WSSV传播途径中的重要因素。     

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.     

Effect of different stock types and dietary protein levels on key enzyme activities of glycolysis‐gluconeogenesis,the pentose phosphate pathway and amino acid metabolism in Macrobrachium rosenbergii

A feeding trial was conducted to study the effect of dietary protein (DP) levels on the enzyme activities of different stocks of Macrobrachium rosenbergii juveniles, fed two diets prawn type diets either at optimum (ODP, 32%) or sub‐optimum (SDP, 27%) dietary protein. The activity of key enzymes: glycolysis, gluconeogenesis, amino acid catabolism, pentose phosphate pathway, and energy metabolism was examined on different stocks of M. rosenbergii juveniles in various tissues (muscle, hepatopancreas, and gill). Wild juveniles of M. rosenbergii were collected from the west coast of India, Gujarat (G), Maharashtra (M) and from the east coast of India, Andhra Pradesh (A), and raised in culture ponds of 200 m² at 1 juvenile m^‐2. The experiment was conducted for a period of 60 days. A Visible Implant Elastomer (VIE) Tag was injected into different prawn juveniles’ stocks A, (3.06–3.10 g), M, (0.80–1.01 g) and G (0.90–1.06 g) for the individual identification of each class. All the animals were acclimatized for 7 days before being released into the pond. Each of the two diets, the first with 27% SDP, termed the sub‐optimum level (S), and the second 32% DP, termed the optimum level (O), was fed in triplicate ponds. The weight gain of stocked prawn was significantly higher for G stock (3.03 ± 0.18 g) compared to M stock (2.53 ± 0.25 g) and A stock (1.33 ± 0.10 g). In addition weight gain in ODP was significantly higher (3.05 ± 0.22 g) compared to SDP (2.11 ± 0.17 g). Furthermore, both protein level and stock type had a significant (p ≤ 0.05) effect on the survival rate, specific growth rate, and feed conversion ratio of the prawn. A significant interaction was recorded between metabolic enzyme activities and the variation of dietary protein levels and differences in M. rosenbergii stocks, in which stock “G” and “M” exhibited healthier enzyme physiological status than stock “A”. The findings presented here may conclude that the enzymes quickly respond to dietary protein manipulations of cultured shrimp. The results can be useful in identifying the optimum stock (genotypes) in a given culture condition and subsequently higher production may be achieved.     

Genetic diversity of the cultured giant freshwater prawn (Macrobrachium rosenbergii) in China based on microsatellite markers

The Giant freshwater prawn (GFP), Macrobrachium rosenbergii, is one of the most important crustacean species cultured in China, but information on the genetic diversity of GFP in China is still limited. In this study we analyzed the genetic diversity of 6 cultured GFP populations collected in China, and 2 wild populations from Viet Nam. The wild populations were included as a reference so that the genetic diversity of the introduced cultured GFP populations could be evaluated. We used six polymorphic primer pairs to estimate genetic diversity. Results showed that the number of alleles per locus ranged from 6 to 19. The mean observed heterozygosity (0.364 ± 0.090) was less than the expected heterozygosity (0.893 ± 0.016); highly significant deficiencies in heterozygotes were detected (100%). The polymorphism information content (PIC) of 8 populations at 6 loci was greater than 0.5 (varying from 0.616 to 0.912). Pair-wise genetic differentiation (F_ST) varied from 0.006 to 0.129 among the eight populations. The dendrogram based on genetic distance showed six populations distributed on the same major branch, suggesting that they have closely genetic distance and two populations (ZJC and GDB) distributed on the other branch, so they also have closely genetic distance. The results of study provide a baseline assessment of genetic diversity in some populations in China and Viet Nam that will be useful for the development of breeding programmes in the future.     

Prophenoloxidase activating enzyme-III from giant freshwater prawn Macrobrachium rosenbergii: characterization, expression and specific enzyme activity

The prophenoloxidase activating system is an important innate immune response against microbial infections in invertebrates. The major enzyme, phenoloxidase, is synthesized as an inactive precursor and its activation to an active enzyme is mediated by a cascade of clip domain serine proteinases. In this study, a cDNA encoding a prophenoloxidase activating enzyme-III from the giant freshwater prawn Macrobrachium rosenbergii, designated as MrProAE-III, was identified and characterized. The full-length cDNA contains an open reading frame of 1110 base pair (bp) encoding a predicted protein of 370 amino acids including an 22 amino acid signal peptide. The MrProAE-III protein exhibits a characteristic sequence structure of a long serine proteases-trypsin domain and an N- and C-terminal serine proteases-trypsin family histidine active sites, respectively, which together are the characteristics of the clip-serin proteases. Sequence analysis showed that MrProAE-III exhibited the highest amino acid sequence similarity (63%) to a ProAE-III from Atlantic blue crab, Callinectes sapidus. MrProAE-III mRNA and enzyme activity of MrProAE-III were detectable in all examined tissues, including hepatopancreas, hemocytes, pleopods, walking legs, eye stalk, gill, stomach, intestine, brain and muscle with the highest level of both in hepatopancreas. This is regulated after systemic infectious hypodermal and hematopoietic necrosis virus infection supporting that it is an immune-responsive gene. These results indicate that MrProAE-III functions in the proPO system and is an important component in the prawn immune system.     

Polymorphic microsatellite loci for species of Australian freshwater cod, <Emphasis Type="Italic">Maccullochella</Emphasis>

The Australian freshwater cod genus, Maccullochella is represented by three species: Murray cod, M. peelii peelii, eastern freshwater cod, M. ikei, and trout cod, M.macquariensis. Seven novel microsatellite loci from M. ikei and six previously published loci from M. peelii peelii were tested on wild populations of Murray, eastern and trout cod. Levels of polymorphism varied between species with 13 loci polymorphic in Murray cod, 9 in trout cod and 7 in eastern cod. Observed heterozygosities ranged from 0.053 to 0.842. This suite of microsatellite loci will facilitate future studies of the genetic status of wild and hatchery bred populations of Maccullochella.