Full‐length cloning and phylogenetic analyses of translationally controlled tumour protein and ferritin genes from the Indian white prawn,Fenneropenaeus indicus (H. Milne Edwards)

Elucidation, through molecular analyses, of bacterial afflictions in commercially important aquaculture‐reared shrimps is pivotal for the prevention and/or control of disease outbreaks. In this study, we examined the phylogenetic relatedness and compared the possible immune‐related functional roles of both translationally controlled tumour protein (TCTP) and ferritin genes with previous studies. Both TCTP and ferritin genes were substantially upregulated in the Indian white prawn, Fenneropenaeus indicus (H. Milne Edwards), post‐larvae following bath challenge with the virulent strain of bacteria, Vibrio harveyi D3. Full‐length cloning of these genes by rapid amplification of complementary DNA ends ‐polymerase chain reaction (RACE‐PCR) yielded 727‐base pair (bp)‐long TCTP and 1212‐bp‐long ferritin gene sequences. Their open reading frames (ORFs) were 507 and 510 bp, respectively. The TCTP‐ORF coded for 168 amino acids with three substitutions at positions 37, 141, 155, and the ferritin ORF coded for 170 amino acids with no species‐specific substitutions. Phylogenetic analysis suggested the closest relatedness of both TCTP and ferritin from F. indicus to Chinese white prawn, Fenneropenaeus chinensis (Osbeck). In addition to reporting the full‐length sequences of these immune‐relevant genes, this study highlighted their conserved natures, which perhaps make them important defence‐related proteins in the innate immune system of F. indicus.     

Dietary fulvic acid effects on survival and expression of immune‐related genes in Litopenaeus vannamei challenged with Vibrio parahaemolyticus

The effects of fulvic acid (FA) on survival and immune‐related gene expression were investigated in Litopenaeus vannamei challenged with Vibrio parahaemolyticus by immersion. Shrimp were fed with different dietary FA concentrations (1, 2, 4 and 6 g/kg feed) for 20 days (first bioassay) or 8 days (second bioassay, 2 g/kg feed of FA added every 2 days) and then challenged with V. parahaemolyticus. In a third bioassay, the expression of three immune‐related genes (translationally controlled tumour protein [TCTP], superoxide dismutase [SOD] and heat‐shock protein 70 [HSP70]) in haemocytes or hepatopancreas of experimental shrimp was measured by real‐time quantitative PCR at 0, 6, 12, 24, 48, 72 and 96 hr after FA (2 g/kg feed) administration. Fulvic acid increased survival at a concentration of 2 g/kg feed supplied every two days. Interestingly, TCTP gene expression was upregulated, whereas gene expression of SOD and HSP70 was downregulated. In conclusion, dietary fulvic acid improves survival in white shrimp challenged with V. parahaemolyticus and modulates the immune response. Therefore, FA merits further evaluation as prophylactic treatment in commercial shrimp farms.     

Characteristics and pathogenicity of a Vibrio campbellii-like bacterium affecting hatchery-reared Penaeus indicus (Milne Edwards, 1837) larvae

In the summer months, epizootics occurred regularly among larvae of Penaeus indicus (Milne Edwards) in a hatchery located at Narakkal near Cochin, India. The clinical signs of infected larvae were expansion of chromatophores. opaqueness of body with twisting and degeneration of appendages. Bacteria were isolated from the infected larvae. These bacteria had morphological, physiological and biochemical characteristics of the genus Vibrio, but these properties did not correspond with those of known Vibrio species. The Vibrio was most related to Vibrio campbellii according to DNA-hybridization experiments. By pathogenicity experiments, the present Vibrio isolate was proved to be pathogenic to larvae, postlarvae and adult shrimp. Shrimp larvae challenged by immersion showed significant mortalities. The bacterial isolate failed to produce the infection in adult shrimp after oral inoculation but it caused significant mortality in adults after intramuscular injection. LC⁵⁰ and LD⁵⁰ values of the Vibrio isolate were determined for larvae and adult shrimps, respectively, at different time intervals.     

Virulence status,viral accommodation and structural protein profiles of white spot syndrome virus isolates in farmed Penaeus monodon from the southeast coast of India

The objective of this study was to investigate the reason for variation in the virulence of white spot syndrome virus (WSSV) from different shrimp farms in the Southeast coast of India. Six isolates of WSSV from farms experiencing outbreaks (virulent WSSV; vWSSV) and three isolates of WSSV from farms that had infected shrimps but no outbreaks (non‐virulent WSSV; nvWSSV) were collected from different farms in the Southeast coast of India. The sampled animals were all positive for WSSV by first‐step PCR. The viral isolates were compared using histopathology, electron microscopy, SDS‐PAGE analysis of viral structural proteins, an in vivo infectivity experiment and sequence comparison of major structural protein VP28; there were no differences between isolates in these analyses. A significant observation was that the haemolymph protein profile of nvWSSV‐infected shrimps showed three extra polypeptide bands at 41, 33 and 24 kDa that were not found in the haemolymph protein profile of vWSSV‐infected shrimps. The data obtained in this study suggest that the observed difference in the virulence of WSSV may not be due to any change in the virus, rather it could be due to the shrimp defence system producing certain factors that help it to accommodate the virus without causing any mortality.     

Cloning,expression and sequence analysis of Macrobrachium rosenbergii nodavirus genes: Indian isolate

RNA‐dependent RNA polymerase (RdRp), B2 and capsid genes of Macrobrachium rosenbergii nodavirus (MrNV) of Indian isolate were polymerase chain reaction amplified, cloned and sequenced. Expression of the MrNV fusion recombinant proteins of RdRp (44.5 kDa), B2 (32.2 kDa) and capsid (58.4 kDa) was confirmed by Western blot analysis using anti‐His mouse monoclonal antibodies. Polyclonal antibodies specific to purified recombinant MrNV capsid protein showed specificity against the capsid protein by Western blot. The protein sequence analysis of the partial RdRp gene of MrNV revealed the signature sequence along with the conserved core residues of the catalytic domain and indicated the presence of active sites, metal ion‐binding site and nucleic acid‐binding site residues. The Indian isolate of MrNV showed high RdRp and capsid gene sequence homology with the other MrNV geographical isolates. However, the Belize isolate was found to be the most distinct among the different geographical prawn nodavirus isolates due to the host specificity. Secondary structure prediction analysis of the MrNV capsid predicted it to be a DNA‐binding protein consisting of α helix (22.91%), extended strand (24.80%), β turn (5.39%) and random coil (46.90%) regions.     

The immune response in rohu,Labeo rohita (Actinopterygii: Cyprinidae) to Argulus siamensis (Branchiura: Argulidae) infection: kinetics of immune gene expression and innate immune response

Argulus siamensis is a major pathogen in freshwater aquaculture. The immune responses of Indian major carp, Labeo rohita to experimental infection of A. siamensis was evaluated by quantitation of immune‐relevant gene expression in head kidney and skin, and serum innate immune parameters through the course of infection. In skin of infected fish, antioxidant genes like natural killer cell enhancing factor (NKEF‐B) and superoxide dismutase (MnSOD) were significantly up‐regulated in addition to lysozyme G and β₂ microglobulin (β₂M). Both tumour necrosis factor α (TNFα) and toll‐like receptor 22 (TLR22) genes were significantly down‐regulated in skin during early phases of the infection. Most of the genes exhibited significant down‐regulation in head kidney; immunoglobulin (IgM) and β₂M genes being the exceptions which were significantly up‐regulated at 12 h and 3 days post infection. Most of the innate immune parameters like serum complement activity and ceruloplasmin levels showed significant reduction in infected fish. The observed results are indicative of A. siamensis modulating the immune response of rohu by down‐regulation of many immune factors which may explain the susceptibility of rohu to A. siamensis infection. The interaction of this parasite with the host need to be further explored to understand its pathogenesis.     

Green fluorescent protein visualization of Vibrio parahaemolyticus infections in Indian white shrimp Fenneropenaeus indicus (H Milne Edwards)

The pathways by which pathogens invade Fenneropenaeus indicus, the potential colonization in various tissues and the disease transmission mechanisms are unclear. The aims of the present study were to visualize the colonization and pathogenesis of GFP‐tagged Vibrio parahaemolyticus, in various tissues of F. indicus to evaluate the pathogen interaction. Among the three strains isolated, a virulent strain VpDAHV2 was tagged with green fluorescent protein (GFP‐VpDAHV2) and validated for both its growth characteristics and its virulence as a genuine model for F. indicus infection. VpDAHV2 was positive for toxR and tlh genes and negative for tdh genes. CLSM images revealed that maximum colonization was observed in the haemolymph of the F. indicus challenged with GFP‐VpDAHV2. The haemolymph was the primary site for the colonization of GFP‐VpDAHV2 in F. indicus. The enteric localization occurred independently of the flagellum or motility of GFP‐VpDAHV2 through the intestinal route. The F. indicus infection model suggests that the haemolymph and the intestine represent the sites of infection by GFP‐VpDAHV2, and hence are the active sites of pathogen interactions. GFP tagging of V. parahaemolyticus is a new and systemic approach to determine the presence of bacteria in vivo for the confirmation of host pathogen interactions in aquaculture studies.     

Pharmacological level of copper induces the immune and antioxidant mechanisms of Fenneropenaeus indicus conferring better protection against white spot syndrome virus infection

Effect of ambient copper on the immune, antioxidant and lipid peroxidation parameters of Fenneropenaeus indicus and its susceptibility to white spot syndrome virus under heavy metal (Cu²⁺) exposure has been studied. Adult shrimps were acclimated to 25‰ salinity for a period of 7?days, and after 12?h of starvation, shrimps were dosed with 0.075, 0.150, 0.225 and 0.30?ppm Cu²⁺ by adding appropriate quantities of copper sulphate solution. After 14?days of metal exposure, the shrimps were challenged with white spot syndrome virus through oral administration and the immune and antioxidant parameters were analysed. Analysis of variance showed that there were significant differences (P?<?0.05) in the immune and antioxidant parameters and lipid peroxidation product in different treatment groups of F. indicus compared to untreated group. The immune parameters and antioxidant enzymes have been significantly lowered (P?<?0.05) in the haemolymph of shrimps dosed with higher concentrations of copper sulphate leading to lipid peroxidation and accumulation of malondialdehyde. However, there was less oxidative stress in shrimps exposed to 0.075?ppm Cu²⁺. The present study showed that high levels of Cu²⁺ enhanced the mortality of F. indicus with concomitant reduction in immune and antioxidant parameters. A concentration of 0.075?ppm Cu²⁺ in the rearing water was found to have beneficial effect in shrimps in terms of immunostimulation and higher survival against WSSV infection. This can be adopted as a pharmacological approach towards shrimp health management in culture systems.     

Production of recombinant capsid protein of Macrobrachium rosenbergii nodavirus (r‐MCP43) of giant freshwater prawn,M. rosenbergii (de Man) for immunological diagnostic methods

White tail disease (WTD) caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) is a serious problem in prawn hatcheries. The gene for capsid protein of MrNV (MCP43) was cloned into pRSET B expression vector. The MCP43 protein was expressed as a protein with a 6‐histidine tag in Escherichia coli GJ1158 with NaCl induction. This recombinant protein, which was used to raise the antiserum in rabbits, recognized capsid protein in different WTD‐infected post‐larvae and adult prawn. Various immunological methods such as Western blot, dot blot and ELISA techniques were employed to detect MrNV in infected samples using the antiserum raised against recombinant MCP43 of MrNV. The dot blot assay using anti‐rMCP43 was found to be capable of detecting MrNV in WTD‐infected post‐larvae as early as at 24 h post‐infection. The antiserum raised against r‐MCP43 could detect the MrNV in the infected samples at the level of 100 pg of total protein. The capsid protein of MrNV estimated by ELISA using anti‐rMCP43 and pure r‐MCP43 as a standard was found to increase gradually during the course of infection from 24 h p.i. to moribund stage. The results of immunological diagnostic methods employed in this study were compared with that of RT‐PCR to test the efficiency of antiserum raised against r‐MCP43 for the detection of MrNV. The Western blot, dot blot and ELISA detected all MrNV‐positive coded samples as detected by RT‐PCR.     

Protein expression profiling in haemocytes and plasma of the Manila clam Ruditapes philippinarum in response to infection with Perkinsus olseni

The protein expression profiling in clam haemocytes and plasma in response to Perkinsus olseni was addressed. Adult Manila clams from a P. olseni‐free bed were experimentally challenged with parasite zoospores to analyse immune response. In another experiment, the effects of longer term infection were assessed in adult clams collected from a P. olseni‐affected bed, by comparing moderate to very heavily infected clams with non‐infected ones. Haemocyte and plasma proteins were separated by two‐dimensional electrophoresis; spot patterns were qualitatively compared between treatments within each experiment and the spots indicating differential protein expression associated with P. olseni challenge or with field infection were processed for protein identification. Fifteen clam proteins (four in haemocytes and eleven in plasma) of which expression was markedly affected by P. olseni were identified. Some of the identified proteins have a well‐known role in clam immune response against the parasite, such as lysozyme and lectins. Rho GTPase‐activating protein 6 could be a marker of resistance against P. olseni, which should be further studied.     

Effect of Indian lotus (Nelumbo nucifera Gaertn.) leaf powder on immune status and disease resistance of Nile tilapia

This study assessed the immune response of Nile tilapia (Oreochromis niloticus L.) after feeding on different levels (0.1%, 0.2% and 0.4%) of dietary Indian lotus (Nelumbo nucifera) leaf powder for 45 days. We evaluated both the nonspecific immune response at the end of the feeding period and the resistance to Aeromonas hydrophila. Exposure to Indian lotus resulted in a significant elevation in serum total globulins, serum lysozyme activity, serum killing percentage and the phagocytic activity (p < 0.05). Total serum protein and albumin showed no remarkable variation between tilapia fed on 0.1% Indian lotus and the control group (p > 0.05). In addition, the relative expressions of immune‐related genes, namely interleukin–1β and tumour necrosis factor–α were significantly up‐regulated in tilapia fed on 0.4% Indian lotus as compared to the control group; their expressions were down‐regulated in the other tested groups (p < 0.05). The survival rate of Nile tilapia postchallenge to A. hydrophila reported a significant and dose‐dependent increase in the Indian lotus‐supplemented groups (p < 0.05). Therefore, dietary incorporation of Indian lotus leaves (0.4%, 0.2% and 0.1%) could strengthen the immunity of Nile tilapia and improve its resistance to A. hydrophila infection. Therefore, Indian lotus leaves could serve as potential feed supplements for Nile tilapia.     

Efficacy of fermented prawn shell waste as a feed ingredient for Indian white prawn, Fenneropenaeus indicus

Prawn shell waste collected from shrimp‐processing plants in Cochin, India, was subjected to fermentation using 20 chitinoclastic and proteolytic/non‐proteolytic bacterial strains. The products generated were analysed for protein, lipid, total sugars, N‐acetyl glucosamine, free amino acids and ash. Shrimp diets were prepared using these 20 fermented products and a control diet using raw prawn shell waste. Feeding experiment was conducted with postlarvae (PL21) of Indian white prawn, Fenneropenaeus indicus for a period of 21 days. Biogrowth parameters such as mean weight gain, feed conversion ratio, specific growth rate and protein efficiency ratio were estimated and the animals were challenged with white spot virus orally via diet. Enhanced growth could be observed in prawns fed F134 and F124, incorporated with the fermentation products generated using Bacillus spp., C134 and C124 respectively. The percentage survival of prawns after 7 days of challenge was found to be highest for groups fed diet F111 incorporated with fermentation product generated using Bacillus sp. These products of bacterial fermentation hold promise as growth enhancers and immunostimulants in aquaculture.     

Identification of a mitochondrial‐targeting secretory protein from Nocardia seriolae which induces apoptosis in fathead minnow cells

Nocardia seriolae is the main pathogen responsible for fish nocardiosis. A mitochondrial‐targeting secretory protein (MTSP) 3141 with an N‐terminal transit peptide (TP) from N. seriolae was predicted by bioinformatic analysis based on the genomic sequence of the N. seriolae strain ZJ0503. However, the function of the MTSP3141 and its homologs remains totally unknown. In this study, mass spectrometry analysis of the extracellular products from N. seriolae proved that MTSP3141 was a secretory protein, subcellular localization research showed the MTSP3141‐GFP fusion protein co‐localized with mitochondria in fathead minnow (FHM) cells, the TP played an important role in mitochondria targeting, and only the TP located at N‐terminus but not C‐terminus can lead to mitochondria directing. Moreover, quantitative assays of mitochondrial membrane potential (ΔΨm) value, caspase‐3 activity and apoptosis‐related gene (Bcl‐2, Bax, Bad, Bid and p53) mRNA expression suggested that cell apoptosis was induced in FHM cells by the overexpression of both MTSP3141 and MTSP3141ΔTP (with the N‐terminal TP deleted) proteins. Taken together, the results of this study indicated that the MTSP3141 of N. seriolae was a secretory protein, might target mitochondria, induce apoptosis in host cells and function as a virulence factor.     

Secretory expression and characterization of xylanase isolated from Bacillus subtilis E20 increase the utilization of plant ingredients in tilapia feed

Bacillus subtilis E20, an isolate from natto – a Japanese fermented soy product – is used in a wide variety of marine and freshwater aquatic species to enhance growth and immunity. This paper describes the cloning, expression and characterization of xylanase isolated from B. subtilis E20 in B. subtilis RIK1285. The BsXynE20 gene was identified to encode a 213‐amino‐acid protein for a glycoside hydrolase family 11 xylanase containing a 28‐residue signal peptide whose cleavage yields a 185‐residue mature protein with a predicted molecular weight of 20.25 kDa. The full length of the BsXynE20 gene was subcloned into the pBE‐S expression vector for secretory production of recombinant BsXynE20 in B. subtilis RIK1285. Western blot and zymogram analysis, respectively, revealed that recombinant BsXynE20 can be secreted into culture medium and is a functional xylanase. The xylanase exhibited optimal activity at pH 5.0–6.0 and 60°C. Using recombinant BsXynE20‐pretreated duckweed meal for feed preparation improved the growth performance of tilapia. Finally, we achieved secretive expression of functional BsXynE20, which can improve the nutrient utilization of a plant‐derived diet. Thus, the use of BsXynE20 may be beneficial for tilapia aquaculture.     

Synergistic osmoregulatory dysfunction during salmon lice (Lepeophtheirus salmonis) and infectious hematopoietic necrosis virus co‐infection in sockeye salmon (Oncorhynchus nerka) smolts

While co‐infections are common in both wild and cultured fish, knowledge of the interactive effects of multiple pathogens on host physiology, gene expression and immune response is limited. To evaluate the impact of co‐infection on host survival, physiology and gene expression, sockeye salmon Oncorhynchus nerka smolts were infected with the salmon louse Lepeophtheirus salmonis (V?/SL+), infectious hematopoietic necrosis virus (IHNV; V+/SL?), both (V+/SL+), or neither (V?/SL?). Survival in the V+/SL+ group was significantly lower than the V?/SL? and V?/SL+ groups (p = 0.024). Co‐infected salmon had elevated osmoregulatory indicators and lowered haematocrit values as compared to the uninfected control. Expression of 12 genes associated with the host immune response was analysed in anterior kidney and skin. The only evidence of L. salmonis‐induced modulation of the host antiviral response was down‐regulation of mhc I although the possibility of modulation cannot be ruled out for mx‐1 and rsad2. Co‐infection did not influence the expression of genes associated with the host response to L. salmonis. Therefore, we conclude that the reduced survival in co‐infected sockeye salmon resulted from the osmoregulatory consequences of the sea lice infections which were amplified due to infection with IHNV.