Resistance of pearlspot larvae,Etroplus suratensis,to redspotted grouper nervous necrosis virus by immersion challenge

Viral nervous necrosis (VNN) affects more than 120 species mostly belonging to the order Perciformes. However, none of the brackishwater species belonging to the family Cichlidae under the order Perciformes are reported to be susceptible. Hence, the present experiment was undertaken to study the susceptibility of the brackishwater cichlid, pearlspot, Etroplus suratensis to NNV. Thirty‐day‐old pearlspot larvae were infected with NNV by immersion. Mortality was recorded till 14 days post‐infection, and the infected larvae were subjected to nested RT‐PCR and histology. The virus was isolated from infected larvae using SSN‐1 cells. To study the replication of the virus in vitro, primary cultured brain cells of E. suratensis and IEK cells were infected with NNV. No mortality was observed in any of the control or experimentally infected larvae. However, the experimentally infected larvae were positive for NNV by nested RT‐PCR and the virus was isolated using SSN‐1 cells. Further, the infected pearlspot brain cells and IEK cells showed cytopathic effect at second and third passage of the virus and they were positive for NNV by nested RT‐PCR. Pearlspot is relatively resistant to VNN although the virus could replicate in the larvae and in cell culture.     

A new species of philometrid parasite (Nematoda,Philometridae) and histopathological lesions in juvenile whitemouth croakers,Micropogonias furnieri (Desmarest)

A new species of nematode parasite, Philometroides tahieli sp. nov. is described. The juvenile whitemouth croaker, Micropogonias furnieri Desmarest, was collected during the spring and summer of the years 2008–2010 from estuarial environments of Argentina. During investigations of the parasite fauna a nematode encapsulated in the musculature of the operculum was found. The nematodes were removed from the host's muscle tissue and slide‐mounted in lactophenol solution to clarify the specimens. Opercular muscle with the parasite was processed for histopathological examination. Philometroides tahieli sp. nov. can be identified by the location of gravid females in the host, presence of anterior oesophageal bulb, bosses on the surface of the body, presence of transversal mounds and 14 cephalic papillae in two circular rows. The pathogenicity of the parasite is low in the natural environment, but lesions are consistent with a chronic process. The appearance of caseous necrosis suggests the presence of a locally acting substance. Philometroides tahieli sp. nov. is the second species of this genera described from brackish waters, and since the discovery of Philometroides maplestoni in 1928, is the first species of this genus recorded for South America.     

Lumpfish (Cyclopterus lumpus,Linnaeus) is susceptible to viral nervous necrosis: Result of an experimental infection with different genotypes of Betanodavirus

In recent years, the use of cleaner fish for biological control of sea lice has increased considerably. Along with this, a number of infectious diseases have emerged. The aim of this study was to investigate the susceptibility of lumpfish (Cyclopterus lumpus) to Betanodavirus since it was detected in asymptomatic wild wrasses in Norway and Sweden. Three betanodaviruses were used to challenge lumpfish: one RGNNV genotype and two BFNNV genotypes. Fish were injected and monitored for 4 weeks. Brain samples from clinically affected specimens, from weekly randomly selected fish and survivors were subjected to molecular testing, viral isolation, histopathology and immunohistochemistry. Reduced survival was observed but was attributed to tail‐biting behaviour, since no nervous signs were observed throughout the study. Betanodavirus RNA was detected in all samples, additionally suggesting an active replication of the virus in the brain. Viral isolation confirmed molecular biology results and revealed a high viral titre in BFNNV‐infected groups associated with typical lesions in brains and eyes of survivor fish. We concluded that lumpfish are susceptible to Betanodavirus, as proven by the high viral titre and brain lesions detected, but further studies are necessary to understand if Betanodavirus can cause clinical disease in this species.     

Host,agent and environment interactions affecting Nervous necrosis virus infection in Australian bass Macquaria novemaculeata

Australian bass Macquaria novemaculeata were challenged by immersion with nervous necrosis virus (NNV) at different ages and under controlled conditions to investigate factors affecting disease expression. Fish challenged at 3 weeks of age with 10³ TCID₅₀/ml and higher doses developed clinical disease; a lower dose of 10² TCID₅₀/ml resulted in incidence below 100% and 10¹ TCID₅₀/ml was insufficient to cause infection. Additionally, fish were challenged at 5, 6 and 13 weeks of age at 17 and 21°C to assess the role of the age of the host and water temperature on disease expression. Although Australian bass challenged at all ages had evidence of replication of NNV, only those challenged at 3 weeks of age (20 and 24 days post?hatch [dph]) developed clinical disease. Higher water temperature had an additive effect on disease expression in larvae challenged at 24 dph, but it did not affect the disease outcome in older fish. Finally, isolates of NNV derived from fish with clinical or subclinical disease presentations caused similar cumulative mortality and clinical signs when larvae at 24 dph were challenged, suggesting that agent variation was not responsible for variation in clinical presentation in these field outbreaks of NNV infection.     

Identification and pathogenicity study of emerging fish pathogens Acinetobacter junii and Acinetobacter pittii recovered from a disease outbreak in Labeo catla (Hamilton, 1822) and Hypophthalmichthys molitrix (Valenciennes, 1844) of freshwater wetland in West Bengal,India

The disease outbreaks in aquaculture system of wetlands are the major cause of fish mortality. Among various bacterial septicaemic diseases, fish mortality caused by Acinetobacter spp. is recently reported in different fish species. Fish disease outbreak was investigated in a wetland of West Bengal, India to identify the aetiological factors involved. The moribund fish were examined and subjected to bacterial isolation. Two bacterial causative agents were identified as Acinetobacter junii and Acinetobacter pittii by biochemical characterization and 16S rRNA gene amplification. Both the isolates were oxidase‐negative, nitrate‐negative, catalase‐positive and indole‐negative. The molecular identification using 16S rRNA gene sequencing and phylogenetic tree analysis further confirmed the two Acinetobacter spp. with 97%–99% similarity. The antibiotic resistance patterns of these two bacteria revealed that both of them were resistant to β‐lactam, cefalexin, cephalothin, amoxyclav, cefuroxime, cefadroxil, clindamycin, vancomycin and penicillin. In addition, A. pittii was also resistant to other antibiotics of cephams group such as ceftazidime and cefotaxime. In the challenge experiment, both A. junii and A. pittii were found to be pathogenic with LD₅₀ of 1.24 × 10⁵ and 1.88 × 10⁷ cfu/fish respectively. Histopathological examination of gill, liver and kidney revealed prominent changes supporting bacterial septicaemia. The investigation reports for the first time on concurrent infection by A. junii and multidrug‐resistant (MDR)‐A. pittii as emerging fish pathogens to cause severe mortality in Labeo catla and Hypophthalmichthys molitrix in a freshwater wetland.     

Effect of Lactobacillus plantarum and Jerusalem artichoke (Helianthus tuberosus) on growth performance,immunity and disease resistance of Pangasius catfish (Pangasius bocourti,Sauvage 1880)

This study evaluated the effects of Jerusalem artichoke (JA) and Lactobacillus plantarum singly or combined on growth, immunity and disease resistance of Pangasius bocourti. In the first experiment, different concentrations of JA (0, 5, 10, 20, 40, 80 and 160 g kg^?1) were administered to determine an optimal concentration on growth of P. bocourti. In the second experiment, the optimal concentration of JA (5 g kg^?1) was combined with 10⁸ cfu g^?1 L. plantarum. In the final experiment, five randomly selected fish from the second experiment were challenged with Aeromonas hydrophila. Treatments for second and third experiments were 0 g kg^?1 JA (Diet 1), 5 g kg^?1 JA (Diet 2), 10⁸ cfu g^?1 L. plantarum (Diet 3) and 5 g kg^?1 JA + 10⁸ cfu g^?1 L. plantarum (Diet 4). Fish fed 5 g kg^?1 JA or 10⁸ cfu g^?1 of L. plantarum significantly improved specific growth rate (SGR), feed conversion ratio (FCR), serum lysozyme activity and postchallenge survival rate (PCSR). Dietary in the combination of JA and L. plantarum showed significantly enhanced SGR, FCR, serum lysozyme, phagocytosis, respiratory burst activities and PCSR compared with control and individual applications. Dietary JA and L. plantarum significantly stimulated growth, immunity and disease resistance of P. bocourti.     

Development of chitosan conjugated DNA vaccine against nodavirus in Macrobrachium rosenbergii (De Man, 1879)

The protective efficacy of a DNA construct containing extra small virus antisense (XSVAS) gene of nodavirus encapsulated with chitosan nanoparticles (NPs) was investigated in giant freshwater prawn Macrobrachium rosenbergii (De Man, 1879). The delivery was carried out using oral and immersion methods. A plasmid concentration of 100 ng μL^?1 when conjugated with chitosan NPs was found to be more effective in increasing the survivability of the infected prawn. The particle mean size, zeta potential and loading efficiency percentage were 297 nm, 27 mV and 85%, respectively. The ability of the chitosan to form a complex with the plasmid was studied by agarose gel electrophoresis. The NPs were characterized by atomic force microscopy (AFM). Persistence study showed the presence of the DNA construct up to 30th day post‐treatment. The oral treatment was found to be better than the immersion treatment for delivery of the chitosan‐conjugated DNA construct. This is probably the first report on the delivery of nanoconjugated DNA construct in M. rosenbergii, against nodavirus.     

Utilization of Caridina nilotica (Roux) meal as a protein ingredient in feeds for Nile tilapia (Oreochromis niloticus)

The effects of replacing fish meal with Caridina nilotica as a protein ingredient on growth performance, nutrient utilization, carcass, proximate composition and economic benefits in Nile tilapia (Oreochromis niloticus) culture was evaluated. Replacement of the FM with C. nilotica was done at 25%, 50%, 75% and 100% (D25, D50, D75 and D100) and the substitution effects was compared with the control diet (D0, 0% C. nilotica). After 140 days of culture, the best growth performance, nutrient utilization and economic benefits occurred in fish groups fed diets with 25% C. nilotica inclusion. However, growth performance in fish fed diets D50 and D75 were comparable with the control (P > 0.05). At 100% substitution level of FM with C. nilotica, the growth performance and fish survival was lower than control. Protein and lipid contents in the fish and their digestibilities were highest in diet D25 and decreased with increasing levels of substitution of FM with C. nilotica. This study demonstrate that utilization of local protein sources (C. nilotica) can be effectively used to replace up to 75% of FM in the diets without compromising growth performance, survival, nutrient utilization and economic benefits in O. niloticus culture.     

Dietary Houttuynia cordata leaf extract and meal enhances the immunity and expression of immune genes in Labeo rohita (Hamilton, 1822)

A feeding trial was conducted to explore the effect of dietary Houttuynia cordata leaf extract (HCLE) and leaf meal (HCLM) on immunological responses and expression of interferon‐gamma (IFN‐γ) and tumour necrosis factor‐alpha (TNF‐α) gene in Labeo rohita fingerlings. Six isonitrogenous (350 g/kg CP) and isocaloric (17 MJ/kg DE) purified experimental diets were formulated with Houttuynia cordata leaf extract and leaf meal comprising control, C (0 g/kg HCLE and HCLM), E2.5 (2.5 g/kg HCLE), E5 (5 g/kg HCLE), E10 (10 g/kg HCLE), M10 (10 g/kg HCLM) and M20 (20 g/kg HCLM). Labeo rohita fingerlings (3.37 ± 0.23 g) were distributed in six experimental groups in triplicates following the complete random distribution. Fish were fed twice daily with respective experimental diets for a period of 60 days. A significantly (p < .05) lower lactate dehydrogenase, malate dehydrogenase, superoxide dismutase and catalase activities were registered in supplemented groups compared with control group, while respiratory burst and lysozyme activities were significantly (p < .05) higher in E10 group compared with other experimental groups. Haemoglobin, total leucocyte count, total erythrocyte count and haematocrit values were significantly (p < .05) higher in E10 group. The expression of IFN‐γ and TNF‐α in both the kidney and liver was significantly up‐regulated in leaf extract and meal supplemented groups with the highest expression in the fish of E10 group. Overall, these results suggest that the dietary supplementation of ethanolic extract of the Houttuynia cordata leaf at 10 g/kg level can enhance the immune response of L. rohita fingerlings.     

Identification and pathogenicity of Vibrio parahaemolyticus isolates and immune responses of Penaeus (Litopenaeus) vannamei (Boone)

Five different Vibrio parahaemolyticus strains (SH8, SH108, SH58, AH5 and GD10) isolated from the hepatopancreas of moribund shrimp in farms of mainland China were identified and capable of inducing massive mortality of Penaeus (Litopenaeus) vannamei. The immersion challenge results with five isolates indicated variance of virulence, while only GD10 caused massive sloughing of tubule epithelial cells which was recognized as the most significant symptom of AHPND. Differences in immune responses were detected of P. vannamei during 48 h post‐infection (p.i.) by injection or immersion challenge with V. parahaemolyticus (SH8, SH108 and GD10) isolates. When injected SH8 and SH108 isolates, the expression of lysozyme (LSZ) showing statistically significant upregulation at 16 and 48 h p.i. and that of Toll‐like receptors (TLR) showed statistically significant upregulation at 48 h p.i. When immersion challenge with the GD10 isolate, TLR were upregulated after 8 h p.i. challenge with 10⁴ cfu mL^?1; however, LSZ was downregulated when challenged with 10³ cfu mL^?1. The results suggested that LSZ and TLR serve as crucial molecular markers of innate immunity in shrimp against V. parahaemolyticus infection. LSZ is a vital marker for acute bacterial infection, while TLR serves as a crucial marker for chronic infection.     

Effects of supplementary fennel (Foeniculum vulgare) essential oil in diet on growth and reproductive performance of the ornamental fish,Convict cichlid (Cichlasoma nigrofasciatum)

This study evaluated the effects of diet containing 0 (C: control), 75 (D₁), 100 (D₂), 125 (D₃) and 150 (D₄) mg kg^?1 F. vulgare essential oil on growth and reproductive performance of C. nigrofasciatum. A total of 225 Convict cichlids female with mean weight (1.65 ± 0.02 g) were distributed into 15 glass aquaria (15 fish tank^?1) in triplicate treatments. During 40 days of experimental period, fish fed at 3% of their body weight daily. At the end of experiment, growth indices, gonadosomatic index and reproductive indices were determined. The best feed conversion ratio was obtained in D₄ (1.19 ± 0.03, P < 0.05). The addition of F. vulgare essential oil did not have any statistical effects on other growth indices (specific growth rate, weight gain and condition factor). Total survival rates in all treatments and control were uniformly high, ranging from 93% to 97% (P > 0.05). F. vulgare essential oil has no negative effect on survival rate of C. nigrofasciatum. There were no significant differences among protein content of fish body (P > 0.05). The lowest value of fat content (26.5 ± 0.5%) was observed in D_2, and it had significant difference with control and other groups (P < 0.05). The ash percentage in treatments fed with F. vulgare essential oil were higher than control fish (P < 0.05). The highest moisture content was detected in D₄ (72.68 ± 0.19%) and D₂ (71.23 ± 2%) groups. Among the used dosage, D₄ was the most effective dosage that could significantly increase GSI (11.06 ± 1.55%), fecundity (340 ± 21) and hatching ratio (92.33 ± 1.63%). There were no significant differences in diameter of eggs among various treatments (P > 0.05).     

Occurrence of trypanosomiasis in net‐cage cultured groupers (Cromileptes altivelis and Epinephelus fuscoguttatus) in Nanshan port of Sanya,Hainan province,China

In the present study, reported was an unidentified trypanosome that caused massive mortalities of cultured humpback grouper, Cromileptes altivelis (Valenciennes, 1828) and brown‐marbled grouper, Epinephelus fuscoguttatus (Forsskål, 1775) in the net‐cages in Sanya, China, and some general pathological features of E. fuscoguttatus after experimental infection of the trypanosome. Before dying in the net cages of diseased fish, listlessness and anorexia were the observable symptoms. The blood of the diseased fish was light‐coloured in comparison with that of healthy one. E. fuscoguttatus experimentally infected with this trypanosome are with different degrees of engorgement in gill lamella, liver, spleen and kidney. Tissue lesion and necrosis were observed in liver and spleen of diseased fish.     

Effect of probiotics on digestive enzyme activities and growth of cichlids,Etroplus suratensis (Pearl spot) and Oreochromis mossambicus (Tilapia)

In aquaculture, exposure to stressful conditions and problems related to diseases often occur resulting in serious economic loss. The use of probiotics that control pathogens through different mechanisms, aiding digestion by exoenzyme supply and establishment of beneficial microflora in the digestive tract has got increased attention recently. Two different bacterial species Micrococcus MCCB 104 and Bacillus MCCB 101 were tested as probiotics for two cichlid species Etroplus suratensis and Oreochromis mossambicus. The strains were administered separately and together, at a rate of 10³ CFU animal^?1 day^?1 for 28 days. The effects on digestive enzyme activity and growth were compared with those fed on control diet. FCR of E. suratensis was high in case of Bacillus alone as well as the mixed culture (Micrococcus and Bacillus together) than that of control. However, the SGR of E. suratensis was slightly lower than that of control during 14 days trial. SGR of both the treatments (Bacillus alone and mixed culture) was higher than that of the control on 28th day, and this might be due to the reason that specific duration is needed for the assimilation and conversion of nutrients into proteins. The intestinal digestive hydrolases of E. suratensis were profoundly enhanced mainly by Bacillus and mixed cultures. The hepatopancreatic versions of major digestive enzymes in E. suratensis were influenced variously by different probiotic treatments. The alpha‐amylase activity of hepatopancreas has been elevated significantly by mixed culture during 14 days experiments while on 28 days trials both Bacillus and mixed culture showed an enhancing effect. During 14 days feeding trials, the intestinal lipase activity has been prominently enhanced by all probiotic treatments. It is found that, in O. mossambicus, both 14 days and 28 days feeding experiments, the pepsin as well as intestinal digestive enzymes such as amylase, total alkaline protease, lipase and their hepatopancreatic counter parts showed a significant elevation in their specific activity. Intestinal alpha‐amylase and total alkaline proteases of O. mossambicus showed an increase in specific activities on both 14 days and 28 days feeding experiments with the Bacillus and Micrococcus combination. The hepatopancreatic enzymes were highly influenced by the Bacillus species in O. mossambicus. The results of this study suggest that live probiotic microorganisms such as Micrococcus MCCB 104 and Bacillus MCCB 101 may be incorporated while formulating the cost‐effective, nutritionally balanced diet of E. suratensis and O. mossambicus for better growth performance and nutrient utilization.     

Effects of dietary supplementation of Astragalus membranaceus,Codonopsis pilosula,and Glycyrrhiza uralensis extract mixture on growth performance,haematological parameters and hepatopancreatic performance in juvenile Pacific white shrimp (Litopenaeus vannamei)

Pacific white shrimp is the major farmed shrimp species in the world. It is known to be very sensitive to the environmental and management changes such as intensification, which is one of the primary necessities to increase shrimp production, but represents a stressful condition, and needs to be managed properly to diminish its negative effects in aquaculture. In this study, juvenile Pacific white shrimp were fed diets supplementing with equal quantities of Astragalus membranaceus, Codonopsis pilosula and Glycyrrhiza uralensis extracts in different concentrations (0, 1.0, 2.0 and 3.0 g/kg) for 8 weeks. We subsequently estimated the effects of supplemented diets on growth performance, haematological parameters, and histological changes of hepatopancreas. Results revealed that the supplemented mixture had no benefits on growth and survival rate, but had favourable effects on haemocyte count, number of granulocytes, and hemocyanin concentration in the haemolymph (p > 0.05). We observed that R cells and B cells were increased in hepatopancreatic tubules of shrimps fed on supplemented diets. Besides, a diet containing 2.0 g/kg of extract mixture maintained the decreased levels of alanine aminotransferase (p < 0.05) and aspartate aminotransferase (p > 0.05). Among the used concentrations, 2.0 g/kg seemed to be the most suitable concentration.     

Increased gonad growth of the purple sea urchin (Strongylocentrotus purpuratus) fed the giant kelp (Macrocystis pyrifera) and the sea lettuce (Ulva lactuca) enriched with nutrients

Adult (Strongylocentrotus purpuratus) purple sea urchins were fed giant kelp (Macrocystis pyrifera) and sea lettuce (Ulva lactuca) macroalgae that were unenriched and enriched with nutrients. Urchins were fed over 10 weeks (5% body weight per day) under laboratory conditions to determine the effect of macroalgae species and treatment (enriched and unenriched) on growth, gonad index and gonad quality. Significant differences were observed when urchins were fed enriched Macrocystis pyrifera (MPE). Gonad weight increased from 0.7 ± 0.1 g (mean ± SD) to 5.9 ± 1 g, and gonad index increase from 3.5 ± 1.5% to 14.9 ± 2.3% over 10 weeks. Significant differences were also noted in gonad quality, and gonad colour and firmness were better in urchins that were fed unenriched M. pyrifera (MPUE) and MPE – whereas texture did not differ between M. pyrifera treatments and unenriched U. lactuca (ULUE). With regard to taste, gonads were sweeter in MPE‐fed individuals. Enriched U. lactuca (ULE) failed to grow gonads significantly; as a result, their quality was unacceptable for the market. These results suggest that S. purpuratus experience increased gonad production and quality in short periods when fed nutrient‐enriched M. pyrifera.