Revealing the compositions of the intestinal microbiota of three Anguillid eel species using 16S rDNA sequencing

Anguilla japonica, A. marmorata and A. bicolor pacifica are important eel species for aquaculture in Taiwan. In the past, seldom studies focused on the intestinal microbiome of them or the probiotics used in their cultivation. Probiotics are microbes that can improve the health of organisms, and the microbes isolated from the intestines of organisms might be the potential probiotics. Therefore, the aim of this study was to collect the intestinal mucus samples of these three eel species for investigating the autochthonous microbiome using 16S rDNA sequencing. The diversity, abundance of the intestinal microbiome and compositions of all the libraries were identified. Moreover, the composition of intestinal microbiome of eel was suggested that it would be affected by the waters and the characteristics of different eel species. Furthermore, most of the identified predominant genera of the intestinal microbiome of these three eel species, such as Cetobacterium, Clostridium, Shewanella, Acinetobacter and Bacteroides, seemed to have great potential as the probiotics. In the future, more studies shall be conducted to promote the practical application of these candidate probiotics for improving the techniques of eel farming.     

Retracted: Effectiveness of carp pituitary extract and luteinizing hormone releasing hormone analogue administration (by either injection or cholesterol pellet implantation) on spawning performance in female sturgeon,Huso huso

The effectiveness of common carp pituitary extract (CPE), luteinizing hormone releasing hormone analogue (LHRH‐A₂) injections and LHRH‐A₂ implants for spawning induction in female sturgeon, Huso huso was examined. In the first trial, fish were injected with 7% physiological saline (control), 50 mg kg^?1 CPE or LHRH‐A₂ at 3.5, 7, 8 or 10 μg kg^?1. In the second trial, fish were treated with LHRH‐A₂ cholesterol pellet implants containing 0, 3.5, 7, 8 and 10 μg kg^?1 LHRH‐A₂. Ovulated eggs were removed using a minimally invasive surgical technique and were artificially fertilized. Injection of CPE and LHRH‐A₂ at doses of 3.5, 7, 8 and 10 μg kg^?1 resulted in the number of ovulated fish more than LHRH‐A₂ implants (similar doses) or controls, although there was no significant difference at doses of 8 and 10 μg kg^?1 (P ≥ 0.05). The latency period of fish receiving CPE and LHRH‐A₂ injections was approximately 20 h, which was significantly lower than in fish receiving LHRH‐A₂ implants (P ≤ 0.05). Furthermore, there were no significant differences in rates of fertilization or hatching among the progeny produced in any of the treatment groups (P ≥ 0.05). In conclusion, the data from this study could be useful for artificial propagation of not‐fully‐matured females of H. huso at sturgeon hatcheries.     

Molecular characterization and interactome analysis of aerolysin (aer) gene from fish pathogen Aeromonas veronii: The pathogenicity inferred from sequence divergence and linked to histidine kinase (cheA)

Aerolysin (aer) is one of the most important and abundant virulence factors in the infection of fish by Aeromonas veronii. A comprehensive study on the molecular characterization and pathogenicity of the aer gene from 34 A. veronii isolates from diseased carp and catfish was carried out and its interactome was analysed to observe the functional correlations between aer and other proteins within the A. veronii network. The PCR‐based amplification of aer from the 34 isolates of A. veronii showed more aer‐positive isolates from catfish with a high pathogenic potential in the in vivo challenge test than the carp fish. The analysis of aer gene sequence from challenged fish revealed significant sequence divergence according to the types and geographical distribution of the fish. The networking analysis of aer from the model A. veronii B565 revealed histidine kinase (cheA) as the most functional interacting partner. The study of the interaction between aer from the experimental A. veronii and cheA demonstrated that the A chain of cheA plays a more important role than the corresponding B chain during contact, and a linker sequence of 15 residues controlled the entire interaction process. Therefore, cheA could be an excellent drug target for controlling A. veronii infection of fish.     

Aeromonas salmonicida infection levels in pre‐ and post‐stocked cleaner fish assessed by culture and an amended qPCR assay

Due to increasing resistance to chemical therapeutants, the use of ‘cleaner fish’ (primarily wrasse, Labridae, species) has become popular in European salmon farming for biocontrol of the salmon louse, Lepeophtheirus salmonis (Krøyer). While being efficient de‐licers, cleaner fish mortality levels in salmon cages are commonly high, and systemic bacterial infections constitute a major problem. Atypical furunculosis, caused by Aeromonas salmonicida A‐layer types V and VI, is among the most common diagnoses reached in clinical investigations. A previously described real‐time PCR (qPCR), targeting the A. salmonicida A‐layer gene (vapA), was modified and validated for specific and sensitive detection of all presently recognized A‐layer types of this bacterium. Before stocking and during episodes of increased mortality in salmon cages, cleaner fish (primarily wild‐caught wrasse) were sampled and screened for A. salmonicida by qPCR and culture. Culture indicated that systemic bacterial infections are mainly contracted after salmon farm stocking, and qPCR revealed A. salmonicida prevalences of approximately 4% and 68% in pre‐ and post‐stocked cleaner fish, respectively. This underpins A. salmonicida's relevance as a contributing factor to cleaner fish mortality and emphasizes the need for implementation of preventive measures (e.g. vaccination) if current levels of cleaner fish use are to be continued or expanded.     

Identification and characterization of a phospholipase A1 activity type three secreted protein,PP_ExoU from Pseudomonas plecoglossicida NB2011, the causative agent of visceral granulomas disease in large yellow croaker (Larimichthys crocea)

Pseudomonas plecoglossicida NB2011, the causative agent of visceral granulomas disease in farmed Larimichthys crocea in China, encodes a predicted type three effector PP_ExoU, a homolog of the cytotoxin ExoU of Pseudomonas aeruginosa. In this study, secretion of PP_ExoU was tested in various broth, the protein was expressed with the pET30a prokaryotic system, the phospholipase A (PLA) activity of the recombinant protein was determined with fluorogenic phospholipid substrates, fusion expression with green fluorescent protein in transfected HeLa cells was investigated, and the lactate dehydrogenase (LDH) level was measured. The results showed the protein was type three secreted in several media; the recombinant protein displayed significant PLA1 activity with ubiquitin. Fluorescence was observed on the cell membrane and scattered in the cytoplasm of HeLa cells expressing catalytic wild‐type PP_ExoU, blebbing and stretching developed in the cell membranes indicating of membrane damage. Fluorescence scattered in the cytoplasm of cells expressing the catalytic inactive protein. A significant LDH level was detected in HeLa cells expressing wild‐type PP_exoU, but not in the Ser/Asp‐mutated protein, suggestion mutation of predicted catalytic residues abolished the PLA activity. This is the first report on the function of a secreted type three protein from P. plecoglossicida.     

Effects of vitamin A inclusion in practical diets on survival and growth of juvenile crayfish (Pacifastacus leniusculus Dana,Astacidae) from the onset of exogenous feeding

The aim of this study was to evaluate the effects of practical diets with different levels of vitamin A on survival and growth of juvenile crayfish (Pacifastacus leniusculus). An 80‐day trial was conducted with stage 2 juveniles from the onset of exogenous feeding. Four diets differing in the level of retinyl acetate were tested: 0, 0.0025, 0.0050 or 0.0100 g kg^?1 diet, equivalent to 0, 2500, 5000 or 10 000 IU vitamin A kg^?1 diet, respectively. Each diet was tested on grouped or individually isolated crayfish. Crayfish fed the diet with 5000 or 10 000 IU vitamin A kg^?1 diet achieved the highest survival (average of grouped and isolated: 88.9%) and growth (grouped and isolated: 13.45 mm carapace length, 533.4 mg weight). Final growth of isolated crayfish was significantly higher than that of grouped crayfish for all diets. This study provides the first data on vitamin A requirements of freshwater crayfish. A level of 5000 IU vitamin A kg^?1 can be recommended for juvenile P. leniusculus during the first period of intensive rearing.     

Enhanced immune responses and effectiveness of refined outer membrane protein vaccines against Vibrio harveyi in orange‐spotted grouper (Epinephelus coioides)

Vibriosis is a severe infection occurring in many commercially important marine fish species. In this study, vaccines containing Vibrio harveyi recombinant outer membrane protein K (rOmpK), outer membrane protein U (rOmpU) and rOmpK‐OmpU fusion protein in addition to the metabolizable Montanide^TM ISA 763 A VG adjuvant were developed and evaluated in the orange‐spotted grouper. The results indicate that recombinant V. harveyi protein‐based vaccines resulted in a remarkably higher expression of IL‐1β and IL‐8 at 24 hr, and greater antibody production, as early as 2 weeks postimmunization. Notably, enhanced immune responses and significant protective efficacy against V. harveyi infections were observed in the fusion protein vaccine‐injected fishes with relative per cent survival value of 81.8%. Additionally, the rOmpK‐OmpU antisera presented a high bactericidal effect on not only V. harveyi, but also Vibrio parahaermolyticus and Vibrio alginolyticus. Our results demonstrated that the fusion protein rOmpK‐OmpU was an effective vaccine candidate that exhibited potentially great versatility for controlling vibrio infections.     

First isolation of Vibrio tapetis and an atypical strain of Aeromonas salmonicida from skin ulcerations in common dab (Limanda limanda) in the North Sea

Skin ulcerations rank amongst the most prevalent lesions affecting wild common dab (Limanda limanda) with an increase in prevalence of up to 3.5% in the Belgian part of the North Sea. A complex aetiology of these ulcerations is suspected, and many questions remain on the exact factors contributing to these lesions. To construct the aetiological spectrum of skin ulcerations in flatfish, a one‐day monitoring campaign was undertaken in the North Sea. Fifteen fish presented with one or more ulcerations on the pigmented and/or non‐pigmented side. Pathological features revealed various stages of ulcerations with loss of epidermal and dermal tissue, inflammatory infiltrates and degeneration of the myofibers bordering the ulceration, albeit in varying degrees. Upon bacteriological examination, pure cultures of Vibrio tapetis were retrieved in high numbers from five fish and of Aeromonas salmonicida in one fish. The V. tapetis isolates showed cross‐reactivity with the sera against the representative strain of serotype O2 originating form a carpet‐shell clam (Ruditapes descussatus). Moreover, the A. salmonicida isolates displayed a previously undescribed vapA gene sequence (A‐layer type) with possible specificity towards common dab. Further research is necessary to pinpoint the exact role of these agents in the development of skin ulcerations in common dab.     

A3α‐peptidoglycan extracted from Bifidobacterium sp. could enhance immunological ability of Apostichopus japonicus

To assess the effects of A3α‐peptidoglycan (A3α‐PG) extracted from Bifidobacterium sp. on the immune response and disease resistance of sea cucumber, different concentrations (0, 0.5, 5 and 50 mg mL^?1) of A3α‐PG suspensions were used to perform hypodermic injection on Apostichopus japonicus, followed by a Vibrio splendidus challenge. Total coelomocyte count (TCC), phagocytosis activity and activities of four immunological enzymes in both cell‐free coelomic fluid (extra‐cellular, EC) and coelomocyte lysate supernatant (intracellular, IC), including acid phosphatase (ACP), alkaline phosphatase (ALP), superoxide dismutase (SOD) and peroxidase (POD), were measured at 2, 6, 14 and 24 h post injection (hpi). The TCC was not significantly affected (P > 0.05) by A3α‐PG, ranging from 1.84 × 10⁶ to 3.53 × 10⁶ cells mL^?1. The coelomocyte phagocytosis activity was significantly activated (P < 0.05) in all the A3α‐PG treatments, whereas no significant difference was observed between them except 24 hpi (P > 0.05). The EC‐ACP activity in the 5.0 mg mL^?1 treatment increased significantly (P < 0.05) at all sampling times, while the IC‐ACP activity in the 50 mg mL^?1 treatment increased significantly (P < 0.05) at 2 hpi. Also, the 5.0 mg mL^?1 treatment had significant (P < 0.05) increase in the EC‐ALP activity within 14 hpi and the EC‐POD activity at 2 hpi, respectively, while significantly (P < 0.05) enhanced IC‐ALP and IC‐POD activities were observed in the 50 mg mL^?1 treatment within 6 hpi and at 2 hpi, respectively. Only the 5.0 mg mL^?1 treatment showed significant (P < 0.05) increase in the EC‐SOD activity at 2 hpi and IC‐SOD activity within 14 hpi, respectively. The challenge test showed that the animals treated with 50 mg mL^?1 of A3α‐PG had notably lower cumulative mortality after 14 days following V. splendidus exposure. All together, these results suggest that A3α‐PG could positively enhance immune response that effectively promotes the health status of A. japonicus against V. splendidus infection.     

Estimation of dietary vitamin A requirement of juvenile soft‐shelled turtle,Pelodiscus sinensis

A feeding trial was conducted to evaluate the dietary vitamin A requirement of the soft‐shelled turtle Pelodiscus sinensis. Turtles with an approximate body weight of 6.8 g were fed diets containing 0–4.58 mg kg^?1 (15 251 IU) vitamin A (retinyl acetate) for 8 weeks. Turtles fed diets without vitamin A showed the lowest growth performance. The animals gradually gained weight when dietary vitamin A increased from 0 to 3.6 mg kg^?1. Liver vitamin A gradually increased with supplementation of dietary vitamin A of up to 2.7 mg kg^?1. Beyond this concentration, the vitamin A in the liver significantly increased. Data analysis using a polynomial regression or exponential model showed that the estimated dietary vitamin A requirement for juvenile soft‐shelled turtles based on weight gain, red blood cell count and liver vitamin A measurements was 3.48, 2.84 and 2.58 mg kg^?1, respectively.     

Hepatoprotective and antioxidant effects of dietary Angelica sinensis extract against carbon tetrachloride‐induced hepatic injury in Jian Carp (Cyprinus carpio var. Jian)

The present study aimed to evaluate the hepatoprotective effects of Angelica sinensis extract (ASE) against carbon tetrachloride (CCl₄)‐induced hepatotoxicity in Jian carp (Cyprinus carpio var.Jian). Fish were fed diets containing four doses of ASE (0%, 0.1%, 0.5% and 1.0%) for 60 days, and then given an intraperitoneal injection of 30% CCl₄ in olive oil at a volume of 0.05 mL/10 g body weight. At 72 h post injection, blood and liver samples were collected for biochemical analysis, comet assay, histopathological examination and CYP3A mRNA expression. Results showed that the increases of glutamate pyruvate transaminase (GPT) and glutamate oxalate transaminase (GOT) induced by CCl₄ were significantly inhibited by pre‐treating the fish with 0.1%, 0.5% and 1.0% ASE in the diets. The elevation of lactate dehydrogenase (LDH) and the reductions of the total protein (TP) and albumin (Alb) in the serum induced by CCl₄ were also inhibited by pre‐treatments with 0.5 and 1.0% ASE. In the liver tissue, pre‐treatment with 1.0% ASE significantly inhibited the malondialdehyde (MDA) formation and the reductions of the total antioxidant capacity (T‐AOC), superoxide dismutase (SOD), glutathione (GSH) and CYP3A mRNA expression induced by CCl₄. Comet assay showed that tail moment, olive tail moment, tail length and tail DNA% were positively changed in fish pretreated with 0.5 and 1.0% ASE. CCl₄‐induced histological changes were obviously reduced by 0.5% and 1.0% ASE. Overall results prove the hepatoprotective effect of ASE in a dose‐dependent manner and support the use of ASE (1.0%) as a hepatoprotective and antioxidant agent in fish.     

Two epizootic Perkinsus spp. events in commercial oyster farms at Santa Catarina,Brazil

Perkinsus spp. have been detected in various bivalve species from north‐east Brazil. Santa Catarina is a South Brasil state with the highest national oyster production. Considering the pathogenicity of some Perkinsus spp., a study was carried out to survey perkinsosis in two oyster species cultured in this State, the mangrove oyster Crassostrea gasar and the Pacific oyster Crassostrea gigas. Sampling involved eight sites along the state coast, and oyster sampling was collected during the period between January 2013 and December 2014. For the detection of Perkinsus, Ray's fluid thioglycollate medium (RFTM) and histology were used, and for the identification of the species, PCR and DNA sequencing were used. Perkinsus spp. was found by RFTM in C. gigas and C. gasar from São Francisco do Sul. This pathology was also detected in C. gasar from Balneário Barra do Sul both, by RFTM and histology. Perkinsus marinus was identified in C. gigas and C. gasar from São Francisco do Sul and Perkinsus beihaiensis in C. gasar from Balneário Barra do Sul. This is the first report of P. marinus in C. gigas from South America. Results of this preliminary study suggest that both oyster species tolerate the species of Perkinsus identified, without suffering heavy lesions.     

Effects of dietary zinc oxide and selenium nanoparticles on growth performance,immune responses and enzyme activity in rohu,Labeo rohita (Hamilton)

A 120‐day feeding trial was conducted to assess the effects of dietary supplementation of zinc oxide (ZnO) and selenium (Se) nanoparticles (NPs) on growth, immunological and enzymatic profiles in rohu, Labeo rohita. Fishes were fed with basal diet incorporated with laboratory‐synthesized nanoparticles, viz., zinc oxide nanoparticles—10 mg/kg and Se nanoparticles—0.3 mg/kg throughout the experimental period. At an interval of every 30 days, treated and control fishes were randomly collected and subjected to estimation of growth, non‐specific immune parameters and serum enzyme assays. It was observed that there was significant (p < 0.05) increase in growth and non‐specific immune parameters like respiratory burst, lysozyme and myeloperoxidase activities as compared to the control group (only basal diet). Serum enzymatic profiles such as lactate dehydrogenase and alkaline phosphatase activities down‐regulated (p < 0.05) in treated group compared to control group, and superoxide dismutase and acetylcholine esterase activity up‐regulated in treated group. The relative percentage survival (RPS) found to be significantly higher in treated groups (60.00 ± 8.82%) as compared to control group (45.00 ± 6.17%) while challenged with virulent bacterial strain Aeromonas hydrophila. These results indicated that dietary supplementation of zinc oxide (ZnO) and selenium (Se) nanoparticles stimulates immunity and enhances resistance to bacterial infection in L. rohita.     

Effects of dietary inclusion of various concentrations of Scutellaria baicalensis Georgi extract on growth,body composition,serum chemistry and challenge test of far eastern catfish (Silurus asotus)

Effects of various concentrations of Scutellaria baicalensis (SB) extract in diets on growth, body composition, serum chemistry and disease challenge test of far eastern catfish (Silurus asotus) were determined and compared with a commercially available immune enhancer. Eight experimental diets were prepared in triplicate: control (Con) diet without supplementation of SB and SB‐0.25, SB‐0.5, SB‐1, SB‐2, SB‐3 and SB‐5 diets containing SB at concentrations of 0.25, 0.5, 1, 2, 3 and 5%, respectively. In addition, 0.1% of a commercial immune enhancer product (CP) was also tested. No significant difference in weight gain of fish was found. Feed consumption, feed efficiency ratio and protein retention of fish were not affected by the experimental diets. At the end of the 8‐week feeding trial, 10 externally normal fish from each tank were infected by Vibrio anguillarum or Strepotococcus iniae. Cumulative mortality of fish fed the Con diet was higher than that of fish fed the all other diets in 10 and 25 days after V. anguillarum or S. iniae infection. Results of this study indicated that dietary inclusion of SB extract was effective in improving survival of eastern catfish after V. anguillarum and S. iniae infection, but the various concentrations of SB did not affect fish performance.     

Functional identification of ToLAAO genes and polymorphism association analysis of Cryptocaryon irritans resistance in Trachinotus ovatus

‘Marine white spot disease’ is caused by Cryptocaryon irritans infection and can lead to high mortality in Trachinotus ovatus. L-Amino acid oxides (LAAOs) play a key role in antibacterial activity and parasitic activity. To investigate the function of the LAAO (ToLAAO) and LAAO-like (ToLAAO-like) genes of T. ovatus, this study explored the sequence characteristics and relationship between polymorphisms and traits of anti-C. irritans. The ToLAAO and ToLAAO-like ORF sequences obtained from the whole genome of T. ovatus were 1563 and 1584 bp, which encoded 520 and 527 amino acids respectively. Both sequences contained a highly conserved flavin adenine dinucleotide-binding domain and a similar amino oxidase domain. Sequence multiple alignment analysis showed that ToLAAO and ToLAAO-like had the highest homology to the LAAO sequence of Larimichthys crocea. Quantitative real-time polymerase chain reaction (qRT-PCR) results showed that ToLAAO and ToLAAO-like mRNA were generally expressed in 10 tissues. ToLAAO mRNA was highly expressed in the testis, while ToLAAO-like mRNA was highly expressed in muscle tissue. After C. irritans infection, ToLAAO and ToLAAO-like mRNA were significantly upregulated in the skin and spleen, while only ToLAAO mRNA was significantly upregulated in the liver and head kidney, and only ToLAAO-like mRNA was significantly upregulated in the gills. Five SNP sites were identified from the ToLAAO and ToLAAO-like genomic sequence fragments, and two sites (6200C/T and 6237G/A) of LAAO were significantly associated with resistance to C. irritans. These results suggest that ToLAAO and ToLAAO-like genes play crucial roles in defending against the immune response to C. irritans.     

Pathogenic threats and probiotic use in larviculture of the scallop,Pecten maximus

During a series of experiments, bacteriological elements in scallop larval rearing were investigated: larvae susceptibility to pathogens as a function of their age, and the use of probiotic bacteria during larviculture. Younger larvae (d5 PF) were highly more susceptible to pathogenic‐challenge than their older siblings, which were challenged at an older age (d15 PF). A challenge with 10⁴ CFU mL^?1 of V. pectenicida killed 100% of d5 PF larvae 7 days following challenge, yet killed only 9% of d15 PF larvae 9 days following challenge. Use of the probiotics Phaeobacter gallaeciensis, Alteromonas macleodii 0444 and Neptunomonas sp. 0536, provided for larger larvae, a high yield of competent larvae and, perhaps more importantly, protection against pathogen‐challenge similar to levels achieved from antibiotic use. When challenged with V. pectenicida, d29 survivals were 20.3%, 85.1% and 75.0% respectively for control (no probiotic), antibiotic treated, and ‘probiotic mix’ administered larvae. Use of potential probiotic Pseudoalteromonas sp. D41 appeared to hinder scallop larvae. Future use of probiotics in scallop larval rearing would benefit from combined use of P. gallaeciensis, A. macleodii 0444 and Neptunomonas sp. 0536.     

Effect of using Spirulina and Chlorella as feed additives for elevating immunity status of Nile tilapia experimentally infected with Aeromonas hydrophila

The present trial was performed to evaluate the effect of dietary incorporation of dried Spirulina, Chlorella and their mixture on the immune status of Nile tilapia (Oreochromis niloticus) before and after artificial infection with pathogenic bacterium Aeromonas hydrophila (A. hydrophila). Two hundred and forty fish were divided into four groups: (a) a control group fed on a basal diet only, (b) a second group fed on a diet containing Spirulina 15%, (c) a third group fed on a diet containing Chlorella 15%, and (d) a fourth group fed on a diet containing a mixture of both Spirulina 15% and Chlorella 15%. At the end of the experiment (9 weeks), the four groups were experimentally infected with A. hydrophila for 7 days. Antioxidant enzymes, lysozyme and bactericidal activities and histopathological changes were determined just before the challenge test and 7 days post‐challenge. Significant (p ≤ 0.05) increases in fish body protein% before the challenge test and increases in serum antioxidant enzymes, lysozyme and bactericidal activity in the Chlorella and algal mixture groups before and after the challenge test were observed. Spirulina, Chlorella and their mixture groups significantly decreased serum malondialdehyde compared to the control group before and after the challenge test. Using Spirulina, Chlorella and their mixture mitigated the necrotic and degenerative changes induced by A. hydrophila and revealed well‐developed and multiple melanomacrophage centres. Thus, dietary Spirulina, Chlorella and their mixture inclusion in Nile tilapia fish proved to have a protective effect against A. hydrophila infection.     

Recent advances of genome mapping and marker‐assisted selection in aquaculture

Aquaculture is the fastest growing sector in agriculture. Substantial genetic gains have been achieved in a few cultured species using conventional selective breeding approaches. However, the majority of fish and shellfish species remain in their wild state. Due to the recognition of the enormous potential of marker‐assisted selection (MAS) to speed up genetic gain through early selection, aquaculture scientists have constructed linkage maps in over 40 species and mapped quantitative trait loci (QTL) for important traits in over 20 species since the 1990s. Although MAS and genomic selection (GS) have not been widely used in aquaculture, their application in breeding programmes is expected to be a fertile area of research. In this paper, I summarized the recent advances of linkage and QTL mapping, as well as MAS in aquaculture species. I also discussed the potentials of genome‐wide association studies (GWAS) and GS in aquaculture species.