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.     

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.     

Hybridization between Haliotis rufescens and Haliotis discus hannai: evaluation of fertilization,larval development,growth and thermal tolerance

Two introduced abalone species are currently produced in Chile, red abalone Haliotis rufescens and Japanese abalone Haliotis discus hannai. However, red abalone accounts for 99% of total production, while the Japanese abalone has not adapted well to Chilean coastal waters. This study reports the hatching, growth and thermal tolerance performance in interspecific hybrids produced between red (R) and Japanese (J) abalone. Our results show that egg age and sperm concentration were critical factors to produce hybrids. The cross R♀ × J♂ showed a fertilization rate of 55.3 ± 3.5% using 20‐min‐old eggs and sperm concentrations of 14 × 10⁶ cells mL^?1, while the reciprocal cross (J♀ × R♂) was not successful. Further, larval development stages were similar in RR, JJ and RJ hybrid abalones. Among the experimental trials, settlement rate varied from 12.3% to 18.6% and final survival from 20.1% to 31.7%, being the RJ hybrid rates intermediate between parental species. The final shell lengths were similar between RR and RJ hybrids, but significantly higher in JJ abalones. In addition, thermal tolerance was ascertained due its pivotal role for the abalone physiology. Thus, RJ hybrids showed the highest HSP70 gene expression and offers new possibilities to expand Chilean abalone production in warm waters zones.     

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.     

Hen egg yolk and skinned krill as possible foods for rearing leptocephalus larvae of Anguilla japonica Temminck & Schlegel

Usual diets for rearing leptocephalus larvae of Japanese eel Anguilla japonica include eggs of the endangered spiny dogfish Squalus acanthias (SE). We investigated the effects of alternative food materials, hen egg yolk (HEY) and exoskeleton‐free (skinned) Antarctic krill (SAK), on the growth and survival of eel larvae. We found that feed comprising whole krill including exoskeleton (WAK) containing higher levels of fluoride (37.89 mg kg^?1) was acutely toxic to eel larvae exposed to this alone. In contrast, extract from SAK containing lower concentrations of fluoride (4.25 mg kg^?1) showed no apparent adverse effects. Growth of larvae fed a mixture of SE and SAK in a feed trial of 58 days [mean body weight (BW), 6.0 mg] was about twofold higher than that of larvae fed a mixture of SE and WAK (3.2 mg) (P < 0.01). A mixture of HEY and SAK also had some dietary benefits for eel larvae, enabling them to survive for up to 58 days and to grow significantly (mean BW, 2.4 mg), compared with their initial weight (mean BW, 0.2 mg) (P < 0.001). Although additional nutritional improvements are needed, the present results suggest that combination diet HEY and SAK may be a good alternative to SE as an effective diet for eel larvae.     

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.     

Time kinetics of fatty acid changes in phospholipids following enrichment and starvation of Artemia franciscana with a main focus on docosahexaenoic acid

The main objective was to study time kinetics of change in important highly unsaturated fatty acids (HUFAs) in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of Artemia franciscana nauplii and juveniles following enrichment and subsequent starvation. Samples of Artemia nauplii were taken at variable times (0.5–24 h) following enrichment and starvation. Samples of Artemia juveniles were taken after 2, 3 and 4 days of cultivation. No docosahexaenoic acid (DHA) was found in PC and PE of Artemia nauplii during the first hour of enrichment, while a significant (P < 0.05) increase was found in total lipids (TLs). The content of DHA in PC and PE increased thereafter steadily from 1 to 8 h of enrichment. DHA in PC and PE during enrichment (1–8 h) and following starvation (8–24 h), respectively, increased and decreased significantly (P < 0.05), but at a lower rate than that in TL. Moreover, juvenile Artemia (2–4 days) contained a relatively low level of DHA in TL compared with enriched Artemia nauplii, but the content of DHA in PC and PE was similar. The results open perspectives for both industry and science. For scientific studies, the lag phase in HUFA enrichment makes it possible to produce Artemia nauplii with variable relative HUFA enrichments in phospholipids and TL.     

Nutrient removal efficiency of Hydropuntia cornea in an integrated closed recirculation system with pink shrimp Farfantepenaeus brasiliensis

In this study, we have tested the effect of seaweed stocking density in an experimental seaweed biofilter using the economically important red seaweed Hydropuntia cornea integrated with the cultivation of the pink shrimp Farfantepenaeus brasiliensis. Nutrient removal efficiency was evaluated in relation to seaweed stocking density (2.5, 4, 6 and 8 g fw L^?1). Total ammonia nitrogen (TAN) was the main nitrogen source excreted by F. brasiliensis, with concentrations ranging from 41.6 to 65 μM of NH₄⁺‐N. H. cornea specific growth rates ranged from 0.8 ± 0.2 to 1.4 ± 0.5% day^?1 with lowest growth rates at higher seaweed stocking density (8 g fw L^?1). Nutrient removal was positively correlated with the cultivation densities in the system. TAN removal efficiency increased from 61 to 88.5% with increasing seaweed stocking density. Changes in the chemical composition of the seaweed were analysed and correlated with nutrient enrichment from shrimp effluent. The red seaweed H. cornea can be cultured and used to remove nutrients from shrimp effluents in an integrated multi‐trophic aquaculture system applied to a closed recirculation system. Recirculation through seaweed biofilters in land‐based intensive aquaculture farms can also be a tool to increase recirculation practices and establish full recirculation aquaculture systems (RAS) with all their known associated benefits.     

Effects of native medicinal herbs (Alternanthera sessilis,Eclipta alba and Cissus quadrangularis) on growth performance,digestive enzymes and biochemical constituents of the monsoon river prawn Macrobrachium malcolmsonii

The medicinal herbs such as Alternanthera sessilis, Eclipta alba and Cissus quadrangularis were used to assess the growth promoting ability, enhancement of muscle biochemical constituents and profiles of amino acid in the freshwater prawn, Macrobrachium malcolmsonii. The herbal powders were separately incorporated with basal diet, and these diets were fed to M. malcolmsonii postlarvae for a period of 90 days. At the end of experiments, the survival of prawn significantly improved in herbal incorporated feed. In addition, the growth parameters, such as weight gain, specific growth rate and protein efficiency rate were significantly (P < 0.05) higher in herbal diets fed groups when compared with control. The activities of digestive enzyme, biochemical constituents, profile of amino acid, energy utilization parameters, concentrations of vitamin C and E were significantly (P < 0.05) elevated in experimental diets fed groups when compared with control. The profiles of essential amino acids were also found elevated in herbal incorporated diets fed PL. Among the three herbs tested, C. quadrangularis incorporated diet fed group showed better performance followed by the E. alba and A. sessilis. The present study suggests that these herbs can be incorporated in artificial feed formulations for Macrobrachium culture.     

The efficacy of MS‐222 as anaesthetic agent in four freshwater aquarium fish species

The efficacy of anaesthetic tricaine methanesulfonate (MS‐222) was evaluated in four freshwater aquarium fish species, Zebrafish (Danio rerio), Guppy (Poecilia reticulata), Discu (Symphysodon discus) and Green swordtail (Xiphophorus helleri). The correct dose of anaesthetic should induce the plane 4 of anaesthesia in less than 180 s, recovery in less than 300 s and must survive when exposed during 30 min to anaesthetic. Fishes were exposed to six concentrations of anaesthetic (75, 100, 125, 150, 200 and 250 mg L^?1) and the time of fish reaching plane 4 of anaesthesia, post exposure recovery, and the percentage of survival when fish were subject to 30 min in the anaesthetic were recorded. The optimal doses varied according to the species: D. rerio – 75, 100 and 125 mg L^?1, P. reticulata – 125, 150 and 200 mg L^?1, S. discus – 75 and 100 mg L^?1 and X. helleri – 125 and 150 mg L^?1. The induction time generally decreased significantly with increasing concentration of MS‐222 for all of the species evaluated. The recovery time had a tendency to increase with the increase of the MS‐222 concentration for D. rerio, P. reticulata and S. discus. On the other hand, X. helleri recovery time decreased with the increase of MS‐222 concentration. MS‐222 proved to be effective in anaesthesia for all the freshwater ornamental species studied. The main results clearly show that the optimal dose to anesthetize is fish species dependent and it is completely wrong to extrapolate optimal anaesthetic concentrations between different species.     

Histopathological changes on the gills of asp (Aspius aspius) and European catfish (Silurus glanis) caused by Lamproglena pulchella and a Lamproglena sp. (Copepoda: Lernaeidae), respectively

In a parasitology survey of Hungarian fishes, heavy infections of parasitic copepods Lamproglena pulchella and a Lamproglena sp. were found in the gills of the asp and the European catfish, respectively. Individuals of both fish species were emaciated and infected with hundreds of Lamproglena. Copepods located close to the tip of gill filaments and formed a depression at the attachment sites. In histological sections, cell degenerations and local haemorrhages were present adjacent to the maxillipeds and where the maxillary claws pierced the gill tissue. Around maxillae and in the midgut of the Lamproglena, damaged piscine blood cells and remains of the gill tissue were observed. Host reaction was expressed by proliferation of epithelioid cells, increase in both number and size of goblet and mast cells and formation of giant cells.     

Co‐Infection with Pseudomonas anguilliseptica and Delftia acidovorans in the European eel,Anguilla anguilla (L.): a case history of an illegally trafficked protected species

Inspections by customs agents at Barcelona airport discovered 420 kg of contraband glass eels prepared for shipment to Hong Kong. After confiscation of these animals by police, they were transported to holding facilities to be maintained until after a judicial hearing. Upon arrival, they were separated into two groups and held under ambient flow‐through conditions in fresh water. During their captivity period, several peaks in mortality occurred and multiple bacterial strains were isolated from moribund animals. Sequencing of 16S rDNA was used to determine specific identity of the isolates. An initial isolation of Pseudomonas anguilliseptica was treated with oxytetracycline. A subsequent isolation of Delftia acidovorans proved resistant to oxytetracycline and was treated with gentamicin in combination with sulphadiazine–trimethoprim. Once the health condition of the animals was stabilized, they were partitioned into groups and subsequently released as part of a restocking effort for the species following the guidelines of Regulation (EC) 1100/2007 (Anon 2007). This represents the first record for both bacterial species in the host Anguilla anguilla in the Spanish Mediterranean.     

Comparison of mudcrab‐based brackishwater polyculture systems with different finfish species combinations in Sundarban,India

To compare production and economic performance of polyculture systems with different species combinations, a 210‐day trial was carried out. In the first combination (T1), milkfish (Chanos chanos) and mudcrabs (Scylla serrata), and in the second (T2), mullets (Mugil cephalus, Liza tade and Liza parsia at 0.5:0.5:0.5 ratio) and mudcrabs were stocked keeping fish and mudcrabs at 15,000 numbers per ha, respectively, in both treatments. The finfish were fed floating pellet at 2%–3% and mudcrabs were fed fresh and farm made feed at 5%–8% body weight. Growth parameters of mudcrabs were similar in both T1 (407.64 ± 105.78 g) and T2 (418.89 ± 105.24 g), with no significant differences. Among finfish, M. cephalus attained highest final body weight, 241.55 ± 26.44 g followed by milkfish, 200.46 ± 11.82 g whereas lowest growth noticed in L. parsia (63.69 ± 6.62 g). Length–weight analysis of fish indicated negatively allometric growth (b < 3) for grey mullets, parsia and milkfish while L. tade recorded perfect cube low (b = 2.99). Male mudcrabs recorded positive allometric (b = 3.3) and female crab exhibited negative allometric growth (b = 2.68). The total productivity was 4,533 and 3,694 kg/ha with mudcrab contributes 53.69% and 60.56% to the total productivity in T1 and T2 respectively. The economic analysis indicated benefit–cost ratio (BCR) of 1.57 and 1.73 in T1 and T2 respectively with 10% and 35% insignificant increase (p > 0.05) in BCR and profit per kg, respectively, in T2 compared with T1. The study elucidates polyculture of mudcrabs with finfish can be a taken up as a profitable venture for sustainable diversification of brackishwater farming in Sundarbans.     

Probiotic activity of bacteria associated with egg capsules of Concholepas concholepas (common name ‘Loco’)

Egg capsules of Concholepas concholepas were analysed microbiologically, showing the presence of bacteria inside the capsules. The bacteria were isolated and identified, and their probiotic potential on early larvae of C. concholepas was evaluated. From 32 capsules that were examined, 45 bacterial strains were isolated, of which 67% belonged to the genus Bacillus. Survival bioassays were carried out with all the isolated bacterial strains on C. concholepas larvae. It was found that 71% of the bacteria had positive effects on larval survival. This research determined that bacteria of the genera Bacillus and Vibrio present in the capsules have probiotic potential. The bacteria that led to the greatest larval survival were Bacillus pumilus, which may be used in the cultivation of C. concholepas, because one of the limiting factors for the cultivation of this species is larval survival; thus, we propose that the probiotic strains identified in this study will be used during the cultivation of C. concholepas to enhance the chances of larval survival.     

First report of Shewanella sp. and Listonella sp. infection in freshwater cultured loach,Misgurnus anguillicaudatus

A novel disease outbreak occurred among cultured loach, Misgurnus anguillicaudatus in China. The diseased fish displayed lethargy, loss of appetite, black discolouration, anasarca and ventral skin hyperaemia. Clinical signs were observed in most of the fish examined, which consisted of discoloured liver, swollen spleen and petechial haemorrhage in the intestine and peritoneal membranes. The causative agent was found to be two species of bacteria by experimental infection, identified as Shewanella sp. and Listonella sp. The result showed that these were likely to be Shewanella putrefaciens and Listonella anguillarum respectively. Bacterial identification consisted of physiological and biochemical tests as well as 16S rRNA sequence analysis. In addition, antibiotic susceptibility testing was performed, revealing that Shewanella putrefaciens and Listonella anguillarum were susceptible to enrofloxacin, ciprofloxacin, levofloxacin, pipemidicacid and norfloxacin. This is the first report of S. putrefaciens and L. anguillarum from cultured loach.     

Acute toxicity of ammonia in Meagre (Argyrosomus regius Asso, 1801) at different temperatures

Argyrosomus regius (3.0 ± 0.9 g) were exposed to different concentrations of ammonia in a series of acute toxicity tests by the static renewal method at three temperature levels (18, 22 and 26°C) at a pH of 8.2. Low temperature clearly increased the tolerance of the fish to total ammonia nitrogen (TAN) and unionized ammonia (NH₃) (P < 0.05). While the 96‐h LC₅₀ values of TAN were 19.79, 10.39 and 5.06 mg L^?1, the 96‐h LC₅₀ of NH₃ were 1.00, 0.70 and 0.44 mg L^?1 at 18, 22 and 26°C respectively. The safe levels of NH₃ for A. regius was estimated to be 0.10, 0.07 and 0.04 mg L^?1 at 18, 22 and 26°C respectively (P < 0.05). This study clearly indicates that A. regius is more sensitive to ammonia than other marine fish species cultured on the Mediterranean and Eastern Atlantic coasts.     

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.     

Identification and characterization of pathogens associated with fin erubescence and ulceration of cultured Scophthalmus maximus

Scophthalmas maximus is a precious and main aquacultural species in northern China with an annual output value of more than 1.5 billion yuan. However, with the expansion of breeding range, increased stocking density and variety degradation, a variety of bacterial, parasitic and viral diseases have often occurred and worsened. Among them, bacterial disease is the most serious in farmed S. maximus. Since 2010, an epidemic bacterial disease with primary symptoms of lighter colour of lateral and caudal fins, spotty bleeding, fin base bleeding, abdominal swelling and redness and lesions of internal organs has put broken in S. maximus cultured in Weihai, Longkou and Haiyang, Shandong Province, China. The disease occurred mostly at the seedling and early development stages and propagated very fast, resulting in high mortality. These S. maximus died with obvious surface ulceration in only 3–4 days, causing great economic losses to the aquaculture industry. Beginning in 2010, we conducted epidemiological investigation in farmed S. maximus and isolated two different pathogens from the body of the diseased S. maximus from different farms. In this paper, we observed their morphology, conducted phylogenetic analysis based on their physiological and conventional biochemical characteristics and 16S rRNA gene sequences, preliminarily analysed their pathogenesis and investigated the histopathological changes of the diseased S. maximus. We believed that Vibrio anguillarum and V. parahaemolyticus were the pathogenic bacteria for the epidemic disease in S. maximus.