A Practical Approach for Sexing Zebrafish,Danio rerio

This study aimed at a simple, rapid, reliable, practical, and non-destructive sexing technique for mature zebrafish (0.61 ± 0.011 g each). In the first experiment, 36 fish of each sex were selected on the basis of the presence of a prominent genital papilla on mature females and bred randomly. All the couples laid fertilized eggs with a mean fecundity of 304 eggs per female, for a 100% sexing accuracy. In the second experiment, 140 mature zebrafish were randomly selected and individually sexed like in the first experiment. Each fish was then dissected in order to confirm the reliability of this sexing technique through the observation of either the ovaries or the testes. Upon dissection, an accuracy of 100% was recorded. This study demonstrates that sexing mature zebrafish only on the basis of the presence of a prominent genital papilla on the mature female is a reliable technique.     

Effect of Dietary N‐acyl Homoserin Lactonase on the Immune Response and the Gut Microbiota of Zebrafish,Danio rerio,Infected with Aeromonas hydrophila

Quorum sensing interference is a novel strategy in bacterial disease control. Degradation of quorum sensing signal molecules, such as N‐acyl homoserine lactones (AHLs), by enzymes is a potential interfering method. In this study, the in vivo effect of an N‐acyl homoserine lactonase from Bacillus sp. B546, AiiA_B546, on the protection of zebrafish, Danio rerio, against Aeromonas hydrophila infection was first investigated by co‐feeding. The short‐term immune responses and the long‐term effects on the gut microbiota of zebrafish were analyzed by quantitative polymerase chain reaction (qPCR) and denaturing gradient gel electrophoresis (DGGE), respectively. The results showed that the expression levels of some anti‐infectious immunity‐related factors, including interleukin 10 (IL‐10), toll‐like receptor 5b (TLR5b), and inducible nitric oxide synthase 2a (iNOS2a) that were related to A. hydrophila virulent factors under regulation of quorum sensing, were more susceptible to the addition of AiiA_B546. It indicated that AiiA_B546 may attenuate the infection of A. hydrophila by interfering with quorum sensing. A. hydrophila was not detected in the enzyme co‐feeding diet group, which might be due to its inability to adhere to the gut with enzyme interference. This study revealed that AiiA_B546 as a feed additive might represent a novel biocontrol agent in aquaculture; however, the topic merits further investigations using fish species of commercial interest.     

Development of a two‐step,non‐probed multiplex real‐time PCR for surveilling Vibrio anguillarum in seawater

Vibrio anguillarum is an aggressive and halophilic bacterial pathogen most commonly originating from seawater. Vibrio anguillarum presence in fisheries and aquaculture facilities causes significant morbidity and mortality among aquaculture species primarily from haemorrhaging of the body and skin of the infected fish that eventually leads to death, collectively recognized as the disease vibriosis. This study served to develop a non‐probe, multiplex real‐time PCR assay to rapidly detect V. anguillarum presence in seawater. Specific primers targeting genes vah1, empA and rpoN of V. anguillarum were selected for multiplex reaction among 11 different primer sets and the extension step was eliminated. Primer concentration, denaturation time as well as annealing time and temperature of DNA amplification were optimized, thus reducing reaction duration. The two‐step, non‐probed multiplex real‐time PCR set forth by this study detects as little as 3 CFU mL^?1 of V. anguillarum presence in sea water, without enrichment cultivation, in 70 min with molecular precision and includes melting curve confirmation.     

Dietary biotin requirement for growth of juvenile zebrafish Danio rerio (Hamilton‐Buchanan)

This study was conducted to estimate the dietary biotin requirement for maximum growth of zebrafish Danio rerio. Six isonitrogenous and isocaloric purified diets containing 0.031 (biotin‐unsupplemented diet), 0.061, 0.263, 0.514, 1.741 and 2.640 mg biotin kg^?1 diet were fed in triplicate tanks for a total of 12 weeks to juvenile zebrafish (initial mean body mass 0.13 ± 0.001 g). From 4 weeks of feeding, fish fed diets with ≤0.061 mg biotin kg^?1 showed biotin deficiency signs, such as retarded growth and skeletal deformity, was observed on some dead fish. At the end of the study, gill disorders were observed on some fish and liver glycogen accumulation were also observed in fish fed these diets. Fish fed the biotin‐unsupplemented diet exhibited a lower final body weight, protein efficiency ratio and feed utilization than the fish fed the biotin‐supplemented diets, whereas the highest values were observed with the diet containing 0.51 mg biotin kg^?1 diet (P < 0.05). A linear relationship (r² = 0.77; P < 0.0001) was observed between whole‐body biotin content and dietary biotin level. A broken‐line analysis indicated that the optimum dietary biotin content for maximal growth expressed as final body weight is 0.51 mg kg^?1 diet.     

Vibrogen‐2 vaccine trial in lumpfish (Cyclopterus lumpus) against Vibrio anguillarum

Lumpfish (Cyclopterus lumpus), a native fish of the North Atlantic Ocean, is utilized as cleaner fish to biocontrol sea lice infestations in Atlantic salmon aquaculture. However, bacterial infections are affecting cleaner fish performance. Vibrio anguillarum, the aetiological agent of vibriosis, is one of the most frequent bacterial infections in lumpfish, and effective vaccine programmes against this pathogen have been identified as a high priority for lumpfish. Vibrogen‐2 is a commercial polyvalent bath vaccine that contains formalin‐inactivated cultures of V. anguillarum serotypes O1 and O2, and Vibrio ordalii. In this study, we evaluated Vibrogen‐2 efficacy in lumpfish against a local isolated V. anguillarum strain. Two groups of 125 lumpfish were bath‐immunized, bath‐boost‐immunized at four weeks post‐primary immunization, and intraperitoneally (i.p.) boost‐immunized at eight weeks post‐primary immunization. The control groups were i.p. mock‐immunized with PBS. Twenty‐seven weeks post‐primary immunization, the fish were i.p. challenged with 10 or 100 times the V. anguillarum J360 LD₅₀ dose. After the challenge, survival was monitored daily, and samples of tissues were collected at ten days post‐challenge. Commercial vaccine Vibrogen‐2 reduced V. anguillarum tissue colonization and delayed mortality but did not confer immune protection to C. lumpus against the V. anguillarum i.p. challenge.     

牙鲆(Paralichthys olivaceus) Hsp90 mRNA在温度刺激和鳗弧菌感染下的表达特征

利用 qPCR 方法,检测 Hsp90α和 Hsp90β mRNA 在不同月龄牙鲆(Paralichthys olivaceus)不同组织的表达水平,进一步检测在不同温度刺激、鳗弧菌感染后的表达变化。结果显示,Hsp90α和 Hsp90β在不同发育期的13种组织(肝脏、脾脏、头肾、后肾、心、肌肉、背皮、胃、肠、鳃、腹鳍、脑和血液)中均有表达。Hsp90β表达水平整体高于 Hsp90α;8月龄牙鲆在肝、鳃、背皮、背肌、腹鳍的 Hsp90α表达量要显著高于其他发育期(1月龄、12月龄、24月龄)同组织的表达量(P<0.05),而 Hsp90β在不同月龄牙鲆的表达量也存在差异,在肝和肠的表达量较高,在鳃、背肌和腹鳍的表达量较低。将8月龄牙鲆在5–32℃刺激1 h,牙鲆肝、脾 Hsp90α在高温(22–32℃)的表达量显著升高(P<0.05),而 Hsp90β在低温（5–10℃）的表达量显著升高(P<0.05);在10℃、28℃连续刺激8 h, Hsp90α和 Hsp90β的 mRNA 的表达量呈先上升后下降的趋势,Hsp90α的表达量变化幅度显著高于Hsp90β(P<0.05)。在鳗弧菌感染72 h 内,牙鲆脾脏 Hsp90α和 Hsp90β的表达量先下降后上升,Hsp90β上升的幅度更加显著(P<0.05)。结果显示,Hsp90α和 Hsp90β在不同月龄、不同组织的表达水平存在差异,Hsp90α在高温时对热应激的反应明显,Hsp90β在低温和对病原菌感染的反应明显,研究结果可为了解牙鲆 Hsp90的作用机制提供参考。     

Effect of water hardness on peracetic acid toxicity to zebrafish, Danio rerio, embryos

The use of peracetic acid (PAA) in aquaculture has been suggested as an alternative therapeutic agent. Few data are available concerning fish toxicity by PAA or factors that modify this toxicity. The aim of this study was to investigate the influence of water hardness on the acute toxicity of PAA products to embryos of zebrafish (Danio rerio). Embryos were exposed to PAA ranging from 0 to 9 mg/L in low-hardness (1.4 °dH or 25 mg/L hardness as CaCO₃), medium-hardness (14 °dH or 250 mg/L hardness as CaCO₃) and high-hardness (140 °dH or 2,500 mg/L hardness as CaCO₃) waters. The lowest LC50 value was 2.24 mg/L PAA in the low-hardness water, and the highest LC50 value was 7.14 mg/L PAA in the high-hardness water. Toxicity of PAA to embryos was found to be negatively correlated with water hardness. The pH decreased with increasing concentrations of PAA, and the test waters were observed to become more acidic in low hardness. In conclusion, aquaculturists using PAA should pay attention to water hardness to avoid acidosis.     

Comparison of Protective Efficacy between Formalin‐killed and aroA Gene‐knockout Vibrio anguillarum Vaccines in Olive Flounder,Paralichthys olivaceus

The aro genes in bacteria encode enzymes needed for the biosynthesis of aromatic amino acids, and mutant bacteria that are defective in the enzymes can replicate only a limited number in vertebrates owing to the lack or scarceness of chorismate, through which the mutant bacteria of the aro genes become attenuated. In the present study, the 5‐enolpyruvylshikimate‐3‐phosphate synthase (aroA) gene‐knockout Vibrio anguillarum (ΔaroA V. anguillarum) were generated by the allelic exchange method, and its vaccine potential was evaluated in the olive flounder, Paralichthys olivaceus, by comparing the protective efficacy of a formalin‐inactivated V. anguillarum. The LD₅₀ (50% lethal dose) value of ΔaroA V. anguillarum was 1000 times higher than that of wild‐type V. anguillarum in olive flounder fingerlings, and the growth of ΔaroA V. anguillarum was significantly suppressed by coincubation with nonimmune olive flounder serum compared with that of wild‐type V. anguillarum. The survival rates and serum agglutination titers of fish immunized with ΔaroA V. anguillarum were significantly higher than those of fish immunized with the same amount of formalin‐inactivated V. anguillarum, suggesting that although the inactivated V. anguillarum vaccine can provide a high protection in olive flounder, the protective efficacy can be enhanced by immunization with an auxotrophic mutant ΔaroA V. anguillarum.     

Bacterial host interaction of GFP‐labelled Vibrio anguillarum HI‐610 with gnotobiotic sea bass,Dicentrarchus labrax (L.), larvae

The location and cell damage caused by Vibrio anguillarum, the causative agent of classical vibriosis, within the developing gut of the newly hatched sea bass, Dicentrarchus labrax (L.), is unknown. A gnotobiotic sea bass model was used to investigate the early interactions of V. anguillarum with sea bass larvae. In the present study, germ‐free sea bass larvae were orally exposed to a V. anguillarum HI‐610 pathogen labelled with the green fluorescent protein (GFP‐HI‐610) and sampled at regular intervals. Pathogenic colonization of gut enterocytes was observed 2 h post‐exposure (p.e.) and onwards, whereas bacteria within the swim bladder were visualized 48 h p.e and onwards. Ultrastructural findings demonstrated direct bacterial contact with the host cell in the oesophageal mucosa and putative attachment to microvilli of mid‐ and hindgut enterocytes. The present findings form a starting point for studies assessing the impact of potential candidates (probiotics, prebiotics, antimicrobial peptides) to mitigate bacterial virulence.     

Immune Responses to Vibrio anguillarum in Yellowtail Kingfish,Seriola lalandi,Fed Selenium Supplementation

Effects of dietary selenium (Se) on immune competence of yellowtail kingfish, Seriola lalandi, were investigated. The fish were fed one of the three experimental diets including a control diet without Se supplementation and two diets supplemented with Se from Se‐yeast (Selplex) at 2 and 4 mg/kg. After feeding for 6 wk, the fish were challenged by injecting Vibrio anguillarum and observed for 2 wk. Dietary Se had no effect on feed intake, feed conversion ratio, and survival over the course of 6‐wk feeding; however, it significantly increased weight gain and Se content in muscle. Following the bacterial infection, the immune‐stimulating effects of Se were observed in antibody, lysozyme, and bactericidal responses, and there was a corresponding increase in survival and hematocrit by Se. Under infectious condition, antioxidant capacity of fish as measured in terms of resistance of red blood cells to peroxidation and glutathione peroxidase activity also increased by supplementation of Se. Liver necrosis and kidney melano‐macrophages were only seen in surviving fish fed the control diet after the challenge. Furthermore, there was evidence of myopathy in fish fed the diet without Se supplementation. This study suggests that Se, supplemented at 2 or 4 mg/kg, can improve growth and health of yellowtail kingfish.     

斑马鱼和鲢鱼对微囊藻毒素与孔雀石绿的行为反应

鱼类行为能准确地反映环境变化,并且随着污染物浓度的变化而发生改变,因此将毒素敏感鱼类的行为作为标记,用以监测环境是可行的。本研究用人工饲养,对环境毒素敏感的斑马鱼和对环境毒素有较强吸收能力的鲢鱼作为指示生物,检测其行为对水体中微囊藻毒素(MC)和孔雀石绿(MG)的反应。结果表明,斑马鱼和鲢鱼在2 µg/L MC浸泡下未出现显著的行为反应,而在5 µg/L的MC浸泡下,斑马鱼摆尾频率在10 min 后,移动速度在40 min后显著性下降;鲢鱼摆尾频率与移动速度均在40 min后显著性下降。另一方面,0.5 mg/L MG处理下,斑马鱼未出现显著性行为变化,而鲢鱼摆尾频率与移动速度均在35 min后显著性下降。     

1,3‐1‐6 ß‐glucans enhance tissue regeneration in zebrafish (Danio rerio): Potential advantages for aquaculture applications

High aquacultural rearing density and handling of fish may frequently result in skin or gills wounds, thereby facilitating the onset of secondary infections. The capacity of the zebrafish to regenerate tissues, as well as fins and other organs, makes it an ideal animal model for studying the mechanisms of tissue regeneration. Since macrophages are involved in tissue regeneration, a diet including ß‐glucans might positively affect the process through activation of macrophages and other immune pathways. Consequently, the aim of this study was to investigate the effects of the oral administration of 1,3‐1,6 β‐glucans on the regeneration process of the caudal fin after its amputation in zebrafish. One hundred and twenty zebrafish were randomly distributed into four groups with three replicates each: an untreated non‐amputated group (CNA) and an untreated amputated group (CA) fed a control diet; two treated and amputated groups (MI and MII) fed for 14 days the same diet with the addition of two differently extracted 1,3‐1,6 ß‐glucans (MacroGard^® and Experimental MacroGard^®, Biorigin^©). ß‐glucans were added to allowed the administration of 12.5 mg/kg of fish body weight (0.35 g/kg of feed). Results showed that 1,3‐1,6 ß‐glucans decreased fish mortality rate and enhanced both daily and cumulative regenerated fin area, independent of the specific ß‐glucan extraction method used. Based on the mechanisms similarities of the innate immune system and tissue regeneration among different teleost species, these results may likely be extended to species of interest for the aquaculture sector.     

Delivering oxytetracycline to first-feeding zebrafish, Danio rerio (Hamilton), and goby, Asterropteryx semipunctata Rüppell, larvae using lipid spray beads

Lipid spray beads (LSB) were evaluated for delivery of the low-molecular weight, water-soluble antibiotic, oxytetracycline·HCl (OTC) to fish larvae. Various OTC core-to-lipid ratios and OTC core concentrations were evaluated to maximize OTC delivery efficiency by LSB. Acceptability and digestion of LSB containing OTC and riboflavin by larval zebrafish, Danio rerio (Hamilton), and larval gobies, Asterropteryx semipunctata Rüppell, were also evaluated. Increasing LSB core-to-lipid ratios from 1:3 to 1:1 v/v resulted in an increase in encapsulation efficiency (EE) from 2.33 to 3.68% w/w. Increasing OTC concentrations of core solutions from 0.1 to 0.5 g OTC mL⁻¹ increased EE from 3.95 to 18.77% w/w, respectively. Although retention efficiency (RE) was unaffected by this increase, delivery efficiency was increased to 7.9 ± 0.7% w/w, after correcting for leakage losses because of the suspension of beads in water for 60 min. Consumption of LSB containing OTC by first-feeding zebrafish and goby larvae was confirmed by analysis of feeding incidence and gut fullness. Visual observations of larvae fed on LSB containing riboflavin indicated that larvae of both species digested LSB. Zebrafish larvae fed on OTC LSB contained 39.3 ± 2.5 ng OTC after purging LSB from their guts. Use of LSB provides an effective means of delivering therapeutics to fish larvae and could greatly reduce required doses compared with current methods of immersing larvae in solutions of therapeutic agents.     

Effects of embryonic exposure to α‐lipoic acid or ascorbic acid on hatching rate and development of zebrafish (Danio rerio)

This study investigates the effects of embryonic exposure to two different antioxidants on growth and development in fish. Zebrafish (Danio rerio) embryos (100 per group) were exposed to lipoic acid (LA, 6–12 μM) or ascorbic acid (AA, 100–200 μM) and the hatching rate, standard lengths (SL) at hatching, development and growth post‐hatching monitored. The SLs at hatching were increased (P<0.05) in both antioxidant‐exposed groups relative to the controls, with no effect on yolk reserves. This enhanced development persisted up to 15 days post hatching. At hatching, cell proliferation rates (P<0.0005) and basic fibroblast growth factor (P<0.001), were greater in the antioxidant‐exposed fish than in the controls (0 μM antioxidant); no oxidative DNA damage was detected (P>0.05). Activity of the endogenous antioxidant enzyme superoxide dismutase was greater (P<0.001) in LA‐treated fish than in the controls. The results suggest that embryonic treatment of zebrafish with LA or AA during embryogenesis enhanced cell proliferation, leading to increased somatic growth in the larval stages, persisting into the juvenile stage. The findings support the treatment of embryonic fish with antioxidants for enhanced results in aquaculture.