Toxicity of a synthetic phenolic antioxidant,butyl hydroxytoluene (BHT), in vertebrate model zebrafish embryo (Danio rerio)

The synthetic antioxidant 3,5‐di‐tert‐butyl‐4‐hydroxytoluene (BHT) is widely used as an additive in the food, cosmetic and plastic industries to increase the tenability of food and plastic for the past 70 years. BHT is degraded to 3,5‐di‐test‐butyl‐4‐hydroxybenzaldehyde (BHT‐CHO) in mammals, as well as in the natural environment such as in river and water. The average daily intake of BHT for human being is estimated to be 0.3 mg/kg body weight. Even though it is considered safe for human at authorized level, but its ubiquitous presence in the aquatic environment and the controversial toxicological data are of great concern for human as well as aquatic life. The experimental findings of zebrafish embryo toxicity test (ZFET) showed that the acute toxicity of 96‐hr (LC₅₀) exposure during the embryogenic stage was found to be 4.388 mg/L and the effective concentration (EC₅₀) was 1.375 mg/L. The reduce heart rate from the sublethal concentrations indicates the chemical to be cardiotoxic but a further review is to be needed. The Teratogenic Index (TI) calculated to be 3.19, which implies the compound may be a potential teratogen in aquatic life. The findings obtained in this study will stretch more evidence regarding developmental toxicity of BHT, which will be of much importance in further risk assessment of ecotoxicological studies.     

Detection and partial characterization of a UV-damaged-DNA binding activity highly expressed in zebrafish (Danio rerio) embryos

The expression of distorted DNA-binding factors was studied in developing zebrafish (Danio rerio) using UV-damaged DNA as the binding target. A strong and high-shifting binding activity was detected in the extracts of zebrafish early embryos (12 h after fertilization), and the expression of this activity dramatically decreased in 60 to 84-h-old zebrafish. The embryonic extracts produced a similar pattern of high-shifting complexes after incubating with a CPD-specific or a 6-4PP-specific probe, while different types of low-shifting complexes were generated by the extracts of 84-h-old larvae. The formation of high-shifting complexes was suppressed in the presence of NaCl at 0.25 M or higher concentrations, yet the production of low-shifting complexes was stimulated by increasing salt concentration. The binding activity expressed in zebrafish embryos was apparently unrelated to NER-associated damage-recognition protein XPA, since two polypeptides recognized by an anti-human XPA antibody were detected only in 84-h-old zebrafish extracts. A competitive binding assay indicated that both CPDs and 6-4PPs were recognized by the same binding activity expressed in 12-h-old zebrafish, and this activity contained at least two protein fractions that were eluted from a DEAE-cellulose column by NaCl at 0.1 M and 0.2 M. UV crosslinking of the two NaCl eluates to a 6-4PP probe produced covalent complexes with the same electrophoretic mobility except one 34-kDa complex generated by the 0.1 M NaCl eluate, suggesting the existence of two multisubunit damage-recognition protein complexes in zebrafish embryos. UV-binding factors found in 12-h-old zebrafish embryos may be involved in processing developmental stage-specific DNA structures similar to UV-damaged DNA. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Experimental exposure of zebrafish, Danio rerio (Hamilton), to Mycobacterium marinum and Mycobacterium peregrinum reveals the gastrointestinal tract as the primary route of infection: a potential model for environmental mycobacterial infection

The natural route by which fish become infected with mycobacteria is unknown. Danio rerio (Hamilton) were exposed by bath immersion and intubation to Mycobacterium marinum and Mycobacterium peregrinum isolates obtained from diseased zebrafish. Exposed fish were collected over the course of 8 weeks and examined for the presence of mycobacteriosis. Mycobacteria were consistently cultured from the intestines, and often from the livers and spleens of fish exposed by both methods. Mycobacteria were not observed in the gills. Histological analysis revealed that fish infected with M. marinum often developed granulomas accompanied by clinical signs of mycobacteriosis, while infection with M. peregrinum infrequently led to clinical signs of disease. Passage of the bacteria through environmental amoebae (Acanthamoeba castellani) was associated with increased growth of M. peregrinum over the course of 8 weeks, when compared to infection with the bacteria not passed through amoebae. The results provide evidence that zebrafish acquire mycobacteria primarily through the intestinal tract, resulting in mycobacterial dissemination.     

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.     

Effects of the wood extractive betulinol and 17beta-oestradiol on reproduction in zebrafish, Danio rerio (Hamilton)--complications due to a bacterial infection

Zebrafish were exposed to the wood extractive betulinol (5 microg L(-1)) and to 17beta-oestradiol (E2, 0.27 microg L(-1)) for 8 weeks in an attempt to study the possible endocrine-disrupting activity of betulinol. Females exposed to betulinol showed increased spawning intensity, while males exposed to betulinol and E2 had increased incidences of structural alterations in the testes. However, histological examination of the fish revealed that they were infected by acid-fast bacteria suspected to be Mycobacterium sp. despite a careful examination of their health state prior to the onset of the experiment. Fish exposed to betulinol and E2 showed more serious consequences of the bacterial infection than control fish indicating that the test chemicals had weakened the immune defence of the fish. When the exposure was repeated with healthy fish, an increase in the proportion of spermatogonia was seen in the testes of betulinol-treated males. A similar alteration, although not statistically significant, was also seen in the first experiment. However, no increase in the incidences of structural alterations in the testes was seen in betulinol- and E2-treated fish in the second experiment. Our study indicates that betulinol might have an endocrine-disrupting effect in zebrafish, but the increase in incidences of structural alterations in the testes might have been caused by a synergistic action between the test compounds and the bacterial infection. Our study stresses the importance of carefully checking the health of experimental fish, not only prior to the onset of an experiment but also upon termination of the experiment, in order to avoid misinterpretation of the results.     

The effects of Coriandrum sativum L. as feed additive on mucosal immune parameters,antioxidant defence and,immune‐related genes expression in zebrafish (Danio rerio)

This study investigates the effects of Coriander (C. sativum) on the growth, antioxidant, and immune‐associated genes and serum and mucosal immune parameters in zebrafish (D. rerio). The experimental fish were treated with 0 [control], 5, 10 and 20 g/kg coriander supplemented diets for 8 weeks. The results revealed that coriander remarkably increased mucosal immune parameters (the total Immunoglobulin, protease and lysozyme activity). The highest levels of the measured parameters were noticed in 20 g/kg CP treatment. Also, the zebrafish fed 20 g/kg coriander powder showed significantly higher expression gene for lysozyme and TNF‐alpha. The same results were noticed in case of IL‐1B gene expression. In case of sod gene expression there was no significant difference between treatments. However, regarding cat gene expression, significant difference was noticed between 20 g/kg CP treatment and other groups. In addition, no significant changes were observed between coriander fed zebrafish and control treatments regarding GH gene expression level, although IGF‐I remarkable up‐regulate in 20 g/kg coriander powder treatment compared other groups. In conclusion, it can be proposed that dietary Coriander powder can improve mucosal immune parameters and immune and antioxidant genes expression.     

Field emission scanning electron microscopy and transmission electron microscopy studies of the chorion, plasma membrane and syncytial layers of the gastrula-stage embryo of the zebrafish Brachydanio rerio: a consideration of the structural and functional relationships with respect to cryoprotectant penetration

The structure of the chorion and plasma membranes of gastrula‐stage zebrafish Brachydanio rerio embryos were studied using field emission scanning electron microscopy (FE‐SEM) and transmission electron microscopy (TEM). These studies confirm the outer chorion membrane complex to be 1.5–2.5 μm in thickness and to consist of three layers, electron‐dense outer and innermost layers (0.2–0.3 and 1.0–1.6 μm in thickness respectively) separated by an electron‐lucent middle layer (0.3–0.6 μm in thickness). The middle and inner layers are pierced by pore canals. A granular to farinaceous nature of the thin outer surface of the outer layer of the chorion has been revealed for the first time. The study provides original TEM images of the plasma membrane structures of gastrula‐stage embryos, and FE‐SEM and TEM images showing the plasma membrane to have three morpohologically distinct regions, being prominently ridged and folded at the surface of the blastoderm, smooth over the syncytial layer at the vegetal pole and with an intermediate region between the animal and vegetal pole where folding develops in advance of the expanding blastodermal disc of cells. FE‐SEM and TEM studies reveal details of the syncytial layer (1–4 μm thick) beneath the smooth plasma membrane at the vegetal pole, containing cytoplasmic organelles and small yolk globules. The significance of the structural detail shown in these studies is considered in the light of the difficulties experienced in cryopreservation of the embryo resulting from the inability of achieving cryoprotectant penetration of the yolk mass.     

Rederivation of a mutant line (prop 1) of zebrafish Danio rerio infected with Pseudoloma neurophilia using in vitro fertilization with eggs from pathogen-free wild-type (AB) females and sperm from prop 1 males

Along with the growing number of laboratories that work with zebrafish (Danio rerio), it is necessary to have animals with good sanitary quality. Specific pathogens can interfere with the experimental results and in the life quality of the animals. Pseudoloma neurophilia is a parasite with high potential for interference in behavioural, morphology, toxicological and genetic research, and is very common in zebrafish facilities. With that, we implemented a protocol for the pathogen elimination in a genetically modified lineage (prop 1) using eggs from specific pathogen-free (SPF) wild-type fish (AB line) for in vitro fertilization, along with water recirculation equipment disinfection, appropriate PCR screening and back crossing protocols. This resulted in SPF prop 1 heterozygotes, which allowed us to move forward with subsequent crossings to develop homozygote prop 1 mutants for our research. Hence, this demonstrates a useful strategy for an individual research laboratory to rederive a specific mutant free line that is not available from other SPF laboratories.     

Evaluation of the immune response in rainbow trout fry,Oncorhynchus mykiss (Walbaum), after waterborne exposure to Flavobacterium psychrophilum and/or hydrogen peroxide

The immune response in rainbow trout fry against Flavobacterium psychrophilum was elucidated using an immersion‐based challenge with or without prior exposure to hydrogen peroxide (H₂O₂). Samples were taken from the head kidney 4, 48, 125 and 192 h after immersion, and the regulation of several genes was examined. Bacterial load was assessed based on the presence of 16S rRNA and correlated with gene expression, and the levels of specific antibodies in the blood were measured 50 days post‐infection. Separately, both H₂O₂ and F. psychrophilum influenced gene expression, and pre‐treatment with H₂O₂ influenced the response to infection with F. psychrophilum. Pre‐treatment with H₂O₂ also affected correlation between gene regulation and pathogen load for several genes. A delay in antibody production in H₂O₂‐treated fish in the early phase of infection was indicated, but H₂O₂ exposure did not affect antibody levels 50 days post‐infection. An increasing amount of F. psychrophilum 16S rRNA was found in the head kidneys of infected fish pre‐treated with H₂O₂ relative to the F. psychrophilum group. The results show that a single pre‐treatment with H₂O₂ impairs the response against F. psychrophilum and may intensify infection.     

Characterization of swim bladder non-inflation (SBN) in angelfish, Pterophyllum scalare (Schultz), and the effect of exposure to methylene blue

Failure to inflate the swim bladder is regarded a major obstacle in the rearing of many fish species. We present a study of swim bladder non-inflation (SBN) in angelfish, Pterophyllum scalare. A normal developing primordial swim bladder was first discernable at the end of the first day post-hatch (p.h.) as a cluster of epithelial cells with a central lumen, surrounded by presumably mesenchymal cells. Initial inflation occurred on the fourth day p.h. Prior to inflation the swim bladder epithelium consisted of an outer squamous and inner columnar layer. Cells of the inner layer were filled at their basal region with an amorphous material, which disappeared upon inflation. A pneumatic duct was absent, and larvae presented no need to reach the water surface for inflation, suggesting that angelfish are pure physoclists. A model for the role of the amorphous material in normal initial inflation is proposed. Abnormal swim bladders were apparent from the fourth day p.h., and methylene blue (MB) at a concentration of 5 ppm significantly increased the prevalence of SBN. Histologically, abnormal swim bladders in larvae hatched in 5 ppm MB could not be distinguished from those in fish raised under routine conditions (0.5 ppm MB). We suggest that MB may have a teratogenic effect in angelfish.     

Viral replication in excised fin tissues (VREFT) corresponds with prior exposure of Pacific herring, Clupea pallasii (Valenciennes), to viral haemorrhagic septicaemia virus (VHSV)

Procedures for a viral replication in excised fin tissue (VREFT) assay were adapted to Pacific herring, Clupea pallasii, and optimized both to reduce processing time and to provide the greatest resolution between naïve herring and those previously exposed to viral haemorrhagic septicaemia virus (VHSV), Genogroup IVa. The optimized procedures included removal of the left pectoral fin from a euthanized fish, inoculation of the fin with >10⁵ plaque‐forming units (PFU) mL^?1 VHSV for 1 h, rinsing the fin in fresh medium six times to remove unadsorbed virions, incubation of the fin in fresh medium for 4 days and enumeration of the viral titre in a sample of the incubation medium by plaque assay. The optimized VREFT assay was effective at identifying the prior exposure history of laboratory‐reared Pacific herring to VHSV. The geometric mean VREFT value was significantly greater (P < 0.01) among naïve herring (1.2 × 10³ PFU mL^?1) than among groups that survived exposure to VHSV (1.0–2.9 × 10² PFU mL^?1); additionally, the proportion of cultures with no detectable virus was significantly greater (P = 0.0002) among fish that survived exposure to VHSV (39–47%) than among naïve fish (3.3%). The optimized VREFT assay demonstrates promise for identifying VHSV exposure history and forecasting disease potential in populations of wild Pacific herring.     

Nutritional quality of amino acid in farmed,farm‐aggregated and wild Axillary seabream (Pagellus acarne) with implications to Human Health

This study evaluated the nutritional quality of farmed and wild axillary seabream (Pagellus acarne R., 1827) focusing on amino acid profiles, with regards to possible interactions with wild fish aggregating around the cage facility. Total amino acids (∑AA), essential amino acids (∑EAA), non‐essential amino acids (∑NEAA) and neutral amino acids (∑NAA) in farmed fish were lower than those in the wild individuals (p > .05). Amino acid pattern in cage‐aggregated fish showed a slight decline from the wild populations, but still higher than the farmed fish. Based on the amino acid scores (AAS), lysine and leucine could be underlined as the ‘first limiting amino acids’, but all other AASs were over ‘1’, in accordance with reference amino acid contents of FAO/WHO (>1.00), showing that farmed axillary seabream provides high nutritional quality and can be considered as a favourable protein source. The ratios of ∑EAA/∑AA (44%–46%) and ∑EAA/∑NEAA (79%–86%) exceeded the minimum recommendation of 40% and >60% by FAO/WHO for all three groups. It can be concluded that axillary seabream either farmed, farm‐aggregated or distant wild fish presented high‐quality protein generating a healthy source for human food.     

Effect of protein and lipid levels in diets for female red claw crayfish Cherax quadricarinatus on quality of offspring (juvenile), with emphasis on growth performance,biochemical composition and stress resistance to low oxygen,high ammonia and salinity

This study measures the effect of protein and lipid levels in broodstock diet on the quality of juvenile Cherax quadricarinatus. Diets with different contents of crude protein (180, 250, 310, 370 g kg^?1) and lipids (30 and 70 g kg^?1) were offered to female crayfish. Juveniles were used to assess initial weight (IW), biochemical composition (BC) and survival to stress tests (ST). After 50 days, growth was estimated. No significant effects of broodstock diet on IW and BC of juveniles were detected. Protein content (PC) significantly influenced growth (P < 0.05), with an optimum level of 250 g kg^?1, representing a weight gain of 1.17 g (98.08% body weight). PC significantly influenced survival of juveniles exposed to ST (P < 0.05). Results from fitting dose–response models indicated that maximum survival was obtained with PC above 261, 258 and 312 g kg^?1 for hypoxia, salinity, ammonia tests. No significant effect of dietary lipid level was observed on growth and survival to ST, except for greater survival to ammonia ST at lipid content of 70 g kg^?1. We recommend feed with PC of 260 g kg^?1 to simultaneously enhance growth and quality of juveniles.