Husbandry stress exacerbates mycobacterial infections in adult zebrafish, Danio rerio (Hamilton)

Mycobacteria are significant pathogens of laboratory zebrafish, Danio rerio (Hamilton). Stress is often implicated in clinical disease and morbidity associated with mycobacterial infections but has yet to be examined with zebrafish. The aim of this study was to examine the effects of husbandry stressors on zebrafish infected with mycobacteria. Adult zebrafish were exposed to Mycobacterium marinum or Mycobacterium chelonae , two species that have been associated with disease in zebrafish. Infected fish and controls were then subjected to chronic crowding and handling stressors and examined over an 8-week period. Whole-body cortisol was significantly elevated in stressed fish compared to non-stressed fish. Fish infected with M. marinum ATCC 927 and subjected to husbandry stressors had 14% cumulative mortality while no mortality occurred among infected fish not subjected to husbandry stressors. Stressed fish, infected with M. chelonae H1E2 from zebrafish, were 15-fold more likely to be infected than non-stressed fish at week 8 post-injection. Sub-acute, diffuse infections were more common among stressed fish infected with M. marinum or M. chelonae than non-stressed fish. This is the first study to demonstrate an effect of stress and elevated cortisol on the morbidity, prevalence, clinical disease and histological presentation associated with mycobacterial infections in zebrafish. Minimizing husbandry stress may be effective at reducing the severity of outbreaks of clinical mycobacteriosis in zebrafish facilities.     

Effect of different feeds on the growth,survival and reproductive performance of zebrafish,Danio rerio (Hamilton, 1822)

Present experiment was conducted to determine the effect of different feeds with varying protein levels on the growth, survival and reproductive performance of zebrafish, Danio rerio. The control diet (T1) was wild‐collected zooplankton from local fish ponds, while test diets with 350 g kg^?1 protein (T2), 400 g kg^?1 protein (T3) and 450 g kg^?1 protein (T4) were formulated and fed to fish for a period of 210 days. The significantly (P < 0.05) highest mean weight gain and specific growth rate were observed in T1, which were similar with T3 and T4. The significantly (P < 0.05) highest number of egg production per female and relative fecundity were found in T1, followed by T4 and T3 while T2 produced lowest number of eggs. No significant (P > 0.05) differences were observed in brood survival rate, fertilization and hatching rate among the dietary treatments. The highest (P < 0.05) fry survival rate was recorded in T1, followed by T3 and T4. Thus, it is suggested that control diet i.e. mixed zooplankton exhibited better growth, reproductive performance and fry survival rate. However, diet containing 400 g kg^?1 crude protein also gave comparable results in terms of growth, survival and reproductive performance of zebrafish.     

Further evaluation of the efficacy of emamectin benzoate for treating Pseudocapillaria tomentosa (Dujardin 1843) in zebrafish Danio rerio (Hamilton 1822)

Pseudocapillaria tomentosa is a pathogenic nematode parasite, causing emaciation and severe inflammatory lesions in the intestines in zebrafish Danio rerio (Hamilton 1822). Emamectin benzoate is commercially available analogue of ivermectin used for treating salmon for sea lice, under the brand name SLICE^®, and we have used this for treating zebrafish with the P. tomentosa. Here, SLICE^®, 0.2 per cent active emamectin benzoate, was used for oral treatments at 0.35 mg emamectin benzoate/kg fish/day for 14 days starting at 7 days post‐exposure (dpe). Another experiment entailed initiating treatment during clinical disease (starting at 28 dpe). Early treatment was very effective, but delaying treatment was less so, presumably due to inappetence in clinically affected fish. We evaluated emamectin benzoate delivered in water, using Lice‐Solve? (mectinsol; 1.4% active emamectin benzoate) in two experiments. Application of four 24‐hr treatments, space over 7 days was initiated at 28 dpe at either 0.168 or 0.56 mg emamectin benzoate/L/bath, and both treatments completely eradicated infections. This was 3 or 10 times manufacture's recommended dose, but was not associated with clinical or histological side effects.     

Induction of DNA damage in the peripheral blood of zebrafish (Danio rerio) by an agricultural organophosphate pesticide,monocrotophos

The use of organophosphate pesticides (OPs) in agricultural practices in India has gained popularity due to their broad spectrum, low cost and high potency. Monocrotophos (an OP) which is a systemic and contact broad-spectrum cholinesterase inhibitor is being widely used in India. However, its use entails the damage to other non-target organisms such as fish. In the present study, zebrafish (Danio rerio) were exposed to various doses of monocrotophos (0.125, 0.625 and 1.25 μL/L) and genotoxicity studies were carried out employing the comet assay and micronucleus test in the peripheral blood of these fish after 24, 48 and 72 h of exposure. Significant DNA damage in the form of % tail DNA and micronuclei was observed in all the treated fish compared to the control. Both % tail DNA and micronuclei frequency were found to increase significantly as the concentration increased. DNA damage was also observed at all the time intervals for all treatments except in the 0.125 μL/L-treated group. A positive correlation was also observed between the comet assay and micronucleus test. The study thus suggests the use of zebrafish as an experimental model to study the genotoxic effects of agricultural pesticides using the comet assay and micronucleus test.

     

Survival of zebrafish, Brachydanio rerio (Hamilton-Buchanan), embryo after immersion in methanol and exposure to ultrasound with implications to cryopreservation

This study examined the viability of embryos after immersion in highly concentrated methanol solutions (40–60%) and exposing embryos to ultrasound to enhance efficient transport of the cryoprotectant. The exposure to ultrasound, methanol concentrations, duration of treatment and the stages of embryonic development was found to have measurable effects on embryo viability. The effect of ultrasound was more evident at high voltage (>440 V) settings and at early developmental stages (30 and 60% epiboly stage). Older embryos were more resistant to cryoprotectant toxicity and ultrasound‐induced mortality. The high concentration of methanol (60%) was more toxic to embryos than the low concentration (40%). When methanol treatment and ultrasound were applied simultaneously the optimum concentration was found to be 45% methanol (45% survival; P<0.05) in a 3 min treatment. Although there was no significant difference between the 2 and 3 min treatments, embryos treated for 4 min had a significantly lower survival rate (P<0.05). These findings provide initial results to select the developmental stage of the embryo, the concentration of methanol for the preparation of a vitrification solution and duration of ultrasound treatment for cryopreservation. Furthermore, it indicates the potential use of ultrasound to enhance the transport of methanol intracellularly with minimum mortality of the developing embryos.     

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.     

Induction of triploidy in Melicertus kerathurus (Forskal, 1775) with temperature shock

Triploidy in fertilized eggs of Melicertus kerathurus was induced by cold (8, 10, 12°C) and heat (34, 36, 38°C) shock for different duration times (2, 4 and 8 min) after 10 min of post spawning. The best individual treatment produced 64.5% triploid nauplii in cold shock application at a temperature of 10°C for a duration of 8 min. Temperature did not have significant effect (P > 0.05) on triploid rate but duration time had a significant effect (P < 0.05) for individual cold or heat shock. This study demonstrates that because of a wide variety of effective parameters, it is essential to optimize shock conditions for each species strain at each location.     

大斑马鱼5种同工酶表达的初步分析

采用垂直聚丙烯酰胺平板电泳技术对大斑马鱼的眼、肌肉、鳃、尾鳍、卵巢5种组织器官的5种同功酶进行了研究分析。结果表明脂酶(EST)、苹果酸脱氢酶(MDH)、苹果酸酶(MEP)在眼、肌肉、鳃、尾鳍、卵巢都有表达;过氧化物酶(POX)仅在眼睛和鳃中表达;三磷酸腺苷酶(ATP)除眼睛外其它4种组织器官中均有表达。     

Ontogeny of sexual differentiation in different strains of zebrafish (Danio rerio)

The zebrafish (ZF) undergoes juvenile hermaphroditism, where in all fish, the gonad first develops as an ovary-like structure, before the sex is fixed and the gonad then differentiates into either a testis or an ovary. Sexual differentiation is being adopted as an endpoint for assessing endocrine disrupting chemicals, yet there is only very limited information on the timing and variability in sexual differentiation process both within and between strains of ZF. In this study, using gonad histology, the ontogeny of sexual differentiation was studied in two strains of ZF, one with a high level of heterozygocity (WIK strain) and another that had been in-bred for several generations. There was a high variability in the timing of sex differentiation between individuals within a specific strain (with no obvious relationship with body mass), but there were no differences between the two strains. Transformation of the immature gonad into testes in males started in week 5 post fertilization (pf) and completion of sexual differentiation for all fish in both study populations occurred by week 11–12 pf. The size of the fish containing transforming gonads in the WIK strain and in-bred strain were similar (ranging from 12 to 23 mm, and from 13 to 22 mm total length, respectively).     

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.