Inflammation is an essential process as a reaction towards infections or wounding. Exposure to hazardous environmental pollutants can lead to chronic inflammations, where the resolving phase is ...delayed or blocked. Very contradictory studies have been reported on the pro- and anti-inflammatory effects of humic substances (HSs) leading to significant disagreements between researchers. To a certain extent, this can be attributed to the chemical heterogeneity of this group of xenobiotics. Here we show for the first time that pro- and anti-inflammatory effects can occur by one HSs. We adapted an assay that uses green fluorescence-labeled zebrafish larvae and CuSO
to indue an inflammation. In wild-type larvae, exposure to 50 µM CuSO
for 2 h activated the production of reactive oxygen species, which can be monitored with a fluorescence dye (H2DCFDA) and a microplate reader. This allows not only the use of wild-type fish but also a temporal separation of copper exposure and inflammatory substance while retaining the high throughput. This modified assay was then used to evaluate the inflammatory properties of a fulvic acid (FA). We found, that the aromatic structure of the FA protects from inflammation at 5 and 50 mg C/L, while the persistent free radicals enhance the copper-induced inflammation at ≥ 300 mg C/L.
Aquaculture has become imperative to cover the demands for dietary animal protein. Simultaneously, it has to overcome prejudices from excessive use of antibiotics and environmental impacts. Natural ...supplements are traditionally applied orally. In this study, we demonstrated another pathway: the gills. Humic substances are immunostimulants and a natural part of every aquatic ecosystem, making them ideal to be used as bath stimulants. Five and 50 mg C/L of a fulvic acid-rich humic substance was added for 28 days to the water of juvenile rainbow trout (Oncorhynchus mykiss). This fulvic acid is characterized by a high content of phenolic moieties with persistent free radicals and a high electron exchange capacity. The high concentration of the fulvic acid significantly increased growth and reduced the food conversion ratio and the response to a handling-stressor. Phagocytosis and potential killing activity of head kidney leukocytes were increased, as well as the total oxyradical scavenging capacity (TOSC) and lysozyme activity in the gills. In conclusion, immunostimulation via gills is possible with our fulvic acid, and the high phenolic content improved overall health and stress resistance of fish.
Many classical therapeutants are going to be banned in Europe, and an urgent need for alternatives is emerging. This issue can be exemplified by one major parasitic disease in aquaculture and ...ornamental fish breeding: velvet disease. This disease, caused by dinoflagellates of the genera Amyloodinium and Piscinoodinium, is an important infection affecting cultured freshwater and marine ornamental and food fish, and consistently causes great financial loss to the associated industries. Therapeutants available contain copper, malachite green, or methylene blue, and which can be toxic to non‐target organisms in the surrounding environment. As a result, these chemicals are banned for use by the aquaculture industry in several countries, and a prohibition for commercial ornamental fishkeeping is likely to follow in most countries. Increasing development of resistance to therapeutants, and growing public awareness for animal welfare and environmental protection, have prompted research in the areas of alternative treatment options and immunostimulants. Hydrogen peroxide and peracetic acid are possible ‘green’ therapeutants which do not contribute residues to the environment. Natural feed supplements such as pre‐ and probiotics can increase animal welfare and prevent stress and/or infections. Humic substances are another promising, natural immunostimulants which will be considered in depth. The aim of this review is to provide an overview of risks and benefits of current treatment options and new approaches to replace harmful therapeutants and minimize the number of toxic residues discharged into the environment. Treatments will be discussed on various parasitic infections and focus, where available, on Amyloodinium and Piscinoodinium.
An 8-week husbandry trial was carried out toexamine the impacts of graded levels of dietary fulvic acid (FA) on growth, stress, and disease resistance of Asian seabass (Lates calcarifer, 38.2 g) ...juveniles. Five levels of FA were included in a basal diet (470 g kg-1 crude protein, 150 g kg-1 crude lipid) to prepare experimental diets, including: 0 (control), 2.5, 5.0, 10, and 15 g kg-1 diet. Two hundred and twenty-five fish (38.2 ± 0.1 g) were stocked randomly into 15 1000-L circular polyethylene tanks (15 fish tank-1) filled with 800 L disinfected seawater and fed with the experimental feeds three times daily. Each dietary treatment had three replicates. Tanks were supplied with seawater (32.2 °C, 45.0 ppt) in a flow-throw system. At the end of the husbandry trial, fish were challenged with Vibrio harveyi and air exposure stress for one min. Fish fed FA-supplemented diets showed a positive quadratic growth response, and those fed with a 5 g FA kg-1 diet had higher final weight and weight gain percentage than the control. Feed intake in fish fed FA-supplemented diets were higher than in control, showing significant linear and quadratic trends (P < 0.05). The highest and lowest skin mucus lysozyme (LYZ) and serum alternative complement pathway (ACH50) activities were in fish fed 10 and 15 g FA kg-1 diets, respectively, exhibiting a quadratic response to dietary FA. Serum total protein level (TP) in fish fed FA-supplemented diets was higher than in the control group. Medium or high levels (10–15 g kg-1) of FA significantly enhanced the survival rate against Vibrio harveyi, which was associated with increased activities of serum lysozyme and ACH50 as well as liver glutathione peroxidase activity in these groups. Air exposure induced oxidative stress in all treatments and gradually reduced antioxidant enzyme activities associated with increased malondialdehyde content in the liver. Furthermore, air exposure suppressed some immune responses, including serum total immunoglobulin and TP levels. Still, LYZ activity was restored after 24 h, with the highest level found in fish fed 5 g FA kg-1 diet. Based on the broken-line regression analysis, the adequate level of FA in the diet for Asian seabass is 4.4 g kg-1.
•Supplementing diet with 5 g kg-1 fulvic acid (FA) enhanced growth in Asian sea bass.•Supplementing diet with FA increased resistance against Vibrio harveyi and air exposure stress.•Dietary FA enhanced antioxidant capacity, humoral and skin mucosal immune responses.•The optimum level of FA in the diet for Asian sea bass is 4.4 g kg-1.
Marine algae release a plethora of organic halogenated compounds, many of them with unknown ecological impact if environmentally realistic concentrations are applied. One major compound is ...dibromoacetic acid (DBAA) which was tested for neurotoxicity in the invertebrate model organism Caenorhabditis elegans (C. elegans). This natural compound was compared with the widespread synthetic xenobiotic tetrabromobisphenol-A (TBBP-A) found in marine sediments and mussels. We found a neuro-stimulating effect for DBAA; this is contradictory to existing toxicological reports of mammals that applied comparatively high dosages. For TBBP-A, we found a hormetic concentration-effect relationship. As chemicals rarely occur isolated in the environment, a combination of both organobromines was also examined. Surprisingly, the presence of DBAA increased the toxicity of TBBP-A. Our results demonstrated that organohalogens have the potential to affect single organisms especially by altering the neurological processes, even with promoting effects on exposed organisms.
This is the first study determining the effects of bath exposure to fulvic acid, a humic substance, on the skin mucosal immunity of rainbow trout (Oncorhynchus mykiss). Humic substances have recently ...been gaining attention for their increasing concentrations in aquatic ecosystems and their use as supplements in sustainable aquaculture. This study demonstrated that water exposure to fulvic acid at concentrations of 5 mg C/L and 50 mg C/L increased lysozyme and alkaline phosphatase activities in the mucus by approximately 2-fold and 2.5 to 3.2-fold, respectively. Furthermore, exposure to 50 mg C/L resulted in a 77.0% increase in mucosal immunoglobulin concentrations compared to the other groups. Importantly, all mucus samples demonstrated significant antibacterial activity against Yersinia ruckeri, with control mucus reducing bacterial growth by 44.5% and exposure to fulvic acid increasing this effect to 26.3%. Although these modulations show promise for application in aquaculture, alterations of the beneficial microbiota from long-term exposure in natural waters can be expected. Monitoring the rising concentrations of humic substances in natural water bodies is therefore urgently needed. Overall, this study represents the first investigation revealing the ability of humic substances to modulate skin mucosal immunity and the capacity to combat microorganisms.
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•Concentrations of humic substances in natural waters are increasing recently.•Unmonitored humic substances pose a risk to fish in aquaculture and the environment.•Fulvic acid increased innate immune response in the skin mucus of rainbow trout.•Lysozyme, alkaline phosphatase, and immunoglobulin content increased after exposure.•Increased antibacterial defenses protect against pathogens, but may alter microbiota.
There is no definitive evidence associated with administration duration of nucleotide on aquaculture organisms. This study evaluates the long-term feeding effects of cytidine monophosphate (CMP) ...nucleotide as functional supplement on growth and health conditions of red sea bream, Pagrus major. A formulated basal diet added with 0% (control), 0.2% (CMP-0.2) and 0.4% (CMP-0.4) individual CMP nucleotide to prepare three experimental feeds. Each experimental feed was fed to triplicate tanks of red sea bream (3.0 ± 0.01 g) for 150 days. At day 50th, growth performances were influenced significantly by dietary CMP inclusion and fish fed CMP-0.4 diet showed significantly higher value. In day 100 and 150 growth performances also non-significantly enhanced by CMP supplementation. Feed utilization performances also showed improved values in nucleotide included diets compared to the non-supplemented control diet (P > 0.05). No significant changes observed on measured hematological parameters by nucleotide administration duration. Lysozyme activity (LA) and total serum protein (TSP) significantly rose to their highest level on 50 days in CMP-0.4 diet group. In 100 and 150th day supplementation also improved TSP and LA values and it reaches maximum in CMP-0.2 diet group (P > 0.05). Oxidative radical production (nitro-blue-tetrazolium assay) and the activity of peroxidase and catalase were not affected significantly with feeding nucleotide in all sampling days. Supplementation of nucleotide improved oxidative stress tolerance capacity. Remarkably, CMP-0.2 diet group showed the least oxidative stress condition in 50th and 100th day. In 150th day fish showed acceptable but reduced oxidative stress resistances. On the basis of the overall findings, finally we concluded that, CMP nucleotide administration regime influences the optimum growth, health performance and immune response of Pagrus major and its mostly after feeding for 50 days, but not after 100 days.
•No previous work has addressed the long-term feeding effects of dietary nucleotide (NT) as functional nutrient for marine fish.•Administration duration of NT for 50 days showed significantly enhanced growth performances but not ≥100 days.•Overall innate immunity enhanced by NT addition, but its intensity decreased with the increasing administration duration (≥100 days).•Administration duration of NT for ≤100 days showed improved oxidative status of P. major.•The administration regime of NT for the optimum response of fish performances was mostly after 50 days of feeding, but not after 100 days.
Aquaculture plays a pivotal role in covering dietary animal protein demands and restocking endangered fish populations. However, high mortality takes place at the earliest life stages: prior and ...immediately after hatching. Improving growth and health parameters by immunostimulants is widely used in older fish, but rarely studied in larvae. Fulvic acids (FAs) are natural substances found in soil and water. Using zebrafish as a model organism, we evaluated the effects of exposure to a FA at concentrations ranging from 1 to 500 mg C/L (mg dissolved organic carbon per liter) on embryonic development. Furthermore, the concentration of reactive oxygen species (ROS) inside the larvae as well as the molecular mechanisms involved in growth, immune response, and antioxidative protection were determined at 5, 50, and 500 mg C/L. 20 to 200 mg C/L accelerated the hatching, which was mediated by increased expression of ifg-1, gh, and he1-α. Furthermore, lyz and mpx were significantly increased at 5 and 50 mg C/L. A concentration of 500 mg C/L induced genes involved in the protection against ROS (nrf-2, keap-1, cat, sod-1), increased the concentration of ROS inside the larvae and caused tissue damage and mortality. Interestingly, 50 mg C/L activated ROS protection as well (nrf-2, sod-2), while no increase of ROS was found in the larvae. Our results show, that FA at low to medium concentrations can increase the health of larvae, but becomes detrimental at higher concentrations.
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•Fulvic acid between 20 and 200 mg C/L accelerates hatching of zebrafish larvae•At ≥300 mg C/L oxidative stress causes tissue damage and mortality•Hatching is mediated by induced expression of gh, igf-1, and he1-α•5 mg C/L and 50 mg C/L of the fulvic acid induced expression of lyz and mpx•The antioxidative protection is activated via the keap1-nrf2 pathway
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