A New Bioassay for the Detection of Paralytic and Amnesic Biotoxins Based on Motor Behavior Impairments of Zebrafish Larvae.

The global concern about the increase of harmful algal bloom events and the possible impacts on food safety and aquatic ecosystems presents the necessity for the development of more accessible techniques for biotoxin detection for screening purposes. Considering the numerous advantages that zebrafish present as a biological model and particularly as a toxicants sentinel, we designed a sensitive and accessible test to determine the activity of paralytic and amnesic biotoxins using zebrafish larvae immersion. The ZebraBioTox bioassay is based on the automated recording of larval locomotor activity using an IR microbeam locomotion detector, and manual assessment of four complementary responses under a simple stereoscope: survival, periocular edema, body balance, and touch response. This 24 h acute static bioassay was set up in 96-well microplates using 5 dpf zebrafish larvae. For paralytic toxins, a significant decrease in locomotor activity and touch response of the larvae was detected, allowing a detection threshold of 0.1-0.2 µg/mL STXeq. In the case of the amnesic toxin the effect was reversed, detecting hyperactivity with a detection threshold of 10 µg/mL domoic acid. We propose that this assay might be used as a complementary tool for environmental safety monitoring.

The Zebrafish Perivitelline Fluid Provides Maternally-Inherited Defensive Immunity.

In the teleost egg, the embryo is immersed in an extraembryonic fluid that fills the space between the embryo and the chorion and partially isolates it from the external environment, called the perivitelline fluid (PVF). The exact composition of the PVF remains unknown in vertebrate animals. The PVF allows the embryo to avoid dehydration, to maintain a safe osmotic balance and provides mechanical protection; however, its potential defensive properties against bacterial pathogens has not been reported. In this work, we determined the global proteomic profile of PVF in zebrafish eggs and embryos, and the maternal or zygotic origin of the identified proteins was studied. In silico analysis of PVF protein composition revealed an enrichment of protein classes associated with non-specific humoral innate immunity. We found lectins, protease inhibitors, transferrin, and glucosidases present from early embryogenesis until hatching. Finally, in vitro and in vivo experiments done with this fluid demonstrated that the PVF possessed a strong agglutinating capacity on bacterial cells and protected the embryos when challenged with the pathogenic bacteria Edwardsiella tarda. Our results suggest that the PVF is a primitive inherited immune extraembryonic system that protects the embryos from external biological threats prior to hatching.

Behavioral effects of triadimefon in zebrafish are associated with alterations of the dopaminergic and serotonergic pathways.

Triadimefon (TDF) is a triazole fungicide extensively used in agriculture that has been found as a pollutant in numerous water sources. In mammals, it inhibits monoamine uptake through binding to the dopamine transporter, with a mechanism of action similar to cocaine, resulting in higher levels of dopamine at the synapse. Dopamine is a neurotransmitter involved in a broad spectrum of processes such as locomotion, cognition, reward, and mental disorders. In this work we have studied, for the first time, the effects of TDF on behavior of both larval and adult zebrafish and its connection with changes in the dopaminergic and serotonergic systems. We evaluated the acute exposure of 5 dpf larvae to different concentrations of TDF, ranging from 5 mg/L to 35 mg/L. The lowest concentration does not alter neither locomotor activity nor dopamine levels but produced changes in the expression of two genes, tyrosine hydroxylase 1 (th1) and dopamine transporter (dat). Besides, it induced a reduction in extracellular serotonin and had an anxiolytic-like effect, supported by a decrease in cortisol production. On the other hand, a high concentration of TDF produced a dose-dependent reduction in locomotion, which was reversed or enhanced by D1 (SCH-23390) or D2 (Haloperidol) dopamine receptor antagonists, respectively. Using in vivo electrochemistry, we show that these changes could be associated with higher levels of dopamine in the brain. Thus, in adult zebrafish, though not in larvae, TDF exposure increases locomotor activity, anxiety and aggressiveness, which coincides with the behaviors observed in mammals.

Triazole Fungicides Inhibit Zebrafish Hatching by Blocking the Secretory Function of Hatching Gland Cells.

In animals, hatching represents the transition point from a developing embryo to a free-living individual, the larva. This process is finely regulated by many endogenous and environmental factors and has been shown to be sensitive to a variety of chemical agents. It is commonly evaluated in bioassays in order to establish the effects of different agents on early development and reproductive capabilities in fish and other aquatic animals. In fish, the breakdown of the chorion is achieved by the secretion of choriolysin by hatching gland cells (HGCs) into the perivitelline space (PVS), coupled with spontaneous movements of the developing larva. In this work, we used zebrafish to assay the effects of a family of widely used agrochemicals-triazoles Triadimefon (FON), Triadimenol (NOL) and free triazole (1,2,4-T)-on hatching success. We found a strong inhibition of hatching by triazole exposure which was correlated with morphological changes and a reduction in the secretory function of the HGCs. As a consequence, the release of choriolytic enzymes by HGCs was reduced. We also found that HGC secretion reduction after exposure to FON can be rescued by co-incubation with a dopamine D2 receptor antagonist but not by antagonists of the D1-like receptors. This suggests a specific pathway through which this family of fungicides may be impairing a critical event in the fish life cycle.

Lactoferrin Decreases the Intestinal Inflammation Triggered by a Soybean Meal-Based Diet in Zebrafish.

Intestinal inflammation is a harmful condition in fish that can be triggered by the ingestion of soybean meal. Due to the positive costs-benefits ratio of including soybean meal in farmed fish diets, identifying additives with intestinal anti-inflammatory effects could contribute to solving the issues caused by this plant protein. This study evaluated the effect of incorporating lactoferrin (LF) into a soybean meal-based diet on intestinal inflammation in zebrafish. Larvae were fed with diets containing 50% soybean meal (50SBM) or 50SBM supplemented with LF to 0.5, 1, 1.5 g/kg (50SBM+LF0.5; 50SBM+LF1.0; 50SBM+LF1.5). The 50SBM+LF1.5 diet was the most efficient and larvae had a reduced number of neutrophils in the intestine compared with 50SBM larvae and an indistinguishable number compared with control larvae. Likewise, the transcription of genes involved in neutrophil migration and intestinal mucosal barrier functions (mmp9, muc2.2, and ß-def-1) were increased in 50SBM larvae but were normally expressed in 50SBM+LF1.5 larvae. To determine the influence of intestinal inflammation on the general immune response, larvae were challenged with Edwardsiella tarda. Larvae with intestinal inflammation had increased mortality rate compared to control larvae. Importantly, 50SBM+LF1.5 larvae had a mortality rate lower than control larvae. These results demonstrate that LF displays a dual effect in zebrafish, acting as an intestinal anti-inflammatory agent and improving performance against bacterial infection.

Formation of posterior cranial placode derivatives requires the Iroquois transcription factor irx4a.

Members of the Iroquois (Irx) homeodomain transcription factor gene family have been implicated in a variety of early developmental processes, including neural pre-patterning, tissue differentiation, neural crest development and cranial placode formation. Here, we report that, in zebrafish, the irx4a gene participates in specification of a number of placode derivatives that arise from the posterior placodal field. Specifically, differentiation of the trigeminal, epibranchial and lateral line placodes are affected when irx4a function is interrupted using antisense morpholino oligonucleotides. We show that both in the trigeminal ganglion and in the lateral line, irx4a is involved in controlling the number of sensory cells that develop. Other phenotypes observed in morphant embryos include misspecification of the heart chambers and failure of retinal ganglion and photoreceptor cell differentiation, functions described previously for Irx4 in other species. We also provide evidence that irx4a regulates the expression of the sox2 gene, both in the neural plate and in progenitor cells of the lateral line system. Our results point to irx4a as a critical gene for numerous developmental processes and highlight its role in the formation of placodal derivatives in vertebrates.