Triadimefon triggers circling behavior and conditioned place preference/aversion in zebrafish in a dose dependent manner.

Triadimefon (TDF) is a pesticide used in agricultural crops to control powdery mildews, rusts and other fungal pests. It exerts its fungicidal activity through the inhibition of ergosterol biosynthesis, impairing the formation of the cell membrane. For vertebrates, one of its side effects is the binding to the dopamine transporter increasing the levels of synaptic dopamine, similarly to cocaine. In addition, it has been demonstrated that TDF affects the abundance of other monoamines in the brain, specifically serotonin. It is well known that drugs which alter the dopaminergic and serotonergic systems produce behavioral changes and participate in the development of addictions in mammals. In this work we have used the conditioned place preference paradigm to assess, for the first time, the rewarding properties of TDF in zebrafish. We found out that TDF triggers both, preference and aversion depending on the dosage used during conditioning. We observed that 5 mg/L produced aversion to the pattern previously paired with TDF. However, 15 mg/L induced the opposite behavior, showing that zebrafish seek out those environments which had previously been paired with the higher dose of TDF. These results are congruent with our previous findings, where we showed that 5 mg/L reduced the levels of serotonin, usually linked to anxious behaviors (a negative cue), whereas higher concentrations of TDF increased extracellular dopamine, the main currency of the reward system. Interestingly, both doses of TDF induced circling behavior, a feature usually seen in glutamatergic antagonists.

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.

CXCL12a/CXCR4b acts to retain neutrophils in caudal hematopoietic tissue and to antagonize recruitment to an injury site in the zebrafish larva.

Neutrophils are a major component of the innate immune response and the most abundant circulating cell type in humans and zebrafish. The CXCL12/CXCR4 ligand receptor pair plays a key role in neutrophil homeostasis, controlling definitive hematopoiesis and neutrophil release into circulation. Neutrophils overexpressing CXCR4 respond by migrating towards sources of CXCL12, which is abundant in hematopoietic tissues. However, the physiological role of CXCL12/CXCR4 signaling during inflammatory responses remains unknown. Here, we show that zebrafish mutants lacking functional CXCL12a or CXCR4b show disrupted granulopoiesis in the kidney and increased number of circulating neutrophils. Additionally, CXCL12a and CXCR4b mutants display exacerbated recruitment of neutrophils to wounds and not to infections, and migrating neutrophils to wounds show increased directionality. Our results show that CXCL12a/CXCR4b signaling antagonizes wound-induced inflammatory signals by retaining neutrophils in hematopoietic tissues as a part of a balance between both inflammatory and anti-inflammatory cues, whose dynamic levels control neutrophils complex migratory behavior.

Macrophage Recruitment Contributes to Regeneration of Mechanosensory Hair Cells in the Zebrafish Lateral Line.

In vertebrates, damage to mechanosensory hair cells elicits an inflammatory response, including rapid recruitment of macrophages and neutrophils. While hair cells in amniotes usually become permanently lost, they readily regenerate in lower vertebrates such as fish. Damage to hair cells of the fish lateral line is followed by inflammation and rapid regeneration; however the role of immune cells in this process remains unknown. Here, we show that recruited macrophages are required for normal regeneration of lateral line hair cells after copper damage. We found that genetic ablation or local ablation using clodronate liposomes of macrophages recruited to the site of injury, significantly delays hair cell regeneration. Neutrophils, on the other hand, are not needed for this process. We anticipate our results to be a starting point for a more detailed description of extrinsic signals important for regeneration of mechanosensory cells in vertebrates. J. Cell. Biochem. 117: 1880-1889, 2016. © 2016 Wiley Periodicals, Inc.