Neurotoxic effect of the herbicide paraquat on ascidian larvae.

Paraquat is an herbicide widely used in agriculture, that proved to have toxic effect on many animal models. Moreover, it is considered a potential etiologic factor of Parkinson's disease. Ascidians are invertebrate chordates, whose larval central nervous system shares basic structural homologies with the vertebrate one. Ascidian larvae exposed to paraquat developed specific alterations of the CNS, that were characterized by histological and immunohistochemical analysis. Tyrosine hydroxylase (TH) expression was examined by "in situ" hybridization. A decrease of dopamine content in anterior CNS of treated larvae was observed. In combined treatments with paraquat and l-ascorbic acid, a common anti-oxidant, the severity of the malformations was significantly reduced, confirming that the oxidative stress is involved in the toxicity mechanism of paraquat on ascidians. For its sensitivity to paraquat and its simple chordate body plan, ascidian larva is a promising animal model to further investigate the molecular mechanism of paraquat neurotoxicity.

Effects of the azole fungicide Imazalil on the development of the ascidian Ciona intestinalis (Chordata, Tunicata): morphological and molecular characterization of the induced phenotype.

Imazalil (IMA) is a fungicide that is used extensively in fruit plantations and post-harvest treatments, but has teratogenic effects on vertebrate development, possibly due to the perturbation of retinoic acid (RA) levels in the embryo. Ascidians are sessile marine invertebrate chordates that develop through a tadpole larva, with a body plan that shares basic homologies with vertebrates. In this work, we tested the effects of IMA on the development of the solitary ascidian Ciona intestinalis by treating two-cell stage embryos with a range of concentrations (0.1, 0.5, 1, 2.5, 5, 10, 20 and 50microThe fungicide significantly altered ascidian development even at low concentrations and its effects were dose-dependent. Probit analysis revealed that the median lethal concentration, LC(50), was 4.87microM and the median teratogenic concentration, TC(50), was 0.73microM. Larvae developing from embryos exposed to IMA showed malformations of the anterior structures, which became more severe as IMA concentration increased. In particular, the anterior nervous system and the sensory vesicle were reduced, and the pigmented organs (the ocellus and the otolith) progressively lost their pigmentation. The larval phenotype induced by 5microM IMA exposure was further characterized by means of molecular analysis, through whole mount in situ hybridization with probes for genes related to the nervous system: Ci-Otp, Ci-GAD, Ci-POU IV, which are markers of the anterior neuro-ectoderm, the central nervous system and the peripheral nervous system respectively, and Ci-Hox-1, a gene specifically activated by RA, and Ci-Aldh2, a gene for aldehyde dehydrogenase, which is involved in RA synthesis. The altered expression of Ci-Otp, Ci-GAD, Ci-POU IV in 5microM IMA-exposed larvae compared to control larvae showed that this fungicide could affect the differentiation of the anterior nervous system, particularly of the sensory vesicle neurons. Recent studies suggest a similarity between IMA- and RA-induced phenotypes in tunicates, indicating that triazoles may also alter RA metabolism in ascidians. The observed Ci-Hox-1 and Ci-Aldh2 expression in control and treated larvae did not allow a direct link between IMA teratogenic potential and RA-dependent morphogenesis to be identified. It is likely that the fungicidal teratogenic mechanism involved RA signalling but that its effects on ascidian development depend on a more complex mechanism.

Developmental expression of glutamic acid decarboxylase and of gamma-aminobutyric acid type B receptors in the ascidian Ciona intestinalis.

We describe Ciona intestinalis gamma-aminobutyric acid (GABA)-ergic neurons during development, studying the expression pattern of Ci-GAD (glutamic acid decarboxylase: GABA synthesizing enzyme) by in situ hybridization. Moreover, we cloned two GABA(B) receptor subunits (Ci-GABA(B)Rs), and a phylogenetic analysis (neighbor-joining method) suggested that they clustered with their vertebrate counterparts. We compared Ci-GAD and Ci-GABA(B)Rs expression patterns in C. intestinalis embryos and larvae. At the tailbud stage, Ci-GAD expression was widely detected in central and peripheral nervous system (CNS/PNS) precursors, whereas Ci-GABA(B)Rs expression was evident at the level of the precursors of the visceral ganglion. GABA was localized by immunohistochemistry at the same developmental stage. In the larva, Ci-GAD transcripts and GABA immunofluorescence were also detected throughout the CNS and in some neurons of the PNS, whereas transcripts of both GABA(B) receptor subunits were found mainly in the CNS. The expression pattern of Ci-GABA(B)Rs appeared restricted to Ci-GAD-positive territories in the sensory vesicle, whereas, in the visceral ganglion, Ci-GABA(B)Rs transcripts were found in ventral motoneurons that did not express Ci-GAD. Insofar as GABAergic neurons are widely distributed also in the CNS and PNS of vertebrates and other invertebrate chordates, it seems likely that GABA signaling was extensively present in the protochordate nervous system. Results from this work show that GABA is the most widespread inhibitory neurotransmitter in C. intestinalis nervous system and that it can signal through GABA(B) receptors both pre- and postsynaptically to modulate different sensory inputs and subsequent swimming activity.

Developmental expression of tryptophan hydroxylase gene in Ciona intestinalis.

To describe the serotonergic system in a tunicate larva, we cloned a gene encoding for tryptophan hydroxylase (TPH), the rate-limiting enzyme in serotonin synthesis, in the ascidian Ciona intestinalis and studied its expression pattern during development. Ci-TPH expression was found from tailbud stage in the precursor cells of the visceral ganglion and in the tail. In the larva, TPH-expressing neurons formed two clusters in the anterior central nervous system at the level of the visceral ganglion. Moreover, we found Ci-TPH expression at the level of the muscle cells of the tail and suggested that this localisation might be at the level of neuro-muscular junctions. Moreover, we discussed the involvement of serotonin in the control of larval locomotory activity.

Fluconazole induces teratogenic effects in the tunicate Phallusia mammillata.

Fluconazole (FLUCO) is an azole derivative used to treat fungal and yeast infections. Embryotoxicity tests on the ascidian Phallusia mammillata were performed to evaluate the effects of this drug. FLUCO proved to have strong consequences on P. mammillata development. Incidence of malformations and of lethality increased in a dose dependent way. Probit analysis showed that FLUCO had a high TI value (Teratogenic Index, LC(50)/TC(50)), thus this substance could be classified as a teratogenic compound for ascidians. Larvae exposed to FLUCO showed a typical phenotype characterized by malformations restricted to the trunk region: the trunk appeared round in shape with flat palps, the sensory vesicle cavity was absent or reduced and the anterior central nervous system failed to correctly differentiate. These anomalies resulted similar to those induced by retinoic acid (RA) treatment. Thus, it could be hypothesized that FLUCO and RA may act with a similar pathogenic mechanism in ascidian larvae, as it has been proposed for mammals.

Toxic effects of two pesticides, Imazalil and Triadimefon, on the early development of the ascidian Phallusia mammillata (Chordata, Ascidiacea).

Azole compounds are fungicides used in agriculture and in clinical area and are suspected to produce craniofacial malformations in vertebrates. Toxicity tests on sperm viability, fertilization and embryogenesis of the solitary ascidian Phallusia mammillata were performed to evaluate the effects of two azole derivatives, Imazalil and Triadimefon. Ascidian (Chordata, Ascidiacea) embryos and larvae could provide biological criteria for seawater quality standards because the larvae are lecitotrophic and have a short pelagic period, allowing to run the larval toxicity tests over a short period of time. Imazalil and Triadimefon proved to have strong consequences on P. mammillata. They could influence the reproductive rate of the animal exerting their effects at different levels: acting as spermiotoxic agents, inhibiting fertilization and impairing embryological development. Fertilization rate significantly decreased after 30min exposure of sperm to 25microM Imazalil (P<0.0001) and after exposure of both gametes to 50microM Imazalil (P<0.05) and 1mM Triadimefon (P<0.0001) as compared to controls. Malformations caused by exposure of embryos to both substances were dose dependent. Imazalil median teratogenic concentration (TC50 concentration, the concentration that resulted in 50% malformed larvae) value was 0.67microM and median lethal concentration (LC50, the concentration that resulted in 50% embryos dead before completing the development) value was 10.23microM while for Triadimefon TC50 value was 29.56 and LC50 value was 173.7microM. Larvae developed from embryos treated with Imazalil and Triadimefon showed alterations of the anterior structures of the trunk: papillary nerves and the anterior central nervous system failed to correctly differentiate, as showed by immunostaining with anti-beta-tubulin antibody. Comparing the anomalies caused by retinoic acid, reported in a previous study, it was possible to hypothesize that malformations induced by Imazalil and Triadimefon could be due to a perturbation of the endogenous retinoid content, as it has been proposed for mammals. Ascidians proved to be good models to study the toxic effects of pesticides since they offered both the convenience of working with an invertebrate species and the tissue sensitivity to chemical compound comparable to vertebrates.

Teratogenic effects of two antifungal triazoles, triadimefon and triadimenol, on Xenopus laevis development: craniofacial defects.

Triadimefon and triadimenol, fungicides used in agriculture, are suspected of producing craniofacial malformations. As the results of FETAX analysis showed that Triadimefon was highly teratogenic, we studied the action of these triazoles on the development of the branchial apparatus in Xenopus, using early molecular markers and analysis of the cartilaginous-muscular elements of tadpoles. Teratogenic effects were observed, after exposure at the neurula stage, at the level of cartilages and muscles of the 1st and 2nd branchial arches. By in situ hybridization, we observed that in exposed specimens the territories of the branchial arches are perturbed. From these results, we considered the craniofacial malformations related to the influence of triazoles on the differentiation of branchial arches. Comparing the anomalies caused by triazoles and by retinoic acid (RA) led us to the conclusion that triazoles can affect endogenous RA content, as has been shown for mammals.

Dopamine and serotonin modulate the onset of metamorphosis in the ascidian Phallusia mammillata.

Neurotransmitters play an important role in larval metamorphosis in different groups of marine invertebrates. In this work, the role of dopamine and serotonin during metamorphosis of the ascidian Phallusia mammillata larvae was examined. By immunofluorescence experiments, dopamine was localized in some neurons of the central nervous system and in the adhesive papillae of the larvae. Dopamine and serotonin signaling was inhibited by means of antagonists of these neurotransmitters receptors (R(+)-SCH-23390, a D(1) antagonist; clozapine, a D(4) antagonist; WAY-100635, a 5-HT(1A) antagonist) and by sequestering the neurotransmitters with specific antibodies. Moreover, dopamine synthesis was inhibited by exposing 2-cell embryos to alpha-methyl-l-tyrosine. Dopamine depletion, obtained by these different approaches, caused early metamorphosis, while serotonin depletion delayed the onset of metamorphosis. The opposite effects were obtained using agonists of the neurotransmitters: lisuride, a D(2) agonist, inhibited metamorphosis, while DOI hydrochloride and 8-OH-DPAT HBr, two serotonin agonists, promoted it. So, it is possible to suppose that dopamine signaling delayed metamorphosis while serotonin signaling triggers it. We propose a mechanism by which these neurotransmitters may modulate the timing of metamorphosis in larvae.

WAY-100635, an antagonist of 5-HT(1A) receptor, causes malformations of the CNS in ascidian embryos.

Serotonin (5-HT) is a neurotransmitter which is supposed to play a key role during development. In the last few years 5-HT receptors have been cloned in many animal species, and there is evidence that different 5-HT receptors are also present in ascidians. Ascidians and vertebrates are both members of the phylum Chordata and both have a dorsal tubular central nervous system. Embryos of the ascidian Phallusia mammillata have been treated with WAY-100635, a potent and selective 5-HT(1A) receptor antagonist. The larvae developed from treated embryos showed a dramatic reduction of their anterior sensory vesicles and the pigment of two sensory organs, the ocellus and the otolith. Immunofluorescence experiments with an anti beta-tubulin monoclonal antibody specific for the neural system showed that the anterior neural system of treated animals was radically altered by the action of the drug in a dose-dependent way. These results suggest that 5-HT plays a role in the development of the neural system in ascidians and its action is mediated by receptors similar to the members of the 5-HT(1A) receptor subtype of mammals.