Roberta Pennati

Morphological Differences between Larvae of the Ciona intestinalis Species Complex: Hints for a Valid Taxonomic Definition of Distinct Species.

The cosmopolitan ascidian Ciona intestinalis is the most common model species of Tunicata, the sister-group of Vertebrata, and widely used in developmental biology, genomics and evolutionary studies. Recently, molecular studies suggested the presence of cryptic species hidden within the C. intestinalis species, namely C. intestinalis type A and type B. So far, no substantial morphological differences have been identified between individuals belonging to the two types. Here we present morphometric, immunohistochemical, and histological analyses, as well as 3-D reconstructions, of late larvae obtained by cross-fertilization experiments of molecularly determined type A and type B adults, sampled in different seasons and in four different localities. Our data point to quantitative and qualitative differences in the trunk shape of larvae belonging to the two types. In particular, type B larvae exhibit a longer pre-oral lobe, longer and relatively narrower total body length, and a shorter ocellus-tail distance than type A larvae. All these differences were found to be statistically significant in a Discriminant Analysis. Depending on the number of analyzed parameters, the obtained discriminant function was able to correctly classify > 93% of the larvae, with the remaining misclassified larvae attributable to the existence of intra-type seasonal variability. No larval differences were observed at the level of histology and immunohistochemical localization of peripheral sensory neurons. We conclude that type A and type B are two distinct species that can be distinguished on the basis of larval morphology and molecular data. Since the identified larval differences appear to be valid diagnostic characters, we suggest to raise both types to the rank of species and to assign them distinct names.

Complex neural architecture in the diploblastic larva of Clava multicornis (Hydrozoa, Cnidaria).

The organization of the cnidarian nervous system has been widely documented in polyps and medusae, but little is known about the nervous system of planula larvae, which give rise to adult forms after settling and metamorphosis. We describe histological and cytological features of the nervous system in planulae of the hydrozoan Clava multicornis. These planulae do not swim freely in the water column but rather crawl on the substrate by means of directional, coordinated ciliary movement coupled to lateral muscular bending movements associated with positive phototaxis. Histological analysis shows pronounced anteroposterior regionalization of the planulas nervous system, with different neural cell types highly concentrated at the anterior pole. Transmission electron microscopy of planulae shows the nervous system to be unusually complex, with a large, orderly array of sensory cells at the anterior pole. In the anterior half of the planula, the basiectodermal plexus of neurites forms an extensive orthogonal network, whereas more posteriorly neurites extend longitudinally along the body axis. Additional levels of nervous system complexity are uncovered by neuropeptide‐specific immunocytochemistry, which reveals distinct neural subsets having specific molecular phenotypes. Together these observations imply that the nervous system of the planula of Clava multicornis manifests a remarkable level of histological, cytological, and functional organization, the features of which may be reminiscent of those present in early bilaterian animals. J. Comp. Neurol. 519:1931–1951, 2011.

Serotonin localization in Phallusia mammillata larvae and effects of 5‐HT antagonists during larval development

The neurotransmitter 5‐hydroxytryptamine (5‐HT, serotonin) plays an important role in a wide range of non‐neural processes. Using immunofluorescence with an antiserotonin antibody, 5‐HT was localized in the brain and in some neurons of the larval tail of Phallusia mammillata. To test the effect of 5‐HT on development, we treated embryos with two different 5‐HT receptor subtype antagonists. Treatment at the gastrula stage with 10 μM ondansetron, an antagonist of the 5‐HT3 receptor, induced anterior truncation and a short tail. At 10 μM, ritanserin, a 5‐HT2B receptor antagonist, induced larval phenotypes characterized by a roundish trunk region with flat papillae. The juveniles developed from these larvae had an abnormal cardiocirculatory system: their heart contractions were ineffective and their blood cells accumulated in the heart cavity. We conclude that an appropriate level of 5‐HT is necessary for correct development and morphogenesis. Moreover, a different key role for multiple receptors in modulating the morphogenetic effects of 5‐HT is suggested.

Spatial segregation among age classes in cave salamanders: habitat selection or social interactions?

Within species, individuals with different sexes, morphs and age classes often show spatial segregation. Both habitat selection and social processes have been proposed to explain intraspecific spatial segregation, but their relative importance is difficult to assess. We investigated spatial segregation between age classes in the cave salamander Hydromantes (Speleomantes) strinatii, and used a hypothetico-deductive approach to evaluate whether social or ecological processes explain segregation pattern. We recorded the location and age class of salamanders along multiple caves; we measured multiple microhabitat features of different sectors of caves that may determine salamander distribution. We assessed age-class segregation, and used generalized mixed models and an information-theoretic framework, to test if segregation is explained by social processes or by differences in habitat selection. We found significant age-class segregation, juveniles living in more external cave sectors than adults. Multiple environmental features varied along caves. Juveniles and adults showed contrasting habitat selection patterns: juveniles were associated with sectors having high invertebrate abundance, while adults were associated with scarce invertebrates and low temperature. When the effect of environmental features was taken into account, the relationship between juveniles and adults was non negative. This suggests that different habitat preferences, related to distinct risk-taking strategies of age classes, can explain the spatial segregation. Juveniles require more food and select more external sectors, even if they may be risky. Conversely, adults may trade off food availability in favour of safe areas with stable micro-climate.

Ci-POU-IV expression identifies PNS neurons in embryos and larvae of the ascidian Ciona intestinalis

Several lines of evidence suggest that members of the POU domain gene family may regulate invertebrate and vertebrate neurogenesis. In particular, POU IV genes appear to be neural genes involved in differentiation of sensory neurons, as demonstrated in mollusc, Drosophila, Caenorhabditis elegans and vertebrates. In the present work, we describe the developmental expression of a homologue of POU IV genes, Ci-POU-IV, in the ascidian Ciona intestinalis. Ci-POU-IV is expressed in the precursor cells of the neural system during development and in the neural system of the larva. In particular, transcripts are prevalent in the peripheral nervous system (PNS), with expression in the central nervous system (CNS) restricted to the posterior sensory vesicle. Therefore, the evolution of a complex sensory system seems to be under the control of a common genetic mechanism.

Developmental expression of glutamic acid decarboxylase and of γ‐aminobutyric acid type B receptors in the ascidian Ciona intestinalis

We describe Ciona intestinalis γ‐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 GABAB receptor subunits (Ci‐GABABRs), and a phylogenetic analysis (neighbor‐joining method) suggested that they clustered with their vertebrate counterparts. We compared Ci‐GAD and Ci‐GABABRs 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‐GABABRs 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 GABAB receptor subunits were found mainly in the CNS. The expression pattern of Ci‐GABABRs appeared restricted to Ci‐GAD‐positive territories in the sensory vesicle, whereas, in the visceral ganglion, Ci‐GABABRs 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 GABAB receptors both pre‐ and postsynaptically to modulate different sensory inputs and subsequent swimming activity. J. Comp. Neurol. 506:489–505, 2008.

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.

Observations on the settlement of phallusia mammillata larvae: Effects of different lithological substrata

Abstract In the sessile marine tunicates, the selection of a suitable substratum by the larvae is an important and critical factor determining the distribution of species. The present paper investigated, under laboratory conditions, the role played by mineral content of the substratum in settlement of the ascidian Phallusia mammillata, using siliceous and carbonaceous stones. Individuals that attached to the different substrata and metamorphosed were scored. The data indicate that larvae could discriminate between the substrata on the basis of their silica content. Under the same laboratory conditions, the larvae that attached to siliceous stones grew faster and had a wider area of contact with the substratum than those that grew on carbonaceous stones. The present study concludes that silica is a mineral factor that can be discriminated by chemosensory palps of ascidian larvae during the choice of substratum. It is suggested that the mineral composition of the habitat can contribute, with other environmental factors, to regulate the spatial distribution of tunicate communities.

The synapsin gene family in basal chordates: evolutionary perspectives in metazoans

BackgroundSynapsins are neuronal phosphoproteins involved in several functions correlated with both neurotransmitter release and synaptogenesis. The comprehension of the basal role of the synapsin family is hampered in vertebrates by the existence of multiple synapsin genes. Therefore, studying homologous genes in basal chordates, devoid of genome duplication, could help to achieve a better understanding of the complex functions of these proteins.ResultsIn this study we report the cloning and characterization of the Ciona intestinalis and amphioxus Branchiostoma floridae synapsin transcripts and the definition of their gene structure using available C. intestinalis and B. floridae genomic sequences. We demonstrate the occurrence, in both model organisms, of a single member of the synapsin gene family. Full-length synapsin genes were identified in the recently sequenced genomes of phylogenetically diverse metazoans. Comparative genome analysis reveals extensive conservation of the SYN locus in several metazoans. Moreover, developmental expression studies underline that synapsin is a neuronal-specific marker in basal chordates and is expressed in several cell types of PNS and in many, if not all, CNS neurons.ConclusionOur study demonstrates that synapsin genes are metazoan genes present in a single copy per genome, except for vertebrates. Moreover, we hypothesize that, during the evolution of synapsin proteins, new domains are added at different stages probably to cope up with the increased complexity in the nervous system organization. Finally, we demonstrate that protochordate synapsin is restricted to the post-mitotic phase of CNS development and thereby is a good marker of postmitotic neurons.

Action of valproic acid on Xenopus laevis development: teratogenic effects on eyes.

Valproic acid (VPA) is an anticonvulsive drug used in the treatment of epilepsy. Teratogenic effects of VPA have been described in different animal species. In this study, we investigate the effects of VPA on the development of Xenopus laevis embryos, by short pulse treatments (4 h) with relation to the dose and the stage of exposure to the drug. We exposed Xenopus embryos from blastula to stage 32 to three different doses of VPA (0.25, 5, and 10 mM) and we allowed these to develop until the controls reached stage 47. The embryos became more sensitive during the stages of neurulation, as observed in mouse and differently from Amblystoma, in which the more severe effects were produced by treatments at blastula stage. The malformations observed were similar to those described in mammals and other amphibians and consisted in developmental delay, perturbation of neural crest migration, and somite segmentation. We also observed abnormal development of the retina, which had never been described for VPA treatments. Therefore we analyzed the relation between VPA-induced eye malformations and the expression of Pax-6. We examined VPA-treated Xenopus embryos by whole mount in situ hybridization for mis-expression of Pax-6 in correlation with eye anomalies. Our results are consistent with the hypothesis that different members of Pax gene family are candidate target of VPA teratogenic action and in particular the decreased level of Pax-6 expression, shown by Northern blot analysis, is responsible for the retinal malformations we observed in VPA-treated Xenopus embryos. Teratogenesis Carcinog. Mutagen. 21:121-133, 2001.