Interspecific competitive interactions affect body size and oxidative status of two nonnative salmonid species.

In fish, interspecific interactions between nonnative and other sympatric species are considered determinants in shaping species assemblages. Such interactions can also arise between nonnative fish species only, including salmonids such as the brown trout (Salmo trutta, Linnaeus, 1758) and the rainbow trout (Oncorhynchus mykiss, Walbaum, 1792), returning contrasting outcomes. The present manipulative experiment was aimed at exploring the effect of interspecific competition on the body growth and the oxidative status of parr (2 + -year-old individuals) of the brown trout and the rainbow trout. Allopatric (intraspecific competition) and sympatric (interspecific competition) populations of these species were experimentally recreated in two wild streams. At the end of a 2-month-long experiment, changes in specific growth rate (SGR), oxidative status (i.e., levels of reactive oxygen species and activity of antioxidant enzymes such as superoxide dismutase - SOD, catalase - CAT and glutathione peroxidase - GPx) and oxidative damage (i.e., lipid peroxidation) were investigated in brown and rainbow trout individuals maintained in allopatric or sympatric populations. Sympatric interactions between rainbow and brown trout parr resulted in a significant decrease in SGR of brown trout individuals only. Moreover, an overall modulation of the oxidative status, in terms of an increase in ROS levels coupled with the activation of SOD and CAT activity, occurred in brown trout individuals under sympatric conditions. These findings might suggest that, under sympatric conditions, parr of the rainbow trout are more competitive than brown trout for food acquisition. However, this competition affected the antioxidant defenses of the brown trout only, probably because of reduced ingestion of dietary antioxidants or increased physical activity and aggressive behavior. Thus, interspecific interactions can induce physiological and phenotypic effects on parr of nonnative salmonids, with potential consequences on the establishment of populations of these species in freshwater ecosystems.

Fluorescence Properties of a Novel Isoquinoline Derivative Tested in an Invertebrate Chordate, Ciona intestinalis.

3-Hydroxyisoquinolines (ISOs) and their tautomeric isoquinolin-3-ones are heterocycles with attractive biological properties. Here we reported the revisited synthesis of a highly functionalized ISO that showed blue fluorescence and the characterization of its biological properties in an invertebrate animal model, the ascidian Ciona intestinalis. Larvae exposed to ISO at concentrations higher than 1 µM showed an intense fluorescence localized in the cell nuclei of all tissues. Moreover, exposure to ISO interfered with larval ability to swim; this neuromuscular effect was reversible. Overall, these results suggested that ISOs can have promising applications as novel fluorescent dyes of the cell nuclei.

Exploring miR-9 Involvement in Ciona intestinalis Neural Development Using Peptide Nucleic Acids.

The microRNAs are small RNAs that regulate gene expression at the post-transcriptional level and can be involved in the onset of neurodegenerative diseases and cancer. They are emerging as possible targets for antisense-based therapy, even though the in vivo stability of miRNA analogues is still questioned. We tested the ability of peptide nucleic acids, a novel class of nucleic acid mimics, to downregulate miR-9 in vivo in an invertebrate model organism, the ascidian Ciona intestinalis, by microinjection of antisense molecules in the eggs. It is known that miR-9 is a well-conserved microRNA in bilaterians and we found that it is expressed in epidermal sensory neurons of the tail in the larva of C. intestinalis. Larvae developed from injected eggs showed a reduced differentiation of tail neurons, confirming the possibility to use peptide nucleic acid PNA to downregulate miRNA in a whole organism. By identifying putative targets of miR-9, we discuss the role of this miRNA in the development of the peripheral nervous system of ascidians.

Luminescent conjugates between dinuclear rhenium complexes and 17α-ethynylestradiol: synthesis, photophysical characterization, and cell imaging.

Three new luminescent conjugates between dinuclear rhenium complexes and an estradiol, namely E2-Re, are described. The derivatives have the general formula [Re2(µ-Cl)2(CO)6(µ-R-pydz-17α-ethynylestradiol)] (R-pydz = functionalized 1,2-pyridazine), where the estradiol moiety is covalently bound to the ß position of the pyridazine ligand. Different synthetic pathways are investigated, including the inverse-type [4 + 2] Diels Alder cycloaddition reaction between the electron poor 1,2,4,5-tetrazine and 17α-ethynylestradiol for the synthesis of E2-Re1. The three E2-Re conjugates are purified on silica gel and isolated in a spectroscopically pure form in moderate to good yields (28-50%). All the E2-Re conjugates are comprehensively characterized from the spectroscopic and photophysical points of view. Cellular internalization experiments on human MCF-7 and 231 cells are also reported, displaying interesting staining differences depending on the nature of the spacer linking the estradiol unit to the organometallic fragment. Furthermore, the suitability of these conjugates to also stain simple multicellular organisms, i.e. Ciona intestinalis embryos and larvae at different stages of development, is reported here for the first time.

miR-7 Knockdown by Peptide Nucleic Acids in the Ascidian Ciona intestinalis.

Peptide Nucleic Acids (PNAs) are synthetic mimics of natural oligonucleotides, which bind complementary DNA/RNA strands with high sequence specificity. They display numerous advantages, but in vivo applications are still rare. One of the main drawbacks of PNAs application is the poor cellular uptake that could be overcome by using experimental models, in which microinjection techniques allow direct delivery of molecules into eggs. Thus, in this communication, we investigated PNAs efficiency in miR-7 downregulation and compared its effects with those obtained with the commercially available antisense molecule, Antagomir (Dharmacon) in the ascidian Ciona intestinalis. Ascidians are marine invertebrates closely related to vertebrates, in which PNA techniques have not been applied yet. Our results suggested that anti-miR-7 PNAs were able to reach their specific targets in the developing ascidian embryos with high efficiency, as the same effects were obtained with both PNA and Antagomir. To the best of our knowledge, this is the first evidence that unmodified PNAs can be applied in in vivo knockdown strategies when directly injected into eggs.

Age- and sex-dependent variation in the activity of antioxidant enzymes in the brown trout (Salmo trutta).

Oxidative stress is defined as the imbalance between pro-oxidant and antioxidant molecules in favor of the former and it represents one of the main driving forces of aging. To counteract the harmful effects of oxidative stress, organisms evolved a complex antioxidant system. According to the free radical theory of aging, while the production of reactive oxygen species (ROS) increases with age, the antioxidant defenses decline. Although this relationship has been investigated in diverse vertebrate taxa, the information in fish is scant and inconsistent, particularly for wild populations. Thus, the aim of the present study was the investigation of age- and sex-related changes of the antioxidant enzymes activity in free-living individuals of the brown trout (Salmo trutta). We measured the activity of the main enzymes involved in antioxidant protection, namely superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione S-transferase (GST), as well as lipid peroxidation, in the gills and the liver dissected from brown trout (1+- to 5+-year-old). A significant age-dependent variation in the activity of antioxidant enzymes was noted, with the exception of CAT. GPx activity followed a significant increasing trend with age in both the organs, while SOD decreased in the liver. Increased GST activity was found in the gills only. Lipid peroxidation levels significantly decreased with age in both the organs. SOD and CAT showed sex-dependent differences in the liver of brown trout, with males showing lower enzymatic activity than females. Our data contribute to improve the knowledge on the relationship between antioxidant enzyme activity, aging, and sex in fish.

Teratogenic potential of nanoencapsulated vitamin A evaluated on an alternative model organism, the tunicate Ciona intestinalis.

Nano-encapsulation is a technology used to pack substances in order to enhance their stability and bioavailability, but this packing may interact with living systems, causing unexpected toxicity. Vitamin A (vit A) is a substance that has received attention, because in developed countries, the increasing availability of supplements is leading to its excessive intake. This study aims to compare teratogenic effects caused by exposure to the traditional formulation of vit A versus nano-encapsulated vit A. We used ascidian embryos as an alternative model. Ascidians are marine organisms closely related to vertebrates that share with them a body plan and developmental programme, including the morphogenetic role of retinoic acid (RA). Our data showed that the adverse effects of exposure to the same concentration of the two formulations were different, suggesting that the nano-encapsulation increased the bioavailability of the molecule, which could be better absorbed and metabolised to RA, the effective teratogenic substance.

Thermal equilibrium and temperature differences among body regions in European plethodontid salamanders.

Information on species thermal physiology is extremely important to understand species responses to environmental heterogeneity and changes. Thermography is an emerging technology that allows high resolution and accurate measurement of body temperature, but until now it has not been used to study thermal physiology of amphibians in the wild. Hydromantes terrestrial salamanders are strongly depending on ambient temperature for their activity and gas exchanges, but information on their body temperature is extremely limited. In this study we tested if Hydromantes salamanders are thermoconform, we assessed whether there are temperature differences among body regions, and evaluated the time required to reach the thermal equilibrium. During summers of 2014 and 2015 we analysed 56 salamanders (Hydromantes ambrosii and Hydromantes italicus) using infrared thermocamera. We photographed salamanders at the moment in which we found them and 1, 2, 3, 4, 5 and 15min after having kept them in the hands. Body temperature was equal to air temperature; salamanders attained the equilibrium with air temperature in about 8min, the time required to reach equilibrium was longer in individuals with large body size. We detected small temperature differences between body parts, the head being slightly warmer than the body and the tail (mean difference: 0.05°C). These salamanders quickly reach the equilibrium with the environment, thus microhabitat measurement allows obtaining accurate information on their tolerance limits.

Bisphenol A affects the development and the onset of photosymbiosis in the acoel Symsagittiferaroscoffensis.

Photosymbiosis indicates a long-term association between animals and photosynthetic organisms. It has been mainly investigated in photosymbiotic cnidarians, while other photosymbiotic associations have been largely neglected. The acoel Symsagittifera roscoffensis lives in obligatory symbiosis with the microalgal Tetraselmis convolutae and has recently emerged as alternative model to study photosymbiosis. Here, we investigated the effects of Bisphenol A, a common plastic additive, on two pivotal stages of its lifecycle: aposymbiotic juvenile development and photosymbiogenesis. Based on our results, this pollutant altered the development of the worms and their capacity to engulf algae from the environment at concentrations higher than the levels detected in seawater, yet aligning with those documented in sediments of populated areas. Data provide novel information about the effects of pollutants on photosymbiotic associations and prompt the necessity to monitor their concentrations in marine environmental matrices.

A feather star is born: embryonic development and nervous system organization in the crinoid Antedon mediterranea.

Crinoids belong to the Echinodermata, marine invertebrates with a highly derived adult pentaradial body plan. As the sister group to all other extant echinoderms, crinoids occupy a key phylogenetic position to explore the evolutionary history of the whole phylum. However, their development remains understudied compared with that of other echinoderms. Therefore, the aim here was to establish the Mediterranean feather star (Antedon mediterranea) as an experimental system for developmental biology. We first set up a method for culturing embryos in vitro and defined a standardized staging system for this species. We then optimized protocols to characterize the morphological and molecular development of the main structures of the feather star body plan. Focusing on the nervous system, we showed that the larval apical organ includes serotonergic, GABAergic and glutamatergic neurons, which develop within a conserved anterior molecular signature. We described the composition of the early post-metamorphic nervous system and revealed that it has an anterior signature. These results further our knowledge on crinoid development and provide new techniques to investigate feather star embryogenesis. This will pave the way for the inclusion of crinoids in comparative studies addressing the origin of the echinoderm body plan and the evolutionary diversification of deuterostomes.

Serotonin Receptors and Their Involvement in Melanization of Sensory Cells in Ciona intestinalis.

Serotonin (5-hydroxytryptamine (5-HT)) is a biogenic monoamine with pleiotropic functions. It exerts its roles by binding to specific 5-HT receptors (5HTRs) classified into different families and subtypes. Homologs of 5HTRs are widely present in invertebrates, but their expression and pharmacological characterization have been scarcely investigated. In particular, 5-HT has been localized in many tunicate species but only a few studies have investigated its physiological functions. Tunicates, including ascidians, are the sister group of vertebrates, and data about the role of 5-HTRs in these organisms are thus important for understanding 5-HT evolution among animals. In the present study, we identified and described 5HTRs in the ascidian Ciona intestinalis. During development, they showed broad expression patterns that appeared consistent with those reported in other species. Then, we investigated 5-HT roles in ascidian embryogenesis exposing C. intestinalis embryos to WAY-100635, an antagonist of the 5HT1A receptor, and explored the affected pathways in neural development and melanogenesis. Our results contribute to unraveling the multifaceted functions of 5-HT, revealing its involvement in sensory cell differentiation in ascidians.

Mixotrophic flagellate ingestion boosts microplastic accumulation in ascidians.

Microplastics are contaminants of global environmental concern. They can be ingested by a variety of organisms when they enter the food web. Several studies have reported trophic transfer of microplastics from low trophic levels to higher ones. Bioaccumulation has been suggested to occur but few studies have demonstrated it for marine environments. In this article, in controlled laboratory conditions, we exposed filter-feeder ascidian juveniles to microplastics in the presence or in absence of mixotrophic cryptomonad flagellates. Cryptomonads can efficiently ingest microbeads, and their presence significantly increased the concentration of microplastics in the digestive tract of the ascidians. Our results demonstrate the occurrence of microplastic bioaccumulation in the lower levels of the marine trophic chain and suggest that unicellular organisms can be key actors in microplastic trophic transfer at the microscale level.

A New Disease Caused by an Unidentified Etiological Agent Affects European Salamanders.

New pathologies are causing dramatic declines and extinctions of multiple amphibian species. In 2013, in one fire salamander population of Northern Italy, we found individuals with undescribed cysts at the throat level, a malady whose existence has not previously been reported in amphibians. With the aim of describing this novel disease, we performed repeated field surveys to assess the frequency of affected salamanders from 2014 to 2020, and integrated morphological, histological, and molecular analyses to identify the pathogen. The novel disease affected up to 22% of salamanders of the study population and started spreading to nearby populations. Cysts are formed by mucus surrounding protist-like cells about 30 µm long, characterized by numerous cilia/undulipodia. Morphological and genetic analyses did not yield a clear match with described organisms. The existence of this pathogen calls for the implementation of biosecurity protocols and more studies on the dynamics of transmission and the impact on wild populations.

Embryotoxicity characterization of the flame retardant tris(1-chloro-2-propyl)phosphate (TCPP) in the invertebrate chordate Ciona intestinalis.

Tris(1-chloro-2-propyl)phosphate (TCPP) is the most common chlorinated organophosphorus flame retardant in seawater. Due to its chemical features and abundance, TCPP has been classified as a high hazard, and restrictions of use have been set in multiple countries. Despite TCPP being highly present in the marine environment, only a few studies have explored the TCPP impact on the development of marine invertebrates. Ascidians are important invertebrate members of benthic marine communities and reliable model systems for ecotoxicological research. The aim of this study was to assess the adverse effects of TCPP exposure on the embryogenesis of the ascidian Ciona intestinalis. Our results showed that this pollutant affected both muscles and nervous system development. Malformations appeared similar to those reported in other animal models for other flame retardants, suggesting that these molecules could share a common mechanism of action and induce a mixture effect when simultaneously present in the aquatic environment even at sub-teratogenic concentrations.

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

BACKGROUND: Synapsins 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. RESULTS: In 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. CONCLUSION: Our 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.

Immunohistochemical analysis of adhesive papillae of Clavelina lepadiformis (Müller, 1776) and Clavelina phlegraea (Salfi, 1929) (Tunicata, Ascidiacea).

Almost all ascidian larvae bear three mucus secreting and sensory organs, the adhesive papillae, at the anterior end of the trunk, which play an important role during the settlement phase. The morphology and the cellular composition of these organs varies greatly in the different species. The larvae of the Clavelina genus bear simple bulbous papillae, which are considered to have only a secretory function. We analysed the adhesive papillae of two species belonging to this genus, C. lepadiformis and C. phlegraea, by histological sections and by immunolocalisation of b-tubulin and serotonin, in order to better clarify the cellular composition of these organs. We demonstrated that they contain at least two types of neurons: central neurons, bearing microvilli, and peripheral ciliated neurons. Peripheral neurons of C. lepadiformis contain serotonin. We suggest that these two neurons play different roles during settlement: the central ones may be chemo- or mechanoreceptors that sense the substratum, and the peripheral ones may be involved in the mechanism that triggers metamorphosis.

Bisphenol A affects neural development of the ascidian Ciona robusta.

Bisphenol A (BPA) is an organic pollutant derived from plastic degradation that has numerous and variable adverse effects on human health and wildlife. In particular, it has been reported that BPA can alter reproductive processes and nervous system development in vertebrates. Considering BPA presence in marine environment and the scant data available on its interaction with nervous system development, we analyzed the effect of BPA exposure on sperm viability, fertilization, embryogenesis, and neural differentiation of the ascidian Ciona robusta. Ascidians are members of the Phylum Tunicata, the sister group of Vertebrata, sharing with them fundamental developmental processes. Our results showed that first cell division was altered starting from 5 µM concentration. Lethal concentration (LC 50 ) was estimated to be 5.2 µM. Larvae developed from treated embryos showed specific malformations to the pigment cells even at 0.1 µM, corresponding to the highest environmental concentration reported so far. Moreover, GABAergic and dopaminergic neurons proved to be target organs of BPA teratogenic action, in accordance with similar results reported in vertebrate animal models. Overall, our results suggest that BPA can exert its effects on nervous system acting on different pathways and underline that C. robusta is a valuable invertebrate animal model for preliminary screenings of effects of pollutants on vertebrates.

Nervous system characterization during the development of a basal echinoderm, the feather star Antedon mediterranea.

Neural development of echinoderms has always been difficult to interpret, as larval neurons degenerate at metamorphosis and a tripartite nervous system differentiates in the adult. Despite their key phylogenetic position as basal echinoderms, crinoids have been scarcely studied in developmental research. However, since they are the only extant echinoderms retaining the ancestral body plan of the group, crinoids are extremely valuable models to clarify neural evolution in deuterostomes. Antedon mediterranea is a feather star, endemic to the Mediterranean Sea. Its development includes a swimming lecithotrophic larva, the doliolaria, with basiepithelial nerve plexus, and a sessile filter-feeding juvenile, the pentacrinoid, whose nervous system has never been described in detail. Thus, we characterized the nervous system of both these developmental stages by means of immunohistochemistry and, for the first time, in situ hybridization techniques. The results confirmed previous descriptions of doliolaria morphology and revealed that the larval apical organ contains two bilateral clusters of serotonergic cells while GABAergic neurons are localized under the adhesive pit. This suggested that different larval activities (e.g., attachment and metamorphosis) are under the control of different neural populations. In pentacrinoids, the analysis showed the presence of a cholinergic entoneural system while the ectoneural plexus appeared more composite, displaying different neural populations. The expression of three neural-related microRNAs was described for the first time, suggesting that these are evolutionarily conserved also in basal echinoderms. Overall, our results set the stage for future investigations that will reveal new information on echinoderm evo-devo neurobiology.

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.

Serotonin involvement in the metamorphosis of the hydroid Eudendrium racemosum.

Hydroid planulae metamorphose in response to an inducing external stimulus, usually a bacterial cue. There is evidence that neurotransmitters participate in the signal transduction pathway of hydroid metamorphosis. Eudendrium racemosum is a colonial hydroid common in the Mediterranean Sea. It lacks the medusa stage and the planulae develop on female colonies during the fertile season. In this work, serotonin (5-HT) was localized in some planula ectodermal cells. Co-localization of serotonin and beta-tubulin suggested that 5-HT was present in sensory nervous cells and in different ectodermal cells. To investigate the role of neurotransmitters in metamorphosis, E. racemosum planulae were treated with serotonin and dopamine and with agonists and antagonists of the corresponding receptors. Serotonin and a serotonin receptor agonist induced metamorphosis, while a 5-HT receptor antagonist inhibited it. Dopamine and all dopaminergic drugs used did not show any significant effect on the onset of metamorphosis. Results from this work showed that 5-HT could stimulate metamorphosis in E. racemosum planulae in the presence of a natural inducer. A mechanism by which this neurotransmitter could act in this phase is proposed.