Environmental factors associated with amphibian breeding in streams and springs : Effects of habitat and fish occurrence

Streams are among the most threatened aquatic habitats for amphibians. Amphibians often demonstrate a complex community structure and investigations are mainly performed on pond-dwelling species, whereas data regarding streamdwelling species is still scarce. The aim of this study was to evaluate the relevance of stream features and the effects of the occurrence of predatory fish on the community structure of amphibians. To assess the occurrence of breeding among amphibians (in Northern Apennine streams), sampling was performed on 57 stream and spring sites between early March and late May 2014. The research process recorded the presence of four breeding species (Salamandra salamandra, Salamandrina perspicillata, Bufo bufo, and Rana italica) and discovered that the community structures of amphibians were significantly connected to different habitat features. The investigation showed that fish presence is the primary habitat feature that affects the communities of amphibians: B. bufo was the only amphibian species not affected by fish presence, while the other species avoided sites with fish. Sun exposure, depth of water and slope inclination played important roles in affecting amphibian breeding. Our study confirms that fish presence in small streams can be detrimental for different amphibian species

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

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 β-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

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

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

Effects of exposure to microplastics on the development and metamorphosis of Ciona robusta

Microplastics are plastic particles produced to be of small size, as sandblasting media, virgin pellet, cosmetics (Fendall and Sewell, 2009) or that originate from degradation of bigger plastic debris, as polyester fibers, polyethylene plastic bags and polystyrene particles from buoys and floats (Browne et al., 2011; Davidson, 2012; O\u2019Brine and Thompson, 2010). An incredible amount of plastic reaches the marine environment and its ecological impacts are of particular interest. In fact, after ingestion, they may cause damage and block of feeding appendages and digestive system, can migrate from alimentary canal to other body tissues, limit the food intake and transfer pollutants in living organisms (Cole et al., 2013). We evaluated microplastics effects on development and metamorphosis of the solitary ascidian Ciona robusta. We used microbeads with 10 \u3bcm diameter made of polystyrene, because it is highly stable with a negligible styrene migration (Cohen et al., 2002). We tested four different concentrations (0.125, 1.25, 12.5 and 25 \u3bcg/ml) and analyzed the effects on larval development and survival. Then we exposed attached larvae for five days to test the effects on metamorphosis. Juvenile survival was calculated exposing metamorphosed individuals to microplastics for eight days. We also estimated the ingestion rate as the number of particles ingested in one minute and the expulsion capability measuring the localization of particles in stomach or fecal pellets after 24 hours. Metamorphosis was the process most affected by the microplastics. In fact, the percentage of individuals that completed metamorphosis was statistically lower in all the exposed groups, compared to control. Feeding individuals were also fed with microalgae. The co-ingestion of algae and beads probably reduced the amount of algae assimilated by the juveniles. Moreover, they might spend energy to expel microplastics thus reducing the energy available for the metamorphosis. After 8 days, juvenile survival was not affected, even if the particles persisted longer in the stomach of individuals exposed to the highest concentration, suggesting that further detrimental effects could be observed after a longer exposure

Factors driving semi-aquatic predator occurrence in traditional cattle drinking pools : conservation issues

In several cases, human impact on water bodies and on their freshwater communities is detrimental, but in some cases the human activity may favour and enhance the biodiversity of small water bodies, as traditional cattle drinking pools. Despite their small size, small water bodies may constitute hot spot of biodiversity often representing the only lentic aquatic biotope in landscapes where superficial water lacks or flows in lotic environments like creeks and streams. Predators are good indicators of biodiversity in ponds and give information of food chain web complexity. In particular, semi-aquatic predators like amphibians and dragonflies may account for a substantial percentage of energy flow between aquatic and terrestrial ecosystems. In this study, we evaluated the conservation value of traditional cattle drinking pools building by assessing the factors determining the occurrence and distribution of the semiaquatic predators. From April to August 2015, we investigated 30 distinct pools recording several abiotic and biotic environmental variables. We detected 4 semi-aquatic predators: Salamandra salamandra larvae, Triturus carnifex, Aeshna sp. larvae and Libellula sp. larvae. Abiotic features played a major role in shaping the predator community that resulted linked to stable, with no dryness period, and large drinking pools. Invertebrate prey biomass was not particularly important, while vegetation cover and occurrence of unpalatable tadpoles were the most important biotic features of the pools. Our study provides novel evidence on the importance of cattle drinking pools management to preserve biodiversity especially in areas where traditional pastoral activity is disappearing

Activity of a trinuclear platinum complex in human ovarian cancer cell lines sensitive and resistant to cisplatin: cytotoxicity and induction and gene-specific repair of DNA lesions

A collateral sensitivity or a very modest cross-resistance to BBR 3464 was found in 2 ovarian cancer cell lines with experimentally induced resistance to cisplatin. Loss of mismatch repair proteins (hMLH1, hPMS2) or overexpression of nucleotide excision repair proteins (ERCC1) was not detrimental for the cellular sensitivity to BBR 3464. Moreover, interesting differences in the kinetics of formation and removal of DNA lesions at the single-gene (N- ras) level were observed between BBR 3464 and CDDP. © 2001 Cancer Research Campaign www.bjcancer.co

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(\u3bc-Cl)2(CO)6(\u3bc-R-pydz-17\u3b1-ethynylestradiol)] (R-pydz = functionalized 1,2-pyridazine), where the estradiol moiety is covalently bound to the \u3b2 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 17\u3b1-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

Effects of bisphenol A on early development of the ascidian Phallusia mammillata (Chordata, Tunicata)

Bisphenol A (BPA) is an organic compound used in the manufacture of polycarbonate plastic and epoxy resins that is released into the environment from sewage treatment effluent, landfill leachate and degradation of plastic products. BPA can act both as a teratogenic substance and as an endocrine disruptor. The phylogenetic position of tunicates as sister group of vertebrates and their cosmopolitan distribution in marine ecosystems coupled with their ecology and easy manipulability make them reliable model organisms for ecotoxicology bioassays. Here we evaluated the effects of different concentration of BPA (0.1, 0.5, 1, 5, 10, and 20 \ub5M) on ontogenetic processes sensitive to environmental pollutants in the ascidian Phallusia mammillata. To test the effects of this substance on sperm viability we fertilized eggs with sperm pre-exposed for 30 minutes to BPA. One hour post fertilization (hpf) we calculated the percentage of eggs that reached the 2-cell stage compared to control. Then we analyzed the consequences of a fertilization performed directly in BPA solutions, counting the percentage of 2 cell-stage embryos 1 hpf. To test the effects on embryogenesis, we analyzed hatched larvae developed from 2-cell embryos exposed to BPA. We evaluated the number and type of malformations under a dissection microscope and we performed immunostaining of central nervous system (CNS). Exposure of sperm to BPA did not influence fertilization rate. Coexposure of eggs and sperm at concentrations higher than 5 \ub5M caused incomplete division of eggs, producing heart shaped embryos that did not develop further. Regarding embryogenesis, 10 \ub5M BPA caused specific malformations at central nervous system, with an increased distance between otolith and ocellus, or an extrusion of ocellus from the sensory vesicle. Embryonic development was ignificantly altered at 20 \ub5M concentration. 97% of the larvae presented a severely affected phenotype with short and kinked tail. Immunostainig with anti \u3b2-tubulin antibody showed an altered pattern of fibers in CNS. These results showed that the most sensitive process to BPA is the first cell division. When the 2-cell stage embryos are treated, higher concentration are required to alter the correct development of CNS. BPA confirmed its teratogenic effects on ascidians and its interference with CNS development even if the mechanism of action in this group is still to be clarified