Landscape-stream interactions and habitat conservation for amphibians.

Semiaquatic organisms depend on the features of both water bodies and landscapes; the interplay between terrestrial and aquatic systems might influence the semiaquatic communities, determining the scale at which management would be more effective. However, the consequences of such interplay are not frequently quantified, particularly at the community level. We analyzed the distribution of amphibians to evaluate whether the influence of landscape features on freshwater ecosystems can have indirect consequences at both the species and community level. We surveyed 74 streams in northern Italy to obtain data on breeding amphibians, water, and microhabitat features; we also measured features of surrounding landscapes. We used an information-theoretic approach and structural equation models to compare hypotheses on causal relationships between species distribution and variables measured at multiple levels. We also used a constrained redundancy analyses to evaluate causal relationships between multivariate descriptors of habitat features and community composition. Distribution of Salamandra salamandra was related to landscape, hydrological, and water characteristics: salamanders were more frequent in permanent streams with low phosphate concentration within natural landscapes. Water characteristics were dependent on landscape: streams in natural landscapes had less phosphates. Landscape influenced the salamander both directly and indirectly through its influence on phosphates. Community structure was determined by both landscape and water characteristics. Several species were associated with natural landscapes, and with particular water characteristics. Landscape explained a significant proportion of variability of water characteristics; therefore it probably had indirect effects on community. Upland environments play key roles for amphibians, for example, as the habitat of adults, but upland environments also have indirect effects on the aquatic life stages, mediated through their influence on water characteristics. Synergistic effects can magnify the negative consequences of landscape alteration on amphibians; landscape management can be particularly effective, as it can also improve wetland features.

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.

Influence of landscape elements in riparian buffers on the conservation of semiaquatic amphibians.

Studies on riparian buffers have usually focused on the amount of land needed as habitat for the terrestrial life stages of semiaquatic species. Nevertheless, the landscape surrounding wetlands is also important for other key processes, such as dispersal and the dynamics of metapopulations. Multiple elements that influence these processes should therefore be considered in the delineation of buffers. We analyzed landscape elements (forest cover, density of roads, and hydrographic network) in concentric buffers to evaluate the scale at which they influence stream amphibians in 77 distinct landscapes. To evaluate whether our results could be generalized to other contexts, we determined whether they were consistent across the study areas. Amphibians required buffers of 100-400 m of suitable terrestrial habitat, but interspecific differences in the amount of habitat were large. The presence of amphibians was related to roads and the hydrographic network at larger spatial scales (300-1500 m), which suggests that wider buffers are needed with these elements. This pattern probably arose because these elements influence dispersal and metapopulation persistence, processes that occur at large spatial scales. Furthermore, in some cases, analyses performed on different sets of landscapes provided different results, which suggests caution should be used when conservation recommendations are applied to disparate areas. Establishment of riparian buffers should not be focused only on riparian habitat, but should take a landscape perspective because semiaquatic species use multiple elements for different functions. This approach can be complex because different landscape elements require different spatial extents. Nevertheless, a shift of attention toward the management of different elements at multiple spatial scales is necessary for the long-term persistence of populations.

Deforestation and the structure of frog communities in the Humedale Terraba-Sierpe, Costa Rica.

Loss of tropical forests is a major cause of biodiversity loss worldwide. Although drastic modification of the habitat has been shown to negatively affect amphibians, we are far from a complete understanding of the response of amphibian communities to deforestation. We studied frog assemblages in a gradient of forest modification in a humid area of Costa Rica, where the primary forest has been partially converted into pasture. The study area is a mosaic of primary palm forest, abandoned pasture covered by secondary forest, and pasture. Species richness was assessed by randomized walk surveys and audio strip transects. We also measured ecological features to evaluate the relationship between landscape alteration and amphibian distribution. The study area hosted a large number of amphibian species. We focused our monitoring on six anurans: Leptodactylus labialis, Eleutherodactylus fitzingeri, E. diastema, Hyla rosenbergi, H. microcephale, and Cochranella granulosa. Three species (L. labialis, H. rosenbergi, and H. microcephala) were most abundant in pasture areas with livestock presence, while E. fitzingeri, E. diastema, and C. granulosa were associated with primary forest. Most of the variation in community structure was explained by the joint effect of forest alteration and presence of livestock. Whereas forest specialists suffer direct negative effect from deforestation, generalist species can take advantage of forest alteration and the presence of farm animals. Species that are able to take advantage of the new environmental characteristics associated with human modifications of landscapes will come to prevail in the new communities.

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.

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.

Rapid selection against inbreeding in a wild population of a rare frog.

Populations that are small and isolated can be threatened through loss of fitness due to inbreeding. Nevertheless, an increased frequency of recessive homozygotes could increase the efficiency of selection against deleterious mutants, thus reducing inbreeding depression. In wild populations, observations of evolutionary changes determined by selection against inbreeding are few. We used microsatellite DNA markers to compare the genetic features of tadpoles immediately after hatch with those of metamorphosing froglets belonging to the same cohort in a small, isolated population of the threatened frog Rana latastei. Within a generation, the inbreeding coefficient (F IS) decreased: at hatch, F IS was significantly >0, whereas F IS was <0 after metamorphosis. Furthermore, heterozygosity increased and allelic frequencies changed over time, resulting in the loss of genotypes at metamorphosis that were present in hatchlings. One microsatellite locus exhibited atypically large F ST values, suggesting it might be linked to a locus under selection. These results support the hypothesis that strong selection against the most inbred genotypes occurred among early life-history stages in our population. Selective forces can promote changes that can affect population dynamics and should be considered in conservation planning.

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.

Genetic diversity, but not hatching success, is jointly affected by postglacial colonization and isolation in the threatened frog, Rana latastei.

Both postglacial colonization and habitat fragmentation can reduce the genetic diversity of populations, which in turn can affect fitness. However, since these processes occur at different spatial and temporal scales, the consequences of either process may differ. To disentangle the relative role of isolation and postglacial colonization in determining genetic diversity and fitness, we studied microsatellite diversity of 295 individuals from 10 populations and measured the hatch rate of 218 clutches from eight populations of a threatened frog, R. latastei. The populations that were affected by fragmentation to a greater extent suffered higher embryo mortality and reduced hatch rate, while no effects of distance from glacial refugium on hatch rate were detected. Altogether, distance from glacial refugium and isolation explained > 90% of variation in genetic diversity. We found that the genetic diversity was lowest in populations both isolated and far from the glacial refugium, and that distance from refugium seems to have the primary role in determining genetic diversity. The relationship between genetic diversity and hatch rate was not significant. However, the proportion of genetic diversity lost through recent isolation had a significant, negative effect on fitness. It is possible that selection at least partially purged the negative effects of the ancestral loss of genetic diversity.

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.

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.

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.

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.