Insights into Emydid Turtle Cytogenetics: The European Pond Turtle as a Model Species.

Our knowledge of Testudines evolution is limited by the lack of modern cytogenetic data. Compared to other reptiles, there is little information even on chromosome banding, let alone molecular cytogenetic data. Here, we provide detailed information on the karyotype of the European pond turtle Emys orbicularis, a model Emydidae, employing both chromosome banding and molecular cytogenetics. We provide a high-resolution G-banded karyotype and a map of rDNA genes and telomeric sequences using fluorescence in situ hybridization. We test hypotheses of sex-determining mechanisms in Emys by comparative genomic hybridization to determine if Emys has a cryptic sex-specific region. Our results provide valuable data to guide future efforts on genome sequencing and anchoring in Emydidae and for understanding karyotype evolution in Testudines.

The Weierstrassian movement patterns of snails.

Weierstrassian Lévy walks are the archetypical form of random walk that do not satisfy the central limit theorem and are instead characterized by scale invariance. They were originally regarded as a mathematical abstraction but subsequent theoretical studies showed that they can, in principle, at least, be generated by chaos. Recently, Weierstrassian Lévy walks have been found to provide accurate representations of the movement patterns of mussels (Mytilus edulis) and mud snails (Hydrobia ulvae) recorded in the laboratory under controlled conditions. Here, we tested whether Weierstrassian Lévy walks and chaos are present under natural conditions in intertidal limpets Patella vulgata and P. rustica, and found that both characteristics are pervasive. We thereby show that Weierstrassian Lévy walks may be fundamental to how molluscs experience and interact with the world across a wide range of ecological contexts. We also show in an easily accessible way how chaos can produce a wide variety of Weierstrassian Lévy walk movement patterns. Our findings support the Lévy flight foraging hypothesis that posits that because Lévy walks can optimize search efficiencies, natural selection should have led to adaptations for Lévy walks.

Serpentine soils affect heavy metal tolerance but not genetic diversity in a common Mediterranean ant.

Natural habitats with serpentine soils are rich in heavy metal ions, which may significantly affect ecological communities. Exposure to metal pollutants results, for instance, in a reduction of population genetic diversity and a diffused higher tolerance towards heavy metals. In this study, we investigated whether chronic exposure to metals in serpentine soils affect accumulation patterns, tolerance towards metal pollutants, and genetic diversity in ants. In particular, we studied colonies of the common Mediterranean ant, Crematogaster scutellaris, along a contamination gradient consisting of two differently contaminated forests and a reference soil with no geogenic contamination. We first evaluated the metal content in both soil and ants' body. Then, we tested for tolerance towards metal pollutants by evaluating the mortality of ants fed with nickel (Ni) solutions of increasing concentrations. Finally, differences in genetic diversity among ants from different areas were assessed using eight microsatellite loci. Interestingly, a higher tolerance to nickel solutions was found in ants sampled in sites with intermediate levels of heavy metals. This may occur, because ants inhabiting strongly contaminated areas tend to accumulate higher amounts of contaminants. Additional ingestion of toxicants beyond the saturation threshold would lead to death. There was no difference in the genetic diversity among ant colonies sampled in different sites. This was probably the result of queen mediated gene flow during nuptial flights across uncontaminated and contaminated areas of limited geographical extent.

The Rules of Aggression: How Genetic, Chemical and Spatial Factors Affect Intercolony Fights in a Dominant Species, the Mediterranean Acrobat Ant Crematogaster scutellaris.

Nest-mate recognition plays a key role in the biology of ants. Although individuals coming from a foreign nest are, in most cases, promptly rejected, the degree of aggressiveness towards non nest-mates may be highly variable among species and relies on genetic, chemical and environmental factors. We analyzed intraspecific relationships among neighboring colonies of the dominant Mediterranean acrobat ant Crematogaster scutellaris integrating genetic, chemical and behavioral analyses. Colony structure, parental relationships between nests, cuticular hydrocarbons profiles (CHCs) and aggressive behavior against non nest-mates were studied in 34 nests located in olive tree trunks. Bayesian clustering analysis of allelic variation at nine species-specific microsatellite DNA markers pooled nests into 14 distinct clusters, each representing a single colony, confirming a polydomous arrangement of nests in this species. A marked genetic separation among colonies was also detected, probably due to long distance dispersion of queens and males during nuptial flights. CHCs profiles varied significantly among colonies and between nests of the same colony. No relationship between CHCs profiles and genetic distances was detected. The level of aggressiveness between colonies was inversely related to chemical and spatial distance, suggesting a 'nasty neighbor' effect. Our findings also suggest that CHCs profiles in C. scutellaris may be linked to external environmental factors rather than genetic relationships.

Pre-exposure to Cadmium or Zinc Alters the Heart Rate Response of the Crayfish Procambarus clarkii Towards Copper.

Exposure to sub-lethal concentrations of a pollutant induces, in some organisms, an acclimation process which increases their resistance to other substances (cross-acclimation). Understanding this phenomenon is important as a basis for a better comprehension of the effects of pollutants in ecosystems. In this paper we investigated whether the exposure to Cd or Zn is able to modify the heart rate response of the crayfish Procambarus clarkii to acute Cu stress. A first set of experiments provided the basis to understand heart rate changes induced by varying Cd or Zn concentrations. In a second set of experiments crayfish were acclimated for 96 h to control water, Cd or Zn enriched water, and then exposed to a 10 mg L(-1) Cu solution, known to induce bradycardia in this species. Bradycardia was suppressed in specimens previously exposed to Cd or Zn but not in those exposed to clean water, providing a clear evidence of a cross-acclimation in the heart rate response of P. clarkii.

When the rule becomes the exception. no evidence of gene flow between two Zerynthia cryptic butterflies suggests the emergence of a new model group.

There is increasing evidence that most parapatric cryptic/sister taxa are reproductively compatible across their areas of contact. Consequently, the biological species concept, which assumes absence of interbreeding, is becoming a not so effective criterion in evolutionary ecology. Nevertheless, the few parapatric sister taxa showing complete reproductive barriers represent interesting models to study speciation processes and the evolution of reproductive isolation. In this study, we examined contact populations in northwestern Italy of two butterfly species, Zerynthia polyxena and Z. cassandra, characterized by different genitalic morphotypes. We studied levels of divergence among 21 populations distributed from Sicily to France using three genetic markers (the mitochondrial COI and ND1 genes and the nuclear wingless gene) and genitalic geometric morphometrics. Moreover, we performed species distribution modelling to estimate different climatic requirements of Z. polyxena and Z. cassandra. We projected climatic data into glacial maximum scenarios in order to verify if and to which extent glacial cycles could have contributed to speciation processes. Genetic and morphometric analyses identified two main groups. All specimens showed a concordant pattern of diversification, including those individuals sampled in the contact area. Haplotype distribution and climatic models showed that during glacial maxima both species experienced a strong range contraction and presumably remained separated into different microrefugia in southern France, in the Italian Peninsula and on the islands of Elba and Sicily. Long term separation was probably favoured by reduced dispersal ability and high phylopatry, while genitalic diversification probably favoured interbreeding avoidance. Conversely, the aposematic wing pattern remained almost identical. We compared our results with those obtained in other species and concluded that Z. polyxena and Z. cassandra represent a valuable model in the study of speciation.

Exploring the effects of seasonality and chemical pollution on the hepatopancreas transcriptome of the Manila clam.

The assessment of marine environmental health is a complex but fundamental task both for ecosystem conservation and food safety related to the human consumption of marine products. Manila clams inhabiting the Venice Lagoon constitute an excellent case study for evaluating the effects of complex mixtures of industrial and urban effluents on aquatic organisms. Clams were collected in different seasons at four locations within the Venice Lagoon. The sampling sites were characterized by a range of pollutant concentrations and included Porto Marghera, a highly polluted industrial area where clam harvesting for human consumption is strictly forbidden. Pooled soft tissues were subjected to mass spectroscopy analysis to measure the concentrations of PCDDs/PCDFs/PCBs-DL, PCBs, PBDEs, HCB and PAHs, and pooled digestive gland samples were used for gene expression profiling. While seasonal variation was found to be responsible for the largest proportion of transcriptional changes, significance analysis of microarrays quantitative correlation analysis identified 162 transcripts that were correlated with at least one class of chemicals measured in the samples from the four different sampling sites. Prediction Analysis of Microarrays (PAM) identified a minimal set of seven genes that correctly assigned samples collected in the restricted polluted area (Porto Marghera), independent of the season in which they were collected. An integrated approach combining transcriptomics and chemical analyses of the Manila clam provided a global picture of how Manila clams respond to complex mixtures of xenobiotics and their interplay with other biotic and abiotic factors. We were also able to identify gene expression signatures for different classes of chemicals and a set of robust biomarkers of exposure to these chemicals.

Transcriptome sequencing and microarray development for the Manila clam, Ruditapes philippinarum: genomic tools for environmental monitoring.

BACKGROUND: The Manila clam, Ruditapes philippinarum, is one of the major aquaculture species in the world and a potential sentinel organism for monitoring the status of marine ecosystems. However, genomic resources for R. philippinarum are still extremely limited. Global analysis of gene expression profiles is increasingly used to evaluate the biological effects of various environmental stressors on aquatic animals under either artificial conditions or in the wild. Here, we report on the development of a transcriptomic platform for global gene expression profiling in the Manila clam. RESULTS: A normalized cDNA library representing a mixture of adult tissues was sequenced using a ultra high-throughput sequencing technology (Roche 454). A database consisting of 32,606 unique transcripts was constructed, 9,747 (30%) of which could be annotated by similarity. An oligo-DNA microarray platform was designed and applied to profile gene expression of digestive gland and gills. Functional annotation of differentially expressed genes between different tissues was performed by enrichment analysis. Expression of Natural Antisense Transcripts (NAT) analysis was also performed and bi-directional transcription appears a common phenomenon in the R. philippinarum transcriptome. A preliminary study on clam samples collected in a highly polluted area of the Venice Lagoon demonstrated the applicability of genomic tools to environmental monitoring. CONCLUSIONS: The transcriptomic platform developed for the Manila clam confirmed the high level of reproducibility of current microarray technology. Next-generation sequencing provided a good representation of the clam transcriptome. Despite the known limitations in transcript annotation and sequence coverage for non model species, sufficient information was obtained to identify a large set of genes potentially involved in cellular response to environmental stress.

Inhibitory effect of Pb2+ on the transport cycle of the Na+,K+-ATPase.

The effect of Pb(2+) on the transport cycle of the Na(+),K(+)-ATPase was characterized in detail at a molecular level by combining electrical and biochemical measurements. Electrical measurements were performed by adsorbing purified membrane fragments containing Na(+),K(+)-ATPase on a solid-supported membrane. Upon adsorption, the Na(+),K(+)-ATPase was activated by carrying out concentration jumps of different activating substrates, for example, Na(+) and ATP. Charge movements following Na(+),K(+)-ATPase activation were measured in the presence of various Pb(2+) concentrations to investigate the effect of Pb(2+) on different ion translocating steps of the pump cycle. These charge measurements were then compared to biochemical measurements of ATPase activity in the presence of increasing Pb(2+) concentration. Our results indicate that Pb(2+) inhibits cycling of the enzyme, but it does not affect cytoplasmic Na(+) binding and release of Na(+) ions at the extracellular side at concentrations below 10 muM. To explain the inhibitory effect of Pb(2+) on the Na(+),K(+)-ATPase, we propose that Pb(2+) may interfere with the hydrolytic cleavage of the phosphorylated intermediate E(2)P, which occurs in the K(+)-related branch of the pump cycle.

Characterization of microsatellite loci in the European pond turtle Emys orbicularis.

A set of eight highly polymorphic microsatellite markers was isolated and characterized from a genomic library enriched for dinucleotide repeats in the European pond turtle, Emys orbicularis. The markers were tested for polymorphism in a total of 33 turtles sampled in two natural ponds in the nature reserve of Kerkini, northern Greece. Number of alleles varied from 10 to 18, and expected heterozygosity ranged between 0.738 and 0.921. This novel set of loci will be particularly useful to assess fine-scale population structure and for parentage analysis in E. orbicularis.

Acclimatable cardiac and ventilatory responses to copper in the freshwater crayfish Procambarus clarkii.

Mortality and physiological tests following exposure to waterborne copper were performed in the red swamp crayfish Procambarus clarkii from a central Italian population. Mortality tests gave an estimated 96 h LC50 value (with 95% confidence limits) of 162 (132-211) mg L(-1) waterborne copper II. Variations in cardiac and ventilatory rates were simultaneously monitored using a non-invasive plethysmographic technique. In experiments with different sub-lethal copper concentrations (control, 0.5, 1 and 10 mg L(-1)) performed at different times (3, 6, 96 h), copper exposure elicited a reduction in both heart and scaphognatite rates. Following exposure to 10 mg L(-1) copper for 96 h, the heart and scaphognatite rates decreased to about 35% of the initial values. The reduction was fully reversible, since crayfish exposed to 0.5, 1 and 10 mg Cu L(-1) for 96 h resumed control rates after a 3-h residence in clean water. In crayfish pre-exposed (96 h) to sub-lethal copper concentrations (0.1 and 1 mg L(-1)) and then held in control water (3 h), the reduction of heart and scaphognatite rates after exposure to 10 mg Cu L(-1) were significantly lower than in specimens pre-exposed to control water. Therefore, copper induces a concentration and time dependent reduction of both cardiac and ventilatory activity in P. clarkii; these responses can be reduced or fully abolished by pre-exposure to sub-lethal levels of the metal.

Neuronal control of the cardiac responses to osmotic stress in the gastropod limpet Patella caerulea.

Mediterranean limpets Patella caerulea were exposed to different salinity conditions and treated with drugs interfering with neuronal control of heartbeat. Heart rate was monitored using a non-invasive method. Limpets were superfused with control (33 g l(-1)), hyposaline (0 and 10 g l(-1)) or hypersaline (56 and 66 g l(-1)) artificial seawater. Under osmotic stress the limpets showed an initial increase of heart rate, followed by acardia, particularly under hyposalinity. The tachycardia observed after exposure to 56 g l(-1) was abolished in the animals injected with a selective sodium channel blocker, tetrodotoxin (TTX), or with a serotoninergic antagonist, methysergide. Injection of TTX also partly prevented the acardia occurring at 0 g l(-1). The acardia was completely prevented after injection with atropine and benzoquinonium, two selective cholinergic antagonists. These findings indicate that cardiac responses of P. caerulea to variations in external salinity are regulated by an extrinsic neuronal control involving the serotoninergic and the cholinergic systems in the tachycardic and acardic responses, respectively.

Tetrodotoxin prevents copper-induced bradycardia in gastropod limpets.

Acute exposure to waterborne copper is followed by a reduction in heart rate in gastropod limpets. In order to understand the mechanism of this effect, exposure to copper (0.25 or 0.5 mg l(-1); for 3 and 6 h) was combined with an injection of tetrodotoxin (TTX, 20 microl, 0.5 or 1 microM), a natural toxin that inhibits the propagation and transmission of impulses in excitable tissues. Experiments were performed on the Mediterranean limpet Patella caerulea, using a non-invasive method for the recording of cardiac activity. TTX did not affect the bradycardic effect of the cholinergic agonist carbachol. However, this toxin significantly antagonized the bradycardia induced by 0.25 and 0.5mg l(-1) of copper exposure and prevented the acardia observed in some limpets exposed to 0.5mg l(-1) of copper for 6h. These results are consistent with the hypothesis that the inhibitory action of copper on limpet cardiac activity involves an extrinsic, cholinergic neuronal control.

Thermal benefits from familiarity with the environment in a reptile.

The tortoise Testudo hermanni Gmelin is non-territorial, frequents non-personal shelters and is generalist in diet, but stays within a home range very stable in time. Analysis of shell temperatures obtained at set intervals by radiothermometers showed that while tortoises resident in the study area behave as homoiotherms, animals imported from a similar environment appear relatively poikilotherms. Moreover, residents are more efficient in basking, reaching the daily thermal maxima systematically earlier than the unfamiliar animals. These observations support the hypothesis that its home range stability and the associated familiarity with the microenvironment facilitates the behavioural thermoregulation in T. hermanni.