Organismi acquatici e ambiente: meccanismi biochimici di interazione, risposta e adattamento

The research is focused on the relationship between some Mg2+-dependent ATPase activities of plasma- and mitochondrial membranes from tissues of cultured marine bivalve molluscs and potentially stressful environmental conditions, such as the exposure to contaminants both of natural origin (ammonia nitrogen, the main contaminant of aquaculture plants) and of anthropic source (alkyltins). The two filter-feeding bivalve species selected colonize different habitats: the common mussel Mytilus galloprovincialis binds to hard substrates and the Philippine clam Tapes philippinarum burrows into sea bottom sandy beds. The choice of typical species of coastal waters, extremely suitable for environmental studies due to their features of poor motility, resistance to transport and great filtering efficiency, may constitute a model to evaluate responses to contaminants of membrane-bound enzyme activities involved in key biochemical mechanisms, namely cell ionic regulation and mitochondrial energy production. In vitro and in vitro approaches have been pursued. In vitro assays were carried out by adding the contaminants (NH4Cl and alkyltins) directly to the ATPase reaction media. In vivo experiments were carried out by exposing mussels to various tributyl tin (TBT) concentrations under controlled conditions in aquaria. ATPase activities were determined spectrophotometrically according to the principles of the method of Fiske and Subbarow (1925). The main results obtained are detailed below. In Tapes philippinarum the interaction of NH4 +, the main form of ammonia nitrogen at physiological and seawater pHs, with the Na,K-ATPase and the ouabaininsensitive Na-ATPase was investigated in vitro on gill and mantle microsomal membranes. The proven replacement by NH4 +of K+ in the activation of the Na,KATPase and of Na+ in the activation of the ouabain-insensitive ATPase displayed similar enzyme affinity for the substituted cation. on the one hand this finding may represent one of the possible mechanisms of ammonia toxicity and, on the other, it supports the hypothesis that NH4 + can be transported across the plasma membrane through the two ATPases. In this case both microsomal ATPases may be involved and co-operate, at least under peculiar circumstances, to nitrogen excretion and ammonia detoxification mechanisms in bivalve molluscs. The two ATPase activities stimulated by NH4 + maintained their typical response to the glycoside ouabain, specific inhibitor of the Na,K-ATPase, being the Na++ NH4 +-activated ATPase even more susceptive to the inhibitor and the ouabain-insensitive ATPase activity activated indifferently by Na+ or NH4 + unaffected by up to 10-2 M ouabain. In vitro assays were carried out to evaluate the response of the two Na-dependent ATPases to organotins in clams and mussels and to investigate the interaction of TBT with mussel mitochondrial oligomycin-sensitive Mg-ATPase. Since no literature data were available, the optimal assay conditions and oligomycin sensitivity of mussel mitochondrial MgATPase were determined. In T. philippinarum the ouabain-insensitive Na-ATPase was found to be refractory to TBT both in the gills and in the mantle, whereas the Na,K-ATPase was progressively inhibited by increasing TBT doses; the enzyme inhibition was more pronounced in the gills than in the mantle. In both tissues of M. galloprovincialis the Na,K-ATPase inhibition by alkyltins decreased in the order TBT>DBT(dibutyltin)>>MBT(monobutyltin)=TeET(tetraethyltin) (no effect). Mussel Na-ATPase confirmed its refractorimess to TBT and derivatives both in the gills and in the mantle. These results indicate that the Na,K-ATPase inhibition decreases as the number of alkyl chains bound to tin decreases; however a certain polarity of the organotin molecule is required to yield Na,K-ATPase inhibition, since no enzyme inhibition occurred in the presence of tetraalkyl-substituted derivatives such as TeET . Assays carried out in the presence of the dithioerythritol (DTE) pointed out that the sulphhydrylic agent is capable to prevent the Na,K-ATPase inhibition by TBT, thus suggesting that the inhibitor may link to -SH groups of the enzyme complex.. Finally, the different effect of alkyltins on the two Na-dependent ATPases may constitute a further tool to differentiate between the two enzyme activities. These results add to the wealth of literature data describing different responses of the two enzyme activities to endogenous and exogenous modulators . Mussel mitochondrial Mg-ATPase was also found to be in vitro inhibited by TBT both in the gills and in the mantle: the enzyme inhibition followed non competitive kinetics. The failed effect of DTE pointed out that in this case the interaction of TBT with the enzyme complex is probably different from that with the Na,K-ATPase. The results are consistent with literature data showing that alkyltin may interact with enzyme structures with different mechanisms. Mussel exposure to different TBT sublethal doses in aquaria was carried out for 120 hours. Two samplings (after 24 and 120 hrs) were performed in order to evaluate a short-term response of gill and mantle Na,K-ATPase, ouabain-insensitive Na-ATPase and Mg-ATPase activities. The in vivo response to the contaminants of the enzyme activities under study was shown to be partially different from that pointed out in the in vitro assays. Mitochondrial Mg-ATPase activity appeared to be activated in TBTexposed mussels with respect to control ones, thus confirming the complexity of evaluating in vivo responses of the enzyme activities to contaminants, due to possible interactions of toxicants with molluscan metabolism. Concluding, the whole of data point out that microsomal and mitochondrial ATPase activities of bivalve molluscs are generally responsive to environmental contaminants and suggest that in some cases membrane-bound enzyme activities may represent the molecular target of their toxicity. Since the Na,K-ATPase, the Na-ATPase and the Mg-ATPase activities are poorly studied in marine bivalves, this research may contribute to enlarge knowledge in this quite unexplored field.

Ricerca, mediante tecniche di proteomica, di biomarcatori proteici utili nella valutazione della qualità degli alimenti di origine animale

In the last decades, the increase of industrial activities and of the request for the world food requirement, the intensification of natural resources exploitation, directly connected to pollution, have aroused an increasing interest of the public opinion towards initiatives linked to the regulation of food production, as well to the institution of a modern legislation for the consumer guardianship. This work was planned taking into account some important thematics related to marine environment, collecting and showing the data obtained from the studies made on different marine species of commercial interest (Chamelea gallina, Mytilus edulis, Ostrea edulis, Crassostrea gigas, Salmo salar, Gadus morhua). These studies have evaluated the effects of important physic and chemical parameters variations (temperature, xenobiotics like drugs, hydrocarbons and pesticides) on cells involved in the immune defence (haemocytes) and on some important enzymatic systems involved in xenobiotic biotransformation processes (cytochrome P450 complex) and in the related antioxidant defence processes (Superoxide dismutase, Catalase, Heat Shock Protein), from a biochemical and bimolecular point of view. Oxygen is essential in the biological answer of a living organism. Its consume in the normal cellular breathing physiological processes and foreign substances biotransformation, leads to reactive oxygen species (ROS) formation, potentially toxic and responsible of biological macromolecules damages with consequent pathologies worsening. Such processes can bring to a qualitative alteration of the derived products, but also to a general state of suffering that in the most serious cases can provoke the death of the organism, with important repercussions in economic field, in the output of the breedings, of fishing and of aquaculture. In this study it seemed interesting to apply also alternative methodologies currently in use in the medical field (cytofluorimetry) and in proteomic studies (bidimensional electrophoresis, mass spectrometry) with the aim of identify new biomarkers to place beside the traditional methods for the control of the animal origin food quality. From the results it’s possible to point out some relevant aspects from each experiment: 1. The cytofluorimetric techniques applied to O. edulis and C. gigas could bring to important developments in the search of alternative methods that quickly allows to identify with precision the origin of a specific sample, contributing to oppose possible alimentary frauds, in this case for example related to presence of a different species, also under a qualitative profile, but morpholgically similar. A concrete perspective for the application in the inspective field of this method has to be confirmed by further laboratory tests that take also in account in vivo experiments to evaluate the effect in the whole organism of the factors evaluated only on haemocytes in vitro. These elements suggest therefore the possibility to suit the cytofluorimetric methods for the study of animal organisms of food interest, still before these enter the phase of industrial working processes, giving useful information about the possible presence of contaminants sources that can induce an increase of the immune defence and an alteration of normal cellular parameter values. 2. C. gallina immune system has shown an interesting answer to benzo[a]pyrene (B[a]P) exposure, dose and time dependent, with a significant decrease of the expression and of the activity of one of the most important enzymes involved in the antioxidant defence in haemocytes and haemolymph. The data obtained are confirmed by several measurements of physiological parameters, that together with the decrease of the activity of 7-etossi-resourifine-O-deetilase (EROD linked to xenobiotic biotransformation processes) during exposure, underline the major effects of B[a]P action. The identification of basal levels of EROD supports the possible presence of CYP1A subfamily in the invertebrates, still today controversial, never identified previously in C. gallina and never isolated in the immune cells, as confirmed instead in this study with the identification of CYP1A-immunopositive protein (CYP1A-IPP). This protein could reveal a good biomarker at the base of a simple and quick method that could give clear information about specific pollutants presence, even at low concentrations in the environment where usually these organisms are fished before being commercialized. 3. In this experiment it has been evaluated the effect of the antibiotic chloramphenicol (CA) in an important species of commercial interest, Chamelea gallina. Chloramphenicol is a drug still used in some developing countries, also in veterinary field. Controls to evaluate its presence in the alimentary products of animal origin, can reveal ineffective whereas the concentration results to be below the limit of sensitivity of the instruments usually used in this type of analysis. Negative effects of CA towards the CYP1A- IPP proteins, underlined in this work, seem to be due to the attack of free radicals resultant from the action of the antibiotic. This brings to a meaningful alteration of the biotransformation mechanisms through the free radicals. It seems particularly interesting to pay attention to the narrow relationships in C. gallina, between SOD/CAT and CYP450 system, actively involved in detoxification mechanism, especially if compared with the few similar works today present about mollusc, a group that is composed by numerous species that enter in the food field and on which constant controls are necessary to evaluate in a rapid and effective way the presence of possible contaminations. 4. The investigations on fishes (Gadus morhua, and Salmo salar) and on a bivalve mollusc (Mytilus edulis) have allowed to evaluate different aspects related to the possibility to identify a biomarker for the evaluation of the health of organisms of food interest and consequently for the quality of the final product through 2DE methodologies. In the seafood field these techniques are currently used with a discreet success only for vertebrates (fishes), while in the study of the invertebrates (molluscs) there are a lot of difficulties. The results obtained in this work have underline several problems in the correct identification of the isolated proteins in animal organisms of which doesn’t currently exist a complete genomic sequence. This brings to attribute some identities on the base of the comparison with similar proteins in other animal groups, incurring in the possibility to obtain inaccurate data and above all discordant with those obtained on the same animals by other authors. Nevertheless the data obtained in this work after MALDI-ToF analysis, result however objective and the spectra collected could be again analyzed in the future after the update of genomic database related to the species studied. 4-A. The investigation about the presence of HSP70 isoforms directly induced by different phenomena of stress like B[a]P presence, has used bidimensional electrophoresis methods in C. gallina, that have allowed to isolate numerous protein on 2DE gels, allowing the collection of several spots currently in phase of analysis with MALDI-ToF-MS. The present preliminary work has allowed therefore to acquire and to improve important methodologies in the study of cellular parameters and in the proteomic field, that is not only revealed of great potentiality in the application in medical and veterinary field, but also in the field of the inspection of the foods with connections to the toxicology and the environmental pollution. Such study contributes therefore to the search of rapid and new methodologies, that can increase the inspective strategies, integrating themselves with those existing, but improving at the same time the general background of information related to the state of health of the considered animal organism, with the possibility, still hypothetical, to replace in particular cases the employment of the traditional techniques in the alimentary field.

Mitochondrial Genomics: structure, inheritance and phylogenetic utility of the Mitochondrial genome in Bivalvia and Insecta

This PhD Thesis is the result of my research activity in the last three years. My main research interest was centered on the evolution of mitochondrial genome (mtDNA), and on its usefulness as a phylogeographic and phylogenetic marker at different taxonomic levels in different taxa of Metazoa. From a methodological standpoint, my main effort was dedicated to the sequencing of complete mitochondrial genomes, and the approach to whole-genome sequencing was based on the application of Long-PCR and shotgun sequences. Moreover, this research project is a part of a bigger sequencing project of mtDNAs in many different Metazoans’ taxa, and I mostly dedicated myself to sequence and analyze mtDNAs in selected taxa of bivalves and hexapods (Insecta). Sequences of bivalve mtDNAs are particularly limited, and my study contributed to extend the sampling. Moreover, I used the bivalve Musculista senhousia as model taxon to investigate the molecular mechanisms and the evolutionary significance of their aberrant mode of mitochondrial inheritance (Doubly Uniparental Inheritance, see below). In Insects, I focused my attention on the Genus Bacillus (Insecta Phasmida). A detailed phylogenetic analysis was performed in order to assess phylogenetic relationships within the genus, and to investigate the placement of Phasmida in the phylogenetic tree of Insecta. The main goal of this part of my study was to add to the taxonomic coverage of sequenced mtDNAs in basal insects, which were only partially analyzed.

Alterazione di risposte fisiologiche in invertebrati marini esposti nella Baia di Cadice (Spagna) come indici della qualità dell'ambiente

La forte crescita nella pescicoltura ha portato ad una significativa pressione ambientale di origine antropica nei sistemi costieri. Il bivalve locale Scrobicularia plana è stato usato come bioindicatore per valutare la qualità ambientale di un ecosistema affetto da scarichi di acque residuali di una piscifattoria nel braccio di mare Rio San Pedro (Spagna sud-occidentale). I bivalvi sono stati raccolti nei sedimenti intertidali nell'ottobre del 2010 da cinque siti del braccio di mare, seguendo un gradiente di inquinamento decrescente dall'effluente al sito di controllo. Per valutare l'esposizione e l'effetto di contaminanti legati alle acque residuali delle piscifattorie è stata selezionata una batteria di biomarker. Sono state misurate nei tessuti delle ghiandole digestive dei bivalvi: l'attività di enzimi del sistema di detossificazione della Fase I (etossiresorufina-O-deetilasi, EROD e dibenzilfluoresceina, DBF) l'attività di un enzima del sistema di detossificazione di Fase II (glutatione S-transferasi, GST), l'attività di enzimi antiossidanti (glutatione perossidasi, GPX e glutatione reduttasi, GR) e parametri di stress ossidativo (perossidazione lipidica, LPO, e danno al DNA). In parallelo sono state misurate in situ, nelle aree di studio, temperatura, pH, salinità e ossigeno disciolto nelle acque superficiali; nelle acque interstiziali sono stati misurati gli stessi parametri con l'aggiunta del potenziale redox. Sono state trovate differenze significative (p<0,05) tra siti di impatto e sito di controllo per quanto riguarda l'attività di EROD e GR, LPO e danno al DNA; è stato osservato un chiaro gradiente di stress riconducibile alla contaminazione, con alte attività di questi biomarker nell'area di scarico delle acque residuali della pescicoltura e livelli più bassi nel sito di controllo. È stata trovata inoltre una correlazione negativa significativa (p<0,01) tra la distanza alla fonte di inquinamento e l’induzione dei biomarker. Sono state analizzate le componenti abiotiche, inserendole inoltre in una mappa georeferenziata a supporto. Ossigeno disciolto, pH, salinità e potenziale redox mostrano valori bassi vicino alla fonte di inquinamento, aumentando man mano che ci si allontana da esso. I dati ottenuti indicano nel loro insieme che lo scarico di acque residuali dalle attività di pescicoltura nel braccio di mare del Rio San Pedro può indurre stress ossidativo negli organismi esposti che può portare ad un'alterazione dello stato di salute degli organismi.

Molecular phylogenetic inferences on the position of the Mollusca within the Lophotrochozoa

Die phylogenetische Position der Mollusken innerhalb der Trochozoa sowie die interne Evolution der Klassen der Mollusca sind weitgehend unbekannt und wurden in meiner Arbeit anhand molekularer Merkmale untersucht. Phylogenomische Analysen zeigten in der Vergangenheit eine gute Auflösung für ursprüngliche Speziationsereignisse. Daher wurden hier drei neue EST Datensätze generiert: für Sipunculus nudus (Sipuncula), Barentsia elongata (Kamptozoa) und Lepidochitona cinerea, (Polyplacophora, Mollusca). Zusätzlich wurden gezielt Gene verschiedener Mollusken mittels RT-PCR amplifiziert. rnSowohl Kamptozoen als auch Sipunculiden wurden aufgrund morphologischer Kriterien bisher als mögliche Schwestergruppe der Mollusken gehandelt, aber die hier erzielten Ergebnisse zur Evolution der Hämerythrine, Gen-Anordnungen der mitochondrialen Genome und phylogenetische Analysen der ribosomalen und der mitochondriellen Proteine stützen diese Hypothese nicht. Die Position der Kamptozoa erwies sich hier generell als unbeständig; phylogenomische Analysen deuten eine Nähe zu den Bryozoen an, aber diese Position wird stark durch die Auswahl der Taxa beeinflusst. Dagegen weisen meine Analysen klar auf eine nähere Beziehung zwischen Annelida und Sipuncula hin. Die ribosomalen Proteine zeigen Sipuncula (und Echiura) sogar als Subtaxa der Anneliden. Wie den Mollusken fehlt den Sipunculiden jegliche Segmentierung und meine Ergebnisse legen hier die Möglichkeit des Verlusts dieses Merkmals innerhalb der Anneliden bei den Sipunculiden nahe. Innerhalb der Mollusken wurden die Solenogastren bereits als Schwestergruppe aller rezenten Mollusken vorgeschlagen. Im Rahmen meiner Arbeit wurden von drei verschiedenen Solenogastren-Arten die ersten zuverlässigen 18S rRNA-Sequenzen ermittelt, und es zeigte sich, dass alle bisher veröffentlichten 18S-Sequenzen dieser Molluskenklasse höchst unvollständig oder fehlerhaft sind. rnRibosomale Proteine sind gute phylogenetische Marker und hier wurden die Auswahl und Anzahl dieser Gene für phylogenetische Analysen optimiert. Über Sonden-basierte Detektion wurde eine sampling-Strategie getestet, die im Vergleich mit standard-phylogenomischen Ansätzen zukünftige molekulare Stammbaumrekonstruktionen mit größerem Taxonsampling ermöglicht.rn

Confronto fra metodi di analisi di comunità bentoniche in differenti spiagge del Nord Adriatico

Il presente studio si colloca nell’ambito del progetto europeo (FP7) THESEUS, fra i cui scopi c’è quello di fornire informazioni su vulnerabilità e resilienza degli habitat costieri in seguito all’aumento di frequenza delle inondazioni dovuto al sea level rise. E’ stata indagata la zona intertidale di spiagge sabbiose, come recettore di cambiamenti climatici. All’interno dell’habitat intertidale le comunità macrobentoniche sono di solito individuate come indicatori delle variazioni dei parametri fisico-chimici e morfodinamici. Lo scopo di questo lavoro è consistito nell’analisi delle comunità macrobentoniche e della loro interazioni con le variabili ambientali lungo tre spiagge del Nord Adriatico sottoposte a fenomeni di erosione e differenti fra di loro per caratteristiche morfodinamiche: Lido di Spina, Bellocchio e la zona della Bassona di Lido di Dante. La risposta delle comunità bentoniche è stata indagata utilizzando i dati tassonomici delle specie e raggruppando le stesse nei rispettivi gruppi trofici. Le variabili ambientali considerate sono state quelle relative alla tipologia del sedimento e quelle relative alla morfodinamica Le comunità macrobentoniche delle spiagge di Lido di Spina e di Lido di Dante sono risultate relativamente più simili tra loro, nonostante i due siti fossero i più distanti. A Lido di Spina e Lido di Dante sono state rinvenute associazioni di specie, come Scolelepis squamata ed Eurydice spinigera, tipiche delle spiagge sabbiose europee esposte al moto ondoso. In questi due siti, è risultato dominante il bivalve Lentidium mediterraneum, la cui ecologia e modalità di distribuzione aggregata permette di evidenziare il maggiore idrodinamismo che caratterizza i due siti. A Bellocchio, invece, è stato riscontrato un maggior numero di specie. Questo sito è caratterizzato dalla presenza di patch di giovanili del bivalve Mytilus galloprovincialis che sembrerebbe determinare il pattern del resto della comunità fungendo da ecosystem engineer. In termini di gruppi trofici, a Lido di Spina e a Lido di Dante prevalgono Filtratori, Carnivori e Detritivori di Superficie mentre Bellocchio è dominato da Filtratori e Misti discostandosi dagli altri siti per le sue condizioni del tutto particolari. Per quanto riguarda i descrittori abiotici, Lido di Spina e Lido di Dante, rispetto a Bellocchio, presentano una fascia intertidale più corta, pendenze maggiori, granulometrie più grossolane e risultando quindi, in generale, meno dissipative.

Effects of temperature and acidity on Mediterranean benthic calcifiers, in the face of globale climate change

The Mediterranean Sea is expected to react faster to global change compared to the ocean and is already showing more pronounced warming and acidification rates. A study performed along the Italian western coast showed that porosity of the skeleton increases with temperature in the zooxanthellate (i.e. symbiotic with unicellular algae named zooxanthellae) solitary scleractinian Balanophyllia europaea while it does not vary with temperature in the solitary non-zooxanthellate Leptopsammia pruvoti. These results were confirmed by another study that indicated that the increase in porosity was accompanied by an increase of the fraction of the largest pores in the pore-space, perhaps due to an inhibition of the photosynthetic process at elevated temperatures, causing an attenuation of calcification. B. europaea, L. pruvoti and the colonial non-zooxanthellate Astroides calycularis, transplanted along a natural pH gradient, showed that high temperature exacerbated the negative effect of lowered pH on their mortality rates. The growth of the zooxanthellate species did not react to reduced pH, while the growth of the two non-zooxanthellate species was negatively affected. Reduced abundance of naturally occurring B. europaea, a mollusk, a calcifying and a non-calcifying macroalgae were observed along the gradient while no variation was seen in the abundance of a calcifying green alga. With decreasing pH, the mineralogy of the coral and mollusk did not change, while the two calcifying algae decreased the content of aragonite in favor of the less soluble calcium sulphates and whewellite (calcium oxalate), possibly as a mechanism of phenotypic plasticity. Increased values of porosity and macroporosity with CO2 were observed in B. europaea specimens, indicating reduces the resistance of its skeletons to mechanical stresses with increasing acidity. These findings, added to the negative effect of temperature on various biological parameters, generate concern on the sensitivity of this zooxanthellate species to the envisaged global climate change scenarios.

Confronto tra composizione tassonomica e struttura trofica delle comunità macrobentoniche di spiagge dell'Emilia Romagna

Lo studio si inserisce all’interno del progetto THESEUS, il cui obiettivo primario è quello di fornire una metodologia integrata per la pianificazione di strategie di difesa sostenibile per la gestione dell’erosione costiera e delle inondazioni. Nel presente lavoro di tesi in particolare sono state esaminate le componenti biotiche e abiotiche della zona intertidale di 6 spiagge (Lido di Spina, Bellocchio, Lido di Dante, Cervia, Cesenatico, Cesenatico sud) lungo la costa emiliano-romagnola, diverse tra loro per caratteristiche morfodinamiche, per grado di antropizzazione, modalità di gestione ed interventi effettuati per contrastare i fenomeni di erosione costiera. Sono state quindi considerate porzioni di litorale in cui non sono presenti opere di difesa costiera rigide, quali pennelli e barriere, e dove sono quasi del tutto assenti strutture turistico-balneari, e tre siti più antropizzati per la presenza di strutture di difesa costiera o per attività inerenti il turismo. Lo scopo di questo lavoro consiste nella: 1) analisi delle comunità macrobentoniche della zona intertidale in termini di composizione tassonomica; 2) analisi delle comunità macrobentoniche in termini di gruppi funzionali o gruppi trofici, cioè specie che, all’interno della comunità, svolgono la stessa funzione ecologica; 3) relazione fra la struttura delle comunità e le caratteristiche abiotiche e morfodinamiche delle spiagge indagate. L’analisi dei dati ha mostrato come le comunità rinvenute a Bellocchio e Lido di Dante, sia in termini di struttura trofica che tassonomica, siano le più diverse fra di loro. Con l’eccezione di Bellocchio, tutte le spiagge sono dominate dal gruppo trofico dei “suspension feeders” a causa dell’elevata abbondanza del bivalve Lentidium mediterraneum. Quando dalla matrice multivariata biotica viene elimina questa specie, si evidenziano differenze più marcate fra i siti definiti naturali e quelli definiti più antropizzati. Considerando le matrici morfo-abiotiche, Lido di Spina, Cesenatico e Cesenatico sud presentano un sedimento medio-fine e moderatamente ben classato, tipico delle spiagge del litorale emiliano-romagnolo; viceversa Lido di Dante e Bellocchio presentano un sedimento rispettivamente più grossolano e molto fine e argilloso a Bellocchio.

Zur Karbonatsedimentologie, Biofazies und sequenzstratigraphischen Architektur eines fossilen Hochenergie-Schelfs aus dem Neogen der Algarve (Miozän, Südportugal)

Die neogene Lagos-Portimão Formation (Unter- bis Mittelmiozän) bildet einen Teil der Steilküste der Algarve (S-Portugal) und besteht aus einer zyklischen Wechsellagerung von Karbonaten und Sand-steinen. Die vorliegende Arbeit bietet ein Modell zur sedimentologischen, faziellen und stratigraphischen Entwicklung dieser Einheit an. Basierend auf Profilen entlang der gesamten lateralen Erstreckung der Einheit wurden verschiedene Gelände- und Labormethoden angewandt, um ein Modell entwickeln zu können. Messungen des Sr87/86-Isotopenverhältnisses sollten Klarheit bezüglich der stratigraphischen Position bringen. Die laterale Korrelation der Profile erfolgte über lithologische und fazielle Ansprachen. Unterstützend wurden einzelne Profile mit einem tragbaren Gammaray-Spektrometer gemessen. Es wurden vier Leithorizonte etabliert, die sich durch fazielle Merkmale und spezielle Fossilführung defi-nieren lassen. Die Mikrofazies wurde qualitativ und quantitativ analysiert. Als statistisches Verfahren wurde unter anderem eine hierarchische Clusteranalyse durchgeführt, über welche fünf Biofaziestypen des warm-temperierten Klimabereichs unterschieden werden. Die Fossilführung wird von Mollusken, Bryozoen und Rotalgen dominiert. Ausnahmen bilden stratigraphisch isolierte Vorkommen von kolo-nialen Korallen, die jedoch keine Riffkörper aufbauen. Die Ergebnisse aller zuvor erwähnten Untersuchungen deuten auf Ablagerungen eines nicht-tropischen Hochenergie-Schelfs hin. Sedimentäre Zyklen sind oftmals unvollständig, es treten Hartgründe und Auf-arbeitungs- bzw- Kondesationshorizonte auf. Die geochemische Altersdatierung weist Alterssprünge und -inversionen auf. Ein Vergleich mit dem SW-australischen Schelf und dem von James et al. (1994) eingeführten Modell des shaved shelf bietet sich aufgrund der Ähnlichkeit der Sedimentgesteine und des ozeanographischen Settings an. Weiterhin werden zeitgleiche bzw. faziell ähnliche Becken vergleichend diskutiert. Das Sedimentationsgeschehen der Lagos-Portimão Formation wird maßgeblich durch eine halokinetisch bedingte unregelmäßige Subsidenz und Hebung beeinflußt. Der Salzdom von Albufeira war während der Sedimentation der Einheit mehrfach in Bewegung. Rutschungspakete, Entlastungsspalten und Sanddikes zeugen davon. Die sequenzstratigraphische Interpretation bietet einen neuen Ansatz, in dem sie von Hochstand-Sandsteinen und Tiefstand-Karbonaten ausgeht.

Gastropod Seed Dispersal: An Invasive Slug Destroys Far More Seeds in Its Gut than Native Gastropods

Seed dispersal is one of the most important mechanisms shaping biodiversity, and animals are one of the key dispersal vectors. Animal seed dispersal can directly or indirectly be altered by invasive organisms through the establishment of new or the disruption of existing seed dispersal interactions. So far it is known for a few gastropod species that they ingest and defecate viable plant seeds and consequently act as seed dispersers, referred to as gastropodochory. In a multi-species experiment, consisting of five different plant species and four different gastropod species, we tested with a fully crossed design whether gastropodochory is a general mechanism across native gastropod species, and whether it is altered by the invasive alien slug species Arion lusitanicus. Specifically, we hypothesized that a) native gastropod species consume the seeds from all tested plant species in equal numbers (have no preference), b) the voracious invasive alien slug A. lusitanicus – similarly to its herbivore behaviour – consumes a higher amount of seeds than native gastropods, and that c) seed viability is equal among different gastropod species after gut passage. As expected all tested gastropod species consumed all tested plant species. Against our expectation there was a difference in the amount of consumed seeds, with the largest and native mollusk Helix pomatia consuming most seeds, followed by the invasive slug and the other gastropods. Seed damage and germination rates did not differ after gut passage through different native species, but seed damage was significantly higher after gut passage through the invasive slug A. lusitanicus, and their germination rates were significantly reduced.

Sediment core fossils in ancient Lake Ohrid: testing for faunal change since the Last Interglacial

Ancient Lake Ohrid is probably of early Pleistocene or Pliocene origin and amongst the few lakes in the world harbouring an outstanding degree of endemic biodiversity. Although there is a long history of evolutionary research in Lake Ohrid, particularly on molluscs, a mollusc fossil record has been missing up to date. For the first time, gastropod and bivalve fossils are reported from the basal, calcareous part of a 2.6 m long sediment succession (core Co1200) from the north-eastern part of Lake Ohrid. Electron spin resonance (ESR) dating of mollusc shells from the same stratigraphic level yielded an age of 130 ± 28 ka. Lithofacies III sediments, i.e. a stratigraphic subdivision comprising the basal succession of core Co1200 between 181.5–263 cm, appeared solid, greyish-white, and consisted almost entirely of silt-sized endogenic calcite (CaCO3>70%) and intact and broken mollusc shells. Here we compare the faunal composition of the thanatocoenosis with recent mollusc associations in Lake Ohrid. A total of 13 mollusc species (9 gastropod and 4 bivalve species) could be identified within Lithofacies III sediments. The value of sediment core fossils for reconstructing palaeoenvironmental settings was evaluated and the agreement between sediment and palaeontological proxies was tested. The study also aims at investigating major faunal changes since the Last Interglacial and searching for signs of extinction events. The combined findings of the ecological study and the sediment characteristics suggest deposition in a shallow water environment during the Last Interglacial. The fossil fauna exclusively included species also found in the present fauna, i.e. no extinction events are evident for this site since the Last Interglacial. The thanatocoenosis showed the highest similarity with recent Intermediate Layer (5–25 m water depth) mollusc assemblages. The demonstrated existence of a mollusc fossil record in Lake Ohrid sediment cores also has great significance for future deep drilling projects. It can be hoped that a more far reaching mollusc fossil record will then be obtained, enabling insight into the early evolutionary history of Lake Ohrid.

Seasonal changes in the behaviour and respiration physiology of the freshwater duck mussel, Anodonta anatina.

For low-energy organisms such as bivalves, the costs of thermal compensation of biological rates (synonymous with acclimation or acclimatization) may be higher than the benefits. We therefore conducted two experiments to examine the effect of seasonal temperature changes on behaviour and oxygen consumption. In the first experiment, we examined the effects of seasonal temperature changes on the freshwater bivalve Anodonta anatina, taking measurements each month for a year at the corresponding temperature for that time of year. There was no evidence for compensation of burrowing valve closure duration or frequency, or locomotory speed. In the second experiment, we compared A. anatina at summer and winter temperatures (24 and 4°C, respectively) and found no evidence for compensation of the burrowing rate, valve closure duration or frequency, or oxygen consumption rates during burrowing, immediately after valve closure or at rest. Within the experimental limits of this study, the evidence suggests that thermal compensation of biological rates is not a strategy employed by A. anatina. We argue that this is due to either a lack of evolutionary pressure to acclimatize, or evolutionary pressure to not acclimatize. Firstly, there is little incentive to increase metabolic rate to enhance predatory ability given that these are filter feeders. Secondly, maintained low energetic demand, enhanced at winter temperatures, is essential for predator avoidance, i.e. valve closure. Thus, we suggest that the costs of acclimatization outweigh the benefits in A. anatina.

Bifurcation and singularity analysis of a molecular network for the induction of long-term memory.

Withdrawal reflexes of the mollusk Aplysia exhibit sensitization, a simple form of long-term memory (LTM). Sensitization is due, in part, to long-term facilitation (LTF) of sensorimotor neuron synapses. LTF is induced by the modulatory actions of serotonin (5-HT). Pettigrew et al. developed a computational model of the nonlinear intracellular signaling and gene network that underlies the induction of 5-HT-induced LTF. The model simulated empirical observations that repeated applications of 5-HT induce persistent activation of protein kinase A (PKA) and that this persistent activation requires a suprathreshold exposure of 5-HT. This study extends the analysis of the Pettigrew model by applying bifurcation analysis, singularity theory, and numerical simulation. Using singularity theory, classification diagrams of parameter space were constructed, identifying regions with qualitatively different steady-state behaviors. The graphical representation of these regions illustrates the robustness of these regions to changes in model parameters. Because persistent protein kinase A (PKA) activity correlates with Aplysia LTM, the analysis focuses on a positive feedback loop in the model that tends to maintain PKA activity. In this loop, PKA phosphorylates a transcription factor (TF-1), thereby increasing the expression of an ubiquitin hydrolase (Ap-Uch). Ap-Uch then acts to increase PKA activity, closing the loop. This positive feedback loop manifests multiple, coexisting steady states, or multiplicity, which provides a mechanism for a bistable switch in PKA activity. After the removal of 5-HT, the PKA activity either returns to its basal level (reversible switch) or remains at a high level (irreversible switch). Such an irreversible switch might be a mechanism that contributes to the persistence of LTM. The classification diagrams also identify parameters and processes that might be manipulated, perhaps pharmacologically, to enhance the induction of memory. Rational drug design, to affect complex processes such as memory formation, can benefit from this type of analysis.

Sensory regulation of network components underlying ciliary locomotion in Hermissenda.

Ciliary locomotion in the nudibranch mollusk Hermissenda is modulated by the visual and graviceptive systems. Components of the neural network mediating ciliary locomotion have been identified including aggregates of polysensory interneurons that receive monosynaptic input from identified photoreceptors and efferent neurons that activate cilia. Illumination produces an inhibition of type I(i) (off-cell) spike activity, excitation of type I(e) (on-cell) spike activity, decreased spike activity in type III(i) inhibitory interneurons, and increased spike activity of ciliary efferent neurons. Here we show that pairs of type I(i) interneurons and pairs of type I(e) interneurons are electrically coupled. Neither electrical coupling or synaptic connections were observed between I(e) and I(i) interneurons. Coupling is effective in synchronizing dark-adapted spontaneous firing between pairs of I(e) and pairs of I(i) interneurons. Out-of-phase burst activity, occasionally observed in dark-adapted and light-adapted pairs of I(e) and I(i) interneurons, suggests that they receive synaptic input from a common presynaptic source or sources. Rhythmic activity is typically not a characteristic of dark-adapted, light-adapted, or light-evoked firing of type I interneurons. However, burst activity in I(e) and I(i) interneurons may be elicited by electrical stimulation of pedal nerves or generated at the offset of light. Our results indicate that type I interneurons can support the generation of both rhythmic activity and changes in tonic firing depending on sensory input. This suggests that the neural network supporting ciliary locomotion may be multifunctional. However, consistent with the nonmuscular and nonrhythmic characteristics of visually modulated ciliary locomotion, type I interneurons exhibit changes in tonic activity evoked by illumination.

Polysensory interneuronal projections to foot contractile pedal neurons in Hermissenda.

A Pavlovian-conditioning procedure may produce modifications in multiple behavioral responses. As an example, conditioning may result in the elicitation of a specific somatomotor conditioned response (CR) and, in addition, other motor and visceral CRs. In the mollusk Hermissenda conditioning produces two conditioned responses: foot-shortening and decreased locomotion. The neural circuitry supporting ciliary locomotion is well characterized, although the neural circuit underlying foot-shortening is poorly understood. Here we describe efferent neurons in the pedal ganglion that produce contraction or extension of specific regions of the foot in semi-intact preparations. Synaptic connections between polysensory type Ib and type Is interneurons and identified foot contractile efferent neurons were examined. Type Ib and type Is interneurons receive synaptic input from the visual, graviceptive, and somatosensory systems. Depolarization of type Ib interneurons evoked spikes in identified tail and lateral foot contractile efferent neurons. Mechanical displacement of the statocyst evoked complex excitatory postsynaptic potentials (EPSPs) and spikes recorded from type Ib and type Is interneurons and complex EPSPs and spikes in identified foot contractile efferent neurons. Depolarization of type Ib interneurons in semi-intact preparations produced contraction and shortening along the rostrocaudal axis of the foot. Depolarization of Is interneurons in semi-intact preparations produced contraction of the anterior region of the foot. Taken collectively, the results suggest that type Ib and type Is polysensory interneurons may contribute to the neural circuit underlying the foot-shortening CR in Hermissenda.