Analisi della struttura genetica del crostaceo cirripede Chthamalus montagui nell'area mediterranea.

Analisi della struttura genetica del crostaceo cirripede Chthamalus montagui nell’area mediterranea. Riassunto della tesi di Laurea Magistrale in Biologia Marina di Fabio Piccolin. Chthamalus montagui (Southward) è un crostaceo cirripede comunemente presente nella fascia intertidale delle coste rocciose dell’area mediterraneo-atlantica. In questo studio sono stati analizzati 355 individui di C.montagui provenienti da 11 località differenti. Una località (Biarritz) si trova lungo la costa atlantica della Francia, 3 località (Palma de Mallorca, Baia Blu e Cala Sinzias) si trovano nel Mediterraneo Occidentale, 3 località (Portonovo, Zaton e Grado) si affacciano sul Mare Adriatico, 3 località (Malta, Volos, Buyukada) si trovano nel Mediterraneo Orientale e l’ultima località (Sozopol) si trova in Mar Nero. L’analisi della struttura genetica è stata condotta utilizzando come marcatori molecolari 6 loci microsatellitari specie-specifici. L’attività di campo svolta è consistita nella raccolta di individui di C. montagui sulle scogliere e nell’immediata fissazione di questi in etanolo. L’attività di laboratorio è consistita nell’estrazione del DNA totale con metodo del salting-out, nella verifica della qualità e della quantità di DNA estratto tramite gel-elettroforesi, nell’opportuna diluizione del DNA, nell’amplificazione tramite PCR dei loci microsatellitari, nella verifica della presenza dell’amplificato tramite gel-elettroforesi e, in caso positivo, nell’analisi dei frammenti mediante sequenziamento automatico. Infine si è proceduto all’elaborazione statistica dei dati ottenuti, con lo scopo di mettere in evidenza le caratteristiche della struttura genetica della specie esaminata all’interno dell’area geografica considerata. A partire dai dati raccolti sono stati effettuati i test statistici per il linkage disequilibrium e per l’equilibrio di Hardy-Weinberg, quindi per ciascun locus e per ciascuna località sono state calcolate l’eterozigosità media osservata, l’eterozigosità media attesa e la ricchezza allelica media. Sono poi state stimate le frequenze alleliche e sulla base di queste è stata condotta l’analisi della statistica F di Weir e Cockerham (1984). Quindi è stata verificata la presenza di correlazione fra le distanze geografiche e le distanze genetiche fra le coppie di località e sono stati prodotti un grafico MDS ed un albero Neighbor Joining per rappresentare graficamente le relazioni genetiche fra le località analizzate. È stata poi condotta un’analisi della varianza molecolare che ha permesso di raggruppare le località analizzate all’interno di tre gruppi geneticamente distinti e geograficamente delimitati: un gruppo del Mediterraneo Occidentale, uno del Mediterraneo Orientale ed un altro del Mare Adriatico. L’esistenza di questi distinti cluster genetici all’interno del Mediterraneo è stata confermata anche attraverso un’analisi bayesiana di assegnazione individuale. I risultati ottenuti sono stati discussi alla luce dei lavori presenti in letteratura sulla biogeografia del Mediterraneo e sulla struttura genetica di altri organismi marini, partendo dagli stessi Ctamali per allargarsi poi anche ad altri invertebrati e vertebrati marini sia mediterranei che non

The role of photoperiod in the entrainment of endogenous clocks and rhythms in Antarctic krill (Euphausia superba)

Antarctic krill (Euphausia superba), hereafter krill, are key players in the ecosystem of the Southern Ocean. They are distributed all around Antarctica, and they are exceptionally abundant, representing the main link between primary producers and the higher trophic levels in the Antarctic marine food web. Due to their high ecological relevance, krill have been extensively studied in the field and in the laboratory, and it is known that their life-cycle is shaped by fundamental daily and seasonal rhythmic events. Actual knowledge about the external and internal factors involved in the regulation of rhythmic functions in krill is still quite limited but pivotal, especially in the context of future environmental changes driven by climate change. One hypothesis is that the daily and seasonal rhythmic functions in krill might be regulated through the activity of so-called “endogenous” clocks. Endogenous clocks are molecular function units, which promote rhythmic oscillations in transcription, physiology and behavior at the daily and seasonal levels. Endogenous clocks can be entrained (i.e. synchronized) by rhythmic environmental cues, like the day/night cycle (i.e. photoperiod = day length) at the daily level, and the seasonal photoperiodic cycle at the seasonal level. The implications of endogenous rhythmicity (i.e. rhythmicity promoted by endogenous clocks) in the regulation of rhythmic biological functions are well documented among terrestrial species, but studies dealing with marine organisms are very scarce. At the daily level, the best studied endogenous clock is the circadian clock, which is based on molecular feedback loops generating a rhythm with a period of approximately 24 h. Specific light-sensitive proteins promote the entrainment of the circadian clock with the day/night cycle, ensuring effective synchronization of rhythmic output functions according to daily recurring environmental changes. In krill, a circadian clock has been recently identified and characterized, and its influence on daily rhythms of metabolism and transcription has been demonstrated in the laboratory and in natural conditions. At the seasonal level, the regulation of rhythmic functions is less well understood, also in terrestrial species. An endogenous circannual clock seems to be involved, but the molecular mechanisms underlying its functioning are still unclear. Due to its ability to measure changes in day length, the circadian clock might contribute to the seasonal entrainment of the circannual clock. In krill, a circannual rhythm (i.e. a rhythm promoted by a circannual clock) might be involved in the regulation of the seasonal shifts in sexual maturity and metabolic activity observed in the field in summer and winter. During this dissertation, I investigated the involvement of endogenous clocks and rhythms in the regulation of rhythmic functions in krill at the daily and seasonal levels. Moreover, I also examined the role played by photoperiod in the entrainment of those clocks and rhythms. The work focused on three main research topics, which resulted in three publications: 1) the impact of the extreme seasonal photoperiodic cycle of the Southern Ocean on the activity of the circadian clock of krill at different times of the year (Publication I); 2) the involvement of an endogenous circannual rhythm and the role played by photoperiod in the regulation of the seasonal metabolic activity cycle of krill (Publication II); and 3) the involvement of the circadian clock and the role played by photoperiod in the regulation of diel vertical migration (DVM) in krill (Publication III). In publication I, I investigated the activity of the circadian clock of krill in different simulated seasonal Antarctic light conditions. The extreme variability displayed by the seasonal photoperiodic cycle in the Southern Ocean might cause a problem for the photoperiodic entrainment of the clock in different seasons. Especially during summer and winter, when overt light/dark cues are missing, the clock might get disrupted and the clock output might become arrhythmic. Indeed, laboratory work demonstrated that under simulated mid-summer and mid-winter conditions, when overt photoperiodic cues were missing, the circadian clock of krill was arrhythmic, and the metabolic output was de-synchronized. Conversely, under simulated early-autumn and late-winter conditions, when overt photoperiodic cues were present, the circadian clock of krill was active, and the metabolic output was synchronized with the light/dark cycle. This suggested that major changes are occurring during the year in the entraining process of the circadian clock of krill, depending on the different seasonal light conditions to which krill are exposed. In publication II, I investigated the involvement of an endogenous circannual rhythm in the regulation of the seasonal metabolic activity cycle of krill. Moreover, I also examined the role played by photoperiod in the entrainment of this rhythm. In response to the strong seasonal variability displayed by light and food availability in the Southern Ocean, krill display seasonal differences in metabolic rates, feeding activity and growth. During summer, when light and food availability is high, krill metabolic and feeding activity is enhanced, and krill growth rates are positive. During winter, when light and food conditions are low, krill metabolic and feeding activity is reduced, and krill show reduced growth or even shrinkage (i.e. reduction of size). It has been hypothesized that an endogenous rhythm entrained by the seasonal Antarctic light regime might be responsible for the regulation of the seasonal metabolic cycle of krill. Krill exposed to different long-term simulated natural seasonal light conditions, showed seasonal patterns of growth, enzyme activity and gene expression of key metabolic genes, which were also observed in krill exposed to constant darkness. The results strongly suggested the involvement of a circannual clock in the regulation of the seasonal metabolic cycle of krill. However, major differences were observed in the seasonal patterns of oxygen consumption, suggesting that exposition of krill to specific seasonal light cues might be necessary for the effective entrainment of the circannual clock. In publication III, I investigated the involvement of an endogenous circadian rhythm in the regulation of krill diel vertical migration (DVM). Moreover, I also examined the role played by photoperiod in the entrainment of krill DVM. DVM is a mass migratory movement displayed by many zooplankton species worldwide. During the night, the animals come to the surface to graze on phytoplankton, while during the day they sink to deeper layers to escape from visual predators. The environmental factors involved in the regulation of DVM are photoperiod, food availability and presence/absence of predators. However, DVM occurs also in constantly dark environments (e.g. the deep sea and the Arctic ocean during the polar night), suggesting the involvement of an endogenous rhythm of regulation. Using krill exposed to different light/dark (LD) and constant darkness (DD) conditions, I found that krill DVM was driven by an endogenous rhythm, with krill moving upward during the light phase and downward during the dark phase. A similar rhythm was found in krill oxygen consumption, confirming the presence of an endogenous rhythm of activity associated with DVM. Rhythmic expression of clock genes related to the circadian clock was found in the eyestalks of krill entrained to similar LD conditions, suggesting that an involvement of the circadian clock in the regulation of krill DVM would be possible. Major differences were observed among individual krill in the rhythmic regulation of DVM and oxygen consumption, suggesting that the circadian system of krill might display high degrees of individual plasticity. In conclusion, this dissertation improves our knowledge about the mechanisms regulating daily and seasonal rhythmic functions in the Antarctic krill, E. superba. The implication of endogenous rhythmicity was demonstrated for krill DVM at the daily level, and for krill seasonal metabolic cycle at the seasonal level. Photoperiod proved to be a most fundamental factor for the entrainment of krill DVM and krill seasonal metabolic cycle, as well as for the modulation of the activity of the circadian clock of krill at different times of the year. This work provides an example of how techniques which have been developed to study the molecular biology and chronobiology of terrestrial model species can be applied to the study of ecologically relevant species in the marine environments. In the future, understanding the regulation of rhythmic functions in ecological key marine species like Antarctic krill will help us to understand how these species will adapt to environmental changes driven by climate change

Photoperiodic modulation of circadian functions in Antarctic krill Euphausia superba Dana, 1850 (Euphausiacea)

An endogenous circadian clock influences metabolic output rhythms in the Antarctic krill (Euphausia superba Dana, 1850), a key species in the Southern Ocean ecosystem. Seasonal changes in photoperiod in Antarctica, ranging from midnight sun (24 h light) during mid-summer to very short days (3\u20134 h light) during mid-winter, represent a challenge for the synchronization of the krill circadian clock. We analyzed clock gene activity and clock output functions in krill exposed to different light conditions during a long-term photoperiodic simulation in the laboratory. In simulated early-autumn (light/dark or LD 16:8) and late-winter (LD 8:16) conditions, the circadian clock of krill was functional and the metabolic output was synchronized to the light/dark cycle, the clock genes Esper and Esclk peaked in antiphase around simulated dusk/dawn and most metabolic-related genes showed upregulation around simulated dusk. In contrast, in simulated mid-summer (light/light or LL) and mid-winter (LD 3:21) conditions, the synchronization of the circadian clock and the metabolic output appeared to be weaker, with clock gene expression becoming arrhythmic and upregulation of metabolic genes occurring at different times during the day. Early-autumn and late-winter photoperiodic cues in the laboratory thus seem to be sufficient to entrain the krill clock and promote metabolic synchronization, whereas midwinter and mid-summer photoperiodic cues seem to be insufficient for krill entrainment. Krill in the field may overcome the seasonal lack of overt photoperiodic cycle occurring during midsummer and mid-winter by using alternative light-related Zeitgebers (i.e., varying light intensity rather than the presence or absence of light) to promote basic homeostatic rhythms over 24 h

The Seasonal Metabolic Activity Cycle of Antarctic Krill (Euphausia superba): Evidence for a Role of Photoperiod in the Regulation of Endogenous Rhythmicity

Antarctic krill (Euphausia superba), a key species in the Southern Ocean, reduce their metabolism as an energy saving mechanism in response to the harsh environmental conditions during the Antarctic winter. Although the adaptive significance of this seasonal metabolic shift seems obvious, the driving factors are still unclear. In particular, it is debated whether the seasonal metabolic cycle is driven by changes in food availability, or if an endogenous timing system entrained by photoperiod might be involved. In this study, we used different long-term photoperiodic simulations to examine the influence of light regime and endogenous rhythmicity on the regulation of krill seasonal metabolic cycle. Krill showed a seasonal cycle of growth characterized by null-to-negative growth rates during autumn-winter and positive growth rates during spring-summer, which was manifested also in constant darkness, indicating strong endogenous regulation. Similar endogenous cycles were observed for the activity of the key-metabolic enzyme malate dehydrogenase (MDH) and for the expression levels of a selection of metabolic-related genes, with higher values in spring-summer and lower values in autumn-winter. On the other side, a seasonal cycle of oxygen consumption was observed only when krill were exposed to simulated seasonal changes in photoperiod, indicating that light-related cues might play a major role in the regulation of krill oxygen consumption. The influence of light-regime on oxygen consumption was minimal during winter, when light-phase duration was below 8 h, and it was maximal during summer, when light-phase duration was above 16 h. Significant upregulation of the krill clock genes clk, cry2, and tim1, as well as of the circadian-related opsins rh1a and rrh, was observed after light-phase duration had started to decrease in early autumn, suggesting the presence of a signaling cascade linking specific seasonal changes in the Antarctic light regime with clock gene activity and the regulation of krill metabolic dormancy over the winter

The Seasonal Metabolic Activity Cycle of Antarctic Krill (Euphausia superba): Evidence for a Role of Photoperiod in the Regulation of Endogenous Rhythmicity

Antarctic krill (Euphausia superba), a key species in the Southern Ocean, reduce their metabolism as an energy saving mechanism in response to the harsh environmental conditions during the Antarctic winter. Although the adaptive significance of this seasonal metabolic shift seems obvious, the driving factors are still unclear. In particular, it is debated whether the seasonal metabolic cycle is driven by changes in food availability, or if an endogenous timing system entrained by photoperiod might be involved. In this study, we used different long-term photoperiodic simulations to examine the influence of light regime and endogenous rhythmicity on the regulation of krill seasonal metabolic cycle. Krill showed a seasonal cycle of growth characterized by null-to-negative growth rates during autumn-winter and positive growth rates during spring-summer, which was manifested also in constant darkness, indicating strong endogenous regulation. Similar endogenous cycles were observed for the activity of the key-metabolic enzyme malate dehydrogenase (MDH) and for the expression levels of a selection of metabolic-related genes, with higher values in spring-summer and lower values in autumn-winter. On the other side, a seasonal cycle of oxygen consumption was observed only when krill were exposed to simulated seasonal changes in photoperiod, indicating that light-related cues might play a major role in the regulation of krill oxygen consumption. The influence of light-regime on oxygen consumption was minimal during winter, when light-phase duration was below 8 h, and it was maximal during summer, when light-phase duration was above 16 h. Significant upregulation of the krill clock genes clk, cry2, and tim1, as well as of the circadian-related opsins rh1a and rrh, was observed after light-phase duration had started to decrease in early autumn, suggesting the presence of a signaling cascade linking specific seasonal changes in the Antarctic light regime with clock gene activity and the regulation of krill metabolic dormancy over the winter

Rapporto tecnico sulle attività di campagna oceanografica “BANSIC 2013”

La campagna oceanografica BANSIC 2013 è stata condotta a bordo della N/O “Urania” dal 26 Giugno al 16 Luglio 2013 nell’ambito delle attività previste dal WP3 del progetto SSD-Pesca, finanziato dal MIUR su fondi MISE, a supporto della pesca italiana nelle Regioni Obiettivo 1, e dal progetto bandiera RITMARE (SP2_WP1_AZ1_UO01 e UO04) Obiettivi generali della campagna oceanografica sono stati lo studio delle relazioni tra le strutture oceanografiche a mesoscala (vortici verticali ed orizzontali, upwelling, ecc.) e le strutture spaziali dei fenomeni biologici relativi ai primi anelli della catena trofica (zooplancton, distribuzione e abbondanza di larve di piccoli pelagici e grandi pelagici), e lo sviluppo del dispositivo di Fishing Vessel Monitoring System denominato FOOS (Fishery Oceanography Observing System) da installare a bordo di imbarcazioni da pesca. In particolare, il campionamento di uova di acciuga è finalizzato all’applicazione del metodo DEPM (Daily Egg Production Method) per la stima dell’abbondanza dello stock riproduttore (SP2_WP1_AZ3_UO04). Il campionamento ittioplanctonico, che ha riguardato anche le acque Maltesi, è inserito anche nel piano di lavoro del progetto regionale MIPAF-FAO “MedSudMed” (“Assessment and Monitoring of the Fishery Resources and the Ecosystems in the Straits of Sicily”). La campagna ha visto anche l’avvio di una linea di ricerca in collaborazione con la Martin-Luther-Universität Halle-Wittenberg, avente come obiettivo i radiolari