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Additional file of High-throughput sequencing and analysis of the gill tissue transcriptome from the deep-sea hydrothermal vent mussel Bathymodiolus azoricus

Trabajo presentado en el 18th International Symposium on Pollutant Responses in Marine Organisms (PRIMO 18), celebrado en Trondheim (Noruega) del 24 al 27 de mayo de 2015., Hydrothermal vents are extreme environments rich in biodiversity. The polychaete Branchipolymoe seepensis subsists in the palleal cavity of the mussel vent Bathymodiolus azoricus. These scale-worms absorb metal particles deposited in mussel gills surface and their presence has been associated with local environmental characteristics. The goal of this work was to establish a host-symbiont relationship between these two species using oxidative stress as an endpoint in response to metal accumulation., This study is included in the project DEEPFun PTDC/MAR/111749/2009 and NANOECOTOX PTDC/AAC- AMB /121650/2010, funded by Foundation of Science and Technology (FCT).

Trabajo presentado en el 18th International Symposium on Pollutant Responses in Marine Organisms (PRIMO 18), celebrado en Trondheim (Noruega) del 24 al 27 de mayo de 2015., Lucky Strike hydrothermal vent field is the largest known vent in the Mid-Atlantic Ridge (MAR), characterized by fluids that reach temperatures of 330ºC at 1,700m deep and by high amounts of metals (e.g. Cd, Cu, Fe, Hg, Mn and Zn). The vent polychaete Branchipolynoe seepensis lives in the palleal cavity of the mussel dominant species Bathymodiolus azoricus, cleaning the gills surface from particles in excess. This scale-worm is found more frequently inside the host mussel compared to other scale-worms, probably related with local environmental characteristics. Polychaetes are a very good trace-metal bioindicators, accumulating metals directly related with local metal bioavailability. The aim of this study was to understand the changes in protein expression of the membranar subproteome of polychaetes B. seepensis in response to metal concentration characteristics of Lucky Strike vent sites. Mussels were collected from two sites from Lucky Strike Hydrothermal vent field, Montségur-MS and Eiffel Tower-ET, during BIOBaz mission in August 2013 and polychaetes separated from the mussels. Subcellular fractionation was performed by sequential centrifugation of polychaetes tissues and 2D-SDS-PAGE applied to the membranar subproteome. Metal concentrations were also determined in polychaetes cellular and subcellular fractions by ICP-OES. Protein expression signatures show significant differences in 471 protein spots for B. seepensis between the two vent sites, with 11 specific for Montségur and 382 specific for Eiffel Tower. Common proteins to both sites with the higher change in expression (2-fold or higher) were selected to be identified by MALDI-TOF. Metal concentrations in the cellular (insoluble) and subcellular (thermo labile and heat-stable compounds) fractions were higher in ET and MS, respectively. Data suggests that metal concentrations from both sites reflect the environmental fluctuations between the two microhabitats within the same vent field and are related with the changes found at the subproteome level.

Trabajo presentado en el 14th Deep-Sea Biology Symposium, celebrado en Aveiro del 31 de agosto al 4 de septiembre de 2015., Lucky Strike is the biggest hydrothermal vent field located in the Mid-Atlantic Ridge (MAR) at 1, 700m de pth, characterized by fluid chemistry with high metal concentrations (e.g. Cu, Fe, Mn and Zn), low pH and temperatures of 330ºC. The polychaete Branchipolynoe seepensis live in the palleal cavity of the mussel dominant species Bathymodiolus azoricus. B.seepensis is known by clean the mussel gills surface from particles in excess and its presence is above 85% in Lucky Strike vent field. The aim of this study was to understand, at a membranar subproteome level, if the presence of B.seepensis in vent mussel B.azoricus is a response to high metal concentrations in Lucky Strike vent sites (Montsegur-MS and Eiffel Tower-ET). Mussels and polychaetes were collected from Lucky Strike hydrothermal vent field (MS and ET), in August 2013, aboard the vessel “Pourquoi Pas?”. Subcellular fractionation of polychaetes and mussel gills was performed and 2D-SDS-PAGE applied to the membranar subproteome. Metal concentrations (e.g. Ag, Cd, Cu, Fe and Zn) of both species were also determined in cellular and subcellular fractions by ICP-OES. Protein expression signatures showed significant differences, suggesting intra variation in Lucky Strike field. For B. seepensis 471 different proteins were detected between the two vent sites, 11 specific for MS and 382 specific for ET and 71 and 67 specific proteins for B.azoricus for MS and ET, respectively, out of a total of 176. Common proteins to both sites with the higher change in expression (2-fold or higher) were identified by MALDI-TOF. Metal concentrations in the cellular (insoluble) and subcellular (heat-stable compounds) fractions were higher for both species in ET and MS, respectively. Data suggests that the different metal concentrations found in these species from both sites reflect the environmental chemical fluctuation between the two microhabitats within the same vent field and are related with the changes at the subproteome level. For B.seepensis protein changes are clear, suggesting that polychaetes from ET contain specific proteins that help to deal with excess of metals, regulating the metal accumulation in themussel gills.

Trabajo presentado en el 14th Deep-Sea Biology Symposium, celebrado en Aveiro del 31 de agosto al 4 de septiembre de 2015., Hydrothermal vent biology has been studied in an attempt to understand the mechanisms involved in vent organisms that permit their survival and reproduction in extreme conditions. Symbiosis is very important for the subsistence of these deep-sea organisms. The scale-worm Branchipolymoe seepensis lives in the palleal cavity of the mussel vent Bathymodiolus azoricus, the dominant species in the MAR. Polychaetes are known to absorb particles in excess deposited in mussel gills and at the same time get protection. The aim of this study was to understand the metal accumulation in this symbiosis relationship. Mussels B. azoricus were collected from two sites from Lucky Strike Hydrothermal vent (1700m), Montségur-MS and Eiffel Tower-ET and from Rainbow-Rb hydrothermal vent (2300m) where B. seepensis is present. To compare the interaction between both species, mussels were also collected from Menez Gwen hydrothermal vent (MG2, MG3 and MG4) (850m) where polychaetes are not present. Induction of metallothionein (MT) and lipid peroxidation (LPO) levels were higher in polychaetes from Rb and MS vent sites, while in mussels were higher in the gills followed by the digestive gland and mantle in all vent fields. Enzymatic activities (CAT and GST) showed that mussels had higher CAT activity in digestive gland, followed by gills and mantle, while GST levels were higher in gills. In polychaetes , both CAT and GST activities were higher in ET. Metal concentrations (Ag, Cd, Co, Cu, Fe, Mn and Zn) in the cellular and subcellular fractions determined in mussels tissues and polychaetes whole body by ICP-OES suggest intra-and inter-variation of fluid chemistry between hydrothermal vent sites. The subcellular metal partitioning in polychaetes was higher in insoluble and soluble (thermolabile and heat-stable) fractions from ET, MS and Rb, respectively. In mussel tissues, me tal partitioning was higher in gills and digestive gland. Overall, the data suggest that the presence of the scale-worm is an adaptation to the elevated metal concentrations present in vent sites.

Parablennius sanguinolentus and P. parvicornis have been classified as either two distinct species or as two sub-species depending on the different criteria used to classify them. An analysis of fragments of mitochondrial 12S and 16S rDNA showed that the genetic distance between samples of P. sanguinolentus and P. parvicornis is similar or higher than those found for other blenniids that are widely recognized as distinct species. These results, together with the distinct geographical distributions and meristic differences, support the conclusion that P. sanguinolentus and P. parvicornis should be considered as two different species. Parablennius sanguinolentus y P. parvicornis han sido clasificados por diferentes autores como especies distintas o bien como subespecies. El análisis de fragmentos de las subunidades 12S y 16S del ADN ribosómico de la mitocondria muestra que la distancia genética entre los dos taxones es comparable o bien superior a las distancias entre especies de blénidos claramente reconocidas como válidas. Este resultado, sumado a las ligeras diferencias merísticas y a distribuciones geográficas bien distintas, soporta el reconocimiento de estos taxa como especies válidas.

Relatório de Cruzeiro CORALFISH:Condor, Voador e Banco Açores 2010a bordo do NRP Almirante Gago Coutinho.(datas 5 a 13 Agosto 2010) Autores: Fernando Tempera, Filipe Porteiro, Marina Carreiro-Silva, Andreia Braga-Henriques, Eva Giacomello, Ana Branco, Pedro Ferreira, Pedro Ribeiro, Telmo Morato, Ricardo Santos, Projectos: CoralFISH (IMAR, FP7), CONDOR (UAc, EEA), CORAZON (IMAR, FCT), Hermione (UAc, FP7) Objectivos: mapear as comunidades bentónicas dos monte submarinos Condor e Voador, identificar novas zonas de corais de águas frias, comparar a comunidade de peixes associada a zonas com e sem corais, recolher amostras biológicas e de água do mar, levantamento multifeixe (batimetria e backscatter) dos dois montes submarinos e outras áreas importantes, colocação de módulos de colonização no banco Condor. Navio: NRP Almirante Gago Coutinho Chefe de cruzeiro: Leg1- Ricardo Serrão Santos (monte submarino Condor); Leg2- Fernando Tempera (monte submarino Voador) Equipa científica: Leg 1- Ricardo Santos, Fernando Tempera, Filipe Porteiro, Marina C. Silva, Andreia Henriques, Eva Giacomello, Ana Branco (LNEG) e Pedro Ferreira (LNEG) Leg 2- Fernando Tempera, Telmo Morato, Marina C. Silva, Andreia Henriques, Pedro Ribeiro, Ana Branco (LNEG) e Pedro Ferreira (LNEG) &nbsp

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Os mares e oceanos têm um papel vital para a vida do planeta Terra. Para além de fonte importante de recursos vivos, minerais e energéticos têm assegurado a reciclagem e o depósito de numerosos contaminantes com que as sociedades modernas, após a revolução industrial, vêm poluindo os ecossistemas do planeta que habitamos. Deste modo, têm vindo a adiar, mas não a eliminar, cenários de catástrofe ambientais. Os estudos actuais em ciências do mar são assim importantes não só em termos estritamente científicos mas também dada a relevância e urgência social, económica, política e de gestão. Portugal está particularmente bem posicionado para o desenvolvimento e aplicação da investigação naquele domínio científico. Portugal, que detém uma das maiores Zonas Económicas Exclusivas da Europa, na qual grande parte é oceano aberto e mar profundo, tem assim condições especiais, mas também obrigações para desenvolver a investigação em ciências do mar. A investigação científica nacional em ciências do mar, apesar de tardia no âmbito do sistema científico mundial e nacional, tem vindo a fazer progressos acentuados em comparação com outros domínios disciplinares. Na última década, o ranking de publicações científicas indexadas internacionalmente em domínios das ciências marinhas passou para o 13ª lugar, quando na década passada ocupava o 30ª lugar entre os diferentes tópicos identificados pelo portal Web of Knowledge. Neste artigo são analisados alguns dos progressos alcançados e encarado com optimismo o progresso científico nacional para o século XXI agora que o país está dotado de uma estratégica nacional para os Oceanos, de uma estratégia nacional para o Mar e que dispõe de dois navios oceanográficos de alto mar e um ROV que pode explorar os oceanos até à profundidade de 6000 metros.

The northeastern Atlantic and the Mediterranean Sea share geological histories and display great faunal affinities. The majority of the Mediterranean species have Atlantic origins, with a few species with tropical affinities. These include the parrotfish Sparisoma cretense and the wrasse Thalassoma pavo that are restricted to the subtropical northeastern Atlantic, the Macaronesian archipelagos (Azores, Madeira, and Canaries) and the southern Mediterranean. The Pleistocene glaciations have been described as having different effects on the fauna of the two regions. During glacial peaks, Mediterranean waters remained warmer than those of the adjacent Atlantic. Within the eastern Atlantic, the effects of Pleistocene glaciations were differentiated. Here, we perform a comparative analysis focusing on T. pavo and S. cretense populations from the northeastern Atlantic and the Mediterranean to assess the effects of Pleistocene glaciations in these two species. Sequences from the mitochondrial control region were obtained and analyzed combining phylogeographic and demographic approaches. Gene flow between Atlantic and Mediterranean populations was shown to be very high. The Mediterranean populations of T. pavo and S. cretense showed high levels of genetic diversity, even in the eastern basin, pointing to an ancient colonization event. This suggests that both species must have been able to persist in the Mediterranean during the cold Pleistocene periods. Historical migration estimates revealed a Mediterranean towards Atlantic trend in the case of T. pavo, which may reflect the re-colonization of areas in the Atlantic by fish that survived the cold phases in relatively warmer Mediterranean refugia. Our data also showed that within the Macaronesian Archipelagos, migrations occurred from Madeira towards the Azores, for both T. pavo and S. cretense, thus supporting a post-glacial colonization of the Azores by fish that persisted in the warmer region of Madeira. Similar geographic distributions, thermal affinities, and means of dispersion for T. pavo and S. cretense resulted in a similar response to the effects of Pleistocene glaciations, as evidenced by identical phylogeographic patterns.

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Coryphoblennius galerita is a small intertidal fish with a wide distribution and limited dispersal ability, occurring in the northeastern Atlantic and Mediterranean. In this study, we examined Atlantic and Mediterranean populations of C. galerita to assess levels of genetic divergence across populations and to elucidate historical and contemporary factors underlying the distribution of the genetic variability. We analyse three mitochondrial and one nuclear marker and 18 morphological measurements. The combined dataset clearly supports the existence of two groups of C. galerita : one in the Mediterranean and another in the northeastern Atlantic. The latter group is subdivided in two subgroups: Azores and the remaining northeastern Atlantic locations. Divergence between the Atlantic and the Mediterranean can be the result of historical isolation between the populations of the two basins during the Pleistocene glaciations. Present-day barriers such as the Gibraltar Strait or the ‘Almeria-Oran jet’ are also suggested as responsible for this isolation. Our results show no signs of local extinctions during the Pleistocene glaciations, namely at the Azores, and contrast with the biogeographical pattern that has been observed for Atlantic– Mediterranean warm-water species, in which two groups of populations exist, one including the Mediterranean and the Atlantic coast of western Europe, and another encompassing the western tropical coast of Africa and the Atlantic islands of the Azores, Madeira and Canaries. Species like C. galerita that tolerate cooler waters, may have persisted during the Pleistocene glaciations in moderately affected locations, thus being able to accumulate genetic differences in the more isolated locations such as the Azores and the Mediterranean. This study is one of the first to combine morphological and molecular markers (mitochondrial and nuclear) with variable rates of molecular evolution to the study of the relationships of the Atlantic and Mediterranean populations of a cool-water species.

The genus Tripterygion (Risso 1826) is restricted to the eastern Atlantic and the Mediterranean, and comprises only three species. T. melanuros and T. tripteronotus are essentially endemic to the Mediterranean, while Tripterygion delaisi occurs in the Atlantic and in the Mediterranean. Two subspecies of T. delaisi have been described (T. d. xanthosoma in the Mediterranean and T. d. delaisi in the Atlantic). Several scenarios have been proposed for the evolution of T. delaisi subspecies, but so far its subspeciation process is not clear. In this study we present a population survey of T. delaisi including specimens from the two recognized subspecies. We combined a phylogeographic approach with estimates of the direction of migration (between the Atlantic and the Mediterranean) and of the coalescence time of the two subspecies, using polymorphic mitochondrial and nuclear genes. The results of this study clearly support the existence of two Tripterygion delaisi clades, one in the eastern Atlantic islands and another in the Atlantic coasts of Europe and in the Mediterranean. Historical migration between the islands and Western Europe plus Mediterranean was restricted, and showed a westbound trend, with a higher number of migrants going from the Western Europe plus Mediterranean into the islands. We estimated the time of coalescence of both groups of T. delaisi to be more recent than the onset of Pleistocene glaciations (1.7 Mya). Our results are consistent with previous hypothesis that consider successive dispersal events of a Tripterygion ancestor from the western African coast colonizing the Atlantic islands and the Mediterranean, promoting the evolutionary divergence between these areas.

DNA sequence analysis confirms the distinction between Parablennius ruber and Parablennius gattorugine, simultaneously validating the presence of the former species in Western Europe where it has been reported for >150 years. A possible scenario involving speciation of P. ruber at the Azores and subsequent transport of larvae to Europe, a process that may be still occurring nowadays, could explain this pattern of occurrence.

Phylogeography of Diplodus sargus from the northeastern Atlantic and the Mediterranean was assessed using sequences from the mitochondrial control region and the first intron of the S7 ribosomal protein gene. The relationship between genetic and geographic distances supported an isolation by distance model, with the Azores having a peripheral position. The geographic distribution of the genetic diversity, together with the historical demography of the populations studied can be explained by the effect of the Pleistocene glaciations in the northeastern Atlantic warm water fauna. D. sargus might have disappeared from western Europe during glacial peaks and suffered considerable demographic reductions in the Canaries and Mauritania, surviving in less affected areas such as Madeira, Azores and the Mediterranean. The mismatch analysis and the Fu's Fs values provide clear evidence of expansion in western Iberia (S. Pedro), Canaries, Mauritania and also in the eastern Mediterranean. Atlantic and Mediterranean populations of D. sargus showed no signs of genetic differentiation. D. sargus are active swimmers that can undergo extensive movements along the shores. This and the presence of planktonic eggs and larvae would allow rapid mixing between Mediterranean and Atlantic fish, erasing signs of population differentiation.

Marine Protected Areas (MPAs) are critically important to the conservation of marine biodiversity and ecological processes and to achieving a sound basis for sustainable use and development of marine environments and resources. This is clearly reflected in the statement from World Summit on Sustainable Development (Johannesburg): "MPAs are the key to achieving at least 10% of each of the world's ecological regions effectively conserved - the target established at the 6th Conference of Parties to the Convention on Biodiversity". At the same time, it is widely considered that MPAs can make a significant contribution to regional economic development and to improving the wellbeing of local communities. Fisheries are one of the main threats to marine biodiversity and ecological processes and MPAs are therefore important for conserving ecosystem services provided by the sea and sustaining tourism that depends on these. By restricting damaging activities, MPAs offer protection for marine natural resources and provide them with critical habitats they need at key times, e.g. for breeding or feeding. MPAs benefit species and habitats of regional interest and may also help to avoid or at least limit the deleterious effects of non-sustainable uses and activities, including pollution and other sources of degradation, in favour of the local economy. This publication has been developed in the framework of the project EMPAFISH (SSP8-006539) supported by the Commission of the European Communities within the Sixth Framework Programme.

Phylogenetic analysis of partial 12S and 16S mitochondrial rDNA regions, together with meristic and ethological data, has revealed that Centrolabrus trutta (Lowe, 1833), a species occurring in the Canaries and Madeira, and its close relative Centrolabrus caeruleus (Azevedo, 1999) from the Azores, are more closely related to most species of the genus Symphodus than to Centrolabrus exoletus (Linnaeus, 1758). Underwater behavioural observations showed that C. trutta, like C. caeruleus, shares with most Symphodus species sexual dichromatism, male nest building and male parental care of the eggs, traits that are absent in C. exoletus. The low number of base substitutions found between C. trutta and C. caeruleus indicates that these species are very closely related. The 16S rDNA data suggest that C. trutta, C. caeruleus and S. roissali form a monophyletic group. The analysis of the 16S rDNA data also showed that S. melanocercus is the sister species of C. exoletus and it is not closely related to the species of Symphodus included in this study. Lack of nest building and parental behaviour in S. melanocercus, and its marked divergence to other members of the genus Symphodus in meristic characters, also stress its affinity with C. exoletus and its separation from the remaining species of Symphodus. Our data and the evidence available in the literature led us to propose the inclusion of C. trutta and C. caeruleus in the genus Symphodus, while S. melanocercus must be transferred to the genus Centrolabrus.

This paper presents an analysis of the distributional patterns of blenniids (Pisces: Blenniidae) in the north-eastern Atlantic. Two peaks of species diversity were found, both in terms of number of species and number of endemics: one in the tropical African coast and another in the Mediterranean Sea. A cluster analysis of similarity values (Jaccard coefficient) among the eastern Atlantic zoogeographical areas, revealed the following groups: a north temperate group, a tropical group formed by the tropical African coast and Mauritania, another group formed by the islands of Cape Verde, a south temperate group (South Africa), and a southern Atlantic group formed by the islands of Ascension and St Helena. Within the north temperate group, the subgroups with higher similarities were: Azores and Madeira, Canary Islands and Morocco, and the Mediterranean and the Atlantic coast of the Iberian Peninsula. Based on affinity indices, the probable directions of faunal flows were inferred. The tropical coast of Africa and the Mediterranean emerged from this analysis as probable speciation centres of the north-eastern Atlantic blenniid fauna. The Mediterranean may have also acted as a refuge during glacial periods.

female visits, were courted more often by females and received more spawnings. Larger males also received a higher number of male intrusions, attacked more conspecifics and defended larger territories. Larger males showed more nest cleaning behaviour and a marginally non-significant trend for higher egg fanning rate. Male courtship, male attack rate against conspecifics and parental behaviour were all correlated with the frequency of female spawnings received by each male even when controlling for male size, suggesting that these behaviour patterns influence male mating success. On the other hand, a positive partial correlation was found between female courtship and the frequency of female spawnings, controlling for the number of female visits, which suggests a role for female mate choice on male mating success. Finally, males nesting in chambers in the bottom of pools received more spawnings than males nesting either in crevices or under boulders. However, nest opening area was associated significantly negatively with male mating success, when controlling for male size. Thus, the present data suggest strongly that male characteristics overrule nest characteristics in determining male mating success in the Azorean rock-pool blenny.