Application of flow cytometry analysis to elucidate the impact of scale-down conditions in Escherichia coli cultivations P. Gil Salvador 2013 Award in Bioengineering category. (November 22, 2013 in the Annual General Assembly of the AIQS)

Inhomogeneities appear in large-scale fed-batch bioprocesses;especially when the distribution of the feedingsolution and of the oxygen is uneven. In order to studythe consequences of these heterogenic conditions on theculture, scale-down bioreactor experiments have beendesigned. These studies have revealed that cells exposedto oscillatory conditions are affected on various cellularlevels of regulation. However, not many of these studieshave been performed on the observation of the behaviourof the single cell level of Escherichia coli. Therefore, flowcytometry (FCM) is chosen as analytical tool to study thecellular viability in the scale-down approaches of a twocompartment reactor (Two-CR) and a newly establishedthree compartment reactor (Three-CR). An optimization ofdifferent staining methods applied in these experiments isalso performed, since staining procedures for flow cytometrystudies of bacterial populations are still not well-established yet

Stable populations in unstable habitats: temporal genetic structure of the introduced ascidian Styela plicata in North Carolina

14 páginas, 3 tablas, 4 figuras.The analysis of temporal genetic variability is an essential yet largely neglected tool to unveil and predict the dynamics of introduced species. We here describe the temporal genetic structure and diversity over time of an introduced population of the ascidian Styela plicata (Lesueur, 1823) in Wilmington (North Carolina, USA, 34°08′24″N, 77°51′44″W). This population suffers important salinity and temperature changes, and in June every year we observed massive die-offs, leaving free substratum that was recolonized within a month. We sampled 12–14 individuals of S. plicata every 2 months from 2007 to 2009 (N = 196) and analyzed a mitochondrial marker (the gene cytochrome oxidase subunit I, COI) and seven nuclear microsatellites. Population genetic analyses showed similar results for both types of markers and revealed that most of the genetic variation was found within time periods. However, analyses conducted with microsatellite loci also showed weak but significant differences among time periods. Specifically, in the samplings after die-off episodes (August–November 2007 and 2008) the genetic diversity increased, the inbreeding coefficient showed prominent drops, and there was a net gain of alleles in the microsatellite loci. Taken together, our results suggest that recruits arriving from neighboring populations quickly occupied the newly available space, bringing new alleles with them. However, other shifts in genetic diversity and allele loss and gain episodes were observed in December–January and February–March 2008, respectively, and were apparently independent of die-off events. Overall, our results indicate that the investigated population is stable over time and relies on a periodic arrival of larvae from other populations, maintaining high genetic diversity and a complex interplay of allele gains and losses.This research was supported by a grant from the United States–Israel Binational Science Foundation (BSF), Jerusalem, Israel (number 2014025), the Spanish Government project CTM2013—48163—and the Catalan Government Grant 2014SGR-336 for Consolidated Research Groups.Peer reviewe

Feeding cessation alters host morphology and bacterial communities in the ascidian Pseudodistoma crucigaster

11 páginas, 2 tablas, 7 figurasBackground: Ascidians can associate with abundant and diverse consortia ofmicrobial symbionts, yet these communities remain unexamined for the majority of host ascidians and little is known about host-symbiont interactions. Methods: We coupled electron microscopy and 16S rRNA gene tag pyrosequencing to investigate the bacterial communities associated with the colonial ascidian Pseudodistoma crucigaster, a species endemic to theMediterranean Sea that has a life cycle with two phases: actively-filtering (active) and non-filtering (resting) forms. Results: Resting colonies exhibited a reduced branchial sac (feeding apparatus) and a thickened cuticle. Electron microscope images also suggested higher abundance of colonizing microorganisms on surfaces of resting colonies. Accordingly, bacterial sequences associated with environmental sources (sediment and biofilms, >99 % similarity) were detected exclusively in resting colonies. Bacterial communities of P. crucigaster colonies (active and resting) were dominated by 3 core taxa affiliated (>94 % similarity) with previously described symbiotic Alphaproteobacteria in marine invertebrates. Shifts in rare bacteria were detected when ascidians entered the resting phase, including the appearance of strictly anaerobic lineages and nitrifying bacterial guilds. Conclusions: These findings suggest that physical (thickened cuticle) and metabolic (feeding cessation) changes in host ascidians have cascading effects on associated bacteria, where modified oxygen concentrations and chemical substrates for microbial metabolism may create anaerobic microhabitats and promote colonization by environmental microorganisms.This research was funded by the Marie Curie International Reintegration Grant FP7-PEOPLE-2010-RG 277038 within the 7th European Community Framework Program, the Spanish Government projects MARSYMBIOMICS CTM2013-43287-P and CHALLENGEN CTM2013-48163, and the Catalan Government grant 2014SGR-336 for Consolidated Research Groups.Peer reviewe

Microsatellite markers reveal shallow genetic differentiation between cohorts of the common sea urchin Paracentrotus lividus (Lamarck) in northwest Mediterranean

Temporal variability was studied in the common sea urchin Paracentrotus lividus through the analysis of the genetic composition of three yearly cohorts sampled over two consecutive springs in a locality in northwestern Mediterranean. Individuals were aged using growth ring patterns observed in tests and samples were genotyped for five microsatellite loci. No reduction of genetic diversity was observed relative to a sample of the adult population from the same location or within cohorts across years. FST and amova results indicated that the differentiation between cohorts is rather shallow and not significant, as most variability is found within cohorts and within individuals. This mild differentiation translated into estimates of effective population size of 90-100 individuals. When the observed excess of homozygotes was taken into account, the estimate of the average number of breeders increased to c. 300 individuals. Given our restricted sampling area and the known small-scale heterogeneity in recruitment in this species, our results suggest that at stretches of a few kilometres of shoreline, large numbers of progenitors are likely to contribute to the larval pool at each reproduction event. Intercohort variation in our samples is six times smaller than spatial variation between adults of four localities in the western Mediterranean. Our results indicate that, notwithstanding the stochastic events that take place during the long planktonic phase and during the settlement and recruitment processes, reproductive success in this species is high enough to produce cohorts genetically diverse and with little differentiation between them. Further research is needed before the link between genetic structure and underlying physical and biological processes can be well established

Too cold for invasions? Contrasting patterns of native and introduced ascidians in subantarctic and temperate Chile

10 páginas, 1 tabla, 2 figuras.We analysed the biodiversity of ascidians in two areas located in southern and northern Chile: Punta Arenas in the Strait of Magellan (53º latitude, subantarctic) and Coquimbo (29º latitude, temperate). The oceanographic features of the two zones are markedly different, with influence of the Humboldt Current in the north, and the Cape Horn Current System, together with freshwater influxes, in the Magellanic zone. Both regions were surveyed twice during 2013 by SCUBA diving and pulling ropes and aquaculture cages. Both artificial structures and natural communities were sampled. A total of 22 species were identified, three of them reported for the first time in Chilean waters: Lissoclinum perforatum, Synoicum georgianum, and Polyzoa minor. The first is an introduced species found here for the first time in the Pacific. No species occurred in both regions, highlighting the very different environmental conditions of subantarctic vs. temperate waters. In spite of exhaustive searches in aquaculture facilities and on artificial structures such as harbour docks and piers, no introduced species were found in the Punta Arenas area. Conversely, 5 out of 11 (45%) species found in northern Chile were introduced. The Coquimbo area has a history of ship traffic dating back at least 150 years, and cultures of native (e.g. scallop) as well as exotic species (e.g. abalone) have been deployed for ca. 35 years. Some of the introduced species, such as Ciona robusta (formerly C. intestinalis sp. A), constitute pests for scallop culture facilities in the area, causing serious losses to local farmers. It is surprising that the Punta Arenas zone, with a history of ship traffic dating back ca. 500 years and over 25 years of sustained mussel and salmon aquaculture activity, is apparently free from introduced species. The ascidian cover on artificial structures is high, but it is made up of native species such as Paramolgula sp., Cnemidocarpa verrucosa, or Polyzoa opuntia. It is hypothesized that cold waters (5 to 11ºC) are the determining factor hindering the development of introduced ascidians, which tend to be temperate-warm water species. The ongoing warming in the Southern Cone may change this picture and continued monitoring is strongly advised.This research was funded by CONICYT Chile (Grant 80122006). Additional funding was obtained by XT from the Spanish Government (project CHALLENGEN CTM2013-48163) and by JIC from University of Magallanes - UMAG/DI&P Grant PR-F2-01CRN- 12, CIMAR 18 & CIMAR 20 Fjords Chilean Navy, and GAIAAntarctic Project (MINEDUC-UMAG). RMR received a research grant from CNPq–National Counsel of Technological and Scientific Development (304768/2010-3).Peer reviewe

Biodiversidad genética de organismos marinos en el Parque Nacional de Cabrera: aplicaciones para la conservación

10 Páginas ; 1 Figura ; 2 TablasSe estudió la diversidad genética de las poblaciones de varias especies representativas del bentos marino (esponjas, cnidarios, ascidias, equinodermos y peces) del Parque Nacional de Cabrera. Se utilizaron marcadores moleculares de tasa de mutación alta y evolutivamente neutros (microsatélites) y, en algunos casos, genes mitocondriales. Los muestreos se realizaron dentro del Parque y varias zonas de las islas de Mallorca e Ibiza y a lo largo de la costa peninsular. Nuestros resultados indican que numerosas especies de invertebrados sésiles y algunos peces, que forman una parte esencial de los ecosistemas rocosos del Parque, están genéticamente aislados de las zonas adyacentes. Ello implica que las fases larvarias o adultas de estas especies no provienen mayoritariamente de las zonas próximas al Parque (ni siquiera de la isla de Mallorca) sino del propio Parque. Es decir hay un elevado nivel de autoreclutamiento. Por lo tanto, la desaparición de estas poblaciones en el Parque tendría una lenta recuperación.Peer reviewe