Phylogenetic characterization and quantification of ammonia-oxidizing archaea and bacteria from Lake Kivu in a long-term microcosm incubation

A microcosm cultivation-based method was set up to investigate the growth of ammonia-oxidizing archaea (AOA), isolated from a water sample acquired at a depth of 50 m from the northern basin of Lake Kivu. For this purpose, both CARD-FISH and qPCR targeting of archaeal 16S rRNA and amoA genes were used. Archaeal cell growth at the end of the 246-day microcosm experiment accounted for 35 % of the SybrGold-stained cells, which corresponded to 6.61 × 106 cells/ml and 1.76 ± 0.09 × 106 archaeal 16S rRNA gene copies/ml. Clone libraries and DGGE fingerprinting confirmed the dominance of AOA phylotypes in the archaeal community microcosm. The majority of the identified archaeal 16S rRNA gene sequences in the clone libraries were affiliated with Thaumarchaeota Marine Group 1.1a. Subsequent cultivation of the AOA community on deep-well microtiter plates in medium containing different carbon sources to stimulate archaeal growth failed to show significant differences in archaeal abundance (ANOVA t14 = –1.058, P = 0.308 and ANOVA t14 = 1.584, P = 0.135 for yeast extract and simple organic acids, respectively). The lack of growth stimulation by organic compounds is in concordance with the oligotrophic status of Lake Kivu. Finally, the addition of antibiotics to the growth medium resulted in archaeal cell counts that were significantly lower than those obtained from cultures in antibiotic-free medium (ANOVA t14 = 12.12, P < 0.001). [Int Microbiol 2013; 16(3):177-189]Keywords: ammonia-oxidizing archaea and bacteria · ammonia monooxygenase alpha subunit (amoA) · Lake Kivu ·microcosm · multi-color CARD-FIS

Controles locales y regionales del metabolismo en ríos de cabecera: aportaciones del primer proyecto colaborativo de la AIL

Streams play a key role in the global biogeochemical cycles, processing material from adjacent terrestrial systems and transporting it downstream. However, the drivers of stream metabolism, especially those acting at broad spatial scales, are still not well understood. Moreover, stream metabolism can be affected by hydrological changes associated with seasonality, and thus, assessing the temporality of metabolic rates is a key question to understand stream function. This study aims to analyse the geographical and temporal patterns in stream metabolism and to identify the main drivers regulating the wholeecosystem metabolic rates at local and regional scales. Using a coordinated distributed experiment, we studied ten headwaters streams located across five European ecoregions during summer and fall 2014.We characterized the magnitude and variability of gross primary production (GPP) and ecosystem respiration (ER) with the open-channel method. Moreover, we examined several climatic, geographical, hydrological, morphological, and physicochemical variables that can potentially control stream metabolic rates. Daily rates of stream metabolism varied considerately across streams, with GPP and ER ranging from 0.06 to 4.33 g O2 m?2 d?1 and from 0.72 to 14.20 g O2 m?2 d?1, respectively. All streams were highly heterotrophic (P/R < 1), except the southernmost one. We found that the drier climates tended to have the highest GPP, while humid regions presented the highest ER. Between the sampling periods no statistical differences were found. Partial-least squares models (PLS) explained ?80% of the variance in GPP and ER rates across headwater streams and included both local and regional variables. Rates of GPP varied primarily in response to the local variables, such as streambed substrate and stream water temperature. In contrast, regional variables, such as the mean annual temperature or the land use of the catchment, had more relevance to explain ER. Overall, our results highlight that stream metabolism depends on both local and regional drivers and show the positive experience of a young network of researchers to assess scientific challenges across large-scale geographic areas.Los ecosistemas fluviales tienen un papel relevante en los ciclos biogeoquímicos globales, ya que procesan el material de la cuenca y lo transportan río abajo. Sin embargo, los factores que afectan al metabolismo, especialmente aquellos que actúan en una escala espacial más amplia, todavía no se conocen completamente. Además, el metabolismo fluvial puede variar a causa de cambios hidrológicos relacionados con la estacionalidad; lo que también hace importante incluir la variabilidad temporal para entender el funcionamiento de los ríos. Mediante el uso de un experimento distribuido y coordinado, estudiamos diez ríos de cabecera localizados a lo largo de cinco ecoregiones europeas durante verano y otoño del 2014. Se midió la magnitud y la variabilidad de la producción primaria bruta (PPB) y la respiración ecosistémica (RE) mediante el método de canal abierto. Así mismo, se examinaron los factores climáticos, hidrológicos y físico-químicos que potencialmente pueden afectar las tasas metabólicas fluviales. Las tasas diarias de metabolismo fluvial variaron considerablemente entre ríos, con valores de PPB de entre 0.06 a 4.33 g O2 m-2 día-1, y de RE de entre 0.72 a 14.20 g O2 m-2 día-1. Todos los ríos fueron altamente heterotróficos (P/R < 1), excepto el río más meridional. Se observó que los climas secos tendían a tener mayor PPB y las regiones húmedas tendían a tener una mayor RE. No se encontraron diferencias estadísticas entre los períodos de estudio. Los modelos de regresión de mínimos cuadrados parciales (PLS) explicaron --80% de la varianza de PPB y RE para los ríos estudiados. Las tasas de PPB variaron principalmente con factores locales como el sustrato del lecho del río y la temperatura del agua. En cambio, las tasas de RE estuvieron estrechamente relacionadas con factores regionales, como la temperatura media anual y los impactos en la cuenca. En conjunto, nuestros resultados resaltan la necesidad de incluir la escala local y regional en el estudio del metabolismo fluvial y muestran la experiencia positiva de una red coordinada de jóvenes investigadores para lograr retos científicos de amplia escala geográfica.We thank the support of the AIL board throughout the development of the project and all the affiliated institutions for the accessibility of laboratory premises, especially the Limnology Department of Uppsala University and the Department d’Ecologia of the Universitat de Barcelona. We greatly thank all the extended team of participants for their valuable contribution in DOMIPEX project. The authors were supported by the following founding: NC by Wenner-Gren foundation stipend (Sweden, 2014-2016) and a Juan de la Cierva grant (FJCI-2014-23064), EE by a pre-doctoral grant from the Basque Government (2014-2017), AGB by a Swedish Research Council grant (SMAREF 2014-2016), PRL by a Ramón Areces Foundation Postdoctoral Scholarship AM by the Univ. of the Basque Country, AMGF and SP by pre-doctoral research grants from the Spanish Ministry of Economy and Competitiveness (Ref: BES-2013-065770, Ref: BES-2012-059743, respectively), and RR by the IMDEA-Water Institute. DOMIPEX project was founded by the First Call of Collaborative Projects among Young Researchers of the Iberian Association of Limnology (AIL; 2013-2015)

Early-Career Coordinated Distributed Experiments: Empowerment Through Collaboration

Este artículo contiene 7 páginas, 1 tabla, 3 figuras.Coordinated distributed experiments (CDEs) enable the study of large-scale ecological patterns in geographically dispersed areas, while simultaneously providing broad academic and personal benefits for the participants. However, the effective involvement of early-career researchers (ECRs) presents major challenges. Here, we analyze the benefits and challenges of the first CDE exclusively led and conducted by ECRs (i.e. ECR-CDE), which sets a baseline for similar CDEs, and we provide recommendations for successful CDE execution. ECR-CDEs achieve most of the outcomes identified in conventional CDEs as well as extensive benefits for the young cohort of researchers, including: (i) receiving scientific credit, (ii) peer-training in new concepts and methods, (iii) developing leadership and communication skills, (iv) promoting a peer network among ECRs, and (v) building on individual engagement and independence. We also discuss the challenges of ECR-CDEs, which are mainly derived from the lack of independence and instability of the participants, and we suggest mechanisms to address them, such as resource re-allocation and communication strategies. We conclude that ECR-CDEs can be a relevant tool to empower ECRs across disciplines by fostering their training, networking and personal well-being.The authors were supported by the following founding: NC the support of the Beatriu de Pinós postdoctoral program of the Government of Catalonia’s Secretariat for Universities and Research of the Ministry of Economy and Knowledge (BP2016- 00215), EE by a predoctoral grant from the Basque Government (2014-2017), AB by a Generalitat de Catalunya—Beatriu de Pinós (BP-00385-2016), AMG-F by a predoctoral research grant (BES-2013-065770) from the Spanish Ministry of Economy and Competitiveness, MAr by a postdoctoral grant from the Basque Government, MIA by a Juan de la Cierva postdoctoral grant (FJCI-2015-26192), PR-L by a Margalida Comas postdoctoral contract (PD/031/2018) funded by the Government of the Balearic Islands and the European Social Fund, AP by a Ramón Areces Foundation Postdoctoral Scholarship, and AL by a Kempe Foundation stipend. DOMIPEX project was founded by the First Call of Collaborative Projects among Young Researchers of the Iberian Association of Limnology (AIL; 2013-2015).Peer reviewe

Insights into the distribution and ecological role of members of the archaeal Phylum Bathyarchaeota. From the global to the local scale

The Phylum Bathyarchaeota is currently one of the most studied uncultured archaeal phyla. Bathyarchaeota are phylogenetically diverse, globally ubiquitous and especially abundant in marine subsurface sediments. Despite their global significance, their ecological role is still unknown due to the lack of cultivated representatives and the paucity of available sequenced genomes. Besides, current distribution patterns provide few clues about their metabolic capabilities and physiological requirements. The current work tackled the problem by combining different phylogenetic and molecular techniques to shed light on the main environmental drivers that affect the distribution patterns of Bathyarchaeota at a global and regional scale. Our results showed that the phylum Bathyarchaeota went across various saline–freshwater transitions during its evolution that caused diversification events that resulted in subgroups specifically adapted to marine and saline habitats. Besides, our approach provided a robust ecological framework in which Bathyarchaeota appear as a core generalist group in the sediment realm, sharing their key role with other uncultured archaeal lineages such as the Thermoplasmata. At a regional scale, a molecular survey carried out in different stratified lakes of the Banyoles Karstic System provided evidences that Bathyarchaeota are a common component of archaeal assemblages thriving in both euxinic water compartments and sediments. Multivariate analyses identified sulfide and dissolved organic carbon as main environmental variables that explain the distribution of Bathyarchaeota subgroups between the planktonic and sedimentary habitats. In the latter, Bathyarchaeota were particularly prevalent in biofilms developed on leaf litter settled in sediments of Lake Cisó, that provided a natural enrichment where to better characterize their habitat segregation, phylogenetic diversity and membrane lipid composition. The application of complementary molecular tools such as massive sequencing of 16S rRNA gene and lipidomics revealed that biofilms were almost solely composed of subgroup Bathyarchaeota-6 and possessed a characteristic, and hitherto unknown, profile of isomeric isoprenoid tetraether lipids GDGT-1, -2 and -3. Altogether, our study recognised Bathyarchaeota as key players in marine and freshwater sediments at both global and regional scales. We also identified the main environmental drivers behind their group diversification and current subgroup segregation, and stablished the co-occurrence and putative synergy between members of the Phylum Bathyarchaeota and the class Thermoplasmata. Besides, our work provides new molecular tools to better address ecological studies focused on the Bathyarchaeota and make an important step forward to finally find a specific lipid biomarker for this widespread and diverse archaeal lineage.El Fílum Bathyarchaeota és un dels llinatges d’arqueus no cultivats més estudiat degut a la seva diversitat filogenètica i ubiqüitat, especialment en sediments marins on són particularment abundants. Tot i així, la manca de representants cultivats i els pocs genomes seqüenciats del que es disposa limita el coneixement sobre el seu paper en l’ecosistema, els factors ambientals que afecten la seva distribució, les seves capacitats metabòliques i requeriments fisiològics. El treball que aquí es presenta combina tècniques moleculars i filogenètiques per conèixer quins factors ambientals són els que afecten la distribució dels Bathyarchaeota a escala global i regional. Els resultats mostren que el Fílum Bathyarchaeota ha patit, al llarg de l’evolució, diverses transicions entre ambients salins i d’aigua dolça que han resultat en una diversificació i adaptació dels diferents subgrups a cadascun d’aquests ambients. Les aproximacions moleculars que hem desenvolupat defineixen un marc ecològic robust en el que Bathyarchaeota apareix com a un grup central, generalista en termes de recursos i que desenvolupa,juntament amb altres llinatges d’arqueus no cultivats com ara els Thermoplasmata, un paper clau en els sediments. A escala regional, hem aplicat un anàlisi molecular en diversos llacs estratificats del Sistema Càrstic de Banyoles que ha permès determinar que els Bathyarchaeota són membres estables de la comunitat d’arqueus en ambients euxínics tant de la columna d’aigua com del sediment. Les anàlisi multivariants han identificat que el sulfhídric i el carboni orgànic dissolt són les principals variables ambientals que expliquen la segregació dels subgrups de batiarqueus entre nínxols planktònics i sedimentaris. Concretament en ambients bentònics, els Bathyarchaeota són particularment abundants en biofilms desenvolupats sobre fulles en descomposició acumulades al sediment del llac Cisó, representant un vertader “enriquiment natural” d’aquests arqueus. Estudis de lipidòmica i seqüenciació massiva del gen del 16S rRNA han mostrat que en aquests biofilms la comunitat d’arqueus està composada gairebé en la seva totalitat per Bathyarchaeota del subgrup–6 i mostren un perfil d’isòmers dels lípids isoprenoides de membrana (GDGT–1, –2 i –3) únic i fins ara desconegut. En conjunt, el nostre estudi identifica als Bathyarchaeota com a grup clau en sediments marins i lacustres tant a escala global com regional. També identifica els principals factors ambientals que han afectat la seva diversificació durant l’evolució així com en la segregació que actualment mostrenen ambients lacustres. També es presenten evidències de la co-ocurrència freqüent entre membres del fílum Bathyarchaeota i la classe Thermoplasmata en sediments, fet que suggereix una possible relació sintrófica entre aquests dos llinatges. El treball aporta, a més, eines moleculars que permetran estudiar els Bathyarchaeota amb més detall i avenços clau per la identificació de biomarcadors lipídics específics per aquest grup d’arqueus tan ubic i divers

Metabolic versatility of freshwater sedimentary archaea feeding on different organic carbon sources.

Members of the phylum Bathyarchaeota and the class Thermoplasmata are widespread in marine and freshwater sediments where they have been recognized as key players in the carbon cycle. Here, we tested the responsiveness of archaeal communities on settled plant debris and sediment from a karstic lake to different organic carbon amendments (amino acids, plant-derived carbohydrates, and aromatics) using a lab-scale microcosm. Changes in the composition and abundance of sediment and biofilm archaeal communities in both DNA and RNA fractions were assessed by 16S rRNA gene amplicon sequencing and qPCR, respectively, after 7 and 30 days of incubation. Archaeal communities showed compositional changes in terms of alpha and beta diversity in relation to the type of carbon source (amino acids vs. plant-derived compounds), the nucleic acid fraction (DNA vs. RNA), and the incubation time (7 vs. 30 days). Distinct groups within the Bathyarchaeota (Bathy-15 and Bathy-6) and the Thermoplasmata (MBG-D) differently reacted to carbon supplements as deduced from the analysis of RNA libraries. Whereas Bathyarchaeota in biofilms showed a long-term positive response to humic acids, their counterparts in the sediment were mainly stimulated by the addition of tryptophan, suggesting the presence of different subpopulations in both habitats. Overall, our work presents an in vitro assessment of the versatility of archaea inhabiting freshwater sediments towards organic carbon and introduces settled leaf litter as a new habitat for the Bathyarchaeota and the Thermoplasmata

Validation of a heuristic set to evaluate the accessibility of statistical charts

A remote user test was performed with two versions (one accessible and another one non-accessible) of three types of web-based charts (horizontal bar chart, vertical stacked bar chart, and line chart). The objectives of the test were a) to validate a set of heuristic indicators for the evaluation of the accessibility of statistical charts presented in a previous work 7; b) to identify new barriers and preferences for users with low vision in the access and use of this content not previously contemplated. 12 users were tested, with a variety of conditions associated with low vision: low visual acuity (6 users), reduced central vision (2 users), reduced peripheral vision (2 users), blurry vision (1 user), sensitivity to light (3 users), Nystagmus (2 users) and color vision deficiency (CVD) (4 users). From a quantitative standpoint, accessible versions of charts were more efficient, effective, and satisfactory. From a qualitative point of view, results verify the relevance of heuristics H2, Legend; H3, Axes; H6, Data source (as data table); H10, Safe colors; H11, Contrast; H12, Legibility; H13, Image quality; H14, Resize; H16, Focus visible; H17, Independent navigation; related to the proposed tasks. As new observations, tooltips were highly valued by all users, but their implementation must be improved to avoid covering up significant parts of the charts when displayed. The data table has also been frequently used by all users, especially in the non-accessible versions, allowing them to carry out tasks more efficiently. The position and size of the legend can be a significant barrier if it is too small or appears in an unusual position. Finally, despite the limitations related to color perception, users prefer color graphics to black and white, so, to target all profiles, it is necessary to redundantly encode categories with colors and patterns as well

Phylogenetic characterization and quantification of ammonia-oxidizing archaea and bacteria from Lake Kivu in a long-term microcosm incubation

A microcosm cultivation-based method was set up to investigate the growth of ammonia-oxidizing archaea (AOA), isolated from a water sample acquired at a depth of 50 m from the northern basin of Lake Kivu. For this purpose, both CARD-FISH and qPCR targeting of archaeal 16S rRNA and amoA genes were used. Archaeal cell growth at the end of the 246-day microcosm experiment accounted for 35 % of the SybrGold-stained cells, which corresponded to 6.61 × 106 cells/ml and 1.76 ± 0.09 × 106 archaeal 16S rRNA gene copies/ml. Clone libraries and DGGE fingerprinting confirmed the dominance of AOA phylotypes in the archaeal community microcosm. The majority of the identified archaeal 16S rRNA gene sequences in the clone libraries were affiliated with Thaumarchaeota Marine Group 1.1a. Subsequent cultivation of the AOA community on deep-well microtiter plates in medium containing different carbon sources to stimulate archaeal growth failed to show significant differences in archaeal abundance (ANOVA t14 = -1.058, P = 0.308 and ANOVA t14 = 1.584, P = 0.135 for yeast extract and simple organic acids, respectively). The lack of growth stimulation by organic compounds is in concordance with the oligotrophic status of Lake Kivu. Finally, the addition of antibiotics to the growth medium resulted in archaeal cell counts that were significantly lower than those obtained from cultures in antibiotic-free mediumThis work was founded by the Spanish Government through projects CRENYC (CGL2006-12058-C02-01), ARKI (CGL2007-29823-E) and ARCANOX (CGL2009-13318-C02-02). A.P.C. and M.F.H. are recipients of pre-doctoral fellowships from the Catalan (2006FI-109) and theSpanish (FPI BES-2010-035225) Governments, respectivel