Seasonal impact of grazing, viral mortality, resource availability and light on the group-specific growth rates of coastal Mediterranean bacterioplankton

Estimation of prokaryotic growth rates is critical to understand the ecological role and contribution of different microbes to marine biogeochemical cycles. However, there is a general lack of knowledge on what factors control the growth rates of different prokaryotic groups and how these vary between sites and along seasons at a given site. We carried out several manipulation experiments during the four astronomical seasons in the coastal NW Mediterranean in order to evaluate the impact of grazing, viral mortality, resource competition and light on the growth and loss rates of prokaryotes. Gross and net growth rates of different bacterioplankton groups targeted by group-specific CARD-FISH probes and infrared microscopy (for aerobic anoxygenic phototrophs, AAP), were calculated from changes in cell abundances. Maximal group-specific growth rates were achieved when both predation pressure and nutrient limitation were experimentally minimized, while only a minimal effect of viral pressure on growth rates was observed; nevertheless, the response to predation removal was more remarkable in winter, when the bacterial community was not subjected to nutrient limitation. Although all groups showed increases in their growth rates when resource competition as well as grazers and viral pressure were reduced, Alteromonadaceae consistently presented the highest rates in all seasons. The response to light availability was generally weaker than that to the other factors, but it was variable between seasons. In summer and spring, the growth rates of AAP were stimulated by light whereas the growth of the SAR11 clade (likely containing proteorhodopsin) was enhanced by light in all seasons. Overall, our results set thresholds on bacterioplankton group-specific growth and mortality rates and contribute to estimate the seasonally changing contribution of various bacterioplankton groups to the function of microbial communities. Our results also indicate that the least abundant groups display the highest growth rates, contributing to the recycling of organic matter to a much greater extent than what their abundances alone would predict.En prensa2,92

Estacionalitat de procariotes marins usant mètodes d’anàlisi taxonòmics i funcionals

Memoria de tesis doctoral presentada por Adrià Auladell Martín para obtener el título de Doctor en Microbiologia por la Universitat Autònoma de Barcelona (UAB), realizada bajo la dirección del Dr. Josep M Gasol del Institut de Ciències del Mar (ICM-CSIC) y de la Dra. Isabel Ferrera Ceada del Centro Oceanográfico de Málaga, Instituto Español de Oceanografía (IEO-CSIC).-- 265 pages, figures, tables.-- The code to produce all the analysis and visualizations for each chapter are available here: Chapter I. https://github.com/adriaaulaICM/bbmo_niche_sea Chapter II. https://gitlab.com/aauladell/AAP_time_series Chapter III. https://figshare.com/s/51e6d1fc462950b1f582 Chapter IV. https://figshare.com/s/65d241e03c6393446e43 The data for Chapter I and II are found in the same repositories, with the location indicated in the repository manual. The data for Chapter III and IV are still unpublished. Both chapters present the summary information (abundance tables, taxonomy, basic environmental data…) but the raw data is still unavailable[EN] The oceans are ecosystems dominated by microbes, in which bacteria and archaea play key roles in biogeochemical cycling. In temperate oceans, seasonal changes in environmental conditions deeply influence the marine microbiome. In this thesis I analyse the seasonality of the marine microbiome of a coastal ocean site, using the long-term time series of the Blanes Bay Microbial Observatory (BBMO) to understand the seasonal changes through several molecular approaches. Using amplicons of the 16S rRNA gene, I evaluate the dynamics of the main bacterial groups in this coastal oligotrophic station during 11 years and test how similar the temporal niches of closely related taxa are, and what are the environmental parameters modulating their patterns of seasonality. I further explore how conserved the niche is at higher taxonomic levels. The community presented recurrent seasonality for 297 out of 6825 amplicon sequence variants (ASVs), which constituted almost half of the total relative abundance (47%). For certain genera, niche similarity decreased as nucleotide divergence in the 16S rRNA gene increased, a pattern compatible with the selection of similar taxa through environmental filtering. Additionally, I observed evidence of seasonal differentiation within various genera as seen by the distinct seasonal patterns of closely related taxa. I then switch the focus to the seasonal patterns of a specific functional group. Using the pufM gene as a marker gene for the aerobic anoxygenic phototrophic bacteria (AAPs) −a relevant photohete-rotrophic functional group in the marine microbial food web− I evaluated their long-term temporal dynamics through multivariate and co-occurrence analyses. Phylogroup K (Gammaproteobacteria) was the greatest contributor to community structure over all seasons, with phylogroups E and G (Alphaproteobacteria) being prevalent in spring. Diversity indices showed a clear seasonal trend, with maximum values in winter, which was inverse to that of AAP abundance. I afterwards extend these analyses to 21 biogeochemical relevant functions through 7 years of metagenomic data from the BBMO. Most genes presented a seasonal abundance trend: photoheterotrophic processes were enriched during spring, phosphorous-related genes were dominant during summer coinciding with phosphate limitation conditions, and assimilatory nitrate reductases correlated negatively with nitrate availability. Additionally, I identified the main taxa driving each function in each season and showed that, for some groups, the seasonality of bacterial families is different than that of their gene repertoire, so that different taxa within the same group present different functional specialization. Finally, I complement this descriptive view of the temporal changes with manipulation experiments to test how bottom-up and top-down processes exert selection on specific bacterial genomic species over the seasons. I experimentally modified the presence of predators, viruses, nutrient limitation (by diluting the samples with filtered seawater) and light availability in seawater from the BBMO in different seasons and assessed the growth of different organisms defined by metagenome assembled genomes (MAGs) under the manipulated conditions. Overall, I recovered 262 MAGs mainly from the Rhodobacterales, Flavobacteriales and Alteromonadales classes. Season and treatment greatly influenced community composition, with 26% of the MAGs indicative of the control treatments, 24% of both the control and predator-reduced treatments, 12.8% indicators of both the virus-reduced and the diluted treatments, and 7.3% of the predator-reduced treatment only. Flavobacteriaceae MAGs developed mostly in the predator-reduced treatment with distinct species in each season, whereas Alteromonadaceae and Sphingomonadaceae taxa developed preferably in the virus-reduced and diluted treatments indistinctively of season. Overall, this dissertation provides new insights into the seasonal patterns of key taxonomic and functional groups in the coastal surface ocean through the integration of information obtained using several molecular techniques and experimental approaches applied to a long-term time series[CAT] Els oceans són ecosistemes dominats per microbis i els bacteris i els arqueus hi juguen papers clau en els cicles biogeoquímics. En oceans temperats, els canvis estacionals determinen la composició del microbioma a través de les adaptacions al nínxol de les diferents espècies. En aquesta tesi analitzo l’estacionalitat del microbioma marí usant una sèrie temporal de llarga durada obtinguda a l’Observatori Microbià de la Badia de Blanes, per entendre els canvis estacionals mitjançant diverses aproximacions moleculars. A partir de seqüències d’amplicons del gen de l’RNA ribosòmic (16S) avaluo la dinàmica estacional dels principals grups bacterians durant onze anys, examinant com de similars són els nínxols temporals de taxons estretament relacionats, i quins són els paràmetres que modulen els seus patrons d’estacionalitat. També he explorat com de conservat és aquest nínxol en els nivells taxonòmics més alts. La comunitat presenta patrons estacionals de recurrència en 297 de les 6725 variants d’amplicons que hi apareixen, la qual cosa suposa gairebé la meitat de l’abundància relativa total (47%) de seqüències. Per a determinats gèneres, la similitud de nínxol disminueix amb l’increment de divergència en nucleòtids del gen del RNAr 16S, un patró compatible amb selecció de taxons similars per mitjà del filtratge ambiental. També he observat diferents patrons estacionals entre taxons del mateix gènere. A continuació vaig centrar l’anàlisi en els patrons estacionals d’un grup funcional concret. Utilitzant el gen pufM com a marcador dels bacteris aeròbics anoxigènics fotoheterotròfics −un grup funcional rellevant a la xarxa tròfica marina− avaluo la seva dinàmica temporal a través d’anàlisis multivariants i de co-ocurrència. El filogrup K (Gammaproteobacteria) és el grup dominant a l’estructura de la comunitat durant totes les estacions de l’any, amb els filogrups E i G (Alphaproteobacteria) dominants durant la primavera. Els índexs de diversitat presenten un patró estacional clar, amb els valors màxims durant l’hivern i presentant una relació inversa amb l’abundància. Després vaig ampliar aquest anàlisi a 21 funcions biogeoquímiques fent ús de set anys de dades metagenòmiques de l’observatori de Blanes. La majoria dels gens presenten un patró estacional d’abundància: els processos fotoheterotròfics enriquits durant la primavera, els gens relacionat amb l’adquisició de fòsfor dominants durant l’estiu coincidint amb una major limitació de fòsfor, i els enzims de reducció assimilatòria de nitrat correlacionant negativament amb la disponibilitat de nitrat. També he identificat els taxons principals que contenen cada gen funcional i he demostrat que, per alguns grups, l’estacionalitat a nivell de família és diferent de la del seu repertori gènic, indicant que els taxons dins del mateix grup presenten especialització funcional. Finalment, complemento la visió descriptiva dels canvis temporals amb experiments de manipulació per avaluar com els processos bottom-up i top-down influencien la selecció d’organismes durant les diferents estacions. He modificat experimentalment la presència de depredadors, de virus, la limitació per nutrients (diluint les mostres amb aigua sense microorganismes) i la llum en mostres de la Badia de Blanes en diferents estacions i he avaluat el creixement de diferents organismes definits a partir de genomes construïts a partir de metagenomes (MAGs, de les sigles en anglès). He recuperat 262 MAGs, principalment de les classes Rhodobacterales, Flavobacteriales i Alteromonadales. L’estació de l’any i el tractament influeixen la composició de la comunitat, amb el 26% dels MAGs identificats com a indicadors dels tractaments control, el 24% indicant tant el tractament control com el de reducció de depredadors, el 12.8% indicant tant el tractament de reducció de virus com el tractament diluït, i el 7.3% indicant el tractament de reducció de depredadors. Els MAGs afiliats a Flavobacteriaceae creixien majoritàriament al tractament amb reducció de depredadors, amb diferents espècies a cada estació, mentre que les especies afiliades a Alteromonadaceae i Sphingomonadaceae creixien preferentment als tractaments de reducció vírica i al tractament diluït indistintament de l’estació. En termes generals, aquesta tesi presenta resultats nous sobre els patrons estacionals de grups taxonòmics i funcionals rellevants a l’oceà costaner superficial per mitjà de la integració d’informació obtinguda usant diverses tècniques moleculars i diverses aproximacions experimentals aplicades a una sèrie temporal de llarga duradaDuring the realization of this thesis, Adrià Auladell Martín held a PhD Fellowship FPI (BES-2016-077820), funded by the Spanish Ministry of Science and Innovation and was a student of UAB’s PhD program in Microbiology. This thesis was also supported by the REMEI (CTM2015-70340-R), ECLIPSE (PID2019-110128RB-I00), and MIAU (RTI2018-101025-B-I00) projectsPeer reviewe

Diversitat de bacteris fotoheterotròfics marins a escala temporal

Seminario Nous Avenços en Ecologia Microbiana desarrollado el 13 de enero de 2017 en BarcelonaPeer reviewe

Interannual seasonal dynamics of aerobic anoxygenic photoheterotrophs in the NW Mediterranean Sea

Trabajo presentado por Joan Martí Carreras para un Máster de la Universitat Pompeu Fabra (UPF), realizada bajo la dirección de la Dra. Isabel Ferrera y del Dr. Pablo Sánchez del Institut de Ciències del Mar (ICM-CSIC)Peer Reviewe

Insights into the ecology of marine aerobic anoxygenic phototrophic bacteria

1st Iberian Ecological Society Meeting (2019); XIV Congreso Nacional de la Asociación Española de Ecología Terrestre (AEET), Ecology: an integrative science in the Anthropocene, 4-7 February 2019, Barcelona, SpainThe study of marine microbes has bloomed thanks to the use of molecular tools opening new windows into ecological studies. Oneof the major advances achieved last decade was the discovery of marine photoheterotrophic bacteria, which challenged our view onmicrobial food webs in the ocean. These organisms are heterotrophs that rely mainly on organic matter but are capable of harvestinglight to supplement their energy requirements. In the last years, we have used various approaches to uncover the ecology of a groupof marine photoheterotrophs, the aerobic anoxygenic phototrophic bacteria (AAPs). In particular, we have combined infrared mi-croscopy, quantitative PCR and Illumina sequencing with large sampling initiatives (i.e., the Blanes Bay Microbial Observatory andthe Malaspina circumnavigation expedition) as well as with manipulation experiments to explore their diversity, seasonality, biogeog-raphy and, more importantly, their functional role in marine ecosystems. Our results confirm that the AAPs are widely distributed inthe global surface oceans and that the different phylogenetic groups present a certain degree of biogeography. Moreover, we havedemonstrated that these bacteria show a clear seasonality both in abundance and community structure. But foremost, we have foundthat the AAPs are fast-growing bacteria contributing significantly to the recycling of organic matter and that they can be stimulatedby light in the marine environment. The results of our ongoing work will be presented and discussed in detail at the conferencePeer Reviewe

El camí científic: igual per tothom?

El dia 10 de març a les 12:00 al pati principal tindrà lloc aquesta taula rodona després del visionat de Picture a Scientist (dia 7 de març a les 15:00h). Tindrem com a ponents les nostres companyes Sara Martínez, Queralt Guerrero, Adrià Auladell i Pere Puig. Reflexionarem sobre els aspectes descrits al documental i sobre les experiències personals dels ponents respecte a les desigualtats a la carrera científicaPeer reviewe

Performance of the melting seawater‐ice elution method on the metabarcoding characterization of benthic protist communities

10 pages, 4 figures, 2 tables, supporting Information https://doi.org/10.1111/1758-2229.12834.-- This is the peer reviewed version of the following article: Performance of the melting seawater‐ice elution method on the metabarcoding characterization of benthic protist communities, A, Reñé, A. Auladell, G. Reboul, D. Moreira, P. López‐García, Environmental Microbiology Reports12(3): 314-323 (2020) which has been published in final form at https://doi.org/10.1111/1758-2229.12834. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived VersionsMassive amplicon sequencing approaches to characterize the diversity of microbial eukaryotes in sediments are scarce and controls about the effects introduced by different methods to recover DNA are lacking. In this study, we compare the performance of the melting seawater‐ice elution method on the characterization of benthic protist communities by 18S rRNA gene metabarcoding with results obtained by direct cell lysis and DNA purification from sediments. Even though the most abundant operational taxonomic units were recovered by both methods, eluted samples yielded higher richness than samples undergoing direct lysis. Both treatments allowed recovering the same taxonomic groups, although we observed significant differences in terms of relative abundance for some of them. Dinoflagellata and Ciliophora strongly dominated the community in eluted samples (> 80% reads). In directly lysed samples, they only represented 37%, while groups like Fungi and Ochrophytes were highly represented (> 20% reads respectively). Our results show that the elution process yields a higher protist richness estimation, most likely as a result of the higher sample volume used to recover organisms as compared to commonly used volumes for direct benthic DNA purification. Motile groups, like dinoflagellates and ciliates, are logically more enriched during the elution processG.R., D.M. and P. L.‐G. were funded by European Research Council Grant ProtistWorld (no. 322669) under the European Union's Seventh Framework Program. A.R. was granted by MINECO Grant COPAS ‘Understanding top‐down control in coastal bloom‐forming protists’ (CTM2017‐86121‐R), and a MECD grant ‘Estancia de Movilidad en el extranjero José Castillejo’ (CAS17/00237)With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI

Patterns of bacterioplankton diversity and community structure along contrasting cross-shore environmental gradients in the northwestern Mediterranean coast

Association for the Sciences of Limnology and Oceanography (ASLO) Summer meeting, Water connects!, 10-15 June 2018, Victoria, CanadaPeer Reviewe

Growth rates of marine prokaryotes are extremely diverse, even among closely related taxa

ASLO Aquatic Sciences Meeting, Resilience and Recovery in Aquatic Systems, 4-9 June 2023, Palma de Mallorca, SpainMarine prokaryotes play crucial roles in ocean’s biogeochemical cycles, being their contribution largely determined by their growth rates. To improve our understanding of microbial communities’ dynamics and structure, we estimated prokaryotic growth rates at high resolution, the amplicon sequence variant (ASV) level, using manipulation experiments in each of the four astronomical seasons with different treatments that reduced limiting growth factors such as predators, nutrient availability, viruses and light. Single-ASV-based growth rate calculations showed a continuous range of values, reaching almost 10 day-1. Mean growth rates per treatment increased as limiting factors were removed, and changed seasonally. Results revealed significant variability in growth rate distribution patterns even within closely related ASVs, and with no general taxonomic coherence observed after removal of each growth limiting factor. Reduction of these factors showed that most responsive ASVs were rare, and formed a pool of taxa with the potential to rapidly respond to environmental changes. In essence, our results highlight the significance of rare-responsive taxa often overlooked in whole-community studies and suggest high dynamism in marine microbial communities with a strong capacity to adapt to changing environmentsPeer reviewe

Growth rates of marine prokaryotes are extremely diverse, even among closely related taxa

pages, 7 figures, 1 table.-- Data Availability Statement: Sequence data have been deposited in ENA under accession number PRJEB60085. The R-scripts used to calculate growth rates, generate figures and statistical analyses are available at: https://github.com/onadeulofeu/Single-ASV-based_prokaryotes_growth_rates.Marine prokaryotes play crucial roles in ocean biogeochemical cycles, being their contribution strongly influenced by their growth rates. Hence, elucidating the variability and phylogenetic imprint of marine prokaryotes' growth rates are crucial for better determining the role of individual taxa in biogeochemical cycles. Here, we estimated prokaryotic growth rates at high phylogenetic resolution in manipulation experiments using water from the northwestern Mediterranean Sea. Experiments were run in the four seasons with different treatments that reduced growth limiting factors: predators, nutrient availability, viruses, and light. Single-amplicon sequence variants (ASVs)-based growth rates were calculated from changes in estimated absolute abundances using total prokaryotic abundance and the proportion of each individual ASV. The trends obtained for growth rates in the different experiments were consistent with other estimates based on total cell-counts, CARD-FISH subcommunity cell-counts or metagenomic-OTUs. Our calculations unveil a broad range of growth rates [0.3-10 d-1] with significant variability even within closely related ASVs. Likewise, the impact of growth limiting factors changed over the year for individual ASVs. High numbers of responsive ASVs were shared between winter and spring seasons, as well as throughout the year in the treatments with reduced nutrient limitation and viral pressure. The most responsive ASVs were rare in the in situ communities, comprising a large pool of taxa with the potential to rapidly respond to environmental changes. Essentially, our results highlight the lack of phylogenetic coherence in the range of growth rates observed, and differential responses to the various limiting factors, even for closely related taxaThis research was supported by grants REMEI (CTM2015-70340-R) and MIAU (RTI2018-101025-B-100) funded by the Spanish Ministry of Science and Innovation. We acknowledge the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S). Adrià Auladell and Ona Deulofeu-Capo were supported by Spanish FPI grantsPeer reviewe