Abstract This research investigated the spatial and temporal distributions of Trichodesmium spp. and Richelia intracellularis in the South China Sea. The surveys covered the period from July 2000 to July 2002. A total of eight cruises, including spring, summer and fall were conducted. The sampling stations located between 18~22o N and 115~122 o E , over the continental shelf, the slope, and the basin of the northern South China Sea. Trichodesmium biomass was higher in summer and fall than spring. There was no significant difference in biomass among shelf , slope and basin. The averaged biomass was 69

A fixação de N2 é uma fonte importante de nitrogênio novo nos ciclos biogeoquímicos, realizado potencialmente por cianobactérias diazotróficas, principalmente em condições de limitação de nitrogênio. O canal de São Sebastião (CSS) é reconhecidamente meso-oligotrófico, porém, eventos episódicos de precipitação podem alterar as concentrações de nutrientes locais interferindo na ocorrência de diazotróficas. Este trabalho investigou o efeito do aumento da pluviosidade na ocorrência e coexistência de diazotróficas no CSS e na baía do Araçá (BA), em 9 cenários distintos. As diazotróficas Trichodesmium spp. e Richelia intracellularis ocorreram após o aumento da pluviosidade nos dois pontos, não co-existindo em dois eventos. R. intracellularis ocorreu somente em endossimbiose com diatomáceas do gênero Hemiaulus, com predomínio em H. membranaceus. Os ventos de SW no CSS indicaram relações diretas na ocorrência das diazotróficas, sugerindo advecção de células provindas de outros locais. A temperatura e o fosfato apresentaram relações significativas com a ocorrência das diazotróficas. As densidades de diazotróficas e da comunidade fitoplanctônica foram maiores na BA em todos os eventos, assim como as concentrações de clorofila-a e nutrientes, ressaltando a importância da interação das águas da BA com o CSS. / The biological nitrogen fixation adds new nitrogen into the biogeochemical cycle. This process is attributed to diazothrophic cyanobacteria, which are known to occur in environments with nitrogen limitation. Although the São Sebastião Channel (CSS) is a meso-oligothrophic environment, local rain can affect nutrients concentrations, hence the occurrence of diazothrophic species. This work addressed how the increase in the precipitation rate alters the occurrence and co-existence of diazothrophic cyanobacteria in nine scenarios in the CSS and Araçá Bay (BA). The diazothrophic species Trichodesmium spp. and Richelia intracellularis occurred after an increase in rainfall, co-occurring in 7 of the 9 scenarios. Richelia intracellularis was always associated symbiotically with Hemiaulus diatoms, specially H. membranaceus. The occurrence of diazothrophic species was highly correlated to intense SW winds, suggesting the allochthonous input of cells in the CSS. The diazothrophic species occurrence was also associated with variations in temperature and phosphate concentration. The abundance of both diazothrophic species and phytoplankton community were higher in the BA in every scenario. The nutrients and chlorophyll-a concentrations were higher in the BA, highlighting the importance of water transport between BA and CSS.

A fixação de N2 é uma fonte importante de nitrogênio novo nos ciclos biogeoquímicos, realizado potencialmente por cianobactérias diazotróficas, principalmente em condições de limitação de nitrogênio. O canal de São Sebastião (CSS) é reconhecidamente meso-oligotrófico, porém, eventos episódicos de precipitação podem alterar as concentrações de nutrientes locais interferindo na ocorrência de diazotróficas. Este trabalho investigou o efeito do aumento da pluviosidade na ocorrência e coexistência de diazotróficas no CSS e na baía do Araçá (BA), em 9 cenários distintos. As diazotróficas Trichodesmium spp. e Richelia intracellularis ocorreram após o aumento da pluviosidade nos dois pontos, não co-existindo em dois eventos. R. intracellularis ocorreu somente em endossimbiose com diatomáceas do gênero Hemiaulus, com predomínio em H. membranaceus. Os ventos de SW no CSS indicaram relações diretas na ocorrência das diazotróficas, sugerindo advecção de células provindas de outros locais. A temperatura e o fosfato apresentaram relações significativas com a ocorrência das diazotróficas. As densidades de diazotróficas e da comunidade fitoplanctônica foram maiores na BA em todos os eventos, assim como as concentrações de clorofila-a e nutrientes, ressaltando a importância da interação das águas da BA com o CSS. / The biological nitrogen fixation adds new nitrogen into the biogeochemical cycle. This process is attributed to diazothrophic cyanobacteria, which are known to occur in environments with nitrogen limitation. Although the São Sebastião Channel (CSS) is a meso-oligothrophic environment, local rain can affect nutrients concentrations, hence the occurrence of diazothrophic species. This work addressed how the increase in the precipitation rate alters the occurrence and co-existence of diazothrophic cyanobacteria in nine scenarios in the CSS and Araçá Bay (BA). The diazothrophic species Trichodesmium spp. and Richelia intracellularis occurred after an increase in rainfall, co-occurring in 7 of the 9 scenarios. Richelia intracellularis was always associated symbiotically with Hemiaulus diatoms, specially H. membranaceus. The occurrence of diazothrophic species was highly correlated to intense SW winds, suggesting the allochthonous input of cells in the CSS. The diazothrophic species occurrence was also associated with variations in temperature and phosphate concentration. The abundance of both diazothrophic species and phytoplankton community were higher in the BA in every scenario. The nutrients and chlorophyll-a concentrations were higher in the BA, highlighting the importance of water transport between BA and CSS.

Marine primary productivity in large parts of the oceans is supported by nitrogen fixers (diazotrophs). Richelia, a genus of multiple closely related species of heterocystous filamentous cyanobacterial diazotrophs, are often found in symbioses with a few genera of diatoms. Several Richelia species: R. euintracellularis, R. intracellularis and R. rhizosoleniae, form stable host specific partnerships and these populations are important for the nitrogen (N) cycle, especially in the oligotrophic open oceans. In general, the gene nifH, which encodes the enzyme nitrogenase for N2 fixation, is used as a molecular marker for presence (DNA level) and activity (RNA level) of diazotrophs. However, evidence of cross-reactivity of the nifH assays between two of the Richelia species shows the risk of incorrect estimations of abundance and activity. Moreover, transcript abundance is rarely normalized to a housekeeping gene.  The aims of this work were to develop and assess new assays for the detection of three symbiotic Richelia species: R. euintracellularis, R. intracellularis and R. rhizosoleniae. These new assays targeted molecular markers that were indicative nutrient acquisition. For example, one assay targets a gene encoding the high affinity phosphate transporter (pstS), a second targets a gene involved in iron (Fe) transport (exbB) (indicator of P transport, Fe transport, respectively), and a third assays a housekeeping gene encoding Ribonuclease P protein (rnpA). All assays were used to estimate abundance and expression in lab and field based samples. The new assays have high species specificity as revealed by BlastN analyses and lab-based cross-hybridization assays. We applied the assays to samples from a lab-based experiment of the facultative symbiont R. rhizosoleniae RrhiSC01 in order to investigate the temporal dynamics of expression. This revealed periodic expression of nifH and pstS related to the photoperiod with higher expression in the early and late photoperiod, respectively. The peak of pstS expression in the late photoperiod is likely to support the high P requirement of both photosynthesis and N2 fixation. Given the temporal regulation of pstS (and nifH) expression shown here, one must consider these results when sampling and/or interpreting field studies. Moreover, a comparison of the Richelia draft genomes and environmental metagenomic assembled genomes (MAGs) show that gene copy number per genome of exbB and pstS varies between strains. Given that Richelia tend to live in low nutrient environments, including N, P, and often Fe, the increased copy number per genome for pstS and exbB could be suggestive/evidence of adaptation to constant nutrient limitation. In addition, the assays were applied to natural samples containing populations of R. intracellularis and R. euintracellularis collected in the North and South Atlantic Ocean from multiple depths (5-90 meters). The DNA based gene copy abundances showed higher estimates when nifH is used compared to rnpA, which is likely due to a higher degree of cross-reactivity in the nifH assays. Gene expression was present but low for all targets. However, expression of exbB and pstS was higher in surface water – where nutrients are expected to be depleted. Lastly, bulk fixation rates of N2 and carbon (C) showed that the activity was low and fixation rates did not increase with the addition of dissolved organic phosphate (DOP) (a 1:4 mixture of 2-aminoethylphosphonic acid and beta-glycerophosphate disodium salt hydrate). This could be due to patchiness and low abundance of diazotrophs and discrepancies between samples, seasonal variation or that the populations were not P limited. In summary, the new assays constitute a better option to interspecific detection of Richelia. However, more work is required to assess how the expression relates to limitation of nutrients and other external factors to better understand the activity of these biogeochemically important populations. / I stora delar av haven stöds marin primärproduktion av kvävefixerare (diazotrofer). Richelia är ett släkte med ett flertal närbesläktade arter av heterocysta, filamentösa och kvävefixerande cyanobakterier som lever i symbios med ett fåtal kiselalger. Dessa är viktiga för kvävecykeln, särskilt i de oligotrofa (kvävefattiga) öppna haven. Genen nifH som kodar för det kvävefixerande enzymet nitrogenas, brukar användas som en molekylär markör för att detektera diazotrofa populationer och dess genuttryck används som en indikator för kvävefixering. Det finns emellertid tecken på att nifH ger felaktiga uppskattningar av dessa populationers abundans och aktivitet. Dessutom, när nifH-uttryck används som ett mått på aktivitet är det endast kvävefixering man observerar. Andra faktorer som begränsar förekomsten av dessa organismer, t.ex. brist på järn eller fosfor, kan inte avläsas. Dessutom har uttrycket av nifH sällan normaliserats till en stabilt uttryckt gen vilket gör jämförelser svåra. Syftet med detta arbete var att utveckla och utvärdera nya kvantitativ realtidspolymeraskedjereaktion (qPCR)-analyser för specifik detektion av identitet, abundans och aktivitet av tre symbiotiska Richelia-arter: R. euintracellularis, R. intracellularis och R. rhizosoleniae. De nya analyserna är inriktade på generna som kodar för högaffinitetsfosfattransportören pstS och biopolymertransportproteinet exbB (indikator för P-transport respektive Fe-transport). Genen som kodar för ribonukleas P-protein, rnpA, en gen med stabilt uttryck, användes för att uppskatta abundans och för att normalisera uttrycket av de andra målgenerna.  BlastN-analyser och labb-baserade korshybridiseringsanalyser visar att de nya analyserna har hög artspecificitet. Vi tillämpade analyserna på en kultur av den fakultativa symbionten R. rhizosoleniae RrhiSC01 för att undersöka det periodiska uttrycksmönster av målgenerna kopplat till tid på dygnet. Resultatet visade på ett periodiskt uttryck av nifH och pstS relaterat till fotoperioden med högre uttryck i den tidiga, respektive sena fotoperioden.  Det ökade uttrycket av pstS i den sena fotoperioden stödjer sannolikt det höga P-kravet för både fotosyntes och N2-fixering. Den tidsmässiga regleringen av pstS-uttryck som visas här, behöver man ta hänsyn till vid provtagning och tolkning av fältstudier. ​​ Dessutom visar en jämförelse av Richelia-genom att genkopiantalet för exbB och pstS varierar mellan stammar av Richelia. Med tanke på att Richelia tenderar att leva i miljöer med låga nivåer av näringsämnen, inklusive kväve, fosfor och ofta järn, kan det ökade antalet kopior per genom av pstS och exbB ett tecken på anpassning till konstant näringsbegränsning. Analyserna tillämpades även på naturliga populationer av R. intracellularis och R. euintracellularis i norra och södra Atlanten. DNA-baserad estimering av abundans (genkopior) visar på en högre uppskattning när nifH används som markör jämfört med rnpA - troligtvis på grund av en högre grad av korsreaktivitet i nifH-analyserna. Genuttryck var närvarande men lågt för alla målgener i norra och södra Atlanten. Uttrycket av exbB och pstS var dock högre i ytvatten – där halter av näringsämnen förväntas vara låga. Slutligen var kväve- och kolfixering i planktonsamhället låg och ökade inte vid tillsats av fosfor, vilket kan bero på ojämn fördelning av celler, låg förekomst av diazotrofer och diskrepanser mellan prover, säsongsvariationer eller att populationerna inte var primärt begränsade av fosfor. Sammanfattningsvis utgör de nya analyserna ett bättre alternativ för specifik detektion av Richelia. Det krävs dock mer arbete för att bedöma hur uttrycket påverkas av  näringsbrist och yttre faktorer och för att bättre förstå aktiviteten hos dessa ovärderliga populationer.