Valentina Valdés
email: vvaldesc@udec.cl

François Carlotti

Ruben Escribano

Katty Donoso

Marc Pagano

Veronica Molina

Camila Fernandez

Nitrogen and phosphorus recycling mediated by copepods in the Western Tropical South Pacific

Zooplankton plays a key role in the regeneration of nitrogen and phosphorus in the ocean through grazing and metabolism. In this study, we explore the role of zooplankton release of organic and inorganic nitrogen and phosphorus compounds on the microbial community, over an oligotrophic gradient in the Western Tropical South Pacific. Three microcosm experiments were performed in the long duration stations (LD) from the Melanesian Archipelago region

x

List of Figures

Figure 1: Particulate organic consumption and metabolism by zooplankton. Prey is consumed by zooplankton. Resulting of sloppy feeding some of dissolved organic compounds in the prey become lost to the surrounding water, while the rest of the prey is ingested. Some of the ingested prey is assimilated and used for growth and reproduction. The rest of the compounds that were not absorbed can be egested as fecal pellets, and released as waste from excretion . The design consisted of four phases done sequentially. Each copepod group was acclimated for at least 6 h (phase 1), followed by feeding (phase 2) under two natural size fraction of food: 20-150 µm and <20 µm during 4 h. Copepod excretion (phase 3) had treatment and control (without copepods) in two replicated chambers. Finally, the microbial response (phase 4) was carried out in glass flasks and each treatment had four replicates. FSW: filtered seawater ................................... 15 Figure 6: Experimental design used in PROMO experiments. The design consisted of four phases done sequentially. Each copepod group was acclimated for at least 6 h (phase 1) inside cylinders isolated from the surrounding water with a 200 µm mesh. Cylinders are then transferred to two glass containers for feeding (phase 2) under two natural size fractions of food: 20-150 µm and <20 µm during 4 h. Copepod excretion (phase 3) had replicated treatments and controls (without copepods), in 10 L chambers equipped with a faucet near the bottom for sampling aliquots. Finally, the microbial response (phase 4) was carried out in glass flasks and each treatment had four replicates .. Figure 7: Experimental design. The design consisted of three phases done sequentially. Each bottle with copepods was acclimated for at least 6 hours (phase 1), followed by feeding (phase 2) with the Deep Chlorophyll-a Maximum (DCM) seawater for 3 hours. Finally, copepod recycling (phase 3) has treatment and control (without copepods). Three bottles were sacrificed at each sampling time . The design consisted of four phases done sequentially. Each copepod group was acclimated for at least 6 h (phase 1), followed by feeding (phase 2) under two natural size fractions of food: 20-150 µm and < 20 µm during 4 h. Copepod excretion (phase 3) had treatment and control (without copepods) in two replicated chambers. Finally, the microbial response (phase 4) was carried out in glass flasks and each treatment had four replicates. FSW, filtered seawater ................................... 28 Figure 8. Conceptual model representing the role of zooplankton for N cycling in the water column of an upwelling system, linked to the autotrophic community and the microbial loop. Three major processes are considered: (1) DON input by zooplankton through sloppy feeding, (2) DON and NH 4 + input via zooplankton excretion, and (3) DON input by leaching of zooplankton fecal pellets. These compounds are then used by heterotrophic and chemoautotrophic bacteria and archaea to transform organic nitrogen into inorganic forms (microbial loop), making them available for new uptake by phytoplankton. It should be noted that nitrification can indeed occur in the upper illuminated layer. In subsurface water, an oxygen deficient zone is present mostly during the spring and summer. Some zooplankton able xii to migrate daily to this zone also contribute to anaerobic ammonium oxidation through processes (2) and(3 The design consisted of four phases done sequentially. Each copepod group was acclimated for at least 6 h (phase 1) inside cylinders isolated from the surrounding water with a 200 µm mesh. Cylinders are then transferred to two glass containers for feeding (phase 2) under two natural size fractions of food: 20-150 µm and <20 µm during 4 h. Copepod excretion (phase 3) had replicated treatments and controls (without copepods), in 10 L chambers equipped with a faucet near the bottom for sampling aliquots. Finally, the microbial response (phase 4) was carried out in glass flasks and each treatment had four replicates ... 102

DISSOLVED COMPOUNDS EXCRETED BY COPEPODS RESHAPE THE ACTIVE MARINE BACTERIOPLANKTON COMMUNITY COMPOSITION

NITROGEN AND PHOSPHORUS RECYCLING MEDIATED BY COPEPODS IN WESTERN TROPICAL SOUTH PACIFIC

Figure 1: Experimental design. The design consisted of three phases done sequentially. Each bottle with copepods was acclimated for at least 6 hours (phase 1), followed by feeding (phase 2) with the Deep Chlorophyll-a Maximum (DCM) seawater for 3 hours. Finally, copepod recycling (phase 3) has treatment and control (without copepods). Three bottles were sacrificed at each sampling time . S3. ANOVA followed by the Tukey´s Honest Significant Difference (HSD), statistical comparison was performed on nutrients turnover rates in treatments and between times throughout the experiments. The mean of HSD is recorded at 95% confidence level with its associated P value for this comparison S3: Consumption and production rates obtained in the treatment incubated with copepods. The turnover rates were obtained for each compound studied between incubation times and were estimated using an end-point approach. A positive value indicates a net accumulation and negative values indicates a net nutrient consumption .............................. 143 Table S4: Consumption and production rates obtained in the control (without copepods). The turnover rates were obtained for each compound studied between incubation times and were estimated using an end-point approach. Supervised by: Fabien Joux -Ingrid Obernosterer.

• Noviembre 2014. Observatorio de Ecología Microbiana, Universidad de Playa Ancha, Valparaíso, Chile. Supervised by: Veronica Molina.

• Febrero-April 2015. Institut Méditerranéen d`Océanologie, Marseille, France.

Supervised by: François Carlotti -Marc Pagano.

• Abril-Junio 2015. Observatoire Océanologique de Banyuls sur Mer, France.

Supervised by: Dr. Pierre Galand.

• Julio 2015. Observatorio de Ecología Microbiana, Universidad de Playa Ancha, Valparaíso, Chile. Supervised by : Veronica Molina.

• Diciembre 2015-Febrero 2016. Institut Méditerranéen d`Océanologie, Marseille, France. Supervised by: François Carlotti.

SCHOLARSHIP

• 2012-2015. CONICYT (Chile) Doctoral graduate fellowship.

• 2014-2015. CONICYT (Chile) Supplementary grant "Support doctoral thesis".

• 2014-2015. CONICYT (Chile) Supplementary grant "Doctoral internship"

• Sept-Dec 2016. Scholarship grant Instituto Milenio de Oceanografía.

• 2015. Scholarship grant "doctoral internship" REDOC CTA-UdeC. The interaction between metazooplankton metabolism and the microbial loop is recognized as one of the principal mechanisms controlling the quantity, composition and cycling of DOM in the sea. Zooplankton and bacteria are usually considered as separate groups despite their temporal and spatial co-existence and close interaction. Zooplankton may release dissolved organic and inorganic matter through sloppy feeding, leaching from fecal pellets and excretion. However, there is a lack of knowledge on the impact of these dissolved compounds released by zooplankton on biogeochemical cycles and on the microbial community structure. An experimental approach with natural food sources and simulated in situ conditions was used to determine the role that trophic interactions involving primary and secondary producers in the recycling of nitrogen and phosphorus in the pelagic system of central-southern Chile. First, we quantified the nitrogen and phosphorus compounds excreted by copepods fed with natural sized-fractioned diets (20-150 µm and <20 µm). Then, we assessed the biogeochemical response of the microbial community to released nitrogen, including the nitrifying community, in terms of total and active cells, to the products excreted by copepods. Secondly, we explored the simultaneous interaction between copepod excretion and the biogeochemical response of active microbial community composition, over an oligotrophic gradient in the western tropical south Pacific (WTSP).

Our results show that DON was the main excretion product, with rates of up to 3.7 µmol L -1 h -1 under autumn/winter conditions in central/southern Chile. Meanwhile, in spring/summer copepods actively excreted nitrogen and phosphorus compounds, mainly in the form of ammonium and dissolved organic phosphorus (DOP), reaching excretion rates of up to 2.6 µmol L -1 h -1 and 0.05 µmol L -1 h -1 , respectively. In both cases the main products excreted xxv were closely related to the main item food available for copepods (nanoplankton and microplankton, respectively). In the autumn/winter experiments, ammonium was rapidly consumed by the microbial community. Ammonium consumption coincided with increased archaea (AOA) and bacteria ammonia-oxidizing (AOB) amoA transcript copies in copepods fed with the larger-sized fraction, whereas a different microbial community, probably heterotrophic, reacted to nitrogen input via excretion by copepods fed with the smaller-sized fraction in autumn. Moreover, during spring/summer a shift in the composition of active bacterial community was observed between periods and treatments, associated with the response of common-opportunistic seawater surface phyla Proteobacteria and Bacteroidetes.

In the WTSP, copepods contributed elevated levels of ammonium, DON and DOP highly labile. Copepod excretion can enhance the remineralization (ammonificationnitrification) process and reshape the diversity and composition of the active bacterial community. These changes were characterized by shifts in Alteromonadales and SAR11 in both study regions (Melanesian archipelago and subtropical gyre) and differed substantially from the in-situ community. Enhanced bacterial activity in presence of copepods would further increase the available substrates for microbial and phytoplankton growth in a highly stratified system and provide an alternative source of phosphorus for diazotrophs blooms in the WTSP.

We concluded that nitrogen and phosphorus excreted by copepods can be used directly by microbial communities, including nitrifying microbes, providing significant remineralized nitrogen for sustaining new and regenerated production in the upper ocean of different marine La interacción entre el metabolismo del metazooplancton y el anillo microbiano es considerado uno de los principales mecanismos que controla la cantidad, composición y el reciclaje de la materia orgánica disuelta en el océano. Zooplancton y microorganismos suelen considerarse como grupos separados a pesar de su coexistencia temporal y espacial, y su estrecha interacción. El zooplancton libera materia orgánica e inorgánica disuelta a través del consumo incompleto de su presa, la disolución de sus pellets fecales y la excreción. Sin embargo, el impacto de estos compuestos liberados por el zooplancton en los ciclos biogeoquímicos y en la estructura de la comunidad microbiana ha sido pobremente estudiado.

En este trabajo, a través de un enfoque experimental, con fuentes de alimentos naturales y condiciones in situ simuladas, se determinó el rol que juegan las interacciones tróficas involucrando productores primarios y secundarios en el reciclaje de nitrógeno y fósforo en el sistema pelágico en la zona de surgencia costera del centro-sur de Chile. En primer lugar, se cuantificaron los productos excretados por copépodos alimentados con dietas naturales de tamaño fraccionado. A continuación, se evaluó la respuesta biogeoquímica de la comunidad microbiana y su estructura, en términos de células totales y activas, incluyendo la comunidad nitrificante en respuesta a los productos excretados por copépodos. En segundo lugar, se exploró la interacción simultánea entre la excreción de copépodos y la comunidad microbiana, en términos de la respuesta biogeoquímica y la composición activa en un gradiente de oligotrofia en el Pacífico Sur Occidental Tropical (WTSP por sus siglas en inglés).

Nuestros resultados muestran que DON fue el principal producto de excreción, con tasas de hasta 3,7 µmol L -1 h -1 durante otoño-invierno en el centro-sur de Chile. Mientras que, en primavera/verano se excretó activamente compuestos de nitrógeno y fósforo, xxvii principalmente en forma de amonio y fósforo orgánico disuelto (DOP), alcanzando tasas de hasta 2,6 µmol L -1 h -1 y 0,05 µmol L -1 h -1 , respectivamente. En ambos casos los principales productos excretados estuvieron estrechamente relacionados con el principal alimento disponible para copépodos (nanoplancton y microplancton, respectivamente). En los experimentos de otoño/invierno, el amonio fue rápidamente consumido por la comunidad microbiana. Este consumo coincidió con el aumento de los transcritos del gen amoA de arqueas (AOA) y bacterias amonio-oxidantes (AOB) en copépodos alimentados con la fracción de tamaño mayor, mientras que una comunidad microbiana diferente, probablemente heterotrófica, reaccionó a nitrógeno aportado vía excreción de copépodos, alimentados con la fracción de tamaño menor en otoño. Por otra parte, durante primavera/verano se observaron cambios en la composición de la comunidad bacteriana activa entre períodos y tratamientos, asociado a bacterias de aguas superficiales con características oportunistas como Proteobacteria y Bacteroidetes.

En el WTSP, los copépodos aportaron niveles elevados de amonio, DON y DOP altamente lábiles. La excreción de copépodos estimula el proceso de remineralización (amonificación-nitrificación) y puede modificar la diversidad y la composición de la comunidad bacteriana activa, caracterizada por cambios principalmente en Alteromonadales y SAR11 en ambas regiones de estudio (archipiélago Melanesio y giro subtropical). La actividad bacteriana en presencia de copépodos se intensificó, incrementando los sustratos disponibles para el crecimiento de microbios y fitoplancton, lo cual podría proporcionar una fuente alternativa de fósforo para la floración de diazotrófos en WTSP. Nous concluons que l'azote et le phosphore excrétés par copépodes peuvent être utilisés directement par des communautés microbiennes, y compris des nitrifiants, fournissant de l'azote reminéralisé pour soutenir la production nouvelle et régénérée dans l'océan supérieur des différents écosystèmes marins.

INTRODUCTION

Dissolved organic matter in marine environments

Large areas in the ocean are deficient in one or more nutrients necessary for life, such N, P, Si and other trace metals, and the distribution of life in the ocean depends on these bioessential nutrient elements. This is especially true in the open ocean, known as oligotrophic zones, which can account for 40% of the global primary production [START_REF] Carlson | DOM source, sinks, reactivity and budgets[END_REF]. Trophic interactions in the pelagic ecosystems depend on primary and secondary production requiring these bio-elements, either as dissolved or as particulate forms. For several decades, research has focused on quantifying the inorganic concentration and on determining the stoichiometry of these bio-elements, which together with the Redfield anomalies are useful to describe the functioning of food webs, and to examine the processes that regulate marine biogeochemical cycles. The organic matter in marine systems is operationally divided into particulate organic matter (>0.7 µm; POM) and dissolved organic matter (<0.7 µm; DOM).

DOM is an important reservoir of nutrients in the upper ocean and is composed by a complex and heterogeneous mixture of molecules. However, only a small fraction has been identified and little is known about their reactivity. Most DOM is immediately respired by marine micro-heterotrophs, oxidized by photochemical process, or permanently buried in sediments (Ridgwell and Arndt, 2015).The euphotic zone is the principal site of organic matter production in the ocean and the main process of DOM production are: extracellular release by phytoplankton, grazers-mediated release and excretion, cell lysis (both viral and bacterial), solubilization of detrital and sinking particles and release from prokaryotes [START_REF] Carlson | DOM source, sinks, reactivity and budgets[END_REF]. All production, removal and transformation processes leave an imprint on the composition of DOM and how it is cycled in the water column. Thus, all the processes that influence the reactivity of organic matter in the ocean, have gained attention in the last decades (Strom et al, 1997;Zubkov et al, 2008;[START_REF] Wawrik | Use of Inorganic and Organic Nitrogen by Synechococcus spp. and Diatoms on the West Florida Shelf as Measured Using Stable Isotope Probing[END_REF].

Nitrogen and phosphorus are fundamental components of DOM. The cycle of nitrogen includes a variety of forms and a series of complex transformations, involving electron shifts between the most oxidized form (nitrate; NO 3 -) and the most reduced one (ammonium; NH 4 + ).

Inorganic nitrogen species include nitrate, nitrite (NO 2 -

) and ammonium, and organic forms are present as amino acids, nucleic acids, amino sugars and urea, as well as their polymers (DNA, RNA, proteins). Primary production in marine ecosystems can be partitioned in according to the source of nitrogen substrate utilized by phytoplankton [START_REF] Dugdale | Uptake of new and regenerated forms of nitrogen in primary productivity[END_REF]. "New" production refers to the part of primary production that is fueled by nutrients inputs from outside the euphotic zone (mainly NO 3 -and N 2 ), while "regenerated" production is the fraction of primary production sustained by reduced compounds of nitrogen (mainly NH 4 + and dissolved organic nitrogen; DON), produced in situ through the food web [START_REF] Dugdale | Uptake of new and regenerated forms of nitrogen in primary productivity[END_REF]. Another element which is part of DOM is phosphorous. This has been much less studied than nitrogen, although it is as essential as N for growth and it can exist as dissolved and particulate forms. Dissolved fractions include inorganic phosphorous, existing in the environment almost exclusively as orthophosphate (PO 4

3-

) and organic phosphorus compounds like P-esters, P-diesters and phosphonates [START_REF] Paytan | The Oceanic Phosphorus Cycle[END_REF].

Release of dissolved organic matter by zooplankton in pelagic system

An important component of the pelagic food web is zooplankton. This group controls the secondary production and hence the transfer of nutrients through the pelagic food web.

Zooplankton is mainly represented by crustacean and gelatinous organisms, among which the microcrustacean copepods appears as the most abundant group for all regions, accounting for up to 55 to 95% of total numerical abundance of zooplankton [START_REF] Harris | ICES Zooplankton Methodology Manual[END_REF]Escribano et al., 2007).

The four main processes by which zooplankton can release DOM (Figure 1) are direct excretory release, egestion, breakage of preys during feeding (sloppy feeding) and leaching from fecal pellets. Further studies on the relative importance of these processes demonstrated that sloppy feeding and excretion are the dominant modes of DOM production, being a minor role the leaching from the fecal pellets [START_REF] Vincent | Net and gross incorporation of nitrogen by marine copepods fed on 15N-labelled diatoms: Methodology and trophic studies[END_REF]Saba et al., 2011).

Mesozooplankton can remove from 10% to 40% of daily primary production in the coastal and open ocean, respectively (Calbet 2001). Complementary to the control exerted on the structure of food webs through direct predator-prey interactions, exerting a control on primary production (PP), mesozooplankton also play an important role in the active transport of DOM, and are capable of fueling the microbial loop (Richardot et al., 2011;Steinberg et al., 2002). In general, it has been reported that ammonium is the primary nitrogenous product excreted by marine zooplankton, comprising up to 75% of the total nitrogen excreted, followed by urea and amino acids which can account for 25% [START_REF] Bidigare | Nitrogen excretion by marine zooplankton[END_REF]. Ammonium supplied by zooplankton in the pelagic ecosystem is believed to represent the largest input of this element in marine ecosystems. A global estimation of rates of ammonium by Hernández-León et al. (2008) showed that excretion rates were highest in equatorial waters and decreased rapidly poleward. Furthermore, other studies on zooplankton excretion have shown that dissolved organic nitrogen (DON) in the form of urea and amino acids is a significant excretion product and can range from 7% to 80% of total dissolved nitrogen release, exceeding even inorganic nitrogen excretion (Steinberg et al. 2000(Steinberg et al. , 2002;;[START_REF] Steinberg | Nitrogen consumption and metabolism in marine zooplankton[END_REF]. This finding has also been reported in oligotrophic zones with a 60% of total dissolved nitrogen excretion as DON (Le Borgne and Rodier, 1997). However, relatively few studies of DON release by excretion support those results [START_REF] Conover | Sources of urea in arctic seas: zooplankton metabolism[END_REF][START_REF] Miller | Nitrogen excretion by the calanoid copepod Acartia tonsa: results of mesocosm experiments[END_REF]. Most of the research has been focused on nitrogen excretion and only a few studies are focused on phosphorus compounds, mainly on orthophosphate [START_REF] Pomeroy | Excretion of phosphate and soluble organic phosphorus compounds by zooplankton[END_REF][START_REF] Ikeda | Metabolic rates of epipelagic marine copepods as a function of body mass and temperature[END_REF]Saba et al., 2009;[START_REF] Alcaraz | The role of arctic zooplankton in biogeochemical cycles: respiration and excretion of ammonia and phosphate during summer[END_REF]. One of the first reported on phosphorus excretion demonstrated that one-third of soluble phosphorus released by zooplankton (amphipods) was in the organic form, also they found a tight coupling between dissolved organic phosphorus (DOP) production and uptake by bacteria (Johannes 1964). The same pattern was found by Satomi and Pomeroy (1965), where zooplankton excreted a high proportion (60%) of organic phosphorus.

Effects of feeding on excretion products

Because of the high variability in the composition of phytoplankton prey items, zooplankton can be affected in their growth and reproduction [START_REF] Jónasdóttir | Diet composition and quality for Calanus finmarchicus egg production and hatching success off south-west Iceland[END_REF]Aguilera and Escribano, 2012), but also the products excreted with consequences in the composition of bioavailable regenerated products. In this regard, it has been reported that composition and concentration of nitrogen excreted by zooplankton are affected by the quantity and quality of the food ingested (Saba et al., 2009). Miller and Roman (2008) reported that the N: C ratio of ingested food in copepods varied drastically in the various diets, ranging from 0.26 to 0.04 for ciliates and detritus respectively, affecting the nutritional status of organisms and with significant effects on nitrogen excretion. Thus, copepods fed food with N: C ratios resembling their own body composition, such as log-phase diatoms and ciliates, excrete more ammonium when fed in higher concentration. In the same context, other studies showed that ammonium was a high portion of total nitrogen release when copepods were feeding carnivorously (Corner et al., 1976), and[START_REF] Bidigare | Nitrogen excretion by marine zooplankton[END_REF] proposed that an increase in the release of urea by zooplankton may reflect the proportion of phytoplankton ingested in their diet, since phytoplankton contains a high content of arginine, a precursor of urea, although Miller and Roman (2008) suggested that urea excretion rates of Acartia tonsa were higher when fed with ciliates compared to a diatom diet.

On the other hand, Saba et al., (2009) showed a high dependency of the prevailing compound being excreted on the food provided to copepods, showing that DOP release was only detectable in diets containing micro-zooplankton. The available information however is contradictory and not always reflect the environmental conditions in terms of the food offered to zooplankton and only there are a few studies that reflect the omnivorous behavior of copepods in the natural environment.

The biogeochemical role of zooplankton in the marine system

Phytoplankton prefer ammonium over other sources of nitrogen, since it is in the reduced form, and therefore requires less energy to assimilate, but ammonium is not always the dominant substrate to be used by phytoplankton. It has long been recognized that urea uptake can support a significant amount of regenerated production in a wide variety of environments [START_REF] Sipler | Dynamics of Dissolved Organic Nitrogen[END_REF]. Furthermore, DON consumption can satisfy a large proportion of the requirements of nitrogen for the phytoplankton [START_REF] Wawrik | Use of Inorganic and Organic Nitrogen by Synechococcus spp. and Diatoms on the West Florida Shelf as Measured Using Stable Isotope Probing[END_REF]Bronk et al., 2006), although little is known about its reactivity. The contribution of nitrogen to phytoplankton community has been more studied than the Phosphorus. Pérez-Aragón et al.

(2011) showed that copepods in a coastal upwelling zone of Chile can sustain up to 26.7% of the phytoplankton ammonium demand. In other upwelling zones, e.g. in northwest of Africa, it was found that zooplankton supplied an average of 44% of the ammonia demand [START_REF] Smith | The role of zooplankton in the regeneration of nitrogen in a coastal upwelling system off northwest Africa[END_REF]. Moreover, in oligotrophic zones, such as the Mediterranean Sea, the nitrogen regenerated by mesozooplankton accounted for 43% of the nitrogen requirements by phytoplankton, and even in some coastal sites the ammonia excreted by zooplankton exceeded the phytoplankton nitrogen demand [START_REF] Alcaraz | Excretion of ammonia by zooplankton and its potential contribution to nitrogen requirements for primary production in the Catalan Sea (NW Mediterranean)[END_REF]. However, on a global basis, nutrient regeneration by mesozooplankton (between 200 to 2000 µm) was estimated to be in the range of 12-23% of the requirements for phytoplankton growth (Hernández-Leon et al., 2008). [START_REF] Alcaraz | The role of arctic zooplankton in biogeochemical cycles: respiration and excretion of ammonia and phosphate during summer[END_REF] studied the contribution to the regeneration of inorganic phosphorus via zooplankton excretion in the Artic zone and found that zooplankton contributed with 53% of the phosphorus requirement by phytoplankton.

On the other hand, the microbial community, as responsible for approximately half of the overall global primary production, plays an important role in nutrient cycling [START_REF] Arrigo | Marine microorganism and global nutrient cycles[END_REF]. Furthermore, it is the dominant biological component in the marine ecosystem exhibiting the most diverse metabolisms. The dominant nitrogen sources and their relative concentrations are important in determining the microbial community structure in aquatic environments. Bacteria and zooplankton are usually considered as separate group despite their temporal and spatial co-existence [START_REF] Tang | Linkage between crustacean zooplankton and aquatic bacteria[END_REF]. However, recent research has established a direct relationship between the microbial and the zooplankton community with a consequent effect on the different biogeochemical pathways linked to nitrogen and phosphorus cycles [START_REF] Calbet | Mesozooplankton influences on the microbial food web: direct and indirect trophic interactions in the oligotrophic open ocean[END_REF][START_REF] Grossart | Bacterial diversity associated with freshwater zooplankton[END_REF]Titelman et al., 2008;Richardot et al., 2011;[START_REF] Tinta | Jellyfish modulate bacterial dynamic and community structure[END_REF]Aristegui et al., 2014). Vargas et al. (2007) showed that the copepod Acartia tonsa play an important role influencing bacterial production through the production of significant amounts of bacterial substrate by means of grazing activity. Furthermore, in addition to the classical contribution of zooplankton to food webs, their role as environmental reservoirs for a high diversity of microorganism, including pathogens, is increasingly recognized [START_REF] Tang | Linkage between crustacean zooplankton and aquatic bacteria[END_REF].

Ammonium is the most commonly regenerated product released during bacterial DOM remineralization [START_REF] Bronk | DON as a source of bioavailable nitrogen for phytoplankton[END_REF], zooplankton excretion [START_REF] Alcaraz | Excretion of ammonia by zooplankton and its potential contribution to nitrogen requirements for primary production in the Catalan Sea (NW Mediterranean)[END_REF], and

photochemical breakdown of DOM (Rain-Franco et al. 2014). It promotes regenerated production in marine ecosystems [START_REF] Dugdale | Uptake of new and regenerated forms of nitrogen in primary productivity[END_REF] and it is rapidly consumed in surface waters through assimilation, by phototrophic and heterotrophic plankton, including microbial communities with different life styles, and also by aerobic ammonia-oxidizing bacteria (AOB) and archaea (AOA) (Fernandez and Farias 2012;[START_REF] Molina | Potential contribution of planktonic components to ammonium cycling in the coastal area off central-southern Chile during non-upwelling conditions[END_REF], and possibly by anaerobic oxidizers by anammox, eventually occurring in surface waters [START_REF] Galán | Temporal variability and phylogenetic characterization of planktonic anammox bacteria in the coastal upwelling ecosystem off central Chile[END_REF]. Nitrifying microorganisms can couple aerobic chemoautotrophic metabolism with ammonia oxidation via nitrite to nitrate conversion in two steps: ammonia-oxidation and nitrite-oxidation, carried out by two functional groups of microorganisms, i.e., ammonia and nitrite oxidizers. Although a recent study showed that a single nitrite oxidizing bacteria belonging to the genus Nitrospira can do both steps and its relevance in marine pelagic environments is still unknown (Daims et al. 2015). This conversion plays an important biogeochemical role in the pelagic system by remineralizing organic matter and then supplying oxidized forms of nitrogen to photosynthesis, affecting our estimation of new vs. regenerated primary production [START_REF] Yool | The significance of nitrification for oceanic new production[END_REF]. In fact, it has been reported that nitrification is active in marine euphotic zone (Wankel et al. 2007;Santoro et al. 2010;Shiozaki et al. 2016) and a recent study found that ammonium excretion and microbial nitrification affect the capacity of epipelagic ecosystems to retain their nutrients (nutrient retention efficiency [NRE]), and thus to maintain high levels of regenerated production (Fernández-Urruzola et al. 2016).

A recent study on the impact of krill excretion on microbial activity demonstrates that the addition of krill excretion products stimulates bacteria growth and production (Arístegui et al., 2014). Also, studies on jellyfish showed that the bloom of these organisms can provide significant amounts of dissolved organic matter (DOM) due to the jellyfish degradation and excretion, facilitating the mineralization of dissolved organic carbon and dissolved organic nitrogen (DON), followed by a high accumulation of ammonium and orthophosphate available for microbial communities [START_REF] West | Influence of decomposing jellyfish on the sediment oxygen demand and nutrient dynamics[END_REF].

On the other hand, recent research has demonstrated that bacterial community composition can change in response to the addition of organic matter (Landa et al., 2013;Sarmento et al., 2013) DOM release during phytoplankton blooms has been associated with an important change in the microbial diversity and their metabolism (Mc Carren et al., 2010;[START_REF] Sarmento | Use of phytoplankton-derived dissolved organic carbon by different types of bacterioplankton: Use of phytoplankton-derived DOC by bacterioplankton[END_REF]. These results, was also found in studies on degradation of jellyfish, showed a rapid shift in community composition from unculturable Alphaproteobacteria to culturable species of Gammaproteobacteria and Flavobacteria [START_REF] Tinta | Jellyfish modulate bacterial dynamic and community structure[END_REF]. However, the response of bacterial community structure to the compounds release by copepod excretion has not been studied yet.

The scientific problem

At present, the processes and mechanisms through which nitrogen and phosphorus compounds released by zooplankton can determine and influence the food web structure of the microbial community are unclear. At one end of the food web, zooplankton nutrition may depend on quality of their food resources, and the interaction between zooplankton organisms and their food may determine the quality of nitrogen and phosphorus organic compounds.

Meanwhile, variable species of organic nitrogen and phosphorus can determine the structure of the microbial community. Although previous studies have shown responses of the bacterial community to input of DOM, the interaction among zooplankton feeding conditions, zooplankton excretion, and the associated microbial loop has not yet been examined in the pelagic system. All these processes however may vary depending on the environmental forcing, of which seasonal and regional effects can be highly significant. The complex relationships among biological components, nutrients and related cycling processes can be sketched for pelagic systems as shown in Figure 2. The type of food ingested (based on size fraction and composition) by zooplankton can determine the main nitrogen and phosphorus compounds to be excreted in a highly productive upwelling system off central-southern Chile.

Hypothesis 2:

Zooplankton excretion in different marine systems, modulated by the type of ingested food, can significantly reshape the microbial community structure and activity.

General goal

This project aims at enhancing our comprehension on the role that trophic interactions involving microbial and metazoan food webs of the ocean can play in the recycling of nitrogen and phosphorus in the pelagic environment.

Specific goals

1) Identifying the nitrogen and phosphorus compounds excreted by zooplankton under different diets (prey composition), depending on seasonal variability in central-southern

Chile and along an oligotrophic gradient in the western tropical south Pacific.

2) Quantifying the contribution of nitrogen and phosphorus excreted by zooplankton to the potential nitrogen and phosphorus demand of phytoplankton community and evaluate the impact of excreted products on local regenerated production.

3) Determining the role of nitrogen and phosphorus compounds excreted by zooplankton on nitrifying communities and in shaping the structure of the microbial community and its seasonal and regional variability in the central-southern Chile and western tropical south Pacific.

MATERIALS AND METHODS

The present study took place in the coastal area of Central Chile and in the oligotrophic region in the Western Tropical South Pacific (WTSP). The thesis work was done in order to evaluate two main issues: the excretion of copepods and the impact of the excreted compounds on microbial communities. The study was developed as based on experimental approaches with a total of seven experiments. The productivity in the coastal zone off southern-central Chile is stimulated by upwelled water that fertilize the photic zone with inorganic nutrients during the springsummer season. In contrast, the productivity is much lower in winter, and the system is mostly sustained by regenerated production (Fernandez and Farias 2012), and the input of new nutrients mainly comes from river runoff and mixing [START_REF] Escribano | The structure and functioning of the coastal upwelling system off central/southern Chile[END_REF][START_REF] Sobarzo | Coastal upwelling and seasonal cycles that influence the water column over the continental shelf off central Chile[END_REF]. sediments and is influenced by multiple ocean currents which promote shearing instabilities and strong eddies [START_REF] Qiu | Source of the 70-Day Mesoscale Eddy Variability in the Coral Sea and the North Fiji Basin[END_REF] reducing their oligotrophic condition in the MA region.

Study area

Central Southern Chile

Furthermore, the WTSP has recently been considered a hotspot for N 2 fixation (Bonnet et al., 2017), which is considered to the largest external nitrogen source to the ocean [START_REF] Sohm | Emerging patterns of marine nitrogen fixation[END_REF].

Chapter I: Nitrogen excretion by copepods and its effect on ammonia-oxidizing communities from a coastal upwelling zone

During autumn and winter 2012, two experiments were performed in order to determine the main nitrogen compounds excreted by dominant species of copepods and its possible effect on the transcriptional activity of ammonia monooxygenase subunit A (amoA), a functional marker for ammonia oxidizing archaea (AOA) and bacteria (AOB), as a response to the input of DON and ammonium by copepod excretion. For this, at each sampling date we monitored the hydrological variables, such as temperature, salinity and dissolved oxygen, using a CTD-O profiler (SeaBird SBE 25 Plus). In order to examine the available food for copepods at this sampling site seawater samples were collected with 10-liter Niskin bottles for total and size fractioned chlorophyll-a (Chl-a), particulate organic carbon (POC), particulate organic nitrogen (PON), microplankton and nanoplankton. See methodological details in the results section in chapter I.

Experimental procedures

Live copepod samples for experiments were collected with a WP2 zooplankton net with a 200 µm mesh size, equipped with a 5 L non-filtering cod-end, between 0 and 50 m depth under daylight conditions. Live samples were immediately transferred to coolers and diluted with surface seawater until arrival (~1 h after sampling) to the laboratory at the Dichato Marine Station of the University of Concepcion. One sample was also taken with a filtering cod-end and preserved immediately with formalin buffered by sodium borate (5% final concentration) for zooplankton composition analysis [START_REF] Postel | Biomass and abundance[END_REF]Turner 1979).

In the laboratory, undamaged zooplankton individuals were sorted from the live samples and identified using a stereomicroscope at low light. The number of copepods used in the two experiments was determined based on the average abundances observed in the coastal area in previous studies [START_REF] Escribano | The structure and functioning of the coastal upwelling system off central/southern Chile[END_REF][START_REF] Hidalgo | Patterns of copepod diversity in the Chilean coastal upwelling system[END_REF]. Copepod samples in the two experiments consisted of Paracalanus cf. indicus (80%) and Acartia tonsa

(20%).
The experimental design consisted of 4 steps: acclimatizing copepods, feeding, excretion and microbial response (Figure 5), as follows: The acclimation phase (1) consisted of maintaining 4 copepod groups (20 individuals per group), for 6 h in filtered seawater (0.7 µm; GF/F Whatman), which had been obtained previously from 10 m depth at the sampling site and filtered with a peristaltic pump. Copepods were isolated from surrounding water using 200 µm mesh-tubes and were acclimatized in darkness at controlled temperatures simulating in situ conditions (ca. 12º C). Acclimatization was followed by the copepod-feeding phase (2), to assess the impact of the type of food ingested by copepods, two size fractions of food were considered: microplankton and nanoplankton which were obtained by filtering natural food through sieves (20-150 µm and < 20 µm, respectively). The feeding phase lasted 4 h under controlled temperature (12º C). During this phase copepods were maintained in 200 µm meshtubes as the acclimation phase. Food quality for these size fractions was determined by analyzing species composition and abundance for nanoplankton and microplankton (see physical, chemical and biological in situ measurements during CopeMOD experiments section).

Thereafter, each copepod group was placed in an incubator for the excretion phase (3).

The incubation system consisted of six chambers filled with 6 L of seawater previously filtered (0.2 µm; Millipore) through a peristaltic pump, to avoid the presence of large microbial communities. Four of the chambers contained groups of copepods each, and two chambers were without copepods and used as controls. The incubation system was previously washed with 10% HCl to remove any residual organic matter. Each chamber was equipped with a sampling faucet to retrieve subsamples for chemical and biological analysis during the incubation period. Water samples were collected in duplicate from each chamber at one-hour intervals (t0= 0h, t1 = 1h and t2 = 2h) to determine NH 4 + , nitrate (NO 3 -), nitrite (NO 2 -) and DON. Additionally, samples for picoplankton abundance were taken to evaluate the potential occurrence of small-sized bacteria, although efforts were made reduce this influence by using filtered seawater (0.22 µm).

The microbial response to copepods excretion (4). To assess the response of ammoniaoxidizing communities to the DON and ammonium excreted by copepods, 8.5 L of seawater were filtered (0.7 µm; GF/F Whatman) evenly distributed into ten 1 L glass bottles under a peristaltic pump (1 L; Duran Schott). The seawater was filtered to avoid the presence of phytoplankton and small protist, that can prey on bacteria, and ensure the presence of natural microbial assemblages. Subsamples (150 mL) were taken from each incubation chamber

Data analysis

Average control concentrations (NH 4 + and DON) during the excretion phase were subtracted from treatment values to obtain the corresponding concentration related to excretion during the incubation. Negative values indicated that control values were higher than those of the treatment, whereas positive values indicated that treatment values were higher than those of the control, indicating that copepods were indeed excreting. Copepod excretion rates for NH 4 + and DON were determined using the endpoint approach for the time periods t0 to t1 and t1 to t2. For the entire incubation time (from t 0 to t2 ) these rates were obtained from the slope of the linear regression of concentration as a function of incubation time. This ) in the microbial response phase were subtracted from treatments to obtain the production or consumption due to microbial activity at the respective sampling times. Finally, a two-way ANOVA and a Tukey´s post hoc test were applied to the copepod excretion and microbial response phases of the experiment.

The responses of AOA and AOB were evaluated as amoA gene copy number transcripts in the different treatments (20-150 µm and <20 µm). This estimation is positive when the AOA and AOB responses increase and negative when they are the opposite compared to the control. , PO 4 3-and bacterioplankton abundance were taken on board, DNA and RNA samples (the latter was taken only in spring) were immediately filtered upon arrival to the laboratory. Also, total and size fractioned chlorophyll-a (Chl-a), microplankton and nanoplankton abundance, were obtained at each site in order to determine the in-situ food available for copepods. See methodological details in the results section, chapter II.

Chapter

Experimental procedures

Zooplankton were captured at each site by means of vertical hauls of a WP-2 net with 200 µm mesh-size. The net was equipped with a non-filtering cod end to capture undamaged individuals. Once onboard, the samples were poured into coolers and diluted with surface seawater (10 m depth) to maintain the organisms alive and in properly conditions until sorting at the laboratory (~1 h).

To explore the interaction between dissolved compounds excreted by copepods and bacteria, we designed an experiment consisting in four phases: (1) Copepods selection and acclimation, (2) Copepods feeding, (3) Copepod excretion and ( 4) Microbial response to dissolved compounds excreted in the previous phase (Figure 6). The experimental design of each phase is as follows:

Phase 1. Live zooplankton samples for each sampling event were sorted under a stereomicroscope at low light. The number of individuals used in both experiments was determined as based on the mean abundance observed in the St. 18 of the COPAS time series (Escribano et al., 2007;[START_REF] Hidalgo | Patterns of copepod diversity in the Chilean coastal upwelling system[END_REF]. In late summer, a mix of two abundant adult copepods, Acartia tonsa (71%) and Paracalanus cf indicus (29%) were sorted, whereas, in spring, a mix of three adult copepods was sorted, P. cf indicus (37.5%), Calanoides patagoniensis (45%) and Spinocalanus sp. (17.5%). In both sampling dates, four equal groups of copepods, containing the same community, were isolated from surrounding water using 200 µm mesh-tubes and maintained in two incubators (previously washed with 10% HCl) filled with 10 L of 0.7 µm filtered seawater (GF/F; Whatman). The acclimation was carried out under dark conditions for 6 hours.

Phase 2. The 4-copepod groups selected were then transferred for feeding purposes into two 10 L incubators containing different size fractions of natural food, <20 µm and 20-150 µm (2 groups for each incubator), obtained earlier. This feeding phase was performed under dark conditions (12º C) and lasted 4 hours.

Phase 3. The copepod groups were transferred to a different set of incubators containing 0.2 µm filtered seawater (Durapore; Millipore). This was done to minimize the presence of microbial assemblages during the excretion phase. The incubator system consisted in six chambers (6 L each), two for the treatment fed with 20-150 µm size fraction, two for the treatment fed with <20 µm size fraction, and two incubators as controls (without copepods).

Copepods were isolated in the chamber in tubes using 200 µm mesh-tubes, as feeding and acclimation phase. Each chamber was equipped with a sampling faucet to retrieve subsamples with minimal disturbance. At time intervals of 0 h (T0), 1 h (T1: T0+1h Phase 4. To evaluate the microbial response to dissolved compounds provided by copepod excretion, 10 glass bottles with 850 mL of seawater with natural microbial assemblages (10 m depth), were enriched with 150 mL aliquots of the excretion products derived from phase 3.

Only one control chamber was used.

Samples were collected in duplicate at time intervals of 0 h (T0), 2 h (T1: T0+2h), 4 h (T2: Bacterial community structure was analyzed in terms of both total and active fraction by Illumina MiSeq sequencing method from 16S rRNA, using cDNA as template, and 16S rDNA (Campbell and Kirchman, 2013). See molecular methods, nutrient analysis, bacterioplankton abundance in the results section in chapter II.

Statistical analyses

Statistical analysis of treatments on chemical parameters associated with excretion products was performed using a paired t-test, and during the microbial response phase a two-way ANOVA was applied using treatments and time as effect levels, after testing for normality assumptions (Kolmogorov-Smirnov test) and homoscedasticity (Levene´s test). Pairwise multiple comparisons were performed using a Tukey test as a posteriori analysis.

Bacterial community structure was compared using ordination Bray-Curtis similarities, visualized in UPGMA dendrograms, whose nodes were further tested using a bootstrap analysis. Multivariate analysis was also applied to relate the variability in biological responses and environmental variables, using PRIMER v.6 and the add-on PERMANOVA+ software package. The permutational multivariate analysis of variance (PERMANOVA) with fixed factors was applied to investigate the differences of bacterial community composition for treatments and control in both experiments. Principal co-ordinate analysis (PCO) was performed to visualize patterns of the bacterial community OTUs in response to different treatments.

Chapter III: Nitrogen and phosphorus recycling mediated by copepods in Western Tropical South Pacific

Live zooplankton samples were captured at night conditions at each LD station (day 2) with a WP2 zooplankton net with a 120 µm mesh size, equipped with a non-filtering cod-end to obtain undamaged individuals, between 0 to 100 m depth under night conditions in the three LD stations. Live samples were immediately transferred to coolers until sorting at the laboratory.

The experiment design involved three steps: (1) Copepod acclimation, (2) feeding and ( 3) copepod recycling (Figure 7). Seawater for incubations (30 L) was collected into clean (10% HCl rinsed) polycarbonate carboys from DCM using Niskin bottles (12 L) arranged on a CTD rosette. For acclimatizing ( 2) and copepod recycling (3) steps, seawater (22.5 L) was immediately filtered onto a 0.7 µm (GF/F; Whatman) using a peristaltic pump. The 7.5 L remaining was used in the feeding phase. Seawater for the different steps was maintained in a cold room (in situ temperature ~25º C) until the beginning of the experiment.

Undamaged individuals were sorted from the live samples and identified using a stereomicroscope at low light. Copepods samples in the three experiments consisted in a mix of the most representative copepods (adults) in the sample (Fig. S1). In acclimatization phase

(1), 15 groups of 10 copepods were incubated in 500 mL Nalgene bottles, maintained for 4-6 h in filtered seawater previously filtered (GF/F Whatman) in darkness and at controlled temperature (in situ). In feeding step (2) copepods were removed from the bottles used in the previous phase, with a sieve (20 µm) and maintained in 500 mL polycarbonate bottles (Nalgene) with <150 µm filtered seawater (polycarbonate membrane) for feeding. This step lasted 4 hours and, as the acclimatization phase, was under controlled temperature. Thereafter, each copepod group (15 groups) was placed in 500 mL polycarbonate bottles (Nalgene) with the seawater previously filtered (0.2 µm; Millipore). Also, other 15 bottles without copepods were incubated as control. In total, 30 bottles were used in this step. The seawater used was filtered to avoid the presence of phytoplankton and small protist, that they can prey on bacteria, and ensure the presence of natural microbial assemblages. At the end of each step copepods were observed and checked by direct observation for ensuring swimming behavior in the bottles.

At time intervals of 0 h (T0), 0.5 h (T1: T0+0.5 h), 1 h (T2: T0+1 h), 2 h (T3: T0+2h bottle with copepods was acclimated for at least 6 hours (phase 1), followed by feeding ( phase2) with the Deep Chlorophyll-a Maximum (DCM) seawater for 3 hours. Finally, copepod recycling (phase 3) has treatment and control (without copepods). Three bottles were sacrificed at each sampling time. Source: elaborated by the author.

Bacterial community structure was analyzed by Illumina MiSeq sequencing method from 16S rRNA (samples from incubation), using cDNA as template, and 16S rDNA (in situ; Campbell and Kirchman, 2013). Methodological details for nutrients, bacterioplankton abundance and molecular methods were provided in the results section in chapter III.

Statistical analysis

Statistical analysis of treatments effects on chemical and biological parameters were performed using a two-way analysis of variance (ANOVA) after checking normality assumptions (Kolmogorov-Smirnov test) and homoscedasticity (Levene´s test). Pairwise multiple comparisons were performed using a Tukey test as a posteriori analysis. Statistical significance was set a p=0.05 and analysis was computed using R software.

The bacterial community structure was compared using the ordination Bray-Curtis similarities which were also used to build dendrograms by the unweighted pair group method with arithmetic averages (UPMG). For multivariate statistical analysis, the software package PRIMER v.6 and the add-on PERMANOVA+ was used. The permutational multivariate analysis of variance (PERMANOVA) with fixed factors was applied to investigate the differences of bacterial community composition for treatments and control in both experiments. Non-metric multidimensional scaling and principal co-ordinate analysis (PCO) was performed to visualize patterns of the bacterial community in response to different treatments. was the main excretion product, with rates up to 3.7 µmol L -1 h -1 . DON production increased in copepods fed with the small-sized food. Ammonium was also excreted, with rates up to 0.08 µmol L -1 h -1 in autumn and 0.4 µmol L -1 h -1 in winter, and rapidly consumed by the microbial community, decreasing down to µmol L -1 between the initial time to 4 h.

RESULTS

Chapter

Ammonium consumption coincided with increased AOB and AOA amoA transcript copies in copepods fed with the larger-sized food, while a different microbial community, probably heterotrophic, reacted to nitrogen input via excretion by copepods fed with the smaller-sized food in autumn. AOA-A was transcriptionally active in winter with nearly zero ammonium concentration suggesting that AOA outcompete AOB when ammonium becomes limited. We conclude that nitrogen excreted by copepods can be used directly by microbial communities, including nitrifying ones. Zooplankton excretion may thus provide significant remineralized nitrogen for new and regenerated production in the upper ocean.

Artificial ammonium enrichment experiments

The predominant ammonia-oxidizers active in the study area were identified and transcriptional activity was assessed by ammonium enrichment experiments with in situ communities in the austral winter and summer (August 2008 and January 2009). To do so, seawater (20 L) was collected at different depths (surface and oxycline) at Station 18 (Fig. 1), considering that ammonia-oxidizing groups could be favored in the oxycline than in surface waters in the study area [START_REF] Molina | High diversity of ammonia-oxidizing archaea in permanent and seasonal oxygen-deficient waters of the eastern South Pacific: AOA in marine oxygen-deficient waters[END_REF]. The oxycline depth was determined based on the oxygen profiler obtained from the COPAS time series at Station 18. Seawater was kept in carboys until reaching the laboratory. Seawater was then distributed into 1 L glass bottles (Duran Schott), with two replicates for controls and ammonium enrichment experiments for each sampling time, consisting of three hours in total t 0 : 0h (carboy subsampling) and then t 1 :1h, t 2 :2h and t 3 :3h. Thus, a total of 12 bottles were used per depth for the entire experiment.

The number of bottles was selected to do the experiment in a timely manner considering the number of filtration units available to stop each experiment time. The ammonium enrichment treatment used a NH 4 Cl solution designed to obtain a final concentration of 0.1 and 1 µmol L -1 in winter and summer, respectively. These values were based on the expected ammonium concentration during the respective seasons considering the data available for the time series at Station 18. In situ ammonium concentrations obtain during our sampling varied between the surface and oxycline, 0.1-0.9 and 0.3-1.2 µmol L -1 in the winter and summer experiments, respectively. The bottles were then incubated in darkness with a controlled temperature (12ºC). The incubation was completed by water filtration using a peristaltic pump. To avoid saturating the filters and to do the filtering in a timely manner, the water was pre-filtered through 1.6 µm GF/A (Whatman) and then filtered onto 0.22 µm Durapore filters (Millipore).

The filters were soaked in 300 µL of RNAlater reagent (Ambion), frozen and kept at -80ºC until analysis (see the Molecular Methods section).

Identification and quantification of ammonia-oxidizing groups in artificial ammonium

enrichment experiments. Specific RNA transcription assays were carried out using the reversed primers for archaeal amoA, Arch-amoAR [START_REF] Francis | Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean[END_REF], and bacterial amoA, amoA2R [START_REF] Rotthauwe | The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations[END_REF], with Sensiscript Reverse Transcriptase (Quiagen). All the reactions included a control without the template. The archaeal and bacterial amoA were then amplified by PCR and qPCR using cDNA as template and the following primers Arch-amoAF and Arch-amoAR [START_REF] Francis | Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean[END_REF], amoA-1F and amoA-2R primers [START_REF] Rotthauwe | The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations[END_REF]. A 25 µL PCR reaction was done using the following reagents: 1 µL template DNA (5- °C, without a final extension. PCR products were visualized on agarose gels using standard electrophoresis procedures. The specificity of the PCR primer products was also evaluated by direct sequencing using the corresponding forward primers, only as a verification of the potential actively transcribed group and not as a community composition analyses since that had been reported previously in the study area [START_REF] Molina | High diversity of ammonia-oxidizing archaea in permanent and seasonal oxygen-deficient waters of the eastern South Pacific: AOA in marine oxygen-deficient waters[END_REF]). These sequences were The qPCR protocol was similar for all the assays based on [START_REF] Mosier | Relative abundance and diversity of ammonia-oxidizing archaea and bacteria in the San Francisco Bay estuary[END_REF], which was as follows: 10 min at 95ºC, then 40 cycles for 30 s at 95ºC, 45s at 56ºC and 1 min at 72ºC with a detection step at the end of each cycle. A melting curve stage was run at the end of all the qPCR assays. The detection limit of the standards was always observed at a CT mean <30.

The specificity of qPCR assays was verified by checking the dissociation curves and running the PCR products in agarose gels.

Plasmids containing cloned and sequenced amoA gene fragments were used as qPCR standards. The plasmids were extracted using Wizard Plus SV Minipreps (Promega) and quantified using the Quant-iTTM Broad Range DNA assay (Qubit Fluorometer, Invitrogen).

The clones ST010550.E7 (FJ615376) and ST080550.F7 (GU066855) archaeal and bacterial amoA, respectively, were used as standards. The standard curves were prepared using 10-fold dilution series from 2 x10 7 -2 x 10 1 . The PCR efficiencies (E) and correlation coefficients (r 2 )

for the standard curves were as follows: archaeal amoA (E=91%, r2=0.989) and bacterial amoA (E=84%, r2= 0.994).

Abstract

Copepods are important suppliers of bio-reactive compounds for marine bacteria through fecal pellet production, sloppy feeding and excretion of dissolved compounds. However, the interaction between copepods and bacteria is poorly understood in the marine environment.

Herein, we investigated the nitrogen and phosphorus compounds excreted by copepods fed with two natural sized-fractioned diets (<20µm and 20-150µm) in the upwelling zone of central/southern Chile in late summer and spring. We then assessed the biogeochemical response of the bacterial community and its structure, in terms of total and active cells, to the enrichment with copepod excreted dissolved compounds. Results revealed that copepods actively excreted nitrogen and phosphorus compounds, mainly in the form of ammonium and dissolved organic phosphorus (DOP), reaching excretion rates up to 2.6 µmol L-1 h-1 and 0.05 µmol L-1 h-1, respectively. Maxima excretion rates were associated in both periods with the 20-150µm size fraction, but particularly during spring, where also a higher organic matter quality was observed in this excretion products compared to late summer. However, dissolved free amino acids (DFAA) excreted revealed higher excretion rates associated with copepods fed with <20µm size fraction, mainly by Histidine in late summer and Glutamic acid in spring.

A shift in the composition of active bacterial community was observed between periods and treatments, associated with the response of common seawater surface phyla Proteobacteria and (LD A and B), which correspond to the oligotrophic region, to the western boundary of the South Pacific Gyre (LD C), which correspond to the ultra-oligotrophic region, during OUTPACE cruise. Using a mix of epipelagic copepods that were feed and then incubated with marine microbial assemblages. We revealed that in the treatment with copepods a significant increase in ammonium, dissolved organic nitrogen (DON) and dissolved organic phosphorus (DOP) was observed and remineralization (ammonification and nitrification) was enhanced in the treatment with copepods compared to controls. A shift in the composition of active bacterial community between the treatment incubated with copepod and control was characterized by an increase in Alteromonadales and SAR11 in LD A and B, respectively, associated with the nutrients concentration at each experiment, higher in LD A and lower in LD B. In the most oligotrophic station, both groups were increased but at different period of incubation. Bacterial remineralization of the dissolved organic matter derived from copepods metabolism could be highly efficient mechanism to maintain the nutrients in the upper layer support the phytoplankton and microbial growth.

Supplementary Tables

T 0 -T 1 -T 2 -T 3 NA NA NA NA T 1 -T 2 -T 2 -T 3 NA NA NA NA T 1 -T 2 -T 0 -T 1 NA NA NA NA DOP T 0 -T 1 -T 2 -

Introduction

The Tropical South Pacific is considered one of the most oligotrophic regions in the world ocean. Its biogeochemical characteristics include nitrogen and phosphate limitation which modulate the low biological productivity [START_REF] Moutin | Phosphate availability and the ultimate control of new nitrogen input by nitrogen fixation in the tropical Pacific Ocean[END_REF]. In addition, the strong thermal stratification, enhanced by current global warming, decreases nutrient supply to the euphotic zone. The Western Tropical South Pacific (WTSP) was recently recognized as a hot spot of N 2 fixation (Bonnet et al., 2017) and the input of new nitrogen to the surface ocean through this process sustain over 50 % of primary productivity [START_REF] Carpenter | Biomass and primary productivity of the cyanobacterium Trichodesmium spp. in the tropical N Atlantic Ocean[END_REF][START_REF] Karl | The role of nitrogen fixation in biogeochemical cycling in the subtropical North Pacific Ocean[END_REF].

However, it has been reported that predator-prey interactions can provide substantial amounts of nitrogen and phosphorus, and supply an alternative substrate for phytoplankton and microbial loop growth in a variety of areas, contributed to the regenerated production (Richardot et al., 2001;Vargas et al., 2007;Arístegui et al., 2014;[START_REF] Valdés | Nitrogen excretion by copepods and its effect on ammonia-oxidizing communities from a coastal upwelling zone[END_REF].

Metazooplankton metabolism is recognized as one of the principal mechanisms controlling abundance, composition and cycling of DOM in the sea (Sipler and Bronk 2014). Zooplankton may release dissolved organic and inorganic matter through sloppy feeding (Møller, 2004[START_REF] Møller | Production of dissolved organic carbon by sloppy feeding in the copepods Acartia tonsa, Centropages typicus, and Temora longicornis[END_REF], leaching of fecal pellet [START_REF] Hasegawa | Dissolved organic nitrogen dynamics in coastal waters and the effect of copepods[END_REF]Steinberg et al., 2002) and excretion (Saba et al., 2011). Along with bacterial remineralization, it determines the amount of regenerated nitrogen and phosphorus available for phytoplankton production (Steinberg and Landry, 2017).Copepods excrete up to 53% of its body nitrogen per day, mainly in the form of ammonium, thereby recycling much of the nitrogen in the water column [START_REF] Steinberg | Nitrogen consumption and metabolism in marine zooplankton[END_REF]. Studies are reported that zooplankton could sustain between 40-50% of the nitrogen requirements of phytoplanktonic oceanic areas as North Pacific central gyre [START_REF] Eppley | A study of plankton dynamics and nutrient cycling in the central gyre of the North Pacific Ocean[END_REF], and in the oligotrophic Sargasso Sea, diel vertical migrating zooplankton can sustain between 2 to 19% of the nitrogen requirements by phytoplankton through ammonium excretion (Steinberg et al., 2002). Furthermore, zooplankton can also excrete substantial amounts of dissolved organic nitrogen (DON) amid total excreted dissolved nitrogen (Steinberg et al., 2002). Even less studied than nitrogen excretion is the phosphorus excretion by zooplankton. It has been reported, that one-third of the phosphors released by zooplankton (amphipods) was in the organic form (Johannes, 1964;Satomi and Pomeroy, 1965).

Furthermore, Johannes, (1964) reported a tight coupling between DOP production and uptake by bacteria. [START_REF] Isla | Size-fractionated mesozooplankton biomass, metabolism and grazing along a 50 N-30 S transect of the Atlantic Ocean[END_REF] estimated that mesozooplankton phosphate excretion can sustain 21.7 % of phytoplankton phosphate requirements in oceanic water of the NW Iberian.

The quantity and quality of the dissolved compounds excreted are highly dependent of the type of food ingested (Miller and Roman, 2008;Saba et al., 2009). It has been documented a direct grazing on Trichodesmium spp for the harpacticoid copepods Macrosetella gracilis, Miracia efferata and Oculosetella gracilis in oligotrophic areas [START_REF] O`neil | Ingestion of the cyanobacterium Trichodesmium spp. by pelagic harpacticoid copepods Macrosetella, Miracia and Oculosetella[END_REF][START_REF] O'neil | Ingestion of 15 N 2-labelled Trichodesmium spp. and ammonium regeneration by the harpacticoid copepod Macrosetella gracilis[END_REF]. Also, [START_REF] Hunt | Contribution and pathways of diazotroph-derived nitrogen to zooplankton during the VAHINE mesocosm experiment in the oligotrophic New Caledonia lagoon[END_REF] recently demonstrated during 15 N 2 labelled grazing experiment in New Caledonia lagoon, that copepods can graze on symbiotic and unicellular cyanobacteria (UCYN-C) and on cyanobacteria associated with diatoms based on. O'Neil et al (1996) estimated that harpacticoid copepods could consume 45% of total colony nitrogen, or 100% of the new nitrogen fixed each day.

The importance of quality and quantity of dissolved organic matter in structuring bacterioplankton community has been increasingly reported (Alonso-Saez and Gasol, 2007).

However, the processes and mechanisms through which N and P compounds released by zooplankton can determine and influence the food web and the structure of the microbial community are unclear. Recent research has demonstrated that bacterial community composition can change in response to the addition of DOM (Landa et al., 2013;Sarmento et al., 2013). Studies on zooplankton-produced DOM by bacterial communities are scarce. Some recent studies are demonstrated that the addition of excretory products released by zooplankton (krill) stimulates bacterial growth and production in the Southern Ocean (Arístegui et al., 2014). [START_REF] Valdés | Nitrogen excretion by copepods and its effect on ammonia-oxidizing communities from a coastal upwelling zone[END_REF] documented that the nitrogen excreted by copepods can be used directly by the nitrifying community in southern/central Chile.

However, studies of the impact nitrogen and phosphorus released derived from copepods metabolism on microbial community can provide valuable information about of the recycling of this nutrients in the ocean.

We studied the role of organic and inorganic dissolved compounds release by copepods along a gradient of oligotrophy in the WTSP, we also explore the role of recycling and the role of zooplankton-derived DOM in structuring the bacterioplankton community.

Materials and methods

Study area and sampling strategy

Mesozooplankton sampling

Live zooplankton samples were captured at night conditions at each LD station (day 2)

with a WP2 zooplankton net with a 120 µm mesh size, equipped with a non-filtering cod-end to obtain undamaged individuals, between 0 to 100 m depth under night conditions in the three LD stations. Live samples were immediately transferred to coolers until sorting at the laboratory.

Preparation of the microcosm and experimental setup

The experiment design consists in three steps: (1) Copepod acclimatizing, (2) feeding and ( 3) copepod recycling (Fig. 1). Seawater for incubations (30 L) was collected into clean (10% HCl rinsed) polycarbonate carboys from DCM using Niskin bottles (12 L) arranged on a CTD rosette. For acclimatizing ( 2) and copepod recycling (3) steps, seawater (22.5 L) was immediately filtered onto a 0.7 µm (GF/F; Whatman) using a peristaltic pump. The 7.5 L remaining was used in the feeding phase. Seawater for the different steps was maintained in a cold room (in situ temperature ~25º C) until the beginning of the experiment.

Figure 1: Experimental design. The design consisted of three phases done sequentially. Each bottle with copepods was acclimated for at least 6 hours (phase 1), followed by feeding (phase

2) with the Deep Chlorophyll-a Maximum (DCM) seawater for 3 hours. Finally, copepod recycling (phase 3) has treatment and control (without copepods). Three bottles were sacrificed at each sampling time Undamaged individuals were sorted from the live samples and identified using a stereomicroscope at low light. Copepods samples in the three experiments consisted in a mix of the most representative copepods (adults) in the sample (Fig. S1). In acclimatization phase

(1), 15 groups of 10 copepods were incubated in 500 mL Nalgene bottles, maintained for 4-6 h in filtered seawater previously filtered (GF/F Whatman) in darkness and at controlled temperature (in situ). In feeding step ( 2) copepods were removed from the bottles used in the previous phase, with a sieve (20 µm) and maintained in 500 mL polycarbonate bottles (Nalgene) with <150 µm filtered seawater (polycarbonate membrane) for feeding. This step lasted 4 hours and, as the acclimatization phase, was under controlled temperature. Thereafter, each copepod group (15 groups) was placed in 500 mL polycarbonate bottles (Nalgene) with the seawater previously filtered (0.2 µm; Millipore). Also, other 15 bottles without copepods were incubated as control. In total, 30 bottles were used in this step. The seawater used was filtered to avoid the presence of phytoplankton and small protist, that they can prey on bacteria, and ensure the presence of natural microbial assemblages. At the end of each step copepods were observed and checked by direct observation of ensuring swimming behavior in the bottles.

At time intervals of 0 h (T 0 ), 0.5 h (T 1 : T 0 +0.5 h), 1 h (T 2 : T 0 +1 h), 2 h (T 3 : T 0 +2h) and were filtered through cellulose ester filters (0.22 µm; Millipore) using a peristaltic pump and stored with RNAlater reagent (Ambion) at -20º C until extraction procedures. DNA extraction was carried out as described in [START_REF] Levipan | Nitrospina -like bacteria are the main drivers of nitrite oxidation in the seasonal upwelling area of the Eastern South Pacific (Central Chile ∼36°S): Nitriteoxidizing bacteria in the seasonal upwelling system off Concepcion[END_REF], quantified by spectrophotometry (NanoDrop ND-1000 Spectrophotometer) and isolated using the PowerSoil DNA Isolation Kit (MoBio Laboratories) in accordance with the manufacturer ś specifications. RNA samples were collected for initial characterization of active bacterial community at DCM at each LD station. Seawater (100 mL) was filtered using a sterilized syringe and 25 mm swinnex through 0.22 µm hydrophilic PVDF filters (Millipore) and the filters were preserved with RNAlater solution (Ambion) and stored at -80º C until RNA extraction procedures. RNA samples were extracted using Mirvana kit (AM1560; Ambion) in accordance with manufacturer´s instructions including a mechanical disruption step and homogenization using 200 µm diameter zirconium beads (Low Binding Zirconium Beads, OPS Diagnostic) and homogenized twice at ~3,000 rpm for 30 s by using Mini-Beadbeater-8 TM (Biospec Products). In order to remove DNA traces, RNA was treated with TURBO DNA-free kit (Ambion). Finally, The 16S rRNA and rDNA gene sequences were processed using Mothur software v1.35.1 [START_REF] Schloss | Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities[END_REF]. Sequencing data sets were curated by quality filtration to minimize the effects of random sequencing errors, by eliminating sequence reads <200 bp and trimming of sequences that contained more than one undetermined nucleotide (N) and sequences with a maximum homopolymer length of 8 nucleotides. After chimeric sequences were identified using the Chimera UCHIME algorithm [START_REF] Edgar | UCHIME improves sensitivity and speed of chimera detection[END_REF] and removed to retain high quality reads.

concentration
The 16S rRNA and rDNA gene sequences retrieved were taxonomically classified using the automatic software pipeline SILVAngs available from https://www.arb-silva.de/ (Quast et al. 2013), and the libraries were deposited in the European Nucleotide Archive (ENA) under study accession PRJEB21648 with the following run access numbers:

ERS1810581-ERS1810616.

The composition was analyzed at Phyla, Class and Order taxonomic level as abundant (>0.5%), semirare (0.1-0.5%) and rare (<0.1%) in total sequences retrieved from each library (Pedrós-Alió, 2012). The Chao, Shannon and Evenness indices were calculated using Past3 software (Hammer et al., 2001). Previously, since the number of sequences per sample was variable, we normalized the different libraries sizes by subsampling routine in Mothur software version 1.36 [START_REF] Schloss | Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities[END_REF], to reflect the lowest number of sequences encountered (LD A:10,831; LD B: 15,260 and LD C: 13,699).

Statistical analysis

Statistical analysis of treatments effects on chemical and biological parameters were performed using a two-way analysis of variance (ANOVA) after checking normality assumptions (Kolmogorov-Smirnov test) and homoscedasticity (Levene´s test). Pairwise multiple comparisons were performed using a Tukey test as a posteriori analysis. Statistical significance was set a p=0.05 and analysis was computed using R software.

Bacterial community structure were compared using ordination Bray-Curtis similarities and used to build dendrograms by the unweighted pair group method with arithmetic averages (UPMG). For multivariate statistical analysis, the software package PRIMER v.6 and the addon PERMANOVA+ was used. The permutational multivariate analysis of variance (PERMANOVA) with fixed factor was applied to investigate the differences of bacterial community composition for treatments and control in both experiments. Non-metric multidimensional scaling and principal co-ordinate analysis (PCO) was performed to visualize patterns of the bacterial community in response to different treatments.

Results

Biogeochemical in situ conditions

Inorganic nutrients concentrations, obtained at the DCM depth, varied substantially between the sampling sites (Table 1). Lower concentrations of NH Suppl. Table 1), and at T4 in LD C experiments (ANOVA and Tukey`s test p=0.009; Suppl.

Table 1), observed a significant increment in the treatment with copepods than controls (Fig. 2b and2c) in nitrate concentrations. DON (Fig. 2) showed significant differences between the treatment and control in LD A and C (LD A: F 1,20 : 8.99, p=0.007; LD C: F 1,20 : 29.0, p<0.0001), and this difference was associated with the increment at T1 in LD A and T0 in LD C in the treatment with copepods. At LD B we did not find significant differences between treatments and controls trough the time (ANOVA and Tukey`s test p<0.01; Suppl. Table 1).

Phosphate in LD A showed a higher concentration during the first hour of incubation, followed by a sharp decrease (from 0.17 to 0.05, approximately; Fig. 3) in the treatment with copepods as well as control (F 1,20 : 17.7, p<0.0001). However, the difference between treatment and control was not significant (F 1,20 : 4.5, p=0.06). The same result was found in LD , nitrate, nitrite and dissolved organic nitrogen variability through th incubation for treatments with copepods and control in the three LD station experiments. 0.44, p=0.51). DOP concentrations (Fig. 3) did 1,20 : 0.3, p=0.87; LD B: : 0.70, p=0.60). Moreover, in the treatment with copepods DOP erences between the treatment with copepods and control in LD B and 1,20 : 7.94, p=0.01).

, nitrate, nitrite and dissolved organic nitrogen variability through th incubation for treatments with copepods and control in the three LD station experiments. 100 0.44, p=0.51). DOP concentrations (Fig. 3) did : 0.3, p=0.87; LD B: : 0.70, p=0.60). Moreover, in the treatment with copepods DOP erences between the treatment with copepods and control in LD B and : 7.94, p=0.01).

, nitrate, nitrite and dissolved organic nitrogen variability through the incubation for treatments with copepods and control in the three LD station experiments. Figure 3 treatments with copepods and control in the three LD experiments. See l inorganic nutrients generally increased through the incubation associated with a decrease in dissolved organic ratios (DON: DOP) and vice versa (Fig. 4). In inorganic N: P ratio hour of incubation followed by an increased from 12.5 to 32.4 at the end of incubation in the treatment with copepods. Meanwhile, in the control end of incubation a decrease in N: P ratios was observed. At the same time DON: DOP ratio (Fig. 4B and4D) decreased from 25 to 16.4. In LD B experiment, lower N: P than Redfield ratio was found through the experime of incubation when a higher N: P ratio was observed (2.6 times higher than Redfield) in treatment with copepods. The DON: DOP ratios was close to treatment and control in both cases Meanwhile, in LD C the organic dissolved nutrients ratio showed a substantial accumulation During the experiments, the resulting N: P ratios (NO: PO inorganic nutrients generally increased through the incubation associated with a decrease in dissolved organic ratios (DON: DOP) and vice versa (Fig. 4). In inorganic N: P ratio hour of incubation followed by an increased from 12.5 to 32.4 at the end of incubation in the treatment with copepods. Meanwhile, in the control end of incubation a decrease in N: P ratios was observed. At the same time DON: DOP ratio (Fig. 4B and4D) decreased from 25 to 16.4. In LD B experiment, lower N: P than Redfield ratio was found through the experime of incubation when a higher N: P ratio was observed (2.6 times higher than Redfield) in treatment with copepods. The DON: DOP ratios was close to treatment and control in both cases Meanwhile, in LD C the organic dissolved nutrients ratio showed a substantial accumulation Phosphate and dissolved organic phosphorus variability through the incubation for treatments with copepods and control in the three LD experiments. See l

During the experiments, the resulting N: P ratios (NO: PO inorganic nutrients generally increased through the incubation associated with a decrease in dissolved organic ratios (DON: DOP) and vice versa (Fig. 4). In inorganic N: P ratios (Fig. 4A and4C) were lower than hour of incubation followed by an increased from 12.5 to 32.4 at the end of incubation in the treatment with copepods. Meanwhile, in the control end of incubation a decrease in N: P ratios was observed. At the same time DON: DOP ratio (Fig. 4B and4D) decreased from 25 to 16.4. In LD B experiment, lower N: P than Redfield ratio was found through the experime of incubation when a higher N: P ratio was observed (2.6 times higher than Redfield) in treatment with copepods. The DON: DOP ratios was close to treatment and control in both cases Meanwhile, in LD C the organic dissolved nutrients ratio showed a substantial accumulation Phosphate and dissolved organic phosphorus variability through the incubation for treatments with copepods and control in the three LD experiments. See l

During the experiments, the resulting N: P ratios (NO: PO inorganic nutrients generally increased through the incubation associated with a decrease in dissolved organic ratios (DON: DOP) and vice versa (Fig. 4). In (Fig. 4A and4C) were lower than hour of incubation followed by an increased from 12.5 to 32.4 at the end of incubation in the treatment with copepods. Meanwhile, in the control end of incubation a decrease in N: P ratios was observed. At the same time DON: DOP ratio (Fig. 4B and4D) decreased from 25 to 16.4. In LD B experiment, lower N: P than Redfield ratio was found through the experiment, suggesting a N of incubation when a higher N: P ratio was observed (2.6 times higher than Redfield) in treatment with copepods. The DON: DOP ratios was close to treatment and control in both cases and Meanwhile, in LD C the organic dissolved nutrients ratio showed a substantial accumulation Phosphate and dissolved organic phosphorus variability through the incubation for treatments with copepods and control in the three LD experiments. See l

During the experiments, the resulting N: P ratios (NO: PO inorganic nutrients generally increased through the incubation associated with a decrease in dissolved organic ratios (DON: DOP) and vice versa (Fig. 4). In (Fig. 4A and4C) were lower than hour of incubation followed by an increased from 12.5 to 32.4 at the end of incubation in the treatment with copepods. Meanwhile, in the control end of incubation a decrease in N: P ratios was observed. At the same time DON: DOP ratio (Fig. 4B and4D) decreased from 25 to 16.4. In LD B experiment, lower N: P than Redfield nt, suggesting a N of incubation when a higher N: P ratio was observed (2.6 times higher than Redfield) in treatment with copepods. The DON: DOP ratios was close to and then to decrease Meanwhile, in LD C the organic dissolved nutrients ratio showed a substantial accumulation Phosphate and dissolved organic phosphorus variability through the incubation for treatments with copepods and control in the three LD experiments. See l

During the experiments, the resulting N: P ratios (NO: PO inorganic nutrients generally increased through the incubation associated with a decrease in dissolved organic ratios (DON: DOP) and vice versa (Fig. 4). In (Fig. 4A and4C) were lower than Redfield hour of incubation followed by an increased from 12.5 to 32.4 at the end of incubation in the treatment with copepods. Meanwhile, in the control we observed the same pattern but at the end of incubation a decrease in N: P ratios was observed. At the same time DON: DOP ratio (Fig. 4B and4D) decreased from 25 to 16.4. In LD B experiment, lower N: P than Redfield nt, suggesting a N deficiency of incubation when a higher N: P ratio was observed (2.6 times higher than Redfield) in treatment with copepods. The DON: DOP ratios was close to then to decreased from 1 h of incubation to the end.

Meanwhile, in LD C the organic dissolved nutrients ratio showed a substantial accumulation Phosphate and dissolved organic phosphorus variability through the incubation for treatments with copepods and control in the three LD experiments. See legends in Figure 2.

During the experiments, the resulting N: P ratios (NO: PO 4 inorganic nutrients generally increased through the incubation associated with a decrease in dissolved organic ratios (DON: DOP) and vice versa (Fig. 4). In LD A experiment the Redfield (N: P= 16) during the first hour of incubation followed by an increased from 12.5 to 32.4 at the end of incubation in the we observed the same pattern but at the end of incubation a decrease in N: P ratios was observed. At the same time DON: DOP ratio (Fig. 4B and4D) decreased from 25 to 16.4. In LD B experiment, lower N: P than Redfield deficiency relative to P, except at 1 h of incubation when a higher N: P ratio was observed (2.6 times higher than Redfield) in treatment with copepods. The DON: DOP ratios was close to the Redfield ratio in both from 1 h of incubation to the end.

Meanwhile, in LD C the organic dissolved nutrients ratio showed a substantial accumulation Phosphate and dissolved organic phosphorus variability through the incubation for egends in Figure 2. ) of the dissolved inorganic nutrients generally increased through the incubation associated with a decrease in LD A experiment the (N: P= 16) during the first hour of incubation followed by an increased from 12.5 to 32.4 at the end of incubation in the we observed the same pattern but at the end of incubation a decrease in N: P ratios was observed. At the same time DON: DOP ratio (Fig. 4B and4D) decreased from 25 to 16.4. In LD B experiment, lower N: P than Redfield relative to P, except at 1 h of incubation when a higher N: P ratio was observed (2.6 times higher than Redfield) in Redfield ratio in both from 1 h of incubation to the end.

Meanwhile, in LD C the organic dissolved nutrients ratio showed a substantial accumulation of N treatment with copepods, showed a substantial deviation from Figure 4: N: P ratios of the organic and inorganic nutrients through the incubation for the three experiments.

3.3.

Changes in bacterioplankton abundance are showed in the Fig. 5. Smallest abundance was found in LD A compared to the B and C experiments. In LD A showed statisti time after 1 hour of incubation, from 100x10 observed significant differences 0.004, p=0.95). In LD B (Fig. 5B), significant differences were found between treatment copepods and control (F decrease in bacterioplankton abundance were observed in the second period of to 1 h) in the treatment with copepods of N with respect to P from the first hour in in treatment with copepods, showed a substantial deviation from Figure 4: N: P ratios of the organic and inorganic nutrients through the incubation for the three experiments.

Changes in bacterioplankton abundance during the experiment evolution

Changes in bacterioplankton abundance are showed in the Fig. 5. Smallest abundance was found in LD A compared to the B and C experiments. In LD A showed statistical differences between the time (F time after 1 hour of incubation, from 100x10 observed significant differences 004, p=0.95). In LD B (Fig. 5B), significant differences were found between treatment copepods and control (F decrease in bacterioplankton abundance were observed in the second period of to 1 h) in the treatment with copepods respect to P from the first hour in in treatment with copepods, showed a substantial deviation from observed significant differences between the treatment with copepods and control 004, p=0.95). In LD B (Fig. 5B), significant differences were found between treatment 1,16 : 7.69, p=0.014) and between decrease in bacterioplankton abundance were observed in the second period of to 1 h) in the treatment with copepods respect to P from the first hour in in treatment with copepods, showed a substantial deviation from between the treatment with copepods and control 004, p=0.95). In LD B (Fig. 5B), significant differences were found between treatment : 7.69, p=0.014) and between decrease in bacterioplankton abundance were observed in the second period of to 1 h) in the treatment with copepods as well as respect to P from the first hour in incubation at the end of incubation in the treatment with copepods, showed a substantial deviation from between the treatment with copepods and control 004, p=0.95). In LD B (Fig. 5B), significant differences were found between treatment : 7.69, p=0.014) and between decrease in bacterioplankton abundance were observed in the second period of as well as control cubation at the end of incubation in the treatment with copepods, showed a substantial deviation from the Redfield ration (> 16:1). between the treatment with copepods and control 004, p=0.95). In LD B (Fig. 5B), significant differences were found between treatment : 7.69, p=0.014) and between time (F 1,16 decrease in bacterioplankton abundance were observed in the second period of control but between 1 h and 2 h of cubation at the end of incubation in the the Redfield ration (> 16:1). Figure 4: N: P ratios of the organic and inorganic nutrients through the incubation for the three Changes in bacterioplankton abundance during the experiment evolution Changes in bacterioplankton abundance are showed in the Fig. 5. Smallest abundance was (Fig. 5A) the abundance , which increase through -1 . However, did not between the treatment with copepods and control 004, p=0.95). In LD B (Fig. 5B), significant differences were found between treatment 1,16 : 33.8, p<0.000). A decrease in bacterioplankton abundance were observed in the second period of incubation (0.5 but between 1 h and 2 h of 102 cubation at the end of incubation in the the Redfield ration (> 16:1). Figure 4: N: P ratios of the organic and inorganic nutrients through the incubation for the three Changes in bacterioplankton abundance are showed in the Fig. 5. Smallest abundance was the abundance increase through . However, did not between the treatment with copepods and control (F 1,20 : 004, p=0.95). In LD B (Fig. 5B), significant differences were found between treatments with : 33.8, p<0.000). A incubation (0.5 but between 1 h and 2 h of incubation p<0.000), and between treatments and control (F 100x10 copepods and control In LD A station, a total of 178,097 sequences were analyzed, ranging between 10,831 and 19,703 sequences for each library (Suppl. Fig. 1). In LD B station a total of 219,019 sequences were analyzed from 15,260 to 20,966, and in LD C a total of 230,805 sequ analyzed, ranging between 13,699 and 22,104 sequences for each library. In the three LD station the higher number of sequences correspond to DNA The alpha diversity derived from normalized 16S rDNA and rRNA libraries is shown in the Fig. 6. The taxonomic richness determined (OTU) and expected on the Chao1 index was higher in DNA experiment. In accordance, Shannon index diversity showed that the bacterial obtained incubation in the three LD station. Through the incubation the differences between treatments with copepods in the three experiments. However, the Shannon index showed higher values at the beginning the incubation, followed by a decrease between T2 incubation. In LD C (Fig. 5C) a strong difference was observed between times (F p<0.000), and between treatments and control (F 100x10 3 cell mL copepods and control

Active bacterial

In LD A station, a total of 178,097 sequences were analyzed, ranging between 10,831 and 19,703 sequences for each library (Suppl. Fig. 1). In LD B station a total of 219,019 sequences were analyzed from 15,260 to 20,966, and in LD C a total of 230,805 sequ analyzed, ranging between 13,699 and 22,104 sequences for each library. In the three LD station the higher number of sequences correspond to DNA The alpha diversity derived from normalized 16S rDNA and rRNA libraries is shown in the g. 6. The taxonomic richness determined (OTU) and expected on the Chao1 index was higher in DNA experiment. In accordance, Shannon index diversity showed that the bacterial ned in situ incubation in the three LD station. Through the incubation the differences between treatments with copepods in the three experiments. However, the Shannon index showed higher values at the beginning the incubation, followed by a decrease between T2-T3, while at T4 an increase was observed in the treatment with copepods (Fig. 6).

. In LD C (Fig. 5C) a strong difference was observed between times (F p<0.000), and between treatments and control (F cell mL -1 between 1 and 4 hours of incubation was observed copepods and control.

Active bacterial community

In LD A station, a total of 178,097 sequences were analyzed, ranging between 10,831 and 19,703 sequences for each library (Suppl. Fig. 1). In LD B station a total of 219,019 sequences were analyzed from 15,260 to 20,966, and in LD C a total of 230,805 sequ analyzed, ranging between 13,699 and 22,104 sequences for each library. In the three LD station the higher number of sequences correspond to DNA The alpha diversity derived from normalized 16S rDNA and rRNA libraries is shown in the g. 6. The taxonomic richness determined (OTU) and expected on the Chao1 index was higher in DNA in situ compared to the cDNA experiment. In accordance, Shannon index diversity showed that the bacterial in situ from 16S rDNA presented higher values than the observed through the incubation in the three LD station. Through the incubation the differences between treatments with copepods in the three experiments. However, the Shannon index showed higher values at the beginning the incubation, followed by a decrease T3, while at T4 an increase was observed in the treatment with copepods (Fig. 6).

. In LD C (Fig. 5C) a strong difference was observed between times (F p<0.000), and between treatments and control (F between 1 and 4 hours of incubation was observed

community composition and their response during the incubation

In LD A station, a total of 178,097 sequences were analyzed, ranging between 10,831 and 19,703 sequences for each library (Suppl. Fig. 1). In LD B station a total of 219,019 sequences were analyzed from 15,260 to 20,966, and in LD C a total of 230,805 sequ analyzed, ranging between 13,699 and 22,104 sequences for each library. In the three LD station the higher number of sequences correspond to DNA The alpha diversity derived from normalized 16S rDNA and rRNA libraries is shown in the g. 6. The taxonomic richness determined (OTU) and expected on the Chao1 index was compared to the cDNA experiment. In accordance, Shannon index diversity showed that the bacterial from 16S rDNA presented higher values than the observed through the incubation in the three LD station. Through the incubation the differences between treatments with copepods in the three experiments. However, the Shannon index showed higher values at the beginning the incubation, followed by a decrease T3, while at T4 an increase was observed in the treatment with copepods (Fig. 6).

. In LD C (Fig. 5C) a strong difference was observed between times (F p<0.000), and between treatments and control (F 1,20 between 1 and 4 hours of incubation was observed

composition and their response during the incubation

In LD A station, a total of 178,097 sequences were analyzed, ranging between 10,831 and 19,703 sequences for each library (Suppl. Fig. 1). In LD B station a total of 219,019 sequences were analyzed from 15,260 to 20,966, and in LD C a total of 230,805 sequ analyzed, ranging between 13,699 and 22,104 sequences for each library. In the three LD station the higher number of sequences correspond to DNA The alpha diversity derived from normalized 16S rDNA and rRNA libraries is shown in the g. 6. The taxonomic richness determined (OTU) and expected on the Chao1 index was compared to the cDNA experiment. In accordance, Shannon index diversity showed that the bacterial from 16S rDNA presented higher values than the observed through the incubation in the three LD station. Through the incubation the differences between treatments with copepods in the three experiments. However, the Shannon index showed higher values at the beginning the incubation, followed by a decrease T3, while at T4 an increase was observed in the treatment with copepods (Fig. 6).

. In LD C (Fig. 5C) a strong difference was observed between times (F 1,20 : 6.22, p=0.02). A higher increase around between 1 and 4 hours of incubation was observed Figure 5: Bacterioplankton abundance through the incubation for the three LD station composition and their response during the incubation In LD A station, a total of 178,097 sequences were analyzed, ranging between 10,831 and 19,703 sequences for each library (Suppl. Fig. 1). In LD B station a total of 219,019 sequences were analyzed from 15,260 to 20,966, and in LD C a total of 230,805 sequ analyzed, ranging between 13,699 and 22,104 sequences for each library. In the three LD station the higher number of sequences correspond to DNA in situ

The alpha diversity derived from normalized 16S rDNA and rRNA libraries is shown in the g. 6. The taxonomic richness determined (OTU) and expected on the Chao1 index was compared to the cDNA in situ and the samples related to the experiment. In accordance, Shannon index diversity showed that the bacterial from 16S rDNA presented higher values than the observed through the incubation in the three LD station. Through the incubation the differences between treatments with copepods in the three experiments. However, the Shannon index showed higher values at the beginning the incubation, followed by a decrease T3, while at T4 an increase was observed in the treatment with copepods (Fig. 6).

. In LD C (Fig. 5C) a strong difference was observed between times (F p=0.02). A higher increase around between 1 and 4 hours of incubation was observed in both treatment with Figure 5: Bacterioplankton abundance through the incubation for the three LD station composition and their response during the incubation In LD A station, a total of 178,097 sequences were analyzed, ranging between 10,831 and 19,703 sequences for each library (Suppl. Fig. 1). In LD B station a total of 219,019 sequences were analyzed from 15,260 to 20,966, and in LD C a total of 230,805 sequ analyzed, ranging between 13,699 and 22,104 sequences for each library. In the three LD in situ.

The alpha diversity derived from normalized 16S rDNA and rRNA libraries is shown in the g. 6. The taxonomic richness determined (OTU) and expected on the Chao1 index was and the samples related to the experiment. In accordance, Shannon index diversity showed that the bacterial from 16S rDNA presented higher values than the observed through the incubation in the three LD station. Through the incubation the alpha diversity showed slight differences between treatments with copepods in the three experiments. However, the Shannon index showed higher values at the beginning the incubation, followed by a decrease T3, while at T4 an increase was observed in the treatment with copepods (Fig. 6).

. In LD C (Fig. 5C) a strong difference was observed between times (F p=0.02). A higher increase around in both treatment with Figure 5: Bacterioplankton abundance through the incubation for the three LD station composition and their response during the incubation In LD A station, a total of 178,097 sequences were analyzed, ranging between 10,831 and 19,703 sequences for each library (Suppl. Fig. 1). In LD B station a total of 219,019 sequences were analyzed from 15,260 to 20,966, and in LD C a total of 230,805 sequences were analyzed, ranging between 13,699 and 22,104 sequences for each library. In the three LD The alpha diversity derived from normalized 16S rDNA and rRNA libraries is shown in the g. 6. The taxonomic richness determined (OTU) and expected on the Chao1 index was and the samples related to the experiment. In accordance, Shannon index diversity showed that the bacterial community from 16S rDNA presented higher values than the observed through the alpha diversity showed slight differences between treatments with copepods in the three experiments. However, the Shannon index showed higher values at the beginning the incubation, followed by a decrease T3, while at T4 an increase was observed in the treatment with copepods (Fig. 6).

103

. In LD C (Fig. 5C) a strong difference was observed between times (F 1,20 : 9.93, p=0.02). A higher increase around in both treatment with Figure 5: Bacterioplankton abundance through the incubation for the three LD station composition and their response during the incubation In LD A station, a total of 178,097 sequences were analyzed, ranging between 10,831 and 19,703 sequences for each library (Suppl. Fig. 1). In LD B station a total of 219,019 sequences ences were analyzed, ranging between 13,699 and 22,104 sequences for each library. In the three LD The alpha diversity derived from normalized 16S rDNA and rRNA libraries is shown in the g. 6. The taxonomic richness determined (OTU) and expected on the Chao1 index was and the samples related to the community from 16S rDNA presented higher values than the observed through the alpha diversity showed slight differences between treatments with copepods in the three experiments. However, the Shannon index showed higher values at the beginning the incubation, followed by a decrease T3, while at T4 an increase was observed in the treatment with copepods (Fig. 6). represented by a higher contribution of Alphaproteobacte in LD C (49.8%) than present in our libraries with a higher contribution in LD C (13.5%) than addition, a high number of abundant phyla were observed in LD A compared to the other stations.

characterized by a higher contribution of Alphaproteobacteria in the three and 7C), as total bacterial composition this class were mainly represented by SAR 11, with higher contribution in LD C (35.5%) than LD A (21.7%) and B (29.1%). The total represented by a higher contribution of Alphaproteobacte in LD C (49.8%) than present in our libraries with a higher contribution in LD C (13.5%) than addition, a high number of abundant phyla were observed in LD A compared to the other stations. On the characterized by a higher contribution of Alphaproteobacteria in the three and 7C), as total bacterial composition this class were mainly represented by SAR 11, with higher contribution in LD C (35.5%) than LD A (21.7%) and B (29.1%). The total in situ bacterial community composition (Fig. 7) at each LD station was represented by a higher contribution of Alphaproteobacte in LD C (49.8%) than in LD A (31.1%) and B (41.1%; Figure 7B). Cyanobacteria were also present in our libraries with a higher contribution in LD C (13.5%) than addition, a high number of abundant phyla were observed in LD A compared to the other On the other hand, the active characterized by a higher contribution of Alphaproteobacteria in the three and 7C), as total bacterial composition this class were mainly represented by SAR 11, with higher contribution in LD C (35.5%) than LD A (21.7%) and B (29.1%). bacterial community composition (Fig. 7) at each LD station was represented by a higher contribution of Alphaproteobacte LD A (31.1%) and B (41.1%; Figure 7B). Cyanobacteria were also present in our libraries with a higher contribution in LD C (13.5%) than addition, a high number of abundant phyla were observed in LD A compared to the other other hand, the active characterized by a higher contribution of Alphaproteobacteria in the three and 7C), as total bacterial composition this class were mainly represented by SAR 11, with higher contribution in LD C (35.5%) than LD A (21.7%) and B (29.1%). bacterial community composition (Fig. 7) at each LD station was represented by a higher contribution of Alphaproteobacte LD A (31.1%) and B (41.1%; Figure 7B). Cyanobacteria were also present in our libraries with a higher contribution in LD C (13.5%) than addition, a high number of abundant phyla were observed in LD A compared to the other other hand, the active in situ characterized by a higher contribution of Alphaproteobacteria in the three and 7C), as total bacterial composition this class were mainly represented by SAR 11, with higher contribution in LD C (35.5%) than LD A (21.7%) and B (29.1%). bacterial community composition (Fig. 7) at each LD station was represented by a higher contribution of Alphaproteobacteria, LD A (31.1%) and B (41.1%; Figure 7B). Cyanobacteria were also present in our libraries with a higher contribution in LD C (13.5%) than addition, a high number of abundant phyla were observed in LD A compared to the other in situ bacterial composition (Fig. 7C) was characterized by a higher contribution of Alphaproteobacteria in the three and 7C), as total bacterial composition this class were mainly represented by SAR 11, with higher contribution in LD C (35.5%) than LD A (21.7%) and B (29.1%). bacterial community composition (Fig. 7) at each LD station was ria, mainly by SAR 11, dominating LD A (31.1%) and B (41.1%; Figure 7B). Cyanobacteria were also present in our libraries with a higher contribution in LD C (13.5%) than addition, a high number of abundant phyla were observed in LD A compared to the other bacterial composition (Fig. 7C) was characterized by a higher contribution of Alphaproteobacteria in the three and 7C), as total bacterial composition this class were mainly represented by SAR 11, with higher contribution in LD C (35.5%) than LD A (21.7%) and B (29.1%). the total and active bacterial community, and through the incubation for the active bacterial community. bacterial community composition (Fig. 7) at each LD station was mainly by SAR 11, dominating LD A (31.1%) and B (41.1%; Figure 7B). Cyanobacteria were also present in our libraries with a higher contribution in LD C (13.5%) than in A and B. In addition, a high number of abundant phyla were observed in LD A compared to the other bacterial composition (Fig. 7C) was characterized by a higher contribution of Alphaproteobacteria in the three LD stations (Fig. 7B and 7C), as total bacterial composition this class were mainly represented by SAR 11, with higher contribution in LD C (35.5%) than LD A (21.7%) and B (29.1%). 104 the total and active in bacterial community, and through the incubation for the active bacterial community. bacterial community composition (Fig. 7) at each LD station was mainly by SAR 11, dominating LD A (31.1%) and B (41.1%; Figure 7B). Cyanobacteria were also A and B. In addition, a high number of abundant phyla were observed in LD A compared to the other bacterial composition (Fig. 7C) was LD stations (Fig. 7B and 7C), as total bacterial composition this class were mainly represented by SAR 11, with Figure 7: Relative abundance of major bacteria phyla and Proteobacteria Alphaproteobacteria order at initial incubation compared to the by the cluster (Fig. 8). During LD A (Fig. 8A) incubation, the bacteria community composition were characterized by an increase of Gammaproteobacteria between 39% to 68% of contribution between T0 and T4 in the treatments w Cyanobacteria, Chloroflexi and other taxa (semirare and rare) were found. In LD B (Fig. 8B) smaller differences in taxa contribution through the incubation was observed.

Gammaproteobacteria, increase 32% at T4. Unlike the LD A and B, LD C experiment (Fig. 8C) showed a decrease in Gammaproteobacteria contribution and an increase in Alphaproteobacteria. The cluster In general, the active bacterial community composition varied significantly during the incubation compared to the by the cluster (Fig. 8). During LD A (Fig. 8A) incubation, the bacteria community composition were characterized by an increase of Gammaproteobacteria between 39% to 68% of contribution between T0 and T4 in the treatments w Cyanobacteria, Chloroflexi and other taxa (semirare and rare) were found. In LD B (Fig. 8B) smaller differences in taxa contribution through the incubation was observed.

Gammaproteobacteria, increase 32% at T4. Unlike the LD A and B, LD C experiment (Fig. 8C) showed a decrease in Gammaproteobacteria contribution and an increase in Alphaproteobacteria. The cluster In general, the active bacterial community composition varied significantly during the incubation compared to the by the cluster (Fig. 8). During LD A (Fig. 8A) incubation, the bacteria community composition were characterized by an increase of Gammaproteobacteria between 39% to 68% of contribution between T0 and T4 in the treatments w Cyanobacteria, Chloroflexi and other taxa (semirare and rare) were found. In LD B (Fig. 8B) smaller differences in taxa contribution through the incubation was observed.

Gammaproteobacteria, increase 32% at T4. Unlike the LD A and B, LD C experiment (Fig. 8C) showed a decrease in Gammaproteobacteria contribution and an increase in Alphaproteobacteria. The cluster

Alphaproteobacteria order at initial in situ

In general, the active bacterial community composition varied significantly during the incubation compared to the in-situ community in the three expe by the cluster (Fig. 8). During LD A (Fig. 8A) incubation, the bacteria community composition were characterized by an increase of Gammaproteobacteria between 39% to 68% of contribution between T0 and T4 in the treatments w Cyanobacteria, Chloroflexi and other taxa (semirare and rare) were found. In LD B (Fig. 8B) smaller differences in taxa contribution through the incubation was observed.

Gammaproteobacteria, increased their contribution th 32% at T4. Unlike the LD A and B, LD C experiment (Fig. 8C) showed a decrease in Gammaproteobacteria contribution and an increase in Alphaproteobacteria. The cluster In general, the active bacterial community composition varied significantly during the community in the three expe by the cluster (Fig. 8). During LD A (Fig. 8A) incubation, the bacteria community composition were characterized by an increase of Gammaproteobacteria between 39% to 68% of contribution between T0 and T4 in the treatments w Cyanobacteria, Chloroflexi and other taxa (semirare and rare) were found. In LD B (Fig. 8B) smaller differences in taxa contribution through the incubation was observed. their contribution th 32% at T4. Unlike the LD A and B, LD C experiment (Fig. 8C) showed a decrease in Gammaproteobacteria contribution and an increase in Alphaproteobacteria. The cluster In general, the active bacterial community composition varied significantly during the community in the three expe by the cluster (Fig. 8). During LD A (Fig. 8A) incubation, the bacteria community composition were characterized by an increase of Gammaproteobacteria between 39% to 68% of contribution between T0 and T4 in the treatments with copepods. Also, a decrease in Cyanobacteria, Chloroflexi and other taxa (semirare and rare) were found. In LD B (Fig. 8B) smaller differences in taxa contribution through the incubation was observed. their contribution through the incubation from 28% at T0 to 32% at T4. Unlike the LD A and B, LD C experiment (Fig. 8C) showed a decrease in Gammaproteobacteria contribution and an increase in Alphaproteobacteria. The cluster In general, the active bacterial community composition varied significantly during the community in the three experiments carried out by the cluster (Fig. 8). During LD A (Fig. 8A) incubation, the bacteria community composition were characterized by an increase of Gammaproteobacteria between 39% to 68% ith copepods. Also, a decrease in Cyanobacteria, Chloroflexi and other taxa (semirare and rare) were found. In LD B (Fig. 8B) smaller differences in taxa contribution through the incubation was observed. rough the incubation from 28% at T0 to 32% at T4. Unlike the LD A and B, LD C experiment (Fig. 8C) showed a decrease in Gammaproteobacteria contribution and an increase in Alphaproteobacteria. The cluster In general, the active bacterial community composition varied significantly during the riments carried out as by the cluster (Fig. 8). During LD A (Fig. 8A) incubation, the bacteria community composition were characterized by an increase of Gammaproteobacteria between 39% to 68% ith copepods. Also, a decrease in Cyanobacteria, Chloroflexi and other taxa (semirare and rare) were found. In LD B (Fig. 8B) smaller differences in taxa contribution through the incubation was observed. rough the incubation from 28% at T0 to 32% at T4. Unlike the LD A and B, LD C experiment (Fig. 8C) showed a decrease in Gammaproteobacteria contribution and an increase in Alphaproteobacteria. The cluster 105 class with a zoom in In general, the active bacterial community composition varied significantly during the as indicated by the cluster (Fig. 8). During LD A (Fig. 8A) incubation, the bacteria community composition were characterized by an increase of Gammaproteobacteria between 39% to 68% ith copepods. Also, a decrease in Cyanobacteria, Chloroflexi and other taxa (semirare and rare) were found. In LD B (Fig. 8B) smaller differences in taxa contribution through the incubation was observed. rough the incubation from 28% at T0 to 32% at T4. Unlike the LD A and B, LD C experiment (Fig. 8C) showed a decrease in Gammaproteobacteria contribution and an increase in Alphaproteobacteria. The cluster analysis (Fig. 8) associated with the Orders contribution in the different libraries indicates that the bacterial community from the experiments was mainly associated according to the incubation time, T0, T1 and T2 versus T3 and T4, in LD A and LD B experiments. However, in LD C experiment was mainly grouped by treatments (with and without copepods; Fig. 8C). In addition, the greatest dissimilarities, based on SIMPER analysis (Fig. 9; Suppl.

Table 2), between copepod treatments and control in LD A (Fig. 9A), was mainly due to an increase in Alteromonadales and Oceanospirillales in the treatment with copepods, whereas SAR11 and Rhodobacterales increase their relative abundance in controls. At LD B experiment (Fig. 9B) smaller dissimilarities between treatment and control (9.5%) were observed and mainly due to a decrease of Alteromonadales, while the contribution of SAR11 increased in the treatment with copepods. In the LD C station (Fig. 9C), the dissimilarities were mainly due to a higher increase in Alteromonadales, however at the end of incubation decrease their contribution in the treatment with copepods and increase in the control, (pseudo-F=12.7, p=0.001). However, differences between treatment with copepods and control, and between sampling points did not showed significant differences (PERMANOVA, pseudo-F=2.1-1.3, p=0.12-0.25).

4.1.

In this study, we did not monitor possible leaching from fecal pellets defecated during the experiment. The leaching is also one of the possible pathways that zooplankton can release dissolved comp demonstrated excretion, less than 6% in the case of the release of DON (Saba et al., 2011;Steinberg et al., 2000) broken up mechanically to release substantial amount of DOM al., 1997) than the leaching In this study, we did not monitor possible leaching from fecal pellets defecated during the experiment. The leaching is also one of the possible pathways that zooplankton can release dissolved compounds, along with the sloppy feeding and excretion. However, it has been demonstrated that the concentration provided by leaching is insignificant compared to excretion, less than 6% in the case of the release of DON (Saba et al., 2011;Steinberg et al., 2000) broken up mechanically to release substantial amount of DOM al., 1997). Thus, we than the leaching metric multidimensional scaling analysis of 16S rRNA libraries based on Bray Curtis similarity for the three experiments carried out in the long durati

Discussion

Methodological considerations

In this study, we did not monitor possible leaching from fecal pellets defecated during the experiment. The leaching is also one of the possible pathways that zooplankton can release ounds, along with the sloppy feeding and excretion. However, it has been that the concentration provided by leaching is insignificant compared to excretion, less than 6% in the case of the release of DON (Saba et al., 2011;Steinberg et al., 2000) broken up mechanically to release substantial amount of DOM s, we believe than the leaching from the fecal pellets.

metric multidimensional scaling analysis of 16S rRNA libraries based on Bray Curtis similarity for the three experiments carried out in the long durati

Methodological considerations

In this study, we did not monitor possible leaching from fecal pellets defecated during the experiment. The leaching is also one of the possible pathways that zooplankton can release ounds, along with the sloppy feeding and excretion. However, it has been that the concentration provided by leaching is insignificant compared to excretion, less than 6% in the case of the release of DON (Saba et al., 2011;Steinberg et al., 2000) broken up mechanically to release substantial amount of DOM believe that our results fecal pellets.

metric multidimensional scaling analysis of 16S rRNA libraries based on Bray Curtis similarity for the three experiments carried out in the long durati

In this study, we did not monitor possible leaching from fecal pellets defecated during the experiment. The leaching is also one of the possible pathways that zooplankton can release ounds, along with the sloppy feeding and excretion. However, it has been that the concentration provided by leaching is insignificant compared to excretion, less than 6% in the case of the release of DON (Saba et al., 2011;Steinberg et al., 2000). Other studies suggest that the fecal pellet must be broken up mechanically to release substantial amount of DOM that our results can be fecal pellets.

metric multidimensional scaling analysis of 16S rRNA libraries based on Bray Curtis similarity for the three experiments carried out in the long durati

In this study, we did not monitor possible leaching from fecal pellets defecated during the experiment. The leaching is also one of the possible pathways that zooplankton can release ounds, along with the sloppy feeding and excretion. However, it has been that the concentration provided by leaching is insignificant compared to excretion, less than 6% in the case of the release of DON, and

. Other studies suggest that the fecal pellet must be broken up mechanically to release substantial amount of DOM can be mostly attributed to the excretion process metric multidimensional scaling analysis of 16S rRNA libraries based on Bray Curtis similarity for the three experiments carried out in the long duration station (LD).

In this study, we did not monitor possible leaching from fecal pellets defecated during the experiment. The leaching is also one of the possible pathways that zooplankton can release ounds, along with the sloppy feeding and excretion. However, it has been that the concentration provided by leaching is insignificant compared to and for ammonium was not detected

. Other studies suggest that the fecal pellet must be broken up mechanically to release substantial amount of DOM (Lampitt et al., 1990;Strom et attributed to the excretion process metric multidimensional scaling analysis of 16S rRNA libraries based on Bray on station (LD).

In this study, we did not monitor possible leaching from fecal pellets defecated during the experiment. The leaching is also one of the possible pathways that zooplankton can release ounds, along with the sloppy feeding and excretion. However, it has been that the concentration provided by leaching is insignificant compared to ammonium was not detected

. Other studies suggest that the fecal pellet must be In this study, we did not monitor possible leaching from fecal pellets defecated during the experiment. The leaching is also one of the possible pathways that zooplankton can release ounds, along with the sloppy feeding and excretion. However, it has been that the concentration provided by leaching is insignificant compared to ammonium was not detected

. Other studies suggest that the fecal pellet must be (Lampitt et al., 1990;Strom et attributed to the excretion process Zooplankton abundance was lower at LD C station compared to LD A and B, which was ~1,400 ind. m -3 , ~1,600ind. m -3 and ~ 500 ind. m -3 , for LD A, B and C, respectively. However, during the three experiments we used the same number of copepods for each incubation, since the lower ammonium concentration found at in situ conditions (Table 1). Our results, in special for the experiments carried out in LD C station, can be overestimated because it did not reflect the real abundance found in the environment. Copepods in the three LD station were mainly represented by Clausocalanus sp, Oncaea sp and Oithona sp. In addition, the higher abundance of Microsetella sp was associated to the MA region, possibly associated whit the Trichodesmium bloom (Carlotti et al., this issue). Copepod incubated in the three experiments consist in a mix of epipelagic copepods (Suppl. Table 5), in which Clausocalanus sp was the genera most represented in our incubation, in contrast to Oncaeasp and Oithona sp which was not used.

Biogeochemical changes in response to the dissolved compounds release by copepods

During our study, the biogeochemical changes observed at each experiment to the addition of compounds release by copepods showed significantly higher ammonium concentration in the treatment incubated with copepods compared to the controls (without copepods).

However, these concentrations not always were associated with DON concentration as we expected. In the experiment carried out in the LD A station, higher DON concentrations were observed at initial times (between 0 and 1 h) in the treatment with copepods, and was actively consumed between 1 and 2 hours of incubation. This higher DON consumption (3.62 µmol L -1 h -1 ; Supplementary Table 3) was not reflected in ammonium concentrations. In addition, at the same time the bacterioplankton abundance increase in 22,8x10 3 cell mL 1 , these results suggest that a higher demand of DON, and could be attributed to heterotrophic bacteria. The second experiment, was carried out in the LD B station, in the same region than LD A station (MA).

At initial time points a decrease in ammonium concentration through the second period of incubation (0.5 and 1 h) could be related to the increase in nitrate concentration (highly variable between the triplicates) in the treatment with copepods, but this was not observed in the control, suggesting that in the treatment with copepods nitrifying communities could be actively used the ammonium. Besides, the increase in ammonium concentration related to the end points of incubations is followed by a decrease in DON concentration in the last period of to hydrolyzing DOP compounds [START_REF] Mulholland | Phosphorus dynamics in cultures and natural populations of Trichodesmium spp[END_REF], so zooplankton metabolism could potentially supply a significant portion of the cellular phosphorus necessary for growth.

Furthermore, during outpace transect we fed copepods with water from the DCM and the depth was different in the three stations, the deepest was LD C at 140 m, followed by LD A (80 m) and LD C (34 m). In the station in MA region, Trichodesmium spp and UCYN-B dominated in the upper photic zone (0-50; Stenegren et al., this issue) and it has been reported that copepods as Macrosetella sp can feeding on both diazotrophs [START_REF] O'neil | Ingestion of 15 N 2-labelled Trichodesmium spp. and ammonium regeneration by the harpacticoid copepod Macrosetella gracilis[END_REF][START_REF] Hunt | Contribution and pathways of diazotroph-derived nitrogen to zooplankton during the VAHINE mesocosm experiment in the oligotrophic New Caledonia lagoon[END_REF].

The food source can affect the relative organic and inorganic nutrient release and the stoichiometry of the resulting DOM. Stoichiometry theory indicated that the grazer and prey elemental composition are crucial parameters influencing ratios of nutrient release [START_REF] Elser | The stoichiometry of consumer-driven nutrient recycling: theory,observations, and consequences[END_REF]. In the three LD station the inorganic N: P ratios in situ revealed a substantial decrease from Redfield ratio, suggesting a P-limitation relative to N under in situ conditions.

This ratio was very lower in LD C station which coincide with the most oligotrophic station.

In LD A station, an accumulation of inorganic nitrogen relative to phosphorus was observed.

In contrast, with LD B and C the inorganic form showed a strong limitation of P relative to N through the incubation, except in the organic form suggest a higher accumulation of N relative to P, this is especially high in the experiment carried out in most oligotrophic station (LD C).

4.3.Changes in bacterial community composition induced by dissolved compounds release by copepods

In general, direct dissolved compounds release by copepods produce a moderate increase in bacterial abundance, which was significantly different from control in the LD B and C.

However, the addition of compounds derived from copepod metabolism can greatly stimulated some bacterioplankton taxa. Through the incubations, the bacterioplankton community were characterized by changes mainly in Gammaproteobacteria and Alphaproteobacteria in all LD stations. A dominance of Gammaproteobacteria is rapidly observed in LD A experiment, mainly by Alteromonadales and Oceanospirillales, increased their contribution in the treatment with copepods compared to the control in 42 % and 7%, respectively (Fig. 9). The faster response in Alteromonadales (Gammaproteobacteria) has been documented and are frequently observed in microcosm studies, due to their high growth rate and their ability to exploit DOM rapidly when is available (Alonso-Saez and Gasol, 2007;Fuchs et al., 2000;Landa et al., 2013). The high demand for DON found in this incubation (LD A) could be related to the increase in Alteromonadales. It has been reported that zooplankton metabolism can release highly labile compound and can be rapidly assimilated by microbial communities (Arístegui et al., 2014;Vargas et al., 2007).

In contrast, in the experiment carried out in LD B station, a substantial decrease in Alteromonadales and Oceanospirillales was observed in the treatment with copepods compared to the control. The higher contribution through the time was due Alphaproteobacteria, in which SAR11 increase their relative abundance (~8%) at 1 hour of incubation and Caulobacterales (<0.1%) at the end of incubation. SAR 11 was one of the major constituent of rRNA (in situ and microcosm incubation) and rDNA in situ. During our incubations SAR11 increased their contribution only in LD B experiment which coincided with the station with lowest nutrients concentrations. Also in this experiment, the bacterioplankton abundance showed a strong decrease in both treatment and control at 1 hour of incubation. SAR11 is the most abundant plankton in the ocean, is well adapted to lower concentration of nutrients, and are specialized to oxidize many labile and low-molecularweight compounds produced by other plankton (Giovannoni, 2017).

By other hand, in the LD C experiment, an increment in Alteromonadales and Vibrionales were observed. Alteromonadales decrease their contribution in the treatment with copepods at the end of incubation, meanwhile increase their contribution in control. The reverse pattern was observed for SAR11 which increase their contribution at the end of incubation. In base with our observations copepods excreted mainly between 0 to 2 h of incubation, time in which Alteromonadales increase rapidly their contribution and when the nutrients were lowest, SAR 11 seem take advantage increase their contribution in the treatment with copepods at the end of incubation. [START_REF] Peduzzi | Zooplankton activity fueling the microbial loop: differential growth response of bacteria from oligotrophic and eutrophic waters[END_REF] observed high monomeric carbohydrate concentration and bacterial activity in experiments were copepods were included.

Furthermore, these authors observed that bacterial communities living in oligotrophic areas can be efficient to utilize the newly available substrate source, in according with our results.

Table S1: ANOVA followed by the Tukey´s Honest Significant Difference (HSD), statistical comparison was performed on nutrients concentration in treatment with copepods and control.

The mean of HSD is recorded at 95% confidence level with its associated P value for this comparison. Significant P values are bolded. about the organic and inorganic phosphorus compounds. This issue is tackled in the Chapter II. Zooplankton excretion is considered a major recycling pathway for nitrogen, phosphorus and trace elements, and along bacterial remineralization determine the amount of regenerated nitrogen and phosphorus available for phytoplankton production [START_REF] Steinberg | Nitrogen consumption and metabolism in marine zooplankton[END_REF].

Our experimental approaches were designed in order to evaluate the compounds excreted by zooplankton and the impact on microbial loop in each experiment performed (7 in total). First, we assessed the dissolved compounds excreted by copepods quantified and identified and second we evaluated the subsequent utilization of these compounds by the microbial community. Also, we evaluated how such effect is conditioned by the type of food available by zooplankton. The impact on microbial community was studied as based on changes in nutrients concentration and shifts in the bacterial community composition throughout the incubation. In Chapter I we evaluated the response of the nitrifying community and in

Chapters II and III we evaluated changes in the composition and the activity of the natural bacterioplankton community.

General considerations on the experimental approaches

Experiments were performed under controlled temperature conditions, corresponding to the in situ temperatures at each sampling site and in darkness to avoid potential interferences by light. The experiments performed in central-southern Chile were designed to evaluate separately the excretion and the subsequent utilization by the microbial community. During the excretion phase, copepods were incubated with filtered seawater (0.22 µm) to avoid the presence of the large-sized microbial community. During this phase, we followed bacterioplankton abundance and the results showed a low and constant number of bacterioplankton (Chapter I and II). However, during the experiments negative excretion rates were found. These negative values are the result from a dynamic process by which compounds excreted by copepods can be rapidly utilized at the same time, since these compounds excreted are highly labile. In other words, a negative value reflects that in our incubations there was a higher consumption of certain compounds compared to the input provided by copepods. This could also be explained by presence of heterotrophic, chemoautotrophic, and also by small photoautotrophic communities. In the sample it could be as a reflect of the carryover of the microbiome associated with the copepod exoskeletons and intestines [START_REF] Gerdts | The microbiome of North Sea copepods[END_REF][START_REF] Tang | Linkage between crustacean zooplankton and aquatic bacteria[END_REF].

During our incubations, we evaluated the excretion of copepods, however we did not monitor possible leaching of dissolved compounds from fecal pellet produced during the experiments. In this regard, Steinberg et al., (2000) found that the leaching of dissolved organic carbon (DOC) from fecal pellets was insignificant compared to changes in DOC observed in excretion experiments. The same result was observed by Saba et al., (2011) which evaluated the relative importance of sloppy feeding, excretion and leaching from fecal pellets in the release of dissolved carbon and nitrogen in Acartia tonsa, and thus demonstrated that the leaching of DON was less than 6% while ammonium was not detected. Thus, we assumed that the leaching from fecal pellets is insignificant compared to the compounds released by excretion for all experiments performed.

The number of copepods incubated was determined as based on the average abundances observed in the coastal area in central-southern Chile in previous studies [START_REF] Hidalgo | Patterns of copepod diversity in the Chilean coastal upwelling system[END_REF]. However, during the OUTPACE cruise the number of copepods was determined in order to obtain a readable signal in ammonium concentrations, since the general concentrations found during the cruise were at nanomolar levels. Zooplankton abundance was lower at LD C station compared to LD A and B, which was ~1,400 ind. m -3 , ~1,600 ind. m -3

and ~ 500 ind. m -3 , for LD A, B and C, respectively (Carlotti et al., unpublished). However, during the three experiments we used the same number of copepods for all incubations.

Therefore, our results, specially for the experiment carried out in LD C station, can be overestimated because it may not reflect the real abundance likely to be found in the field.

Nitrogen and phosphorus excretion by copepods

Zooplankton excretion has been studied for decades for different taxa and environments.

Ammonium has been recognized as the primary nitrogenous product excreted, comprising up to 75% of the total dissolved nitrogen (TDN) excreted, followed by the organic forms (urea and amino acids; [START_REF] Bidigare | Nitrogen excretion by marine zooplankton[END_REF]. However, there is increasing evidence that DON excretion is an important excretory product. The few measurements of DON excretion showed a high variability, from 7% to 80% of TDN, even exceeding inorganic nitrogen excretion [START_REF] Conover | Sources of urea in arctic seas: zooplankton metabolism[END_REF][START_REF] Miller | Nitrogen excretion by the calanoid copepod Acartia tonsa: results of mesocosm experiments[END_REF]Steinberg et al., 2000Steinberg et al., , 2002)). Our results

showed a high excretion of DON during the austral autumn with rates of up to 3.7 µmol L -1 h -1 (Chapter I). However, during the spring higher ammonium excretion rates were observed, nearly 2.6 µmol L -1 h -1 (Chapter II). The excretion of DON in winter is within the range reported in this area [START_REF] Pérez-Aragón | Nitrogen excretion by mesozooplankton in a coastal upwelling area: Seasonal trends and implications for biological production[END_REF]. However, our ammonium excretion rates were higher than those reported in this area in austral spring and for other upwelling zones [START_REF] Kiko | Ammonium excretion and oxygen respiration of tropical copepods and euphausiids exposed to oxygen minimum zone conditions[END_REF][START_REF] Pérez-Aragón | Nitrogen excretion by mesozooplankton in a coastal upwelling area: Seasonal trends and implications for biological production[END_REF]. The effect of the diet of zooplankton on the main product excreted has been studied by several authors [START_REF] Miller | Nitrogen excretion by the calanoid copepod Acartia tonsa: results of mesocosm experiments[END_REF]Miller and Roman, 2008;Saba et al., 2009). Changes on diet may be related to seasonality of the food sources (autotrophic and heterotrophic components). Indeed, our excretion rates have a clearly seasonal component in this zone. In this regard, [START_REF] Vargas | Phytoplankton food quality determines time windows for successful zooplankton reproductive pulses[END_REF] reported that in the upwelling area of central-southern Chile copepods are capable of shifting their diet over the year cycle, feeding on heterotrophic flagellates in winter and on a diet based in diatoms in spring. Furthermore, Miller and Roman (2008) demonstrated that copepods excreted more ammonium when they had fed on a diet based on diatoms, which is in accordance with our results. This impact of the diet provided to copepods in the excreted compounds was also observed in the amino acids composition (Chapter II). During late summer experiment the predominant amino acid excreted was Histidine, while in spring was Glutamic acid. However, the composition of amino acids excreted by zooplankton are poorly studied for comparative analysis.

Phosphorus excretion has been much less studied that nitrogen excretion. Our results indicate that between inorganic and organic phosphorus excretion, DOP was the main product excreted, reaching the 93% of the total release of phosphorus under late summer conditions (Chapter II).

In spite of the fact that during OUTPACE experiments (Chapter III) we could not obtain an estimate of the excretion rate, a higher accumulation of organic compounds than inorganic compounds was observed in the three experiments over different incubation periods (Chapter III, supplementary information). We estimated that copepods could release substantial amounts of DON, which was the main compound released by copepods in the three experiments, with rates of 4.0 µmol L -1 h -1 , 0.9 µmol L -1 h -1 and 0.7 µmol L -1 h -1 for LD A, B and C, respectively.

Effects of DOM derived from copepods excretion on the microbial community: implication for coastal and open ocean regions

The release of dissolved compounds excreted by copepods is considered one of the principal mechanism controlling the quantity, composition and cycling of DOM in the sea (Carlson and Hansel 2014). The impact of copepod excretion on the microbial community is highly dependent on the bacterial community structure and on the biogeochemical characteristic prevailing at each sampling site. Firstly, central-southern Chile is a highly heterogeneous system, characterized by seasonal wind-driven upwelling of subsurface nutrient-rich and poor-oxygen waters, which influence the chemical and biological components of the upper layer. By contrast, the WTSP is largely recognized as the most oligotrophic region of the world ocean, characterized by a limitation of nitrogen and phosphorus, which determine a low biological productivity. Our nutrient concentrations found at each zone agreed with those previously reported, with higher concentrations in the coastal area of central-southern Chile (Chapter I and II) and lower (nanomolar concentrations) in the WTSP (Chapter III). Furthermore, bacterial community composition differed among sampling sites. Thus, the biogeochemical role of zooplankton is highly dependent on the prevailing conditions in the field.

Central-Southern Chile

Results demonstrated that copepods excretion can provide a highly labile substrate for bacterioplankton growth, as evidenced by the rapid utilization (<2 h) through the incubation in the 4 experiments. The results showed coupled uptake-remineralization of ammonium and DON. In chapter I, the treatment derived from copepods fed with the smaller sized-fraction resulted in changes in DON accumulation-consumption that were observed over time, and this resulted in the accumulation of ammonium at the end of the incubation (Chapter I, Figure 5).

These results were also found during the austral spring/summer (Chapter II). In fact, in the late summer experiment, ammonium was consumed throughout the incubation (treatment derived from copepods fed 20-150 µm), meanwhile DON was accumulated at the end, along with nitrate and nitrite. Other studies in the same area have revealed a tight coupling of ammonium production and consumption in winter, including ammonia oxidation and potential uptake by eukaryotic organisms [START_REF] Molina | Ammonium cycling under a strong oxygen gradient associated with the Oxygen Minimum Zone off northern Chile (∼ 23 S)[END_REF][START_REF] Molina | Potential contribution of planktonic components to ammonium cycling in the coastal area off central-southern Chile during non-upwelling conditions[END_REF], which is in agreement with our results.

These results have been also demonstrated for phosphorus compounds. Johannes (1964), reported a coupled DOP production and uptake by bacteria, being capable to utilize the 80% of the DOP released by amphipods, based on radionuclide P 32 . During the incubations, bacterioplankton rapidly uses the excreted phosphorus which was rapidly recycled (Chapter II;

Figure 3). The coupled DOP production-utilization has been also reported in other studies with algae and bacteria using labeled phosphorus experiments in marine environments [START_REF] Dolan | Phosphate transfer between microbial size-fraction in Villefranche Bay (N. W. Mediterranean Sea), France in autumn[END_REF][START_REF] Lovdal | Algal-bacterial competition for phosphorus from dissolved DNA, ATP, and orthophosphate in a mesocosm experiment[END_REF].

During the incubations, nitrifying communities were transcriptionally active and could respond to the addition of ammonium derived from copepod excretion (Chapter I), both AOA and AOB were active in our incubations. However, in the inoculum derived from copepods fed with the smaller sized-fraction of food, presented lower amoA gene counts when ammonium was consumed (Chapter I; Fig. 7). In addition, lower accumulation of nitrite and no visible accumulation of nitrate (Chapter I; Fig. 5), suggesting that a distinct microbial community contributed to ammonium consumption, probably heterotrophic. Our study demonstrated and provided additional evidence that copepods make nitrogen compounds available to nitrifying communities in the surface layer [START_REF] Yool | The significance of nitrification for oceanic new production[END_REF][START_REF] Zehr | Nitrogen Cycling in the Ocean: New Perspectives on Processes and Paradigms[END_REF] in the central-southern Chile in autumn/winter, which in turn contribute with oxidized inorganic nitrogen to primary productivity in the system (Chapter I). One implication of this is overestimating regenerated production and underestimating new production in this system.

To determine the potential contribution of ammonium excretion by copepods to primary production we compared our results with the ammonium demand by phytoplankton at St. 18 in the euphotic layer [START_REF] Fernandez | Assimilation and regeneration of inorganic nitrogen in a coastal upwelling system: ammonium and nitrate utilization[END_REF]. Excreted ammonium could potentially sustain 10% and 30% of phytoplankton ammonium demand in autumn/winter and spring/summer, respectively. Notably, it could also sustain up to 216% and 129% of surface (20-30 m depth) nitrification at St. 18 during autumn/winter and spring/summer, respectively [START_REF] Fernandez | Assimilation and regeneration of inorganic nitrogen in a coastal upwelling system: ammonium and nitrate utilization[END_REF][START_REF] Molina | Potential contribution of planktonic components to ammonium cycling in the coastal area off central-southern Chile during non-upwelling conditions[END_REF]. Copepod excretion could also sustain heterotrophic prokaryote ammonium demand. However, there is scarce information [START_REF] Allen | Importance of heterotrophic bacterial assimilation of ammonium and nitrate in the Barents Sea during summer[END_REF][START_REF] Bradley | Inorganic and organic nitrogen uptake by phytoplankton and heterotrophic bacteria in the stratified Mid-Atlantic Bight[END_REF] for the study area. This prevents us from estimating the possible percentage sustained by copepod excretion.

Recent research has demonstrated that bacterial community composition can change in response to the addition of different sources of DOM (Landa et al., 2013;Sarmento et al., 2013). This has been studied mainly experimentally with amendments of DOM from phytoplankton bloom [START_REF] Mccarren | Microbial community transcriptomes reveal microbes and metabolic pathways associated with dissolved organic matter turnover in the sea[END_REF][START_REF] Sarmento | Use of phytoplankton-derived dissolved organic carbon by different types of bacterioplankton: Use of phytoplankton-derived DOC by bacterioplankton[END_REF] and a few studies are focused on the impact of DOM derived from the jellyfish degradation on bacterial composition [START_REF] Blanchet | Changes in bacterial community metabolism and composition during the degradation of dissolved organic matter from the jellyfish Aurelia aurita in a Mediterranean coastal lagoon[END_REF][START_REF] Tinta | Jellyfish modulate bacterial dynamic and community structure[END_REF][START_REF] Tinta | Microbial transformation of jellyfish organic matter affects the nitrogen cycle in the marine water column -A Black Sea case study[END_REF]. These studies have demonstrated that the DOM derived from jellyfish can promote changes in microbial diversity and their metabolism. During summer/spring, a shift in the composition of active bacterial community was observed between periods and treatments, associated with the response of common seawater surface phyla Proteobacteria and Bacteroidetes (Chapter II). Moreover, specific bacterial activity (16S rRNA: rDNA) suggested a differential response associated with the two size fraction diets provided to copepods. Betaproteobacteria and Bacteroidetes were stimulated in the treatment enriched with excretion products derived from larger and smaller sizedfraction diets, respectively, in the late summer experiment. In spring, Alphaproteobacteria were active in the treatment enriched with excretion products of copepods fed with <20 µm sized-fraction, while they were inhibited in the treatment enriched with excretion products of 20-150 µm sized-fraction. The bacterial response is probably associated with commonopportunistic sea surface microbes adapted to rapidly react to the environmental offer. In this sense, Alphaproteobacteria use preferentially monomers, such as amino acids, and so being important competitors in the ocean and largely responsible for the uptake of low molecular weight DOM [START_REF] Cottrell | Natural assemblages of marine Proteobacteria and members of the Cytophaga-Flavobacter cluster consuming low-and high-molecularweight dissolved organic matter[END_REF]. Besides, Bacteroidetes seem to be specialized in uptake of high molecular weight DOM. In our experiment carried out in late summer, we expected the presence of complex structural biopolymers (cell wall components) associated with the larger size of the microbial community used (1.6 µm). The decay of this community by either predation or autolysis could provide complex substrates in our incubation, enhancing Bacteroidetes activity, in contrast with the spring experiment, in which Alphaproteobacteria were active.

The biogeochemical impacts of copepod excretion are not limited to the upper layers, as zooplankton can move through the water column by diel vertical migration. Le Borgne and Rodier, (1997) estimated that the 60% of the total nitrogen excreted by vertical migrators is in the form of DON. In this context, zooplankton can be an important source of DON for microbial communities at greater depths stimulating ammonium regeneration by bacteria [START_REF] Miller | Effects of grazer and substrate amendments on nutrient and plankton dynamics in estuarine enclosures[END_REF]Steinberg et al., 2002). Furthermore, in central-southern Chile some copepods species are able to cross and inhabit the oxygen deficient zone [START_REF] Escribano | Zooplankton associated with the oxygen minimum zone system in the northern upwelling region of Chile during March 2000[END_REF]. Some authors have suggested that zooplankton provide a missing source of ammonium to anoxic waters, fueling anaerobic ammonia-oxidation and decoupling it from denitrification [START_REF] Bianchi | Enhancement of anammox by the excretion of diel vertical migrators[END_REF]. In the oxygen minimum zone of the eastern south Pacific, daily ammonium excretion by copepods, on occasions, could sustain up to 86% of the ammonium demand by the anammox process, reaching values close to 500 nmol L -1 d -1 in spring near St.

18 [START_REF] Galán | Temporal variability and phylogenetic characterization of planktonic anammox bacteria in the coastal upwelling ecosystem off central Chile[END_REF].

Western Tropical South Pacific

The high demand for DON found in the central-southern Chile was also observed in a contrasting area, as the oligotrophic zone in the WSTP (Chapter III). Our results showed a strong recycling of nutrients, in special in the experiment carried out in the LD A station where DON was accumulated at higher rates in the first period of incubation (~4 µmol L -1 h -1 ).

However, this was not reflected in the ammonium concentration, suggesting a high heterotrophic bacterioplankton demand for DON remineralization/ammonification. The bacterioplankton community was characterized by a change mainly in Gammaproteobacteria and Alphaproteobacteria in all LD stations. A dominance of Gammaproteobacteria was rapidly observed in LD A experiment, mainly accounted by Alteromonadales and Oceanospirillales.

In contrast, in the experiment carried out in LD B station, a higher contribution through the time was due Alphaproteobacteria, in which SAR11 increased their relative abundance (~8%)

at 1 hour of incubation and Caulobacterales (<0.1%) at the end of incubation. In the third experiment (LD C station), which corresponded to the most oligotrophic station, a higher increase in Alteromonadales and Vibrionales was observed. Bacterial degradation of dissolved compounds enrichments is carried out by phylogenetically diverse communities, some generalist and other more specialized. The faster response in Gammaproteobacteria (Alteromonadales; Chapter III) has been documented and are frequently observed in high abundance in incubation experiment, due to their high growth rate and their ability to exploit DOM rapidly when is available (Alonso-Saéz and Gasol, 2007;Fuchs et al., 2000;Landa et al., 2013). The high demand of DON in the experiment carried out in the LD A station could be associated to the high increase in the relative abundance of Alteromonadales through the incubation.

On the other hand, SAR11 is the most abundant plankton in the ocean, is well adapted to lower concentration of nutrients, and are specialized to oxidize many labile and lowmolecular-weight compounds produced by other plankton, being an excellent competitor at ultralow nutrient concentration (Giovannoni, 2017). SAR 11 was one of the major constituent of rRNA (in situ and microcosm incubation) and rDNA in situ in the WTSP and during our incubations, SAR11 increased their contribution only in LD B station which coincided with the lowest concentration of inorganic nitrogen and phosphorus (Table 1; Chapter III). This station coincides with a two-month-old Trichodesmium bloom which explain the lower inorganic in situ nutrients concentration observed. Which provide to SAR11 a competitive advantage over other microbes.

Our results suggest that copepods can greatly stimulate remineralization process, providing substantial amounts of DON and DOP. In addition, during all incubations ammonium was several times higher in the treatment with copepods than the concentrations found in situ and in controls. Steinberg et al. (2002) demonstrated that migrating zooplankton can contribute substantial amounts of DON through active transport to deeper layers and make it available for use by the microbial community. This could be especially important in stratified systems, such as the SG in the WTSP, in which the ongoing global warming has enhanced the stratification and decreased the nutrient availability. Furthermore, bacterial remineralization of the dissolved compounds derived from copepods metabolism could be a highly efficient mechanism to maintain the nutrients in the upper layer supporting the phytoplankton and microbial growth. In this sense, the response associated with the different regions under study in the WTSP suggests that copepods in the MA region could provide substantial amounts of DOP as an alternative substrate for growth or extending the life time of the diazotrophs bloom.

Perspectives for future research

There has been a considerable progress in the last decade on our understanding of the role of marine zooplankton in nitrogen cycling. However, gaps still remain about the organic nitrogen compounds excreted by zooplankton and even more on phosphorus excretion. Further studies are needed to understand the use of specific compounds which are part of DON and DOP release. Studies on both composition and lability of this material are needed to understand the different pathways by which DOM becomes utilized. The preferential uptake of some compounds by microbial community, in special for amino acids, indicates that they are selectively used. For this, a microcosm experimental approach designed to explore this issue and how it can affect the nutrient recycling in the pelagic system should be necessary.

Phosphorus cycle investigation, conducted in the coastal areas, are rare, especially when comparing with the relatively high level of knowledge on nitrogen cycling. Studies on all the processes that impact the phosphorus cycling in coastal areas are necessary too.

CONCLUSION

This thesis work concludes that the role that the pelagic trophic interactions, involving the microbial and classical food webs of the ocean, can play in the recycling of nitrogen and phosphorus in the central-southern Chile and the western tropical south Pacific can be stated as follows:

1. Planktonic copepods can provide substantial amount of DON in winter with copepods fed with the smaller sized-fraction diet and during spring/summer copepods actively excreted ammonium and DOP in copepods fed with higher sized-fraction. The prevailing excreted compounds by copepods is highly dependent to the type of food available for copepods, suggesting a seasonal influence on the excretion by copepods in central-southern Chile. 5. The impact of copepod excretion on biogeochemical cycles, mediated by microbial community, is highly dependent on the environmental characteristic at each site. In central-southern Chile copepods can be an important source of nitrogen and phosphorus for regenerated production, complementing the in-situ nitrate uptake during spring/summer season, which is mostly upwelled, and such impact could be more important during non-active upwelling. Bacterial remineralization of DOM derived from copepods metabolism could be a highly efficient mechanism to maintain the nutrients in the upper layer in a highly stratified system, as in the subtropical Gyre region in the western tropical south Pacific, contributing to phytoplankton and microbial growth.

In summary, it is concluded that the results obtained from the study provide sufficient support for the proposed hypotheses, such that they cannot be rejected.

3 Figure 2 :Figure 3 :Figure 4 :Figure 5 :

 32345 Figure1: Particulate organic consumption and metabolism by zooplankton. Prey is consumed by zooplankton. Resulting of sloppy feeding some of dissolved organic compounds in the prey become lost to the surrounding water, while the rest of the prey is ingested. Some of the ingested prey is assimilated and used for growth and reproduction. The rest of the compounds that were not absorbed can be egested as fecal pellets, and released as waste from excretion ..................................................................................................................................................... 3

  Figure6: Experimental design used in PROMO experiments. The design consisted of four phases done sequentially. Each copepod group was acclimated for at least 6 h (phase 1) inside cylinders isolated from the surrounding water with a 200 µm mesh. Cylinders are then transferred to two glass containers for feeding (phase 2) under two natural size fractions of food: 20-150 µm and <20 µm during 4 h. Copepod excretion (phase 3) had replicated treatments and controls (without copepods), in 10 L chambers equipped with a faucet near the bottom for sampling aliquots. Finally, the microbial response (phase 4) was carried out in glass flasks and each treatment had four replicates ................................................................. 19

Figure 2 .

 2 Figure 7: Experimental design. The design consisted of three phases done sequentially. Each bottle with copepods was acclimated for at least 6 hours (phase 1), followed by feeding (phase 2) with the Deep Chlorophyll-a Maximum (DCM) seawater for 3 hours. Finally, copepod recycling (phase 3) has treatment and control (without copepods). Three bottles were sacrificed at each sampling time .............................................................................................. 22

Figure 3 .Figure 4 .Figure 5 .Figure 6 .

 3456 Figure 3. Temperature (ºC), salinity (‰), and dissolved oxygen (mL L -1 ) in CopeMOD experiments: May 2012 (a) and September 2012 (b). Vertical profiles were obtained by continuous CTD-O measurement. The horizontal dashed lines indicate the collection depth of water samples (10 m depth) ..................................................................................................... 30

  Figure S1: Budget of nitrate and nitrite compounds over time in the excretion phase. The errors bars are standard deviations (n=4) ................................................................................. 42

Figure 1 :Figure 2 :Figure 3 Figure 4 Figure 5 :Figure 6 :

 123456 Figure 1: Ammonium (A-B), dissolved organic nitrogen (C-D), phosphate (E-F) dissolved organic phosphorus (G-H) and dissolved free amino acids (I-J) concentrations through incubation in the excretion phase for both treatments and controls in late summer and spring. Errors bars show standard deviation (n=4) .............................................................................. 90

Figure S3 :

 S3 Figure S3: Bacterioplankton abundance through excretion phase in both experiments, (A) late summer and (B) spring ........................................................................................................... 103

Figure 2 :Figure 3 :Figure 4 :Figure 5 :Figure 6 :Figure 9 :

 234569 Figure 1: Experimental design. The design consisted of three phases done sequentially. Each bottle with copepods was acclimated for at least 6 hours (phase 1), followed by feeding (phase 2) with the Deep Chlorophyll-a Maximum (DCM) seawater for 3 hours. Finally, copepod recycling (phase 3) has treatment and control (without copepods). Three bottles were sacrificed at each sampling time ............................................................................................ 146

  (DFAA) concentration, photosynthetic pigments, nanoplankton, microphytoplankton, bacterioplankton, cyanobacteria and picoeukaryotes abundances in late summer and spring (mean ± standard deviation). n.d. not determined ......................................................................................................................... 88 Table 2. Total excretion rates for ammonium (NH 4 + ), dissolved organic nitrogen (DON), dissolved free amino acid (DFAA), phosphate (PO 4 3-), dissolved organic phosphorus (DOP), in late summer and spring ........................................................................................................ 89 Table S1. DNA and cDNA concentration and quality (260:280 ratio) for all samples obtained from the experiments ............................................................................................................. 111 Table S2. DFAA composition in station 12 at 10 m depth during spring (December). Each value represents two replicates (mean ± standard deviation) ................................................ 112 Table

  A positive value indicates a net accumulation and negative values indicates a net nutrient consumption ............................................................ 144 Table S5: Epipelagic copepod composition used at each experiment ................ 145 xix Curriculum vitae Valentina Paz Valdés Castro Nacida el 10 de octubre, 1986, en San Fernando, Chile 2010: Bachelor degree in Marine Biology 2010: College degree in Marine Biology. Thesis dissertation: Herbivory and evacuation rates of gut content of dominant zooplankton in the upwelling system of Chile. Supervised by Dr. Rubén Escribano, Universidad of Concepción, Chile. 2017: Candidate of PhD in Oceanography and PhD in Sciences d l´Environment D´Ile de France (ED129). Thesis project: Biogeochemical role of zooplankton for nitrogen and phosphorus recycling in the ocean. Thesis in cotutelle, supervised by Dr. Rubén Escribano, Universidad of Concepción, Chile and Dr. Fabien Joux, Université Pierre et Marie Curie, France. PUBLICACIONES • Escribano, R., Hidalgo, P., Valdés, V and Frederick, L. 2014. Temperature effects on development and reproduction of copepods in the Humboldt Current: the advantage of rapid growth. Journal of Plankton Research, 36(1): 104-116 • Valdés, V., Escribano, R and Vergara, O. 2017. Scaling copepod grazing in a coastal upwelling system: the importance of community size structure for phytoplankton C flux. Latin American Journal of Aquatic Research, 45(1): 41-54 • Valdés, V., Fernandez, C., Molina, V. and Escribano, R. 2017. Excretion of nitrogen by zooplankton under different diets and its effect on ammonia oxidizing communities from a coastal upwelling one off Chile. Limnology and Oceanography. doi:10.1002/lno.10629 • Valdés, V., Fernandez, C., Molina, V., Escribano and Joux F. Dissolved compounds excreted by copepods reshape the diversity and the activity of marine bacterioplankton community. Frontiers in Marine Science, 4: 343. doi: 10.3389/fmars.2017.00343 xx • Valdés, V., Carlotti, F., Escribano, R., Donoso, K., Pagano, M., Molina V., and Fernandez C. Nitrogen and phosphorus recycling mediated by copepods in Western Tropical South Pacific. Biogeosciences (in preparation)• Carlotti, F., Pagano, M., Guilloux, L., Donoso, K., and Valdés, V. Mesozooplankton structure and functioning across the Western Tropical South Pacific Ocean. Assistant research. Proyecto FONDECYT 1110539 "Ocean stratification and its impact on the meta-zooplanktonic system: an experimental approach". Supervised by: Dr. Rubén Escribano. TEACHING EXPERIENCE • March-July 2008: Teaching assistant undergraduate course "Zoología I" for students of Marine Biology. Universidad of Concepción. Chile. • August-December 2012: Teaching assistant undergraduate course "Zoología general II" for students of Marine Biology. University of Concepción. Chile. • August-December 2010 and 2013: Teaching assistant undergraduate course "Ecología de sistemas acuáticos" for students of Marine Biotechnology and aquaculture Engineering. Universidad of Concepción. Chile. • March-July 2010-2015: Teaching assistant undergraduate course "Sistemas pelágicos" for students of Marine Biology. Universidad of Concepción. Chile. OCEANOGRAPHIC CAMPAIGNS • 2010-2011. Oceanographic campaign "Biozoo 07" FONDECYT 1080037, a bordo de L/C Kay-Kay, Universidad de Concepción. xxi • 2010-2011. Oceanographic campaingn in Mejillones Bay-Chile, FONDECYT 110539 y FONDECYT 1130051. • 2014. Oceanographic campaign PROMO I y II (Provenance and reactivity of dissolved organic matter) in the Southern central Chile, Station 18 (36°S 75°30´´W) Proyecto ECOS-CONICYT. • 2015. Oceanographic campaign OUTPACE (Oligotrophic to ultra-oligotrophic Pacific experiments) in the Western Tropical South Pacific (Nueva Caledonia-Tahiti), Institut Méditerranéen d´Océanologie, Marseille, France. INTERNSHIP • Septiembre-Octubre 2014. Observatoire Océanologique de Banyuls sur Mer, France.

•

  2016. Scholarship grant FONDECYT 1150891. xxii OUTREACH EXPERIENCE • 2014. Instructor of Oceanography science diffusion, Instituto Milenio de Oceanografía, Universidad de Concepción, Chile. • 2014-2015. Instructor in scientific camp: Explora Chile Va!, awarded byCentro Interactivo de Ciencias, Artes y Tecnologías (CICAT). Dichato, Pinto and Tome, Chile. xxiii Acknowledgement First of all, I would like to express my gratitude to my supervisor Dr. Ruben Escribano and Dr. Camila Fernandez, for being excellent scientific advisors, for their guidance and support throughout my PhD study. I would like to acknowledge to my PhD thesis committee, namely Dr. François Carlotti and Dr. Fabien Joux for great supervision and valuable input. In special to Dr. Veronica Molina for their kind support all the time, fruitful discussions and giving helpful advice to my study. In particular, I would like to thank Dr. François Carlotti and Marc Pagano for giving me the great opportunity to work in OUTPACE cruise. I´m very grateful to Dr. Fabien Joux, Dr. Camila Fernandez and Dr. Ruben Escribano to give me the opportunity to do this PhD in Chile and France, it was a great life experience. I would like to thanks to Dr. Pierre Galand and Dr. Ingrid Obernosterer four your help and training in molecular analysis during the internships in the OBB. Also, I would like to thanks to all the people who made that all the stays in France were amazing: Katty, Anthony, Daniel, Claudia and Marine. I would like to thank the Comisión Nacional de Investigación Científica y Tecnológica for funding during these years. Also, I would like to thanks to Instituto Milenio de Oceanografía, project FONDECYT Nº 1150891 and ECOS project 12026 for funding this study. Many thanks to Claudia Muñoz and Maria Jesus Galvez for technical support and constant instruction in the laboratory. Thanks to the International Associated Laboratory LIA MORFUN and your people to facilitating the materials and equipment during this study. Many thanks to ECOP laboratory colleagues and friends: Leissing, Ramiro, Daniel, Liliana and Johana Many thanks to all my friends for being an important part during these years: Claudia, Benjamin, Paz, Gabriela, Einer and Fabiola. Al final, pero no menos importante a mi familia por siempre darme su vital apoyo para cada proyecto que me he propuesto y a Pablo por su infinito amor y paciencia. xxiv Abstract "The biogeochemical role of zooplankton for nitrogen and phosphorus recycling in the ocean"

  del zooplancton en el reciclamiento de nitrógeno y fósforo en el océano"

  Concluimos que el nitrógeno y el fósforo excretados por los copépodos son utilizados directamente por las comunidades microbianas, incluidas las nitrificantes, proporcionando nitrógeno remineralizado para producción nueva y regenerada en el océano superficial en diferentes ecosistemas marinos. xxviii Résumé "Rôle biogéochimique du zooplancton sur le recyclage d ´azote et phosphore dans l´océan" Valentina Paz Valdés Castro Science d l´Environnement (ED129) Université Pierre et Marie Curie, 2017 L'interaction entre le métabolisme du méso-zooplancton et la boucle microbienne est reconnue comme l'un des principaux mécanismes de contrôle de la quantité, la composition et le recyclage de la matière organique dissoute (DOM) dans l`océan. Le zooplancton et les bactéries sont généralement considérés comme de groupes distincts malgré leur coexistence temporelle et spatiale et leur étroite interaction. Le zooplancton peut libérer de la matière organique et inorganique par une consommation incomplète des proies, lessivage des pelotes fécales et l'excrétion. Cependant, il existe un manque de connaissances sur l'impact de ces composés dissous libérés par le zooplancton sur les cycles biogéochimiques ainsi que sur la structure de la communauté microbienne. Dans ce travail, on a utilisé une approche expérimentale pour déterminer le rôle que les interactions trophiques, impliquant les producteurs primaires et secondaires peuvent agir dans le recyclage de l'azote et du phosphore dans le système pélagique. Pour ce faire, nous avons utilisé des sources alimentaires naturelles et in situ simulées se rapprochant des conditions environnementales. Premièrement, nous avons quantifié l'azote et les composés phosphorés excrétés par des copépodes alimentés de régimes naturels fractionnés en taille. Ensuite, nous avons évalué la réponse biogéochimique de la communauté microbienne et sa structure, en termes de biomasse cellulaire totale et active (y compris la communauté nitrifiante) aux produits excrétés par les copépodes dans la région d'upwelling du Chili central. Deuxièmement, nous avons exploré l'interaction simultanée entre l'excrétion de copépodes et la communauté microbienne, en termes de la réponse biogéochimique et les changements de la composition de la communauté microbienne active, le long d` un gradient oligotrophique dans le Pacifique Sud tropical occidental (WTSP). xxix Nos résultats montrent que l'azote organique dissous (NOD) est le principal produit d'excrétion, avec des taux pouvant atteindre 3.7 µmol L-1 h-1 dans des conditions automne/hiver dans le centre-sud du Chili. Pendant le printemps et l'été, les populations de copépodes excrètent activement l'azote et les composés phosphorés, principalement sous forme d'ammonium et de phosphore organique dissous (DOP), atteignant des taux d'excrétion allant jusqu'à 2.6 µmol L -1 h -1 et 0.05 µmol L -1 h -1 , respectivement. Dans les deux cas, les produits principaux excrétés étaient étroitement liés à la qualité de l'aliment principal disponible pour les copépodes (nanoplancton et microplancton, respectivement). Pendant les expériences d'automne/hiver, l'ammonium a été rapidement consommé par la communauté microbienne. La consommation d'ammonium excrété par des copépodes alimentés avec la fraction de plus grande taille a coïncide avec une augmentation des copies de transcription d'archaeal (AOA) et bactéries (AOB) ammonium oxydatrices alors qu'une communauté microbienne différente, probablement hétérotrophique, a réagi à l'entrée d'azote par excrétion par des copépodes nourris avec une fraction de taille plus basse. Au cours du printemps/été, un changement dans la composition de la communauté bactérienne active a été observé entre les périodes et les traitements, associé à la réponse du phylum bactérien opportuniste Proteobacteria et Bacteroidetes. Dans le WTSP, les copépodes ont contribué avec des niveaux élevés d'ammonium, DON et DOP à forte labilité. L'excrétion des copépodes a favorisé le processus de réminéralisation (nitrification) et modifié la diversité et la composition de la communauté bactérienne active caractérisée principalement par les Alteromonadales et SAR11 dans les deux régions étudiées (archipel Mélanésien et gyre subtropical), différant ainsi considérablement de la communauté in situ. Une activité bactérienne améliorée en présence de copépodes augmenterait encore les substrats disponibles pour la croissance microbienne et du phytoplancton dans un système hautement stratifié et fournirait une source alternative de phosphore pour les proliférations de diazotrophes dans le WTSP.

Figure 1 :

 1 Figure 1: Particulate organic consumption and metabolism by zooplankton. Prey is consumed by zooplankton. Resulting of sloppy feeding some of dissolved organic compounds in the prey become lost to the surrounding water, while the rest of the prey is ingested. Some of the ingested prey is assimilated and used for growth and reproduction. The rest of the compounds that were not absorbed can be egested as fecal pellets, and released as waste from excretion. Extracted from Steinberg and Landry 2017.

Figure 2 :

 2 Figure 2: Pathways of cycling and export of carbon, nitrogen and phosphor in the ocean.

Figure 2 :

 2 Figure 2: Pathways of cycling and export of carbon, nitrogen and phosphor in the ocean. Extracted

Figure 2 :Figure 2 :Figure 2 :Figure 2 :

 2222 Figure 2: Pathways of cycling and export of carbon, nitrogen and phosphor Extracted from Steinberg and Landry 2017 Figure 2: Pathways of cycling and export of carbon, nitrogen and phosphor from Steinberg and Landry 2017 Figure 2: Pathways of cycling and export of carbon, nitrogen and phosphor from Steinberg and Landry 2017 Figure 2: Pathways of cycling and export of carbon, nitrogen and phosphor from Steinberg and Landry 2017.

Figure 2 :

 2 Figure 2: Pathways of cycling and export of carbon, nitrogen and phosphor Figure 2: Pathways of cycling and export of carbon, nitrogen and phosphorus by zooplankton

Four

  oceanographic campaigns were conducted during 2012 and 2014. Two campaigns, named as CopeMOD (Copepods and Dissolved Organic Matter) during austral autumn/winter season (10 th May 2012 and 9 th September 2012, respectively), and two named as PROMO (Provenance and Reactivity of Dissolved Organic Matter), were conducted during the austral spring/summer season (23 rd March 2014 and St 18 on 12 th December 2014), onboard of the R/V Kay Kay II. The sampling sites were station 12 and station 18, located at ~22 km and ~33 km from shore, as indicated in Figure 3.

Figure 3 :

 3 Figure 3: The eastern south Pacific and the coastal upwelling zone off Concepcion, illustrating the sampling stations. St 12 for CopeMOD and PROMO (late summer) experiments and St. 18 for PROMO experiment (spring). The solid contour line indicates 100 m depth and the longdashed contour line indicates 50 m depth. Source: elaborated by the author.

Figure 4 :

 4 Figure 4: Transect during OUTPACE cruise with the sampling stations. The long duration stations are A, B and C. LD A and B are located in the Melanesian archipelago (MA) and LD C in the Subtropical Gyre (SG). Extracted from Moutin et al., 2018

( 3 -

 3 excretion phase) to inoculate microbial assemblages in the 1 L glass bottles. In total 10 bottles were inoculated and incubated at 12° C for 6 h. Every two hours (t0 = 0h, t1 = 2h, t2 = 4h and t3 = 6h) subsamples were collected for NH 4 sample was collected in situ during winter experiment. Quantitative measurements of AOA and AOB were determined by using complementary DNA (cDNA) templates and analyzed by quantitative polymerase chain reaction (qPCR).Methodological details of nutrients analysis (NO molecular analysis were provided in the results section in chapter I.

Figure 5 :

 5 Figure 5: Experimental design used in CopeMOD experiments. The design consisted of four phases done sequentially. Each copepod group was acclimated for at least 6 h (phase 1), followed by feeding (phase 2) under two natural size fraction of food: 20-150 µm and <20 µm during 4 h. Copepod excretion (phase 3) had treatment and control (without copepods) in two replicated chambers. Finally, the microbial response (phase 4) was carried out in glass flasks and each treatment had four replicates. FSW: filtered seawater. Source: elaborated by the author.

  II: Dissolved compounds excreted by copepods reshape the active marine bacterioplankton community composition 2.3.1 Field sampling At each sampling station, an autonomous oceanographic profiler CTD-O SeaBird 19 was deployed to obtain temperature, salinity and dissolved oxygen measurements. Seawater samples were obtained at 10 m depth with a Niskin bottle (10L) for incubations and for chemical-biological measurements. Incubation seawater was maintained in carboys, in dark conditions, until arriving to the Dichato Marine Station of University of Concepcion (~1 h).Seawater samples for NH 4

  ) and 2 h (T2: T0+2h) samples were collected in duplicate for NH 4 ) and dissolved free amino acids (DFAA) from the sampling faucet with 0.5 L glass flask (acid-washed and autoclaved; Duran Schott). Bacterioplankton abundance was monitored during all incubation time to evaluate potential influence of small size microbial communities.

  T0+4h) and 6 h (T3: T0+6h) in duplicate for NH4 + DOP, DFAA, RNA, bacterioplankton, cyanobacteria and picoeukaryotes abundance. DNA and RNA samples were taken to assess the changes in the bacterial community structure, so that the assessment included the whole bacterial assemblages and the active one. The same protocol was applied for DNA samples (duplicated), but in 4 L polycarbonate bottles (Nalgene). In that case, 3.4 L of natural microbial assemblages was inoculated with 600 mL aliquots of dissolved compounds excreted by copepods from each chamber of the previous phase. DNA samples were taken at initial (T0) and final incubation time (T3: T0+6h) and RNA samples during all time intervals.

Figure 6 :

 6 Figure 6: Experimental design used in PROMO experiments. The design consisted of fourphases done sequentially. Each copepod group was acclimated for at least 6 h (phase 1) inside cylinders isolated from the surrounding water with a 200 µm mesh. Cylinders are then transferred to two glass containers for feeding (phase 2) under two natural size fractions of food: 20-150 µm and <20 µm during 4 h. Copepod excretion (phase 3) had replicated treatments and controls (without copepods), in 10 L chambers equipped with a faucet near the bottom for sampling aliquots. Finally, the microbial response (phase 4) was carried out in glass flasks and each treatment had four replicates. Source: elaborated by the author.

  ) and 4 h (T4: T0+4 h) samples were collected for each bottle for NH 4 abundance and RNA. An additional time at 15 min (T0´: T0+0.25h) was added for NH 4 + in LD B and C station. Six bottles were sacrificed at each time, three for treatment with copepods and three without copepods (control). The three bottles sacrificed per time were triplicates in the treatment with copepods and control. At the end of the experiment copepod samples were preserved immediately with formalin buffered by sodium borate (5% final concentration) for zooplankton composition analysis and biomass measurements.

Figure 7 :

 7 Figure 7: Experimental design. The design consisted of three phases done sequentially. Each

  I: Nitrogen excretion by copepods and its effect on ammonia-oxidizing communities from a coastal upwelling zone Manuscript published in Limnology and Oceanography Valentina Paz Valdés Castro PhD in Oceanography Universidad de Concepción Abstract The role of zooplankton in microbial nitrogen turnover in marine environments is poorly understood. Here, we present results from two experiments designed to determine the excretion rate of ammonium and dissolved organic nitrogen (DON) by dominant copepods, Acartia tonsa and Paracalanus cf indicus, fed with two natural sized-fractioned diets (20-150 µm and <20 µm), and its possible effects on the transcriptional activity of ammonia monooxygenase subunit A (amoA), a functional marker for ammonia-oxidizing archaea (AOA) and bacteria (AOB), as a response to the input of ammonium and DON by copepod excretion, during autumn and winter in central/southern Chile. Our results reveal that DON

  10 ng) and a final concentration of 1X PCR buffer minus Mg, 1.25 and 4 mmol L -1 of MgCl 2 (archaeal amoA and bacterial amoA, respectively), 200 µmol L -1 of deoxynucleotide triphosphate, 0.1 µmol L -1 of each primer (forward and reverse), and 1 U of Taq DNA polymerase (Promega). The thermocycler program consisted of the following steps and was used for archaeal amoA gene amplifications: 5 min at 95 °C, then 35 cycles of 1 min at 95 °C, 1 min at 53 °C or 56 ºC (archaeal amoA and bacterial amoA, respectively), and 1.5 min at 72

  aligned and phylogenetic relationship analyses were conducted using the Bosque program available from http://bosque.udec.cl (Ramírez-Flandez and Ulloa 2008). The phylogenetic analyses were based on maximum likelihood using the Fast Tree 2.0 method[START_REF] Price | FastTree 2 -Approximately Maximum-Likelihood Trees for Large Alignments[END_REF] with bootstrap set of 1000 in Bosque. A consensus tree was generated using branches with bootstrap values higher than 70%. For AOA and AOB tree reconstruction N. multiformis and N. maritimus were used as outgroups, respectively. Interactive Tree of Life iTOL (http://itol.embl.de; Letunic and Bork 2016) was used to enhance tree visualization. All the sequences generated were deposited in the GeneBank public database under the accession numbers KU940408-KU949419 for AOA and KX449552 for AOB.Reactions for archaeal amoA qPCR assays were carried out with the primers Arch-amoAF and Arch-amoAR. The primers amoA-1F and amoA-2R were used for experiments to quantify bacterial amoA genes. The qPCR reactions were done in a StepOnePlusTM Real-Time PCR System (Applied Biosystems), using a 20 µL reaction mixture with 5-10 ng of template DNA, (quantified in a NanoDrop ND-1000 Spectophotometer and the Quant-iTTM High-Sensitivity DNA assay with the Qubit Fluorometer (Invitrogen)), 0.4 M of primers and with the Power SYBR® Green PCR Master Mix (Applied Biosystems). All reactions were run in triplicate.

Figure S2 :

 S2 Figure S2: Bacterial and archaeal amoA transcript counts in RNA ammonium enrichment experiment during non-upwelling (August 2008) and upwelling periods (January 2009) from the surface and oxycline layers. Average control values were subtracted from the treatments. <D.L correspond to low detected limit. The errors bars are standard deviations (n=3).

Figure S3 :

 S3 Figure S3: Consensus tree showing the phylogenetic relationship of archaeal amoA sequences. The sequences were obtained from RNA in situ and expression assays from non-upwelling and upwelling periods and sequences representatives from other marine water column environments. All branches are supported by >70% bootstrap values. The scale bar represents NJ branch length.

  Bacteroidetes. Moreover, specific bacterial activity (16S rRNA: rDNA) suggested a differential response associated with the two size fraction diets; in late summer, Betaproteobacteria and Bacteroidetes were stimulated in the treatment enriched with excretion products derived from 150-20µm and <20µm size fraction, respectively. In spring, 0.1%) and (C) rare conditions and throughout the experiments. 0.1%) and (C) rare conditions and throughout the experiments.

  Sampling was conducted in Western Tropical South Pacific during austral summer 2015 (18 February -3 April), on board the R/V L`Atalante. The transect beginning in west of New Caledonia (18º S-159.9º E) and ending near Tahiti (17.56º S-149.05º W). Experiments was conducted in three long duration stations: LD A, B and C, which are located in two geographic regions: LD A and B in the Malanesian archipelago (MA) and LD C in the subtropical gyre (SG). These stations chosen based on the sea surface chlorophyll-a concentration, which was maxima in LD A and LD B, and minimum in LD C station. At each sampling station seawater samples at the Deep Chlorophyll Maximum (DCM), for ammonium, nitrate, nitrite, phosphate, DNA and RNA were immediately obtained for chemical and biological initial characterization. The DCM depths correspond to 80 m, 34 m and 140 m in LD A, B and C, respectively, and seawater samples were obtained using Niskin bottles (12 L) arranged on a CTD rosette.

4 +- 5 .

 45 4 h (T 4 : T 0 +4 h) samples were collected for each bottle for NH abundance and RNA. An additional time at 15 min (T 0 ´: T 0 +0.25h) was added for NH4 + in LD B and C station. Six bottles were sacrificed at each time, three for treatment with copepods and three without copepods (control). The three bottles sacrificed per time were triplicates in the treatment with copepods and control. At the end of the experiment copepod samples were preserved immediately with formalin buffered by sodium borate (5% final concentration) for zooplankton composition analysis and biomass measurements. in duplicate in 20 mL HDPE bottles, filtered through 0.7 µm filters (GF/F; Whatman) and poisoned with HgCl 2 to a final concentration of 20 µg L -1 . Samples were stored at -20 °C and analyzed on board determined by standard colorimetric techniques (Aminot and Kérouel 2007), using a SEAL Analytical AA3 HR system (SEAL Analytica, Serblabo Technologies, Entraugues Sur La Sorgue, France). Samples for NH 4 + determination (40 ml) were taken in duplicate and analyzed on board according to Holmes et al., (1999) using a Turner Designs fluorometer. Samples for DON and DOP (30 mL) were filtered through pre-combusted (450º C, 6h) GF/F filters (Whatman). Samples were collected in Teflon bottles and analyzed immediately on board by the wet oxidation method(Pujo-Pay and Raimbault, 1994). DON and DOP concentrations were determined by sample oxidation (30 min, 120º C) and corrected for NO 3 Bacterioplankton abundance, DNA and RNA extraction. Bacterioplankton abundance was determined by flow cytometry. From each bottle 1350 µL samples were fixed in sterile cryovials with glutaraldehyde (at 0.1% final concentration). The samples were stored at -80º C until laboratory analysis. Samples were analyzed by flow cytometry (FACScan, Becton Dickinson). The abundance of non-fluorescent picoplankton was estimated from samples previously stained with SYBR green I (Molecular probes) according Marie et al., (1997). DNA samples for the initial characterization of bacterial communities were collected at DCM depth of each sampling site. Samples (9-10 L)

Figure 2 :Figure 2 :Figure 2 :

 222 Figure 2: Ammonium incubation for treatments with copepods and control in the three LD station experiments.

Figure 3 :

 3 Figure 3: Phosphate and dissolved organic phosphorus variability through the incubation for treatments with copepods and control in the three LD experiments. See l

  increased through the incubation associated with a decrease in LD A experiment the (N: P= 16) during the first hour of incubation followed by an increased from 12.5 to 32.4 at the end of incubation in the we observed the same pattern but at the end of incubation a decrease in N: P ratios was observed. At the same time DON: DOP ratio (Fig.4B and 4D) decreased from 25 to 16.4. In LD B experiment, lower N: P than Redfield relative to P, except at 1 h of incubation when a higher N: P ratio was observed (2.6 times higher than Redfield) in Redfield ratio in both from 1 h of incubation to the end.Meanwhile, in LD C the organic dissolved nutrients ratio showed a substantial accumulation 101 Phosphate and dissolved organic phosphorus variability through the incubation for egends in Figure2.

Figure 4 :Figure 4 :

 44 Figure 4: N: P ratios of the organic and inorganic nutrients through the incubation for the three

Figure 4 :

 4 Figure 4: N: P ratios of the organic and inorganic nutrients through the incubation for the three

Figure 4 :

 4 Figure 4: N: P ratios of the organic and inorganic nutrients through the incubation for the three

Figure 4 :

 4 Figure 4: N: P ratios of the organic and inorganic nutrients through the incubation for the three

Figure 5 :

 5 Figure 5: Bacterioplankton abundance through the incubation for the three LD station experiments:

Figure 5 :

 5 Figure 5: Bacterioplankton abundance through the incubation for the three LD station experiments: (A) LD A

Figure 5 :

 5 Figure 5: Bacterioplankton abundance through the incubation for the three LD station (A) LD A, (B)

Figure 5 :

 5 Figure 5: Bacterioplankton abundance through the incubation for the three LD station (B) LD B and (C) LD C.

Figure 5 :

 5 Figure 5: Bacterioplankton abundance through the incubation for the three LD station (C) LD C.

Figure 6 :

 6 Figure 6: Richness, Chao1, Diversity (Shannon H´) and Evenness from situ bacterial community, and through the incubation for the active bacterial community.

Figure 6 :

 6 Figure 6: Richness, Chao1, Diversity (Shannon H´) and Evenness from bacterial community, and through the incubation for the active bacterial community.

Figure 6 :

 6 Figure 6: Richness, Chao1, Diversity (Shannon H´) and Evenness from bacterial community, and through the incubation for the active bacterial community.

Figure 6 :

 6 Figure 6: Richness, Chao1, Diversity (Shannon H´) and Evenness from bacterial community, and through the incubation for the active bacterial community.

Figure 6 :

 6 Figure 6: Richness, Chao1, Diversity (Shannon H´) and Evenness from bacterial community, and through the incubation for the active bacterial community.

Figure 6 :

 6 Figure 6: Richness, Chao1, Diversity (Shannon H´) and Evenness from bacterial community, and through the incubation for the active bacterial community.

Figure 6 :

 6 Figure 6: Richness, Chao1, Diversity (Shannon H´) and Evenness from the total and active bacterial community, and through the incubation for the active bacterial community.

Figure 7 :

 7 Figure 7: Relative abundance of major bacteria phyla and Proteobacteria Alphaproteobacteria order at initial

Figure 7 :

 7 Figure 7: Relative abundance of major bacteria phyla and Proteobacteria Alphaproteobacteria order at initial

Figure 7 :

 7 Figure 7: Relative abundance of major bacteria phyla and Proteobacteria

Figure 7 :

 7 Figure 7: Relative abundance of major bacteria phyla and Proteobacteria in situ conditions for the three

Figure 7 :

 7 Figure 7: Relative abundance of major bacteria phyla and Proteobacteria conditions for the three

Figure 7 :

 7 Figure 7: Relative abundance of major bacteria phyla and Proteobacteria conditions for the three-sampling site.

Figure 7 :

 7 Figure 7: Relative abundance of major bacteria phyla and Proteobacteria class with a zoom in sampling site.

Figure 8 :

 8 Figure 8: Similarity dendrograms based on the 16S rRNA and relative contribution of the abundant bacteria phyla and Proteobacteria subclasses for the initial in situ community, treatments with copepods and controls (left panel) through the incubation for the three experiments. Clustering in one the basis of a distance matrix computed using the Bray-Curtis index of similarity. The dendrogram was inferred with the unweighted pair-group average

  meanwhile the contribution of SAR11 increased in the treatment with copepods at the end of incubation. The specific order contribution for the three experiments are shown in Figures S2, S3 and S4.

Figure 9 :Figure 9 :

 99 Figure 9: Contribution of active Orders through the incubation based on the SIMPER results.List of the OTUs explaining between treatment with copepods and control for each LD experiment.

Figure 10 :

 10 Figure 10: Principal component ordination (PCO) based on Bray taxonomic level, of treatment with copepods and control (A) LD A and (B) LD B experiments. Vectors indicate the best environmental variables (normalized transformed) correlated with ordinations and vector lengths correspond with the correlation v

Figure 10 :

 10 Figure 10: Principal component ordination (PCO) based on Bray taxonomic level, of treatment with copepods and control (A) LD A and (B) LD B experiments. Vectors indicate the best environmental variables (normalized transformed) correlated with ordinations and vector lengths correspond with the correlation v

Figure 10 :

 10 Figure 10: Principal component ordination (PCO) based on Bray taxonomic level, of treatment with copepods and control (A) LD A and (B) LD B experiments. Vectors indicate the best environmental variables (normalized transformed) correlated with ordinations and vector lengths correspond with the correlation v

Figure 10 :

 10 Figure 10: Principal component ordination (PCO) based on Bray taxonomic level, of treatment with copepods and control (A) LD A and (B) LD B experiments. Vectors indicate the best environmental variables (normalized transformed) correlated with ordinations and vector lengths correspond with the correlation v

Figure 10 :

 10 Figure 10: Principal component ordination (PCO) based on Bray taxonomic level, of treatment with copepods and control (A) LD A and (B) LD B experiments. Vectors indicate the best environmental variables (normalized transformed) correlated with ordinations and vector lengths correspond with the correlation v

Figure 10 :

 10 Figure 10: Principal component ordination (PCO) based on Bray taxonomic level, of treatment with copepods and control (A) LD A and (B) LD B experiments. Vectors indicate the best environmental variables (normalized transformed) correlated with ordinations and vector lengths correspond with the correlation v

Figure 10 :

 10 Figure 10: Principal component ordination (PCO) based on Bray-Curtis similarity taxonomic level, of treatment with copepods and control (A) LD A and (B) LD B experiments. Vectors indicate the best environmental variables (normalized transformed) correlated with ordinations and vector lengths correspond with the correlation values.

Figure 11 :

 11 Figure 11: Principal component ordination (PCO) based on Bray-Curtis similarity at order taxonomic level, of treatment with copepods and control in LD C experiment. Vectors indicate the best environmental variables (normalized transformed) correlated with ordinations and

Figure 12 :

 12 Figure 12: Non-metric multidimensional scaling analysis of 16S rRNA libraries based on Bray Curtis similarity for the three experiments carried out in the long durati

(

  Lampitt et al., 1990; Strom et attributed to the excretion process 111 metric multidimensional scaling analysis of 16S rRNA libraries based on Bray on station (LD).

  Figure S2FigureS2

2 .

 2 The excreted nitrogen compounds can be directly used by microbial communities in a short response time and revealing a tight coupling of ammonium production and consumption associated with a high demand for DON by heterotrophic bacteria in central-southern Chile and in western tropical south Pacific.3. Archaea and bacteria ammonia-oxidizing (AOA-AOB) can respond to the ammonium excreted by the dominant copepods in central-southern Chile, in which AOA may compete more successfully under limited ammonium concentration than AOB.4. The addition of copepod excreted products can stimulate bacterioplankton community, resulting in an increase in their abundance, and can reshape the diversity and the bacterial community composition. Proteobacteria and Bacteroidetes were the main phyla that respond to the release of compounds by copepods, and they are associated with common-opportunistic sea surface microbes in central-southern Chile and in the western tropical south Pacific.

Table 1 .

 1 Initial contents of photosynthetic pigments, POC, PON concentration, and phytoplankton abundances in duplicate during autumn (May 2012) and winter (September 2012), respectively. n.d., not determined ................................................................................. 30

Table 2 .

 2 Changes in concentration of nitrogen compounds, for controls and treatments, during microbial response phase in CopeMOD experiments. Average control values were subtracted from the treatments, and values were presented with the standard deviation (n=4) ................ 33

Table 3 .

 3 Bacterial and archaeal amoA gene counts (ecotype AOA-A and N. maritimus) in winter experiment. Average values were presented with the standard deviation (n=3). < D.L, low detection limit ................................................................................................................... 35

DISSOLVED COMPOUNDS EXCRETED BY COPEPODS RESHAPE THE ACTIVE MARINE BACTERIOPLANKTON COMMUNITY COMPOSITIONTable 1 .

 1 Initial nutrient concentration, dissolved free amino acid

Table S4 .

 S4 . Significant P values are bolded .............................. 112 Similarity percentage analysis (SIMPER) showing the contribution and taxonomic affiliation of OTUs explaining 70% of the dissimilarity between treatments through time for present and active bacterial community in both sampling date .............................................

113 xviii NITROGEN AND PHOSPHORUS RECYCLING MEDIATED BY COPEPODS IN WESTERN TROPICAL SOUTH PACIFICTable 1 .

 1 Ammonium, nitrate, nitrite, phosphate in situ concentration and N: P ratio at each sampling site .......................................................................................................................... 136

Table S1 :

 S1 ANOVA followed by the Tukey´s Honest Significant Difference (HSD), statistical comparison was performed on nutrients concentration in treatment with copepods and control. The mean of HSD is recorded at 95% confidence level with its associated P value for this comparison. Significant P values are bolded ......................................................................... 139

Table S2 :

 S2 Similarity percentage analysis (SIMPER) showing the contribution and taxonomic affiliation of OTUs explaining 60% of the dissimilarity between treatments through time for present and active bacterial community at each LD station experiments .............................. 140

	Table

Table 2 .

 2 DFAA composition in station 12 at 10 m depth during spring (December). Each values represent two replicates (mean ± standard deviation).

	Supplementary Table 1. DNA and cDNA concentration and quality (260:280 ratio) for all
	samples obtained from the experiments.
	Experiment DNA/cDNA Samples	Concentration (ng µL -1 ) 260:280 ratio
	Late summer DNA	T0_20-150 µm 20.80 ± 1.42	1.70 ± 0.17
			T0_<20 µm	29.58 ± 0.69	1.80 ± 0.07
			T0_Control	27.95 ± 1.92	1.59 ± 0.20
			T3_20-150 µm 29.35 ± 0.60	1.93 ± 0.04
			T3_<20 µm	24.52 ± 0.35	1.81 ± 0.04
			T3_Control	21.95 ± 0.65	1.69 ± 0.10
			In situ	7.19 ± 0.01	2.13 ± 0.14
		cDNA	T0_20-150 µm 10.27 ± 0.19	2.33 ± 0.16
			T0_<20 µm	5.27 ± 0.56	1.51 ± 0.16
			T0_Control	9.16 ± 0.09	1.71 ± 0.14
			T1_20-150 µm 8.39 ± 0.23	1.71 ± 0.12
			T1_<20 µm	6.94 ± 0.54	1.65 ± 0.08
			T1_Control	4.24 ± 0.14	1.18 ± 0.05
			T2_20-150 µm 7.99 ± 0.82	1.58 ± 0.10
			T2_<20 µm	6.72 ± 0.35	1.61 ± 0.06
			T2_Control	9.28 ± 1.69	1.99 ± 0.48
			T3_20-150 µm 8.07 ± 0.83	4.24 ± 2.87
			T3_<20 µm	9.51 ± 0.56	2.23 ± 0.09
			T3_Control	6.53 ± 0.25	2.21 ± 0.43
	Spring	DNA	T0_20-150 µm 10.4	-
			T0_<20 µm	4.79	-
			T0_Control	5.56	-
			T3_20-150 µm 6.4	-
			T3_<20 µm	4.14	-
			T3_Control	15.2	-
			In situ	17.7	-
		cDNA	T0_20-150 µm 6.36	-
			T0_<20 µm	7.8	-
			T0_Control	5.84	-
			T1_20-150 µm 6.96	-
			T1_<20 µm	3.22	-
			T1_Control	7.8	-
			T2_20-150 µm 5.32	-
			T2_<20 µm	7.56	-
			T2_Control	11.5	-
			T3_20-150 µm 6.44	-
			T3_<20 µm	5.44	-
			T3_Control	12.3	-
			In situ	25.2	-

Table 3 .

 3 ANOVA followed by the Tukey´s Honest Significant Difference

(HSD), statistical comparison was performed on nutrients turnover rates in treatments and between times throughout the experiments. The mean of HSD is recorded at 95% confidence level with its associated P value for this comparison. Significant P values are bolded.

Table 4 .

 4 Similarity percentage analysis (SIMPER) showing the contribution and taxonomic affiliation of OTUs explaining 70% of the dissimilarity between treatments through time for present and active bacterial community in both sampling date.

	T 3	HSD=-0.32	HSD=0.03	NA	NA
		P=0.00	P=0.99
	T 1 -T 2 -T 2 -T 3	HSD=2.7E-17	HSD=0.01	NA	NA
		P=1	P=0.99
	T 1 -T 2 -T 0 -T 1	HSD=0.32	HSD=0.02	NA	NA
		P=0.00	P=0.99
	Supplementary Dissimilarity: Present bacterial community, late summer experiment.
	Factors	Av. Diss	Phyla/Class	Diss/SD	Contribution (%)
	20-150 µm &<20 µm	9.50	Alteromonadales	2.33	11.58
			Flavobacteriales	1.26	11.09
			Cellvibrionales	1.51	5.81
			Oceanospirillales	1.00	3.86
			Rhodobacterales	1.81	3.57
			Enterobacteriales	1.39	3.44
			Salinisphaerales	1.27	2.96
			Derived from ARKICE-90	2.23	2.88
			Cytophagales	1.88	2.71
			Burkholderiales	1.29	2.49
			KI89A clade	1.20	2.27
			Caulobacterales	1.41	2.25
			Hydrogenophilales	0.86	2.20
			SAR11 clade	2.28	2.08
			Desulfobacterales	2.04	2.02
			Campylobacterales	1.15	1.87
			Derived from Bacteroidetes	0.87	1.76
			VC2.1 Bac22
			Sphingomonadales	1.33	1.70
			Derived from Chloroplast	1.28	1.60

Figure S4: Consensus tree showing the phylogenetic relationship of bacterial amoA sequences. The sequences were obtained from RNA assays from the oxycline experiment carried out during upwelling period and sequences representatives from other marine water column, soil and active sludge environments. The scale bar represents NJ branch length.

Supplemental Information

Nitrogen excretion by copepods and its effect on ammonia-oxidizing communities from a coastal upwelling zone Valentina Valdés, Camila Fernandez, Veronica Molina and Alphaproteobacteria were active in the treatment enriched with excretion products of copepods fed with <20µm size fraction while they were inhibited in the treatment enriched with excretion products of 20-150µm size fraction. Our findings indicate that different diets provided to copepods can have a significant impact on the quantity and quality of their excretion compounds, which can generate shifts in the active bacterial composition. The bacterial response is probably associated with common-opportunistic sea surface microbes adapted to rapidly react to environmental offers. Supplementary Figure 2. The design consisted of four phases done sequentially. Each copepod group was acclimated for at least 6 h (phase 1), followed by feeding (phase 2) under two natural size fractions of food: 20-150 µm and <20 µm during 4 h. Copepod excretion (phase 3) had treatment and control, without copepods, in two replicated chambers. Finally, the microbial response (phase 4) was carried out in glass flasks and each treatment had four replicates. For this, using a mix of epipelagic copepods were feed and then incubated with marine microbial assemblages. Results revealed that in the treatment with copepods a significant increase in ammonium, DON and DOP was observed, stimulated remineralization (ammonification and nitrification) in the treatment with copepods than control. A shift in the composition of active bacterial community between the treatment incubated with copepod and control and increase in Alteromonadales and SAR11 in LD A and B, respectively, associated with the nutrients concentration at each experiment, higher in LD A and lower in LD B and SAR11. In the most oligotrophic station, both groups were increased but at different period of incubation. Bacterial remineralization of the dissolved organic matter derived from copepods metabolism could be highly efficient mechanism to maintain the nutrients in the upper layer support the phytoplankton and microbial growth.

Supplementary Material

Dissolved Compounds

incubation (1 and 2 h) at a rate of 0.12 µmol L -1 h -1 (Supplementary Table 3), as LD A this demand could be probably associated to heterotrophic bacterioplankton, however, the demand in considerably lower in this experiment than LD A.

The highest differences in ammonium concentrations between copepods treatments and control was observed at the LD C experiment, up to 0.3 µmol L -1 , several times higher than in situ concentration. This increase through the incubation could be associated the consumption of DON observed through the first 2 h of incubation in the treatment with copepods, suggest a higher remineralization/ammonification of DON. In addition, at the end of incubation nitrate concentration increase in the treatment with copepods to the control. Our results, suggest that the release of DON from copepods can stimulate ammonium regeneration by bacteria and also the increment in nitrate concentration suggest strong nitrification occurring, providing an additional substrate for microbial and phytoplankton growth in LD C station, which correspond to the ultra-oligotrophic station in the SG.

The highest differences in DON concentration between the treatment with copepods and the control were found at initial time points, whit a higher DON concentration in the treatment with copepods. Zooplankton can excreted DON in highly concentrated spurts lasting up to an hour (Gardner and Paffenhöfer, 1982;Steinberg et al., 2002).

On the other hand, in the treatment with copepods DOP showed significantly higher concentrations respect to the control in LD B and C experiment at the end and at the initial point of incubation, respectively. In addition, in LD A experiment phosphate was found in higher concentration compared to the control among 0 and 1 h of incubation. Our results suggest that copepods could potentially contribute with substantial amount of DOP and Bacterial remineralization of the dissolved organic matter derived from copepods metabolism could be highly efficient mechanism to maintain the nutrients in the upper layer support the phytoplankton and microbial growth. In this sense, the response associated to the different regions in study suggest that copepods in MA region could provide substantial amounts of DOP as an alternative substrate for growth or extend the life time of the diazotrophs bloom. The enhanced remineralization by copepods could be more important in the SG region, which is the most oligotrophic area.

Acknowledgement

This is a contribution of the OUTPACE (Oligotrophy from Ultra-oligoTrophy PACific S3: Consumption and production rates obtained in the treatment incubated with copepods. The turnover rates were obtained for each compound studied between incubation times and were estimated using an end-point approach. A positive value indicates a net accumulation and negative values indicates a net nutrient consumption.

Period of incubation NH