Production and mortality of phytoplankton and sea-ice microalgae in the Southern Ocean By Dr. philos. Knut Yngve Børsheim and dr. scient. Geir Johnsen^* The Norwegian University of Science and Technology, Laboratory of Biotechnology, N-7491 Trondheim, Norway. ^*Trondhjem Biological Station.

Contents Project summary Introduction Description of woork Figure 1. Major routes of flow of organic material from primary producers to secondary producers in a Southern Ocean pelagic food web Literature cited Agusti, S., Satta, M.P., Mura, M.P., and Benavent, E (1998) Dissolved esterase activity as a tracer of phytoplankton lysis: Evidence of high phytoplankton lysis rates in the northwestern Mediterranean. Limnol. Oceanogr. 43:1836-1849. Børsheim, K.Y. (1990) Bacterial biomass and production rate in the Gulf Stream front regions. Deep-Sea Research 37:1297-1309. Børsheim, K.Y., Gerdts, G., and Schütt, C. (submitted). Proteolytic activity in experimental temperature gradients measured in four size fractions along a trophic gradient in the North Sea. Mar. Ecol. Progr. Ser. Børsheim, K.Y (submitted) Bacterial production rates and concentrations of organic carbon at the end of the productive season in the Greenland and the Norwegian Sea. Aquatic Microb. Ecol. Børsheim, K. Y. and Myklestad, S. M. (1997) Dynamics of DOC in the Norwegian Sea inferred from monthly profiles collected during three years at 66^oN,2^oE. Deep-Sea Research 37:1297-1309 . Christian, J.R. and Karl, D. M. (1995) Bacterial ectoenzymes in marine waters: Activity ratios and temperature responses in three oceanographic provinces. Limnol. Oceanogr. 1042-1049. Johnsen, G., Prézelin, B. B. and Jovine, R. V. M. (1997) Fluorescence excitation spectra and light utilization in two red tide dinoflagellates. Limnol. Oceanogr. 42:1166-1177. Kirchman, D.L. (1999) Phytoplankton death in the sea. Nature 398:293-294. Kroon, B., Prézelin, B. B. and Schofield, O. (1993) Chromatic regulation of quantum yield for photosystem II charge separation, oxygen evolution and carbon fixation in Heterocapsa pygmaea (Pyrrophyta). J. Phycol. 29:453-462. Wheeler, P.A., Gosselin, M., Sherr, E., Thibault, D., Kirchman, D.L., Benner, R. and Whitledge, T.E. (1996) Active cycling of organic carbon in the central Arctic Ocean. Nature 380:697-699.

Project summary During the NARE 2000/2001 expedition, the project will measure primary production, mortality of microalgae and microbial loop variables in samples from open ocean water profiles and samples of algal communities collected from sea ice. Microalgal production and mortality will be investigated by the introduction of two new methods. A Pulse Amplitude Modulated fluorometer will be used to measure primary production and pigment variables. Measurements of the concentration and turnover of enzymes released from microalgae at death will be used to estimate mortality. In addition, DOC and bacterial production, which are identified as important microbial loop variables, will be measured using already established methodology. Introduction The primary production in the open ocean in the Antarctic regions is low compared to other ocean areas, whereas sea ice to some extent represent sites of enhanced productivity. Still, due to the vast extension of these regions, the Antarctic water ecosystem as a whole supports large populations of pelagic animals such as krill and marine mammals, and seabirds. These secondary producers rely to a large extent on a healthy microbial food web, where particulate production enter the higher levels by routes indicated in Figure 1. Microalgal primary production is a key component, but only part of this production is directly transferred into the food chain by grazing. Part is transferred to DOC, either by excretion from healthy cells, or from lysis of cells by other mortality factors. Also other parts of the biota adds to the DOC pool through losses of organic material , and the reutilization of DOC by heterotrophic bacteria is an important component in the production scenario. Heterotrophic bacteria are the principal consumers of DOC, and bacterial production represents the pathway whereby the material in the DOC-pool can be transferred into particulate form which can be available to the food web by micro-zooplankton grazing. Present evidence demonstrates that the flow of material through this microbial loop is especially important in oligotrophic waters such as the Southern Ocean, and we will therefore include bacterial production in our selection of measurements. The presence of a high contribution of the microbial loop input to the grazing food chain, suggest that microalgal mortality induced by factors other than grazing may have a proportionally high impact in the oligotrophic waters of the Southern Ocean. For this reason alone, it is very interesting to introduce methods which can be used to quantify death rates. In the present project we want to introduce new methods/approaches for on site measurement of mortality and primary production of microalgae (phytoplankton and sea-ice microalgae). Knowledge of mortality and production rates is crucial for the understanding of population dynamics. A new method for the detection of esterases released by algal lysis, will be used on board. Likewise, a new method/approach for the estimation of primary production will carried out using a Pulse Amplitude Modulated fluorometer (PAM) instead of the traditional ^14C incubation technique. The PAM technique measures the electron transfer rate to photosystem II, the site of oxygen production of the algae. The amount of electrons generated can be transformed to oxygen units based additional irradiance measurements, the chl a-specific absorption coefficients and the fraction of light received by photosystem II [scaled fluorescence excitation spectra, unit: m^2 mg (chl a)^-1 , cf. Johnsen et al. 1997]. The pigment composition will also be measured to discriminate between different algal groups, pigment functionality (photosynthetic vs. photoprotective) and the corresponding degradation status which then can be related to mortality. As a pilot experiment, the PAM technique will also be used to measure the gut (algal) content of crustacean zooplankton by detecting chlorophyll a fluorescence using fiber optics. To the best of our knowledge, this has never been tried before. Description of work We propose to study primary and secondary production rates, and the fate of microalgal production in the food web by combining a set of new methods with classical methods. New methods & approaches: PAM A submersible Pulse Amplitude Modulated fluorometer (DIVING-PAM) containing irradiance (Photosynthetic Active Radiance, 400-700nm, ×mmol quanta m^-2 s^-1), temperature (ºC), pressure (atm), and fluorescence yield of photosystem II for estimation of photosynthetic electron transfer rate (ETR) will be used in situ (under ice) and on deck. This submersible «mini-laboratory» can be controlled by a two-way communication cable connected to a computer allowing time-series (minutes, hours, days) of changes in photosynthesis, irradiance, temperature, and pressure (depth). Samples will be collected by means of Niskin bottles for open water phytoplankton, and preferably by scuba diving for ice algal communities. For later measurements in the laboratory of chlorophyll a specific absorption coefficients, light transferred to photosystem II (scaled fluorescence excitation spectra) and pigment composition (high pressure liquid chromatography), preparations will be stored in liquid nitrogen. The SCUBA diver in this project hold a class S certificate (certified scientific diver issued from the Directorate of labour inspection, Norway). If SCUBA diving will not be allowed, we will collect ice algae by ice core drilling and eventual scraping of under ice floes. Measurements of the gut turnover of krill by means of PAM fluorescence kinetics will be performed using a 2 mm thin optical nylon fiber. Microalgae mortality by enzyme assay Direct measurements of phytoplankton loss rates have been obstacled by the lack of methods, although the importance of measurements of this process have been recognised for decades (Kirchman 1999). Recently a new method has been published that facilitates simple and reliable measurements of the rate of release of intracellular products from phytoplankton cell death (Agusti et al. 1998). Death of a cell, such as caused by senescence, UV radiation, viral lysis and to some extend handling by gracing zooplankton will release intracellular material into solution in the surrounding medium. Some of the intracellular enzymes are measurable by extremely sensitive techniques which can easily be handled under shipboard conditions (Christian and Karl 1995). Agusti et al. (1998) demonstrated that intracellular esterases are suitable as tracers of phytoplankton cell death, because they are present in stable amounts in all phytoplankton, are released by death, and they are easy to measure. Moreover, they showed that the turnover of esterases, once released into the medium, is of a suitable time scale, so that turnover also is measurable, and consequently the combination of concentration measurement and turnover measurement yield production rates, i.e. cell death rate for each sample investigated. Additional variables In order to establish a set of essential data within the framework depicted in Figure 1, we will include measurements using a small selection of conventional methods which we have used successfully in a large variety of environments. Concentrations of DOC will be measured by high temperature combustion on samples which will be frozen and analysed ashore (Børsheim and Myklestad 1997). Bacterial production rates will be measured using ^3H-thymidine incorporation rates (Børsheim 1990). Project size Manpower needed are two persons on the cruise. With this we will be able to process 2 profiles per day during the active part of the oceanographic expedition. With one profile we mean 12 discrete water samples, either taken as vertical profiles (by Niskin samplers mounted on a rosette) from the euphotic zone at open water locations), or from samples collected in the viscinity of floes, including samples of intrusion brine, floe pore water, and vertical profiles below ice floes of selected age and condition. Selection of floes and the amount of time spent at such sites could be co-ordinated with other projects during the cruise, ideally the investigation of 7-12 floes would be appropriate for the purposes of our study. Methods Production rates Pulse Amplitude Modulated fluorometry (PAM) and bio-optical measurements: The Pam will be used to measure the fluorescence yield from Photosystem II and absorbed quanta to estimate photosynthetic rate, PO2, mg O2 produced mg Chl a ^-1h^-1) according to Kroon et al. (1993) and Johnsen et al. (1997) where: PO2 = AQ · FII · IIe· AQ = Absorbed quanta (unit: mmol m^-3 h^-1) FII = Fraction of AQ directed to Photosystem (PS) II (included its Light Harvesting Complexes), unit: dimensionless. IIe = PAM yield [(Fm’-Fo’)/Fm’] = Operational quantum yield for stable charge separation at PS II (mol charge separation mol quanta^-1). IIe (ratio of oxygen evolved per electron generated at PS II ): Since 4 stable charge separations are needed at PS II to evolve 1 O2-molecule, i.e.: A: IIe (the PAM yield) must be divided by 4, i.e. to give mmol O2 mmol quanta^-1 B: Multiply A with 0.032 to give mg O2 mmol quanta^-1. Finally: [AQ (mmol m^-3 h^-1) (mg O2 mmol quanta^-1) / mg Chl a m^-3 FII] gives us: PO2 (mg O2 produced mg Chl a ^-1h^-1) Chlorophyll a specific absorption coefficients (total amount of light received by the algae) and the corresponding fraction utilized by photosystem II (oxygen production site) will be measured by means of spectrophotometry (Hitachi 150 spectrophotometer) and spectrofluorometry (Hitachi F3000 spectrofluorometer, respectively (Johnsen et al. 1997). Pigment isolation will be carried out using a Hewlett Packard Series 1100 high performance liquid chromatograph using the method outlined in Johnsen et al. (1997). Phytoplankton mortality Assays of enzymes in seawater depend on artificial substrates that leaves a fluorescent product after cleavage of specific bonds in the substrate. There are several such substrates that yield products which are easily measured in a spectrofluorometer. For the measurement of esterases, fluorescein diacetate (FDA) is highly suitable for use in seawater (van Boekel et al. 1992, Agusti et al. 1998). One important refinement introduced by Agusti et al. (1998) was the added protocol for the estimation of esterase turnover in each sample. With this information the utility of the activity measurement is transcended, because production rate then can be calculated, and this will in the case of a strictly intracellular component be proportional to cell lysis (death) rate. The measurement of esterases has not before been reported in Antarctic investigations, however several studies have shown that similar assays (targeting proteases and lipases) have proven very useful and informative when applied in microbial studies in the area (e.g. Christian and Karl 1995). One of us has first hand experience with the shipboard use of such methods (Børsheim et al. submitted). DOC and bacterial production There are several reports that suggest that a large part of the production in Antarctic waters are channeled through the microbial loop (Christian and Karl 1995). With our approach we will be able to demonstrate if gradients in phytoplankton cell death are parallelled by gradients in bacterial production, or whether these processes are uncoupled in time and space. Another important gradient can be found in the viscinity of ice-floes. Cell death and release of intracellular material is one source of DOC. Recent studies have shown estimates of turnover of semilabile DOC in polar regions (Wheeler et al. 1996, Børsheim submitted). We therefore plan to measure DOC and consumption of DOC. Bacterial production rate will be measured useing thymidine incorporation rates according to Børsheim (1990), and DOC will be measured using high temperature combustion of samples which will be frozen and analyzed ashore according to Børsheim and Myklestad (1997).