Edited by: Sonya Dyhrman, Woods Hole Oceanographic Institution, USA
Reviewed by: Lisa Moore, University of Southern Maine, USA; Bethany Jenkins, University of Rhode Island, USA; Jason B. Sylvan, University of Southern California, USA
*Correspondence: Katherine R. M. Mackey, Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA. e-mail:
This article was submitted to Frontiers in Aquatic Microbiology, a specialty of Frontiers in Microbiology.
This is an open-access article distributed under the terms of the
This study explores the cycling of phosphorus (P) in the euphotic zone following upwelling in northeastern Monterey Bay (the Red Tide Incubator region) of coastal California, with particular emphasis on how bacteria and phytoplankton that form harmful algal blooms mediate and respond to changes in P availability.
Coastal regions cover less than 15% of the ocean’s surface, yet they contribute nearly half of the ocean’s primary productivity (Wollast,
Historically much emphasis has been placed on the availability of nitrogen (N) in controlling marine productivity (Dugdale and Goering,
Recent research has also highlighted the importance of dissolved organic P (DOP) as a nutrient source for phytoplankton. Many coastal phytoplankton species can grow on DOP as their sole source of P (Bjorkman and Karl,
Phosphorus in the DOP pool is made accessible to phytoplankton and other microbes via enzyme-mediated hydrolysis reactions that liberate orthophosphate from organic molecules. The enzyme alkaline phosphatase (AP) is one such enzyme that hydrolyzes phosphomonoester bonds, generating a free phosphate group, and an alcohol as products. In many organisms AP is synthesized in response to low ambient DIP levels, a characteristic that has led to its use as an indicator of DIP limitation in microbial communities (Dyhrman and Palenik,
The second category of AP activity assays involve the direct labeling of cells at the location of the AP enzyme. These include staining with azo dyes (Barka,
This study uses ELF labeling to identify AP activity in microbial communities from Monterey Bay, California following an upwelling event that gave rise to a red tide. The red tide incubator (RTI) is a persistent feature of northeastern Monterey Bay, where dense dinoflagellate populations frequently develop (Ryan et al.,
In this study, we use water chemistry data collected during three sampling events to understand the fate of upwelled P and its partitioning between inorganic and organic reservoirs. Observations of phytoplankton relative abundance and AP activity are used to understand how P availability affects community composition and contributes to HAB formation in this dynamic environment. This study increases our understanding of the cell-specific P status of red tide phytoplankton during bloom formation by examining of a broad range of species and measuring their responses to P dynamics following upwelling.
Samples were collected from an along shore transect located within the RTI (Ryan et al.,
The sampling transect stretched between the outlets of Elkhorn Slough and Pajaro Creek, extending northwest toward the San Lorenzo River outlet with the following six stations (Figure
Sample water was syringe filtered (0.45 μm) into acid washed, sample rinsed plastic bottles and frozen until analysis. Seawater concentrations of soluble reactive phosphorus (SRP), nitrate (NO3, including trace amounts of nitrite), and silicate were analyzed using colorimetric methods described by Hansen and Koroleff (
Total dissolved P and N samples were analyzed following persulfate digestion as described in D’Elia et al. (
Samples for DOC analysis were filtered through a sterile 0.2-μm filter and collected into acid washed and combusted borosilicate glass scintillation vials with teflon lined screw caps. Samples were acidified with HCl to pH < 2 and purged to remove inorganic (and purgeable organic) carbon, stored cold (4°C) in the dark until analysis. Samples were analyzed using a Shimadzu TOC analyzer (EPA method 415.1). The detection limit was 1 μmol L−1.
The endogenous phosphatase detection kit (Molecular Probes E 6601) was used to detect cell-specific phosphatase activity. The ELF 97 phosphatase substrate, ELF-P, [2-(5′-ochloro-2′-phosphoryloxyphenol)-6-chloro-4-(3H)-quinazolinone] is a soluble molecule that forms an insoluble alcohol precipitate following cleavage of the phosphate group by the AP enzyme. One liter seawater samples were filtered at low pressure unto 0.45 μm filters, gently eluted with 800 μL 70% ethanol, and stored at 0°C until analysis. Dimethyl sulfoxide (DMSO) was added to a final concentration of 10% to enhance cell penetration by the ELF-P substrate and improve labeling clarity (Lomas et al.,
An incubation experiment was conducted with water collected near the southern edge of the RTI region (Figure
Water chemistry was monitored during three sampling events that occurred immediately before, during, and 1 week after an upwelling event in October 2008. Upwelling brought colder, more saline water to the surface (Figures
An upwelling event introduced cold, nutrient rich water into the RTI region between October 10–15 (Figures
The phytoplankton population initially was a complex assemblage composed of roughly equal densities of diatoms and dinoflagellates, but transitioned toward a higher relative abundance of dinoflagellates following upwelling (Figure
Dinoflagellate relative abundance was ∼53% before upwelling occurred, but increased dramatically to up to 92% in the weeks following upwelling in stations 1–4 (Figures
A large, localized bloom of
A smaller localized bloom of
Alkaline phosphatase (AP) activity was assessed via the ELF assay. Throughout the sampling period, diatom AP activity showed a higher degree of spatial variability and sensitivity to DIP and DOP availability compared to dinoflagellates (Figures
In contrast to diatoms, the dinoflagellate population had higher proportions of ELF labeled cells (62–92%, average 79%, Figure
The combined ELF labeling of cells from all
Enzyme-labeled fluorescence labeling was also observed in bacteria affixed to the surfaces of intact cells, as well as associated with lysed cell debris and aggregates of particulate organic material (Figure
An incubation experiment was conducted to gage the response of phytoplankton from the RTI region of Monterey Bay to availability of
Monterey Bay is a dynamic, spatially heterogeneous region that is strongly influenced by wind-driven upwelling that varies seasonally in intensity and intermittency (Pennington and Chavez,
Upwelling dramatically increased the concentration of DIP in surface waters in October 2008. DIP was then quickly consumed as upwelling relaxed and the phytoplankton bloom developed. The response of phytoplankton to the upwelling pulse was evidently variable over relatively small scales. In the middle to outer northern bay, toxigenic diatoms dominated the response to upwelling (Ryan et al.,
Like many coastal upwelling regions, phytoplankton productivity in Monterey Bay responds strongly to
While N availability appears to be the primary factor controlling phytoplankton abundance in Monterey Bay, several lines of evidence suggest that P availability also regulates the growth and physiological status of cells. First, the moderate increase in phytoplankton growth relative to the untreated control in the incubation experiment suggests that some components of the RTI phytoplankton community are indeed P limited. Second, the high yet variable amount of AP activity revealed through ELF labeling suggests that different species have diverse strategies for coping with P limitation (e.g., inducible AP activity versus high basal AP activity) and, consequently, are likely to have different physiological P statuses despite being exposed to identical ambient concentrations of DIP (Mackey et al.,
It is interesting that despite the large input of
In some strains of phytoplankton phosphatase activity is induced when ambient DIP concentrations are low, however, in other species phosphatase activity may be constitutive or less sensitive to ambient DIP levels (Kuenzler and Perras,
Ruttenberg and Dyhrman (
We suggest that in contrast to inducible AP activity, high basal AP activity may have different underlying causes. The high basal expression of AP activity, as observed in the HAB-forming dinoflagellates in the RTI, may offer a competitive advantage to cells when large pulses of
During our sampling, the RTI region was populated by a number of localized, intense blooms, the largest of which was dominated by
The extensive amount of AP activity observed in bacterial cells suggests three potentially important roles for bacteria in facilitating the turnover of organic P in Monterey Bay. First, their association with particulate material and cell debris demonstrates the critical function they perform in aggregating and breaking down organic materials during upwelling relaxation, and the sharp, rapid decline in DOC observed during upwelling relaxation (Figure
Second, in a decaying bloom, bacteria could support additional primary and secondary production and re-inject P into the food web through their AP activity. Bacteria may employ AP activity to access P or C from organic molecules, and it is not clear if the bacteria in our samples expressed the enzyme to satisfy their needs for P, C, or both. In the microbial loop, bacteria take up and incorporate organic C into their cellular biomass, thereby returning it to the classical grazing food web (Azam et al.,
The third possible role for bacteria in the P cycle of Monterey Bay is based on our observation that some bacteria affixed to the surfaces of intact phytoplankton cells expressed AP activity (Figures
The RTI of Monterey Bay is a region with dynamic changes in nutrient inventories and where diverse phytoplankton species co-exist. Following upwelling, a pulse of DIP and
Diatom and dinoflagellate populations were identified in similar abundances prior to upwelling, but dinoflagellates became more abundant during upwelling and relaxation. Diatom alkaline phosphatase (AP) activity was responsive to ambient DIP and DOP concentrations. In contrast, dinoflagellates showed high basal rates of AP activity regardless of DIP (or DOP) levels, which may have conferred a competitive advantage by helping them avoid secondary limitation for P when N levels were high following upwelling. Diatom species were distributed uniformly among the stations, whereas some dinoflagellate species formed spatially localized blooms within the RTI region. The largest of these blooms comprised
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank the Bhaya and Grossman Labs (Carnegie Institution for Science) for use of their microscope, L. Smolenska (Invitrogen) for technical advice pertaining to phytoplankton ELF labeling, R. Franks for assistance with instrumentation, and E. Rienecker for piloting the boat and provided assistance at sea. This manuscript benefited from thoughtful review comments from L. Moore, B. Jenkins, and J. B. Sylvan. Thanks also to G. Pitcher (Department of Agriculture, Forestry and Fisheries, South Africa), J. Lane (University of California, Santa Cruz), and R. Kudela (University of California, Santa Cruz) for discussions of