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Use of dissolved inorganic and organic phosphorus by axenic and nonaxenic clones of Karenia brevis and Karenia mikimotoi
Harmful Algae 48 (2015) 30–36
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Use of dissolved inorganic and organic phosphorus by axenic and nonaxenic clones of Karenia brevis and Karenia mikimotoi Bill Richardson, Alina A. Corcoran * Florida Fish and Wildlife Conservation Commission, Fish and Wildlife Research Institute, 100 8th Avenue SE, St. Petersburg, FL 33701, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 May 2015 Received in revised form 19 May 2015 Accepted 11 June 2015 Available online
Nearly annual blooms of the marine dinoflagellate Karenia brevis, which initiate offshore on the West Florida Shelf in oligotrophic waters, cause widespread environmental and economic damage. The success of K. brevis as a bloom-former is partially attributed to its ability to use a diverse suite of nutrients from natural and anthropogenic sources, although relatively little is known about the ability of K. brevis and the closely related Karenia mikimotoi to use a variety of organic sources of phosphorus, including phosphomonoesters, phosphodiesters, and phosphonates. Through a series of bioassays, this study characterized the ability of axenic and nonaxenic K. brevis and K. mikimotoi clones isolated from Florida waters to use a variety of organic phosphorus compounds as the sole source of phosphorus for growth, comparing this utilization to that of inorganic sources of phosphate. Differing abilities of axenic and nonaxenic K. brevis and K. mikimotoi cultures to use phosphorus from the compounds evaluated were documented. Specifically, growth of axenic cultures was greatest on inorganic phosphorus and was not supported on the phosphomonoester phytate, or generally on phosphodiesters or phosphonates. The nonaxenic cultures were able to use organic compounds that the axenic cultures were not able to use, often after lags in growth, highlighting a potential role of co-associated bacterial communities to transform nutrients to bioavailable forms. Given the ability of K. brevis and K. mikimotoi to use a diverse suite of inorganic and organic phosphorus, bloom mitigation strategies should consider all nutrient forms. ß 2015 Elsevier B.V. All rights reserved.
Keywords: Harmful algal bloom Nutrient utilization Bacteria Bioassay
1. Introduction In the eastern Gulf of Mexico, the marine dinoflagellate Karenia brevis blooms nearly annually, causing fish kills, marine mammal mortalities, and human illnesses (Flewelling et al., 2005; Landsberg et al., 2009; Fleming et al., 2011). Blooms initiate offshore on the West Florida Shelf in oligotrophic waters (Steidinger and Haddad, 1981; Tester and Steidinger, 1997) often following nitrogen (N) fixation by Trichodesmium species (Lenes and Heil, 2010; Sipler et al., 2013). During the early stages of blooms, the closely related species Karenia mikimotoi can be abundant, but it is ultimately dominated by K. brevis, which can reach monospecific abundances of greater than 106 cells L1. The success of K. brevis as a bloomformer is partially attributed to its ability to use a diverse suite of nutrients from natural and anthropogenic sources, including estuarine outflows, coastal upwelling, atmospheric deposition, remineralization, and benthic fluxes (Heil et al., 2014a,b).
* Corresponding author. Tel.: +1 727 892 4156. E-mail address: [email protected] (A.A. Corcoran). http://dx.doi.org/10.1016/j.hal.2015.06.005 1568-9883/ß 2015 Elsevier B.V. All rights reserved.
Compared to N, less is known about the utilization of phosphorus (P) by K. brevis and related species, despite the important role of differential nutrient utilization abilities in influencing phytoplankton community composition and harmful algal bloom dynamics. Given the low concentrations of dissolved inorganic phosphorus (DIP) in offshore West Florida Shelf waters and the large P requirements needed to support K. brevis blooms, it is likely that organic forms of P are important to bloom initiation and maintenance (Steidinger et al., 1998). The major classes of dissolved organic P (DOP) in marine waters include phosphoesters, which comprise greater than 75% of the high-molecular-weight marine DOP, and phosphonates (Clark et al., 1998; Karl and Bjo¨rkman, 2002). Marine phytoplankton can obtain P from the larger DOP pool by altering P uptake kinetics and inducing the production of enzymes such as alkaline phosphatase (Cembella et al., 1984; Flynn et al., 1986; Yamaguchi et al., 2005, 2014; Dyhrman and Ruttenberg, 2006; Dyhrman et al., 2012), although utilization of the smaller DOP pool via phosphonate hydrolysis appears limited to bacteria and cyanobacteria (Schowanek and Verstraete, 1990; Dyhrman et al., 2006; Yamaguchi and Adachi, 2010). Karenia brevis and K. mikimotoi generally have low half
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saturation constants for sources of DIP (Gentien, 1998; Vargo, 2009) and are able to use sources of DOP. In laboratory studies, Wilson (1996) reported utilization of phosphomonoester compounds by an axenic strain of K. brevis, and Yamaguchi and Itakura (1999) reported the utilization of these compounds by a Japanese strain of K. mikimotoi, as would be expected given phosphatase production by these species (Huang et al., 2007; Lin et al., 2012). However, to date, no studies have compared the abilities of axenic and nonaxenic strains of K. brevis and K. mikimotoi to directly utilize a variety of DIP and DOP sources for growth. This study evaluates the abilities of axenic and nonaxenic strains of K. brevis and K. mikimotoi clones isolated from Florida waters to use DIP and DOP compounds known or likely to be present in seawater. This work provides a better understanding of nutrient utilization by Karenia strains, ultimately contributing to our understanding of nutrient dynamics in these harmful algal bloom species. 2. Materials and methods To evaluate the abilities of axenic and nonaxenic K. brevis and K. mikimotoi to utilize different sources of P for growth, clones were grown on 29 different P compounds (Table 1) and final yields (maximum in vivo chlorophyll fluorescence, chlorophyll a content
Table 1 Compounds evaluated as the sole sources of phosphorus for growth of Karenia brevis and Karenia mikimotoi. The chemical formulas and abbreviations used in Figs. 1 and 2 are also listed. Compound Inorganic P Orthophosphate Sodium pyrophosphate Sodium tripolyphosphate Sodium trimetaphosphate Organic P Phosphomonoesters Glucose-6-phosphate Ribose-5-phosphate Fructose 1,6-bisphosphate b-Glycerophosphate Phosphoenolpyruvic acid Nicotinamide adenine dinucleotide phosphate Phytate (myo-inositol hexakisphosphate) Adenosine-5-diphosphate Adenosine-5-triphosphate Cytidine-5-monophosphate Uridine-5-monophosphate Adenosine-5-monophosphate Guanosine-5-monophosphate Uridine-5-diphosphoglucose Phosphodiesters Adenosine 30 ,50 -cyclic monophosphate Guanosine 30 ,50 -cyclic monophosphate Ribonucleic acid Deoxyribonucleic acid Bis (p-nitrophenyl) phosphate Phosphonates 2-Aminoethylphosphonic acid Methylphosphonic acid Aminomethylphosphonic acid Phosphonoacetic acid Phosphonoacetaldehyde 2-Amino-3phosphonopropionic acid
Chemical formula
Abbreviation
NaH2PO4 Na2H2P2O7 Na5P3O106H2O Na3O9P3
Pi PP TPP TMP
C6H11Na2O9P2H2O C5H9O8Na2P2H2O C6H11O12P2Na3 C3H9O6Na2P5H2O C3H4NaO6PH2O C21H84N7O17P3XH2O
G6P R5P FBP GYP PEP NADP
C6H16O24P6K2
PHY
C10H15N5O10P2 C10H14N5O13P3Na2 C9H12N3Na2O8P C9H11N2O9PNa2 C10H14N5O7PH2O C10H12N5Na2O8P C15H24N2O17P2Na2
ADP ATP CMP UMP AMP GMP UDPG
C10H11N5O6PNa
cAMP
C10H11N5O7PNa
cGMP
– – C12H9N2O8P
RNA DNA bNPP
C2H8NO3P
AEP
CH5O3P NH2CH2P(O)(OH)2
MP AMPA
C2H5O5P C10H23O5P C3H8NO5P
PAA PAC APP
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and cell abundance) were compared. Clones of K. brevis (CCFWC257) and K. mikimotoi (CCFWC67) isolated from Florida and in culture at the Florida Fish and Wildlife Conservation Commission were used during these bioassays. Axenic cultures established via antibiotic treatment were confirmed to be free of bacteria by epifluorescence microscopy (Porter and Feig, 1980; Guillard, 2005). Prior to bioassays, stock cultures were maintained for several weeks in 3-L Fernbach flasks of GSe medium minus soil extract in 0.22 mm filter-sterilized, autoclaved Gulf of Mexico seawater (Blackburn et al., 2001) at a salinity of 35 and temperature of 20 0.5 8C. Light was supplied by full spectrum and cool white fluorescent lights (Ecolux F40C50, Philips F40T12) on a 12-h light/dark cycle at a saturation irradiance of 80 mmol photons m2 s1. To deplete P stores in inocula, stocks were grown in batch culture in P-limited media (GSe minus soil extract with 500 mM NaNO3 and 5 mM NaH2PO4) until cultures reached stationary phase and total dissolved phosphorus concentrations were undetectable. Total dissolved P concentrations in the stocks were verified following Soloranzo and Sharp (1980) using a Beckman Coulter DU 530 spectrophotometer. Bioassays were conducted in triplicate in 25 150 mm Pyrex test tubes. Tubes were inoculated with P-depleted stock cultures, resulting in an initial cell abundance of 2000 cells mL1. Experimental treatments were obtained by adding each of the P compounds at a final concentration of 3 mM P, and no P was added to the control tubes. Culture conditions (e.g., light, P-limited media) were otherwise the same as those described for the maintenance of stock cultures. Growth was monitored daily, four hours into the light cycle, by measuring in vivo chlorophyll fluorescence of the actively growing cultures with a Turner Designs 10 AU fluorometer. When maximum in vivo fluorescence was attained, after one and a half to two weeks, samples for subsequent quantification of chlorophyll a content and cell abundance were taken from each of the tubes. Chlorophyll a was extracted with methanol and concentrations were determined fluorometrically (Holm-Hansen and Riemann, 1978). Cell abundance was estimated using a Beckman Coulter Z2 Coulter Counter and verified by microscopy. Maximum growth rates were determined by a linear least squares regression of the natural log of in vivo fluorescence versus time. No measurements of P substrates were made during the experiments. Differences in growth responses to the P compounds were evaluated separately for axenic and nonaxenic cultures of each species. Differences in average maximum growth rate (mmax), in vivo fluorescence, chlorophyll a content, and cell abundance between the treatments were examined using 1-way ANOVA with post hoc Tukey tests using Minitab 17. Data were rank-transformed to meet the assumptions of normality and equal variance. Because multiple tests were conducted, a (initially 0.05) was adjusted to 0.002 using a Bonferroni correction to reduce Type I error rates (Quinn and Keough, 2002). 3. Results The axenic and nonaxenic K. brevis and K. mikimotoi cultures exhibited differences in their abilities to use DIP and DOP (Figs. 1 and 2, Supplementary Tables 1 and 2). As exhibited by final yields in maximum in vivo fluorescence, cell abundance, and chlorophyll a content, the axenic cultures did not generally grow on the phosphomonoester phytate, the phosphodiesters, or the phosphonates. One exception was the axenic K. brevis culture, which exhibited significantly higher chlorophyll a concentrations and cell abundance when grown on RNA as the sole P source, compared to the control (Fig. 1, Supplementary Table 1). The nonaxenic K. brevis culture did not grow on the phosphodiester guanosine 30 ,50 -cyclic monophosphate and four of the six phosphonates, whereas the nonaxenic K. mikimotoi culture did not grow on five of the six
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Fig. 1. Maximum growth rates (mmax, assessed by in vivo fluorescence), maximum in vivo fluorescence, cell abundance, and chlorophyll a concentrations of axenic (left) and nonaxenic (right) K. brevis cultures. Bars represent means standard deviations of three replicates. See Table 1 for full names of compounds and Supplementary Table 1 for results of statistical comparisons of treatment differences.
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Fig. 2. Maximum growth rates (mmax, assessed by in vivo fluorescence), maximum in vivo fluorescence, cell abundance, and chlorophyll a concentrations of axenic (left) and nonaxenic (right) K. mikimotoi cultures. Bars represent means standard deviations of three replicates. See Table 1 for full names of compounds and Supplementary Table 2 for results of statistical comparisons of treatment differences. ‘nd’ indicates that no data were collected for these treatments.
phosphonates (Figs. 1 and 2). Average maximum chlorophyll a content per cell in the axenic and nonaxenic K. brevis cultures, respectively, ranged from 11 to 13 pg cell1 and 12 to 16 pg cell1 for P compounds that supported growth and from 8 to 9 pg cell1 and 7 to 10 pg cell1 for P compounds on which no growth occurred. A similar pattern was seen for the axenic and nonaxenic
K. mikimotoi cultures, respectively, with average maximum chlorophyll a content per cell ranging from 20 to 24 pg cell1 and 16 to 22 pg cell1 when growth occurred and from and 12 to 14 pg cell1 and 9 to 11 pg cell1 when no growth occurred. Overall, yields (maximum in vivo fluorescence, cell abundance, chlorophyll a content) of all cultures tended to be greater on DIP
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than DOP compounds (Figs. 1 and 2, Supplementary Tables 1 and 2). Yields of the nonaxenic K. brevis culture were significantly reduced on most of the phosphodiesters that supported growth, compared to yields on DIP, phosphomonoesters or phosphonates that supported growth (Fig. 1, Supplementary Table 1). This pattern was not observed in K. mikimotoi. Lags in growth on a number of the organic P compounds were observed in both of the nonaxenic cultures – in K. brevis on the phosphomonoester phytate (PHY); phosphodiesters adenosine 30 ,50 -cyclic monophosphate (cAMP) and guanosine 30 ,50 -cyclic monophoshpate (cGMP); and phosphonates 2-aminoethylphosphonic acid (AEP) and phosphonoacetic acid (PAA); and in K. mikimotoi on the phosphomonoester phytate (PHY); phosphodiesters adenosine 30 ,50 -cyclic monophosphate (cAMP), guanosine 30 ,50 -cyclic monophosphate (cGMP), and bis (p-nitrophenyl) phosphate (bNPP); and the phosphonate phosphonoacetic acid (PAA) (Fig. 3). 4. Discussion 4.1. Inorganic nutrient sources This work illustrated that growth of K. brevis and K. mikimotoi varied with the source of P used as a substrate. It confirmed that K. brevis and K. mikimotoi use sodium pyrophosphate, sodium trimetaphosphate, and sodium tripolyphosphate, and that these compounds, as well as orthophosphate, produce some of the highest yields (Figs. 1 and 2). Prior reports of Karenia growth on DIP compounds have largely been limited to orthophosphate (Wilson,
1996; Vargo, 2009; Steidinger, 2009), with the exception of work on a Japanese clone of K. mikimotoi, which also reported higher yields on DIP compared to DOP (Yamaguchi and Itakura, 1999). Greater yields on DIP is likely due to the simplicity of orthophosphates, the ease with which sodium phosphates are hydrolyzed into orthophosphate, and the natural hydrolysis of polyphosphates in water (Cembella et al., 1984; Bjorkman et al., 2000; TorresDorante et al., 2005). 4.2. Phosphomonoesters Among the phosphomonoesters examined, phytate was unique in that it could not be used by the axenic K. brevis or K. mikimotoi cultures (Figs. 1 and 2) – consistent with observations for other eukaryotic algae and the hydrolysis of phytate by phytase rather than alkaline phosphatase, found in algae (Turner et al., 2002). Microorganisms known to hydrolyze phytate include species of bacteria, cyanobacteria, and protists (Whitton et al., 1991; Ziemkiewicz et al., 2002; Lim et al., 2007). Cembella et al. (1984) reported use of phytate by various species including Phaeocystis pouchetti, Phaeodactylum tricornutum and Pyrocystis noctiluca, although axenic cultures were not always used – highlighting a potential role of co-occurring bacteria in nutrient utilization. In this study, the nonaxenic cultures grew on phytate after a pronounced initial lag in growth (Fig. 3), suggesting that cooccurring bacteria may have degraded the phytate and released orthophosphase, which in turn supported Karenia growth. It should be noted, however, that for this and other compounds, no attempt was made to track the bacterial communities associated with strains or nutrient concentrations through time. To define the role of bacterial communities associated with Karenia strains in nutrient utilization (or as a potential food source, given the potential for mixotrophy), directed studies focused on bacterial dynamics are needed. The similar yields and maximum growth rates of the axenic K. brevis and K. mikimotoi grown on the phosphomonoesters that supported growth (Figs. 1 and 2) indicates no strong preference for any specific compound or type, consistent with previous findings (Wilson, 1996; Yamaguchi and Itakura, 1999) and the production of alkaline phosphatase by K. brevis (Vargo and Shanley, 1985; Craney et al., 2004; Vargo, 2009). Note that the higher growth rate of the nonaxenic culture of K. mikimotoi (0.20 da1) on phytate is likely an experimental artifact, a result of growth rate determination immediately following a prolonged initial lag. 4.3. Phosphodiesters
Fig. 3. In vivo fluorescence of nonaxenic K. brevis and K. mikimotoi cultures in which lags were observed. For comparison, the treatments that did not exhibit lags (‘others’) and control are shown. See Table 1 for full names of compounds.
The inability of the axenic cultures and ability of the nonaxenic cultures to grow on phosphodiesters as the sole source of P suggests a potential role of co-associated bacterial communities in rapidly hydrolyzing phosphodiesters and freeing P for growth by Karenia species. The use of some but not all of phosphodiesters in the nonaxenic cultures suggests that different bacterial communities may produce different phosphodiesterases, which are used to hydrolyze phosphodiesters (Ammerman and Azam, 1981, 1991; Suzumura et al., 1998; Karl and Bjo¨rkman, 2002), and that distinct bacterial assemblages will affect nutrient utilization in phytoplankton communities. Little is known about the ability of marine microalgae without associated bacteria to access biomolecules with phosphodiester bonds (e.g., nucleic acids and cyclic nucleotides), although the use of phosphodiesters as the sole source of P for growth have been reported in Phaeodactylum tricornutum and Chaetoceros ceratosporus (Flynn et al., 1986; Yamaguchi et al., 2005). Moreover, our work is consistent with the previous finding that axenic K. mikimotoi was unable to use bis p-nitrophenyl phosphate to grow (Yamaguchi et al., 2005).
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4.4. Phosphonates
References
The discovery of Trichodesmium species capable of phosphonate mineralization has led to the reconsideration of phosphonates as potential sources of bioavailable P (Dyhrman et al., 2006). The inability of axenic cultures of K. brevis to grow on any of the phosphonates indicates that this strain does not produce either C– P hydrolase or C–P lyases needed to access phosphonate-P, consistent with other studies that show phosphonates are unavailable to eukaryotic phytoplankton and are mineralized primarily by cyanobacteria and bacteria (Kononova and Nesmeyanova, 2002; Dyhrman et al., 2009). In contrast, growth of the nonaxenic K. brevis on the phosphonates 2-aminoethylphosphonic acid (AEP) and phosphonoacetic acid (PAA) and nonaxenic cultures of K. mikimotoi on phosphonoacetic acid, but with pronounced initial growth lags (Figs. 1–3), suggests that bacteria in the cultures may have produced the hydrolytic enzymes needed for these phosphonates. Gilbert et al. (2009) reported that marine bacteria in coastal waters use phosphonoacetic acid as a sole source of P, and Cook et al. (1978) found that orthophosphate was liberated when bacteria were grown on 2-aminoethylphosphonic acid as the sole carbon source in the presence of adequate N and P. The inability of K. mikimotoi to grow on 2-aminoethylphosphonic acid suggests that no 2-aminoethylphosphonic acid-mineralizing bacteria were present in the culture. Presumably, no bacteria capable of hydrolyzing the other phosphonates were present in either of the nonaxenic cultures.
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5. Conclusions This work demonstrated that K. brevis and K. mikimotoi are able to utilize a diverse variety of P sources, some without preconditioning (e.g., phosphomonoesters); this ability may offer a competitive advantage to these species when P is limiting to growth. In addition, this work demonstrated that bacteria may play a key role in nutrient utilization by Karenia species and that associated bacterial communities can differentially permit access to different nutrient pools. The indirectly bacteria-mediated supply of P by DOP mineralization may be particularly important for larger blooms that often closely follow the decay of Trichodesmium blooms. Consequently, bloom development of K. brevis and K. mikimotoi on the west Florida shelf would be expected to be influenced at times by the co-occurring bacterial assemblage’s exploitation and mineralization of constituents of the DOP pool not directly accessible to Karenia. Since natural and anthropogenic nutrient sources may vary widely in both type and amount of DIP and DOP compounds, bloom mitigation efforts that include land-based nutrient management strategies should consider the ability of both K. brevis and K. mikimotoi to access a wide range of DIP and DOP forms. Author contributions B.R. designed and performed the research. A.A.C. and B.R. analyzed the data and wrote the paper. Acknowledgments This study was funded by the Florida Fish and Wildlife Conservation Commission. [SS] Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.hal.2015.06.005.
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Lenes, J.M., Heil, C.A., 2010. A historical analysis of the potential nutrient supply from the N2 fixing marine cyanobacterium Trichodesmium spp. to Karenia brevis blooms in the eastern Gulf of Mexico. J. Plankton Res. 32 (10), 1421–1431. Lim, B.L., Yeung, P., Cheng, C., Hill, J.E., 2007. Distribution and diversity of phytatemineralizing bacteria. ISME J. 1 (4), 321–330. Lin, X., Zhang, H., Huang, B.Q., Lin, S.J., 2012. Alkaline phosphatase gene sequence characteristics and transcriptional regulation by phosphate limitation in Karenia brevis (Dinophyceae). Harmful Algae 17, 14–24. Porter, K.G., Feig, Y.S., 1980. The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr. 25 (5), 943–948. Quinn, G.P., Keough, M.J., 2002. Experimental Design and Data Analysis for Biologists. Cambridge University Press, Cambridge. Schowanek, D., Verstraete, W., 1990. Phosphonate utilization by bacterial cultures and enrichments from environmental samples. Appl. Environ. Microbiol. 56 (4), 895–903. Sipler, R., Bronk, D., Seitzinger, S., Lauck, R., McGuinness, L., Kirkpatrick, G., Heil, C., Kerkhof, L., Schofield, O., 2013. Trichodesmium-derived dissolved organic matter is a source of nitrogen capable of supporting the growth of toxic red tide Karenia brevis. Mar. Ecol. Prog. Ser. 483, 31–45. Soloranzo, L., Sharp, J.H., 1980. Determination of total dissolved phosphorus and particular phosphorus in natural waters. Limnol. Oceanogr. 25 (4), 754–758. Steidinger, K.A., 2009. Historical perspective on Karenia brevis red tide research in the Gulf of Mexico. Harmful Algae 8 (4), 549–561. Steidinger, K.A., Haddad, K., 1981. Biologic and hydrographic aspects of red tides. Bioscience 31 (11), 814–819. Steidinger, K.A., Vargo, G.A., Tester, P., Tomas, C.R., 1998. Bloom dynamics and physiology of Gymnodinium breve with emphasis on the Gulf of Mexico. In: Anderson, D., Cembella, A., Hallegraeff, G. (Eds.), Physiological Ecology of Harmful Algal Blooms. Springer-Verlag, Berlin, pp. 133–153. Suzumura, M., Ishikawa, K., Ogawa, H., 1998. Characterization of dissolved organic phosphorus in coastal seawater using ultrafiltration and phosphohydrolytic enzymes. Limnol. Oceanogr. 43 (7), 1553–1564. Tester, P.A., Steidinger, K.A., 1997. Gymnodinium breve red tide blooms: initiation, transport, and consequences of surface circulation. Limnol. Oceanogr. 42 (5), 1039–1051.
Torres-Dorante, L.O., Claasen, N., Steingrobe, B., Olfs, H.W., 2005. Hydrolysis rates of inorganic polyphosphates in aqueous solution as well as in soils and effects on P availability. J. Plant Nutr. Soil Sci.[[n]]Z. Pflanzenernahr. Bodenkd. 168 (3), 352–358. Turner, B.L., Paphazy, M.J., Haygarth, P.M., McKelvie, I.D., 2002. Inositol phosphates in the environment. Philos. Trans. R. Soc. Lond. B: Biol. Sci. 357 (1420), 449–469. Vargo, G.A., 2009. A brief summary of the physiology and ecology of Karenia brevis Davis (G. Hansen and Moestrup comb.nov.) red tides on the West Florida Shelf and of hypotheses posed for their initiation, growth, maintenance, and termination. Harmful Algae 8 (4), 573–584. Vargo, G.A., Shanley, E., 1985. Alkaline phosphatase activity in the red tide dinoflagellate Ptychodiscus brevis. Mar. Ecol. 6 (3), 251–264. Whitton, B.A., Grainger, S.L.J., Hawley, G.R.W., Simon, J.W., 1991. Cell-bound and extracellular phosphatase activities of cyanobacterial isolates. Microb. Ecol. 21 (2), 85–98. Wilson, W.B., 1996. The Suitability of Sea-water for the Survival and Growth of Gymnodinium breve Davis and Some Effects of Phosphorus and Nitrogen on its Growth, Professional Paper Series. Florida Board of Conservation, Marine Research Laboratory, , pp. 1–42. Yamaguchi, H., Adachi, M., 2010. The utilization of organic phosphorus by eukaryotic phytoplankton in marine environments. Bull. Plankton Soc. Jpn. 57 (1), 1–12. Yamaguchi, H., Arisaka, H., Otsuka, N., Tomaru, Y., 2014. Utilization of phosphate diesters by phosphodiesterase-producing marine diatoms. J. Plankton Res. 36 (1), 281–285. Yamaguchi, H., Yamaguchi, M., Fukami, K., Adachi, M., Nishijima, T., 2005. Utilization of phosphate diester by the marine diatom Chaetoceros ceratosporus. J. Plankton Res. 27 (6), 603–606. Yamaguchi, M., Itakura, S., 1999. Nutrition and growth kinetics in nitrogen- or phosphorus-limited cultures of the noxious red tide dinoflagellate Gymnodinium mikimotoi. Fish. Sci. 65 (3), 367–373. Ziemkiewicz, H.T., Johnson, M.D., Smith-Somerville, H.E., 2002. Phytate as the sole phosphate source for growth of Tetrahymena. J. Eukaryot. Microbiol. 49 (5), 428–431.
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Use of dissolved inorganic and organic phosphorus by axenic and nonaxenic clones of Karenia brevis and Karenia mikimotoi Bill Richardson, Alina A. Corcoran * Florida Fish and Wildlife Conservation Commission, Fish and Wildlife Research Institute, 100 8th Avenue SE, St. Petersburg, FL 33701, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 May 2015 Received in revised form 19 May 2015 Accepted 11 June 2015 Available online
Nearly annual blooms of the marine dinoflagellate Karenia brevis, which initiate offshore on the West Florida Shelf in oligotrophic waters, cause widespread environmental and economic damage. The success of K. brevis as a bloom-former is partially attributed to its ability to use a diverse suite of nutrients from natural and anthropogenic sources, although relatively little is known about the ability of K. brevis and the closely related Karenia mikimotoi to use a variety of organic sources of phosphorus, including phosphomonoesters, phosphodiesters, and phosphonates. Through a series of bioassays, this study characterized the ability of axenic and nonaxenic K. brevis and K. mikimotoi clones isolated from Florida waters to use a variety of organic phosphorus compounds as the sole source of phosphorus for growth, comparing this utilization to that of inorganic sources of phosphate. Differing abilities of axenic and nonaxenic K. brevis and K. mikimotoi cultures to use phosphorus from the compounds evaluated were documented. Specifically, growth of axenic cultures was greatest on inorganic phosphorus and was not supported on the phosphomonoester phytate, or generally on phosphodiesters or phosphonates. The nonaxenic cultures were able to use organic compounds that the axenic cultures were not able to use, often after lags in growth, highlighting a potential role of co-associated bacterial communities to transform nutrients to bioavailable forms. Given the ability of K. brevis and K. mikimotoi to use a diverse suite of inorganic and organic phosphorus, bloom mitigation strategies should consider all nutrient forms. ß 2015 Elsevier B.V. All rights reserved.
Keywords: Harmful algal bloom Nutrient utilization Bacteria Bioassay
1. Introduction In the eastern Gulf of Mexico, the marine dinoflagellate Karenia brevis blooms nearly annually, causing fish kills, marine mammal mortalities, and human illnesses (Flewelling et al., 2005; Landsberg et al., 2009; Fleming et al., 2011). Blooms initiate offshore on the West Florida Shelf in oligotrophic waters (Steidinger and Haddad, 1981; Tester and Steidinger, 1997) often following nitrogen (N) fixation by Trichodesmium species (Lenes and Heil, 2010; Sipler et al., 2013). During the early stages of blooms, the closely related species Karenia mikimotoi can be abundant, but it is ultimately dominated by K. brevis, which can reach monospecific abundances of greater than 106 cells L1. The success of K. brevis as a bloomformer is partially attributed to its ability to use a diverse suite of nutrients from natural and anthropogenic sources, including estuarine outflows, coastal upwelling, atmospheric deposition, remineralization, and benthic fluxes (Heil et al., 2014a,b).
* Corresponding author. Tel.: +1 727 892 4156. E-mail address: [email protected] (A.A. Corcoran). http://dx.doi.org/10.1016/j.hal.2015.06.005 1568-9883/ß 2015 Elsevier B.V. All rights reserved.
Compared to N, less is known about the utilization of phosphorus (P) by K. brevis and related species, despite the important role of differential nutrient utilization abilities in influencing phytoplankton community composition and harmful algal bloom dynamics. Given the low concentrations of dissolved inorganic phosphorus (DIP) in offshore West Florida Shelf waters and the large P requirements needed to support K. brevis blooms, it is likely that organic forms of P are important to bloom initiation and maintenance (Steidinger et al., 1998). The major classes of dissolved organic P (DOP) in marine waters include phosphoesters, which comprise greater than 75% of the high-molecular-weight marine DOP, and phosphonates (Clark et al., 1998; Karl and Bjo¨rkman, 2002). Marine phytoplankton can obtain P from the larger DOP pool by altering P uptake kinetics and inducing the production of enzymes such as alkaline phosphatase (Cembella et al., 1984; Flynn et al., 1986; Yamaguchi et al., 2005, 2014; Dyhrman and Ruttenberg, 2006; Dyhrman et al., 2012), although utilization of the smaller DOP pool via phosphonate hydrolysis appears limited to bacteria and cyanobacteria (Schowanek and Verstraete, 1990; Dyhrman et al., 2006; Yamaguchi and Adachi, 2010). Karenia brevis and K. mikimotoi generally have low half
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saturation constants for sources of DIP (Gentien, 1998; Vargo, 2009) and are able to use sources of DOP. In laboratory studies, Wilson (1996) reported utilization of phosphomonoester compounds by an axenic strain of K. brevis, and Yamaguchi and Itakura (1999) reported the utilization of these compounds by a Japanese strain of K. mikimotoi, as would be expected given phosphatase production by these species (Huang et al., 2007; Lin et al., 2012). However, to date, no studies have compared the abilities of axenic and nonaxenic strains of K. brevis and K. mikimotoi to directly utilize a variety of DIP and DOP sources for growth. This study evaluates the abilities of axenic and nonaxenic strains of K. brevis and K. mikimotoi clones isolated from Florida waters to use DIP and DOP compounds known or likely to be present in seawater. This work provides a better understanding of nutrient utilization by Karenia strains, ultimately contributing to our understanding of nutrient dynamics in these harmful algal bloom species. 2. Materials and methods To evaluate the abilities of axenic and nonaxenic K. brevis and K. mikimotoi to utilize different sources of P for growth, clones were grown on 29 different P compounds (Table 1) and final yields (maximum in vivo chlorophyll fluorescence, chlorophyll a content
Table 1 Compounds evaluated as the sole sources of phosphorus for growth of Karenia brevis and Karenia mikimotoi. The chemical formulas and abbreviations used in Figs. 1 and 2 are also listed. Compound Inorganic P Orthophosphate Sodium pyrophosphate Sodium tripolyphosphate Sodium trimetaphosphate Organic P Phosphomonoesters Glucose-6-phosphate Ribose-5-phosphate Fructose 1,6-bisphosphate b-Glycerophosphate Phosphoenolpyruvic acid Nicotinamide adenine dinucleotide phosphate Phytate (myo-inositol hexakisphosphate) Adenosine-5-diphosphate Adenosine-5-triphosphate Cytidine-5-monophosphate Uridine-5-monophosphate Adenosine-5-monophosphate Guanosine-5-monophosphate Uridine-5-diphosphoglucose Phosphodiesters Adenosine 30 ,50 -cyclic monophosphate Guanosine 30 ,50 -cyclic monophosphate Ribonucleic acid Deoxyribonucleic acid Bis (p-nitrophenyl) phosphate Phosphonates 2-Aminoethylphosphonic acid Methylphosphonic acid Aminomethylphosphonic acid Phosphonoacetic acid Phosphonoacetaldehyde 2-Amino-3phosphonopropionic acid
Chemical formula
Abbreviation
NaH2PO4 Na2H2P2O7 Na5P3O106H2O Na3O9P3
Pi PP TPP TMP
C6H11Na2O9P2H2O C5H9O8Na2P2H2O C6H11O12P2Na3 C3H9O6Na2P5H2O C3H4NaO6PH2O C21H84N7O17P3XH2O
G6P R5P FBP GYP PEP NADP
C6H16O24P6K2
PHY
C10H15N5O10P2 C10H14N5O13P3Na2 C9H12N3Na2O8P C9H11N2O9PNa2 C10H14N5O7PH2O C10H12N5Na2O8P C15H24N2O17P2Na2
ADP ATP CMP UMP AMP GMP UDPG
C10H11N5O6PNa
cAMP
C10H11N5O7PNa
cGMP
– – C12H9N2O8P
RNA DNA bNPP
C2H8NO3P
AEP
CH5O3P NH2CH2P(O)(OH)2
MP AMPA
C2H5O5P C10H23O5P C3H8NO5P
PAA PAC APP
31
and cell abundance) were compared. Clones of K. brevis (CCFWC257) and K. mikimotoi (CCFWC67) isolated from Florida and in culture at the Florida Fish and Wildlife Conservation Commission were used during these bioassays. Axenic cultures established via antibiotic treatment were confirmed to be free of bacteria by epifluorescence microscopy (Porter and Feig, 1980; Guillard, 2005). Prior to bioassays, stock cultures were maintained for several weeks in 3-L Fernbach flasks of GSe medium minus soil extract in 0.22 mm filter-sterilized, autoclaved Gulf of Mexico seawater (Blackburn et al., 2001) at a salinity of 35 and temperature of 20 0.5 8C. Light was supplied by full spectrum and cool white fluorescent lights (Ecolux F40C50, Philips F40T12) on a 12-h light/dark cycle at a saturation irradiance of 80 mmol photons m2 s1. To deplete P stores in inocula, stocks were grown in batch culture in P-limited media (GSe minus soil extract with 500 mM NaNO3 and 5 mM NaH2PO4) until cultures reached stationary phase and total dissolved phosphorus concentrations were undetectable. Total dissolved P concentrations in the stocks were verified following Soloranzo and Sharp (1980) using a Beckman Coulter DU 530 spectrophotometer. Bioassays were conducted in triplicate in 25 150 mm Pyrex test tubes. Tubes were inoculated with P-depleted stock cultures, resulting in an initial cell abundance of 2000 cells mL1. Experimental treatments were obtained by adding each of the P compounds at a final concentration of 3 mM P, and no P was added to the control tubes. Culture conditions (e.g., light, P-limited media) were otherwise the same as those described for the maintenance of stock cultures. Growth was monitored daily, four hours into the light cycle, by measuring in vivo chlorophyll fluorescence of the actively growing cultures with a Turner Designs 10 AU fluorometer. When maximum in vivo fluorescence was attained, after one and a half to two weeks, samples for subsequent quantification of chlorophyll a content and cell abundance were taken from each of the tubes. Chlorophyll a was extracted with methanol and concentrations were determined fluorometrically (Holm-Hansen and Riemann, 1978). Cell abundance was estimated using a Beckman Coulter Z2 Coulter Counter and verified by microscopy. Maximum growth rates were determined by a linear least squares regression of the natural log of in vivo fluorescence versus time. No measurements of P substrates were made during the experiments. Differences in growth responses to the P compounds were evaluated separately for axenic and nonaxenic cultures of each species. Differences in average maximum growth rate (mmax), in vivo fluorescence, chlorophyll a content, and cell abundance between the treatments were examined using 1-way ANOVA with post hoc Tukey tests using Minitab 17. Data were rank-transformed to meet the assumptions of normality and equal variance. Because multiple tests were conducted, a (initially 0.05) was adjusted to 0.002 using a Bonferroni correction to reduce Type I error rates (Quinn and Keough, 2002). 3. Results The axenic and nonaxenic K. brevis and K. mikimotoi cultures exhibited differences in their abilities to use DIP and DOP (Figs. 1 and 2, Supplementary Tables 1 and 2). As exhibited by final yields in maximum in vivo fluorescence, cell abundance, and chlorophyll a content, the axenic cultures did not generally grow on the phosphomonoester phytate, the phosphodiesters, or the phosphonates. One exception was the axenic K. brevis culture, which exhibited significantly higher chlorophyll a concentrations and cell abundance when grown on RNA as the sole P source, compared to the control (Fig. 1, Supplementary Table 1). The nonaxenic K. brevis culture did not grow on the phosphodiester guanosine 30 ,50 -cyclic monophosphate and four of the six phosphonates, whereas the nonaxenic K. mikimotoi culture did not grow on five of the six
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Fig. 1. Maximum growth rates (mmax, assessed by in vivo fluorescence), maximum in vivo fluorescence, cell abundance, and chlorophyll a concentrations of axenic (left) and nonaxenic (right) K. brevis cultures. Bars represent means standard deviations of three replicates. See Table 1 for full names of compounds and Supplementary Table 1 for results of statistical comparisons of treatment differences.
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Fig. 2. Maximum growth rates (mmax, assessed by in vivo fluorescence), maximum in vivo fluorescence, cell abundance, and chlorophyll a concentrations of axenic (left) and nonaxenic (right) K. mikimotoi cultures. Bars represent means standard deviations of three replicates. See Table 1 for full names of compounds and Supplementary Table 2 for results of statistical comparisons of treatment differences. ‘nd’ indicates that no data were collected for these treatments.
phosphonates (Figs. 1 and 2). Average maximum chlorophyll a content per cell in the axenic and nonaxenic K. brevis cultures, respectively, ranged from 11 to 13 pg cell1 and 12 to 16 pg cell1 for P compounds that supported growth and from 8 to 9 pg cell1 and 7 to 10 pg cell1 for P compounds on which no growth occurred. A similar pattern was seen for the axenic and nonaxenic
K. mikimotoi cultures, respectively, with average maximum chlorophyll a content per cell ranging from 20 to 24 pg cell1 and 16 to 22 pg cell1 when growth occurred and from and 12 to 14 pg cell1 and 9 to 11 pg cell1 when no growth occurred. Overall, yields (maximum in vivo fluorescence, cell abundance, chlorophyll a content) of all cultures tended to be greater on DIP
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than DOP compounds (Figs. 1 and 2, Supplementary Tables 1 and 2). Yields of the nonaxenic K. brevis culture were significantly reduced on most of the phosphodiesters that supported growth, compared to yields on DIP, phosphomonoesters or phosphonates that supported growth (Fig. 1, Supplementary Table 1). This pattern was not observed in K. mikimotoi. Lags in growth on a number of the organic P compounds were observed in both of the nonaxenic cultures – in K. brevis on the phosphomonoester phytate (PHY); phosphodiesters adenosine 30 ,50 -cyclic monophosphate (cAMP) and guanosine 30 ,50 -cyclic monophoshpate (cGMP); and phosphonates 2-aminoethylphosphonic acid (AEP) and phosphonoacetic acid (PAA); and in K. mikimotoi on the phosphomonoester phytate (PHY); phosphodiesters adenosine 30 ,50 -cyclic monophosphate (cAMP), guanosine 30 ,50 -cyclic monophosphate (cGMP), and bis (p-nitrophenyl) phosphate (bNPP); and the phosphonate phosphonoacetic acid (PAA) (Fig. 3). 4. Discussion 4.1. Inorganic nutrient sources This work illustrated that growth of K. brevis and K. mikimotoi varied with the source of P used as a substrate. It confirmed that K. brevis and K. mikimotoi use sodium pyrophosphate, sodium trimetaphosphate, and sodium tripolyphosphate, and that these compounds, as well as orthophosphate, produce some of the highest yields (Figs. 1 and 2). Prior reports of Karenia growth on DIP compounds have largely been limited to orthophosphate (Wilson,
1996; Vargo, 2009; Steidinger, 2009), with the exception of work on a Japanese clone of K. mikimotoi, which also reported higher yields on DIP compared to DOP (Yamaguchi and Itakura, 1999). Greater yields on DIP is likely due to the simplicity of orthophosphates, the ease with which sodium phosphates are hydrolyzed into orthophosphate, and the natural hydrolysis of polyphosphates in water (Cembella et al., 1984; Bjorkman et al., 2000; TorresDorante et al., 2005). 4.2. Phosphomonoesters Among the phosphomonoesters examined, phytate was unique in that it could not be used by the axenic K. brevis or K. mikimotoi cultures (Figs. 1 and 2) – consistent with observations for other eukaryotic algae and the hydrolysis of phytate by phytase rather than alkaline phosphatase, found in algae (Turner et al., 2002). Microorganisms known to hydrolyze phytate include species of bacteria, cyanobacteria, and protists (Whitton et al., 1991; Ziemkiewicz et al., 2002; Lim et al., 2007). Cembella et al. (1984) reported use of phytate by various species including Phaeocystis pouchetti, Phaeodactylum tricornutum and Pyrocystis noctiluca, although axenic cultures were not always used – highlighting a potential role of co-occurring bacteria in nutrient utilization. In this study, the nonaxenic cultures grew on phytate after a pronounced initial lag in growth (Fig. 3), suggesting that cooccurring bacteria may have degraded the phytate and released orthophosphase, which in turn supported Karenia growth. It should be noted, however, that for this and other compounds, no attempt was made to track the bacterial communities associated with strains or nutrient concentrations through time. To define the role of bacterial communities associated with Karenia strains in nutrient utilization (or as a potential food source, given the potential for mixotrophy), directed studies focused on bacterial dynamics are needed. The similar yields and maximum growth rates of the axenic K. brevis and K. mikimotoi grown on the phosphomonoesters that supported growth (Figs. 1 and 2) indicates no strong preference for any specific compound or type, consistent with previous findings (Wilson, 1996; Yamaguchi and Itakura, 1999) and the production of alkaline phosphatase by K. brevis (Vargo and Shanley, 1985; Craney et al., 2004; Vargo, 2009). Note that the higher growth rate of the nonaxenic culture of K. mikimotoi (0.20 da1) on phytate is likely an experimental artifact, a result of growth rate determination immediately following a prolonged initial lag. 4.3. Phosphodiesters
Fig. 3. In vivo fluorescence of nonaxenic K. brevis and K. mikimotoi cultures in which lags were observed. For comparison, the treatments that did not exhibit lags (‘others’) and control are shown. See Table 1 for full names of compounds.
The inability of the axenic cultures and ability of the nonaxenic cultures to grow on phosphodiesters as the sole source of P suggests a potential role of co-associated bacterial communities in rapidly hydrolyzing phosphodiesters and freeing P for growth by Karenia species. The use of some but not all of phosphodiesters in the nonaxenic cultures suggests that different bacterial communities may produce different phosphodiesterases, which are used to hydrolyze phosphodiesters (Ammerman and Azam, 1981, 1991; Suzumura et al., 1998; Karl and Bjo¨rkman, 2002), and that distinct bacterial assemblages will affect nutrient utilization in phytoplankton communities. Little is known about the ability of marine microalgae without associated bacteria to access biomolecules with phosphodiester bonds (e.g., nucleic acids and cyclic nucleotides), although the use of phosphodiesters as the sole source of P for growth have been reported in Phaeodactylum tricornutum and Chaetoceros ceratosporus (Flynn et al., 1986; Yamaguchi et al., 2005). Moreover, our work is consistent with the previous finding that axenic K. mikimotoi was unable to use bis p-nitrophenyl phosphate to grow (Yamaguchi et al., 2005).
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4.4. Phosphonates
References
The discovery of Trichodesmium species capable of phosphonate mineralization has led to the reconsideration of phosphonates as potential sources of bioavailable P (Dyhrman et al., 2006). The inability of axenic cultures of K. brevis to grow on any of the phosphonates indicates that this strain does not produce either C– P hydrolase or C–P lyases needed to access phosphonate-P, consistent with other studies that show phosphonates are unavailable to eukaryotic phytoplankton and are mineralized primarily by cyanobacteria and bacteria (Kononova and Nesmeyanova, 2002; Dyhrman et al., 2009). In contrast, growth of the nonaxenic K. brevis on the phosphonates 2-aminoethylphosphonic acid (AEP) and phosphonoacetic acid (PAA) and nonaxenic cultures of K. mikimotoi on phosphonoacetic acid, but with pronounced initial growth lags (Figs. 1–3), suggests that bacteria in the cultures may have produced the hydrolytic enzymes needed for these phosphonates. Gilbert et al. (2009) reported that marine bacteria in coastal waters use phosphonoacetic acid as a sole source of P, and Cook et al. (1978) found that orthophosphate was liberated when bacteria were grown on 2-aminoethylphosphonic acid as the sole carbon source in the presence of adequate N and P. The inability of K. mikimotoi to grow on 2-aminoethylphosphonic acid suggests that no 2-aminoethylphosphonic acid-mineralizing bacteria were present in the culture. Presumably, no bacteria capable of hydrolyzing the other phosphonates were present in either of the nonaxenic cultures.
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5. Conclusions This work demonstrated that K. brevis and K. mikimotoi are able to utilize a diverse variety of P sources, some without preconditioning (e.g., phosphomonoesters); this ability may offer a competitive advantage to these species when P is limiting to growth. In addition, this work demonstrated that bacteria may play a key role in nutrient utilization by Karenia species and that associated bacterial communities can differentially permit access to different nutrient pools. The indirectly bacteria-mediated supply of P by DOP mineralization may be particularly important for larger blooms that often closely follow the decay of Trichodesmium blooms. Consequently, bloom development of K. brevis and K. mikimotoi on the west Florida shelf would be expected to be influenced at times by the co-occurring bacterial assemblage’s exploitation and mineralization of constituents of the DOP pool not directly accessible to Karenia. Since natural and anthropogenic nutrient sources may vary widely in both type and amount of DIP and DOP compounds, bloom mitigation efforts that include land-based nutrient management strategies should consider the ability of both K. brevis and K. mikimotoi to access a wide range of DIP and DOP forms. Author contributions B.R. designed and performed the research. A.A.C. and B.R. analyzed the data and wrote the paper. Acknowledgments This study was funded by the Florida Fish and Wildlife Conservation Commission. [SS] Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.hal.2015.06.005.
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