Vol. 416: 69–78, 2010 MARINE ECOLOGY PROGRESS SERIES Published October 14 doi: 10.3354/meps08788 Mar Ecol Prog Ser OPENPEN ACCESSCCESS Allelopathic compounds of a red tide dinoflagellate have species-specific and context-dependent impacts on phytoplankton Kelsey L. Poulson1,*, R. Drew Sieg1,*, Emily K. Prince1, Julia Kubanek1, 2,** 1School of Biology, Georgia Institute of Technology, 310 Ferst Drive, Atlanta, Georgia 30332–0230, USA 2School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive, Atlanta, Georgia 30332–0400, USA ABSTRACT: The use of chemical compounds to suppress the growth of competitors is a competitive strategy known as allelopathy that can be readily observed with many phytoplankton species in lab- oratory studies. However, it is unclear how these allelopathic interactions are altered when the com- plexity of the system is increased to more closely mimic natural conditions. In the present study, we conducted laboratory experiments to decipher how the identity, abundance, and growth stage of competitors affect the outcome of allelopathic interactions with the red tide dinoflagellate Karenia brevis. Multiple chemical compounds produced by K. brevis were found to inhibit the growth of 4 phytoplankton competitors, although these competitors were susceptible to different combinations of compounds. We found that physiological state and cell concentration of competitors were important determinants of allelopathy, with early-stage (lag phase) cells more vulnerable to allelopathic effects than later growth stages for the diatom Skeletonema grethae. Despite being allelopathic to multiple competitors in the laboratory, in a microcosm experiment using plankton field assemblages, extracel- lular extracts of 2 strains of K. brevis had no effects on some taxa although they stimulated the growth of some diatoms. This suggests that in a species-rich ecological community under oligotrophic condi- tions, the relative importance of K. brevis allelopathy may not be as high as most laboratory studies predict. KEY WORDS: Allelopathy · Competition · Phytoplankton · Red tide Resale or republication not permitted without written consent of the publisher INTRODUCTION by both biotic and abiotic factors, making it difficult to draw conclusions from simplified laboratory studies. In planktonic systems, the use of chemical com- The presence of specific competitor species can induce pounds to kill or slow the growth of competitors, a pro- the production of allelopathic compounds (Vardi et al. cess known as allelopathy, may confer a selective 2002) or undermine the effectiveness of allelopathy advantage to phytoplankton that are weak exploitation (Prince et al. 2008b). Abiotic influences, such as nutri- competitors (Adolf et al. 2006, Tillmann et al. 2008, ent concentrations, salinity, light intensity, and temper- Poulson et al. 2009). Many groups of phytoplankton, ature, can affect allelopathic potency, as found for the including dinoflagellates (Kubanek et al. 2005, Adolf et haptophyte Prymnesium parvum (Graneli & Salomon al. 2006, Tillmann & Hansen 2009), haptophytes (Uro- 2010). Additionally, allelopathic potency can vary nen et al. 2007), raphidophytes (Yamasaki et al. 2009), among strains, as shown with the dinoflagellates diatoms (Hansen & Eilertsen 2007, Ribalet et al. 2007), Alexandrium ostenfeldii (Tillmann et al. 2008), Karenia and cyanobacteria (Suikkanen et al. 2006) are allelo- brevis (Kubanek et al. 2005), and Karlodinium venefi- pathic towards co-occurring species. However, the cum (Adolf et al. 2006), making it difficult to predict effectiveness of phytoplankton allelopathy is affected allelopathic outcomes of genetically diverse blooms. **These authors contributed equally to this paper © Inter-Research 2010 · www.int-res.com ** Corresponding author. Email: [email protected] 70 Mar Ecol Prog Ser 416: 69–78, 2010 Although allelopathy is becoming increasingly ap- ural, non-bloom Florida plankton assemblage. Addi- preciated based upon laboratory results, the relevance tionally, since K. brevis allelopathy can be undermined of allelopathy in ecological settings has been chal- by other competitor species (Prince et al. 2008b), we lenged, particularly in the process of bloom formation investigated how species composition, growth stage, (Flynn 2008, Jonsson et al. 2009). A few micro- and and cell concentration of competitors influence allelo- mesocosm studies have shown that allelopathic effects pathic potency. can be observed within complex plankton communi- ties, but these effects may be dampened depending on environmental conditions (Fistarol et al. 2004, Suikka- MATERIALS AND METHODS nen et al. 2005). When studying allelopathy, pair-wise interactions between competitors are often investi- Phytoplankton culturing. Non-axenic clones of the gated using extracellular extracts or cell-free filtrates diatoms Asterionellopsis glacialis (strain CCMP 137), (e.g. Kubanek et al. 2005, Prince et al. 2008a, Yamasaki Skeletonema grethae (CCMP 775), and Amphora sp. et al. 2009), but it is unlikely that outcomes in a diverse (CCMP 129); the dinoflagellates Akashiwo sanguinea plankton community will be accurately predicted from (CCMP 1740), Prorocentrum minimum (CCMP 695), the sum of these pair-wise interactions. Additionally, and Karenia brevis (CCMP 2228, hereinafter ‘2228’) most studies have utilized crude extracts or cell-free were obtained from the Provasoli-Guillard National filtrates, which do not allow researchers to test for the Center for Culture of Marine Phytoplankton (CCMP). presence of multiple allelopathic compounds of vary- An additional strain of K. brevis (TxB3, hereinafter ing potency released by cells. However, in order to ‘TxB3’) was obtained from Texas A&M University. All efficiently identify allelopathic compounds or the cultures were maintained at 22°C with a 12 h light:12 h mechanism(s) by which these compounds affect com- dark cycle in a Percival incubator with Philips petitors, simpler pair-wise laboratory experiments are F32T8/TL741 Universal/Hi-Vision fluorescent bulbs invaluable. Overall, more field-based and multi- mounted vertically, producing irradiance of 100 to species studies of allelopathy and its role in community 145 μmol m–2 s–1 (Biospherical Instruments, model and bloom dynamics are needed in order to comple- QSL2100). All phytoplankton cultures were grown in ment mechanistic laboratory-based investigations. L1 + silicate media made with 0.2 μm filtered natural Karenia brevis is an allelopathic red-tide dinoflagel- seawater from Boothbay Harbor, Maine, USA (salinity late that blooms most years in the Gulf of Mexico 35, CCMP). Seawater was stored in the dark at 5°C (Tester & Steidinger 1997). Neurotoxic brevetoxins until use. Growth curves and cell concentrations were produced by K. brevis cause massive fish kills (Lands- generated using an Olympus IX-50 inverted micro- berg et al. 2009) and have been shown to accumulate scope with a Palmer-Maloney settling chamber on cul- in shellfish (Plakas et al. 2002), and trophic transfer of ture samples preserved with acidified Lugol’s solution. these compounds can result in marine mammal mortal- Generation of extracellular extracts. To obtain ity (Flewelling et al. 2005). Nevertheless, these toxins extracellular extracts of Karenia brevis cultures used in do not appear to be responsible for allelopathy towards high-performance liquid chromatography (HPLC) and most phytoplankton competitors (Kubanek et al. 2005, subsequent species-specificity experiments (see ‘Test- Prince et al. 2008a). Previous studies showed that ing species-specificity of allelopathy’), a mixture of 3 extracellular extracts and filtrates from both K. brevis adsorbent resins that remove lipophilic organic mole- cultures and blooms were allelopathic to multiple com- cules from aqueous media were added to K. brevis cul- petitors, although some species were resistant tures that were in exponential growth stage (3.5 × 103 (Kubanek et al. 2005, Prince et al. 2008a). K. brevis to 1.6 × 104 cells ml–1; sensu Prince et al. 2006). This produces multiple allelopathic compounds (Prince et method allows for the extraction of lipophilic com- al. in press) which may allow K. brevis to suppress sev- pounds released by cells into the media without eral competitors simultaneously. To further explore extracting intracellular material. For all other experi- this hypothesis, we investigated the species-specificity ments, a modified protocol was used to reduce the of allelopathic compounds against a suite of suscepti- potential for false positives. These extracellular ble phytoplankton. extracts were obtained by adding 2 resins (XAD -7 and Although multiple studies have demonstrated the HP-20, Supelco) to cultures of K. brevis while they allelopathic effects of Karenia brevis against individual were in mid- to late exponential growth stage (9.8 × 103 competitor species, the relative importance of K. brevis to 3.0 × 104 cells ml–1), and incubated for 12 to 15 h. To allelopathy against a backdrop of other competitive clean the resins prior to addition into phytoplankton interactions is unknown. To investigate this, a micro- cultures, both resins were rinsed once with HPLC- cosm experiment was conducted in which the in- grade acetone and 8 times with HPLC-grade methanol. hibitory effect of K. brevis extracts was tested on a nat- Residual solvents were removed from resins with Poulson et al.: Allelopathic interaction of Karenia brevis with phytoplankton 71 deionized