Dataset: Dinoflagellate Evo
Deployment: lab_Hutchins_09-10_plankton

Comparing short vs. long term acclimation of a dinoflagellate community

Principal Investigator:

David Caron (University of Southern California, USC)

Co-Principal Investigator:

Feixue Fu (University of Southern California, USC)

David A. Hutchins (University of Southern California, USC)

Astrid Schnetzer (University of Southern California, USC)

Contact:

Avery Tatters (University of Southern California, USC)

BCO-DMO Data Manager:

Robert C. Groman

Project:

Experimental studies to understand and evaluate acclimation of marine plankton assemblages to increased CO2 and temperature (Plankton acclimation)

Current State:

Preliminary and in progress

Version:

11-20-2012

Version Date:

2012-11-20

Data URL:

https://www.bco-dmo.org/dataset-deployment/455549/data

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Description

Experimental design
A flow chart of the experimental design is shown in Figure 1, including collection and incubation of the natural bloom in a short-term (2 week) pCO2 experiment, isolation of cells of all four species from each pCO2 treatment, conditioning of the isolates at the pCO2 from which they were isolated for one year, and recombining isolates into artificial communities to compete for 2-week periods following 4, 8 and 12 months of conditioning. Growth rates of each species were assessed during conditioning in unialgal cultures at the 8-month timepoint, as well as in mixed communities during the initial bloom experiment and the 12-month artificial community experiment. “Switch” competition experiments in which clones conditioned at each pCO2 were competed in artificial communities at the other two pCO2 levels were also performed after 12-months. The switch competition data are in a separate data set.

Methods & Sampling

Dataset acquisition description

Initial pCO2 incubation experiment

A mixed natural dinoflagellate bloom dominated by Lingulodinium polyedrum, Prorocentrum micans, Alexandrium sp. and Gonyaulax sp. at a total cell density of ~700 cells per ml was collected off Venice Beach, California in September, 2009. This large regional bloom extended throughout the Southern California Bight region. Samples were collected near shore for both the initial incubations and all experimental dilution water used throughout the 12-month experiment.

The experiment was incubated in the laboratory at 18 degrees C under 90 photons per meters squared per second of cool white fluorescent illumination on a 14-h light: 10-h dark cycle. Triplicate sterilized 1 L polycarbonate bottles were gently bubbled (60 bubbles per minute) using commercially prepared air/CO2 mixtures (230 ppm, 433 ppm and 765 ppm, Praxair Gas). Preliminary experiments verified that growth rates of cultures bubbled at this rate were not significantly different from those of unbubbled cultures (data not shown), and these methods have been employed for other CO2 experiments (Hutchins et al. 2007; Fu et al. 2007), including dinoflagellate studies (Fu et al. 2008; 2010). Filtered seawater was amended with L1/20 nutrient, vitamin and trace metal concentrations (Guillard and Hargraves 1993), except NH4Cl+ was substituted for NaNO3- and silicate was omitted. Nutrient concentrations at the Redfield ratio (by atoms) of 16 N: 1 P (Redfield 1958) were added initially to the incubation bottles, and replenished once at the one week dilution (below).

The CO2-amended treatments were maintained in active growth using semi-continuous culture methods (Tatters et al. 2012). Each bottle was diluted to the original time-zero in vivo chlorophyll a fluorescence value after one week with nutrient-amended filtered seawater. Aliquots were removed initially, and after one and two weeks for examination of carbonate buffer system parameters and community structure using microscopic cell counts. Samples for cell counts were obtained at the 1 week time point (after dilution), and after the 2 week incubation, in order to calculate acclimated growth rates (1-2 week rates) and final abundances of all species.

Clonal culture isolations

Three individual cells representing the four dominant genera were isolated from each incubation bottle at the end of the two-week incubation of the natural community, and maintained in long-term culture (52 weeks) at the pCO2 from which they were obtained under conditions of temperature, light, nutrients, CO2 bubbling, etc., identical to the 2-week natural community experiment. Cultures were maintained in exponential phase using autoclave-sterilized enriched seawater growth medium with semi-continuous weekly dilutions based on specific growth rates within each bottle, calculated as in Tatters et al. (2012). The approximate number of generations during this time period was: L. polyedrum (48-62), P. micans (58-71), Alexandrium sp. (34-38) and Gonyaulax sp. (75-126).

Artificial community competition experiments

The conditioned clonal cultures were recombined into artificial communities after 4, 8 and 12 months in the same relative proportions and cell densities as the original natural bloom assemblage. The 8- and 12-month experiments used triplicate communities of all four species, but due to logistical limitations, the 4-month experiment used only L. polyedrum, P. micans, and Alexandrium sp. in duplicate communities. Because Gonyaulax sp. was not included in this preliminary 4-month experiment and replication was different, it is not fully comparable to the other experiments. The dinoflagellates in the artificial community trials were allowed to compete under identical experimental conditions of light, temperature, nutrient availability, and pCO2 for the same time period and diluted exactly as in the original natural bloom incubation. Samples were collected for cell counts and carbonate system parameters (Table 1) in all experiments.

The initial bloom counts, per mL, are as follows:

Lingulodinium: 464
Prorocentrum: 50.5
Alexandrium: 20.5
Gonyaulax: 27

Three samples (A, B, C) were taken for each partial pressure of CO2. The partial pressures of CO2 were targeted at 190, 390, and 750 ppm but the biology can interfere a bit with the actual values achieved.

Literature Cited

Fu, F.-X., M. E. Warner, Y. Zhang, Y. Feng, and D. A. Hutchins. 2007. Effects of increased temperature and CO2 on photosynthesis, growth and elemental ratios of marine Synechococcus and Prochlorococcus (Cyanobacteria). J. Phycol. 43:485-496.

Fu, F.-X., A. R. Place, N. S. Garcia, and D. A. Hutchins. 2010. CO2 and phosphate availability control the toxicity of the harmful bloom dinoflagellate Karlodinium veneficum. Aquat. Mcrob. Ecol. 59: 55-65.

Fu, F.-X., Y. Zhang, M. E. Warner, Y. Feng, and D. A. Hutchins. 2008. A comparison of future increased CO2 and temperature effects on sympatric Heterosigma akashiwo and Prorocentrum minimum. Harmful Algae. 7:76-90

Hutchins, D. A., F.-X. Fu, Y. Zhang, M. E. Warner, Y. Feng, K. Portune, P. W. Bernhardt, and M. R. Mulholland. 2007. CO2 control of Trichodesmium N2 fixation, photosynthesis, growth rates, and elemental ratios: Implications for past, present and future ocean biogeochemistry. Limnol. Oceanogr. 52:1293-1304.

Redfield, A. C. 1958. The biological control of chemical factors in the environment. Am. Sci. 46:205-222.

Tatters, A. O., F.-X. Fu, and D. A. Hutchins. 2012. High CO2 and silicate limitation synergistically increase the toxicity of Pseudonitzschia fraudulenta. PLoS ONE 7(2):e32116.doi:10.1371/journal.pone.0032116.

Data Processing Description

Dataset Processing Description

Analytical methods

Cell counts and growth rates. Growth rates were measured for each species in mixed communities during the second week of the initial natural community incubation, and during the 12-month and switch artificial community experiments. Because of logistical limitations and the fact that dinoflagellates cannot be cryopreserved for subsequent growth rate measurements, growth rates were determined immediately for all isolates in unialgal culture only at the 8-month timepoint. These values are representative of their in long-term steady-state exponential growth rates throughout the conditioning period. Final cell abundances of each species were measured in every natural or artificial community competition experiment. Algal cells were preserved in acidified Lugol’s solution and enumerated using an Accu-Scope 3032 inverted microscope using the Utermöhl method (Utermöhl, 1931).

Carbonate buffer system. Dissolved inorganic carbon analysis used a CM5230 CO2 coulometer (UIC) (King et al. 2011). pH was determined on freshly collected samples using a calibrated Orion 5-star plus pH meter using an NBS buffer system with three-point calibration. Experimental pCO2 was calculated using CO2SYS software as in Tatters et al. (2012) (Table 1). Due to unavoidable minor variability in calculated pCO2 levels between experiments (largely from differences in batches of commercial gas mixtures), for clarity, throughout the text pCO2 values of 230-336 ppm are referred to as “low”, values of 433-506 ppm are referred to as “medium”, and values of 709-792 ppm are referred to as “high”.

Multivariate statistical methods

Multivariate analyses used the PRIMER v6 statistics package (Clarke and Warwick, 2001) with the PERMANOVA add-on. Final cell abundances for each species from replicate bottles were square-root transformed prior to community structure comparisons based on Bray-Curtis similarities and log-transformed species growth rates compared based on Euclidean distance measures. ANOSIM permutation tests were used to test the impact of differing pCO2 competition levels on overall community structure and on relative abundance of the four species at the end of all incubations, and on their growth rates in the initial natural community, 12-month and switch experiments. These tests resulted in R values and significance levels where R = 0 implies no difference among groups, and R = 1 suggests that group separation is so large that all dissimilarities among groups are larger than any dissimilarity within them (Clarke and Warwick, 2001).

Data from the natural community experiment and from competition trials after 8 and 12 months were further combined for PERMANOVA analyses to test for significant differences among and within predefined groups in response to both the pCO2 competition levels and the differing periods of conditioning to these pCO2 concentrations. PERMANOVA resulted in Pseudo-F and significance levels, where Pseudo-F = 1 implies a large overlap among sample groups being compared while Pseudo-F > 1 indicates little or no overlap between them (Anderson et al., 2008). PERMANOVA also allowed us to test for interaction between the factors pCO2 competition level and conditioning period in forcing overall community structure.

For the 12-month switch competition trials, we used final cell abundances from all treatments to examine the comparative effects of differing pCO2 competition levels and differing pCO2 conditioning levels. Using a two-way crossed design for the ANOSIM routine, we tested the average effect on overall community structure and on the four individual species separately of pCO2 levels during competition removing differences in conditioning pCO2, and the average effect of conditioning pCO2 removing differences in competition pCO2 (Clarke and Warwick, 2001).

Literature Cited:

Anderson M. J., R. N. Gorley, and K. R. Clarke. 2008. PERMANOVA+ for PRIMER: Guide to software and statistical methods. PRIMER-E: Plymouth, UK.

Clark, K. R., and R. M. Warwick. 2001. Change in marine communities: an approach to statistical analysis and interpretation. 2nd edition. PRIMER-E, UK.

King, A.L., S. A. Sañudo-Wilhelmy, K. Leblanc, D. A. Hutchins, and F.-X. Fu. 2011. CO2 and vitamin B12 interactions determine bioactive trace metal requirements of a subarctic Pacific diatom. Intl. Society of Microb. Ecol. J.5(8):1388-1396.

Tatters, A. O., F.-X. Fu, and D. A. Hutchins. 2012. High CO2 and silicate limitation synergistically increase the toxicity of Pseudo-nitzschia fraudulenta. PLoS ONE 7(2):e32116.doi:10.1371/journal.pone.0032116.

Utermöhl, H.1931. Neue Wege in der quantitativen Erfassung des Planktons. (Mit besonderer Beriicksichtigung des Ultraplanktons). Verh. Int. Verein. Limnol. 5:567-596.

More information about this dataset deployment

Funding

Award Number	Funding Source
OCE-0962309	NSF Division of Ocean Sciences

Instruments

None available yet

Parameters

Supplied Name	Supplied description	Supplied Units	Standard Name
incubation_period	Period of incubation	months	no_bcodmo_term
species	Species name	n/a	species
pCO2	Partial pressure of carbon dioxide		no_bcodmo_term
sample	Sample name, A, B, or C	n/a	no_bcodmo_term
growth_rate	Growth rate	divisions per day	no_bcodmo_term
count	Count of the cells	cell counts per mL	count_species

Database

Contribute Data

Dataset: Dinoflagellate Evo
Deployment: lab_Hutchins_09-10_plankton

Dataset acquisition description

Dataset Processing Description

Database

Contribute Data

Dataset: Dinoflagellate EvoDeployment: lab_Hutchins_09-10_plankton

Dataset acquisition description

Dataset Processing Description

Dataset: Dinoflagellate Evo
Deployment: lab_Hutchins_09-10_plankton