CRYPTIC DIVERSITY IN PHYTOPLANKTON CULTURES IS REVEALED USING A SIMPLE PLATING TECHNIQUE¹

Abstract

A simple technique has been developed to probe the occurrence of cryptic genetic diversity in populations of laboratory-maintained phytoplankton cultures. This agarose-based method allows the investigator to plate a broad range of fragile algae, including representatives of the Raphidophyceae, Synurophyceae, Prymnesiophyceae, and Dinophyceae. Heterosigma akashiwo (Hada) Hada ex Y. Hara et Chihara was selected as a model system for our genetic diversity study. Further optimization of the plating technique for this alga demonstrated that colony formation was independent of the physiological state of the parent culture and yet dependent on incident light intensity. The density at which cells were plated affected colony formation and the rate of growth, with intermediate densities (10 3 cells per plate) performing best. High-metal stress was used as the selective screen for assessing genetic variability within a single H. akashiwo culture. To this end, ''clonal'' lines (quotation marks used to indicate that within-clone diversity was expected) were generated from individual plated colonies, and their tolerance was measured by plating on selective medium. Results of these experiments suggest the following: (i) ''clonal'' lines generated from a single H. akashiwo culture displayed a significant variation in stress tolerance; (ii) ''clonal'' lines chosen for their ability to grow on selective plates retained this tolerance in the absence of stress, indicating that the observed variation is heritable; and (iii) genetic variation is continually generated in growing cultures. Our results are consistent with a conceptual model, presented here, in which stress tolerance among the individuals in a culture has a genetic basis that varies over a continuous spectrum.

Semi-quantitative assay for polyketide prymnesins isolated from Prymnesium parvum (Haptophyta) cultures

“…In a sterile transfer hood, 0.5e1.0 mL of the irradiated sample was transferred to a 50-mL tube, and the total volume was brought to 30 mL with growth medium containing 50 mg mL À1 streptomycin sulfate. Plating essentially followed the protocol of Lakeman and Cattolico (2007), whereby this mixture was added to 6.6 mL prewarmed (70 C) 1.0% (w/v in growth medium) Omnipure agarose (EMD Millipore Chemicals, Billerica, Massachusetts, USA), swirled and immediately poured into a sterile Petri dish that was subsequently wrapped with aluminum foil and left in the dark for 3e4 days before being exposed to light. After 3 months of growth under our standard illumination conditions, individual colonies were isolated by coring the plate with a micropipette and transferring clones to 96-well microtiter plates containing 250 mL of growth medium with 50 mg mL À1 streptomycin.…”

The strain concept in phytoplankton ecology

“…Spontaneous mutation is an inevitable consequence of DNA replication and is thus unavoidable in any growing culture. Spontaneous mutants have been shown to arise at non-trivial rates in cultures of phytoplankton (Reboud and Bell, 1997;Costas et al, 1998;Kassen and Bell, 1998;Goho and Bell, 2000;Colegrave et al, 2002;Collins and Bell, 2004;Lakeman and Cattolico, 2007). Most mutations are expected to have no effect on the phenotype.…”

“…However, because of rapid growth rates and high population densities in laboratory cultures, rare mutations that do affect the phenotype are likely to arise. For example, mutations affecting stress tolerance accumulated when sub-clones of a clonal Heterosigma akashiwo culture were grown in parallel for approximately a month (Lakeman and Cattolico, 2007). Such spontaneous mutations may have significant impacts on fitness.…”

Intra- and interspecies differences in growth and toxicity of Pseudo-nitzschia while using different nitrogen sources

“…However, Wood and Leatham (1992) argue that difficulties arise when culture studies involving only a few clones are used in an attempt to define interspecies differences without assessing within species variation, not necessarily as the result of a fault in the species definition. The number of isolates necessary to accurately define within species variation using statistical methods is difficult to define and restricts the number of studies able to include such an analysis (Lakeman and Cattolico 2007).…”