Strong and widespread cycloheximide resistance in Stichococcus-like eukaryotic algal taxa

Abstract

This study was initiated following the serendipitous discovery of a unialgal culture of a Stichococcus-like green alga (Chlorophyta) newly isolated from soil collected on Signy Island (maritime Antarctica) in growth medium supplemented with 100 µg/mL cycloheximide (CHX, a widely used antibiotic active against most eukaryotes). In order to test the generality of CHX resistance in taxa originally identified as members of Stichococcus (the detailed taxonomic relationships within this group of algae have been updated since our study took place), six strains were studied: two strains isolated from recent substrate collections from Signy Island (maritime Antarctica) ("Antarctica" 1 and "Antarctica" 2), one isolated from this island about 50 years ago ("Antarctica" 3) and single Arctic ("Arctic"), temperate ("Temperate") and tropical ("Tropical") strains. The sensitivity of each strain towards CHX was compared by determining the minimum inhibitory concentration (MIC), and growth rate and lag time when exposed to different CHX concentrations. All strains except "Temperate" were highly resistant to CHX (MIC > 1000 µg/mL), while "Temperate" was resistant to 62.5 µg/mL (a concentration still considerably greater than any previously reported for algae). All highly resistant strains showed no significant differences in growth rate between control and treatment (1000 µg/mL CHX) conditions. Morphological examination suggested that four strains were consistent with the description of the species Stichococcus bacillaris while the remaining two conformed to S. mirabilis. However, based on sequence analyses and the recently available phylogeny, only one strain, "Temperate", was confirmed to be S. bacillaris, while "Tropical" represents the newly erected genus Tetratostichococcus, "Antarctica 1" Tritostichococcus, and "Antarctica 2", "Antarctica 3" and "Arctic" Deuterostichococcus. Both phylogenetic and CHX sensitivity analyses suggest that CHX resistance is potentially widespread within this group of algae.

Figure 1

Mean (± standard error) of cell density achieved by each of six strains of Stichococcus-like algae and single strains of Chlorella and Coccomyxa in BBM liquid medium supplemented with different CHX concentrations after two weeks’ incubation. Means sharing the same letter (a, b or c) are not significantly different (Tukey’s HSD, p < 0.05).

Figure 2

Mean (± standard error) of the growth rate of each of five strains of Stichococcus-like algae in control (BBM) versus BBM + 1000 µg/mL CHX. Means sharing the same letter (a, b) are not significantly different (Tukey’s HSD).

Figure 3

Mean (± standard error) of the lag time for each of five strains of Stichococcus-like algae to reach constant growth rate in control BBM, BBM + 0.5 µg/mL CHX and BBM + 1000 µg/mL CHX. Means with different letters (a, b, c) are significantly different (Tukey’s HSD, p < 0.05).

Figure 4

Maximum-likelihood tree based on concatenated 18S rDNA and ITS 2 sequences obtained in this study (in boldface). Numbers next to branches indicate statistical support value (maximum likelihood bootstrap/Bayesian posterior probabilities). Chlorella-like algal strains including Pseudochlorella, Koliella and Edaphachlorella were used as the outgroup. Scale bar represents 0.2 changes per site.