Growth and Grazing Kinetics of the Facultative Anaerobic Nanoflagellate, Suigetsumonas clinomigrationis

The functional and numerical responses of the facultative anaerobic heterotrophic nanoflagellate, Suigetsumonas clinomigrationis NIES-3647 to prey density were examined under oxic and anoxic conditions. S. clinomigrationis grew at temperatures between 10 and 30°C and in the salinity range of 3.9–36.9 psu. The maximum specific growth and ingestion rates of S. clinomigrationis were lower under anoxic conditions than under oxic conditions. Half-saturation constants for the growth of S. clinomigrationis were within or greater than the range of bacterial densities in the water column of Lake Suigetsu, suggesting that its growth rate is limited by bacterial prey densities in natural environments.

Supplementary Information for "Growth and Grazing Kinetics of the Facultative Anaerobic Nanoflagellate, Suigetsumonas clinomigrationis"

Strains
In the present study, we used S. clinomigrationis NIES-3647, isolated from the dissolved-oxygen depleted water just below the oxic-anoxic interface of the meromictic Lake Suigetsu (2). A facultative anaerobic Arcobacter sp. strain co01 was isolated from the polyxenic culture of S. clinomigrationis by plating on 1.5% (w/v) agar plate of YE100oxi medium (2). Phylogenetic analysis of 16S rRNA gene of this bacterial strain showed that the nearest neighbour of the isolate was the Arcobacter cloacae strain SW28-13 (Accession no. NR_117570), with sequence similarity of 99% (data not shown). The 16S rRNA gene sequence of Arcobacter sp. co01 was deposited in DDBJ under accession number LC198182.

Establishment of monoxenic culture
To avoid the effect of changes in the bacterial community on the physiology of S. clinomigrationis in the polyxenic culture, a monoxenic culture of S. clinomigrationis with Arcobacter sp. was established as follows. The polyxenic culture of S. clinomigrationis was filtered through a 10-µm membrane filter (Nuclepore, Whatman, Tokyo, Japan) without suction to remove the bacterial frocks and filamentous bacteria. The filtrate was then passed through a 2.0-µm pore membrane filter (Nuclepore, Whatman) without suction, and the filter was washed with autoclaved lake water three times. The culture on the filter was transferred to a plastic tube. An aliquot (1 mL) of the S. clinomigrationis culture was inoculated into a 9-mL culture of Arcobacter sp. containing streptomycin at a final concentration of 100 µg mL −1 and incubated at 20 °C in the dark. After 2 days of incubation, 1 mL of the culture was again inoculated into a 9-mL culture of Arcobacter sp. containing streptomycin. These inoculations were repeated four times until the establishment of monoxenic culture. The monoxenic culture was checked by epifluorescence microscopic examination of 4'-6-diamidino-2-phenylindole (DAPI)-stained bacterial cells and sequence analysis of PCR-amplified 16S rRNA gene using the DNA extracted from the culture as a template. The monoxenic culture of S. clinomigrationis and Arcobacter sp. was maintained in the YE100ppm medium (2) at 20 °C in the dark.
Growth temperature and salinity S. clinomigrationis was cultured in YE100oxi medium for one week at 20 °C in the dark until the late exponential growth phase. Arcobacter sp. was cultured in YE1000oxi medium (1000 mg L −1 yeast extract in the water taken from oxic-anoxic interface of Lake Suigetsu) for three days. The Arcobacter sp. cells were collected by centrifugation at 2,330 × g for 15 min at room temperature and resuspended in diluted (4×) Daigo's artificial seawater SP (Nihon Pharmaceutical, Tokyo, Japan). Then, 1 mL of each culture was inoculated into 18 mL of YE100oxi medium to obtain the initial S. clinomigrationis density of 10 2 to 10 3 cells mL −1 and the initial Arcobacter sp. density of ca. 10 8 cells mL −1 . This bacterial density did not limit the growth of S. and Arcobacter sp. were counted in the same way as in the growth temperature experiment.
Numerical response Arcobacter sp. was aerobically cultured in YE1000ppm medium (lake water in the YE1000oxi medium was

Functional response
For the analysis of the grazing kinetics of S. clinomigrationis, the pre-culture and experimental cultures were prepared in the same way as for the growth kinetics experiment. Bacteria ingestion rates were estimated by the short-term tracer method using 0.5-µm diameter fluorescently labelled beads, FLBeads (Fluoresbrite ® Microparticles, Polysciences, PA, USA). The FLBeads were added to the each tube at ca. 10% of the inoculated Arcobacter sp. density.
In the preliminary experiments, the total number of FLBeads ingested by S. clinomigrationis increased linearly within 60 min of incubation (data not shown). Thus, the test tubes were incubated for 60 min at 25 °C in the dark. After incubation, a 1-mL subsample was taken and fixed immediately with an equal volume of ice-cold 4% (v/v) glutaraldehyde buffered with SCB to stop the egestion of surrogates taken into the food vacuoles of the nanoflagellate (4). To account for FLBeads adsorbed on the cell surfaces of S. clinomigrationis, a zero-time control was taken and fixed as described above. The fixed samples were filtered on 0.8-µm black Nuclepore membrane filters and stained with primulin solution (250 µg mL −1 ; 1). At least 100 cells in each sample were inspected using epifluorescence microscopy under UV excitation. The FLBeads in the food vacuoles were counted under blue-light excitation (1). The specific ingestion rate (I; bacteria flagellate −1 h −1 ) of S. clinomigrationis was calculated as follows: where G f is the number of FLBeads ingested by the flagellate, and N b and N f are the total bacterial and FLBead densities, specific ingestion rates and the initial bacterial densities fitted to a hyperbolic function I = Imax × B / (Ki + B), where Imax is the maximum specific ingestion rate, Ki is the half-saturation constant and B is the initial density of Arcobacter sp. The parameters Imax and Ki were calculated using Michaelis-Menten model in the KaleidaGraph programme. These parameters were expressed as Imax oxi and Ki oxi under oxic conditions, and Imax anoxi and Ki anoxi under anoxic conditions.

Statistical analysis
Bartlett's test of equal variance was followed by one-way ANOVA or Kruskal-Wallis one-way ANOVA for the data on growth rates and maximum cell yields under different conditions of temperature and salinity. Differences were assessed by post-hoc Bonferroni's multiple comparison test and the threshold of significance was set at p = 0.05 for all the analyses.