The B-Vitamin Mutualism Between the Dinoflagellate Lingulodinium polyedrum and the Bacterium Dinoroseobacter shibae

Recent research has shown that in aquatic systems pairs of prokaryote and eukaryote species exercise symbiotic exchanges of metabolites that are essential for the proliferation of either species. Using dinoflagellate Lingulodinium polyedrum cultures and a factorial design, we examined its growth at different concentrations of vitamin B₁ (thiamine) and B₁₂ (cobalamin). When both vitamins were at their lowest concentrations tested, 0.033 pM of B₁ and 0.053 pM of B₁₂ the growth was limited. When axenic L. polyedrum was co-cultured with the bacterium Dinoroseobacter shibae, a known B₁ and B₁₂ producer, then L. polyedrum grew at the same rate as in culture media supplemented with B₁ and B₁₂. In the L. polyedrum vitamin—limited culture (V-L), the abundance of attached and free-living D. shibae was higher than in the vitamin—replete (V-R) culture. In the V-R and V-L co-cultures the measured particulate B₁₂ (pB₁₂) concentration of attached and free-living D. shibae were in the range of 4.7 × 10⁻¹⁹ to 3 × 10⁻¹⁸ and 8.4 × 10⁻²¹ to1.2 × 10⁻¹⁹ (mol cell⁻¹), respectively. Without B₁₂ or B₇ (biotin) added to the culture medium of a co-culture of L. polyedrum and D. shibae, the measured dissolved B₁₂ (dB₁₂) concentration was more than 60 pM higher than necessary for un-limited growth rates of L. polyedrum. In the same culture we measured B₇ in the L. polyedrum particulate fraction (pB₇; 4.7 × 10⁻¹⁹ to 9.4 × 10⁻¹⁹ mol cell⁻¹). We suggest that in response to the production of B₁ and B₁₂ by D. shibae to supply L. polyedrum requeriments, the latter produced B₇, which is required by D. shibae, and in our culture was only produced by L. polyedrum when D. shibae was present. We propose that D. shibae can control L. polyedrum through the release of B₁ and B₁₂, and L. polyedrum can control D. shibae through the release of B₇. D. shibae is also auxotroph for niacin and 4-amino-benzoic acid, not provided by the culture medium. Therefore, L. polyedrum might affect a similar control through the release of these specific compounds and organic substrate necessary for the growth of D. shibae.

Introduction

Eukaryotic phytoplankton and bacteria have developed a number of interkingdom metabolic interactions. These include growth stimulators (Ferrier et al., 2002), potential toxin inducers (Green et al., 2004, 2006), cyst inducers (Adachi et al., 2003, 2004; Mayali et al., 2007), growth inhibitors (Hare et al., 2005), algicids (Doucette et al., 1999), and chemosensors (Miller et al., 2004). The interactions also include mutualistic trade-off of soluble factors, for example, iron siderophores (Amin et al., 2009) and vitamins (Croft et al., 2005; Wagner-Döbler et al., 2010; Kazamia et al., 2012; Cruz-López and Maske, 2016). B vitamins are essential growth factors for most marine eukaryotic phytoplankton because they are required for the activity of several enzymes in central metabolism. Vitamin B₁₂ (cobalamin; hereafter B₁₂) is essential for the biosynthesis of deoxyriboses from riboses, the reduction and transfer of single carbon fragments in many biochemical pathways, porphyrin and methionine biosynthesis, which is dominant in eukaryotic phytoplankton (Tang et al., 2010; Sañudo-Wilhelmy et al., 2014). Vitamin B₁ (thiamin; hereafter B₁) plays a major role for enzymes involved in primary carbon metabolism like the Krebs and Calvin-Benson cycles, and for branch-chain amino acid metabolism, like alanine, leucine and valine (Sañudo-Wilhelmy et al., 2014). Vitamin B₇ (biotin; hereafter B₇) is essential for several carboxylase enzymes, including acetyl coenzyme A (CoA) carboxylase, which is involved in the fatty acid biosynthesis, and pyruvate carboxylase, which catalyzes the first step in gluconeogenesis by converting pyruvate to oxaloacetate, hence universally required (Croft et al., 2006; Tang et al., 2010; Sañudo-Wilhelmy et al., 2014). Many phytoplankton species are natural B vitamin auxotrophs, meaning they lack the biosynthetic pathways to produce B vitamins and thus must acquire them from exogenous sources. Of the phytoplankton surveyed to date, 54% are B12 auxotrophs, 27% are B1 auxotrophs, and 8% are B7 auxotrophs (Croft et al., 2005; Tang et al., 2010). Because these auxotrophs are common in natural waters, they are useful to study to determine the roles that niche partitioning and species succession play in phytoplankton blooms (Helliwell, 2017).

Although B vitamin auxotrophy is not systematically distributed across the algal lineages, in particular, dinoflagellates have evolved obligate dependence on vitamins (Croft et al., 2006; Tang et al., 2010; Helliwell et al., 2011). Dinoflagellates form part of the eukaryotic phytoplankton and contribute significantly to the primary production of coastal areas (Moustafa et al., 2010), many dinoflagellates form blooms that can represent a potential threat to coastal ecosystems, public health, economies, and fisheries (Tang et al., 2010). Tang et al. (2010) reported that of the surveyed dinoflagellate species that are involved in harmful algal bloom events, 100% required B₁₂, 78% required B₁ and 32% B₇.

Recent culture studies showed that for auxotrophic dinoflagellates a minimum of 2 × 10⁻⁵-5 × 10⁻⁴ pM of B₁₂ and from 1.4 × 10⁻¹¹ to 0.55 × 10⁻⁶ nM of B₁ are required for growth (Tang et al., 2010). These concentrations are typically found in coastal waters where dinoflagellates thrive (Gobler et al., 2007; Panzeca et al., 2009; Sañudo-Wilhelmy et al., 2012). During bloom conditions, the B₁ and B₁₂ concentrations are variable, from 5 to 20 pM for both vitamins; these concentrations should be sufficient to support dinoflagellate growth (Gobler et al., 2007; Koch et al., 2011).

Most experimental physiology studies related to vitamins have focused on single-vitamin limitation, for example, the role of B₁₂ at different concentrations (reviewed in Droop, 2007; Tang et al., 2010). Droop (1974) found threshold type limitation for phosphate and B₁₂ in phytoplankton cultures. Other authors have reported concurrent limitation of vitamins with nitrogen (Gobler et al., 2007; Koch et al., 2011; Bertrand and Allen, 2012), iron (Panzeca et al., 2006; Bertrand et al., 2007, 2011; Koch et al., 2011) or CO₂ (King et al., 2011). Although Panzeca et al. (2006), reported primary and secondary production limitation by Fe, B₁, and B₁₂ in coastal communities off the Antarctic, they did not investigate the potential for co-limitation of B₁₂ with other essential vitamins.

Heterotrophic bacteria, archaea, and marine cyanobacteria are the only known source of B vitamins (Bonnet et al., 2010; Sañudo-Wilhelmy et al., 2014; Doxey et al., 2015), although, in early studies, some phytoplankton species have also been reported to produce B vitamins (Bednar and Holm-Hansen, 1964; Carlucci and Bowes, 1970a,b; Aaronson et al., 1971, 1977). Hence dinoflagellates would have to acquire their B vitamin from the environment either through phagotrophy (Jeong et al., 2005), active uptake from the dissolved fraction (Bertrand et al., 2007; Tang et al., 2010; Kazamia et al., 2012), or episymbiosis (Croft et al., 2005; Wagner-Döbler et al., 2010; Kazamia et al., 2012; Kuo and Lin, 2013; Xie et al., 2013; Cruz-López and Maske, 2016). Little is known currently about the relative contribution of these different mechanisms to acquire the different vitamins by auxotrophic dinoflagellates.

Previously we reported Lingulodinium polyedrum vitamin auxotrophy for B₁ and B₁₂ (Cruz-López and Maske, 2016), where a high concentration of one vitamin cannot overcome the lack of the other. This research left several questions open: At very low, ecologically relevant concentrations of both vitamins, can increasing the concentration of one support increased algal growth without an increase the in the other vitamin? Also, what is provided by the dinoflagellate to the bacteria in exchange for the necessary B-vitamins? Here we report the effect of vitamin B₁ and B₁₂ under multiple vitamin concentrations on growth rate. We also report the B₇ and B₁₂ cellular quotas for L. polyedrum in co-culture with the bacterium Dinoroseobacter shibae, a well-known B₁ and B₁₂ producing bacterium. Differently from our previous work concerning a co-culture of L. polyedrum with a bacterial community, the present work did not include auxotrophic bacteria that could compete with L. polyedrum for B₁ and B₁₂.

Materials and Methods

L. polyedrum Growth Conditions

Oceanic seawater was collected off Ensenada (31.671°N, 116.693°W; Ensenada, México) treated with activated charcoal, filtered through GF/F, and 0.22-mm pore-size cartridge filter (Pall Corporation) and stored in the dark at room temperature to age for at least 2 months. Aged seawater was sparged with CO₂ (5 min per 1 L of seawater), autoclaved for 15 min and then equilibrated with air. L. polyedrum strain was isolated from Venice beach California and kindly provided by Avery Tatters (University of Southern California). Xenic L. polyedrum culture was grown in L1 medium (National Center for Marine Algae and Microbiota, NCMA, Maine, USA) prepared with aged oceanic water under 12:12 h light/dark cycle at an irradiance of 100 μmol m² s⁻¹, and a temperature of 20°C. To make the culture axenic, L. polyedrum culture was incubated with 1 mL of antibiotic solution (Penicillin, 5,000 U; Streptomycin, 5 mg mL⁻¹; Neomycin, 10 mg mL⁻¹. Sigma-Aldrich, P4083-100ML) for 50 mL of culture during 24 h, rinsed with the L1 medium, and repeated three times each step. Bacterial presence in the L. polyedrum culture was checked by staining with the nucleic acid-specific stain 4′,6-diamino-2-phenylindole (DAPI,1 μg mL⁻¹) and quantification using epifluorescence microscopy (Axioskope II plus, Carl Zeiss, Oberkochen, Germany) connected by liquid light guide to a 175W xenon arc lamp (Lambda LS, Sutter), with optical filtering (Excitation, 360 nm/Dichroic, 395 nm/Emission, >397 nm; Semrock & Zeiss) under X100 objective lens (Plan-Apochromat, Carl Zeiss). The axenic status was documented after three antibiotic rounds, and sterile medium washes when we could not detect bacteria in the culture using epifluorescence microscopy.

Assessment of L. polyedrum Vitamin B₁ and B₁₂ Limiting Concentrations

To test the vitamin B₁ and B₁₂ limiting concentrations of L. polyedrum, cultures were grown in 50 mL cell culture flasks (BD Falcon™). Cultures were acclimated by six semi-continuous transfers during 6 weeks with an initial cell concentration of 1,300 cell L⁻¹. The medium for L. polyedrum axenic cultures was supplemented with L1 and B₁, B₁₂ vitamins (Sigma-Aldrich) using a factorial experimental design combining 1) two vitamins (B₁ + B₁₂) and five vitamin concentrations ranging from 3.33 × 10⁻² to 3.33 × 10¹ pM of B₁ and 5.25 × 10⁻² to 5.25 × 10¹ pM of B₁₂ (Table 1). After acclimation, each culture and corresponding vitamin amendment were transferred and grown in 15 mL glass test tubes with silicon stoppers (n = 3) and placed randomly in transparent test tube holders to minimize bias in the data due to a heterogeneous light field in the incubator or shading by other cultures tubes. Cell growth rate was monitored by first mixing the cultures with an inclined rotating test tube holder (10 rpm) and then measuring in vivo Chlorophyll a (Chl a) fluorescence using a Turner Designs 10-000 fluorometer at the midpoint of the light phase. The specific growth rate (μ) was calculated according to the equation μ = ln (N2/N1)/(t2-t1): where N1 and N2 was in vivo Chl a fluorescence in relative units (r. u.) at time 1 (t1) and time 2 (t2), respectively. To calculate μ, we used the data obtained during the exponential phase of each growth curve on days 8, 10 and 12 after inoculation. in vivo Chl a fluorescence and cell counts were well correlated (Figure S1, r² = 0.98). The specific growth rate was adjusted to a Monod-type model of specific growth rate vs. the initial B₁ and B₁₂ concentrations in the bioassays. A least square estimate of a Michaelis-Menten type kinetic was calculated with the PRISM 6.07v software (GraphPad, CA, USA).

TABLE 1

Table 1. Half-saturation constants (Ks) and maximum specific growth rates (μ) of L. polyedrum for B₁ and B₁₂ amendments.

Dinoflagellate-Bacterial Co-culture

The axenic bacterium culture D. shibae DFL-12^T (NCMA, Maine, USA) was grown in oceanic seawater supplemented with yeast extract (2 g L⁻¹) and peptone (1.25 g L⁻¹). The co-culture was started by inoculating D. shibae cells to a final concentration of 10⁶ cells mL⁻¹ of an axenic, early exponential phase L. polyedrum culture in the L1 medium that was B₁B₇B₁₂-supplemented (hereafter vitamin-replete, V-R) or B₁B₇B₁₂ non-supplemented (hereafter vitamin-limited, V-L) medium and incubated under 12:12 h light/dark cycle, irradiance of 100 μmol m² s⁻¹, and a temperature of 20°C. The cultures were transferred weekly to fresh L1 medium at a ratio of 1:10 during 4 weeks before the experiment.

Sampling and Cell Fixation

All cultures were monitored for 15 d, and sampled every 2 days for cell counting by light microscopy. One milliliter of L. polyedrum cells from V-R and V-L cultures were harvested at lag (d2), exponential (d8) and stationary (d15) phase and fixed with paraformaldehyde-PBS at a final concentration of 1% for 12 h at 4°C. For attached bacteria, fixed cells were immobilized onto an 8.0 μm pore size, 25 mm-diameter Nucleopore filters (Whatman International, Ltd., Maidstone, England), and rinsed with phosphate-buffered saline (PBS, 0.1 M NaCl, 2 mM KCl, 4 mM Na₂HPO₄, pH 8.1; Palacios and Marín, 2008). For free-living bacteria, the fraction which passed through an 8.0 μm pore size filter was collected on 0.2 μm pore size, 25 mm-diameter Nucleopore filters (Whatman International, Ltd., Maidstone, England) and rinsed with PBS. The cells collected on the 8.0 μm filter were covered with 13 μL of low-melting-point agarose (0.05%, LMA) (BioRad, 161-3111) at 55°C, dried for 15 min at 37°C, then LMA was added again and the filter dried as previously. For all filtration steps, the pressure difference was <3.3 kPa to minimize cell damage.

Visual Observations

To visualize D. shibae cells attached to L. polyedrum cells, we used an epifluorescence microscope (Axioskope II plus, Carl Zeiss, Oberkochen, Germany; oil immersion X100 objective, Plan-Apochromat, Carl Zeiss; 175W xenon arc lamp; Lambda LS, Sutter connected through a liquid light guide) with a triple Sedat filter, a dichroic filter with three transmission bands. Excitation and emission spectra were controlled by filter wheels (Lambda 10-3, Sutter). Images were captured with a cooled CCD camera (Clara E, Andor) with 10 ms integration time. Optical stacks, 2.0 μm focal distance between images, were acquired with a computer controlled focusing stage (Focus Drive, Ludl Electronic Products, Hawthorne, NY, USA) and Micro-Manager (version 1.3.40, Vale Lab, UCSF) that controlled the filter selection and the focusing stage. The images were processed in ImageJ (Schneider et al., 2012).

Quantification of Dissolved and Particulate B₁₂ (dB₁₂, pB₁₂)

For quantification of dB₁₂ and pB₁₂ we prepared four different fractions: 20 mL of L. polyedrum cells from V-R and V-L cultures were harvested at time zero, mid-exponential and stationary phase (n = 3). These samples were (a) pre-filtered (8.0 μm pore size, 25 mm-diameter Nuclepore filters) to harvest the fraction of pB₁₂ of the L. polyedrum and attached D. shibae; (b) The filtrate of (a) was filtered again (0.4 μm pore size, 25 mm-diameter polycarbonate filter (Whatman International, Ltd., Maidstone, England) to recover the free-living D. shibae fraction. (c) The first filter (a) containing the L. polyedrum cells with attached D. shibae were treated with 10 mM N-acetyl cysteine (NAC; Sigma) (PBS−0.2 μM calcium chloride, 0.5 mM magnesium chloride, 15 mM glucose) for 1 h with agitation (70 rpm) at room temperature to detach adhered bacteria (Barr et al., 2013). Filtering this sample again with 0.8 μm collected L. polyedrum, cells without attached D. shibae. (d) The detached D. shibae cells were collected onto a 0.4 μm pore size, 25 mm-diameter polycarbonate filter.

Vitamin B₁₂ was quantified by Enzyme-Linked Immunosorbent Assay, ELISA (Immunolab GmbH, B12-E01. Kassel, Germany) according to Zhu et al. (2011). These authors tested the ELISA with seawater samples and found close to 100 percent recovery of cyanocobalamin, methylcobalamin, hydroxycobalamin and coenzyme B₁₂, but the method cannot distinguish between these different forms of B₁₂.

For dB₁₂, the filtered sample (0.2 μm) was pre-concentrated by solid phase (RP-C18) extraction according to Okbamichael and Sañudo-Wilhelmy (2004); the column was eluted with 5 mL methanol, the methanol was evaporated at 60°C with low vacuum, the sediment dissolved in ddH₂0 with a final volume of 1.5 mL, and 50 μL of the concentrate was injected into the ELISA well plate for quantification. Villegas-Mendoza et al. (in preparation) report a recovery efficiency of 85 percent using the above method for dissolved B₁₂.

For the pB₁₂, filters were cut for easier disintegration and transferred to bead beating tubes using 0.5 mm zirconia beads (biospec.com) with 1 mL of methanol (10:1 methanol:beads). Bead beating was conducted for 5 min followed by 5 min at −20°C, and repeated twice. The tubes were centrifuged at 5,000 rpm for 10 min and the supernatant transferred to a fresh tube. The methanol was evaporated at 60°C with vacuum, the sediment dissolved in ddH₂0 with a final volume of 1.5 mL, and 50 μL of the concentrate was injected into the ELISA well plate for quantification.

Quantification of Particulate B₇ (pB₇)

The samples were prepared for the solid extraction method described in Suffridge et al. (2017), with a slight modification. One hundred milliliters of axenic L. polyedrum culture (n = 3) was harvested at exponential phase using 8.0 μm pore size, 25 mm-diameter Nuclepore filters (Whatman International, Ltd. Maidstone, England). 100 mL of L. polyedrum + D. shibae culture (n = 3) was harvested at mid-exponential phase, and pre-filtered (a) to harvest the fraction of particulate B₇ of the L. polyedrum and attached D. shibae using 8.0 μm pore size, 25 mm-diameter Nuclepore filter; (b) to collect the free-living D. shibae fraction the filtrate from (a) was filtered with 0.4 μm pore size, 25 mm-diameter polycarbonate filter (Whatman International, Ltd., Maidstone, England). The first filter (a) containing L. polyedrum cells with attached D. shibae was rinsed with 10 mL of Milli-Q water to detach adhered D. shibae. (c) Filtering this fraction again through 0.8 μm collected L. polyedrum cells without attached D. shibae. (d) The detached D. shibae cells passing through 8.0 μm filter were collected onto a 0.4 μm pore size, 25 mm-diameter polycarbonate filter.

Frozen filters containing the different fractions mentioned above were processed according to Suffridge et al. (2017) with slight modifications. In brief, frozen filters were placed in an autoclaved 1.5 mL Eppendorf tube with 1 mL of diluent (50% LC-MS grade acetonitrile, 50% water, 0.1% formic acid). The cells were lysed by repeated vortexing for 5 min followed by 1 min cooling on ice for a total period of 30 min. The cells were further sonicated for 5 min. The cell lysate was passed through 0.2 μm pore size, 25 mm-diameter polycarbonate filter (Whatman International, Ltd, Maidstone, England) to remove large particles and cell debris. The filters were washed with 0.5 mL of diluent to collect residual cell metabolites. An equal volume of chloroform was added to the filtrate; the mix was vigorously shaken for 1 min and then centrifuged for liquid phase extraction. The top aqueous phase was transferred to a new Eppendorf tube. The liquid phase extraction with chloroform was repeated twice. The residual aqueous phase was adjusted to 300–500 μL with the diluent, and then the sample was injected in triplicate in a LC-MS (Applied Biosystems API4000 SCIEX) for pB₇ quantification. To obtain the efficiency of recovery, 100 ng mL⁻¹ of B₇ standard (Sigma-Aldrich-B4501) was added to three different samples.

D. shibae Frequency of Attachment on L. polyedrum

The frequency was established using the co-culture in exponential phase with modifications as follows: An initial V-R (V-R₀) culture was followed for 15 d, then, the culture was transferred weakly to fresh L1 medium under semi-continuous culture conditions during 4 weeks; each consecutive culture was termed V-R₁, V-R₂, and V-R_3. A parallel culture was started from the V-R₀ culture under V-L conditions, this was termed V-L₀, then the culture was transferred to a second V-L condition, and termed V-L₁, and for the next transferred, add-back to a V-R condition, and termed V-L₁ to V-${\text{R}}_{1^{\prime }}$. In each culture, a sample at mid-exponential phase was taken, and processed as described in Sampling and cell fixation, and Visual observations.

Statistical Analysis

One-way ANOVA was used to compare the specific growth rate during exponential phase at day 8 of L. polyedrum under V-R vs. V-L cultures, to compare the dB₁₂ in V-R vs. V-L cultures, and to compare pB₁₂ from attached and free-living D. shibae at lag, exponential and stationary phase. Since the distribution of the number of attached D. shibae on L. polyedrum cell had no Gaussian distribution, we used a Mann–Whitney test to assess the significance of the number of attached bacteria on L. polyedrum during mid-exponential phase at day 8. All analysis were performed using STATISTICA 7.1v software (Stat Soft Inc., USA).

Results

Quantitative Vitamin B₁ and B₁₂ Requirements

L. polyedrum has recently been shown to be vitamin B₁ and B₁₂ auxotroph but not to be B₇ auxotroph (Cruz-López and Maske, 2016), following the pattern of the majority of the examined dinoflagellate species requiring these two vitamins (Tang et al., 2010). In a new set of batch culture experiments, we explored the growth limiting concentrations for this species using a factorial design for both vitamins. In these experiments, cultures exhibited a μ_max of 0.18 ± 0.003 d⁻¹ (n = 3) in the ranges of 0.33–3.3 pM for B₁, and 0.053–0.53 pM for B₁₂ (Figure 1). Vitamin half-saturation constants (Ks) for μ_max were 3.3 pM for B₁, and 0.53 pM for B₁₂ (Table 1). Growth rates and biomass were strongly dependent on both vitamins and did not grow at concentrations of 0.033 and 0.053 pM of B₁ and B₁₂, respectively (Tables S1, S2).

FIGURE 1

Figure 1. Vitamin B₁ vs. B₁₂ growth kinetics curve for L. polyedrum. The data plotted are from 16 (n = 3) independent experiments. L. polyedrum growth under (A) 0.0526, (B) 0.526, (C) 5.26, (D) 52.6 pM of B₁₂, and 0.0333, 0.333, 3.33, and 33.3 pM of B₁. Error bars represented one standard deviation (n = 3). A hyperbolic Michaelis–Menten model is plotted, Ks and μmax are summarized in Table 1.

dB₁₂ and Cell Quotas of L. polyedrum and D. shibae

Dissolved B₁₂ varied between cultures. During the lag phase, in the V-R culture, dB₁₂ was 42.7 (±1.8) pM, whereas in the V-L culture dB₁₂ was 29.9 (±5.3) pM. In exponential phase, in the V-R culture the dB₁₂ was 43.1 (±8.6) pM, and in the V-L 65.3 (±24.3) pM (Table 1). During stationary phase, in the V-R culture dB₁₂ was 37.6 (±3.1) pM, and in the V-L culture 372 (±2.4) pM. The dB₁₂ was higher during lag phase in the V-R than the V-L culture, dB₁₂ concentration in the V-R and V-L cultures were similar during stationary phase, but unexpectedly during exponential phase, the dB₁₂ in the V-R culture was much lower than in the V-L culture.

The B₁₂ cell quotas (mol cell⁻¹) of L. polyedrum, attached and free-living D. shibae were calculated by dividing the pB₁₂ concentration by cell abundance. Samples were taken at the lag, exponential and stationary phase of the V-R and V-L cultures. For L. polyedrum, during the lag phase, in the V-R and V-L culture, the pB₁₂ were both the same, 2.7 × 10⁻¹⁷ mol cell⁻¹, respectively (Table 2). During the exponential phase, in the V-R and V-L cultures, the cell quotas were similar at 8.9 × 10⁻¹⁸ (±1.05 × 10⁻¹⁸) and 9.9 × 10⁻¹⁸ (±1.1 × 10⁻¹⁸) mol cell⁻¹, decreasing during the stationary phase to 5.8 × 10⁻¹⁸ (±5.6 × 10⁻¹⁹) and 5.0 × 10⁻¹⁸ (±7.1 × 10⁻¹⁹) mol cell⁻¹ in V-R and V-L cultures, respectively. The cell quotas of L. polyedrum in both types of culture were similar and showed maximum dB₁₂ concentrations during exponential phase.

TABLE 2

Table 2. Dissolved and B₁₂ quotas expressed per cell (mol cell⁻¹) for L. polyedrum and D. shibae in co-culture.

For free-living D. shibae, the estimates for the lag, exponential and stationary phase in the V-R culture, cell quotas were 1.2 × 10⁻¹⁹, 6.5 × 10⁻²⁰ (±1.7 × 10⁻²⁰) and 1.6 × 10⁻²⁰ (2.1 × 10⁻²¹), and for the V-L culture, they were 9.7 × 10⁻²⁰, 2.9 × 10⁻²⁰ (±3.2 × 10⁻²¹) and 8.5 × 10⁻²¹ (3.3 × 10⁻²²) mol cell⁻¹. For the attached D. shibae, the estimates for the lag, exponential and stationary phase in V-R culture were 3.1 × 10⁻¹⁸, 5.9 × 10⁻¹⁹ (±1.3 × 10⁻¹⁹) and 4.9 × 10⁻¹⁹ (±1.3 × 10⁻²⁰) mol cell⁻¹, and for the V-L culture were 3.0 × 10⁻¹⁸, 5.3 × 10⁻¹⁹ (±4.4 × 10⁻²⁰), 4.7 × 10⁻¹⁹ (±9.2 × 10⁻²²) mol cell⁻¹. The D. shibae B₁₂ cell quotas of attached D. shibae were always much higher than those for free-living D. shibae by a factor 10 to 55 (Table 2).

B₇ Cell Quotas of L. polyedrum and D. shibae

We could not detect pB₇ in L. polyedrum axenic cultures, while in the xenic culture; L. polyedrum contained an average of 7.07 × 10⁻¹⁹ mol cell⁻¹ of B₇. As reported previously, D. shibae can thrive in two phenotypes, in attached mode or free-living; when we tried to quantify the free-living fraction, we could not detect any trace of pB₇, whereas, in the attached fraction, we quantified an average of 1.8 × 10⁻¹⁹ (n = 2) mol cell⁻¹ (Table 3).

TABLE 3

Table 3. B₇ quotas expressed per cell (mol cell⁻¹) for axenic L. polyedrum and with D. shibae in co-culture.

D. shibae Frequency of Attachment on L. polyedrum

We quantified the attachment of D. shibae to L. polyedrum cells by counting microscopically the number of attached bacteria to each L. polyedrum cell. Because 100 L. polyedrum cells were observed the frequency of bacteria per host cell in Figure 2 gives the relative frequency in percent. Figure 2 shows that in the V-R culture the average number of D. shibae bound to L. polyedrum was lower, compared with the V-L culture (p < 0.05, Mann-Whitney). In addition, the abundance of free-living D. shibae in the V-R culture was lower than in the V-L culture (Figure 3; p < 0.05, ANOVA).

FIGURE 2

Figure 2. Frequency of D. shibae cells attached to single L. polyedrum cell in semi-continuous culture conditions. L. polyedrum in V-R culture (), and V-L culture ().

FIGURE 3

Figure 3. Growth of L. polyedrum and D. shibae under V-R (-•-) and V-L (-°-) cultures. (A) L. polyedrum abundance; red arrows denoted time-points of sampling. (B) Free-living D. shibae abundance. *p < 0.05 (ANOVA).

Discussion

Limiting Concentrations of B₁ and B₁₂

Our results show that B₁ and B₁₂ at the lowest concentrations tested limited the growth rate of L. polyedrum. The L. polyedrum strain examined in this study must possess high-affinity uptake systems for B₁ and B₁₂ to explain their low Ks values (Table 1). Our Ks estimate for B₁₂ of 0.53 pM is in the range of values reported by Tang et al. (2010; Table 2) for six different phytoplankton species including two dinoflagellate species reported to have the lowest (0.02 pM) and highest Ks values (13.1 pM) for B₁₂ in the list. Our Ks estimate for B₁ of 3.3 pM is lower than the values reported by Tang et al. (2010; Table 2) for five species. In their list, the values of the three dinoflagellates ranged from 86 to 131 pM.

The uptake of B₁ and B₁₂ by phytoplankton communities has been documented in coastal areas (Gobler et al., 2007; Koch et al., 2011, 2012), but the potential for colimitation of B₁ and B₁₂ has not been reported. Given the slow growth rate of dinoflagellates, it is difficult to imagine that traditional bioassay experiments with natural populations probing vitamin limitation would yield conclusive results for dinoflagellates. But concurrent limitation of vitamins with other compounds have been reported (Panzeca et al., 2006; Bertrand et al., 2007, 2011; Gobler et al., 2007; King et al., 2011; Koch et al., 2011; Bertrand and Allen, 2012).

The limiting concentrations of B₁ and B₁₂ supporting maximum growth rates of L. polyedrum can be compared with measured in situ concentrations. In coastal systems dB₁₂ ranges from undetectable to 87 pM (Sañudo-Wilhelmy et al., 2006, 2012; Panzeca et al., 2009) and for dB₁ from undetectable to 200 pM (Gobler et al., 2007; Koch et al., 2012, 2013; Sañudo-Wilhelmy et al., 2012; Suffridge et al., 2017). This comparison suggests that B₁ or B₁₂ might limit the growth rate of L. polyedrum during certain stages of bloom formation. Koch et al. (2014), measured dissolved B₁ and B₁₂ concentrations inside and outside of dinoflagellate blooms, and found concentrations higher than the limiting concentrations reported in our study. They also reported that vitamin concentrations inside dinoflagellate blooms were lower than outside of blooms, which pointed to active uptake and the possibility of vitamin limitation. Further field data will have to show if the dinoflagellate bloom development can be limited by vitamin availability in coastal waters that are less eutrophic than their study area.

L. polyedrum and D. shibae Co-culture

Our experiments were planned after having shown B₁ and B₁₂ auxotrophy of L. polyedrum, including experimental evidence that co-cultured bacterial communities could overcome the vitamin limitation without supplying the bacteria with additional organic substrate in the culture medium (Cruz-López and Maske, 2016). Our previous experimental approach could not separate the vitamin auxotrophy of part of the bacterial community from the auxotrophy of L. polyedrum. In a new set of experiments, we co-cultured L. polyedrum with D. shibae, a bacterium reported to produce B₁ and B₁₂ (Wagner-Döbler et al., 2010). D. shibae has been shown to be B₇ auxotroph (Biebl et al., 2005; Wang et al., 2014a) which combined well with L. polyedrum that was shown not to be B₇ auxotroph (Cruz-López and Maske, 2016).

In the co-cultures, we calculated B₁₂ cell quotas based on pB₁₂ and cell abundances for L. polyedrum ranging from 5 × 10⁻¹⁸ to 2 × 10⁻¹⁷ mol cell⁻¹ (Table 2). These cell quotas are comparable to 4 × 10⁻²² and 1.7 × 10⁻¹⁸, the values reported for dinoflagellates by Tang et al. (2010; Table 2). Our measured cell quotas show between one and two orders higher cellular B₁₂ content for L. polyedrum than for both types of D. shibae phenotypes, attached and free-living. In our results, L. polyedrum had lower Ks but higher cell quotas compared with other dinoflagellate species (Tang et al., 2010); the former might provide an ecological advantage to L. polyedrum or partially compensate for the disadvantage of a high cell quota. The cell quota concentrations based on cell volumes are 2–3 orders of magnitude lower for L. polyedrum cells than for D. shibae cells (Table 1). Panzeca et al. (2009) quantified the concentration of B₁₂ during a L. polyedrum bloom in a coastal, ranging from 2 to 61 pM, concentrations that should not be limiting for L. polyedrum. L. polyedrum abundance was not quantified at the time of B₁₂ measurements, but at nearby locations at a later date the abundance was shown to be high, ranging from 1 × 10⁶ to 6 × 10⁶ (cell L⁻¹) (Peña-Manjarrez et al., 2009).

According to Croft et al. (2005), a minimum of 10 ng L⁻¹ of B₁₂ is required for growth in culture by most phytoplankton species, while Tang et al. (2010) showed that HAB species need higher concentrations than non-bloom forming species. Most of the B₁₂ quantification in the field or in culture studies measured the dissolved form, and only few studies related to dinoflagellates in the field (Carlucci, 1970; Gobler et al., 2007; Panzeca et al., 2009) or in cultures (Croft et al., 2005; Tang et al., 2010; Cruz-López and Maske, 2016). Recently Suffridge et al. (2017), quantified the particulate fraction for several B-type vitamins in the field and cultured marine bacteria, given previous cell quotas for marine phytoplankton or heterotrophic bacteria are based on uptake rates (reviewed in Droop, 2003; Bonnet et al., 2010).

To quantify the B₁₂ in the different fractions, we used a highly sensitive enzyme-linked immunosorbent assay (ELISA) able to recognize all B₁₂ forms (cyanocobalamin, methylcobalamin, hydroxocobalamin, and coenzyme B₁₂), but unable to distinguish them individually. The cyanocobalamin produced by D. shibae can be used directly by the algal auxotroph without post-modifications (Helliwell et al., 2016). A likely explanation for our co-culture experimental results is that L. polyedrum acquired B₁ and B₁₂ vitamins from D. shibae in sufficient quantity to sustain the maximum growth over many generations; however, it had not been shown what D. shibae receives in return from L. polyedrum. For D. shibae to grow together with L. polyedrum, it would need organic substrate for biomass formation but in addition also specific compounds because D. shibae is auxotroph for B₇, niacin and 4-amino-benzoic acid (Biebl et al., 2005). The production and release of niacin and 4-amino-benzoic acid by a dinoflagellate host was previously suggested by Wagner-Döbler et al. (2010) and Wang et al. (2014a) to explain their co-cultures of Prorocentrum minimum CCMP1329 with D. shibae in medium lacking B₁₂ or any of the other essential vitamins mentioned above. However, the dinoflagellate P. minimum CCMP1329 is B₇ auxotroph (Wagner-Döbler et al., 2010) and therefore B₇ had to be supplied to the medium. In the case of L. polyedrum, B₇ is not required (Cruz-López and Maske, 2016) and the culture medium was not supplemented with B₇, niacin or 4-amino-benzoic acid. We assume that these organic growth factors could not be provided by the prepared seawater medium. We suggest that these three compounds were provided by L. polyedrum in the form of exudates in sufficient quantity to support the growth of the D. shibae population. In return, L. polyedrum thus promoted the production of B₁ and B₁₂. Due to technical limitations, we only measured B₇ in the particulate fraction, but not in the dissolved fraction. Future research should quantify B₇, niacin or 4-amino-benzoic acid in the dissolved fraction to better define the mutualistic relationship between D. shibae and L. polyedrum (Figure 4).

FIGURE 4

Figure 4. Proposed mutualism of L. polyedrum and D. shibae. Depending on the degree of B₁ and B₁₂ limitation L. polyedrum liberates growth promoters for D. shibae, for example, B₇, niacin and 4-amino-benzoic acid. D. shibae responds with an increased tendency to attach to L. polyedrum cells and to produce B₁ and B₁₂. The red arrows indicate the kinetic between free-living and attached lifestyle of D. shibae based on the dissolved pool of B₁ and B₁₂.

On the other hand, the release of B₇ from different phytoplankton species has been documented previously (Bednar and Holm-Hansen, 1964; Carlucci and Bowes, 1970a,b; Aaronson et al., 1971, 1977), cell quotas for dinoflagellates are based on uptake rates (Tang et al., 2010). Our pB₇ results showed no pB₇ in the L. polyedrum axenic culture, whereas in co-culture with D. shibae, L. polyedrum we found from 4.74 × 10⁻¹⁹ to 9.41 × 10⁻¹⁹ mol cell⁻¹. Adjusting to pmol cell⁻¹, our results are in the range (4.74 × 10⁻⁷ to 9.41 × 10⁻⁷) for the cell quotas reported for Gymnodinium instriatum L3, and two orders of magnitude higher than those reported for P. minimum CCMP696 and P. minimum PB3 (Tang et al., 2010).

For marine heterotrophic bacteria, the B₇ production has been inferred, based on their available genomes, or in situ transcriptional studies (Luo and Moran, 2014; Sañudo-Wilhelmy et al., 2014; Gómez-Consarnau et al., 2018). Among the Roseobacter clade, of the 52 Roseobacter genomes analyzed by Luo and Moran (2014), 30 species were found to be auxotrophs for B₇, including D. shibae (Biebl et al., 2005; Wienhausen et al., 2017; Gómez-Consarnau et al., 2018). Of these 30 Roseobacter species, 13 produce B₁ and B₁₂, and 17 only B₁₂ (Table 4). In the case of D. shibae in co-culture with L. polyedrum, we could only detect B₇ in the attached D. shibae phenotype, with cell quotas ranging from 1.72 × 10⁻⁷ to 1.9 × 10⁻⁷ pmol cell⁻¹.

TABLE 4

Table 4. Select genes for B-vitamin biosynthesis in 30 B₇ auxotroph Roseobacter isolate genomes.

In a recent study, Suffridge et al. (2017), developed a method to quantify B-type vitamins in the dissolved and particulate fraction using cultures of marine bacteria. They obtained cell quotas of B₇ for Synechococcus strain CC9311 (9.9 × 10⁻⁶ ± 8.1 × 10⁻⁶ mol cell⁻¹), and for Vibrio AND4 (2.47 × 10⁻⁶ ± 1.36 × 10⁻⁶ mol cell⁻¹). In our cultures, surprisingly the free-living D. shibae did not contain pB₇. As was shown by Luo and Moran (2014), the B₇ auxotrophy is a widespread phenomenon among the Roseobacter clade, although this auxotrophy is not always related to a symbiotic lifestyle with phytoplankton, which needs to be clarified with further culture and genomic studies.

D. shibae Frequency of Attachment on L. polyedrum

Eukaryotic phytoplankton and heterotrophic bacteria are interacting to fulfill the need for vitamins of the former and the need of excreted organic substrate of the latter. Different modes of exchange of vitamins and organic substrate are possible; vitamin-producing bacterial epibionts can be attached to the vitamin-auxotroph cells, or the vitamin-producing and consuming cells can be freely suspended, and the exchange of the dissolved compounds is maintained by turbulent and molecular diffusion at sufficiently high rates to supply the metabolic demand of the auxotrophs (Croft et al., 2005; Droop, 2007).

We used vitamin-replete and vitamin-limited cultures of L. polyedrum to produce contrasting host-bacterial culture conditions assuming that the V-L condition would promote the cellular attachment of bacteria and host. The frequency of D. shibae attached to L. polyedrum was higher under V-L condition compared to V-R condition (Figure 2; Table S3; Mann-Whitney, p < 0.005). When V-L cultures returned to V-R condition, the frequency of attachment reduced to average abundance. Higher attachment frequency under V-L condition suggests a more intense exchange of metabolites under growth culture conditions where this exchange would be essential to the growth of either population.

Because no carbon substrates were added to the culture medium that could have supported the growth of D. shibae, our results suggest that D. shibae was able to use dinoflagellate photosynthates as a carbon source in return for supplying the host with vitamin B₁ and B₁₂. As pointed out above L. polyedrum might have provided more specific growth enhancing substrates to D. shibae. Although in media without added vitamin D. shibae was found to be attached more frequently to L. polyedrum cells suggesting a synergy between both species, it leaves the question open about the mechanism of delivery. Vitamin could have either been exchanged in the dissolved form originating from free-living partners or by a more direct exchange of metabolites when both cell types are physically attached. Interestingly, in similar experiments but with a natural community of microbes instead of D. shibae, the frequency of attachment of microbes to L. polyedrum did not increase but the abundance of free-living bacteria in the co-culture increased (Cruz-López and Maske, 2016). For co-cultures of L. polyedrum with bacterial communities, the vitamin exchange and balance is more difficult to analyze because an unknown part of the bacterial community is also B₁₂ auxotroph.

During the co-culture of L. polyedrum and D. shibae, we quantified the free-living and attached D. shibae cells in V-R and V-L cultures, and observed that the abundance of free-living D. shibae in the V-R culture was lower than in V-L culture (Figure 3B; ANOVA, p > 0.05), and that L. polyedrum in the V-R culture was lightly colonized in comparison with the V-L culture (Figure 2; Table S3; Mann-Whitney, p < 0.005). These two alternative lifestyles of D. shibae have been documented before (Biebl et al., 2005; Wagner-Döbler et al., 2010; Wang et al., 2014a), and are controlled by quorum sensing (QS) via CtrA signal transduction protein (Patzel et al., 2013; Wang et al., 2014b). Note, however, that increased bacterial attachment frequency under vitamin limitation coincided with an increased abundance of free-living bacteria, further work will be required to confirm that this phenotype is driven by nutrient exchange requirements.

Conclusion

Our data show that the B₁ and B₁₂ limiting concentrations for L. polyedrum growth are lower than typical concentrations found in coastal waters. In vitamin-depleted cultures of L. polyedrum, the limitation could be overcome by co-culture with D. shibae, because the bacterium was likely able to provide sufficient quantities of B₁ and B₁₂ to support the growth of L. polyedrum. In return, D. shibae was likely provided sufficient exudates by L. polyedrum to support its growth. Also, the mutualism between both partners may have extended beyond the supply of B₁ and B₁₂ and dissolved organic carbon. Since D. shibae is auxotrophic for B₇, niacin and 4-amino-benzoic acid, L. polyedrum needed to provide sufficient quantities of these compounds to overcome D. shibae's auxotrophy. This mutualism was probably helped by the phenotypic ability of D. shibae to choose between a free-living or attached lifestyle, where the attached lifestyle could serve as a reservoir of B₇ within this isogenic population.

Author Contributions

RC-L conceived, performed, and wrote the article. HM conceived and wrote the article. KY and NH performed and wrote the article.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

This work was partially supported by the National Council of Science and Technology (CONACyT-México) project CB-2008-01 106003 (to HM), a Ph.D. scholarship, and a Postdoctoral fellowship for overseas to RCL (CONACyT-México). Additionally, we would like to thank Avery O. Tatters (University of Southern California) for kindly providing the L. polyedrum strain and Prof. Carl J. Carrano (San Diego State University) for allowing us to use his laboratory to prepare samples. We would like to show our gratitude to Sharon and Christopher A. Rhodes, the owners of Drug Delivery Experts LLC., for allowing us to use their facility to generate LC-MS data. Finally, we would like to thank the reviewers for constructive comments and suggestions.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmars.2018.00274/full#supplementary-material

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