Abstract

   Shewanella sp. IRI-160 is an algicidal bacterium that secretes an
   algicide, IRI-160AA. This algicide specifically targets
   dinoflagellates, while having no adverse effects on other algal species
   tested. Dinoflagellates exposed to IRI-160AA exhibited increased
   production of reactive oxygen species (ROS), DNA damage, and cell cycle
   arrest, implying a programmed pathway leading to cell death (PCD).
   Here, a metabolomic analysis was conducted on dinoflagellate
   Karlodinium veneficum and a control cryptophyte species Rhodomonas
   exposed to IRI-160AA to investigate the cellular mechanisms behind the
   physiological effects and the specificity of this algicide. Results of
   this research supported previous observations about physiological
   responses to the algicide. A suite of metabolites was identified that
   increased in the cell pellets of K. veneficum but not in Rhodomonas,
   including oxidative stress biomarkers, antioxidants, and compounds
   involved in DNA damage and PCD. Overall, the results of this study
   illustrated the metabolomic mechanisms underlying the algicidal effects
   of IRI-160AA on dinoflagellates. This research also provided insights
   and future directions for studies on the cellular response of
   dinoflagellates exposed to antagonistic bacteria in the environment.

   Keywords: Shewanella, Karlodinium veneficum, dinoflagellate, algicide,
   programmed cell death, DNA damage, reactive oxygen species,
   metabolomics

1. Introduction

   Phytoplankton are responsible for almost half of the photosynthesis on
   earth and play essential roles in regulating global biogeochemical
   cycles [[26]1,[27]2,[28]3]. The microbes surrounding and/or associated
   with phytoplankton are also key players in this process; heterotrophic
   bacteria recycle up to 90% of organic matter derived from phytoplankton
   [[29]4]. Interactions between phytoplankton and bacteria in the aquatic
   ecosystems are diverse and complex [[30]5,[31]6]. These interactions
   include mutualism, commensalism, antagonism, parasitism, and
   competition, governed by the metabolites and other chemicals exchanged
   by these microbes [[32]3]. Relationships between phytoplankton and
   bacteria are also dynamic in that interactions can be altered by the
   physical state of these microbes [[33]3].

   Dinoflagellates are among the most dominant groups of phytoplankton in
   the marine ecosystem, and are the second largest group of primary
   producers in the ocean, behind diatoms [[34]7]. They also form
   important symbiotic relationships with other organisms, such as
   reef-building corals [[35]8]. Nevertheless, dinoflagellates can also
   cause massive and persistent harmful algal blooms (HABs) that pose a
   threat to other organisms in the environment [[36]9]. In fact,
   dinoflagellates are among the most toxigenic HAB species [[37]9]; they
   are able to produce a wide range of compounds that can be
   bioaccumulated through the food web to cause disease and death of
   marine organisms and negatively impact human health [[38]10,[39]11].
   The HAB dinoflagellate Karlodinium veneficum is a mixotrophic species
   that has caused harmful algal blooms worldwide for decades [[40]12].
   This organism can produce a suite of toxic compounds, termed
   karlotoxins, that are hemolytic, ichthyotoxic, and cytotoxic [[41]12].
   Karlotoxins can decrease the grazing rates of predatory phytoplankton
   and zooplankton, increasing the survival of K. veneficum in the
   environment [[42]13,[43]14]. These toxins have also been associated
   with massive fish kills worldwide [[44]15].

   It has been demonstrated that bacteria surrounding and/or associated
   with dinoflagellates play a critical role in the biology and ecology of
   these algal species. For instance, research demonstrated that some
   dinoflagellates are vitamin B1 and B12 auxotrophs [[45]16] and rely on
   their bacterial partners to provide these micronutrients [[46]17]. In
   addition to the beneficial effects, several species of bacteria have
   shown algicidal activity against dinoflagellates that can either kill
   or inhibit the growth of these algal species
   [[47]4,[48]18,[49]19,[50]20,[51]21,[52]22]. Algicidal bacteria may
   exhibit specific activity against dinoflagellates [[53]21], while
   others also affect the growth of other species [[54]18,[55]19]. Two
   modes of action have been involved in the activity of algicidal
   bacteria: these bacteria can either attach to the targeted algal
   species and attack them directly, or they may secrete algicidal
   compounds to control the growth of targeted algal species indirectly
   [[56]4,[57]22]. For instance, two strains of bacteria designated S03
   and 41-DBG2 both exhibited algicidal effects on the dinoflagellates
   Karenia brevis and Karenia mikimotoi [[58]20]. While S03 required
   direct contact to lyse the cells, 41-DBG2 could kill these
   dinoflagellates by secreting water-soluble compounds [[59]20]. Little
   is known about algicidal compounds produced by bacteria, but those that
   have been identified are diverse, spanning a wide range of structures,
   polarity, and size [[60]4].

   Shewanella sp. IRI-160 is an algicidal bacterium isolated from the
   Delaware Inland Bays, DE, USA [[61]21]. This bacterium can secrete
   water-soluble algicidal compounds, collectively referred to as
   IRI-160AA, that specifically inhibit the growth of dinoflagellates,
   including K. veneficum [[62]23]. No negative effects have been observed
   upon exposure of other algal species, zooplankton, invertebrates, or
   juvenile finfish to IRI-160AA [[63]21,[64]23,[65]24]. Research on K.
   veneficum physiology after exposure to IRI-160AA demonstrated induction
   of reactive oxygen species (ROS), DNA damage, and cell cycle arrest,
   along with caspase-like protease activity, suggesting that the algicide
   induces a programmed pathway leading to cell death (PCD)
   [[66]25,[67]26]. Studies on the photobiology of K. veneficum and other
   dinoflagellates exposed to IRI-160AA also indicated disruption of
   photosynthetic electron transport [[68]27]. Preliminary analysis
   demonstrated that the algicidal fraction is polar [[69]23], and
   identified ammonium among the active algicidal compounds in IRI-160AA
   [[70]28]. There were significant differences, however, between exposure
   to IRI-160AA produced by Shewanella vs. ammonium alone in terms of
   dinoflagellate photobiology [[71]29] and gene level regulation
   [[72]30]. Recent transcriptomics studies of K. veneficum exposed to
   IRI-160AA revealed a global response to the algicide, including
   processes affecting gene expression, protein activity, and cellular
   morphology [[73]30]. Genes involved in ROS response, DNA damage, and
   PCD were also differentially expressed in K. veneficum exposed to
   IRI-160AA compared to controls where the algicide was not added
   [[74]30]. Amines, including the polyamine putrescine, have also been
   identified as algicidal components of IRI-160AA [[75]28]. A negative
   synergistic effect was reported by ammonium and putrescine on
   dinoflagellates including K. veneficum, and molecular data indicated
   ammonium may disrupt polyamine homeostasis, while putrescine may
   sensitize these species to ammonium toxicity [[76]30].

   Recent improvements in metabolomics analysis now provide researchers
   with an opportunity to explore algae–bacteria interactions at a
   systemic level. For example, recent studies used metabolomics analysis
   in co-cultivation systems to investigate the effects of bacteria on
   algae [[77]31,[78]32], and on bacterial algicides produced under
   different environmental conditions [[79]33]. A metabolomics study of
   diatom Thalassiosira pseudonana co-cultured with bacterium
   Dinoroseobacter shibae exhibited an upregulation of various critical
   pathways involving intracellular amino acids compared to the algal
   monocultures [[80]32], suggesting an adaptive response of the diatom to
   the presence of this bacterium [[81]32]. Other research also
   investigated changes in the metabolomics profile of cyanobacteria
   exposed to multiple chemical algicides, including copper sulfate,
   hydrogen peroxide, and sodium carbonate peroxide [[82]34]. So far, no
   research has been conducted to investigate changes in the metabolome by
   algal species in response to bacterial algicides.

   In this research, metabolomics analysis was conducted on dinoflagellate
   K. veneficum exposed to IRI-160AA to explore the effects of this
   algicide at the metabolomics level. Metabolomics analysis of a
   non-target cryptophyte species, Rhodomonas sp., was also conducted as a
   control to elucidate mechanisms underlying the specificity of this
   algicide for dinoflagellates. Finally, an exometabolomics approach
   distinguished those metabolites that were present in the cell pellet
   from those that may have been introduced to the culture by the algicide
   itself. In addition to a number of diverse processes related to
   ammonium metabolism and detoxification in both species, results of this
   study identified a suite of metabolites that was upregulated in K.
   veneficum but not Rhodomonas. Metabolites that were specific to K.
   veneficum included biomarkers for oxidative stress, antioxidants, as
   well as vitamins and metabolites known to induce DNA damage and
   participate in PCD in other organisms, supporting previous research
   that algicidal compounds in IRI-160AA may act synergistically with
   ammonium to induce cell death in dinoflagellates.

2. Results

   The in vivo fluorescence (as a proxy for cell density; [[83]23]) of K.
   veneficum treated with the algicide was significantly lower (61.78%)
   than the control after 19 h exposure (p < 0.05; [84]Figure 1 and
   [85]Figure S1). No significant difference in in vivo fluorescence was
   observed between the treatment and the control for Rhodomonas sp. (p >
   0.05).

Figure 1.

   [86]Figure 1
   [87]Open in a new tab

   In vivo fluorescence of Karlodinium veneficum and Rhodomonas sp. in the
   algicide treatments (with IRI-160AA) relative to controls after 19 h of
   exposure. Error bars indicate standard deviations of 3 replicates.
   Asterisks indicate a significant difference of relative in vivo
   fluorescence of the indicated treatments compared to the controls (p <
   0.05).

   The metabolomics analysis detected 1140 polar and semi-polar compounds
   in the samples, and a total of 139 compounds that were above the
   detection limits across all samples were annotated ([88]Table S1,
   [89]Figure 2A). Among these annotated compounds, 122 were identified in
   the cell pellets, and 107 were in the cell filtrate ([90]Figure 2B,C).

Figure 2.

   [91]Figure 2
   [92]Open in a new tab

   Heatmaps of the annotated compounds in the cell pellets and filtrate of
   Karlodinium veneficum and Rhodomonas treated with the algicide (Alg.)
   compared to the control (Con.; with no algicide addition). All
   annotated compounds were included in the heatmap. (A) Heatmap of all
   annotated compounds; (B) heatmap of compounds identified in the cell
   pellets; (C) heatmap of the compounds identified in the cell filtrate.
   Relative concentrations of each compound (RC) were log10 (RC+1)
   transformed first, and the row z-scores represent the normalized log10
   (RC+1) across each row. The heatmap was created using Heatmapper
   [[93]35].

   Only the annotated compounds with a relative concentration altered by
   the algicide addition by at least 1.5-fold were selected for further
   analysis. In this dataset, 93 compounds were detected in the K.
   veneficum cell pellets and 71 compounds were detected in the K.
   veneficum cell filtrate samples that exhibited higher relative
   concentrations in the treatment compared to the control. For
   Rhodomonas, the relative concentrations of 51 compounds were higher in
   the treatment compared to the control in the cell pellets, and 33
   metabolites had higher relative concentrations in the treatment
   compared to the control for the cell filtrate.

   Additionally, three metabolites had lower relative concentrations in
   the treatment compared to the control in the cell pellets and six in
   the filtrate of K. veneficum. Four compounds had lower relative
   concentrations in the treatment compared to the control in the
   Rhodomonas cell pellet and twenty-four in the filtrate samples.

2.1. Metabolomics Profile of Cell Pellets

   A total of 74 compounds that exhibited higher relative concentrations
   in the algicide treatment compared to the control were included for the
   metabolite enrichment analysis for K. veneficum cell pellets
   ([94]Figure 3 and [95]Figure S2). These compounds were mapped to 31
   KEGG pathways, with almost half of these pathways involved in amino
   acid metabolism (45%). Vitamin metabolism contributed an additional 16%
   to all mapped pathways. Four pathways were enriched by these compounds
   (FDR < 0.05), including the aminoacyl-tRNA biosynthesis, glutamine and
   glutamate metabolism, as well as biosynthesis of branched chain amino
   acids (BCAA; valine, leucine, and isoleucine) and arginine. Though not
   significantly enriched, the metabolite sets’ enrichment revealed the
   potential involvement of B vitamin metabolism (riboflavin, thiamine,
   and pyridoxal) in K. veneficum cell pellets treated with the algicide
   (FDR > 0.05).

Figure 3.

   [96]Figure 3
   [97]Open in a new tab

   Metabolites that increased in the cell pellets in the algicide
   treatment compared to the control (>1.5-fold) were mapped to 31 KEGG
   pathways in Karlodinium veneficum and 26 pathways in Rhodomonas
   [[98]36]. A. The percentage of amino acid and vitamin metabolism in the
   mapped pathways, as well as other processes among these mapped pathways
   in each species (calculated as the number of pathways in
   categories/number of all pathways × 100%). “*” indicates the
   significance of the metabolite sets’ enrichment of the indicated
   pathway (FDR < 0.05). “°” indicates the vitamin metabolism pathways
   that were only mapped by the increased metabolites in the cell pellets
   stimulated by the algicide compared to the control for K. veneficum but
   not Rhodomonas. See [99]Figure S2 for the detailed enrichment analysis.

   Forty-three compounds that had higher relative concentrations in the
   algicide treatment compared to the control were included in the
   metabolite sets’ enrichment analysis for Rhodomonas sp. cell pellets
   ([100]Figure 3 and [101]Figure S2). These compounds were mapped to 26
   KEGG pathways. Similar to the enrichment analysis for K. veneficum cell
   pellets, the majority of these mapped pathways belong to amino acid
   metabolism (54%). Vitamin metabolism contributed 8% of all mapped
   pathways, but notably, the pathways involved in the metabolism of
   riboflavin, thiamine, and pyridoxal that had been identified for K.
   veneficum cell pellets were not observed here. These metabolites were
   enriched in six pathways (FDR < 0.05), including three pathways shared
   with K. veneficum (aminoacyl-tRNA biosynthesis, metabolism of glutamate
   and glutamine, and biosynthesis of BCAA), as well as metabolism of
   purine nucleotides and biosynthesis of alanine and aspartate.

   The compounds that had lower relative concentrations in the algicide
   treatment compared to the control were not included in the metabolite
   sets’ enrichment analysis due to the low numbers of compounds.

2.2. Metabolites Related to Vitamins

   Several B vitamins, including riboflavin, thiamine, pyridoxal,
   nicotinic acid, and nicotinamide, increased in the cell pellets of K.
   veneficum treated with the algicide compared to the control
   ([102]Figure 4). None of these vitamins were enriched in cell pellets
   of Rhodomonas. Pyridoxal and nicotinamide increased in the cell
   filtrate of K. veneficum treatment as well, while the other B vitamins
   were not enriched in the cell filtrate of this species. In Rhodomonas
   cell filtrate, thiamine decreased in algicide treatments while
   nicotinamide increased in concentration compared to the control.

Figure 4.

   [103]Figure 4
   [104]Open in a new tab

   Relative changes in B vitamins, metabolites involved in ROS production,
   DNA damage, and/or PCD, and antioxidants that increased in the cell
   pellets of Karlodinium veneficum but not Rhodomonas. Figures represent
   the log10 transformed fold-change of relative concentrations of these
   compounds in the cell pellets (upper) and the filtrate (lower) of the
   algicide treatment over the control (with no algicide addition). The
   analysis was based on the averaged peak areas (normalized to cell
   densities for the cell pellets) of duplicate samples; error bars were
   not included.

2.3. Metabolites Related to ROS Production, DNA Damage, and Programmed Cell
Death

   Five compounds that could be potentially involved in ROS production,
   DNA damage, and/or PCD were identified: methionine sulfoxide (MetO),
   formylkynurenine (N′-formylkynurenine; NFK), N-methyl-d-aspartic acid
   (NMDA), cyclo(phenylalanine-proline) (cFP), and phenylalanine
   ([105]Figure 4; phenylalanine not shown). MetO, NMDA, and cFP only
   increased in the cell pellets of the algicide treatment of K. veneficum
   but not their filtrate compared to the control. The relative
   concentrations of these compounds did not change in the cell pellets or
   filtrate of the algicide treatment of Rhodomonas. NFK increased in the
   cell pellets of K. veneficum but not Rhodomonas exposed to the algicide
   compared to the respective controls. It also increased in the cell
   filtrate of both K. veneficum and Rhodomonas algicide treatments
   compared to the respective controls. Phenylalanine increased in cell
   pellets and filtrate of the treatments for both K. veneficum and
   Rhodomonas compared to the respective controls.

   Antioxidants, including salicylic acid (SA), 3-hydroxybenzoic acid
   (3-HBA), and proline increased only in the cell pellets of the algicide
   treatment of K. veneficum compared to the control ([106]Figure 4),
   while the levels of these compounds did not change (SA and 3-HBA) or
   decreased (proline) in the cell pellets of Rhodomonas treatment
   compared to the control. In the cell filtrate of K. veneficum
   treatment, the level of SA did not change while 3-HBA and proline
   increased compared to the control. In the cell filtrate of Rhodomonas
   treatment, the level of SA decreased, while concentrations of 3-HBA and
   proline did not change.

2.4. Ammonium Concentrations

   Results indicated that the ammonium concentration in the stock algicide
   was 3.20 mM. At the initial time point, algicide was added to each of
   the treatment cultures at 4.97% (v/v; EC50) of the total volume,
   leading to a calculated addition of 159 μM ammonium in the treatment
   compared to the control (no algicide addition).

   After 19 h, significant differences were observed in ammonium
   concentrations between the treatments and controls of both species (p <
   0.05; [107]Figure 5). Ammonium concentrations in the treatment and
   control of K. veneficum were 216 and 104 μM, respectively, and 203 and
   104 μM in the Rhodomonas treatment and control, respectively. No
   significant differences in ammonium concentrations between the two
   species were observed in the control or the treatment (p > 0.05).

Figure 5.

   [108]Figure 5
   [109]Open in a new tab

   Ammonium concentrations in Karlodinium veneficum and Rhodomonas in
   controls (with no algicide addition) and the algicide treatment
   measured after 19 h incubation. Error bars indicate standard deviations
   of 3 replicates. Asterisks indicate a significant difference of
   ammonium concentrations in the indicated treatment compared to the
   control (p < 0.05).

3. Discussion

   Previous research demonstrated that the algicide IRI-160AA produced by
   Shewanella sp. IRI-160 specifically targets dinoflagellates
   [[110]21,[111]23]. Exposure to the algicide impaired photosynthesis,
   increased ROS production, and induced DNA damage and cell cycle arrest,
   along with other markers indicating a programmed pathway leading to
   cell death (PCD) [[112]25,[113]26,[114]27]. Molecular evidence
   supporting these physiological effects was also identified in
   transcriptomics analysis [[115]30]. Research on IRI-160AA indicated
   that ammonium was among the active algicidal compounds, and that other
   compounds in the algicide contributed to the observed algicidal effects
   [[116]28]. In this study, a metabolomics approach was used to identify
   changes in metabolites for the dinoflagellate K. veneficum after
   exposure to IRI-160AA compared to control cultures. Metabolomics
   analysis was also conducted on a non-target control species, the
   cryptophyte Rhodomonas sp., to elucidate mechanisms behind the
   selectivity of IRI-160AA for dinoflagellate species.

   Results of bioassays ([117]Figure 1) were consistent with previous
   studies showing the specificity of the algicide IRI-160AA to
   dinoflagellates, including K. veneficum [[118]21,[119]23]. The
   differences in response to the algicide were supported by metabolomics
   analysis, in which a suite of compounds was found to increase only in
   the cell pellets of K. veneficum but not Rhodomonas when exposed to the
   algicide ([120]Figure 4 and [121]Figure 6). These compounds included
   antioxidants, vitamins, and other metabolites that can potentially
   contribute to oxidative stress, DNA damage, and PCD, highlighting the
   specific effects of IRI-160AA on dinoflagellates in relationship with
   previously observed physiological responses [[122]25,[123]26,[124]30].

Figure 6.

   [125]Figure 6
   [126]Open in a new tab

   Schematic representation of a model depicting the cellular impact of
   IRI-160AA on Karlodinium veneficum revealed by this study and
   previously reported molecular and physiological data
   [[127]25,[128]26,[129]27,[130]30]. In this model, IRI-160AA stimulated
   ROS production, inducing the formation of oxidative stress markers,
   methionine sulfoxide (MetO), and formylkynurenine (N′-formylkynurenine;
   NFK) in the cell pellets of the algicide treatment of K. veneficum.
   Cellular antioxidants also increased in response to ROS, including
   salicylic acid (SA), 3-hydroxybenzoic acid (3-HBA), proline, and B
   vitamins (riboflavin, thiamine, pyridoxal, nicotinic acid, and
   nicotinamide). Other compounds indicative of reactive oxygen species
   (ROS), DNA damage, and programmed cell death (PCD) that increased in
   the cell pellets included N-methyl-d-aspartic acid (NMDA),
   phenylalanine, and cyclo(phenylalanine-proline) (cFP). cFP can enter
   the cells by simple diffusion, or by the biosynthesis from
   phenylalanine and proline, where the pathway has not been fully
   characterized (indicated by “?” in the figure). The figure was created
   with BioRender.com.

   Previous research observed higher intra- and extracellular ROS
   production in dinoflagellates exposed to the algicide IRI-160AA
   compared to the control [[131]25], suggesting the potential for
   increased levels of antioxidants and oxidation products in cells
   exposed to the algicide. Accumulation of metabolites that serve as
   biomarkers for oxidative stress has been observed in plants
   [[132]37,[133]38] and algae [[134]39,[135]40,[136]41]. One such marker,
   MetO, is the oxidation product from methionine, the most susceptible
   amino acid to ROS oxidation [[137]42,[138]43]. MetO has been used as a
   reliable biomarker for oxidative stress as well as an indicator for
   ineffective repair mechanisms [[139]42].

   The identification of MetO in this study is consistent with previous
   reports demonstrating ROS production and induction of PCD in
   dinoflagellates exposed to IRI-160AA [[140]25] ([141]Figure 4 and
   [142]Figure 6). MetO inactivates proteins, and detoxification of this
   compound through the reduction of MetO by MetO reductase is crucial to
   alleviate the adverse effects of ROS [[143]43]. For example, MetO
   reductase mutants of plants are associated with an increase in ROS
   concentrations and induction of PCD [[144]43]. Administration of MetO
   to animals through subcutaneous injection also induced DNA damage and
   apoptosis, accompanied by increased ROS and antioxidant enzyme
   activities [[145]44]. The selective accumulation of MetO in the cell
   pellets of K. veneficum treated with IRI-160AA compared to controls in
   this study implies a conserved role of MetO among different kingdoms of
   organisms. The accumulation of MetO in K. veneficum but not in
   Rhodomonas also suggests that K. veneficum was not able to efficiently
   detoxify this compound.

   Similar to MetO, formylkynurenine (N′-formylkynurenine; NFK) is an
   oxidative stress biomarker and also an amino acid oxidation product
   [[146]45,[147]46,[148]47]. NFK is a post-translational modification of
   tryptophan that can be formed during the oxidation of its side-chain
   exposed to multiple ROS, including singlet oxygen, ozone, and hydroxyl
   radicals [[149]45,[150]46]. NFK has been identified as a biomarker for
   oxidative stress in plants exposed to high light, and may play a role
   in photosystem II (PSII damage and repair signaling [[151]45,[152]46].
   Research has also suggested that NFK may serve as a protein antioxidant
   and protect mitochondria from oxidative stress [[153]47]. The selective
   increase of NFK in the cell pellets of K. veneficum is consistent with
   the observed ROS production stimulated by the algicide [[154]25]
   ([155]Figure 4 and [156]Figure 6). To be noted, NFK is also a
   photosensitizer; research has indicated its production may stimulate
   its further accumulation in the cells [[157]47]. Here, the increase of
   NFK in the cell filtrate of both Rhodomonas and K. veneficum exposed to
   the algicide compared to the control implies NFK may also be
   (partially) provided by the algicide itself. However, the effect of
   extracellular NFK application is not clear and requires future
   research.

   An increase in antioxidant levels also serves as a marker for oxidative
   stress [[158]48,[159]49]. In this study, there was an increase in
   antioxidant levels in the cell pellets of K. veneficum treated with
   IRI-160AA but not in Rhodomonas ([160]Figure 4 and [161]Figure 6).
   These antioxidants included salicylic acid (SA; 2-hydroxybenzoic acid),
   a ubiquitous phytohormone that plays a central role in the resistance
   and tolerance of plants in response to various abiotic and biotic
   stresses [[162]50]. As an antioxidant, SA can either directly scavenge
   ROS or indirectly regulate the redox balance by enhancing antioxidant
   defense in plants [[163]51,[164]52], including increasing the activity
   of antioxidant enzymes [[165]53]. Both intracellular accumulation and
   exogenous application of SA have been demonstrated to enhance
   antioxidant defense and tolerance to stresses in plants
   [[166]51,[167]53]. In plant cells, the concentration of SA may tip the
   balance from survival to death. For example, accumulation of SA at high
   concentrations at the microbial infection site in Arabidopsis thaliana
   triggered a local immune response, favoring PCD, while at low
   concentrations, SA induced a systemic immune response, enhancing cell
   survival at locations that were distant from the infection site
   [[168]54]. As evidence has been provided for PCD in dinoflagellates
   exposed to the algicide IRI-160AA [[169]25], the increased SA in the
   cell pellets of K. veneficum treatment compared to the control implied
   that this metabolite could serve a function similar to that observed in
   plants. A dose-dependent effect of SA on the growth of chlorophyte
   Chlorella vulgaris [[170]55] and the cyanobacterium Microcystis
   flos-aquae [[171]56] has also been demonstrated. The differential
   response of K. veneficum and Rhodomonas to the algicide may reflect
   their varied tolerance to SA, or species-specific SA requirements to
   cope with stresses induced by other compounds in IRI-160AA. Other
   studies have reported that extracellular application of SA increases
   the intracellular level of this compound [[172]51,[173]53]. Although
   there was a small increase of SA in the filtrate of K. veneficum, the
   decrease of SA in the cell filtrate of Rhodomonas suggests the
   potential for uptake of this compound if it was a component of the
   algicide.

   Other antioxidants were also identified in K. veneficum cell pellets
   that were present in higher concentrations in treatments compared to
   the control ([174]Figure 4 and [175]Figure 6). The SA isomer
   3-hydroxybenzoic acid (3-HBA), for example, exhibited antioxidant
   activities that could alleviate lipid peroxidation [[176]57] and
   protect iron from autoxidation [[177]58]. This metabolite was present
   in K. veneficum cell pellets as well as the culture filtrate of this
   species, but was not elevated for Rhodomonas. It is unclear if the
   increase of 3-HBA in the algicide treatment of K. veneficum was
   synthesized by this species in response to the oxidative stress caused
   by IRI-160AA [[178]25], or was taken up from the algicide.

   Proline was also enriched in K. veneficum cell pellets after exposure
   to the algicide ([179]Figure 4 and [180]Figure 6). This amino acid also
   has antioxidant properties [[181]59,[182]60]. Accumulation of proline
   is a common response of plants under a variety of stresses, where it
   plays a major role as a metal chelator, ROS scavenger, and signaling
   molecule [[183]59,[184]60]. Despite its beneficial role in stress
   tolerance, studies have also indicated that ROS production could be
   stimulated by both endogenous proline accumulation and exogenous
   proline application, leading to either cell survival or death,
   including through PCD, in a concentration-dependent manner [[185]61].
   The selective accumulation of proline in the cell pellets of K.
   veneficum exposed to the algicide was consistent with its role as an
   antioxidant, while higher concentrations may have played a role in
   initiating PCD in this species.

   Other metabolites involved in DNA damage and PCD identified in this
   research included N-Methyl-d-aspartic acid (NMDA) and
   cyclo(phenylalanine-proline) (cFP); they were among the metabolites
   that only increased in the cell pellets of K. veneficum treatment
   compared to the control but not Rhodomonas ([186]Figure 4 and
   [187]Figure 6). NMDA is a known inducer of apoptosis in mammalian cells
   [[188]62]; the apoptosis was accompanied by DNA nick and fragmentation
   [[189]63]. Here, the increase of NMDA in the cell pellets of K.
   veneficum treated with the algicide compared to the control implied it
   may have a comparable role in dinoflagellates. Similar to the other
   metabolites that had a selective increase in the cell pellets of K.
   veneficum over Rhodomonas revealed in this research, its source (e.g.,
   exogenous vs. endogenous) could not be identified with the current
   data.

   Cyclo dipeptides (aka 2,5-diketopiperazines, CDPs) are ubiquitous
   compounds with prominent biological functions [[190]64]. Among the
   CDPs, cyclo(phenylalanine-proline) (cFP; [191]Figure 4 and [192]Figure
   6) is well-known for its quorum-sensing property in various bacterial
   species [[193]65]. Though its function in algal species is not clear,
   cFP is able to pass biological membranes via simple diffusion [[194]66]
   and induce apoptosis in mammalian cells [[195]66], accompanied by
   increased caspase-3 activity [[196]67], ROS production [[197]68], and
   DNA damage [[198]68]. cFP-induced apoptosis was mediated through the
   ATR-CHK2 pathway involved in the DNA damage response (DDR)
   [[199]68,[200]69]. The increase of cFP in K. veneficum cell pellets
   treated with the algicide compared to the control implied cFP could
   have a similar function in dinoflagellates. This was supported by
   previous transcriptomic data that suggested the possible involvement of
   an ATR-CHK2 DDR pathway in this species in response to IRI-160AA
   exposure [[201]30].

   Though cFP could have diffused into cells from IRI-160AA added to the
   culture medium [[202]66], the selective increase of cFP contents in the
   cell pellets of K. veneficum over Rhodomonas implied the contribution
   of other sources, possibly the biosynthesis from phenylalanine and
   proline [[203]65]. This was supported by the evidence that
   phenylalanine increased in the cell pellets of the algicide treatments
   of both K. veneficum and Rhodomonas compared to their respective
   controls (also indicated by the pathway enrichment analysis;
   [204]Figure S2), while the intracellular proline only increased in the
   K. veneficum algicide treatment ([205]Figure 4 and [206]Figure 6).

   B vitamins, including riboflavin (vitamin B2, [[207]70]), thiamine
   (vitamin B1, [[208]71]), pyridoxal (vitamin B6, [[209]72]), nicotinic
   acid (vitamin B3, [[210]73]), and nicotinamide (vitamin B3, [[211]74])
   are antioxidants that have essential protective roles in organisms. The
   increase of these B vitamins in the cell pellets of K. veneficum in the
   algicide treatment compared to the control ([212]Figure 4 and
   [213]Figure S2) may serve as additional biomarkers for oxidative
   stress, and their accumulation in cells of K. veneficum may help this
   species cope with the oxidative stress induced by the algicide
   [[214]25]. The culture filtrate of algicide-treated K. veneficum was
   also enriched with pyridoxal and nicotinamide, suggesting the
   possibility that they were components of the bacterial filtrate
   IRI-160AA.

   Comparison between the metabolomic profiles of the cell pellets of K.
   veneficum and the control cryptophyte Rhodomonas ([215]Figure 2) also
   revealed a number of similar processes stimulated by the algicide
   ([216]Figure 3 and [217]Figure S2) that were likely related to the
   presence of ammonium in the algicide. The shared enriched processes
   included glutamine and glutamate metabolism, as well as biosynthesis of
   branched chain amino acids (BCAAs) and arginine (FDR < 0.05;
   [218]Figure 3).

   Enrichment of glutamine and glutamate metabolism in both K. veneficum
   and Rhodomonas correlated with increased ammonium concentrations
   ([219]Figure 5), introduced to the culture with the algicide. Glutamine
   synthetase/glutamate synthase (GS/GOGAT) pathway is the primary
   ammonium assimilation route in higher plants and algae, and plays an
   essential role in ammonium detoxification
   [[220]75,[221]76,[222]77,[223]78] ([224]Figure 7). Glutamate produced
   here is the precursor for other nitrogen-rich metabolites, including
   proline, γ-aminobutyric acid (GABA), and arginine, among others
   [[225]79]. Arginine can then be utilized to produce polyamines
   [[226]79,[227]80]. Nitrogen flow to polyamines has been identified as a
   major nitrogen sink for excess ammonium and is critical to alleviate
   ammonium toxicity [[228]79,[229]80]. In addition to the assimilation
   from ammonium, glutamate can also be produced from other sources in the
   cells, including from BCAA catabolism
   [[230]81,[231]82,[232]83,[233]84]. In fact, BCAAs are essential
   nitrogen donors for glutamate biosynthesis in animals, and play a
   critical role in ammonia detoxification through the production of
   glutamine from glutamate and ammonia [[234]81,[235]82]. The role of
   BCAAs in algae is not clear, while the pathways involved in BCAA’s role
   in ammonia detoxification are shared among animals, plants, and algae,
   implying a conserved function of these amino acids
   [[236]81,[237]82,[238]83].

Figure 7.

   [239]Figure 7
   [240]Open in a new tab

   Simplified schematic demonstrating the relationship between ammonium
   assimilation and metabolism of glutamine, glutamate, branched chain
   amino acids, and arginine. These metabolites are indicated by a red
   font. Glutamate is the precursor of other amino acids in the cells, and
   a number of pathways related to their metabolism were identified in the
   enrichment analysis (see [241]Figure 3 and [242]Figure S2). The figure
   was created with BioRender.com.

   Comparison of ammonium concentrations for algicide-treated K. veneficum
   and Rhodomonas suggests similar uptake by these species ([243]Figure
   5), while metabolic analysis shows a similar response through ammonium
   detoxification pathways involving BCAAs, glutamine/glutamate, and
   arginine discussed above. Previous studies, however, have demonstrated
   that dinoflagellates have greater sensitivity to ammonium toxicity
   [[244]85]. Polyamines present in the algicide [[245]28] may also
   intensify the effects of ammonium on dinoflagellates [[246]30]. A
   recent study demonstrated the synergistic negative effects of ammonium
   and putrescine on K. veneficum, and transcriptomic analysis revealed
   the role of polyamines by enhancing ammonium toxicity in K. veneficum
   [[247]30].

   Chemical exchanges between bacteria and algae in the microenvironment
   immediately surrounding an algal cell, termed the phycosphere, play an
   important role in algal growth and physiology (reviewed by [[248]3]).
   Dinoflagellates and other algal taxa, for example, rely on bacterial
   communities to provide critical nutrients [[249]86], and there is
   substantial evidence that bacteria–algal interactions have a
   significant effect on the growth of algae [[250]87]. The choice to use
   non-axenic cultures in experiments to examine the effects of IRI-160AA
   on dinoflagellates is similar to other studies
   [[251]23,[252]25,[253]26], and provides a more realistic and
   environmentally relevant understanding of dinoflagellate response to
   the algicide. Metabolomic analyses on algal species in non-axenic
   culture are also fairly common; for example, to evaluate metabolomic
   response due to allelopathic interactions between dinoflagellates and
   diatoms [[254]88], to profile the metabolomic changes of diatoms during
   different growth stages [[255]89], and other studies (see for example
   [[256]90,[257]91]). Similar to the rationale presented here, Vidoudez
   and Pohnert [[258]89] pointed out that using non-axenic diatom cultures
   may better reflect metabolomic responses of diatoms in nature, and
   excluding bacterial communities in the cultures may cause additional
   stress that would influence the results. In the study presented here,
   the algal cell pellets were captured on filters, limiting the
   metabolomics analysis of the cell pellets to the algae and those
   bacteria that were attached to the cells, and eliminating free-living
   bacteria from the analysis. The limited effect of co-cultured bacteria
   on the algal cell pellets was evident by the fact that very few
   metabolites were shared by the cell pellets and filtrate from the same
   culture for either K. veneficum or Rhodomonas, even in the controls
   ([259]Table S1). In summary, it was likely that bacterial cohorts in
   non-axenic cultures of K. veneficum or Rhodomonas made a (limited)
   contribution to the changes in the metabolomic profiles presented here,
   but the results obtained from analysis of non-axenic cultures are more
   representative of these species in the natural environment.

   It should also be noted, this analysis only included polar and
   semi-polar metabolites. The metabolomic profiles of non-polar
   metabolites (e.g., lipids) of algae exposed to the algicide remain
   unknown. Furthermore, enriched metabolites within the algicide
   treatment compared to the control may also be from the addition of the
   algicide itself, although few of the enriched metabolites discussed
   here were found in both the cell pellet and the culture filtrate
   ([260]Figure 4), and only one of these was also enriched in the culture
   filtrate from Rhodomonas. Instead, the stimulation of these compounds
   in the cell pellets by IRI-160AA likely reflects the cellular response
   of K. veneficum to the algicide. Future research should be directed to
   identify the sources of these metabolites through
   stable-isotope-labeling studies, for example.

   In conclusion, this research identified the metabolomic response of
   dinoflagellate K. veneficum to the bacterial algicide IRI-160AA. A
   suite of metabolites was revealed to selectively increase in the cell
   pellets of K. veneficum but not Rhodomonas exposed to IRI-160AA. These
   metabolites included the oxidative stress biomarkers methionine
   sulfoxide and N′-formylkynurenine, a number of antioxidants (salicylic
   acid, 3-hydroxybenzoic acid, proline, and B vitamins), and compounds
   involved in DNA damage and PCD (N-methyl-d-aspartic acid and
   cyclo[phenylalanine-proline]). Pathways related to ammonium
   detoxification, including glutamine/glutamate metabolism, as well as
   biosynthesis of branched amino acids and arginine, were also
   upregulated in K. veneficum. This was consistent with the contribution
   of ammonium to the algicidal effect of IRI-160AA. At the same time, the
   observation that these processes were also upregulated in Rhodomonas
   implied that the specificity of IRI-160AA for dinoflagellates is not
   only related to the addition of ammonium, as noted in Ternon et al.
   [[261]28]. This research provided insights into the algicidal effect of
   IRI-160AA on K. veneficum at the metabolomic level and expanded our
   knowledge of the metabolic response of dinoflagellates to bioactive
   substances produced by bacteria. Future research should be conducted to
   continue to identify the active algicidal compounds in the algicide
   IRI-160AA, and also explore the metabolomic effects of these compounds,
   individually or in combination, on dinoflagellates.

4. Materials and Methods

4.1. Algal Stock Culture

   Non-axenic stock cultures of Karlodinium veneficum (CCMP 2936 [National
   Center for Marine Algae and Microbiota, Bigelow Laboratory for Ocean
   Sciences, East Boothbay, USA]; dinoflagellate) and Rhodomonas sp. (CCMP
   757; cryptophyte) were maintained in natural seawater with f/2
   nutrients (-Si) [[262]92] and a salinity of 20, at 25 °C, and with a
   light intensity of approximately 130 µmol photons m^−2 s^−1. The
   cultures were kept under a 12 h:12 h light:dark cycle and
   semi-continuously in the exponential growth phase.

4.2. Algicide Preparation

   IRI-160AA was prepared as described previously [[263]23] with slight
   modification. Briefly, a single colony of Shewanella IRI-160 was
   transferred to LM medium [[264]93]. The culture was incubated at 25 °C
   with shaking overnight at 100 rpm. The Shewanella IRI-160 culture was
   centrifuged at 6000 rpm for 5 min. The supernatant was discarded, and
   the cell pellet was resuspended in f/2 medium (-Si) [[265]92]. The
   suspension was centrifuged again as above. The supernatant was
   discarded, and cell pellets were resuspended in f/2 medium (-Si)
   [[266]92] and incubated at room temperature for 7 days. To prepare the
   cell-free algicide, the culture was filtered through a 0.2 µm nylon
   syringe filter (Corning, Corning, NY, USA).

4.3. Ammonium Concentration

   Ammonium concentration in the algicide was measured using an API^®
   Ammonia Test Kit (Mars Fishcare Inc., Chalfont, PA, USA). The method
   was modified from the manufacturer’s instructions and was described
   previously [[267]94]. Briefly, each of 250 μL hypochlorite solution and
   salicylate/catalyst solution was added to the 2.5 mL diluted sample and
   incubated at room temperature for 10 min. The absorbance of the sample
   at 690 nm was measured using a NanoDrop 2000 Spectrophotometer
   (ThermoFisher Scientific, Waltham, MA, USA). A standard curve was made
   with a series of diluted ammonium standards (Sigma-Aldrich St. Louis,
   MO, USA). The ammonium concentration in the sample was then calculated
   by linear regression analysis.

4.4. Metabolomic Response of K. veneficum and Rhodomonas sp. to Algicide
IRI-160AA

4.4.1. Culture Treatments

   Algicide IRI-160AA was prepared as described above, and EC50
   (half-maximal effective concentration) was determined on K. veneficum
   cultures (N = 3; 4.97% [v/v]). IRI-160AA at the EC50 level was added to
   50 mL cultures of K. veneficum and Rhodomonas sp. in 250 mL
   polycarbonate Erlenmeyer flasks (N = 3). The control cultures received
   the same amount of f/2 medium without algicide (N = 3). After 19 h, in
   vivo fluorescence was measured for each culture. Microscopy cell counts
   were also performed (data not shown). The relative in vivo fluorescence
   of the treatment was calculated as: [in vivo fluorescence of the
   treatment/average in vivo fluorescence of the control] [[268]23].

4.4.2. Sample Preparation for Metabolomics Analysis

   Cell pellets were collected by filtering each culture through GF/F
   filters (Whatman, Maidstone, UK). Metabolites were extracted from the
   filters using cold 60% methanol (v/v with MilliQ water) immediately
   after filtration. The mixture was vortexed for 30 s, and the filters
   were discarded. The culture filtrate was further filtered through 0.2
   μm nylon syringe filters (Corning). The extracted cell pellets and
   culture filtrate were lyophilized, and duplicates of each sample were
   sent to MS-Omics (Vedbæk, Denmark) on dry ice for analysis.

4.4.3. Sample Analysis for Polar and Semi-Polar Metabolites

   Metabolomics analysis was carried out by MS-Omics using a Vanquish LC
   coupled to Thermo Q Exactive HF MS (Thermo Fisher Scientific, Waltham,
   MA, USA). An electrospray ionization interface was used as an
   ionization source. The analysis was performed in negative and positive
   ionization mode. Ultra Performance Liquid Chromatography (UPLC) was
   performed for semi-polar [[269]95] and polar [[270]96] compounds using
   slightly modified versions of the protocols.

   Peak areas were extracted using Compound Discoverer 2.0 (Thermo Fisher
   Scientific). Annotation was performed at four levels; Level 1:
   identification by retention times (compared against in-house authentic
   standards), accurate mass (with an accepted deviation of 3 ppm), and
   tandem mass spectrometry (MS/MS) spectra; Level 2a: identification by
   retention times (compared against in-house authentic standards) and
   accurate mass (with an accepted deviation of 3 ppm); Level 2b:
   identification by accurate mass (with an accepted deviation of 3 ppm)
   and MS/MS spectra; Level 3: identification by accurate mass alone (with
   an accepted deviation of 3 ppm).

4.4.4. Data Analysis

   Averaged peak areas of compounds detected in the cell pellets were
   normalized to the cell density of each species at the sampling time
   point. The normalized peak areas were considered as relative
   concentrations (RC) of each compound. A heatmap was constructed using
   Heatmapper [[271]35] from the log10 (RC+1) transformed values of
   compounds that were annotated (including all annotation levels).

   The fold-change of each compound comparing the treatment and control
   was calculated as: [Average peak area (treatments) + 1]/[Average peak
   area (controls) + 1]. Only compounds that were altered by the algicide
   by at least 1.5-fold (>1.5 to call “increase” or <0.67 to call
   “decrease”) were included in the metabolite enrichment analysis using
   MetaboAnalyst [[272]36] with the KEGG pathway database and the default
   settings. The compounds that could not be matched in the MetaboAnalyst
   pathway library and metabolite set database were filtered out
   [[273]36]. The pathways with an FDR < 0.05 were considered enriched.

   Additionally, antioxidants and metabolites related to ROS production,
   DNA damage response, and programmed cell death that increased in the
   cell pellets of K. veneficum but not Rhodomonas were identified by
   searching the literature. The same thresholds, >1.5-fold and <0.67-fold
   changes, were used to consider a compound to be increased or decreased,
   respectively, in the treatment compared to the control.

4.5. Statistical Analysis

   One-way ANOVA was used to evaluate the significant differences in the
   relative in vivo fluorescence or ammonium concentrations between the
   controls and treatments of K. veneficum and Rhodomonas (p < 0.05). The
   same test was also conducted to assess the significant differences in
   ammonium concentrations of the controls or the algicide treatments
   between these species (p < 0.05).

5. Conclusions

   This investigation provided insights into the metabolic responses of
   two phytoplankton species after exposure to algicide IRI-160AA,
   produced by naturally-occurring marine bacterium Shewanella sp.
   IRI-160. Results of this research expanded our knowledge of the effects
   of microbially-produced algicides on algal taxa with varying
   sensitivities, and revealed underlying metabolic changes in response to
   these bioactive substances. The suite of metabolites that was
   selectively increased in dinoflagellate Karlodinium veneficum included
   several biomarkers for oxidative stress, and a number of antioxidants,
   as well as compounds involved in DNA damage and PCD. Future research
   may be directed to determination of the synergistic effects of
   compounds identified in IRI-160AA on the dinoflagellate metabolome.

Supplementary Materials

   The following supporting information can be downloaded at:
   [274]https://www.mdpi.com/article/10.3390/metabo12040317/s1, Table S1:
   Log10 (RC+1) transformed relative concentrations (RC) of compounds
   detected in the cell pellets and filtrate of Karlodinium veneficum and
   Rhodomonas; Figure S1: In vivo fluorescence (FLR) of the harmful
   dinoflagellate Karlodinium veneficum and the control cryptophyte
   Rhodomonas treated with f/2 medium without the algicide (control) or
   the algicide for 19 h. Error bars represent standard deviations of 3
   replicates. “*” indicates significant differences between the control
   and the treatment (p < 0.05); Figure S2: Metabolite sets’ enrichment
   analysis on metabolites increased (>1.5-fold comparing the treatment
   and the control) in cell pellets of Karlodinium veneficum (A) and
   Rhodomonas sp. (B).
   [275]Click here for additional data file.^ (396.8KB, zip)

Author Contributions

   Conceptualization, Y.W. and K.J.C.; methodology, Y.W. and K.J.C.;
   software, Y.W.; validation, Y.W. and K.J.C.; formal analysis, Y.W.;
   investigation, Y.W.; resources, K.J.C.; data curation, Y.W.;
   writing—original draft preparation, Y.W.; writing—review and editing,
   Y.W. and K.J.C.; visualization, Y.W.; supervision, K.J.C.; project
   administration, K.J.C.; funding acquisition, Y.W. and K.J.C. All
   authors have read and agreed to the published version of the
   manuscript.

Funding

   This project was funded by the National Oceanic and Atmospheric
   Association (NOAA) Prevention, Control and Mitigation of HABs (PCMHAB)
   program (Grant # NA15NOS4780176 to K.J. Coyne, M.E. Warner, T. Targett
   and J. Cohen; Grant # NA20NOS4780185 to K.L. Pokrzywinski, K.J. Coyne,
   Y. Wang, and A.J. Kennedy. PCMHAB contribution #60).

Institutional Review Board Statement

   Not applicable.

Informed Consent Statement

   Not applicable.

Data Availability Statement

   All metabolomics data for this research can be found in the
   [276]Supplementary Materials (Table S1).

Conflicts of Interest

   The authors declare no conflict of interest.

Footnotes

   Publisher’s Note: MDPI stays neutral with regard to jurisdictional
   claims in published maps and institutional affiliations.

References