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. Author manuscript; available in PMC: 2020 Nov 1.
Published in final edited form as: Limnol Oceanogr. 2019 Aug 19;64(6):2709–2724. doi: 10.1002/lno.11309

Molecular and biochemical basis for the loss of bioluminescence in the dinoflagellate Noctiluca scintillans along the west coast of the USA

Martha Valiadi 1,5, Tristan de Rond 2, Ana Amorim 3, John R Gittins 1, Chrysoula Gubili 4, Bradley S Moore 2, M Debora Iglesias-Rodriguez 1,6, Michael I Latz 2
PMCID: PMC7351363  NIHMSID: NIHMS1603780  PMID: 32655189

Abstract

The globally distributed heterotrophic dinoflagellate Noctiluca scintillans (Macartney) Kofoid & Swezy is well known for its dense blooms and prominent displays of bioluminescence. Intriguingly, along the west coast of the USA its blooms are not bioluminescent. We investigated the basis for the regional loss of bioluminescence using molecular, cellular and biochemical analyses of isolates from different geographic regions. Prominent differences of the non-bioluminescent strains were: (1) the fused luciferase and luciferin binding protein gene (lcf/lbp) was present but its transcripts were undetectable; (2) lcf/lbp contained multiple potentially deleterious mutations; (3) the substrate luciferin was absent, based on the lack of luciferin blue autofluorescence and the absence of luciferin derived metabolites; (4) although the cells possessed scintillons, the vesicles that contain the luminescent chemistry, electron microscopy revealed additional scintillon-like vesicles with an atypical internal structure; (5) cells isolated from the California coast were 43% smaller in size than bioluminescent cells from the Gulf of Mexico. Phylogenetic analyses based on the large subunit of rDNA did not show divergence of the non-bioluminescent population in relation to other bioluminescent N. scintillans from the Pacific Ocean and Arabian Sea. Our study demonstrates that gene silencing and the lack of the luciferin substrate have resulted in the loss of a significant dinoflagellate functional trait over large spatial scales in the ocean. As the bioluminescence system of dinoflagellates is well characterized, non-bioluminescent N. scintillans is an ideal model to explore the evolutionary and ecological mechanisms that lead to intraspecific functional divergence in natural dinoflagellate populations.

Keywords: Luciferase, Luciferin, Scintillon, Functional diversity

Introduction

Since first described by Henry Baker in 1753 as ‘animalcules’ (Harvey 1957), the globally distributed marine dinoflagellate Noctiluca scintillans, whose Latin name means ‘glowing bright by night’ and has the common name ‘sea sparkle’, is known for its brightly bioluminescent blooms with abundances as high as 106 cells L−1 (Staples 1966; Daniel et al. 1979; Mohamed and Mesaad 2007; Kopuz et al. 2014). N. scintillans plays important roles in food webs (Fock and Greve 2002; Yilmaz et al. 2005) as a food source (Sulkin et al. 1998; Vargas and Madin 2004; Zhang et al. 2017a), a voracious predator of phytoplankton and zooplankton (Kimor 1979; Buskey 1995; Kiørboe and Titelman 1998; Nakamura 1998b; Johnson and Shanks 2003; Zhang et al. 2016; Stauffer et al. 2017), an important competitor of zooplankton for phytoplankton prey (Umani et al. 2004; Yilmaz et al. 2005), and a contributor to recycling of organic material through the ingestion of fecal pellets (Kiørboe 2003) and excretion of inorganic nutrients that become available to primary producers (Zhang et al. 2017b).

Although the genus Noctiluca Suriray comprises only one globally distributed species, N. scintillans, there are regional varieties with distinct characteristics. The “green” Noctiluca variety contains the photosynthetic green flagellate symbiont Pedinomonas noctilucae (e.g. Elbrachter and Qi 1998) within its vacuoles, which provides about 70% of its energy requirements (Sweeney 1978), and is endemic to waters of southeast Asia and the Arabian Sea region (Harrison et al. 2011). Otherwise the ”red” Noctiluca variety, which lacks green endosymbionts, is widely distributed in temperate and subtropical waters (Harrison et al. 2011); its orange-red color is due to carotenoid pigments obtained from food prey or synthesized de novo (Balch and Haxo 1984).

Whereas all other dinoflagellates harbor separate luciferase (LCF) and luciferin binding protein (LBP), Noctiluca scintillans contains a fused LCF-LBP, encoded by the lcf/lbp gene. This is likely to be the ancestral gene arrangement for dinoflagellates, as N. scintillans is a basal taxon among dinoflagellates based on phylotranscriptome analyses (Janouskovec et al. 2017), having a typical dinokaryotic nucleus only during the gamete life stage (Hansen et al. 2004) and lacking mitochondrial mRNA editing that is typical of more recent dinoflagellate species (Chang 1960; Sweeney 1963). It is thought that lcf/lbp split into two separate genes in more recent dinoflagellates (Liu and Hastings 2007; Valiadi and Iglesias-Rodriguez 2014), potentially to allow for individual regulation of each protein (Valiadi and Iglesias-Rodriguez 2013).

Light emission by dinoflagellate cells originates from scintillons, organelles distributed throughout the peripheral cytoplasm that contain luciferin and LCF. N. scintillans has on the order of 104 scintillons 0.5-1.5 µm in diameter (Eckert 1966; Eckert and Reynolds 1967), within a cytoplasmic layer that can be as thin as 0.11 µm (Nawata and Sibaoka 1979). Thus, scintillons project into the vacuolar space that takes up a large proportion of the N. scintillans cell volume and contains sap of high acidity (Nawata and Sibaoka 1976). Light flashes are triggered by mechanical stimulation of the cell (Nicol 1958), prompting a propagating action potential across the vacuole membrane (Eckert 1965a; Eckert 1965b; Eckert and Sibaoka 1968) that opens voltage-gated proton channels (Rodriguez et al. 2017). The subsequent flux of protons from the vacuole into the scintillon causes a decrease in pH that dissociates luciferin from LBP and activates luciferase, resulting in the oxidation of luciferin releasing energy in the form of visible light (Fogel and Hastings 1972; Nawata and Sibaoka 1979).

Dinoflagellate bioluminescence acts as a predator defense behavior to reduce grazing (Esaias and Curl 1972; White 1979), through flash responses to predator contact serving to startle predators (Buskey et al. 1983; Buskey and Swift 1985) and acting as a light alarm to attract secondary visual predators of the dinoflagellate predators (Morin 1983; Mensinger and Case 1992; Abrahams and Townsend 1993; Fleisher and Case 1995; Cusick and Widder 2013). Cells also increase their bioluminescence when chemical cues from predators are present (Lindström et al. 2017). Despite the ecological significance of bioluminescence, there are bioluminescent and non-bioluminescent strains within some species (Valiadi et al. 2012). Characterizing patterns in the expression of lcf and biosynthesis of luciferin will aid in understanding the evolution of dinoflagellate bioluminescence and in identifying environmental conditions that favor the maintenance or loss of light production.

The aim of this study was to investigate the molecular, cellular and biochemical basis for the lack of bioluminescence in N. scintillans from the west coast of the USA. This red form of N. scintillans differs from those of other regions by its smaller size (Eckert and Findlay 1962; Balch and Haxo 1984; Tada et al. 2000; Liu and Hastings 2007) and lack of bioluminescence (Esaias 1973; Hoppenrath and Leander 2010). By comparison to a bioluminescent variety from the Gulf of Mexico, we assessed cell size, existence of scintillons, presence of luciferin and its derived metabolites and the presence, sequence, and expression of lcf/lbp. Results confirmed a lack of luciferin fluorescence but surprisingly show that scintillons are present in the cells. Furthermore, lcf is present in the genome but its expression is repressed. Therefore, the bioluminescence system of N. scintillans from the west coast of the USA has become non-functional. Phylogenetic analyses of the LSU rDNA gene showed that the loss of bioluminescence in N. scintillans has not resulted in a divergent population, or a different species. We therefore propose that this is a good model for further studies on the eco-evolutionary and oceanographic processes driving functional divergence in natural plankton populations.

Materials

Cell culturing and bioluminescence tests

Strains of N. scintillans were obtained from three locations off the west coast of the USA (strain abbreviations LJ - La Jolla, CA; SC - Santa Cruz, CA; SPMC - Shannon Point Marine Centre, Puget Sound, WA), as well as the Gulf of Mexico (strain abbreviation GM – Port Aransas, TX) (Figure 1, Table 1). As cell cultures remain viable for a limited time (Sato et al. 1998), in some cases it was necessary to re-establish cultures from the same location. Cells were cultured in filter-sterilized (0.22-µm membrane filter, Steritop, Millipore, UK) or Whatman GF/F (GE Healthcare Bio-Sciences, Pittsburgh, PA USA) filtered seawater supplemented with appropriate amounts of prey culture as required; subculturing was conducted every 10-14 days. A low irradiance of 15 µmol m−2 s−1 was used to prevent overgrowth of the prey. Cells of the phytoplankton prey Dunaliella tertiolecta Butcher CCMP 1320 (Chlorophyceae) and Prorocentrum micans Ehrenberg CCMP 691 (Dinophyceae) were cultured in f/2 (Guillard and Ryther 1962) and L1 (Guillard and Hargraves 1993) seawater media, respectively, without silicate. The dinoflagellate Pyrocystis lunula (Schütt) Schütt, which was used for luciferin extractions, was cultured in half strength f/2 medium. All cultures were maintained at 19-20ºC on a 12:12 h light:dark cycle.

Figure 1.

Figure 1.

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Map of the USA showing major ocean currents along the western seaboard where non-bioluminescent Noctiluca scintillans is found. The geographic origins of the three strains used in this study. LJ = La Jolla (non-bioluminescent), California; SPMC = Shannon Point Marine Center, Washington (non-bioluminescent); GM = Gulf of Mexico (bioluminescent). Information on currents is re-drawn from Sverdrup et al. (Sverdrup et al. 1942).

Table I.

Noctiluca scintillans cultures originating from collections in the USA. Bioluminescence (BL) was assayed by stirring (Jin et al. 2013) or manual agitation. Molecular, cellular and biochemical tests on a subsets of these. BL, production of bioluminescence; Luciferin fluorescence is based on presence of blue autofluorescence; Luciferin metabolites, presence of luciferin derived metabolites based on LC-MS analysis; Lcf in gDNA, presence of the luciferase gene in genomic DNA; Lcf in cDNA, presence of the luciferase gene in cDNA obtained from mRNA; Scintillons, presence of scintillons in electron micrographs.

Collection date Origin BL Time BL tested after collection (months) ID Luciferin fluorescence Luciferin metabolites Lcf in gDNA Lcf in cDNA Scintillons
30 Nov 1998 Gulf of Mexico, Port Aransas, TX + 0.3 GM + + + +
20 Feb 2000 Scripps Pier, La Jolla, CA – 2, 6
21 Jan 2009 Fidalgo Island, WA – 14 SPMC136 – + – +
30 Aug 2010 Scripps Pier, La Jolla, CA – 0 LJ + – +
21 Nov 2011 Scripps Pier, La Jolla, CA – 1
22 Mar 2012 Scripps Pier, La Jolla, CA – 2
3 Jul 2012 Santa Cruz, CA – 0.3
10 Jul 2012 Scripps Pier, La Jolla, CA – 0.3
12 Apr 2017 Gulf of Mexico, Port Aransas, TX + 0.5, 13 GM2017 + +
9 May 2017 Scripps Pier, La Jolla, CA – 0 LJ2017 – –
26 Apr 2018 Santa Cruz, CA – 0.5
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The bioluminescence of N. scintillans strains was tested visually. When no bioluminescence was observed, dense cultures were tested using a luminometer as described in Valiadi et al. (2012). Cell size was measured as equivalent spherical diameter using a Beckman Multisizer 3 with 1000 µm aperture tube. Cell cultures were first sieved through 100 µm Nitex mesh and then resuspended in GF/F filtered seawater prior to measurement. The instrument noise level was 36 µm, well below the expected average cell size of non-bioluminescent cells (approx. 250 µm).

Nucleic acid extraction and reverse transcription

Noctiluca scintillans cells were harvested at stationary phase when the cultures were dense and most of the prey cells had been consumed. Approximately 400 mL of culture were filtered gently onto 25-mm diameter, 5-µm pore size Nuclepore polycarbonate membranes (Whatman, U.K.) and stored at −80 ºC. The filter-bound cells were disrupted using a micropestle while still frozen in liquid nitrogen and DNA extraction was performed as described previously (Valiadi et al. 2012). RNA was isolated using the Nucleospin RNA II kit (Macherey-Nagel, Germany) which includes an integrated on-column DNase digestion step. The quantity and purity of the DNA and RNA were assessed using a Nanodrop spectrophotometer (ND-3000, Nanodrop, USA). The RNA was reverse transcribed using the High Capacity RNA-to-cDNA kit (Applied Biosystems, USA), which utilizes random primers. A control reverse transcription reaction was included for each RNA sample, omitting the reverse transcriptase enzyme (-RT reaction). These samples were used to assess the PCR quality of the DNA and cDNA, as well as the absence of genomic DNA carryover contamination in the latter, using primers for the amplification of the small subunit of the rDNA gene (Lin et al. 2006).

PCR

The oligonucleotide primers used in this study and their gene targets are detailed in Table S1. Detection of lcf was achieved using “universal” primers for this gene as described by (Valiadi et al. 2012), with either DNA or cDNA as the template. Primers to amplify and sequence nearly the whole open reading frame (ORF) of the lcf/lbp gene, were designed using the program Primer3 (http://frodo.wi.mit.edu/primer3/) and the published full lcf/lbp gene of N. scintillans accession no. JF838193 (Liu and Hastings 2007). The 25 µL reactions consisted of 0.1 µM each primer, 250 µM each dNTP, 1x standard PCR buffer, 0.5U GoTaq DNA polymerase (Promega, UK) and 50-100 ng of DNA or cDNA template. PCR amplification was achieved using the following program: 5 min at 95 ºC for initial denaturation, followed by 30 cycles of 95 ºC for 45 s, 55 ºC for 30 s, 68 ºC for 3 min and a final extension at 68 ºC for 10 min.

The LSU rDNA gene was amplified using specific primers designed to prevent amplification of rDNA from the P. micans prey in strain SPMC 136. The PCRs were conducted using the high fidelity and high yield Advantage 2 PCR kit and Polymerase mix (Clontech, USA). Each 25 µL reaction contained 0.02 µM each primer, 200 µM each dNTP, 1x Advantage 2 PCR buffer and 1x Advantage 2 polymerase mix, as recommended by the manufacturer. PCR amplification was achieved using the following program: 3 min at 95 ºC for initial denaturation followed by 30 cycles at 95 ºC for 20 s, 55 ºC for 30 s, 72 ºC for 2 min and a final extension at 72 ºC for 10 min.

Sequence and phylogenetic analyses

We compared lcf/lbp in N. scintillans with differing bioluminescence ability by cloning amplicons from the genomic DNA of two non-bioluminescent strains LJ and SPMC, and the bioluminescent strain GM. We also used the LSU rDNA gene to explore the phylogenetic relationship of non-bioluminescent N. scintillans to bioluminescent strains from other ocean regions. Three clones were sequenced from each LSU rDNA amplicon. Sequences were trimmed to remove vector and primer sequences and assembled using the CAP3 sequence assembly program (http://doua.prabi.fr/software/cap3). For both genes, the BLASTn tool (NCBI) was used to confirm that the correct gene had been amplified and to retrieve similar sequences in the GenBank database for phylogenetic analyses. Additionally, we conducted searches for both lcf/lbp and LSU rDNA in TaraOceans metagenome datasets using the Ocean Gene Atlas platform (http://tara-oceans.mio.osupytheas.fr/ocean-gene-atlas/) to help understand the variability of N. scintillans genes in natural populations. Nucleotide sequence alignments were carried out using ClustalW (Thompson et al. 1994) implemented in MEGA v7.0 (Kumar et al. 2016) with manual refinement. Where multiple sequences from a single strain were identical, only one was retained as a reference in further analyses.

For LSU rDNA, Bayesian phylogenetic analyses were performed using MrBayes 3.2.6 (Ronquist et al. 2012) following the selection of the most appropriate model of evolution (TIM1+G) by jModelTest 2.1.9 (Darriba et al. 2012) based on Bayesian information criterion (BIC). Analyses were performed in two independent runs of 5,000,000 generations and four Markov chains, with sampling every 100 generations resulting in 50,000 trees and a burn-in of 25%. The final phylogenetic tree was generated using FigTree 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree).

Confocal laser scanning microscopy

Cells were collected individually during the mid-dark phase using a plastic Pasteur pipette and placed on a glass slide with a raised circular well, constructed using double sided tape that could accommodate the large N. scintillans cells. The cells were immobilized by adding a drop of viscous Protoslow quieting solution (Blades Biological Ltd, UK). Imaging of the cells was performed using a Leica TCS SP5 laser scanning confocal microscope (Leica Microsystems Inc. USA). Optical slices of 0.5 µm were obtained for part of the cell, stopping when the focal distance of the microscope would cause the objective to contact the raised coverslip. Luciferin auto-fluorescence was induced by a UV-laser with an excitation wavelength of 405 nm and then recorded and imaged at 416-520 nm.

Transmission electron microscopy

Cultures of the bioluminescent N. scintillans strain GM and of non-bioluminescent strain SPMC136 were cooled on ice and then fixed with 2% glutaraldehyde. Fixed cells were concentrated in small baskets made of a 40-µm plankton net. To avoid further disturbance to the cells, all the following steps until embedding in Spurr resin were performed in these baskets. The cells were washed free of glutaraldehyde with 0.1 M PIPES buffer containing 1% NaCl (added to raise the osmolarity of the buffer). They were then post-fixed in 1% osmium tetroxide in the same buffer for 1 h at room temperature. After rinsing in distilled water, the cells were stained in 2% aqueous uranyl acetate for 20 min followed by standard dehydration in an ethanol series and a final 10-min wash in acetonitrile, before embedding overnight in Spurr resin. Individual specimens were then picked and placed in separate polymerizing molds with fresh resin, and left to polymerise at 60ºC for 24h. Sectioning was performed with an ultramicrotome (Reichert-Jung Ultracut E). The sections were mounted on uncoated copper grids and stained with lead citrate. Grids were viewed and photographed with a Hitachi H-7000 electron microscope.

Luciferin and luciferase extractions

Luciferin was extracted following established methods (Nakamura et al. 1989). Approximately 4-10 × 103 cells of N. scintillans GM, 21-108 × 103 cells of N. scintillans LJ, or 500-950 × 103 cells of P. lunula were collected by vacuum filtration onto Whatman GF/F paper, and the filter paper resuspended in 10 mL of pre-heated, anaerobic extraction buffer (5 mM potassium phosphate, 5 mM beta-mercaptoethanol, pH 8) under an argon atmosphere in a 50 mL conical vial. The samples were vortexed for 5 s, boiled for 1 min, and then cooled on ice, all while under an argon atmosphere, followed by centrifugation for 20 min at 20,000 × g at 4°C. The supernatant was passed through a 0.2 µm filter and directly analyzed on an Agilent 1290 infinity liquid chromatography system with a diode array UV/Vis detector and Agilent 6500 series Q-TOF mass spectrometer with an Electrospray Ionization source using only ultrahigh purity nitrogen (99.999%) as drying gas. A Phenomenex Luna 5 µm C18(2) 100 Å column (150 × 4.6 mm, 5 µm particle size) was employed. Mobile phase: A: 0.1% formic acid in water, B: 0.1 % formic acid in acetonitrile. Gradient: 5% B for 5 minutes, ramp to 100% B in 10 minutes, 100% B for 5 minutes, ramp to 5% B in 1 minute, 5% B for 3 minutes. Flow rate: 0.7 mL/min. Source parameters: Drying gas: 11 liters per minute, 300 °C; Nebulizer: 35 psig; Capillary: 3000 V; Fragmentor: 100 V; Skimmer: 65 V; OCT 1 RF Vpp: 750. Tandem MS (Collision Induced Dissociation) parameters: Isolation width: Narrow; Collision energy: 20 V.

In an attempt to detect bona fide dinoflagellate luciferin in N. scintillans, we adopted some approaches aimed at suppressing potential luciferin-LBP binding and oxidation of the luciferin. We extracted using isopropanol: water to unfold LBP. We attempted to avoid oxidation through extended anaerobic incubation of N. scintillans followed by anaerobic extraction. We performed direct LC-MS analysis of N. scintillans luciferase extracts as well as anaerobic ultrafiltration at pH 6, to encourage release from LBP, followed by LC-MS analysis.

Luciferase extracts were prepared based on established protocols (Schmitter et al. 1976). Approximately 7-33 × 103 cells of N. scintillans were collected using vacuum filtration on Whatman GF/F filter paper, which was plunged into ice-cold buffer containing 50 mM Tris-HCl, 10 mM EDTA, and 5 mM 2-mercaptoethanol. After vortexing for 30 s, cells were disrupted in a glass homogenizer, cell debris was removed by collecting the supernatant after centrifuging at 5000 x g for 10 min; following high speed centrifuging at 27,000 x g for 15 min, the supernatant was stored at 4˚C or −80˚C until testing.

Extract cross-reactions

Bioluminescence cross-reactions were measured by spotting 10 µL volumes of ice-cold N. scintillans luciferase extract and P. lunula luciferin extract in different places at the bottom of a test tube, which was placed in a Sirius luminometer (Berthold Detection Systems). The reaction was initiated by injection of 250 µL of 5 mM phosphate buffer pH 6.0 with 1 min light measurement. The blank, representing background, consisted of buffer injection alone. Values for a representative experiment of 14 May 2018 are for means with standard deviations.

Results

Geographic distribution of bioluminescent and non-bioluminescent N. scintillans

The environmental ranges of the bioluminescent and non-bioluminescent varieties of N. scintillans in the northeastern Pacific Ocean do not appear to overlap, as the west coast of the USA is the only known region where non-bioluminescent N. scintillans are present. The observed lack of mechanically stimulated bioluminescence in strains from the coasts of California and estuarine waters of Washington (Figure 1, Table 1) was confirmed using a luminomenter and low pH treatment (Valiadi et al. 2012) to activate the luminescent chemistry independent of mechanotransduction. Furthermore, numerous isolates of N. scintillans from California have been tested over the years and also have invariably been found to lack bioluminescence (Table 1).

Morphological differences

Cell size was the most obvious morphological difference between isolates; the non-bioluminescent isolates were approximately half the size of bioluminescent cells. Cells of the bioluminescent N. scintillans GM2017 had an equivalent spherical diameter of 468 ± 49 µm (N = 1126 cells), while that for the non-bioluminescent strain LJ2017 was 266 ± 37 µm (N = 2872) (Figure S1), representing a 43% smaller size. Otherwise the strains were indistinguishable except for their ability to produce light when mechanically disturbed.

Detection and partial characterization of luciferase gene remnants

We tested one bioluminescent and two non-bioluminescent strains of N. scintillans for the presence and expression of lcf using “universal” PCR primers for dinoflagellate lcf (Valiadi et al. 2012). Fragments of the correct size (~ 270 bp) were amplified from genomic DNA isolated from all strains (Figure S2).

Sequencing of a large section of the lcf/lbp open reading frame revealed the non-bioluminescent strains contain lcf/lbp pseudogenes with multiple mutations (Figure S3). These were mainly deletions ranging from 2 to 96 bp. The highest number of mutations was found in strain SPMC originating from Washington (USA), where sequences contained deletions of at least 36 bp over several loci. Sequences of lcf/lbp from bioluminescent N. scintillans did not show any deleterious mutations in this study. Sequence alignments with another bioluminescent isolate from Gulf of Mexico tested previously (Valiadi and Iglesias-Rodriguez 2014), and bioluminescent environmental N. scintillans samples from the southwest Atlantic (Valiadi et al. 2014), show that while bioluminescent N. scintillans lcf/lbp exhibit deletions too, they were only up to 3 bp. There were no sequences of N. scintillans lcf/lbp present in TaraOceans metagenome from the northeast Pacific area.

Reverse Transcription-PCR only detected lcf in cDNA derived from RNA isolated from the bioluminescent strain (Table 1, Figure S2). The successful amplification of the dinoflagellate Small Subunit (SSU) rDNA gene as a control PCR from the cDNAs of the two non-bioluminescent strains, LJ2000 and SPMC136, verified the quality of the cDNA and therefore the negative result obtained using the lcf primers. Therefore, while lcf was present in the genome of both bioluminescent and non-bioluminescent strains, its transcript was undetectable in the non-bioluminescent cells.

Identification of scintillons and luciferin by microscopy

The presence of scintillons can typically be determined by the blue autofluorescence of dinoflagellate luciferin to UV light excitation (Johnson et al. 1985; Fritz et al. 1990). All strains were examined by confocal laser scanning microscopy in order to determine the presence and cellular location of luciferin. Cells of the bioluminescent strain exhibited blue fluorescent sources around the cell periphery (Figure S4), consistent with the presence of luciferin in scintillons. The number of scintillons in each cell was so high that due to their small size the fluorescence merged into a nearly continuous glow around the periphery of the cell. As scintillons were small in comparison to the large cell size it was not possible to determine their abundance within the cell from the confocal microscope images. Cells of non-bioluminescent strains LJ2000 and SPMC136 did not exhibit any blue autofluorescence, suggesting they lacked luciferin.

The presence of scintillons in the cells of the bioluminescent strain GM and in the non-bioluminescent strain SPMC136 was further investigated using transmission electron microscopy (Figure 2). The bioluminescent strain GM contained organelles appearing as smooth membrane-bound electron dense organelles, 0.7 to 0.9 µm in size, around the periphery of the cells near the cell wall, identical in size and appearance to scintillons (Nicolas et al. 1985; Nicolas et al. 1987), occurring in clusters with or near trichocysts. Organelles of equivalent electron density, morphology, and size, and similar in appearance to the scintillons of the bioluminescent strain were present in the non-bioluminescent strain SPMC136. Additionally, there were several structures of smaller size that also occurred near the cell covering and were surrounded by a membrane that resembled scintillons. However, their contents were granular, often with a very regular pattern. These scintillon-like structures were unique to the non-bioluminescent strain.

Figure 2.

Figure 2.

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Transmission electron micrographs of Noctiluca scintillans cell sections. Upper left shows detail of the cell periphery of bioluminescent N. scintillans GM with a scintillon surrounded by membrane and trichocysts along the cell wall. Lower left shows a slice of the cell surface of N. scintillans GM with a large number of scintillons. Upper right shows detail of the cell periphery of the non-bioluminescent N. scintillans strain SPMC136 with a scintillon surrounded by a membrane near the cell wall and an additional smaller scintillon-like structure with a granular content. Lower right shows this scintillons like structure in higher magnification with detail of the internal granular structure. S = scintillons, S? = scintillon-like structure with lower density of content, T = trichocyst, CW = cell covering, C = cytoplasm.

Luciferin and luciferase extracts

As previously reported by Liu et al. (2007), cross-reactions of luciferin (‘hot’) and luciferase (‘cold’) extracts confirmed that P. lunula luciferin strongly reacted with N. scintillans GM2017 luciferase (3.9 ± 0.2 × 107 RLU s−1, N = 3), and that the luciferase extract also contained co-extracted endogenous luciferin, based on light emission without the addition of P. lunula luciferin (3.3 ± 0.4 × 105 RLU s−1, N = 3) (Table S2). The non-bioluminescent strain LJ2017 luciferase extract had weak but nonzero luciferase activity in the presence of P. lunula luciferin (1.1 ± 0.1 × 104 RLU s−1, N = 3), but unlike strain GM did not emit light of its own accord (23.7 ± 4.0 RLU s−1, N = 3), indicating that the LJ2017 extract did not contain luciferin.

We employed Liquid Chromatography-Mass Spectrometry (LC-MS) to examine the presence of luciferin metabolites in N. scintillans. While P. lunula luciferin can cross-react with luciferases from other dinoflagellates (Schmitter et al. 1976; Liu and Hastings 2007), it is unknown whether the endogenous luciferins are identical. Despite considerable efforts, we were unable to detect luciferin in its reduced (native) form in bioluminescent N. scintillans strain GM2017. We confirmed that this was not due to a methodological failure by successful detection of reduced luciferin in P. lunula extracts, provided ultrapure nitrogen was used as drying gas in the mass spectrometer source. As the presence of endogenous luciferin in strain GM2017 is corroborated by microscopy, luminometry, and the observation that luciferase extracted from this strain exhibits luminescence even in the absence of added luciferin, we suspect that the reduced luciferin is either tightly bound to the LBP domain of the LCF-LBP protein, or enzymatically or non-enzymatically oxidized during the luciferin extraction procedure. These results suggest that alternative methods are needed to detect native luciferin in LBP-containing dinoflagellates.

Nevertheless, we were able to detect enzymatically oxidized luciferin (oxyluciferin) and spontaneously air-oxidized luciferin in aqueous extracts of strain GM2017, as indicated by exact masses, tandem MS fragmentation patterns and UV-Vis absorbance maxima consistent with previous reports (Figure 3, S5) (Nakamura et al. 1989). The presence of these luciferin-derived metabolites suggests that bioluminescent N. scintillans GM2017 may harbor the same luciferin as P. lunula, supporting our suggestion that the reduced form was not detected due to it being bound to LBP. In contrast, no luciferin metabolites were detected in the non-bioluminescent N. scintillans strain LJ2017.

Figure 3.

Figure 3.

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LC-MS analysis of dinoflagellate luciferin and its metabolites in Noctiluca scintillans and Pyrocystis lunula. Shown are summed extracted ion chromatograms for the m/z values of for luciferin, oxyluciferin and air-oxidized luciferin (m/z of 589.3, 603.3, and 605.3, respectively).

Phylogenetic analyses based on LSU rDNA

Bayesian phylogenetic analyses of N. scintillans based on a 595 bp alignment of partial LSU rDNA sequences did not resolve non-bioluminescent N. scintillans as a separate group. Rather, they clustered with sequences from the South China Sea and were not significantly different from other isolates originating from other Chinese seas or from the Arabian Sea; indeed different sequenced clones were intermingled among isolates (Figure 4). Both these regions contain bioluminescent N. scintillans, the red variety in China and the green variety in the Arabian Sea. The bioluminescent strain from the Gulf of Mexico, which was the only representative of the Atlantic Ocean, was distinct from all other sequences.

Figure 4.

Figure 4.

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Bayesian phylogenetic tree based on a nucleotide alignment of partial LSU (large subunit) rDNA sequences. Gaps were excluded in the analysis. Values shown at major nodes are posterior probabilities; values lower than 0.7 are excluded. Sequences from GenBank are labelled with accession numbers, isolate and clone information. The geographic origin of N. scintillans sequences are indicated: CA & WA = California and Washington; CS = Chinese Seas; AS = Arabian Sea; GM = Gulf of Mexico. The scale of branch lengths shows 0.3 substitutions per 10 sites.

To better characterize the high intra-strain variability of LSU rDNA in natural N. scintillans populations, we retrieved 5 N. scintillans LSU rDNA Unigenes from the TaraOceans dataset of approximately 400 bp length. None of these were located within our study region in coastal waters of the north east Pacific, but rather showed with highest abundances in the open northeast and southeast Pacific Ocean, Arabian Sea and Mediterranean Sea. Nevertheless, we performed an alignment and clustering of these new sequences based on genetic distance, to all those included in the phylogenetic analyses. This truncated dataset contained only 185 base positions where all sequences overlapped. Despite the limited phylogenetic resolution, this analysis showed a significantly different group in the TaraOceans dataset, unrelated to any other N. scintillans LSU rDNA sequences known to date (Figure S6).

Discussion

The globally distributed dinoflagellate N. scintillans, known for centuries for its bright bioluminescence (Harvey 1957), has non-bioluminescent populations along the west coast of the USA (Chang 1960; Eckert and Findlay 1962; Sweeney 1963; Esaias 1973; Balch and Haxo 1984; Sulkin et al. 1998); this study). The results of this study indicate that the bioluminescence system of non-bioluminescent N. scintillans has been “silenced” and that the lack of bioluminescence in these populations is due to two factors – a mutated lcf with undetectable expression and lack of luciferin.

Non-bioluminescent N. scintillans cells do not express lcf

The presence of lcf in the genome of non-bioluminescent N. scintillans suggests that they were bioluminescent in the past, but that the bioluminescence system has been silenced at the molecular level by numerous deletions of more than 36 bp (in strains LJ and SPMC), suggesting that this gene has lost its coding potential. Dinoflagellate gene families contain multiple copies with variable sequences; for bioluminescence genes this variability may be as high as 12% base differences (Valiadi and Iglesias-Rodriguez 2014). Long stretches of deletions have been found previously in the lcf of non-bioluminescent strains of normally bioluminescent dinoflagellate species (Valiadi et al. 2012). Pseudogenes containing frameshift deletions are also found in the lcf of bioluminescent N. scintillans cultured isolates and environmental samples (Valiadi and Iglesias-Rodriguez 2014; Valiadi et al. 2014); however, the maximum deleted length has been 3 bp. Thus, large scale deletions in N. scintillans lcf/lbp appear to be confined to the non-bioluminescent strains.

While in dinoflagellates there is no clear relationship between the presence of pseudogenes and genetic function, our observations of large-scale deletions in the non-bioluminescent N. scintillans is in agreement with loss of gene function. A slight amount of luciferase activity in one of the non-bioluminescent strains tested suggests that some of the gene copies may still be functional, and therefore that the loss of bioluminescence may not be complete. The genetic mechanisms that transiently or permanently switch off genes in dinoflagellates, including those coding for bioluminescence, are unknown. The mutations present in lcf of non-bioluminescent N. scintillans may be a cause for lack of gene expression or a result of it by a lack of selection on the protein structure and function. Deep sequencing of lcf/lbp loci will be informative in understanding the functional genomic silencing process within dinoflagellates.

Non-bioluminescent N. scintillans cells lack luciferin

Luciferins are known to be widespread in food webs, being transferred from prey to predator, where they can accumulate in tissue even in non-bioluminescent animals (Shimomura 1987; Shimomura 2006). Thus, bioluminescent organisms may either synthesize luciferin or obtain it through the diet. For example, manipulations that deplete stores of luciferin in laboratory-maintained animals have demonstrated that the lophograstrid Gnathophausia ingens Dohrn requires a dietary source of coelenterazine (Frank et al. 1984), and the midshipman fish Porichthys notatus Girard requires cipridinid ostracod luciferin (Tsuji et al. 1972; Barnes et al. 1973). Populations of P. notatus found north of San Francisco Bay (Strum 1969b; Warner and Case 1980; Thompson and Tsuji 1989) are non-bioluminescent due to lack of cipridinid luciferin (Tsuji et al. 1972; Barnes et al. 1973), presumably because the ostracod prey serving as the dietary source of luciferin is restricted to southern waters (Kornicker and Baker 1977). Despite their lack of bioluminescence, the photophores of the northern P. notatus are structurally identical to those of bioluminescent fish (Strum 1969a; Strum 1969b), and these populations exist in large numbers, suggesting that the lack of bioluminescence is a recent event that has not had a deleterious effect on species physiology or survival.

The lack of luciferin metabolites in non-bioluminescent N. scintillans cells, as well as the lack of fluorescent particles, supports earlier evidence that they lack luciferin (Eckert and Reynolds 1967); non-bioluminescent strains of other bioluminescent dinoflagellates do not display luciferin fluorescence (Johnson et al. 1985). Dinoflagellate luciferin is structurally related to chlorophyll (Dunlap et al. 1981) and is thought to be synthesized de novo in photosynthetic dinoflagellates as part of the chlorophyll biosynthetic pathway (Wu et al. 2003). The origin of luciferin in heterotrophic dinoflagellates is unknown. Although non-bioluminescent N. scintillans in southern California feed on bioluminescent dinoflagellate prey (Torrey 1902; Stauffer et al. 2017; Busch et al. 2019), this does not restore bioluminescence under laboratory conditions (M. Latz, personal obs.), suggesting that N. scintillans does not obtain luciferin from its diet. Furthermore, the bioluminescence of another heterotrophic dinoflagellate, Protoperidinium crassipes (Kofoid) Balech, persists when maintained on a non-bioluminescent non-algal diet composed solely of rice flour (Yamaguchi and Horiguchi 2008). As Noctiluca contains a plastid tetrapyrrole pathway consistent with the endogenous production of luciferin (Janouskovec et al. 2017), the possibility of de novo synthesis of luciferin in Noctiluca and other non-photosynthetic dinoflagellates needs further investigation.

Significance of non-bioluminescent N. scintillans cell architecture

Non-bioluminescent N. scintillans cells contain organelles that are morphologically similar in size and appearance to scintillons in bioluminescent dinoflagellates (Nicolas et al. 1985; Nicolas et al. 1987), despite lacking both luciferin and LCF. Similarly, non-bioluminescent northern west coast USA populations of the midshipman fish P. notatus contain photophores that are morphologically identical to those of bioluminescent southern populations (Strum 1969a; Strum 1969b), despite lacking luciferin. As only bioluminescence related proteins have been found in scintillons (Desjardins and Morse 1993), the chemical composition of the smooth electron dense contents of scintillons from non-bioluminescent cells is unclear. Further investigation of the contents of scintillons as well as the newly described scintillon-like granular organelles is required in order to establish their origin and role in non-bioluminescent cells.

Strains of the non-bioluminescent N. scintillans that originated from the west coast of the USA differed morphologically from bioluminescent cells from other locations only by their smaller size. The diameter of bioluminescent cells in this study averaged 468 µm, within the range of 300 to 900 µm previously reported for other locations (Nawata and Sibaoka 1976; Nakamura 1998a; Tada et al. 2000; Liu and Hastings 2007; Mohamed and Mesaad 2007), while the average diameter of non-bioluminescent cells was 266 µm, within the range of 100-400 µm (Balch and Haxo 1984; Sulkin et al. 1998; Stauffer et al. 2017) reported for N. scintillans collected along the west coast of the USA. Similarly, cells from non-bioluminescent strains of the dinoflagellate P. lunula are also 50% smaller than cells from bioluminescent strains (Swift et al. 1973). This correlation between dinoflagellate cell size and bioluminescence ability is not universal in intraspecific comparisons, as non-bioluminescent Alexandrium catenella (Whedon & Kofoid) Balech cells are of similar size to bioluminescent cells (Schmidt et al. 1978). Factors that can regulate cell size include top down control through size-selective predation, bottom up control through environmental conditions and food supply, and genetic control related to changes in gene expression associated with the lack of bioluminescence. Although the cell size of bioluminescent N. scintillans varies with growth conditions, being inversely proportional to growth rate for cultured cells (Buskey 1995; Kiørboe and Titelman 1998) and water temperature for field populations (Tada et al. 2000; Yilmaz et al. 2005), the factors responsible for the smaller size of non-bioluminescent cells are unknown.

On an intraspecific level, the bioluminescence emission per cell of dinoflagellates is proportional with cell size (Buskey 1995); larger cells may benefit more from the predator protection of bioluminescence than would smaller cells if their brighter light emission is more effective in predator defense. However, the role of bioluminescence in reducing predation on N. scintillans is unknown. The predators of N. scintillans are difficult to determine because the fragile nature of the cells does not allow them to persist in gut contents. Although they are considered too large to be grazed upon by most zooplankton (Yilmaz et al. 2005), known predators of vegetative cells include large copepods (Petipa 1960), crab larvae (Lehto et al. 1998; Sulkin et al. 1998) and gelatinous zooplankton (Daan 1989; Fock and Greve 2002; Vargas and Madin 2004; Gomes et al. 2014), and progametes are consumed by ciliates (Zhang et al. 2017a). Both non-bioluminescent and bioluminescent isolates of N. scintillans are capable of high population growth rates (Buskey 1995; Nakamura 1998b; Busch et al. 2019), which may be effective in overcoming predation pressure, negating the need for bioluminescence as a predator deterrent.

If gelatinous zooplankton exert major predation pressure on N. scintillans along the west coast of the USA, then there would be no ecological advantage in producing bioluminescence, which would be ineffective in deterring grazing by these nonvisual predators. However, gelatinous zooplankton in other regions prey on bioluminescent N. scintillans, and in Californian waters blooms of the bioluminescent dinoflagellate, Lingulodinium polyedra (F.Stein) J.D.Dodge, can immediately precede those of the non-bioluminescent N. scintillans (Sweeney 1975; Hayward et al. 1995; Gregorio and Pieper 2000; John et al. 2003). These observations suggest that the factors that regulate light production in N. scintillans and L. polyedra differ, and may reflect a different physiological or ecological niche for each species.

Phylogenetic and functional variation in dinoflagellates

Phylogenetic analyses of LSU rDNA gene sequences did not resolve N. scintillans according to bioluminescence function, but rather grouped non-bioluminescent strains from the northwest USA with bioluminescent Pacific strains from China and the Arabian Sea. Only the bioluminescent strain from the Gulf of Mexico (southern USA, Atlantic Ocean) was clearly different. The LSU and ITS regions of rDNA have been used to distinguish species complexes with distinct functional groups in other dinoflagellates like Alexandrium Halim based on thresholds of genetic distance (p-distance 0.02 for ITS) (Litaker et al. 2007). Our phylogenetic analyses agree with previous work comparing red N. scintillans populations from various Chinese Seas and an American population from the Gulf of Mexico (Pan et al. 2016), and the green N. scintillans from the Arabian Sea (Wang et al. 2016); despite significant functional differentiation these “varieties” appear to belong to the same species. Intraspecific functional variation is very common in dinoflagellates, particularly in toxin production in morphologically indistinguishable but phylogenetically distinct species of Alexandrium (Lilly et al. 2007; John et al. 2014) and in regionally distinct varieties of Pyrodinium bahamense L.Plate (Steidinger et al. 1980; Azanza 1997). Our findings show that loss of gene function and biochemical pathways are important in creating functional diversity within dinoflagellate species, and may be relevant to other important functions like toxicity. We do note, however, that sequences retrieved from the TaraOceans datasets are different to all others previously sequenced, even when comparing the same region, suggesting a bias in the sequences retrieved by PCR from cultured isolates. A robust phylogeographic study of N. scintillans, particularly in the context of functional diversity, will best use alternative PCR-independent methods, or an alternative phylogenetic marker.

Ecological drivers of functional divergence in N. scintillans

Non-bioluminescent N. scintillans forms isolated blooms over a large area along the entire west coast of the USA. Several observations have confirmed that N. scintillans occurring in this region is exclusively non-bioluminescent. We speculate that the California Current, a dominant oceanographic feature of the west coast of the US (reviewed by Wyrtki 1967), may define the southern part of the geographic range of non-bioluminescent N. scintillans populations. The California Current is part of the North Pacific Gyre (Sverdrup et al. 1942), which transfers waters eastwards from the North Pacific as part of the Subarctic Current to the Washington coast, at an interannually varying latitude approximately 45-50 ºN (Sydeman et al. 2011) (Figure 1). This current bifurcates, with part of it flowing northwards into the Gulf of Alaska, while the other part flows southward towards the equator as the California Current, and then veers west when it reaches the southern tip of the Baja California coast, becoming the North Equatorial Current. The cell isolates in this study from La Jolla, California, are possibly at the southern-most end of the non-bioluminescent N. scintillans range, and within the California Current. In contrast, the bioluminescent variety is found in the hydrographically disconnected water of Baja California (Lapota and Losee 1984) and further south off the Galapagos and Cocos Islands (Staples 1966). There has been only one report of low numbers of bioluminescent N. scintillans cells in California waters (Herren et al. 2004), suggesting that if these cells are transported to this area or are present in the background community, they are outnumbered by the non-bioluminescent variety. Thus, these functionally distinct varieties of N. scintillans may have distinct environmental niches. Geographic separation and local ecological adaptation of non-bioluminescent N. scintillans in California may be augmented by closed circulation in coastal bays due to upwelling-associated nearshore fronts (Barth et al. 2000; Di Lorenzo 2003; Lynn et al. 2003).

The northern and western extent of non-bioluminescent N. scintillans is unknown. The SPMC strain used here originates from Puget Sound in Washington (approximately 50ºN) which, depending on the year, may be aligned or sit north of the Subarctic Current bifurcation and hence in changing proximity to the California Current formation location (Sydeman et al. 2011). The non-bioluminescent population does not extend to the Chuckchi Sea, where blooms of N. scintillans off the north coast of Alaska (McInnes et al. 2015) are bioluminescent (Staples 1966; Tibbs 1967). The northwest Pacific harbours blooms of the red variety of N. scintillans, while in tropical waters further south (e.g., Indonesia), the bioluminescent green variety is present (Harrison et al. 2011). Furthermore, although the bioluminescence characteristics of red N. scintillans blooms in Japan, Korea, and China have not been studied, there are published and anecdotal reports of N. scintillans bioluminescence in this region (Haneda 1955; Nawata and Sibaoka 1979; Sato and Hayashi 1998; Han et al. 2012). A large-scale ecological and (phylo)genetic study on the different morphological and functional varieties of N. scintillans could provide significant insight into the ecological niche of dinoflagellate bioluminescence and mechanisms of evolutionary functional adaptation in dinoflagellates.

Conclusions

Our findings represent the most complete study to date on the molecular, biochemical and cellular basis for the intraspecific functional diversity that is often observed in dinoflagellates, as well as other plankton. We show that bioluminescence, an important predator defense strategy in dinoflagellates, can be “silenced” at the molecular level over large spatial scales in the ocean, perhaps related to distinct oceanographic provinces. As N. scintillans is well-known for its bioluminescence globally, its loss of bioluminescence along the northeast Pacific coast suggests that the environmental factors that influence light production in dinoflagellates differ between the large heterotrophic N. scintillans and smaller photosynthetic species that flourish in the same waters. The ecological role of bioluminescence in heterotrophic dinoflagellates requires further investigation.

Supplementary Material

Supporting Information

Acknowledgments

We thank K. Bright and S. Strom (SPMC), E. Buskey (GM), and K. Hayashi (Santa Cruz collection) for assistance in collecting live samples, D. Johnston for assistance with confocal microscopy, A. Page for assistance with transmission electron microscopy, M. Pinover and M. Lum for assistance with luciferase extractions and cross-reactions, R. Reynolds for assistance with cell size measurements, and J. Lindström for helpful discussions. MV and DIR were funded by the Luminescence and Marine Plankton project (Defence Science and Technology Laboratory and Natural Environment Research Council joint grant scheme proposal. ref 1166) and Office of Naval Research (ONR award number N000140410180) awarded to MDIR, AA was funded by a sabbatical grant SFRH/BSAB/931/2009 and strategic project MARE - UID/MAR/04292/2013, and TdR by NIH/NIGMS award F32GM129960.

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