Harmful Algae 98 (2020) 101850

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Harmful Algae
journal homepage: www.elsevier.com/locate/hal

Review

Bioluminescence and toxicity as driving factors in harmful algal blooms: T

Ecological functions and genetic variability
Kathleen D. Cusicka, , Edith A. Widderb
⁎

a
University of Maryland Baltimore County, Department of Biological Sciences, 1000 Hilltop Circle, Baltimore, MD 21250, United States
b
Ocean Research and Conservation Association, 1420 Seaway Dr, Fort Pierce, FL 34949, United States

ARTICLE INFO ABSTRACT

Keywords: Dinoflagellates are an ecologically important group of marine microbial eukaryotes with a remarkable array of
Bioluminescence adaptive strategies. It is ironic that two of the traits for which dinoflagellates are best known, toxin production
Dinoflagellates and bioluminescence, are rarely linked when considering the ecological significance of either. Although dino-
Harmful algal blooms flagellate species that form some of the most widespread and frequent harmful algal blooms (HABs) are bio-
Luciferase
luminescent, the molecular and eco-evolutionary associations between these two traits has received little at-
Saxitoxin
tention. Here, the major themes of biochemistry and genetics, ecological functions, signaling mechanisms, and
Toxins
evolution are addressed, with parallels and connections drawn between the two. Of the 17 major classes of
dinoflagellate toxins, only two are produced by bioluminescent species: saxitoxin (STX) and yessotoxin. Of these,
STX has been extensively studied, including the identification of the STX biosynthetic genes. While numerous
theories have been put forward as to the eco-evolutionary roles of both bioluminescence and toxicity, a general
consensus is that both function as grazing deterrents. Thus, both bioluminescence and toxicity may aid in HAB
initiation as they alleviate grazing pressure on the HAB species. A large gap in our understanding is the genetic
variability among natural bloom populations, as both toxic and non-toxic strains have been isolated from the
same geographic location. The same applies to bioluminescence, as there exist both bioluminescent and non-
bioluminescent strains of the same species. Recent evidence demonstrating that blooms are not monoclonal
events necessitates a greater level of understanding as to the genetic variability of these traits among sub-
populations as well as the mechanisms by which cells acquire or lose the trait, as sequence analysis of STX+ and
STX- species indicate the key gene required for toxicity is lost rather than gained. While the extent of genetic
variability for both bioluminescence and toxicity among natural HAB sub-populations remains unknown, it is an
area that needs to be explored in order to gain greater insights into the molecular mechanisms and environ-
mental parameters driving HAB evolution.

1. Introduction discussing the ecological significance of either. Although it is well

Dinoflagellates are a common component of the marine plankton,
where they are best known for the roles they play in biogeochemical
cycling, toxin production, and bioluminescence (Hackett et al., 2004).
Autotrophic and heterotrophic species contribute to cycling of key
elements such as C, N and P. Toxin-producing species can significantly
alter food webs and impact both human and ecosystem health via the
bioaccumulation and transfer of toxins through the food chain. Biolu-
minescent species are responsible for brilliant, eye-catching displays
that capture intense public interest and are frequently a focus of eco-
tourism.
It is ironic that the two features for which dinoflagellates are best
known, light emission and toxin production, are rarely linked when
known that many of the dinoflagellates that form harmful algal blooms
(HABs) are bioluminescent, the significance of light emission on the
generation and persistence of HABs has received little attention com-
pared to an ever expanding array of approaches focused on under-
standing these complex phenomena (Anderson et al., 2012b). This
paper examines the relationship between bioluminescence and toxicity
in dinoflagellates under the major themes of: Occurrence; Biochemistry
and Genetics; Energetic Costs; Ecological Functions, including the ge-
netic variability of both traits within natural populations; Signal
Transduction; and Evolution.

⁎
Corresponding author.
E-mail addresses: kcusick@umbc.edu (K.D. Cusick), ewidder@teamorca.org (E.A. Widder).

https://doi.org/10.1016/j.hal.2020.101850
Received 15 September 2019; Received in revised form 29 May 2020; Accepted 2 June 2020
Available online 29 July 2020
1568-9883/ © 2020 Elsevier B.V. All rights reserved.
K.D. Cusick and E.A. Widder Harmful Algae 98 (2020) 101850

Table 1
List of known bioluminescent dinoflagellate species.a and associated toxin production. Presence of luciferin binding protein (LBP), when known, and corresponding
GenBank accession number are indicated.
Species Toxic Toxin produced LBP GenBank LBP

Alexandrium ostenfeldii Y saxitoxin, spirolides, gymnodimines n/a

Alexandrium acatenella Y Saxitoxin n/a
Alexandrium affine Y Saxitoxin Y AFN26992.1
Alexandrium cf. catenella Y Saxitoxin Y ABY78836.1
Alexandrium fundyense Y Saxitoxin n/a
Alexandrium monilatum Y Saxitoxin Y AFN26995.1
Alexandrium tamarense Y Saxitoxin Y AFN27013.1
Ceratium breve N/A
Ceratium furca N
Ceratium fusus N
Ceratium gibberum N
Ceratium horridum N
Ceratium longipes N
Ceratocorys horrida N
Dissodinium lunula N
Fragilidium heterolobum N
Gonyaulax catenata N
Gonyaulax digitale N
Gonyaulax excavata Y Saxitoxin n/a
Gonyaulax hyalina N/A
Gonyaulax monacantha N/A
Gonyaulax parva N
Gonyaulax ploygramma N
Gonyaulax scrippsae N
Gonyaulax sphaeroides N/A
Gonyaulax spinifera Y Yessotoxin Y JQ946866.1
Gymnodinium flavum N
Gymnodinium sanguineum N
Lingulodinium polyedrum Y Yessotoxin Y AAA29166.1
Noctiluca scintillans N
Peridinium elegans N
Polykrikos kofoidii N
Polykrikos schwartzii N
Protoceratium reticulatum Y Yessotoxin Y AFN27016.1
Protoperidinium antarcticum N/A
Protoperidinium brevipes/breve N
Protoperidinium brochii N/A
Protoperidinium ceraseus N/A
Protoperidinium claudicans N
Protoperidinium concoides N
Protoperidinium conicum N
Protoperidinium crassipes N
Protoperidinium curtipes N/A
Protoperidinium depressum N
Protoperidinium divergens N
Protoperidinium excentricum N
Protoperidinium exiquipes N/A
Protoperidinium globulus N/A
Protoperidinium granii N/A
Protoperidinium huberi N/A
Protoperidinium leonis N
Protoperidinium minutum N
Protoperidinium oceanicum N
Protoperidinium ovatum N/A
Protoperidinium pallidum N
Protoperidinium pellucidum N
Protoperidinium pentagonum N
Protoperidinium punctulatum N
Protoperidinium pyriforme N/A
Protoperidinium saltans N/A
Protoperidinium sinaicum N/A
Protoperidinium sournia N/A
Protoperidinium steinii N/A
Protoperidinium subinerme N
Protoperidinium tubum N/A
Pyrocystis acuta N
Pyrocystis fusiformis N
Pyrocystis lunula N
Pyrocystis noctiluca N
Pyrodinium bahamense Y Saxitoxin Y GAIO01054099.1
Pyrophacus steinii N

a
Species summarized from (Cusick and Widder, 2014) and (Marcinko et al., 2013). N/A in Toxic column indicates no information available on whether species is
toxic; n/a in LBP column indicates no information available on whether the species possesses an LBP.

2
K.D. Cusick and E.A. Widder
2. Occurrence
2.1. Bioluminescence
The phenomenon of bioluminescence is widespread in the marine
environment. Bioluminescence is the chemical generation of light by
organisms and occurs across diverse taxa from bacteria to fish in more
than 700 genera (Haddock et al., 2010; Widder, 2010). Although the
number of bioluminescent species is relatively rare, most sampling
schemes have revealed that the total number of light emitters in the
ocean significantly outnumber non-bioluminescent individuals
(Martini and Haddock, 2017; Widder et al., 1999). The relative success
of overall bioluminescent species is also evident in the high degree of
convergent evolution of the trait with multiple origins (>40) of dif-
ferent light producing systems distributed across phyla (Haddock et al.,
2010; Widder, 2010).
Dinoflagellates are the primary source of bioluminescence in surface
waters. The overall number of described dinoflagellate species is esti-
mated to be between 2000 and 2500, assigned to >30 genera, with
approximately 1555 recognized as marine species (Gomez, 2005).
However, only a small percentage are bioluminescent: 70 species
(Table 1), or less than 3%. Yet these include some of the best known
and extensively researched of the dinoflagellata. These species have
attracted attention due to their ability to form HABs (often referred to
as “red tides”), their toxic impacts on shellfish and humans, and their
production of brilliant displays of bioluminescence that illuminate
every disturbance in the water: outlining the forms of swimming fish
and dolphins, illuminating breaking waves and leaving shimmering
wakes behind ocean vessels.
2.2. Toxicity
There are approximately seventeen major types of dinoflagellate
toxins (Table 2). As with bioluminescence, only a small portion of
marine dinoflagellates are toxic - ca. 100 species (Table 3). These
“toxins” are comprised of an array of structurally and mechanistically
diverse secondary metabolites. A general trend is that within the same
species there are toxic and non-toxic strains. Additionally, toxin profile
and composition frequently differ among toxic strains of the same
species, and can be influenced by many parameters (Hoppenrath et al.,
2013). There is also the unique case of Alexandrium: among HAB
genera, Alexandrium is one of the most well-known in terms of bloom
severity, diversity, and frequency. Of its ca. 30 species, at least half are
confirmed toxin-producers. It is the only HAB genus with three different
families of toxins produced by its species: saxitoxin, spirolides, and
goniodomins (Anderson et al., 2012a).
Of the 17 major classes of dinoflagellate toxins, only two are pro-
duced by bioluminescent species: saxitoxin and yessotoxin (Table 1),
and so are briefly expanded upon here. Saxitoxin (STX) (Table 2) is the
parent molecule in a class of compounds known as paralytic shellfish
toxins (PSTs) (Table 4). STX and its derivatives target voltage-gated ion
channels in humans: sodium, potassium, and calcium, though the me-
chanism of action differs among the three (Cusick and Sayler, 2013). It
is the causative agent of paralytic shellfish poisoning (PSP) and sax-
itoxin pufferfish poisoning (SPFP) (Landsberg et al., 2006). Filter-
feeding mollusks and crustaceans ingest the toxic cells, concentrating
the toxins within their organs and tissues. Currently, three genera of
marine dinoflagellates are established STX producers: Alexandrium spp.,
Pyrodinium bahamense, and Gymnodinium catenatum (Cusick and
Sayler, 2013). Of these, Alexandrium spp. and P. bahamense are biolu-
minescent (Table 1).
Yessotoxin (YTX) and its ca. 100 analogues are polycyclic ether
polyketides (Table 2); the basic structure of YTX is that of a disulfated
polyether, with the characteristic ladder-shape comprised of 11 ad-
jacent ether rings of different sizes and a terminal acyclic unsaturated
side chain comprised of 9 carbons and 2 sulfate ethers
3
Harmful Algae 98 (2020) 101850
(Dominguez et al., 2010; Paz et al., 2008). The majority of YTXs syn-
thesized from dinoflagellates were first identified in Protoceratium re-
ticulatum (Alfonso et al., 2016). YTXs were originally included as
Diarrhetic Shellfish Poisoning (DSP) Toxins, as they were commonly
found with okadaic acid during DSP-screening assays (Tubaro et al.,
2010). However, they have recently been re-classified as they do not
share the same mechanism of action as okadaic acid. No human toxicity
has been reported for YTXs (Tubaro et al., 2010). The precise me-
chanism of action of YTX remains unknown (Paz et al., 2008), though in
vitro studies indicate it modulates calcium homeostasis and is able to
inhibit entry of Ca+ ions (Paz et al., 2008). The majority of YTX-pro-
ducing dinoflagellates are bioluminescent and include P. reticulatum
(syn Gonyaulax grindleyi), Gonyaulax spinifera, and Lingulodinium poly-
edra (Draisci et al., 1999; Howard et al., 2009; Paz et al., 2008;
Paz et al., 2004) (Table 1). In keeping with other toxin-producing di-
noflagellate species, there exist both toxic and non-toxic strains of all
three YTX-producers as well (Armstrong and Kudela, 2006;
Howard et al., 2008, 2009; Ramstad et al., 2001; Rhodes et al., 2005).
2.3. Toxic, bioluminescent dinoflagellates
Neither bioluminescence nor toxicity are confined to a specific order
or genus; when considered individually, both are scattered among the
dinoflagellate tree of life (Fig. 1). However, the two traits occur to-
gether in a more defined cluster of genera, all within the order Go-
nyaulacales. Additionally, a general trend is that toxic, bioluminescent
dinoflagellates all occur in the similar environmental niche of coastal
areas. Of the ca. 70 bioluminescent dinoflagellate species, 12 are es-
tablished toxin producers (Table 1). Of the bioluminescent toxin-pro-
ducers, Alexandrium sp. and P. bahamense are notorious STX-producing
HAB species, while L. polyedra, G. spinifera, and P. reticulatum produce
YTX. Most of these toxic bioluminescent species have caused significant
HAB outbreaks worldwide.
3. Biochemistry and genetics
3.1. Biochemistry: bioluminescence
Dinoflagellate light emission occurs as a flash, triggered by a
membrane deformation that activates an action potential in the va-
cuolar membrane (Eckert and Sibaoka, 1968; Widder and Case, 1981a).
In general, the reaction occurs through the oxidation of the luciferin
substrate by molecular oxygen via the luciferase enzyme. In dino-
flagellates, the reaction occurs in many (~400 per cell) small (~0.4
um) organelles known as scintillons (DeSa and Hastings, 1968), which
contain the luciferin and luciferase (LCF) enzyme, and in some genera,
a luciferin-binding protein (LBP) (Johnson et al., 1985; Nicolas et al.,
1991; Schmitter et al., 1976). Mechanical stimulation of the cells cre-
ates an action potential that opens membrane channels, resulting in a
rapid and transient drop in pH within the scintillons due to a rapid
influx of protons from the acidic vacuole. The pH decrease activates the
enzyme, and the oxidation of the luciferin results in a flash of light with
peak emission in the 472–475 nm wavelength range (Hastings, 1983;
Widder et al., 1983; Wilson and Hastings, 1998). The entire mechan-
otransductive process from stimulus to the initiation of the flash is
rapid, requiring only 12–20 ms (Eckert, 1965; Latz et al., 2008;
Widder and Case, 1981a). Flash kinetics and photon flux cover a re-
markably wide range depending on species. The smaller (~20–40 µm)
armored dinoflagellates like L. polyedra emit brief (~100 ms), often
solitary dim flashes of between 3.1 × 107 and 1.2 × 108 photons cell−1
(Biggley et al., 1969; Seliger et al., 1969; Swift et al., 1973;
Widder et al., 1993) while the brightest dinoflagellates, such as Pyr-
ocystis noctiluca (~350 µm) and Pyrocystis fusiformis (up to 1000 µm),
can emit multiple longer (200–500 ms) and brighter flashes of between
3.7 × 1010 to 6.5 × 1011 photons cell−1 (Swift et al., 1973; Widder and
Case, 1981b; Widder et al., 1993).
K.D. Cusick and E.A. Widder Harmful Algae 98 (2020) 101850

Table 2
List of major dinoflagellate toxins.

(continued on next page)

4
K.D. Cusick and E.A. Widder Harmful Algae 98 (2020) 101850

Table 2 (continued)

Spirolide (MW = 694)

macrocyclic Alexandrium neuronal competitive binding to unknown
imine ostenfeldii, A. nicotinic nAChR
peruvianum acetylcholine
receptors
(nAChR)

Pinnatoxin (MW = 711.9)

macrocyclic Vulcanodinium neuronal competitive binding to unknown
imine rugosum nicotinic nAChR
acetylcholine
receptors
(nAChR)

Palytoxin (MW = 2680.1)

linear Ostreopsis cf. Na:K ATPase channel modifier, Palytoxin Poisoning
superchain ovata allowing non-selective - toxic in humans
polyether ion passage; binds to following ingestion,
extracellular portion of inhalation, or
protein cutaneous contact

Amphidinol (MW = 1327.7)

linear Amphidinium cell pore antifungal and
superchain klebsii membranes formation/disruption of hemolytic; no human
polyether biomembranes - apolar illnesses
side chain penetrates
lipid bilayer

(continued on next page)

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K.D. Cusick and E.A. Widder Harmful Algae 98 (2020) 101850

Table 2 (continued)

(continued on next page)

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K.D. Cusick and E.A. Widder Harmful Algae 98 (2020) 101850

Table 2 (continued)

Yessotoxin (MW = 1143.4)

ladder-frame Protoceratium unknown exact mechanism Neurotoxic Shellfish
polyether reticulatum unknown; potentially Poisoning via
targets cardiac muscle consumption of
cells, cytoskeleton contaminated
shellfish

Gymnocins A (top) and B

ladder-frame Karenia mikimotoi unknown unknown unknown
polyether

Maitotoxin (MW = 3425.9)

super-sized Gambierdiscus cation/calcium channel modifier, allowing unknown
ladder-like toxicus channels non-selective ion passage
polyether

Brevisculan F (2054.39)
super-sized Karenia unknown unknown lethal to mice; fish
ladder-like brevisulcata and humans
polyether unknown
linear Coolia monotis unknown unknown symptoms similar to
polyether NSP
(structure
unknown)
linear Ostreopsis unknown inactivation gating symptoms similar to
polyether lenticularis modifier NSP
(structure
unknown)

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K.D. Cusick and E.A. Widder Harmful Algae 98 (2020) 101850

Table 3
List of toxic dinoflagellate species.
Toxin Species Ref

Amphidinol −17, −18 Amphidinium carterae (Meng et al., 2010; Nuzzo et al., 2014)
Amphidinol −2, −3 Amphidinium klebsii (Iwamoto et al., 2017; Paul et al., 1995)
Azaspiracid Azadinium spinosum (Tillmann et al., 2009)
Azaspiracids Azadinium poporum (Luo et al., 2016)
Brevetoxins Karenia bicuneiformis (Brand et al., 2012)
Brevetoxins Karenia brevis (Brand et al., 2012)
Brevetoxins Karenia papilonacea (Brand et al., 2012)
Brevetoxins Karenia selliformis (Harju et al., 2016; Brand et al., 2012)
Ciguatoxins Gamberdiscus australes (Parsons et al., 2012)
Ciguatoxins Gamberdiscus pacificus (Parsons et al., 2012)
Ciguatoxins Gamberdiscus polynesiensis (Parsons et al., 2012)
Ciguatoxins Gamberdiscus toxicus (Parsons et al., 2012)
Ciguatoxins Gambierdiscus excentricus (Fraga et al., 2011)
Cooloatoxin Coolia monotis (Holmes et al., 1995)
Dinophysistoxin derivatives Prorocentrum maculosm (Hoppenrath et al., 2013; Lee et al., 2016)
Goniodomins Alexandrium hiranoi (Anderson et al., 2012a)
Goniodomins Alexandrium monilatum (Anderson et al., 2012a)
Gymnocin-A, -B Karenia mikimotoi (Brand et al., 2012)
Gymnodimine Alexandrium ostenfeldii (Molgo et al., 2017)
Gymnodimine Alexandrium peruvianum (Molgo et al., 2017)
Gymnodimine Karenia selliformis (Harju et al., 2016; Brand et al., 2012)
Karlotoxins Karlodinium veneficum (Place et al., 2012)
Maitotoxins Gambierdiscus excentricus (Fraga et al., 2011)
Neurotoxins Prorocentrum borbinucum (Hoppenrath et al., 2013; Lee et al., 2016; Glibert et al., 2012)
Okadaic Acid Prorcentrum hoffmannianum (Hoppenrath et al., 2013; Lee et al., 2016; Glibert et al., 2012)
Okadaic Acid Prorocentrum belizeanum (Hoppenrath et al., 2013; Lee et al., 2016; Glibert et al., 2012)
Okadaic Acid Prorocentrum caipirignum (Luo et al., 2017)
Okadaic Acid Prorocentrum cf. maculosum (Luo et al., 2017; Glibert et al., 2012)
Okadaic Acid Prorocentrum concavum (Hoppenrath et al., 2013; Glibert et al., 2012; Lee et al., 2016)
Okadaic Acid Prorocentrum faustiae (Hoppenrath et al., 2013; Lee et al., 2016; Glibert et al., 2012)
Okadaic Acid Prorocentrum lima (Hoppenrath et al., 2013; Lee et al., 2016; Luo et al., 2017; Glibert et al.,
2012)
Okadaic Acid Prorocentrum rhathymum (Hoppenrath et al., 2013; Lee et al., 2016; Glibert et al., 2012)
Okadaic Acid/Dinophysistoxins Phalacroma mitra (Reguera et al., 2014, 2012)
Okadaic Acid/Dinophysistoxins Phalacroma rotundatum (Reguera et al., 2012; Rugeura et al., 2014)
Okadaic Acid/Dinophysistoxins Dinophysis acuminata (Reguera et al., 2012; Rugeura et al., 2014)
Okadaic Acid/Dinophysistoxins Dinophysis acuta (Reguera et al., 2012; Rugeura et al., 2014)
Okadaic Acid/Dinophysistoxins Dinophysis caudata (Reguera et al., 2012; Rugeura et al., 2014)
Okadaic Acid/Dinophysistoxins Dinophysis fortii (Reguera et al., 2012; Rugeura et al., 2014)
Okadaic Acid/Dinophysistoxins Dinophysis miles (Reguera et al., 2012; Rugeura et al., 2014)
Okadaic Acid/Dinophysistoxins Dinophysis norvegica (Reguera et al., 2012; Rugeura et al., 2014)
Okadaic Acid/Dinophysistoxins Dinophysis ovum (Reguera et al., 2012; Rugeura et al., 2014)
Okadaic Acid/Dinophysistoxins Dinophysis sacculus (Reguera et al., 2012; Rugeura et al., 2014)
Ostreotoxin 3 Ostreopsis lenticularis (Parsons et al., 2012)
Palytoxin/ostreopsis toxins Osteropsis siamensis (Parsons et al., 2012)
Palytoxin/ostreopsis toxins Ostreopsis mascarenensis (Parsons et al., 2012)
Palytoxin/ostreopsis toxins Ostreopsis cf. ovata (Parsons et al., 2012)
Pinnatoxins Vulcanodinium rugosum (Molgo et al., 2017)
Pinnatoxins Vulcanodinium rugosum (Molgo et al., 2017)
Prorocentrolide Prorocentrum caipirignum (Nascimento et al., 2017)
Prorocentrolides A,B; Spiro-prorocentrimine Prorocentrum lima (Molgo et al., 2017)
Prorocentrolides A,B; Spiro-prorocentrimine Prorocentrum maculosum (Molgo et al., 2017)
Saxitoxin, spirolides, gymnodimines Alexandrium ostenfeldii (Kremp et al., 2014; Tillmann et al., 2014)
Saxitoxins Alexandrium acatenella (Anderson et al., 2012a)
Saxitoxins Alexandrium affine (Anderson et al., 2012a)
Saxitoxins Alexandrium andersonii Balech (Anderson et al., 2012a)
Saxitoxins Alexandrium angustitabulatum (Anderson et al., 2012a)
Saxitoxins Alexandrium catenella (Anderson et al., 2012a)
Saxitoxins Alexandrium cf. catenella (Tillmann and John, 2002)
Saxitoxins Alexandrium cohorticula (Anderson et al., 2012a)
Saxitoxins Alexandrium fundyense (Anderson et al., 2012a)
Saxitoxins Alexandrium minutum (Anderson et al., 2012a)
Saxitoxins Alexandrium tamarense (Andeson et al., 2012a)
Saxitoxins Alexandrium tamiyavanichii (Anderson et al., 2012a)
Saxitoxins Alexandrium taylori (Anderson et al., 2012a)
Saxitoxins Gonyaulax digitale (Swift et al., 1995)
Saxitoxins Gonyaulax excavata (White, 1986)
Saxitoxins Gymnodinium catenatum (Hallegraeff et al., 2012)
Saxitoxins Pyrodinium bahamense (Lewitus et al., 2012)
Spirolides Alexandrium ostenfeldii (Molgo et al., 2017)
Spirolides, saxitoxins Alexandrium peruvianum (Molgo et al., 2017; Anderson et al., 2012a; Tomas et al., 2012)
Yessotoxin Gonyaulax spinifera (Paz et al., 2008)
Yessotoxin Gonyaulax taylorii (Alvarez et al., 2016)
Yessotoxin Lingulodinium polyedra (Paz et al., 2008)
Yessotoxin Protoceratium reticulatum (syn.:)Gonyaulax grindleyi (Paz et al., 2008; Satake et al., 1997)

8
K.D. Cusick and E.A. Widder Harmful Algae 98 (2020) 101850

Table 4 was also sequenced in the heterotrophic dinoflagellate Noctiluca scin-

Saxitoxin and major derivatives, which collectively are referred to as Paralytic
Shellfish Toxins (PSTs).
tillans, a bloom-forming species that is phylogenetically very distant
from the seven photosynthetic species. This is reflected in its LCF gene,
which codes for a polypeptide containing two distinct domains: that of
the LCF found in photosynthetic species, and the LBP, which occurs as a
separate gene in the photosynthetic species (Liu and Hastings, 2007).

3.3. Biochemistry: saxitoxin

Saxitoxin is unique in that it is produced by organisms encom-

passing two kingdoms of life inhabiting different aquatic systems: eu-
karyotic dinoflagellates in marine systems and cyanobacteria in fresh-
water systems (Cusick and Sayler, 2013). The route of biosynthesis is
similar between the two (Shimizu, 1993), and was initially proposed
based on extensive studies using labeled precursors with the dino-
flagellate Alexandrium tamarense and the cyanobacterium Aphanizo-
menan flos-aquae (Shimizu, 1993, 1996). However, more recent genetic
and bioinformatic analyses (Kellmann et al., 2008b; Mihali et al., 2011,
Division Namea R1 R2 R3 R4
STX H H H OCONH2 2009), coupled with screening of the biosynthetic intermediates and in-
NeoSTX OH H H OCONH2 vitro biosynthesis of saxitoxin (Kellmann and Neilan, 2007), has re-
GTX1 OH OSO3− H OCONH2 sulted in modifications of the original pathway. The gene cluster coding
Carbamate GTX2 H OSO3− H OCONH2
for STX biosynthesis, spanning approximately 35 kb and encompassing
GTX3 H H OSO3− OCONH2
GTX4 OH H OSO3− OCONH2
26 proteins, was first identified in the toxic cyanobacterium Cylin-
GTX5 (B1) H H H OCONHSO3− drospermopsis raciborskii T3 (Kellmann et al., 2008b) followed by find-
GTX6 (B2) OH H H OCONHSO3− ings of homologous gene clusters in several other cyanobacteria (Mihali
C1 H OSO3− H OCONHSO3− et al., 2011, 2009). The findings with C. raciborskii T3 revealed that
N-sulfocarbamoyl C2 H H OSO3− OCONHSO3−
saxitoxin biosynthesis is initiated by SxtA, a novel polyketide synthase
C3 OH OSO3− H OCONHSO3−
C4 OH H OSO3− OCONHSO3− (described in detail in the Genetics section below) (Kellmann et al.,
dcSTX H H H OH 2008b). The original model proposed condensation of arginine to
dcNeoSTX OH H H OH acetate as the initial step, with the methyl side chain introduced later.
dcGTX1 OH OSO3− H OH
However, in the revised reaction scheme (Fig. 3), the acyl carrier pro-
Decarbamoyl dcGTX2 H OSO3− H OH
dcGTX3 H H OSO3− OH
tein (ACP) is loaded with acetate from acetyl Co-A, followed by me-
dcGTX4 OH H OSO3− OH thylation of acetyl-ACP by SxtA3. SxtA4 then performs the Claisen
doSTX H H H H condensation (steps 1–2 in Fig. 3). The presence of the putative inter-
Deoxydecarbamoyl doGTX2 H H OSO3− H mediate, designated A’, was confirmed via LC-MS-MA, while the ori-
doGTX3 H OSO3− H H
ginally-proposed intermediate was not detected. The third step in the
a
Abbreviations: STX, saxitoxin, GTX, gonyautoxin. pathway, the amidino transfer from arginine to Ɐ-amino A’ group (B’)
occurs via SxtG. This is followed by the first cyclicization via retroaldol
Differences exist among dinoflagellate species with regards to cleavage of ammonia from cytidine via SxtB (step 4). Hydroxylation
physiological characteristics such as the circadian rhythm and bio- and bicyclicization occur via SxtD, SxtS, and SxtU (steps 5–8). The final
chemistry of bioluminescence. In L. polyedra, one of the best-studied reactions (steps 9–10) involve carbamoyl transfer and dihydroxylation,
dinoflagellate species in terms of both toxicity and bioluminescence, though the exact order remains unknown (Kellmann et al., 2008b).
both LCF and LBP are broken down at the end of the night phase and Intracellular STX quotas are influenced by environmental and nu-
then synthesized again in the next cycle (Wilson and Hastings, 1998). tritional factors. Phosphorus limitation results in an increase in cellular
The scintillons themselves are also broken down and reformed each day levels, while nitrogen limitation results in a decrease (Van de Waal
(Fritz et al., 1990). Additionally, LBP has been shown to be a necessary et al., 2014). Cellular toxin quotas and composition also fluctuate based
component in the biochemical reaction. However, in other species, such on growth phase; in general, higher levels and more diverse profiles
as the non-toxic Pyrocystis species, the daily levels of LCF remain con- have been recorded in mid-exponential and stationary phase (Lim and
stant, there is no LBP (Knaust et al., 1998) and the scintillons do not Ogata, 2005; Parkhill and Cembella, 1999). Salinity has also been
break down, but they do exhibit rhythmic changes in excitability and shown to influence both toxin profile and overall toxin content, though
cellular distribution (Widder and Case, 1982). results vary among species; for example, an inverse correlation was
shown to exist between salinity and toxin content in A. minutum, while
in A. tamiyavanichii, toxin content was greatest at salinities optimal for
3.2. Genetics: bioluminescence growth (Lim and Ogata, 2005). Toxin analyses with batch cultures of a
Malaysian isolate of P. bahamense showed both toxin content and pro-
The full-length lcf gene has been sequenced in seven photosynthetic file were influenced by factors (temperature, salinity, light intensity)
species of dinoflagellates: L. polyedra, P. lunula, P. fusiformis, P. nocti- that alter growth rate, and that toxin content was likely to be de-
luca, A. affine, A. tamarense, and P. reticulatum (Liu et al., 2004). The termined by the manner in which the cells allocate their nitrogen and
predicted proteins all possess the same unique organization: a single carbon to various cellular components and processes under various
polypeptide consisting of an N-terminal region of unknown function growth conditions (Usup et al., 1994). Overall, toxin content and profile
followed by three homologous domains (“D1,” “D2,” “D3”), each with is influenced by growth conditions (Anderson et al., 1990a, 1990b;
catalytic activity (Liu et al., 2004; Okamoto et al., 2001). The individual Lim et al., 2006; Lim and Ogata, 2005), nutrient limitation (Boyer et al.,
domains among species (i.e. D1 A. tamarense, D1 A. affine) are more 1987; John and Flynn, 2000), and intracellular arginine concentration
similar than among the three different domains of the same species (i.e., (Anderson et al., 1990b; John and Flynn, 2000).
D1, D2, D3 of A. tamarense) (Liu et al., 2004) (Fig. 2). The full-length lcf

9
K.D. Cusick and E.A. Widder Harmful Algae 98 (2020) 101850

Fig. 1. Representative distribution of toxic (box) and bioluminescent (blue star) species across the dinoflagellate tree of life. Transparent blue flashes indicate
bioluminescence in a closely related species not included in tree. Orders are designated as vertical lines. Modified from Orr et al. (2012). (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)

10
K.D. Cusick and E.A. Widder Harmful Algae 98 (2020) 101850

3.5. Biochemistry: yessotoxin

Yessotoxins are polyketides. Great structural diversity exists among

the dinoflagellate polyketides. This diversity can be achieved through
different mechanisms, including alternative extender units (acetate,
malonate, propionate, etc.); modification or omission of different re-
actions within the overall biosynthetic pathway; and the integration of
enzymes from other metabolic pathways (Kellmann et al., 2010).
Polyketides are synthesized by polyketide synthases (PKS). PKS en-
zymes are multifunctional complexes that act on acyl monomers; the
minimal set of catalytic domains required for function includes ketoa-
cylsynthase, acyl transferase, and acyl carrier protein. The presence of
three additional domains accounts for the variation in polyketide
structure: ketoacylreductases, dehydrases, and enolreductases
(Gokhale and Dipika, 2001). Polyether ladders are specific to dino-
flagellates, and their close structural similarities indicate shared bio-
synthetic mechanisms (Kellmann et al., 2010). Most biosynthesis stu-
dies have been conducted with brevetoxin (BTX); however, the single
Fig. 2. Maximum likelihood tree of the catalytic domains of the dinoflagellate
study done with YTX demonstrated that the incorporation of acetate
luciferase. The corresponding domains of different LCFs group together more
closely than the individual domains of each species. At = A. tamarense, Aa = A. units, which form the backbone of the structure, was similar to that of
affine, Pr = P. reticulatum, Pf = P. fusiformis, Pn = P. noctiluca, Pl = P. lunula, BTX. The cyclic trans-polyethers were suggested to be formed via a
Lp = L. polyedra. D= Domain. Modified from Liu et al. (2004). polyepoxide cascade mechanism (Fig. 4). The little information avail-
able on YTX biosynthesis has shown that a portion of the ring fragment
can be obtained with a suitable polyepoxide system (Jamison and
3.4. Genetics: saxitoxin
Vilotijevic, 2007), supporting previous findings that suggested YTX and
other dinoflagellate polyether toxins may all be synthesized in a similar
Saxitoxin biosynthesis genes have been identified in all three di-
fashion from a polyepoxide (Nakanishi, 1985).
noflagellate genera (Alexandrium, Pyrodinium, Gymnodinium) capable of
toxin production (Hackett et al., 2013; Stuken et al., 2011), albeit with
3.6. Genetics: yessotoxin
varying degrees of coverage. The core genes of cyanobacterial STX
biosynthesis have been confirmed in dinoflagellates, along with genes
Recent sequencing efforts have identified genes coding for PKS-re-
related to toxin transport and modification (Hackett et al., 2013).
lated enzymes in multiple species of dinoflagellates that produce
Overall, 14 “core” genes have been identified (with the designation as
polyether-based toxins ((Beedessee et al., 2015; Kimura et al., 2015;
“core” based on characterization of STX biosynthesis in cyanobacteria);
Kohli et al., 2017, 2015; Meyer et al., 2015; Pawlowiez et al., 2014).
along with approximately 10 genes involved in transport and/or ana-
Even with these advances, no sequence data currently exist as to the
logue modification and two potential regulators involved in signal
genes and/or proteins involved in YTX biosynthesis.
transduction (as summarized in (Akbar et al., 2020)). Of the 14 core
biosynthesis genes, nine have been putatively identified in STX-pro-
3.7. Correlating bioluminescence and toxicity
ducing dinoflagellate species, though with the exceptions of sxtA and
sxtG, their roles in dinoflagellate STX biosynthesis remain to be de-
The potential molecular and/or biochemical links between toxin
termined (Akbar et al., 2020). SxtA is the first gene in the pathway
production and bioluminescence remain unexplored and so are con-
(Stuken et al., 2011), coding for a novel polyketide synthase comprised
sidered here. As the genes and pathways involved in YTX biosynthesis
of four catalytic domains (SxtA1-SxtA4) (Kellmann et al., 2008a). In
are not known, the link is explored with STX and bioluminescence. One
dinoflagellates, SxtA occurs as two isoforms: SxtA1-4, and SxtA1-3
of the first steps of STX biosynthesis is the claisen condensation of ar-
(Stuken et al., 2011). The gene coding for the SxtA1 domain is wide-
ginine and acetate, performed by a previously unrecognized type of
spread, occurring in both toxic and non-toxic strains (Hackett et al.,
polyketide synthase (Kellmann et al., 2008b). Claisen condensations are
2013; Murray et al., 2015). SxtA4 appears to be specific for toxin
rare on amino acids, but also do occur in the synthesis of tetrapyrroles
synthesis, as screening of over 40 isolates (35 Alexandrium, four Gym-
(Kellmann et al., 2008b). The substrate in the bioluminescence reaction
nodinium, and a single Pyrodinium) demonstrated its presence and high
of dinoflagellates is luciferin, which is an open-chain tetrapyrrole si-
sequence conservation in toxic strains; conversely, it was not found in
milar in structure to chlorophyll. While the synthesis pathway for di-
67 strains of 54 species of non-STX producing species (Murray et al.,
noflagellate luciferin is not known, the structural similarities between
2014, 2011b; Orr et al., 2013a; Stuken et al., 2011). SxtG codes for an
dinoflagellate luciferin and chlorophyll indicate luciferin formation via
amidinotransferase that putatively encodes the second step in STX
chlorophyll catabolism: P. lunula was shown to incorporate radio-
biosynthesis. While sxtG has been detected in Alexandrium species not
actively labeled chlorophyll precursors into chlorophyll and luciferin,
known to synthesize STX, it has not been found in numerous non-STX-
suggesting biosynthesis of the two are linked (Topalov and Kishi, 2001;
producing dinoflagellate species other than those of the genus Alexan-
Wu et al., 2003). Nonphotosynthetic dinoflagellates contain the com-
drium or G. catenatum (Orr et al., 2013a). Overall, transcriptome ana-
ponents of the plastid tetrapyrrole pathway, which accounts for the
lyses from multiple toxin-producing dinoflagellate spp. suggest that the
mechanism of luciferin biosynthesis in nonphotosynthetic biolumines-
genes involved in the first three steps of saxitoxin biosynthesis (sxtA,
cent dinoflagellates (Janouškovec et al., 2017). In any case, both STX
sxtG, sxtB) in dinoflagellates and cyanobacteria are derived from
synthesis and what may be considered the start of the bioluminescence
common ancestral proteins not involved in STX synthesis, as they are
reaction – that of luciferin formation – require an enzyme with a do-
also found in organisms that do not produce the toxin (Hackett et al.,
main that has a somewhat unique catalytic activity. It is also worth
2013).
noting that the sxtA gene encoding polyketide synthase contains an
additional domain specific to the toxin-producing strains that occurred
likely as a gene duplication event (Murray et al., 2015). Could there be
a shared enzyme or associated molecular/genetic element between STX

11
K.D. Cusick and E.A. Widder Harmful Algae 98 (2020) 101850

Fig. 3. Saxitoxin biosynthetic pathway with putative gene functions. The original pathway, derived from radioisotope tracing experiments, has been revised based on
collective genetic, biochemical, and bioinformatic analyses. Core and accessory genes are as initially identified in cyanobacteria; thus far, sxtA and sxtG have been
characterized in dinoflagellates. Modified from Cusick and Sayler (2013) and Kellmann et al. (2008b).

12
K.D. Cusick and E.A. Widder
Fig. 4. Polyepoxide reaction proposed to occur during YTX synthesis. Image redrawn from Kellmann et al. (2010).
biosynthesis and bioluminescence? Intriguingly, a proteomics analysis
between a toxic A. catenella strain and a toxicity-lost lab mutant showed
that multiple proteins similar to the LBP were down-regulated in the
non-toxic mutant (Wang et al., 2012). More recently, proteomic ana-
lysis of a toxic Alexandrium strain revealed bioluminescence-associated
proteins were upregulated during toxin production (Zhang et al., 2018).
The large and complex nature of dinoflagellate genomes presents a
formidable challenge for the identification of potential shared mole-
cular elements in toxic, bioluminescent dinoflagellates, but is a link that
requires further analysis in light of the contributions by both biolumi-
nescence and toxin production on HAB dynamics.
4. Energetic costs
4.1. Bioluminescence
Bioluminescent reactions are energetically costly, requiring the
equivalent of as many as 60 ATP molecules per photon
(Hastings, 1978). The extreme energy demands of light production are
evident in mixed cultures of luminescent and dark mutants of the same
species of bacteria (Vibrio harveyi) where the dark mutants rapidly
overrun the culture. By contrast, the bioluminescent strain dominates if
the mixture is irradiated with ultraviolet light of sufficient energy to
damage the microbial DNA. The selective advantage of the biolumi-
nescent strain in this case is apparently that it initiates DNA repair via
the enzyme photolyase, which is activated by blue light emitted by the
bioluminescent strain (Czyż et al., 2003; Czyz et al., 2000). It appears
that a strong selective advantage is necessary to overcome the high-
energy demands associated with light production.
Even when food deprived, organisms that employ bioluminescence
as the first line of defense will continue to invest energy in light pro-
duction at the expense of other functions. For example, starvation of
adult female copepods (Metridia longa) had no significant effect on
13
Harmful Algae 98 (2020) 101850
bioluminescence potential, while release of eggs dropped to zero
(Buskey and Stearns, 1991). In dinoflagellates, it was found that when
the heterotrophic dinoflagellate Protoperidinium cf. divergens was de-
prived of food it still exhibited high bioluminescence potential even as
growth rate dropped to zero (Latz and Jeong, 1996). This points to a
strong selective advantage for light emission.
4.2. Toxicity
The costs of toxin production are influenced by multiple factors,
including the concentration of grazers and their cues (Selander et al.,
2006) and resource (nitrogen, light) availability. Multiple studies trying
to quantify STX costs produced similar results, in that cells with in-
creased toxin production grew as fast as uninduced cells, and toxic and
non-toxic strains of the same species grow at the same rate (Pančić and
Kiørboe, 2018). As for the role of nutrients in toxin production, when
nitrogen or light is limiting, STX production is reduced, while nutrient-
replete or phosphorus-limited cells produce toxins at a high rate
(Anderson et al., 1990b; John and Flynn, 2000). Toxin levels have also
been shown to increase with direct or indirect exposure to grazers.
Overall, there exists much variability as to the responses of the grazers
to the toxic dinoflagellates, with variability stemming from both the
particular predator-prey combination as well as the strain involved
(Pančić and Kiørboe, 2018). The influence of predator and nutrient are
not mutually exclusive: waterborne cues from Acartia sp. were shown to
result in increased STX production in A. minutum in N-replete but not
low-N environments. Thus, the cost of defense may be neglible under
nutrient-rich conditions, but could be a factor in environments where
nitrogen is limited.
Extrapolating lab findings to environmental settings, the potential
exists for toxic species to gain an advantage under high nitrogen and
high grazer biomass - a scenario typical of coastal environs – as growth
rates can double due to the benefit of toxin production at a minimal
K.D. Cusick and E.A. Widder
production cost. The environmental conditions that promote cell toxi-
city, i.e., high grazer biomass, coincide with the time of the year when
nitrogen levels are low; thus ecological modeling predicts that the cost
of toxin production (quantified as a reduction in cell division rate of
defended relative to undefended cells) is substantial, leading to >20%
decrease in cell division rate. However, the investment in defense pays
off since defended cells experience lower grazing mortality, and the net
growth rates can be up to twice or more of that of undefended cells
(Chakraborty et al., 2019).
5. Ecological functions
5.1. Bioluminescence
The function of the flash has been long debated. While early in-
vestigators could imagine no selective advantage for bioluminescence
in dinoflagellates (Biggley et al., 1969), the energy requirements asso-
ciated with light production and the existence of complex cellular
machinery, which insures that light production occurs at night and is
inhibited during the day, suggests otherwise. Early experiments showed
that bioluminescence reduces grazing by zooplankton predators
(Buskey and Swift, 1983; Esaias and Curl, 1972) and that nocturnally
grazing copepods consumed fewer dinoflagellates when these were
offered in their bioluminescent night phase compared to their non-
bioluminescent day phase (Esaias and Curl, 1972; White, 1979). More
recently, bioluminescence has been shown to increase upon exposure to
the chemical cues of predators (Lindstrom et al., 2017a). The nature of
the flash is such that the light is emitted both as brief flashes (~100 ms)
and as a low intensity glow (Wilson and Hastings, 1998) depending
upon both species and stimulus. Additionally, there exists within single
dinoflagellate species both bioluminescent and non-bioluminescent
strains (Marcinko et al., 2013). Collectively, these findings are evidence
for the adaptive value of light emission.
Three major hypotheses have been proposed regarding the role of
bioluminescence in dinoflagellate ecology. All three are rooted in the
premise that bioluminescence acts as an anti-grazing strategy
(Lindstrom et al., 2017a). The difference among the three hypotheses
lies in the function of the flash. The flash may act as (A) a startle re-
sponse display, (B) a burglar alarm, or (C) an aposematic warning, in
which the flash serves as a warning of toxicity or unpalatability. As a
startle response display, the flash is hypothesized to stimulate an innate
photophobic response in the grazer (Esaias and Curl, 1972). However,
while flash-induced burst swimming occurs in marine copepods
(Buskey et al., 1983; Buskey and Swift, 1983, 1985) it is absent in co-
pepods from freshwater environments, where planktonic biolumines-
cence is not found (Buskey et al., 1987). Also, without any direct benefit
to the marine copepods of avoiding the flash, there is no selective ad-
vantage to retaining the photophobic behavior. For the dinoflagellate
flash and the copepod burst swimming response to be retained, a se-
lective advantage must accrue to both.
According to the burglar alarm hypothesis the adaptive value of
copepod burst swimming is to evade higher order visual predators
alerted to the copepod's presence by dinoflagellate flashing
(Burkenroad, 1943). Laboratory studies have demonstrated an in-
creased susceptibility of zooplankton to visual predators such as fish
and cephalopods in the presence of bioluminescent dinoflagellates,
thereby supporting the adaptive value of flash avoidance by the pri-
mary predator (Abrahams and Townsend, 1993; Fleisher and
Case, 1995; Mensinger and Case, 1992).
For bioluminescence to function as a burglar alarm, sufficient light
must be emitted by the dinoflagellates to alert visual predators. It is
therefore noteworthy that the dinoflagellate used in the experiments by
Mensinger and Case (1992) and Fleisher and Case (1995) was P. fusi-
formis, which is one of the largest (~1 mm long) and brightest (peak
photon flux >1011 photons s − 1) of all known bioluminescent dino-
flagellates. P. fusiformis is not a bloom-forming species and the cell
14
Harmful Algae 98 (2020) 101850
concentrations used in these studies were one to two orders of magni-
tude less than those used in experiments like those of Esaias and
Curl (1972) that used the much smaller (~20–50 µm) and dimmer cells
(~108 photons s−1) associated with blooms. Cusick and Widder (2014)
demonstrated that the foraging efficiency of a nocturnal fish predator
was enhanced in the presence of the bright emitter P. noctiluca at low
cell concentrations (5–40 cells ml−1) but not at similar low cell con-
centrations of the dim emitter L. polyedra. Only at cell concentrations
greater than 500 cells ml−1 did the bioluminescence of L. polyedra
function as a burglar alarm. However, the grass shrimp Palaemonetes
pugio rather than copepods served as the primary predator in these
experiments, and chemical cues (such as the copepodamides produced
by copepods) were not measured, nor was the flash intensity of single
cells at different cell concentrations. Overall, the results from that study
suggest the function of the flash is cell-concentration dependent, and
that in “dim-emitting” bloom-forming bioluminescent dinoflagellates,
high cell concentrations are required for a predator to be able to trace
the path of a copepod through the water (Cusick and Widder, 2014).
Thus, it is plausible that flash function is dependent on both cell
number and the main dinoflagellate predators within that specific
ecosystem. While earlier experimental studies provide clear support for
the selective advantage of flash avoidance by primary predators, recent
work with the copepodamide-induced bioluminescence of L. polyedra
demonstrated the advantage to the dinoflagellate as well, in that the
cells are seemingly rejected unharmed by the copepod predator.
Since many bloom-forming species are also toxin producers an al-
ternative hypothesis is that the bioluminescent flash in these species
functions as an aposematic warning, signaling unpalatability to grazers.
Bioluminescence has been shown to function as an aposematic warning
in firefly larvae (de Cock and Matthysen, 1999, 2002; Underwood et al.,
1997), millipedes (Marek et al., 2011), and brittle-stars (Grober, 1988a,
1988b). In a dark environment such as the ocean at night, the light
substitutes for aposematic coloration, with the flash warning potential
predators of toxicity or some other unpalatable trait. It is worth noting
that most copepod feeding occurs during the night, coinciding with the
bioluminescent phase. The basis for deterrence is that predators learn to
associate the flash with the undesirable trait, resulting in decreased
predation on the bioluminescent prey (Schantz, 1971).
Support for the aposematic function of the flash emerged from a
series of experiments that looked at copepod grazing on toxic and non-
toxic bioluminescent dinoflagellates in the presence of non-luminescent
diatoms (Hanley and Widder, 2017). Although the existence of a non-
toxic bioluminescent dinoflagellate might seem to undermine the
aposematic hypothesis, it is important to recognize the likely existence
of Batesian mimics, palatable prey species that mimic the flash of un-
palatable species, thereby benefitting from the learned avoidance be-
havior of the predator (Lindström et al., 1997). A Batesian mimic does
not long remain aversive to predators when in the absence of the or-
ganisms whose threat it mimics (Waldbauer and Sternbury, 1987).
Evidence that this was in fact the case for the non-toxic bioluminescent
dinoflagellate (L. polyedra), came from the fact that copepods consumed
this dinoflagellate in its non-luminescent phase, but not during its lu-
minescent phase. By contrast the toxic dinoflagellate (P. bahamense)
was not consumed in either its bioluminescent or non-bioluminescent
phase. It was also shown that the feeding efficiency on the alternative
prey (diatoms) was lower in the presence of the non-bioluminescent
phase of toxic dinoflagellates than in the presence of the non-biolumi-
nescent phase of non-toxic dinoflagellates, presumably because of the
increased time needed to handle and reject the toxic dinoflagellates.
In all likelihood, toxic dinoflagellates employ multiple deterrents
thereby improving effectiveness in warding off attacks; however, when
toxic dinoflagellates can be rejected based on the flash alone the
grazing rate on alternative prey is increased. Hanley and Widder (2017)
also corroborated the previously demonstrated concentration depen-
dence of the burglar alarm effect for dim emitters (Cusick and
Widder, 2014), because while the co-occurring diatom was consumed
K.D. Cusick and E.A. Widder
at low cell concentrations of the bioluminescent dinoflagellates, at high
(bloom-level) concentrations virtually all grazing ceased, apparently
because of the escape responses of the grazers.
5.2. How bioluminescence can drive HAB development
A recent study documenting avoidance or rejection of biolumines-
cent cells by predators provides strong support for the role of biolu-
minescence in HAB initiation and persistence. In this study, high-speed,
low-light video recordings of individual copepod behavior showed L.
polyedra cells flashed on contact with the copepod and was then re-
jected unharmed (Prevett et al., 2019). In this study L. polyedra was
induced to brighter bioluminescence by the addition of copepodamides.
When the dinoflagellates were not induced to the brighter biolumi-
nescence, then in mixed populations of phytoplankton where L. poly-
edra was only dimly luminescent and contributed 25% of the available
prey biovolume, the grazer consumed it in proportion to its fraction in
the community (24%). However, with copepodamide induction of
brighter bioluminescence the proportion of L. polyedra in the grazer's
diet dropped to 2%. An important point in this regard is the observation
that after the copepod rejected the flashing cell it exhibited “forceful
beating with its swimming appendages” which is a behavior associated
with the burglar alarm response. While this study did not determine the
mechanism (aposematism, startle response, or burglar alarm) by which
the flash may function in the initial stages of a bloom, it demonstrated
the means by which bioluminescence can facilitate bloom development.
Given that the doubling rate of dinoflagellates is one third that of
diatoms (Banse, 1982), selective feeding by grazers on non-luminescent
prey would favor bioluminescent dinoflagellates, thereby contributing
to bloom initiation. These “windows of opportunity” afforded by
openings or relief from grazing pressure are postulated to play an im-
portant role in dinoflagellate bloom formation (Stoecker et al., 2008).
However, the function of the flash among bioluminescent species may
differ based on the dinoflagellate predators within that ecosystem. Once
flash intensities and/or flash number reaches a threshold value, the
burglar alarm response would remove grazers from the vicinity of the
bloom thereby contributing to its persistence. In the situation described
above, copepodamide-induced bioluminescence protects L. polyedra
from copepod grazers, who seek alternate prey in the presence of bio-
luminescent cells. This allows L. polyedra to successfully compete with
faster-growing phytoplankton competitors. Monitoring of natural po-
pulations of L. polyedra over a ten-year period showed a positive cor-
relation with copepod biomass (Prevett et al., 2019), suggesting that
bioluminescence serves as a defense that provides an advantage to the
slower-growing dinoflagellate species, allowing them to successfully
compete with faster-growing phytoplankton competitors and poten-
tially achieve bloom numbers.
5.3. Toxicity: saxitoxin
The ecological role of both STX and YTX in the species that produce
them remains unknown. Numerous studies have been done to try and
ascertain the function of STX. As yet no studies exist as to the potential
function of YTX, and so STX is discussed here. The well-established
target of STX is the voltage-gated sodium channel in humans and other
mammals, where it binds with high affinity and can result in death due
to respiratory paralysis (Catterall, 1985). Hence, due to its detrimental
impacts on human health, STX and its congeners are typically referred
to as potent neurotoxins. However, it is highly unlikely that toxin
biosynthesis evolved with the purpose of blocking mammalian sodium
channels. The sxt genes are estimated to have emerged at least 2.1
billion years ago (Murray et al., 2011a), pre-dating the evolution of
voltage-gated sodium channels.
Multiple hypotheses have been put forward regarding the eco-evo-
lutionary role of STX and its congeners. Studies on Alexandrium popu-
lations dynamics led to the suggestion that they function as
15
Harmful Algae 98 (2020) 101850
pheromones, with roles in mating and cyst formation (Wyatt and
Jenkinson, 1997). They possess similar characteristics as established
pheromones, including (1) profiles akin to those in terrestrial animal
species, in which a mix of compounds rather than a single congener is
produced and (2) low levels of secretion (10−9–10−10 M). Most of the
toxin is retained within the cells during exponential growth, and re-
leased during senescence, which, in natural populations, would coin-
cide with the induction of sexuality during bloom decline
(Cembella, 2003). This hypothesis explains the diversity in toxin pro-
files among strains and geographical locations; what is not accounted
for, however, is the mechanism by which mating is mediated in non-
toxic strains of the same species (Cembella, 2003). Immunofluorescent
labeling studies with toxic A. fundyense demonstrated chromosomal
localization of the toxin (Anderson and Cheng, 1988; Doucette and
Anderson, 1993); this close proximity to the chromosomes led to the
hypothesis that it may play a role in chromosome structural organiza-
tion, with the two positively-charged guanidinium groups on the mo-
lecule binding in a manner similar to that of polyamines. The large
number of nitrogen atoms contained within the structure has led some
to suggest it functions as a form of nitrogen storage, though a coun-
terargument is that nitrogen storage could be achieved in a more
bioenergetically efficient manner via lower molecular weight com-
pounds such as urea or amino acids (Cembella, 1998).
Two additional theories as to the ecological function of STX in di-
noflagellates are derived from findings demonstrating its role in copper
homeostasis. A combination of genomic, transcriptomic, physiological
and bioinformatic analyses showed that STX inhibited copper uptake
across multiple microbial species, including yeast and photosynthetic
algae, while not affecting their growth (Cusick et al., 2009, 2012,
2013). These studies identified the copper transporter (CTR), a hybrid
ion channel/transporter protein, as an additional target of STX. Copper
is an essential trace metal required for key metabolic and physiological
processes in most organisms, including marine phytoplankton. Thus,
one theory is that it targets membrane proteins (via their “selectivity
filter”) involved in trace metal assimilation in co-occurring phyto-
plankton species. Copper is also toxic to living cells through a variety of
mechanisms (Rademacher and Masepohl, 2012). It has become pre-
valent in marine systems due to its use as an algaecide/biocide and as
an anti-fouling (AF) agent in marine vessel coatings (Borkow and
Gabbay, 2005; Yebra et al., 2004). Studies with non-toxic Alexandrium
spp. demonstrated that exposure to copper compromised membrane
integrity, resulting in leakage and cell death (Li et al., 2008). Thus, the
second theory is that it alleviates copper stress in toxin-producers by
inhibiting copper entry. However, both of these theories remain to be
experimentally verified.
The predominant hypothesis is that the molecule functions as a
grazing deterrent. Similar to bioluminescence, toxin production may
aid in HAB initiation as it alleviates grazing pressure on the HAB spe-
cies. Numerous studies have been conducted with organisms from
multiple trophic levels – including micro- and mesozooplankton, fish,
and macroinvertebrates - and results vary as to the effects on health and
whether the organisms actively reject the toxic cells (Barreiro et al.,
2006; da Costa et al., 2005; Frangoulos et al., 2000; Oikawa et al., 2005;
Robineau et al., 1991; Teegarden, 1999; Zimmer and Ferrer, 2007). One
of the most well-studied interactions is that of copepod grazing on di-
noflagellates. However, as with other organisms, grazing studies with
copepods have produced contradictory results: experiments with toxic
Alexandrium spp, range from no adverse effects upon consumption of
the toxic cells to increased mortality of the grazer (Bagøien et al., 1996;
Teegarden and Cembella, 1996). These results indicate that both the
strain and grazer must be considered when examining the role of toxin
function. Additionally, natural populations of Alexandrium are com-
prised of a mix of strains, differing in both toxin content and profiles
(Alpermann et al., 2010). The copepod species and its co-occurrence
with toxic dinoflagellates also influences the effects of the toxin, as
some copepods have been demonstrated to detoxify STX
K.D. Cusick and E.A. Widder
(Teegarden et al., 2003). Additionally, copepod populations previously
exposed to toxic blooms are less affected than naïve populations
(Colin and Dam, 2004). These results indicate that toxin induction and
function are a combined result of both the strain and grazer. Collec-
tively, these studies point towards a predator-prey co-evolution, and in
interpreting the potential role of the toxin, as with bioluminescence,
one must remain cognizant of the ecosystem view: strain of dino-
flagellate, type of grazer, and environmental parameters.
While a large number of lab studies have examined the ecophysio-
logical requirements of toxin production, these results are not sufficient
to predict the succession of HABs in the environment, as the net growth
performance of a species is affected by complex interactions with other
organisms (Zingone and Oksfeldt Enevoldsen, 2000). Indeed, the pre-
sence of copepods and their waterborne cues have also been shown to
stimulate toxin production (Selander et al., 2006; Wohlrab et al., 2010;
Yang et al., 2011) (as discussed in detail below). Thus, it is also plau-
sible that the toxin may serve multiple ecological functions. When
taking into account the metabolic demands associated with toxin
synthesis and modification, as well as the fluctuations in both in-
tracellular content and profile based on environmental and nutritional
influences, it is logical to propose that natural selection would favor
multi-use compounds (Wink, 2003).
5.4. Toxicity: yessotoxin
As with STX, the eco-evolutionary role of YTX remains unknown. As
yet no studies exist as to the potential function of YTX. However, in
vitro studies with various cell models indicate YTX modulates calcium
homeostasis, and is able to inhibit entry of Ca+ ions (Paz et al., 2008).
Based on the emerging evidence indicating a role for calcium in dino-
flagellate signal transduction mechanisms (discussed below), one in-
triguing hypothesis as to the eco-evolutionary role of YTX is a role in
signal transduction, be it within the cells that produce it or a means of
inhibiting the ability to sense and respond to environmental stimuli in
co-occurring species.
5.5. Saxitoxin resistance in predators
While one of the most studied hypotheses as to the function of STX
is as an anti-predator mechanism, it is also noteworthy that some pre-
dator species are able to build resistance to the toxin. Natural popula-
tions of organisms consistently exposed to STX-producing cells have
been shown to build resistance, resulting in inter-population variation
within a species due to genetic adaptations (Bricelj et al., 2005). Soft-
shell clams (Mya arenaria, a vector for toxin transfer) routinely exposed
to “red tides” are more resistant and accumulate the toxin at a greater
rate than clams from unexposed areas. The genetic basis was found to
be a single amino acid mutation in the sodium channel, resulting in a
1000-fold decrease in toxin binding affinity (Bricelj et al., 2005).
Adaption to STX has also been shown to occur in the copepod A. hud-
sonica, which frequently co-occurs with toxic Alexandrium spp. Inges-
tion and egg production rates, as well as overall population fitness,
were greater in exposed populations than in naïve populations
(Colin and Dam, 2005, 2002, 2007). However, resistance was not due to
sodium channel mutations as with clams (Chen et al., 2015;
Finiguerra et al., 2014, 2015), and the genetic basis for resistance in
this species remains to be identified.
5.6. The role of genetic diversity
When considering the ecological functions of both bioluminescence
and toxin production, one feature that is often overlooked is the genetic
variability for either trait within sub-populations of a natural bloom of
the species – i.e. do all the cells possess the genes necessary for that
trait, or only some of the cells? The theories as to the functions of both
bioluminescence and toxicity have been derived from lab studies, which
16
Harmful Algae 98 (2020) 101850
typically utilize a strain possessing that phenotype. In general, a strain
is obtained by establishing clonal cultures, each of which are derived
from a single-cell isolate from an environmental water sample
(Menden-Deuer and Montalbano, 2015). Thus, each isolate is an in-
dependent, clonal strain of the species under investigation, and in
theory the genetic composition of that cell dictates the genetic make-up
of the cells used in subsequent lab-based studies.
The advancement of molecular tools in dinoflagellate ecology has
resulted in population studies that show blooms are not monoclonal
events. The use of genetic markers to link genetic diversity with trait
diversity is emerging as a valuable mechanism in phytoplankton
ecology. Microsatellite markers, long used to assess population genetics
of macroorganisms (Jarne and Lagoda, 1996), are now being used as a
tool to examine the ecology and evolution of microorganisms
(Pettay and LaJeunesse, 2013). Microsatellite markers have shown ge-
netic diversity in field populations of phytoplankton, and this clonal
diversity has been shown to be associated with differences in genome
size (Whittaker et al., 2012), morphology (Saravanan and
Godhe, 2010), and growth rate (Rynearson and Virginia
Armbrust, 2004). Genetic diversity and bloom dynamics of A. fundyense
blooms assessed using rapidly evolving microsatellite markers revealed
a high level of genetic diversity in a geographically restricted area
(Richlen et al., 2012). The combined phenotypic and genetic diversity
occurring within the same HAB population is further exemplified in
studies with the HAB dinoflagellate species Akashiwo sanguinea. Mul-
tiple strains of A. sanguinea were obtained from a single water sample in
which a bloom was occurring, and broad intra-specific variability in
multiple traits was shown to exist among strains all isolated from the
same water sample (Menden-Deuer and Montalbano, 2015).
In the experiments by Abrahams and Townsend (1993) and
Mensinger and Case (1992), the authors proposed that even if the di-
noflagellate was consumed, its genetic clones would still benefit from
the reduction in grazing pressure. However, evidence demonstrating
that dinoflagellates are not monoclonal, even in a monospecific bloom
(Menden-Deuer and Montalbano, 2015; Richlen et al., 2012) under-
mines this hypothesis, as does the fact that bioluminescence occurs in
non-bloom-forming species. Without any direct selective advantage to
the individual dinoflagellate among the dim emitters, there is no clear
evolutionary path to flash production. Based on the recent work de-
monstrating the flash emitted by L. polyedra upon predator contact al-
lows the cell to escape unharmed, it seems logical for individual cells to
harbor and control the LCF genes, as it results in a direct benefit to the
individual cell.
Minimal field data exist on bioluminescence capacity on strains
from the same geographic area and so studies on lab isolates can only be
considered here in relation to genetic diversity and phenotypic differ-
ences. Within a single dinoflagellate species, there exist bioluminescent
and non-bioluminescent strains, such as has been demonstrated for
Gonyaulax excavata (Schmidt et al., 1978) and N. scintillons
(Valiadi et al., 2019). The genetic and biochemical basis for loss of
bioluminescence in N. scintillons strains has recently been identified.
These included the presence of the lcf gene but undectable transcripts,
and potentially deleterious mutations within the gene sequence
(Valiadi et al., 2019). While it remains to be determined whether these
same mechanisms are the reason for differences in light and dark strains
across species, it is intriguing to consider the influences that initiated
and maintained these mutations. Did bioluminescence no longer confer
an advantage to that population? How frequently does this occur
among sub-populations?
The question of genetic diversity among sub-populations also ap-
plies to toxicity. Twenty years ago, it was feasible to hypothesize that
toxicity functioned via a kin- or group-selection mechanism to decrease
grazing predation; in this scenario, toxin production by a cell, while
decreasing that cell's chance of survival, would increase the survival
rate of other cells in the population. However, in order for this type of
strategy to continue requires the other cells in the population to be
K.D. Cusick and E.A. Widder
capable of toxin production.
While the core genes in the dinoflagellate STX biosynthetic pathway
are known (Hackett et al., 2013; Stuken et al., 2011), the genetic basis
for variability in STX production within natural populations is un-
defined. SxtA4 has been found to occur in multiple, slightly different
genomic copies in multiple Alexandrium strains, suggesting adaptive
plasticity and the potential basis for phenotypic diversity in terms of
STX profile and toxicity (Alpermann et al., 2010; Kremp et al., 2014.).
Differences in toxicity in natural populations highlight the need to
examine the genetic variability among sub-populations and the me-
chanisms by which cells acquire or lose the trait. The factors driving
genetic variability in toxicity, and, to a lesser extent, bioluminescence
in natural HAB populations remain unexplored. Sxt gene duplication
and loss appear to be very common in dinoflagellates (Murray et al.,
2015) and, in keeping with other dinoflagellate genes, are present in
numerous copies due to processes such as the recycling of processed
cDNAs back into the genome through reverse transcription, retro-
position, (Bachvaroff and Place, 2008; Slamovits and Keeling, 2008;
Zhang et al., 2007) and potentially environmental stress (Verma et al.,
2019). In recent years horizontal gene transfer has been shown to be a
significant driver in dinoflagellate genome evolution (Nosenko and
Bhattacharya, 2007; Wisecaver et al., 2013).
One striking example as to the influences of genetic diversity in
relation to ecological functions of toxicity is an event that occurred with
P. bahamense. This species occurs in both the Atlantic-Caribbean and
the Indo-Pacific (Phlips et al., 2011; Usup et al., 2012), and toxic out-
breaks are well-documented from P. bahamense in the Indo-Pacific
(Azanza and Miranda, 2001; Azanza and Taylor, 2001; Gacutan et al.,
1985; Harada et al., 1982; Llewellyn et al., 2006; Montojo et al., 2006).
In contrast, P. bahamense bloomed along the east coast of Florida for
years with no known record of STX production. However, in the mid-
2000s, STX-contaminated seafood outbreaks across the United States
were traced back to Florida, and P. bahamense was identified as the
source (Bodager, 2002; Landsberg et al., 2006), marking the first oc-
currence of toxin production in the Western Atlantic. The mystery as to
the variability for toxin production among natural HAB populations
extends beyond the events in Florida. Analysis of numerous strains of A.
minutum in Irish coastal waters showed strains were indistinguishable
both morphologically and via sequencing of common phylogenetic
markers; however, strains from southern areas proved to be toxic via
HPLC analysis, while no toxins were detected in west coast strains
(Touzet et al., 2007). Studies covering a large range of Gymnodinium
strains indicated a high level of intra-population and regional variation
in both the presence and amounts of STX congeners (Hallegraeff et al.,
2012). Additionally, in screening numerous strains of P. bahamense
from the same geographical area in the Gulf of Mexico, toxicity was
confirmed in only one isolate (Morquecho, 2019). A very recent study
with L. polyedra found high intra-specific variability in YTX composi-
tion among strains from the same geographical region (Peter et al.,
2018). This is further illustrated by findings of broad intra-specific
variability in multiple traits among strains of a HAB species all isolated
from the same water sample in which a bloom was occurring (Menden-
Deuer and Montalbano, 2015). Could this discrepancy in toxin pro-
duction be linked to bioluminescence? StxA4 appears to be a necessary
component for cells to have the genetic capacity for toxin production.
Toxic and non-toxic bioluminescent strains from the same species (e.g.
Table 5
Flash characteristics among toxic and non-toxic bioluminescent dinoflagellates. Flash values obtained from (Latz et al., 2004).
Species Max flash intensity p/s Flash durastion (ms)
Ceratium fusus 1.10E+09 239
Ceratocorys horrida 9.20E+09 184
Lingulodinium polyedra 1.90E+08 100–150
Pyrocystis fusiformis 6.90E+11 210
17
Harmful Algae 98 (2020) 101850
P. bahamense, L. polyedra) have been isolated from the same geographic
area. Linking this back to bioluminescence, could these non-toxic iso-
lates be a modified form of Batesian mimics?
The presence and frequency of the sxtA4 gene in natural populations
of dinoflagellate species capable of STX production remains unknown.
It is well-established that high levels of genetic diversity exist among
toxic (Alexandrium) dinoflagellate species from the same geographic
area (Anderson et al., 2012a; Richlen et al., 2012). Additionally, toxin
production differs in genetically similar populations: population genetic
studies with Alexandrium showed substantial variation in a range of
traits including toxin production both within and across populations,
with differences in trait variation between genetically similar popula-
tions. This trait variation may promote HAB formation and persistence
under fluctuating environmental conditions (Brandenburg et al., 2018).
All the studies examining the genetic basis of toxin potential with STX-
producing species thus far have been based on lab cultures. The fre-
quency and potential for toxin production among natural sub-popula-
tions of HABs remains unknown yet is an area that needs to be explored
in order to gain greater insights into the genetic mechanisms driving
HAB evolution.
5.7. Correlating bioluminescence and toxicity
No data currently exist as to the correlation between biolumines-
cence intensity and genetic/biochemical features and toxin production.
Complicating factors in attempting an analysis of intensity among
species include not only the existence of different strains and Batesian
mimics, but also different measurement protocols for measuring light
output. A common methodology involves stirring a population of cells
to exhaustion, dividing the integrated light output by the number of
cells and then reporting the results as photons per cell. This is not a
meaningful measure when considering the eyes that the flash impacts.
In terms of visibility, photon flux at the peak of the flash is the most
pertinent value, followed by decay time and number of flashes. In a
study where all of these variables were measured using the same pro-
tocols (Latz et al., 2004), it is noteworthy how much these different
parameters varied between species (Table 5). L. polyedra, which is be-
lieved to be a Batesian mimic of toxic species, is the dimmest of the
four, producing only 2 or 3 short duration flashes. By contrast, P. fu-
siformis, which is non-toxic and evidence suggests employs its biolu-
minescence as a burglar alarm (Fleisher and Case, 1995; Mensinger and
Case, 1992), produces a flash intensity almost 3 orders of magnitude
greater, a longer flash duration and many more flashes per cell. In
keeping with the burglar alarm hypothesis it is also noteworthy how
much brighter the first flash is to subsequent flashes in this species
(Widder and Case, 1981b). All aspects of light production in this species
seem optimized for detection over long distance. The other two species,
Ceratium fusus and Ceratocorys horrida produce less light and fewer
flashes than P. fusiformis but more than L. polyedra. Evidence suggests
that they are non-toxic however both species exhibit atypical mor-
phology with spines that may be aversive to predators.
The genetic diversity of lcf within HAB sub-populations has recently
been examined at the single-cell level with P. bahamense (Cusick et al.,
2016). This study utilized single-cell PCR targeting the LCF conserved
catalytic region of D3 on P. bahamense cells from various locations.
Sequences from the Western Atlantic formed two distinct clusters, while
# flashes/cell Toxic Cell surface
2 N thecate
7 N thecate
2–3 Y thecate
23–62 N non thecate
K.D. Cusick and E.A. Widder
Fig. 5. (A.) Neighbor-joining tree of lcf sequences obtained from D3 of P. bahamense cells collected from the Western Atlantic. Available lcf sequences from the Indo-
Pacific were included in the analysis. (B.) Amino acid differences between cluster Lcf-WA1 and Lcf-WA2. Reprinted from Cusick et al. (2016).
sequences from an Indo-Pacific strain formed a third distinct cluster
(Fig. 5A). The clusters were defined by a set of core non-synonymous
substitutions (Fig. 5B). Sequence results obtained from environmental
samples encompassing multiple dinoflagellate species revealed a similar
trend as that obtained for the P. bahamense LCF D3: significant discrete
lcf clusters were observed for both L. polyedra and Protoceratium re-
ticulatum (albeit from the D2 domain) (Valiadi et al., 2014), while those
from non-toxic species clustered together. A. ostenfeldii is a toxic bio-
luminescent dinoflagellate with a wide geographic distribution and
extensive bloom formation in coastal waters of Europe
(Almandoz et al., 2014; Gribble et al., 2005; MacKenzie et al., 1996).
Lcf gene sequences and bioluminescence were examined from toxic
bloom populations in the Baltic and North Seas of Europe, and the re-
sulting phylogenetic analysis demonstrated that lcf sequences from
Baltic Sea strains clustered separately from North Sea strains (Le
Tortorec et al., 2016) (Fig. 6).
Assimilating the collective data on flash characteristics (intensity,
duration), lcf sequence data, and toxicity leads to the question as to the
influence of toxicity on bioluminescence. The clusters obtained for P.
bahamense lcf sequences (Fig. 5A) are defined by non-synonymous
substitutions resulting in a set of core amino acid differences (Fig. 5B)
that occur within the active site of the D3 domain in P. bahamense.
These differences were compared with the crystal structure of the L.
polyedra luciferase D3, which has been resolved at 1.8-Å (Schultz et al.,
2005). Briefly, the region is comprised of seven α-helices and 16 β-
strands, organized into two subdomains: a regulatory domain defined
by a three-helix bundle at the top followed by a β-barrel below
18
Harmful Algae 98 (2020) 101850
containing β-strands 5–14 (Fig. 7A). Comparisons between the two
types of LCF retrieved from the D3 of P. bahamense with this model
demonstrated that ca. 67% of these entailed substitutions that yielded
different physical properties (i.e., polar to non-polar, polar to basic,
etc.), and more importantly, that nearly all of the amino acid changes
occurred within a β-strand (5, 7, 8, and 11) or α-helix 5 – i.e. active
sites of the domain. Additionally, these non-synonymous substitutions
were specific to the Lcf-WA1 sequence cluster, and did not occur among
Lcf-WA2, P. lunula LcfA and LcfB, L. polyedra, A. tamarense, A. affine,
and the Indo-Pacific strain of P. bahamense (Fig. 7B) (Cusick et al.,
2016).
While the biochemical ramifications of the active site domain dif-
ferences found in P. bahamense remain to be determined, this may allow
for the cell to control the level of light produced. This concept of amino
acid variation altering the functional properties is in keeping with
findings of the light-harvesting complex proteins of the Symbiodinium,
in which molecular diversity may translate into functionally distinct
proteins that enable the cells to utilize the different light conditions of
their environment (Boldt et al., 2012). While the overall ecological role
of bioluminescence in a single HAB species may be to alleviate preda-
tion pressure, this may be achieved in different ways based on the toxin
capabilities of the cells and reflected in differences in LCF corre-
sponding to light output.
K.D. Cusick and E.A. Widder Harmful Algae 98 (2020) 101850

Fig. 6. Lcf sequences cluster by geographic location in A. ostenfeldii. Lcf sequences amplified from A. ostenfeldii isolates from the Baltic and North Sea. Sequences were
amplified using primers targeting the N-terminal and initial portion of the D1 domain. Figure reprinted from (Le Tortorec et al., 2016).

6. Signaling
A signal is the transfer of information between two organisms
through a biogenic stimulus that can be perceived by a sensory system
and elicit an adaptive response (Wolfe, 2000). Within this context sig-
naling refers to communication between individuals and includes both
visual and chemical signals. Visual signaling was discussed in the last
section with regard to ecological functions. In this section the focus is
on chemical signals.
The main mechanisms involved in cell surface sensing and trans-
duction include ion-channel-linked receptors, G protein-linked
receptors, and enzyme-linked receptors, while calcium and cyclic AMP
are key molecules used in mediating these processes (Wolfe, 2000).
While some work has been done on cell-surface signal receptors in the
protozoans (mainly ciliates and flagellates), little is known about such
signaling mechanisms in marine dinoflagellates (Wolfe, 2000).
6.1. Signal transduction in bioluminescence
As reviewed in the Biochemistry section, bioluminescence is trans-
duced via mechanical stimulation of the cells. The flash is the culmi-
nation of intricate signal transduction events that begins with an

19
K.D. Cusick and E.A. Widder Harmful Algae 98 (2020) 101850

Fig. 7. (A) L. polyedrum LCF D3 sequence with alpha-helix (α) and B-strand (β) noted. The two underlined six-residue sequences correspond to the beginning and end
of the highly conserved catalytic domain region. Red box designates approximate region amplified by degenerate lcf primers. Redrawn from Schultz et al. (2005). (B)
Amino acid sequence logo of highlighted D3 region. Included are LCF sequences from P. bahamense clusters Lcf-WA1, Lcf-WA2, an Indo-Pacific strain, P. lunula LcfA
and LcfB, A. tamarense, and A. affine. Blue arrows designate amino acid changes in Lcf-WA1.

increase in membrane fluidity due to a high mechanical stress.
Deformation of the membrane creates an action potential that opens
membrane channels and the rapid influx of protons from the acidic
vacuole results in an intracellular pH drop that activates LCF and, in
dinoflagellates with an LBP, dissociates luciferin from the LBP. In 1972,
J. Woodland Hastings and colleagues proposed the existence of a proton
selective channel that opens in response to depolarizing voltage across
the vacuole membrane of bioluminescent dinoflagellates, conducting
protons into the scintillons and resulting in a pH drop that triggers light
emission (Fogel and Hastings, 1972). The discovery and analysis of a
voltage-gated proton channel in the toxic species Karlodinium veneficum
revealed that while it contained enough signature elements to be
identified as a proton channel, its expression exhibited novel properties,
suggesting that these channels in dinoflagellates serve very different
functions than those in mammalian and other systems (Smith et al.,
2011). However, Hastings’ early hypothesis was recently confirmed
with the discovery of a voltage-gated proton channel in L. polyedra (LpH
(v)1) (Rodriguez et al., 2017). Hv1 sequences have also been identified
in A. tamarense and N. scintillans, providing strong evidence that Hv1, in
addition to LCF and LBP, is part of the signal transduction pathway
(Rodriguez et al., 2017). Upon stimulation via shear stress, this signal
transduction system is likely initiated by stretch activated channels
(Jin et al., 2013) at the surface of L. polyedra, and relays a signal
through intracellular calcium signaling via G-proteins (Chen et al.,
2007; von Dassow and Latz, 2002). However, the other members of this
signal transduction system remain unknown.
The ability of cells to sense and respond to their physical environ-
ment involves mechanically activated ion channels as a part of me-
chanotransduction signaling pathways. In prokaryotes, these are
stretch-activated ion channels that regulate cell turgor for osmor-
egulation, while eukaryotes can sense varied environmental mechanical
stresses in addition to stretch. Transient receptor potential (TRP) ion
channels are common elements of mammalian mechanosensing path-
ways. Plasma membrane mechanical stress was previously shown to
activate a TRP family protein in mammalian cells (Shen et al., 2015).
TRP-like channels have recently been identified as components of the
mechanotransduction (bioluminescent) signaling pathway in L. poly-
edra (Lindstrom et al., 2017b).
6.2. Copepodamides enhance bioluminescence
Recent studies revealed that copepodamides influence dino-
flagellate bioluminescence, as both L. polyedra and A. tamarense were
found to increase their total bioluminescence capacity in response to
copepodamides. Additional experiments with L. polyedra showed bio-
luminescence increased in a dose-dependent manner over time in re-
sponse to the copepod cues resulting in a brighter flash upon stimula-
tion and markedly affecting grazing preference by the copepod predator
A. tonsa (Lindstrom et al., 2017a; Prevett et al., 2019). L. polyedra is the
preferred prey when non-bioluminescent; however, when biolumines-
cent, the cells flash upon contact with the A. tonsa and are rejected,
without apparent harm to the cell (Prevett et al., 2019). How are these
chemical cues being sensed and transduced by the cells to enhance
bioluminescence? The study by Lindstrom (Lindstrom et al., 2017a)
exposed the dinoflagellate cells to cell-free copepodamides (i.e. no co-
pepods in treatment vessels), thus removing the mechanical stimula-
tion. However, bioluminescence was measured following acidification
of the cultures; therefore, while these data demonstrate the ability of
copepodamides to enhance bioluminescence, based on the methods, it
appears that bioluminescence is augmented after entry of the copepo-
damides into the cell, such that bioluminescence is not enhanced via a
cell-surface signaling network.

20
K.D. Cusick and E.A. Widder
6.3. Copepodamides enhance toxin production
The presence of copepods and their waterborne cues have also been
shown to boost toxin production (Selander et al., 2006; Wohlrab et al.,
2010; Yang et al., 2011). Comparisons among multiple factors, in-
cluding nitrogen sources, conspecific and interspecific species alarm
cues, and grazer (copepod) presence in a toxic strain of A. catenella,
showed grazer presence enhanced toxin production by an order of
magnitude greater than any other variable (Griffins et al., 2019). A.
minutum has been shown to produce STX in response to waterborne
cues from the calanoid copepod A. tonsa, with toxin production corre-
lated to an increased resistance to further grazing (Selander et al.,
2006). Intriguingly, it has been shown that grazer-induced toxin pro-
duction is a grazer-specific response, as exposure of toxic A. minutum
strains to different species of calanoid copepods elicited significantly
increased cell-specific toxicity to two of the three copepod species (A,
clausi, Centropages typicus, but not Pseudocalanus sp.) tested
(Bergkvist et al., 2008). These species-specific responses have also been
shown to occur in a toxic A. tamarense strain exposed to three different
copepod species (Calanus helgolandicus, A. clausii, and Oithona similis)
(Wohlrab et al., 2010), indicating that co-evolutionary processes may
be involved in these responses. The chemical basis for this grazer-spe-
cific response can be explained by copepodamides (taurine connected
via an amide to an isoprenoid fatty acid conjugate of varying compo-
sition), a suite of lipids produced by copepod species. Toxic A. minutum
was shown to increase its toxin production 20-fold in response to pico-
to nanomolar concentrations of copepodamides. Furthermore, the spe-
cies-specific defense response of toxic cells can be accounted for by the
species-specific blends emitted by the different copepod species
(Selander et al., 2015). Since their initial discovery in three species of
calanoid copepods (Selander et al., 2015), more than 20 additional
copepodamides have been identified, (Grebner et al., 2019), providing
further support as to the findings that the species-specific defense re-
sponse of toxic cells is accounted for by the species-specific blends
emitted by the different copepod species (Selander et al., 2015).
The signal transduction systems by which dinoflagellate cells sense
and respond to these chemical cues are only beginning to be elucidated.
Using functional genomics, serine/threonine kinase signaling pathways
were identified as significant elements in the response to copepod cues,
and in most cases, serine/threonine kinase pathway regulation was
specific to the copepod species (Wohlrab et al., 2010). Additionally,
transcripts for proteins dependent on calcium ions as second messen-
gers were differentially expressed (Wohlrab et al., 2010), in keeping
with other findings as to the role of calcium signaling in the response of
marine phytoplankton to environmental cues (Jingwen et al., 2006;
Verret et al., 2010) as well as bioluminescence (von Dassow and
Latz, 2002). Genomic analyses have found that conserved channel types
likely to be involved in calcium signaling are widely distributed among
algal species, including dinoflagellates (Verret et al., 2010). RNA-Seq
profiling of Alexandrium species (A. fundyense) upon exposure to para-
site-associated (Amoebophrya) waterborne cues showed differential
regulation of signal-transduction-related genes, including serine/
threonine kinases and genes involved in calcium, mitogen-activated
protein kinase and Ras signaling (Lu et al., 2016). Copepodamides have
also been shown to stimulate domoic acid production in the toxic
diatom Pseudo-nitzschia seriata (Selander et al., 2019). RNA-sequencing
results indicate P. seriata transduce copepod cues via G protein path-
ways (Hardardottir et al., 2019). Could dinoflagellates utilize similar
pathways in response to the copepodamides? Recent comprehensive
transcriptomic analyses identified G protein-coupled receptors (GPCRs)
in 81 diatom and 36 dinoflagellate transcriptomes (Mojib and
Kubanek, 2020). The GPCRs of both groups were highly divergent from
known metazoan GPCRs and, unlike other eukaryotes that express
multiple classes of GPCRs, both were found to express only a single
major class with greatest similarity to class C and rhodopsin‐like
GPCRs, respectively. Based on the transcriptome data, dinoflagellate
21
Harmful Algae 98 (2020) 101850
GPCRs are more divergent from known GPCRs than are diatom GPCRs
(Mojib and Kubanek, 2020) and while this divergence may present a
challenge it also provides a platform for future studies to examine the
role of these proteins in dinoflagellate signal transduction.
7. Evolution
7.1. Bioluminescence
Characterization of the LCF from seven species of photosynthetic
dinoflagellates demonstrated that they share a common origin
(Liu et al., 2004). While LCF domain organization is similar among all,
differences exist in the distribution of the synonymous substitution
rates across the lcf gene, as well as the intergenic regions (Liu and
Hastings, 2006; Liu et al., 2004; Okamoto et al., 2001). Synonymous
substitutions rates were found to be greatly reduced in L. polyedra and
P. reticulatum but not the other five (A. affine, A. tamarense, P. fusiformis,
P. noctiluca, P. lunula) (Liu et al., 2004).The significance of these dif-
ferences remains unknown, and how they relate to the evolutionary
constraints of this system in dinoflagellates. The luciferase of L. polyedra
likely diverged early from the other six based on lcf and ribosomal DNA
analysis (Liu et al., 2004). Comparison of the LCF among photo-
synthetic species indicate ancient triplication of one of the domains
occurred in an ancestor common to all that was then carried forward
during the evolution of the different photosynthetic species (Liu et al.,
2004), with the entire lcf gene retained in descendants in which bio-
luminescence provided a selective advantage under strong selective
pressure for maintaining catalytic activity (Liu et al., 2004). However,
the differences in synonymous substitution rates indicate a second se-
lective pressure must have occurred, likely after the divergence of L.
polyedra and P. reticulatum, to account for the very low rate of synon-
ymous substitutions within these species (Liu et al., 2004). The func-
tional implications of this constraint, as evidenced by enhanced stabi-
lity of RNA secondary structure in the LCF of L. polyedra and P.
reticulatum, may be associated with a regulatory mechanism (Liu et al.,
2004), such as the translational control of the circadian rhythm in L.
polyedra. As discussed in the Biochemistry section, L. polyedra has an
LBP and an LCF, both of which are under circadian control. Biolumi-
nescent measurements with P. bahamense, which also possesses an LBP,
also suggest its bioluminescence is under circadian control
(Biggley et al., 1969). This is in contrast to P. lunula, which contains the
greatest rate of synonymous substitutions (Liu et al., 2004): there is no
LBP, and the daily levels of LCF remain constant (Knaust et al., 1998).
Sequencing of lbp genes across multiple genera, including
Alexandrium, Protoceratium, Gonyaulax, and Ceratocorys, revealed a very
diverse gene family comprised of several gene types (Valiadi and
Iglesias-Rodriguez, 2014). Phylogenetic analysis showed that, for mul-
tiple genera, the evolution of the lbp gene differed from the species’
general phylogenies, demonstrating the complex evolutionary history
of dinoflagellate bioluminescence (Valiadi and Iglesias-
Rodriguez, 2014). In N. scintillons, it occurs as a fusion with LCF
(Liu and Hastings, 2007). An additional LBP has since been identified in
N. scintillans and shown to be actively transcribed in addition to the one
attached to LCF, supporting the notion that a significant re-structuring
of the system has taken place in this species (Valiadi and Iglesias-
Rodriguez, 2014). Collectively, the evolutionary analysis of the biolu-
minescent system in multiple dinoflagellate species illustrates the
adaptive value of this trait.
The light produced by toxic species is dim in comparison to that of
many of the non-toxic species. All toxic, bioluminescent species for
which sequence data are available possess a separate LBP in addition to
the LCF (Table 1). It is worth noting that lbp gene sequences have not
been detected in the gonyaucaloids Ceratium and Fragilidium nor in
Protoperidinium and Pyrocystis (Colepicolo et al., 1993; Valiadi and
Iglesias-Rodriguez, 2014). Protoperidinium and Pyrocystis are both large,
bright-emitting, non-toxic cells; Ceratium and Fragilidium are also non-
K.D. Cusick and E.A. Widder
toxic. Additionally, the lbp gene sequences of A. monilatum and G. spi-
nifera, both toxic, displayed increased diversity from those of other
Gonyaulacales. This correlation between LBP evolution and toxicity is a
link that requires further investigation.
7.2. Saxitoxin
Three theories have been proposed as to the origin of STX produc-
tion in dinoflagellates: convergent evolution, horizontal gene transfer,
or autonomous production by associated bacteria (Orr et al., 2013b).
STX biosynthesis genes were first identified in several genera of fresh-
water cyanobacteria; 14 genes were shared among the genera, leading
to designation of 14 “core” genes (Murray et al., 2011a; Wiese et al.,
2010). Due to the complexity of their genomes, identification of these
homologs in dinoflagellates took several more years to achieve. Tran-
scriptomic sequencing of toxin-producing Alexandrium strains resulted
in the discovery of nuclear-encoded sxt homologs in dinoflagellates
(Hackett et al., 2013; Stuken et al., 2011). SxtA transcripts were found
to be closely related to a clade of cyanobacteria sxtA sequences, and
possessed the same domain structure (Stuken et al., 2011). However,
unlike their bacterial homologs, dinoflagellate transcripts contained a
higher GC content, occurred in multiple copies, and displayed the ty-
pical dinoflagellate spliced-leader sequences and eukaryotic polyA-tails
(Stuken et al., 2011) confirming their dinoflagellate origin and pro-
viding strong evidence of autonomous STX production by dino-
flagellates (Orr et al., 2013b). Subsequent characterization of sxtG, the
second “core” gene, confirmed the previous results, as transcripts also
possessed spliced-leader sequences and poly-A tails (Orr et al., 2013a).
While the findings of dinoflagellate STX biosynthesis homologs lessened
the support for the co-culture bacteria theory it is intriguing to note that
half of the core cyanobacterial sxt genes have their origin from pro-
teobacterial genomes, and half of the dinoflagellate sxt homologs also
have a putative proteobacterial origin. While no proteobacteria have
been shown to produce STX, many strains are found in close association
with STX-producing dinoflagellate strains (Orr et al., 2013b).
Based on the initial findings of dinoflagellate-specific sxt homologs,
it was proposed that a horizontal gene transfer (HGT) event between
ancestral STX-producing bacteria and dinoflagellates occurred (Stuken
et al., 2011). Additional sequence analysis has led to a more refined
theory: STX evolution in dinoflagellates was initiated via a massive
gene transfer event from cyanobacteria into an ancestor of Alexandrium
and Pyrodinium, who then acquired additional genes from multiple
prokaryotic sources. The sxt genes were then extensively modified, with
some homologs lost or replaced. The pathway was lost in some lineages,
and transferred to Gymnodinium via an endosymbiotic gene transfer.
Genes and domains were re-shuffled, resulting in loss of sxtA4 in some
species (Orr et al., 2013b).
The presence and high sequence conservation of the sxtA4 domain
was confirmed in 40 strains (35 from Alexandrium species, a single
Pyrodinium, and four Gymnodinium) of STX-producing species, and ab-
sent in non-toxin producing species from these genera (Murray et al.,
2015). Screening for the stxA1 domain found it was widely distributed
in non-STX producing dinoflagellates and occurred as three paralogs,
with one of the paralogs seemingly closely associated with STX synth-
esis (Murray et al., 2015). SxtG was also found to be generally specific
for STX-producing strains (Murray et al., 2015). While detected in
Alexandrium species not known to synthesize STX, its presence was not
detected in non-STX-producing species outside the Alexandrium genus,
P. bahamense, or G. catenatum (Hackett et al., 2013; Orr et al., 2013a).
Phylogenetic analysis of sxtA1 and sxtG indicates that the evolution
of these genes has been complex, with duplication events involved in
the evolution of both sxtA and sxtG (Murray et al., 2015). Analysis of
sxtG in multiple strains of Alexandrium along with a G. catenatum strain
indicate multiple independent acquisitions and losses of this gene
(Orr et al., 2013a). Widespread horizontal gene transfer does not ap-
pear to occur for sxtA4 based on analysis of Alexandrium sxtA4
22
Harmful Algae 98 (2020) 101850
nucleotide sequences; this is further supported by analysis of sxtA and
sxtG in multiple strains of G. catenatum. Although highly conserved,
sxtA4 phylogeny mirrors that of species phylogeny (Mendoza-Flores
et al., 2018; Murray et al., 2015), indicating that the gene may have
evolved in a common dinoflagellate ancestor of G. catenatum, P. baha-
mense and Alexandrium and a secondary loss of these genes occurred in
non-toxic Alexandrium species (Murray et al., 2015). These collective
findings with sxtA1, sxtA4, and sxtG support the theory that the ability
for STX synthesis has been secondarily lost in some species, with sxtA
being lost independently on multiple occasions (Orr et al., 2013b).
Selection (positive or negative) analysis of sxtA1 and sxtG showed
that the clade of sxtA1 putatively associated with STX synthesis is under
negative selection, indicating pressure to constrain function
(Murray et al., 2015). This differs from the evolution of the cyano-
bacterial sxt gene cluster, in which positive selection was found to play
a role (Murray et al., 2011a). Similarly, sxtG also showed negative se-
lection, indicating functional constraint for this gene as well. The pre-
sence of multiple paralogs in different clades of both sxtA1 and sxtG
indicate these genes may perform different functions, as both display
positive selection in clades associated with a lack of toxin production
facilitating the development of different functions (Murray et al.,
2015).
In general, negative selection is the selective removal of (seemingly)
deleterious genes, and removal of these genes can be achieved on the
population genetics level, with as little as a single point mutation.
Linking the findings of potential negative selection of sxt genes with
genetic diversity, how then can this be translated into toxic and non-
toxic HAB populations occurring both spatially and temporally in the
same general geographic area (as discussed in the section on Genetic
Diversity)? If the toxin serves to protect cells from predation pressure,
one would image all cells in a population would retain sxtA4. Here, the
concept of bioluminescence as Batesian mimicry may come into play: if
all cells in the population are bioluminescent, sxtA4 may be lost in a
portion of the sub-population over time, as the dinoflagellate predator
learns to associate the flash with toxicity, thereby avoiding all biolu-
minescent (and at that point, toxic) cells. While this theory requires
additional genetic data on sub-populations from the same geographical
area in order to develop appropriately, it presents a viable and testable
hypothesis as to potential links between bioluminescence and toxicity
in HAB species based on the findings of negative selection for sxt genes.
8. Conclusions
Both bioluminescence and toxin production have the potential to
greatly influence HAB initiation and persistence. Some of the most
notorious HABs consist of species with the capacity for both traits, and
it is this combination for which many knowledge gaps remain, at both
the ecological and genetic levels. The function, and thus adaptive value,
of the flash have been long debated. Multiple hypotheses have been
proposed, all rooted in the premise that bioluminescence acts as an anti-
grazing strategy and this relief from grazing pressure plays an im-
portant role in bloom formation. The same holds for toxin production;
there are multiple hypotheses, with one of the main being that the toxin
functions as a defense from grazers. Combining these traits leads to the
hypothesis that the flash functions as an aposematic signal, and that
Batesian mimicry, which often conjures up images of monarch butter-
flies, may well be occurring within single-celled dinoflagellates as well.
The genetic machinery of bioluminescence has been defined.
Sequencing of lcf and lbp genes across genera has shown a complex
evolutionary history, supporting, at the genomic level, the adaptive
value of this trait. However, several areas remain unexplored that may
provide additional insights into the eco-evolutionary role of dino-
flagellate bioluminescence, especially with regards to HAB species.
These include: the mechanism behind the general trend of LBP presence
in toxic, dim-emitting species and lack of LBP in non-toxic, bright-
emitting species; and the effects of key amino acid differences within
K.D. Cusick and E.A. Widder
the LCF catalytic domain in toxic species. The core genetic machinery in
STX biosynthesis has also recently been determined, and with it the
finding that the sxtA4 gene appears specific for toxin production.
However, the identification of genes involved in both these traits were
derived from lab-based experiments with clonal isolates. A large gap in
our understanding of both these traits is their distribution among nat-
ural bloom populations, especially with regards to toxin production.
When considering the ecological function of toxin production, one
feature that is often overlooked is the variability of this trait within sub-
populations of a natural bloom of the species. It remains unknown
whether all the cells possess the genes necessary for toxin production,
or only a portion of the sub-population. Extending that reasoning leads
to further questions as to how the genes are maintained within that
population, as both toxic and non-toxic strains have been isolated from
the same geographic area. Can toxic cells become non-toxic, and vice
versa? This can further be extended to bioluminescence, for just as there
exist toxic and non-toxic strains within a species, so too are there light
and dark strains. The frequency and potential for both toxin production
and bioluminescence among natural HAB sub-populations remains
unknown yet is an area that needs to be explored in order to gain
greater insights into the genetic and environmental mechanisms driving
HAB evolution.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
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