INFLUENCE OF TEMPERATURE, SALINITY AND NUTRIENTS ON GROWTH AND
TOXIN OF Karenia brevis CLONES
Danelle Kara Lekan
A Thesis Submitted to the
University of North Carolina Wilmington in Partial Fulfillment
of the Requirements for the Degree of
Master of Science
Center for Marine Science
University of North Carolina Wilmington
2008
Approved by
Advisory Committee
Susan J. Simmons
Daniel G. Baden
Carmelo R. Tomas, Chair
Accepted by
Robert D. Roer
Digitally signed by Robert D. Roer
DN: cn=Robert D. Roer, o=UNCW, ou=Dean
of the Graduate School & Research,
email=roer@uncw.edu, c=US
Date: 2008.09.16 09:05:30 -04'00'
Dean, Graduate School
i
TABLE OF CONTENTS
ABSTRACT...................................................................................................................... iii
ACKNOWLEDGMENTS ................................................................................................ iv
LIST OF TABLES............................................................................................................. v
LIST OF FIGURES ......................................................................................................... vii
INTRODUCTION ............................................................................................................. 1
MATERIALS AND METHODS..................................................................................... 11
Overview.................................................................................................................. 11
Cultivation................................................................................................................ 12
Growth Study ........................................................................................................... 12
Toxin Study.............................................................................................................. 15
RESULTS ........................................................................................................................ 20
Growth Patterns ....................................................................................................... 20
Growth Rates ........................................................................................................... 23
Toxin Analyses ........................................................................................................ 25
DISCUSSION .................................................................................................................. 30
REFERENCES ................................................................................................................ 40
TABLES .......................................................................................................................... 48
FIGURES......................................................................................................................... 57
APPENDIX A.................................................................................................................. 65
ii
ABSTRACT
The toxic dinoflagellate Karenia brevis forms extensive, annual blooms in the Gulf of
Mexico releasing potent neurotoxins having significant impacts on human health, mortalities of
marine mammals, birds and fish. This study focuses on the factors affecting growth and cellular
virulence. Effects of environmental factors such as temperature (20-30oC), salinity (20-39) and
differing nutrient environments expressed as N:P ratios of 16:1 (balanced), 1:1 (nitrogen limited)
and 80:1 (phosphorus limited) were explored with three clones of Karenia. The Wilson clone,
historically the source of brevetoxin (PbTx) standards for more than 30 years, was used along
with the SP3 N-tox and SP3 S-tox. Growth as measured by in vivo fluorescence was used to
assess the effects of temperature, salinity and nutrients on growth. At a salinity of 20, none of the
clones in any of the temperature-nutrient treatments grew. At a temperature of 30oC the Wilson
clone did not grow in any salinity-nutrient treatment. The SP3 clones grew only in salinities of 35
and 39 at 30oC. Growth rates for all clones in all treatments ranges from 0.05-0.29 div day-1.
ANOVA results showed growth rates were highly significant based on clone, salinity and
temperature, and significantly different based on nutrients. Growth rates generally decreased at
extremes of low salinity and high temperature. The effects of salinity and nutrients at 20oC on
brevenal and brevetoxin profiles of PbTx-1, 2, 3, 6 and 9 in stationary phase were examined with
LC-MS/MS. The effects of nutrient ratios on toxin profiles did not show a clear pattern. The
clone type studied significantly influenced the amount of brevenal and PbTx-1, 2, 6 and 9. The
salinity studied significantly affected the amount of PbTx-1 and 3 produced. The average cellular
quota of PbTx-2 in all salinity-nutrient treatments was 19340 pg-toxin cellsx10-3 for SP3 N-tox,
11030 for SP3 S-tox and 20180 for Wilson. The Wilson clone produced a significantly greater
amount of brevenal than either SP3 clone in all treatments. The results support clonal differences
in growth and toxin production, and raise the question of inherent genetic variation leading to
these differences.
iii
ACKNOWLEDGMENTS
I greatly appreciate the guidance of my advisor and mentor, Dr. Carmelo Tomas,
for sharing his knowledge and love of phytoplankton and for his continued support and
contribution in my research endeavors. I would like to thank my committee members,
Drs. Daniel G. Baden and Susan J. Simmons, for their enthusiasm and assistance in this
research.
My deepest gratitude goes to my friends for their encouragement and their new
found understanding and interest in toxic phytoplankton. I also owe a great amount of
thanks to my family for their understanding and support throughout my academic career.
This work was supported by the Center for Disease Control and Prevention grant
# 0150407 awarded to principal investigator Carmelo R. Tomas, through the North
Carolina Department of Health and Human Services. Travel awards from the UNCW
Graduate School and the UNCW Center for Marine Science allowed me to travel to
international and national meetings to share this research and gain helpful insights.
I would like to thank Dr. Andrea Bourdelais for teaching me the methodology of
liquid-liquid organic extractions of brevetoxins and brevenal and LC-MS/MS techniques
to analyze brevetoxins and brevenal. I would also like to thank the Baden Lab for the
Wilson clone used in this study, as well as for the Wilson culture to use for dialysis
experiments to determine in cell versus out of cell toxin content.
iv
LIST OF TABLES
Table
Page
1. The names and masses of parent and derivative brevetoxins (PbTx) from Karenia
brevis used as standards in analysis with LC-MS/MS.................................................... 48
2. Growth rates (k) for the SP3 N-tox, SP3 S-tox and Wilson clones at temperatures of
20, 25 and 30oC, salinities of 25, 30, 35 and 39, in balanced P-limited and N-limited
nutrient ratios .................................................................................................................. 49
3. Results for the three, two and one-way ANOVA for growth rates by the variables of
clone (SP3 N-tox, SP3 S-tox and Wilson), temperature (20, 25 and 30oC), nutrient
(Balanced, P-lim and N-lim) and salinity (20, 25, 30, 35 and 39). P-values were
considered significant if less than 0.05. .......................................................................... 50
4. ANOVA results showing where the salinity studied significantly affected the growth
rate for all three clones of K. brevis (SP3 N-tox, SP3 S-tox and Wilson) in salinities of
25, 30, 35 and 39 at temperatures of 20 and 25oC for all clones, and salinities of 35 and
39 at 30oC for the SP3 N-tox and SP3 S-tox clones. P-values were significant if less than
0.05.................................................................................................................................. 51
5. ANOVA results showing where the temperature studied significantly affected growth
rates of three clones of K. brevis (SP3 N-tox, SP3 S-tox and Wilson) in salinities of 25,
30, 35 and 39 at temperatures of 20 and 25oC for all clones, and salinities of 35 and 39 at
30oC for the SP3 clones. P-values were significant if less than 0.05. ............................ 51
6. ANOVA results showing where the clone studied significantly affected growth rates of
three clones of K. brevis (SP3 N-tox, SP3 S-tox and Wilson) at temperatures of 20 and
25oC in salinities of 25, 30, 35 and 39, for all clones, and salinities of 35 and 39 at 30oC
for the SP3 clones. P-values were significant if less than 0.05....................................... 52
7. Cell density of K. brevis clones (SP3 N-tox, SP3 S-tox and Wilson) at the time of
extraction for toxin analysis in stationary phase grown at 20oC under defined nutrientsalinity conditions. .......................................................................................................... 52
8. LC-MS/MS analysis of pg-toxin cell-1 for three clones of Karenia brevis in stationary
phase, grown at 20oC in salinities of 25, 35 and 39 and nutrient ratios of balanced, Plimited and N-limited...................................................................................................... 53
9. Results for the three, two and one-way ANOVA for brevenal and brevetoxin
concentrations of treatments based on variables of clone (SP3 N-tox, SP3 S-tox and
Wilson), temperature (20, 25 and 30oC), nutrient (balanced, P-limited and N-limited) and
salinity (25, 35 and 39). P-values were significant if less than 0.05............................... 54
v
10. A Tukey’s pair wise comparison of total toxin production of PbTx-2+3+9, PbTx1+2+3+9 and total brevenal, between grouped treatments of clone-salinity, clone-nutrient
and salinity-nutrient. P-values were significant is less than 0.05. .................................. 55
11. MANOVA results of total PbTx-2+3+9 and total PbTx-1+2+3+9 for variables of
clone (SP3 N-tox, SP3 S-tox and Wilson), salinity (25, 35 and 39) and nutrient (balanced,
P-limited and N-limited) on toxin production. Wilks’ Lambda values were significant if
less than 0.05.................................................................................................................. .56
vi
LIST OF FIGURES
Figure
Page
6
-1
1. Calibration of cell density (x 10 cell L ) versus relative fluorescence of three clones
of K. brevis. A.) SP3 N-tox B.) SP3 S-tox and C.) Wilson ............................................. 57
2. Flowchart describing the extraction method used to extract brevenal and brevetoxin
from Karenia brevis........................................................................................................ 58
3. Relative fluorescence over time of three clones of Karenia brevis displaying similar
moribund growth patterns of a steady decline after inoculation on day 0. (Bal 16:1 N:P;
P-lim 80:1; N-lim 1:1) .................................................................................................... 59
4. Relative fluorescence over time of three clones of Karenia brevis (SP3 N-tox, SP3Stox and Wilson) displaying a similar lag phase in initial growth patterns after inoculation
on day 0. (Bal 16:1 N:P; P-lim 80:1; N-lim 1:1) ............................................................ 60
5. Relative fluorescence over time of three clones of Karenia brevis (SP3 N-tox, SP3 Stox and Wilson) displaying similar growth patterns of a steady increase in growth after
inoculation without a lag phase. (Bal 16:1 N:P; P-lim 80:1; N-lim 1:1) ........................ 61
6. Relative fluorescence over time of three clones of Karenia brevis (SP3 N-tox, SP3 Stox and Wilson) displaying similar growth patterns of a gradual or rapid decrease in Nlimited treatments in mid or late growth. (Bal 16:1 N:P; P-lim 80:1; N-lim 1:1) .......... 62
7. Brevetoxin profiles in pg-toxin ml-1 from extracts of whole culture (cells and media)
and media only from dialysis tubing suspended in a culture of Karenia brevis for 0, 6, 12,
24 and 48 hour intervals.................................................................................................. 63
8. Percent of total PbTx-1+2+3+9 (A) and brevenal (B) in P-lim and N-lim nutrient
treatments relative to balanced nutrients for three clones of Karenia brevis (SP3 N-tox,
SP3 S-tox and Wilson) in salinities of 25, 35 and 39 at a temperature of 20oC. (Bal 16:1
N:P; P-lim 80:1; N-lim 1:1) ............................................................................................ 64
vii
INTRODUCTION
The toxic dinoflagellate Karenia brevis (formerly Gymnodinium breve Davis
1948; Ptychodiscus brevis Steidinger 1978) forms extensive, annual red tide blooms in
the Gulf of Mexico. In the Gulf of Mexico K. brevis is common at background
concentrations of ~1 to 1000 cells L-1 year round (Geesey & Tester 1993). Blooms
typically initiate 18-74 km offshore of the central western coast of Florida in oligotrophic
waters at depths of 13-40 m (Steidinger 1973, 1975a; Steidinger & Haddad 1981),
reaching fish killing concentrations of 1 to 2.5 x 105 cells L-1 within a month (Tester &
Steidinger 1997; Steidinger et al. 1998). As a positively phototactic species, K. brevis
congregates in surface waters during the day (Steidinger 1975b; Heil 1986), making
established offshore populations easily susceptible to transport by winds and the Loop
Current system to the Gulf Stream (Tester et al. 1991).
Karenia brevis becomes a major concern when it is transported near coastal
waters where its potent brevetoxins can cause mass mortalities of fishes and marine
mammals, human neurotoxic shellfish poisoning (NSP) and human respiratory irritation
(Rounsefell & Nelson 1966; Kirkpatrick et al. 2004). Brevetoxins (PbTx’s) produced by
K. brevis are a major concern because very low concentrations induce toxicity (Naar et al.
2002). Human NSP results from the ingestion of neurotoxins from shellfish contaminated
with K. brevis toxins, causing debilitating, but usually non-fatal shellfish poisoning
(Magana et al. 2003). Brevetoxins in shellfish are tasteless, colorless, heat and acid stable
and cannot be removed in food preparation (Baden & Mende 1982; Baden & Trainer
1993; Baden et al. 1995; Sakamoto et al. 1987). Karenia brevis blooms are also a concern
in coastal areas, because the fragile, unarmored dinoflagellate easily lyses from wave
1
action, releasing cell fragments with brevetoxins (Kirkpatrick et al. 2004). Wind carries
the brevetoxin aerosols inland (Pierce et al. 1989; Pierce et al. 1990), where human
inhalation of the air-borne toxins results in respiratory irritation, stinging eyes, stinging
nose and a dry, choking cough (Magana et al. 2003). The major route of human exposure
to brevetoxins is through inhalation, whereupon 80% of PbTx-3 is rapidly absorbed from
the lungs to the blood and distributed to all tissues (Benson et al. 1999). Brevetoxins are
potent respiratory toxins that act through cholinergic and histamine related mechanisms,
posing particular threats to people with underlying respiratory problems and diseases
(Fleming et al. 2005). The tourism and recreation industry on the West coast of Florida
are adversely affected by bloom events through the closure of beaches and other
establishments located near beaches (Anderson et al. 2000). It was estimated by
Anderson et al. (2000) that $18 million was lost to the commercial fishing industry due to
red tides alone over a five year period.
Red tides in the Gulf of Mexico not only affect the human population in nearby
coastal areas, but marine animals as well. In fish and marine mammals, exposure to the
toxin causes damage by sodium channel activation and death results from respiratory
failure (Baden & Trainer 1993). Unusually high mortality rates of threatened marine
species including manatees, bottlenose dolphins and sea turtles have coincided with red
tide events (Trainer & Baden 1999). In 1996, 149 manatees (Trichechus manatus
latirostris) died in an unprecedented epizootic event along the southwest coast of Florida
during the presence of a nearby K. brevis bloom (Bossart et al. 1998). All animals
displayed severe nasopharyngeal, pulmonary, hepatic, renal and cerebral congestion.
Analysis later showed intense positive staining for PbTx in tissue samples.
2
In 1998, Tester et al. reported the occurrence of a K. brevis bloom during 26 of
the past 27 years along the coast of Florida. In addition to this, mounting evidence
supports the trend of an increase in frequency of phytoplankton blooms worldwide over
the past two decades (Smayda 1990; Hallegraeff 1993). Elevated scientific awareness of
this natural phenomenon may in part be the reason for this apparent global increase but
other factors such as extensive aquaculture in coastal waters, eutrophication, unique
climate conditions, as well as transport of cysts in ballast water of ships may also be to
blame (Hallegraeff 1993).
Aldrich and Wilson (1960) first examined the salinity tolerance of K. brevis, and
found it grew well in salinities from 27 to 37 with less than optimum growth at salinities
below 24 or above 44. This narrow salinity range led to a suggested “salinity barrier” of
24 or less for K. brevis. However, within the past fifty years, K. brevis blooms have
occurred more frequently in low salinity waters (Maier Brown et al. 2006). Several
blooms occurred near the mouth of the Mississippi River and the Florida Panhandle,
where there is a significantly lower salinity due to the freshwater outflow (Dortch et al.
1998; Maier Brown et al. 2006).
Evidence of blooms occurring in low salinity waters questions whether salinity is
truly a barrier in the bloom development of Karenia spp., and how lower salinities affect
growth and toxin production. Blooms of K. brevis are transported in the Gulf of Mexico
by the Loop Current, and may at times encounter water masses of a lower salinity.
However, the mixing of these water masses of high and low salinities may allow for K.
brevis to acclimate to lower salinities. Initial salinity studies that did not acclimate K.
brevis suggested that K. brevis has a narrow salinity tolerance (Aldrich & Wilson 1960).
3
However, a recent study by Maier Brown et al. (2006) acclimated K. brevis at increments
of 5 or less and various clones were able to grow at salinities below 24. The Mexico
Beach clone from Northern Florida grew at a salinity of 17.5, the Wilson clone grew at
20, and the Charlotte Harbor clone at 18.5 (Maier Brown et al. 2006). Magana and
Villareal (2006) also acclimated the SP3 clone of K. brevis to lower salinities, but could
not grow it below salinities of 25. The bloom of K. brevis cited by Dortch et al. (1998) in
the mouth of the Mississippi River may have been a different clone of K. brevis, which
may have survived due to differences in clonal variation which may result in the ability to
acclimate to lower salinities.
Growth rates appear to be strongly affected by salinity. Maier Brown et al. (2006)
reported growth rates for K. brevis when examining the Mexico Beach, Wilson, and
Charlotte Harbor clones, and each clone varied in growth rates. At 20oC and a salinity of
25 Magana and Villareal (2006) found a growth rate of 0.30 divisions day-1, 0.36 at a
salinity of 30, 0.32 at a salinity of 35, and 0.26 at a salinity of 40. Wilson (1966) reported
rates between 0.2 and 0.5 divisions day-1. However, Shanely and Vargo (1993) reported
elevated growth rates of 0.2-1.0 divisions day-1 with the Wilson clone. Loret et al. (2002)
examined maximum growth rates between five clones of K. brevis isolated from the coast
of Texas and from the Coast of Florida, and found differences among the strains.
Between each of these clones there was variability in growth rates, supporting the
suggestion that different clones may inherently have different growth rates.
While salinity is the primary factor for determining bloom initiation, the critical
factor that contributes to ultimate bloom initiation, development and duration is the
nutrient supply (Liu et al. 2001). Blooms of K. brevis initiated offshore in oligotrophic
4
waters must be maintained by sufficient amounts of nitrogen, phosphorus, vitamins and
amino acids. When transported near shore, blooms are often supported by nutrient run-off
from anthropogenic influences from point and non-point sources; however, it must be
noted that red tide blooms occurred in Florida prior to significant human pollution and
development (Kirkpatrick et al. 2004).
In the 1950’s Redfield measured the nutrient composition of carbon, nitrogen and
phosphorus in the open ocean. He discovered a 106:16:1, C:N:P ratio per atom in the
water column and in living cells (Redfield 1958). This ratio, considered the optimal ratio
for balanced growth, is reflected in cells when not stressed. Nutrient stress results when
one or more nutrient(s) is not sufficient in the open ocean. According to Leibig’s Law of
the Minimum, that constituent present in the least amount relative to the requirement for
growth of organisms, will be the limiting factor for growth. Carbon is present in vast
quantities in the ocean in the form of carbonate and therefore is rarely a limiting factor.
Other trace elements required for phytoplankton growth, such as sulfur, calcium,
magnesium and potassium, are generally not limiting relative to nitrogen and phosphorus
(Redfield 1958). Nitrogen or phosphorus however, can often be a limiting factor when
considering phytoplankton growth in the open ocean. Of note, the relative amounts of
nitrogen to phosphorus may limit or encourage the growth and/or toxin production of K.
brevis.
In marine waters nitrogen is considered to be the limiting nutrient, while
phosphorus is considered to be the limiting nutrient in freshwater environments (Heckey
& Kilham 1988). Vargo et al. (2007), examined the magnitude and variety of the
complicated sources of nitrogen and phosphorus available to support blooms of K. brevis.
5
These nutrients act to regulate growth rate, biomass and length of a bloom. As cited in
Vargo et al. (2007) there are several probable sources of nitrogen and phosphorus to
support blooms; N2 fixation from Trichodesmium blooms supply nitrogen (Walsh &
Steidinger 2001; Mulholland et al. 2004, 2006), shelf-break upwelling supplies
remineralized nutrients from diatom blooms (Walsh et al. 2006), brevetoxins from bloom
events kill fish, vertebrates and other invertebrates, ultimately leading to the
remineralization of nutrients from decaying organisms (Walsh et al. 2006), and estuarine
flux of nitrogen and phosphorus also supplies coastal blooms with nutrients (Vargo et al.
2004). However, no single source provides flux rates sufficient to support and maintain
high biomass blooms (Vargo et al. 2007). Along the West Coast of Florida, phosphorus is
presumably supplied by large deposits of phosphorite (Brand & Compton 2007).
Comparison of K. brevis bloom abundance from 1954-1963 and 1994-2002
showed a 13-18 fold increase in the highest concentrations achieved, possibly implying
an increase in nutrient availability (Brand & Compton 2007). No natural nutrient sources
have increased 13-18 fold, but human activity along the coast may be involved in the
increase in nutrient availability. Population increase along the coast of Florida led to
increased sewage, more disturbances in terrestrial and wetland ecosystems and more land
surface runoff (Brand & Compton 2007). There has also been an increase in mining of
phosphate deposits, oxidation of nitrogen-rich organic peat, and fertilizer use (McPherson
& Halley 1996).
Nutrient inputs from runoff vary in quantity, and composition is suspected to lead
to important implications for harmful algal bloom species (HABs) developments
(Anderson et al. 2002). Fertilizer production has recently shifted to an increased
6
proportion of urea, roughly 40% of all nitrogen fertilizers produced (Constant &
Sheldrick 1992), a form more easily assimilated by phytoplankton than nitrate or nitrite
(McCarthy 1980). This could potentially favor some HAB species (Anderson et al. 2002),
as nitrogen is typically the limiting nutrient in marine waters (Heckey & Kilham 1988).
Increasing the amount of nitrogen to the point where it is no longer limiting or where it is
in excess, would create a P-limiting environment. Nutrient limitation has been shown to
stimulate toxin production in other HAB species, but the role of nutrient limitation on
toxin production in K. brevis is unclear, particularly perplexing the role of anthropogenic
nutrient inputs. Supporting the idea that nutrient limitation may not affect toxin
production or bloom formation are historical records over the past 350 years where
blooms of K. brevis were cited to occur in the GoMex prior to significant human impact
on nutrient levels in the marine environment.
In many harmful algal species nutrients play a strong role in toxin production. Plimited nutrient stress was shown to stimulate toxin production in other harmful algal
species, such as Prorocentrum lima and Chrysochromulina polylepsis (Tomas & Baden
1993; Graneli et al. 1993). Other environmental factors, such as salinity impact toxin
production. Kim and Martin (1974) showed that concentration of toxin per cell in K.
brevis was highest at low and high salinities, rather than intermediate salinities. Maier
Brown et al. (2006) cite salinity and growth phase as significant factors affecting toxin
concentration, with the highest concentrations at a salinity of 20 then 40 then 30. Low
cell concentrations at a salinity of 20 could account for the high concentrations. While the
findings show that salinity affects toxin production, the role of salinity in toxin
production was unclear.
7
Brevetoxin levels were shown to be greatest during the stationary phase of
growth, leading to the conclusion that high density blooms may pose the biggest public
health threat (Maier Brown et al. 2006). In stationary phase, the growth rate is minimal
because cell growth relatively equals cell death allowing toxins to accumulate. Cells also
maintain large amounts of storage products, accumulate fats, oils and fatty acids in
stationary phase. This accumulation, as well as the possible ability of K. brevis to
structurally modify sterols rendering them non-nutritious to marine invertebrates, may
reduce predation and enhance survival (Giner et al. 2003).
Maier Brown et al. (2006) examined the toxin profile of the Mexico Beach clone
at salinities of 20, 30 and 40. The maximum concentrations during stationary phase were
from the lower salinity of 20, with a total of 22 pg-toxin cell-1. Baden and Tomas (1988)
found differences in the toxic fractions of brevetoxins between various clones, and that
the toxin profile is directly related to the potency of the clone analyzed.
Brevetoxins are fused-ring polyether compounds that most notably bind actively
to site 5 on VSSC’s (voltage-gated sodium channels; Poli et al. 1986). Brevetoxins are
characterized by two skeletal types with a number of congeners that result from variation
in side chains of the K-Ring (Type B) or the J-Ring (Type A) (Purkerson-Parker et al.
2000). The variation of side chains creates over thirteen different derivatives of
brevetoxins, each with their own degree of toxicity and their own binding affinity on
VSSC’s (Baden et al. 2005). Normally VSSC’s open only in response to membrane
depolarization then return to their closed configuration after membrane repolarization
(Taylor 1994); however all natural brevetoxins interact with receptor site 5 on VSSC’s
and display a dose-dependant depolarization of the membrane. The brevetoxin orients
8
itself across the sodium channel membrane parallel to the lipid bilayer and alpha helices,
with the K-ring or J-ring pointing outward (Purkerson-Parker et al. 2000). This binding
causes four distinct responses: 1) a shift in the activation potential for the sodium channel
to more negative values due to a conformational change, 2) inhibition of activation, 3)
increased mean channel open times by stabilizing the open conformation, 4) and an
induction of sub-conductance states (Purkserson-Parker et al. 2000).
The severity of adverse effects from brevetoxin exposure depends on the amount
of specific brevetoxin derivatives present as well as the health of the individual. Bloom
toxicity of K. brevis depends on the toxic fractions present, the strain, growth phase,
salinity, nutrients and temperature (Baden & Tomas 1988; Bourdelais et al. 2002). The
intensity of the toxic effects vary from bloom to bloom, allowing for small blooms with
low cell counts to be highly toxic, and large blooms with high cell counts to be only
slightly toxic (Bourdelais et al. 2002). Brevenal, an antagonist to brevetoxin, was
discovered in the early 2000’s (Bourdelais et al. 2004). Brevenals are thought to act on
site 1 of the sodium channel and brevetoxins on site 5 of the sodium channel. Even
though brevenal and brevetoxins act on different sites of the sodium channel, the effects
of brevenal take precedence over the competing brevetoxin (Purkerson-Parker et al.
2000). Brevenal does not inhibit brevetoxin binding; instead brevenal inhibits the effects
of brevetoxin after it binds to site 5 on the sodium channel. Brevenal counteracts the
effects of brevetoxin, and may cause variation in bioassays of bloom toxicity by
alleviating the harmful effects of brevetoxin (Bourdelais et al. 2002). Brevetoxin causes
DNA damage in lymphocytes, however application of brevenal beforehand protects DNA
9
(Sayer et al. 2005). Brevenal production varies within a single bloom depending on the
growth phase (Bourdelais et al. 2005).
The scope of this research explores the effects of environmental conditions such
as salinity, nutrients and temperature on the growth of K. brevis and the effects of varying
salinity and nutrients on toxin profiles of K. brevis. A multitude of physical, biological,
environmental and chemical factors interact to determine the time and location of bloom
development (Liu et al. 2001), and it is hypothesized that changing environmental
conditions such as differing salinities, nutrient ratios and temperatures may regulate K.
brevis’ ability to initiate a bloom, induce toxin production and elevate growth rate. In this
study, K. brevis was cultured in N:P balanced conditions (N:P = 16:1), P-limited
conditions (80:1), and N-limited conditions (1:1), for each salinity of 20, 25, 30, 35 and
39, and temperatures of 20, 25 and 30oC, in order to observe growth and toxin production
in environmental conditions realistic to the Gulf of Mexico.
Three clones of K. brevis examined in this study were the SP3 N-tox, SP3 S-tox
and Wilson. Three clones of K. brevis were selected examined because of a noted
variability in growth and toxin per cell concentration at various environmental conditions
for different clones (Baden & Tomas 1988).
Examining how the environmental factors of temperature, salinity and nutrients
relate to the growth and toxin production in the toxic marine dinoflagellate, K. brevis,
will help to understand conditions where some blooms are more toxic than others and
determine critical factors that influence toxicity. In terms of regulation, these experiments
will help to define conditions when and where blooms may be the most virulent. By
10
identifying toxic conditions these experiments will contribute to understanding factors
influencing bloom toxicity and hopefully help to prevent human exposure.
MATERIALS AND METHODS
Overview
The effects of temperature, salinity and nutrients on the growth and toxin profiles
of three strains of K. brevis were observed. The clones used for these studies were the
Wilson clone, originally obtained from Florida Marine Research Institute and two SP3
clones labeled SP3 S-tox and SP3 N-tox obtained from Dr. Ed Buskey, UTMSI Port
Aransas, Texas, both originally isolated in Dr. Tracy Villareal’s laboratory. The Wilson
clone was originally isolated from the coast of Florida in 1953 (Loret et al. 2002; Aldrich
& Wilson 1960; Shanely & Vargo 1993) and the SP3 clones were originally isolated from
the coast of Texas in 1999 (Loret et al. 2002; Magana & Villareal 2006). Test treatments
included salinities of 20, 25, 30, 35 and 39, and nitrogen:phosphorus (N:P) nutrient
conditions of balanced 16:1, P-limited 80:1, and N-limited 1:1, at temperatures of 20, 25
and 30oC. The first experiments for growth of the three clones tested salinity-nutrient
treatments at various temperatures, where growth phases and rates were defined. For
toxin experiments, the growth experiments at 20oC were repeated using larger volumes of
500 mL, from which samples for toxin analysis were taken. Samples were collected for
liquid-liquid organic extractions in preparation for liquid chromatography and tandem
mass spectrometry (LC-MS/MS). LC-MS/MS yielded toxin profiles of PbTx-1, 2, 3, 6, 9,
carboxylic acid (CBA) and brevenal for each treatment. From these analyses, toxin
concentration per cell was calculated.
11
Cultivation
Growth and toxin experiments used three clonal cultures of Karenia brevis grown
in a salinity of 33 in basal L1 media (-Si) (Gulliard & Havarges 1993) and V8 vitamins
(Provasoli et al. 1957), at 20oC on a 14:10 hour light:dark cycle with a light intensity of
66 µEm-2s-1. Seawater having a salinity of 39, used for this media was collected >27
miles off the North Carolina coast from the Gulf Stream. The seawater was filtered using
a 47 mm Whatman GF/F filter to remove plankton and debris, and specific salinities for
experiments were made by adding pyrogen free deionized water (18.3 megaohms at
25oC) to dilute filtered seawater to obtain desired salinities of 20, 25, 30 and 35. Salinities
were checked using a Fisher handheld refractometer. Fifteen hundred milliliters of
filtered seawater at the desired salinity was transferred to 2 L Teflon bottles and sterilized
for 20 minutes at 121oC and 15 lbs. pressure.
Media for growth studies consisted of salinities of 20, 25, 30, 35 and 39, while
media for toxin studies consisted of salinities of 25, 35 and 39. Three nutrient ratios were
made for each salinity; balanced, (N:P 16:1, 58 µM NO3- + 3.63 µM PO4-), P-limited
(N:P 80:1, 80 µM NO3- + 1 µM PO4-), and N-limited (N:P 16:16, 16 µM NO3- + 16 µM
PO4-).
Growth Study
Triplicate culture tubes (25x160 mm) with 30 mL of media of each nutrient
treatment were made. Excess media for each salinity was combined and 30 mL was
poured into a test tube labeled “Media Blank.” The media blank tube was placed with
experimental test tubes. Thirty milliliters of sterile media at a salinity of 33 was poured
12
into six additional test tubes labeled, “Fluorometer Test.” All test tubes were placed in an
incubator overnight at the appropriate temperature (20, 25 or 30oC) in order to
equilibrate. Once equilibrated, an inoculum of the parent culture in stationary phase was
added to each tube. To determine the desired inoculate concentration, several
“Fluorometer Test” tubes were inoculated with various amounts of parent culture and
measured on using a calibrated Turner Designs 10-AU Fluorometer to measure in vivo
chlorophyll flouresence. For this study, a relative fluorescence reading of ~0.500 allowed
for the best observations of growth for the SP3 N-tox and SP3 S-tox clones, while a
fluorometer reading of ~2.50 allowed for the best observations of growth for the Wilson
clone. Once the amount of inoculate was determined, each experimental media was
inoculated and initial fluorometric readings were taken of experimental treatments as was
the media blank (Day 0).
Growth, as indicated by an increase in relative in vivo fluorescence, was measured
at the same time daily until experiments reached stationary phase. Each day, tubes were
removed from the water bath, caps tightened, gently inverted to mix thoroughly, then
wiped with a Kimwipe prior to reading. After measuring fluorescence, caps were
loosened and tubes were placed back in the racks and racks returned to the water bath at
the appropriate experimental temperature (20, 25 or 30o C) (+/- 1o C), and a photoperiod
of 16:8 hour light:dark cycle and fluence flux of 66 µEm-2s-1.
To confirm the use of fluorescence as an indicator of growth, the relationship
between increased fluorescence and with cell number was established. A series of
dilutions made of the original culture in stationary phase and sterile media were measured
for fluorescence and 20 mL aliquots of each dilution were preserved with seven drops of
13
Lugols Iodine solution in scintillation vials. To calculate the number of cells per liter, cell
counts from preserved samples were performed using the Utermöhl method (Utermöhl
1931). A minimum of 300 cells were counted on a calibrated Nikon Diaphot inverted
microscope to ensure a 95% confidence interval with +/-11.5% accuracy (Guillard 1973).
Cell concentrations were determined using the equation: (Nt*fn*1*n-1) * 1000*mL-1.
Where Nt is the total number of cells counted, fn is the number of fields per chamber, n is
the number of fields counted, and mL is the number of mL used in the settling chamber.
Three counts were performed for each sample. Data of average cell number vs.
fluorescence was plotted, and a linear regression analysis yielded an equation for each
clone (Fig. 1).
Daily relative fluorescence data was collected for replicates of each experimental
treatment. From the fluorescence data growth curves were created by plotting the day of
growth on the x-axis versus the log2 (fluorescence + 1) on the y-axis. The value of one
was added to all average fluorescence values so that all log2 values were positive. Growth
curves were generated in the program SigmaPlot. From the growth curves, growth phases
(i.e. lag, log and stationary) were identified for each clone at each specific salinity,
nutrient and temperature treatment.
Growth rates as calculated from the steepest slope of the log growth phase using a
least squares regression equation with the Program LM in the statistical software R (R
Development Core Team 2008). The slope was defined by identifying start and end days
of log growth with the steepest slope having a duration of three days or more. The slope
of the least squares regression equation for each treatment was the growth rate as div day1
(k). To test whether or not there was a significant effect (p<0.05) in growth rates due to
14
clones and treatments, an Analysis of Variance (ANOVA) was performed. In the model
all one-way, two-way and three-way interactions were included and assessed for
significance. Assumptions such as normality of residuals and homogeneous variances
were verified through residual plots and normal probability plots. The ANOVA results
were analyzed for significant effects in growth rates at the 0.05 level. If an interaction
was not found to be significantly different (p>0.05), the data was collapsed to remove
that variable and increase the power of detecting other significant differences.
Toxin Study
For the toxin study, growth experiments were repeated in larger volumes and
samples were collected during stationary growth phases for 20oC. Four hundred
milliliters of experimental media was made for each treatment and transferred to a sterile
500 mL erlynmeyer flask under a laminar flow hood and allowed to equilibrate at the
appropriate temperature. The SP3 S-tox and SP3 N-tox clones were inoculated to yield a
fluorescence reading of ~0.500 in a test tube (25x160 mm) in the fluorometer, while the
Wilson clone was inoculated to yield a fluorescence reading of ~2.50. Once the amount
of inoculate was determined for 30 mL in the test tube the amount of parent culture to
inoculate into 400 mL was calculated. After inoculation an initial fluorescence reading
(Day 0) was taken by thoroughly swirling the erlynmeyer and collecting 4.7 mL of each
treatment under a laminar flow hood and dispensed to a labeled cuvette. Erlynmeyers
were then placed into a constant temperature water bath at the appropriate temperature in
the same conditions as described for the growth study. Daily fluorescence was measured
15
by pouring a subsample into a cuvette. Cuvettes were gently inverted, wiped with a
Kimwipe, fluorescence was measured and recorded.
When relative fluorescence readings indicated the treatment had reached
stationary phase, erlynmeyers were thoroughly swirled, 10 mL was collected in a
scintillation vial and preserved with Lugol’s Iodine solution for cell counts to determine
cell density, while 50-100 mL was collected for use in toxin extractions. Three cell
counts of at least 300 cells each were performed for each sample using a calibrated
inverted Nikon Diaphot microscope with the same calculation as described in the growth
study.
One hundred milliliters of each treatment was collected to perform a liquid-liquid
organic extraction in preparation for LC-MS/MS (Fig. 2). For a 100mL sample, thirty
milliliters of HPLC grade ethyl acetate (EtOAc) was added and sonicated with an
Autotune series high intensity ultrasonic processor, set at an amplitude of 50 and an
output of 12 for 90 seconds. The lysed culture was poured into a glass separatory funnel
with a Teflon stopcock. The beaker used for sonication was rinsed with EtOAc three
times and poured into the separatory funnel in order to collect any remaining toxin or
lysed cells that adhered to the glassware. The funnel was shaken vigorously, then placed
on a ring stand undisturbed until the water layer and the organic layer were completely
separated. After separation, the water layer was drawn off from the bottom and the ethyl
acetate layer was poured off from the top of the funnel.
The water layer was extracted again with ~50 mL of fresh EtOAc solvent. The
water layer was drawn off from the bottom and the EtOAc layers were combined. DIW
was added to the combined EtOAc layers and extracted. DIW was drawn from the bottom
16
of the funnel while the ethyl acetate layer was poured out from the top of the funnel into a
glass round bottom flask. The funnel was rinsed with EtOAc, residual water was drained
from the bottom and the EtOAc layer was poured off from the top into the round bottom
flask.
The round bottom flask was attached to a bump trap on a Heidolph Laboratory
4000 efficient rotary evaporator system (Germany) to evaporate the solvent and leave
toxins in the round bottom flask. The water bath used in this system was set to 37oC to
keep the solvent from cooling or freezing during the evaporation process. The rotation
ranged from 70 to 170 rpm. Vacuum pressure was manually controlled by covering the
vacuum tube and allowing the system to vent if the sample neared a boiling point, this
helped to prevent loss of sample into the bump trap. Vapor from the extraction process
was routed through a Tygon rubber hose and released into a chemical hood. Antifreeze
running through the condenser was set to -10oC in order to condense the evaporated
solvent.
After evaporation of the solvent, 5 mL of certified acetone (Ac) was added to the
extract to precipitate any waxes, fats, lipids or proteins and placed in a -4oC freezer for
thirty minutes. A glass microanalysis filtration unit was assembled with a magna nylon
plain 0.22 um filter and the sample was filtered. The round bottom flask was rinsed a
twice more with 2-4 mL of acetone and filtered, then the filtration chimney was rinsed
with acetone to obtain the entire sample. The filtrate was poured into a 20 mL glass
scintillation vial and acetone was evaporated in the rotary evaporator system as described
above.
17
The dried sample was brought up in 1 mL of mobile phase (98% ACN, 2% H2O
and 0.1% Formic Acid), and the entire sample was transferred to a 1.8 mL amberglass
vial. The vial was placed in a clean scintillation vial and dried down using the rotary
evaporator system. The dried sample was then brought up in mobile phase. Depending on
the cell density of the original sample, a calculated portion of the sample was transferred
to a 300 uL polyspring insert inside a 1.8 mL amberglass vial with a PTFE/rubber seal
polypropylene cap in preparation for LC-MS/MS.
LC-MS/MS analyses were performed in positive ion mode, using a Q TRAP 2000
Tandem Mass Spectrometer (ABI MDS/SCIEX, Ontario, Canada) in conjunction an
Agilent Technologies 1100 LC Binary Pump liquid chromatography system using a
Varian HPLC Column (Microsorb 100-3 C18, s100x2 COL). Samples were run using an
injection volume of 4µL on an elution gradient of 40% stationary phase (98% H2O, 2%
ACN) and 60% mobile phase (98% ACN, 2% H2O, 0.1% FA) for 0.1 to 5 minutes at a
flow rate of 110 µL minute-1, 1% stationary phase and 99% mobile phase for 5.1-12
minutes at a flow rate of 140 µL minute-1, and 40% stationary phase 60% mobile phase
for 12.1-20 minutes at a flow rate of 110 µL minute-1. Settings for the mass spectrometer
included a turbo spray ion source, ion spray voltage of 5kV, an interface heater
temperature of 300oC, with a curtain gas of nitrogen.
Samples were run against known concentrations of five brevetoxin standards,
PbTx-1, 2, 3, 6 and 9, brevenal and carboxylic acid (CBA) (Table 1). CBA was run as a
standard, because the mass of PbTx-6 and CBA are equal. By running both standards we
demonstrated that the concentration of PbTx-6 and CBA can be individually
distinguished in samples. Samples were examined for masses of PbTx-1, 2, 3, 6, 9, CBA
18
and brevenal, as well as for the masses of common derivatives of these compounds which
included PbTx-1, 2, 3, 6, 9, brevenal and CBA with the loss of H2O (Table 1). Total
concentration for each compound equaled the sum of the concentration of the compound
in addition to the concentration of the compound with the loss of H2O. The concentration
of each compound was calculated with Bioanalyst Software for use with ABI
MDS/SCIEX for the 2000 Q TRAP, using a manually optimized protocol for the
brevetoxin compounds in question. Standard concentrations for each brevetoxin were
calibrated in a linear regression through zero with a 1/x weighting (as determined from
residual plots) where all R2 values were >0.99. Calibrations of each brevetoxin were used
to determine the pg-brevetoxin µL-1 in each sample. Concentration of pg-toxin cell-1 was
found by dividing the concentration in pg-toxin µL-1 calculated from BioAnalyst by the
calculated cells µL-1 from preserved samples.
Brevenal, total PbTx-2+3+9 and total PbTx-1+2+3+9 were compared between
clones, nutrient ratios and salinities to determine how each environmental condition
affected toxin profiles and total brevetoxin produced. Total PbTx-2+3+9 was chosen
because these brevetoxins are a measure of the synthesis of the Type B backbone (Kring). Total PbTx-1+2+3+9 was also chosen to better measure total brevetoxin
concentration for potential biological exposure. An ANOVA was run to determine
significant (p<0.05) effects on toxin production of total PbTx-1+2+3+9, PbTx-2+3+9 and
brevenal in the three clones at 20oC, salinities of 25, 35 and 39 and nutrient conditions of
balanced, P-limited and N-limited. A Tukey’s pair wise comparison between variables
was run to more closely examine where toxin production was significantly affected by
the clone and/or environmental factors. MANOVAs of PbTx-1, 2, 3, 9 and PbTx-2, 3, 9
19
from combined effects of salinity-clone, clone-nutrient and nutrient-salinity, were run to
determine if the environmental variables studied significantly affected total toxin
production.
Toxin profile within cells versus that in external media was tested. Spectra/Por®
7 Dialysis Membrane tubing with a molecular weight cut off of 3500 (daltons)
conditioned in wet 0.1% sodium azide soluation was rinsed with Wilson’s NH-15
synthetic seawater media (Gates & Wilson 1960). Four dialysis membranes were filled
with 50 mL of NH-15 media, securely closed off, and suspended in a four week old 8 L
culture of the Wilson clone of K. brevis grown in NH-15 media. After hours 0, 6, 12, 24
and 48, a dialysis tube was harvested and media content was extracted in a liquid-liquid
organic extractions as described in Figure 2. A media blank of NH-15 was also extracted
using the procedure from Figure 2. An extraction of 50 mL of the whole culture was also
performed. To describe the brevetoxin and brevenal profiles in versus out of the cell,
extracts were analyzed for PbTx-1, 2, 3, 6, 9, brevenal and CBA on LC-MS/MS as
described above.
RESULTS
Growth Patterns
Salinity and temperature appeared to have a strong influence on the growth
patterns observed in the three clones of K. brevis (SP3 N-tox, SP3 S-tox and Wilson).
Nutrient regimes also affected growth in mid to late growth phases in some treatments.
Four main patterns of growth were observed in culture treatments. One pattern (type 1)
observed was a decrease in cells after inoculation on day 0 (Fig. 3). Cells rapidly declined
20
in some cultures after inoculation on day 0 (Fig. 3 B, D, E), while others showed some
initial survival followed by a precipitous decline (Fig. 3 A, F), or an intermediate
response observed (Fig. 3 C). The second pattern (type 2) had a measurable lag period
after inoculation with growth commencing after a short period of time and continuous
growth until the end of the experimental period (Fig. 4). In some cultures the lag phase
was only 1-2 days (Fig. 4 E, F), while in others this period lasted 7-8 days (Fig. 4 A, B,
D), and the Wilson clone displayed an intermediate lag response of 3-4 days followed by
a brief period of growth (Fig. 4 C). A third pattern (type 3) reflected a steady increase in
growth immediately after inoculation with continued strong growth into log phase with a
decline in some of the nutrient stressed cultures (Fig. 5 A-F). A fourth pattern (type 4)
displayed growth followed by a stationary phase with and immediate decline in cell
number, most notably in N-limited nutrient treatments, after log growth (Fig. 6). Some
cultures displayed a typical stationary phase (Fig. 6 A-C), others showed a leveling off
for a few days followed by a slow decline (Fig. 6 F), still some others displayed an
immediate, rapid decline in cell number after log growth (Fig. 6 D, E). Representative
growth patterns of each growth type were combined into Figures 3-6. All growth patterns
were displayed in Appendix A in Appendix Figures 1-15.
The type 1 growth pattern was observed in all treatments at a salinity of 20 for all
clones. In addition, all clones at 30oC in salinities of 25 and 30 displayed this decline in
cell densities. The Wilson clone in salinities of 35 and 39 at a temperature of 30oC also
showed this pattern.
The type 2 growth pattern displaying a measurable lag phase was observed at the
lower temperature of 20oC in the Wilson clone at all salinities. This pattern was not
21
observed for any clone at 25oC. At 30oC, only the SP3 N-tox and SP3 S-tox clones
displayed a lag phase in salinities of 35 and 39.
The type 3 growth pattern of an immediate increase in cell density without a lag
phase was observed in the SP3 N-tox and SP3 S-tox clones at 20oC. At 20oC the SP3 Ntox clone displayed an immediate log growth in salinities of 25-39, while the SP3 S-tox
clone showed this pattern in salinities of 30-39. At the intermediate temperature of 25oC
the SP3 clones displayed this growth patterns in salinities of 30-39. At 25oC, the Wilson
clone showed a steady increase in growth at all salinities. None of the clones showed this
pattern at 30oC.
Type 4 patterns were observed mainly in the N-limited nutrient treatments. At
20oC in a salinity of 30, the SP3 N-tox and Wilson clone showed a leveling off to
stationary phase in N-limited treatments, and at salinities of 35 and 39 all clones
displayed this trend. A drastic decline in cell density after log growth was observed for
the SP3 N-tox clone at 25oC in a salinity of 25 for all nutrient treatments. A drastic
decline in cell density was also observed in N-limited nutrient treatments of the SP3
clones at a temperature of 25oC in salinities of 30-39 in. A gradual decline in cell density
occurred in N-limited nutrient treatments immediately after log growth in the SP3 S-tox
clone in salinities of 25 and 30 at 20oC and in a salinity of 35 at 30oC. In N-limited
nutrient treatments of the Wilson clone grown at 25oC in salinities of 25-39 also showed
a gradual decline in cell density after log phase growth.
22
Growth Rates
Growth rates from 0.05 to 0.29 div. day-1 were measured (Table 2). The highest
growth rates, >0.25 div. day-1, were found exclusively in the SP3 clones. The Wilson
clone had particularly low growth rates, all <0.18 div. day-1, for all treatments. The
highest growth rate of all treatments and clones was 0.29 div. day-1, for the SP3 N-tox
clone in N-limited conditions at 25oC in a salinity of 30 (Table 2). This high growth rate
was followed by a rate of 0.28 div. day-1, observed in both the SP3 N-tox clone in
balanced conditions at 25oC in a salinity of 35 and in the SP3 S-tox clone in P-limited
conditions in a salinity of 39 at 25oC (Table 2). The next highest growth rates all occurred
in the SP3 S-tox clone in a salinity of 39 at 25oC in N-limited and balanced conditions
with 0.27 and 0.25 div. day-1, respectively, and in the SP3 N-tox clone in P-limited and
balanced conditions in a salinity of 30 at 25oC with 0.26 and 0.25 div. day-1, respectively
(Table 2).
The lowest growth rates were generally found in the lower salinity treatments for
each clone. The lowest growth rates occurred in a salinity of 25 at 25oC in the SP3 S-tox
clone with 0.07, 0.06 and 0.05 div. day-1 in balanced, P-limited and N-limited nutrients
treatments respectively and in the Wilson clone in a salinity of 25 at 20oC with 0.07, 0.05
and 0.05 div. day-1 in balanced, P-limited and N-limited treatments respectively (Table
2). The exception to observations of decreased growth rates in lower salinities occurs in
high temperature conditions of 30oC for the SP3 N-tox clone where the growth rate is
slightly compressed relative to high salinity treatments at 20 and 25oC (Table 2).
An ANOVA was run to test significant difference in growth rates based upon
clone, temperature, salinity and nutrients (Table 3). In the ANOVA the only significant 3-
23
way interaction was the combination of the variables salinity-temperature-clone (p<0.05);
while all 3-way interactions with the variable of nutrient were not significantly different.
All 2-way interactions of the variables that included nutrients were not significant, while
interactions between salinity, clone and temperature were found to be significant. The
main effect of nutrient was significant with a p-value of 0.04 (Table 3). Due to highly
significant interactions among salinity clone and temperature, slicing was used to further
investigate significance among these variables.
Tables 4-6 display results from slicing the data. The salinity studied significantly
affected growth rates for all clones in salinities of 25 and 30 at temperatures of 20 and
25oC (Table 4). At 30oC, no significant difference was found in growth rates between the
two salinities of 35 and 39 for the SP3 clones. ANOVA results examining the effects of
the temperature studied on growth rates showed that growth rates of the SP3 N-tox clone
were significantly affected by temperatures of 20 and 25oC in salinities of 25 and 30, and
by temperatures of 20, 25 and 30oC in salinities of 35 and 39 (Table 5). Growth rates of
the SP3 S-tox clone were significantly affected by temperature in a salinity of 25 at
temperatures of 20 and 25oC, and in a salinity of 39 at temperatures of 20, 25 and 30oC
(Table 5). The Wilson clone growth rates in salinities of 25 and 35 at temperatures of 20
and 25oC were significantly affected by temperature (Table 5). The SP3 S-tox clone’s
growth rates were not affected by temperatures of 20 or 25oC in a salinity of 30 or by
temperatures of 20, 25 or 30oC in a salinity of 35. The Wilson clone’s growth rates were
not affected by temperatures of 20 or 25oC in salinities of 30 and 39 (Table 5). ANOVA
results examining growth rates between all three clones indicated that the clone examined
24
significantly influenced the growth rate in all salinity and temperature treatments of the
study (Table 6).
Toxin Analyses
For the toxin study, whole culture samples were extracted for toxin profile
analysis and quantification on a cellular basis. The use of whole cell culture samples for
cellular toxin content was shown to have a strong representation of actual toxin content
for PbTx-1, 2 and brevenal. (Fig. 7). These results show that the concentration of PbTx-2
in whole cell cultures was very high relative to other PbTx’s by 5-10 fold, and that 99.5%
of PbTx-2 from whole culture extracts was located inside the cell (Fig. 7). PbTx-1 and
brevenal from whole culture extractions accounts for 100% of all PbTx-1 and brevenal,
as none was found in media extractions. However, only 19% of PbTx-3 and 0.4% of
PbTx-6 found in whole culture extractions was from inside of the cell (Fig. 7). PbTx-9
and CBA were not detected in the whole culture sample or any of the media samples. The
results suggest that whole culture extractions for toxin profiles and cellular toxin
quantification are representative of toxin content inside K. brevis cells for PbTx-1, 2 and
brevenal, whereas PbTx-3 and 6 are mostly located outside of the cell.
Treatments of each clone at 20oC were harvested for brevetoxin extraction in
stationary phase for salinities of 25, 35 and 39 and nutrient regimes of balanced 16:1 N:P,
P-limited 80:1 and N-limited 1:1. Cell density of each treatment (Table 7) was used to
determine brevetoxin cell-1 and brevenal cell-1 for LC-MS/MS results (Table 8).
Cell density was highest in balanced nutrient treatments for all clones at all
salinities with the exception of the Wilson clone at a salinity of 39. The highest cell
25
density for N-tox was 2.10 x 104 cells mL-1 occurring at a salinity of 35 in balanced
nutrient conditions. The highest cell density for all clones and treatments occurred in the
S-tox clone at a salinity of 39 in balanced nutrient conditions with 3.85 x 104 cells mL-1.
The highest cell density for the Wilson clone occurred under P-limited nutrient conditions
at a salinity of 39 with 3.10 x 104 cells mL-1.
N-limited treatments yielded the lowest cell density in stationary phase for all
clones at all salinities, with the exception of the Wilson clone at a salinity of 35. The
salinity yielding the highest cell density for balanced nutrient treatments was 35 for N-tox
and Wilson clones, and 39 for S-tox clones. The lowest cell density of all clones was
observed at a salinity of 25. Under balanced, P-limited and N-limited conditions the S-tox
clone exhibited a low range in density from 6.27 to 6.53 x 103 cells mL-1, with the lowest
density occurring in N-limited conditions. The Wilson clone showed low densities in Nlimited and P-limited conditions with 6.31 and 8.87 x 103 cells mL-1 respectively, with a
greater than two-fold higher density in balanced conditions. The N-tox clone exhibited
low cell density in N-limited conditions at a salinity of 25, with 6.52 x 103 cells mL-1.
However, densities of balanced and P-limited treatments at this salinity were higher at
1.42 and 1.12 x 104 cells mL-1 respectively. The N-tox clone exhibited low density
outside of the salinity of 25 in the N-limited treatment at a salinity of 39 with 7.54 x 103
cells mL-1.
The greatest combined toxin production on a per cell basis for both total PbTx2+3+9 and for total PbTx-1+2+3+9 was observed in the Wilson clone at a salinity of 25
in N-limited conditions with 36740 and 36890 pg-toxin cellsx10-3 respectively; the
second highest toxin production was observed in the N-tox clone at a salinity of 39 in N-
26
limited nutrient conditions with 32140 and 36110 pg-toxin cellsx10-3 in PbTx-2+3+9 and
PbTx-1+2+3+9 respectively (Table 8). The greatest toxin production for each clone based
on nutrient regime for PbTx-1+2+3+9 relative to balanced treatments for each clone
generally occurred in N-limited conditions for the SP3 N-tox clone, P-limited conditions
for the SP3 S-tox clone and N-limited conditions for the Wilson clone (Fig. 8 A; Table
8). Greatest toxin elevation relative to balanced nutrient treatments occurred for Nlimited treatments at a salinity of 25 for all clones, P-limited conditions at a salinity of 35
for all clones and N-limited conditions at a salinity of 39 for the SP3 S-tox and Wilson
clones, while P-limited conditions for the SP3 N-tox clone at a salinity of 39 yielded the
greatest relative toxin production (Fig. 8 A; Table 8).
The least total toxin production occurred in the S-tox clone at a salinity of 25 in Plimited conditions with 5620 pg-toxin cellsx10-3 for total PbTx-2+3+9, and 5920 pg-toxin
cellsx10-3 for total PbTx-1+2+3+9. In the majority of treatments, the lowest toxin
production for PbTx-1+2+3+9 based on nutrient treatments occurred in balanced
conditions. A few exceptions occur in the SP3 clones at a salinity of 25 and the Wilson
clone at a salinity of 39 where P-limited conditions showed the lowest toxin
concentration (Fig. 8 A) Another exception occurs in the SP3 S-tox clone at a salinity of
39 where the N-limited treatment was found to have the lowest toxin concentration (Fig.
8A).
Analyses of individual PbTx-1, 2, 3, 6, 9, brevenal, total PbTx-2+3+9 and total
PbTx-1+2+3+9 showed that clone type was significant in brevetoxin production with the
exception of PbTx-3 (Table 9). Salinity significantly influenced toxin and brevenal
production except for PbTx-3 and PbTx-1 (Table 9). Nutrients did not significantly
27
influence toxin production in any PbTx’s, brevenal or total PbTx-2+3+9 or PbTx1+2+3+9 (Table 9).
A Tukey’s pair wise comparison of total PbTx-2+3+9 and PbTx-1+2+3+9 showed
significant interactions in the examination of clone-salinity, clone-nutrient and salinitynutrient combinations (Table 10). For total PbTx-2+3+9 and PbTx-1+2+3+9, the SP3 Ntox clone in all salinities of 25, 35 and 39 differed in toxin production from the SP3 S-tox
clone at a salinity of 25. Toxin production was also significantly different for the SP3 Stox clone at a salinity of 25 from that of the SP3 S-tox clone at a salinity of 35 and the
Wilson clone from the Wilson clone at a salinity of 25 (Table 10). For total PbTx-2+3+9
the SP3 S-tox clone in a salinity of 25 also significantly differed from the Wilson clone at
a salinity of 35 and 39 for clone-salinity comparison. For total PbTx-1+2+3+9, toxin
production for the SP3 S-tox clone at a salinity of 39 significantly differed from the SP3
N-tox clone at a salinity of 39 (Table 10).
The clone-nutrient comparison for total PbTx-2+3+9 and PbTx-1+2+3+9 showed
that both had significant differences in toxin production between SP3 S-tox balanced
nutrient conditions and SP3 N-tox N-limited and P-limited nutrients conditions, in
additions SP3 S-tox N-limited conditions were found to be significantly different from
SP3 N-tox N-limited and P-limited conditions (Table 10). In PbTx-2+3+9 significant
differences were also found between clone-nutrient comparisons for the SP3 S-tox clone
in N-limited conditions and the SP3 N-tox clone in balanced nutrient conditions as well
as the Wilson clone in N-limited and P-limited conditions and the SP3 S-tox clone in
balanced and N-limited conditions (Table 10).
28
For clone-salinity comparisons of total PbTx-2+3+9 and PbTx-1+2+3+9 P-limited
conditions in a salinity of 35 significantly differed from P-limited conditions in a salinity
of 25, and a salinity of 39 with P-limited nutrient conditions varied significantly for a
salinity of 25 with balanced nutrient conditions (Table 10). For total PbTx-2+3+9 a
salinity of 35 with P-limited conditions significantly differed from a salinity of 25 with
balanced conditions. And for total PbTx-1+2+3+9 a salinity of 39 with P-limited
conditions significantly differed from a salinity of 25 with P-limited conditions (Table
10).
MANOVA results comparing production of total PbTx-2+3+9 and total PbTx1+2+3+9 under the effects of clone, salinity and nutrients also showed that clone and
salinity, but not nutrients, were significant in altering the toxin production (Table 11). It
should be noted that nutrients altered toxin production in some manner relative to toxin
production in balanced conditions (Fig. 8).
Examination of brevenal production showed that the Wilson clone produced the
greatest concentration of brevenal of all clones in all nutrient-salinity conditions (Table
8). The greatest brevenal concentration occurred in the Wilson clone under N-limited
conditions in salinities of 25 and 39 with 5270 and 4060 pg cellsx10-3, respectively.
Results of the brevenal ANOVA (Table 9) show that a significant difference in
brevenal production occurs between clones, however, not between salinity or nutrient
treatments. However, examination of brevenal production in the Wilson clone based on
nutrients relative to balanced treatments showed elevated production in N-limited
conditions at salinities of 25, 35 and 39 (Fig. 8 B). For the SP3 clones at a salinity of 25,
P-limited and N-limited conditions appear to decrease toxin production, however at
29
salinities of 35 and 39 brevenal production in the SP3 N-tox clone appears to be
stimulated by N-limited conditions while the SP3 S-tox clone appears to be stimulated by
P-limited conditions (Fig. 8 B). While the ANOVA (Table 9), did not show a significant
difference in brevenal production between nutrient treatments, it appears that nutrients
affect brevenal production in some way relative to balanced conditions and suggest
further investigation (Fig. 8 B).
A Tukey’s pair wise comparison of brevenal productions between clone-salinity,
clone-nutrient and salinity-nutrient shows that significant difference occurred between
clone-salinity and clone-nutrient conditions for all treatments of the Wilson clone
differed from all conditions of the SP3 clones, while brevenal production between
salinity-nutrient conditions was not significant for any clone at any condition (Table 11).
DISCUSSION
The experimental results of the three Karenia brevis clones (SP3 N-tox, SP3 Stox and Wilson) demonstrated variable growth responses to changing temperature,
salinity and nutrient ratios. Aldrich and Wilson (1960) reported an optimal salinity range
for growth of K. brevis to be 27-37, with no growth below salinities of 24 or above 44.
However, salinity decreases in the field with mixing water masses may be more gradual
and permit successful acclimation to lower salinities (Aldrich & Wilson 1960). Salinities
of 37-39 are common in the GoMex, however near river outflow along the coast salinity
may be lower. The ability of K. brevis to acclimate to lower salinities was studied by
Maier Brown et al. (2006) after a bloom of K. brevis occurred in the lower salinity waters
of the Northern GoMex. Maier Brown et al. (2006) found the lowest salinities where
30
growth occurred ranged from 17.5 to 22.5 and varied by the clone of K. brevis studied. In
this study, all three clones exhibited a precipitous decline in cell number in low salinities
of 20 at all temperatures, suggesting this lower salinity is suboptimal for growth of this
species. This study agrees with previous findings in treatments of a salinity of 20 that
were not gradually acclimated.
Magana & Villareal (2006) showed that adaptation by K. brevis to an
environmental stressor, such as salinity, occurs best under otherwise optimal conditions.
The Wilson clone was unable to grow at a temperature of 30oC in any of the salinities
from 20-39. The SP3 N-tox and S-tox clones were unable to grow at 30oC in salinity of
20, 25 or 30, but were able to grow at salinities of 35 and 39 at this temperature.
Compared to the SP3 clone, the less favorable tolerance displayed by the Wilson clone at
30oC in salinities of 35 and 39 suggests a lower temperature tolerance of the Wilson
clone. By including an additional stressor of high temperature at 30oC in this study, the
Wilson clone responded differently in growth than the two SP3 clones at the same
salinity suggesting a decreased ability to adapt to higher salinities. A temperature of 30oC
is rare in open waters of the GoMex, however, temperatures of 30oC may occur in
shallow water areas near the coast. This study confirms the differences in growth between
clones inferring that in a given salinity-temperature environment a bloom of K. brevis
may or may not occur depending on the clone type present.
Growth rates of clones in identical conditions showed that salinity, temperature
and clone significantly affected the growth rate. For example, the SP3 N-tox clone had
significantly higher growth rates at salinities of 35 and 39 than those at a lower salinity of
25 (Table 2). Maier Brown et al. (2006) found that K. brevis displayed better growth at
31
higher salinities, supporting the idea that K. brevis is more likely to bloom at high
salinities.
Comparison of growth rates between clones revealed slightly elevated growth
rates for the SP3 S-tox and Wilson clones at 25oC, whereas the SP3 N-tox clone had
elevated rates at 20 and 25oC. Significant differences in growth rates between all clones
studied were found, with the SP3 N-tox clone having the highest overall growth rates,
followed by the SP3 S-tox then the Wilson. Loret et al. (2002) also found growth of five
clones of K. brevis from Texas and Florida differed significantly from one another when
grown under identical conditions. Within the Texas clones SP1, SP2 and SP3, Loret et al.
(2002) found ~2-fold range in growth rates.
The ANOVA analyses of growth rates (Table 3) showed nutrients significantly
influenced growth rate (p-value = 0.0369), but factors such as the clone type, temperature
and salinity (p-value = 0.0001) have a stronger influence on growth. N-limitation strongly
affects growth patterns in stationary phase where cell density greatly decreased (Fig. 6).
Revealing effects of nutrient limitation on growth rates may assist in understanding
effects of nutrient limitation on toxin production in K. brevis.
In the harmful algal bloom species, Prorocentrum lima, a correlation was found
between decreased growth rate and increased toxin production (Sohet et al. 1995). This
trend was found in this study where toxin production of the Wilson clone was high with
23.67 and 36.74 pg-toxin cell-1 in treatments of 20oC at a salinity of 25 where growth rate
of the P- and N-limited treatments were both low with 0.05 div day-1 (Table 2), and cell
density was low (Table 7).
32
Brevetoxin production varied according to clone and salinity in some cases.
Overall, the Wilson clone produced the most PbTx-2, followed by the SP3 N-tox clone,
and the SP3 S-tox clone produced the least. Brevenal production also varied by the clone
studied. The Wilson clone produced a significantly greater amount of brevenal than either
SP3 clone in any condition studied. Salinity effects on brevenal production showed that
the Wilson clone produced a greater amount of brevenal at lower salinities, and the SP3
clones produced a greater amount at higher salinities. Clonal variability in toxin content
of K. brevis was previously observed (Baden & Tomas 1988). Loret et al. (2002) also
studied the toxin content of five clones of K. brevis and found significant variation
between clones. In this study, individual and total toxin production between clones differs
significantly (Tables 9-10), supporting previous findings.
ANOVA results of brevetoxin production showed significant effects of salinity on
toxin production for PbTx-1 and 3 only. However, the SP3 clones contained a relatively
higher toxin content at higher salinities, while the Wilson clone contained higher toxin
content at lower salinities. Maier Brown et al (2006) also concluded that the role of
salinity in toxin production, if any, is unclear.
Brevenal production varied significantly by the clone studied. The Wilson clone
produced a significantly greater amount of brevenal than either SP3 clone in any
condition studied. Salinity affects on brevenal production, although not significant,
showed that the Wilson clone produced a greater amount of brevenal at lower salinities,
while the SP3 clones produced a greater amount at higher salinities. These trends
similarly reflect those of toxin production in the clones.
33
Brevenal production by the Wilson clone was significantly higher than production
in the SP3 N-tox or S-tox clones. The mean values of brevenal produced by the SP3 Ntox clone in response to salinity, independent of nutrients, varied from 310 to 880 pg
cellsx10-3. Mean values for the SP3 S-tox clone for the same variables, varied from 0.22
to 0.82 pg cell-1. Under these same conditions, the mean values for the Wilson clone
varied from 2990 to 3540 pg cellsx10-3.
Understanding factors leading to the increased production of brevenal are of
concern as it is postulated the effects of airborne aerosols on human populations on or
near the beach could be alleviated in the presence of a greater amount of brevenal in the
aerosol. This study shows clone type present in a bloom could greatly influence the
toxicity effects on human, marine mammal and fish populations in the vicinity. Further
study of the clonal and environmental effects on brevenal production may shed light as to
why some blooms are less toxic than others.
Examining toxin concentration per cell for nutrient and clone without the effects
of salinity showed an increase in toxin for the SP3 N-tox in N-limited conditions, while
P-limited conditions yielded higher toxins in the Wilson clone (Fig. 8). In the SP3 S-tox
clone, toxin concentration decreased in N-limited treatments, but greatly increased from
in P-limited treatments. The effects of nutrient limitation on toxin production are variable
and there is no clear pattern as to how nutrient limitation affects toxin production.
Analyzing the amount of toxin per cell present in stationary phase provides a way
to compare toxin on a per cell basis between clones, however it does not describe the
absolute toxin production potential of a bloom. P-limitation appears to allow blooms to
persist longer than N-limited conditions and may therefore pose a greater threat in the
34
long term as they can persist longer. The nutrient ratios studied did not influence growth
until the cultures reached stationary phase, where the effects of N-limitation terminated
the culture bloom more quickly than P-limited conditions or balanced conditions (Fig. 6).
It is hypothesized that if a bloom is formed under N-limited conditions in the
environment, the bloom may crash more quickly than a bloom in balanced or P-limited
conditions. To better understand the dynamics of N- and P-limitation in K. brevis nutrient
uptake experiments are needed to determine intake of N or P in an endeavor to
understand absolute N or P-limitation and the subsequent effects on growth and toxin
production.
The interactions of iron availability may also play a role in nitrogen limitation in
the GoMex. Deposition of iron rich Saharan dust in the GoMex was suggested to allow
the diazotrophic cyanophyte, Trichdesmium, to bloom (Lenes et al. 2001). Iron trace
metal enrichment that occurs in the GoMex may act synergistically with nitrogen to
stimulate coastal production (Paerl 1997), Trichodesmium fixes N2 into DON and NO3-,
forms more easily assimilated by HABs (Gilbert & Bronk 1994). It is hypothesized that
Trichodesmium supports offshore blooms of K. brevis in the GoMex as K. brevis blooms
often follow blooms of Trichodesmium (Walsh & Stedinger 2001; Lenes et al. 2001;
Mulholland et al. 2006). Management/regulation of offshore nitrogen enrichment by
Trichodesmium may not be possible; however regulations of inshore nutrient supply are
practical and may be the only feasible management application to bloom control for
inshore waters (Paerl 1997).
In Alexandrium spp. P-limitation leads to a greater cellular toxin concentration
compared to N-limitation (Graneli et al. 1998). Toxins produced by Alexandrium have a
35
high nitrogen content (Graneli et al. 1998), possibly explaining why N-limitation does not
lead to greater toxin concentration. Prorocentrum lima was also reported to increase
toxin production in P-limited conditions (Tomas & Baden 1993). The prymnesiophytes,
Chrysochromulina polylepsis and Prymnesium parvum, have a higher toxin concentration
with nutrient limitation, regardless of whether it is N- or P-limitation (Graneli et al.
1998). For Dinophysis acuminata, okadaic acid cellular concentration is increased in both
N- and P-limited conditions (Graneli et al. 1998). Brevetoxins produced by K. brevis are
not known to contain nitrogen or phosphorus. To understand how N- or P-limitation
affects toxin production in K. brevis, the biosynthetic pathways used by the cell to
produce brevetoxin must be understood. Any possible involvement of nitrogen containing
enzymes used in toxin production or high energy pathways producing brevetoxin
requiring phosphorus may shed light on as to why N or P-limitation may or may not
affect toxin production in this organism.
To determine if the responses observed in this study truly reflect N- or P-limited
conditions in cells, the physiological characteristics of the cell need to be studied, where
enzymes may be used as physiological markers. For example, increased alkaline
phosphatase activity may be indicative of P-limited environments. And for N-limitation,
nitrogen stress may be indicated by an increase in nitrogen reductase, indicating cells are
using nitrate, and not the preferred ammonia.
Other environmental conditions affecting growth and toxicity in previous studies
examined the effects of temperature on growth and bloom formation in K. brevis, but
only a few have focused on the relationship between temperature and toxin production
(Vargo et al. 2001). Generally K. brevis can tolerate temperatures in the field and in the
36
lab from 15-30oC (Kusek et al. 1999). However, blooms of K. brevis do not thrive well at
temperatures less than 19oC (Lamberto et al. 2002), and at 15oC, maximum growth rates
for the SP3 clone were 50% less than at higher, optimal temperatures (Magana &
Villareal 2006). It can be expected that changes in conditions may also have an effect on
toxin production (Lamberto et al. 2002). Lamberto et al. (2002), found that low
temperature stress of 15oC led to a decrease in cell number but an increase in production
of toxin per cell in comparison to the control at 24oC. At 15oC slow cell division reflects
a lower cell density over time, where toxin may accumulate in cells.
All clones used are various isolates of K. brevis (e.g. the same species), however
they behaved drastically different under identical conditions. Loret et al. (2002) reported
an approximate 3-fold difference in toxin production between the SP1 and SP3 clones
under identical conditions. Toxin production is assumed to be a biological process
intrinsic to some toxin producing dinoflagellates such as Alexandrium (Anderson 1990),
with a genetic basis encoded by the algal genome (Ishida et al. 1993). Therefore, the
physiology and variability observed in the field and experimental data suggests that
isolates of K. brevis may remain genetically distinct. Future work is needed to determine
the genetic similarities or differences among K. brevis clones. Work with 18s rDNA did
not yield a marker for intraspecific probes, and the ITS region seems to be unsuitable for
this endeavor as well (Loret et al. 2002). Possible work with AFLP fingerprinting may
yield a way to identify intraspecific variation in K. brevis.
Whole culture extractions were used in this study to calculate pg-toxin and pgbrevenal cell-1. These extractions accurately reflect concentrations of PbTx-1, 2 and
brevenal inside the cell. Whole culture extractions account for 99.95% of the PbTx-2
37
inside the cell and provide an accurate comparison of total PbTx-2 production.
Concentrations of brevenal and PbTx-1 were found entirely in whole culture extractions,
suggesting that brevenal and PbTx-1 are contained inside the cell. Brevenal may be
converted to brevenol outside of the cell and future analyses should include brevenol in
LC-MS/MS identification techniques (Dr. Andrea Bourdelais, personal communication).
Concentrations of PbTx-3 and 6 were most likely all outside of the cell, and the minute
differences between amounts in dialysis tubing extractions and whole culture extractions
may be due to diffusion rates.PbTx-9 was not detected in dialysis tubing or whole culture
extractions. As reported by Schulman et al. (1990), the amount of PbTx-9 is very small
compared to PbTx-2 or 3. Total toxin concentration of PbTx-2+3+9 and PbTx-1+2+3+9
used in this study were almost entirely influenced by the amount of PbTx-2 present. The
amount of PbTx-2 inside the cell is 5-10 fold higher than brevenal or any other PbTx.
When PbTx-2 is released from the cell it is chemically reduced to form PbTx-3 or 9 along
one of two separate pathways (Schulman et al. 1990). A bloom with a high PbTx-3 to
PbTx-2 ratio indicates a mature bloom or a bloom with many lysed cells (Pierce et al.
2001). Brevetoxins are incorporated into marine aerosols through bubble-mediated
transport to the surface (Pierce et al. 2001, 2006), and it is the extra-cellular brevetoxins,
most notably PbTx-3, that pose the biggest threat to public health and marine mammals
(Pierce et al. 2008). The amount of cellular PbTx-2 may reflect the potential degree of
toxicity of a bloom, however, further research is needed to relate the amount of cellular
PbTx-2 with the amount of PbTx-3 in the water and in aerosols.
The Wilson clone produced a significantly higher amount of total PbTx than the
SP3 clones. PbTx-2 values for the Wilson clone ranged from 12060-31700 pg cellsx10-3.
38
The concentration of PbTx-2 for the SP3 N-tox clone ranged from 10550-28860 pg
cellsx10-3, and the SP3 S-tox clone had the lowest amounts of PbTx-2 with 3060-25450
pg cellsx10-3. The results for the PbTx-2 concentration of the Wilson clone differ greatly
from those reported by McNabb et al. (2006), who analyzed the K. brevis Wilson clone
for brevetoxin content using solid phase extraction techniques in conjunction with LCMS/MS. They claimed to find a range of <0.0003-<0.0008 pg PbTx-2 cell-1, speculating
that extended time in culture may have caused the clones to not produce PbTx’s.
However, the Wilson clone in this study has been in culture for over 30 years and still
produces a large amount of PbTx’s. The inability to find PbTx-2 in the McNabb et al.
(2006) study may relate to their use of methanol to analyze PbTx in LC-MS/MS.
Methanol reacts with PbTx to form an acetal that has a different molecular weight than
that of the original compound and the molecular weights programmed into LC-MS/MS
for analysis (Dr. Andrea Bourdelais, personal communication).
The significant findings of this study are as follows;
•
•
•
•
•
•
•
Karenia brevis clones were variable in growth and toxin production.
The clone studied significantly influenced growth rates, and the amount of
brevetoxin and brevenal produced.
Salinity and temperature significantly influenced growth rates.
Nutrient stress significantly influenced growth rate, but not toxin production.
Whole culture extractions are accurate for calculating pg PbTx-1, 2 and brevenal
per cell.
Low salinity media of 20 inhibited growth of all K. brevis clones studied.
Acclimation of blooms to low salinity waters may allow for blooms to persist in
low salinity waters, but a sudden drop in salinity does not.
From this study, the role of nutrient stress in relation to growth and toxin
production was found to be a complex interplay that requires a better understanding
of the biosynthetic pathway of brevetoxin.
39
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47
Table 1: The names and masses of parent and derivative brevetoxins (PbTx)
from Karenia brevis used as standards in analysis with LC-MS/MS
Brevetoxin
Mass (amu)
Brevetoxin Derivatives Mass (amu)
PbTx-1
867.5
PbTx-1-H2O
889.5
877.3
PbTx-2
895.5
PbTx-2-H2O
PbTx-3
897.5
PbTx-3-H2O
879.5
893.5
PbTx-6
911.5
PbTx-6-H2O
881.5
PbTx-9
899.5
PbTx-9-H2O
Brevenal
657.5
Brevenal-H2O
639.5
893.5
CBA
911.5
CBA-H2O
48
Table 2: Growth rates (k) for the SP3 N-tox, SP3 S-tox and Wilson
clones at temperatures of 20, 25 and 30oC, salinities of 25, 30, 35
and 39, in balanced, P-limited and N-limited nutrient ratios.
Growth Rates (k)
Clone
Temp
Salinity Balanced* P-limited* N-limited*
N-tox
20
25
0.15
0.16
0.15
N-tox
20
30
0.17
0.18
0.17
N-tox
20
35
0.23
0.23
0.20
N-tox
20
39
0.20
0.18
0.16
N-tox
25
25
0.13
0.11
0.13
N-tox
25
30
0.25
0.26
0.29
N-tox
25
35
0.28
0.22
0.24
N-tox
25
39
0.23
0.24
0.22
N-tox
30
35
0.16
0.11
0.13
N-tox
30
39
0.19
0.14
0.14
S-tox
20
25
0.14
0.15
0.17
S-tox
20
30
0.16
0.15
0.19
S-tox
20
35
0.20
0.19
0.21
S-tox
20
39
0.18
0.17
0.20
S-tox
25
25
0.07
0.06
0.05
S-tox
25
30
0.17
0.20
0.18
S-tox
25
35
0.21
0.13
0.24
S-tox
25
39
0.25
0.28
0.27
S-tox
30
35
0.18
0.16
0.20
S-tox
30
39
0.19
0.20
0.19
Wilson
20
25
0.07
0.05
0.05
Wilson
20
30
0.15
0.12
0.14
Wilson
20
35
0.10
0.12
0.13
Wilson
20
39
0.13
0.09
0.10
Wilson
25
25
0.12
0.13
0.10
Wilson
25
30
0.14
0.14
0.15
Wilson
25
35
0.18
0.14
0.16
Wilson
25
39
0.14
0.10
0.13
*Balanced = 16:1 N:P, P-limited = 80:1, N-limited = 1:1
49
Table 3: Results for the three, two and one-way ANOVA for
growth rates by the variables of clone (SP3 N-tox, SP3 S-tox
o
and Wilson), temperature (20, 25 and 30 C), nutrient (Balanced,
P-lim and N-lim) and salinity (20, 25, 30, 35 and 39). P-values
were considered significant if less than 0.05.
Treatment Variables
p-value
clone
<.0001
*
nutrient
0.0369
*
temp
<.0001
*
salinity
<.0001
*
temperature-clone
0.0001
*
nutrient-clone
0.1975
nutrient-temp
0.9117
salinity-temp
salinity-clone
<.0001
<.0001
salinity-nutrient
0.1496
salinity-temp-clone
<.0001
salinity-nutrient-temp
0.0779
nutrient-temp-clone
0.2690
salinity-nutrient-clone
0.1561
*=significant difference
50
Significance
*
*
*
Table 4: ANOVA results showing where the salinity studied significantly
affected the growth rate for all three clones of K. brevis (SP3 N-tox, SP3
S-tox and Wilson) in salinities of 25, 30, 35 and 39 at temperatures of 20
o
o
and 25 C for all clones, and salinities of 35 and 39 at 30 C for the SP3
N-tox and SP3 S-tox clones. P-values were significant if less than 0.05.
Clone
Temperature # of Salinities
p-value
Significance
N-tox
20
4
0.0005
*
N-tox
25
4
<.0001
*
N-tox
30
2
0.1020
S-tox
20
4
0.0100
*
S-tox
25
4
<.0001
*
S-tox
30
2
0.2122
Wilson
20
4
<.0001
*
Wilson
25
4
0.0207
*
*=significant difference
Table 5: ANOVA results showing where the temperature studied significantly affected
growth rates of three clones of K. brevis (SP3 N-tox, SP3 S-tox and Wilson) in salinities
o
of 25, 30, 35 and 39 at temperatures of 20 and 25 C for all clones, and salinities of
o
35 and 39 at a temperature of 30 C for the SP3 clones. P-values were significant
if less than 0.05.
Clone
Salinity
# of Temperatures
p-value
Significance
N-tox
25
2
0.0335
*
N-tox
30
2
<.0001
*
N-tox
35
3
<.0001
*
N-tox
39
3
0.0001
*
S-tox
25
2
<.0001
*
S-tox
30
2
0.1551
S-tox
35
3
0.2156
S-tox
39
3
<.0001
*
Wilson
25
2
0.0001
*
Wilson
30
2
0.4993
Wilson
35
2
0.0033
Wilson
39
2
0.1881
*=significant difference
51
*
Table 6: ANOVA results showing where the clone studied significantly affected
growth rates of three clones of K. brevis (SP3 N-tox, SP3 S-tox and Wilson) at
o
temperatures of 20 and 25 C in salinities of 25, 30, 35 and 39, for all clones,
o
and salinities of 35 and 39 at 30 C for the SP3 clones. P-values were significant
if less than 0.05.
Temperature
Salinity
# of Clones
p-value
Significance
20
25
3
<.0001
*
20
30
3
0.0096
*
20
35
3
<.0001
*
20
39
3
<.0001
*
25
25
3
0.0001
*
25
30
25
35
25
39
30
35
30
39
*=significant difference
3
3
3
2
2
<.0001
<.0001
<.0001
0.0020
0.0048
*
*
*
*
*
Table 7: Cell density of K. brevis clones (SP3 N-tox,
SP3 S-tox and Wilson) at the time of extraction in
o
stationary phase grown at 20 C under defined
nutrient-salinity conditions.
3
-1
cellsx10 ml
Clone
Salinity
Bal*
P-lim*
Wilson
25
17.25
8.87
N-tox
25
14.15
11.18
S-tox
25
6.53
6.48
Wilson
35
26.50
15.09
N-tox
35
20.99
13.54
S-tox
35
36.89
15.16
Wilson
39
23.29
31.04
N-tox
39
18.34
12.61
S-tox
39
38.49
19.98
*Bal = 16:1 N:P; P-lim = 80:1; N-lim = 1:1
52
N-lim*
6.31
6.52
6.27
15.59
10.40
10.09
11.58
7.54
15.84
-3
Table 8: LC-MS/MS analysis of pg-brevetoxin or brevenal cellsx10 for three clones of Karenia brevis (SP3 N-tox, SP3 S-tox and Wilson) in stationary phase, grown
o
at 20 C in slainities of 25, 35 and 39, and nutrient ratios of balanced, P-limited and N-limited.
-3
Clone Salinity Nutrient*
brevenal
N-tox
25
Bal
330 +/- 10
N-tox
25
P-lim
280 +/- 10
N-tox
25
N-lim
310 +/- 10
N-tox
35
Bal
550 +/- 10
N-tox
35
P-lim
680 +/- 20
N-tox
35
N-lim
790 +/- 20
N-tox
39
Bal
810 +/- 20
N-tox
39
P-lim
890 +/- 20
N-tox
39
N-lim
960 +/- 30
S-tox
25
Bal
250 +/- 10
S-tox
25
P-lim
190 +/- 10
S-tox
25
N-lim
240 +/- 10
S-tox
35
Bal
620 +/- 20
S-tox
35
P-lim
1120 +/- 30
S-tox
35
N-lim
710 +/- 20
S-tox
39
Bal
480 +/- 10
S-tox
39
P-lim
1200 +/- 30
S-tox
39
N-lim
400 +/- 10
Wilson
25
Bal
2160 +/- 50
Wilson
25
P-lim
3190 +/- 80
Wilson
25
N-lim
5270 +/- 130
Wilson
35
Bal
2990 +/- 70
Wilson
35
P-lim
2910 +/- 70
Wilson
35
N-lim
3080 +/- 70
Wilson
39
Bal
2920 +/- 70
Wilson
39
P-lim
2030 +/- 50
Wilson
39
N-lim
4060 +/- 100
*Bal = 16:1 N:P; P-lim = 80:1; N-lim = 1:1
*LOD = Limit of Detection
PbTx-1
1380 +/- 60
950 +/- 40
800 +/- 30
1010 +/- 40
2120 +/- 100
2460 +/- 110
1470 +/- 70
2800 +/- 140
3970 +/- 190
370 +/- 10
310 +/- 10
360 +/- 10
460 +/- 20
2300 +/- 110
1380 +/- 60
500 +/- 20
2390 +/- 130
830 +/- 30
40 +/- 1
170 +/- 4
150 +/- 4
50 +/- 1
70 +/- 2
110 +/- 3
60 +/- 2
30 +/- 1
110 +/- 10
PbTx-2
14840 +/- 220
10550 +/- 180
15620 +/- 270
14100 +/- 200
24110 +/- 340
22150 +/- 330
16920 +/- 240
27030 +/- 370
28860 +/- 440
4160 +/- 90
3060 +/- 70
4900 +/- 110
9470 +/- 130
24140 +/- 320
11680 +/- 200
10410 +/- 140
25450 +/- 320
5980 +/- 100
12060 +/- 180
20020 +/- 310
31700 +/- 490
18290 +/- 240
20990 +/- 290
20220 +/- 280
17170 +/- 230
14950 +/- 190
26250 +/- 370
pg cellsx10
PbTx-3
5200 +/- 230
3520 +/- 140
6920 +/- 280
2480 +/- 100
2620 +/- 100
1060 +/- 30
2020 +/- 80
2970 +/- 110
3270 +/- 120
2080 +/- 70
2520 +/- 80
2870 +/- 100
1220 +/- 50
6250 +/- 290
2140 +/- 80
1730 +/- 80
4480 +/- 210
1190 +/- 40
2080 +/- 80
3610 +/- 130
5020 +/- 190
2350 +/- 100
1710 +/- 60
610 +/- 20
1150 +/- 40
1420 +/- 60
1250 +/- 40
53
PbTx-6
860 +/- 40
440 +/- 30
720 +/- 40
420 +/- 20
420 +/- 30
240 +/- 20
340 +/- 20
330 +/- 20
550 +/- 40
410 +/- 30
490 +/- 30
270 +/- 20
240 +/- 10
1610 +/- 60
<LOD
350 +/- 20
760 +/- 30
280 +/- 20
380 +/- 30
16 +/- 30
500 +/- 40
230 +/- 10
70 +/- 10
30 +/- 10
180 +/- 10
10 +/- 2
40 +/- 10
CBA
<LOD
<LOD
30 +/- 2
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
10 +/- 1
<LOD
<LOD
PbTx-9
<LOD
10 +/- 2
<LOD
10 +/- 1
10 +/- 2
10 +/- 2
10 +/- 1
10 +/- 2
20 +/- 2
30 +/- 4
40 +/- 4
50 +/- 5
20 +/- 2
160 +/- 8
20 +/- 3
30 +/- 2
60 +/- 4
10 +/- 1
20 +/- 2
40 +/- 4
20 +/- 3
20 +/- 2
10 +/- 1
10 +/- 1
60 +/- 4
<LOD
10 +/- 4
PbTx-2+3+9
20040
14080
22540
16590
26740
23220
18950
30010
32150
6270
5620
7820
10710
30550
13840
12170
29990
7180
14160
23670
36740
20660
22710
20540
18380
16370
27510
PbTx-1+2+3+9
21420
15030
23340
17600
28860
25680
20420
32810
36120
6640
5930
8180
11170
32850
15220
12670
32380
8010
14200
23840
36890
20710
22780
20650
18440
16400
27620
Table 9: Results for the three, two and one-way ANOVA for brevenal and brevetoxin
concentrations of treatments based on variables of clone (SP3 N-tox, SP3 S-tox
o
and Wilson), temperature (20, 25 and 30 C), nutrient (Balanced, P-limited and
N-limited) and salinity (25, 35 and 39). P-values were significant if less than 0.05.
Brevenal
PbTx-6
Treatment Variables p-value Significance
Treatment Variables p-value Significance
Clone
<.0001
*
Clone
0.0187
*
Salinity
0.7970
Salinity
0.3082
Nutrient
0.1811
Nutrient
0.2223
Clone-Salinity
0.3610
Clone-Salinity
0.2559
Clone-Nutrient
0.1200
Clone-Nutrient
0.0193
Salinity-Nutrient
0.6519
Salinity-Nutrient
0.0873
PbTx-1
Treatment Variables p-value Significance
Clone
0.0004
*
Salinity
0.0268
*
Nutrient
0.0736
Clone-Salinity
0.1331
Clone-Nutrient
0.2232
Salinity-Nutrient
0.3189
PbTx-9
Treatment Variables p-value Significance
Clone
0.0498
*
Salinity
0.8564
Nutrient
0.2954
Clone-Salinity
0.5918
Clone-Nutrient
0.2401
Salinity-Nutrient
0.5664
PbTx-2
Treatment Variables p-value Significance
Clone
0.0097
*
Salinity
0.0661
Nutrient
0.0721
Clone-Salinity
0.2162
Clone-Nutrient
0.1488
Salinity-Nutrient
0.4365
PbTx-2, 3 and 9
Treatment Variables p-value Significance
Clone
0.0196
*
Salinity
0.2538
Nutrient
0.0736
Clone-Salinity
0.2046
Clone-Nutrient
0.1413
Salinity-Nutrient
0.3106
PbTx-3
Treatment Variables p-value Significance
Clone
0.0857
Salinity
0.0154
*
Nutrient
0.1717
Clone-Salinity
0.0717
Clone-Nutrient
0.1170
Salinity-Nutrient
0.0698
*=significant difference
PbTx-1, 2, 3 and 9
Treatment Variables p-value Significance
Clone
0.0257
*
Salinity
0.2068
Nutrient
0.0705
Clone-Salinity
0.1981
Clone-Nutrient
0.1559
Salinity-Nutrient
0.3505
54
Table 10: A Tukey’s pair wise comparison of total toxin production of PbTx-2+3+9, PbTx-1+2+3+9 and total brevenal, between grouped
treatments of salinity-clone, nutrient-clone and nutrient-salinity. P-values were significant if less than 0.05.
Salinity-Clone
PbTx-2+3+9
1
2
3
1
0.92
0.07
2
0.32
3
4
5
6
7
8
9
PbTx-1+2+3+9
1
2
3
1
0.98
0.10
2
0.34
3
4
5
6
7
8
9
Brevenal
1
2
3
1
*<0.01 *<0.01
2
1.00
3
4
5
6
7
8
9
*=significant difference
Salinity-Clone:
Salinity
Clone
25 Wilson
25 Non-tox
25 S-tox
35 Wilson
35 Non-tox
35 S-tox
39 Wilson
39 Non-tox
39 S-tox
4
1.00
1.00
0.17
5
1.00
1.00
0.14
1.00
6
0.89
1.00
0.36
1.00
0.99
7
0.99
1.00
0.20
1.00
1.00
1.00
8
1.00
0.73
*0.04
0.94
0.97
0.68
0.90
9
0.70
1.00
0.54
0.97
0.93
1.00
0.99
0.47
4
1.00
1.00
0.24
5
1.00
0.99
0.13
1.00
6
0.97
1.00
0.35
1.00
0.99
7
0.99
1.00
0.28
1.00
1.00
1.00
8
0.98
0.63
*0.03
0.78
0.96
0.61
0.72
9
0.87
1.00
0.53
1.00
0.93
1.00
1.00
0.41
4
0.93
*0.01
*<0.01
5
*<0.01
0.99
0.98
*0.01
6
*0.01
0.95
0.90
*0.02
1.00
7
0.94
*0.01
*<0.01
1.00
*0.01
*0.02
8
*0.01
0.91
0.85
*0.02
1.00
1.00
*0.02
9
*<0.01
0.99
0.97
*0.01
1.00
1.00
*0.01
1.00
Number
1
2
3
4
5
6
7
8
9
Nutrient-Clone:
Nutrient
Clone
Bal
Wilson
Bal
Non-tox
Bal
S-tox
P-lim
Wilson
P-lim
Non-tox
P-lim
S-tox
N-lim
Wilson
N-lim
Non-tox
N-lim
S-tox
Number
1
2
3
4
5
6
7
8
9
Nutrient-Clone
PbTx-2+3+9
1
2
1
1.00
2
3
4
5
6
7
8
9
PbTx-1+2+3+9
1
2
1
1.00
2
3
4
5
6
7
8
9
Brevenal
1
2
1
*0.02
2
3
4
5
6
7
8
9
3
0.74
0.66
4
1.00
1.00
0.41
5
0.93
0.97
0.21
1.00
6
0.99
1.00
0.32
1.00
1.00
7
0.47
0.55
0.06
0.80
0.98
0.90
8
0.72
0.80
0.12
0.97
1.00
0.99
1.00
9
0.73
0.65
1.00
0.40
0.21
0.31
0.06
0.11
3
0.84
0.64
4
1.00
1.00
0.53
5
0.83
0.96
0.20
0.99
6
0.95
1.00
0.30
1.00
1.00
7
0.54
0.74
0.10
0.85
1.00
0.98
8
0.55
0.75
0.10
0.86
1.00
0.99
1.00
9
0.86
0.68
1.00
0.56
0.21
0.32
0.10
0.11
3
*0.02
1.00
4
1.00
*0.02
*0.02
5
*0.03
1.00
1.00
*0.03
6
0.05
1.00
0.99
0.05
1.00
7
0.15
*<0.01
*<0.01
0.16
*<0.01
*<0.01
8
*0.03
1.00
1.00
*0.03
1.00
1.00
*<0.01
9
*0.02
1.00
1.00
*0.02
1.00
0.99
*<0.01
1.00
Nutrient-Salinity:
Nutrient
Salinity
Bal
Bal
Bal
P-lim
P-lim
P-lim
N-lim
N-lim
N-lim
Number
25
35
39
25
35
39
25
35
39
1
2
3
4
5
6
7
8
9
55
Nutrient-Salinity
PbTx-2+3+9
1
2
1
1.00
2
3
4
5
6
7
8
9
PbTx-1+2+3+9
1
2
1
1.00
2
3
4
5
6
7
8
9
Brevenal
1
2
1
0.97
2
3
4
5
6
7
8
9
3
1.00
1.00
4
1.00
1.00
1.00
5
0.26
0.46
0.51
0.33
6
0.34
0.59
0.64
0.43
1.00
7
0.65
0.89
0.93
0.75
0.99
1.00
8
0.93
1.00
1.00
0.97
0.81
0.91
1.00
9
0.66
0.90
0.93
0.76
0.98
1.00
1.00
1.00
3
1.00
1.00
4
1.00
1.00
1.00
5
0.27
0.45
0.51
0.32
6
0.33
0.54
0.61
0.40
1.00
7
0.74
0.93
0.96
0.82
0.97
0.99
8
0.92
0.99
1.00
0.96
0.84
0.91
1.00
9
0.63
0.86
0.90
0.71
0.99
1.00
1.00
1.00
3
0.96
1.00
4
1.00
1.00
1.00
5
0.85
1.00
1.00
0.99
6
0.97
1.00
1.00
1.00
1.00
7
0.44
0.93
0.94
0.78
0.99
0.92
8
0.89
1.00
1.00
1.00
1.00
1.00
0.98
9
0.59
0.98
0.99
0.91
1.00
0.98
1.00
1.00
Table 11: MANOVA results of total PbTx-2+3+9 and total PbTx-1+2+3+9 for variables of clone
(SP3 N-tox, SP3 S-tox and Wilson), salinity (25, 35 and 39) and nutrient (balanced, P-limited
and N-limited) on toxin production. Wilks' Lambda values were significant if less than 0.05.
PbTx-2+3+9
PbTx-1+2+3+9
Treatment Variable
Wilks' Lambda
Significance
Wilks' Lambda
Significance
clone
0.0012
*
<0.0001
*
salinity
0.0148
*
0.0387
*
nutrient
0.2841
0.5000
clone-salinity
0.1526
0.2313
clone-nutrient
0.3035
0.1757
salinity-nutrient
0.4744
0.2486
*=significant
56
30
25
A
y = 1.61x
R2 = 0.99
B
y = 1.86x
2
R = 0.98
C
y=1.89x
R2 = 0.98
20
15
10
5
0
Relative Fluorescence
30
25
20
15
10
5
0
30
25
20
15
10
5
0
0
2
4
6
8
10
12
6
14
16
18
-1
Cell Density (x10 cells L )
Figure 1: Calibration of cell density (x 106 cells L-1) versus
relative fluorescence of three clones of K. brevis. A) SP3
N-tox B) SP3 S-tox and C) Wilson.
57
20
100 mL sample + ~30 mL EtOAc
Sonicate sample + EtOAc
Add ~20 mL EtOAc to sonicated sample and
extract
Draw off H2O layer
Pour off EtOAc layer and save
Add ~50 mL to H2O layer
and extract
Draw off H2O layer
Combine EtOAc layers
Pour off EtOAc
Extract EtOAc layers with 80mL DIW
Discard H 2O layer
Discard H 2O layer Evaporate EtOAc layer to dryness
in a RotoVap system
Bring up in 7 mL Ac and freeze >1 hr.
Filter using a 0.22 mm nylon filter
Figure 2: Flowchart describing the
extraction method used to extract
brevetoxins from Karenia brevis.
*EtOAc: Ethyl Acetate; Ac: Acetone;
DIW: Deionized Water; Mobile Phase:
98% ACN, 2% H2O, 0.1% Formic Acid
Evaporate filtrate to dryness in a RotoVap
system
Bring up in 1 mL mobile phase and transfer
to HPLC vial
Evaporate contents of HPLC Vial in
RotoVap system
Bring up in 200 µL mobile phase
and transfer to HPLC vial for
analysis with LC-MS/MS
58
0.8
o
A: SP3 N-tox 20 20 C
o
D: SP3 N-tox 30 30 C
Bal
P-lim
N-lim
0.6
0.4
0.2
Relative Fluorescence
0.0
Day
o
B: SP3 S-tox 20 20 C
0.8
o
E: SP3 S-tox 30 30 C
0.6
0.4
0.2
0.0
Day
o
C: Wilson 20 20 C
2.0
o
F: Wilson 30 30 C
1.5
1.0
0.5
0.0
0
5
10
15
20 0
Day
2
4
6
8
10
Day
Figure 3: Relative fluorescence over time of three clones of Karenia brevis
displaying similar moribund growth patterns of a steady decline after
inoculation on day 0. (Bal 16:1 N:P; P-lim 80:1; N-lim 1:1).
59
12
3.5
o
3.0
o
A: SP3 N-tox 35 30 C
2.5
D: SP3 N-tox 39 30 C
Bal
P-lim
N-lim
2.0
1.5
1.0
0.5
0.0
4.5
Relative Fluorescence
4.0
o
o
B: SP3 S-tox 35 30 C
3.5
E: SP3 S-tox 39 30 C
3.0
Bal
P-lim
N-lim
2.5
2.0
1.5
1.0
0.5
0.0
4.5
o
4.0
o
C: Wilson 25 20 C
F: Wilson 30 20 C
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
5
10
15
20
25
30
35 0
Day
5
10
15
20
25
Day
Figure 4: Relative fluorescence over time of three clones of Karenia brevis
(SP3 N-tox, SP3 S-tox and Wilson) displaying a similar lag phase in initial
growth patterns after inoculation on day 0. (Bal 16:1 N:P; P-lim 80:1;
N-lim 1:1).
60
30
35
o
4.5
o
A: SP3 N-tox 30 20 C
4.0
D: SP3 N-tox 30 25 C
Bal
P-lim
N-lim
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
5.0
5.0
o
B: SP3 S-tox 30 25 C
4.5
o
E: SP3 S-tox 35 25 C
4.5
4.0
4.0
3.5
3.5
3.0
3.0
2.5
2.5
2.0
2.0
1.5
1.5
1.0
1.0
0.5
0.5
0.0
0.0
0
o
5.5
C: Wilson 25 25 C
5
10
o
F: Wilson 30 25 C
15
20
25
30
20
25
30
Days
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
0
5
10
15
20
25
30
Day
0
5
10
15
Day
Figure 5: Relative fluorescence over time of three clones of Karenia brevis
(SP3 N-tox, SP3 S-tox and Wilson) displaying similar growth patterns
of a steady increase in growth after inoculation without a lag phase. (Bal
16:1 N:P; P-lim 80:1; N-lim 1:1).
61
5.0
o
o
A: SP3 N-tox 35 20 C
4.5
4.0
D: SP3 N-tox 30 25 C
Bal
P-lim
N-lim
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
4.5
o
o
B: SP3 S-tox 35 20 C
4.0
E: SP3 S-tox 30 25 C
3.5
3.0
2.5
2.0
1.5
1.0
0.5
5.5
o
o
C: Wilson 30 20 C
5.0
F: Wilson 30 25 C
4.5
4.0
3.5
3.0
2.5
2.0
1.5
0
5
10
15
20
25
30 0
Day
5
10
15
20
25
Day
Figure 6: Relative fluorescence over time of three clones of Karenia brevis
(SP3 N-tox, SP3 S-tox and Wilson) displaying similar growth patterns
of a gradual or rapid decrease in N-limited treatments in mid or late growth.
(Bal 16:1 N:P; P-lim 80:1; N-lim 1:1).
62
30
510
Brevenal
PbTx-1
PbTx-2
PbTx-3
PbTx-6
CBA
PbTx-9
495
480
120
pg-toxin µl
-1
105
90
75
60
45
30
15
0
0
6
12
24
48
whole culture
Hour
Figure 7: Brevetoxin profiles in pg-toxin µl-1 from extracts of whole culture
(cells and media) and media only from dialysis tubing suspended in a
culture of Karenia brevis for 0, 6, 12, 24 and 48 hour intervals.
63
Brevenal % Relative to Balanced Treatments
Toxin % Relative to Balanced Treatments
200
175
A
150
P-lim
N-lim
125
100
75
50
25
0
-25
-50
150 Wilson 25 N-tox 25 S-tox 25 Wilson 35 N-tox 35 S-tox 35 Wilson 39 N-tox 39 S-tox 39
125
Treatment
B
100
75
50
25
0
-25
-50
Wilson 25
N-tox 25
S-tox 25
Wilson 35
N-tox 35
S-tox 35
Wilson 39
N-tox 39
S-tox 39
Figure 8: Percent of total PbTx-1+2+3+9 (A) and brevenal (B) in P-lim and
N-lim nutrient treatments relative to balanced nutrients for three clones of
Karenia brevis (SP3 N-tox, SP3 S-tox and Wilson) in salinities of 25, 35
o
and 39 at a temperature of 20 C. (Bal 16:1 N:P; P-lim 80:1; N-lim 1:1).
64
Appendix A: Growth curves of three clones of K. brevis (SP3 N-tox, SP3 S-tox
and Wilson) in salinities of 20, 25, 25, 30, 35 and 39 with N:P nutrient ratios of
balanced (16:1), P-lim (80:1) and N-lim (1:1) at temperatures of 20, 25 and 30oC
0.8
A
●
○
▼
Bal
P-lim
N-lim
0.6
0.4
0.2
0.0
Day
Relative Fluorescence
0.8
B
0.6
0.4
0.2
0.0
Day
C
1.5
1.0
0.5
0.0
0
5
10
15
20
Day
Figure A.1: Relative fluoresence for three clones of Karenia brevis grown
o
at a salinity of 20 and temperature of 20 C in three N:P ratios (Bal 16:1 N:P;
P-lim 80:1; N-lim 1:1). A) SP3 N-tox, B) SP3 S-tox and C) Wilson.
65
4.0
3.5
A
3.0
●
○
▼
Bal
P-lim
N-lim
2.5
2.0
1.5
1.0
0.5
0
5
10
15
20
25
30
20
25
30
4.0
Day
Relative Fluorescence
B
3.5
3.0
2.5
2.0
1.5
1.0
Day
C
2.0
1.5
1.0
0.5
0
5
10
15
Day
Figure A.2: Relative fluoresence for three clones of Karenia brevis grown
o
at a salinity of 25 and temperature of 20 C in three N:P ratios (Bal 16:1 N:P;
P-lim 80:1; N-lim 1:1). A) SP3 N-tox, B) SP3 S-tox and C) Wilson.
66
5.0
●
A ○
▼
4.5
4.0
3.5
Bal
P-lim
N-lim
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Relative Fluorescence
4.0
B
3.5
3.0
2.5
2.0
1.5
4.5
C
4.0
3.5
3.0
2.5
2.0
1.5
0
5
10
15
20
25
30
Day
Figure A.3: Relative fluoresence for three clones of Karenia brevis grown
o
at a salinity of 30 and temperature of 20 C in three N:P ratios (Bal 16:1 N:P;
P-lim 80:1; N-lim 1:1). A) SP3 N-tox, B) SP3 S-tox and C) Wilson.
67
5.0
●
A ○
▼
4.5
4.0
Bal
P-lim
N-lim
3.5
3.0
2.5
2.0
1.5
1.0
0.5
Relative Fluorescence
4.5
B
4.0
3.5
3.0
2.5
2.0
1.5
4.5
C
4.0
3.5
3.0
2.5
2.0
1.5
0
5
10
15
20
25
30
Day
Figure A.4: Relative fluoresence for three clones of Karenia brevis grown
o
at a salinity of 35 and temperature of 20 C in three N:P ratios (Bal 16:1 N:P;
P-lim 80:1; N-lim 1:1). A) SP3 N-tox, B) SP3 S-tox and C) Wilson.
68
5.0
●
A ○
▼
4.5
4.0
3.5
Bal
P-lim
N-lim
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Relative Fluorescence
5.0
B
4.5
4.0
3.5
3.0
2.5
2.0
1.5
4.0
C
3.5
3.0
2.5
2.0
1.5
0
5
10
15
20
25
30
Day
Figure A.5: Relative fluoresence for three clones of Karenia brevis grown
o
at a salinity of 39 and temperature of 20 C in three N:P ratios (Bal 16:1 N:P;
P-lim 80:1; N-lim 1:1). A) SP3 N-tox, B) SP3 S-tox and C) Wilson.
69
0.8
A
0.6
●
○
▼
Bal
P-lim
N-lim
0.4
0.2
0.0
Relative Fluorescence
0.8
B
0.6
0.4
0.2
0.0
2.0
1.8
C
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
5
10
15
20
25
30
Day
Figure A.6: Relative fluoresence for three clones of Karenia brevis grown
o
at a salinity of 20 and temperature of 25 C in three N:P ratios (Bal 16:1 N:P;
P-lim 80:1; N-lim 1:1). A) SP3 N-tox, B) SP3 S-tox and C) Wilson.
70
●
A ○
▼
2.0
Bal
P-lim
N-lim
1.5
1.0
0.5
0.0
Relative Fluorescence
2.0
B
1.5
1.0
0.5
0.0
5.0
C
4.5
4.0
3.5
3.0
2.5
2.0
1.5
0
5
10
15
20
25
30
Day
Figure A.7: Relative fluoresence for three clones of Karenia brevis grown
o
at a salinity of 25 and temperature of 25 C in three N:P ratios (Bal 16:1 N:P;
P-lim 80:1; N-lim 1:1). A) SP3 N-tox, B) SP3 S-tox and C) Wilson.
71
5.0
4.5
A
4.0
3.5
●
○
▼
Bal
P-lim
N-lim
3.0
2.5
2.0
1.5
1.0
0.5
0.0
4.5
Relative Fluorescence
4.0
B
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
5.5
5.0
C
4.5
4.0
3.5
3.0
2.5
2.0
1.5
0
5
10
15
20
25
30
Day
Figure A.8: Relative fluoresence for three clones of Karenia brevis grown
o
at a salinity of 30 and temperature of 25 C in three N:P ratios (Bal 16:1 N:P;
P-lim 80:1; N-lim 1:1). A) SP3 N-tox, B) SP3 S-tox and C) Wilson.
72
4.0
●
A ○
▼
3.5
3.0
Bal
P-lim
N-lim
2.5
2.0
1.5
1.0
0.5
0.0
4.5
B
Relative Fluorescence
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
5.0
C
4.5
4.0
3.5
3.0
2.5
2.0
1.5
0
5
10
15
20
25
30
Day
Figure A.9: Relative fluoresence for three clones of Karenia brevis grown
o
at a salinity of 39 and temperature of 25 C in three N:P ratios (Bal 16:1 N:P;
P-lim 80:1; N-lim 1:1). A) SP3 N-tox, B) SP3 S-tox and C) Wilson.
73
5.0
●
A ○
▼
4.5
4.0
3.5
Bal
P-lim
N-lim
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0
5
10
15
20
25
30
20
25
30
20
25
30
4.5
Relative Fluorescence
Days
B
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0
5
10
4.5
15
X Data
C
4.0
3.5
3.0
2.5
2.0
1.5
0
5
10
15
Day
Figure A.10: Relative fluoresence for three clones of Karenia brevis grown
o
at a salinity of 39 and temperature of 25 C in three N:P ratios (Bal 16:1 N:P;
P-lim 80:1; N-lim 1:1). A) SP3 N-tox, B) SP3 S-tox and C) Wilson.
74
●
A ○
▼
0.6
Bal
P-lim
N-lim
0.4
0.2
0.0
0
1
2
3
4
5
4
5
4
5
0.6
Relative Fluorescence
B
0.4
0.2
0.0
2.0
0
1
2
3
C
1.5
1.0
0.5
0.0
0
1
2
3
Day
Figure A.11: Relative fluoresence for three clones of Karenia brevis grown
o
at a salinity of 20 and temperature of 30 C in three N:P ratios (Bal 16:1 N:P;
P-lim 80:1; N-lim 1:1). A) SP3 N-tox, B) SP3 S-tox and C) Wilson.
75
0.6
●
○
▼
A
0.5
Bal
P-lim
N-lim
0.4
0.3
0.2
0.1
0.0
Relative Fluorescence
0.6
B
0.4
0.2
0.0
2.0
C
1.5
1.0
0.5
0.0
0
2
4
6
8
Day
Figure A.12: Relative fluoresence for three clones of Karenia brevis grown
o
at a salinity of 25 and temperature of 30 C in three N:P ratios (Bal 16:1 N:P;
P-lim 80:1; N-lim 1:1). A) SP3 N-tox, B) SP3 S-tox and C) Wilson.
76
0.6
A
●
○
▼
Bal
P-lim
N-lim
0.4
0.2
0.0
Relative Fluorescence
0.6
B
0.4
0.2
0.0
2.0
C
1.5
1.0
0.5
0.0
0
2
4
6
8
10
12
Day
Figure A.13: Relative fluoresence for three clones of Karenia brevis grown
o
at a salinity of 30 and temperature of 30 C in three N:P ratios (Bal 16:1 N:P;
P-lim 80:1; N-lim 1:1). A) SP3 N-tox, B) SP3 S-tox and C) Wilson.
77
3.5
3.0
●
A ○
▼
Bal
P-lim
N-lim
2.5
2.0
1.5
1.0
0.5
0.0
Relative Fluorescence
4.0
3.5
B
3.0
2.5
2.0
1.5
1.0
0.5
0.0
2.2
C
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
Day
Figure A.14: Relative fluoresence for three clones of Karenia brevis grown
o
at a salinity of 35 and temperature of 30 C in three N:P ratios (Bal 16:1 N:P;
P-lim 80:1; N-lim 1:1). A) SP3 N-tox, B) SP3 S-tox and C) Wilson.
78
4.0
3.5
A
3.0
●
○
▼
Bal
P-lim
N-lim
2.5
2.0
1.5
1.0
0.5
0.0
Relative Fluorescence
4.0
B
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
2.0
C
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0
5
10
15
20
25
30
35
Day
Figure A.15: Relative fluoresence for three clones of Karenia brevis grown
o
at a salinity of 39 and temperature of 30 C in three N:P ratios (Bal 16:1 N:P;
P-lim 80:1; N-lim 1:1). A) SP3 N-tox, B) SP3 S-tox and C) Wilson.
79