THE EFFECT OF NUTRIENT LIMITATION ON THE GROWTH AND TOXICITY OF THE
DINOFLAGELLATE GYRODINIUM INSTRIATUM
Joann Kelly
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
2009
Approved by
Advisory Committee
Dr. Wilson Freshwater
______________________________
Dr. Lawrence Cahoon
______________________________
Dr. Stephen Skrabal
______________________________
Dr. Carmelo Tomas
______________________________
Chair
Accepted by
DN: cn=Robert D. Roer, o=UNCW,
ou=Dean of the Graduate School &
Research, email=roer@uncw.edu, c=US
Date: 2010.02.04 10:50:44 -05'00'
______________________________
Dean, Graduate School
JOURNAL PAGE
This thesis was formatted according to the requirements for the Journal of Phycology.
ii
TABLE OF CONTENTS
JOURNAL PAGE ........................................................................................................................... ii
ABSTRACT ................................................................................................................................... iv
ACKNOWLEDGMENTS ...............................................................................................................v
LIST OF TABLES ......................................................................................................................... vi
LIST OF FIGURES ...................................................................................................................... vii
INTRODUCTION ...........................................................................................................................1
OBJECTIVES ................................................................................................................................11
METHODS ....................................................................................................................................11
Cultivation..........................................................................................................................11
Growth Measurements .......................................................................................................15
Growth Determinations ......................................................................................................15
Erythrocyte Lysis Assay ....................................................................................................16
Natural Samples .................................................................................................................17
RESULTS ......................................................................................................................................18
DISCUSSION ................................................................................................................................41
CONCLUSIONS............................................................................................................................47
REFERENCES ..............................................................................................................................48
APPENDIX ....................................................................................................................................52
iii
ABSTRACT
Gyrodinium instriatum Freudenthal et Lee is an unarmored photosynthetic dinoflagellate
with two ventrally inserted flagella, that was first described by Freudenthal and Lee in 1963. It
is able to grow at a variety of temperatures and salinities, but no research thus far has
investigated its growth in various nutrient concentrations. Gyrodinium instriatum is widely
distributed around the world, including North Carolina, making this research useful both locally
and internationally. While reported as a harmful algal species in Japan, its toxicity is inferred but
not proven chemically. Since the organism occurs year-round in the coastal waters of North
Carolina, investigating the effect of nutrients on growth and toxin production is critical to
evaluating blooms in local waters. Its toxicity must be characterized, along with the factors, such
as nutrient limitation, that can regulate its virulence. Growth studies and hemolytic assays were
conducted in order to better understand the dynamics of this species and to begin to describe its
toxicity. Natural samples from the New River, NC were examined to determine if the organism
was potentially capable of reaching toxic concentrations in its natural environment. Findings
indicate that G. instriatum is a variable organism, as it did not show consistent responses for
growth or hemolytic activity. It survived in nutrient stressed environments and was better able to
deal with P-limited conditions. Hemolysis levels were variable, but the nutrient condition
generally caused increased hemolytic activity in the order P-limited > replete > N-limited. The
hemolytic compounds were only found within the cell and they behaved like secondary
metabolites by accumulating over time. In the natural samples from 2007 and 2008, G.
instriatum usually did not reach the necessary concentrations for substantial hemolytic activity.
There was one exception in August 2008 when the organism reached higher cell numbers, but
nutrient information suggests that it did not have high enough activity to achieve toxic levels.
iv
ACKNOWLEDGMENTS
I would like to thank my advisor Dr. Carmelo Tomas for his support and guidance during
my time here at UNCW. I would also like to thank the members of my committee, Dr. Wilson
Freshwater, Dr. Lawrence Cahoon, and Dr. Stephen Skrabal, for their help with my thesis.
Thank you to all of my past and present labmates including Brooke Stuercke, Harris Muhlstein,
Ian Misner, Avery Tatters, Bob York, Jen Klinkner, and Chris Holland, for their friendship, as
well as their advice and help throughout my project. Thanks also to Harris for taking excellent
photographs and videos of G. instriatum, and to Dr. Hugh Crews and Dr. Karl Jacocks for
statistical assistance. Thanks to Stephanie Pettergarrett with the North Carolina Department of
Environment and Natural Resources for the monthly New River samples and nutrient data. I
would like to acknowledge the funding I received from the Center for Disease Control and
Prevention with the North Carolina Department of Health and Human Services grant number
0150407, as well as the UNCW Graduate School and Center for Marine Science. I would like to
thank my family and especially my parents for their continued love, support, encouragement, and
faith in me, as well as Jason Holaday for constantly being there for me and making me happy.
Also, thank you to my VA friends Rachel Roberts, Jennifer Eudy, and Beckie Dashiell for their
loyalty, and my NC friends for their encouragement, dependability, and for always keeping
things fun, particularly Alex Lee, Brooke Stuercke, Nadya Mamoozadeh, and Samantha Schmitt.
v
LIST OF TABLES
Table
Page
1.
Nutrient concentrations for each treatment ........................................................................14
2.
Hemolytic activity and estimated EC50 values for G. instriatum in replete
and P-limited media for each day that an assay was performed during
September 2008 experiment ..............................................................................................24
3.
Estimated EC50 values for G. instriatum in replete and N-limited media for
each day that an assay was performed during November 2008 experiment ......................28
4.
Estimated EC50 values for G. instriatum in replete, N-limited, and
P-limited media for each day that was assay was performed during
March 2009 experiment .....................................................................................................33
5.
Estimated EC50 values for G. instriatum in replete, N-limited, and
P-limited media for each day that was assay was performed during
June 2009 experiment ........................................................................................................37
vi
LIST OF FIGURES
Figure
Page
1.
Line drawing of a Gyrodinium instriatum cell ....................................................................2
2.
Map of the New River Estuary, showing Northeast Creek and Brinson Creek .................12
3.
Comparison of relative fluorescence values to actual cell count numbers ........................19
4.
Graph of chlorophyll a/cell over time ................................................................................20
5.
Relative fluorescence growth curves and hemolytic activity for G. instriatum
in replete and P-limited media during September 2008 experiment..................................22
6.
Growth rates (k) of G. instriatum in replete and P-limited media during
September 2008 experiment ..............................................................................................23
7.
Relative fluorescence growth curves and hemolytic activity for G. instriatum
in replete and N-limited media during November 2008 experiment .................................26
8.
Growth rates (k) of G. instriatum in replete and N-limited media during
November 2008 experiment ...............................................................................................27
9.
Relative fluorescence growth curve for G. instriatum in nutrient replete
media during February 2009 experiment ...........................................................................29
10.
Relative fluorescence growth curves and hemolytic activity for G. instriatum
in replete, N-limited, and P-limited media during March 2009 experiment ......................31
11.
Growth rates (k) of G. instriatum in replete, N-Limited, and P-limited
media during March 2009 experiment ...............................................................................32
12.
Relative fluorescence growth curves and hemolytic activity for G. instriatum
in replete, N-limited, and P-limited media during June 2009 experiment .........................35
13.
Growth rates (k) of G. instriatum in replete, N-Limited, and P-limited
media during June 2009 experiment ..................................................................................36
14.
Cell counts of G. instriatum in monthly New River samples at the
Northeast Creek site for 2007 and 2008 ............................................................................39
15.
Cell counts of G. instriatum in monthly New River samples at the
Brinson Creek site for 2007 and 2008 ...............................................................................40
vii
INTRODUCTION
The phytoplankton species Gyrodinium instriatum Freudenthal et Lee is a photosynthetic
dinoflagellate that is widely distributed around the world and is capable of growing in a variety
of conditions. First described by Freudenthal and Lee in 1963 from Long Island Sound in New
York, G. instriatum was maintained axenically and it was discovered that the organism changes
size and shape as the cultures age (Freudenthal and Lee 1963). Gyrodinium instriatum is found
worldwide, as it has been observed in the waters of Japan (Kojima and Kobayashi 1992, Uchida
et al. 1995, Matsuoka 1999), China (Wang et al. 2001), Taiwan (Su and Chiang 1991), Ecuador
(Jimenez 1993), and Portugal (Silva 1982), as well as along the Atlantic coast of the United
States including New York, Delaware, Maryland, North Carolina, South Carolina, and Florida
(Tomas et al. 2004). In North Carolina, it occurs in both the New River and the Cape Fear River;
the clone used for this research was isolated from the New River. Blooms in temperate East
Coast environments can reach 106 cells•L-1 (Tomas et al. 2004).
As an unarmored dinokont, G. instriatum has an outer membrane called the amphiesma,
does not have cellulosic thecal plates, and the cell is divided into an anterior epicone and a
posterior hypocone, separated by a transverse girdle, or cingulum (Fig. 1). A narrow apical
groove, located on the uppermost region of the epitheca, is an elliptical structure circling the
apical end (Fig. 1). In G. instriatum, the epitheca at the apex of the cell tapers to a flattened end
and is usually devoid of pigment, while the hypotheca at the anapex of the cell is bi-lobed. G.
instriatum has two ventrally inserted flagella, allowing the species to swim with a forward,
rolling motion. Its transverse flagellum encircles the middle of the cell in the cingulum, provides
forward propulsion, and is helical shaped, causing the cell to rotate in a distinctive spiral motion.
Figure 1. Line drawing of a Gyrodinium instriatum cell, showing the described features.
2
The longitudinal flagellum which steers the cell and controls direction, projects from the
posterior end of the cell and is inserted in the sulcus, a longitudinal groove on the ventral surface
of the cell that is perpendicular to the cingulum. At 20°C, G. instriatum swims at an average
linear rate of 319 µm•second-1 (Lee 1999), allowing it to swim approximately 13.8 meters in
twelve hours and indicating that it is easily capable of vertical migration in a 10 meter water
column.
In the past, the main criterion used to distinguish the genus Gyrodinium was displacement
of the cingulum more than one-fifth the cell length. In accordance, one end of the Gyrodinium
instriatum cingulum is displaced about one-third the body length lower in a descending spiral
(Fig. 1). However, Daugbjerg et al. (2000) state that the cingulum displacement criterion is
unsatisfactory because the separation distance can vary within a clonal culture. Using scanning
electron microscopy (SEM), they determined that the characteristic feature of the genus
Gyrodinium is the morphology of the apical groove, rather than the cingulum. They concluded
that many species previously called Gyrodinium should be classified as Gymnodinium
(Daugbjerg et al. 2000). Although they did not specifically deliberate about Gyrodinium
instriatum, the organism has a horseshoe-shaped apical groove; thus, it has recently been called
Gymnodinium instriatum (Coats and Park 2002). However, this name is not fully accepted
because further research, including molecular studies, is needed to resolve the issue for G.
instriatum in particular, so the original name of Gyrodinium is used here.
G. instriatum is a medium to large sized species with a width of 23-46 µm and a length of
29-66 µm (Freudenthal and Lee 1963, Handy et al. 2007). The cell is typically oval-shaped, so it
is broadest near the cingulum and tapers at each end. However, it is able to stretch and change
shape in different conditions and the various morphologies can make identification difficult. G.
3
instriatum returns to its characteristic shape when isolated and grown in culture in the laboratory
(Tomas et al. 2004). The cell is also somewhat difficult to identify when preserved with Lugol’s
potassium iodine solution because it stains dark brown and its distinctive features cannot be
clearly seen.
Gyrodinium instriatum has mixotrophic capabilities, fuctioning as either an autotroph
conducting photosynthesis, a heterotroph consuming loricated ciliates, or both (Uchida et al.
1997). While the number of chloroplasts within G. instriatum varies and its cells can be lightly
or darkly pigmented, the organism is usually a yellow-brown color (Tomas et al. 2004). When
consuming ciliates, G. instriatum is able to change its feeding style according to the size of its
prey. It feeds on smaller ciliates, such as Favella azorica and Eutintinnus tubulosus by direct
engulfment through the posterior end of the sulcus and maintains its normal cell shape after
consuming them (Uchida et al. 1997). However, when it feeds on Favella taraikaensis, G.
instriatum draws the cell out of the lorica and absorbs its cell contents. Since this ciliate is
several times larger than G. instriatum, the dinoflagellate is forced to expand its body size,
temporarily losing its original shape. The fact that this photosynthetic dinoflagellate feeds on
consumers such as ciliates demonstrates a reversal of energy flow in the food chain (Uchida et al.
1997).
Uchida et al. (1995) found that when cultured together, the red tide dinoflagellate
Heterocapsa circularisquama immobilizes G. instriatum, resulting in cell death. This was the
first report of inhibition of phytoplankton growth by cell contact. After contact with
Heterocapsa, G. instriatum becomes elliptical, loses its flagella, and eventually dies by cell lysis.
While intact Heterocapsa cells kill G. instriatum upon contact, a filtrate of the Heterocapsa
culture has no effect, so it is likely that the source of toxicity is localized on the cell surface
4
(Matsuyama et al. 1997). As expected, higher Heterocapsa cell densities increase the chance of
contact between the two organisms and decrease the time required for immobilization and lysis.
In addition, the diatom Skeletonema costatum was shown to have an allelopathic effect on G.
instriatum (Nagasoe et al. 2006a) and the parasitic dinoflagellate Amoebophrya ceratii caused
mortality in G. instriatum populations (Coats and Park 2002). The haptophyte Prymnesium
parvum releases potent exotoxins called prymnesins, which break down the membrane of G.
instriatum. Thus, when exposed to P. parvum, G. instriatum quickly becomes immobilized and
begins to lyse, releasing its cell contents on which P. parvum then feeds (Tomas, unpublished
data).
Gyrodinium instriatum can reproduce both sexually and asexually, and the nucleus
always contains condensed chromosomes (Fig. 1). Gametes of G. instriatum are haploid and are
usually isogamous, although anisogametes sometimes occur as well. The gametes have a
tendency to clump together, which increases their chances of successfully pairing, particularly
when there are low gamete densities (Uchida et al. 1996); however, the cause of this clumping
behavior is uncertain. Studies found that the fusion of gametes is completed 60 to 75 minutes
after they pair (Uchida et al. 1996). The diploid planozygote that forms from the fusion of two
haploid gametes resembles a vegetative cell, except it has two longitudinal flagella. It only has
one transverse flagellum because the other is absorbed. This planozygote enlarges and within six
days of the gamete fusion, it divides into two normal vegetative cells, each with one longitudinal
flagellum. These haploid daughter cells continue multiplying asexually through binary fission,
and sometimes the vegetative cell undergoes a resting period by forming a non-motile temporary
cyst. As an alternative to meiotic division, the diploid planozygote can form a permanent cyst
5
called a hypnozygote if its surroundings are too harsh. This non-motile cyst is durable and forms
when the planozygote absorbs its flagella and thickens its cell wall.
Cyst formation results in a dormant stage that protects the organism, allowing it to
withstand difficult conditions until the environment becomes more favorable. Excystment
begins in a less severe environment with increased temperatures and light levels. A motile cell is
released from the cyst by exiting through an opening at the anterior end of the cyst called the
tremic archeopyle. The cell exits as a diploid planomyocyte with two longitudinal flagella. Like
the planozygote, the planomyocyte undergoes meiosis to produce two haploid vegetative cells.
These daughter cells then continue to asexually reproduce, and all divisions are mitotic until a
cellular cue signals that it is time for gamete formation again, and then gamete pairing and fusion
generate another planozygote.
Gyrodinium instriatum is known to have at least two different cyst morphotypes.
Morphotype I is ovoidal, has a smooth surface with no ornaments, and is sometimes covered
with a mucus layer (Matsuoka 1999). Morphotype II, discovered by Kojima and Kobayashi
(1992) in sediments of Lake Hamana, Japan, has an external form similar to that of the motile
cell, so morphological characteristics such as a paracingulum and parasulcus are present. It is
thought that these cell-like cysts form only in extremely reducing environments (Kojima and
Kobayashi 1992).
Upon examination of sediment cores from the coastal waters in the Yokohama Port in
Tokyo Bay, Japan, Matsuoka (1999) found that cysts of G. instriatum began to appear in that
area around the year 1900. In the dinoflagellate cyst assemblages from these sediments, the
relative frequency of G. instriatum cysts was ≤ 5% until approximately 1995 when their cysts
became much more dominant and the frequency rose to 53% (Matsuoka 1999). This high
6
abundance of G. instriatum cysts infers that there was likely a large G. instriatum bloom in
Yokohama Port.
Strains of Gyrodinium instriatum can contain endocytoplasmic and endonuclear gramnegative bacteria (Biegala et al. 2002). Depending on the origin of the strain, the bacteria may
be present only in the cytoplasm, or they may grow in both the cytoplasm and the nucleus (Silva
and Franca 1985). Bacteria are able to enter the dinoflagellate cells when the culture medium is
inoculated with bacteria prior to transfer or during the log phase of growth (Silva 1982). The
bacteria are able to divide intracellularly during the host cell division and are transmitted to
daughter cells. In addition, the intracellular bacteria can either be expelled or digested as needed.
The relationship between G. instriatum and endocellular bacteria has been observed for over
twenty years, and this long term association suggests a symbiotic relationship; however, the
specific benefits are unknown (Alverca et al. 2002). The bacteria may influence the host
toxicity, as G. instriatum clones with intensive growth of endonuclear bacteria were found to be
toxic. Yet, clones with only endocytoplasmic bacteria and no endonuclear bacteria were not
found to be toxic (Silva 1982).
In natural systems, competition between phytoplankton species is influenced by
numerous factors, such as varying nutrient concentrations, salinities, temperatures, allelopathy,
and grazing, all of which make it difficult to predict the competitive outcome. A study in the
Indian River Bay, a part of the Delaware Inland Bays (Handy et al. 2007), examined competition
between three dinoflagellates (Karlodinium veneficum, Prorocentrum minimum, and Gyrodinium
instriatum) and the raphidophyte Heterosigma akashiwo. The study site was located in the lower
portion of the bay and had low dissolved phosphate concentrations. G. instriatum was
substantially larger than the other three species and was almost always present at the site;
7
however, it was the least dominant and never reached cell densities higher than 104 cells•L-1
(Handy et al. 2007), suggesting growth was inhibited by nutrient competition. It was assumed
that the low phosphate concentrations restrained the growth of G. instriatum. In addition, there
may have been some inhibition from organisms such as Skeletonema (Nagasoe et al. 2006a) and
Amoebophrya (Coats and Park 2002), which were also present at the site. Another concept to
consider is biomass. Although the cell densities of G. instriatum were lower than the other
species, G. instriatum may have actually comprised a greater amount of biomass because of its
larger cell size.
Nagasoe et al. (2006b) consider G. instriatum a harmful species, so they studied the
effects of temperature, salinity, and irradiance on the growth of G. instriatum in order to explain
the mechanisms responsible for red tides of this species. When G. instriatum isolated from
Hakozaki Harbor, Japan was exposed to forty five combinations of temperatures and salinities,
the relative contributions of temperature and salinity to growth were determined to be almost
equal and the effects of temperature, salinity, and the temperature-salinity interaction on the
growth rates were highly significant (Nagasoe et al. 2006b).
Saturated irradiance for G. instriatum growth was found to be 70 µmol photons•m-2•s-1
(Nagasoe et al. 2006b). As irradiance increased, growth rates leveled off showing no
photoinhibition, even at the maximum irradiance of 180 µmol photons•m-2•s-1. The growth
saturated irradiance of 70 µmol photons•m-2•s-1 is lower than that of several other harmful
dinoflagellates, providing an advantage in turbid environments (Nagasoe et al. 2006b). Even in
water with high light attenuation, G. instriatum can outcompete other organisms because it
requires less light.
8
Growth of G. instriatum occurred at temperatures from 15 to 30°C and salinities from 5
to 40 (Nagasoe et al. 2006b). The maximum growth rate of 0.7 divisions•day-1 occurred at 25°C
and a salinity of 30. Optimum growth rates exceeding 0.5 divisions•day-1 occurred at
temperatures from 20 to 30°C and salinities from 10 to 35. No growth occurred at temperatures
< 10°C or at a salinity of 0. Growth only occurs at a salinity of 5 if the temperature is between
20 and 25°C. Equations developed from this study, along with in situ temperature and salinity
data, were used to estimate the growth rate of G. instriatum and to predict the outbreak of
blooms in the future (Nagasoe et al. 2006b).
The production of hemolysins is a characteristic used to indicate the toxicity of an algal
species (Eschbach et al. 2001). Hemolysins are compounds that lyse red blood cells and even
cause fish kills by rupturing the gill capillaries. Hemolytic activity is often the result of the
production of secondary metabolites, such as long chain polyunsaturated fatty acids, and
secondary metabolites generally accumulate with time. These fatty acids are known to be
hemolytic (Bodansky 1932), as they are able to penetrate cell membranes, tearing them apart and
causing cell lysis.
Some investigators consider Gyrodinium instriatum to be a harmful algal species (Uchida
et al. 1995, Matsuoka 1999, Nagasoe et al. 2006b), but some scientists do not share this view
(Eschbach et al. 2001, Biegala et al. 2002). Eschbach et al. (2001) consider G. instriatum a
harmless dinophyte, despite finding that it had minor to intermediate hemolytic activity. Even
with these differences in opinion, the possible toxicity of G. instriatum has not been fully
studied. It is necessary to clarify this discrepancy and to determine if the organism is noxious or
not, by using hemolytic activity as a measure of toxin production.
9
Gyrodinium instriatum is relevant globally, as well as specifically in North Carolina, yet
it has not been extensively examined and little is known about its potentially toxic effects. It is a
eurythermal and euryhaline organism capable of tolerating a wide range of conditions (Nagasoe
et al. 2006b), but there is little or no published research studying the impact of nutrients. Since
the organism is common inshore where there are variable nutrient inputs, it is necessary to
investigate if they impact its hemolytic activity because the degree of toxicity may depend on the
nutrient conditions. Research has found that other organisms show increased hemolytic activity
and environmental effects such as fish kills when exposed to nitrogen and phosphorus limitation,
so perhaps G. instriatum cell toxicity is similarly influenced by nitrogen and phosphorus stress.
For example, research with Prymnesium parvum (Johansson and Graneli 1999a) and
Chrysochromulina polylepis (Johansson and Graneli 1999b) found that while a small amount of
hemolytic activity was present in non-limited conditions, activity was significantly higher when
cellular growth was limited by nitrogen and phosphorus. Additionally, other species that are
known to be hemolytic produce polyunsaturated fatty acids (Fu et al. 2004), so these secondary
metabolites could possibly be involved in the hemolytic activity of G. instriatum as well. In
order to more completely understand the dynamics of this species, its hemolytic patterns must be
examined and the features that control its virulence defined. Since there is little information
published about G. instriatum and this species is present year-round in both the New River and
the Cape Fear River, as well as around the world (Tomas et al. 2004), the results of this research
benefit both local and international communities by providing valuable information that can be
used when deciding which toxic algal species should be monitored for blooms and the expected
toxicity of G. instriatum according to the current environmental conditions.
10
OBJECTIVES
1) Use clonal cultures of G. instriatum to conduct growth studies in order to investigate its
nutrient utilization and growth patterns.
2) Test the effect of nutrient stress on the hemolytic activity of G. instriatum and explore the
dynamics and patterns of its toxicity. The treatments include nitrogen limited cells and
phosphorus limited cells while the control is nutrient replete cells.
3) Relate laboratory findings to field observations by examining natural G. instriatum
abundance and distribution in archived monthly samples from sites in the New River
Estuary. Compare environmental nutrient information and cell counts to experimental
hemolytic data to discern if G. instriatum can potentially reach toxic concentrations in its
natural environment.
METHODS
Cultivation
Clonal cultures of Gyrodinium instriatum were isolated by Dr. Carmelo Tomas from the
New River Estuary in southeastern North Carolina (Fig. 2) and maintained in a modified (minus
Si) L1 media (Guillard and Hargraves 1993) at a salinity of 30, temperature of 20°C, and
irradiance of 70 µmol photons•m-2•s-1 similar to the optimal conditions reported by Nagasoe et
al. (2006b). Aliquots of the culture were exposed to nutrient stressed conditions of nitrogen and
phosphorus limitation, and compared to a nutrient sufficient or “replete” environment, to assess
the effect on the organism and its toxicity. The treatment groups included cells grown in
11
Figure 2. Map of the New River Estuary in southeastern North Carolina, showing
Northeast Creek and Brinson Creek, where monthly samples were taken for subsequent G.
instriatum cell counts.
12
nitrogen limited media containing an N:P ratio of 4:1, cells grown in phosphorus limited media
with a ratio of 80:1, and the control group of cells grown in nutrient replete media at the 16:1
Redfield ratio.
Oligotrophic offshore Gulf Stream seawater containing < 1 µM nitrogen as NO3- or
phosphorous as PO43- was filtered and adjusted to a salinity of 30 with pyrogen-free water prior
to autoclaving at 15 lbs pressure and 121°C for 20 minutes in Teflon bottles. The sterilized
seawater was used to prepare the media by adding a constant 1.0 ml•L-1 of a 1 µM phosphate
(NaH2PO4) stock solution to each medium and varying amounts of nitrate (NaNO3) to create the
different nutrient ratios for each treatment. The replete 16:1 medium received 1.0 ml•L-1 of a 16
µM nitrate (NaNO3) stock solution, while the N-limited 4:1 medium only received 0.25 ml•L-1 of
the 16 µM nitrate stock. The P-limited 80:1 medium received 1.0 ml•L-1 of an 80 µM nitrate
(NaNO3) stock solution, as the excess nitrogen produced phosphorus limited conditions. (The
nutrient concentrations for each treatment ratio are shown in Table 1.) In addition, 0.5 ml•L-1 of
L1 vitamins and 1.0 ml•L-1 of L1 trace metals were added to each medium. The media were
allowed to acclimate prior to adding 100-300 ml of G. instriatum culture to each Fernbach flask
containing 1.5 liters of the N-limited, P-limited, or replete L1 media. Prior to each experiment,
the inoculating volume was tested to give similar fluorescence readings for each experimental
start. The Fernbachs were kept at 20°C and 70 µmol photons•m-2•s-1 for the duration of each
experiment. Experiments were run from 18 to 25 days after the cells in each treatment reached
stationary phase.
13
Table 1. Nutrient concentrations* for each treatment.
Treatment
N-Limited
Replete
P-Limited
NaNO3
4 µM
16 µM
80 µM
NaH2PO4
1 µM
1 µM
1 µM
* concentrations per liter
14
Growth Measurements
The cultures of G. instriatum were sampled each day and growth was measured using
three methods. The first was monitoring the daily relative in vivo fluorescence of the cultures
using a Turner Designs 10-AU fluorometer. The second method was measuring chlorophyll a,
which involved filtering culture subsamples onto 25 mm GF/F glass fiber filters, extracting the
chlorophyll overnight with 90% acetone, centrifuging, and determining the concentration of
chlorophyll in the supernatant on a Turner Designs 10-AU fluorometer equipped with the
Welschmeyer filter system (Welschmeyer 1994). The third method was conducting cell counts
by fixing 20 ml aliquots of culture with Lugol’s iodine solution and then making direct counts
with a Motic AE21 inverted light microscope using the settling chamber, or sedimentation
method (Utermöhl 1931), in which the samples were settled for at least one hour in 10 ml
sedimentation chambers. Images of G. instriatum were captured using a Zeiss Axio Imager.Z1
compound microscope with Spot RT and a Zeiss Axio cam MRc5 digital camera system. All
calculations and graphing were accomplished using Microsoft Excel and SigmaPlot.
Growth Determinations
Growth curves were generated by plotting the relative fluorescence (log2 of in vivo) over
time. Fluorescence based growth rates were calculated when cultures were in the log growth
phase by using the Guillard method (1973) involving a least squares regression of the linear
portion of the growth curve. The slope of that trendline was equal to the growth rate (k =
divisions•day-1), and both the mean growth rate during the log phase and the maximum growth
rate of the steepest linear portion of the curve were calculated. Statistical significance between
the growth rates was determined by an analysis of covariance using the software program JMP.
15
The cell counts were conducted under the 4x, 10x, 20x, or 40x objective lens, depending
on the cell density of the sample. For sparse samples, the entire chamber was counted using the
4x objective. Otherwise, 10 fields in each chamber were counted in triplicate, making a total of
30 fields. The count values were adjusted for microscope calibrations and for the volume that
was settled to give the total number of cells•ml-1, which could be converted to cells•L-1. The cell
numbers were compared to the relative fluorescence values to verify that a linear relationship
existed between the two measures, allowing fluorescence to be used as a valid estimate of
changes in cell concentration (Fig. 3). Additionally, the chlorophyll a measurements were
compared to the cell count data to ensure that the amount of chlorophyll per cell (pg•cell-1)
remained constant throughout each experiment (Fig. 4).
Erythrocyte Lysis Assay
Analysis of the hemolytic potential of cultures in this study was accomplished using a
modified erythrocyte lysis assay as described by Tatters et al. (2009). This method employs
washed human erythrocytes incubated with dilutions of either cell pellet or cell free supernatant
for 24 hours in the dark at 4°C with ELA buffer as the negative control and saponin
(20µg•125µl-1) as the positive control. The ELA was performed in a 96-well V-bottom
microtitre plate and after incubation the plate was centrifuged in the Hermle Labnet Z383K
centrifuge at 2,500 x g and 4°C for 10 minutes. The resulting supernatant from each well was
transferred to a 96-well flat-bottom microtitre plate and optical density readings were made at a
wavelength of 415 nm using the BioTek PowerWave X plate reader. Data were processed using
the KC Junior software, the optical density readings were corrected for the controls, and the
results for each sample dilution were expressed as a percent of the full lysis control.
16
The direct cell counts were used to normalize the hemolytic assays, and the software
program GraphPad Prism analyzed the data by plotting the percent lysis versus the log of
cells•well-1 according to each dilution (Appendix A). The program then used the relationship
between the points on the curve and a sigmoidal dose-response equation to calculate an EC50,
which is the effective concentration of cells required to lyse 50% of the erythrocytes, or the cell
concentration that provoked a response halfway between the baseline and maximum response. If
the samples failed to meet a 50% lysis, the software program computed the expected EC50 by
estimating the amount of cells that would result in 50% lysis. Since increased biomass can cause
increased lytic activity, it was necessary to standardize the responses according to cell number,
so these estimated EC50 values allowed for a more accurate comparison between treatments and
across experiments. The GraphPad Prism program also calculated 95% confidence intervals for
each EC50 value, and these intervals were compared between treatments to determine if the
differences between the EC50 values were significant. If the intervals did not overlap, then the
result was considered significant (p < 0.05).
Natural Samples
Monthly samples from stations along the New River were obtained from the North
Carolina Department of Environment and Natural Resources (NC-DENR) and were preserved by
the addition of Lugol’s solution. These archived natural samples were explored to identify a
seasonal distribution of where and when Gyrodinium instriatum occurred in the river, in order to
make comparisons between laboratory findings and environmental data. The sedimentation
method described above (Utermöhl 1931) was used to conduct cell counts with the 2007 and
2008 monthly natural samples from Northeast Creek and Brinson Creek (Fig. 2). These sites
17
were chosen because previous samples from this estuary indicated that G. instriatum was
consistently present there. The monthly G. instriatum cellular concentrations at each site were
compared to the laboratory experimental EC50 values obtained from the hemolytic assays, along
with nutrient data obtained from NC-DENR, to determine the likelihood of bloom mediated
hemolysis in the natural waters.
RESULTS
Since in vivo fluorescence of G. instriatum was to be used as an estimate of cell number,
the relationship between actual cell concentration and relative fluorescence was examined (Fig.
3). A two-fold increase in the fluorescence reading was observed over a four-fold increase in
cell number, and the fluorometric scale of 1.5 to 3.5 relative units at cellular concentrations from
0.5 to 2.0x106 cells•L-1 was within the range of planned experiments. The direct relationship
measured between these two variables provided a regression coefficient (R2) of 0.96, supporting
the use of fluorescence as a valid estimator of cellular concentration changes. Another
requirement for using in vivo fluorescence to estimate cell number was that the cellular
chlorophyll must be relatively constant. For the trial experiment lasting 18 days (Fig. 4), cellular
chlorophyll a (pg•cell-1) varied evenly between 35 and 65 pg•cell-1, and mainly hovered around
the 52 pg•cell-1 mean. Although variable, the chlorophyll a appeared to be consistent enough to
support the use of fluorometric measurements as a reliable proxy for growth and cell numbers.
For the hemolytic assays, both the cells and supernatant were tested each time, but the hemolytic
activity always occurred in the cell pellet, while the supernatant portion never showed any
18
3.5
Relative Fluorescence
y = 1.0032x + 1.4937
R² = 0.9551
3.0
2.5
2.0
1.5
0.0
0.5
1.0
1.5
2.0
2.5
6
Cells/L (x 10 )
Figure 3. Comparison of relative fluorescence values to actual cell count numbers,
confirming that the two are linearly correlated, so G. instriatum fluorescence can be used as
a proxy for cell number.
19
90
80
Chl a/cell (pg/cell)
70
60
50
40
30
20
10
0
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18
Day
Figure 4. Graph of chlorophyll a/cell over time, showing that the amount remained
approximately constant, varying evenly around the average. Dotted line indicates the mean
chl/cell.
20
activity, indicating that the hemolytic agents were only present within the cells. Therefore, all of
the given EC50 values are for the cell portion of the culture.
Most experiments were done on monthly intervals and are presented in sequence by the
month in which they were conducted. The September 2008 experiment (Fig. 5) examined the
growth of G. instriatum over a 22 day period by measuring the relative fluorescence and
hemolytic activity of the cells in a culture with a complete nutrient addition (replete), as
compared to a culture that was phosphorus limited (P-limited). Both cultures were in
logarithmic growth from day 0 to day 13, after which the replete culture began a gradual cellular
decline, while the P-limited culture stabilized and remained in stationary phase for a few days
longer before showing a similar decline (Fig. 5). Both the mean and maximum growth rates of
the P-limited treatment exceeded that of the replete (Fig. 6), and the mean growth rate over the
log phase of the P-limited cells was significantly higher than the replete (p < 0.005). Regarding
hemolytic activity, the values for the P-limited cells exceeded 60% of the control (Fig. 5B),
while the replete cells reached around 40% lysis (Fig. 5A). In both treatments, the cellular
hemolytic activity was low at the beginning of the experiment and then increased drastically after
day 12 as the cultures entered stationary phase, which is characteristic of secondary metabolites
that build up over time. The highest percent lysis for both was on day 16 and then the activity
began to decline toward the end of the experiment (Fig. 5). There was an inverse relationship
between hemolytic activity and the EC50 values (Table 2). As time passed, the hemolytic activity
of the cells became stronger and the percent lysis increased, so the EC50 decreased because it
required fewer cells to reach 50% lysis. Consequently, the lowest EC50 value of around 3.8x103
cells•ml-1 was reached at the peak of hemolytic activity on day 16 (Table 2). After that, the
hemolytic activity began to decrease, so the EC50 values started going back up because more
21
4.5
90
A) Replete
80
4.0
3.5
60
50
3.0
40
2.5
% Lysis
Relative Fluorescence
70
30
20
2.0
10
1.5
0
4.5
90
B) P-Limited
80
4.0
3.5
60
50
3.0
40
2.5
% Lysis
Relative Fluorescence
70
30
20
2.0
10
1.5
0
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Day
Figure 5. Relative fluorescence growth curves (line graphs) and hemolytic activity (bar
graphs) for G. instriatum in A) replete media and B) P-limited media during September
2008 experiment. Error bars indicate standard deviation.
22
0.30
Growth Rate (k)
0.25
0.20
0.15
0.10
0.05
0.00
Replete
P-Limited
Treatment
Figure 6. Growth rates (k) of G. instriatum in replete and P-limited media during
September 2008 experiment. Black = mean k during log phase, White = maximum k
reached.
23
Table 2. Hemolytic activity (% lysis) and estimated EC50 values (cells•ml-1)
for G. instriatum in replete and P-limited media for each day that an assay
was performed during September 2008 experiment.
Day
Replete % Lysis
Replete EC50
P-Limited % Lysis
P-Limited EC50
6
1.7
3
78.9x10
1.5
3
90.7x10
12
9.5
3
28.8x10 *
16.6
3
15.4x10 *
* = significant differences
24
14
20.8
3
21.1x10
40.0
3
7.6x10
16
39.9
3
9.7x10
62.6
3
3.8x10
20
26.6
3
17.2x10
61.8
3
4.7x10
22
6.9
3
71.6x10 *
21.0
3
8.4x10 *
cells were needed to cause 50% lysis. The P-limited culture had higher hemolytic activities and
therefore lower EC50 values than the replete culture, and the significant difference between the Plimited and nutrient replete EC50 values on days 12 and 22 (Table 2) indicated that phosphorus
deficiency caused the cells to become significantly more hemolytic.
During the November 2008 experiment, which compared nutrient replete and N-limited
cultures of G. instriatum, both failed to thrive, and stationary phase was established by day 9
after a short log phase (Fig. 7). The replete culture reached slightly higher fluorescence values
and also had a significantly higher mean growth rate during the log phase (p < 0.005), while
oddly, the N-limited culture was able to reach a significantly larger maximum growth rate (p <
0.005) (Fig. 8). The cellular hemolytic activities barely reached 10% lysis by the end of the
experiment (Fig. 7), and these low activities gave high EC50 values ranging from 22.0x103 to
374.0x103 cells•ml-1 (Table 3). The N-limited EC50 value on day 8 was significantly higher than
the replete (Table 3), implying that insufficient nitrogen significantly decreased the hemolytic
activity.
To investigate the unusual results of the November experiment, an experiment was begun
with a lower initial inoculum to see if this would extend the log phase. This February 2009
experiment examined a nutrient replete culture and found that the smaller inoculum did in fact
facilitate a more complete growth curve by allowing a more apparent lag phase and a longer log
phase (Fig. 9). The mean growth rate for the log phase was 0.17 and the maximum growth rate
reached was 0.25 divisions•day-1. Based on these results, a March 2009 experiment was
conducted with lowered inoculating volumes, comparing nutrient replete, P-limited, and Nlimited G. instriatum cultures.
25
4.5
90
A) Replete
80
4.0
3.5
60
50
3.0
40
2.5
% Lysis
Relative Fluorescence
70
30
20
2.0
10
1.5
4.5
0
90
B) N-Limited
80
4.0
3.5
60
50
3.0
40
2.5
% Lysis
Relative Fluorescence
70
30
20
2.0
10
1.5
0
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19
Day
Figure 7. Relative fluorescence growth curves (line graphs) and hemolytic activity (bar
graphs) for G. instriatum in A) replete media and B) N-limited media during November
2008 experiment. Error bars indicate standard deviation.
26
0.30
Growth Rate (k)
0.25
0.20
0.15
0.10
0.05
0.00
Replete
N-Limited
Treatment
Figure 8. Growth rates (k) of G. instriatum in replete and N-limited media during
November 2008 experiment. Black = mean k during log phase, White = maximum k
reached.
27
Table 3. Estimated EC50 values (cells•ml-1) for G. instriatum
in replete and N-limited media for each day that an assay
was performed during November 2008 experiment.
Day
Replete EC50
N-Limited EC50
2
3
374.0x10
3
257.1x10
8
3
58.3x10 *
3
103.0x10 *
* = significant difference
28
14
3
164.7x10
3
134.9x10
18
3
26.4x10
3
22.0x10
4.5
Relative Fluorescence
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22
Day
Figure 9. Relative fluorescence growth curve for G. instriatum in nutrient replete media
during February 2009 experiment. Error bars indicate standard deviation.
29
Each of the cultures in the March experiment experienced a lag phase until approximately
day 3 (Fig. 10). However, the log phase did not extend as long as in the February experiment,
but was more similar to the September experiment. The log phase of the P-limited culture lasted
the longest, until around day 13, when it leveled out into the stationary phase plateau and then
eventually began to decline (Fig. 10C). The N-limited growth curve was similar to the replete,
except the N-limited cells reached stationary phase a little earlier, but the cell numbers in both
quickly diminished (Figs. 10A and 10B). The mean growth rates of all three treatments were
between 0.18 and 0.19 divisions•day-1, and the P-limited cells reached the largest maximum
growth rate of 0.26 (Fig. 11). Once more, the hemolytic activity in all treatments became
stronger as the cells began to enter stationary phase, and the activity continued to increase
throughout, so the highest values were at the end of the experiment on day 22 (Fig. 10). In
accordance, the lowest EC50 values around 1.3x103 cells•ml-1 were reached on day 22 (Table 4).
Throughout the experiment, the N-limited culture had the lowest hemolytic activity, reaching
approximately 40% lysis at its peak (Fig. 10B) and an EC50 of 3.3x103 cells•ml-1 (Table 4). The
P-limited cells reached hemolysis levels between 60 and 70% (Fig. 10C), while the activity of
the replete cells was around 55% lysis and then reached 85% lysis on day 22 (Fig. 10A). A
sudden decrease in hemolytic activity occurred in all the treatments on day 15 (Fig. 10), making
the EC50 values higher for that day (Table 4), and this abnormality may have been due to a
human error with the assay. The significant difference between the day 20 P-limited and Nlimited EC50 values (Table 4) suggests that phosphorus stress significantly increased the
hemolytic strength of the cells as compared to nitrogen stress.
The sample dilution curves (Appendix A) show the relationships between the full
strength cells and their dilutions, which contained fewer cells and therefore caused less
30
4.5
90
A) Replete
80
4.0
3.5
60
50
3.0
40
2.5
% Lysis
Relative Fluorescence
70
30
20
2.0
10
1.5
4.5
0
90
B) N-Limited
80
70
3.5
60
50
3.0
40
2.5
% Lysis
Relative Fluorescence
4.0
30
20
2.0
10
1.5
4.5
0
90
C) P-Limited
80
4.0
3.5
60
50
3.0
40
2.5
% Lysis
Relative Fluorescence
70
30
20
2.0
10
1.5
0
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Day
Figure 10. Relative fluorescence growth curves (line graphs) and hemolytic activity (bar
graphs) for G. instriatum in A) replete media, B) N-limited media, and C) P-limited media
during March 2009 experiment. Error bars indicate standard deviation.
31
0.30
Growth Rate (k)
0.25
0.20
0.15
0.10
0.05
0.00
Replete
N-Limited
P-Limited
Treatment
Figure 11. Growth rates (k) of G. instriatum in replete, N-Limited, and P-limited media
during March 2009 experiment. Black = mean k during log phase, White = maximum k
reached.
32
Table 4. Estimated EC50 values (cells•ml-1) for G. instriatum in replete,
N-limited, and P-limited media for each day that an assay was performed
during March 2009 experiment.
Day
Replete EC50
N-Limited EC50
P-Limited EC50
1
3
36.7x10
3
60.5x10
3
31.6x10
8
3
7.9x10
3
6.7x10
3
7.0x10
12
3
3.8x10
3
5.6x10
3
3.6x10
* = significant differences
33
15
3
10.1x10
3
14.6x10
3
4.7x10
20
3
2.8x10
3
7.5x10 *
3
2.2x10 *
22
3
1.3x10
3
3.3x10
3
1.4x10
hemolysis because they were dilutions. The relationship between these points allowed the
software program to estimate the EC50 value for each culture (Appendix A). The hemolytic
activity of each culture increased from day 1 to day 20, while the estimated EC50 values
decreased, again illustrating that inverse relationship. For example, the hemolytic activity of the
P-limited cells increased from approximately 1% to 60% lysis, so the EC50 values decreased
from 3.2x104 to 2.2x103 cells•ml-1 (Appendix A).
The final experiment was conducted in June 2009 and again compared the three nutrient
treatments for growth and hemolytic activity (Fig. 12). The growth curves showed brief lag
phases, and the log phase of both the replete and N-limited cultures lasted through day 13 and
then the cells began to decline rapidly (Figs. 12A and 12B). The P-limited culture again had the
longest log phase and a broad stationary phase that lasted until decline began around day 21 (Fig.
12C). Once again, similar mean growth rates between 0.19 and 0.20 divisions•day-1 were found
for all three treatments, and the maximum growth rates were quite high ranging from 0.25 to
0.30 divisions•day-1, with the N-limited culture having the largest maximum rate (Fig. 13). The
hemolytic results of the June experiment were similar to the November experiment, as the
activities were much lower in all of the treatments (Fig. 12). The P-limited culture again showed
the highest hemolytic activity, but it only reached a hemolysis level of 10% of the control lysis
(Fig. 12C). The replete cells had around 8% lysis (Fig. 12A) and the N-limited again had the
lowest activity with 5% lysis (Fig. 12B). There was still an increase in the hemolytic activities
as the cultures entered stationary phase, but this increase was very slight because the hemolysis
levels were so low (Fig. 12). It was not surprising that the EC50 values for this experiment were
much higher (Table 5), reflecting the weak activity in all treatments. Even at the peak
hemolysis, 38.1x103 cells•ml-1 were required to reach 50% lysis (Table 5), while in previous
34
4.5
90
A) Replete
80
4.0
3.5
60
50
3.0
40
2.5
% Lysis
Relative Fluorescence
70
30
20
2.0
10
1.5
4.5
0
90
B) N-Limited
80
4.0
3.5
60
50
3.0
40
2.5
% Lysis
Relative Fluorescence
70
30
20
2.0
10
1.5
4.5
0
90
C) P-Limited
80
4.0
3.5
60
50
3.0
40
2.5
% Lysis
Relative Fluorescence
70
30
20
2.0
10
1.5
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Day
Figure 12. Relative fluorescence growth curves (line graphs) and hemolytic activity (bar
graphs) for G. instriatum in A) replete media, B) N-limited media, and C) P-limited media
during June 2009 experiment. Error bars indicate standard deviation.
35
0.30
Growth Rate (k)
0.25
0.20
0.15
0.10
0.05
0.00
Replete
N-Limited
P-Limited
Treatment
Figure 13. Growth rates (k) of G. instriatum in replete, N-Limited, and P-limited media
during June 2009 experiment. Black = mean k during log phase, White = maximum k
reached.
36
Table 5. Estimated EC50 values (cells•ml-1) for G. instriatum in replete, N-limited, and
P-limited media for each day that an assay was performed during June 2009 experiment.
Day
Replete EC50
N-Limited EC50
P-Limited EC50
3
3
259.0x10
3
278.8x10
3
301.9x10
8
3
71.9x10
3
101.2x10
3
84.9x10
14
3
38.1x10 *°
3
71.4x10 *†
3
44.5x10 †°
* † ° = significant differences for each day
37
16
3
55.0x10 *
3
70.3x10 *†
3
38.3x10 †
21
3
57.2x10 *°
3
100.4x10 *†
3
42.8x10 †°
24
3
42.7x10 *
3
100.0x10 *†
3
41.3x10 †
experiments with stronger hemolytic activities, the EC50 values were as low as 1.3x103 cells•ml-1
(Table 4). From day 14 to 24, the P-limited EC50 values were consistently between 38.3 and
44.5x103 cells•ml-1. On days 14, 16, 21, and 24, both the P-limited and nutrient replete EC50
values were significantly lower than the N-limited values (Table 5), which showed that
phosphorus limitation again significantly increased the hemolytic activity of the cells over
nitrogen limitation, and also signified that nitrogen limitation again caused cells to have
significantly less activity than the replete cells. There were some variable results between the Plimited and nutrient replete EC50 values (Table 5) because on day 14 the replete culture had a
significantly lower EC50 and therefore higher hemolytic activity than the P-limited while on day
21 the P-limited culture had a significantly lower EC50 and higher activity than the replete.
In order to assess the meaning of the EC50 values relative to natural bloom populations,
archived samples from two stations in the New River Estuary, North Carolina were examined
and counted for G. instriatum cells. The two sites, Northeast Creek and Brinson Creek, were
noted in previous observations to have considerable G. instriatum populations (Tomas,
unpublished data). During the 2007 to 2008 period, G. instriatum was present each month at
both the Northeast Creek and Brinson Creek sites, as there was always at least one cell•ml-1
(Figs. 14 and 15). In general, there were higher cell concentrations in 2008 than in 2007 for both
sites. At the Northeast Creek, there were some elevated values in the spring and summer of both
years, but G. instriatum never occurred at concentrations above 600 cells•ml-1 (Fig. 14). Brinson
Creek had low G. instriatum numbers of under 20 cells•ml-1 throughout 2007 (Fig. 15A). In
2008 at Brinson Creek there were some elevated concentrations, but the organism did not rise
above 700 cells•ml-1, except in an August 2008 bloom, when it reached a concentration of
approximately 1,300 cells•ml-1 (Fig. 15B).
38
100
A) 2007
Cells/ml
80
60
40
20
0
600
B) 2008
500
Cells/ml
400
300
200
100
0
1
2
3
4
5
6
7
8
9
10
11
12
Month
Figure 14. Cell counts of G. instriatum in monthly New River samples at the Northeast
Creek site for A) 2007 and B) 2008.
39
100
A) 2007
Cells/ml
80
60
40
20
0
1400
B) 2008
1200
Cells/ml
1000
800
600
400
200
*
0
1
2
3
4
5
6
7
8
9
10
11
12
Month
Figure 15. Cell counts of G. instriatum in monthly New River samples at the Brinson Creek
site for A) 2007 and B) 2008. * indicates a month when no sample was taken.
40
DISCUSSION
The behavior of Gyrodinium instriatum in culture showed variable responses for growth
and hemolytic activity. The growth rate values provided unclear patterns, as the culture with the
highest maximum growth rate was not necessarily the most successful culture because the
elevated rate was not maintained throughout the entire log phase. This disparity was exemplified
during the November 2008 experiment when the N-limited culture was able to reach the highest
growth rate over a three day period but the replete culture actually reached greater fluorescence
values and maintained a higher mean growth rate over the course of the log phase. The G.
instriatum growth rates found in this study were modest when compared to the growth rates of
other dinoflagellates (Smayda 1997), although growth is rarely normalized for biomass.
Gyrodinium instriatum can survive nutrient stressed environments. The fact that it was
able to grow in both nutrient sufficient and limited conditions without large variation in growth
rate suggests the high tolerance of this organism. The P-limited cultures seemed to reach slightly
higher fluorescence values and then remained in the stationary phase plateau the longest, while
the N-limited and nutrient replete cultures quickly died off, indicating that the organism was
more capable of dealing with phosphorus limitation. Phytoplankton are known for their luxury
consumption of phosphorus, where in replete environments they accumulate internal pools of
phosphate to be utilized under limiting conditions (Kimura et al. 1999). In some species this
luxury storage occurs as polyphosphate while others simply store it as orthophosphate (Kimura
et al. 1999). The G. instriatum cells were not acclimated to the nutrient limited conditions before
the start of each experiment, so it is possible that they were taking up excess phosphorus from
their regular replete media. When introduced to the P-limited media, these cells were able to use
41
their phosphorus stores to keep growing. It was unusual that the growth of the nutrient replete
cultures declined so rapidly because these cells should have had sufficient nutrients available,
enabling them to remain in the log and stationary growth phases longer than the limited cultures.
Since this was not the case, these cells may have lacked some unknown element, or perhaps G.
instriatum simply prefers higher nitrogen concentrations above the 16:1 ratio.
Since the toxin was contained within the cells and the organism did not actively discharge
it, it must cause lysis either by actual contact such as cells passing through the gills of fish, or the
hemolysins are released as stressed G. instriatum cells die and break apart thereby inhibiting
other organisms and helping the remaining G. instriatum cells succeed. Though the organism
can be hemolytic, it is not necessarily harmful because fish kills do not occur every time G.
instriatum is present.
Gyrodinium instriatum cells that were P-limited generally tended to have a higher
hemolytic activity than nutrient replete cells, consistent with results from other species, such as
P. parvum and C. polylepis (Johansson and Graneli 1999a, Johansson and Graneli 1999b). It is
feasible that in nutrient limiting environments where cells are not growing optimally, increased
toxin production is a response to cellular physiological stress and serves as a mechanism to lyse
other cells, not only eliminating competitors, but also allowing the organism to obtain the
nitrogen and phosphorus that are subsequently released and utilize these nutrients for its own
growth. However, the enhanced growth under P-limited conditions implies that these G.
instriatum cells were not as stressed as the replete so it is interesting that the P-limited usually
became more hemolytic.
The P-limited G. instriatum cultures showed greater hemolytic activity than N-limited
cultures, which is also consistent with the C. polylepis research (Johansson and Graneli 1999b).
42
N-limited G. instriatum cells had lower hemolytic activities than replete cells, differing from the
other research showing that N-limited P. parvum and C. polylepis demonstrate increased
hemolytic activity, rather than decreased (Johansson and Graneli 1999a, Johansson and Graneli
1999b). In addition, these other species only exhibit a small amount of hemolytic activity when
grown with sufficient nutrients, whereas considerable activity was often found in the nutrient
replete G. instriatum. As previously suggested, G. instriatum appeared to prefer alternative
nutrient concentrations, implying that maybe the traditional replete ratio of 16:1 was not optimal
for this organism and therefore caused increased hemolysis.
The replete cells occasionally had a slightly higher activity than the P-limited and the
difference between the EC50 values of the various treatments was not always significant,
suggesting that other factors were involved in the dynamics of its toxicity. Overall, the
hemolytic activity of the organism was variable and it was not always strongly toxic. Its toxicity
certainly appeared to increase as the cultures aged and the hemolytic compounds accumulated,
indicating the behavior of a secondary metabolite. Long chain polyunsaturated fatty acids are
secondary metabolites that are known to be hemolytic (Bodansky 1932), and there are many
hemolytic species that produce these compounds, including Gyrodinium aureolum, Fibrocapsa
japonica, Chattonella marina, and C. polylepis (Yasumoto et al. 1990, Marshall et al. 2002), so it
is possible that fatty acids are also involved in the hemolytic activity of G. instriatum, although
fatty acids were not measured in this study. The fact that the N-limited cultures always showed
the least hemolysis implies that perhaps the hemolytic compounds are somehow dependent on
nitrogen. Since fatty acids are composed of carbon, hydrogen, and oxygen, it is unlikely that
nitrogen is involved in their synthesis but it could influence their activity in some other way, if
fatty acids are in fact the active hemolytic compound in G. instriatum.
43
Some experiments showed drastically lower hemolytic activities (Figs. 6 and 11).
Although G. aureolum is known to be ichthyotoxic and hemolytic, it did not show any lytic
activity in an ELA performed by Eschbach et al. (2001), suggesting that the strain lost activity
after being in culture for so long or that there is a certain stimulus in nature that is not present in
the lab setting. For G. instriatum, it is unlikely that the strain simply lost its hemolytic activity
because the amount of hemolysis dropped off suddenly and then returned, rather than slowly
declining over time. Similarly, there appeared to be no absent stimulus because at times the
activity was quite high in the lab cultures. All growth methods were identical and there were no
notable changes in the techniques used from one assay to the next. While the organism has been
in culture for a long time, perhaps it still follows a natural cycle, which somehow influenced its
hemolytic activity. Several raphidophytes, including C. marina, F. japonica, H. akashiwo, and
Olisthodiscus luteus contained completely light-dependent hemolytic agents, as no hemolytic
activity was detected when the assays and incubations were conducted in the dark (Kuroda et al.
2005). This does not seem to be the case with G. instriatum because all of the assays were
performed in the light and were always incubated in the dark as described in the Eschbach
method (2001), and cultures showed both high and low hemolytic activities.
Gyrodinium instriatum was continuously found in the New River Estuary throughout the
two year period at both the Northeast Creek site and the Brinson Creek site (Figs. 13 and 14),
demonstrating its high tolerance to temperature, salinity, and nutrients. Cysts of G. instriatum
were observed in the cold month samples, explaining how it persists year after year. While G.
instriatum cells in the environments examined were possibly hemolytic, their numbers were
generally not high enough to cause substantial toxicity. In the laboratory experiments, high
hemolytic activity accompanied EC50 values of thousands of cells•ml-1, but for the most part
44
throughout 2007 and 2008 at Northeast Creek and Brinson Creek, G. instriatum did not have
concentrations higher than 700 cells•ml-1, suggesting that the organism did not reach toxic levels
at these New River sites. However, the G. instriatum concentration at these two sites was not
necessarily representative of the New River as a whole.
An exception occurred when the G. instriatum concentration rose above 700 cells•ml-1,
reaching 1.3 x 103 cells•ml-1 in Brinson Creek in August of 2008 (Fig. 14). This concentration is
comparable to the EC50 values found at the end of the March 2009 experiment when the
hemolytic activity of both the P-limited and nutrient replete cells was very high (Table 3). Thus,
G. instriatum numbers in the August 2008 Brinson Creek sample were high enough that if the
cells had extremely elevated hemolytic activity because of phosphorus limitation or other
reasons, they would have been capable of causing toxic effects. However, research shows that
the New River Estuary actually has strong year-round nitrogen limitation due in part to the
sewage treatment plant effluent loading, which contains excessive phosphorus levels, thereby
causing low N:P ratios (Mallin et al. 1997). Accordingly, nutrient data from the Northeast and
Brinson Creek sites on each sampling day consistently showed nitrogen limited waters.
Excluding the Brinson Creek samples from February and March 2007, the N:P ratios did not
exceed 10:1 and reached as low as 0.4:1 (Appendix B). On the August 2008 sampling day when
the Brinson Creek cell count was so high, the N:P ratio was only 0.5:1 (Appendix B). This
extreme nitrogen limitation should cause the G. instriatum cells to be less hemolytic, so cell
concentrations much higher than 1.3 x 103 cells•ml-1 would be needed to produce toxic effects.
The cell density and nutrient conditions of each individual environment must be considered
when predicting the toxic capabilities of G. instriatum.
45
The nutrient preferences of G. instriatum need to be studied further to see if the organism
continues to respond favorably to nitrogen concentrations above the 16:1 ratio and to learn more
about its ability to store phosphorus. It would be interesting to see if different results are
obtained from growth experiments in which the organism is acclimated to the nutrient limited
conditions prior to the start of the experiment. Future research could involve the growth and
hemolytic responses to trace metal limitation and to other nutrient sources, such as organic
nitrogen and phosphorus. It would be useful to explore the effects of increased nutrient
limitation to determine if the hemolytic activity of G. instriatum becomes more pronounced.
Additionally, it would be interesting to consider seasonality and investigate if the time of year
has any effect on the level of hemolysis. Further research is also needed to identify precisely
what hemolytic compounds are produced by Gyrodinium instriatum and to examine the fate of
these hemolysins in order to determine whether they degrade or accumulate in the sediments,
possibly causing toxicity. Also, future studies could examine additional environments to see if
the organism is able to reach toxic levels, and more frequent sampling would be beneficial, in
order to obtain a better understanding of how the cell concentrations and potential toxicity vary
in the natural environment.
46
CONCLUSIONS
1) Gyrodinium instriatum shows variable responses for growth and hemolytic activity. The
growth rates found in this study are modest compared to other dinoflagellate species.
2) The organism is able to survive and grow in various nutrient environments, including
phosphorus and nitrogen stress, and is better able to deal with phosphorus limited
conditions.
3) In general, its hemolytic activity is increased by phosphorus limitation and decreased by
nitrogen limitation as compared to the nutrient replete control.
4) The hemolysins are not released into the supernatant, but are retained in the cell, and they
build up over time like secondary metabolites.
5) In the two natural New River samples that were examined, the cell concentrations and
nutrient information indicate that G. instriatum is unlikely to reach substantial toxicity at
those specific sites.
47
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51
APPENDIX
70
1.5
A) Replete
EC50 = 36,668 cells/ml
1.0
A) Replete
EC50 = 2,750 cells/ml
60
50
40
30
0.5
20
10
0
70
0.0
1.5
% Lysis
B) N-Limited
EC50 = 60,467 cells/ml
1.0
B) N-Limited
EC50 = 7,517 cells/ml
60
50
40
30
0.5
20
10
0
70
0.0
1.5
C) P-Limited
EC50 = 31,644 cells/ml
1.0
C) P-Limited
EC50 = 2,215 cells/ml
60
50
40
30
0.5
20
10
0.0
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Log of Cells/well
0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Log of Cells/well
Appendix A. Sample dilution curves from hemolytic assays, used to estimate EC50 values
for G. instriatum in A) replete media, B) N-limited media, and C) P-limited media during
March 2009 experiment. Left column = Day 1 and right column = Day 20.
52
Appendix B. NC-DENR New River nitrogen and phosphorus data (µM) from A) Brinson
Creek and B) Northeast Creek with N:P ratios (µM) showing that the sites were mainly Nlimited. N:P was calculated by combining the NH3 and NO2 + NO3 values and dividing by
the P value.
A) Brinson Creek
B) Northeast Creek
Sample
Date
1/11/07
2/22/07
3/13/07
4/23/07
5/29/07
6/27/07
7/11/07
8/6/07
9/17/07
10/9/07
11/14/07
12/12/07
1/8/08
2/7/08
3/18/08
4/24/08
5/27/08
6/18/08
7/16/08
9/3/08
9/30/08
10/14/08
11/6/08
12/4/08
NH3
NO2+NO3
Total P
N:P
2.1
2.9
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
5.0
1.4
1.4
1.4
1.4
6.4
1.4
2.9
2.9
46.4
70.7
68.6
15.0
1.4
1.4
1.4
1.4
1.4
2.9
1.4
11.4
4.3
7.1
35.0
33.6
1.4
1.4
1.4
1.4
7.9
5.7
17.9
8.6
4.8
3.9
3.5
4.5
6.1
6.5
7.1
4.8
4.8
5.2
2.3
5.5
5.2
2.9
5.2
6.1
4.5
7.7
8.1
5.8
3.5
3.9
3.2
5.8
10.0
19.0
19.7
3.6
0.5
0.4
0.4
0.6
0.6
0.8
1.3
2.3
1.1
3.0
7.1
6.3
0.6
0.4
0.4
0.5
4.0
1.8
6.4
2.0
Sample
Date
1/11/07
2/22/07
3/13/07
4/23/07
5/29/07
6/27/07
7/11/07
8/6/07
9/17/07
10/9/07
11/14/07
12/12/07
1/8/08
2/7/08
3/18/08
4/24/08
5/27/08
6/18/08
7/16/08
9/3/08
9/30/08
10/14/08
11/6/08
12/4/08
53
NH3
NO2+NO3
Total P
N:P
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
10.0
7.9
1.4
2.9
1.4
1.4
1.4
1.4
1.4
2.1
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
2.9
1.4
2.9
2.6
2.6
2.6
2.6
5.2
6.5
3.2
3.2
2.3
1.6
1.9
1.9
3.2
3.5
2.9
3.2
4.5
5.8
2.6
1.6
1.9
2.3
1.9
3.9
3.6
1.1
1.7
1.1
0.6
0.4
0.9
0.9
1.6
1.8
1.5
1.5
0.9
0.8
1.0
0.9
0.6
0.5
1.1
1.8
1.5
1.9
1.5