AN
ABSTRACT
OF
THE
THESIS
OF
Aaron
J.
Hartz
for
the
degree
of
Master
of
Science
in
Oceanography
presented
on
May
26,
2010.
Title:
Investigating
the
Ecological
Role
of
Cell
Signaling
in
Free‐Living
Marine
Heterotrophic
Protists.
Abstract
approved:
Barry
F.
Sherr
Phagotrophic
protists
are
major
consumers
of
microbial
biomass
in
aquatic
ecosystems.
However,
biochemical
mechanisms
underlying
prey
recognition
and
phagocytosis
by
protists
are
not
well
understood.
We
investigated
the
potential
roles
of
cell
signaling
mechanisms
in
chemosensory
response
to
prey,
and
in
capture
of
prey
cells,
by
a
marine
ciliate
(Uronema
sp.)
and
a
heterotrophic
dinoflagellate
(Oxyrrhis
marina).
Inhibition
of
protein
kinase
signal
transduction
caused
a
decrease
in
both
chemosensory
response
and
predation.
Inhibition
of
G
protein
coupled
receptor
signaling
pathways
significantly
decreased
chemosensory
response
but
had
no
effect
on
prey
ingestion.
Inhibitor
compounds
did
not
appear
to
affect
general
cell
health,
but
had
a
targeted
effect.
In
addition,
upon
finding
rhodopsin
in
protein
extracts
from
the
plasma
membrane
of
O.
marina,
an
investigation
into
the
photosensory
behavior
of
O.
marina
was
made.
This
organism
has
a
positive
phototactic
response
to
moderate
levels
of
white
light
and
a
wavelength‐specific
phototactic
response.
O.
marina
response
to
light
was
affected
by
culture
conditions
and
feeding
state.
In
the
presence
of
blue
light,
phytoplankton
emit
red
fluorescence;
results
indicate
that
in
the
presence
of
blue
light,
O.
marina
exhibited
a
positive
phototactic
response
to
a
simulated
“patch”
of
phytoplankton
suggesting
the
use
of
the
photoreceptor
to
detect
the
red
phytoplankton
fluorescence.
These
results
support
the
idea
that
cell
signaling
pathways
known
in
other
eukaryotic
organisms
are
involved
in
feeding
behavior
of
free‐living
protists.
As
this
field
of
study
makes
advances
and
more
information
relating
to
biochemical
signaling
in
free‐living
protists
becomes
available,
it
will
give
researchers
new
tools
and
approaches
in
the
study
of
feeding
behavior
in
marine
protists.
©
Copyright
by
Aaron
J.
Hartz
May
26,
2010
All
Rights
Reserved
Investigating
the
Ecological
Role
of
Cell
Signaling
in
Free‐Living
Marine
Heterotrophic
Protists.
by
Aaron
J.
Hartz
A
THESIS
submitted
to
Oregon
State
University
in
partial
fulfillment
of
the
requirements
for
the
degree
of
Master
of
Science
Presented
May
26,
2010
Commencement
June
2011
Master
of
Science
thesis
of
Aaron
J.
Hartz
presented
on
May
26,
2010.
APPROVED:
Major
Professor,
representing
Oceanography
Dean
of
the
College
of
Oceanic
and
Atmospheric
Sciences
Dean
of
the
Graduate
School
I
understand
that
my
thesis
will
become
part
of
the
permanent
collection
of
Oregon
State
University
libraries.
My
signature
below
authorizes
release
of
my
thesis
to
any
reader
upon
request.
Aaron
J.
Hartz,
Author
ACKNOWLEDGEMENTS
The
author
expresses
appreciation
to
the
following
people:
My
major
professor
and
committee,
Barry
Sherr,
Evelyn
Sherr,
Rick
Colwell,
Theresa
Filtz,
and
Mario
Magaña.
I
would
also
like
to
thank
Ricardo
Letellier
for
the
assistance
he
provided.
My
office
mates
Wiley
Evans,
Brandon
Briggs,
Maria
Kavanaugh,
and
Brie
Lindsay.
Robert
Allan,
Lori
Hartline,
and
Robert
Duncan
from
the
student
programs
office.
Additional
friends
and
family,
Norman
Hartz,
Karen
Hartz,
Jeremy
Hartz,
Johnny
Anderson,
Amy
Simmons,
Chris
Holm,
Paul
Walczak,
Jon
Ellenger,
Faron
Anslow,
John
Stevenson,
and
Nate
Meehan.
CONTRIBUTION
OF
AUTHORS
Dr.
Barry
Sherr
and
Dr.
Evelyn
Sherr
were
involved
in
the
design
and
writing
of
manuscripts
in
Chapters
2
and
3.
TABLE
OF
CONTENTS
Page
1
General
Introduction……………………………………………………………………
1
1.1
Overview………………………………………………………………………………….
1
1.2
Receptor
activation/signal
transduction……………………………………..
2
1.3
Behavioral
response
of
phagotrophic
protists
to
environmental
stimuli………………………………………………………………………
1.3.1
Chemosensory
behavior…………………………………………………………..
4
1.3.2
Photosensory
behavior……………………………………………………………
7
1.4
Organisms
used
in
this
study………………………………………………………
9
1.5
Goals
of
this
study………………………………………………………………………
9
2
Using
Inhibitors
to
Investigate
the
Involvement
of
Cell
Signaling
in
Predation
by
Marine
Phagotrophic
Protists………………………………………
2.1
Abstract……………………………………………………………………………………
11
2.2
Introduction………………………………………………………………………………
13
2.3
Materials
and
methods………………………………………………………………
14
2.4
Results
and
discussion………………………………………………………………
17
2.5
Acknowledgements…………………………………………………………………….
24
2.6
References…………………………………………………………………………………
24
3
Photoresponse
in
the
colorless
marine
dinoflagellate
Oxyrrhis
marina
3.1
Abstract……………………………………………………………………………………
27
3.2
Introduction………………………………………………………………………………
31
3.3
Materials
and
Methods………………………………………………………………
32
3.3.1
Cultures…………………………………………………………………………………
32
3.3.2
Flow
cytometric
analysis…………………………………………………………
32
3.3.3
Extraction
and
characterization
of
O.
marina
membrane
33
5
12
29
proteins
3.3.4
Phototaxis
experiments
…………………………………………………………
35
3.3.5
Evaluation
of
cAMP
induction
by
light
……………………………………..
37
3.3.6
Inhibition
of
Rhodopsin
…………………………………………………………
38
TABLE
OF
CONTENTS
(Continued)
Page
3.4
Results
………………………………………………………………………………………
39
3.4.1
Characterization
of
O.
marina
membrane‐associated
proteins
…..
39
3.4.2
O.
marina
response
to
light
………………………………………………………
39
3.4.3
O.
marina
response
to
Chl‐a
fluorescence
…………………………………
40
3.4.4
Light
stimulation
of
intracellular
downstream
signaling
……………
40
3.4.5
Inhibition
of
rhodopsin
function
by
hydroxylamine
…………………..
41
3.5
Discussion
………………………………………………………………………………….
41
3.6
Conclusions
………………………………………………………………………………..
45
3.7
References
…………………………………………………………………………………
47
4
General
Conclusions
……………………………………………………………………
59
4.1
Overview
…………………………………………………………………………………
59
4.2
Chemosensory/inhibitor
study
…………………………………………………
59
4.3
Photosensory
behavior
study
……………………………………………………
61
4.4
What
does
this
mean
for
the
field
of
aquatic
microbial
ecology?
…
65
4.5
Future
studies
…………………………………………………………………………
67
4.6
Closing
remarks
………………………………………………………………………
69
5
Bibliography
………………………………………………………………………………
71
LIST
OF
FIGURES
Figure
Page
1
Flow
cytometry
plot
…………………………………………………………………
52
2
Configurations
for
phototaxis
experimental
setup
……………………
53
3
Maximum
likelihood
tree
of
rhodopsin
proteins
……………………….
54
4
O.
marina
phototaxis
to
white
light
……………………………………………
55
5
O.
marina
phototaxis
to
specific
wavelengths
……………………………
56
6
O.
marina
photoresponse
to
phytoplankton
auto‐fluorescence
…
57
7
Intracellular
cAMP
……………………………………………………………………
58
1 Introduction
1.1 Overview
Phagotrophic protists are important components of the marine ecosystem. They
are significant grazers of bacteria and phytoplankton (Jurgens and Matz 2002; Quevedo
and Anadon 2001). Predation by bacterivorous protists can be sufficient to control
bacterial biomass (Gasol et al. 2002) and to play a role in shaping the genotypic and
phenotypic composition of planktonic bacteria (Jurgens and Matz 2002). Herbivorous
protists, including ciliates and heterotrophic dinoflagellates, also consume a significant
fraction of phytoplankton standing stocks and biomass production in the ocean (Strom et
al. 2001, Calbet & Landry 2004, Calbet 2008). This has implications for the marine
ecosystem since bacteria and phytoplankton influence the major biogeochemical cycles in
the ocean (Azam 1998, Falkowski and Raven 2007). In addition, heterotrophic protists
excrete nitrogen and phosphorus compounds, as well as trace elements such as iron, that
can be utilized by primary producers (Caron and Goldman 1990, Barbeau et al. 1996) and
contribute to the flow of energy to higher trophic levels by serving as a food source for
metazooplankton (Sherr and Sherr 2007; Stoecker and Capuzzo 1990; Calbert and Saiz
2005). Most heterotrophic protists ingest prey via phagocytosis, an ancient mode of
nutrition, most likely predating eukaryotic photosynthesis (Dyer & Obar 1994).
Phagocytosis is widespread among the major phylogenetic branches of protistans (Sherr
and Sherr 2002). Currently the study of heterotrophic protists in aquatic food webs is a
frontier in marine microbial ecology research (Wolfe 2000, Boenigk and Arndt 2002,
Sherr and Sherr 2002, Weiss 2002, Caron et al. 2009). Questions of current interest in
marine microbial ecology are: What determines prey location and selection by
2
phagotrophic protists? How do environmental stimuli affect protist swimming and
feeding behavior?
Heterotrophic protists respond to surrounding stimuli and motile cells can change
their position to find optimal conditions. Protistan response to stimuli can be complex
and may be affected by the physiological state of the cells (Fenchel 2002). In addition,
physiological and behavioral responses to signal molecules via their binding to
transmembrane receptor molecules on the cell surface is a ubiquitious characteristic of
microbes (Hunter 2000, Gomperts et al. 2004, Rosales 2005). These mechanisms consist
of ligand-receptor molecule binding on the cell surface, which initiates an intracellular
signal transduction cascade resulting in a change in behavior as such as engulfment and
digestion of a prey cell, or a change in swimming velocity and direction in response to a
signal (i.e. chemical cues).
Ligand-receptor binding and intracellular signal transduction mechanisms have
been studied extensively in metazoan cells and in a small number of protist species
(Greenberg 1995, Hunter 2000, Dodd and Drickamer 2001, Alberts et al. 2002, Renaud et
al. 2004, Rosales 2005). Although the biochemical basis of the complex feeding
behavior of marine protists has not yet been fully explored, there is good background
evidence that the mechanisms described below are involved in prey ingestion by
phagotrophic protists.
1.2 Receptor activation/signal transduction
Various types of receptor molecules are associated with cell membranes. The
largest family of cell-surface receptors is that of the G protein-coupled receptors (GPCRs;
3
a class of seven-transmembrane receptors) (Alberts et al. 2002). G-protein coupled
receptors are a ubiquitous part of the cellular machinery across a range of eukaryotes,
including protists (Van Haastert and Devreotes 2004) and have the ability to transduce a
variety of signals into the cell including light, amino acids, peptides, sugars, and large
proteins. GPCR’s have been shown to be part of the chemosensory machinery used by
immune cells in mammalian systems (Devreotes and Janetopoulos 2003; Haribabu et al.
1999) and in the protist Dictyostelium discoideum (Manahan et al. 2004; Traynor et al.
2000).
These trans-membrane receptors are activated when stimulated by the binding of
specific signal compounds. This induces a conformational change in the GPCR molecule
that sets off guanine-triphosphate (GTP)-activated phosphorylation pathways in the cell,
leading to changes in cell physiology and behavior. GPCRs are the basic biochemical
machinery for chemosensory and other stimulus-linked responses in eukaryotes (Fawzi et
al. 2001, Alberts et al. 2002). There is evidence that the fundamental elements for GPCRmediated signal transduction are present in phagotrophic protists (Renaud et al. 2004).
Another important class of receptor molecules are lectins, which bind to sugar
moieties of larger biomolecules on the surfaces of prey cells. Such binding can initiate
prey ingestion in phagotrophic cells. There is a large body of work on biochemical
control of phagocytosis in laboratory cultured protists, such as Tetrahymena and
Paramecium (Almagor et al. 1981; Hellung-Larsen et al. 1986; Renaud et al. 1995, 2004;
Leick et al. 1996, 1997; Bell et al. 1998), euglenoid flagellates in the genera Euglena and
Astacia (Levandowsky 2001), parasitic protists such as trypanosomes and Leishmania
(Mosser &, Rosenthal 1993, Ratcliff et al. 1996, Basseri et al. 2002), and in metazoan
4
white blood cells (Greenberg 1995, Ballarin et al. 2002). These studies include
demonstrations of the effect on cell behavior of ligand binding by membrane-bound
receptor molecules, and of signal transduction mechanisms within the cell that are
switched on by ligand-receptor binding (Greenberg 1995, Alberts et al. 2002, Renaud et
al. 2004).
As mentioned above, ligand-receptor binding at the cell surface activates
intracellular signal transduction, in which phosphorylation of specific proteins leads to
molecular processes in the cell that can result in changes in cell behavior, e.g. change in
motility or phagocytosis of a prey cell. The signal transduction activator molecules are
protein kinases (Stoka 1999), which facilitate phosphorylation (adding phosphate
molecules) of compounds in cells. Phosphorylation of specific proteins is known to be a
widely used molecular mechanism involved in switch-like outputs in cell behavior
(Rasmussen et al. 1996, Hunter 2000, Alberts et al. 2002, Gomperts et al. 2004). Tyrosine
kinase proteins in particular are important in phagocytosis in both animal and protist cells
(King et al. 2003, Renaud et al. 2004, Rosales 2005). Components of tyrosine kinase
signaling pathways appear highly conserved from sponges to vertebrates, and have been
found in two choanoflagellates (King and Carroll 2001, King et al. 2003).
1.3 Behavioral response of phagotrophic protists to environmental stimuli
Examples of stimuli that can result in a change in protist behavior include:
detection and orientation with respect to chemical stimuli (chemotaxis, Fenchel and
Blackburn 1999); magnetic fields (magnetotaxis, Bazylinski et al 2000); gravity
(geotaxis, Fenchel and Finlay 1984; Noever, Cronise, and Matsos 1994), and light
5
(phototaxis, Song 1983). To date, the study of chemotaxis has probably received most
attention among the sensory systems in phagotrophic protists.
Protistan response to potential prey can be complex, resulting in selective
ingestion or avoidance of some prey items (Verity 1991, Boenigk and Arndt 2000, Weiss
2002). Previous studies suggest several factors that may be involved in response to
potential prey, including prey size (Fenchel 1987, Gonzalez et al. 1990, Simek and
Chrzanowski 1992, Strom 2000), prey motility (Monger and Landry 1992, Gonzalez et
al. 1993, Matz and Jurgens 2002) and growth state (Gonzalez et al. 1993, Flynn et al.
1996), prey cell surface hydrophobicity (Monger et al. 1999, Matz and Jurgens 2001,
Matz et al. 2002), and chemical composition which affects ‘taste’ of prey cells (Verity
1991, Wolfe 2000). A combination of these factors is likely involved. Prey selection or
rejection is also likely to involve cell signaling events which are initiated by binding of
specific ligand compounds to transmembrane receptor molecules (Hartz et al. 2008,
Wooten et al. 2007).
1.3.1 Chemosensory behavior
Several studies have shown heterotrophic protists to utilize chemosensory
mechanisms to move towards an attractant as a proxy for prey cells (e.g. Fenchel and
Blackburn 1999; Fenchel and Jonsson 1988; Snyder 1991; Manahan et al. 2004).
However, these studies do not provide details on what type of receptors are involved or
the resulting intracellular signaling pathways that lead to a change in motility (speed
and/or direction) or initiation of prey ingestion (phagocytosis).
6
A variety of chemicals have been shown to affect the movement and, in some
cases, the grazing rate, of phagotrophic protists, both positively and negatively (Wolfe
2000). For example, chemicals that have higher concentrations in patches of
decomposing organic matter such as marine snow aggregates or decaying fecal pellets
could signal a bacterial food patch (Sibbald et al. 1987, Fenchel and Blackburn 1999).
Previously demonstrated chemo-attractants for protists include ammonium, amino acids,
fatty acids, peptides, and bacterial and phytoplankton prey (Hellung-Larsen et al. 1986;
Sibbald et al. 1987; Bennett et al. 1988; Van Houten and Preston 1988; Verity 1988,
1991; Fenchel and Blackburn 1999, Martel 2006). Repellant molecules can also cause
changes in protistan behavior. Several studies show that compounds associated with
phytoplankton sulfur chemistry, DMS and DMSP, act as sublethal chemical defenses
against protist grazers and can decrease prey ingestion rates (Wolfe and Steinke 1996,
Wolfe et al. 1997, Strom et al. 2003 a&b).
A study by Wootton et al. (2007) has shown that a cell surface mannose-binding
lectin is directly involved in prey recognition by the heterotrophic dinoflagellate Oxyrrhis
marina. Furthermore, Strom et al. (2007) suggested that dissolved free amino acids (Lenantiomers) serve as signal molecules that can differentially affect feeding responses in
phagotrophic protists. This finding is quite possibly related to GPCR signaling; GPCR’s
are known to be effective receptors for a variety of peptides (Cooper and Hausman 2007)
and L-amino acids (Conigrave et al 2000).
7
1.3.2 Photosensory behavior
Light is an important physical property of aquatic ecosystems, and has diverse
effects on microbes. Light is of fundamental importance to photosynthetic microbes.
Heterotrophic microbes have also been shown to benefit from light. It is now well known
that many prokaryotes have proteorhodopsins, some of which may act as a light-activated
proton pump, a process termed phototrophy (Beja et al. 2001, Furhman et al. 2008).
Rhodopsin-based proton pumps are not limited to prokaryotes; they have also been found
in the marine macro-alga Acetabularia acetabulum (Tsunoda et al. 2006) and in the
fungus Leptosphaeria maculans (Waschuk et al. 2005). In addition, sensory rhodopsins
are found in many species in Domains Archaea, Bacteria, and Eukarya (Sharma et al.
2006, Spudich, 2006) and are involved in photosensory behavior such as phototaxis.
Photosensory behavior in algae is relatively well studied (e.g. review of Hegemann
2008), but is less well known for heterotrophic protists.
Phototaxis has been investigated in only a handful of heterotrophic protist species,
and even less attention has been given to the ecological advantages phototaxis might
entail for a non-photosynthetic protist. Responses to light by heterotrophic protists are
diverse and can vary even among species of the same genus. Some examples of
response to light include the following observations: Amoeba proteus locomotion can be
oriented in the direction of a light gradient (Mast 1910) and the rate of movement of the
amoeba is dependent on light intensity (Mast and Stahler 1937). The ciliate
Chlamydodon mnemosyne exhibits positive phototaxis during starvation and negative
phototaxis when well fed (Selbach, Hader, and Kuhlmann 1999). The ciliate Stentor
coeruleus moves away from light while Stentor niger moves toward light and Stentor
8
polymorphus exhibits no response to light. Another ciliate Blepharisma japonicum is
positively phototactic to blue light and photophobic to red light (Song 1983). Zoospores
of Ulkenia sp., which consume bacteria, exhibit positive phototaxis to bioluminescence
produced by the bacteria Vibrio fischeri (Amon and French 2004).
In order for phototaxis to occur, a photoreceptor must receive the initial light
stimuli, leading to an intracellular signal transduction cascade, and ending in the
photoresponse (Song 1983). Classes of photoreceptors suggested to be involved in
protist photoresponse include flavins (the ciliates Loxodes sp., Finlay and Fenchel 1986,
and Chlamydodon mnemosyne; Selbach and Kuhlmann 1999), hypericins (heterotrich
ciliates; Lobban et al. 2007), and rhodopsins (in the ciliates Blepharisma japonicum,
Stentor coeruleus; Fabczak et al. 2008, Fabrea salina; Podesta et al. 1994, Paranema
trichophorum; Saranak and Foster 2005).
Rhodopsin is a seven-transmembrane photoreceptor in the class of G-protein
couple receptors (GPCR) (Palczewski 2006). Rhodopsin consists of an opsin (transmembrane protein) and a chromophore (retinal). The 11-cis-retinal is located in the
transmembrane region of the opsin and is covalently bound by a protonated Schiff base.
When the chromophore absorbs a photon, it changes form to all-trans-retinylidene,
leading to a conformational change in the opsin (i.e. inactive to active). The active opsin
then binds to intracellular G-proteins and a signal transduction cascade begins
(Palczewski 2006); in the case of a photosensitive protist this would allow the cell to
‘perceive’ the light and could lead to a change in behavior.
9
1.4 Organisms used in this study
The marine phagotrophic organisms used in these studies include a small (15 µm long)
marine hymenostome ciliate, Uronema sp., and the marine dinoflagellate Oxyrrhis
marina. These protists were chosen because they are robust and can be grown to high
density in laboratory culture. The ciliate was isolated from Oregon coastal seawater and
grown on a bacterial isolate cultured from the same seawater. The heterotrophic
dinoflagellate (CCMP 604), obtained from the national marine phytoplankton collection
at the Bigelow Laboratory for Ocean Sciences, West Boothbay Harbor, ME, was
originally isolated from Friday Harbor, WA, and was cultured using an algal prey,
Dunaliella tertiolecta (CCMP 1320) grown in f/2 medium.
1.5 Goals of this study
The goals of this thesis changed throughout the course of the study. This was
partly due to the availability of existing protocols, the ability to modify those protocols,
and the development of new methods. Originally a large portion of the proposed study
was aimed toward an investigation of predator/prey attachment and what cell surface
molecules on the predator and prey were involved. The study quickly changed to an
investigation of intracellular signal transduction following a chemotactic response.
Subsequently, an attempt was made to isolate chemo-receptor molecules from the cell
surface of O. marina, but the only receptors recovered were rhodopsins. After finding a
report by Zhang et al. (2007) that Oxyrrhis marina (strain: CCMP 1795; Long Island
sound, CT, USA) had at least 7 genes encoding for rhodopsins, and realizing that the
capacity of O. marina to respond to light stimuli had not yet been explored, the focus of
10
this study shifted to the photosensory behavior of O. marina as a model for how light can
be used by heterotrophic marine protists to orient in their environment.
The studies reported in the following manuscripts seek to bridge the gap between
cell and molecular biology and ecology. Much is known, for example, about how signals
are transferred from one molecule to the other at the receptor and intracellular level.
However, relatively little is understood about how this relates to actual physiological and
behavioral response at the microbial level in marine ecosystems. This is especially true
in the context of feeding behavior by marine heterotrophic protists. Results from the
following studies provide evidence that sensory responses are involved in the behavior of
both O. marina and Uronema sp., and that O. marina exhibits photosensory response that
may aid in detection of algal prey.
11
Using
Inhibitors
to
Investigate
the
Involvement
of
Cell
Signaling
in
Predation
by
Marine
Phagotrophic
Protists.
Aaron
Hartz,
Barry
Sherr,
Evelyn
Sherr
Journal
of
Eukaryotic
Microbiology
Blackwell
Publishing
Inc.
350
Main
Street
Malden,
MA
02148
USA
Volume
55,
Issue
1,
2008
12
Using
Inhibitors
to
Investigate
the
Involvement
of
Cell
Signaling
in
Predation
by
Marine
Phagotrophic
Protists
AARON
J.
HARTZ,
BARRY
F.
SHERR
and
EVELYN
B.
SHERR
College
of
Oceanic
and
Atmospheric
Sciences,
Oregon
State
University,
Corvallis,
Oregon
97331
Journal
of
Eukaryotic
Microbiology,
55(1),
2008
pp.
18–21
2.1
Abstract
Phagotrophic
protists
are
major
consumers
of
microbial
biomass
in
aquatic
ecosystems.
However,
biochemical
mechanisms
underlying
prey
recognition
and
phagocytosis
by
protists
are
not
well
understood.
We
investigated
the
potential
roles
of
cell
signaling
mechanisms
in
chemosensory
response
to
prey,
and
in
capture
of
prey
cells,
by
a
marine
ciliate
(Uronema
sp.)
and
a
heterotrophic
dinoflagellate
(Oxyrrhis
marina).
Inhibition
of
protein
kinase
signal
transduction
biomolecules
caused
a
decrease
in
both
chemosensory
response
and
predation.
Inhibition
of
G
protein
coupled
receptor
signaling
pathways
significantly
decreased
chemosensory
response
but
had
no
effect
on
prey
ingestion.
Inhibitor
compounds
did
not
appear
to
affect
general
cell
health,
but
had
a
targeted
effect.
These
results
support
the
idea
that
cell
signaling
pathways
known
in
other
eukaryotic
organisms
are
involved
in
feeding
behavior
of
free‐living
protists.
Key
Words.
Cell
signaling,
chemosensory
response,
heterotrophic
protist,
Oxyrrhis
marina,
prey
ingestion,
Uronema
sp.
13
2.2
Introduction
Response
of
free‐living
phagotrophic
protists
to
potential
prey
can
be
complex,
resulting
in
selective
ingestion
or
avoidance
of
prey
cells
(Boenigk
and
Arndt
2000a,
b;
Fenchel
1987;
Strom
2000;
Verity
1991;
Wolfe
2000).
Phagotrophic
protists
also
exhibit
chemosensory
response
to
prey
cells
or
to
attractant/repellant
compounds
(Fenchel
and
Blackburn
1999;
Fenchel
and
Jonsson
1988;
Sibbald,
Albright,
and
Sibbald
1987).
However,
little
is
known
regarding
specific
biochemical
mechanisms
underlying
protistan
feeding
behavior.
At
the
cellular
level,
prey
selection
or
chemosensory
response
is
likely
to
be
based
on
cell
signaling
events
initiated
by
binding
of
specific
ligand
compounds
to
receptor
molecules
associated
with
cell
membranes.
Ligand–receptor
binding
at
the
cell
surface
can
activate
intracellular
signal
transduction,
in
which
phosphorylation
of
specific
proteins
(e.g.
G
proteins
or
protein
kinases)
leads
to
processes
in
the
cell
that
can
result
in
changes
in
cell
behavior,
such
as
change
in
motility
or
phagocytosis
of
a
prey
cell.
Phosphorylation
of
specific
proteins
is
known
to
be
a
widely
used
molecular
mechanism
involved
in
switch‐like
outputs
in
cell
behavior
(Alberts
et
al.
2002;
Gomperts,
Kramer,
and
Tatham
2004;
Hunter
2000;
Rasmussen
et
al.
1996).
Tyrosine
kinase
proteins
in
particular
are
important
in
phagocytosis
in
both
animal
and
protist
cells
(King,
Hittinger,
and
Caroll
2003;
Renaud
et
al.
2004;
Rosales
2005).
G‐protein
coupled
receptors
(GPCRs)
are
a
ubiquitous
part
of
the
cellular
machinery
in
a
range
of
eukaryotes,
including
protists
(Van
Haastert
and
Devreotes
14
2004).
GPCRs
have
been
show
to
be
part
of
the
chemosensory
machinery
used
by
immune
cells
in
mammalian
systems
(Devreotes
and
Janetopoulos
2003;
Haribabu
et
al.
1999)
and
in
the
slime
mold
Dictyostelium
discoideum
(Manahan
et
al.
2004;
Traynor
et
al.
2000).
Components
of
G‐protein
and
tyrosine
kinase
signaling
pathways
appear
highly
conserved
from
sponges
to
vertebrates,
and
have
been
found
in
choanoflagellates
(King
and
Carroll
2001;
King
et
al.
2003).
The
genomes
of
two
ciliates
(Tetrahymena:
www.ciliate.org;
Paramecium:
http://paramecium.cgm.cnrs
gif.fr/db/index)
include
genes
involved
in
G‐protein
pathway
signaling.
We
used
specific
inhibitor
compounds
to
investigate
the
extent
to
which
cell
signaling
mechanisms
may
be
involved
in
chemosensory
response
and
predation
by
two
free
living
marine
protists
‐
a
bacterivorous
ciliate,
Uronema
sp.,
and
a
herbivorous
dinoflagellate,
Oxyrrhis
marina.
2.3
Materials
and
Methods
Cultures.
Cultures
were
grown
and
maintained
in
autoclaved
0.2
µm
filtered
seawater
(FSW)
at
~20
°C.
A
small
(15
µm
long)
hymenostome
ciliate,
Uronema
sp.
was
isolated
from
Oregon
coastal
seawater
and
grown
on
a
bacterial
isolate
cultured
from
the
same
seawater.
The
bacterial
isolate
was
maintained
on
agar
slants
(marine
agar;
2216,
BD
Difco,
Franklin
Lakes,
NJ)
and
grown
in
marine
broth
(5.0
g
peptone11.0
g
yeast
extract,
BD
Difco,
L‐1
FSW)
before
experiments.
The
heterotrophic
dinoflagellate
O.
marina
(CCMP
604)
was
cultured
using
an
algal
prey,
15
Dunaliella
tertiolecta
(CCMP
1320)
grown
in
f/2
medium.
Chemical
inhibitors.
Inhibitors
of
specific
cell
signaling
mechanisms
were:
(1)
genistein—an
inhibitor
of
protein
tyrosine
kinase
(Sigma‐Aldrich,
St.
Louis,
MO);
(2)
staurosporine—an
inhibitor
of
phospholipid/calcium‐dependent
protein
kinase
(Sigma‐
Aldrich);
(3)
NF023—an
antagonist
for
G‐protein
a‐subunits
(Calbiochem,
San
Diego,
CA);
(4)
guanosine—a
GDP
analog
that
competitively
inhibits
G‐protein
activation
by
GTP
(Calbiochem);
(5)
GP­ant—an
inhibitor
of
G‐protein
activation
by
GPCRs
(Tocris
Bioscience,
Ellisville,
MO);
and
(6)
SCH­202676—an
inhibitor
of
ligand
binding
to
G
protein
coupled
receptor
(Tocris
Bioscience).
Initial
concentrations
(see
Table
1)
were
chosen
based
on
manufacturers
recommendation
of
IC50
(concentration
required
for
50%
inhibition).
Compounds
(1),
(2),
and
(6)
were
dissolved
in
DMSO
and
(3),
(4),
and
(5)
were
dissolved
in
0.2
µm
FSW.
Ranges
of
concentrations
of
inhibitors
were
added
to
2ml
of
protist
cells
in
late
log
phase
of
growth,
and
equivalent
volume
of
FSW
or
DMSO
(final
concentration
<1
µl/ml)
were
added
to
control
treatments.
All
protist
treatments
were
preincubated
for
30
min
before
testing
for
chemosensory
or
predation
response.
Cells
were
observed
at
200X
magnification
for
irregularities
in
swimming
behavior
as
a
qualitative
assessment
of
general
cell
health
with
and
without
inhibitors.
Chemosensory
assay.
Based
on
the
protocols
of
Sjoblad,
Chet,
and
Mitchell
(1978),
triplicate
1.5‐ml
microcentrifuge
tubes
(Fisher
Scientific,
Waltham,
MA)
containing
0.5‐ml
pre
incubated
protist
cells
(104
cells/ml)
were
laid
horizontally
and
a
15‐µl
glass
16
capillary
(Drummond
Scientific
Co.,
Broomall,
PA)
filled
with
either
FSW
or
FSW+attractant
was
inserted
into
each
tube.
Attractants
used
were
25‐mg
peptone
(BD
Difco)
l‐1
FSW
for
Uronema
sp.
and
D.
tertiolecta
lysate
(concentrated
by
centrifugation,
350
g
for
5
min,
lysed
by
sonication,
vortexed,
and
filtered
through
a
0.2‐µm
filter)
for
O.
marina.
After
1
h,
the
capillary
contents
were
dispensed
into
a
microcentrifuge
tube
containing
0.75
ml
FSW,
fixed
with
paraformaldehyde
(final
concentration
~1%
w/v),
stained
with
DAPI
(Sigma‐Aldrich;
25
µg/ml;
Sherr,
Caron,
and
Sherr
1993),
and
filtered
onto
a
black
membrane
filter
(0.8‐µm
pore
size).
Filters
were
mounted
on
slides
and
protist
cells
on
the
whole
filter
enumerated
via
epifluorescence
microscopy.
Results
were
reported
as
a
chemosensory
index
(cells
in
test
capillaries/cells
in
control
capillaries)
in
which
values
<
1.0
indicate
reduced
chemosensory
response.
Predation
assay.
Prey
cells
were
concentrated
by
centrifugation
(bacteria:
6,400
g
for
5min;
D.
tertiolecta:
350
g
for
5min),
re‐suspended
in
1ml
of
FSW,
and
added
to
duplicate
subsamples
of
protist
cells
at
concentrations
of
107
ml‐1
for
bacteria,
and
105
ml‐1
for
D.
tertiolecta
and
incubated
at
~
20
°C.
Protist
cells
were
allowed
to
graze
on
prey
items
for
3
h.
At
time
0
and
3
h,
0.5
ml
samples
were
transferred
to
2.0‐ml
cryovials,
fixed
with
paraformaldehyde
(final
concentration
~
1%
w/v)
for
10
min,
and
stored
at
‐80
°C.
Thawed
samples
were
analyzed
using
a
FACSCalibur
flow
cytometer
(BD
Bioscience,
Franklin
Lakes,
NJ)
using
protocols
of
Sherr,
Sherr,
and
Longnecker
(2006).
Remaining
prey
items
were
counted
at
time
0
and
3
h
to
evaluate
number
of
prey
consumed.
Bacteria
were
stained
with
SYBR
Green
I
(Invitrogen
Corp.,
17
Carlsbad,
CA;
Marie
et
al.
1997)
before
flow
cytometric
analysis;
bacteria
counts
were
based
on
SYBR
Green
fluorescence
and
D.
tertiolecta
were
detected
based
on
Chl‐a
fluorescence.
Results
were
reported
as
an
index
of
prey
survival
(prey
in
treatment/prey
in
control)
in
which
values
>1
indicate
decreased
prey
ingestion
by
protists.
Apoptosis
test.
The
Vybrant
Apoptosis
Kit
#2
(Invitrogen
Corp.)
containing
propidium
iodide,
which
stains
DNA
in
membrane‐
compromised
cells,
and
AnnexinV‐
AlexaFluor488
(Invitrogen
Corp.),
which
binds
to
externalized
phosphatidylserine
and
is
indicative
of
early
stages
of
programmed
cell
death
(van
Engeland
et
al.
1998)
were
used
to
evaluate
the
possible
artifact
of
the
protein
kinase
inhibitors
initiating
programmed
cell
death
(Elliott,
Scarpello,
and
Morgan
2002).
Protist
cells
were
incubated
with
either
1
µl/ml
DMSO,
50–100
µM
genistein,
250–500
nM
staurosporin,
or
1–2mM
hydrogen
peroxide,
a
known
inducer
of
apoptosis
(Das,
Mukherjee,
and
Shaha
2001).
Flow
cytometry
was
used
to
analyze
protist
cells
for
cell‐specific
change
in
green
and
red
fluorescence
indicating
Annexin
and
PI
binding,
respectively.
2.4
Results/Discussion
Effect
of
inhibitors
on
general
cell
health.
We
evaluated
whether
the
inhibitor
compounds
used
might
have
non‐
specific
effects
on
the
physiology
of
phagotrophic
protists.
Cell
swimming
behavior
did
not
appear
to
be
qualitatively
affected
by
incubation
with
DMSO
or
inhibitor
18
compounds,
except
for
1
µM
SCH‐
202676,
which
resulted
in
complete
cessation
of
movement.
Genistein
and
staurosporine
did
not
induce
programmed
cell
death‐like
symptoms
in
O.
marina
and
Uronema
sp.
at
the
concentrations
used
in
this
study
based
on
AnnexinV/PI
binding
(data
not
shown).
Effects
of
inhibitors
on
chemosensory
response
and
prey
ingestion.
The
protein
kinase
inhibitor
staurosporine
decreased
chemosensory
response
in
Uronema
sp.
(t‐test;
n=3;
P<0.005)
and
O.
marina
(t‐test;
n=3;
P<0.05)
and
predation
in
both
protists
(Table
1).
In
the
ciliate,
genistein
inhibited
the
chemosensory
response
(t‐test;
n=3;
P<0.005)
and
prey
ingestion,
but
not
in
O.
marina
(Table
1A).
SCH‐202676,
an
inhibitor
of
GPCRs
effectively
reduced
the
chemosensory
response
in
O.
marina
(t‐test;
n=3;
P<0.05)
and
Uronema
sp.
(t‐test;
n=3;
P<0.02)
(Table
1).
Two
inhibitors
of
G
proteins
(NF023
and
guanosine)
resulted
in
a
moderate
decrease
in
chemosensory
ability
in
both
protists,
but
had
little
effect
on
predation.
GPant‐2
(25
µM)
decreased
the
chemosensory
response
in
the
ciliate
by
~
70%
(t‐test;
n=3;
P<0.005)
but
had
no
effect
on
the
chemosensory
ability
in
O.
marina
and
no
effect
on
predation
in
either
protist.
Results
from
predation
experiments
(n=2)
were
not
assigned
a
statistical
comparison.
Our
study
is
the
first
to
use
selective
inhibitor
compounds
to
examine
the
role
of
cell
signaling
pathways
in
chemosensory
behavior
and
prey
ingestion
by
free‐living
of
marine
phagotrophic
protists
and
provides
evidence
that
biochemical
mechanisms
involving
cell
membrane‐bound
GPCR
molecules
and
intracellular
signal
transduction
molecules
are
likely
to
be
involved
in
feeding
behavior
of
phagotrophic
protists.
Other
studies
provide
additional
evidence
suggesting
19
biochemical
signaling
mechanisms
are
involved
in
chemosensory
response
and
feeding
behavior
of
free‐living
aquatic
protists.
Leick
et
al.
(1997)
found
that
protein
kinase
inhibitors
eliminated
chemosensory
behavior
in
the
freshwater
hymenostome
ciliate
Tetrahymena
sp.
In
addition,
a
cell
surface
mannose‐binding
lectin
appears
to
be
directly
involved
in
prey
recognition
by
O.
marina
(Wootton
et
al.
2007)
and
Strom,
Wolfe,
and
Bright
(2007)
suggested
that
dissolved
amino
acids
(L‐enantiomers)
may
serve
as
signal
molecules
that
can
affect
feeding
responses
in
a
marine
tintinnid
ciliate.
Ligand
receptor
binding
at
the
cell
surface,
and
signal
transduction
pathways
are
likely
to
control
grazing
behavior
of
marine
phagotrophic
protists.
These
mechanisms
have
been
shown
to
operate
in
‘‘model’’
cells
ranging
from
protists
to
mammalian
white
blood
cells
(Alberts
et
al.
2002;
Devreotes
and
Janetopoulos
2003;
Gomperts
et
al.
2004;
Haribabu
et
al.
1999;
Hunter
2000;
King
and
Carroll
2001;
Manahan
et
al.
2004;
Rasmussen
et
al.
1996;
Renaud
et
al.
2004;
Rosales
2005;
Traynor
et
al.
2000;
Van
Haastert
and
Devreotes
2004).
It
is
likely
that
such
cellular
processes
are
vitally
important
in
determining
prey
selection,
capture
success,
and
rates
of
ingestion
among
heterotrophic
protists
in
the
sea.
Understanding
the
extent
to
which
cell
surface
ligand–
receptor
binding
and
consequent
signal
transduction
pathways
operate
in
protist
prey
selection,
ingestion
rates,
and
other
aspects
of
feeding
behavior
may
result
in
the
development
of
novel
approaches
to
study
of
predator
prey
interactions
in
microbial
communities.
Our
study
suggests
at
least
two
phagotrophic
marine
protists
are
utilizing
20
GPCRs
to
detect
molecules
that
evoke
a
chemosensory
response.
Once
a
stimulus
is
encountered,
intracellular
signaling,
mediated
by
protein
kinase
phosphorylation,
is
probably
involved
in
producing
a
physiological
response
leading
the
protist
to
move
toward
an
attractant
or
ingesting
a
prey
item.
Although
the
results
of
this
study
are
preliminary,
they
do
demonstrate
that
signaling
pathways
studied
in
other
cell
lines
are
involved
in
the
chemosensory
ability
and
phagocytosis
in
marine
protozoa
as
indicated
by
experiments
utilizing
inhibitors.
Additional
studies
are
needed
to
further
elucidate
the
role
of
cell
signaling
in
protist
feeding
behavior.
21
Table
1.
Summary
of
results
for
signal
transduction
inhibition
of
prey
ingestion
and
chemosensory
response
in:
A)
the
heterotrophic
ciliate
Uronema
sp.
feeding
on
a
marine
bacterial
isolate,
and
B)
the
heterotrophic
dinoflagellate
Oxyrrhis
marina
feeding
on
the
marine
alga
Dunaliella
tertiolecta.
Data
for
prey
survival
index
and
chemotaxis
index
are
given
as
mean
values
±
1
standard
error
(SE).
A
prey
survival
index
>
1
indicates
decreased
protist
grazing
on
prey
cells;
a
chemotaxis
index
<
1
indicates
decreased
chemosensory
response,
compared
to
control
treatments
without
the
inhibitor.
Control
values
=
1.
22
(A)
Ciliate,
Uronema
sp.
Cellular
target
Protein
kinase
(general)
Inhibitor
Predation
response
Inhibitor
Conc.
Prey
survival
index
(SE)
G‐protein
G‐protein
G‐protein
coupled
receptor
Conc.
Chemotaxis
Index
(SE)
10
nM
1.4
(0.03)
10nM
0.65
(0.21)
50
nM
4.3
(0.02)
50nM
0.54
(0.22)
100
nM
4.5
(0.2)
Genistein
25
µM
2.2
(0.03)
100
µM
4.0
(0.1)
Guanosine
50
nM
0.84
(0.03)
100
nM
1.6
(0.5)
150
nM
1.4
(0.2)
G‐protein
Inhibitor
Staurosporine
250
nM
4.3
(0.4)
Protein
kinase
(general)
Chemosensory
response
GPant‐2
NF023
25
µM
1.0
(0.01)
500nM
0.82
(0)
1
µM
0.83
(0.05)
SCH‐202676
‐‐‐‐‐‐‐‐‐‐
‐‐‐‐‐‐‐‐‐‐‐
500
nM
1.18
(0.1)
100nM
0.04
(0.014)
‐‐‐‐‐‐‐‐
‐‐‐‐‐‐‐‐‐‐‐‐
25
µM
0.10
(007)
‐‐‐‐‐‐‐‐‐‐
‐‐‐‐‐‐‐‐‐‐‐‐
250
nM
1.1
(0.15)
500
nM
0.84
(0.05)
60
µM
0.61
(0.048)
25
µM
0.31
(0.02)
500
nM
0.81
(0.04)
1
µM
0.89
(0.06)
100
nM
0.92
((0.15)
500
nM
0.10
(0.04)
23
B.
Heterotrophic
dinoflagellate,
Oxyrrhis
marina
Cellular
target
Protein
kinase
(general)
Inhibitor
Predation
response
Inhibitor
Conc.
Prey
survival
index
(SE)
Chemotaxis
response
Inhibitor
Chemotaxis
Index
(SE)
Conc.
Staurosporine
100
nM
1.2
(0.1)
100
nM
0.62
(0.2)
250
nM
1.4
(0.1)
250
nM
0.20
(0.07)
500
nM
1.9
(0.2)
500
nM
0.16
(0.09)
Protein
kinase
(general)
Genistein
G‐protein
Guanosine
50
µM
1.1
(0.1)
100
µM
0.76
(0.28)
NF023
500
nM
1.1
(0.1)
10
µM
0.77
(0.08)
G‐protein
25
µM
1.1
(0.2)
25
µM
1.0
50
µM
1.0
(0.04)
50
µM
0.75
(0.28)
G‐protein
GPant‐2
25
µM
1.0
(0.1)
25
µM
1.0
(0.05)
G‐protein
coupled
receptor
SCH‐202676
‐‐‐‐‐‐‐
‐‐‐‐‐‐‐‐‐‐‐
100
nM
0.93
(0.17)
500nM
1.0
(0.1)
500
nM
0.40
(0.01)
24
2.5
Acknowledgements
Funding
for
this
study
was
provided
by
NSF
grant
OCE‐0647593
to
B.
and
E.
Sherr.
2.6
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27
3
Photoresponse
in
the
Colorless
Marine
Dinoflagellate
Oxyrrhis
Marina.
Aaron
Hartz,
Barry
Sherr,
Evelyn
Sherr
TO
BE
SUBMITTED
Journal
of
Eukaryotic
Microbiology
Blackwell
Publishing
Inc.
350
Main
Street
Malden,
MA
02148
USA
28
Photoresponse
in
the
colorless
marine
dinoflagellate
Oxyrrhis
marina
Aaron
Hartz*
Barry
Sherr
Evelyn
Sherr
College
of
Oceanic
and
Atmospheric
Sciences
Oregon
State
University
29
3.1
Abstract
As
phagotrophic
protists
are
the
major
consumers
of
phytoplankton
and
bacterioplankton
in
the
sea,
it
is
important
to
understand
the
capabilities
of
these
unicellular
predators
to
respond
to
environmental
stimuli
which
may
aid
in
orientation
in
the
environment
and
in
detecting
prey
cells.
In
an
investigation
of
potential
signal
receptor
proteins
in
the
cell
membrane
of
a
strain
of
the
marine
colorless
dinoflagellate
Oxyrrhis
marina,
we
discovered
two
expressed
rhodopsins
which
matched
two
of
seven
rhodopsin‐coding
genes
reported
in
the
NCBI
database
for
another
strain
of
O.
marina.
A
BLASTp
search
based
on
the
rhodopsins
of
O.
marina
showed
them
to
be
most
closely
related
to
a
rhodopsin
of
the
autotrophic
dinoflagellate
Pyrocystis
lunula,
and
to
rhodopsins
of
Bacteria
rather
than
of
Archaea
or
other
eukaryotic
microbes.
We
inferred
these
to
be
sensory
rhodopsins,
a
type
of
G‐protein
coupled
receptor
(GPCR)
trans‐membrane
signaling
molecules.
Since
phototaxic
behavior
based
on
sensory
rhodopsins
has
been
reported
in
other
protists,
we
investigated
the
photosensory
response
of
O.
marina.
The
dinoflagellate
exhibited
strongest
positive
phototaxis
at
low
levels
(2
–
3
µE
m‐2
s‐1)
of
white
light
when
the
cells
were
previously
light
adapted
and/or
starved.
Positive
phototaxis
was
also
found
for
blue
(450
nm),
green
(525
nm)
and
red
(680
nm)
wavelengths.
In
a
further
test,
O.
marina
showed
significantly
greater
phototaxis
toward
concentrated
algal
food,
compared
to
bleached
algae,
illuminated
by
blue
light
to
stimulate
red
chlorophyll‐a
(Chl‐a)
autofluorescence.
Concentration
of
a
cytoplasmic
downstream
messenger
molecule,
cyclic
adenosine
monophosphate
30
(cAMP),
a
component
of
the
signaling
pathway
of
GPCRs,
increased
in
O.
marina
cells
after
exposure
to
white
light.
In
addition,
treatment
with
hydroxylamine,
a
rhodopsin
signaling
inhibitor,
significantly
decreased
phototaxic
response
in
the
dinoflagellate.
Our
results
demonstrate
that
a
colorless
marine
dinoflagellate
can
orient
to
light
based
on
rhodopsin
present
in
the
outer
cell
membrane
and
may
be
able
to
use
photosensory
response
to
detect
algal
prey
based
on
Chl‐a
autofluorescence.
31
3.2
Introduction
How
phagotrophic
protists
orient
in
their
environment
and
locate
prey
is
a
research
topic
of
longstanding
interest.
Chemosensory
response
to
prey
by
marine
heterotrophic
protists
is
well
known
(Sibbald
et
al.
1987,
Verity
1988,
Fenchel
and
Blackburn
1999).
Studies
of
microbial
response
to
light
stimuli
have
an
even
longer
history,
dating
to
the
19th
century.
Verworn
(1889)
noted:
“we
find
that
among
unicellular
organisms,
so
far
as
they
are
irritable
at
all
to
light,
phototaxis
is
a
wide‐
spread
phenomenon.”
Since
that
time,
significant
advances
have
been
made
in
understanding
the
biochemical
basis
of
photosensory
response
in
unicellular
algae
(Kateriya
et
al.
2004,
Hegemann
2008).
Photosensory
capability
in
heterotrophic
protists
has
received
less
attention.
Prior
studies
have
found
variable
photosensory
response
in
an
amoeba
(Mast
and
Stahler
1937),
a
colorless
euglena
(Saranak
and
Foster
2005),
and
several
species
of
ciliates
(Song
1983,
Fenchel
and
Finlay
1986,
Podesta
et
al.
1994,
Selbach
et
al
1999,
Cadetti
et
al.
2000,
Sobierajska
et
al.
2006,
Lobban
et
al.
2007,
Fabczak
et
al.
2008).
The
marine
heterotrophic
dinoflagellate
Oxyrrhis
marina
is
widely
distributed
in
coastal
habitats,
ranging
from
tidal
pools
to
open
coastal
water,
exhibits
a
high
degree
of
genetic
and
functional
diversity,
and
has
been
used
as
a
model
marine
protist
in
laboratory
experiments
(Lowe
et
al.
2005,
Menden‐Deuer
and
Grumbaum
2006,
Wooten
et
al.
2007,
Hartz
et
al.
2008).
The
findings
we
report
here:
that
sensory
rhodopsins
present
in
the
outer
cell
membrane
of
a
strain
of
O.
marina
may
be
the
basis
of
observed
photosensory
response,
expands
the
scope
of
32
behavioral
abilities
of
this
species
and
of
heterotrophic
dinoflagellates
in
general.
Although
sensory
rhodopsins
have
been
described
in
a
few
species
of
ciliates,
fungi,
and
algae
(Ridge
2002,
Ruiz‐Gonzalez
&
Marin
2004,
Sharma
et
al.
2006,
Fabczak
et
al.
2008,
Klare
et
al.
2008),
our
study
is
the
first
to
report
phototaxic
behavior
due
to
a
putative
sensory
rhodopsin
expressed
by
a
colorless
dinoflagellate.
3.3
Materials
and
Methods
3.3.1
Cultures:
Algal
food,
Dunaliella
tertiolecta
(CCMP
1320),
was
grown
on
f/2
medium
made
with
0.2
µm
filtered
autoclaved
seawater
(FASW)
obtained
from
the
Oregon
coast.
Oxyrrhis
marina
(CCMP
604;
originally
isolated
from
Friday
Harbor,
WA,
USA)
was
grown
in
FASW
on
D.
tertiolecta
as
the
sole
food.
All
cultures
were
maintained
at
room
temperature
(20
‐
25
oC)
with
a
12
hour
light/dark
cycle
under
natural
daylight
fluorescent
lamps.
For
photosensory
experiments
that
tested
response
based
on
light
history,
light
adapted
O.
marina
were
grown
with
a
12
hour
light/dark
cycle,
while
dark
adapted
O.
marina
were
grown
for
3
transfer
cycles,
with
approximately
4
days
between
transfers,
in
the
dark
at
room
temperature.
3.3.2
Flow
cytometric
analysis:
The
abundances
and
cell‐specific
Chl‐a
fluorescence
of
the
dinoflagellate
and
algal
prey
were
determined
with
a
Becton‐Dickinson
FACSCaliber
Flow
Cytometer
using
a
488
nm
laser.
Depending
on
cell
abundances,
from
100
to
300
μl
of
sample
were
processed
during
run
times
of
3
‐5
minutes.
BD
CellQuest
software
was
used
to
set
flow
cytometer
photomultiplier
tubes
to
optimize
detection
of
O.
marina
and
33
D.
tertiolecta
cells
based
on
side
scatter
and
FL‐3
(red)
fluorescence
properties
(Figure
1).
In
preliminary
tests,
we
determined
that
preservation
of
O.
marina
with
buffered
formaldehyde
resulted
in
cell
loss,
and
that
preservation
even
with
low
final
concentration
of
acid
Lugol
solution
interfered
with
flow
cytometric
analysis,
so
for
experiments
live
cells
were
promptly
assayed.
The
relative
cell‐specific
red
(FL‐3)
fluorescence
of
O.
marina
was
used
to
assess
feeding
state.
Dinoflagellate
cells
with
ingested
algal
prey
had
FL‐3
fluorescence
signatures
equivalent
to
amount
of
fluorescence
observed
in
one
to
several
algal
cells
(upper
right
box
in
Figure
1).
Cultures
that
had
consumed
algal
prey
to
undetectable
abundance,
but
still
had
traces
of
prey
in
food
vacuoles,
based
on
average
cell‐specific
FL‐3
fluorescence
2‐3
fold
higher
than
that
of
completely
starved
cells,
were
considered
to
be
well fed.
Two
days
after
all
prey
were
consumed,
O.
marina
cells
had
negligible
FL‐3
fluorescence
and
were
considered
to
be
starved.
3.3.3
Extraction
and
characterization
of
O.
marina
membrane
proteins:
Initial
protein
extractions
were
done
using
the
Mem-PER
protein
extraction
kit
(Pierce,
Thermo
Fisher
Scientific,
Rockford,
IL).
As
this
detergent‐based
protocol
lyses
cells,
all
membrane
proteins,
including
those
from
intracellular
organelles,
are
extracted.
One‐liter
cultures
of
O.
marina
were
grown
under
light
until
negligible
red
fluorescence
of
ingested
prey
remained.
The
entire
liter
of
culture
was
concentrated
via
centrifugation
at
2200
RPM
(~500
x
g)
for
a
yield
of
approximately
1‐2
x
107
O.
marina
cells.
After
membrane
extraction,
detergent
was
cleaned
from
proteins
with
34
a
SDS-PAGE
sample
preparation
kit
(Pierce,
Thermo
Fisher
Scientific).
Extracted
proteins
were
concentrated
using
a
Model
SPD111V
Savant
SpeedVac.
Proteins
in
aliquots
of
the
concentrated
sample
were
separated
via
SDS-PAGE
gel
electrophoresis
(Ready
Gel
Tris-HCL
4-15%;
Bio-Rad,
Hercules,
CA),
and
resulting
protein
bands
were
stained
with
BioSafe
Coomassie
stain
(Bio-Rad)
or
Coomassie
Fluor
Orange
(Invitrogen
Corp.,
Carlsbad,
CA).
Individual
gel
bands
were
excised
and
digested
using
an
In-Gel
Tryptic
Digestion
Kit
(Pierce,
Thermo
Fisher
Scientific.
Digested
protein
samples
were
concentrated
by
vacuum
centrifugation
and
then
analyzed
on
an
LC‐MS/MS
mass
spectrometer
at
the
Oregon
State
University
Mass
Spectrometry
Facility.
Since
the
Mem-PER
extraction
protocol
non‐specifically
targets
all
membrane
proteins,
an
additional
protein
extraction
was
done
with
the
Hook
Cell
Surface
Protein
Isolation
Kit
(G-Biosciences,
Maryland
Heights,
MO).
This
protocol
uses
a
biotin-tag/streptavidin
affinity
column
to
extract
only
proteins
exposed
on
the
outer
plasma
cell
membranes
of
intact
cells.
O.
marina
culture
conditions,
cell
concentration,
and
protein
separation
and
analysis
were
as
described
above.
Protein
data
were
searched
against
databases
of
known
proteins
using
Mascot
software
(Matrix
Science,
Boston,
MA.)
The
rhodopsin
proteins
identified
in
our
study
were
used
as
a
query
in
a
BLAST
search
(NCBI,
BLASTp
using
preset
parameters).
From
the
BLASTp
output
we
selected
the
top
30
sequences
(E
values
ranging
from
3e-145
to
2e-20).
The
protein
sequences
were
imported
into
the
Geneious
software
(Biomatters,
Ltd.,
Auckland,
NZ)
based
on
an
algorithm
35
developed
by
Guindon
and
Oliver
(2003).
The
sequences
were
aligned
and
a
tree
produced
using
maximum
likelihood
(bootstrapped
1000
times).
3.3.4
Phototaxis
experiments:
To
determine
O.
marina
response
to
light,
rectangular
four-well
polystyrene
dishes
(Nunc,
Rochester,
NY)
were
cut
and
separated
into
individual
rectangular
dishes.
In
a
darkroom,
light
emitting
diodes
(LED’s)
were
placed
6
cm
away
from
individual
dishes
and
connected
to
a
13.8
volt
power
supply
(RadioShack
Corporation,
Fort
Worth,
TX)
(Figure
2‐A).
For
each
experiment,
20
ml
of
well
mixed
O.
marina
culture
were
added
to
each
of
four
replicate
dishes,
and
each
dish
was
exposed
to
a
LED
light
for
30
minutes.
Various
phototaxis
experiments
used
either
white
light
LED’s
(DigiKey
Corp.,
Thief
River
Falls,
NM)
or
red
(680
nm),
green
(525
nm)
or
blue
(430,
450
nm)
LED’s
(Roithner
Lasertechnik,
Vienna,
Austria).
Light
level
(μE
m2
s‐1)
was
varied
by
placing
neutral
density
film
(GamProducts,
Inc.,
Hollywood,
CA)
in
front
of
the
LED
lights.
Light
levels
were
measured
using
a
PAR
sensor
(Heinz
Walz
GmbH,
Effeltrich,
Germany).
After
30
minutes
of
exposure
of
O.
marina
cells
to
light,
samples
were
taken
from
the
proximal
(P)
and
distal
(D)
ends
of
the
dish
relative
to
the
light.
Plastic
transfer
pipettes
cut
diagonally
at
the
tip
were
used
to
draw
sample
from
the
entire
depth
of
the
water
column
(about
1cm)
at
the
P
and
D
ends
of
the
dish.
Cell
per
ml
abundances
were
determined
immediately
via
flow
cytometry.
A
phototaxis
index:
(P-D)/(P+D),
with
values
ranging
from
0
to
1,
was
calculated
from
the
cell
abundance
data.
Positive
index
values
indicated
that
the
cells
moved
towards
the
36
light,
values
near
0
indicated
no
photoresponse,
and
negative
values
indicated
that
the
cells
moved
away
from
the
light.
Additional
phototaxis
experiments
were
carried
out
to
investigate
the
possibility
that
O.
marina
can
detect
red
Chl‐a
fluorescence
induced
by
blue
light.
The
dishes
described
above
were
used
in
preliminary
experiments.
200
ml
of
late
log
phase
D.
tertiolecta
culture
was
centrifuged
at
350
g
for
5
minutes
and
resuspended
in
10
ml
FASW.
Three
2
ml
glass
vials
were
filled
with
concentrated
D.
tertiolecta
and
sealed
to
prevent
potential
chemosensory
cues.
The
sealed
vials
were
placed
at
one
end
of
three
experimental
dishes.
As
a
control,
sealed
vials
filled
with
concentrated
algae
photo‐bleached
under
intense
white
light
for
approximately
90
minutes
were
positioned
similarly
in
three
other
dishes.
All
dishes
were
illuminated
with
blue
450
nm
light
under
the
sealed
vials
of
algae
(Figure
2‐B).
The
rest
of
the
experimental
protocol
was
as
described
for
the
initial
phototaxis
experiments.
At
the
end
of
the
experiment,
photo‐bleached
cells
were
examined
using
an
Olympus
BX61
epifluorescence
microscope
to
confirm
that
the
algal
cells
remained
intact.
The
relative
cell‐specific
FL‐3
(red)
fluorescence
in
bleached
and
in
unbleached
algal
cells
used
in
the
experiments
was
assessed
by
flow
cytometry.
In
order
to
further
evaluate
O.
marina
photoresponse
to
chlorophyll
autofluorescence
of
algal
prey,
an
additional
linear
tube
experimental
design
was
devised
(Figure
2‐C).
Two
6
cm
sections
of
clean
10
mm
I.D.
glass
tubing
were
connected
with
silicon
tubing.
A
sealed
glass
vial
containing
either
concentrated
unbleached
or
bleached
D.
tertiolecta,
prepared
as
described
above,
was
connected
37
to
one
end
of
each
tube
with
silicon
tubing.
The
opposite
end
contained
a
reservoir
of
~2
x
104
cells
ml‐1
of
O.
marina
open
to
the
glass
tube.
The
glass
tubing
was
filled
with
FASW,
allowing
the
dinoflagellate
cells
to
swim
freely
through
the
connected
glass
tubes.
The
sealed
vial
with
concentrated
algae
was
illuminated
with
a
450
nm
LED
positioned
at
a
90°
angle
with
respect
to
the
glass
tubing.
The
suspension
of
concentrated
unbleached
algae
was
sufficiently
dense
to
be
visibly
red
when
illuminated
with
blue
light.
Each
treatment
was
done
in
triplicate.
After
1.5
hours,
the
6
cm
sections
of
glass
tubing
adjacent
to
the
vials
of
algae
were
removed
and
abundances
of
O.
marina
in
these
tube
sections
were
immediately
analyzed
by
flow
cytometry.
3.3.5
Evaluation
of
cAMP
induction
by
light:
Cyclic
adenosine
monophosphate
(cAMP)
is
a
downstream
cytoplasmic
component
of
membrane‐bound
receptor
signaling.
If
the
photoresponse
of
O.
marina
were
due
to
a
membrane
bound
GPCR
such
as
rhodopsin,
exposure
to
light
should
result
in
a
detectable
increase
in
intracellular
cAMP
concentration.
The
cAMP-Glo
assay
(Promega
Corp.,
Madison,
WI)
was
used
to
investigate
the
potential
for
such
a
response.
Dark‐cultured
O.
marina
cells
were
either
left
in
the
dark
or
stimulated
by
exposure
to
a
white
light
LED
for
periods
of
1
to
90
seconds.
Following
light
exposure,
cells
were
lysed
and
the
cAMP‐Glo
reaction
mix
added.
In
this
assay,
reaction
with
cAMP
results
in
a
decrease
in
concentration
of
ATP,
assessed
by
decreased
luciferase
light
production.
Thus
there
is
an
inverse
relationship
between
the
luminescent
output
of
the
assay
and
cAMP
concentration.
38
Sample
aliquots
were
placed
in
white
96
well
plates
(Greiner
Bio-One
North
America,
Monroe,
NC)
and
relative
luminescence
units
recorded
using
a
microplate
luminometer
(SpectraMaxL;
Molecular
Devices,
Sunnyvale,
CA.).
3.3.6
Inhibition
of
Rhodopsin:
Hydroxylamine,
an
inhibitor
of
rhodopsin
GCPR
signaling,
was
used
to
investigate
whether
O.
marina
photoresponse
could
be
based
on
rhodopsin.
Sensory
rhodopsin
consists
of
a
retinal
chromophore
covalently
linked
to
the
rhodopsin
protein
by
a
protonated
Schiff
base
bond
(Palczewski
2006).
Hydroxylamine
cleaves
the
retinal
from
the
protein,
rendering
the
sensory
rhodopsin
disfunctional
(Zadok
et
al.
2005).
Hydroxylamine
(Sigma-Aldrich
Inc.,
Saint
Louis,
MO)
was
dissolved
in
autoclaved
0.2
μm
filtered
seawater.
To
check
whether
hydroxylamine
might
have
a
non‐specific
effect
on
cell
physiology,
aliquots
of
O.
marina
light
adapted/well
fed
cells
were
incubated
with
hydroxylamine
at
final
concentrations
of
0.0,
0.25,
0.5,
0.75,
1,
and
2
mM
for
2
hours
at
room
temperature
in
the
dark.
Approximately
104
cells
ml‐1
of
algal
prey
were
then
added
to
all
treatments.
After
a
further
two
hour
incubation
in
the
dark,
all
treatments
were
sampled
for
O.
marina
cell
abundance
and
cell‐specific
prey
ingestion
using
flow
cytometry.
To
test
the
effect
of
hydroxylamine
on
O.
marina
phototactic
behavior,
the
dinoflagellate
was
incubated
with
hydroxylamine
at
final
concentrations
of
0.0,
0.1,
0.12,
0.5,
and
1
mM
for
30
minutes
in
bright
light,
followed
by
90
minutes
in
the
dark
prior
to
the
start
of
the
phototaxis
experiments.
Phototaxis
experiments
were
set
up
in
rectangular
dishes
with
white
light
LEDs
as
previously
described
(Figure
2‐
39
A).
The
phototaxis
index
of
cells
incubated
with
hydroxylamine
was
compared
to
control
cells
with
no
hydroxylamine
exposure.
3.4
Results
3.4.1
Characterization
of
O.
marina
membrane­associated
proteins
Mascot
search
with
protein
analysis
data
from
the
Mem‐PER
membrane
protein
extraction
kit
samples
resulted
in
the
identification
of
an
expressed
rhodopsin
(O.
marina;
gi|157093543).
A
similar
search
of
proteins
from
gel
bands
from
the
Hook
outer
membrane
extraction
kit
identified
two
expressed
rhodopsins
(O.
marina;
gi|157093543
and
gi|157093545).
No
other
putative
signal
receptor
proteins
were
detected
in
our
samples.
A
maximum
likelihood
tree
of
the
top
30
matches
to
the
two
O.
marina
rhodopsins
recovered
showed
that
the
rhodopsins
found
in
this
study
were
most
closely
related
to
other
O.
marina
rhodopsins
and
to
the
rhodopsin
of
an
autotrophic
dinoflagellate,
Pyrocystis
lununa.
These
dinoflagellate
rhodopsins
grouped
with
rhodopsins
present
in
Domain
Bacteria,
Phylum
Proteobacteria
(Figure
3).
3.4.2
O.
marina
response
to
light
O.
marina
showed
variable
response
to
light
depending
on
light
level
and
wavelength,
as
well
as
on
growth
history
of
the
dinoflagellate
cells.
Based
on
a
phototaxis
index,
the
strongest
positive
photoresponse
was
observed
at
low
light
intensity,
2
or
3
μE
m‐2
s‐1
(Figure
3).
The
phototaxis
index
decreased
at
the
upper
and
lower
light
levels
and
became
negative
in
the
case
of
light
adapted‐well
fed
cells
40
at
the
highest
light
level
tested.
However,
cells
that
were
dark
adapted‐well
fed
showed
negative
photoresponse
to
all
light
levels
tested.
In
experiments
to
test
photoresponse
to
individual
light
wavelengths,
significant
positive
phototaxis
occurred
in
treatments
with
450
nm,
525
nm,
and
680
nm
light
(Figure
4).
In
general,
the
phototaxis
index
values
for
individual
light
wavelengths
were
within
the
range
of
those
from
experiments
with
white
light.
3.4.3
O.
marina
response
to
Chl­a
fluorescence
In
the
initial
dish
experiment,
results
were
suggestive
of
phototaxis
toward
Chl‐a
fluorescence,
but
not
definitive.
In
the
linear
tube
experimental
configuration,
O.
marina
phototaxis
was
significantly
greater
(P=0.006;
t
test,)
in
response
to
concentrated
unbleached
algae
compared
to
photo‐bleached
algae
illuminated
with
450
nm
light
(Figure
5).
Microscopic
inspection
showed
that
the
photo‐bleaching
process
left
algal
prey
cells
intact.
However,
after
photo‐bleaching,
the
algal
cells
visually
changed
color
from
green
to
white,
and
based
on
relative
cell‐specific
red
(FL3)
fluorescence
units
as
measured
by
flow
cytometry,
they
emitted
significantly
less
Chl‐a
fluorescence
per
cell
(average
of
7
FL3
units)
compared
to
normal
algal
cells
(average
of
854
FL3
units,
P
<
0.001;
t‐test).
3.4.4
Light
stimulation
of
intracellular
downstream
signaling
A
one
second
exposure
of
intact
O.
marina
cells
to
light
resulted
in
a
sharp
intracellular
increase
(measured
as
a
decrease
in
relative
luminescence
units,
RLU)
in
cAMP
concentration
(Figure
6).
With
further
light
exposure
up
to
90
seconds,
41
cAMP
levels
in
O.
marina
cells
were
also
elevated
(significantly
lower
RLU)
compared
to
cAMP
concentrations
in
control
cells
with
no
light
exposure.
3.4.5
Inhibition
of
rhodopsin
function
by
hydroxylamine
After
exposure
to
hydroxylamine
concentrations
of
0.12,
0.5,
and
1.0
mM,
photosensory
response
was
significantly
decreased
(P
<0.0003
to
0.028;
t‐test)
compared
to
controls
with
no
exposure
to
hydroxylamine.
However
it
was
observed
that
treatment
of
O.
marina
with
as
little
as
0.25
mM
hydroxylamine
resulted
in
a
significant
decrease
in
O.
marina
cells
(t‐test;
p=0.014).
Treatment
with
hydroxylamine
did
not
affect
prey
ingestion.
3.5
Discussion
Although
heterotrophic
dinoflagellates
are
ubiquitous
and
important
in
marine
food
webs
(Sherr
and
Sherr
2007),
to
date
there
has
been
little
study
of
the
behavior
of
these
protists.
Buskey
(1997)
investigated
the
feeding
behavior
of
a
heterotrophic
armored
dinoflagellate,
Protoperidinium
pellucidum,
which
captures
and
digests
prey
extracellularly
via
a
feeding
veil.
He
found
that
the
dinoflagellate
used
chemoreception
to
judge
the
location
of
a
prey
cell,
and
fed
preferentially
on
diatoms
over
dinoflagellate
prey.
Menden‐Deuer
&
Grünbaum
(2006)
reported
that
O.
marina
could
detect
and
aggregate
in
patches
of
algal
prey.
Wooten
et
al.
(2007)
showed
that
chemoreception
of
algal
prey
by
O.
marina
was
due
in
part
to
a
mannose‐binding
lectin
present
on
the
outer
cell
membrane
of
the
dinoflagellate.
We
have
previously
reported
that
intracellular
signal
transduction
pathways
are
involved
in
the
chemosensory
response
of
O.
marina
(Hartz
et
al.
2008).
42
While
the
heterotrophic
dinoflagellate
O.
marina
was
previously
noted
by
Droop
(1954)
to
move
toward
light
in
culture,
our
study
is
the
first
to
report
details
of
photosensory
behavior
and
a
potential
biochemical
basis
of
light
response
in
this
species.
Our
results
showed
that
O.
marina
has
the
ability
to
detect
white
light
as
well
as
individual
wavelengths
of
light.
The
direction
and
strength
of
phototaxic
response
in
O.
marina
varied
with
light
intensity.
The
strongest
positive
phototaxis
occurred
at
light
levels
of
2‐3
μE
m‐2
s‐1
for
white
light
and
4
μE
m‐2
s‐1for
450
nm
light.
This
may
represent
light
intensities
preferred
by
O.
marina
in
its
natural
habitat,
the
coastal
ocean.
These
values
approximate
the
light
level
found
at
the
base
of
the
euphotic
zone,
about
1
%
of
incident
light.
For
example,
1
%
of
the
average
surface
photosynthetically
active
radiation
of
the
Oregon
coastal
ocean,
~
35
mol
quanta
m‐2
day‐1
(Wetz
et
al.
2004),
is
equivalent
to
~
4
μE
m‐2
s‐1.
O.
marina
that
were
grown
in
the
dark
and
well
fed
exhibited
negative
phototaxis
to
all
levels
of
white
light
tested,
while
dark
adapted
and
starved
cells
were
positively
phototaxic
(Figure
4).
These
results
mirror
those
of
Selbach
et
al.
(1999),
who
found
that
the
ciliate
Chlamydodon
mnemosyne
exhibited
positive
phototaxis
during
starvation
and
negative
phototaxis
when
well
fed.
Ability
to
sense
a
change
in
light
level
may
aid
phagotrophic
protists
in
orienting
in
their
environment,
i.e.
as
a
cue
to
the
direction
of
a
different
location
if
food
is
scarce,
or
to
remain
in
a
location
where
food
conditions
have
been
favorable.
O.
marina
exhibited
positive
phototaxis
to
low
light
levels
of
at
least
three
distinct
wavelengths
of
light:
blue
(450
nm),
green
(525
nm)
and
red
(680
nm).
43
Blue
and
green
wavelengths
of
light
penetrate
farthest
in
coastal
ocean
water,
while
red
wavelength
radiation
is
rapidly
diminished
in
the
upper
few
meters
of
the
water
column.
However,
chlorophyll
pigments
of
phytoplankton
produce
red
fluorescence
when
illuminated
with
blue
light.
This
suggested
the
possibility
that
the
capacity
to
detect
red
light
might
be
used
by
O.
marina
to
locate
patches
of
phytoplankton
prey.
We
did
find
that
O.
marina
exhibited
significantly
greater
photoresponse
toward
unbleached
compared
to
bleached
algae
illuminated
by
blue
light.
In
these
experiments,
the
sealed
vials
of
unbleached
algae
were
visibly
dark
red
due
to
Chl‐a
autofluorescence
when
illuminated
with
450
nm
light,
while
no
visible
red
color
was
emitted
from
bleached
algae.
Flow
cytometric
analysis
confirmed
a
more
than
100‐
fold
decrease
in
cell‐specific
red
fluorescence
of
algae
after
bleaching.
This
result
indicated
that
O.
marina
was
attracted
to
the
red
fluorescence
emitted
from
algal
cells.
While
the
idea
of
photosensory
prey
detection
by
marine
protists
needs
further
study,
we
note
that
Amon
and
French
(2004)
showed
that
phagotrophic
zoospores
of
the
marine
thraustochitrid
Ulkenia
sp.
were
attracted
to
bioluminescence
from
the
bacterial
prey
species
Vibrio
fisheri.
Furthermore,
they
found
Ulkenia
sp.
zoospore
phototaxis
toward
blue
and
white
light
peaked
at
3.5
μE
m‐2
s‐1,
similar
to
light
levels
found
by
us
to
induce
maximum
phototaxic
response
in
O.
marina.
Several
types
of
biomolecules
have
been
shown
to
serve
as
photosensory
receptors
in
marine
heterotrophic
protists,
including
flavins,
hypericins,
and
rhodopsins
(Selbach
and
Kuhlmann
1999,
Cadetti
et
al.
2000,
Saranak
and
Foster
2005,
Lobban
et
al.
2007).
While
the
first
two
classes
of
sensory
pigments
are
44
mainly
found
in
ciliates,
genes
coding
for
rhodopsins
are
widespread
in
Archaea,
Bacteria,
and
Eukarya.
Rhodopsins
are
known
to
have
photosensory
functions
in
microbes
in
all
three
Domains
(Sharma
et
al.
2006,
Fuhrman
et
al.
2008,
Klare
et
al.
2008).
We
found
two
different
rhodopsin
proteins
expressed
in
the
outer
cell
membrane
of
the
strain
of
O.
marina
(CCMP
604)
used
in
this
study.
Seven
rhodopsin‐coding
genes
of
O.
marina
(strain
CCMP
1795)
are
posted
in
the
NCBI
database
(www.ncbi.nlm.nih.gov).
The
strain
of
O.
marina
we
used
might
have
a
different
complement
of
rhodopsin
genes,
although
the
proteins
we
isolated
matched
two
of
the
O.
marina
rhodopsin
genes
in
the
database.
It
is
also
possible
that
other
expressed
O.
marina
rhodopsins
were
not
detected,
as
the
dinoflagellate
responded
to
three
distinct
wavelengths
of
light.
A
BLAST
search
using
the
rhodopsin
protein
sequences
identified
in
our
study
as
a
query
revealed
that
these
and
the
other
O.
marina
rhodopsins
in
the
database
had
a
closest
match
to
a
type
I
(microbial)
rhodopsin‐related
sequence
discovered
in
the
marine
autotrophic
dinoflagellate,
Pyrocystis
lunula
(Figure
3).
These
dinoflagellate
rhodopsins
are
related
to
Bacterial
rhodopsins
(Figure
3),
as
has
previously
been
reported
for
the
P.
lunula
rhodopsin
(Ruiz‐Gonzalez
and
Marin
2004;
Sharma
et
al.
2006).
In
contrast,
sensory
type
1
rhodopsins
present
in
other
eukaryotic
microbes,
including
fungi,
green
algae,
and
cryptophyte
algae,
are
most
closely
related
to
Archaeal
rhodopsins
(Ruiz‐Gonzalez
and
Marin
2004;
Sharma
et
al.
2006).
The
scattered
distribution
of
eukaryotic
rhodopsin‐related
genes
has
been
suggested
by
these
authors
as
indicative
of
early
and
diverse
origins,
including
45
lateral
transfer
between
species
in
separate
domains,
for
this
universal
class
of
proteins.
Internal
cell
signal
cascades
triggered
by
activation
of
membrane‐bound
GPCR
molecules
such
as
rhodopsin
have
been
well
described
in
eukaryotic
cells.
Cyclic
adenosine
monophosphate
(cAMP)
is
an
important
downstream
signal
transduction
messenger
and
is
catalyzed
by
adenylyl
cyclases
which
are
activated
by
GPCRs
(Gomparts
et
al.
2004).
After
exposure
to
white
light
for
one
second
or
longer,
cAMP
concentration
increased
in
O.
marina
cells.
We
also
investigated
the
role
of
rhodopsin
in
O.
marina
phototaxis
by
exposing
the
dinoflagellate
to
hydroxylamine,
which
prevents
the
retinylidene
chromophores
from
re‐binding
to
the
opsin
component
of
sensory
rhodopsin
(Hegemann
et
al.
1988,
Saranak
and
Foster
1997).
We
found
that
phototaxis
in
O.
marina
was
significantly
decreased
by
treatment
with
hydroxylamine.
It
was
also
observed
that,
although
hydroxylamine
caused
a
decrease
in
O.
marina
cell
count
at
concentrations
of
0.25
mM,
hydroxylamine
did
not
affect
prey
ingestion
or
normal
swimming
behavior
suggesting
the
remaining
cells
were
relatively
healthy.
These
results
support
the
idea
that
rhodopsin
may
be
the
basis
of
the
observed
photosensory
behavior
of
O.
marina.
3.6
Conclusions
To
date
there
has
been
little
investigation
of
the
ecological
role
of
photoreceptors
in
predatory
protists.
We
found
that
the
colorless
dinoflagellate
O.
marina
responded
both
to
white
light
and
specific
wavelengths
of
light
and
46
exhibited
both
positive
and
negative
phototaxis.
O.
marina
may
be
able
to
locate
patches
of
algal
prey
by
detection
of
Chl‐a
autofluorescence.
Finding
of
expressed
rhodopsins
in
the
cell
membrane,
as
well
as
results
of
experimental
tests,
indicated
that
rhodopsin
is
likely
to
be
a
sensory
photoreceptor
in
this
marine
dinoflagellate.
The
capacity
to
respond
to
light
as
well
as
to
chemical
cues
in
the
environment
expands
the
behavioral
repertoire
of
phagotrophic
protists;
this
research
topic
deserves
increased
attention.
47
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50
Figure
Legends
Figure
1.
Flow
cytometer
dot
plot
of
red
fluorescence
versus
side
scatter
parameters
showing
locations
of
the
algal
prey,
D.
tertiolecta,
O.
marina
cells
with
ingested
algal
prey,
and
O.
marina
cells
without
ingested
algal
prey.
Figure
2.
Phototaxis
experiment
configurations:
A)
Experimental
configuration
used
to
measure
O.
marina
photoresponse
to
white
and
specific
wavelengths
of
light,
using
LED’s
to
create
a
light
gradient
through
a
rectangular
dish.
B)
As
in
A),
but
a
sealed
glass
vial
of
concentrated
algae,
or
a
bleached
algal
control
at
one
end
of
the
rectangular
dish
were
illuminated
with
a
450
nm
LED
placed
under
the
dish
to
stimulate
Chl‐a
autofluoresence.
C)
Linear
tube
configuration
used
in
an
additional
test
of
response
to
Chl‐a
autofluorescence.
Figure
3.
Maximum
likelihood
tree
of
rhodopsins
most
closely
related
to
those
found
in
O.
marina.
Numbers
on
nodes
represent
bootstrap
values.
Figure
4.
Photoresponse
of
O.
marina
to
a
range
of
intensities
of
white
light,
quantified
as
a
phototaxis
index
comparing
cell
abundances
in
the
proximal
and
distal
ends
of
a
rectangular
dish
after
a
30
min
exposure
to
each
light
level.
Phototaxis
index
values
represent
the
average
of
three
replicates,
error
bars
=
standard
deviation.
The
dotted
line
indicates
an
index
value
of
0,
which
represents
no
response
to
light.
Figure
5.
Photoresponse
of
O.
marina
to
a
range
of
intensities
of
red,
green
and
blue
wavelengths
of
light
in
dish
experiments
as
in
Figure
4.
Phototaxis
index
values
represent
average
of
four
replicates,
error
bars
=
one
standard
deviation.
The
dotted
line
indicates
an
index
value
of
0,
which
represents
no
response
to
light.
Figure
6.
Comparison
of
O.
marina
photoresponse
to
unbleached
and
bleached
concentrated
algae
illuminated
with
450
nm
light
in
the
linear
tube
experiment.
Data
represent
average
of
triplicate
samples,
error
bars
are
one
standard
deviation.
51
Figure
7.
Cyclic
adenosine
monophosphate
(cAMP)
activation
in
response
to
a
light
stimulus.
cAMP
concentration
has
an
inverse
relationship
to
Relative
Luminiscence
Units
(RLU).
Data
points
represent
average
of
duplicate
measurements
and
error
bars
are
one
standard
error.
52
Hartz
et
al.
Figure
1
53
Hartz
et
al.
Figure
2
54
Hartz
et
al.
Figure
3
55
Hartz
et
al.
Figure
4
56
Hartz
et
al.
Figure
5
57
Hartz
et
al.
Figure
6
58
Hartz
et
al.
Figure
7
59
4
Conclusions
4.1
Overview
The
manuscripts
in
this
thesis
add
new
information
to
the
scant
literature
on
how
membrane
receptors
and
intracellular
signaling
are
linked
to
behavior
in
marine
phagotrophic
protists.
The
results
of
these
studies
are
supported
by
previous
work
on
biochemical
control
of
phagocytosis
in
laboratory
cultured
protists
such
as
ciliates
in
the
genera
Tetrahymena
and
Paramecium
(Almagor
et
al.
1981;
Hellung‐Larsen
et
al.
1986;
Renaud
et
al.
1995,
2004;
Leick
et
al.
1996,
1997;
Bell
et
al.
1998),
and
euglenoid
flagellates
in
the
genera
Euglena
and
Astacia
(Levandowsky
2001);
in
parasitic
protists
such
as
trypanosomes
(cause
of
malaria)
and
Leishmania
(cause
of
skin
lesions)
parasites
(Mosser
&,
Rosenthal
1993,
Ratcliff
et
al.
1996,
Basseri
et
al.
2002);
and
in
metazoan
white
blood
cells
(Greenberg
1995,
Hill
&
Rowley
1998,
Ballarin
et
al.
2002).
These
studies
include
demonstrations
of
the
effect
on
cell
behavior
of
ligand
binding
by
membrane‐bound
receptor
molecules,
and
of
signal
transduction
mechanisms
within
the
cell
that
are
switched
on
by
ligand‐receptor
binding
(Greenberg
1995,
Alberts
et
al.
2002,
Renaud
et
al.
2004).
4.2
Chemosensory/inhibitor
study
Data
reported
in
Hartz
et
al.
(2008)
provided
evidence
that
signaling
pathways
found
in
other
types
of
eukaryotic
cells
are,
in
fact,
involved
in
swimming
and
feeding
behavior
of
marine
heterotrophic
protists.
The
effects
of
the
two
protein
kinase
signal
transduction
inhibitors
varied
between
the
heterotrophic
60
dinoflagellate
O.
marina
and
the
Uronema
sp.
ciliate.
One
of
these
inhibitors,
staurosporine,
decreased
chemosensory
response
and
predation
in
both
protists,
suggesting
that
protein
kinase
signaling
pathways
are
involved
in
at
least
two
behavioral
responses.
The
second
protein
kinase
inhibitor,
genistein,
inhibited
chemotaxis
and
prey
ingestion
in
the
ciliate,
but
not
in
the
dinoflagellate.
This
could
reflect
a
difference
in
intracellular
signaling
pathways
in
the
two
protists,
or
in
the
ability
of
the
cells
to
take
up
the
chemical
inhibitor.
An
inhibitor
of
G‐protein
coupled
receptors
(GPCR’s)
effectively
reduced
the
chemosensory
response
in
O.
marina
and
Uronema
sp.,
but
had
no
affect
on
prey
ingestion.
Two
inhibitors
of
intracellular
G‐proteins
resulted
in
a
moderate
decrease
in
chemosensory
ability
in
both
protists,
but
had
little
effect
on
predation.
In
the
natural
environment,
protist
feeding
would
most
likely
be
affected
by
inhibition
of
chemosensory
ability.
The
conditions
used
for
the
prey
ingestion
experiments
in
this
study
provided
the
protists
with
prey
abundances
one
or
two
orders
of
magnitude
greater
than
what
would
be
found
in
situ.
It
is
possible
that
protists
may
not
need
chemosensory
signals
to
locate
prey
at
high
abundance.
These
findings
suggest
that
while
G‐
protein
signaling
systems
are
involved
in
chemotaxis
in
these
two
marine
protozoa,
they
are
probably
not
directly
involved
in
prey
ingestion
(phagocystosis).
This
study
was
the
first
to
use
selective
inhibitor
compounds
to
examine
the
role
of
cell
signaling
pathways
in
chemosensory
behavior
and
prey
ingestion
by
free‐living
marine
phagotrophic
protists
and
provided
evidence
that
biochemical
mechanisms
involving
cell
membrane‐bound
GPCR
molecules
and
intracellular
signal
transduction
molecules
are
likely
to
be
involved
in
feeding
behavior
of
61
phagotrophic
protists.
Many
enzymes
involved
in
signal
transduction
cascades
are
regulated
by
protein
kinase
phosphorylation/dephosphorylation.
Knowing
this,
it
comes
as
no
surprise
that
general
inhibitors
of
protein
kinases
had
a
significant
effect
on
the
chemosensory
ability
and
grazing
in
both
protists.
This
work
supports
the
results
of
other
studies
which
indicated
that
biochemical
signaling
mechanisms
are
involved
in
chemosensory
response
and
feeding
behavior
of
‘model
cell’
protists.
Leick
et
al.
(1997)
found
that
protein
kinase
inhibitors
eliminated
chemosensory
behavior
in
the
freshwater
hymenostome
ciliate
Tetrahymena
sp.
In
addition,
Strom,
Wolfe,
and
Bright
(2007)
suggested
that
dissolved
amino
acids
(L‐enantiomers)
may
serve
as
signal
molecules
that
can
affect
feeding
responses
in
a
marine
tintinnid
ciliate.
It
is
likely
that
such
cellular
processes
are
important
in
determining
prey
selection,
capture
success,
and
rates
of
ingestion
among
heterotrophic
protists
in
the
sea.
Understanding
the
extent
to
which
cell
signaling
pathways
operate
in
protist
prey
selection,
ingestion
rates,
and
other
aspects
of
feeding
behavior
may
result
in
the
development
of
novel
approaches
to
study
of
predator
prey
interactions
in
microbial
communities.
4.3
Photosensory
behavior
study
It
has
been
over
half
a
century
since
Droop
(1954)
noted
that
O.
marina
moved
toward
light
during
culturing
studies.
However
Hartz
et
al.
(ms
in
preparation)
is
the
first
study
to
provide
details
of
O.
marina
photoresponse.
The
heterotrophic
dinoflagellate
Oxyrrhis
marina
was
clearly
able
to
respond
62
behaviorally
to
white,
blue,
green,
and
red
light.
The
strongest
positive
phototaxis
occurred
between
2‐3
µE/m2/s
for
white
light
and
4
µE/m2/s
for
450
nm
light.
This
may
represent
an
optimum
light
level
for
O.
marina
in
its
natural
habitat,
the
coastal
ocean.
Although
this
value
would
most
likely
fluctuate,
our
results
suggest
that
O.
marina
seeks
out
low
light
levels
that
may
be
similar
to
those
found
at
the
base
of
the
euphotic
zone.
Culture
conditions
and
physiological
state
affected
O.
marina
photo‐behavior.
For
example,
the
dark
adapted
starved
cells
may
have
used
light
as
a
cue
to
find
more
favorable
conditions.
The
dark
adapted
well
fed
cells
were
already
experiencing
optimum
conditions
with
respect
to
food
availability,
and
may
have
perceived
light
as
a
cue
to
less
favorable
conditions
(i.e.
they
may
not
gain
an
advantage
by
moving
from
their
current
location
if
they
already
have
a
sufficient
food
source).
Additionally,
our
study
showed
that
O.
marina
has
the
ability
to
respond
to
individual
wavelengths
of
light;
this
ability
may
enable
O.
marina
to
use
light
cues
to
find
optimum
conditions
no
matter
what
habitat
the
cell
is
in.
The
results
from
this
study
are
also
the
first
to
suggest
a
herbivorous
protist
has
the
ability
to
detect
phytoplankton
auto‐fluorescence.
These
results
showed
that
O.
marina
was
attracted
to
the
induced
red
fluorescence
emitted
from
Dunaliella
cells
illuminated
with
blue
light.
This
could
have
ecological
implications
since
in
the
ocean
most
light
below
10
m
is
blue
due
to
attenuation
of
other
wavelengths.
Dense
patches
of
phytoplankton
in
the
blue‐lit
water
column
may
emit
sufficient
numbers
of
photons
to
serve
as
a
signal
for
photoreceptive
63
herbivorous
protists.
This
could
serve
as
an
additional
sensory
response
used
in
finding
photosynthetic
prey
and
could
aid
the
predator
in
finding
patches
of
prey
when
chemical
signals
are
insufficient.
Chemical
cues
from
prey
will
be
limited
by
the
rate
of
diffusion,
whereas
fluorescence
emitting
from
prey
would
be
almost
instantaneous.
These
results
are
supported
by
Amon
and
French
(2004)
who
showed
that
Ulkenia
sp.
zoospores
were
attracted
to
bioluminescence
from
Vibrio
fisheri.
Furthermore,
they
found
Ulkenia
sp.
phototaxis
toward
blue
and
white
light
to
peak
at
3.5
µE/m2/s,
a
similar
finding
to
results
reported
in
our
current
study.
The
Amon
and
French
study
along
with
results
from
our
study
could
point
toward
a
new
area
of
focus
within
the
broader
topic
of
feeding
behavior
at
the
microbial
level.
The
results
from
these
two
studies
suggest
photo
detection
systems
in
predatory
microbes
may
be
used
for
more
than
sensing
the
surrounding
environment,
and
could
be
used
to
aid
in
prey
location.
Two
different
rhodopsin
proteins
were
recovered
from
the
O.
marina
strain
used
in
this
study.
There
are
seven
rhodopsin
genes
from
O.
marina
in
the
NCBI
database
(www.ncbi.nlm.nih.gov).
This
suggests
either
that
we
were
not
successful
in
isolating
all
O.
marina
rhodopsin
proteins,
or
that
O.
marina
differentially
expresses
rhodopsin
proteins
depending
on
conditions.
Or
perhaps
different
strains
of
O.
marina
contain
different
suites
of
rhodopsin
genes.
The
rhodopsin
genes
available
in
the
NCBI
database
are
all
from
strain
CCMP
1795,
whereas
in
our
study
O.
marina
strain
CCMP
604
was
used
Further
studies
are
needed
to
valuate
if
64
variations
in
culture
conditions
(i.e.
light
levels,
light
cycles,
and
wavelength)
contribute
to
expression
of
different
rhodopsins.
Rhodopsins
are
found
in
eukaryotic
and
prokaryotic
microbes
(Spudich
2006),
therefore
it
is
not
suprising
that
rhodopsin
genes
are
found
in
O.
marina.
A
BLAST
search
using
the
rhodopsin
proteins
from
O.
marina
identified
in
our
study
as
a
query
revealed
they
were
closely
related
to
rhodopsin
from
the
dinoflagellate
Pyrocystis
lunula.
However,
unlike
other
eukaryote
rhodopsins,
the
next
most
closely
matched
proteins
were
rhodopsins
from
prokaryotes
rather
than
rhodopsins
present
in
other
eukaryotes.
There
is
evidence
that
Pyrocystis
lunula
rhodopsin
is
a
result
of
lateral
gene
transfer
from
Bacteria
(Ruiz‐Gonzalez
and
Marin
2004;
Sharma
et
al.
2006).
Since
the
rhodopsin
genes
of
O.
marina
are
also
within
the
Bacterial
rhodopsin
lineage,
it
maybe
that
the
acquisition
of
the
rhodopsin
gene
occurred
early
in
dinoflagellate
evolution.
If
so,
then
other
dinoflagellate
rhodopsins
should
also
fall
into
this
group.
Results
from
this
study
suggest
that
cAMP
signaling
is
involved
in
transmitting
a
light
stimulus
signal
in
O.
marina,
which
supports
the
involvement
of
rhodopsin
signaling.
Most
likely
there
are
multiple
signal
transduction
pathways
involved
in
downstream
signaling
following
a
light
stimulus
in
O.
marina.
It
has
been
well
over
a
hundred
years
since
researchers
began
seriously
studying
microbial
photoresponse.
Verworn
(1889)
wrote,
“we
find
that
among
unicellular
organisms,
so
far
as
they
are
irritable
at
all
to
light,
phototaxis
is
a
wide‐
spread
phenomenon.”
Since
then
study
of
light
detection
in
photosynthetic
protists
65
has
made
large
advances
(for
reviews:
Hegemann
2008,
Kateriya
et
al
2004),
however
photosensory
mechanisms
and
behavior
in
heterotrophic
protists
have
received
less
attention.
Light
is
an
important
factor
affecting
the
marine
ecosystem
and
will
have
an
affect
on
the
behavior
of
any
organism
containing
a
photo‐
detection
system.
This
will
likely
be
linked
to
behavioral
changes
including
circadian
rhythms,
position
in
the
water
column,
and
possibly
feeding
behavior.
Although
evidence
from
this
study
points
to
O.
marina
using
rhodopsin
receptors
to
respond
to
light,
other
photoreceptors
could
also
be
involved.
4.4
What
does
this
mean
for
the
field
of
aquatic
microbial
ecology?
The
results
presented
in
this
thesis
build
upon
previous
studies.
However
there
is
still
a
lack
of
information
in
this
area
of
research.
The
biochemical
signaling
occurring
within
a
single
celled
predator
is
complex
and
is
no
doubt
integrated
into
the
behavior
of
the
cell.
Until
the
cellular
processes
controlling
protist
behavior
are
better
comprehended
there
will
be
gray
areas
in
the
understanding
of
aquatic
microbial
ecology.
Bulk
sampling
and
counting
of
field
samples,
and
incubation
experiments
can
provide
information
on
species
composition,
cell
abundances,
and
grazing
rates,
but
they
offer
little
or
no
information
on
why
certain
species
are
being
grazed
or
why
some
species
are
found
in
higher
abundances
at
specific
depths.
To
make
sense
of
field
measurements,
it
is
necessary
to
have
a
better
understanding
of
how
protist
cells
(and
other
microbes)
sense
and
respond
with
behavioral
changes
to
environmental
stimuli
and
potential
prey.
This
gap
in
information
has
existed
for
66
too
long
and
only
relatively
recently
has
available
technology
allowed
for
this
to
become
widely
studied.
Prior
efforts
to
elucidate
the
role
of
biochemistry
in
prey
selectivity
by
protists
in
marine
systems
have
also
included:
evaluation
of
the
role
of
cell
surface
electrostatic
charge
or
hydrophobicity
(Hammer
et
al.
1999,
Monger
et
al.
1999,
Matz
and
Jurgens
2001);
role
of
prey
nutritional
status
(Gonzalez
et
al.
1993,
Flynn
et
al.
1996,
Martel
2006);
evaluation
of
relative
ingestion
rates
of
artificial
particles
coated
with
various
chemicals
(Pace
and
Bailiff
1987;
Matz
et
al.
2002);
and
the
phylogenetic
affiliation
or
phenotypic
characteristics
of
bacterial
cells
(Van
Hannen
et
al.
1999,
Jurgens
et
al
1999,
Simek
et
al.
1999,
Suzuki
1999,
Jurgens
and
Matz
2002,
Matz
et
al.
2002).
Martel
(2006)
found
that
chemosensory
response
and
prey
ingestion
in
the
heterotrophic
dinoflagellate
Oxyrrhis
marina
did
not
appear
to
be
closely
coupled,
and
suggested
that
the
biochemical
mechanisms
of
chemosensory
behavior
and
of
prey
capture
may
differ.
Studying
the
response
of
a
herbivorous
protist
to
thin
layers
of
phytoplankton,
Menden‐Deuer
and
Grumbaum
(2006)
reported
that
the
swimming
speed
of
O.
marina
increased
significantly
in
response
to
intact
Isochrysis
galbana
cells,
but
not
in
response
to
I.
galbana
exudates.
Behavioral
responses
elicited
by
receptor‐ligand
binding
on
the
cell
surface
will
depend
on
signal
transduction
mechanisms
in
the
protistan
cell,
analogous
to
the
better‐known
biochemical
signal
mechanisms
of
bacteria
and
of
the
cells
of
multicellular
organisms
(Alberts
et
al.
2002,
Rogales
2005).
As
an
example
of
the
universality
of
this
mechanism,
chemical
signal
receptors
on
the
surface
of
protistan
67
membranes
are
known
to
bind
compounds,
including
insulin
and
opiates,
which
act
as
signaling
hormones
in
metazoans
(Renaud
et
al.
1995).
Once
a
signal
molecule
binds
to
a
receptor
site,
the
release
of
secondary
messenger
compounds
within
the
cell
is
triggered,
which
then
may
result
in
a
change
in
motility,
or
in
initiation
of
phagocytosis
(Van
Houten
1994,
Renaud
et
al.
1995).
As
a
demonstration
of
such
a
signaling
mechanism
in
protists,
Leick
et
al.
(1997)
were
able
to
eliminate
chemosensory
behavior
of
Tetrahymena
by
exposing
the
ciliates
to
protein
kinase
inhibitors.
Leick
et
al.
(1997)
showed
that
addition
of
inhibitors
of
tyrosine
kinase
did
not
disrupt
swimming
in
the
ciliates,
but
did
block
chemosensory
response
of
the
ciliates
to
a
chemo‐attractant,
proteose
peptone.
Limited
evidence
already
exists
that
free‐living
flagellated
protists
have
such
signaling
systems:
there
is
molecular
genetic
evidence
for
the
presence
of
a
protein
kinase
in
a
photosynthetic
flagellate
(Kiriyama
et
al.
1999),
and
for
a
receptor
for
tyrosine
kinase
in
choanoflagellates
(King
&
Carroll
2001).
4.5
Future
studies
As
more
genetic
information
becomes
available
for
free
living
phagotrophic
protists,
it
will
be
possible
to
see
what
specific
genes,
encoding
for
signaling
pathway
components,
are
included
in
each
species’
genome.
This
will
also
allow
researchers
to
design
experiments
that
make
use
of
gene
knockout
or
knockdown
techniques
using
RNA
interference.
In
this
way,
an
experiment
could
be
designed
that
either
changes
or
reduces
the
expression
of
a
protein
involved
in
cell
signaling,
either
at
the
receptor
level
or
downstream
signal
transduction.
Following
gene
68
knockdown
the
behavioral
response
of
a
marine
protist
could
be
evaluated
in
response
to
environmental
stimuli
(e.g.
light),
or
in
response
to
potential
prey
items.
More
work
needs
to
be
done
on
predator/prey
contact,
since
this
is
likely
to
be
the
point
when
prey
will
be
accepted
or
rejected
by
the
predator.
Wootton
et
al.
(2007)
have
shown
a
cell
surface
mannose‐binding
lectin
to
be
directly
involved
in
prey
recognition
by
O.
marina.
It
is
possible
that
the
matchup
of
lectins
and
sugars
on
the
cell
surface
of
predators
and
prey
determines
if
the
prey
is
accepted
or
rejected.
Lectins
present
in
the
cell
membranes
of
phagotrophic
protists
could
be
investigated
using
the
same
approach
used
in
Hartz
et
al.
(ms
in
preparation)
to
detect
rhodopsins
in
the
cell
membranes
of
O.
marina:
extraction
of
cell
membrane
proteins,isolation
by
SDS‐Page
gel
electrophoresis,
and
mass
spectroscopic
identification.
This
could
allow
for
a
‘survey’
approach
to
screening
protists
and
their
prey
for
lectin
molecules.
Additional
experiments
could
be
developed
to
better
simulate
“field”
conditions.
For
example,
studies
could
be
designed
to
determine
if
the
phytoplankton
autofluorescence
under
blue
light
conditions
results
in
a
higher
prey
ingestion
rate
by
O.
marina.
Experiments
could
be
set
up
in
large
tanks
(or
columns)
to
investigate
if
O.
marina
is
orienting
to
an
optimum
light
level
in
the
water
column
to
find
prey
that
might
also
be
attracted
to
a
similar
light
level.
O.
marina
appears
to
be
widely
distributed
in
coastal
habitats
ranging
from
tidal
pools
to
open
coastal
water
and
exhibits
a
high
degree
of
genetic
and
functional
diversity
(Lowe
et
al.
2005).
Future
studies
could
use
strains
of
O.
marina
collected
69
from
different
habitat
types
and
determine
if
they
respond
differently
to
a
variety
of
stimuli
and
prey
type.
More
work
can
be
done
to
extract
and
identify
chemical
and
photoreceptors
from
O.
marina.
A
range
of
O.
marina
strains
could
be
used
to
determine
if
the
expression
of
receptors
changes
when
the
cells
are
grown
in
different
culture
conditions
and
on
different
prey.
These
experiments
could
also
be
done
with
other
protist
species.
In
general,
there
is
much
work
to
be
done
to
investigate
the
role
of
cell
signaling
in
the
ecological
behavior
of
phagotrophic
protists.
4.6
Closing
remarks
Phagotrophic
protists
have
essential
roles
in
marine
systems,
as
consumers
of
biomass
at
the
base
of
food
webs
and
as
regenerators
of
inorganic
nutrients
from
the
organic
matter
of
their
prey.
Previous
laboratory
studies
using
model
cells
have
provided
limited
information
on
how
biochemical
receptor
binding
and
intracellular
signaling
mechanisms
might
regulate
protistan
behavior
and
predator‐prey
interactions
in
microbial
food
webs
under
natural
conditions.
In
addition,
very
little
is
known
about
the
role
of
photosensory
mechanisms
in
heterotrophic
protists
and
how
they
might
play
a
part
in
ecological
interactions
and
more
specifically
feeding
behavior.
Understanding
the
importance
of
these
mechanisms,
and
to
what
extent
they
affect
feeding
behavior
in
natural
microbial
food
webs
is
essential
to
understanding
the
functional
ecology
of
phagotrophic
protists.
As
this
field
of
study
makes
advances
and
more
information
relating
to
biochemical
signaling
in
free‐
living
protists
becomes
available,
it
may
give
researchers
new
tools
and
approaches
70
in
the
study
of
in
situ
grazing
rates
and
prey
selectivity
by
marine
protists.
71
5
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