AN ABSTRACT OF THE THESIS OF
Lynne M. Fessenden for the degree of Doctor of Philosophy in Oceanography
presented on September 25, 1995. Title: Calanoid Copepod Diet in an Upwelling
System: Phagotrophic Protists vs. Phytoplankton
Redacted for privacy
Abstract approved:
J.
Calanoid copepod diet was investigated in Oregon coastal waters to determine
the trophic significance of phagotrophic protists as copepod prey within an ecosystem
where the microplankton biomass is typically dominated by large diatoms. Prior to and
during the 1991 upwelling season, clearance rates on phytoplankton and phagotrophic
ciliates were measured for Calanus pacificus, Centropages abdominalis, Acartia
ion giremis and Pseudocalanus sp.. During the 1992 upwelling season, clearance rates
were measured for Calanus marshailae and Pseudocaianus sp. on phytoplankton and
both phagotrophic ciliates and dinoflagellates. Copepods cleared ciliates at higher
mean rates than they cleared phytoplankton during both years, whereas dinoflagellates
were cleared at similar rates to phytoplankton in 1992. C. marshaliae cleared large
ciliates at higher rates than equivalently sized diatoms in the midst of diatom bloom
conditions, suggesting that C. marshaliae may select ciliates over diatoms.
Phagotrophic protists typically comprised <3% of the carbon ingested by
copepods during diatom blooms, but 16 to 100% between upwelling induced bloom
events and during winter months. Ingestion of ciliates alone provided enough carbon to
meet the basic respiration requirements of copepods in the winter of 1991. Copepods
are estimated to graze 50 to 100% of the phagotrophic protist biomass per day, thus
linking protist production to the carbon flux from surface waters.
The lipid content of Calanus pacificus CV's fed diets of diatoms vs.
phagotrophic protists was investigated in the laboratory during their maturation process.
Dietary inclusion of ciliates and dinoflagellates did not enhance the lipid content or
affect the lipid class composition of male copepods. Both diatom and dinoflagellate
diets supported a similar level of lipid in the females, whereas females fed diets of
ciliates had significantly lower levels of total lipid and no measurable wax ester. The
ratio of neutral to phospholipid (WE+TAG I PL) was >1 for males and <1 for females
for all treatments. The results of this preliminary lipid study suggest the question of
copepod lipid enhancement via dietary inclusion of protists should be addressed at the
level of specific fatty acids rather than lipid class.
©Copyright by Lynne M. Fessenden
September 25, 1995
All Rights Reserved
Calanoid Copepod Diet in an Upwelling System:
Phagotrophic Protists vs. Phytoplankton
by
Lynne M. Fessenden
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Completed September 25, 1995
Commencement June 1996
Doctor of Philosophy thesis of Lynne M. Fessenden presented on September 25, 1995
Redacted for privacy
Oeanography
Major Professor, represnti
Redacted for privacy
Dean of the College of Ocean
dAospecSciences
Redacted for privacy
Dean of GraduaQt 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.
Redacted for privacy
Lynne M. Fessenden, Author
ACKNOWLEDGMENTS
The majority of this work was supported by a three year Patricia Roberts Harris
Fellowship for Women and Minorities in Science from the U.S. Department of
Education. Thanks to Jeff Gonor for lining me up for that funding, it has definitely
made my life and work easier.
I am indebted to Charlie Miller for the kick in the pants at the critical moment,
for the continuum of sound advice and constructive criticism, and for the in-depth
editorial reviews of earlier drafts. Thanks to my major advisor, Tim Cowles, for his
generous patience with my alternative time line, for encouraging me to publish prior to
finishing, and for including me in two multi-investigator projects that allowed me to
gain research experience while getting to know several oceanographers from around the
country. Dian Gifford was one of those scientists I met at sea and it was her work that
inspired my approach to the investigation of copepod diet in Oregon waters. She has
coached me from afar throughout this project and constructively reviewed the
manuscript which is chapter two.
I am sincerely grateful to Ev and Barry Sherr for employing me in this final
year. They have been graciously flexible in allowing me time to finish this thesis, as
well as instrumental in educating me on protist biology. Thanks also to Pat Wheeler for
moral support and constructive comments on manuscripts.
Margaret Sparrow and Cheryl Morgan were both patient and good humored
while offering invaluable technical support with the lipid analyses. Fellow student and
sea going comrade Mary Lynn Dickson shared nitrogen and primary productivity data
from the first field season. John Rogers relentlessly volunteered his services as a deck
hand during my second field season. Nathan Potter, fellow lab mate, was always there
and always useful. Dr. David Montagnes, alias Ciliate Man, shared his knowledge and
expertise on culturing ciliates.
There are a few people without whom I would not have made it to this point of
completion. One is (former) fellow student Susanne Neuer, who kept me on track
during my roughest hours, and who has worn confidently and compassionately the
shoes of mentor, advisor, friend and colleague. Another, Mark Lorang, has the
steadiest pair of sea legs to grace the deck of the IVY Sacajawea, and has been the
steadiest of friends to grace my graduate career. I am deeply grateful for his "big
picture" perspective on life and science and the grad school game. My parents and
sisters have offered constant support and encouragement in the face of much whining.
And thank you Peder, for seeing me through this final leg.
This work is dedicated to the spirit of
Reginald Aubrey Fessenden
four generations later,
and to the memory of my grandparents,
Ken and Rita Fessenden,
to whom the completion of this thesis would have meant the most.
CONTRIBUTION OF AUTHORS
Dr. Timothy J. Cowles, serving as academic advisor, aided in interpretation of
data and editing of manuscripts.
TABLE OF CONTENTS
Page
1. INTRODUCTION ............................................................................................
1
The Physical Setting: Oregon's Upwelling System .................................... 8
The Ecological Setting ..............................................................................
13
Trophic Dynamics in Upwelling Systems ................................................... 16
The Role of Protists in Copepod Diet ........................................................ 20
Rationale for Experimental Technique ....................................................... 25
2. COPEPOD PREDATION ON PHAGOTROPHIC CILIATES IN
OREGON COASTAL WATERS .........................................................................
30
Introduction..............................................................................................
31
Methods....................................................................................................
33
Field Collections ............................................................................
Grazing Experiments .....................................................................
Quantification of Copepod Predation .............................................
UpwellingIndex ............................................................................
Results......................................................................................................
33
34
37
37
39
Phytoplankton and Protist Biomass ................................................ 39
Copepod Clearance Rates .............................................................. 42
Discussion................................................................................................
Ciliate Contribution to Copepod Diet ............................................
Possible Experimental Complications ............................................
Copepod Impact on Ciliate Population ..........................................
Altered View of Carbon Flux ........................................................
46
46
50
51
52
TABLE OF CONTENTS (Continued)
Page
3. COPEPOD DIET IN OREGON COASTAL WATERS
DURING UPWELLING SEASON ......................................................................
54
Introduction..............................................................................................
55
Methods...................................................................................................
57
Field Collections ........................................................................... 57
Grazing Experiments ..................................................................... 59
Microplankton Biomass Estimates ................................................. 60
Quantitative and Statistical Analysis of Copepod Feeding .............. 61
Egg Production Experiments ......................................................... 62
Results......................................................................................................
UpwellingIndices ..........................................................................
Phytoplankton Biomass ..................................................................
Protist Assemblage and Biomass ....................................................
Copepod Feeding Rates .................................................................
Copepod Egg Production ..............................................................
Discussion................................................................................................
63
63
63
66
70
73
75
Copepod Clearance Rates and Evidence of Prey Selection ............. 75
Composition of Copepod Diet ....................................................... 79
Impact of Copepod Predation on Protist Community ..................... 81
Summary and Conclusions ........................................................................
84
TABLE OF CONTENTS (Continued)
Page
4. LIPIDS IN CALANUS PACIFICUS FED A PHAGOTROPHIC
PROTIST VS. DIATOM DIET ..........................................................................
85
Introduction.............................................................................................
86
Methods.................................................................................................
91
Experimental Design and Cultures ...............................................
Lipid Class Analysis .....................................................................
91
95
Results....................................................................................................
96
Discussion..............................................................................................
103
Addendum..............................................................................................
107
5. CONCLUSIONS ............................................................................................
108
BIBLIOGRAPHY...............................................................................................
112
APPENDICES....................................................................................................
129
Appendix A Discussion of Ingestion Rate Calculations ......................... 130
Appendix B
Abundance and Composition of Phagotrophic Ciliate and
Dinoflagellate Assemblages in Oregon Coastal Waters ....... 133
LIST OF FIGURES
Pag
Figure
1.1
The Newport Hydrographic Line on the Oregon coast .............................
7
1.2
Cross-section of the water column off Oregon depicting the hydrography
and position of the permanent pycnocline .................................................
10
1.3
Two general models of the cross-shelf circulation in the Oregon upwelling
12
zone........................................................................................................
2.1
Bakun upwelling indices for 1991 ............................................................
2.2
Initial phytoplankton biomass for grazing experiments conducted in 1991.. 41
3.1
Bakun upwelling indices for April through September 1992 ...................... 64
3.2a
1992 in situ Chlorophyll a concentrations from 8m depth .......................... 65
3.2b
Initial Chlorophyll a concentrations for 1992 copepod grazing
experiments.................................................................................................. 65
3.3
Percent composition of copepod diet ......................................................... 72
3.4
Clearance rates of Calanus marshallae on large phagotrophic protists
plotted against clearance rates on diatoms .................................................. 78
4.1
The wax ester (WE), triacylgylceride (TAG), and phospholipid (PL)
content of Calanus pacficus adult males and females for each prey
treatment..................................................................................................
38
98
4.2
Mean lipid class composition on a per weight basis for Calanus pacificus
CV's, females, and males .......................................................................... 100
4.3
Ratio of neutral lipid content (WE+TAG) vs. phospholipid content (PL) for
Calanuspacificus adult males and females ................................................ 101
LIST OF TABLES
Page
Table
2.1.
Initial conditions for copepod grazing experiments conducted in 1991
40
2.2
Measured and calculated results of copepod grazing experiments:
phytoplankton and ciliate growth rate(k ±SD), decline rate (g) in the
presence of copepods, and copepod clearance rates ..................................
44
Copepod ingestion rates (±SD) on phytoplankton and ciliate prey
in Oregon coastal waters, total carbon ingested per day, and carbon
required for respiration .............................................................................
47
Initial phytoplankton and phagotrophic protist biomass for copepod
feeding experiments ..................................................................................
67
Initial numerical abundance of phagotrophic ciliates and dinoflagellates
in copepod feeding experiments ................................................................
68
2.3
3.1
3.2
3.3
Copepod clearance rates on phytoplankton and phagotrophic ciliates and
dinoflagellates during the summer of 1992 ................................................. 71
3.4
Copepod ingestion of phytoplankton and phagotrophic protists in ig
carbon per copepod per day ....................................................................... 74
3.5
Phagotrophic protist (ciliates and dinoflagellate) carbon as percentage
of total carbon measured in the food supply and in copepod diet ............... 80
3.6
Length, dry weight, and respiratory requirement of Calanus marshallae
and Pseudocalanus sp ..............................................................................
81
Mean cell density and estimated carbon concentration for each prey
treatment.................................................................................................
93
4.1
4.2
Per cell carbon estimates for autotrophic and heterotrophic microplankton
94
prey.........................................................................................................
4.3
Total lipid and percent composition of lipid classes, (WE, TAG, and PL) for
Calanus pacificus adult males and females fed each prey treatment ........... 99
4.4
Length, weight, and % lipid of Calanus pacificus initial CV's and adult
males and females ......................................................................................
102
CALANOID COPEPOD DIET IN AN UPWELLING SYSTEM:
PHAGOTROPHIC PROTISTS VS. PHYTOPLANKTON
CHAPTER 1
INTRODUCTION
Calanoid copepods historically were considered the major herbivores of the
plankton. They were classified as such primarily due to their abundance and
geographical association with blooms of phytoplankton, especially diatoms (Bigelow
1926, Fleming 1939, Bainbridge 1953). Copepods were often observed with
phytoplankton pigments in their guts (e.g. Lebour 1922, Marshall 1924, Gauld 1953)
and seemed morphologically suited for herbivory (e.g. Cannon 1928, Anraku and
Omori 1963). In laboratory studies of copepod feeding behavior, cultured
phytoplankton were used to provide unialgal diets on which the copepods were able to
grow and reproduce (e.g. Marshall and On 1955, Mullin and Brooks 1967, Paffenhöfer
1971). Carnivorous feeding by copepods was characterized as predation on copepod
eggs and nauplii and was thought to be confined to a few species (e.g. Dagg 1977,
Paffenhöfer and Knowles 1980, Landry 1981, Conley and Turner 1985).
Recognition of zooplankton grazing as one of the major processes influencing
the fate of primary production in the ocean also contributed to this overly simplified
view of copepod diet. The focus on a direct link between important primary producers
such as diatoms, and abundant herbivorous crustaceans such as copepods played a large
2
role in descriptons of productive pelagic food chains. In the past two decades, as our
understanding of the magnitude and importance of the microbial community has grown,
our concept of trophic energy exchange has shifted from simplistic, linear food chains
to complex, highly interactive food webs (e.g. Pomeroy 1974, Sieburth et al. 1978,
Williams 1981, Azam et al. 1983, Sherr and Sherr 1988, 1992), requiring that the
diatom*copepod link be re-examined. There is now ample evidence that copepod
diets are diverse, reflecting the variety of phytoplankton and microzooplankton taxa
present in the environment (reviewed by Kleppel 1993). Since the distribution of prey
taxa in copepod diet often differs from that in their environment, it has become
apparent that the nutritional needs of copepods must play a role in what was once
considered a simple carbon balance (Kieppel 1993). Pelagic microbes themselves can
be major consumers of primary production (e.g. Gifford 1988, Burkhill et al. 1993,
Verity et al. 1993, Neuer and Cowles 1994), and phagotrophic protists are recognized
as a trophic link between cells <5 im in diameter (pico- and nano-phytoplankton and
bacteria) and metazoans such as copepods (e.g. Sherr et al. 1986, Stoecker and Capuzzo
1990, Gifford 1991). The inclusion of phagotrophic protists in copepod diets offers a
link to carbon pools previously thought to be unavailable to copepods.
Upwelling ecosystems present a compelling case for examination of this trophic
link. We know that diatoms figure prominently in copepod diets during upwelling
events (Schnack and Elbrächter 1981, Smith 1982), however, a recent study by Neuer
and Cowles (1994) has revealed that large phagotrophic dinoflagellates are also major
grazers of diatoms during Oregon's upwelling season. These herbivorous protists
utilized 16 to 52% of the primary production during upwelling-induced diatom blooms.
What we don't know is whether copepods prey on the protists during these events. The
fate of protist secondary production depends on whether they are eaten by higher
consumers. Due to the small size of their fecal material (Stoecker 1984, Gowing and
Silver 1985), protists contribute to the recycling of organic matter within surface
waters, whereas copepods contribute to the flux of carbon to deeper water or sediments
because their fecal pellets are large and dense (e.g. Dagg and Walser 1986). Upwelling
regions are the most productive pelagic areas in the world ocean; therefore a link
between protist and copepod grazers within these systems could influence the fate of a
significant amount of both primary and secondary production.
The purpose of this study was to evaluate the trophic link between protists and
copepods within the Oregon upwelling system with the following questions in mind.
What is the fate of protist secondary production in Oregon waters? Is it recycled within
the surface waters or linked to higher trophic levels via consumption by copepods?
Will copepods eat protists when surrounded by dense aggregations of phytoplankton
prey? If copepods are eating protists in the midst of diatom blooms is it because of
some nutritional benefit? Calanoid copepods are the dominant mesozooplankton in
Oregon's nearshore waters (Pearcy 1976, Peterson and Miller 1976). A diverse
assemblage of flagellated and ciliated protists, in the appropriate size range to be
copepod prey, is also present and reaches its highest density following upwellinginduced diatom blooms (Neuer and Cowles 1994, Fessenden and Cowles 1994).
4
My primary research objective was:
1) To investigate the diet of calanoid copepods in Oregon's upwelling system
and to determine whether, and under what conditions, phagotrophic protists are
consumed by copepods.
A secondary objective was:
2) To explore a possible nutritional benefit to copepods from inclusion of
protists in their diet, via examination of copepod lipid content when fed cultured
diatoms, ciliates, and dinoflagellates in the laboratory.
A total of thirteen grazing experiments were conducted during two field seasons
(1991 and 1992). The field experiment on any given date was conducted with the most
prevalent copepod species and the natural microplankton assemblage at hand, so even
when a particular condition was replicated in time, such as a diatom bloom, the same
copepod species was not necessarily used. The results presented are therefore more of
an overview of possible trophic interactions than statements about the feeding behavior
of a particular species of copepod. The grazing experiments during both years were
conducted with adult females or Stage V copepodites of Calanus marshailae, Calanus
paccus, Centropages abdominalis, Acartia ion giremis, or Pseudocalanus sp..
Field sampling and copepod collection were conducted five miles off Newport,
Oregon (Fig 1.1). Station NH-5 is on the Newport Hydrographic line, which has a
historical record of hydrographic and zooplankton population data. During the 1991
field season, copepod grazing experiments were conducted monthly or bi-monthly
throughout the winter, spring, and summer and were done in conjunction with Susanne
Neuer's (1992) investigation of protist herbivory and MaryLynn Dickson's (1994)
investigation of nitrogen dynamics. In 1992, grazing experiments were purposefully
concentrated during the upwelling season with bi-weekly sampling throughout July and
August.
In introducing this study, I first discuss the physical and ecological setting of
Oregon's upwelling system, along with the traditional view of trophic dynamics in
upwelling ecosystems and the ways in which that view has begun to change. I review
what is known of copepod predation on protists and the protist role in copepod diet,
and, as there are many approaches to investigating copepod diet, I discuss the rationale
behind my experimental technique. Chapter two reports the results of an investigation
during the winter, spring, and summer of 1991 of copepod predation on pelagic
phagotrophic ciliates, and compares ciliate and phytoplankton contribution to copepod
diet. Chapter three presents an in depth investigation of copepod diet during an
upwelling season (1992), and includes copepod grazing rates on phytoplankton as well
as phagotrophic ciliates and dinoflagellates. In Chapter four I present the results of a
laboratory investigation of copepod lipid content when fed diets of diatoms versus
phagotrophic ciliates and dinoflagellates. The appendices include a discussion of the
equations used to calculate copepod ingestion rates (Appendix A), and a list of the
phagotrophic protists identified during the study along with a comparison of the in situ
abundance and biomass estimates of ciliates and dinoflagellates during 1992 upwelling
season (Appendix B).
Much of the literature refers to all non-autotrophic protists as heterotrophic.
However, recent studies have shown that many heterotrophic ciliates (40 to 50% in
neritic waters) sequester chloroplasts from their food and are capable of photosynthesis
(reviewed by Stoecker et al. 1989). Also, some bloom-forming autotrophic
dinoflagellates are capable of phagocytosis (Stoecker 1992), and a photosynthetic
dinoflagellate from Arctic waters has been observed with pico-phytoplankton in cell
vacuoles (pers. obs.). Due to this prevalence of mixotrophy among protists, I prefer to
use the term "phagotrophic" when referring to cells that ingest food.
7
=
I
I
I
47*
WASH
Mlora
46*
I200
-
0
30
II
=
124
l24
I
I
I
/
o0,_
-
o
=
Newport
,
,
50
60
40
30
20
10
44*
I
44*
-;
30
=
I
I
I25
I
I24
I24
30
Coos 8y
-
43*
25
0
omi
BrookinOs
---- CAL
128*
I III
I
HI
12
126*
125*
124*
II II Hull H 11111!! 111111111111 IHI HIM H
Figure 1.1. The Newport Hydrographic Line on the Oregon coast.
From Oceanography: Perspectives on a Fluid Earth by Steve Neshyba. Copyright ©
1987, by John Wiley & Sons, Inc. Reprinted by permission of John Wiley & Sons, Inc.
The Physical Setting: Oregon's Upwelling System
Coastal upwelling is a mesoscale response of the ocean to large-scale wind
forcing, a process in which subsurface water rises to the surface alongshore and moves
offshore at the surface (Barber and Smith 1981). The world's four maj or coastal
upwelling regions are located along eastern ocean boundaries where predominantly
equatorward winds circle mid-ocean atmospheric high pressure systems. Upwelling
along the Oregon coast is the northern limit of a larger system extending along the west
coast of North America from Baja, California. In the northern hemisphere the mean
alongshore flow is southward at the surface with a jet like structure in the offshore
direction and a poleward undercurrent. The divergence in the surface current at the
coastal boundary is due to offshore Ekman transport induced by the alongshore wind
stress.
On the Oregon coast, wind stress is predominantly southward (favorable for
upwelling) from April through September (Bakun 1973). The high pressure system in
the Gulf of Alaska shifts south during the winter months, causing a reversal in the
predominant alongshore wind stress. The seasonality of the Oregon system is in
contrast to the year-round continuous upwelling off Peru and Northwest Africa. Even
during summer months coastal upwelling occurs as intermittent events off Oregon due
to fluctuations in wind speed and direction followed by the immediate response (less
than one day) of the upwelling circulation (Barber and Smith 1981). An upwelling
event may persist for 3-10 days (Huyer 1976, Small and Menzies 1981). There are
9
usually four or five major events during a season, in addition to many of lesser intensity
(Huyer 1976). Wind reversals lead to relaxation of upwelling, and if persistent,
downwelling. Huyer (1976, 1977, 1983) has described the hydrodynamic features of
Oregon's upwelling region. Stratification of the water colunm over the narrow
continental shelf is strong, and the mixed layer depth is limited to about 20 m. Active
upwelling primarily affects the circulation and hydrography of this shallow surface
layer. The offshore flow occurs in the upper 20 m, and does not extend farther than 50
km from the coast. During upwelling events the permanent pycnocline, characterized
by the 25.5-26.0
interval, slopes upward and intersects the surface to form an
upwelling front within 15 km of shore (Fig 1.2). Changes in the spatial distribution of
density due to wind fluctuations are greatest between the upwelling front and the shore.
The maximum onshore flow is just below the pycnocline. The source water for the
vertical transport typically comes from 20 to 35 km offshore and from a depth between
100 and 200 m. Temperature isopleths follow the upward sloping pattern of the
isopycnals, and the coldest surface waters lie adjacent to the coast. During the summer
months, outflow from the Columbia River is carried southward along the Oregon coast
and this warm, low salinity water forms a lens at the surface which often determines the
seaward limit of the upwelling flow.
Freshly upwelled water is characterized by nitrate concentrations between 15
and 25 jiM (Dickson 1994). The amount of nitrate available to phytoplankton depends
on the duration of an upwelling event. According to Small and Menzies (1981), the
10
NtUTICAL MILES OFFSHORE
80
60
40
20
00
20
40
60
80-E
KD0
l20
140
ISO
200
Figure 1.2. Cross-section of the water column off Oregon depicting the hydrography
and the position of the permanent pycnocline. During a summer upwelling event, the
pycnocline slopes upward and transects the surface to form the upwelling front. During
winter, when the predominant winds are reversed, downwelling causes the pycnocline
to slope downward. Reprinted from Deep Sea Research, Vol. 28A, Small, L.F. and
Menzies, D.W., Patterns of primary productivity and biomass in a coastal upwelling
region, pp. 123-149, Copyright (1981), with kind permission from Elsevier Science Ltd,
The Boulevard, Langford Lane, Kidlington 0X5 1GB, U.K.
11
mesoscale patterns of primary production are highly dependent on the upwelling state
and have a response time of less than a day to changes in circulation. In early summer,
a single band of dense phytoplankton forms a few miles offshore running parallel to
bathymetric contours. In late summer there are typically two bands of high
productivity, one inshore of the pycnocline and the other seaward of the upwelling
front. The simultaneous development of both bands suggests a two-cell zonal
circulation pattern, in contrast to the single cell circulation earlier in the season (Fig
1.3). The nearshore cell is separated from the offshore cell by the steep density gradient
of the frontal region. Wroblewski (1977) modeled phytoplankton biomass dynamics
using the two-cell circulation scheme and obtained agreement with late summer field
observations. Zooplankton distributions during active upwelling also suggest a two-cell
zonal circulation pattern (Peterson et al. 1979). Under weak upwelling conditions the
biomass bands are closer to the coast, and are often twice as productive as when
upwelling is strong (Small and Menzies 1981). Less intense upwelling allows
phytoplankton cells a longer residence time in the photic zone and thus more effective
utilization of available nutrients, although the cessation of upwelling ultimately results
in the depletion of nitrate in the surface waters.
12
100
.
E
200
60
40
kilometers
20
0
100
E
200
60
40
20
0
kilometer3
Figure 1.3. Two general models of the cross-shelf circulation in the Oregon upwelling
zone. The one-cell model was proposed by Huyer (1976), the two-cell model by
Wroblewski (1977). Reprinted from Deep Sea Research, Vol. 26A, Peterson, W.T.,
Miller, C.B. and Hutchinson A., Zonation and maintenance of copepod populations in
the Oregon upwelling zone, pp. 467-494, Copyright (1979), with kind permission from
Elsevier Science Ltd, The Boulevard, Langford Lane, Kidlington 0X5 1GB, U.K.
13
The Ecological Setting
The highest biomass of both phytoplankton and zooplankton is usually found in
the upper 20 m of the frontal region. Historically, the highest chlorophyll a
concentrations reported in the Oregon upwelling system were 20 .tg per liter (Peterson
et al.1979, Small and Menzies 1981). However, in our two year investigation of this
region, chlorophyll a concentrations as high as 55 ig per liter were observed during
upwelling induced phytoplankton blooms, and Dickson's (1994) estimate of maximum
primary production, 1650 g C m2y', derived from both nitrogen uptake and 14C
experiments, was three to six-fold higher than previous measurements.
Neuer and Cowles (1994) and Fessenden and Cowles (1994, i.e. Chapter Two of
this thesis) have published the only data to date on protist abundance in Oregon coastal
waters. Heterotrophic protist biomass is low in newly upwelled water and highest
during phytoplankton blooms, ranging from 3 to 74 ig carbon per liter throughout the
year. The assemblage consists of choreotrichous ciliates, thecate and athecate
dinoflagellates, and non-pigmented nanoflagellates. Cell densities span an order of
magnitude,
iO3
to
iO4 per
liter. The ciliate fauna is dominated by aloricate oligotrichs,
while tintinnids typically represent less than 10% of the count. The autotrophic ciliate
Myrionecta rubra (formerly Mesodinium rubrum) was more common in the nonupwelling season during this study and never reached bloom forming abundances (10
cells per liter; Smith and Barber 1979). Autotrophic dinoflagellates are comparable in
abundance to the heterotrophic forms during non-upwelling periods, but the
heterotrophic dinoflagellates dominate, often by an order of magnitude, following
14
upwelling-induced diatom blooms.
Peterson and Miller (1976) surveyed zooplankton abundance and distribution
over Oregon's continental shelf between 1969 and 1972. Copepods were the most
frequently occurring and the most abundant members of the zooplankton conmiunity in
nearshore waters (within 10 miles). Six species of copepods dominated the community
during the upwelling season (in order of decreasing average abundance):
Pseudocalanus sp., Acartia clausii, Acartia ion giremis, Calanus marshailae,
Centropages abdominaiis, and Oithona similis. Although 32 other copepod species
were identified in samples taken during summer months, none approached the numbers
consistently reached by these six species (500 to 20,000 per m3). Variations in species
assemblages typically reflect seasonal changes in advection. Flow from the south
during the fall and winter months brings copepods with southern affinities into the
region, whereas spring and summer populations have northern affinities.
Five of the dominant copepod species interact with the circulation patterns in
ways that maintain their population within the upwelling zone (Peterson et al. 1979).
Acartia clausii is almost completely restricted to the upper 5 to 10 m of the water
column and the first 5 km from shore. Pseudocaianus sp. is abundant from 0 to 15 km
and between 10 and 20 m depth, and reproduces only within a few kilometers of the
shore. Acartia ion giremis lives and reproduces 10 km offshore in the surface mixed
layer. Oithona similis is also abundant offshore but slightly deeper (20 m). Calanus
marshaliae lives offshore as older copepodite stages, but the females return shoreward
(10 km) to lay their eggs and the nauplii and younger copepodites develop in the very
15
nearshore zone.
Other common, although less abundant, holoplankton are: euphausiids,
chaetognaths, larvaceans, ctenophores and pteropods (Peterson and Miller 1976).
Common meroplankton include larval forms of barnacles, bivalves, gastropods, crabs
and pink shrimp (Lough 1975, Peterson and Miller 1976, Rothlisberg and Miller 1983).
Thysanoessa spinfera is the common coastal euphausiid, and adults reach densities of
15 per m3. Sagitta elegans is the predominant chaeotognath during summer months (10
per m3), yet no relationship was observed between their abundance and upwelling
events (Peterson and Miller 1976). A larvacean, Oikopleura sp., is most commonly
found offshore in the low salinity water associated with the Columbia River plume (20
per m3). Ctenophores are abundant during early upwelling season and range from 2 to 8
per m3. The pteropod, Limacina helicina, is most frequently found in nearshore waters
during weak upwelling events. Peterson et al. (1979) reported a meso- and macro-
zooplankton (everything >120 .tm) dry weight biomass of 50 to 200 ig per liter.
The seasonal abundance peaks of the larvae of crab, shrimp, and fish are similar
but do not correspond with the peak abundance of zooplankton (Richardson and Pearcy
1977). Larval fish common in coastal waters during the spring and early summer
months are smelt, Pacific tomcod, butter sole, sand sole, and flounder (Richardson and
Pearcy 1977, Mundy 1984). All of these fish spawn between February and June, and by
August very few of their larvae are present. Juvenile salmon also frequent coastal
waters in June, feeding on eupausiids, hyperiid amphipods and larval fish (Peterson et
al. 1982). The only coastal fish that actually spawns during the peak upwelling season
16
is the northern anchovy, and it does not spawn in the upwelling front but offshore in the
Columbia river plume (Richardson 1980).
Trophic Dynamics in Upwelling Systems
Upwelling regions have the highest annual mean productivity of any pelagic
areas of the world ocean (Ryther 1969). This distinction in primary production is due
to the maintenance of optimal nutrient and light conditions for phytoplankton growth
within a stabilized horizontal flow (Barber and Smith 1981). Upwelling induced
phytoplankton blooms have the quantitative impact of annual spring blooms with
regard to biomass, primary production and export of organic material. In addition to
supporting high levels of primary production (>300 g carbon m2 year'), upwelling
regions are responsible for almost half of the world's fish catch (Ryther 1969).
Ryther' s classic essay attributed this elevated fish production to the short length and
high trophic efficiency of the food chains in upwelling ecosystems. He associated
fewer trophic steps with the presence of large primary producers and phytophagous fish,
and high trophic efficiency in part with the dense aggregations of phytoplankton prey.
Many of the diatoms common to upwelling regions form large gelatinous masses or
long filamentous chains which can reach several millimeters in size (Ryther 1969,
Elbrachter and Boje 1978), and many of the indigenous clupeoid fishes, such as the
Peruvian anchoveta, have modified gill rakers for filtering large phytoplankton from the
water (Cushing 1978).
17
Margalef (1978) argued, from a somewhat different perspective, that food
chains are kept shorter in upwelling areas due to the variability in the vertical
circulation. Thus, in the upwelling front, opportunistic species will have the advantage.
The response to the physical event of upwelling is an ecological event in which the
nutrient enriched waters support blooms of primary producers followed by blooms of
primary consumers. The pattern of intermittent bloom events results in a less mature
ecosystem, that is, one with abundant populations but low diversity.
The combination of short food chains and efficient trophic transfers between a
few dominant species was generally believed to be sufficient to explain the high
productivity of clupeoid fish (sardines and anchovies), which form the bulk of the
commercial catch in upwelling regions. However, in the last two decades the shift in
perception of how pelagic foodwebs function has forced us to reconsider Ryther's
hypothesis. Microheterotrophic pathways cannot be overlooked in upwelling systems
since nano- and pico-phytoplankton can be major contributors to primary production
(e.g. Probyn et al. 1990), bacterial production is high (e.g. Painting et al. 1992 and
1993), heterotrophic protists are abundant (e.g. Sorokin and Kogelschatz 1979, Beers et
al. 1980, Neuer and Cowles 1994), and regenerated nitrogen is plentiful (Whitledge
1978, Probyn 1985 and 1987, Kokkinakis and Wheeler 1988, Dickson 1994). Also, the
assumptions regarding the traditionally phytophagous habit of clupeoid fish have been
challenged. While it has been known for some time that small diatoms are nutritionally
inadequate for survival of larval anchovy, and that the larvae feed on dinoflagellates
and small zooplankton (Lasker 1975, Cushing 1978, Richardson 1980), recent work
18
indicates that adult anchovy in both the Benguela and the California system feed
predominantly and most efficiently on zooplankton, rather than on phytoplankton
(James 1987 and 1988, Chiappa-Carrara et al. 1989).
It is now apparent that the length of the average food chain to a pelagic fish in
an upwelling system is at least two steps, as opposed to Ryther's assignment of 1.5.
From simulation studies of trophic flow in the Benguela system, which incorporate
microplanktonic processes and near-exclusive carnivory by the anchovy, Moloney
(1992) asserts that longer food chains require a higher trophic efficiency to account for
the observed fish production. In contrast to the value of 20% assumed by Ryther
(1969), she estimates trophic efficiencies of 40 to 50% for the first trophic transfer, 15
to 20% for the second, and 5 to 10% for remaining transfers. Moloney et al.'s (1991)
simulations also illustrate that because pico-phytoplankton are ubiquitous components
of marine plankton communities, the number of potential trophic pathways will
generally increase with the size of the largest autotroph. Thus a number of different
length trophic paths will exist alongside of the diatom
fish path, implying that
productive ecosystems may have more complex food webs than unproductive ones.
A review of fishery yields in the four major upwelling areas by Hutchings
(1992) revealed that fish production in the Humboldt (or Peru) system is an order of
magnitude higher than in the Canary, Benguela, or California systems. Some of the
reasons advanced for this difference are lower turbulence, increased stability, and
restricted advective losses due to Peru's concave coastline, wider shelf and shallower
mixed layer. This stability may favor the growth of dinoflagellates (Huntsman et al.
19
1982), which are eminently suitable as food for first-feeding larvae (Lasker 1975), and
because dinoflagellates can form much denser concentrations than crustacean larvae,
survival of fish larvae may be enhanced in the Peru system.
In the Benguela system, adults of the Cape anchovy acquire the bulk of their
daily ration feeding on calanoid copepods and euphausiids (James and Findlay 1989).
Painting et al. (1993) found mesozooplankton production, and thus also fish production,
to be suppressed in the Benguela system due to the episodic nature of the upwelling
pulses which result in an overall dominance of small phytoplankton cells (<5 rim).
They concluded that herbivorous copepods become food-limited during the quiescent
phase of the upwelling cycle when the phytoplankton community is dominated by small
cells. They did not examine the possibility of a trophic link between microzooplankton
and copepods; however, a study by Matthews (1991) concluded that in maturing
upwelled water only 3% of the nitrogen ingested by protists was transferred to
mesozooplankton in the Benguela system. Probyn et al. (1990) estimated a 14%
contribution of protist nitrogen to mesozooplankton production in aged upwelled waters
(which translates to approximately twice Matthew's value), by applying measurements
of nitrogen uptake to a regeneration-based model with small cells dominating primary
production. Microzooplankton grazers are estimated to remove 3-50% of the primary
production per day in the system, but their impact is confined almost entirely to
phytoplankton cells <10 im (Painting et al. 1993).
Although diatoms dominate the autotrophic biomass and production during
Oregon upwelling events (Dickson 1994), the microbial community is also important.
20
New and regenerated production are of similar magnitude (Dickson 1994), and
phagotrophic protists are responsible for grazing 16-52% of the primary production
(Neuer and Cowles 1994). The rates of protist herbivory were closely linked to the
abundance of large (>20 pm), athecate, gymnodinoid dinoflagellates. The abundance of
these cells covaried with the abundance of diatoms, indicating the diatoms were being
utilized as a dinoflagellate food source. Thus for copepods in Oregon waters, protist
grazers are both a link to smaller phytoplankton as well as direct competitors for diatom
prey.
The Role of Protists in Copepod Diet
Four recent reviews of protozoan-metazoan trophic interactions in the plankton
cover research in marine (Sherr et al. 1986, Stoecker and Capuzzo 1990, Gifford 1991)
and freshwater environments (Sanders and Wickham 1993). Each emphasizes common
points with regard to protists as prey for copepods: the size range and frequently high
abundances of heterotrophic protists argue for their utilization as food by planktonic
copepods; copepod clearance rates on protists are typically as great as, or greater than
those on phytoplankton cells; and since inclusion of protists in copepod diets enhances
growth and fecundity, protists may be an important source of essential nutrients. The
following is a brief synopsis of the evidence for copepod ingestion of protists, with
additional recent results not included in the reviews.
The examination of copepod gut contents (e.g. Pierce and Turner 1992) and
fecal pellets (Turner 1984, Urban et al. 1992) has demonstrated the inclusion of various
21
protists (tintinnine ciliates, choanoflagellates, and thecate dinoflagellates) in their diets.
However, the majority of planktonic ciliates and at least half of the heterotrophic
dinoflagellates do not have hard coverings (loricae or thecae), and thus they produce no
recognizable remains. Urban et al. (1992) noted abundant amorphous mucous blobs in
the fecal pellets of Calanusfinmarchicus which they assumed to be "soft" celled
protists. This observation was made in the fall of their seasonal study, when C.
finmarchicus fecal pellets also contained tintinnine ciliates, choanoflagellates and
thecate gymnodinoid dinoflagellates. Although a seasonal succession of
microplankton, from diatoms to heterotrophic microbes, can be observed in copepod
fecal pellets, the presence of aloricate or athecate protists in any season is completely
missed.
Several laboratory studies have demonstrated predation by calanoid copepods
on planktonic ciliates (Robertson 1983, Ayukai 1987, Jonsson and Tiselius 1990).
Copepods, including Centropages typicus and several species of Acartia, when offered
a mixture of ciliates and phytoplankton, cleared ciliates at rates 2 to lOx higher than
they cleared phytoplankton (Turner and Anderson 1983, Stoecker and Sanders 1985,
Stoecker and Egloff 1987, Wiadnyana and Rassoulzadegan 1989). Ciliates were
preferentially selected by Acartia tonsa when an autotrophic dinoflagellate prey source
was more abundant in terms of carbon and nitrogen than the ciliate (Stoecker and
Sanders 1985), and by Acartia clausi and Centropages lypicus when offered
equiproportional amounts (as biovolume) of an oligotrichous ciliate, a dinoflagellate,
and a diatom (Wiadnyana and Rassoulzadegan 1989). Sheldon et al. (1986) observed a
22
preference by Euterpina acutifrons for an oligotrichous ciliate over diatoms and
phytoflagellates in mesocosm experiments.
There is some evidence that copepods can capture heterotrophic nanoflagellates.
Acartia tonsa cleared small flagellates (3-5 tim) at lower rates than larger cells but at
comparable rates to autotrophic cells of the same size (Caron 1984). Kopylov et al.
(1981) documented ingestion of heterotrophic nanoflagellates associated with detrital
particles by Acartia clausi. Very few studies have quantified the predation rates of
copepods specifically on heterotrophic dinoflagellates. Oxyrrhis marina has been used
as a food source for 4 copepod species in continuous culture (Klein Breteler et al.
1990). The copepod Temora longicornis ingested Oxyrrhis at a higher rate than the cooccurring phytoplankton, but at a lower rate than two co-occurring oligotrichous ciliates
(Hansen et al. 1993). Acartia tonsa cleared the heterotrophic dinoflagellate
Protoperidinium cf. divergens at higher rates than the co-occurring autotrophic
dinoflagellate (Jeong 1994).
Recent studies of calanoid copepods feeding on natural microplankton
assemblages, from estuarine to open ocean to polar waters, suggest heterotrophic
protists supply from 1-70% of total carbon ingested (Gifford and Dagg 1988, 1991,
Tiselius 1989, Dolan 1991, Atkinson 1994). Acartia tonsa from Terrebonne Bay, on
the Gulf of Mexico, cleared protists at rates an order of magnitude higher than they
cleared phytoplankton (Gifford and Dagg 1991). Aloricate ciliates dominated the
protist population in the bay, and they contributed 50% of the carbon ingested by A.
tonsa when the chlorophyll standing stock was <5 jim in size, and only 2% of ingested
23
carbon when the chlorophyll standing stock was dominated by large diatoms, >20 im
(Gifford and Dagg 1988, 1991). In the subarctic Pacific, where the low phytoplankton
standing stock is dominated by cells < 5 rim, 60-70% of the carbon ingested by
Neocalanus plumchrus was from ciliate and dinoflagellate prey (Gifford and Dagg
1991). Atkinson (1994) found heterotrophic ciliates and dinoflagellates comprised
43%, on average, of the carbon ingested by pseudocalanids during the austral
midsummer near South Georgia.
Both laboratory and field data have shown copepod fecundity to be enhanced by
the dietary inclusion of heterotrophic protists. Acartia tonsa increased egg production
by 25% in the lab when tintinnid ciliates were added to its unialgal diet (Stoecker and
Egloff, 1987). During egg production experiments with Calanus pacificus, conducted
with natural prey assemblages from Puget Sound, Runge (1985) found that in two
instances when egg production rates were high, there were no phytoplankton >7 im
present, but there were high concentrations of ciliates. White and Roman (1992) found
egg production of Acartia tonsa in the Chesapeake Bay to be significantly correlated
with protist biomass, while not correlated with any measure of phytoplankton biomass,
production or ingestion. A similar finding for Acartia tonsa off southern California
was made by Kleppel et al. (1991). Copepod egg production was highly correlated with
both microplankton biomass and dietary concentrations of ciliates and dinoflagellates,
whereas diatom biomass was not systematically associated with egg production.
Ohman and Runge (1994) found Calanusfinmarchicus egg production in the Gulf of
24
St. Lawrence was sustained by heterotrophic ciliates and dinoflagellates when ambient
phytoplankton concentrations were low.
Other evidence suggests copepod growth is enhanced by a protist diet. Adult
body size and rate of development of Temora Ion gicornis and Pseudocalanus elongatus
were increased when the standard algal diet was supplemented with a large
heterotrophic flagellate (Klein Bretler 1980). Estuarine copepods grew poorly when fed
detritus with small amounts of microbiota, but grew well when the detritus was rich in
ciliates (Heinle et al. 1977).
Protists may supply essential nutrients to copepod diet. Heterotrophic protists
are a protein rich prey source, with a C:N ratio of 3-5 compared to a C:N of 6-14 for
phytoplankton (reviewed in Stoecker and Capuzzo 1990). Along with being rich in
proteins, protists are a source of essential fatty acids and sterols (Stoecker and Capuzzo
1990). While some phytoplankton are rich sources of total lipids, many lack the specific
lipids critical to certain functions in copepods (Stottrup and Jensen 1990).
Docosahexaenoic acid (22:6) is a crucial component of the phospholipid in copepod
cell membranes (Benson and Lee 1975) and along with eicopentaenoic acid (20:5) is
effective in promoting growth and normal metabolism (Kanazawa et al. 1979, Phillips
1984). Copepods are capable of synthesizing these two long-chain polyunsaturated
fatty acids (PUFAs) from a shorter-chained precursor, linolenic acid (18:3) (Benson and
Lee 1975, Sargent and Falk-Peterson 1981). Although linolenic acid is found in most
algae and higher plants, it is a minor constituent of diatoms (Kates and Volcani 1966,
Kattner et al. 1983, Mayzaud et al. 1989). Diatoms are rich in C-16 and C-20 length
25
PUFAs, but typically have >50% saturated and monosaturated short chains (Kates and
Volcani 1966, Lee et al. 1971, Kattner et al. 1983, Sargent et al. 1985, Claustre et al.
1989). From the little work that has been done regarding the lipid composition of
marine protists, we know that tintinnine ciliates and both autotrophic and heterotrophic
dinoflagellates are rich in the long-chain PUFAs that are essential to copepod growth
(Harrington et al. 1971, Dikarev et al. 1982, Claustre et al. 1989).
In conclusion, heterotrophic protists are consumed, often selectively, by
copepods, and may offer specific nutritional value as well as a link to bacterial and
picoplankton production. Protists are qualitatively and quantitatively important prey
sources for copepods in environments and seasons in which primary production is
dominated by cells <5 .im, such as nearshore environments between phytoplankton
blooms, oligotrophic waters, and in detritus dominated food webs. However, we do not
know the extent to which protists contribute to copepod diets when the phytoplankton
assemblage is dominated by large diatoms, as is frequently the case in coastal upwelling
systems.
Rationale for Experimental Technique
As previously mentioned, a few field studies and several laboratory studies have
documented ingestion of protists by copepods. Typically, laboratory incubation
experiments have been conducted with a single phytoplankton source and an easily
cultured heterotrophic protist. My objective was to expand upon the field
26
investigations using natural prey assemblages to determine the role of protists in
copepod diets in an episodic, diatom rich upwelling system.
In order to examine copepod predation on phagotrophic protists, most of which
contain no chlorophyll, I incubated copepods in their natural prey assemblage and
monitored the changes in protist numbers over time. I chose to do microscopic counts
of all phagotrophic cells, which allowed me to see the range of prey that copepods
confront in their natural environment. Ciliates and dinoflagellates vary greatly in size
and shape, and in this system are in the same size range as the prevalent diatoms.
Electronic particle counters size particles on the basis of their volume, not their linear
dimensions (Harbison and McAlister 1980), making it impossible in this type of
experiment to separate autotrophic from heterotrophic prey on the basis of size alone.
Along with trophic distinctions, the data from microscopic counts offer taxon specific
classification. Copepod grazing of autotrophic microplankton was also measured
during the experiments by monitoring disappearance of chlorophyll a. During the 1992
field season, copepod egg production was also measured as an independent estimate of
ingestion.
The use of natural particle assemblages does not free incubation experiments
from bottle effects. The longer seawater is enclosed in a bottle, the greater is the
deviation of the quantity and quality of the suspended particulate material from its
original state (Roman and Rublee 1980). Possible containment effects in bottles with
copepods include nutrient enrichment and crowding. Phytoplankton growth may be
enhanced in experimental bottles due to excretion of ammonia by copepods, resulting in
27
a masking of the true differences in pigment concentrations and an underestimate of
grazing rates (Roman and Rublee 1980). Copepods may be inhibited by crowding or by
contact with the vessel walls, and entrainment may hinder vertical migration. Peters
and Downing (1984) found the volume allowed per animal to be a positive factor on
feeding rates, and along with Roman and Rublee (1980) noted feeding rates are
negatively correlated with incubation time. Minimizing containment effects requires
large containers, minimal copepod density, and the shortest incubation time permitting
the detection of a grazing difference between experimental and control treatments. I
used one and two liter bottles for my incubations, and did not crowd the copepods (5-10
per liter, natural densities: 1-20 per liter). Incubation times were 8 to 12 hours.
Nutrient limitation was not a problem in these experiments. Nitrate concentrations
were very high on the sampling dates during both upwelling seasons (10 to 20 iM), and
were above limiting concentrations on the winter sampling dates (6 to 9 jiM, Dickson
1994).
An indirect estimate of copepod ingestion can be made from measuring egg
production during incubation experiments (KiØrboe et al. 1985, Peterson et al. 1990).
The carbon content of the eggs is converted to carbon ingested via an estimate of gross
production efficiency. As egg production is a measure of previous feeding, it is less
likely to be biased by possible reductions in grazing due to bottle effects. It is also a
measure of all prey ingested, both heterotrophic and autotrophic.
Obtaining feeding rates from pigment analyses or incorporation rates of radio-
labeled substances limits the measure to grazing on fluorescent or labeled particles. The
28
gut fluorescence technique (Mackas and Bohrer 1976) has been particularly useful for
obtaining grazing rates in situ, as it avoids artifacts associated with incubations and
electronic particle counters. Gut pigment content is measured upon capture and a gut
evacuation rate is determined by monitoring over time the gut pigment of copepods
placed in filtered sea water. Like egg production, it is actually a measure of what
copepods have been eating prior to capture, however unlike egg production, it is only a
measure of ingestion of pigmented food. In order to differentiate between ingestion of
heterotrophic versus autotrophic cells, the heterotrophic cells must be observed
microscopically or labeled in such a way as to separate them from autotrophic cells.
Kieppel et al. (1988) have addressed this problem by extracting carotenoid pigments
specific to both plants and animals from copepod guts. They identified the pigments
using high performance liquid chromatography (HPLC), and were able to determine the
percent contribution of both phytoplankton and microzooplankton to copepod diet.
Carotenoids are commonly used as taxon-specific biomass analogs for planktonic
organisms (Smith et al. 1987) and show promise as tracers in copepod ingestion
experiments.
Several investigators have measured copepod ingestion of radio-labeled prey,
using '4C-bicarbonate for autotrophic particles and tritiated thymidine ([methyl-3H]-
thymidine) for bacteria (Haney 1971, 1973, Daro 1978, 1980, Roman and Rublee 1981,
White and Roman 1991). Tritiated thymidine is not taken up directly by heterotrophic
dinoflagellates (Lessard and Swift 1985), and although many flagellates and ciliates are
bacterivorous, there is no way of knowing if detected thymidine label within a copepod
29
is from an ingested bacterivorous protist or a piece of bacterial coated detritus.
Phagotrophic protists ingest the labeled phytoplankton too, complicating the
interpretation of the autotrophic label within copepods.
Simultaneous comparisons of grazing estimates made using the gut fluorescence
method, disappearance of chlorophyll, disappearance of cells, and egg production have
resulted in good agreement (Kiørboe et al. 1985, Peterson et al. 1990). Although each
method has its strengths and limitations, they all yield reliable estimates of in situ
feeding rates in phytoplankton bloom situations. By monitoring the disappearance of
both chlorophyll and phagotrophic protists in the presence of copepods I have
quantified copepod ingestion on autotrophic and heterotrophic prey sources and have
determined the magnitude of the protist component of copepod diet in a system
characteristically dominated by large autotrophs. I have also separated the
heterotrophic prey component taxonomically, and report copepod grazing rates on both
ciliates and dinoflagellates. The results of the grazing experiments, along with
estimates of the impact of copepod predation on protist standing stocks, are discussed
in Chapters 2 and 3.
30
CHAPTER 2
COPEPOD PREDATION ON PHAGOTROPHIC CILIATES
IN OREGON COASTAL WATERS (1991)
Lynne M. Fessenden and Timothy J. Cowles
Marine Ecology Progress Series
Vol. 107: 103-111, 1994
31
Introduction
Phagotrophic protists are now recognized as a trophic link between <5 tm cells
(bacteria, picophytoplankton and nanoflagellates) and metazoans such as copepods
(Sherr and Sherr 1988, Stoecker and Capuzzo 1990, Gifford 1991). Bactivory by
phagotrophic protists has been well documented (reviewed by Sherr and Sherr 1989),
and the quantitative importance of protists as consumers of phytoplankton may equal or
exceed that of metazoan grazers (Sherr and Sherr 1992). Neuer and Cowles (1994)
recently measured protist herbivory in the upwelling region off the Oregon coast and
found that the protist assemblage of heterotrophic and mixotrophic ciliates, and
heterotrophic dinoflagellates and nanoflagellates, grazed 16 to 52% of the potential
primary production. The ultimate fate of the primary production grazed by protists
depends on whether the protists themselves are eaten by higher consumers. Due to the
small size of their fecal material (Stoecker 1984, Gowing and Silver 1985), protists
contribute to the recycling and increased residence time of organic matter within
surface waters (Welschmeyer and Lorenzen 1985). It is not clear if the protist-copepod
trophic link operates under the diatom bloom conditions typical of coastal upwelling
regions. Determining the magnitude of this link in productive coastal waters is relevant
not only to trophic studies but also to the quantification of carbon flux.
Phagotrophic protists, including heterotrophic nanoflagellates, heterotrophic
dinoflagellates and heterotrophic and mixotrophic ciliates, often dominate marine nano-
(2 to 20 jim) and microplankton (20 to 200 jim) assemblages (Sherr et al. 1986,
32
Stoecker and Capuzzo 1990, Gifford 1991). The C:N ratio of the heterotrophic forms
(3-5:1) is lower than that of phytoplankton (6-15:1), suggesting that protists are a
protein rich prey source for zooplankton predators (reviewed by Stoecker and Capuzzo
1990). Kieppel et al. (1991) have found the variability in egg production of copepods
in the field (coastal and open ocean) to be better correlated with changes in
microzooplankton biomass than with diatom biomass. They argue that protists may be
responsible for providing a major portion of copepod nutrition, especially that
associated with reproductive energy. In the lab, the calanoid copepod Acartia tonsa
increases its egg production by as much as 25% when tintinnine ciliates are included in
its diet (Stoecker and Egloff, 1987).
Aloricate, choreotrichous ciliates are commonly the dominant phagotrophic
protists in the size range most efficiently grazed by adult copepods (Sherr et al. 1986,
Berggreen et al. 1988). Several laboratory studies have demonstrated predation by
calanoid copepods on both loricate and aloricate ciliates (Robertson 1983, Ayukai 1987,
Jonsson and Tiselius 1990). When offered a ciliate/phytoplankton mixture, copepod
clearance rates are found to be significantly higher on ciliates (Stoecker and Sanders
1985, Stoecker and Egloff 1987, Wiadnyana and Rassoulzadegan 1989). Recent
studies of copepods feeding on natural microplankton assemblages, from estuarine to
oceanic waters, suggest ciliates supply from 1 to 80% of total carbon ingested (Tiselius
1989, Dolan 1991, Gifford and Dagg 1991, Gifford 1993a). Generally, a greater
percentage of ingested carbon is supplied by ciliates when phytoplankton stocks are
low or dominated by small cells. Kleppel et al. (1988) found phagotrophic protist
33
carbon (detected by the presence of specific carotenoid pigments) dominated gut
contents of copepods off southern California when phytoplankton biomass and
productivity were relatively low.
This paper presents results from a series of copepod grazing experiments
conducted with natural microplankton assemblages from Oregon coastal waters
between January and August of 1991. We found that calanoid copepods grazed
phagotrophic, aloricate ciliates on most sampling dates, with exceptions occurring in
late July and August during upwelling-induced diatom blooms. The highest copepod
clearance rates on ciliates were measured when the phytoplankton standing stock was
<5 ig Chl-a F', which was the case during the winter months and between upwelling
blooms. The results suggest that the degree of trophic linking between ciliates and
copepods in these waters may vary as a function of timing and intensity of upwelling
events.
Methods
Field Collections
Copepods and microplankton assemblages were collected 5 miles off Yaquina
Head, Oregon, in 70 m of water. The station is within the upwelling zone on the
Newport Hydrographic line (44°40 N), and was chosen for its historical record of
hydrographic and zooplankton population data. Sampling cruises were conducted
monthly during January, February, April and June to August of 1991 (twice in July).
Copepods were collected in gentle oblique tows from 40 m with a 0.75 m ring net of
34
335 pm mesh and a 4 liter cod end. Water for the grazing experiments was collected
from 8 m in Niskin bottles with interior silicon springs. This depth was commonly
within the chlorophyll and primary productivity maxima, and was the depth from which
water was obtained for protistan herbivory experiments conducted on the same dates
(Neuer and Cowles 1994). The microplankton assemblage was collected by gently
filtering water through a submerged 200 pm mesh to remove macrozooplankton. The
filtrate was stored in a water bath under a neutral density screen during transport to the
shore-based laboratory. A 200 ml aliquot was preserved with 10% (v/v) acid Lugols
solution for microzooplankton enumeration. Samples for in situ chlorophyll analysis
were split into three size-classes (<3 pm, 3-20 pm, >20 pm), collected on GFJF or 3
pm nucleopore filters, and extracted in 90% acetone for 24 hours at 0°C. Temperature
and salinity were recorded using a portable CTD.
Grazing Experiments
Copepod clearance rates were determined by measuring the change in
chlorophyll concentration and ciliate density during bottle incubations (Gifford 1 993b).
Copepods were kept in a cooler and sorted under a dissecting scope immediately upon
returning to the lab. The most abundant calanoid copepod, >335 pm, was chosen for
the grazing experiment on each sampling date. Adult females or copepodite stage CY's
were placed in 1 liter beakers containing the microplankton assemblage, kept in the
dark at 100 C, and allowed to acclimate for 24 h prior to experimental incubations.
Since the counting variation typically encountered when enumerating protists is ±20%,
it is necessary to separate the copepod feeding signal from the variation (Gifford
35
1993b). Therefore, body size and known phytoplankton clearance rates of the copepod
species selected for each experiment were taken into consideration when determining
how many copepods to add to the experimental bottles (10 to 25 copepods bottle-1).
The microplankton assemblage was kept in the dark at 100 C for 24 h while
copepods acclimated, and at the start of an experiment was mixed gently and siphoned
into 2 liter polycarbonate bottles using silicon tubing. Three control (microplankton
assemblage only) and three or four experimental (microplankton assemblage plus
copepods) bottles were filled along with one "initial assemblage" bottle. Aliquots for
chlorophyll analysis were taken immediately from the "initial assemblage" bottle and
processed as above. A 200 ml sample (preserved in 10% acid Lugols solution) was also
taken from the initial assemblage for enumeration of ciliates. As treatment bottles were
not sampled until the end of an experiement, they were free of air space or bubbles
which are known to cause protist mortality (Gifford 1993b). The bottles were secured
to a plankton wheel rotating at 0.3 rpm. Experimental incubations were conducted at
10°C (range of in situ temperature at 8 m: 8 to 11°C). The winter and spring
experiments were incubated for 8 hours, at night, in the dark. The summer incubations
ran for 12 h (evening and night) and were exposed to the local light regime, resulting in
5 h of light (100 FIE) and 7 h of darkness.
At the end of the experiments, chlorophyll samples were taken in triplicate from
each bottle, filtered onto GFIF filters, and extracted in 90% acetone. Chlorophyll and
phaeopigment were analyzed using a Turner Designs Model-lO fluorometer (Strickland
and Parsons 1972). Microzooplankton samples were collected from all bottles and
36
preserved in 10% Lugols solution (as above). Preserved microzooplankton were
concentrated by settling 50 to 100 mIs sample', for 24 h. The entire settling chamber
was enumerated for all ciliates >10 jim using an inverted microscope at 200X
magnification, so that >100 ciliates were counted for each replicate. Assuming the
ciliate distribution in the sample is Poisson, confidence intervals can be obtained for
single counts (Lund et al. 1958, Venrick 1978). A count of 100 cells will give a 95%
confidence interval of the estimate within ±20% of the mean (Lund et al. 1958).
Ciliates were measured and categorized by geometrical shape in order to convert
to biovolume, and identified to genus when possible. Five to ten size categories were
used for each of 2 shapes: spheres and cones. Aloricate ciliate carbon was estimated
using the ratio of 0.19 pg carbon
jim3
(Putt and Stoecker 1989). As this relationship
was determined for ciliates preserved in 2% Lugols solution, an additional shrinkage
factor of 1.5 was applied to the ciliate biovolumes in these experiments (D. Stoecker,
pers. comm.). It should be mentioned that the ciliate carbon estimates presented here
are underestimates, as oral membranelles were not always included in the determination
of biovolume. A carbon:chlorophyll ratio of 50:1 was used for estimating
phytoplankton biomass. Dickson (1994) measured particulate nitrogen at the same
station, on the same dates, and estimated carbon:chlorophyll ratios were 45 to 50 during
both the winter and summer of 1991. Landry and Lorenzen (1989) also used a ratio of
50:1 when calculating zooplankton grazing estimates in Washington coastal waters.
37
Quantification of Copepod Grazing
Copepod clearance rates were calculated according to Frost (1972). Clearance
rates on phytoplankton were determined only when the difference in chlorophyll
concentration between control and experimental bottles proved significant (t-test, p <
0.05). The coefficient of variation between replicate chlorophyll samples was 5%. In
order to determine clearance rates on ciliates the observed changes in ciliate density
needed to exceed 20%. Differences in ciliate density were based on the entire ciliate
count (ciliates ranging in size from 10 to 100 jim). Copepod clearance rates on specific
size categories of ciliates and chlorophyll were not analyzed in these experiments.
Ingestion rates were calculated by multiplying clearance rate by the initial standing
stock (Mann et al. 1986). Ingestion rates calculated in this way will be slightly higher
than those calculated using the logarithmic mean prey concentration during a given
experiment (Frost 1972).
Upwelling Index
The Bakun upwelling index (Bakun 1973; made available by the NOAAINMFS
Pacific Fisheries Environmental Group, Monterey, CA.) was used as a measure of the
strength of the wind-induced coastal upwelling on all sampling dates (Figure 2.1). The
index, presented as weekly averages at 45° N, 125° W, has units of m3
(100 m of
coastline)'. A positive index indicates water is driven offshore, while a negative index
indicates downwelling. Indices >50 reflect strong upwelling along this coastline (Small
and Menzies 1981).
C)
1 50
100
G)
-C
50
0)
C
0.
-50
C
-100
co
-150
-200
Figure 2.1. Bakun upwelling indices for 1991 (weekly averages at 45° N, 125° W).
Units are m2 s (100 m of coastline)1. An index >50 indicates strong upwelling. Arrows
indicate sampling dates.
0
39
Results
The rnicroplankton assemblage used in the experiments was collected on the
same day as the copepods, due to constraints on boat time, and thus sat for 24 hours (in
the dark at 100 C), while the copepods were acclimating. The phytoplankton biomass
(chlorophyll) increased on average by 50% in the absence of large grazers. The
increase occurred in both the 3 to 20 tm and >20 tm chlorophyll size ranges. The
ciliate density increased on average by 43%, but showed little change in size
distribution. On the April and late July dates, ciliate mortality occurred in the
microplankton assemblage and ciliate density was ca. 45% lower in the experimental
assemblage than in the field.
Phytoplankton and Protist Biomass
Chlorophyll concentrations ranged from 2 to 45 tg 11 in the experimental
microplankton assemblages (2 to 33 tg 11
in situ)
between January and August of 1991
(Table 2.1). The late July and August sampling dates coincided with strong upwelling
events (Figure 1.1). On these dates, chlorophyll concentration in the experimental
assemblage was ca. 45 jig 1' and more than 80% of the phytoplankton biomass was
composed of diatoms >20 jim (Figure 2.2). On all other dates, with the exception of
June (11.2 jig chlorophyll
11),
the chlorophyll concentration was <5 jig 1'. During
January, February, and early July the majority of the phytoplankton biomass was in the
<20 jim fraction (Figure 2.2).
Table 2.1.
Date
Initial conditions for copepod grazing experiments conducted in 1991.
Chlorophyll a
(pg F')
Aloricate
ciliates> 10 pm
(ind.l )
Phytoplankton
carbon
(pgl
)
Ciliate
carbon
(pg 1')
22Jan
3.4
4810
170
11
26 Feb
2.0
7370
100
32
14 Apr
4.6
1200
230
3
24 Jun
11.2
1700
560
3
09 Jul
4,7
2200
235
6
23 Jul
44.3
3500
2215
8
20 Aug
44.6
7700
2230
15
C
45
40
>20gm
35 +
3-2O.tm
30
U<3.tm
P 25 ±
>%
a-
o
20
o
o 15
22-Jan
26-Feb
14-Apr
24-Jun
9-Jul
23-Jul
20-Aug
Figure 2.2. Initial phytoplankton biomass for grazing experiments conducted in 1991
(in terms of size fractions of chlorophyll a).
42
Phagotrophic ciliate carbon was <6.5% of the phytoplankton carbon on all but
the February sampling date, when it was 32%. Ciliate densities ranged from 1200 to
7700 cells 11(1100 to 6500 in situ), with highest abundances in February and during
the August upwelling bloom (Table 2.1). Aloricate choreotrichous genera were most
prominent. Tintinnids were present but not abundant (<10011). The prominent
mixotrophs were Laboea spp. and Tontonia spp., and their contribution to total
numbers was greatest in January and February (ca. 50%). No large predatory ciliates,
such as Dindinium spp., were observed on any of the sampling dates.
Growth of ciliates in the controls was observed in January, June and late July,
and ranged from 0.2 to 1 doubling per day (Table 2.2). Mortality was observed in the
controls during the February, April, July and August experiments. There was no
apparent response of copepod clearance rates to either mortality or growth of the
ciliates.
Copepod Clearance Rates
Copepods cleared ciliates during the 5 grazing experiments conducted between
January and early July, when chlorophyll concentrations did not exceed 12 tg 11 (Table
2.2). In late July and August, when upwelling events induced diatom bloom conditions
(44 jig chlorophyll
11),
no significant clearance of ciliates was measured.
The highest clearance rates on ciliates were measured for Calanus pacificus in
January, 12.6 to 32.4 ml copepod' h' (Table 2.2). However, no clearance of
phytoplankton by C. pacificus was detected. In February, Pseudocalanus sp. cleared
43
Table 2.2. Measured and calculated results of copepod grazing experiments:
phytoplankton (Chi) and ciliate growth rate (k ±SD), decline rate (g) in the presence of
copepods, and copepod clearance rates. All experimental replicates presented.
(ns: no significant difference between control and experimental prey densities, p >0.05)
Table 2.2. (Legend on previous page)
Date
Copepod Species
g
k
(h1)
(ha)
22 Jan
Calanuspacficus
(female)
26Feb
Pseudocalanussp.
(female)
Clearance rates
(ml copepod' h1)
Chi
Ciliates
Chi
Ciliates
Chi
Ciliates
-0.0169
(0.0000)
+0.0442
(0.0035)
ns
ns
ns
ns
0.1318
0.1392
0.0992
0.0541
ns
ns
ns
ns
32.4
30.7
23.1
12.6
-0.0239
(0.0055)
-0.0248
(0.0260)
0.0228
0.0151
0.0052
0.0114
0.0312
0.0306
0.0474
0.0461
3,5
2.3
0.8
1.8
4.8
4.7
7.4
7.2
14 Apr
Centropages abdominalis
(female)
-0.0010
(0.0116)
-0.0578
(0.0230)
ns
ns
ns
0.0459
0.0443
0.0443
ns
ns
ns
7.1
6.9
6.9
24 Jun
Centropages abdominalis
(female)
+0.0096
(0.0015)
+0.0065
(0.0147)
ns
ns
ns
0.0360
0.0250
0.0128
ns
ns
ns
3.4
2.3
1.2
09 Jul
Cent ropages abdominalis
(female'
+0.0146
-0.0052
(0.0043')
0.0033
0.0099
0.0542
0.0559
0.4
(0.0020')
6.3
6.5
Acartia longiremis
(female)
+0.023 8
+0.0270
(0.0253)
0.0063
0.0035
-0.0002
ns
ns
ns
0.7
0.4
0.0
ns
0.0065
0.0065
0.0022
ns
ns
ns
0.9
0.9
0.3
ns
ns
ns
23 Jul
20 Aug
Ca1anuspacficus
(CV)
(0.0006)
+0.0025
(0.0033)
-0.0076
(0.0217)
1.2
ns
ns
45
ciliates at rates of 4.7 to 7.4 ml copepod1
-i
copepod h
-1
.
h1
and chlorophyll at rates of 0.8 to 3.5 ml
Centropages abdominalis was the most abundant mesozooplankton
grazer available for experiments in April, June and early July. This copepod cleared
aloricate ciliates at rates of 6.3 to 7.1 ml copepod' h1 in April and early July, and at
somewhat lower rates, 1.2 to 3.4 ml
h', in June. C. abdominalis cleared
-1
-1
phytoplankton at measurable rates only on the July date (0.4 to 1.2 ml copepod h ).
Only two replicates are reported for 9 July due to copepod mortality in one of the
bottles.
No detectable clearance of ciliates was measured for Acartia ion giremis or
Calanus pacificus during the diatom blooms in late July and August respectively.
Clearance rates based on chlorophyll removal were 0.0 to 0.7 ml
ion giremis and 0.3 to 0.9 ml
copepod1 h1
copepod1 h1 for A.
for C. pacficus (Table 2.2).
Ingestion of ciliate carbon, in jig C copepod1 d, averaged 6.5 (± 2.4) for
Calanus pacificus, 4.6 (± 1.1) for Pseudocaianus sp. and 0.7 (±0.4) for Centropages
abdominalis (Table 2.3). The highest weight-specific ingestion rate of ciliates was
found for Pseudocaianus sp., which ingested 58% of its body carbon per day. This was
an order of magnitude higher than the weight specific ingestion of ciliates measured for
C. pacifIcus (6% of body carbon per day) or C. abdominalis (ito 7% body carbon per
day).
46
Discussion
Ciliate Contribution to Copepod Diet
Omnivory is especially apparent among copepods living in highly dynamic
environments which exhibit numerous phytoplankton bloom cycles (Landry 1981). Our
results reinforce previous observations of protist-copepod trophic links occurring within
such cyclic environments (Gifford and Dagg 1991). We found that calanoid copepods
in Oregon coastal waters cleared aloricate, phagotrophic ciliates from natural
microplankton assemblages at considerably higher rates than they cleared
phytoplankton, except during upwelling-induced diatom blooms. During nonupwelling months and between bloom events, ciliates contributed 16 to 100% of the
estimated carbon ingested by copepods, even though ciliate biomass was significantly
less than phytoplankton biomass.
Ciliates comprised 100% of the estimated carbon ingested by Calanus pacficus
in January when the entire phytoplankton assemblage was <20 .tm in size and about
half of it was <3 im (see Figure 2.2). In February, Pseudocalanus sp. averaged 52%
phytoplankton and 48% ciliate carbon in its daily ration (Table 2.3). The chlorophyll
biomass was still low, but diatoms >20 tm accounted for 42% of it. Pseudocalanus sp.
cleared ciliates at rates 1.3 to 9.3 times higher than rates measured for clearance of
chlorophyll. On 9 July, 16% of the carbon ingested by Centropages abdominalis came
from ciliates and 84% from phytoplankton. On that date, 20% of the chlorophyll
Table 2.3. Copepod ingestion rates (± SD) on phytoplankton (Chl) and ciliate prey in Oregon coastal waters,
total carbon ingested per day, and carbon required for respirationa. (ns: no significant clearance rate measured, p >0.05)
Date
Copepod species
Ingestion rates
(pg C copepod1 d')
Chi
Ciliates
Total carbon ingested
(Mg C copepod'1
d1)
Respiration
requirementa
(pg C copepod1
22 Jan
Calanuspac4ficus
ns
6.5 (2.4)
6.5 (2.4)
6.5
26 Feb
Pseudocalanus sp.
5.0 (2.7)
4.6 (1.1)
9.6 (1.9)
0.6
14 Apr
Centropages abdominalis
ns
0.5 (0.01)
0.5 (0.01)
1.1
24 Jun
Cent ropages abdominalis
ns
0.2 (0.06)
0,2 (0.06)
1.1
9 Jul
Centropages abdominalis
4.6 (3.2)
0.9 (0.02)
5.5 (3.0)
1.1
23 Jul
Acartia longiremis
23.1 (16.2)
ns
23.1 (16.2)
0.3
20 Aug
Calanuspaqficus
37.3 (18.5)
ns
37.3 (18.5)
6.5
0884
a: R O.1O1Wc
(Dagg et al. 1982)
d)
48
biomass was > 20 jim. C. abdominalis cleared ciliates at rates 5 to 15 times higher than
they cleared chlorophyll.
Similar results were found for Acartia tonsa in Terrebonne Bay, on the inshore
Gulf of Mexico (Gifford and Dagg 1991). A. tonsa cleared protists at rates an order of
magnitude higher than those measured for chlorophyll. Aloricate ciliates dominated the
protist population in the Gulf, and they contributed 50% of the carbon ingested by A.
tonsa when the phytoplankton standing stock averaged <5 jim in cell size (August).
Ciliates comprised only 2% of the carbon ingested by A. tonsa in January, when the
chlorophyll standing stock was dominated by diatoms >20 jim. During both seasons in
Terrebonne Bay, the chlorophyll biomass was high (30 jig 11). Our results are also
consistent with the observed grazing behavior of 3 species of Calanus in open and ice-
edge areas of the Greenland Sea (Barthel 1988). In the diatom bloom conditions of the
shelf polynya, copepods ingested diatoms, although ciliate biomass was substantial. In
pack-ice regions where the phytoplankton was dominated by small flagellates, copepods
ate primarily ciliates. In open waters, where diatom and ciliate biomass were roughly
equal, copepods ingested both diatoms and ciliates.
We observed an exception to this trend in the April and June experiments, when
Centropages abdominalis did not clear chlorophyll at detectable rates even though large
diatoms were present. Ingestion of ciliates alone did not meet the estimated respiration
requirements (Dagg et al. 1982) of C. abdominalis on these dates (Table 2.3).
Copepods in all other experiments satisfied their respiration requirements with the
ingestion of ciliates or phytoplankton or the combination of both. Centropages is
49
known to tend toward carnivory (Paffenhöffer and Knowles 1980), and it is possible
that they were feeding on protists or nauplii that weren't accounted for in these
experiments.
Clearance rates on ciliates for Centropages abdominalis ranged from 28 to 170
-1
-1
ml copepod d
.
These rates are in the same range as those measured for C. hamatus
(32 to 125 ml copepod1 d') feeding on ciliates in a natural microplankton assemblage
in coastal Sweden (Tiselius 1989). Clearance rates on ciliates for C. abdominalis were
much lower in June (25 to 50 ml copepod1 d1) than in April and early July (150 to 170
-1
-1
ml copepod d ). The lower clearance rates in June may be due to a shift in the prey
size distribution: 50% of the ciliates were <20 im in size. With the exception of June,
cell size of the aloricate ciliate populations averaged 30 to 60 jim.
Neuer and Cowles (1994), using epifluorescence microscopy, found
heterotrophic gymnodinoid dinoflagellates were more numerous than ciliates in these
waters on the same sampling dates, although ciliate biomass equaled or exceeded the
dinoflagellate biomass on all but the August date. Assuming copepods clear
heterotrophic dinoflagellates at the same rate as they clear ciliates, the additional protist
carbon ingested would come close to balancing the respiration requirements of
Centropages abdominalis.
Copepod omnivory is likely to be unnecessary during diatom bloom conditions.
In contrast to the high clearance rates of Calanus pacificus on ciliates in January, no
clearance on ciliates was detected during the upwelling-induced diatom blooms for
either C. pacificus in August or Acartia ion giremis in late July. Clearance rates on
50
chlorophyll were low for both species (see Table 2.2), but the chlorophyll biomass was
so high (44 jig 1') that phytoplankton carbon ingested averaged 37 jig copepod1
for C. pacficus (33% of body carbon), and 23 jig
copepod1
d1
d' for A. ion giremis (578%
of body carbon) (Table 2.3).
Possible Experimental Complications
It is important to restate that the experimental food concentrations in our study
did not exactly replicate field conditions. Because of the 24 hour acclimation period,
the experimental microplankton assemblages often had higher concentrations of both
ciliates and phytoplankton than the in situ assemblages on the actual sampling dates.
The composition of the assemblage was not greatly changed, however, and the
increased prey concentrations still fell in the range that copepods experience in these
waters. The largest increase between experimental and in situ phytoplankton biomass
occurred during the June experiment (138%), but, as no clearance rate on
phytoplankton for Centropages abdominalis was detected, results were not affected. A
complete doubling of the ciliate population occurred between the August sampling and
experimental incubation. In this case, no clearance on ciliates was detected for
Calanus pacificus so the extreme increase again did not sway experimental results. In
the 3 experiments done with Centropages abdominalis, similar clearance rates on
ciliates were measured on 2 dates (April and early July) when the ciliate density
differed by a factor of 2 (Table 2.1). Although the prey densities in the incubations
51
differed somewhat from the field, we feel the results can still be safely applied to field
conditions.
The presence of predatory ciliates (eating smaller ciliates), and mixotrophic
ciliates (counted twice, once as chlorophyll, once as protist) in the natural assemblages
could bias the outcome of this type of grazing experiment. We did not observe large
predatory ciliate species on any of our sampling dates. Phytoplankton biomass was
much greater than ciliate biomass, with the exception of the February date, so that the
mixotroph contribution to the total chlorophyll was probably small. In February,
however, mixotrophs represented 50% of the phagotrophic ciliate count (according to
epifluorescence counts by Neuer 1992), and since chlorophyll biomass was low, the
clearance rates on chlorophyll for Pseudocalanus sp. may be biased by the presence of
mixotrophs. The autotrophic ciliate Mesodinium rubrum was also abundant in February
(40001', Neuer 1992), and some individuals may have been mistakenly included in the
phagotrophic protist counts.
Copepod Impact on Ciliate Population
Ciliate densities observed in this study are in the range of those previously
reported for coastal waters (Sherr et al. 1986, Burkill et al. 1987, Gifford 1988,
Montagnes et al. 1988, Tiselius 1989), although somewhat lower than the high end of
the range (2700 to 27,800) reported by Landry and Hassett (1982) in Washington
coastal waters. An estimate of copepod predation on ciliate biomass was obtained by
applying average clearance rates to historical average abundance estimates of
52
Pseudocalanus spp., Calanus marshailae (assume similar rate to Calanuspacijicus),
and Centropages abdominalis at the same station (Peterson and Miller 1976). Ignoring
fine-scale vertical differences, and considering only these 3 species, the estimated
grazing impact is 25% of the ciliate population removed per day. This is likely an
underestimate, since Acartia clausii and A. ion giremis both play a numerically
important role in the copepod assemblage at this location during the summer months
(Peterson and Miller 1976). Including them in the estimate would no doubt increase the
impact. Also, copepod nauplii are known to consume phagotrophic protists. Dolan
(1991) measured clearance rates of 0.4 ml
nauplius1 h1
for A. tonsa nauplii on
phagotrophic protists in Chesapeake Bay. Applying this clearance rate to abundance
estimates of the feeding stages of copepod nauplii (Peterson and Miller 1976, Landry
and Lorenzen 1989) adds an additional impact of 10 to 20% clearance per day on the
ciliate population.
Altered View of Carbon Flux
An altered view of pelagic carbon flux is suggested by the concept of the
microbial food web serving as the principal food resource for metazooplankton (Sherr
and Sherr 1988). In productive coastal waters, the consumption of primary production
by heterotrophic protists (versus rapid sinking of a diatom bloom) implies carbon is
available in the water column to metazoan consumers, or for remineralization, for a
longer period of time. If protists are consuming as much as 50% of the primary
production during upwelling-induced diatom blooms (Neuer and Cowles 1994), the
53
existence of a protist-metazoan link would greatly influence the fate of that carbon.
Copepods can contribute a disproportionately large percentage of the carbon flux from
surface waters due to the rapid sinking of their fecal pellets. Grazing and sediment trap
measurements made in Washington shelf waters show micro-crustaceans contributing
up to 21% of the grazing pressure on phytoplankton, but 65% of the vertical flux of
organic carbon (Landry and Lorenzen, 1989).
There is no evidence for mass sinking of diatom blooms in the Washington and
Oregon coastal upwelling system. Landry and Lorenzen (1989) maintain that the vast
majority of Washington shelf production is utilized locally and that up to 50% is grazed
by protists. Neuer and Cowles (1994) found the protist grazing impact to be of similar
magnitude on the Oregon shelf. Our results illustrate the potential for a substantial
portion of protist secondary production to be consumed by copepods between diatom
bloom events, and therefore to be linked to the carbon flux to deeper water or shelf
sediments.
54
CHAPTER 3
COPEPOD DIET IN OREGON COASTAL WATERS
DURING UPWELLING SEASON (1992)
Lynne M. Fessenden and Timothy J. Cowles
College of Oceanic and Atmospheric Sciences
Oregon State University
Corvallis, Oregon
55
Introduction
Coastal upwelling areas have been classified as regions of high trophic
efficiency where fewer trophic levels result from the presence of large plant cells
(Ryther 1969). Diatoms have long been considered the predominant food source for
copepods in upwelling ecosystems (Ryther 1969, Cowles 1979, Schnack and Elbrachter
1981, Smith 1982), and as evidenced by frustules in copepod fecal pellets, diatom
bloom conditions in any waters can provide a large percentage of copepod diet (Turner
1984, 1985). Throughout the last two decades, exceptions to the classic diatom-
copepod-fish food chain have emerged as our understanding of the magnitude and
importance of the microbial community has grown (Pomeroy 1974, Sieburth et al.
1978, Sorokin 1981, Williams 1981, Azam et al. 1983, Sherr and Sherr 1988, 1992),
and as copepod omnivory has been reported to be widespread (Reviews: Stoecker and
Capuzzo 1990, Gifford 1991, Kleppel 1993, Sanders and Wickham 1993). Dietary
diversity in copepods is now seen as the rule rather than the exception (Kleppel 1993).
The ability to switch between herbivory and carnivory allows copepods to respond to
substantial changes in food composition (Gifford and Dagg 1988, Kleppel et al. 1988),
and increases the probability that they will obtain nutritionally complete rations.
Recent field documentation of copepod predation on protists, from coastal and
open ocean waters, shows a general trend for copepod diet to follow seasonal shifts in
water column particulates, with a larger percentage of their diet consisting of protists
when phytoplankton stocks are low or dominated by small cells (Barthel 1988, Kieppel
56
et al. 1988, Tiselius 1989, Dolan 1991, Gifford and Dagg 1991, Urban et al. 1992,
Gifford 1993a, Atkinson 1994). A prior investigation (Fessenden and Cowles 1994) of
copepod predation on phagotrophic ciliates in Oregon coastal waters reached a similar
conclusion: calanoid copepods ingested ciliates during winter months, when
phytoplankton biomass was low and dominated by small cells, and in the summer
months, between the upwelling-induced diatom blooms. Phytoplankton was the only
detectable prey ingested by copepods during the diatom blooms.
The grazing impact of phagotrophic protists on phytoplankton production in
Oregon coastal waters during upwelling season is 16-52% (Neuer and Cowles 1994).
Athecate dinoflagellates were responsible for more grazing than ciliates, due to higher
cell specific clearance rates on phototrophic food sources and to their ability to ingest
large diatom prey. If these herbivorous protists are being eaten by copepods, the flux of
primary production from the surface waters will be enhanced via copepod fecal matter.
The alternative to this trophic link is the remineralization of protist secondary
production within the surface waters. Thus it is important to quantify the presence of
phagotrophic protists in copepod diet, especially in upwelling systems where, due to the
high level of primary production, the magnitude of the protist-copepod link will
substantially influence the magnitude of the carbon flux from the surface waters.
The study described here is an extension of our previous investigation
(Fessenden and Cowles 1994) of copepod predation on ciliates in Oregon coastal
waters, the only study to date of a protist-copepod trophic link within an upwelling
system. We focused this investigation on the diatom bloom conditions of the upwelling
57
season in an effort to evaluate whether diatom-grazing dinoflagellates serve as prey for
copepods. The experiments were designed to quantify copepod predation on both
phagotrophic dinoflagellates and ciliates in conjunction with grazing on diatoms. In
addition, copepod fecundity was simultaneously measured as an independent estimate
of ingestion and to relate variations in diet to an aspect of copepod productivity.
Methods
Field Collections
Six copepod grazing experiments and four egg production experiments were
conducted within a five week period during the 1992 upwelling season. Copepods and
microplankton were collected five miles off Yaquina Head, Oregon, twice in late July
and four times in August. The sampling station was within the upwelling zone on the
Newport Hydrographic line (44° 40' N). Depth at the station was 70 m. Copepods
were collected in oblique tows from 50 m, with a 335 im net. The microplankton
assemblage was obtained by gently filtering water collected at 8 m depth through a
submerged 200 jim Nitex mesh. The position of the chlorophyll maximum typically
varies between the surface and 20 m in these waters (Dickson 1994). We chose the 8 m
sampling depth as representative of the mixed layer and to maintain consistency with
the 1991 experiments. Other details of sampling protocol, i.e. handling of copepods
and microplankton assemblage, can be found in Fessenden and Cowles (1994). A
temperature profile in 5 m intervals was obtained with a thermistor. Samples for nitrate
58
determination were taken at 0, 8, and 20 m, frozen at -20°C, and analyzed within two
months using a standard Technicon Autoanalyzer. Nitrate values were used as an
indicator of upwelling along with the Bakun upwelling index (Bakun 1973), a measure
of the strength of wind-induced coastal upwelling, made available by the NOAA/NMFS
Pacific Fisheries Environmental Group, Monterey, CA.
Samples for chlorophyll analysis were taken from 0, 8, and 20m. Size-fractions
of <3 .im, 3-20 jim, >20 jim were filtered in triplicate onto GFIF filters, and extracted
in 90% acetone for 24 hours at -20°C. Chlorophyll a and phaeopigment fluorescence
were measured using a Turner Designs Model-lO fluorometer according to Strickland
and Parsons (1972).
A 200 ml aliquot of the microplankton assemblage was preserved with 10%
(vlv) acid Lugols solution (Throndsen 1978) for later enumeration by inverted
microscopy. Protists (especially flagellates) preserved with the Lugols fixitive cannot
easily be identified as autotrophs or heterotrophs, so a 50 ml aliquot of the
microplankton assemblage was preserved in 2% gluteraldehyde. They were kept on ice
until staining with DAPI, and filtering onto a black nucleopore filter (0.2 jim). Filters
were mounted on slides, stored at -20°C, and examined (within a year) by
epifluorescence microscopy to qualitatively evaluate the trophic status of the
dinoflagellates.
59
Grazing Experiments
Copepod clearance rates on chlorophyll and protists were measured as described
by Gifford (1993b). The experimental design utilizes two treatments: the experimental
treatment, in which the microplankton prey assemblage (protists and phytoplankton) is
incubated in bottles with copepods, and the control treatment, in which bottles contain
only the prey assemblage. Experimental and control assemblages were incubated in 1
liter polycarbonate bottles for 12 hours in the dark, rotating on their long axes at 0.3
rpm. Three replicates were incubated for each treatment. Experimental temperatures
ranged from 10 to 12°C (which was the in situ temperature range at 8 m).
Calanus marshallae CY's were abundant on both the July and the August 20th
sampling dates. C. marshallae females were used in the August 3rd experiment.
Calanus was not present in significant numbers on August 24 and 28, and on those
dates experiments were run with Pseudocalanus sp. females. Copepods were sorted
using a dissecting microscope and were acclimated to experimental conditions for 24
hours. For the Calanus experiments, copepod densities were 12 per liter, and for
experiments using Pseudocalanus, 20 per liter.
A subsample of the initial microplankton assemblage was taken at the beginning
of each experiment and subsamples of each replicate were taken at the end. Air spaces
in the bottles, a possible cause of protist mortality, were avoided in this way. Sizefractions of samples for chlorophyll were analyzed as above. Aliquots for quantitative
protist enumeration were preserved with acid Lugols solution, and aliquots for
60
qualitative evaluation via epifluorescence were preserved with 2% gluteraldehyde and
processed as above.
Microplankton Biomass Estimates
Microplankton samples preserved with acid Lugols solution were concentrated
by settling 50 ml aliquots for 24 hours. All protists (>10 rim) in the settling chamber
were enumerated under an inverted microscope at 200X magnification. Confidence
intervals were calculated around these single counts assuming a Poisson distribution of
randomly distributed organisms (Lund et al. 1958, Venrick 1978). A count of 100 cells
gives a 95% confidence interval of the estimate within ±20% of the mean, while a count
of 400 cells gives a precision of 10% of the mean (Lund et al. 1958). Estimates of
protist abundance were made from counts of >100 cells for all dates, and on July 22nd
and August 24th >400 cells were counted.
Loric ate (tintinnine) and aloricate ciliates, and thecate and athecate
dinoflagellates were identified to genus when possible, and categorized by size and
geometry. A shrinkage factor of 1.5 was applied to the ciliate biovolumes (D. Stoecker
pers. comm.), and a factor of 1.3 was applied to dinoflagellate cell volumes
(Montagnes et al. 1994). Aloricate ciliate carbon was determined from the empirical
volume-to-carbon relationship of Putt and Stoecker (1989), and tintinnid carbon
according to Verity and Langdon (1984). Dinoflagellate carbon was determined
according to Lessard (1991). A carbon:chlorophyll ratio of 50:1 was used for
estimating phytoplankton biomass (Dickson 1994). Protist and phytoplankton net
61
growth rates during the experiments were calculated from the difference between initial
and control concentrations, assuming exponential growth.
Quantitative and Statistical Analysis of Copepod Feeding
The success of this experimental design depends on separating the copepod
feeding signal from the variation in microscopic counts of protists (Gifford 1993b). As
mentioned above, a single count of 100 cells will give an estimate within ±20% of the
mean concentration in 95% of trials (Lund et al. 1958). Thus, if counts of 100 cells are
used, the observed changes in protist density resulting from the presence of copepods
need to exceed 20% to be statistically significant. Changes in prey abundance were
accepted as significant in categories of protists whose confidence limits for
experimental and control counts did not overlap.
Copepod clearance rates on phytoplankton were considered valid when the
observed difference in chlorophyll concentrations between control and experimental
treatments was significant (t-test, p <0.05). The coefficient of variation between
replicate chlorophyll samples in these experiments was <5%. In all six experiments,
the difference between the >20 im chlorophyll concentration in the control and the
experimental bottles exceeded 20%.
Copepod clearance rates on protists and phytoplankton were calculated from
Frost's (1972) equations. Ingestion rates were calculated by multiplying clearance rates
for a given category by the initial standing stocks of phagotrophic dinoflagellates,
ciliates, or >20 im phytoplankton (Mann et al. 1986). This method results in a
62
maximal estimate of ingestion (see Appendix A). Daily ingestion rates were calculated
based on the assumption of continuous feeding, which also maximizes our estimates.
Strong diel vertical migration patterns are not the rule for copepods in Oregon's coastal
waters (Peterson et al. 1979). In Peterson's study, Ca/anus marshallae was rarely
observed to be exclusively at depth during the day. Boyd et al. (1980) found vertical
migration patterns of copepods in the Peru upwelling system varied with the
concentration of available food; when food was abundant copepods did not migrate
and fed continuously.
Egg Production Experiments
Egg production was determined using the incubation method (Beckman and
Peterson 1986, Tiselius et al. 1991). Experiments using Calanus marshallae females
were conducted on the first four dates, in conjunction with the Calanus grazing
experiments. Copepods for egg production experiments were sorted immediately upon
arrival at the lab (about 1 hour after collection). Six sets of five Ca/anus females were
placed in 500 ml polycarbonate bottles containing 64 im pre-screened water from 8 m.
The screen of 64 tm was chosen to exclude C. marshallae nauplii and eggs from the
field while still allowing a prey assemblage through. C. marshallae eggs are
approximately 220 tm in size (Peterson 1980). Copepods were incubated for 16 hours
in the dark at in situ temperatures (10 to 12°C). At the end of the experiments, copepod
vitality was observed, and the entire volume was screened through a 64 tm sieve,
rinsed into a vial and preserved with 5% buffered formalin. Eggs were measured and
enumerated at a later date under a dissecting scope and cannibalized eggs (crumpled or
63
dented shells) were included in the count. Egg production was calculated as number of
eggs per female per 16 hours. Egg production was not measured for Pseudocalanus.
Results
Upwelling Indices
Bakun upwelling indices >50 reflect strong upwelling along this coastline
(Small and Menzies 1981). In 1992 (Figure 3.1), the July 27th and August 20th
sampling dates had indices >50. The lowest index during the five week sampling
period was 19, on August 3rd, a period of low winds near the end of a relaxation event.
On July 22nd, and August 24th and 28th, although the index was between 25 and 50, it
was on the decline. At no time during July and August did the index fall below 0, an
indication of complete wind reversal and the onset of downwelling. Nitrate was
plentiful (12 to 26 tiM) in the surface waters on all dates, excepting July 22nd and
August 3rd, when the surface waters were depleted but concentrations at 20 m reached
20 tiM. Temperature profiles indicate that on both of those dates the water column was
stratified, with the upper 10 m ranging from 12 to 15°C. On all other dates the water
column was relatively isothermal, with surface temperatures between 10 and 11°C and
temperatures at 50 m between 8 and 9°C.
Phytoplankton Biomass
Phytoplankton biomass spanned an order of magnitude, ranging from 195 to
2,165 jig carbon per liter during the five weeks sampled (Table 3.1). Chlorophyll
125
100
C)
C)
.c
75
C)
i5025
A
M
J
J
A
Figure 3.1. Bakun upwelling indices for April through September 1992 (weekly averages
at 45°N 125° W). Units are m2 s (lOOm of coastline)'. An index> 50 indicates strong
upwelling. Arrows indicate sampling dates.
S
45
40
>2Oum
35 +
30
25
>.
Q. 20
0
0
0 15
10
5
0
22-Jul
27-Jul
3-Aug
20-Aug
24-Aug
28-Aug
45
40
35
30
0)
25
0
0
20
15
10
5
0
22-Jul
27-Jul
3-Aug
20-Aug
24-Aug
28-Aug
Figure 3.2. A) 1992 in situ chlorophyll a concentrations from 8 m depth.
B) Initial chlorophyll a concentrations for 1992 copepod grazing experiments.
concentrations ranged from 3.9 to 43.3 rig/liter. Phytoplankton >20 urn (diatoms) were
present on all dates, and always composed more than 50% of the biomass. The three
dates on which phytoplankton <3 um were detected (Figure 3.2a) coincided with the
relaxation of upwelling winds (July 22, August 24 and 28).
The microplankton assemblage used in the grazing experiments sat for 24 hours
in the dark at in situ temperature prior to the start of the experiments. Figures 3.2a and
3.2b show the changes in chlorophyll concentrations which occurred in this 24 hour
period. Although the initial experimental chlorophyll concentrations were sometimes
greater and sometimes less than the in situ concentrations, there was very little
difference in the size distribution of the phytoplankton cells. The July 27 experiment,
however, displayed an increase in the <3 tm category relative to the initial distribution.
Protist Assemblage and Biomass
Phagotrophic ciliates and dinoflagellates were numerically equal in the protist
assemblage on most dates (Table 3.2), excepting July 27 and August 24 when ciliates
dominated. However, with regard to biomass, ciliates were often the dominant protists
(see Appendix B). Phagotrophic protist carbon ranged from 19 to 74 pg/liter, and was
>10% of the phytoplankton biomass on the three dates when algal carbon was at its
lowest (Table 3.1).
Aloricate choreotrichous ciliate numbers spanned an order of magnitude,
ranging from 2,882 to 22,682 cells per liter (Table 3.2). The genera most commonly
represented were Strombilidium, Strombidinopsis, and Strombidium (according to
Table 3.1. Initial phytoplankton and phagotrophic protist biomass for copepod
feeding experiments. All units are pg per liter (±SD). (Carbon:Chlorophyll = 50)
Chlorophyll a
Phytoplankton
Carbon
Phagotrophic
Protist Carbon
Date
>2O.tm
<20im
22 July
3.1 (±0.3)
2.9 (±0.1)
300
74
27 July
4.2 (±0.6)
1.7 (±0.2)
295
30
03 Aug
37.4 (± 1.9)
5.9 (±0.4)
2165
29
20 Aug
17.1 (±0.2)
1.4 (±0.1)
925
19
24 Aug
2.0 (±0.3)
1.9 (±0.1)
195
73
28 Aug
13.0 (±0.6)
3.0 (±0.2)
800
33
68
descriptions by Montagnes and Lynn 1991). Mixotrophic ciliates such as Laboea and
Tontonia contributed 3% of the total aloricate ciliate count. Tintinnine ciliates were
present in low numbers also, typically <10% of total ciliate count, with the exception of
July 27, where they contributed 34% of the count. The most common tintinnid was
Parafavella. Predatory ciliates were not observed in any of the samples. The
autotrophic ciliate Merionecta rubrum (formerly Mesodinium rubra) was present on the
last three August dates, at concentrations of 4,000 to 10,000 cells per liter. They were
not included in protist carbon estimates due to their inclusion as pigmented carbon.
Table 3.2. Initial numerical abundances of phagotrophic ciliates
and dinoflagellates in copepod feeding experiments.
All units are cells per liter. [95% confidence interval of the estimate]
Dinoflagellates
Date
Ciliates
22 July
18,744 [17516-20057]
15,092 [13993-16276]
27 July
4466 [3882-5 136]
990 [730-1337]
03 Aug
2,882 [2419-3432]
2,728 [2278-3264]
20 Aug
4,224 [3657-4877]
4,686 [4087-537 1]
24 Aug
22,682 [21328-24121]
3,388 [2883-3979]
28 Aug
7,436 [6674-8284]
7,282 [6528-8 122]
69
The phagotrophic dinoflagellates were dominated by the genera Gymnodinium,
Gyrodinium, and Protoperidinium. Their total numbers also spanned an order of
magnitude, ranging from 990 to 15,092 cells per liter (Table 3.2). Large, athecate
dinoflagellates were the most common, however they were always fewer in number
than ciliates, a finding that differs from the observation of Neuer and Cowles (1994). It
is likely that their ciliate numbers were underestimated, since their counts were based
on samples filtered for epifluorescence microscopy. Filtering samples, versus settling,
is known to damage aloricate ciliates and can result in a 30-50% underestimate (pers.
obs.). Examination of the microplankton assemblage via epifluorescence revealed that
there were far more heterotrophic than autotrophic dinoflagellates present on all dates
(typically >70% were heterotrophic). Neuer and Cowles (1994) also observed fewer
autotrophic than heterotrophic dinoflagellates during the 1990 and 1991 upwelling
seasons. Autotrophic dinoflagellate genera observed were Ceratium, Dinophyses,
Gonyaulax, and Prorocentrum.
As previously mentioned, the microplankton assemblage sat for 24 hours in the
dark at in situ temperature prior to the experiments, therefore the protist concentration
available to the copepods during the experiments was slightly different from the time of
capture. Protist growth in the assemblage prior to the experiments ranged from 10 to
40%, and on some dates there was mortality (10 to 50%). However, there was no
apparent shift in the size distribution of cells, and the range of protist concentrations in
the experiments was the same as that of the in situ range over the five week span.
70
Growth rates of aloricate ciliates during the experiments ranged from -0.3 to 1.7 per
day. The range of dinoflagellate growth was similar, -0.5 to 1.2 per day.
Copepod Feeding Rates
Copepod clearance rates on phytoplankton and phagotrophic ciliates and
dinoflagellates are given in Table 3.3. No significant clearance of the <20 jim
chlorophyll fraction was measured in any of the experiments, although all copepods
cleared phytoplankton >20 jim. Calanus females and CV's cleared phytoplankton at
similar rates (2.1 ±0.5 and 2.8 ±0.8
mis
copepod
h
respectively), while the mean
clearance on phytoplankton for Pseudocalanus females was lower (0.9 ±0.8 mis
h'). Calanus CV's grazed both large and small protists. They cleared
copepod1
ciliates and dinoflagellates >30 jim at higher rates than those <30 jim, and also at
higher rates on average than phytoplankton. Calanus CV's cleared large ciliates at
twice the rate they cleared similarly sized dinoflagellates (6.0 ±2.1 versus 2.9 ±1.6 mIs
copepod
1
h
1)
Large tintinnine ciliates were only prevalent on July 27th, when
Calanus CV's cleared them at a rate of 6.6 ±1.6 mls copepod1 h1. Tintinnids were not
included in the large ciliate category for determining copepod clearance rates on any of
the other dates. Calanus females cleared large ciliates at more than twice the rate they
cleared phytoplankton (6.9 ±1.9 versus 2.1 ±0.5 mis copepod1 h1), but did not clear
the large dinoflagellates or smaller ciliates that were also available on August 3rd.
Pseudocalanus females cleared large dinoflagellates on August 24th at the same rate
they cleared phytoplankton, but did not clear any protists on August 28th.
Table 3.3. Copepod clearance rates on phytopiankton (as Chi a), and phagotrophic ciliates and dinoflagellates during
the summer of 1992. All units are mis copepod' h1 (±SD), n 3.
(nd: no data, i.e. not enough protists in a given category to detect a clearance rate)
Date
22 July
Species
C.
marshallae
Chi a:
Ciliates:
> 20j.tm
<3Oim
> 3Ojim
Dinoflagellates:
>30
<30 im
2.4 (±0.9)
0.8 (±0.2)
5.3 (±2.7)
1.8 (± 1.0)
3.3 (±2.5)
2.3 (±1.0)
2.9 (±2.5)
6.6 (±1.6)
nd
nd
jim
(CV)
27 July
C.
marshallae
(CV)
03
Aug
C. marshallae
(CVI femaie)
2.1 (±0.5)
0.0 (±0.0)
6.9 (±1.9)
nd
0.0 (±0.0)
20
Aug
C. marshallae
3.8 (±0.9)
1.3 (± 1.2)
nd
nd
2.5 (±0.4)
sp.
1.6 (±0.5)
0.0 (±0.0)
0.0 (±0.0)
nd
1.4 (±1.2)
Pseudocalanus sp.
0.2 (±0.1)
0.0 (±0.0)
nd
nd
0.0 (±0.0)
(CV)
24
Aug
28 Aug
Pseudocalanus
(CVI female)
(CVI female)
100
90
80
70
U
a,
C
C
0
e
[1
LJ
50
U
0
:
40
0
30
20
10
0
22-Jul
27-Jul
3-Aug
20-Aug
24-Aug
28-Aug
Figure 3.3. Percent composition of copepod diet. Protist and phytoplankton carbon ingested
as percent of total carbon ingested.
-4
73
The contribution of protists to the total carbon ingested by copepods ranged
from 0 to 36% (Figure 3.3). Calanus ingested a higher percentage of protist carbon on
the dates when phytoplankton biomass was lowest. Protists ingested by Calanus
amounted to 0.8 5.0 ig carbon per day (Table 3.4), or approximately 1- 7% of their
body carbon. Calanus ingested phytoplankton at 8.9 to 94.2 tg carbon per day, a range
of 12 to 96% of their body carbon. Phagotrophic dinoflagellates ingested by
Pseudocalanus represented only 1% of its body carbon, whereas ingested
phytoplankton contributed 3.8 5.8 ..tg carbon per copepod per day, or 48 to 73% of its
body carbon.
Copepod Egg Production
Egg production for Calanus marshallae ranged from 0 to 31 eggs per copepod
(mean =10 ± 8) per 16 hour incubation on the first three dates, and on August 20th they
laid no eggs. When extrapolated to a daily mean of 16 eggs, this rate is lower than
other estimates made at similar temperatures and food concentrations (C. ,narshallae,
24, Peterson 1988; C. chiliensis, 20, Peterson et al. 1988; C. pacificus, 62, Runge
1985). Instances of cannibalism were <3% in these experiments. Egg production was
not well correlated with phytoplankton or protist biomass.
Table 3.4. Copepod ingestion of phytoplankton and phagotrophic protists in ig carbon per
copepod per day (±SD).
Date
Species
Phytoplankton
j.tgC copepod1 day1
Phagotrophic Protists
jigC copepod' day
22 July
C. marshallae
8.9
27 July
C. marshallae
11.6 (5.0)
1.9 (1.2)
03 Aug
C. marshallae
94.2 (22.4)
2.2 (0.6)
20 Aug
C. marshallae
59.3 (18.5)
0.8 (0.5)
24 Aug
Pseudocalanus sp.
3.8 (1.2)
0.1 (0.1)
28 Aug
Pseudocalanus sp.
5.8 (1.6)
0.0 (0.0)
(
3.3)
5.0 (4.3)
-.1
75
Discussion
Copepod Clearance Rates and Evidence of Prey Selection
Copepods did not ingest a detectable amount of small phytoplankton cells
(<20tm) during any of the experiments, even though on the July dates, and also August
24th, small phytoflagellates represented 20-50% of the total phytoplankton biomass.
Based on Berggreen et al.'s (1988) formula of 2-5% of a copepod's prosome length
corresponding to its range of optimum prey size, in these experiments Ca/anus
marshallae females would have optimally grazed particles of 50 to 130 pm, Ca/anus
CV's, 40 to 100 pm, and Pseudocalanus females, 20 to 60 pm. The lower limit of prey
size for Calanus is about 4 to 11 im (Fernandez 1979), and for Pseudocalanus, 2 to 4
tm (Poulet 1974). The size spectrum of available prey was optimal during all of the
experiments. Single diatom cells ranged from 30 to 90 tm, with the exception of
pennate forms which averaged 120 pm in size. The phagotrophic ciliates and
dinoflagellates ranged from 15 to 100
pm.
The large phytoplankton (>20 pm) and
protists (>30 jim) are considered equivalently sized prey categories, however, the
protist cells in the <30 jim category were always >10 jim and averaged 20 jim, so they
were much larger than the 2 to 5 jim phytoflagellates which composed most of the <20
jim category for phytoplankton.
The range of clearance rates on phytoplankton for Pseudocalanus in this study,
0.1 to 2.1
mis copepod1
h', was similar to the 0.8 to 3.5 mis copepod1
h1
measured
during the winter of 1991 (Fessenden and Cowles 1994). The fact that Pseudocalanus
76
cleared phytoplankton at rates an order of magnitude lower on August 28th than on
August 24th could be attributed not only to the dramatic increase in phytoplankton
biomass, but also to the abundance of diatom chains on August 28th. These chains,
ranging in length from 100 to 200 jim, were well above the optimum prey size for
Pseudocalanus. It is not clear why Pseudocalanus did not graze ciliates on August
24th, since ciliates were an order of magnitude more abundant than the dinoflagellates
which Pseudocalanus did graze. Almost 90% of the ciliates on that date were < 30 pm,
the low end of the optimum range in prey size for Pseudocalanus. In the winter of
1991, when phytoplankton biomass was low and the ciliate assemblage was dominated
by 30-60 jim cells, Pseudocalanus did clear ciliates at high rates (Fessenden and
Cowles 1994).
Calanus cleared phytoplankton at rates of 1.6 to 4.8 mis copepod1 h', a similar
range to that measured by Peterson (1980) for C. marshallae in Oregon coast waters
(1.7 to 3 mIs copepod'). Combined results from the four Calanus experiments show
that mean clearance rates were higher on large protists than on phytoplankton or small
protists (4.9 ±2.0 ml copepod'
h1
versus 2.7 ±0.8 and 1.7 ±0.9 respectively). As the
large protists and phytoplankton were of similar size, the higher clearance rates on the
protists vs. phytoplankton could be the result of a difference in nutritional content of the
prey, or a difference in copepod capture mechanisms. Jonsson and Tiselius (1990)
found that Acartia tonsa captures microflagellates and ciliates in two fundamentally
different ways, and suggested this difference may explain why clearance rates for
Acartia sp. are typically higher on ciliates than on microflagellates and small diatoms.
77
Although in our experiments the phytoplankton grazed were large diatoms, the mobility
of the protists, especially the ciliates, may have made them easier to detect.
To examine the evidence that Calanus may have been selecting protist over
phytoplankton prey, we used a ratio of clearance rates as an index of selection:
Ratio =
clearance rate on protists
clearance rate on phytoplankton of equivalent size
where a value >1 indicates selection of protist over phytoplankton prey. Clearance rates
on protists >30 tm were plotted against rates on phytoplankton for the four Calanus
experiments (Figure 3.4). The rates for clearance on ciliates were plotted separately
from those on dinoflagellates. The results indicate that Calanus was selectively grazing
ciliates over phytoplankton of equivalent size, but was not exhibiting this selectivity for
dinoflagellates.
The selection of ciliates over phytoplankton by Calanus CV's during the first
two experiments is not as surprising as the selection of ciliates by Calanus females on
August 3rd in the midst of an intense diatom bloom (43 jig chlorophyll per liter) when
ciliate abundance was at its lowest. In the diatom bloom conditions of August 20th (18
jig chlorophyll per liter) large ciliates were scarce, but Calanus did clear dinoflagellates
and smaller ciliates at rates somewhat lower than they cleared diatoms. It appears that
these grazers are behaviorally adapted to feed on protists even when their food supply is
seemingly swamped with algal biomass. Copepod predation on protists in the midst of
78
4-i
0
s-i
,-;'-J-o
)
4-i
0
)
0
6.0
1)
3.0
3.0
6.0
Clearance Rate On Diatoms
ml copepod' h'
Figure 3.4. Clearance rates of Calanus marshallae on >30 jim phagotrophic protists
plotted against clearance rates on equivalently sized diatoms. See Table 3 for
experimental dates. (I ciliates, 0 dinoflagellates)
79
diatom blooms was not observed in similar experiments conducted during the 1991
upwelling season with Calanus pacificus and Acartia Ion giremis (Fessenden and
Cowles 1994). It is likely that the copepods were actually feeding on protists during the
1991 diatom blooms, but the grazing signal may not have been large enough to be
detected due to the use of a lower concentration of copepod grazers in those
experiments.
Composition of Copepod Diet
The composition of the diet of Calanus typically reflected water column shifts
in phytoplankton biomass, as protist contribution was highest (14 and 36%) on those
dates when phytoplankton biomass was at its lowest. During diatom blooms,
phagotrophic protists represented 1 to 5% of the total microplankton biomass, and 0 to
2% of copepod diet (Table 3.5). The copepods in these experiments were ingesting 18
to 96% of their body carbon per day, albeit mostly in the form of phytoplankton. The
respiration requirement, determined according to Dagg et al. (1982), was 6% of body
carbon for Ca/anus, and 7% for Pseudocalanus sp. (Table 3.6). As phagotrophic
protists made only a small contribution to the copepods' daily carbon ration, the fact
that copepods were feeding on protists in the midst of diatom blooms, and possibly
selecting ciliates over diatoms, suggests that ingestion of protists is of some nutritional
benefit to the copepods or that protists are simply easier prey to detect.
80
Table 3.5. Phagotrophic protist (ciliate and dinoflagellate)
carbon as percentage of total carbon measured in the food
supply and in copepod diet. Mean percentage (±SD).
Date
Phagotrophic protist carbon as % of total carbon:
Food Supply
Copepod Diet
22 July
32
36 (±30)
27 July
13
14(±9)
03 Aug
2
2(±1)
2OAug
2
1(±1)
24 Aug
42
3 (±3)
28Aug
5
0(±O)
Egg production is considered a measure of recent food ingestion. By converting
the carbon produced in eggs to carbon ingested, using a carbon to volume ratio of 0.14
x 106 .ig C tm3 for the eggs and a gross efficiency of egg production of 0.33 (Kiørboe
et al. 1985), an independent measure of ingestion was obtained for Calanus on the first
three dates. Ingested carbon estimated from egg production was 38% of body carbon
for Calanus females. This estimate falls between those measured in the grazing
experiments, 18% body carbon per day for Calanus CV's on the July dates, and 96%
for Calanus females in the diatom bloom on August 3rd.
81
Table 3.6. Length, dry weight, and respiratory requirement (jigC per day) of Calanus
marshallae and Pseudocalanus sp. Dry weights account for 30% shrinkage due to
preservation in formalin. Carbon biomass is 40% dry weight. Respiratory requirement
calculated from R=0. 101 W°884 (Dagg et.al. 1982). (±SD)
Calanus marshallae
CV
Female
Pseudocalanus sp.
Female
1.7 (±0.1)'
Body Length (mm)
3.4 (±O.2)a
2.9 (±0.3y
Dry Weight (jig)
246 (±10)c
191
jig carbon
98
76
14
5.8
4.6
1.0
R (jigC
d')
(34)C
33
(+)d
a: n=10
b: n=20
c: n = 5 (15 copepods each)
d: n =2 (20 copepods each)
Impact of Copepod Predation on Protist Community
Due to the dominance of phytoplankton in the overall microplankton biomass
during the upwelling season, protists comprise a small portion of copepod diet.
However, since copepods clear protists at high rates, the impact of copepod predation
on the protist community is substantial. We have estimated copepod impact on the
protist standing stock in the Oregon upwelling system by applying the clearance rates
determined in these experiments to estimates of copepod abundance (Peterson and
Miller 1976, Fessenden unpublished). Our estimate of copepod predation on ciliates in
82
1991 was 25% of the standing stock per day (Fessenden and Cowles 1994). However
this was admittedly an underestimate as it included only three of the six major copepod
species. The six major copepod species found in Oregon coastal waters during the
summer months are, in order of decreasing average abundance, Pseudocalanus sp.,
Acartia clausii, Acartia ion giremis, Calanus marshailae, Centropages abdominalis,
and Oithona similis (Peterson and Miller 1976). Clearance rates from the 1991
upwelling season were applied for Centropages abdominalis and Acartia Ion giremis
(Fessenden and Cowles 1994). A. longiremis was assumed to clear protists at an
equivalent rate to that on phytoplankton, and this rate was also applied to Acartia clausi
and Oithona simiiis. The mean clearance rate for each species was multiplied by both
the average and maximum copepod abundance estimates (# of copepods per m3) to
obtain a rate equivalent to the % of water colunm cleared per day.
These calculations indicate that during upwefling season, copepods are capable
of clearing from 45 to 74% of the protist population from the surface waters per day.
Pseudocalanus sp. numerically dominates Oregon and Washington near shore waters in
the summer, especially during upwelling events, and also accounts for the largest
fraction of copepod grazing potential (Peterson and Miller 1976, Landry and Lorenzen
1989). Peterson and Miller (1976) documented one summer population of
Pseudocalanus to be an order of magnitude more abundant than in other years.
Applying this high abundance estimate we obtain a daily copepod predation potential of
120% of the protist standing stock. We estimated another 10 to 20% impact for
copepod nauplii by applying Dolans (1991) rate estimate for Acartia tonsa nauplii
83
clearing phagotrophic ciliates to abundance estimates of the feeding stages of copepod
nauplii in Oregon coastal waters (Peterson and Miller 1976, Landry and Lorenzen
1989). Including the grazing potential of naupiliar forms in this estimate suggests that
with average copepod abundance, >50% of the phagotrophic protist standing stock may
be consumed per day.
Both phagotrophic ciliates and dinoflagellates were being consumed during
diatom bloom conditions, albeit by different copepod species and at different times. If
Pseudocalanus, like Calanus CV's, cleared ciliates at higher rates than dinoflagellates,
there would be a substantial difference in the impact of copepods on the two protist
groups. We have measured high clearance rates for Pseudocalanus on ciliates in the
winter, but do not know if the rates are applicable during upwelling season (Fessenden
and Cowles 1994). The results from these experiments suggest ciliates experience a
greater loss to copepod predation than dinoflagellates. Ciliates typically grow twice as
fast as dinoflagellates (Banse 1982), and it is evident that copepods are taking
advantage of those higher growth rates.
84
Summary and Conclusions
In summary, Calanus marshallae cleared large ciliates at higher rates than
phytoplankton of equivalent size, suggesting that Calanus is selecting ciliates over
diatoms, even in the midst of diatom bloom conditions. Although Calanus did not
show a preference for large phagotrophic dinoflagellates over diatoms, CV's did
consume dinoflagellates during diatom blooms. Phagotrophic ciliates and
dinoflagellates typically comprised <3% of the carbon ingested by Calanus and
Pseudocalanus sp. during upwelling induced diatom blooms, but as much as 36%
during periods when phytoplankton biomass was <8 jig chlorophyll per liter.
Regardless of their small contribution to copepod diet, predation on protists by
copepods can have a large impact on the protist community, possibly more so on
ciliates than dinoflagellates. With the potential of >50%, and at times 100% of the
protist biomass being consumed on a daily basis by copepods, it is possible that much
of the protist production in this system is transferred to deeper waters or sediments via
copepod fecal pellets.
85
CHAPTER 4
LIPIDS IN CALANUS PACIFICUS FED A
PHAGOTROPHIC PROTIST VS. DIATOM DIET
Lynne M. Fessenden and Timothy J. Cowles
College of Oceanic and Atmospheric Sciences
Oregon State University
Corvallis, Oregon
86
Introduction
The enhancement of copepod growth and fecundity by the inclusion of
heterotrophic protists in the diet suggests protists may be a qualitatively important
source of essential nutrients (Klein Bretler 1980, Stoecker and Egloff 1987, Kleppel et
al. 1991, White and Roman 1992). Heterotrophic protists are considered a particularly
rich source of protein and amino acids, because their C:N ratio is generally lower than
that of phytoplankton (reviewed by Stoecker and Capuzzo 1990). Although the protein
content in copepod diet clearly affects the rate of egg production (Checkley 1980), a
recent study of the diet of Acartia spp. indicated the chemical constituents providing the
best correlation with egg production rates were in the lipid fraction (Jónasdóttir 1994).
Ahlgren et al. (1990) also suggested that lipid composition is more important than
protein or carbohydrates in determining the nutritional quality of algal prey for
cladoceran zooplankters. Ohman and Runge (1994) have recently compiled field
evidence that heterotrophic microplankton provide a prey resource sufficient for net
lipid synthesis, as well as for egg production, of Calanusfinmarchicus in the Gulf of St.
Lawrence, and Klein Bretler et al. (1990) noticed the size of the oil sacs of laboratory
reared Pseudocalanus elongatus increased when their algal diet was supplemented with
a heterotrophic dinoflagellate.
Lipids serve several important functions in marine invertebrates: they are
critically involved in the storage and mobilization of energy, as well as in egg
production and molting, and they function both as structural components of membranes
87
and as metabolic regulators (Phillips 1984). Lipids are grouped into neutral and polar
classes with regard to structure and function. Wax esters and triacylglycerides comprise
the bulk of the neutral lipids, or energy storage compounds. The major role of the polar
or phospholipids is in membrane structure and function. Calanoid copepods contain far
more reserve neutral lipid than any other group of zooplankton and are the main
producers of wax esters in marine food webs (Benson and Lee 1975). Seasonal changes
in the total lipid content of copepods are generally well correlated with changes in their
neutral lipids, as wax esters are utilized in times of starvation, and during maturation
and egg production (Lee et al. 1971). Approximately one-half of the wax ester reserve
in Calanus helgolandicus is utilized when stage V copepodites (CV) molt into adult
females and form ovaries (Gatten et al. 1980). For copepods in a reduced metabolic
state, wax ester catabolism may extend several weeks to months (Lee 1974, Ohman
1988). Triacyiglycerides are considered a short term energy source as they are
preferentially mobilized over wax esters due to the relative activity of triglyceride lipase
(Benson and Lee 1975, Hakanson 1984, Sargent and Henderson 1986). Copepods
which experience short periods of enhanced food supplies tend to store predominantly
wax esters, where as triacylglycerides compose the largest percent of neutral lipid in
copepods which feed more or less continuously (Lee et al. 1971, Lee and Hirota 1973,
Sargent and Henderson 1986). Thus a high percentage of wax ester in copepod lipid
composition is associated with herbivory, and a high percentage of triacyiglyceride is
associated with omnivory (e.g. Lee and Hirota 1973, Graeve et al. 1994).
88
Fatty acids are the aliphatic hydrocarbon components of lipids. Marine
crustaceans characteristically have high levels of two long-chain polyunsaturated fatty
acids (PUFA): 20:5, eicopentaenoic acid, and 22:6, docosahexaenoic acid (reviewed by
Phillips 1984) which are effective in promoting growth and normal metabolism
(Kanazawa et al. 1979). These long-chain PUFA can be synthesized from a shorterchained precursor, linolenic acid (18: 3w3). Although most metazoans can synthesize
unbranched, even-length fatty acid chains from acetate and have some ability to
desaturate and elongate fatty acids, they cannot desaturate beyond the 9th carbon
toward the methyl end of the chain (e.g. o3 series), and therefore PUFA of the linolenic
series are referred to as essential fatty acids and are required in the diet. Dietary
deficiencies in PUFA can reduce growth in marine invertebrates (Langdon and
Waldock 1981). Both Kanazawa et al. (1979) and Read (1981) found that marine
prawns showed a limited capacity to chain elongate and desaturate linolenic acid to
20:5 and 22:6. Although supplementing the diet of the prawns with linolenic acid did
result in a slight weight gain, supplementing the diet with PUFA 20:5 and 22:6
significantly enhanced both growth and survival. It is not known whether copepods can
convert linolenic acid, 18:3, to 20:5 and 22:6 at rates sufficient to meet their growth
requirements, or whether they require substantial amounts of pre-formed 20:5 and 22:6
in their diet (Sargent and Henderson 1986).
Linolenic acid is biosynthesized
de novo by
photosynthetic organisms which
contain high concentrations of lipids in their chioroplast structures (Kates and Volcani
1966, Sargent and Whittle 1981). Fatty acids in the linolenic series, while a major
89
component of most algae and higher plants, are a minor component of diatoms (Kates
and Volcani 1966). Cryptomonads (Ahlgren et al. 1990, Jónasdóttir 1994) and
Phaeocystsis sp. (Sargent et al. 1985, Al-Hasan et al. 1990) are reportedly rich in C-18
PUFA. Diatoms typically consist of 50% saturated and monosaturated short chains
(14:0 and 16:1), but also 10-40% of the long-chain PUFA 20:5.
Claustre et al. (1988) found that the fatty acid composition of tintinnine ciliates
in a coastal microplankton assemblage had a much higher unsaturation index than that
of diatoms. The unsaturation index of dinoflagellates in the assemblage fell in between
diatoms and ciliates. Very little is known of the lipid composition of other marine
pelagic ciliates. A benthic, bacterivorous scuticociliate (Pleuronema sp.) was found to
be low in PUFA and high in the uneven length and branched saturated fatty acids
characteristic of bacteria (Ederington et al. 1995). Fresh water ciliates of the genus
Tetrahymena have a dietary sterol requirement but can synthesize all of their own
PUFA (Koll and Erwin 1990). The most characteristic PUFA of both autotrophic and
heterotrophic marine dinoflagellates is 22:6, docosahexaenoic acid (Harrington et al.
1970). The heterotrophic dinoflagellate Noctiluca has been characterized as a rich
source of both 20:5 and 22:6 PUFA (Dikarev et al. 1982).
In a study of the biotransformation and assimilation of dietary lipids by Calanus
helgolandicus, Harvey et al. (1987) found that copepods preferentially removed PUFA
20:5 and 22:6 from ingested algal fatty acids. Calanus helgolandicus raised on the
dinoflagellate Gymnodinium splendens had more reserve lipid than when fed either of
two species of diatoms (Lee et al. 1971). The components of an algal diet best
correlated with egg production in Acartia hudsonica were the 20:5 and 22:6 PUFA
(Jónasdóttir 1994), and in both A. hudsonica and A. tonsa egg production was
negatively correlated with the 20:22 fatty acid ratio indicating the importance of the
22:6 PUFA in the biosynthesis of eggs (Støttrup and Jensen 1990, Jónasdóttir 1994).
The neutral lipids invariably contain PUFA, but at a much lower concentration than the
phospholipid (Sargent and Whittle 1981). If heterotrophic protists are indeed
enhancing copepod growth and fecundity due to their PUFA content, one might expect
to find an increase in phospholipid relative to neutral lipids in copepods fed
heterotrophic protists, especially during periods of rapid growth and gonadal maturation
when copepods use both dietary and storage lipids as fuel.
I hypothesized that feeding copepods heterotrophic protists versus diatoms
would possibly increase the amount of wax ester or triacylglyceride per copepod, or at
least shift the ratio of copepod neutral to phospholipid. During an active phase of
copepod growth, enhancement of membrane (phospholipid) formation would cause the
ratio to decrease. However, if the copepods were well enough fed during such a phase,
they may actually increase storage lipid too, balancing out the newly formed
phospholipid and causing the ratio to remain the same or to possibly increase. The
experiment was conducted in March with Calanuspacificus CV's, which were actively
feeding and molting into adults. Total lipid content and lipid class composition were
monitored for copepods fed diets of a diatom, a mixture of diatoms and
phytoflagellates, and mixtures of phytoflagellates with both a heterotrophic
dinoflagellate and a ciliate.
91
Methods
Experimental Design and Cultures
Copepods were collected five miles off Newport, Oregon in late March 1995.
Sea surface temperature was 11.5°C. A 335 im mesh net with a 4 liter cod end bucket
was towed obliquely from 50 m, and the contents were diluted with surface water and
stored in insulated coolers for transport to the lab. Copepods were kept on ice while
being examined under a dissecting scope at 60X. Several sets of five Calanus pacificus
CV's were placed into 500 ml polycarbonate bottles of filtered seawater (0.2 jim).
Three sets of 10 CV's were dried at 60°C for two days and weighed. Another three sets
were immersed in 2:1 chloroform:methanol (vol/vol) for two days, dried and weighed
to determine lipid-free dry mass. CV's were also frozen in liquid nitrogen for lipid
analysis.
Copepod incubation bottles were rotated on their long axes on a plankton wheel
at 0.5 rpm in a 10°C cold room on a 14 hour light, 10 hour dark cycle. All copepods
were fed a mixture of the diatom, Thalassiosira weisflogii, and the phytoflagellate,
Chroomonas sauna for two days. Copepods were then placed into fresh filtered sea
water and the following prey treatments were added: Diatom, Thalassiosira weissflogii
only; Phytoplankton Mixture, Thalassiosira weissflogii plus Chroomonas sauna and
the autotrophic dinoflagellates, Prorocentrum minimum and Gymnodinium simplex;
Ciliate, Strombidinopsis acuminatum plus Gymnodinium, Prorocentrum, and
Chroomonas; Dinoflagellate, Oxyrrhis marina plus Chroomonas and Gymnodinium
(Table 4.1). Because it is difficult to get cultured choreotrichous ciliates, such as S.
acuminatum, to survive at low phytoplankton concentrations, and also in the interest of
keeping the total carbon available in all prey treatments between 200-300 pig/liter,
phytoplankton were included in both heterotrophic protist treatments (Ciliate and
Dinoflagellate), and the Phytoplankton Mix treatment was added as a control. Three
replicate bottles for each prey treatment were maintained for four days, with sea water
and prey exchanged daily. Aliquots of each prey treatment were preserved daily
according to Sherr and Sherr (1993), stained with DAPI, filtered onto black nucleopore
filters, and mounted on slides and frozen for later enumeration of cell concentrations by
epifluorescence microscopy. A separate aliquot of the Ciliate treatment was preserved
with 10% acid Lugol's solution for settling and enumeration of S. acuminatum with an
inverted microscope. The average cell density and estimated carbon concentration for
each prey treatment are reported in Table 4.1. Per cell carbon estimates and references
are given in Table 4.2.
The diatom and phytoflagellates were cultured on IMR/2 medium (Eppley et al.
1967) at 16°C under continuous light. The heterotrophic ciliate and dinoflagellate were
kept in double screened flasks of protist media (Gifford 1985) at 16°C, on a 14/10 hour
light/dark cycle, and fed the above mentioned phytoplankton.
There was no copepod mortality during the experiment, however, all of the
Cv's molted to CVI females or males. Treatment replicates were then sorted according
to sex. Copepods were removed from the bottles, rinsed in filtered sea water, blotted
Table 4.1. Mean cell density and estimated carbon concentration for each prey treatment.
Prey Treatment
cells / ml
Diatom
Thalassiosira weissflogii
7000
245
Phytoplankton Mixture
Thalassiosira weissflogii
Prorocentrum minimum
Gymnodinium simplex
Chroomonas salina
ug C / liter*
310
3300
600
670
850
Ciliate
300
Strombidium acumentum
4
plus:
Prorocentrum minimum
Gymnodinium simplex
Chroomonas sauna
Dinoflagellate
Oxyrrhis marina
500
2000
1500
200
300
plus:
Chroomonas sauna
Gymnodinium simplex
* See Table 4.2 for per cell carbon estimates
1500
1500
Table 4.2 Per cell carbon estimates for autotrophic and heterotrophic microplankton prey. Size measured
from actual cultures. References for carbon estimates include shrinkage factor for fixation in Lugols.
Prey
Autotrophs:
Diatom
Thalassiosira weisfioggi
Cryptomonad
Chroomonas sauna
Dinoflagellates
Pro rocentrum minimum
Gymnodinium simplex
Size
(jim)
Volume
(jim3)
Carbon per Cell
Reference
(ng)
4x16
340
0.035
Montagnes et al. 1995
6x11
275
0.029
Montagnes et al. 1995
0.240
Montagnes et al. 1995
8x10
2350
356
14x25
2566
0.360
Lessard 1991
50x120
78540
15
Putt
15
0.03 8
Heterotrophs:
Dinoflagellate
Oxyrrhismarina
Ciliate
Strombidinopsis acuminatum
& Stoecker 1989
95
briefly on a kimwipe, and placed into cryo vials (3-4 per vial) and immersed in liquid
nitrogen. Lipid analyses were run within two weeks of freezing. Several CY's were
kept in a 4 liter beaker in the same environmental chamber as the plankton wheel
during the experiment. These copepods were fed a mixture of all prey types, and were
available for weight measurements at the end of the experiment.
Lipid Class Analysis
Copepod lipids were extracted following the micromethod of Gardner et al.
(1985), modified somewhat by Miller and Morgan (1995). Lipids from 2 to 4 whole
animals were extracted in 50 jil of 2:1 chloroform:methanol (vol/vol) while
homogenizing in a glass tissue grinder suspended in an ice bath. The extract was
partitioned against 0.9% NaCl solution to remove protein. Extracted lipids were
analyzed by TLC-FID (thin-layer chromatography-flame ionization detection) with an
Iatroscan Th-10 with SIll chromarods. The silica rods were spotted with 1-2 il of
extracted sample, dried for 5 minutes at 80-90°C, and rehumidified for 5 minutes in a
closed chamber with a 60% saturated CaC1 solution. Lipid classes were separated
according to Ohman's (1988) double development technique: 76:4 (vol:vol, 8Oml)
hexane:ethyl ether for 20 minutes, dry for 5 minutes, then 20 minutes in 66:14:0.1
(vol:vol:vol, 80m1) hexane:ethyl ether:formic acid. Rods were then scanned by the
Tatroscan FID. Three lipid classes were measured: wax ester (WE), triglyceride (TAG),
and phospholipid (PL). There were no detectable sterols in these copepods. The
compounds used for calibration were palmitic acid stearyl ester (WE), tripalmitin
(TAG), and L- phosphatidylcholine dipalmitoyl (PL). Two to three standard curves
covering a range of concentrations were determined for each compound. The response
of the FID is non-linear at very low concentrations (<1 jig), and as the TAG
concentrations were highly variable and often in the low range, two different linear
equations were used.
The extract solvent contained a known amount of 1-nonadecanol as an internal
standard, and solvent losses during sample processing were accounted for by
monitoring recovery of that initial mass. Each sample was spotted, developed, and run
through the FID twice, and the resulting means applied to the treatment average. The
coefficient of variation between replicate latroscan runs averaged 7% (n= 60) WE 4%,
TAG 9%, and PL 7%. The C.V. between replicate samples within treatments was 16%
for WE, 75% for TAG, and 23% for PL.
Results
A diet of heterotrophic protists vs. diatoms and phytoflagellates did not change
the lipid composition of Calanus pacficus during this experiment. The lipid contents
of males and females fed each treatment are shown in Figure 4.1, along with the
composition of the initial CV's. There was no significant difference in the lipid levels
of the males between treatments. (Note: there are no error bars for the male
Dinoflagellate prey treatment because there was one sample). For females, the only
between treatment difference was the lack of wax ester in those fed the Phytoplankton
97
Mix or Ciliate. Males and females had a similar amount of total lipid, excepting the
females with no WE, although the lipid class composition differed between sexes
(Table 4.3). In all treatments, %WE increased and %PL decreased in the males, while
the exact opposite occurred in the females. By examining the changes in lipid class
content on a ig lipid per tg copepod dry weight basis (Figure 4.2), it is evident that the
real change between CV and adult was the decrease in WE content of the females and
the increase in WE content of the males. PL stayed within the same range, although
that range was narrower for males, and there was no change in the TAG content
between the CV's and the adults in any of the treatments. The ratio of neutral lipids to
phospholipids (WE+TG / PL) was thus >1 for males, and <1 for females in all
treatments (Figure 4.3).
All Calanus pacificus Cv's increased in length and dry weight during the
experiment as they molted to adult males and females (Table 4.4). Females were longer
on average than males, although they weighed less. The lipid content as % of dry
weight was slightly higher for males (16% vs. 13% for females) and actually higher in
the Cv's (18%) than the adults. The lipid content of the initial Cv's determined by
measuring lipid-free dry weight was slightly higher (23%) than the value obtained by
the extraction and latroscan technique.
98
16
14
12
10
OWE
STAG
0
0
PL
a,
6
4
2
C
Initial (CS)
Diatom
Phyto mix
Ciliate
Dinoflagellate
16
14
FEMALES
12
10
0
OWE
0
I
a,
TAG
UPL
6
T
2
0
Initial (CS)
Diatom
Phylo mix
Ciliate
Dinoulagellate
Figure 4.1. The wax ester (WE), triacylglyceride (TAG), and phospholipid (PL) content
of Calanus pacificus adult males and females for each prey treatment. The lipid class
content of the initial CV's is included in both plots for comparison.
Table 4.3. Total lipid and percent composition of lipid classes: Wax ester (WE), Triacyiglyceride (TAG),
and Phospholipid (PL) for Calanus pacificus adult males and females fed each prey treatment.
(±SD, no SD for male dinoflagellate treatment as there was one sample). Values for initial CV's:
l4(±3.5) .ig Total, 30(±2.5)% WE, 15(±3.5)% TAG, 55(±5.3)% PL.
Diatom
Phytoplankton Mix
Ciliate
Total Lipid
(.tg/copepod)
22 (±1.1)
20 (±15)
24 (±1.2)
22
% WE
48 (±7.4)
48 (±2.7)
50 (±13.1)
38
% TAG
10 (±2.6)
13 (±18.4)
12 (±9.9)
24
% PL
42 (±4.7)
39 (±16.4)
38 (±2.8)
36
Total Lipid
21 (±1.2)
13 (±1.8)
10 (±5.4)
19 (±2.0)
% WE
23 (±3.4)
0 (±0)
0 (±0)
18 (±5.1)
% TAG
Il (±3.8)
17 (±24.4)
8 (±11)
18 (±6.3)
% PL
66 (±1.4)
83 (±24.7)
92 (±12.0)
65 (±5.6)
Dinoflagellate
Males:
Females:
(g/copepod)
100
0 14
0.12
0.1
C)
w
0
0.08
0
0
ITAG
0
PL
C)
w
a-
0.06
aC)
0.04
0.02
Initial (C5)
Female
Male
Figure 4.2. Mean lipid class composition on a per weight basis for Calanus pacificus
CV's, females, and males.
101
Males
14
U
12
10
0
0.
w
0.
0
U
+
w6
Females
4
2
0
0
2
4
6
6
10
12
14
16
PL (ug per copepod)
Figure 4.3. Ratio of neutral lipid content (WE+TAG) vs. phospholipid content (PL) for
Ca/anus pacificus adult males and females.
Table 4.4. Length, weight, and % lipid (±SD) of Calanuspacificus, initial CV's and adult males and females.
CV
CVI Male
CVI Female
Overall Length (mm)
2.8 (±O.10)a
2.9 (+0.13)'
3.2 (+013)b
Prosome Length (mm)
1.9(±O.l0)a
2.1 (+016)b
2.3 (i011)b
Dry Weight (tg)
79 (±18)c
134 (+29)d
122
Lipid Content (% dry mass)
18% (±4.0)
16% (±1.3)
13% (±4.2)
(35)d
a: n=20
b: n = 10
C:
fl = 3 (10 copepods each)
d: n = 3 (6 copeods each)
C
NJ
103
Discussion
The lipid levels of Calanus pacficus determined in this study are well within
the range found by both Hakanson (1984, 1987) and Ohman (1988) for C. pacificus
caught in March and April off southern California. The range in total lipid for CV's of
11-18 jig is in the middle of Hakanson's range (7-24 jig) and close to Ohman's (13-21
jig) for CV's of similar size and dry weight. The total lipid per female in this study
ranged from 6-21 jig per copepod, while Ohman (1988) reported a range of 16-20 jig
for females that were somewhat heavier. Lipid as percent dry weight was 15.8% for
CV's and 8.7% for females in Ohman's study, and in Hakanson's combined work lipid
ranged from 10-31% of the dry weight of CV's. Lipid class composition was very
similar in Ohman's study, as females had less WE and more PL than the CV's.
Hakanson's CV's were also consistently low in TAG (4-6%), but more variable in WE
(29-65%) and PL (3 1-67%).
The increase in PL and decrease or lack of WE in the females is consistent with
the observation that they were maturing and developing ovaries during the experiment.
A smaller amount of storage lipid is also consistent with the lower average weight of
females than males, although they had longer prosome lengths (Table 4.4). Female
copepods are known to convert wax ester in forming ovaries and eggs (Lee et al. 1971,
Kattner and Krause 1987). Copepod eggs have been reported to be composed primarily
of triacylglyceride (C. helgolandicus: Gatten et al. 1980, Euchaeta japonica: Lee et al.
1974) or phospholipid (Neocalanus tonsus: Ohman et al. 1989, C. finmarchicus:
104
Ohman and Runge 1994), with very little or no wax ester. As mentioned earlier, Gatten
et al. (1980) noted that half of the WE reserves in C. helgolandicus were utilized during
the final molt and maturation from CV to female. The energetic cost of forming the
male reproductive system must be less, as negligible lipid was lost from CV's molting
into adult males. The ratio of storage to membrane lipids (>1 for males, <1 for females)
determined in this study also illustrates the difference between sexes in lipid
composition during maturation.
To date, no information on lipids in Calanus pacficus males has been reported.
The results of this study are consistent with those of both Kattner and Krause (1987)
and Gatten et al. (1979, 1980) who found males of newly matured populations of C.
finmarchicus and C. helgolandicus Claus had more lipid and a higher percentage of
wax esters than Cv's or females. They also observed a decrease in WE with increasing
age of the males, due to reduced feeding and increased physical activity during the
mating season.
Diatom, phytoflagellate, and heterotrophic protist prey appeared to be of equal
value with regard to the lipid nutrition of Calanus pacficus males. It is possible that
four days may not have been enough time for differences in lipid composition based on
dietary input to manifest themselves. However, the length of this experiment was
chosen based on time frames reported in the literature. Ohman and Runge (1994)
documented changes in female Calanusfinmarchicus TAG and WE content when field
collected animals were fed diatoms for 3.5 days. Hakanson (1984) found both TAG
and WE in Calanus pacificus decreased during three days of starvation. Differences in
105
egg production of Acartia species fed different diets were seen within 24 hours
(Stoecker and Egloff 1987, Jónasdóttir 1994), and Ederington et al. (1995) documented
differences in lipid classes within 4 days for A. tonsa fed a bacterivorous ciliate and a
diatom.
Four days proved to be enough time to see substantial changes in lipid
composition between the initial CV's and the resulting adults (increased WE for males
and increased PL for females), and, therefore, changes in adult lipid composition due to
dietary input would have been seen. It is unlikely that the molting process hampered
copepod feeding for much of the experiment. The actual time required to slip out of
their exoskeleton is just a matter of minutes, and none of the copepods in this
experiment were observed to be "stuck" shedding their exoskeletons. Fecal pellets
were abundant in all bottles each day, indicating that the copepods were actively
feeding.
The lack of WE in the females in the Phytoplankton Mix and Ciliate treatments
is puzzling. Some proportion of the females in the experiment were likely to have
utilized their entire WE store during maturation, but the fact that they were not
randomly distributed throughout all the treatments and were found only in the
Phytoplankton Mix and Ciliate treatments suggests a dietary effect. However, a better
balancing of the treatments may have eliminated this "effect". Both the Phytoplankton
Mix and the Ciliate treatments actually had a higher amount of total carbon than the
Diatom and Dinoflagellate treatments (Table 4.1). The carbon contribution of the
ciliate, Strombidinopsis acuminatum, in the Ciliate treatment was only 20% of the total,
106
whereas the carbon contribution of Oxyrrhis marina was 50% of the total carbon in the
Dinoflagellate treatment. This could aid in the explanation of the difference between
the WE content of the females fed ciliates and dinoflagellates, but it does not explain
why the females fed the mixed phytoplankton also had lower WE. To be completely
unbiased, the Dinoflagellate treatment should have included Prorocentrum too. CV's
fed the ciliate and mixed phytoplankton apparently catabolized their entire WE reserve
while maturing to adult females, whereas CV' s fed diatom and dinoflagellate prey lost
no WE during maturation. The mean PL content was also lower for females in the
Ciliate and Phytoplankton Mix treatments. The variation around the PL mean was
extremely large, indicating that perhaps a few females weren't feeding and therefore
biased the samples (each replicate for lipid analysis contained three females).
The lipid composition of heterotrophic protists and their subsequent
contribution to copepod lipid nutrition needs to be investigated beyond the level of lipid
class. Heterotrophic protists may vary in lipid content to a large degree based on their
own dietary lipid input. The results of this study suggest the biochemistry surrounding
the lipid physiology of female maturation needs to be investigated at the level of
specific fatty acids. Without knowing the exact fatty acid composition of the prey,
conclusions regarding changes in the lipid class composition during maturation of the
females remain speculative. If this study had been conducted with Cv's in preparation
for over-wintering, i.e. increasing their neutral lipid reserves, it is quite possible that
dietary input would have affected larger changes at the level of lipid class.
107
Addendum
A very recent finding by Miller and Morgan (unpublished) is that the palmitic
acid stearyl ester used as a WE standard in the Jatroscan method gives a very different
response from that of purified WE from Calanusfinmarchicus. The response is
curvilinear, but in general the standard extracted from the copepods gives a value 1.5 to
2 times higher than the palmitic stearyl ester. Although we can not assume the
difference would be the same for C. pacficus, if the WE values in this study were
doubled, the total lipid content would increase, as would the ratio of WE+TAG / PL,
but the conclusions reached would not change.
108
CHAPTER 5
CONCLUSIONS
Oregon's coastal upwelling system is a highly variable environment with regard
to both phytoplankton and phagotrophic protist biomass. The phytoplankton always
dominate, but both fluctuate rapidly, on a scale of days, and thus there are times during
the upwelling season when protists can contribute significantly to the assemblage of
available copepod prey.
In the 1991 experiments, copepods cleared ciliates at higher rates than they
cleared phytoplankton, except during upwelling-induced diatom blooms when they
ingested only phytoplankton. Ciliates contributed approximately 16-100% of the
carbon ingested by copepods during the winter months and between diatom blooms
during upwelling season. Ingestion of ciliates alone provided enough carbon to meet
the basic respiration requirements of Calanus pacficus and Pseudocalanus sp. during
winter months.
During the 1992 upwelling season copepod clearance rates on both ciliates and
dinoflagellates were measured during diatom bloom conditions. Ca/anus marshallae
cleared large ciliates at higher rates than equivalently sized diatoms in the midst of
bloom conditions, suggesting that C. marshallae was selecting ciliates over diatoms.
Although C. marshallae did not show the same preference for large dinoflagellates
over diatoms, they did consume dinoflagellates during bloom conditions, at rates
109
equivalent to those on phytoplankton. Phagotrophic ciliates and dinoflagellates
comprised <3% of the carbon ingested by C. marshallae and Pseudocalanus sp. during
diatom blooms, but as much as 36% during periods when phytoplankton biomass was
low (<8tg chlorophyll per liter). Carbon ingestion for C. marshallae estimated
independently from egg production rates was the equivalent of 36% of copepod body
carbon per day, which is within the range of total ingested carbon determined in the
grazing experiments (18 to 96%).
Due to their high clearance rates on protists, copepod grazers are likely to have a
large impact on the protist biomass. When the clearance rates determined in this study
are applied to estimates of copepod abundance in Oregon coast waters during upwelling
season, the estimated impact is 50 to 120% of the phagotrophic protist biomass cleared
per day by copepods. Copepod predation on ciliates may be greater than that on
dinoflagellates if the numerically dominant copepod, Pseudocalanus sp., clears ciliates
at higher rates than dinoflagellates, as Calanus marshallae did in this study. This may
indeed be the case as is evident from the high clearance rates on ciliates measured for
Pseudocalanus in the winter of 1991. Because the growth rate of pelagic ciliates is
approximately twice that of dinoflagellates, the higher grazing rate of copepods on
ciliates may be keeping the ciliate biomass in check.
Copepod grazing pressure on phytoplankton production was determined by
applying the estimated daily clearance rate on chlorophyll (per m3) to Dickson's (1994)
range of mean primary production per m3 at 8m, assuming a carbon:chlorophyll ratio of
50. The copepods in this system are estimated to graze 20 to 40% of the primary
110
production per day. Phagotrophic protists consume an equivalent, if not greater,
amount of phytoplankton during the upwelling season (Neuer and Cowles 1994). If
phagotrophic protists consume 50% of the primary production during a diatom bloom
event (Neuer and Cowles 1994), at a 40% trophic efficiency, and if copepods then
consume 50 to 100% of these herbivorous protists, 10 to 20% of the initial primary
production is reaching copepods via the path of phagotrophic protists.
Although diatoms make up the bulk of copepod diet during upwelling season,
phagotrophic protists may be a qualitatively important source of essential nutrients. I
examined the possibility that the lipid composition of copepods is enhanced by
inclusion of protists in the diet, and found that dietary inclusion of phagotrophic ciliates
and dinoflagellates did not affect the lipid content or the lipid class composition of
Calanus pacificus males. A similar level of lipid was supported in females fed diatom
and dinoflagellate diets, whereas females fed ciliates catabolized their entire wax ester
store during the experiment. The ratio of neutral or storage lipid to membrane or
phospholipid (WE+TAG I PL) was >1 for males and <1 for females for all prey
treatments, illustrating the difference in the energetic cost of the maturation process
between males and females. The results of this preliminary lipid study suggest the
question of copepod lipid enhancement via dietary inclusion of protists should be
addressed at the level of specific fatty acids rather than lipid class, and that more
information is needed on the lipid composition of marine pelagic protists.
In summary, although phagotrophic protists make up a relatively small
percentage of copepod diet during the upwelling season in Oregon coast waters, they
111
are consumed by copepods even in the midst of diatom blooms. It is evident that some
copepods are selecting ciliates over diatoms of equivalent size, although it is not clear if
this selection is due to ciliates being a source of essential nutrients, such as lipids, or to
some other factor, such as being easier to detect. Regardless, copepod predation is
estimated to have a large impact on the standing stock of phagotrophic protists in
Oregon's upwelling system, and thus the protist-copepod link is substantial enough to
influence the flux of carbon from the surface waters.
112
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129
APPENDICES
130
APPENDIX A
DISCUSSION OF INGESTION RATE CALCULATIONS
The index of feeding used to calculate copepod ingestion rates is the "volume
swept clear" or "clearance rate", which is defined as the volume of ambient medium
from which cells are completely removed by copepods to achieve the measured
ingestion rate (Frost 1972). Harvey (1937) introduced the model describing the
changes in prey cell concentrations (or chlorophyll concentrations) in the presence of
copepod grazers. However, Frost's (1972) landmark paper provides the version of
those equations most commonly used for calculation of copepod feeding rates today:
The prey growth coefficient, k, is calculated by
C2 = C1 e
where
C1
and
C2 are
k(t2-rl)
prey concentrations in the control treatments at the beginning
(t1)
and end (t2) of the experiment.
For each replicate of the treatment with grazers, the grazing coefficient, g, is
calculated by
C2
where
ci *
Ci
(k)(t2tI)
and C2 are the prey concentrations in the experimental treatment at the
beginning and end of the experiment.
The mean prey concentration, {C], is calculated by (Frost's original)
[C] = Ci
((k)(t2tl)
1)! (t2 t1)(k -g)
131
APPENDIX A (Continued)
The clearance rate,
F,
is calculated by
F=VgIN
where V is the volume of the incubation chamber and N is the number of copepods in
the chamber.
Ingestion rate, I, is calculated by
1= F[C]
The units of k and g are inverse time, [C] may be expressed as numbers or
biomass (carbon), F is volume cleared per copepod per unit time, and I is numbers or
biomass consumed per copepod per unit time.
A central assumption in the use of this model is that the grazing coefficient, g
(and F since it is a function of g), is constant.
Rigler (1961) pointed out that this
central assumption will be violated above a certain critical prey concentration at which
copepod ingestion remains constant, requiring the clearance rate to decrease. He first
suggested the use of the arithmatic mean of C1 and C2 (1971), and Frost (1972)
expanded the method to account for the growth of prey cells during the experiment,
arriving at the presently used [C].
In 1986, Mann, Huntley, and Frost re-evaluated the statistical procedures used
to determine the relationship between copepod feeding rates and prey concentration,
and found the use of [C], or the "mean concentration", in the ingestion equation is
statistically erroneous. The main problem being that [C] is not an independent variable,
132
APPENDIX A (continued)
but depends upon g, the variable being measured. The use of [C] produces an artificial
increase in the degrees of freedom that may result in the acceptance of non-significant
regression lines, and also negates the value of replication. They recommend the use of
C1, the initial prey concentration.
I have used the initial prey concentration in calculating copepod ingestion rates,
recognizing that the resulting rates may be maximal, or even overestimates. Protist
biomass was always less than Frost's (1972) critical concentration, and also in those
experiments in which the initial phytoplankton concentration was <300 .tg carbon per
liter it is likely that the grazing coefficient during the experiment did not change.
Typically, in experiments in which the initial prey concentration is greater than the
critical concentration, a decrease in cell concentration during the experiment will
produce an increase in the grazing coefficient. Frost (1972) illustrates that the change
in the grazing coefficient is most dramatic directly around the critical prey
concentration and then it levels off. Because of this pattern, I would argue that in my
experiments with diatom bloom conditions the situation was in such extreme, i.e. C
(initial) >> C (critical), that even after copepods grazed for the duration of the
experiment that C (final) was still >> than C (critical), and therefore any change in the
grazing coefficient would be very small.
133
APPENDIX B
ABUNDANCE AND COMPOSITION OF PHAGOTROPHIC CILIATE AND
DINOFLAGELLATE ASSEMBLAGES IN OREGON COASTAL WATERS
Ciliates:
Choreotrichs
Laboea
Tontonia
Strombidium
Strombilidium
Strombidinopsis
Lee gaardiella
Tintinnids
Parafavella
Favella
Tiarina
Urotricha
Dinoflagellates:
Athecate
Gymnodinium
Gyrodinium
Thecate
Protoperidinium
APPENDIX B (Continued)
1992 Taxonomic Division of Protists. All units cells per liter. (Note: dinoflagellate numbers refer to
heterotrophic forms only)
Date
Ciliates
Aloricate
Tintinnids
22 July
18,282
462
27 July
2,926
1,540
04 Aug
2,618
20 Aug
Autotrophic
Dinoflagellates
Thecate
Athecate
9,219
5,873
484
462
528
264
220
1,408
1,320
4,136
88
4,334
506
4,180
24 Aug
22,682
0
9,922
1,650
1,738
28 Aug
7,370
66
8,426
2,904
4,378
APPENDIX B (COfltifl)
80
10
61
C
C
at8
t.)
22-JUt
Ciliate and jflOflagete Biofflass in 1992
tJl
APPENDIX B (Continued)
25000
20000
15000
o Ciliat
Dino
I
10000
5000
22-Jul
27-Jul
3-Aug
20-Aug
24-Aug
Ciliate and Dinoflagellate Abundance in 1992
28-Aug
U)